Design, synthesis and biological evaluation of thiazole- and indole-based derivatives for the treatment of type II diabetes

Article abstract of DOI:10.1016/j.ejmech.2012.03.006

Present studies have shown that the lipid carrier has a significant role in several aspects of metabolic syndrome in A-FABP/ap2-deficient mice, including type 2 diabetes and atherosclerosis. 38 Thiazole- and indole-based derivatives were synthesized and investigated for their inhibitory effects on the production of LPS-stimulated TNF-α. Among them, 12b exhibited an excellent inhibitory efficiency compared to BMS309403 (95% vs. 85%) at the concentration of 10 μM and a binding affinity for ap2 with the apparent K_i values 33 nM. Oral administration of 12b at a dosage of 50 mg/kg effectively reduced the levels of plasma blood glucose, triglycerides, insulin, total cholesterol and alanine aminotransferase in high-fat/diet-induced obesity model. The results highlighted that 12b was a potent anti-diabetic agent.

Full text of DOI:10.1016/j.ejmech.2012.03.006

European Journal of Medicinal Chemistry 52 (2012) 70e81
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of thiazole- and indole-based
derivatives for the treatment of type II diabetes
Qinyuan Xu, Li Huang, Juan Liu, Liang Ma, Tao Chen, Jinying Chen, Fei Peng, Dong Cao, Zhuang Yang,
Neng Qiu, Jingxiang Qiu, Guangcheng Wang, Xiaolin Liang, Aihua Peng, Mingli Xiang, Yuquan Wei,
*
Lijuan Chen
State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Present studies have shown that the lipid carrier has a significant role in several aspects of metabolic
syndrome in A-FABP/ap2-deficient mice, including type 2 diabetes and atherosclerosis. 38 Thiazole- and
indole-based derivatives were synthesized and investigated for their inhibitory effects on the production
Received 25 November 2011
Received in revised form
29 February 2012
Accepted 2 March 2012
Available online 13 March 2012
of LPS-stimulated TNF-
a. Among them, 12b exhibited an excellent inhibitory efficiency compared to
BMS309403 (95% vs. 85%) at the concentration of 10
m
M and a binding affinity for ap2 with the apparent
Ki values 33 nM. Oral administration of 12b at a dosage of 50 mg/kg effectively reduced the levels of
plasma blood glucose, triglycerides, insulin, total cholesterol and alanine aminotransferase in high-fat/
diet-induced obesity model. The results highlighted that 12b was a potent anti-diabetic agent.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Ap2
Thiazole- and indole-based
TNF-
a
Diet-induced obesity
1. Introduction
Considerable studies have focused on the drug target of T2D,
including glucagon-like Peptide-1 (GLP-1), dipeptidyl peptidase-4
Type II diabetes (T2D) is the most common disorder of meta-
bolic homeostasis characterized by insulin resistance and dyslipi-
demia, accounting for 90e95% of all cases of diabetes. In 2010,
approximately 285 million people suffer from diabetes, corre-
sponding to 6.4% of the world’s population, and the figure is pre-
dicted to increase to 438 million by 2030. Insulin resistance and
dyslipidemia are closely associated with catabolic or lipodystrophic
conditions and with pathological states of over-nutrition (e.g.,
obesity-related diabetes). The main feature of these metabolic
disorders is the abnormal elevation of serum levels of pro-
(DPP-4), peroxisome proliferators activated receptor g (PPAR g),
protein tyrosine phosphatase 1B (PTP1B) etc. Recently, adipocyte
fatty acid binding protein (A-FABP or ap2), a 14.6 kDa cytosolic
protein that bind with high affinity to saturated and unsaturated
long-chain fatty acids, has become a pivotal target with more
attention. Ap2 is highly expressed in adipocytes and macrophages,
and could be regulated by PPAR
g agonists, insulin, and fatty acids.
Ap2 played significant roles in many aspects of metabolic syndrome
(e.g., glucose and lipid homeostasis), and the over-expression of
ap2 invariably occurred to obese mice or human. The ap2-deficient
mice access to a high-fat diet were protected against glucose
tolerance and insulin resistance in contrast to the wild type ones
inflammatory cytokines such as tumor necrosis factor-
a (TNF-a),
interleukin-1 (IL-1 ), and IL-6, etc. [1,2]. TNF- protein is secreted
b
b
a
by activated monocyte/macrophage and highly expressed in
adipose tissue of obese animal and human with T2D [3,4]. Specif-
ically, it has been reported that obese mice with the lack of either
[8]. Furthermore, the production of TNF-a in response to lipo-
polysaccharide (LPS) was down-regulated in aP2 knockout mice [9].
Up to date, several potent small-molecule inhibitors of ap2 such
as BMS309403, HTS01037, and 1 have been rationally designed and
developed for the treatment of T2D (Fig. 1) [10e12]. Inspection of
their structural features highlighted that the five-membered
heterocyclic ring and carboxylic group seemed to be crucial for
anti-ap2 potency. Furthermore, there were also literature reported
that some compounds with indole had anti-inflammatory activity
[13,14]. Thus, the results encouraged us to develop more potent
drug-like candidates.
TNF-
development of insulin resistance [5,6]. Therefore, blockage of the
over-expression of TNF- might be an effective strategy for the
a production or TNF-a receptor could be protected against the
a
prevention and treatment of T2D [7].
* Corresponding author. Tel.: þ86 28 85164063; fax: þ86 28 85164060.
E-mail address: lijuan17@hotmail.com (L. Chen).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.006

Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
71
Scheme 1. General synthesis of thiazole-based derivatives. Reagents and conditions:
(a) CuBr2, EtOAc-CHCl3; (b) thiourea, EtOH; (c) 4-methoxy-4-oxobutanoic acid,DMAP,
EDCI, room temperature; (d) NaOH, HCl; (e) 2-chloroacetyl chloride, Et3N; (f) 2-
mercaptoacetic acid, Et3N.
has been identified from 1H NMR spectra. The coupling constants (J)
of these compounds were all between 15.6 and 16.4 Hz in the range
from 6.25 to 6.48 ppm in dimethylsulfoxide (DMSO)-d6 solution,
while the values of the Z-isomer were commonly between 7 and
12 ppm.
The formyl group was added at C3 of 13 by the Vielsmeyer
reaction utilizing POCl3 and DMF (Scheme 3) [17]. A similar
sequence of reactions was carried out to prepare the intermediates
15 and final products 16, starting from the appropriately
substituted derivatives. Benzylation of the amide 17 was achieved
by deprotonation of the amide with sodium hydride and then
reacted with the benzylation agent (Scheme 4).
Fig. 1. Structure of BMS309403, HTS01037, 1 and 12b.
In this study, a series of thiazole- and indole-based derivatives
has been synthesized and their inhibitory effects on the production
of TNF-
cells. At a concentration of 10
indol-3-yl)acrylic acid (12b) was found to possess an excellent
inhibitory rate against the production of TNF- in contrast to
a
were subsequently performed in LPS-induced RAW 264.7
mM, (E)-3-(1-(2-fluorobenzyl)-1H-
a
BMS309403 (95% vs. 85%), leading to the establishment of a SAR.
12b was also evidenced as a potent ap2 inhibitor with the apparent
Ki value of 33 nM by a fluorescent 1,8-anilino-8-naphthalene
sulfonate (ANS) assay. Importantly, oral administration of 12b at
a dosage of 50 mg/kg improved the weight gain and serum levels of
blood glucose, TG, insulin, total cholesterol and ALT in diet-induced
obesity HF, highlighting that 12b was an orally active anti-diabetic
candidate.
3. Results and discussion
3.1. Inhibition of LPS-induced TNF-a production in RAW 264.7 cells
All synthesized compounds were evaluated for their ability to
inhibit the production of LPS-induced TNF- in RAW 264.7 cells.
a
2. Chemistry
The inhibitory rate was depicted in Table 1. BMS309403 was used as
positive control. In the series of thiazole-based compounds, we
started from halogen substitution on the phenyl ring. We can find
the compounds with a para-substitution demonstrated moderate
inhibitory potency (e.g., 6a, 6e and 6k), while compound 6b and 6f
that contained ortho or meta substitutions showed poor inhibitory
effect. It is interesting to note that the 3,4-difluoride analogue 6g
showed more potent inhibitory activity as compared to the 3,4-
dichloride 6f. It is surprising that methyl substitution on the para
position of the phenyl ring showed good activity, while methoxyl
didn’t work. The introduction of mercaptoacetic acid to the side did
not result in increased activity (see 8aed).
The thiazole analogues 6aem and 8aed were prepared starting
from the reaction of various acetophenones 2 and copper (II)
bromide to give 3 (Scheme 1) [15]. The ɑ-bromoketones 3 were
then reacted with thioureas to give the corresponding thiazoles 4
via cyclization reactions. To prepare the final products 6 and 8,
intermediates 4 were divided into two routes as shown in Scheme
1. When treated with 4-methoxy-4-oxobutanoic acid in the pres-
ence of DMAP and EDCI, yielded 5, and then the hydrolysis of 5 with
sodium hydroxide provided corresponding carboxylic acids 6.
When treated with chloroacetyl chloride catalyzed by Et3N,
provided 7, and then reacted with 2-mercaptoacetic acid in anhy-
drous THF to yielded 8.
As shown in Table 1, introduction of phenyl ring which con-
tained various substitutions replaced the hydrogen on the nitrogen.
Based on our previous SAR of the thiazole-based compounds, they
The indole analogues 12aek, 16aeg and 18aee were outlined in
Schemes 2e4. The coupling of N-unsubstituted 1H-indole-3-
carbaldehydes
9 with benzyl bromides 10, using potassium
hydroxide as base, converted to intermediates 11. The synthesis of
the final products 12 were carried out by adding malonic acid to
a solution of 11 dissolved in pyridine in the presence of piperidine
catalyst (Scheme 2). In theory, E and Z geometrical isomers around
the exocyclic double bond (CH]C) are possible for 3-(1-benzyl-1H-
indol-3-yl)acrylic acid derivatives. However, on the basis of litera-
ture data for similar compounds, these compounds belong to the E-
configuration [16]. The E-configuration of similar compounds also
Scheme 2. General synthesis of 12aek. Reagents and conditions: (a) KOH, EtOH,
acetone; (b) malonic acid, pyridine, piperidine, 100 ꢀC.

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Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
Scheme 3. General synthesis of 16aeg. Reagents and conditions: (a) POCl3, DMF, NaOH; (b) KOH, EtOH, acetone; (c) malonic acid, pyridine, piperidine, 100 ꢀC.
had a similar law that the compounds which had ortho- or para-
substitutions on the phenyl ring showed good activity (e.g., 12b,
12e, 12f and 12k), while the meta ones had a little or no activity
(e.g., 12c, 16d, 16e, 16g and 18d). We also observed that the
compounds which had strongly electron-withdrawing groups only
showed moderate inhibitory activity (e.g., 12h and 12i). Further, the
5-substituted indole analogues (e.g., 16a, 16b, 16def) were
synthesized. As the results, the methoxyl substituted had higher
inhibition rate (e.g., 16b and 16f) while the methyl was poor
(16a). However, compound had methyl at the 2-position (16c)
indicated good effect. More important, fluorine played a crucial role
3.3. In vivo activity in diet-induced obesity (DIO) rats
For further investigate in vivo studies of compound 12b, we
established a high-fat/diet-induced obesity (DIO) rat model. Male
C57BL/6J rats (age range: 7e8 weeks) were purchased from
Western China Experimental Animal Center. To get a fully devel-
oped insulin-resistant diet-induced obese (DIO) animal phenotype,
the animals were fed with a normal diet or a high-fat diet (kcal: 60%
of energy derived from fat, 20% from protein and 20% from carbo-
hydrates, D12494 Research Diet, USA) for 8 weeks. Then, the rats
which were fed with a high-fat diet were divided into two groups
fed with the high-fat diet as before (HF) or with the same food and
oral administration of 12b once a day at a dose of 50 mg/kg. The
body weight of the two groups were respectively from
22.42 ꢁ 0.72 g and 24.08 ꢁ 0.61 g at the beginning to 32.26 ꢁ 4.71 g
and 29.18 ꢁ 2.54 g after 8 weeks. The weight gain rate of 12b-
treated was 21.18% compared with HF groups 43.89% (Fig. 3A and
B). While there was no significant difference in food intake between
HF and treated groups (Fig. 4). In contrast, blood glucose levels had
obviously decreased after treatment with 12b (Fig. 5A). Throughout
the in vivo experiment, decrements in levels of triglyceride (TG),
total cholesterol (TC), alanine aminotransferase (ALT) were 27.13%,
38.52% and 26.66% respectively (Fig. 5B, C and D).
to the inhibitors of TNF-a. Almost all the compounds which had
ortho- or para- fluorine showed excellent activity (see 12b,12k,16b,
16c,16f and 18e). The indole acetic acid analogues (see 18aee) were
also evaluated in vitro, there were the similar SAR as discussed
above.
3.2. Ki values and docking study of 12b
The compound 12b was selected for further study because of its
strongest inhibitory activity among these derivatives. In a fluores-
cent 1,8-anilino-8-naphthalene sulphonate (ANS) binding
displacement assay, 12b exhibited similar Ki values to BMS309403
for mouse ap2 (33 nM vs. 29 nM). Meanwhile, we docked the 12b to
the protein of ap2 (Fig. 2). The X-ray crystal structure of fatty acid-
binding protein 4 (A-FABP) was obtained from protein data bank
(PDB ID: 2QM9) with 2.31 Å resolution. Ligand molecules drawn
with ChemBio3D were docked to the binding site of A-FABP by the
aid of a protein-ligand docking program FRED 2.2.5 (OpenEye
Scientific Software, Santa Fe, NM 87508) [18,19]. The binding site of
the protein was prepared by employing FRED RECEPTOR 2.2.5
(OpenEye Scientific Software, Santa Fe, NM 87508). AM1-BCC
partial charges were assigned to ligand molecules while Merck
Molecular Mechanics Force Field (MMFF) partial charges were
assigned to protein [20e22]. In the figure, we can find that 12b
Meanwhile, glucose tolerance tests revealed
a significant
improvement in glucose metabolism in the 12b-treated group
(Fig. 6A). Similarly, insulin tolerance tests showed significantly
increased insulin sensitivity in the DIO rats treated with 12b
(Fig. 6B). To evaluate whether 12b improved fat deposition in
C57BL/6J rat HF with diet-induced obesity, liver tissures from HF
and 12b-treated rats were processed for H&E staining assay (Fig. 7).
We can find many fat balloons in the liver tissure of the HF group
(Fig. 7A), while the fat droplets significantly decreased after 12b-
treated as evidenced by H&E staining. In general, 12b demonstrated
an excellent effect for the treatment of diabetes and obesity.
4. Conclusion
formed hydrogen bonds to Tyr 19 and Arg 78. The
p-caterion
interaction between the indole moiety of the compound and the
side chain of Arg 126 is shown with orange mark.
We performed the synthesis and SAR of thiazole-based and
indo-based compounds. Several exhibited significantly inhibitory
effect in LPS-induced TNF-
a production. The most effective
compound 12b was also a high affinity ligand of A-FABP/ap2 with
apparent Ki values 33 nM. In the diet-induced obesity (DIO) rodent
model, 12b not only control effectively the growth of the rat’s
weight which were fed high-fat diet, but also obviously decreased
the serum levels of GLU, TC, TG, ALT. In glucose and insulin toler-
ance tests, it also improved glucose metabolism and increased
insulin sensitivity. In general, it was worth for further investigation
as a drug for treatment of diabetes.
Scheme 4. General synthesis of 18aee. Reagents and conditions: (a) NaH, DMF.

Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
73
Table 1
Inhibitory effects of all compounds on LPS-induced TNF-
a
production.
Compd
R1
R2
H
R3
Inhibition %a
6a
41.22
8.56
6b
6c
H
H
36.67
6d
6e
H
H
12.15
27.24
6f
H
H
12.38
92.45
6g
6h
6i
H
H
NI
35.15
6j
eCH3
eCH3
25.23
27.14
6k
6l
H
58.75
(continued on next page)

74
Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
Table 1 (continued )
Compd
R1
R2
R3
Inhibition %a
23.45
8a
8b
43.24
18.19
8c
8d
37.26
12a
12b
H
H
H
H
18.17
95.47
12c
H
H
36.36
12d
12e
H
H
H
H
NI
66.56
12f
H
H
H
H
92.24
50.18
12g
12h
12i
H
H
H
H
32.34
31.46
12j
H
H
93.28

Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
75
Table 1 (continued )
Compd
R1
R2
H
R3
H
Inhibition %a
12k
16a
16b
16c
86.78
11.18
86.67
93.27
H
eCH3
eOCH3
H
H
eCH3
16d
16e
H
H
eCH3
NI
NI
eOCH3
16f
H
eOCH3
94.28
16g
eCH3
H
NI
18a
18b
18c
52.17
14.29
24.72
18d
NI
18e
92.24
85.13
BMS309403
a
Values are expressed as the concentration of TNF-a in the medium of the compounds dosing group divided by it of the LPS-induced group. The concentration of all
compounds are 10
mmol/mL (NI ¼ not inhibition).

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Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
Fig. 4. The food intake in C57BL/6J rats treated with vehicle (HF) or 12b at the state of
feeding with high-fat diet for 8 weeks. The arrow bars were determined by Excel.
5.1.2. General procedure for the synthesis of 4-(2-chlorophenyl)
thiazol-2-amine
Fig. 2. The interaction mode of compound 12b within the binding site of FABP4 (PDB
ID: 2QM9).
The crude 2-bromo-1-(2-chlorophenyl) ethanone (0.64 mL,
4.28 mmol) was dissolved in anhydrous ethanol (10 mL) and
treated with thiourea (344 mg, 4.51 mmol). After heating at reflux
for 2 h, the reaction mixture was cooled to room temperature, with
a yellow precipitation. The solid obtained was filtered, washed with
water and ethanol and vacuum dried to afford 4b as a yellow solid
5. Experimentals
5.1. Chemistry
(884 mg, 98.0%). 1H NMR (CDCl3, 400 MHz):
d 7.12 (s, 1H), 7.46e7.55
Purchased reagents and anhydrous solvents were used as
received. NMRs were obtained with a Bruker 400 MHz in the indi-
(m, 2H), 7.62 (s, 1H), 7.63 (d, 1H, J ¼ 1.2 Hz), 8.94 (s, 1H), 9.15 (s, 1H).
cated solvent with chemical shifts (d) reported in ppm vs. tetrame-
5.1.3. General procedure for the synthesis of methyl 4-(4-(2-
chlorophenyl)thiazol-2-yla-mino)-4-oxobutanoate
thylsilane and coupling constants (J) in Hz. The multiplicity of the
signal is indicated as s, 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 (MS) were measured by Q-TOF Priemier mass spec-
trometer utilizing electrospray ionization (ESI) (Micromass, Man-
chester, UK). Room temperature (RT) is within the range 20e25 ꢀC.
A
mixture of 4-(2-chlorophenyl)thiazol-2-amine (500 mg,
2.37 mmol), DMAP (13.27 mg, 0.12 mmol), EDCI (682.43 mg,
3.55 mmol) and 4-methoxy-4-oxobutanoic acid (470.3 mg,
3.55 mmol) in chloroform (20 mL) was stirred at room temperature
for 24 h. The solution was filtered, and filtrate was washed with
water. The organic phase was dried (Na2SO4) and evaporated to
dryness. The residue was purified by flash chromatography over
silica gel eluting with CH2Cl2:EtOAC (80:1) yielded 280 mg (36.3%
yield) to 5b.
5.1.1. General procedure for the synthesis of 2-bromo-1-(2-
chlorophenyl)ethanone
A
solution of 1-(2-chlorophenyl)ethanone (2b) (0.84 mL,
6.5 mmol) in anhydrous ethyl acetate (25 mL) and chloroform
(25 mL) was treated with copper (II) bromide (4.33 g, 19.5 mmol)
and ethanol (6 mL), stirred at 65 ꢀC for 0.5 h. The reaction was
extracted with chloroform (40 mL). The organic extracts were
washed with water (3 ꢂ 30 mL), dried (NaSO4), filtered, and
concentrated in vacuo to give a colourless oil. 1H NMR (CDCl3,
5.1.4. General procedure for the synthesis of 4-(4-(2-chlorophenyl)
thiazol-2-ylamino)-4-oxobutanoic acid
A mixture of methyl 4-(4-(2-chlorophenyl)thiazol-2-ylamino)-
4-oxobutanoate (100 mg, 0.31 mmol) and NaOH (123 mg,
3.1 mmol) in 1,4-dioxane (6 mL) was stirred at 60 ꢀC for 2.5 h. After
cooling to room temperature, the mixture was added water (5 mL),
acidification with 4 N HCl which resulted in the formation of
a white solid. The solid was washed with water and vacuum dried
400 MHz):
d 4.53 (s, 2H), 7.36e7.41 (m, 1H), 7.44e7.46 (m, 2H), 7.56
(d, 1H, J ¼ 8.0 Hz).
Fig. 3. Body weight and body weight gain rate of untreated and 12b-treated DIO rat model access to HF diet. A, The body weight of treatment in C57BL/6J rats with vehicle (HF) or
12b at the state of feeding with high-fat diet for 8 weeks. B, The body weight gain rate of feeding with high-fat diet (HF), high-fat diet added 12b (50 mg/kg) and normal food for 8
weeks. *P < 0.05; **P < 0.01; vs. HF group by ANOVA.

Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
77
Fig. 5. Metabolic studies in 12b-treated and untreated DIO rat model access to HF diet. A, Blood glucose levels after 8 weeks of treatment in C57BL/6J rats with vehicle (HF) or 12b at
the state of feeding with high-fat diet. B, Serum triglyceride levels after 8 weeks of treatment in C57BL/6J rats with vehicle (HF) or 12b at the state of feeding with high-fat diet. C,
Serum total cholesterol levels after 8 weeks of treatment in C57BL/6J rats with vehicle (HF) or 12b at the state of feeding with high-fat diet. D, Serum alanine aminotransferase levels
after 8 weeks of treatment in C57BL/6J rats with vehicle (HF) or 12b at the state of feeding with high-fat diet. *P < 0.05; **P < 0.01; ***P < 0.001 vs. HF group by ANOVA.
to afford 6b (65.6 mg, 68.4% yield). 1H NMR (DMSO-d6, 400 MHz):
d
2.57 (t, 2H, J ¼ 6.0 Hz), 2.69 (t, 2H, J ¼ 6.0 Hz), 7.36e7.45 (m, 2H),
7.54 (d, 1H, J ¼ 8.0 Hz), 7.59 (s, 1H), 7.82 (d, 1H, J ¼ 7.6 Hz), 12.23 (s,
1H), 12.33 (s, 1H); MS (ESI), m/z: 309.30 [M ꢃ H]ꢃ.
5.1.4.1. 4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)-4-oxobutanoic acid
(6a). Yield 72.5%. 1H NMR (DMSO-d6, 400 MHz):
d 2.57 (t, 2H,
J ¼ 6.8 Hz), 2.69 (t, 2H, J ¼ 6.8 Hz), 6.48e7.51 (m, 2H), 7.67 (s, 1H),
7.79 (d, 1H, J ¼ 8.8 Hz), 7.90 (d, 1H, J ¼ 8.4 Hz), 12.32 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz):
d 28.80, 30.33, 109.05, 127.82, 129.22, 132.67,
133.62, 147.95, 158.54, 171.14, 174.06; MS (ESI), m/z: 309.27
[M ꢃ H]ꢃ.
5.1.4.2. 4-Oxo-4-(4-p-tolylthiazol-2-ylamino)butanoic acid (6c). Yield
62.8%. 1H NMR (DMSO-d6, 400 MHz):
d 2.30 (s, 3H), 2.56 (t, 2H,
J ¼ 6.4 Hz), 2.68 (t, 3H, J ¼ 6.4 Hz), 7.22 (d, 2H, J ¼ 8.0 Hz), 7.52 (s,1H),
7.77 (d, 2H, J ¼ 8.0 Hz), 12.15 (s, 1H), 12.27 (s, 1H); MS (ESI), m/z:
289.33 [M ꢃ H]ꢃ.
5.1.4.3. 4-Oxo-4-(4-(4-(trifluoromethyl)phenyl)thiazol-2-ylamino)
butanoic acid (6d). Yield 75.2%. 1H NMR (DMSO-d6, 400 MHz):
d
2.58 (t, 3H, J ¼ 6.4 Hz), 2.70 (t, 3H, J ¼ 6.4 Hz), 7.79 (d, 2H,
J ¼ 8.4 Hz), 7.85 (s, 1H), 8.10 (d, 2H, J ¼ 8 Hz), 12.38 (s, 1H); MS (ESI),
m/z: 343.30 [M ꢃ H]ꢃ.
5.1.4.4. 4-((4-(4-Fluorophenyl)thiazol-2-yl)amino)-4-oxobutanoic
acid (6e). Yield 82.5%. 1H NMR (DMSO-d6, 400 MHz):
d 2.57 (t, 2H,
J ¼ 6.4 Hz), 2.69 (t, 2H, J ¼ 6.4 Hz), 7.27 (t, 2H, J ¼ 8.8 Hz), 7.60 (s,1H),
7.91e7.95 (m, 2H), 12.22 (s, 1H), 12.30 (s, 1H); MS (ESI), m/z: 293.27
[M ꢃ H]ꢃ.
Fig. 6. Glucose and insulin tolerance tests in 12b-treated and untreated DIO rat model
access to HF diet. A, Glucose tolerance tests after 8 weeks of treatment in C57BL/6J rats
with vehicle (HF) or 12b at the state of feeding with high-fat diet. B, Insulin tolerance
tests after 8 weeks of treatment in C57BL/6J rats with vehicle (HF) or 12b at the state of
feeding with high-fat diet. *P < 0.05 vs. HF group by ANOVA.
5.1.4.5. 4-(4-(3,4-Dichlorophenyl)thiazol-2-ylamino)-4-oxobutanoic
acid (6f). Yield 43.7%. 1H NMR (DMSO-d6, 400 MHz):
d
2.57 (t, 2H,
J ¼ 6.4 Hz), 2.69 (t, 2H, J ¼ 6.4 Hz), 7.69 (d, 1H, J ¼ 8.4 Hz), 7.83 (s,

78
Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
Fig. 7. H&E staining of liver sections of untreated and 12b-treated DIO rat model access to HF diet. A, H&E staining of liver sections of untreated C57BL/6J rats access to high-fat diet.
B, H&E staining of liver sections of 12b-treated C57BL/6J rats access to high-fat diet.
1H), 7.87 (dd, 1H, J1,2 ¼ 2.0 Hz, J1,3 ¼ 8.4 Hz), 8.13 (d, 1H, J ¼ 2.0 Hz),
12.24 (s, 1H),12.38 (s, 1H); MS (ESI), m/z: 343.24 [M ꢃ H]ꢃ.
(466 mL, 5.88 mmol). After 4 h, the reaction quenched with water,
filtered, and filtrate extracted with EtOAc. The combined organics
were washed with water and dried (NaSO4). The solution was
evaporated to give compound 7b as yellow solid (830 mg, 61.7%
5.1.4.6. 4-(4-(3,4-Difluorophenyl)thiazol-2-ylamino)-4-oxobutanoic
acid (6g). Yield 48.5%. 1H NMR (DMSO-d6, 400 MHz):
d
2.57 (t, 2H,
Yield). 1H NMR (DMSO-d6, 400 MHz):
d 4.44 (s, 2H), 8.07 (s, 1H),
J ¼ 6.4 Hz), 2.69 (t, 2H, J ¼ 6.4 Hz), 7.48 (q, 1H, J1,2 ¼ 8.4 Hz,
J1,3 ¼ 10.4 Hz), 7.72 (s, 1H), 7.74e7.77 (m, 1H), 7.88e7.93 (m, 1H),
12.24 (s, 1H), 12.34 (s, 1H); MS (ESI), m/z: 311.26 [M ꢃ H]ꢃ.
8.16 (d, 2H, J ¼ 8.8 Hz), 8.31 (d, 2H, J ¼ 9.2 Hz), 12.78 (s, 1H); MS
(ESI), m/z: 296.52 [M ꢃ H]ꢃ.
5.1.6. General procedure for the synthesis of 2-(2-(4-(4-nitrophenyl)
thiazol-2-ylamino)-2-oxoethylthio)acetic acid
5.1.4.7. 4-(4-(4-Methoxyphenyl)thiazol-2-ylamino)-4-oxobutanoic
acid (6h). Yield 55.8%. 1H NMR (DMSO-d6, 400 MHz):
d
2.56 (t, 2H,
A mixture of 2-chloro-N-(4-(4-nitrophenyl)thiazol-2-yl)-acet-
J ¼ 6.4 Hz), 2.68 (t, 2H, J ¼ 6.4 Hz), 5.98 (d, 2H, J ¼ 8.4 Hz), 7.44 (s,
1H), 7.81 (d, 2H, J ¼ 8.4 Hz), 12.22 (s, 1H), 12.26 (s, 1H); MS (ESI), m/
z: 305.22 [M ꢃ H]ꢃ.
amide (830 mg, 2.79 mmol), 2-mercaptoacetic acid (966.95 mL,
13.95 mmol) and Et3N (1.06 mL, 0.5.58 mmol) in anhydrous THF
(42 mL) stirred at 35 ꢀC overnight. The reaction quenched with
water, filtered, and filtrate extracted with EtOAc. The combined
organics were washed with water and dried (NaSO4). The solution
was evaporated to give compound 8b as yellow solid (450 mg,
5.1.4.8. 4-(4-(2,4-Dichlorophenyl)thiazol-2-ylamino)-4-oxobutanoic
acid (6i). Yield 45.6%. 1H NMR (DMSO-d6, 400 MHz):
d 2.58 (t, 2H,
J ¼ 6.4 Hz), 2.70 (t, 2H, J ¼ 6.4 Hz), 7.53 (dd, 1H, J1,2 ¼ 2.0 Hz,
J1,3 ¼ 8.4 Hz), 7.66 (s, 1H), 7.33 (d, 1H, J ¼ 2.0 Hz), 7.86 (d, 1H,
J ¼ 8.4 Hz), 12.37 (s, 1H); MS (ESI), m/z: 343.20 [M ꢃ H]ꢃ.
38.1% Yield). 1H NMR (DMSO-d6, 400 MHz):
d 3.44 (s, 2H), 3.66 (s,
2H), 8.03 (s, 1H), 8.15 (d, 2H, J ¼ 8.4 Hz), 8.30 (d, 2H, J ¼ 8.4 Hz),
12.54 (s, 1H), 12.72 (s, 1H); MS (ESI), m/z: 352.02 [M ꢃ H]ꢃ.
5.1.4.9. 4-(5-Methyl-4-phenylthiazol-2-ylamino)-4-oxobutanoic acid
5.1.6.1. 2-(2-(4-(4-Chlorophenyl)thiazol-2-ylamino)-2-oxoethylthio)
(6j). Yield 66.5%. 1H NMR (DMSO-d6, 400 MHz):
d
2.46 (s, 3H), 2.56
acetic acid (8a). Yield 45.8%. 1H NMR (DMSO-d6, 400 MHz):
d 3.56
(t, 2H, J ¼ 6.4 Hz), 2.66 (t, 2H, J ¼ 6.4 Hz), 7.34 (d, 1H, J ¼ 7.6 Hz), 7.45
(t, 2H, J ¼ 7.6 Hz), 7.63 (d, 2H, J ¼ 7.6 Hz), 12.13 (s, 1H), 12.22 (s, 1H);
MS (ESI), m/z: 289.35 [M ꢃ H]ꢃ.
(d, 2H, J ¼ 5.6 Hz), 3.68 (s, 2H), 7.49 (d, 2H, J ¼ 8.8 Hz), 7.72 (s, 1H),
7.90 (d, 2H, J ¼ 8.8 Hz), 12.44 (s, 1H), 12.68 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz):
d 34.25, 34.70, 109.45, 127.82, 129.23, 132.75, 133.54,
148.11, 158.39, 168.49, 171.36; MS (ESI), m/z: 341. 82 [M ꢃ H]ꢃ.
5.1.4.10. 4-((4-(4-Chlorophenyl)-5-methylthiazol-2-yl)amino)-4-
oxobutanoic acid (6k). Yield 54.8%. 1H NMR (DMSO-d6, 400 MHz):
5.1.6.2. 2-(2-(4-(4-Fluorophenyl)thiazol-2-ylamino)-2-oxoethylthio)
d
2.46 (s, 3H), 2.55 (t, 2H, J ¼ 6.4 Hz), 2.65 (t, 2H, J ¼ 6.4 Hz), 7.49 (d,
acetic acid (8c). Yield 38.7%.1H NMR (DMSO-d6, 400 MHz):
d 3.68 (s,
2H, J ¼ 8.8 Hz), 7.65 (d, 2H, J ¼ 8.4 Hz),12.15 (s, 1H),12.25 (s,1H); 13C
2H), 3.82 (s, 2H), 7.27 (t, 2H, J ¼ 8.8 Hz), 7.64 (s, 1H), 7.91 (dd,
J1,2 ¼ 5.2 Hz, J1,3 ¼ 8.0 Hz), 12.42 (s, 1H). 12.69 (s, 1H); MS (ESI), m/z:
325.10 [M ꢃ H]ꢃ.
NMR (DMSO-d6, 100 MHz):
d 12.28, 28.84, 30.27, 121.94, 128.86,
130.05, 132.25, 134.22, 143.15, 154.52, 170.79, 174.05; MS (ESI), m/z:
323.27 [M ꢃ H]ꢃ.
5.1.6.3. 2-((2-Oxo-2-((4-(4-(trifluoromethyl)phenyl)thiazol-2-yl)
amino)ethyl)thio)acetic acid (8d). Yield 32.4%. 1H NMR (DMSO-d6,
5.1.4.11. 4-((4-(Naphthalen-2-yl)thiazol-2-yl)amino)-4-oxobutanoic
acid (6l). Yield 76.4%. 1H NMR (DMSO-d6, 400 MHz):
d
2.59 (t, 2H,
400 MHz):
d
3.44 (s, 2H), 3.56 (s, 2H), 7.79 (d, 2H, J ¼ 8.4 Hz), 7.89 (s,
J ¼ 6.4 Hz), 2.71 (t, 2H, J ¼ 6.4 Hz), 7.50e7.56 (m, 2H), 7.77 (s, 1H),
7.92e7.98 (m, 3H), 8.0 (d, 1H, J ¼ 8.4 Hz), 12.26 (s, 1H), 12.39 (s, 1H);
MS (ESI), m/z: 325.34 [M ꢃ H]ꢃ.
1H), 8.10 (d, 2H, J ¼ 8.0 Hz), 12.49 (s, 1H), 12.68 (s, 1H); MS (ESI), m/
z: 375.00 [M ꢃ H]ꢃ.
5.1.7. General procedure for the synthesis of 1-benzyl-1H-indole-3-
carbaldehyde
A mixture of 1H-indole-3-carbaldehyde(1 g, 6.9 mmol) and KOH
(570.86 mg, 10.2 mmol) in EtOH (66 mL) was stirred at room
temperature when the solid was dissolved. The solvent was evap-
orated in vacuo, then added acetone (66 mL) and 1-(bromomethyl)-
5.1.5. General procedure for the synthesis of 2-chloro-N-(4-(4-
nitrophenyl)thiazol-2-yl)-acetamide
A mixture of 4-(4-nitrophenyl)thiazol-2-amine (1 g, 4.52 mmol)
and Et3N (1.28 mL, 6.72 mmol) in the DMF (30 mL) stirred below
10 ꢀC, then followed by slow addition of 2-chloroacetyl chloride

Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
79
2-fluorobenzene (1.22 mL, 10.2 mmol). The mixture was stirred at
5.1.8.7. (E)-3-(1-(4-Nitrobenzyl)-1H-indol-3-yl)acrylic acid (12h).
Yield 45.6%. 1H NMR (DMSO-d6, 400 MHz):
5.65 (s, 2H), 6.34 (d,
1H, J ¼ 16 Hz), 7.19e7.24 (m, 2H), 7.44 (d, 2H, J ¼ 8.4 Hz), 7.49 (dd,
1H, J1,2 ¼ 2.8 Hz, J1,3 ¼ 7.2 Hz), 7.77 (d, 1H, J ¼ 16 Hz), 7.89 (dd, 1H,
J1,2 ¼ 2.0 Hz, J1,3 ¼ 5.6 Hz), 8.12 (s, 1H), 8.18 (d, 1H, J ¼ 8.8 Hz), 12.05
(s, 1H); MS (ESI), m/z: 321.27 [M ꢃ H]ꢃ.
room temperature for 20 min, filtered, the filtrate was concentrated
under reduced pressure. The residue was added a little water and
put it in the refrigerator overnight. The precipitate was washed
with water and vacuum dried to afford 11a (636 mg, 39.2%). 1H
d
NMR (DMSO-d6, 400 MHz):
d 5.36 (s, 2H), 7.17e7.20 (m, 2H),
7.29e7.37 (m, 5H), 7.71 (s, 1H), 8.32e8.34 (m, 1H), 10.00 (s, 1H); MS
(ESI), m/z: 234.28 [M ꢃ H]ꢃ.
5.1.8.8. (E)-3-(1-(4-Cyanobenzyl)-1H-indol-3-yl)acrylic acid (12i).
Yield 58.0%. 1H NMR (DMSO-d6, 400 MHz):
d 5.60 (s, 2H), 6.33 (d,
5.1.8. General procedure for the synthesis of (E)-3-(1-benzyl-1H-
indol-3-yl)acrylic acid
1H, J ¼ 16 Hz), 7.19e7.25 (m, 2H), 7.37 (d, 2H, J ¼ 8.0 Hz), 7.50 (d, 1H,
J ¼ 8.4 Hz), 7.80 (t, 3H, J ¼ 8.0 Hz), 7.89 (d, 1H, J ¼ 8.0 Hz), 8.12 (s,
1H), 11.98 (s, 1H); MS (ESI), m/z: 301.34 [M ꢃ H]ꢃ.
A mixture of 1-benzyl-1H-indole-3-carbaldehyde (300 mg,
1.28 mmol) and malonic acid (332.28 mg, 3.19 mmol) in pyridine
(9 mL) and piperidine (0.12 mL) was stirred at 100 ꢀC for 12 h. After
cooling to room temperature, the solution was added water (9 mL)
and acidified with 6 N HCl while the precipitate produced. The
reaction mixture was filtered, the solid was washed with water and
EtOH. The product was recrystallised from acetone and PE as yellow
5.1.8.9. (E)-3-(1-(3-Fluorobenzyl)-2-methyl-1H-indol-3-yl)acrylic
acid (12j). Yield 78.2%. 1H NMR (DMSO-d6, 400 MHz):
d 5.98 (s, 2H),
6.31 (d, 1H, J ¼ 16 Hz), 6.88 (d, 1H, J ¼ 6.8 Hz), 7.20e7.25 (m, 2H),
7.41 (t, 1H, J ¼ 8.0 Hz), 7.55e7.63 (m, 3H), 7.77 (d, 1H, J ¼ 16 Hz),
7.88e7.93 (m, 2H), 7.98 (d, 1H, J ¼ 1.6 Hz), 8.00 (s, 1H), 8.16 (d, 1H,
J ¼ 8.4 Hz), 11.94 (s, 1H); MS (ESI), m/z: 326.00 [M ꢃ H]ꢃ.
solid. Yield 72.5%. 1H NMR (DMSO-d6, 400 MHz):
d 5.47 (s, 2H), 6.31
(d,1H, J ¼ 16.4 Hz), 7.19 (t, 2H, J ¼ 7.2 Hz), 7.25 (t, 2H, J ¼ 7.2 Hz), 7.28
(s, 1H), 7.33 (t, 2H, 7.2 Hz), 7.54 (d, 1H, J ¼ 8.0 Hz), 7.78 (d, 1H,
J ¼ 16 Hz), 7.87 (d, 1H, J ¼ 7.6 Hz), 8.12 (s, 1H), 11.95 (s, 1H); 13C NMR
5.1.8.10. (E)-3-(1-(4-Fluorobenzyl)-1H-indol-3-yl)acrylic acid (12k).
Yield 68.4%. 1H NMR (DMSO-d6, 400 MHz):
d 5.40 (s, 2H), 6.25 (d,
(DMSO-d6, 100 MHz):
d
49.85, 111.64, 111.80, 113.26, 120.55, 121.67,
1H, J ¼ 16.0 Hz), 6.83 (dd, 1H, J1,2 ¼ 2.4 Hz, J1,3 ¼ 8.8 Hz), 7.15 (t, 2H,
J ¼ 8.8 Hz), 7.28e7.31 (m, 3H, JHH ¼ 6.0 Hz, JHF ¼ 7.6 Hz), 7.43 (d, 1H,
J ¼ 8.8 Hz), 7.76 (d, 1H, J ¼ 16.0 Hz), 8.06 (s, 1H); MS (ESI), m/z:
294.36 [M ꢃ H]ꢃ.
123.13, 126.22, 127.61, 128.07, 129.11, 134.61, 137.56, 137.79, 138.26,
169.00; MS (ESI), m/z: 276.41 [M ꢃ H]ꢃ.
5.1.8.1. (E)-3-(1-(2-Fluorobenzyl)-1H-indol-3-yl)acrylic acid (12b).
Yield 66.7%. 1H NMR (DMSO-d6, 400 MHz):
d
5.53 (s, 2H), 6.31 (d,
5.1.9. General procedure for the synthesis of 5-methyl-1H-indole-3-
carbaldehyde
1H, J ¼ 16 Hz), 7.14e7.19 (m, 2H, JHH ¼ 7.6 Hz, JHF ¼ 8.0 Hz),
7.21e7.27 (m, 3H), 7.34e7.37 (m, 1H), 7.56 (d, 1H, J ¼ 8.0 Hz), 7.77 (d,
1H, J ¼ 16 Hz), 7.88 (d, 1H, J ¼ 7.6 Hz), 8.05 (s, 1H), 11.97 (s, 1H); MS
(ESI), m/z: 294.36 [M ꢃ H]ꢃ.
To a 100 mL three-necked round flask was introduced anhydrous
N,N-dimethylformamide (4.2 mL, 34.35 mmol) at 0 ꢀC under argon
followed by slow addition of phosphorus oxychloride (1.3 mL,
13.62 mmol). Solution was mixed at 0 ꢀC for 40 min. A solution of 5-
methyl-1H-indole (1.627 g,12.41 mmol) in 2.5 mL of DMF was slowly
added maintaining the temperature below 10 ꢀC. The solution was
stirred for 40 min at 0 ꢀC and then 35 ꢀC for additional 40 min. Then
ice was added to the flask and a solution of sodium hydroxide (5.5 g,
137.25 mmol dissolved in 14.6 mL of water) was introduced using
a dropping funnel. Solution was vigorously stirred during the addi-
tion, then heated to 100 ꢀC for 30 min and left to reach room
temperature. The brown precipitate was filtered off and washed
with large volumes of water. The powder was dried in vacuum. Yield
5.1.8.2. (E)-3-(1-(3-Fluorobenzyl)-1H-indol-3-yl)acrylic acid (12c).
Yield 78.8%.1H NMR (DMSO-d6, 400 MHz):
d 5.30 (s, 2H), 6.44 (d,1H,
J ¼ 15.6 Hz), 6.82 (d,1H, J ¼ 9.2 Hz), 6.91 (d,1H, 7.6 Hz), 6.98e7.01 (m,
1H), 7.28e7.32 (m, 4H, JHH ¼ 8.0 Hz, JHF ¼ 9.2 Hz), 7.46 (s, 1H),
7.95e7.98 (m, 1H), 8.01 (s, 1H); MS (ESI), m/z: 294.36 [M ꢃ H]ꢃ.
5.1.8.3. (E)-3-(1-(2-Methylbenzyl)-1H-indol-3-yl)acrylic acid (12d).
Yield 80.5%. 1H NMR (DMSO-d6, 400 MHz):
d 2.36 (s, 3H), 5.47 (s,
3H), 6.32 (d, 1H, J ¼ 16 Hz), 6.58 (d, 1H, J ¼ 7.6 Hz), 7.07 (t, 1H,
J ¼ 7.2 Hz), 7.16e7.22 (m, 4H), 7.46e7.49 (m, 1H), 7.78 (d, 1H,
J ¼ 16 Hz), 7.9e7.92 (m, 2H), 11.96 (s, 1H); MS (ESI), m/z: 290.08
[M ꢃ H]ꢃ.
71.5%. 1H NMR (DMSO-d6, 400 MHz):
d 2.41 (s, 3H), 7.07 (d, 1H,
J ¼ 8.4 Hz), 7.38 (d,1H, J ¼ 8.0 Hz), 7.90 (s,1H), 8.32 (s,1H), 9.89 (s,1H),
12.01 (s, 1H); MS (ESI), m/z: 158.10 [M ꢃ H]ꢃ.
5.1.8.4. (E)-3-(1-(4-Methylbenzyl)-1H-indol-3-yl)acrylic acid (12e).
5.1.10. General procedure for the synthesis of 1-(2-fluorobenzyl)-5-
methyl-1H-indole-3-carbaldehyde
Yield 60.0%. 1H NMR (DMSO-d6, 400 MHz):
d
2.24 (s, 3H), 5.40 (s,
2H), 6.29 (d,1H, J ¼ 16 Hz), 7.11e7.23 (m, 6H), 7.53 (d,1H, J ¼ 8.0 Hz),
7.77 (d, 1H, 16 Hz), 7.85 (d, 1H, J ¼ 7.2 Hz), 8.09 (s, 1H), 11.96 (s, 1H);
MS (ESI), m/z: 290.07 [M ꢃ H]ꢃ.
A mixture of 5-methyl-1H-indole-3-carbaldehyde (500 mg,
3.14 mmol) and KOH (263.85 mg, 4.71 mmol) in EtOH (35 mL) was
stirred at room temperature when the solid was dissolved. The solvent
was evaporated in vacuo, then added acetone (35 mL) and 1-(bromo-
5.1.8.5. (E)-3-(1-(4-Isopropylbenzyl)-1H-indol-3-yl)acrylic acid (12f).
methyl)-2-fluorobenzene (568 mL, 4.71 mmol). The mixture was stirred
Yield 65.8%. 1H NMR (DMSO-d6, 400 MHz):
d
1.14 (s, 3H), 1.15 (s, 3H),
at room temperature for 20 min, filtered, the filtrate was concentrated
under reduced pressure. The residue was added a little water and put it
in the refrigerator overnight. The precipitate was washed with water
and vacuum dried to afford 15a (580 mg, 69.0% Yield).1H NMR (DMSO-
2.83 (t,1H, J ¼ 6.8 Hz), 5.41 (s, 2H), 6.30(d,1H, J ¼ 16 Hz), 7.17e7.25 (m,
6H),7.57(d,1H, J¼ 7.6Hz),7.77(d,1H, J¼ 16 Hz), 7.86 (d, 1H, J¼ 7.6 Hz),
8.10 (s, 1H), 11.94 (s, 1H); MS (ESI), m/z: 318.03 [M ꢃ H]ꢃ.
d6, 400 MHz): d 2.40 (s, 3H), 5.57 (s, 2H), 7.10e7.18 (m, 2H), 7.20e7.27
5.1.8.6. (E)-3-(1-(4-(tert-Butyl)benzyl)-1H-indol-3-yl)acrylic
acid
(m, 2H), 7.36 (d, 1H, J ¼ 7.2 Hz), 7.46 (d, 1H, J ¼ 8.0 Hz), 7.93 (s, 1H),
(12g). Yield 77.6%. 1H NMR (DMSO-d6, 400 MHz):
d
1.22 (s, 9H), 5.41
8.34 (s, 1H), 9.91 (s, 1H); MS (ESI), m/z: 266.20 [M ꢃ H]ꢃ.
(s, 2H), 6.30 (d, 1H, J ¼ 16 Hz), 7.17e7.25 (m, 4H), 7.32 (d, 1H, 8.0 Hz),
7.75 (d, 1H, J ¼ 16 Hz), 7.86 (d, 1H, J ¼ 7.6 Hz), 8.10 (s, 1H), 11.94 (s,
5.1.11. General procedure for the synthesis of (E)-3-(1-(2-
fluorobenzyl)-5-methyl-1H-in-dol-3-yl)acrylic acid
1H); 13C NMR (DMSO-d6, 100 MHz):
d
31.49, 34.64, 49.49, 111.65,
111.76, 113.20, 120.52, 121.62, 123.10, 125.84, 126.20, 127.41, 134.54,
A
mixture of 1-(2-fluorobenzyl)-5-methyl-1H-indole-3-
137.56, 138.26, 150.47, 168.99; MS (ESI), m/z: 332.07[M ꢃ H]ꢃ.
carbaldehyde (580 mg, 2.17 mmol) and malonic acid (565 mg,

80
Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
5.42 mmol) in pyridine (16 mL) and piperidine (0.25 mL) was stirred
at 100 ꢀC for 12 h. After cooling to room temperature, the solution
was added water (16 mL) and acidified with 6 N HCl while the
precipitate produced. The reaction mixture was filtered, the solid
was washed with water and EtOH. The product was recrystallised
from acetone and PE as yellow solid. Yield 44.6%.1H NMR (DMSO-d6,
for 30 min. The solution was stirred at 0 ꢀC followed by slow
addition of 1-(bromomethyl)benzene (1.80 mL, 13.7 mmol) for
another 1 h. After the reaction was completed, put the mixture
into water (150 mL) and acidified with 4 N HCl while the
precipitate produced. The white precipitate was filtered off,
washed with water and EtOH, vacuum dried to afford 18a (2.49 g,
400 MHz):
d
2.42 (s, 3H), 5.48 (s, 2H), 6.29 (d,1H, J ¼ 16.0 Hz), 7.06 (d,
82.3% Yield). 1H NMR (CDCl3, 400 MHz):
d 3.66 (s, 2H), 5.38 (s, 2H),
1H, J ¼ 8.4 Hz), 7.09e7.16 (m, 2H, JHH ¼ 6.0 Hz, JHF ¼ 7.6 Hz), 7.23 (t,1H,
J ¼ 9.2 Hz), 7.34 (t,1H, J ¼ 6.8 Hz), 7.42 (d,1H, J ¼ 8.8 Hz), 7.68 (s,1H),
7.74 (d,1H, J ¼ 16.0 Hz), 7.97 (s,1H),12.01 (s,1H); 13C NMR (DMSO-d6,
7.01 (t, 1H, J ¼ 7.2 Hz), 7.10 (t, 1H, J ¼ 7.6 Hz), 7.19 (d, 2H,
J ¼ 7.6 Hz), 7.23 (d, 1H, J ¼ 7.2 Hz), 7.30 (t, 2H, J ¼ 7.2 Hz), 7.41 (t,
2H, J ¼ 8.8 Hz), 7.50 (d, 1H, J ¼ 7.6 Hz), 12.22 (s, 1H); 13C NMR
100 MHz):
d
21.59, 43.99, 111.05, 111.55, 113.09, 115.88, 120.38,
(DMSO-d6, 100 MHz): d 31.32, 49.38, 108.14, 110.51, 119.22, 119.45,
124.53, 125.15, 125.18, 126.33, 130.00, 134.69, 135.97, 138.29, 148.79,
121.78, 127.56, 127.79, 128.12, 128.99, 136.39, 138.78, 173.52; MS
159.20, 161.64, 169.02; MS (ESI), m/z: 308.00 [M ꢃ H]ꢃ.
(ESI), m/z: 264.31 [M ꢃ H]ꢃ.
5.1.11.1. (E)-3-(1-(2-Fluorobenzyl)-5-methoxy-1H-indol-3-yl)acrylic
5.1.12.1. 2-(1-(2-Methyl)-1H-indol-3-yl)acetic acid (18b). Yield 85.2%.
acid (16b). Yield 56.2%. 1H NMR (DMSO-d6, 400 MHz):
d
(d, 3H,
1H NMR (DMSO-d6, 400 MHz):
d 2.30 (s, 3H), 3.66 (s, 2H), 5.37 (s, 2H),
J ¼ 9.6 Hz), 5.48 (s, 2H), 6.25 (d, 1H, J ¼ 16.0 Hz), 6.86 (dd, 1H,
J1,2 ¼ 2.0 Hz, J1,3 ¼ 9.2 Hz), 7.11e7.16 (m, 2H, JHH ¼ 7.6 Hz,
JHF ¼ 7.6 Hz), 7.23 (t, 1H, J ¼ 9.2 Hz), 7.29 (d, 1H, J ¼ 2.4 Hz),
7.32e7.36 (m,1H), 7.44 (d, 1H, J ¼ 8.8 Hz), 7.76 (d, 1H, J ¼ 16.0 Hz),
8.00 (s, 1H), 11.91 (s, 1H); MS (ESI), m/z: 324.00 [M ꢃ H]ꢃ.
6.57 (d, 1H, J ¼ 7.6 Hz), 7.01e7.10 (m, 3H), 7.11e7.19 (m, 2H), 7.21 (s,
1H), 7.35 (d, 1H, J ¼ 8.4 Hz), 7.53 (d, 1H, J ¼ 7.6 Hz), 12.28 (s, 1H); MS
(ESI), m/z: 278.45 [M ꢃ H]ꢃ.
5.1.12.2. 2-(1-(2-Fluorobenzyl)-1H-indol-3-yl)acetic acid (18c). 1H
NMR (DMSO-d6, 400 MHz):
d 3.65 (s, 2H), 5.44 (s, 2H), 7.03 (T, 1H,
5.1.11.2. (E)-3-(1-(2-Fluorobenzyl)-2-methyl-1H-indol-3-yl)acrylic
J ¼ 7.6 Hz), 7.07e7.14(m, 3H, JHH ¼ 7.6 Hz, JHF ¼ 9.2 Hz), 7.22 (t, 1H,
J ¼ 9.2 Hz), 7.30e7.34 (m, 1H), 7.35 (s, 1H), 7.44 (d, 1H, J ¼ 8.4 Hz),
7.51 (d, 1H, J ¼ 7.6 Hz), 12.23 (s, 1H); MS (ESI), m/z: 282.30 [M ꢃ H]ꢃ.
acid (16c). Yield 74.9%. 1H NMR (CDCl3, 400 MHz):
d 2.51 (s, 3H),
5.35 (s, 2H), 6.48 (d, 1H, J ¼ 16.0 Hz), 6.68 (d, 1H, J ¼ 9.2 Hz), 6.75 (d,
1H, J ¼ 8.0 Hz), 6.93e6.98 (m, 1H), 7.24e7.30 (m, 4H, JHH ¼ 8.0 Hz,
JHF ¼ 8.4 Hz), 7.96 (d, 1H, J ¼ 7.6 Hz), 8.09 (d, 1H, J ¼ 16.0 Hz), 12.01
5.1.12.3. 2-(1-(3-Fluorobenzyl)-1H-indol-3-yl)acetic acid (18d).
(s,1H); 13C NMR (DMSO-d6, 100 MHz):
d
10.81, 46.05,108.94,110.92,
Yield 88.6%. 1H NMR (DMSO-d6, 400 MHz):
d 3.67 (s, 2H), 5.41 (s,
112.67, 113.50, 114.55, 120.11, 121.94, 122.62, 124.36, 125.72, 131.26,
137.47, 140.81, 142.66, 150.05, 161.57, 164.60, 169.16; MS (ESI), m/z:
308.04 [M ꢃ H]ꢃ.
2H), 7.00e7.05 (m, 3H, JHH ¼ 7.6 Hz, JHF ¼ 7.2 Hz), 7.07 (d, 1H,
J ¼ 1.6 Hz), 7.11 (t, 1H, J ¼ 7.2 Hz), 7.32 (q, 1H, J1,2 ¼ 8.0 Hz,
J1,3 ¼ 14.4 Hz), 7.42 (s, 1H), 7.44 (s, 1H), 7.51 (d, 1H, J ¼ 8.0 Hz), 12.25
(s, 1H); MS (ESI), m/z: 282.30 [M ꢃ H]ꢃ.
5.1.11.3. (E)-3-(1-(3-Fluorobenzyl)-5-methyl-1H-indol-3-yl)acrylic
acid (16d). Yield 68.3%. 1H NMR (DMSO-d6, 400 MHz):
d
2.42 (s,
5.1.12.4. 2-(1-(4-Fluorobenzyl)-1H-indol-3-yl)acetic
acid
3.65 (s, 2H),
3H), 5.45 (s, 2H), 6.30 (d, 1H, J ¼ 16.0 Hz), 7.03e7.12 (m, 4H,
JHH ¼ 6.8 Hz, JHF ¼ 7.2 Hz), 7.33 (q, 1H, J1,2 ¼ 7.6 Hz, J1,3 ¼ 14.0 Hz),
7.42 (d, 1H, J ¼ 8.4 Hz), 7.68 (s, 1H), 7.75 (d, 1H, J ¼ 16.0 Hz), 8.05 (s,
1H), 11.96 (s, 1H); MS (ESI), m/z: 308.04 [M ꢃ H]ꢃ.
(18e). Yield 80.5%. 1H NMR (DMSO-d6, 400 MHz):
d
5.37 (s, 2H), 7.01 (t, 1H, J ¼ 7.2 Hz), 7.08 (d, 1H, J ¼ 1.6 Hz), 7.10e7.16
(m, 2H, JHH ¼ 8.8 Hz, JHF ¼ 9.2 Hz), 7.24e7.28 (m, 2H), 7.40 (s, 1H),
7.43 (d,1H, J ¼ 8.4 Hz), 7.50 (d,1H, J ¼ 7.6 Hz),12.23 (s,1H); MS (ESI),
m/z: 282.30 [M ꢃ H]ꢃ.
5.1.11.4. (E)-3-(1-(3-Fluorobenzyl)-5-methoxy-1H-indol-3-yl)acrylic
acid (16e). Yield 76.7%. 1H NMR (DMSO-d6, 400 MHz):
d 3.79 (s,
3H), 5.44 (s, 2H), 6.25 (d, 1H, J ¼ 16.0 Hz), 6.84 (dd, 1H, J1,2 ¼ 2.4 Hz,
J1,3 ¼ 8.8 Hz), 7.03e7.12 (m, 3H, JHH ¼ 8.0 Hz, JHF ¼ 8.0 Hz), 7.29 (d,
1H, J ¼ 2.0 Hz), 7.33 (q, 1H, J1,2 ¼ 7.6 Hz, J1,3 ¼ 14. 0 Hz), 7.43 (d, 1H,
J ¼ 8.8 Hz), 7.77 (d, 1H, J ¼ 16.0 Hz), 8.08 (s, 1H), 11.91 (s, 1H); MS
(ESI), m/z: 324.00 [M ꢃ H]ꢃ.
5.2. Biological methods
5.2.1. TNF-a inhibition assay
RAW 264.7 cells were seeded in wells and incubated for 24 h.
After incubation, the cells were incubated with all compounds of
different concentrations in the presence of LPS (1
mg/mL) for 24 h.
5.1.11.5. (E)-3-(1-(4-Fluorobenzyl)-5-methoxy-1H-indol-3-yl)acrylic
The TNF- concentration in the culture medium was determined by
ELISA kit (R&D Systems, Minneapolis, MN).
a
acid (16f). Yield 66.4%. 1H NMR (DMSO-d6, 400 MHz):
d
3.79 (s, 3H),
5.40 (s, 2H), 6.25 (d, 1H, J ¼ 16.0 Hz), 6.83 (dd, 1H, J1,2 ¼ 2.4 Hz,
J1,3 ¼ 8.8 Hz), 7.15 (t, 2H, J ¼ 8.8 Hz), 7.28e7.31 (m, 3H, JHH ¼ 6.0 Hz,
JHF ¼ 7.6 Hz), 7.43 (d, 1H, J ¼ 8.8 Hz), 7.76 (d, 1H, J ¼ 16.0 Hz), 8.06 (s,
1H); MS (ESI), m/z: 324.00 [M ꢃ H]ꢃ.
5.2.2. Apparent Ki values
1,8-ANS was dissolved in absolute ethanol and diluted with
25 mM TriseHCl (pH 7.4) to a final concentration of 5 M (final
EtOH concentration of 0.05%). Rat ap2 protein was titrated into
500 L 1,8-ANS and the fluorescence enhancement was measured
m
5.1.11.6. (E)-3-(1-(3-Fluorobenzyl)-2-methyl-1H-indol-3-yl)acrylic
m
acid (16g). Yield 65.3%. 1H NMR (CDCl3, 400 MHz):
d
2.53 (s, 3H),
using a PerkineElmer 650-10S fluorescence spectrophotometer
with 4 nm excitation and emission slit widths. 12b (diluted from
a 10 mM stock in absolute ethanol) was added to the ap2/1,8-ANS
complex, mixed for 45 s, and the fluorescence signal was record.
The decay in corrected fluorescence intensity as a function of
competitor concentration was used to determine the midpoint of
the competition (I50).
An apparent Ki value was calculated using Ki ¼ [I50]/(1 þ [L]/Kd),
where [L] ¼ free concentration of 1,8-ANS, and Kd ¼ apparent
dissociation constant of ap2 for 1,8-ANS.
5.41 (s, 2H), 6.48 (d, 1H, J ¼ 16.0 Hz), 6.51 (d, 1H, J ¼ 14.4 Hz), 6.97 (t,
1H, J ¼ 7.6 Hz), 7.12 (t,1H, J ¼ 8.8 Hz), 7.22e7.29 (m, 3H, JHH ¼ 7.2 Hz,
JHF ¼ 8.0 Hz), 7.96 (m, 2H), 8.09 (d, 1H, J ¼ 16.0 Hz), 8.86 (d, 1H,
J ¼ 4.8 Hz); MS (ESI), m/z: 308.05 [M ꢃ H]ꢃ.
5.1.12. General procedure for the synthesis of 2-(1-benzyl-1H-indol-
3-yl)acetic acid
A mixture of 2-(1H-indol-3-yl)acetic acid (2 g, 11.4 mmol) and
NaH (1.1 g, 45.8 mmol) in anhydrous DMF (20 mL) stirred at 0 ꢀC

Q. Xu et al. / European Journal of Medicinal Chemistry 52 (2012) 70e81
81
[2] P.C. William, K.S. Jaswinder, TNF-
a
and adipocyte biology, FEBS Lett. 582
5.2.3. Animals
(2008) 117e131.
Male C57BL/6J mice (age range: 7e8 weeks) were purchased
from Western China Experimental Animal Center. The animals were
fed a normal diet or a high-fat diet (kcal: 60% of energy derived from
fat, 20% from protein and 20% from carbohydrates, D12494 Research
Diet, USA) throughout the study, which was used to establish the HF
of diet-induced obesity (DIO). After 8 weeks, the DIO mice were
divided into two groups (control and 12b-treated; n ¼ 8 per group).
The 12b-treated groups were administeredwith 12b orallyeveryday
at a dose of 50 mg/kg. The compound 12b suspended in Tween 80
and 5% saline (SigmaeAldrich, St. Louis, MO). Body weight was
recorded during the study period. After the experimental period,
animals were sacrificed, photographed for morphology of abdomen
adipose, while their inguinal white adipose tissue (WAT), muscle
and whole body tissue composition was extracted for evaluation of
other parameters.
[3] G.S. Hotamisligil, Mechanisms of TNF-alpha-induced insulin resistance, Exp.
Clin. Endocrinol. Diabetes 107 (1999) 119e125.
[4] H. Xu, J. Hirosumi, K.T. Uysal, A.D. Guler, G.S. Hotamisligil, Exclusive action of
transmembrane TNF-a in adipose tissue leads to reduced adipose mass and
local but not systemic insulin resistance, Endocrinology 143 (2002)
1502e1511.
[5] K.T. Uysal, S.M. Wiesbrock, M.W. Marino, G.S. Hotamisligil, Protection from
obesity-induced insulin resistance in mice lacking TNF-a function, Nature 389
(1997) 610e614.
[6] K.T. Uysal, S.M. Wiesbrock, G.S. Hotamisligil, Functional analysis of tumor
necrosisfactor (TNF) receptors in TNF- -mediated insulin resistance in genetic
a
obesity, Endocrinology 139 (1998) 4832e4838.
[7] L. Scheja, L. Makowski, K.T. Uysal, S.M. Wiesbrock, D.R. Shimshek, D.S. Meyers,
M. Morgan, R.A. Parker, G.S. Hotamisligil, Altered insulin secretion associated
with reduced lipolytic efficiency in ap2 ꢃ/ꢃ mice, Diabetes 48 (1999)
1987e1994.
[8] L. Makowski, K.C. Brittingham, J.M. Reynolds, J. Suttles, G.S. Hotamisligil,
The fatty acid-binding protein, aP2, coordinates macrophage cholesterol
trafficking and inflammatory activity, J. Biol. Chem. 280 (2005)
12888e12895.
[9] G.S. Hotamisligil, R.S. Johnson, R.J. Distel, R. Ellis, V.E. Papaioannou,
B.M. Spiegelman, Uncoupling of obesity from insulin resistance through
a targeted mutation in aP2, the adipocyte fatty acid binding protein, Science
274 (1996) 1377e1379.
[10] M. Furuhashi, G. Tuncman, C.Z. Görgün, L. Makowski, G. Atsumi,
E. Vaillancourt, K. Kono, V.R. Babaev, S. Fazio, M.F. Linton, R. Sulsky, J.A. Robl,
R.A. Parker, G.S. Hotamisligil, Treatment of diabetes and atherosclerosis by
inhibiting fatty-acid-binding protein aP2, Nature 447 (2007) 959e965.
[11] A.V. Hertzel, K. Hellberg, J.M. Reynolds, A.C. Kruse, B.E. Juhlmann, A.J. Smith,
M.A. Sanders, D.H. Ohlendorf, J. Suttles, D.A. Bernlohr, Identification and
characterization of a small molecule inhibitor of fatty acid binding proteins,
J. Med. Chem. 52 (2009) 6024e6031.
5.2.3.1. Oral glucose tolerance test. Mice were fasted for 12 h before
the OGTT. At 0 min, a drop of blood was taken via a tail nick before
giving the oral glucose load. Mice were then given 5% glucose
(0.5 g/kg). Blood samples were obtained at 15, 30, 60, 90 and
120 min following glucose ingestion. The blood glucose was
determined by a glucose meter.
5.2.3.2. Insulin tolerance test. Mice were fasted for 12 h and then
given an intraperitoneal injection of insulin (2 U/Kg) in saline.
Blood samples were taken at 0, 15, 30, 60, 90 and 120 min for the
determination of blood glucose with a glucose meter.
[12] L. Fredik, H. Saba, A. Eva, M. Carmen, U. Jonas, S. Stefan, L. Thomas, R. Lena,
B. Tjeerd, Discovery of inhibitors of human adipocyte fatty acid-binding
protein,
a potential type 2 diabetes target, Bioorg. Med. Chem. Lett. 14
(2004) 4445e4448.
[13] D. Alexandra, B. Anne, E. Johan, D. Muriel, L.B. Guillaume, G. Nicole, P. Jean-
Yves, New N-pyridinyl(methyl)-indolalkanamides acting as topical inflam-
mation inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 5441e5444.
[14] A. Andreanil, M. Rambaldil, A. Locatellil, G. Pifferi, Synthesis and anti-
flammatory activity of indolylacrylic and methylacrylic acids, Eur. J. Med.
Chem. 29 (1994) 903e906.
[15] S. PengAn, W. Binli, Synthesis ofpmethoxy bromoacetophenone by substituting
cupric bromide for liquid bromine, Liaoning Chem. Ind. 32 (2003) 17e18.
[16] A. Andreani, M. Rambaldi, A. Locatelli, F. Andreani, R. Bossa, I. Galatulas,
M. Ninci, Synthesis and analgesic activity of 1-(N-methylanilinoethyl)indoles,
Eur. J. Med. Chem. 3 (1991) 125e129.
[17] G. Francis, L. Cedric, P. Fabrice, C. Damien, B. Sophie, P. Carine, L.P. Patrice,
L.B. Marc, Design, synthesis and evaluation of 3-(imidazol-1-ylmethyl)
indoles as antileishmanial agents, J. Enzym. Inhib. Med. Chem. 24 (2009)
1067e1075.
[18] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, The protein data bank, Nucleic Acids Res. 28
(2000) 235e242.
5.2.3.3. Plasma measurements. After 8 weeks treatment, blood was
collected from the heart under anaesthesia. Samples were centri-
fuged at 5,000 g for 15min at 4 ꢀC. Plasma was analyzed for total
cholesterol (TC), triglycerides (TG), alanine transaminase (ALT) and
glucose, which were measured with a multifunctional biochemistry
analyzer Olympus AU2700 (Olympus, Tokyo, Japan).
5.2.3.4. Histopathological examination. Liver samples were fixed in
4% buffered formalin and embedded in Tissue-Tek OCT compound
and paraffin for histological analysis. Formalin-fixed and paraffin-
embedded section was processed routinely for H&E staining. The
OCT-embedded samples were serially sectioned at 4 mm.
Acknowledgments
[19] R.E. Gillilan, S.D. Ayers, N. Noy, Structural basis for activation of fatty acid-
binding protein 4, J. Mol. Biol. 372 (2007) 1246e1260.
This study was supported by National Key Technologies R&D
Program (grant number: 2012ZX09103-101-033).
[20] A. Jakalian, B.L. .Bush, D.B. Jack, C.I. Bayly, Fast, efficient generation of high-
quality atomic charges. AM1-BCC HF: I. Method, J. Comput. Chem. 21 (2000)
132e146.
[21] A. Jakalian, D.B. Jack, C.I. Bayly, Fast, efficient generation of high-quality
atomic charges. AM1-BCC HF: II. Parameterization and validation, J. Comput.
Chem. 23 (2002) 1623e1641.
References
[22] T.A. Halgren, Merck molecular force field. I. Basis, form, scope, parame-
terization, and performance of MMFF94, J. Comput. Chem. 17 (1996)
490e519.
[1] F. Masakazu, H. Tetsuya, I. Naoko, Design, synthesis and bioactivities of novel
diarylthiophenes: inhibitors of tumor necrosis factor-a (TNF-a) production,
Bioorg. Med. Chem. 10 (2002) 3113e3122.