Synthesis, Activity, and Docking Study of Novel Phenylthiazole-Carboxamido Acid Derivatives as FFA2 Agonists

Article abstract of DOI:10.1111/cbdd.12729

Free fatty acid receptor 2 (FFA2), also known as GPR43, is activated by short-chain fatty acids (SCFAs) that are mainly produced by the gut microbiota through the fermentation of undigested carbohydrates and dietary fibers. FFA2 currently appears to be a

Full text of DOI:10.1111/cbdd.12729

Chem Biol Drug Des 2016; 88: 26–37
Research Article
Synthesis, Activity, and Docking Study of Novel
Phenylthiazole-Carboxamido Acid Derivatives as FFA2
Agonists
Liang Ma1,2,#,*, Taijin Wang2,#, Min Shi1, Ping Fu1,
is highly expressed in adipose tissue and gastrointestinal
tract and mediates SCFA-promoted GLP-1 release. FFA2
Heying Pei2 and Haoyu Ye2
agonists promote GLP-1 release, and they furthermore
increase glucose uptake in adipocytes, thus providing sup-
1Division of Nephrology, Kidney Research Institute, West
China Hospital, West China Medical School of Sichuan
University, Chengdu 610041, China
port for the notion that FFA2 agonists could be of interest
for the treatment of obesity and diabetes. Additionally,
FFA2 is also expressed in immune cells especially in neu-
trophils and mediates the chemotactic effects of SCFAs
on neutrophils. Therefore, FFA2 currently appears to be a
potential target in the management of several pathological
conditions such as obesity, diabetes, and inflammatory
diseases (6,7).
2State Key Laboratory of Biotherapy and Cancer Center/
Collaborative Innovation Center of Biotherapy, West China
Hospital, West China Medical School of Sichuan
University, Chengdu 610041, China
*Corresponding author: Liang Ma, Liang_m@scu.edu.cn
#Equally contributed to the study.
Free fatty acid receptor
2 (FFA2), also known as
The activated potencies of SCFAs for FFA2 are low, in
the high micromolar to millimolar concentrations (4). Previ-
ously, Amgen Inc. has discovered phenylacetamide
derivative AMG7703 as the first class of non-SCFA allos-
teric agonist of FFA2 which induced positive cooperativity
with natural SCFAs (8). Euroscreen S.A. has patented a
compound series of agonists including compounds 1 and
2 with potent FFA2-activated potency and claimed their
uses in treating metabolic disorders (9). Additionally, com-
GPR43, is activated by short-chain fatty acids (SCFAs)
that are mainly produced by the gut microbiota
through the fermentation of undigested carbohydrates
and dietary fibers. FFA2 currently appears to be a
potential target in the management of obesity, dia-
betes, inflammatory diseases, and cancer. In the study,
a
series of novel phenylthiazole-carboxamido acid
derivatives has been synthesized and evaluated as
potential orthosteric FFA2 ligands for the study of
structure–activity relationships. Compound 6e was
found to exhibit the twofold potent agonistic activity
in the stable hFFA2-transfected CHO-K1 cells
(EC50 = 23.1 lM) as that of positive control propionate
(EC50 = 43.3 lM). We also reported the results of muta-
genesis studies based on the crystal structure of
pound
3 as a constrained lactam analog has been
explored to increased GLP-1 secretion from a rat lower
intestinal cell preparation (10). These reported agonists all
possessed the moiety of thiazol-2-amine group, which
gave us a guideline to the structural design of FFA2 mod-
ulators.
ꢀ
hFFA1 bound to TAK-875 at 2.3 A resolution to identify
important residues for orthosteric agonist 6e inducing
FFA2 activation.
Here, a series of novel phenylthiazole-carboxamido acid
derivatives as potential orthosteric FFA2 ligands has been
synthesized and evaluated for the study of structure–activ-
ity relationship (SAR). In the study, based on the crystal
Key words: agonist activity, FFA2, flexible docking, phenylthi-
azolecarboxamido acids
ꢀ
structure of hFFA1 bound to TAK-875 at 2.3 A resolution,
Received 3 September 2015, revised 6 December 2015 and
accepted for publication 30 December 2015
we also reported the results of mutagenesis studies to
identify important residues for novel orthosteric FFA2 ago-
nist 6e-induced receptor activation (11) (Figure 1).
Free fatty acid receptor 2 (FFA2), also known as GPR43,
is a member of G protein-coupled receptor (GPCR) super-
family (1,2). FFA2 is activated by short-chain fatty acids
(SCFAs, carbon number ≤6) that are mainly produced by
the gut microbiota through the fermentation of undigested
carbohydrates and dietary fibers (3,4). Acetate, propionate,
and butyrate are the most potent natural ligands for FFA2
that could induce coupling to both Gai and Gaq (5). FFA2
Methods and Materials
FFA2 assay in stably transfected CHO-K1 cells
CHO-K1 cells expressing human recombinant FFA2 grown
prior to the test in media without antibiotic are detached
by gentle flushing with PBS–EDTA (5 mM EDTA), recov-
ered by centrifugation, and resuspended in assay buffer
26
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12729

Phenylthiazole-carboxamido Acids as FFA2 Agonists
Figure 1: Selected small-molecule FFA2 agonists and compound 6e in this study.
(KRH: 5 mM KCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM
HEPES, 13.3 mM glucose, 1.25 mM KH2PO4, 1.45 mM
CaCl2, 0.5 g/L BSA). Dose–response curves are per-
formed in parallel with the reference compounds. For ago-
nist test (96-well): 12 lL of cells was mixed with 6 lL of
the test compound at increasing concentrations and 6 lL
of forskolin and then incubated for 30 min at room tem-
perature. After the addition of the lysis buffer and incuba-
tion for 1 h, cAMP concentrations are estimated according
to the manufacturer specification with the HTRF kit (Cisbio
International, Paris, France). Agonist activity of test com-
pound will be expressed as a percentage of the activity of
the reference agonist at its EC100 concentration.
Synthesis of benzothioamides
A Schlenk tube was charged under N2 atmosphere with
the corresponding benzamide (0.2 mmol, 2.0 eq), Lawes-
son’s reagent (0.1 mmol, 1.0 eq), and toluene (4 mL) and
stirred at 60 °C. After 6 h, when the reaction was com-
pleted as determined by TLC, neutral aluminum oxide
(2.0 g) was added and the solvent was evaporated. The
resulting solid was placed on a short column packed with
silica. The column was eluted with EtOAc (20–50 mL) to
give benzothioamides without further purification.
Synthesis of 2-phenylthiazole-4-carboxylic acids
(1–3)
To
a solution of the corresponding benzothioamides
Chemical synthesis
(5.0 mmol) in MeOH (~10 mL) was added ethyl 3-bromo-2-
oxopropanoate (0.77 mL, 5.5 mmol) at room temperature.
The reaction mixture was stirred for 4 h at 80 °C and then
cooled to room temperature. The resulting precipitate was
isolated by filtration and washed with Et2O to give a solid
product. The solid was dissolved in EtOH (~15 mL), and
aqueous lithium hydroxide (2 M, 10 mL) was added. The
mixture was stirred overnight at room temperature and neu-
tralized by 1 N HCl to pH 4–5, resulting in the formation of a
solid precipitate. The solid product was washed with water
and dried in vacuo to give a pure solid product.
Chemical reagents of analytical grade were purchased
from Chengdu Changzheng Chemical Factory (Sichuan, P.
R. China). TLC was performed on 0.20 mm silica gel
60 F254 plates (Qingdao Ocean Chemical Factory, Shan-
dong, China). NMR was recorded at 400 MHz on a
Waters spectrometer and reported in parts per million.
Chemical shifts (d) are quoted in ppm relative to TMS as
an internal standard, where (d) TMS = 0.00 ppm. The mul-
tiplicity of the signal is indicated as s, singlet; brs, broad
singlet; d, doublet; t, triplet; q, quartet; m, multiplet,
defined as all multipeak signals where overlap or complex
coupling of signals makes definitive descriptions of peaks
difficult. Mass spectra were measured by Q-TOF Premier
mass spectrometer utilizing electrospray ionization (ESI)
(Micromass, Manchester, UK). Room temperature (RT)
was within the range 20–25 °C. The purity was analyzed
by HPLC system (Waters 2695, Separations Module) with
a photodiode array detector (Waters 2996, Milford, MA,
USA), and the chromatographic column was a reversed-
phase C18 column (Waters, 150 mm 9 4.6 mm, i.d.
5 lm). All compounds were supplied in HPLC-degree
methanol with 10 lL, which were injected on a partial loop
fill with isocratic elution from 90% methanol and 10%
water to 10% methanol and 90% water (containing 0.1%
formic acid) within 30 min at a flow rate of 1 mL/min. The
purity of all tested compounds was ≥95% according to
our analytical HPLC method.
2-(2-Chlorophenyl)thiazole-4-carboxylic acid (1). Yield:
78.9% for two steps; 1H NMR (400 MHz, DMSO-d6) d
13.23 (s, 1H), 8.66 (s, 1H), 8.21–7.19 (m, 1H), 7.70–7.68
(m, 1H), 7.58–7.53 (m, 2H). 13C NMR (100 MHz, DMSO-
d6) d 162.73, 162.03, 146.95, 131.66, 130.90, 130.71,
130.09, 127.84. MS (ESI), m/z: 238.2 [MÀH]À.
2-(4-Chlorophenyl)thiazole-4-carboxylic acid (2). Yield:
61.2% for two steps; 1H NMR (400 MHz, CDCl3) d 10.32
(s, 1H), 8.32 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.46 (d,
J = 8.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
d
166.07, 161.91, 148.15, 135.31, 131.30, 129.36, 129.18,
128.10. MS (ESI), m/z: 238.1 [MÀH]À.
2-(2,4-Dichlorophenyl)thiazole-4-carboxylic acid (3). Yield:
74.1% for two steps; 1H NMR (400 MHz, DMSO-d6) d
Chem Biol Drug Des 2016; 88: 26–37
27

Ma et al.
13.26 (s, 1H), 8.67 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.88
(s, 1H), 7.64 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) d 161.95, 161.66, 147.00, 135.38, 132.02,
131.76, 130.29, 130.12, 129.69, 128.14. MS (ESI), m/z:
272.1 [MÀH]À.
tion mixture was stirred overnight at room temperature.
When the reaction was completed as determined by TLC,
the mixture was poured into the ice water and then the
precipitate was formed. The resulting precipitate was iso-
lated by filtration and washed with water to give a crude
product. The solid (0.3 mmol) was dissolved in EtOH
(~5 mL), and aqueous lithium hydroxide (2 M, 0.6 mL) was
added. The mixture was stirred overnight at room temper-
ature and neutralized by 1 N HCl to pH 4–5, resulting in
the formation of a solid precipitate. The solid product was
washed by water and EtOH and dried in vacuo to give a
pure solid product.
Synthesis of 2-phenylthiazole-5-carboxylic acids
(4–6)
To a suspension of the corresponding benzothioamides
(5.0 mmol) in EtOH (~10 mL) was added ethyl 2-chloro-
3-oxobutanoate (0.69 mL, 5.0 mmol). The solution was
stirred and refluxed for 4 h, and then, the solvent was
removed under reduced pressure. The solid was stirred
in cooled hexane (15 mL) for 30 min, filtered, and
washed with hexane (2 9 10 mL) to give a crude pro-
duct without any further purification. To a cooled solution
of crude solid (10 mmol) in EtOH (~40 mL) was added a
solution of lithium hydroxide (2 M, 20 mL). The mixture
was stirred overnight at room temperature, and after the
evaporation of half of EtOH solvent, the solution was
neutralized by 1 N HCl to pH 4–5, resulting in the for-
mation of a solid precipitate. The solid product was
washed with water and dried in vacuo to give a pure
solid product.
2-(2-(2-Chlorophenyl)thiazole-4-carboxamido)acetic
acid (1a). Yield: 31.9% for five steps from the starting
1
chloro-substituted benzamide; H NMR (400 MHz, DMSO-
d6) d 12.69 (s, 1H), 8.87 (s, 1H), 8.49 (s, 1H), 8.43 (s, 1H),
7.69–7.56 (s, 3H), 3.98 (d, J = 4.0 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) d 171.09, 162.47, 160.59, 149.00,
131.68, 130.97, 130.92, 130.74, 130.56, 127.70, 125.92,
40.87. MS (ESI), m/z: 295.1 [MÀH]À.
2-(2-(4-Chlorophenyl)thiazole-4-carboxamido)acetic
acid (2a). Yield: 44.0% for five steps from the starting
chloro-substituted benzamide; 1H NMR (400 MHz,
DMSO-d6) d 12.72 (s, 1H), 8.82 (t, J = 6.0 Hz, 1H), 8.37
(s, 1H), 8.09 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz,
2H), 3.97 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) d 171.07, 165.88, 160.50, 150.20, 135.36,
131.23, 129.33, 128.11, 124.86, 40.88. MS (ESI), m/z:
295.1 [MÀH]À.
2-(2-Chlorophenyl)-4-methylthiazole-5-carboxylic acid
(4). Yield: 89.3% for two steps; 1H NMR (400 MHz,
DMSO-d6) d 13.49 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.58
(d, J = 8.4 Hz, 1H), 2.68 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 166.93, 162.80, 159.51, 135.80, 131.10,
129.37, 129.21, 128.15, 127.60, 123.45, 17.01. MS (ESI),
m/z: 252.2 [MÀH]À.
2-(2-(2,4-Dichlorophenyl)thiazole-4-carboxamido)
acetic acid (3a). Yield: 34.1% for five steps from the
1
2-(4-Chlorophenyl)-4-methylthiazole-5-carboxylic acid
(5). Yield: 86.6% for two steps; 1H NMR (400 MHz,
DMSO-d6) d 13.53 (brs, 1H), 8.28 (d, J = 8.0 Hz, 1H),
7.83 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 2.69 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) d 162.79, 162.08, 158.00,
135.64, 131.91, 131.68, 130.23, 129.34, 128.03, 124.64,
16.83. MS (ESI), m/z: 252.3 [MÀH]À.
starting chloro-substituted benzamide; H NMR (400 MHz,
DMSO-d6) d 12.78 (s, 1H), 8.90 (s, 1H), 8.51–8.47 (m,
2H), 7.89 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 3.97 (d,
J = 4.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
d
171.09, 161.39, 160.48, 149.04, 135.40, 132.07, 131.76,
130.16, 129.56, 128.00, 126.15, 40.94. MS (ESI), m/z:
329.2 [MÀH]À.
2-(2,4-Dichlorophenyl)-4-methylthiazole-5-carboxylic
1
2-(2-(2-Chlorophenyl)-4-methylthiazole-5-
acid (6). Yield: 92.9% for two steps; H NMR (400 MHz,
carboxamido)acetic acid (4a). Yield: 36.3% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 12.71 (s, 1H), 8.68 (t,
J = 7.2 Hz, 1H), 8.26 (dd, J = 7.2, 2.0 Hz, 1H), 7.69–
7.67 (m, 1H), 7.57–7.51 (m, 1H), 3.92 (d, J = 5.2 Hz,
1H), 2.67 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d
171.44, 161.75, 161.55, 154.72, 132.19, 131.35, 131.11,
130.99, 128.33, 127.68, 41.78, 17.37. MS (ESI), m/z:
309.1 [MÀH]À.
DMSO-d6) d 12.71 (s, 1H), 8.68 (t, J = 7.2 Hz, 1H), 8.26
(dd, J = 7.2, 2.0 Hz, 1H), 7.69–7.67 (m, 1H), 7.57–7.51
(m, 1H), 3.92 (d, J = 5.2 Hz, 1H), 2.67 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) d 171.44, 161.75, 161.55, 154.72,
132.19, 131.35, 131.11, 130.99, 128.33, 127.68, 41.78,
17.37. MS (ESI), m/z: 286.1 [MÀH]À.
Synthesis of phenylthiazole-carboxamido acids
(1a-6f)
2-(2-(4-Chlorophenyl)-4-methylthiazole-5-
To a solution of the corresponding 2-phenylthiazole-4(5)-
carboxylic acids (1.00 mmol) and amino acid ester
(1.05 mmol) in DMF (~5 mL) were added EDCI (287.6 mg,
1.50 mmol) and DMAP (244.3 mg, 2.00 mmol). The reac-
carboxamido)acetic acid (5a). Yield: 29.9% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 12.71 (s, 1H), 8.59 (t,
J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.59 (d,
28
Chem Biol Drug Des 2016; 88: 26–37

Phenylthiazole-carboxamido Acids as FFA2 Agonists
J = 8.0 Hz, 2H), 3.91 (d, J = 8.0 Hz, 1H), 2.64 (s, 2H).
13C NMR (100 MHz, DMSO-d6) d 170.91, 161.14, 159.92,
154.23, 135.39, 131.67, 130.21, 129.45, 128.08, 127.48,
41.30, 16.83. MS (ESI), m/z: 309.1 [MÀH]À.
1H NMR (400 MHz, DMSO-d6) d 12.30 (s, 1H), 8.35 (t,
J = 4.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.56 (d,
J = 8.0 Hz, 2H), 3.44 (q, J = 8.0 Hz, 2H), 2.60 (s, 3H),
2.54–2.52 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d
172.80, 164.41, 160.82, 154.83, 135.39, 131.22, 129.39,
127.90, 126.80, 35.67, 33.62, 16.96. MS (ESI), m/z:
323.4 [MÀH]À.
2-(2-(2,4-Dichlorophenyl)-4-methylthiazole-5-
carboxamido)acetic acid (6a). Yield: 48.1% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 12.86 (brs, 1H), 8.68 (s, 1H),
8.28 (s, 1H), 7.85 (s, 1H), 7.61 (s, 1H), 3.93 (m, 2H), 2.67
(s, 3H). 13C NMR (100 MHz, DMSO-d6) d 170.93, 164.76,
161.16, 155.44, 135.48, 131.17, 129.41, 127.98, 126.23,
41.31, 17.02. MS (ESI), m/z: 343.5 [MÀH]À.
3-(2-(2,4-Dichlorophenyl)-4-methylthiazole-5-
carboxamido)propanoic acid (6b). Yield: 33.9% for
five steps from the starting chloro-substituted benzamide;
1H NMR (400 MHz, DMSO-d6) d 12.59 (s, 1H), 8.42 (s,
1H), 8.29–8.25 (m, 1H), 7.84 (s, 1H), 7.59 (d, J = 8.0 Hz,
1H), 3.45 (d, J = 8.0 Hz, 2H), 2.62 (s, 3H), 2.53 -2.51 (m,
2H). 13C NMR (100 MHz, DMSO-d6) d 172.80, 160.79,
159.57, 153.69, 135.30, 131.63, 131.59, 130.19, 129.49,
128.07, 35.68, 33.59, 16.83, 16.77. MS (ESI), m/z: 357.2
[MÀH]À.
3-(2-(2-Chlorophenyl)thiazole-4-carboxamido)
propanoic acid (1b). Yield: 41.2% for five steps from
the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6) d 12.29 (s, 1H), 8.63 (t, J = 8.0 Hz,
1H), 8.45 (s, 1H), 8.41–8.38 (m, 1H), 7.69–7.67 (m, 1H),
7.56–7.54 (m, 2H), 3.52 (q, J = 8.0 Hz, 2H), 2.56 (t,
(R)-2-(2-(2-Chlorophenyl)thiazole-4-carboxamido)
butanoic acid (1c). Yield: 23.1% for five steps from the
J = 8.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
d
1
172.98, 162.37, 160.29, 149.39, 131.63, 131.02, 130.90,
130.71, 130.59, 127.68, 125.52, 34.98, 33.81. MS (ESI),
m/z: 310.0 [MÀH]À.
starting chloro-substituted benzamide; H NMR (400 MHz,
DMSO-d6) d 12.83 (s, 1H), 8.55 (s, 1H), 8.44 (s, 1H), 7.68
(s, 1H), 7.57 (s, 1H), 4.41 (s, 1H), 1.90 (d, J = 2.4 Hz,
2H), 0.94 (m, 3H). 13C NMR (100 MHz, DMSO-d6) d
173.18, 162.55, 160.29, 149.00, 131.66, 131.13, 130.90,
130.71, 130.56, 127.75, 126.00, 53.38, 24.11, 10.41. MS
(ESI), m/z: 323.5 [MÀH]À.
3-(2-(4-Chlorophenyl)thiazole-4-carboxamido)
propanoic acid (2b). Yield: 22.6% for five steps from
the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6)
d
12.30 (s, 1H), 8.63 (t,
J = 8.0 Hz, 1H), 8.45 (s, 1H), 8.40 (s, 1H), 7.68–7.67
(m, 1H), 7.55 (m, 2H), 3.52 (q, J = 8.0 Hz, 2H), 2.56 (t,
(R)-2-(2-(4-Chlorophenyl)thiazole-4-carboxamido)
butanoic acid (2c). Yield: 35.9% for five steps from the
starting chloro-substituted benzamide; H NMR (400 MHz,
1
J = 8.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
d
172.98, 162.37, 160.29, 149.39, 131.63, 131.02,
130.90, 130.71, 130.59, 127.68, 125.52, 34.98, 33.81.
MS (ESI), m/z: 309.8 [MÀH]À.
DMSO-d6) d 12.82 (s, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.38
(s, 1H), 8.11 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H),
4.40 (q, J = 5.6 Hz, 1H), 1.90 (dq, J = 12.0, 4.0 Hz, 2H),
0.94 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) d
173.17, 165.95, 160.22, 150.16, 135.37, 131.21, 129.29,
128.21, 124.94, 53.36, 24.13, 10.41. MS (ESI), m/z:
323.2 [MÀH]À.
3-(2-(2,4-Dichlorophenyl)thiazole-4-carboxamido)
propanoic acid (3b). Yield: 29.1% for five steps from
the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6) d 8.66 (s, 1H), 8.44 (s, 1H), 7.85 (s,
1H), 7.63 (s, 1H), 3.51 (m, 2H), 2.55 (m, 3H). 13C NMR
(100 MHz, DMSO-d6) d 173.01, 161.36, 160.30, 149.32,
135.38, 132.11, 131.74, 130.11, 129.54, 127.96, 125.81,
35.00, 33.79. MS (ESI), m/z: 343.2 [MÀH]À.
(R)-2-(2-(2,4-Dichlorophenyl)thiazole-4-carboxamido)
butanoic acid (3c). Yield: 37.1% for five steps from the
1
starting chloro-substituted benzamide; H NMR (400 MHz,
DMSO-d6) d 12.83 (s, 1H), 8.52 (m, 3H), 7.88 (s, 1H), 7.66
(d, J = 8.0 Hz, 1H), 4.40 (s, 1H), 1.90 (d, J = 2.4 Hz, 2H),
0.94 (m, 3H). 13C NMR (100 MHz, DMSO-d6) d 173.16,
161.47, 160.23, 149.03, 135.39, 132.27, 131.75, 130.13,
129.57, 128.02, 126.28, 53.41, 24.10, 10.46. MS (ESI),
m/z: 357.2 [MÀH]À.
3-(2-(2-Chlorophenyl)-4-methylthiazole-5-
carboxamido)propanoic acid (4b). Yield: 46.5% for
five steps from the starting chloro-substituted benzamide;
1H NMR (400 MHz, DMSO-d6) d 12.30 (s, 1H), 8.41 (s,
1H), 8.24 (m, 1H), 7.66 (m, 1H), 7.52 (m, 2H), 3.44 (m,
2H), 2.63 (s, 3H), 2.51 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) d 172.80, 160.90, 160.72, 153.64, 131.61,
130.83, 130.79, 130.57, 127.81, 127.74, 35.67, 33.61,
16.81. MS (ESI), m/z: 323.6 [MÀH]À.
(R)-2-(2-(2-Chlorophenyl)-4-methylthiazole-5-
carboxamido)butanoic acid (4c). Yield: 28.3% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 12.69 (s, 1H), 8.58 (d,
J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.67 (d,
J = 4.0 Hz, 1H), 7.54 (m, 2H), 4.27 (d, J = 4.0 Hz, 1H),
2.71 (s, 3H), 1.87–1.77 (m, 2H), 0.97 (t, J = 6.0 Hz,
3H). 13C NMR (100 MHz, DMSO-d6) d 173.20, 161.31,
3-(2-(4-Chlorophenyl)-4-methylthiazole-5-
carboxamido)propanoic acid (5b). Yield: 25.5% for
five steps from the starting chloro-substituted benzamide;
Chem Biol Drug Des 2016; 88: 26–37
29

Ma et al.
160.97, 153.82, 131.61, 130.83, 130.60, 130.57,
127.81, 127.54, 54.27, 23.79, 16.82, 10.77. MS (ESI),
m/z: 337.1 [MÀH]À.
129.51, 128.51, 128.11, 127.94, 127.71, 126.70, 56.16.
MS (ESI), m/z: 405.2 [MÀH]À.
(R)-2-(2-(2-Chlorophenyl)-4-methylthiazole-5-
carboxamido)-2-phenyl-acetic acid (4d). Yield: 42.5%
for five steps from the starting chloro-substituted benza-
(R)-2-(2-(4-Chlorophenyl)-4-methylthiazole-5-
carboxamido)butanoic acid (5c). Yield: 45.2% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 12.75 (s, 1H), 8.51 (d,
J = 4.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.59 (d,
J = 8.0 Hz, 2H), 4.26 (d, J = 4.0 Hz, 1H), 2.62 (s, 3H),
1.87–1.76 (m, 2H), 0.96 (t, J = 8.0 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) d 173.20, 164.63, 161.20, 155.03,
135.41, 131.23, 129.39, 127.93, 126.54, 54.31, 23.81,
16.97, 10.71. MS (ESI), m/z: 337.3 [MÀH]À.
1
mide; H NMR (400 MHz, DMSO-d6) d 13.05 (s, 1H), 9.09
(d, J = 8.0 Hz, 1H), 8.25 (dd, J = 8.0, 4.0 Hz, 1H), 7.67–
7.66 (m, 1H), 7.56–7.49 (m, 4H), 7.42–7.33 (m, 3H), 5.54
(d, J = 8.0 Hz, 1H), 2.65 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 171.54, 161.14, 160.99, 154.14, 136.67,
131.63, 130.85, 130.58, 130.53, 128.46, 128.09, 128.00,
127.82, 127.30, 57.07, 16.91. MS (ESI), m/z: 385.2
[MÀH]À.
(R)-2-(2-(2,4-Dichlorophenyl)-4-methylthiazole-5-
carboxamido)butanoic acid (6c). Yield: 33.5% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 12.91 (brs, 1H), 8.59 (m,
1H), 8.29 (m, 1H), 7.86 (m, 1H), 7.61 (m, 1H), 4.28 (s,
1H), 2.65 (s, 3H), 1.86–1.77 (m, 2H), 0.97 (m, 3H). 13C
NMR (100 MHz, DMSO-d6) d 173.20, 164.63, 161.20,
155.03, 135.41, 131.23, 129.39, 127.93, 126.54, 54.31,
23.81, 16.97, 10.71. MS (ESI), m/z: 371.2 [MÀH]À.
(R)-2-(2-(4-Chlorophenyl)-4-methylthiazole-5-
carboxamido)-2-phenyl-acetic acid (5d). Yield: 21.9%
for five steps from the starting chloro-substituted benza-
1
mide; H NMR (400 MHz, DMSO-d6) d 13.06 (s, 1H), 9.00
(d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.58 (d,
J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.42–7.33 (m,
3H), 5.52 (d, J = 8.0 Hz, 1H), 2.62 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) d 171.52, 164.83, 160.90, 155.37,
136.66, 135.44, 131.20, 129.40, 128.47, 128.02, 127.94,
126.32, 57.07, 17.05. MS (ESI), m/z: 385.2 [MÀH]À.
(R)-2-(2-(2-Chlorophenyl)thiazole-4-carboxamido)-2-
phenylacetic acid (1d). Yield: 13.8% for five steps
from the starting chloro-substituted benzamide; 1H NMR
(R)-2-(2-(2,4-Dichlorophenyl)-4-methylthiazole-5-
carboxamido)-2-phenyl-acetic acid (6d). Yield: 18.1%
for five steps from the starting chloro-substituted benza-
(400 MHz, DMSO-d6)
d
13.26 (s, 1H), 8.85 (d,
1
J = 8.0 Hz, 1H), 8.53 (s, 1H), 8.38 (m, 1H), 7.70 (m,
1H), 7.57 (m, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.41–7.34
(m, 3H), 5.61 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) d 171.53, 162.78, 159.65, 148.63, 137.40,
131.74, 131.13, 130.90, 130.75, 130.49, 128.53,
127.94, 127.85, 127.67, 126.43, 56.15. MS (ESI), m/z:
371.1 [MÀH]À.
mide; H NMR (400 MHz, DMSO-d6) d 13.16 (s, 1H), 9.11
(d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.87 (s, 1H),
7.61 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.42–
7.35 (m, 3H), 5.54 (d, J = 4.0 Hz, 1H), 2.64 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) d 171.51, 160.89, 160.01,
154.22, 136.62, 135.37, 131.77, 131.69, 130.25, 129.51,
128.47, 128.12, 128.09, 128.01, 127.56, 57.08, 16.87.
MS (ESI), m/z: 419.2 [MÀH]À.
(R)-2-(2-(4-Chlorophenyl)thiazole-4-carboxamido)-2-
phenylacetic acid (2d). Yield: 56.9% for five steps from
the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6) d 13.29 (s, 1H), 8.80 (d, J = 8.0 Hz,
1H), 8.41 (s, 1H), 8.09 (d, J = 8.0 Hz, 2H), 7.63 (d,
J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.41–7.34 (m,
3H), 5.60 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) d 171.52, 166.17, 159.57, 149.78, 137.40,
135.42, 131.11, 129.35, 128.53, 128.24, 127.94, 127.66,
125.38, 56.15. MS (ESI), m/z: 371.1 [MÀH]À.
(R)-2-(2-(2-Chlorophenyl)thiazole-4-carboxamido)-3-
phenylpropanoic acid (1e). Yield: 43.9% for five steps
from the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6)
d 13.30 (brs, 1H), 8.48 (d,
J = 8.0 Hz, 1H), 8.44 (s, 1H), 8.34 (m, 1H), 7.68 (m, 1H),
7.56 (m, 2H), 7.27–7.18 (m, 5H), 4.68–4.67 (m, 1H), 3.23
(m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 172.62,
162.48, 159.90, 149.00, 137.80, 131.67, 130.93, 130.91,
130.76, 130.49, 129.19, 128.14, 127.77, 126.35, 125.86,
53.56, 36.38. MS (ESI), m/z: 385.2 [MÀH]À.
(R)-2-(2-(2,4-Dichlorophenyl)thiazole-4-carboxamido)-
2-phenylacetic acid (3d). Yield: 51.3% for five steps
from the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6) d 13.26 (s, 1H), 8.89 (d, J = 8.0 Hz,
1H), 8.55 (s, 1H), 8.45 (d, J = 8.0 Hz, 2H), 7.90 (d,
J = 4.0 Hz, 1H), 7.67 (dd, J = 8.0, 4.0 Hz, 1H), 7.50 (d,
J = 8.0 Hz, 2H), 7.41–7.34 (m, 3H), 5.62 (d, J = 8.0 Hz,
1H). 13C NMR (100 MHz, DMSO-d6) d 171.52, 161.69,
159.62, 148.67, 137.34, 135.44, 132.29, 131.77, 130.16,
(R)-2-(2-(4-Chlorophenyl)thiazole-4-carboxamido)-3-
phenylpropanoic acid (2e). Yield: 23.1% for five steps
from the starting chloro-substituted benzamide; 1H NMR
(400 MHz, DMSO-d6)
d 13.02 (brs, 1H), 8.49 (d,
J = 8.0 Hz, 1H), 8.32 (s, 1H), 8.06 (d, J = 8.0 Hz, 2H),
7.63 (d, J = 8.0 Hz, 2H), 7.28–7.20 (m, 5H), 4.71–4.70
(m, 1H), 3.23 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d
172.63, 165.90, 159.99, 150.01, 137.63, 135.39, 131.17,
30
Chem Biol Drug Des 2016; 88: 26–37

Phenylthiazole-carboxamido Acids as FFA2 Agonists
129.34, 129.10, 128.24, 128.13, 126.47, 124.95, 53.44,
J = 8.0 Hz, 1H), 4.53 (d, J = 8.0 Hz, 1H), 3.40–3.27 (m,
2H). 13C NMR (100 MHz, DMSO-d6) d 172.92, 162.30,
159.43, 149.65, 135.90, 131.57, 130.84, 130.80, 130.71,
130.46, 127.87, 127.75, 125.30, 123.50, 120.56, 118.51,
118.04, 111.10, 110.48, 54.15, 27.07. MS (ESI), m/z:
424.2 [MÀH]À.
36.25. MS (ESI), m/z: 385.1 [MÀH]À.
(R)-2-(2-(2,4-Dichlorophenyl)thiazole-4-carboxamido)-
3-phenylpropanoic acid (3e). Yield: 18.1% for five
steps from the starting chloro-substituted benzamide; 1H
NMR (400 MHz, DMSO-d6) d 13.47 (brs, 1H), 8.50 (d,
J = 8.0 Hz, 1H), 8.46 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),
7.87 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.26–7.18 (m, 5H),
4.65 (m, 1H), 3.22 (m, 2H). 13C NMR (100 MHz, DMSO-
d6) d 172.56, 161.41, 159.78, 149.10, 137.88, 135.39,
132.03, 131.76, 130.18, 129.51, 129.19, 128.11, 128.05,
126.31, 126.09, 53.69, 36.43. MS (ESI), m/z: 419.2
[MÀH]À.
(R)-2-(2-(4-chlorophenyl)thiazole-4-carboxamido)-3-
(1H-indol-3-yl) propanoic acid (2f). Yield: 18.9% for
five steps from the starting chloro-substituted benzamide;
1H NMR (400 MHz, DMSO-d6) d 12.93 (s, 1H), 10.92 (s,
1H), 8.33 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.97 (d,
J = 8.0 Hz, 2H), 7.61 (m, 3H), 7.35 (d, J = 8.0 Hz, 1H),
7.21 (m, 1H), 7.09 (t, J = 8.0 Hz, 1H), 6.97 (t, J = 8.0 Hz,
1H), 4.73 (dd, J = 16.0, 8.0 Hz, 1H), 3.36–3.35 (m, 2H).
13C NMR (100 MHz, DMSO-d6) d 172.91, 165.86, 159.94,
149.94, 136.09, 135.38, 131.05, 129.29, 128.11, 127.30,
124.89, 123.72, 121.00, 118.51, 118.22, 111.44, 109.43,
52.90, 26.53. MS (ESI), m/z: 424.3 [MÀH]À.
(R)-2-(2-(2-Chlorophenyl)-4-methylthiazole-5-
carboxamido)-3-phenyl propanoic acid (4e). Yield:
40.0% for five steps from the starting chloro-substituted
benzamide; 1H NMR (400 MHz, DMSO-d6) d 12.90 (s,
1H), 8.65 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 4.0 Hz, 1H),
7.66 (m, 1H), 7.52 (m, 2H), 7.31–7.22 (m, 5H), 4.61 (m,
1H), 3.23–3.02 (m, 2H), 2.48 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 172.70, 161.05, 153.71, 137.88, 131.63,
130.82, 130.61, 130.53, 129.08, 128.19, 127.80, 127.48,
126.41, 54.32, 36.05, 16.66. MS (ESI), m/z: 435.9
[M + Cl]À.
(R)-2-(2-(2,4-Ddichlorophenyl)thiazole-4-
carboxamido)-3-(1H-indol-3-yl)propanoic
acid
(3f). Yield: 25.1% for five steps from the starting chloro-
substituted benzamide; 1H NMR (400 MHz, DMSO-d6) d
10.82 (s, 1H), 8.44 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.17
(d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 7.57 (t, J = 8.0 Hz, 2H),
7.29 (s, J = 8.0 Hz, 2H), 7.14 (s, 1H), 7.01 (t, J = 8.0 Hz,
1H), 6.87 (t, J = 8.0 Hz, 1H), 4.53 (d, J = 4.0 Hz, 1H),
3.40–3.27 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d
172.83, 161.23, 159.37, 149.65, 135.93, 135.27, 131.88,
131.71, 130.13, 129.46, 128.01, 127.82, 125.62, 123.51,
120.59, 118.50, 118.06, 111.14, 110.43, 54.11, 27.05.
MS (ESI), m/z: 458.3 [MÀH]À.
(R)-2-(2-(4-Chlorophenyl)-4-methylthiazole-5-
carboxamido)-3-phenyl propanoic acid (5e). Yield:
35.0% for five steps from the starting chloro-substituted
benzamide; 1H NMR (400 MHz, DMSO-d6) d 12.90 (s,
1H), 8.57 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H),
7.58 (d, J = 8.0 Hz, 2H), 7.31–7.22 (m, 5H), 4.58 (m, 1H),
3.23–3.00 (m, 2H), 2.44 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) d 171.52, 164.83, 160.90, 155.37, 136.66,
135.44, 131.20, 129.40, 128.47, 128.02, 127.94, 126.32,
57.07, 17.05. MS (ESI), m/z: 399.3 [MÀH]À.
(R)-2-(2-(2-Chlorophenyl)-4-methylthiazole-5-
carboxamido)-3-(1H-indol-3-yl)propanoic
acid
(4f). Yield: 33.9% for five steps from the starting chloro-
substituted benzamide; 1H NMR (400 MHz, DMSO-d6) d
10.83 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.21 (dd, J = 8.0,
2.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz,
1H), 7.54–7.48 (m, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.18 (s,
1H), 7.04 (t, J = 8.0 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H),
4.55 (d, J = 4.0 Hz, 1H), 3.37–3.18 (m, 2H), 2.49 (s, 3H).
13C NMR (100 MHz, DMSO-d6) d 172.99, 160.85, 160.62,
153.35, 136.03, 131.57, 130.81, 130.56, 128.03, 127.76,
127.45, 123.56, 120.76, 118.30, 118.22, 111.26, 110.44,
54.45, 26.60, 16.66. MS (ESI), m/z: 438.3 [MÀH]À.
(R)-2-(2-(2,4-Dichlorophenyl)-4-methylthiazole-5-
carboxamido)-3-phenylpropanoic acid (6e). Yield:
41.0% for five steps from the starting chloro-substituted
benzamide; 1H NMR (400 MHz, DMSO-d6) d 13.85 (s,
1H), 9.47 (d, J = 8.0 Hz, 1H), 9.05 (d, J = 8.0 Hz, 1H).
8.67 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.12–8.02 (m, 5H),
5.42 (m, 1H), 4.04–3.82 (m, 2H), 3.28 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) d 173.98, 162.24, 161.24, 155.06,
139.17, 136.67, 133.02, 132.96, 131.53, 130.81, 130.38,
129.49, 129.41, 129.07, 127.72, 55.64, 37.36, 17.93. MS
(ESI), m/z: 433.3 [MÀH]À.
(R)-2-(2-(4-Chlorophenyl)-4-methylthiazole-5-
carboxamido)-3-(1H-indol-3-yl)propanoic
acid
(R)-2-(2-(2-Chlorophenyl)thiazole-4-carboxamido)-3-
(1H-indol-3-yl) propanoic acid (1f). Yield: 28.5% for
five steps from the starting chloro-substituted benzamide;
1H NMR (400 MHz, DMSO-d6) d 10.80 (s, 1H), 8.42
(s, 1H), 8.31 (d, J = 4.0 Hz, 1H), 8.13 (dd, J = 8.0,
4.0 Hz, 1H), 7.65 (dd, J = 8.0, 4.0 Hz, 1H), 7.56 (d,
J = 8.0 Hz, 1H), 7.53–7.48 (m, 2H), 7.29 (d, J = 8.0 Hz,
1H), 7.14 (m, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.87 (t,
(5f). Yield: 39.0% for five steps from the starting chloro-
substituted benzamide; 1H NMR (400 MHz, DMSO-d6) d
12.88 (s, 1H), 10.87 (s, 1H), 8.46 (d, J = 4.0 Hz, 1H), 7.94
(d, J = 8.0 Hz, 2H), 7.58–7.57 (m, 3H), 7.35 (d,
J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.07 (t, J = 8.0 Hz, 1H),
6.99 (t, J = 8.0 Hz, 1H), 4.63 (m, 1H), 3.34–3.18 (m, 2H),
2.47 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 173.05,
164.65, 160.97, 155.05, 136.13, 135.43, 131.20, 129.38,
Chem Biol Drug Des 2016; 88: 26–37
31

Ma et al.
127.92, 127.11, 126.47, 123.67, 120.95, 118.38, 118.13,
111.41, 110.05, 53.86, 26.48, 16.85. MS (ESI), m/z:
438.2 [MÀH]À.
I-TASSER (16), Phyre2 (17), IntFOLD2 (18), RaptorX (19).
The quality of models was assessed by Verify Models
implemented by DISCOVERY STUDIO 3.1 and PROCHECK (20).
Finally, based on the reported mutational analysis and the
model evolution results, the most rational structure was
recruited for molecular docking (21–23).
(R)-2-(2-(2,4-Dichlorophenyl)-4-methylthiazole-5-
carboxamido)-3-(1H-indol-3-yl)propanoic
acid
(6f). Yield: 33.2% for five steps from the starting chloro-
substituted benzamide; 1H NMR (400 MHz, DMSO-d6) d
13.02 (s, 1H), 10.87 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.25
(d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 7.61–7.60 (m, 2H), 7.35
(d, J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.07 (t, J = 8.0 Hz, 1H),
6.99 (t, J = 8.0 Hz, 1H), 4.66 (m, 1H), 3.35–3.19 (m, 2H),
2.50 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 173.02,
160.96, 159.86, 153.86, 136.12, 135.34, 131.67, 130.27,
130.20, 129.50, 128.07, 127.74, 127.13, 123.63, 120.93,
118.38, 118.15, 111.40, 110.09, 53.87, 26.46, 16.66. MS
(ESI), m/z: 472.2 [MÀH]À.
Flexible docking was performed using the Flexible Docking
protocol implemented in DISCOVERY STUDIO 3.1. In the pro-
cess of docking, the following residues were selected as
flexible residues: Tyr90, Arg180, Tyr238, His242, and
Arg255. Residue Arg180 was used to define the binding
ꢀ
site, and the sphere radius was set as 12 A. The best
docked conformations of small agonists were selected
according to docking score and the binding action of
short-chain fatty acids and other small carboxylic modula-
tors of FFA2.
Modeling simulation
Results and Discussion
To further investigate the bound mode of our compound,
FFA2 model was built by using multithreading alignments.
To search the similar sequence of human FFA2, the PSI-
BLAST program was employed directed from its Web site at
http://blast.be-md.ncbi.nlm.nih.gov/Blast.cgi (12). EASYMO-
DELLER 4.0 was used manually built homology models of
human FFA2 from human FFA1 (PDB code: 4PHU) and
other GPCR protein structures (13). In addition, the follow-
ing web server also were implemented from Protein Model
Portal (http://www.proteinmodelportal.org/) (14): M4T (15),
Chemistry
The preparation of a library of 2-phenylthiazole-carboxylic
acids (1–6) and their amido acid derivatives (1a–6f) has
been carried out in a tandem five-step sequence from the
commercially available corresponding benzamides as
described in Scheme 1. Treatment of benzamides with
Lawesson’s reagent in the solvent of toluene afforded
benzothioamides in a good yield without any further purifi-
cation for the next step (24–26). 2-Phenylthiazole-4-car-
a
.
Scheme 1: Synthesis of phenylthiazole-carboxamido acid derivatives
aReagent and conditions: (a) Lawesson’s reagent, toluene, 60 °C,
6 h; (b) ethyl 3-bromo-2-oxopropanoate, MeOH, reflux, 4 h, then 2 M LiOH, EtOH, rt; (c) ethyl 2-chloro-3-oxobutanoate, EtOH, reflux, 4 h;
(d) amino acid ester, EDCI, DMAP, DMF, rt, then 2 M LiOH, EtOH, rt;
32
Chem Biol Drug Des 2016; 88: 26–37

Phenylthiazole-carboxamido Acids as FFA2 Agonists
Table 1: In vitro agonistic activity of test compounds 1–6 for
boxylic acids (1–3) were synthesized via a condensation of
ethyl 3-bromo-2-oxopropanoate with respective benzoth-
ioamides in refluxed methanol and then the hydrolysis
using 2 M LiOH in ethanol at room temperature (27–29).
The 2-phenylthiazole-5-carboxylic acids (4–6) were pre-
pared via a similar chemical condensation of ethyl 2-
chloro-3-oxobutanoate with appropriate benzothioamides
in refluxed ethanol and then hydrolysis by the solution of
2 M LiOH at room temperature (28, 30). The desired
phenylthiazole-carboxamido acid derivatives 1a–6f were
obtained by the condensation of 1–6 with appropriate
amino acid ester (a–f) using EDCI and DMAP as efficient
condensing agents, followed by the hydrolysis in the LiOH/
EtOH solution (2 M), which was further neutralized by 1 N
HCl to pH 4–5 (28, 31). At this stage, all final products
were fully analyzed and characterized by NMR, MS and
HPLC before submitted to the biological screening.
hFFA2a
R2
R2
O
S
O
N
N
S
OH
OH
R1
R1
1~3
4~6
Compounds
R1
R2
H
% Activation (at 100 lM)
l
C
1
10.56 Æ 5.93
*
*
*
2
3
H
H
8.17 Æ 1.05
Cl
Cl
Cl
13.73 Æ 2.41
Agonistic activity in vitro
In this study, we evaluated the FFA2 agonistic activity of
test compounds by the assay of cAMP concentrations
with the homogeneous time-resolved fluorescence (HTRF)
kits in stable human recombinant FFA2-transfected CHO-
K1 cells. Natural potent ligand propionate was selected as
positive control in the assay, and agonistic activity of the
test compounds was expressed as a percentage of the
activity of the reference agonist at the EC100 concentra-
tion.
Cl
*
4
5
-CH3
-CH3
31.25 Æ 0.90
10.93 Æ 2.72
*
*
Cl
Cl
Cl
6
-CH3
41.49 Æ 4.54
As shown in Table 1, we initially accessed the agonistic
potency of six chloro-substituted phenylthiazole-5-car-
boxylic acid and chloro-substituted phenyl-4-methylthia
zole-5-carboxylic acid derivatives, which have been
designed on the basis of previously reported thiazol-2-
amine agonists. By the inspection of their structural fea-
tures and agonistic potency, compounds of 2,4-dichloro-
aIn vitro agonist activity of test compounds at 100 lM was per-
formed in a cAMP assay for human Gi-coupled FFA2. Agonistic
activity of the test compound was expressed as a percentage of
the activity of the reference agonist at the EC100 concentration.
The activity of propionate is 85.35 Æ 5.89% at a concentration of
100 lM.
Figure 2: In vitro agonistic activity of test compounds 1a–6f for hFFA2. The agonist activity of 1a–6f at 100 lM was performed in a
cAMP assay for human Gi-coupled FFA2. Agonistic activity of test compound was expressed as a percentage of the activity of the
reference agonist at the EC100 concentration.
Chem Biol Drug Des 2016; 88: 26–37
33

Ma et al.
Table 2: EC50 values of selected compounds for hFFA2.
5 > 2 or 6 > 3). Herein, we concluded that the number
and substituent site of chloro atom and 4-methylthiazole
group seemed to be crucial for in vitro hFFA2 activation
(Figure 2).
Compounds
Chemical structures
EC50 (lM)
Cl
4e
60.6 Æ 1.98
HN
O
S
COOH
As shown in Table 2, three selected compounds were fur-
ther investigated for the hFFA2 agonistic activity. We found
that compounds (4e, 5e and 6e) exhibited EC50 values of
60.6, 89.6 and 23.1 lM, respectively, compared to
43.3 lM of the positive control propionate. These results
were in keeping with the aforementioned screening data
and SAR (compound 6e > 4e > 5e) and were also prof-
itable to structural design and exploration for orthosteric
FFA2 agonists.
N
Cl
Cl
5e
89.6 Æ 10.4
23.1 Æ 2.46
43.3 Æ 1.71
HN
S
S
COOH
COOH
N
O
Cl
6e
HN
O
N
Propionate
O
Mutagenesis analysis and molecular simulation
As for the orthosteric FFA2 agonist 6e, we further investi-
gated the mechanism of action using Flexible Docking
protocol implemented in DISCOVERY STUDIO 3.1. In the pro-
cess of docking, the following residues were selected as
flexible residues: Tyr90, Arg180, Tyr238, His242, Arg255
(11). Residue Arg180 was used to define the binding site,
ONa
phenyl group exhibited more potent activity than those of
2-chlorophenyl group and compounds of 4-chlorophenyl
group were weak (compound 3 > 1 > 2). By comparison
with thiazole (1–3) and 4-methylthiazole derivatives (4–6),
the introduction of methyl group to the phenylthiazole moi-
ety contributed to agonistic activity (compound 4 > 1 or
ꢀ
and the sphere radius was set as 12 A. The best docked
conformations of small agonists were selected according
to the docking score and the binding action of short-
chain fatty acids and other small carboxylic modulators of
Figure 3: Structure alignment of the crystal structure of FFA1 (4PHU) and FFA2. (A) The conserved seven-transmembrane helical bundle
fold of I-FFA2 was represented as rainbow, while seven-transmembrane helical bundle fold of FFA1 was painted gray. TAK-875, bound
between TM3 and TM4 in FFA1 crystal structure, was shown as sphere; (B) Arg90, Arg255, His242, Tyr90 and Tyr238 were shown as
rainbow stick, and the corresponding residues in the FFA1 were represented as gray lines. The part of TAK-875 was displayed as gray
stick, and its carboxylate moiety was highly co-ordinated by these critical residues. Hydrogen bonds are represented by yellow dashes.
34
Chem Biol Drug Des 2016; 88: 26–37

Phenylthiazole-carboxamido Acids as FFA2 Agonists
Figure 4: The binding mode of 6e in orthosteric binding sites of FFA2. (A) The interaction between I-FFA2 and compound 6e on 2D
diagram; (B) The binding site of 6e in the surface of I-FFA2.
FFA2. By site-directed mutagenesis, Arg180 and Arg255
in FFA2 (the two corresponding residues in FFA1: Arg183
and Arg258) were found to be critical for the recognition
of SCFAs. Thus, the two arginine residues functioned as
conserved anchoring residues of the fatty acid carboxy-
late group in FFA1 and FFA2. Moreover, site-directed
mutagenesis of Tyr90 and His242 exhibited detrimental
effects on the binding affinity of SCFAs (21–23). There-
fore, rational conformation of these residues was the first
thing to be assessed after the FFA2 models were con-
structed. There are five FFA2 models that generated by
above-mentioned methods. Among these methods, I-
FFA2 model, generated by I-TASSER web server, showed
the lowest DOPE score with the value of À42259.32.
Ramachandran plot of phi and psi angles revealed the
stereochemical quality of the models, and the model gen-
erated by I-TASSER showed that 80% of the residues are
in core regions and 15.1% in allowed regions; 95.1% of
allowed regions were sufficient for GPCR to conform the
reliability of the model.
model, Arg180, Arg255 and His242 formed charge center
and hydrogen bond network. In addition to the arginine
residues, Tyr90 and Tyr238 may be involved in the stabi-
lization of the carboxylate moiety (Figure 3B).
Mutagenesis analysis and molecular simulation revealed
that our carboxylic modulators functioned as agonists by
binding in the orthosteric site. So compound 6e was
docked into the canonical binding site, and the rational
conformation was singled out based on docking scores
and mutagenesis experiments. Three hydrophilic residues
Arg180, Arg255 and His242 acted as anchors for the
carboxylate group, while phenyl rings of Phe89, Tyr90
and Tyr238 were involved in the formation of p-p stack-
ing interactions. 2, 4-Dichlorophenyl moiety formed a
cation–p interaction with the side chain of Lys65 (Fig-
ure 4A). As shown in Figure 4B, the conformation of 6e
was exhibited in a crescent shape and reached into the
binding pocket. These results may be a right guideline
for our further structural modification and exploration for
potent agonists.
The FFA1 crystal structure shared 32% sequence identify
and 73% query coverage with query sequence of FFA2.
Superimposing I-FFA2 and FFA1 (4PHU) yielded an RMSD
Conclusion
ꢀ
of 2.98 A, indicating that FFA2 approximately adopted the
same conformation. The seven-transmembrane helices
bundle (TM), typical of GPCR structures, remained con-
served in the I-FFA2 model. Notably, I-FFA2 model had a
conserved hairpin loop between the transmembrane helix
3 (Cys82) and the C-terminal portion of the ECL2 loop
(Cys164). The gap between TM3 and TM4 in the FFA1
was wider than in the I-FFA2 model. So the modulators of
FFA1 accessed into the binding pocket through lipid inter-
face from the side between TM3 and TM4. However,
FFA2 possessed canonical solvent-accessible binding
pocket and the modulator could enter into the binding
pocket from extracellular surface (Figure 3A). In the I-FFA2
In the study, a series of novel phenylthiazole-carboxamido
acid derivatives has been synthesized and evaluated as
potential orthosteric FFA2. Compound 6e was found to
exhibit more potent agonistic activity in the stable hFFA2-
transfected CHO-K1 cells (EC50 = 23.1 lM) in contrast to
positive control propionate (EC50 = 43.3 lM). As for the
study of structure–activity relationships, we concluded that
the number and substituent site of chloro atom and 4-
methylthiazole group were crucial for in vitro hFFA2 activa-
tion. Based on the crystal structure of hFFA1 bound to
ꢀ
TAK-875 at 2.3 A resolution, we also reported the results
of mutagenesis studies to identify important residues for
Chem Biol Drug Des 2016; 88: 26–37
35

Ma et al.
orthosteric agonist 6e inducing the activation of FFA2.
These results may be a guideline for next structural modifi-
cation and exploration for potent agonists.
11. Srivastava A., Yano J., Hirozane Y., Kefala G., Grus-
witz F., Snell G., Lane W., Ivetac A., Aertgeerts K.,
Nguyen J. (2014) High-resolution structure of the
human GPR40 receptor bound to allosteric agonist
TAK-875. Nature;513:124–127.
Acknowledgments
12. Camacho C., Coulouris G., Avagyan V., Ma N., Papa-
dopoulos J., Bealer K., Madden T.L. (2009) BLAST+:
architecture and applications. BMC Bioinformat-
ics;10:421.
13. Kuntal B.K., Aparoy P., Reddanna P. (2010) EasyMo-
deller: a graphical interface to MODELLER. BMC Res
Notes;3:226.
This research was supported by Natural Science Founda-
tion of China (No. 81402782) and China Postdoctoral
Science Foundation (No. 2015T80985).
Conflict of Interest
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