Discovery of HWL-088: A highly potent FFA1/GPR40 agonist bearing a phenoxyacetic acid scaffold

Article abstract of DOI:10.1016/j.bioorg.2019.103209

Based on a previously reported phenoxyacetic acid scaffold, compound 7 (HWL-088) has been identified as a superior free fatty acid receptor 1 (FFA1) agonist by comprehensive structure-activity relationship study. Our results indicated that the introduction of ortho-fluoro greatly increased the activity of phenoxyacetic acid series, and the unique structure-activity relationship in biphenyl moiety is different from previously reported FFA1 agonists. Moreover, the modeling study was also performed to better understand the binding mode of present series. Compound 7 significantly improved glucose tolerance both in normal and diabetic models, and even exerted greater potential on glucose control than that of TAK-875. These findings provided a novel candidate HWL-088, which is currently in preclinical study to evaluate its potential for the treatment of diabetes.

Full text of DOI:10.1016/j.bioorg.2019.103209

Journal Pre-Proof
Discovery of HWL-088: A Highly Potent FFA1/GPR40 Agonist Bearing A
Phenoxyacetic Acid Scaffold
Zheng Li, Qiang Ren, Xuekun Wang, Zongtao Zhou, Lijun Hu, Liming Deng,
Li Guan, Qianqian Qiu
PII:
S0045-2068(19)30880-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103209
YBIOO 103209
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2019
16 July 2019
15 August 2019
Please cite this article as: Z. Li, Q. Ren, X. Wang, Z. Zhou, L. Hu, L. Deng, L. Guan, Q. Qiu, Discovery of HWL-088:
A Highly Potent FFA1/GPR40 Agonist Bearing A Phenoxyacetic Acid Scaffold, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.103209
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Discovery of HWL-088: A Highly Potent FFA1/GPR40 Agonist Bearing A Phenoxyacetic
Acid Scaffold
Zheng Li1, *, Qiang Ren1, Xuekun Wang2, Zongtao Zhou1, Lijun Hu1, Liming Deng1, Li Guan1, *
Qianqian Qiu3, *
,
1 School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
2 College of pharmacy, Liaocheng University, Liaocheng 252059, China.
3 School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and
Environmental Protection, Yancheng Teachers’University, Yancheng, PR China.
Abstract
Based on a previously reported phenoxyacetic acid scaffold, compound 7 (HWL-088) has been
identified as a superior free fatty acid receptor 1 (FFA1) agonist by comprehensive
structure-activity relationship study. Our results indicated that the introduction of ortho-fluoro
greatly increased the activity of phenoxyacetic acid series, and the unique structure-activity
relationship in biphenyl moiety is different from previously reported FFA1 agonists. Moreover, the
modeling study was also performed to better understand the binding mode of present series.
Compound 7 significantly improved glucose tolerance both in normal and diabetic models, and
even exerted greater potential on glucose control than that of TAK-875. These findings provided a
novel candidate HWL-088, which is currently in preclinical study to evaluate its potential for the
*
li.zheng.sky@163.com (Z. Li), guanli@gdpu.edu.cn (L. Guan), qianqianqiu_cpu@126.com (Q. Qiu).
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treatment of diabetes.
Keywords: Diabetes; FFA1; GPR40; Hyperglycemia; Phenoxyacetic acid.
1. Introduction
Type 2 diabetes mellitus (T2DM) is mainly displayed as sustained hyperglycemia due to insulin
insufficient and/or resistance.[1, 2] Current insulin secretagogues such as sulfonylureas are widely
used,[3] but these agents usually cause the side effect of hypoglycemia due to the
glucose-independent insulin secretion.[4] Moreover, most of current anti-diabetic drugs are
associated with lactic acidosis, cardiovascular risk, and weight gain.[5-8] Hence, there still have
many unmet needs to provide new anti-diabetic drugs. The free fatty acid receptor 1 (FFA1) has
attracted considerable attention as a potential anti-diabetic target.[9] In pancreatic β-cells, the
activation of FFA1 promotes insulin secretion in a glucose dependent manner, which provides a
lower risk of hypoglycemia than sulfonylureas.[10-12] Moreover, FFA1 is mainly expressed in
pancreas islet, which reduces the possibility of target-induced side effects in other tissues.[13]
As described in review[14], many FFA1 agonists containing phenylpropionic acid scaffold
have been disclosed (Figure 1).[15-25] Among them, several candidates including AMG-837,
LY2881835 and TAK-875 have reached in clinical trials.[9, 14] More recently, a series of novel
FFA1 agonists were also explored based on hybrid of quercetin and oleic acid.[26, 27] In our
laboratory, several novel scaffolds were also comprehensively explored to extend the chemical
space of FFA1 agonists.[28-34] To avoid β-oxidation of phenylpropanoic acid, a phenoxyacetic
acid scaffold has been used to replace the phenylpropanoic acid of FFA1 agonists. However, the
obtained phenoxyacetic acid analog significantly reduced agonistic activity compared to
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phenylpropanoic acid analog (Figure 2A and B).[22, 35] Herein, we describe structural
optimization of phenoxyacetic acid series and our efforts toward the discovery of superior agonist
(Figure 2C). All of these efforts ultimately led to the identification of a new superior agonist 7
(HWL-088, EC50 = 19 nM), which revealed better in vitro and in vivo activity than TAK-875, the
most advanced candidate of FFA1 agonists.
2. Results and Discussion
2.1. Chemistry
The synthetic route of compounds 4-11 and 14-17 is illustrated in Scheme 1. Treatment of methyl
bromoacetate with phenol 1a-b provided 2a-b, which were converted to intermediates 3a-b by
Baeyer–Villiger oxidation. Hydrolysis of intermediates 3a-b in the presence of sodium methoxide
provided 4a-b. Treating commercially available 5a with thionyl chloride yielded intermediate 6a,
which was condensed with 4a-b by Williamson ether synthesis, followed by hydrolysis to furnish
target compounds 4 and 5. The isoxazole 8a was synthesized using Suzuki coupling by treating
(3-formylphenyl) boronic acid with commercially available isoxazole borate 7a. The intermediate
13a was synthesized by alkylation of 11a or 11b, which were derived from Suzuki coupling
reaction of (3-formylphenyl) boronic acid with 10a or 10b. Reduction of aldehyde 8a or 13a in the
presence of sodium borohydride, followed by treating with thionyl chloride yielded intermediate
9a or 14a. Condensation of 9a or 14a with 4a-b by Williamson ether synthesis, and then basic
hydrolysis provided target compounds 6-11 and 14-17.
The target compounds 12-13 were synthesized as shown in Scheme 2. Intermediate 17a-b
was produced from reduction of nitro 16a-b, which was prepared by Williamson ether synthesis of
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methyl chloroacetate or chloroacetamide with phenols 15a. Compound 17a or 17b was connected
with 18a by reductive amination reaction in the presence of NaBH3CN to provide target
compound 13 and intermediate 19a. The ester hydrolytic reaction of intermediate 19a afforded
target compound 12.
2.2. Structure-activity relationship study
All of target compounds were evaluated in a cell-based functional calcium mobilization assay. As
shown in Table 1, we firstly explored the importance of ortho-fluoro at C ring, a substituent
introduced to improve metabolic stability and slightly increase potency on phenylpropionic acid
series in previous report.[15] Indeed, the introduction of ortho-fluoro (compound 4) significantly
improved potency compared to parent compound 2, while ortho-methyl analog 5 turned out to be
more than 3-fold reduced activity compared to analog 4. Similar results were observed in biphenyl
scaffold (such as compound 7 vs 8, and 9 vs 10), which suggested that ortho-fluoro is crucial to
maintain high potency of phenoxyacetic acid derivative. The 3,5-dimethylisoxazole has been
identified as a potent scaffold of FFA1 agonists with high activity,[36] however, hybrid of
3,5-dimethylisoxazole scaffold with phenoxyacetic acid (compound 6) resulted in a lower potency
than that of biphenyl scaffold. Moreover, mono-methyl analog (9) revealed better potency than
dimethyl analog (11), indicating that mono-methyl group is preferable to replace in the
ortho-position of A ring. Further exploration exhibited ortho-chlorine was also tolerated in A ring
(compound 17).
Replacement of oxygen atom with amine provided compound 12, which maintained the
potency on FFA1. Introducing an amide (compound 13), the bioisosteres of acid, resulted in a
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significant drop of activity. This result indicated that the carboxylic acid is crucial to activity of
FFA1, which is consistent with previous report.[35] Next, various substituents were explored in
para-position of A ring based on the optimal compound 7. For para-substituted analogs, the
potency of compound 9 (MeO) > 14 (EtO) > 15 (PrO) > 16 (cyclopropyl methoxyl), indicating
that the steric effect of para-substituent in A ring might be an influence factor on potency.
2.3. Molecular modeling study
To better understand the binding mode of present series, we performed docking studies of
compounds 7 and 15 based on X-ray structure of FFA1.[37] As shown in Figure 3A, the
carboxylic acid of compound 7 formed four hydrogen bonds with key residues Arg183, Arg2258,
and Tyr2240. However, only three hydrogen bonds were formed for the propoxy analog 15
(Figure 3B), which reasonably explained that the potency of compound 15 was inferior to
compound 7. Moreover, the orientation of biphenyl scaffold in compound 7 was completely
different from that of compound 15, which might be attributed to the steric effect of propoxy in
compound 15. Furthermore, an additional ionic bond could be formed between basic residues
(Arg183, Arg2258) and carboxylic acid, but amide could not. Therefore, the amide analog 13
revealed a lower potency than carboxylic acid derivatives.
2.4. Oral glucose tolerance test in mice
The target compounds with EC50 value less than 50 nM (compounds 7, 9, 12 and 14) were
selected to evaluate their oral glucose tolerance test in normal mice. As shown in Figure 4, all of
these tested compounds revealed significant glucose-lowering effects. Gratifyingly, the optimal
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compound 7 exerted the best effect in the improvement of glucose tolerance, and its glucose
AUC0−120min was significantly lower than that of TAK-875. Besides, the plasma glucose curve in
compound 7-treated group tends to stabilize after 60 minutes rather than decreased continually,
suggesting its low risk of hypoglycemia.
2.5. Glucose-lowering effects in HF/STZ mice
To evaluate anti-diabetic effects, the oral glucose tolerance test of optimal compound 7 was
performed in HF/STZ mice, a high-fat fed and streptozotocin-treated diabetic model with insulin
resistance and insulin insufficient.[38] As shown in Figure 5, compound 7 significantly improved
the glucose tolerance in HF/STZ diabetic mice, and glucose AUC0−120min was slightly better than
that of TAK-875, the most advanced candidate of FFA1 agonists. Our results suggested that
compound 7 might be a potential choice for the control of hyperglycemia in T2DM.
3. Conclusion
In conclusion, we identified a superior FFA1 agonist 7 (HWL-088, EC50 = 19 nM) based on
comprehensive structure-activity relationship study. The important aspects resulted in the
discovery of compound 7 were as follows: 1) the introduction of ortho-fluoro at acid head greatly
increased the activity on FFA1; 2) fine tuning of biphenyl scaffold further explored the optimal
substituent with improved potency. Compound 7 exhibited significantly glucose-lowering effects
both in normal mice and diabetic model, and even exerted greater potential on the improvement of
hyperglycemia than that of TAK-875. This study extended the chemical space of FFA1 agonists
bearing phenoxyacetic acid scaffold, and provided us a novel superior agonist HWL-088 for the
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treatment of T2DM.
4. Experimental section
4.1. General chemistry
All starting materials, reagents and solvents were obtained from commercial sources. Purifications
of chromatography were performed by silica gel and detected by thin layer chromatography using
UV light at 254 and 365 nm. Melting points were measured on RY-1 melting-point apparatus.
NMR spectra were recorded on a Bruker ACF-300Q instrument (300 MHz for 1H NMR and 75
MHz for 13C NMR spectra), chemical shifts are expressed as values (ppm) relative to
tetramethylsilane as internal standard, and coupling constants (J values) were given in hertz (Hz).
LC/MS spectra were recorded on a Waters LC-MS system (ESI). Elemental analyses were
performed by the Heraeus CHN-O-Rapid analyzer and were within 0.4% of the theoretical values.
TAK-875 was synthesized by published procedures.[16]
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
methyl bromoacetate (2 equiv). The mixture was stirred at 45 °C for 12 h, and filtered. The filtrate
was concentrated 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 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
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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 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 and the residue was purified by 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, CDCl3) δ: 9.35
(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]+.
Methyl 2-(2-fluoro-4-hydroxyphenoxy)acetate (4b). 1H NMR (300 MHz, CDCl3) δ: 9.44 (s,
1H), 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),
3.69 (s, 3H). ESI-MS m/z: 201.1 [M+H]+.
General procedure for 11a-b. The bromobenzene (1 equiv) and (3-formylphenyl)boronic acid (1
equiv) were dissolved in a mixture of 1 M sodium carbonate solution (15 mL), EtOH (5mL) and
toluene (15 mL). After nitrogen substitution, Pd(PPh3)4 (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 ethyl acetate (15 mL), and the
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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 column chromatography using a mixture of petroleum ether/ethyl acetate (10:1, v/v) as
eluent to afford the desired product as a solid.
1
4’-Hydroxy-2’-methyl-3-biphenylcarbaldehyde (11a). Yield: 92%; white solid; H NMR
(300 MHz, CDCl3) δ: 10.04 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 1.7 Hz, 1H), 7.59 – 7.53
(m, 2H), 7.42 (d, J = 7.4 Hz, 1H), 6.89 (d, J = 1.5 Hz, 1H), 6.76 – 6.72 (m, 1H), 4.67 (s, 1H), 1.98
(s, 3H).
1
4’-Hydroxy-2’,6’-dimethyl-3-biphenylcarbaldehyde (11b). Yield: 83%; white solid; H
NMR (300 MHz, CDCl3) δ: 10.05 (s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 1.5 Hz, 1H), 7.61
– 7.55 (m, 2H), 6.62 (s, 2H), 4.69 (s, 1H), 1.97 (s, 6H).
General synthetic procedure for 13a. To a solution of 11a-b (1 equiv) and alkyl halide (1.2
equiv) in acetone was added K2CO3 (2 equiv) and a catalytic amount of KI at room temperature.
The reaction mixture was heated to reflux with stirring overnight. Then reaction mixture was
cooled followed by filtration and the filtrate was concentrated under vacuum. The residue was
purified by column chromatography using a mixture of petroleum ether/ethyl acetate (10:1, v/v) as
eluent to afford a white solid.
1
4'-ethoxy-2',6'-dimethyl-[1,1'-biphenyl]-3-carbaldehyde. Yield: 78%; H NMR (300 MHz,
CDCl3) δ: 10.06 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.69 (t, J = 1.7 Hz, 1H), 7.63 – 7.57 (m, 2H),
6.63 (s, 2H), 4.02 (q, J = 6.7 Hz, 2H), 1.96 (s, 6H), 1.31 (t, J = 6.7 Hz, 3H).
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General synthetic procedure for 14a. To a solution of 13a (1 equiv) in MeOH (10 mL) and 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 × 15 mL), the organic fractions were combined, washed with saturated brine (2 × 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 further 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 product.
3'-(chloromethyl)-4-ethoxy-2,6-dimethyl-1,1'-biphenyl. Yield: 59%; 1H NMR (300 MHz,
CDCl3) δ: 7.44 – 7.39 (m, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.14 (s, 1H), 7.05 – 7.10 (m, 1H), 6.82 (s,
2H), 4.52 (s, 2H), 4.02 (q, J = 6.7 Hz, 2H), 1.96 (s, 6H), 1.31 (t, J = 6.7 Hz, 3H).
3-(3,5-dimethylisoxazol-4-yl)benzaldehyde (8a). To
a
mixture of 3,5-dimethyl-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (0.50 g, 2.2 mmol), 3-bromobenzaldehyde
(0.50 g, 2.7 mmol), Pd(PPh3)4 (0.13 g, 0.12 mmol) and sodium carbonate (0.71 g, 6.7 mmol) in
toluene/ethanol/H2O (35 mL, 3/1/3) was refluxed under nitrogen atmosphere for 24 h. Then the
mixture 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.
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The residue was purified by column chromatography using a mixture of petroleum ether/ethyl
acetate (10 : 1, v/v) as eluent to afford the desired product 8a (0.37 g, 77%) as a white solid. 1H
NMR (300 MHz, DMSO-d6) δ: 10.08 (s, 1H), 7.99 – 7.85 (m, 2H), 7.81 – 7.63 (m, 2H), 2.43 (s,
3H), 2.25 (s, 3H).
4-(3-(chloromethyl)phenyl)-3,5-dimethylisoxazole (9a). To a solution of 8a (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 × 15 mL), the organic fractions were
combined, washed with saturated brine (2 × 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 further purification. To a solution of the obtained solid in dichloromethane (20 mL) was
slowly added thionyl chloride (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 concentrated under reduced pressure.
The residue was purified by silica gel column chromatography using a mixture of petroleum
1
ether/ethyl acetate (20:1, v/v) as eluent to afford the 9a (0.27 g, 82%) as a white solid. H NMR
(300 MHz, DMSO-d6) δ: 7.57 – 7.43 (m, 3H), 7.36, 7.34 (dt, J = 6.9, 1.8 Hz, 1H), 4.82 (s, 2H),
2.41 (s, 3H), 2.23 (s, 3H).
1-(chloromethyl)-3-phenoxybenzene (6a). The title compound was prepared as described for
compound 9a as white solid. Yield 82%; 1H NMR (300 MHz, DMSO-d6) δ: 7.43 – 7.35 (m, 3H),
7.31 – 7.15 (m, 5H), 7.04 – 6.92 (m, 1H), 4.61 (s, 2H).
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General synthetic procedure for target compounds 4-11 and 14-17.
To a solution of chlorine intermediates (1 equiv) and intermediates 4a-b (0.8 equiv) in acetone
was added K2CO3 (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 temprature followed by filtration and the filtrate 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 a solution of the obtained solid (1
equiv) in 2:3:1 THF/MeOH/H2O (18 mL) was added LiOH·H2O (3 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 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.
2-(2-fluoro-4-((3-phenoxybenzyl)oxy)phenoxy)acetic acid (4)
Yield: 61%; m.p. 120-122 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.52 – 7.35 (m, 3H), 7.27 – 7.11
(m, 2H), 7.10 – 6.91 (m, 6H), 6.81 – 6.68 (m, 1H), 5.05 (s, 2H), 4.67 (s, 2H). 13C NMR (75 MHz,
DMSO-d6) δ: 170.51, 157.26, 156.89, 153.11, 141.67, 139.64, 130.61, 130.56, 124.06, 122.98,
119.21, 118.33, 117.98, 116.27, 110.66, 104.44, 69.78, 66.23. ESI-MS m/z: 367.1 [M-H]-. Anal.
calcd. For C21H17FO5: C, 68.47; H, 4.65; Found: C, 68.63; H, 4.57.
2-(2-methyl-4-((3-phenoxybenzyl)oxy)phenoxy)acetic acid (5)
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Yield: 47%; m.p. 107-109 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.51 – 7.33 (m, 3H), 7.27 – 7.10
(m, 2H), 7.09 – 6.90 (m, 4H), 6.87 – 6.78 (m, 1H), 6.75 – 6.66 (m, 2H), 5.01 (s, 2H), 4.53 (s, 2H),
2.16 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 170.18, 157.23, 157.05, 152.83, 150.96, 140.87,
128.67, 128.26, 121.85, 120.67, 118.63, 117.25, 116.85, 112.43, 112.05, 65.87, 62.59, 15.63.
ESI-MS m/z: 363.1 [M-H]-. Anal. calcd. For C22H20O5: C, 72.51; H, 5.53; Found: C, 72.77; H,
5.65.
2-(4-((3-(3,5-dimethylisoxazol-4-yl)benzyl)oxy)-2-fluorophenoxy)acetic acid (6)
1
Yield: 43%; m.p. 86-88 °C; H NMR (300 MHz, DMSO-d6) δ: 7.76 – 7.61 (m, 1H), 7.49 – 7.43
(m, 2H), 7.34 (d, J = 7.2 Hz, 1H), 7.08 – 6.99 (m, 2H), 6.78 (d, J = 9.0 Hz, 1H), 5.13 (s, 2H), 4.67
(s, 2H), 2.39 (s, 3H), 2.22 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 170.57, 165.66, 158.55,
153.20, 138.03, 131.97, 130.46, 129.48, 129.13, 128.80, 127.31, 116.36, 116.18, 110.66, 104.73,
69.97, 66.27, 11.75, 10.90. ESI-MS m/z: 370.1 [M-H]-. Anal. calcd. For C20H18FNO5: C, 64.69; H,
4.89; N, 3.77; Found: C, 64.53; H, 4.76; N, 3.62.
2-(2-fluoro-4-((2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)acetic acid (7)
Yield: 59%; m.p. 125-126 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.57 – 7.37 (m, 3H), 7.34 – 7.13
(m, 5H), 7.11 – 6.91 (m, 2H), 6.78 (d, J = 8.6 Hz, 1H), 5.13 (s, 2H), 4.66 (s, 2H), 2.21 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δ: 170.39, 154.10, 153.51, 141.91, 140.43, 137.35, 135.15, 130.79,
129.90, 128.88, 128.83, 128.67, 127.83, 126.64, 126.37, 116.74, 110.80, 104.82, 70.41, 66.64,
20.50. ESI-MS m/z: 365.1 [M-H]-. Anal. calcd. For C22H19FO4: C, 72.12; H, 5.23; Found: C, 72.37;
H, 5.15.
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2-(2-methyl-4-((2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)acetic acid (8)
1
Yield: 57%; m.p. 96-98 °C; H NMR (300 MHz, DMSO-d6) δ: 7.50 – 7.37 (m, 3H), 7.34 – 7.01
(m, 5H), 6.96 – 6.45 (m, 3H), 5.09 (s, 2H), 4.58 (s, 2H), 2.21 (s, 3H), 2.19 (s, 3H). 13C NMR (75
MHz, DMSO-d6) δ: 170.92, 152.92, 150.92, 141.85, 141.59, 137.95, 135.15, 130.79, 129.90,
128.77, 128.70, 128.53, 127.91, 127.81, 126.50, 126.38, 118.27, 113.11, 112.75, 70.10, 66.23,
20.50, 16.57. ESI-MS m/z: 361.1 [M-H]-. Anal. calcd. For C23H22O4: C, 76.22; H, 6.12; Found: C,
76.45; H, 6.28.
2-(2-fluoro-4-((4'-methoxy-2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)acetic acid (9)
Yield: 53%; m.p. 142-143 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.52 – 7.32 (m, 3H), 7.30 – 7.20
(m, 1H), 7.12 (d, J = 6.6 Hz, 1H), 7.08 – 6.91 (m, 2H), 6.89 – 6.66 (m, 3H), 5.11 (s, 2H), 4.60 (s,
2H), 3.78 (s, 3H), 2.19 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 170.58, 158.35, 153.37, 150.74,
141.65, 137.38, 136.56, 133.87, 130.85, 129.12, 128.76, 128.75, 126.28, 116.73, 116.58, 112.65,
110.82, 104.76, 70.48, 69.46, 55.85, 20.83. ESI-MS m/z: 395.1 [M-H]-. Anal. calcd. For
C23H21FO5: C, 69.69; H, 5.34; Found: C, 69.45; H, 5.48.
2-(4-((4'-methoxy-2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)-2-methylphenoxy)acetic acid (10)
1
Yield: 61%; m.p. 96-98 °C; H NMR (300 MHz, DMSO-d6) δ: 12.64 (brs, 1H), 7.51 – 7.07 (m,
5H), 6.96 – 6.68 (m, 5H), 5.08 (s, 2H), 4.59 (s, 2H), 3.79 (s, 3H), 2.20 (s, 6H). 13C NMR (75 MHz,
DMSO-d6) δ: 170.99, 167.43, 158.93, 152.80, 150.71, 141.55, 137.81, 136.57, 134.04, 131.05,
128.92, 128.72, 127.75, 126.20, 118.15, 116.15, 112.82, 112.54, 111.89, 69.92, 65.86, 55.51, 20.87,
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16.67. ESI-MS m/z: 391.1 [M-H]-. Anal. calcd. For C24H24O5: C, 73.45; H, 6.16; Found: C, 73.71;
H, 6.33.
2-(2-fluoro-4-((4'-methoxy-2',6'-dimethyl biphenyl-3-yl)methoxy)phenoxy)acetic acid (11)
1
Yield: 65%; m.p. 152-154 °C; H NMR (300 MHz, DMSO-d6) δ: 12.83 (s, 1H), 7.46 – 7.37 (m,
2H), 7.15 (s, 1H), 7.05 (d, J = 8.8 Hz, 1H), 7.00 – 6.94 (m, 2H), 6.75 (d, J = 9.0 Hz, 1H), 6.68 (s,
2H), 5.09 (s, 2H), 4.67 (s, 2H), 3.74 (s, 3H), 1.91 (s, 6H); 13C NMR (75 MHz, DMSO-d6) δ: 170.1,
157.9, 153.3, 152.7, 140.3, 139.7, 136.8, 133.5, 128.9, 128.6, 128.3, 125.9, 115.8, 112.6, 110.2,
104.1, 69.7, 65.7, 54.8, 20.6; ESI-MS m/z: 409.1 [M – H]-; Anal. calcd. For C24H23FO5: C, 70.23;
H, 5.65; Found: C, 70.20; H, 5.66.
2-(4-((4'-ethoxy-2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)-2-fluorophenoxy)acetic acid (14)
Yield: 58%; m.p. 105-106 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.41 – 7.34 (m, 3H), 7.23 (d, J =
6.2 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H), 7.03 – 6.93 (m, 2H), 6.84 – 6.75 (m, 3H), 5.09 (s, 2H), 4.60
(s, 2H), 4.03 (q, J = 5.7 Hz, 2H), 2.18 (s, 3H), 1.32 (t, J = 5.7 Hz, 3H). 13C NMR (75 MHz,
DMSO-d6) δ: 170.63, 158.26, 153.36, 153.23, 141.70, 140.61, 137.25, 136.54, 133.94, 130.99,
129.05, 128.83, 128.72, 126.24, 116.77, 116.63, 112.41, 110.74, 104.75, 70.45, 67.04, 63.47,
20.76, 15.12. ESI-MS m/z: 409.1 [M-H]-. Anal. calcd. For C24H23FO5: C, 70.23; H, 5.65; Found: C,
70.55; H, 5.83.
2-(2-fluoro-4-((2'-methyl-4'-propoxy-[1,1'-biphenyl]-3-yl)methoxy)phenoxy)acetic acid (15)
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1
Yield: 49%; m.p. 80-82 °C; H NMR (300 MHz, DMSO-d6) δ: 7.53 – 7.33 (m, 3H), 7.25 (d, J =
5.1 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.06 – 6.91 (m, 2H), 6.88 – 6.67 (m, 3H), 5.10 (s, 2H), 4.59
(s, 2H), 3.95 (t, J = 6.8 Hz, 2H), 2.19 (s, 3H), 1.84 – 1.62 (m, 2H), 1.00 (t, J = 6.4 Hz, 3H). 13C
NMR (75 MHz, DMSO-d6) δ: 170.60, 158.44, 153.30, 150.80, 141.70, 137.27, 136.53, 133.94,
130.99, 129.04, 128.84, 128.71, 126.24, 116.82, 116.62, 112.43, 110.76, 104.73, 70.44, 69.46,
67.08, 22.56, 20.76, 10.81. ESI-MS m/z: 423.1 [M-H]-. Anal. calcd. For C25H25FO5: C, 70.74; H,
5.94; Found: C, 70.51; H, 5.78.
2-(4-((4'-(cyclopropylmethoxy)-2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)-2-fluorophenoxy)
acetic acid (16)
Yield: 43%; m.p. 116-118 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.49 – 7.32 (m, 3H), 7.25 (d, J =
7.1 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 7.02 – 6.91 (m, 2H), 6.89 – 6.63 (m, 3H), 5.09 (s, 2H), 4.53
(s, 2H), 3.83 (d, J = 7.0 Hz, 2H), 2.18 (s, 3H), 1.27 – 1.19 (m, 1H), 0.66 – 0.47 (m, 2H), 0.38 –
0.25 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ: 170.63, 158.47, 153.35, 150.83, 141.78, 137.25,
136.64, 133.87, 130.83, 129.08, 128.87, 128.73, 126.26, 116.88, 116.58, 112.64, 110.73, 104.69,
78.64, 70.48, 67.07, 20.76, 10.63, 2.87. ESI-MS m/z: 435.1 [M-H]-. Anal. calcd. For C26H25FO5: C,
71.55; H, 5.77; Found: C, 71.73; H, 5.64.
2-(4-((2'-chloro-4'-methyl-[1,1'-biphenyl]-3-yl)methoxy)-2-fluorophenoxy)acetic acid (17)
Yield: 59%; m.p. 118-120 °C; 1H NMR (300 MHz, DMSO-d6) δ: 7.54 – 7.19 (m, 7H), 7.06 – 6.89
(m, 2H), 6.77 (d, J = 8.7 Hz, 1H), 5.11 (s, 2H), 4.56 (s, 2H), 2.36 (s, 3H). 13C NMR (75 MHz,
DMSO-d6) δ: 170.55, 158.47, 153.35, 139.58, 139.31, 137.39, 131.63, 131.43, 130.55, 129.18,
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128.89, 128.76, 128.64, 127.27, 116.60, 110.77, 104.46, 70.34, 67.17, 20.72. ESI-MS m/z: 399.1
[M-H]-. Anal. calcd. For C22H18ClFO4: C, 65.92; H, 4.53; Found: C, 65.76; H, 4.41.
General procedure for 17a-b. To a solution of 2-fluoro-4-nitrophenol (1 equiv) in 30 mL
acetonitrile was added methyl chloroacetate or chloroacetamide (1.5 equiv) and K2CO3 (3 equiv)
at room temperature. The solution was heated to reflux for 6 h. The reaction mixture was filtered
and evaporated to afford a colorless oil product. To a solution of the obtained product in ethanol
was added Pd/C (5%) and the mixture was stirred for 18 h at room temperature in a hydrogen
atmosphere. Insoluble matters were removed using Celite, and the filtrate was concentrated in
vacuo to give the desired product as a white solid.
1
Methyl 2-(4-amino-2-fluorophenoxy)acetate (17a). H NMR (300 MHz, DMSO-d6) δ: 6.94 –
6.90 (m, 1H), 6.79 – 6.77 (m, 1H), 6.54, 6.50 (dd, J = 2.2, 12.5 Hz, 1H), 5.28 (s, 2H), 4.76(s, 2H),
3.75 (s, 3H).
General procedure for target compounds 12 and 13. To a solution of 18a (1 equiv) and 17a-b
(1 equiv) in MeOH (10 mL) and THF (20 mL) was added portionwise NaBH3CN (3 equiv) and
the mixture was stirred for 12 h. The reaction mixture was pouring into ice water (10 mL), and
extracted with ethyl acetate (3 × 15 mL), washed with saturated brine (2 × 15 mL) prior to drying
over anhydrous sodium sulfate. After filtration and concentrate, the residue was purified by
column chromatography using a mixture of petroleum ether/ethyl acetate (5:1, v/v) as eluent to
afford a white solid.
2-(4-(((4'-ethoxy-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methyl)amino)-2-fluorophenoxy)acetic
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acid (12). The title compound was obtained from 19a according to the method of basic hydrolysis
1
described for the synthesis of compound 4. Yield: 67%; m.p. 102-104 °C; H NMR (300 MHz,
DMSO-d6) δ: 7.43 – 7.26 (m, 2H), 7.04 (s, 1H), 6.95 (d, J = 7.3 Hz, 1H), 6.81 (t, J = 9.3 Hz, 1H),
6.65 (s, 2H), 6.42, 6.37 (dd, J = 14.1, 2.5 Hz, 1H), 6.29 (d, J = 8.9 Hz, 1H), 4.51 (s, 2H), 4.26 (s,
2H), 4.01 (q, J = 7.0 Hz, 2H), 1.87 (s, 6H), 1.31 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
δ: 170.78, 157.55, 144.89, 140.61, 140.49, 136.93, 134.27, 128.87, 128.77, 128.07, 125.92, 117.63,
113.55, 108.23, 101.25, 66.90, 63.16, 47.14, 21.17, 15.23. ESI-MS m/z: 422.2 [M-H]-. Anal. calcd.
For C25H26FNO4: C, 70.91; H, 6.19; N, 3.31; Found: C, 70.84; H, 6.08; N, 3.16.
2-(4-(((4'-ethoxy-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methyl)amino)-2-fluorophenoxy)Acetam
ide (13)
1
Yield: 73%; m.p. 96-97 °C; H NMR (300 MHz, DMSO-d6) δ: 7.47 – 7.23 (m, 4H), 7.13 – 7.03
(m, 1H), 7.01 – 6.83 (m, 2H), 6.66 (s, 2H), 6.53 – 6.26 (m, 2H), 6.15 (s, 1H), 4.30 (s, 4H), 4.02 (q,
J = 7.0 Hz, 2H), 1.91 (s, 6H), 1.33 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 169.32,
156.35, 152.93, 142.59, 141.36, 137.16, 136.23, 130.78, 129.12, 128.63, 125.93, 126.24, 116.73,
111.47, 110.75, 105.35, 70.23, 67.26, 62.67, 20.73, 14.72. ESI-MS m/z: 423.1 [M+H]+. Anal.
calcd. For C25H27FN2O3: C, 71.07; H, 6.44; N, 6.63; Found: C, 71.31; H, 6.28; N, 6.56.
4.2. FLIPR Assay
CHO cells stably expressing FFA1 were seeded into 96-well plates and incubated 16 h at 37 °C.
After, the culture medium was removed and washed with 100 μL of Hank’s Balanced Salt
Solution. Then, cells were incubated in loading buffer (containing 2.5 μg/mL Fluo 4-AM, 2.5
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mmol/L probenecid and 0.1% fatty acid-free BSA) for 1 h at 37 °C. Various concentrations of
compounds or γ-linolenic acid (Sigma) were added and the signals of calcium flux were monitored
by FLIPR Tetra system (Molecular Devices). The agonistic activities were calculated as
[(A−B)/(C−B)]×100 (increased concentration of calcium (A) in the compound group and (B) in
vehicle group, and (C) in 10 μM γ-linolenic acid group). The EC50 value was calculated using
GraphPad InStat version 5.00 (GraphPad software, San Diego, CA, USA).
4.3. Molecular Docking
Molecular docking simulations were performed using AutoDock vina1.1.2. The Pymol 2.3.1 was
employed to analyze the docking results. The crystal structures of FFA1 (PDB code: 4PHU) was
obtained from Protein Data Bank. Before the docking process, the structure of protein was treated
by adding polar hydrogen atoms, deleting water molecules, adding gasteiger charge, and assigning
type of atoms as AD4 using AutoDockTools package. Docking calculations were performed with
full flexibility of the ligand inside the binding site. Docking was performed using the bound
crystallographic ligand as the centroid of the search space. Other docking parameters were kept to
the default values.
4.4. Animals and Statistical Analysis
10 weeks old male ICR mice and C57BL/6 mice were purchased from Guangdong Medical
Laboratory Animal Center (Guangdong, China) and acclimatized for one week. Animal room was
keep constant 12 h light/black cycle at 23 ± 2 °C with relative humidity 50 ± 10%. Animals were
allowed ad libitum. All animal experimental protocols were approved by the ethical committee at
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Guangdong Pharmaceutical 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).
Statistical analyses were carried out by GraphPad InStat 5.00 (San Diego, CA, USA), and
analyzed by one-way ANOVA with Tukey’s multiple-comparison post hoc test.
4.5. Oral glucose tolerance test in mice
ICR mice were fasted for 12 h, weighted, and randomized into 6 groups (n = 6). Mice were
administrated orally with vehicle, TAK-875 (20 mg/kg), or selected compounds (20 mg/kg) and
subsequently dosed orally with 3 g/kg glucose solution after 30 min. Blood samples were
collected before drug administration (-30 min), before glucose load (0 min), and at 15, 30, 60 and
120 min post-dose. The plasma glucose was measured by blood glucose test strips (SanNuo
ChangSha, China).
4.6. Glucose-lowering effects in HF/STZ Model
C57BL/6 mice were fed with high-fat diet (45% calories from fat, Mediscience Ltd., Yangzhou,
China) ad libitum for 4 weeks and then injected intraperitoneally (i.p.) with STZ (80 mg/kg). The
mice were fed with high-fat-diet for another 4 weeks, and diabetes was verified by glucose
tolerance. The obtained type 2 diabetic HF/STZ mice were fasted for 12 h, weighted, and
randomized into 3 groups (n = 6 per group). Mice were administrated orally with vehicle,
TAK-875 (20 mg/kg), or compound 7 (20 mg/kg) and subsequently dosed orally with 2 g/kg
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glucose solution after 30 min. Blood samples were collected immediately before drug
administration (-30 min), before glucose load (0 min), and at 15, 30, 60 and 120 min post-dose.
The blood glucose was measured by blood glucose test strips (SanNuo ChangSha, China).
Declaration of interest
The authors declare no competing financial interest.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grant 81803341),
the Natural Science Foundation of Guangdong Province, China (Grant 2018A030313445),
Innovation and Strong School Project of Guangdong Pharmaceutical University (Grant
2017KQNCX111), and Research Projects of the Chinese Medicine Council of Guangdong
Province (Grant 20191199).
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Table 1: Structure-activity relationship of target compounds
Compd.
R1
R2
R3
R4
R5
X
EC50 (nM) a
53.8
TAK-875
36.3
320.7
2065.3
381.6
19.0
1
F
Me
F
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CONH2
COOH
COOH
COOH
O
O
4
5
O
6
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
F
O
7 (HWL-088)
H
Me
F
O
1387.2
43.6
8
MeO
MeO
MeO
EtO
EtO
EtO
PrO
H
O
9
H
Me
F
O
1895.3
64.5
10
11
12
13
14
15
16
Me
Me
Me
H
O
F
NH
NH
O
34.6
F
615.4
47.5
F
H
F
O
73.5
H
F
O
79.8
Me
Cl
H
F
COOH
O
56.9
17
a EC50 values for FFA1 activities represent the mean values of three independent determinations.
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Figure 1: Typical FFA1 agonists containing phenylpropionic acid scaffold (blue).
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Figure 2: Reported examples of FFA1 agonists bearing phenoxyacetic acid scaffold (A and B), and the design of
structure-activity relationship in this paper (C).
29