Discovery of first-in-class thiazole-based dual FFA1/PPARδ agonists as potential anti-diabetic agents

Article abstract of DOI:10.1016/j.ejmech.2018.12.069

The free fatty acid receptor 1 (FFA1 or GPR40) and peroxisome proliferator-activated receptor δ (PPARδ) have attracted a lot of attention due to their role in promoting insulin secretion and sensibility, respectively, which are two major features of diabetes. Therefore, the dual FFA1/PPARδ agonists would increase insulin secretion and sensibility by FFA1 and PPARδ activation. In this study, we hybrid FFA1 agonist AM-4668 with PPARδ agonist GW501516, leading to the identification of orally bioavailable dual agonist 32, which revealed high selectivity over other PPARs. Moreover, compound 32 exhibited good pharmacokinetic profiles with high plasma concentration, sustained half-life and low clearance in vivo. During the hypoglycemic test, a dual agonist 32 enhanced the tolerance of ob/ob mice for glucose loading in a dose-dependent manner. Our results suggest that dual FFA1/PPARδ agonist could be a valuable therapy for type 2 diabetes.

Full text of DOI:10.1016/j.ejmech.2018.12.069

Accepted Manuscript
Discovery of first-in-class thiazole-based dual FFA1/PPARδ agonists as potential anti-
diabetic agents
Zheng Li, Yueming Chen, Zongtao Zhou, Liming Deng, Yawen Xu, Lijun Hu, Bing Liu,
Luyong Zhang
PII:
S0223-5234(18)31115-2
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.069
EJMECH 11003
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 November 2018
Revised Date: 26 December 2018
Accepted Date: 26 December 2018
Please cite this article as: Z. Li, Y. Chen, Z. Zhou, L. Deng, Y. Xu, L. Hu, B. Liu, L. Zhang, Discovery
of first-in-class thiazole-based dual FFA1/PPARδ agonists as potential anti-diabetic agents, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.069.
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ACCEPTED MANUSCRIPT
Discovery of first-in-class thiazole-based dual FFA1/PPARδ agonists as potential
anti-diabetic agents
1
, 2*
1
1
1
1
1
1,
Zheng Li , Yueming Chen , Zongtao Zhou , Liming Deng , Yawen Xu , Lijun Hu , Bing Liu
2
,3*
1, 2, 3, 4*
,
Luyong Zhang
1
2
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
Key Laboratory of New Drug Discovery and Evaluation of ordinary universities of Guangdong
province, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
3
Guangzhou Key Laboratory of Construction and Application of New Drug Screening Model
Systems, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
4
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009,
PR China.
Graphical Abstract
Aiming to develop potent dual FFA1/PPARδ agonists, we have hybrid the FFA1 agonist AM-4668
with PPARδ agonist GW501516 based on their structural similarity, exemplified by the orally
bioavailable dual FFA1/PPARδ agonist 32.
*
Corresponding author.
E-mail addresses: li.zheng.sky@163.com (Z. Li), liubing52000@163.com (B. Liu), lyzhang@cpu.edu.cn (L.
Zhang).

ACCEPTED MANUSCRIPT
Discovery of first-in-class thiazole-based dual FFA1/PPARδ agonists as potential
anti-diabetic agents
1
, 2*
1
1
1
1
1
1,
Zheng Li , Yueming Chen , Zongtao Zhou , Liming Deng , Yawen Xu , Lijun Hu , Bing Liu
2
,3*
1, 2, 3, 4*
,
Luyong Zhang
1
2
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
Key Laboratory of New Drug Discovery and Evaluation of ordinary universities of Guangdong
province, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
3
Guangzhou Key Laboratory of Construction and Application of New Drug Screening Model
Systems, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
4
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009,
PR China.
Abstract
The free fatty acid receptor 1 (FFA1 or GPR40) and peroxisome proliferator-activated receptor δ
(PPARδ) have attracted a lot of attention due to their role in promoting insulin secretion and
sensibility, respectively, which are two major features of diabetes. Therefore, the dual
FFA1/PPARδ agonists would increase insulin secretion and sensibility by FFA1 and PPARδ
activation. In this study, we hybrid FFA1 agonist AM-4668 with PPARδ agonist GW501516,
leading to the identification of orally bioavailable dual agonist 32, which revealed high selectivity
*
Corresponding author.
E-mail addresses: li.zheng.sky@163.com (Z. Li), liubing52000@163.com (B. Liu), lyzhang@cpu.edu.cn (L.
Zhang).
1

ACCEPTED MANUSCRIPT
over other PPARs. Moreover, compound 32 exhibited good pharmacokinetic profiles with high
plasma concentration, sustained half-life and low clearance in vivo. During the hypoglycemic test,
a dual agonist 32 enhanced the tolerance of ob/ob mice for glucose loading in a dose-dependent
manner. Our results suggest that dual FFA1/PPARδ agonist could be a valuable therapy for type 2
diabetes.
Keywords: Dual agonist; FFA1; Hybrid; PPAR; Diabetes.
1
. Introduction
Type 2 diabetes is usually caused by insufficient insulin production and resistance to insulin.[1, 2]
Agents with pharmacological mechanism on improving the pathogenesis of diabetes are used
singly or combined, such as sulfonylureas, metformin and thiazolidinediones. However, there are
several potential risks for most of current drugs, including the limited efficacy, hypoglycemia and
weight gain.[3-6] Hence, there still have many unmet clinical needs to obtain novel anti-diabetic
drugs.[7-9]
In pancreatic β-cells, the free fatty acid receptor 1 (FFA1/GPR40) promotes insulin secretion
dependent on blood glucose levels.[10, 11] Based on its physiological mechanism, FFA1 is
considered as a novel hypoglycemic target because of its low possibility of hypoglycemia.[12] At
present, many groups reported FFA1 agonists with acid moieties (Figure 1), and many clinical
trials of candidates (e.g. TAK-875) have been completed. Readers want to know more details on
the related research progress may refer to the reviews.[13, 14] In this field, we also did some
preliminary exploration on FFA1 agonists to extend its chemical space.[15-23]
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The peroxisome proliferator-activated receptors (PPARs) have received significant attention
due to their function on regulating energy metabolism.[24] There are three subtypes for PPARs:
including PPARα, PPARγ, and PPARδ. Among them, PPARα and PPARγ exhibited considerable
advantages on improvement of insulin sensibility and lipid disorder.[25-27] The physiological
mechanisms of PPARδ include anti-inflammatory, adipocyte differentiation, insulin sensibilization
and lipid metabolism.[28-31] Notably, PPARδ activation protects β cells from apoptosis,[32]
up-regulating GLP-1 receptor,[33] and increasing mitochondrial function in β cells.[34] Therefore,
PPARδ has been considered as a potent target for the treatment of various metabolic disorders.[35]
Based on their complementary mechanisms, the dual FFA1/PPARδ agonists would increase
insulin secretion and sensibility by FFA1 and PPARδ activation, respectively. Interestingly, the
pharmacophoric features of FFA1 and PPARδ agonists are very similar,[36] and we have explored
several hits as FFA1/PPARδ agonists, though the potency is limited to micromole levels.[37] As
shown in Figure 1, AM-4668 (FFA1 agonist)[38] has the same thiazole moiety (red lable in
Figure 1) with GW501516 (a selective PPARδ agonist).[39] Thus, it will be a feasible strategy by
hybrid AM-4668 with GW501516 to provide dual FFA1/PPARδ agonist. Furthermore, PPARδ
agonist GW501516 has the same acid moiety (blue lable in Figure 1) with our previous reported
FFA1 agonist CP-1.[40, 41] In this study, FFA1 agonists AM-4668 and CP-1 are hybrid with
PPARδ agonist GW501516 to maintain the common scaffolds as much as possible (Figure 2).
After comprehensive exploration, compound 32 was identified as a dual FFA1/PPARδ agonist
with high selectivity over other PPARs. Herein, we focus on the description of hybrid strategy,
structural optimization, and the hypoglycemic effects in ob/ob mice.
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2
. Results and Discussion
2
.1. Chemistry
Preparative process of compounds 1-8 are described in Scheme 1. Condensation of raw materials
a-b with methyl bromoacetate provided 2a-b, which were then converted to compounds 3a-b
1
through Baeyer–Villiger oxidation. Hydrolysis of acetates 3a-b with sodium methoxide afforded
phenols 4a-b. Cyclization of various thiobenzamides 5a-f with ethyl 2-chloroacetoacetate
generated intermediates 6a-f, followed by ester reduction using sodium borohydride,[42] and
further treated with thionyl chloride under catalytic condition yielded chlorinated intermediates
7
a-f. Condensation of 7a-f with 4a-b using classical Williamson ether synthesis with potassium
carbonate as base, followed by basic hydrolysis, furnished compounds 1-8. Compounds 9-14 were
synthesized as summarized in Scheme 2. Condensation of ethyl 2-chloroacetoacetate with 8a
formed oxazole 9a, which was further converted to intermediates 10a. Condensing raw materials
11a with 12a, followed by region-selective reaction in the presence of N-bromobutanimide
afforded 14a.[43] The intermediate 10a or 14a was connected with 4b or 15a, and then hydrolysis
using lithium hydroxide, provided target compounds 9-11. The thiazole ester 16a or 20a was
prepared by cyclization reaction, and convert to chlorinated intermediate 17a or 21a.
Condensation of halogenate intermediates with phenols 4a-b, followed by hydrolysis under basic
condition, afforded the carboxylic acids 9-14.
The compounds 15-20 were synthesized as shown in Scheme 3. The condensation of
2
-aminoacetophenone with chloroacetyl chloride provided 23a, which was further converted to
2
4a or 25a by cyclization reaction in the condition of phosphorus oxychloride or Lawesson’s
reagent, respectively.[44, 45] Similar reaction process furnished 28a or 29a from the starting
material benzoylhydrazide. Condensation of these chlorinated intermediates with 4a-b, then
4

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hydrolysis to provide the target compounds 15-20. Scheme 4 describes the synthesis of
compounds 21-32. The phenols 30a,[21] 31a[22] and 32a[46] were synthesized via reported
procedures. The optically pure 35a was separated from racemate 33a in the presence of
R-phenethylamine. Condensation of phenols (30a-32a, 35a) with 7a-h, and then ester hydrolysis,
provide 21-32.
2
.2. SAR study
The potency of synthesized compounds on FFA1 was measured using FFA1-expressed Chinese
hamster ovary cells, and the potency of PPARδ was evaluated in Gal4 receptor cell-based assay.
First, we explore the hybrids of FFA1 agonist AM-4668 and PPARδ agonist GW501516 (Table 1),
and briefly investigated the tolerability of substituents at terminal B ring. Compound 1, a direct
analog of GW501516, exhibited an approximately 130-fold reduced potency of PPARδ compared
to positive control GW0742. Replacement of methyl group at A ring with fluorine (compound 2)
hardly affected potency on PPARδ, while the agonistic activity of FFA1 has significant
improvement compared to compound 1. These results indicated that fluorine at A ring might be a
better substituent to explore the dual FFA1/PPARδ agonists. Introduction of trifluoromethyl group
at the B ring (compounds 3 and 4) drastically increased potency on PPARδ, suggesting that small
hydrophobic substituent in B ring is very important for matching with the hydrophobic pocket of
PPARδ. Based on previously reported SAR studies on PPARδ,[39] the 4-position and 3-position
substituents at B ring were incorporated to evaluate their potential on dual FFA1/PPARδ agonists
in this study. Fluorine and methyl scanning in B ring (compounds 5-8) indicated that hydrophobic
substitution is preferred in 4-position and tolerated in 3-position for the potency on FFA1 and
5

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PPARδ.
Meanwhile, we focused on the exploration of other heterocycles to replace the thiazole
scaffold (Table 2). Replacement of sulphur (compound 2) with oxygen (compound 9) resulted in
remarkable reduction of activity on FFA1 and PPARδ, might attributing to these differences of
sulphur and oxygen between hydrophobic effect and electronic distribution. Indeed, the potency of
oxazole derivatives (15 and 16) was inferior to that of the corresponding thiazole analog 17. The
exchange of sulphur and nitrogen atom in compound 2 provided analog 10 appears to diminishing
agonistic activities both in FFA1 and PPARδ, implying that there is a strict ligand-receptor
interaction in binding pockets. Incorporation of nitrogen-containing linker (compound 11)
improved agonistic activity of FFA1 to some extent, while significantly decreased potency on
PPARδ. Compounds 12-14 revealed lower potencies on FFA1 and PPARδ compared to compound
1
0, which might be attributed to the reduced hydrophobic interaction due to the absence of methyl
group at thiazole moiety. To decrease the lipophilicity of ligand, the thiadiazole and oxdiazole
derivatives (18-20) were designed. However, none of them (compounds 18-20) exhibited desired
agonistic activity, probably because of the highly hydrophobic pockets in FFA1 and PPARδ.
Based on SAR studies above, thiazole scaffold of compound 3 was selected as our starting
point for further optimization. In this part, our efforts were directed to explore whether other acid
scaffolds could also retain dual potency on FFA1 and PPARδ (Table 3). The sulfone acid (21 and
2
2) and deuterated acid (23-25), two reported scaffolds of FFA1 agonists,[21, 22] were
incorporated to investigate the potency on FFA1 and PPARδ. The sulfone acid analogs 21 and 22
revealed moderate activity for FFA1, but their potential to activate PPARδ was found to be rather
low (> 10 µM). The deuterated acid derivatives 23-25 indicated improved PPARδ activities back
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to the level of phenoxyacetic acid analog 4 but, nevertheless, led to reduced potency on FFA1
(compound 4 vs 25). Interestingly, introduction of dihydrobenzofuran carboxylic acid of TAK-875
provided compounds 26-32, which revealed better balance between FFA1 and PPARδ compared to
phenoxyacetic acid series. In this series, different substituents at left benzene ring have little effect
on agonistic activity of FFA1, while 4-methoxy group (compound 30) decreased potency on
PPARδ. Notably, compound 31 (4-CF ) exhibited the best balance between FFA1 and PPARδ,
3
which was selected for further evaluation by separating its optimal enantiomer. As expected, the
optimal enantiomer 32 (FFA1: 68 nM; PPARδ: 102 nM) revealed marked improvement compared
with racemate 31 (FFA1: 113 nM; PPARδ: 175 nM). Moreover, compound 32 exhibited high
selectivity for FFA1 and PPARδ over other nuclear receptors of PPAR family (for which EC >
5
0
1
0 µM, Table 4).
2
.3. Docking study
Based on co-crystal structure of PPARδ (PDB number: 1GWX) and FFA1 (4PHU), the induced-fit
docking study was performed to explore the interactional mode of the dual FFA1/PPARδ agonist
3
2. As shown in Figure 3, the dual agonist 32 fitted well with these two receptors in binding site.
In binding site of PPARδ (Figure 3A), carboxylic acid of agonist 32 formed interactions with
residues His449, Tyr473 and His323 by hydrogen-bonding network. Moreover, trifluoromethyl
group at the left benzene is fitted well with the small hydrophobic pocket surrounded by Ile249,
Leu255 and Ala258. Therefore, the un-substituted derivative 26 and compound 30 with bulkier
substituent revealed significantly reduced activities on PPARδ. In binding site of FFA1 (Figure
3
B), carboxylic acid of 32 formed anchor point with Arg2258 and Arg183. Furthermore, an
7

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edge-on interaction was generated between the plane of dihydrobenzofuran and Trp174. The
trifluoromethyl group was exposed to the outside of receptor, rationally explained that the left
phenyl ring could tolerate various types of substituents while little effect for FFA1 activity.
2
.4. Pharmacokinetic study
The pharmacokinetic (PK) profiles of compound 32 were evaluated in fasted rats. As shown in
Table 5, compound 32 exhibited good PK profiles with high maximum plasma concentration
(
Cmax = 9.45 µg/mL), which was 200-fold higher than its EC50 values on FFA1 and PPARδ.
Moreover, compound 32 showed low metabolic clearance (CL = 16.73 mL/h/kg) and sustained
half-life (T1/2 = 4.31 h) suitable for twice daily, resulting in high exposure in vivo (AUC0−24h
=
9
5.62 µg/mL·h).
2
.5. Hypoglycemic effects of 32
To investigate glucose-lowering effect in vivo, different doses of compound 32 (10, 30 and 100
mg/kg) were evaluated in ob/ob mice, a genetic defect model with metabolic disorders such as
diabetes-related obese and insulin resistance.[47, 48] As shown in Figure 4, the dual agonist 32
(10, 30 and 100 mg/kg) exhibited hypoglycemic effect in a dose-dependent manner, with changes
in plasma glucose AUC0−120min of +1.1%, -6.1%, and -10.9%, respectively. Notably, 32 (100 mg/kg)
exhibited significantly glucose-lowering effect though it is inferior to the typical agonist TAK-875
(-15.7% AUC0−120min).
3
. Conclusion
In this paper, FFA1 agonist AM-4668 was hybrid with PPARδ agonist GW501516 to design dual
8

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FFA1/PPARδ agonists. After comprehensive SAR exploration, we identified the orally
bioavailable dual FFA1/PPARδ agonist 32 (FFA1: 68 nM; PPARδ: 102 nM), which has high
selectivity over other PPARs. The dual agonist 32 exhibited good PK profiles with high plasma
exposure, which ensured the enough effective concentration to simultaneously activate FFA1 and
PPARδ in vivo. Moreover, compound 32 reduced the levels of glucose in a dose-response manner.
These interesting findings indicated that the dual agonist 32 is meaningful as a tool compound,
which will help us to seek better dual FFA1/PPARδ agonist.
4
. Experimental section
4
.1. General chemistry
Reagents and solvents were purchased from commercial sources. The progress of reaction was
monitored by thin layer chromatography analysis on GF254 plates at 254 and 365 nm.
Chromatographic purification was performed on silica gel (200-300 mesh). Melting points were
1
carried out on a RY-1 melting-point apparatus. Nuclear magnetic resonance spectra ( H NMR at
1
3
3
00 MHz and C NMR at 75 MHz) were measured by Bruker ACF-300Q instrument, coupling
constants (J values) are expressed as hertz (Hz), and chemical shifts are expressed as parts per
million (ppm) relative to internal standard (tetramethylsilane). Liquid chromatography-tandem
mass spectrometry (Waters) was used to determine LC-MS spectra. Elemental analysis was
recorded on Heraeus CHN-O-Rapid analyzer within 0.4% of theoretical values. Experimental
protocols for TAK-875 were disclosed in the supporting information of previous literature.[46]
General synthetic procedure for intermediates 4a and 4b
To a stirred solution of 1a or 1b (1 equiv) in acetone was added potassium carbonate (3 equiv) and
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methyl bromoacetate (2 equiv). The mixture was stirred at 45 °C for 12 h, and filtered. The filtrate
was concentrated under vacuum and the residue was dissolved in ethyl acetate. The organic layers
was washed with brine (2 × 20 mL), dried over anhydrous sodium sulfate and filtered. The filtrate
was concentrated under vacuum to give intermediate 2a or 2b as colorless oil, which was used for
the next reaction without further purification. To a solution of intermediate 2a or 2b (1 equiv) in
dichloromethane was added p-toluenesulfonic acid (0.1 equiv) and 3-chloroperoxybenzoic acid (2
equiv) at 0 °C. The mixture was stirred at room temperature for 24 h and then poured into
saturated sodium bisulfite solution (25 mL) stirred for 20 min. The aqueous layer was separated
and extracted with dichloromethane (3 × 10 mL). The combined organic phases were washed with
water (15 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated
under vacuum and the residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate, 20:1, v/v) to afford intermediates 3a and 3b as white solid. To a solution of 3a
or 3b (1 equiv) in methanol was added sodium methoxide (3 equiv). The mixture was stirred at
room temperature for 4-6 h and then quenched with 1 N hydrochloric acid (20 mL). The mixture
was extracted with ethyl acetate (3 × 10 mL), and the combined organic phases were washed with
brine (15 mL), dried and filtered. The filtrate was evaporated under vacuum and the residue was
purified by silica gel column chromatography (petroleum ether/ethyl acetate, 10:1, v/v) to afford
intermediates 4a and 4b as white solid.
1
Methyl 2-(4-hydroxy-2-methylphenoxy)acetate (4a). H NMR (300 MHz, CDCl ) δ: 9.35
3
(s, 1H), 7.06 – 7.01 (m, 1H), 6.84 – 6.63 (m, 2H), 4.75 (s, 2H), 3.67 (s, 3H), 2.18 (s, 3H). ESI-MS
+
m/z: 197.1 [M+H] .
1
Methyl 2-(2-fluoro-4-hydroxyphenoxy)acetate (4b). H NMR (300 MHz, CDCl ) δ: 9.44 (s,
3
1
0

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1
3
H), 6.92 – 6.85 (m, 1H), 6.63, 6.59 (dd, J = 13.1, 2.8 Hz, 1H), 6.51-6.47 (m, 1H), 4.73 (s, 2H),
+
.69 (s, 3H). ESI-MS m/z: 201.1 [M+H] .
General synthetic procedure for intermediates 6a-h
A solution of 5a-h (1 equiv) and ethyl 2-chloroacetoacetate (1.2 equiv) in ethanol (25 mL) was
heated to reflux for 6 h, then the mixture was allowed to stand at 0 °C for 10 hrs, and a white
needle crystal was precipitate out. The reaction mixture was filtered and the filter cake was
washed with ethanol (10 mL), dried to give the title compounds.
1
Ethyl 4-methyl-2-phenylthiazole-5-carboxylate (6a). Yield 75%; white powder; H NMR
(
300 MHz, DMSO-d ) δ: 7.95 – 7.86 (m, 2H), 7.45 – 7.40 (m, 3H), 4.30 (q, J = 7.1 Hz, 2H), 2.68
6
+
(
s, 3H), 1.31 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 248.1 [M+H] .
Ethyl 4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carboxylate (6b). Yield 89%;
1
white powder; H NMR (300 MHz, DMSO-d ) δ: 7.92 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.1 Hz,
6
+
2
H), 4.29 (q, J = 7.1 Hz, 2H), 2.67 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 316.1 [M+H] .
Ethyl 2-(4-fluorophenyl)-4-methylthiazole-5-carboxylate (6c). Yield 72%; white powder;
1
H NMR (300 MHz, DMSO-d ) δ: 8.02 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 4.30 (q, J =
6
+
7
.1 Hz, 2H), 2.90 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 266.1 [M+H] .
Ethyl 2-(3-fluorophenyl)-4-methylthiazole-5-carboxylate (6d). Yield 67%; white powder;
1
H NMR (300 MHz, DMSO-d ) δ: 7.84 – 7.79 (m, 2H), 7.60 – 7.56 (m, 1H), 7.44 – 7.40 (m, 1H),
6
+
4
.29 (q, J = 7.1 Hz, 2H), 2.67 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 266.1 [M+H] .
1
Ethyl 4-methyl-2-(p-tolyl)thiazole-5-carboxylate (6e). Yield 85%; white powder; H NMR
(
300 MHz, DMSO-d ) δ: 7.87 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 4.29 (q, J = 7.1 Hz,
6
1
1

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+
2
H), 2.67 (s, 3H), 2.36 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 262.1 [M+H] .
1
Ethyl 4-methyl-2-(m-tolyl)thiazole-5-carboxylate (6f). Yield 68%; white powder; H NMR
(
(
[
300 MHz, DMSO-d ) δ: 7.86 – 7.81 (m, 1H), 7.76 (d, J = 7.2 Hz, 1H), 7.41 – 7.38 (m, 2H), 4.28
6
q, J = 7.1 Hz, 2H), 2.69 (s, 3H), 2.38 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 262.1
+
M+H] .
Ethyl 2-(4-chlorophenyl)-4-methylthiazole-5-carboxylate (6g). Yield 84%; white powder;
1
H NMR (300 MHz, DMSO-d ) δ: 8.01 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 4.30 (q, J =
6
+
7
.1 Hz, 2H), 2.69 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 282.1 [M+H] .
Ethyl 2-(4-methoxyphenyl)-4-methylthiazole-5-carboxylate (6h). Yield 67%; white
1
powder; H NMR (300 MHz, DMSO-d ) δ: 7.95 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 4.29
6
(q, J = 7.1 Hz, 2H), 3.83 (s, 3H), 2.67 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 278.1
+
[
M+H] .
General synthetic procedure for intermediates 7a-h
To a stirred mixture of sodium borohydride (3 equiv), 6a-h (1 equiv) in reflux tetrahydrofuran (20
mL) was added methanol (1 mL). After refluxing for futher 30 min, the reaction mixture was
pouring into water (20 mL), and extracted with ethyl acetate (3 × 10 mL), the organic fractions
were combined, washed with saturated brine (2 × 10 mL) prior to drying over anhydrous sodium
sulfate. After filtration and concentrate, the residue was dissolved in dichloromethane (20 mL),
thionyl chloride (6 equiv) and catalytic DMF were added at room temperature. After stirring at
4
0 °C for 4 h, the reaction was concentrated. The residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate, 20:1, v/v) to afford the title compounds.
1
2

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1
5
-(chloromethyl)-4-methyl-2-phenylthiazole (7a). Yield 83%; yellow semisolid; H NMR
(
300 MHz, DMSO-d ) δ: 7.93 – 7.85 (m, 2H), 7.43 – 7.38 (m, 3H), 5.12 (s, 2H), 2.68 (s, 3H).
6
+
ESI-MS m/z: 224.1 [M+H] .
1
5
-(chloromethyl)-4-methyl-2-(p-tolyl)thiazole (7e). Yield 89%; yellow solid; H NMR (300
MHz, DMSO-d ) δ: 7.85 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 5.13 (s, 2H), 2.67 (s, 3H),
6
+
2
.36 (s, 3H). ESI-MS m/z: 238.1 [M+H] .
General synthetic procedure for target compounds 1-8
To a solution of 4a-b (1 equiv) and 7a-f (1.1 equiv) in acetonitrile was added potassium carbonate
(2 equiv) and catalytic potassium iodide. The mixture was heated at 45 °C for 12 h. Then the
mixture was cooled followed by filtration and the filtrate was concentrated. The residue was
purified by silica gel column chromatography (petroleum ether/ethyl acetate, 10:1, v/v) to afford a
white solid, which was dissolved in the mixed solvent of THF/MeOH/H O (18 mL, 2:3:1), and
2
added lithium hydroxide (1.5 equiv). After stirring at room temperature for 4 h, the volatiles were
removed under reduced pressure. The residue was acidified with 1N hydrochloric acid solution,
and then filtered and the filter cake was washed with cold water (5 mL), dried in vacuum to afford
a white powder. Recrystallization from 75% ethanol provides pure target compounds.
2
-(2-methyl-4-((4-methyl-2-phenylthiazol-5-yl)methoxy)phenoxy)acetic acid (1)
1
Yield 53%; white solid; m.p. 154-156 °C; H NMR (300 MHz, DMSO-d ) δ: 12.74 (brs, 1H), 7.90
6
(d, J = 4.0 Hz, 2H), 7.56 – 7.38 (m, 3H), 6.89 (s, 1H), 6.86 – 6.70 (m, 2H), 5.22 (s, 2H), 4.62 (s,
1
3
2
1
H), 2.43 (s, 3H), 2.21 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.95, 165.63, 152.19, 151.64,
6
51.09, 133.46, 130.63, 129.65, 128.10, 127.85, 126.36, 118.48, 112.96, 112.76, 65.79, 62.53,
1
3

ACCEPTED MANUSCRIPT
-
1
6.66, 15.54. ESI-MS m/z: 367.5 [M-H] . Anal. calcd. For C H NO S: C, 65.02; H, 5.18; N, 3.79;
2
0
19
4
Found: C, 65.27; H, 5.06; N, 3.73.
2
-(2-fluoro-4-((4-methyl-2-phenylthiazol-5-yl)methoxy)phenoxy)acetic acid (2)
1
Yield 65%; white solid; m.p. 106-108 °C; H NMR (300 MHz, DMSO-d ) δ: 8.08 – 7.81 (m, 2H),
6
7
.60 – 7.39 (m, 3H), 7.17 – 6.94 (m, 2H), 6.80 (d, J = 8.6 Hz, 1H), 5.27 (s, 2H), 4.68 (s, 2H), 2.44
1
3
(
s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.39, 165.85, 152.82, 152.05, 140.85, 133.48, 130.64,
6
1
29.62, 127.44, 126.41, 116.66, 111.25, 105.17, 104.88, 66.59, 62.97, 15.51. ESI-MS m/z: 371.5
-
[
M-H] . Anal. calcd. For C H FNO S: C, 61.12; H, 4.32; N, 3.75; Found: C, 61.35; H, 4.21; N,
1
9
16
4
3
.58.
2
-(2-methyl-4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methoxy)phenoxy)acetic
acid (3)
Yield 61%; white solid; m.p. 160-162 °C; H NMR (300 MHz, DMSO-d ) δ: 12.75 (brs, 1H), 8.09
1
6
(d, J = 7.3 Hz, 2H), 7.82 (d, J = 7.3 Hz, 2H), 7.06 – 6.64 (m, 3H), 5.25 (s, 2H), 4.61 (s, 2H), 2.45
1
3
(
s, 3H), 2.20 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.92, 163.64, 152.12, 152.06, 151.12,
6
1
36.90, 129.81, 127.86, 126.99, 126.57, 118.43, 112.94, 112.76, 65.77, 62.51, 16.64, 15.52.
-
ESI-MS m/z: 435.6 [M-H] . Anal. calcd. For C H F NO S: C, 57.66; H, 4.15; N, 3.20; Found: C,
2
1
18
3
4
5
7.45; H, 4.27; N, 3.12.
2
-(2-fluoro-4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methoxy)phenoxy)acetic
acid (4)
1
4

ACCEPTED MANUSCRIPT
1
Yield 57%; white solid; m.p. 148-150 °C; H NMR (300 MHz, DMSO-d ) δ: 12.99 (brs, 1H), 8.10
6
(d, J = 6.7 Hz, 2H), 7.83 (d, J = 6.7 Hz, 2H), 7.13 – 6.77 (m, 3H), 5.30 (s, 2H), 4.69 (s, 2H), 2.46
1
3
(
s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.34, 163.89, 152.81, 152.47, 140.86, 136.94, 129.09,
6
-
1
27.05, 126.53, 116.65, 111.23, 105.16, 104.87, 66.48, 62.94, 15.47. ESI-MS m/z: 439.8 [M-H] .
Anal. calcd. For C H F NO S: C, 54.42; H, 3.43; N, 3.17; Found: C, 54.67; H, 3.55; N, 3.26.
2
0
15
4
4
2
-(2-fluoro-4-((2-(4-fluorophenyl)-4-methylthiazol-5-yl)methoxy)phenoxy)acetic acid (5)
1
Yield 59%; white solid; m.p. 142-144 °C; H NMR (300 MHz, DMSO-d ) δ: 12.72 (brs, 1H), 7.93
6
(d, J = 7.8 Hz, 2H), 7.29 (d, J = 7.8 Hz, 2H), 7.13 – 6.92 (m, 2H), 6.79 (d, J = 8.7 Hz, 1H), 5.25 (s,
1
3
2
H), 4.69 (s, 2H), 2.42 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.44, 164.70, 153.87, 152.68,
6
1
50.63, 140.70, 130.03, 128.73, 127.49, 116.80, 116.31, 111.06, 105.05, 104.77, 66.18, 62.74,
-
1
5.47. ESI-MS m/z: 389.6 [M-H] . Anal. calcd. For C H F NO S: C, 58.31; H, 3.86; N, 3.58;
1
9
15
2
4
Found: C, 58.53; H, 3.79; N, 3.46.
2
-(2-fluoro-4-((2-(3-fluorophenyl)-4-methylthiazol-5-yl)methoxy)phenoxy)acetic acid (6)
1
Yield 52%; white solid; m.p. 128-130 °C; H NMR (300 MHz, DMSO-d ) δ: 7.77 – 7.63 (m, 2H),
6
7
4
1
6
3
.61 – 7.46 (m, 1H), 7.36 – 7.21 (m, 1H), 7.10 – 6.90 (m, 2H), 6.79 (d, J = 8.8 Hz, 1H), 5.27 (s, 2H),
1
3
.63 (s, 2H), 2.43 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.46, 164.73, 153.85, 152.69,
6
50.65, 140.72, 130.06, 127.65, 126.33, 116.81, 115.93, 115.46, 111.03, 105.05, 104.77, 66.18,
-
2.74, 15.45. ESI-MS m/z: 389.7 [M-H] . Anal. calcd. For C H F NO S: C, 58.31; H, 3.86; N,
1
9
15
2
4
.58; Found: C, 58.58; H, 3.74; N, 3.67.
1
5

ACCEPTED MANUSCRIPT
2
-(2-fluoro-4-((4-methyl-2-(p-tolyl)thiazol-5-yl)methoxy)phenoxy)acetic acid (7)
1
Yield 69%; white solid; m.p. 152-153 °C; H NMR (300 MHz, DMSO-d ) δ: 7.79 (d, J = 8.1 Hz,
6
2
H), 7.30 (d, J = 8.1 Hz, 2H), 7.11 – 6.97 (m, 2H), 6.81, 6.78 (dd, J = 9.0, 1.4 Hz, 1H), 5.26 (s,
1
3
2
H), 4.70 (s, 2H), 2.42 (s, 3H), 2.35 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.35, 164.83,
6
1
53.81, 152.59, 150.65, 141.85, 140.31, 130.06, 129.45, 129.07, 127.49, 116.85, 110.87, 105.05,
-
6
6.16, 62.78, 21.46, 15.45. ESI-MS m/z: 386.2 [M-H] . Anal. calcd. For C H FNO S: C, 62.00;
2
0
18
4
H, 4.68; N, 3.62; Found: C, 62.27; H, 4.56; N, 3.71.
2
-(2-fluoro-4-((4-methyl-2-(m-tolyl)thiazol-5-yl)methoxy)phenoxy)acetic acid (8)
1
Yield 54%; white solid; m.p. 138-140 °C; H NMR (300 MHz, DMSO-d ) δ: 13.02 (brs, 1H), 7.79
6
–
7.60 (m, 2H), 7.45 – 7.19 (m, 2H), 7.14 – 6.94 (m, 2H), 6.80 (d, J = 9.1 Hz, 1H), 5.27 (s, 2H),
1
3
4
.70 (s, 2H), 2.43 (s, 3H), 2.37 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.47, 165.99, 154.36,
6
1
53.64, 151.99, 139.06, 133.34, 131.41, 129.59, 127.25, 126.78, 123.64, 116.25, 111.08, 66.12,
-
6
2.77, 21.33, 15.53. ESI-MS m/z: 385.6 [M-H] . Anal. calcd. For C H FNO S: C, 62.00; H, 4.68;
2
0
18
4
N, 3.62; Found: C, 62.15; H, 4.73; N, 3.75.
Ethyl 4-methyl-2-phenyloxazole-5-carboxylate (9a). A solution of benzamide (1.0 g, 8.3
mmol) and ethyl 2-chloroacetoacetate (1.6 g, 9.9 mmol) in ethanol (20 mL) was heated to reflux
for 16 h, the solvent was removed under reduced pressure, and the residue was washed with water
(
25 mL) and extracted with ethyl acetate (3 × 15 mL). The organic layer was dried over anhydrous
sodium sulfate and concentrated. The residue was purified by silica gel column chromatography
petroleum ether/ethyl acetate, 25:1, v/v) to afford the title compound (0.8 g, 42%) as a white solid.
(
1
6

ACCEPTED MANUSCRIPT
1
H NMR (300 MHz, DMSO-d ) δ: 8.07 – 8.01 (m, 2H), 7.43 – 7.38 (m, 3H), 4.34 (q, J = 7.11 Hz,
6
+
2
H), 2.57 (s, 3H), 1.35 (t, J = 7.11 Hz, 3H). ESI-MS m/z: 232.1 [M+H] .
2
-(2-fluoro-4-((4-methyl-2-phenyloxazol-5-yl)methoxy)phenoxy)acetic acid (9)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 9a as starting material. Yield 37%; white solid; m.p. 159-161 °C; H NMR (300
MHz, DMSO-d ) δ: 12.87 (brs, 1H), 8.12 – 7.97 (m, 2H), 7.56 – 7.42 (m, 3H), 7.19 – 6.96 (m, 2H),
6
1
3
6
.85 (d, J = 8.4 Hz, 1H), 5.31 (s, 2H), 4.67 (s, 2H), 2.43 (s, 3H). C NMR (75 MHz, DMSO-d ) δ:
6
1
70.42, 158.34, 154.65, 153.16, 141.63, 138.13, 130.52, 129.65, 128.73, 127.56, 125.47, 116.92,
-
1
10.97, 103.42, 66.57, 62.95, 15.23. ESI-MS m/z: 356.1 [M-H] . Anal. calcd. For C H FNO : C,
1
9
16
5
6
3.86; H, 4.51; N, 3.92; Found: C, 63.58; H, 4.43; N, 3.76.
4
-(bromomethyl)-5-methyl-2-phenylthiazole (14a). A solution of thiobenzamide (1.0 g, 7.3
mmol) and 3-chloro-2-butanone (1.2 g, 8.5 mmol) in ethanol (20 mL) was heated at reflux for 6 h.
The reaction mixture was concentrated, diluted with 1N sodium bicarbonate solution (15 mL) and
ethyl acetate (25 mL), and then washed with water (1 × 15 mL) and brine (1 × 15 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced
1
pressure to provide 4,5-dimethyl-2-phenyl-1,3-thiazole (13a). H NMR (300 MHz, DMSO-d ) δ:
6
7
1
.93, 7.91 (dd, J = 7.6, 1.3 Hz, 2H), 7.45-7.40 (m, 3H), 2.38 (s, 3H), 2.21 (s, 3H). ESI-MS m/z:
+
90.1 [M+H] .
To a solution of 13a (1 g, 5.3 mmol) in acetonitrile (8 mL) was added N-bromosuccinimide
(0.9 g, 5.3 mmol) and the mixture was stirred at room temperature for 2 h. To the mixture, water
1
7

ACCEPTED MANUSCRIPT
(10 mL) was added dropwise at room temperature to give colourless to pale yellow precipitation.
The mixture was stirred at 0–5 °C for 1 h. The precipitation was filtered and washed with cold
1
water (8 mL) to afford the title compound 14a (0.7 g, 50%). H NMR (300 MHz, DMSO-d ) δ:
6
7
2
.93, 7.91 (dd, J = 7.6, 1.3 Hz, 2H), 7.45-7.40 (m, 3H), 4.56 (s, 2H), 2.36 (s, 3H). ESI-MS m/z:
+
68.1 [M+H] .
2
-(2-fluoro-4-((5-methyl-2-phenylthiazol-4-yl)methoxy)phenoxy)acetic acid (10)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 14a as starting material. Yield 75%; white solid; m.p. 119-121 °C; H NMR (300
MHz, DMSO-d ) δ: 7.98 – 7.83 (m, 2H), 7.57 – 7.45 (m, 3H), 7.13 – 6.87 (m, 2H), 6.76 (d, J = 8.8
6
1
3
Hz, 1H), 5.28 (s, 2H), 4.65 (s, 2H), 2.43 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.25, 168.12,
6
1
54.37, 153.62, 152.09, 143.37, 141.87, 130.96, 129.23, 128.74, 123.13, 116.93, 110.94, 103.42,
-
6
8.73, 62.58, 12.35. ESI-MS m/z: 372.1 [M-H] . Anal. calcd. For C H FNO S: C, 61.12; H, 4.32;
1
9
16
4
N, 3.75; Found: C, 61.31; H, 4.24; N, 3.58.
2
-(2-fluoro-4-(((5-methyl-2-phenylthiazol-4-yl)methyl)amino)phenoxy)acetic acid (11)
To a solution of 14a (0.20 g, 0.75 mmol) and 15a (0.15 g, 0.75 mmol) in ethanol (20 mL) was
added sodium bicarbonate (0.19 g, 2.25 mmol) at room temperature. The reaction mixture was
stirred at room temperature for 24 h. Then the reaction mixture was filtrated and the filtrate was
concentrated. The residue was purified by silica gel column chromatography (petroleum
ether/ethyl acetate, 8:1, v/v) to afford a white solid (0.18 g, 64%), which was dissolved in the
mixed solvent of THF/MeOH/H O (18 mL, 2:3:1), and added lithium hydroxide (0.02 g, 0.73
2
1
8

ACCEPTED MANUSCRIPT
mmol). After stirring at room temperature for 4 h, the volatiles were removed under reduced
pressure. The residue was acidified with 1N hydrochloric acid solution (pH = 5 – 6), then filtered
and the filter cake was washed with cold water (5 mL), dried to afford a white powder.
Recrystallization from 75% ethanol gave the title compound 11 (0.12 g, 67%) as white solid, m.p.
1
1
36-138 °C; H NMR (300 MHz, DMSO-d ) δ: 7.95 – 7.81 (m, 2H), 7.54 – 7.43 (m, 4H), 7.06 –
6
13
6
.82 (m, 1H), 6.62 (d, J = 14.1 Hz, 1H), 6.44 (s, 1H), 4.54 (s, 2H), 4.25 (s, 2H), 2.49 (s, 3H). C
NMR (75 MHz, DMSO-d ) δ: 170.60, 164.53, 152.95, 152.07, 143.56, 141.59, 137.43, 130.93,
6
-
1
29.08, 128.76, 123.15, 116.69, 111.47, 103.58, 67.12, 42.85, 14.35. ESI-MS m/z: 371.1 [M-H] .
Anal. calcd. For C H FN O S: C, 61.28; H, 4.60; N, 7.52; Found: C, 61.57; H, 4.48; N, 7.36.
1
9
17
2
3
Ethyl 2-phenylthiazole-4-carboxylate (16a). A solution of ethyl bromopyruvate (0.68 g, 3.5
mmol) and thiobenzamide (0.40 g, 2.9 mmol) in ethanol (20 mL) was heated at reflux for 4 h. The
solvent was removed, and the residue was added water (15 mL) and extracted with ethyl acetate (3
×
15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The
residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 20:1, v/v)
1
to afford the title compound (0.52 g, 76%) as a white solid. H NMR (300 MHz, DMSO-d ) δ:
6
8
.96 (s, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.31 (m, 3H), 4.39 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz,
+
3
H). ESI-MS m/z: 234.1 [M+H] .
2
-(2-fluoro-4-((2-phenylthiazol-4-yl)methoxy)phenoxy)acetic acid (12)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 16a and 4b as starting material. Yield 46%; white solid; m.p. 150-152 °C; H
1
9

ACCEPTED MANUSCRIPT
NMR (300 MHz, DMSO-d ) δ: 12.74 (brs, 1H), 8.01 – 7.90 (m, 2H), 7.77 (s, 1H), 7.58 – 7.43 (m,
6
1
3
3
H), 7.11 – 6.99 (m, 2H), 6.89 – 6.76 (m, 1H), 5.18 (s, 2H), 4.70 (s, 2H). C NMR (75 MHz,
DMSO-d ) δ: 170.15, 168.95, 159.31, 154.55, 153.12, 143.25, 141.67, 130.96, 129.63, 128.59,
6
-
1
16.93, 115.62, 110.92, 103.46, 66.67, 62.74. ESI-MS m/z: 357.6 [M-H] . Anal. calcd. For
C H FNO S: C, 60.16; H, 3.93; N, 3.90; Found: C, 60.39; H, 3.78; N, 3.82.
1
8
14
4
2
-(2-methyl-4-((2-phenylthiazol-4-yl)methoxy)phenoxy)acetic acid (13)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 16a and 4a as starting material. Yield 53%; white solid; m.p. 134-136 °C; H
NMR (300 MHz, DMSO-d ) δ: 12.74 (brs, 1H), 8.12 – 7.86 (m, 2H), 7.71 (s, 1H), 7.61 – 7.43 (m,
6
1
3
3
H), 6.91 (s, 1H), 6.87 – 6.70 (m, 2H), 5.15 (s, 2H), 4.61 (s, 2H), 2.20 (s, 3H). C NMR (75 MHz,
DMSO-d ) δ: 170.81, 167.76, 153.91, 152.78, 150.98, 133.47, 130.75, 129.70, 127.95, 126.61,
6
-
1
18.48, 118.24, 113.10, 112.69, 66.47, 66.12, 16.59. ESI-MS m/z: 353.7 [M-H] . Anal. calcd. For
C H NO S: C, 64.21; H, 4.82; N, 3.94; Found: C, 64.47; H, 4.68; N, 3.75.
1
9
17
4
Ethyl 4-phenylthiazole-2-carboxylate (20a). A solution of ethyl thiooxamate (0.40 g, 3.0
mmol) and 2-Bromoacetophenone (0.60 g, 3.0 mmol) in ethanol (15 mL) was heated to reflux for
h. The mixture was concentrated, then diluted with ethyl acetate (20 mL), and washed with 1N
6
sodium bicarbonate solution (2 × 15 mL) and brine (15 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product was
purified by silica gel column chromatography (petroleum ether/ethyl acetate, 20:1, v/v) to afford
1
the title compound (0.52 g, 74%) as a white solid. H NMR (300 MHz, DMSO-d ) δ: 8.55 (s, 1H),
6
2
0

ACCEPTED MANUSCRIPT
8
.03 (d, J = 1.2 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.52-7.39 (m, 3H), 4.42 (q, J = 7.1 Hz, 2H),
+
1
.37 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 234.1 [M+H] .
2
-(2-fluoro-4-((4-phenylthiazol-2-yl)methoxy)phenoxy)acetic acid (14)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 20a as starting material. Yield 48%; white solid; m.p. 174-176 °C; H NMR (300
MHz, DMSO-d ) δ: 8.11 (s, 1H), 8.03 – 7.90 (m, 2H), 7.56 – 7.30 (m, 3H), 7.17 – 7.00 (m, 2H),
6
1
3
6
1
6
3
.86 (d, J = 8.9 Hz, 1H), 5.46 (s, 2H), 4.66 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 170.41,
6
66.72, 154.75, 152.42, 134.42, 129.27, 128.59, 126.51, 116.63, 115.52, 111.04, 105.10, 104.81,
-
8.02, 66.65. ESI-MS m/z: 357.6 [M-H] . Anal. calcd. For C H FNO S: C, 60.16; H, 3.93; N,
1
8
14
4
.90; Found: C, 60.27; H, 3.83; N, 3.87.
2
-chloro-N-(2-oxo-2-phenylethyl)acetamide (23a). The choroacetylchloride (1.71 mL, 22.7
mmol) dissolved in dichloromethane (15 mL) was added dropwise to a solution of
-aminoacetophenonehydrochloride (3 g, 17.5 mmol) and triethylamine (7.27 mL, 52.4 mmol) in
2
dichloromethane (30 mL), keeping at 0 °C for 2 h, and then stirred at room temperature for 16 h.
The reaction solution was washed with 1N hydrochloric acid (2 × 15 mL), saturated sodium
carbonate solution (2 × 15 mL), and brine (1 × 15 mL) in sequence. The organic layer was dried
over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product
was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 5:1, v/v) to
1
afford the title compound (2.3 g, 62%) as a white solid. H NMR (300 MHz, DMSO-d ) δ: 8.58 (s,
6
1
H), 7.99 (d, J = 7.2 Hz, 2H), 7.71-7.53 (m, 3H), 4.69 (d, J = 5.5 Hz, 2H), 4.21 (s, 2H). ESI-MS
2
1

ACCEPTED MANUSCRIPT
+
m/z: 212.1 [M+H] .
2
-(chloromethyl)-5-phenyloxazole (24a). To a solution of 23a (1.0 g, 4.8 mmol) in
acetonitrile (20 mL) was added phosphorus oxychloride (0.86 mL, 9.4 mmol) at ambient
temperature. After addition was complete, the solution was heated to reflux for 4 h. The reaction
mixture was concentrated, then diluted with ethyl acetate (25 mL), and washed with water (2 × 15
mL), 1N sodium bicarbonate solution (2 × 15 mL) and brine (25 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product
was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 20:1, v/v) to
1
afford the title compound (0.71 g, 77%) as a white solid. H NMR (300 MHz, DMSO-d ) δ: 8.21
6
(s, 1H), 7.70, 7.69 (dd, J = 7.1, 0.9 Hz, 2H), 7.48-7.36 (m, 3H), 5.13 (s, 2H). ESI-MS m/z: 194.1
+
[
M+H] .
2
-(chloromethyl)-5-phenylthiazole (25a). To a solution of 23a (1 g, 4.7 mmol) in
tetrahydrofuran (20 mL) was added Lawesson’s reagent (1.14 g, 2.8 mmol), and the mixture was
heated to reflux for 4 h. The reaction mixture was concentrated, then diluted with ethyl acetate (30
mL), and washed with 1N sodium bicarbonate solution (2 × 20 mL) and brine (20 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced
pressure. The crude product was purified by silica gel column chromatography (petroleum
1
ether/ethyl acetate, 30:1, v/v) to afford the title compound (0.75 g, 75%) as a white solid. H NMR
(
300 MHz, DMSO-d ) δ: 8.23 (s, 1H), 7.70, 7.69 (dd, J = 7.1, 0.9 Hz, 2H), 7.49-7.37 (m, 3H),
6
+
5
.14 (s, 2H). ESI-MS m/z: 210.0 [M+H] .
2
2

ACCEPTED MANUSCRIPT
2
-(2-fluoro-4-((5-phenyloxazol-2-yl)methoxy)phenoxy)acetic acid (15)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 24a and 4b as starting material. Yield 42%; white solid; m.p. 156-158 °C; H
NMR (300 MHz, DMSO-d ) δ: 7.80 – 7.62 (m, 3H), 7.57 – 7.34 (m, 3H), 7.16 – 6.96 (m, 2H),
6
1
3
6
.89 – 6.79 (m, 1H), 5.25 (s, 2H), 4.70 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 170.41, 159.21,
6
1
54.26, 152.00, 150.13, 129.63, 129.30, 127.61, 124.55, 123.29, 116.29, 110.82, 104.99, 104.70,
-
6
6.15, 62.99. ESI-MS m/z: 341.5 [M-H] . Anal. calcd. For C H FNO : C, 62.97; H, 4.11; N, 4.08;
1
8
14
5
Found: C, 62.83; H, 4.23; N, 4.15.
2
-(2-methyl-4-((5-phenyloxazol-2-yl)methoxy)phenoxy)acetic acid (16)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 24a and 4a as starting material. Yield 47%; white solid; m.p. 154-156 °C; H
NMR (300 MHz, DMSO-d ) δ: 7.80 – 7.65 (m, 3H), 7.56 – 7.30 (m, 3H), 6.95 – 6.70 (m, 3H),
6
1
3
5
.19 (s, 2H), 4.63 (s, 2H), 2.18 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.87, 159.68, 152.08,
6
1
51.88, 151.22, 129.62, 129.25, 127.87, 127.68, 124.52, 123.24, 118.34, 112.74, 65.78, 62.83,
-
1
6.66. ESI-MS m/z: 338.1 [M-H] . Anal. calcd. For C H NO : C, 67.25; H, 5.05; N, 4.13; Found:
1
9
17
5
C, 67.46; H, 5.12; N, 4.25.
2
-(2-fluoro-4-((5-phenylthiazol-2-yl)methoxy)phenoxy)acetic acid (17)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 25a and 4b as starting material. Yield 57%; white solid; m.p. 123-125 °C; H
2
3

ACCEPTED MANUSCRIPT
NMR (300 MHz, DMSO-d ) δ: 7.83 – 7.67 (m, 2H), 7.52 – 7.38 (m, 3H), 7.16 – 7.05 (m, 1H),
6
1
3
7
.04 (s, 1H), 6.95 – 6.76 (m, 2H), 5.27 (s, 2H), 4.72 (s, 2H). C NMR (75 MHz, DMSO-d ) δ:
6
1
70.41, 168.95, 154.56, 153.13, 141.86, 141.32, 132.97, 132.46, 129.31, 128.76, 126.47, 116.27,
-
1
10.85, 103.76, 67.85, 66.43. ESI-MS m/z: 358.1 [M-H] . Anal. calcd. For C H FNO S: C, 60.16;
1
8
14
4
H, 3.93; N, 3.90; Found: C, 60.34; H, 3.78; N, 3.85.
N'-(2-chloroacetyl)benzohydrazide (27a). To the slurry of benzoylhydrazide (3 g, 22.0
mmol) in acetonitrile (20 mL) were added simultaneously choroacetylchloride (2 mL, 26.4 mmol)
and 50% sodium hydroxide (26.4 mmol) while maintaining temperature below 0˚C. After 1h, the
resulting slurry was filtered and the solid was washed with water (2 × 5 mL). The filter cake was
1
dried to provide the title compound (3.2 g, 68%) as a white solid. H NMR (300 MHz, DMSO-d )
6
δ: 10.50 (s, 1H), 10.38 (s, 1H), 7.80-7.90 (m, 2H), 7.56-7.63 (m, 1H), 7.40-7.55 (m, 2H), 4.21 (s,
+
2
H). ESI-MS m/z: 213.1 [M+H] .
2
-(chloromethyl)-5-phenyl-1, 3, 4-thiadiazole (28a). The title compound was obtained from
2
7a according to the method described for the synthesis of the compound 25a in 63% yield as a
1
white solid. H NMR (300 MHz, DMSO-d ) δ: 8.04, 8.02 (dd, J = 7.6, 1.3 Hz, 2H), 7.76-7.57 (m,
6
+
3
H), 5.26 (s, 2H). ESI-MS m/z: 211.0 [M+H] .
2
-(2-fluoro-4-((5-phenyl-1,3,4-thiadiazol-2-yl)methoxy)phenoxy)acetic acid (18)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 28a and 4b as starting material. Yield 65%; white solid; m.p. 172-174 °C; H
2
4

ACCEPTED MANUSCRIPT
NMR (300 MHz, DMSO-d ) δ: 8.03 – 7.90 (m, 2H), 7.67 – 7.48 (m, 3H), 7.18 – 6.99 (m, 2H),
6
1
3
6
.86 (d, J = 8.7 Hz, 1H), 5.58 (s, 2H), 4.67 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 170.30,
6
1
69.66, 166.78, 152.24, 152.11, 131.93, 129.91, 129.85, 128.18, 116.65, 111.11, 105.21, 66.55,
-
6
5.42. ESI-MS m/z: 358.6 [M-H] . Anal. calcd. For C H FN O S: C, 56.66; H, 3.64; N, 7.77;
1
7
13
2
4
Found: C, 56.43; H, 3.57; N, 7.56.
2
-(2-methyl-4-((5-phenyl-1,3,4-thiadiazol-2-yl)methoxy)phenoxy)acetic acid (19)
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 28a and 4a as starting material. Yield 47%; white solid; m.p. 135-137 °C; H
NMR (300 MHz, DMSO-d ) δ: 8.11 – 7.94 (m, 2H), 7.69 – 7.48 (m, 3H), 7.04 – 6.70 (m, 3H), 5.55 (s,
6
1
3
2
H), 4.63 (s, 2H), 2.19 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 170.89, 169.46, 167.62, 151.65,
6
1
51.44, 131.95, 129.95, 129.86, 128.18, 128.03, 118.42, 112.87, 112.79, 65.81, 65.16, 16.65.
-
ESI-MS m/z: 354.6 [M-H] . Anal. calcd. For C H N O S: C, 60.66; H, 4.53; N, 7.86; Found: C,
1
8
16
2
4
6
0.89; H, 4.65; N, 7.73.
2
-(chloromethyl)-5-phenyl-1, 3, 4-oxadiazole (29a). The title compound was obtained from
2
7a according to the method described for the synthesis of the compound 24a in 75% yield as a
1
white solid. H NMR (300 MHz, DMSO-d ) δ: 8.03, 8.01 (dd, J = 7.6, 1.3 Hz, 2H), 7.65-7.60 (m,
6
+
3
H), 5.16 (s, 2H). ESI-MS m/z: 195.1 [M+H] .
2
-(2-fluoro-4-((5-phenyl-1,3,4-oxadiazol-2-yl)methoxy)phenoxy)acetic acid (20)
2
5

ACCEPTED MANUSCRIPT
The title compound was prepared by the method similar to that described for compound 1 using
1
the intermediate 29a and 4b as starting material. Yield 53%; white solid; m.p. 186-188 °C; H
NMR (300 MHz, DMSO-d ) δ: 8.08 – 7.95 (m, 2H), 7.69 – 7.46 (m, 3H), 7.16 – 6.97 (m, 2H),
6
1
3
6
.84 – 6.73 (m, 1H), 5.43 (s, 2H), 4.58 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 170.30, 165.43,
6
1
63.15, 154.52, 153.16, 141.85, 129.35, 128.16, 127.47, 123.45, 116.92, 110.96, 103.42, 66.18,
-
6
2.93. ESI-MS m/z: 343.1 [M-H] . Anal. calcd. For C H FN O : C, 59.31; H, 3.81; N, 8.14;
17
13
2
5
Found: C, 59.56; H, 3.65; N, 8.27.
4
.2. FLIPR Assay
Human FFA1-expressed CHO cells were plated and incubated in the condition of 5% CO for 16 h
2
at 37 °C. After, removed the medium and washed, then, loading cells with buffer (containing 2.5
µg/mL Fluo 4-AM, 0.1% fatty acid-free bovine serum albumin and 2.5 mmol/L probenecid) for 1
h at 37 °C. During the test, γ-linolenic acid (Sigma) or compounds with different concentrations
were added and the signals of intracellular calcium flux after addition were measured using FLIPR
(
Molecular Devices). The potency of compound on hFFA1 was calculated as the equation of
(A−B)/(C−B)]×100%. Among them, the raise of calcium level in compound-treated cells was
[
expressed as A, the increase of calcium level in vehicle-treated cells was expressed as B, and the
increase of calcium level in 10 µM γ-linolenic acid-treated cells was expressed as C. EC values
5
0
were obtained from GraphPad 5.00 (San Diego, USA).
4
.3. Evaluation for PPARα, PPARγ and PPARδ
Detailed descriptions on transfection and cell-based evaluation for PPARα, PPARγ and PPARδ
were given in our previously reported literature.[37] Briefly, HepG2 or HEK293 cells were
2
6

ACCEPTED MANUSCRIPT
transfected with pBIND-PPARα, PPARδ or PPARγ according to the manufacturer’s protocol.
After transfection, positive controls or compounds with different concentrations were added and
incubated for 18 h, then lysed with lysis buffer, and added Luciferase Assay Reagent II. The
luciferase signals of firefly and renilla were measured using Dual Luciferase Reporter Assay
System (Promega). EC values were obtained from GraphPad 5.00 (San Diego, USA).
5
0
4
.4. Docking studies
The induced-fit docking study was performed on MOE 2014.0901 (Montreal, Canada) based on
structure of PPARδ (PDB number: 1GWX) and FFA1 (4PHU). Before docking study, removing
water and other ligands from co-crystal structure, and then performed by Protonate 3D to add
hydrogen at co-crystal complex. After that, Gaussian Contact surface was analyzed around ligand
to identify property of binding site, and then isolated. In the binding pocket, compound 32 was
docked by using the induced-fit mode and set as the London dG scoring. Moreover, the Forcefield
Refinement was performed and set as London dG scoring for energy minimization. Docking
image was drawn by Pymol-1.5.0.3 (DeLano Scientific LLC).
4
.5. Animals
Male SD rats (180-200 g) were obtained from Guangdong Medical Laboratory Animal Center
Guangzhou, China), and male ob/ob mice (six weeks old) were obtained from Model Animal
(
Research Center of Nanjing University (Nanjing, China). Before experimental operations, rats and
mice were acclimatized for one week. Under circadian rhythm of 12 h light/black, animal room
was maintained relative humidity 50 ± 10% at 23 ± 2 °C throughout experimental period. The
2
7

ACCEPTED MANUSCRIPT
standard food and water were allowed ad libitum access for animals unless otherwise stated, and
0
.5% Carboxy Methyl Cellulose solution was used as vehicle for drug administration. The ethical
committee of Guangdong Pharmaceutical University has approved all experimental procedures
involved in animal, and these experimental procedures were performed based on Laboratory
Animal Management Regulations in China and adhered to the Guide for the Care and Use of
Laboratory Animals (NIH publication, 2011).
4
.6. Pharmacokinetic studies
SD rats acclimatized for seven days were fasted for 12 h, and weighted (n = 4). After oral
administration of compound 32 (3 mg/kg), collect blood samples at 5, 15, 30, 45 min and 1, 2, 4, 6,
8
, 12, 18, 24 h. By centrifuging, the plasma samples were separated, and acetonitrile containing
internal standard was used to precipitate proteins. The supernatant was diluted and determined by
LC-MS/MS (Waters) to obtain the plasma concentration of compound 32. PK parameters were
analyzed by DAS 2.1.1.
4
.7. Hypoglycemic effects of 32
ob/ob mice were fasted for 12 h, weighted, and randomized into 5 groups (n = 6). Mice were
orally administrated with single doses of vehicle, TAK-875 (40 mg/kg), or compound 32 (10, 30,
1
00 mg/kg) and then orally dosed with 3 g/kg glucose solution after 30 min. Blood were collected
before drug administration (-30 min), before glucose loading (0 min), and at 15, 30, 60 and 120
min after glucose challenge. The levels of glucose were measured by glucometer (SanNuo
ChangSha, China). Data were performed using GraphPad 5.00 (San Diego, USA).
2
8