Identification of highly potent and orally available free fatty acid receptor 1 agonists bearing isoxazole scaffold

Article abstract of DOI:10.1016/j.bmc.2017.12.030

The free fatty acid receptor 1 (FFA1) is being considered to be a novel anti-diabetic target based on its role in amplifying insulin secretion. We have previously identified several series of FFA1 agonists with different heterocyclic scaffolds. Herein, we describe the structural exploration of other heterocyclic scaffolds directed by drug-like physicochemical properties. Further structure-based design and chiral resolution provided the most potent compound 11 (EC₅₀ = 7.9 nM), which exhibited improved lipophilicity (LogD_7.4: 1.93), ligand efficiency (LE = 0.32) and ligand lipophilicity efficiency (LLE = 6.2). Moreover, compound 11 revealed an even better pharmacokinetic property than that of TAK-875 in terms of plasma clearance, maximum concentration, and plasma exposure. Although robust agonistic activity and PK profiles for compound 11, the glucose-lowering effects in vivo is not ideal, and the exact reason for in vitro/in vivo difference was worthy for further exploration.

Full text of DOI:10.1016/j.bmc.2017.12.030

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of highly potent and orally available free fatty acid
receptor 1 agonists bearing isoxazole scaffold
a,b
b
b
b
b
b
b,c,
⇑
Zheng Li , Chunxia Liu , Wei Shi , Xingguang Cai , Yuxuan Dai , Chen Liao , Wenlong Huang
Hai Qian
a
,
c,
⇑
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The free fatty acid receptor 1 (FFA1) is being considered to be a novel anti-diabetic target based on its role
in amplifying insulin secretion. We have previously identified several series of FFA1 agonists with differ-
ent heterocyclic scaffolds. Herein, we describe the structural exploration of other heterocyclic scaffolds
directed by drug-like physicochemical properties. Further structure-based design and chiral resolution
provided the most potent compound 11 (EC50 = 7.9 nM), which exhibited improved lipophilicity
Received 23 November 2017
Revised 16 December 2017
Accepted 22 December 2017
Available online xxxx
(
LogD7.4: 1.93), ligand efficiency (LE = 0.32) and ligand lipophilicity efficiency (LLE = 6.2). Moreover, com-
Keywords:
Diabetes
FFA1
Lipophilicity
Ligand efficiency
Pharmacokinetic property
pound 11 revealed an even better pharmacokinetic property than that of TAK-875 in terms of plasma
clearance, maximum concentration, and plasma exposure. Although robust agonistic activity and PK pro-
files for compound 11, the glucose-lowering effects in vivo is not ideal, and the exact reason for in vitro/
in vivo difference was worthy for further exploration.
Ó 2017 Elsevier Ltd. All rights reserved.
1
. Introduction
arylalkanoic acids (Fig. 1),11–18 and the clinical trials were
performed to evaluate the potential of TAK-875, LY2881835 and
5
,10
Type 2 diabetes mellitus (T2DM), a most common type of dia-
AMG-837 as anti-diabetic agents.
In addition, the chemical
betes, is characterized by impaired insulin secretion and/or sensi-
space of FFA1 agonists with different scaffolds has also been
1
,2
17–24
tization.
Various available oral insulin secretagogues, such as
explored by our colleagues.
In particular, the common biphe-
3
sulfonylureas, are widely used for the treatment of T2DM. How-
ever, these available therapies are often related to side effect of
hypoglycemia because insulin secretion induced by them are inde-
nyl scaffold has been systematically replaced by various heterocy-
cles in our laboratory (eg., compounds 1 and 2 in Fig. 1) to reduce
the lipophilicity, and the lipophilicity of candidate is usually
related to a higher promiscuity, metabolic instability, and failure
4
pendent of glucose. Thus, there are unmet needs for new oral
2
5–29
insulin secretagogues without the risk of hypoglycemia.
rates in research and development.
Herein, we describe our
The free fatty acid receptor 1 (FFA1, or GPR40), has emerged as
an attractive target in the last decade for the treatment of T2DM.
FFA1 is predominantly expressed in the pancreatic b-cell and aug-
efforts toward discovering preferable heterocycle scaffold with
better drug-like physicochemical properties directed by lipophilic-
ity, ligand efficiency (LE) and ligand lipophilicity efficiency (LLE)
(Fig. 2). These efforts ultimately led to the identification of
compound 11, a potent agonist with improved physicochemical
properties and excellent pharmacokinetic (PK) profiles.
5
ments insulin secretion dependent on the levels of glucose, provid-
6–8
ing a huge advantage of reducing incidence rate of hypoglycemia.
Moreover, the limited tissue distribution of FFA1 suggests that less
9
possibilities of adverse effects related to FFA1 in other tissues.
As summarized in the most recent review,10 many literatures
have reported structurally diverse FFA1 agonists based on
2
. Results and discussion
2.1. Chemistry
⇑
Corresponding authors at: Centre of Drug Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
The synthesis of heterocycle derivatives 3–5 and 8–11 is shown
2
10009, China.
in Scheme 1. Compound 2 was synthesized via our previous pub-
lished procedures. The intermediates 3a or 7a were provided
E-mail addresses: ydhuangwenlong@cpu.edu.cn (W. Huang), qianhai24@cpu.
edu.cn (H. Qian).
1
8
https://doi.org/10.1016/j.bmc.2017.12.030
0
968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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2
Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Synthetic FFA1 agonists.
followed by reduction and chlorination. The dihydrobenzofuran
1
1
1
3a was prepared via previous reported procedures. Williamson
ether synthesis of intermediates 4a, 8a or 12a–d with 13a,
followed by basic hydrolysis with lithium hydrate, afforded the
desired heterocycle derivatives 3–5 and 8–10. A new and simple
chiral resolution was developed to convert the racemate 13a to
optically pure 15a by using R-phenethylamine. Further condensa-
tion of intermediate 12b with 15a, followed by hydrolysis, fur-
nished the target compound 11.
The deuterated compounds 6 and 7 were synthesized according
to the route summarized in Scheme 2. The key intermediates 17a
and 17b were afforded by Knoevenagel reaction with Meldrum’s
acid and 4-hydroxybenzaldehyde 16a–b, followed by treating with
Fig. 2. Design strategy of FFA1 agonists bearing various heterocycles.
NaBH
4
. The intermediates 17a and 17b were further converted to
O and then
deuterated 18a and 18b by decarboxylation with D
2
esterification. Compounds 6 and 7 were synthesized from deuter-
ated intermediates 18a–b and 12a by condensation followed by
basic hydrolysis.
by reduction of intermediates 2a or 6a, which were prepared from
Suzuki-Miyaura cross-coupling of 3-formylphenylboronic acid
with commercially available aryl bromide 1a or 5a in the presence
of Pd(PPh
chloride catalyzed by DMF to afford chlorine intermediates 4a or
a. The isoxazole intermediates 12a–d were synthesized by
3 4
) . The intermediates 3a or 7a were treated with thionyl
2.2. FFA1 agonistic activity and structure-based optimization
8
palladium-catalyzed Suzuki coupling between commercially avail-
able isoxazole borate 9a and appropriate bromobenzene 10a–d,
To counteract the negative factors associated with high
lipophilicity, the LE, LLE, and LogD7.4 were monitored to screen
Scheme 1. Synthesis of target compounds 3–5 and 8–11. Reagents and conditions: (a) Pd(PPh
)
3 4
2
, Na CO
3
, toluene, ethanol, H
2 4 3
O, 80 °C, 12 h; (b) NaBH , CH OH, THF, 0 °C, 1 h;
(
c) SOCl
2
, CH
2
Cl
2
, DMF, 40 °C, 4 h; (d) 12b or 13a, K
2
CO
3
, KI, acetone, reflux, 12 h; and then LiOHÁH
2
O, THF/MeOH/H
2
O, rt, 4 h; (e) R-Phenethylamine, acetone, reflux; (f) 1 M
HCl; (g) H
2
SO
4
, MeOH, reflux.
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Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 2. Synthesis of target compounds 6 and 7. Reagents and conditions: (a) Meldrum’s acid, water, 75 °C, 2 h; (b) NaBH
4
, r.t.; (c) D
2
O, DMF, 100 °C, 12 h, then MeOH,
H
2
SO
4
, reflux, 3 h; (d) 12a, K
2
CO
3
, KI, acetone, reflux, 12 h; and then LiOHÁH
2 2
O, THF/MeOH/H O, rt, 4 h.
the heterocyclic analogs.30,31 As shown in Table 1, the thiophene
derivative 3 revealed a significantly reduced agonistic activity on
FFA1 compared with 2, 5-dimethyl pyrrole analog 2. As expected,
more than 100-fold decrease of lipophilicity (cLogP value: 3.348
vs 5.556) was afforded by replacing terminal pyrrole with parazole
dimethylisoxazole scaffold, we next directed efforts to explore
the acid head moiety and the deuterated phenylpropionic acid
moiety (compound 6 and 7) was introduced to reduce the b-oxida-
3
2,33
tion of phenylpropanoic acid.
Interestingly, the ortho-fluoro
(analog 7), a substituent increases potency in previous phenylpro-
3
4
(
compound 4 vs 2) but unfortunately also markedly reduced ago-
pionic acid series, turned out to be a slight lower potency than
un-substituted analog 6. This result indicated that the previous
structure-activity relationship (SAR) cannot be transferred directly
to deuterated series despite the difference between hydrogen and
deuterium is quite small.
nistic activity on FFA1. Gratifyingly, the 3,5-dimethylisoxazole
derivative 5 (EC50 = 27.5 nM), derived from bioisosterism of para-
zole analog 4, exhibited an even stronger potency than compound
2
(EC50 = 53.6 nM). Moreover, compound 5 (LogD7.4: 1.57) had
lipophilicity decreased by more than two log unit than compound
(LogD7.4: 3.95), and thereby provided a marked advantage on LLE
To better explore the SAR of potent compound 5 series, a molec-
ular docking study was performed using the crystal structure of
FFA1 (PDB code: 4PHU). As shown in Fig. 3, the 6-position of
2
value (6.0 vs 3.3). Having identified the preferable 3, 5-
Table 1
In vitro activities of target compounds.
Compd.
R
1
Het
EC50 (nM)a
cLogPb
LogD c7 .4
LE (LLE)d
TAK-875
2
31.8
53.6
4.697
5.556
2.43
3.95
0.20 (5.1)
0.24 (3.3)
H
H
H
3
4
157.1
103.7
5.35
ND
ND
3.348
5
6
7
8
9
H
27.5
38.4
45.7
18.6
39.7
53.7
7.9
3.212
3.395
3.678
3.706
3.956
3.661
3.706
1.57
1.83
2.05
1.96
2.13
2.07
1.93
0.27 (6.0)
0.27 (5.6)
0.27 (5.3)
0.28 (5.8)
0.25 (5.3)
0.23 (5.2)
0.32 (6.2)
4-Cl
6-Cl
6-Me
4-Cl
1
1
0
1 (S-isomer)
ND = Not determined. a EC50 values for FFA1 activities represent the mean of three independent determinations. b clogP values were estimated with ChemDraw Ultra, version
2.0. c LogD7.4 values were determined by shake-flask procedure. LE values were calculated by À
d
D
g = RT lnKD, presuming K
ꢀ EC50, and the LLE values were calculated by
1
D
the formula pEC50 À LogD7.4
.
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Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 3. The frontal view (A) and side-view (B) in docking model of compound 5 (silvery) in crystal structure of hFFA1. Key residues are labeled in white, TAK-875 is
represented by gray, and hydrogen bonding interactions are represented by yellow dashed lines, the unoccupied subpocket is highlighted by a red circle.
middle benzene points to an unoccupied hydrophobic subpocket
the red circle), so a better potency might be obtained by occupy-
2.3. Effect of selected compounds on glucose tolerance
(
ing this subpocket. Furthermore, the 4-position of middle benzene
was exposed to the outside of receptor, which could tolerate a vari-
ety of structural optimizations. Therefore, we next focus on
incorporating small substituent at the middle phenyl 6-position
and 4-position (Table 1). Interestingly, the 4-chlorine derivative 8
exhibited a significant improvement on potency in comparison
with compound 5, but the corresponding 6-position substituted
analogs 9 and 10 resulted in a slight reduce of activity. One reasonable
explanation is that the steric bulkiness of 6-position substituent
might disturb the binding interaction due to the unoccupied sub-
pocket is quite small. Subsequently, we separated the most potent
racemic compound 8 by a new chiral resolution with R-phenethy-
lamine and provided the more active (S)-enantiomer 11 (EC50 = 7.9
nM), which revealed desired lipophilicity (LogD7.4: 1.93), LE and
LLE values (0.32 and 6.2, respectively). As shown in Fig. 4, com-
pound 11 fitted to the binding site of TAK-875 very well, and the
acid moiety was coordinated by hydrogen bonding interaction
with Arg183, Arg258 and Tyr91. Moreover, the 4-chlorine of com-
pound 11 was exposed to the outside of FFA1, and could maintain
the preferential conformation with 3,5-dimethylisoxazole oriented
nearly perpendicularly to middle benzene.
Based on these positive in vitro results, these compounds with
considerable potency (compounds 5, 6, 8, 9 and 11) were selected
to evaluate the glucose-lowering effects in mice. As shown in Fig. 5,
all of these compounds exhibited glucose-lowering effects to some
extent during oral glucose tolerance test (OGTT). Gratifyingly, com-
pounds 6 and 11 (40 mg/kg) significantly lowered the levels of
plasma glucose. Notably, the glucose AUC0À120 min of compound 6
was lower than that of compounds 5 and 8 despite the latter have
stronger agonistic activity on FFA1. Besides, the in vitro potency of
compound 11 (EC50 = 7.9 nM) was better than TAK-875 (EC50
=
31.8 nM), but the glucose-lowering effect of compound 11
(À26.2%) was still inferior to that of TAK-875 (-37.5%).
2.4. Pharmacokinetic evaluation of compound 11
In order to determine whether the difference of PK properties is
the main reason for their in vivo pharmacodynamic differences, the
oral PK profiles of TAK-875 and compound 11 were evaluated in
fasted SD rats (Table 2). Surprisingly, compound 11 (3 mg/kg)
revealed an excellent PK profile in terms of plasma clearance (CL
= 18.51 mL/h/kg), maximum concentration (Cmax = 14.09 lg/mL),
Fig. 4. The docking model of compound 11 in the complex of FFA1 (PDB code: 4PHU). Key residues are labeled in white. Hydrogen bonds are represented by yellow dashed
lines. TAK-875 is represented by gray, and compound 11 is represented by silvery.
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Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 5. Effects of selected compounds during OGTT in fasting male ICR mice. (A) represented time-dependent changes of blood glucose levels after oral administration of
*
**
tested compounds, followed by 3 g/kg oral glucose challenge. (B) represented the AUC0À120min of blood glucose levels. Values are mean ± SD (n = 6 per group). p ꢁ .05, p ꢁ
01 and * p ꢁ .001 was analyzed using a one-way ANOVA with Tukey’s multiple-comparison post hoc test.
**
.
Table 2
PK parameters for TAK-875 and compound 11 in fasted SD rats.
Compd
Dose (po)a
CL (mL/h/kg)
C
max
(lg/mL)
AUC0-24 h
(lg/mL h)
T1/2 (h)
TAK-875
Compound 11
3 mg/kg
3 mg/kg
37.06 ± 5.81
18.51 ± 7.63
6.46 ± 1.52
14.09 ± 5.28
79.52 ± 5.37
142.02 ± 35.65
4.29 ± 0.56
4.65 ± 0.74
a
po = oral administration. Results are expressed as mean ± SD for four male SD rats (fasted for 12 h) in each group. Test compounds were suspended in 0.5% methyl-
cellulose aqueous solution.
and plasma half-life (T1/2 = 4.65 h), which resulted in a more than
otherwise indicated. Column chromatography was carried out on
silica gel (200–300 mesh) and monitored by thin layer chromatog-
raphy performed on GF/UV 254 plates and were visualized by
using UV light at 365 and 254 nm. Melting points were measured
using a RY-1 melting-point apparatus, which was uncorrected.
All of the NMR spectra were recorded on a Bruker ACF-300Q instru-
1
.8-fold drug exposure (AUC0À24 h = 142.02
lg/mL h) than that of
TAK-875 (3 mg/kg, AUC0À24h = 79.52 g/mL h). In other words,
l
there must be other factors for the in vivo pharmacodynamic dif-
ferences between TAK-875 and compound 11, such as the differ-
3
5
ences of drug–target residence time confirmed by Eli Lilly.
. Conclusion
In the continued SAR study of our previous heterocycle scaffolds
1
13
ment (300 MHz for H NMR and 75 MHz for C NMR spectra),
chemical shifts are expressed as values relative to tetramethylsi-
lane as internal standard, and coupling constants (J values) were
given in hertz (Hz). LC/MS spectra were recorded on a Waters liq-
uid chromatography-mass spectrometer system (ESI). Elemental
analyses were performed by the Heraeus CHN-O-Rapid analyzer.
3
represented by compound 2, we focused especially on improving
their drug-like physicochemical properties directed by lipophilic-
ity, LE and LLE. After explored several heterocyclic scaffolds, the
potent FFA1 agonists bearing 3, 5-dimethylisoxazole scaffold was
identified. Further structure-based optimization and chiral resolu-
tion, leading to the discovery of (S)-enantiomer 11, the most potent
agonist (EC50 = 7.9 nM) in this series with improved lipophilicity
1
1
TAK-875 was synthesized via published procedures.
4.1.1. General synthetic procedure for compounds 2a and 6a
The starting materials 1a or 5a (1 equiv) and (3-formylphenyl)
boronic acid (1 equiv) were dissolved in a mixture of 1 M sodium
carbonate aqueous solution (15 mL), EtOH (5 mL) and toluene
(
LogD7.4: 1.93), LE and LLE values (0.32 and 6.2, respectively). PK
evaluation in rats indicated excellent PK profiles and >1.8-fold
higher plasma exposure of 11 compared to TAK-875, the most
promising candidate in this field. Interestingly, the in vitro activity
and PK properties of compound 11 were better than TAK-875, but
the anti-diabetic effect of compound 11 was inferior to that of TAK-
(
3 4
15 mL). After nitrogen substitution, Pd(PPh ) (0.05 equiv) was
added. The reaction mixture was stirred at 80 °C under nitrogen
atmosphere for 12 h. The reaction mixture was cooled, and water
(
15 mL) was added. The mixture was diluted with AcOEt (15 mL),
and the insoluble material was filtered off through Celite. The
organic layer of the filtrate was washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo. The residue
was purified by silica gel column chromatography using a mixture
of petroleum ether/ethyl acetate (10:1, v/v) as eluent to afford the
desired product 2a or 6a as a solid.
8
75. Further studies on the identification of in vitro/in vivo correla-
tion are currently in progress and will be presented in due course.
4
. Experimental section
4.1. Chemistry
1
All starting materials, reagents and solvents were obtained from
4.1.1.1. 3-(3-methylthiophen-2-yl)benzaldehyde (2a). Yield: 56%; H
commercial sources and used without further purification unless
6
NMR (300 MHz, DMSO-d ) d: 10.08 (s, 1H), 7.97 (d, J = 1.3 Hz, 1H),
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6
Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
1
.88 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 1.3 Hz, 1H), 7.70 (d, J = 7.6 Hz,
H), 7.54 (d, J = 5.1 Hz, 1H), 7.05 (d, J = 5.1 Hz, 1H), 2.32 (s, 3H).
The following procedures described the synthesis of compound
12a, and these procedures can also be applied to the synthesis of
compounds 12b–d.
4
4
2
.1.1.2. 3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzaldehyde (6a). Yield:
Step 1: 3-(3,5-dimethylisoxazol-4-yl)benzaldehyde (11a). To a
mixture of 3,5-dimethyl-4- (4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)isoxazole (0.50 g, 2.2 mmol), 3-bromobenzaldehyde
1
8%; H NMR (300 MHz, DMSO-d
6
) d: 10.05 (s, 1H), 7.87–7.75 (m,
H), 7.70–7.56 (m, 2H), 3.72 (s, 3H), 2.24 (s, 3H), 2.15 (s, 3H).
(
0.50 g, 2.7 mmol), Pd(PPh
3
)
4
(0.13 g, 0.12 mmol) and sodium car-
O (35 mL, 3/1/3)
4.1.2. General synthetic procedure for target compounds 3 and 4
bonate (0.71 g, 6.7 mmol) in toluene/ethanol/H
2
Step 1: To a solution of 2a or 6a (1 equiv) in MeOH (10 mL) and
was refluxed under nitrogen atmosphere for 24 h. Then the mix-
ture was diluted with saturated ammonium chloride solution
and ethyl acetate, and the insoluble material was filtered through
Celite. The organic layer of the filtrate was washed with water
(25 mL) and brine (25 mL), dried over anhydrous sodium sulfate,
and evaporated in vacuo. The residue was purified by column chro-
matography using a mixture of petroleum ether/ethyl acetate (10:
THF (20 mL) was added portionwise sodium borohydride (3 equiv)
at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction
mixture was pouring into ice water (10 mL), and extracted with
ethyl acetate (3 Â 15 mL), the organic fractions were combined,
washed with saturated brine (2 Â 15 mL) prior to drying over
anhydrous sodium sulfate. After filtration and concentrate using
a rotary evaporator, the residue was used in next step without fur-
ther purification. To a solution of the obtained solid (1 equiv) in
dichloromethane (20 mL) was slowly added thionyl chloride (6
equiv) and a catalytic amount of DMF at room temperature. After
stirring at 40 °C for 4 h, the reaction was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography using a mixture of petroleum ether/ethyl acetate
20:1, v/v) as eluent to afford the desired intermediates 4a or 8a.
Step 2: To a solution of intermediates 4a or 8a (1 equiv) and
2 3
intermediate 13a (0.8 equiv) in acetone was added K CO (2 equiv)
and a catalytic amount of KI at room temperature. The reaction
mixture was heated to reflux with stirring overnight. Then the
reaction mixture was cooled to room temperature followed by fil-
tration and the filtrate was concentrated under vacuum. The resi-
due was purified by silica gel column chromatography using a
mixture of petroleum ether/ethyl acetate (4:1, v/v) as eluent to
afford a white solid. To a solution of the obtained solid (1 equiv)
1, v/v) as eluent to afford the desired product 11a (0.37 g, 77%) as a
1
white solid. H NMR (300 MHz, DMSO-d
6
) d: 10.08 (s, 1H), 7.99–
7.85 (m, 2H), 7.81–7.63 (m, 2H), 2.43 (s, 3H), 2.25 (s, 3H).
Step 2: 4-(3-(chloromethyl)phenyl)-3,5-dimethylisoxazole (12a).
To a solution of 11a (0.3 g, 1.49 mmol) in MeOH (10 mL) and THF
(20 mL) was added portionwise sodium borohydride (0.17 g,
4.47 mmol) at 0 °C and the mixture was stirred at 0 °C for 1 h.
The reaction mixture was pouring into ice water (10 mL), and
extracted with ethyl acetate (3 Â 15 mL), the organic fractions
were combined, washed with saturated brine (2 Â 15 mL) prior
to drying over anhydrous sodium sulfate. After filtration and con-
centrate using a rotary evaporator, the residue was used in next
step without further purification. To a solution of the obtained
solid in dichloromethane (20 mL) was slowly added thionyl chlo-
ride (1.06 g, 8.94 mmol) and a catalytic amount of DMF at room
temperature. After stirring at 40 °C for 4 h, the reaction was con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography using a mixture of petroleum
(
in 2:3:1 THF/MeOH/H
After stirring at room temperature for 4 h, the volatiles were
removed under reduced pressure. The residue was acidified with
2
O (18 mL) was added LiOHÁH
2
O (3 equiv).
ether/ethyl acetate (20:1, v/v) as eluent to afford the 12a (0.27 g,
1
82%) as a white solid. H NMR (300 MHz, DMSO-d
6
) d: 7.57–7.43
1
N hydrochloric acid solution, and then filtered and the filter cake
(m, 3H), 7.36, 7.34 (dt, J = 6.9, 1.8 Hz, 1H), 4.82 (s, 2H), 2.41 (s,
was washed with 5 mL of water, dried in vacuum to afford a white
powder. The white powder was purified by column chromatogra-
phy using a mixture of petroleum ether/ethyl acetate (2:1–1:2, v/
v) as eluent to afford the target compounds 3 or 4 as white solid.
3H), 2.23 (s, 3H).
4.1.3. General synthetic procedure for target compounds 5 and 8–10
To a solution of 12a–d (1 equiv) and intermediate 13a (0.8
2 3
equiv) in acetone was added K CO (2 equiv) and a catalytic
4
.1.2.1. 2-(6-((3-(3-methylthiophen-2-yl)benzyl)oxy)-2,3-dihydrobenzo-
amount of KI at room temperature. The reaction mixture was
heated to reflux with stirring overnight. Then the reaction mixture
was cooled to room temperature followed by filtration and the fil-
trate was concentrated under vacuum. The residue was purified by
silica gel column chromatography using a mixture of petroleum
ether/ethyl acetate (4:1, v/v) as eluent to afford a white solid. To
1
furan-3-yl)acetic acid (3). Yield 43%; white solid, m.p. 87–89 °C. H
NMR (300 MHz, DMSO-d ) d: 7.51 (s, 1H), 7.47–7.32 (m, 4H), 7.12
d, J = 7.8 Hz, 1H), 6.98 (d, J = 5.1 Hz, 1H), 6.56–6.43 (m, 2H), 5.11
s, 2H), 4.69 (t, J = 9.0 Hz, 1H), 4.31–4.17 (m, 1H), 3.72, 3.68 (dd,
6
(
(
J = 13.7, 7.3 Hz, 1H), 2.73, 2.68 (dd, J = 16.6, 5.6 Hz, 1H), 2.48, 2.43
dd, J = 16.6, 7.3 Hz, 1H), 2.26 (s, 3H). 13C NMR (75 MHz, DMSO-
) d: 173.10, 160.71, 159.06, 137.78, 136.54, 134.23, 133.15,
31.45, 128.86, 127.76, 127.43, 126.67, 126.36, 124.59, 124.16,
(
a solution of the obtained solid (1 equiv) in 2:3:1 THF/MeOH/H
2
O
d
6
(18 mL) was added LiOHÁH O (3 equiv). After stirring at room tem-
2
1
1
perature for 4 h, the volatiles were removed under reduced pres-
sure. The residue was acidified with 1N hydrochloric acid
solution, and then filtered and the filter cake was washed with 5
mL of water, dried in vacuum to afford a white powder. The white
powder was purified by column chromatography using a mixture
of petroleum ether/ethyl acetate (2:1–1:2, v/v) as eluent to afford
the target compounds as white solid.
22.00, 106.84, 96.80, 77.11, 69.05, 38.85, 37.08, 14.63. ESI-MS
À
m/z: 379.5 [MÀ1] . Anal. calcd. For C22
20 4
H O S: C, 69.45; H, 5.30;
Found: C, 69.49; H, 5.31.
4
.1.2.2. 2-(6-((3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzyl)oxy)-2,3-
dihydrobenzofuran-3-yl)acetic acid (4). Yield 59%; white solid, m.
p. 195–197 °C. H NMR (300 MHz, DMSO-d
1
6
) d: 7.51–7.38 (m,
1
1
4
1
H), 7.36–7.24 (m, 2H), 7.20 (d, J = 7.1 Hz, 1H), 7.11 (d, J = 7.8 Hz,
4.1.3.1. 2-(6-((3-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)-2,3-dihydrobenzo-
H), 6.61–6.37 (m, 2H), 5.09 (s, 2H), 4.68 (t, J = 8.9 Hz, 1H), 4.28–
.10 (m, 1H), 3.70 (s, 3H), 3.68–3.64 (m, 1H), 2.72, 2.66 (dd, J =
6.6, 5.2 Hz, 1H), 2.46 (d, J = 9.3 Hz, 1H), 2.19 (s, 3H), 2.11 (s, 3H).
furan-3-yl)acetic acid (5). Yield 61%; white solid, m.p. 105–107 °C.
1
6
H NMR (300 MHz, DMSO-d ) d: 7.53–7.30 (m, 4H), 7.10 (d, J =
7.9 Hz, 1H), 6.55–6.42 (m, 2H), 5.10 (s, 2H), 4.66 (t, J = 9.0 Hz,
1H), 4.21–4.12 (m, 1H), 3.68–3.64 (m, 1H), 2.71, 2.66 (dd,
J = 16.6, 5.5 Hz, 1H), 2.49–2.40 (m, 1H), 2.36 (s, 3H), 2.19 (s, 3H).
1
3
6
C NMR (75 MHz, DMSO-d ) d: 173.03, 160.66, 159.07, 143.07,
1
1
9
7
37.37, 136.19, 133.77, 128.55, 128.21, 128.04, 125.17, 124.57,
21.91, 106.87, 96.79, 77.08, 69.18, 40.00, 37.05, 35.69, 12.16,
1
3
6
C NMR (75 MHz, DMSO-d ) d: 173.00, 165.13, 160.65, 158.99,
À
.78. ESI-MS m/z: 391.2 [MÀ1] . Anal. calcd. For C23
24
H N
2
O
4
: C,
158.04, 137.81, 129.87, 128.94, 128.65, 128.13, 127.92, 126.64,
124.59, 121.99, 117.74, 115.68, 106.84, 96.80, 77.08, 68.98, 40.19,
0.39; H, 6.16; N, 7.14; Found: C, 70.35; H, 6.17; N, 7.15.
Please cite this article in press as: Li Z., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.030

Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
À
3
C
7.04, 11.22, 10.36. ESI-MS m/z: 378.2 [MÀH] . Anal. calcd. For
4.76 (t, J = 9.11 Hz, 1H, -OCH
1H, -OCH
2.68 (dd, J = 5.72, 16.41 Hz, 1H, -COCH
6.41 Hz, 1H, -COCH ).
2
), 4.26, 4.24 (dd, J = 5.72, 9.11 Hz,
22
H
21NO
5
: C, 69.65; H, 5.58; N, 3.69; Found: C, 69.68; H, 5.57;
2
), 3.74–3.84 (m, 1H, ArCH), 3.72 (s, 3H, -OCH
3
), 2.73,
N, 3.68.
2
), 2.56, 2.51 (dd, J = 9.11,
1
2
4
2
.1.3.2.
2-(6-((4-chloro-3-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)-
,3-dihydrobenzofuran-3-yl)acetic acid (8). Yield 31%; white solid,
4.1.3.5. (S)-2-(6-((4-chloro-3-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)-
2,3-dihydrobenzofuran-3-yl)acetic acid (11). The target compound
was prepared as described for compound 5 by using intermediates
1
m.p. 168–169 °C. H NMR (300 MHz, DMSO-d
Hz, 1H), 7.54–7.40 (m, 2H), 7.11 (d, J = 8.2 Hz, 1H), 6.55–6.43 (m,
6
) d: 7.61 (d, J = 8.2
2
3
=
H), 5.08 (s, 2H), 4.67 (t, J = 9.0 Hz, 1H), 4.24–4.12 (m, 1H), 3.78–
.59 (m, 1H), 2.67, 2.61 (dd, J = 16.3, 5.4 Hz, 1H), 2.43, 2.38 (dd, J
16.3, 9.1 Hz, 1H), 2.24 (s, 3H), 2.06 (s, 3H). 13C NMR (75 MHz,
15a and 12b as starting material. Yield 36%; white solid, 98.2% ee.
1
m.p. 165–166 °C. H NMR (300 MHz, DMSO-d
6
) d: 7.63 (d, J = 8.2
Hz, 1H), 7.54–7.41 (m, 2H), 7.11 (d, J = 7.9 Hz, 1H), 6.54–6.42 (m,
2H), 5.09 (s, 2H), 4.68 (t, J = 9.1 Hz, 1H), 4.24–4.16 (m, 1H), 3.78–
3.59 (m, 1H), 2.72, 2.66 (dd, J = 16.6, 5.5 Hz, 1H), 2.43, 2.38 (dd, J
DMSO-d
6
) d: 173.57, 166.14, 160.66, 158.71, 158.54, 136.82,
1
1
32.51, 131.40, 129.76, 129.24, 128.29, 124.60, 122.54, 113.88,
06.77, 96.83, 77.34, 68.20, 39.95, 37.31, 11.26, 10.11. ESI-MS m/
1
3
= 16.3, 9.1 Hz, 1H), 2.25 (s, 3H), 2.07 (s, 3H). C NMR (75 MHz,
DMSO-d ) d: 173.56, 166.13, 160.65, 158.72, 158.54, 136.82,
132.51, 131.40, 129.76, 129.24, 128.29, 124.60, 122.54, 113.88,
À
z: 412.2 [MÀH] . Anal. calcd. For C22
H
20ClNO
5
: C, 63.85; H, 4.87;
6
N, 3.38; Found: C, 63.81; H, 4.88; N, 3.37.
1
06.77, 96.83, 77.34, 68.20, 39.95, 37.31, 11.26, 10.11. ESI-MS m/
À
4.1.3.3.
2-(6-((2-chloro-5-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)-
z: 412.1 [MÀH] . Anal. calcd. For C22
5
H20ClNO : C, 63.85; H, 4.87;
2
,3-dihydrobenzofuran-3-yl)acetic acid (9). Yield 45%; white solid,
N, 3.38; Found: C, 63.88; H, 4.86; N, 3.37.
1
m.p. 127–128 °C. H NMR (300 MHz, DMSO-d
6
) d: 7.67–7.49 (m,
2
2
3
H), 7.40 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 8.1 Hz, 1H), 6.57–6.38 (m,
4.1.4. General synthetic procedure for intermediates 18a–b
H), 5.15 (s, 2H), 4.68 (t, J = 9.0 Hz, 1H), 4.29–4.11 (m, 1H), 3.73–
.64 (m, 1H), 2.71, 2.66 (dd, J = 16.5, 5.5 Hz, 1H), 2.46 (d, J = 9.0
A mixture of Meldrum’s acid (1.1 equiv) and substituted ben-
zaldehyde 16a–b (1 equiv) in water (10 mL) was stirred at 75 °C
for 2 h. After cooling to room temperature the solid product was
filtered on Buchner and dried under vacuum. The purification from
traces of starting aldehyde, when present, was performed by crys-
tallization with ethanol. The obtained intermediate (1 equiv) was
13
Hz, 1H), 2.30 (s, 3H), 2.17 (s, 3H). C NMR (75 MHz, DMSO-d
6
) d:
1
1
6
73.55, 165.98, 161.17, 159.24, 158.46, 135.30, 132.35, 130.96,
30.53, 130.34, 129.42, 125.16, 122.85, 107.16, 97.32, 77.65,
7.29, 40.74, 37.53, 11.70, 10.79. ESI-MS m/z: 412.2 [MÀH] . Anal.
À
calcd. For C22
H
20ClNO
5
: C, 63.85; H, 4.87; N, 3.38; Found: C, 63.87;
stirred in methanol (20 mL) and NaBH
the temperature between 23 and 28 °C. After stirring for another
5 min, the reaction mixture was acidified (PH: 5–6) with 1N
4
was added slowly to keep
H, 4.86; N, 3.38.
1
4
.1.3.4.
2-(6-((5-(3,5-dimethylisoxazol-4-yl)-2-methylbenzyl)oxy)-
hydrochloric acid solution, and extracted with ethyl acetate (4 Â
25 mL), the organic fractions were combined, washed with satu-
rated brine (2 Â 15 mL) prior to drying over anhydrous sodium sul-
fate. After filtration and concentrate using a rotary evaporator, the
residue was used in the next step without further purification. To a
solution of the obtained solid in DMF (10 mL) was added 0.5 mL
2,3-dihydrobenzofuran-3-yl)acetic acid (10). Yield 32%; white solid,
1
m.p. 115–116 °C. H NMR (300 MHz, DMSO-d
3
6
) d: 7.43–7.19 (m,
H), 7.12 (d, J = 8.7 Hz, 1H), 6.57–6.45 (m, 2H), 5.09 (s, 2H), 4.68
(
(
t, J = 9.0 Hz, 1H), 4.20, 4.17 (dd, J = 8.9, 6.8 Hz, 1H), 3.74–3.67
m, 1H), 2.72, 2.66 (dd, J = 16.6, 5.6 Hz, 1H), 2.46 (d, J = 9.0 Hz,
1
1
1
7
H), 2.34 (s, 6H), 2.17 (s, 3H). 13C NMR (75 MHz, DMSO-d
73.05, 164.86, 160.68, 159.08, 158.08, 135.99, 135.53, 130.62,
28.90, 128.28, 127.15, 124.58, 122.03, 115.62, 106.80, 96.81,
6
) d:
2
D O at room temperature. After stirring at 100 °C for 12 h, the mix-
ture was diluted with water (60 mL), and extracted with ethyl
acetate (4 Â 25 mL), washed with saturated brine (2 Â 15 mL) prior
to drying over anhydrous sodium sulfate and concentrated in
vacuo. The residue was dissolved in MeOH (20 mL), and then conc.
7.12, 67.81, 40.27, 37.07, 18.13, 11.16, 10.34. ESI-MS m/z: 392.2
À
[
MÀH] . Anal. calcd. For C23
5
H23NO : C, 70.21; H, 5.89; N, 3.56;
Found: C, 70.25; H, 5.88; N, 3.57.
2 4
H SO (2 mL) was added slowly at ambient temperature. After stir-
ring at reflux for 3 h, the volatiles were removed under reduced
pressure. The residue was extracted with ethyl acetate (3 Â 25
mL), and the combined organic phases were washed with brine
(2 Â 15 mL), dried and filtered. The residue was purified by silica
gel column chromatography using a mixture of petroleum ether/
ethyl acetate (10:1, v/v) as eluent to afford the desired product
as a solid.
4
.1.3.4. Methyl (S)-2-(6-hydroxy-2,3-dihydrobenzofuran-3-yl)acetate
(15a). A solution of 13a (9.32 g, 48 mmol) in acetone (50 mL) was
heated to reflux, and the solution of R-Phenethylamine (2.91 g, 24
mmol) in acetone (15 mL) was slowly drip into the solution above,
then the mixture was allowed to stand at room temperature for 10
h, and a white crystal was precipitate out. The mixture was filtered
and the filter cake was treated with 1 M HCl and adjusted pH < 2,
which was extracted with ethyl acetate (3 Â 20 mL), the organic
fractions were combined, washed with saturated brine (2 Â 15
4.1.4.1. Methyl 3-(4-hydroxyphenyl)propanoate-2,2-d
2
(18a). Yield:
) d: 9.32 (s, 1H), 7.16 (d, J = 7.9
Hz, 2H), 6.93 (d, J = 7.9 Hz, 2H), 3.63 (s, 3H), 2.74 (s, 2H).
1
56%; H NMR (300 MHz, DMSO-d
6
2 4
mL) prior to drying over anhydrous Na SO . After filtration and
concentrate using a rotary evaporator, the residual white solid
was dissolved in acetone (30 mL) and repeated the procedure
above for three times, the obtained residue was dissolved in MeOH
4.1.4.2.
Methyl
3-(2-fluoro-4-hydroxyphenyl)propanoate-2,2-d
2
1
(18b). Yield: 48%; H NMR (300 MHz, DMSO d
6
) d: 9.35 (s, 1H),
(
15 mL), and then conc. H
2
SO
4
(1 mL) was added slowly at ambient
7.23–7.15 (m, 2H), 7.05 (d, J = 6.8 Hz, 1H), 3.63 (s, 3H), 2.74 (s, 2H).
temperature. After stirring at reflux for 3 h, the volatiles were
removed under reduced pressure. The residue was extracted with
ethyl acetate (3 Â 25 mL), and the combined organic phases were
washed with brine (2 Â 15 mL), dried, filtered and the filtrate
was concentrated under vacuum. The residue was purified by silica
gel column chromatography using a mixture of petroleum ether/
ethyl acetate (10:1, v/v) as eluent to afford 15a (0.32 g, 3.3%) as a
4.1.5. General synthetic procedure for target compounds 6 and 7
To a solution of 12a (1 equiv) and intermediate 18a–b (0.8
equiv) in acetone was added K CO (2 equiv) and a catalytic
2 3
amount of KI at room temperature. The reaction mixture was
heated to reflux with stirring overnight. Then the reaction mixture
was cooled to room temperature followed by filtration and the fil-
trate was concentrated under vacuum. The residue was purified by
silica gel column chromatography using a mixture of petroleum
white solid, 99.6% ee. 1H NMR (300 MHz, CDCl
.72 Hz, 1H, ArH), 6.35–6.32 (m, 2H, ArH), 4.83 (brs, 1H, ArOH),
3
) d: 6.98 (d, J =
8
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8
Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
ether/ethyl acetate (4:1, v/v) as eluent to afford a white solid. To a
solution of the obtained solid (1 equiv) in 2:3:1 THF/MeOH/H
18 mL) was added LiOHÁH O (3 equiv). After stirring at room tem-
4.4. Biological methods
2
O
(
2
4.4.1. FFA1 agonistic activity (FLIPR Assay)
CHO cells stably expressing human FFA1 were seeded into 96-
well plates at a density of 15 K cells/well and incubated 16 h in
perature for 4 h, the volatiles were removed under reduced pres-
sure. The residue was acidified with 1N hydrochloric acid
solution, and then filtered and the filter cake was washed with 5
mL of water, dried in vacuum to afford a white powder. The white
powder was purified by column chromatography using a mixture
of petroleum ether/ethyl acetate (2:1–1:2, v/v) as eluent to afford
the target compounds as white solid.
5% CO
washed with 100
quently, cells were incubated in loading buffer (containing 2.5
g/mL fluorescent calcium indicator Fluo 4-AM, 2.5 mmol/L probe-
necid and 0.1% fatty acid-free BSA) for 1 h at 37 °C. Various concen-
trations of test compounds or -linolenic acid (Sigma) were added
2
at 37 °C. Then, the culture medium was removed and
lL of Hank’s Balanced Salt Solution. Subse-
l
c
into the well and the intracellular calcium flux signals were mea-
sured by FLIPR Tetra system (Molecular Devices). The agonistic
activities of test compounds on human FFA1 were expressed as
4
.1.5.1. 3-(4-((3-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)phenyl)pro-
panoic-2,2-d acid (6). Yield 43%; white solid, m.p. 121–123 °C.
H NMR (300 MHz, DMSO-d ) d: 12.08 (brs, 1H), 7.51–7.37 (m,
H), 7.33 (d, J = 6.6 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H), 6.93 (d, J =
.0 Hz, 2H), 5.13 (s, 2H), 2.74 (s, 2H), 2.37 (s, 3H), 2.20 (s, 3H).
2
1
6
2+
[
(A À B)/(C À B)] Â 100 (increase of the intracellular Ca concen-
tration (A) in the test compounds-treated cells and (B) in vehicle-
treated cells, and (C) in 10 -linolenic acid-treated cells).
EC50 values were obtained with Prism 5 software (GraphPad).
3
8
1
3
lM c
6
C NMR (75 MHz, DMSO-d ) d: 174.25, 165.60, 158.52, 156.99,
1
6
38.41, 133.55, 130.40, 129.54, 128.53, 127.15, 116.19, 115.16,
À
9.27, 29.86, 11.72, 10.86. ESI-MS m/z: 352.1 [MÀH] . Anal. calcd.
4
.4.2. Animals and statistical analysis of the data
weeks old SD rats (male, 180–200 g, batch number: SCXK
Jiangsu)2017-0001) and 10 weeks old ICR mice (male, 18–22 g,
19 2 4
For C21H D NO : C, 71.37; H, 6.56; N, 3.96; Found: C, 71.33; H,
6
8
.57; N, 3.95.
(
batch number: SCXK(Jiangsu)2016-0011) were purchased from
Comparative Medicine Centre of Yangzhou University (Jiangsu,
China), acclimatized for 1 week before experiments. The breeding
room was keep on a constant 12 h light/black cycle with tempera-
ture at 23 ± 2 °C and relative humidity 50 ± 10% throughout the
experimental period. Mice were allowed ad libitum access to stan-
dard pellets and water unless otherwise stated, and the vehicle
used for drug administration was 0.5% Caboxy Methyl Cellulose
aqueous solution for all animal studies. All animal experimental
protocols were approved by the ethical committee at China Phar-
maceutical University and conducted according to the Laboratory
Animal Management Regulations in China and adhered to the
Guide for the Care and Use of Laboratory Animals published by
the National Institutes of Health (NIH Publication NO. 85-23,
revised 2011).
4
.1.5.2. 3-(4-((3-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)-2-fluorophenyl)
acid (7). Yield 36%; white solid, m.p. 129–131 °C.
H NMR (300 MHz, DMSO-d ) d: 7.50 (d, J = 6.7 Hz, 1H), 7.46 – 7.42
m, 2H), 7.33 (d, J = 6.9 Hz, 1H), 7.20 (t, J = 8.7 Hz, 1H), 6.91–6.75
propanoic-2,2-d
2
1
6
(
(
1
3
m, 2H), 5.15 (s, 2H), 2.76 (s, 2H), 2.37 (s, 3H), 2.20 (s, 3H).
) d: 173.52, 165.12, 159.18, 157.92,
37.40, 130.85, 129.98, 128.96, 128.17, 126.74, 115.66, 110.85,
C
NMR (75 MHz, DMSO-d
1
1
6
02.44, 102.10, 69.19, 22.95, 11.17, 10.32. ESI-MS m/z: 370.1
À
[
3
MÀH] . Anal. calcd. For C21
18 2 4
H D FNO : C, 67.91; H, 5.97; N,
.77; Found: C, 67.94; H, 5.96; N, 3.78.
4
.2. Molecular modeling
Docking simulations were performed using MOE (version
008.10, The Chemical Computing Group, Montreal, Canada). The
2
Statistical analyses were performed using specific software
crystal structure of FFA1 (PDB ID: 4PHU) was downloaded from
the Protein Data Bank (PDB). Prior to ligand docking, the structure
was prepared with Protonate 3D and a Gaussian Contact surface
was draw around the binding site. Subsequently, the active site
was isolated and the backbone was removed. The ligand poses
was filtered using Pharmacophore Query Editor. The compound
structures were placed in the site with Pharmacophore method
and then ranked with the London dG scoring function. For the
energy minimization in the pocket, MOE Forcefield Refinement
was used and ranked with the London dG scoring function.
(
GraphPad InStat version 5.00, GraphPad software, San Diego, CA,
USA). Unpaired comparisons were analyzed using the two-tailed
Student’s t-test, unless otherwise stated.
4
.4.2.1. Oral glucose tolerance test. Normal ICR mice 10 weeks old
after 1 week adaptation were fasted overnight (12 h), weighted,
bled via the tail tip, and randomized into 7 groups (n = 6). Mice
were administrated orally with a single dose of vehicle, TAK-875
(
20 mg/kg), or selected compounds (40 mg/kg) and subsequently
dosed orally with 30% aqueous glucose solution (3 g/kg) after half
an hour. Blood samples were collected immediately before drug
administration (À30 min), before glucose challenge (0 min), and
at 15, 30, 60 and 120 min post-dose. The blood glucose was mea-
sured by blood glucose test strips (SanNuo ChangSha, China). Data
and statistical analyses were performed using GraphPad InStat ver-
sion 5.00 (GraphPad software, San Diego, CA, USA). General effects
were analyzed using a one-way ANOVA with Tukey’s multiple-
comparison post hoc test.
4.3. Determination of logD7.4
In 10 mL glass vial, 40 mL of 10 mM stock solution in DMSO was
added 1980 mL phosphate buffer solution (0.01 M, pH = 7.4) and
980 mL 1-octanol (Sigma), obtaining 100 mM final concentration
1
of the test compounds. The glass vials were shaken at 700 rpm
for 24 h and left for 1 h to allow the phases to separate. The 1-octa-
nol phase was pipetted out and diluted Â10 with a mixture of
methanol (containing 0.1% formic acid) and MilliQ H
2
O (4:1) prior
4.4.2.2. Pharmacokinetic evaluation in SD rats. 8 weeks old normal
male SD rats after 1 week adaptation were fasted overnight (12
h), weighted, and randomized into 2 groups (n = 4 for each group).
The pharmacokinetic profiles of TAK-875 and compound 11 were
determined in fasted male SD rats following single oral dosing (3
mg/kg suspended in 0.5% methylcellulose aqueous solution). At
5, 15, 30, 45 min and 1, 2, 4, 6, 8, 12, 18, 24 h after oral administra-
tion, blood samples were collected from tail vein into heparin-
containing microcentrifuge tubes. Then centrifuged at 6000 rpm
to analysis on HPLC with 60 mL injections. The buffer phase was
analyzed directly in 120 mL injections. Each HPLC analysis was per-
formed in duplicates by the method described above. The logD7.4
values were calculated by dividing the peak area (mAU * min) at
2
54 nm of the 1-octanol phase by the corresponding peak area of
the buffer phase. Peak areas were corrected for systematic errors
using two calibration points per compound per solvent. All test
compounds were analyzed in three independent experiments.
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Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
for 10 min to separate the plasma and stored at À20 °C until anal-
ysis. Plasma proteins were precipitated with two volumes of
methanol containing an internal standard, mixed by vortexing,
and centrifuged at 14,000 rpm for 14 min. The supernatant was
9. Rayasam GV, Tulasi VK, Davis JA, Bansal VS. Expert Opin Ther Targets.
007;11:661–671.
2
10. Li Z, Xu X, Huang W, Qian H. Med Res Rev. 2017. https://doi.org/10.1002/
med.21441.
11. Negoro N, Sasaki S, Mikami S, et al. Chem Lett. 2010;1:290–294.
12. Mikami S, Kitamura S, Negoro N, et al. J Med Chem. 2012;55:3756–3776.
13. Negoro N, Sasaki S, Mikami S, et al. J Med Chem. 2012;55:3960–3974.
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diluted and centrifuged again, and 5 lL of supernatant was
analyzed by Waters LC-PDA-MS/MS to determine plasma drug
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Acknowledgement
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This study was supported by grants from the National Natural
Science Foundation of China (Grants 81673299 and 81273376).
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Please cite this article in press as: Li Z., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.030