Design, synthesis, and evaluation of a series of novel phenylpropanoic acid derivatives agonists for the FFA1

Article abstract of DOI:10.1111/cbdd.13480

Free fatty acid 1 (FFA1/GPR40) has attracted extensive attention as a novel target for the treatment of type 2 diabetes for its role in the enhancement of insulin secretion with glucose dependency. Aiming to develop novel potent FFA1 agonists, a new series of phenylpropionic acid derivatives were designed and synthesized on the basis of the modification of chemical cement of TAK-875, AMG-837, and LY2881835. Among them, most promising compounds 7, 14, and 15 were obtained with EC₅₀ values of 82, 79, and 88?nM, exhibiting a powerful agonistic activity compared to TAK-875 (95.1?nM). During Oral glucose tolerance test in normal mice, compound 7, 14, and 15 had significant glucose-lowering effect at the dose of 50?mg/kg. Furthermore, compound 15 (50?mg/kg) also significantly improved in glucose tolerance in type 2 diabetic mice. Herein, we reported the discovery and optimization of a series of potent FFA1 agonists. The discovery supported further exploration surrounding this scaffold.

Full text of DOI:10.1111/cbdd.13480

PROFESSOR BAINIAN FENG (Orcid ID : 0000-0003-3024-2599)
PROFESSOR CHUNLEI TANG (Orcid ID : 0000-0002-8908-1075)
Article type
: Research Article
Design, synthesis and evaluation of a series of novel phenylpropanoic acid derivatives agonists
for the FFA1
Authors
1,
1,
2
1
1
1,*
†
†
Jiaju Yang , Enke Gu , Ting Yan , Daoming Shen , Bainian Feng and Chunlei Tang
Affiliation and Address
1School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122,
China
2Jiangyin Tianjiang pharmaceutical Co.Ltd No. 1, Xinsheng Road, Jiangyin hi tech Zone, Jiangsu
214400,China
†These authors contributed equally to this work.
* Corresponding Author: Chunlei Tang
School of Pharmaceutical Science, Jiangnan University 1800 Lihu Road, Wuxi, Jiangsu 214122,
China
E-mail: tangcl@jiangnan.edu.cn
Tel: +86-510-8519-7052; Fax: +86-510-8519-7052
The work was supported by the National First-class Discipline Program of Food Science and
Technology (JUFSTR20180101), the National Natural Science Foundation of China (21305051).
RUNNING TITLE
A series of agonists for the FFA1
KEYWORDS
FFA1, agonistic activity, synthesis, type 2 diabetes
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13480
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ABSTRACT
Free fatty acid 1 (FFA1/GPR40) has attracted extensive attention as a novel target for the treatment of type
2 diabetes for its role in the enhancement of insulin secretion with glucose dependency. Aiming to develop
novel potent FFA1 agonists, a new series of phenylpropionic acid derivatives were designed and
synthesized on the basis of the modification of chemical cement of TAK-875, AMG-837 and LY2881835.
Among them, most promising compounds 7, 14 and 15 were obtained with EC50 values of 82, 79 and 88
nM, exhibiting a powerful agonistic activity compared to TAK-875(95.1 nM). During Oral glucose
tolerance test (OGTT) in normal mice, compound 7, 14 and 15 had significant glucose-lowering effect at
the dose of 50 mg/kg. Furthermore, compound 15 (50mg/Kg) also significantly improved in glucose
tolerance in type 2 diabetic mice. Herein, we reported the discovery and optimization of a series of potent
FFA1 agonists. The discovery supported further exploration surrounding this scaffold.
1 Introduction
Type 2 diabetes mellitus (T2DM), a worldwide epidemic syndrome characterized by an inability to
maintain glucose homeostasis due to insufficient insulin secretion and/or insulin desensitization, has
become a serious global health burden. [1-3] Though some medications are available for treatment of T2DM,
they are often associated with series of side effects such as body weight gain, high risk of hypoglycemia
[4-6]
and gastrointestinal discomfort or edema.
Therefore, we are still searching for a novel drug that
improves glycemic control with safety and durability. [7,8]
Free fatty acid 1 (FFA1, also known as GPR40), preferentially expressed in pancreatic β-cells, plays a key
role in amplifying glucose-stimulated insulin secretion (GSIS) without the associated risk of
hypoglycemia[9-11]. When activated by its endogenous ligands (medium/long chain fatty acids), FFA1
elicits insulin secretion only in the presence of elevated glucose levels, without affecting insulin secretion
at low glucose levels. [12-13]
Recently, a variety of synthetic FFA1 agonists were reported and many of them were progressed to clinical
trials such as TAK-875, AMG-837 and LY2881835. [14-18] Unfortunately, these compounds were terminated
for liver toxicity. [19-20] By analyzing their structures, we found that these compounds commonly have high
molecular weight and high ClogP values, and they all have the benzene ring in the hydrophobic area and
[21-22]
the phenylalanine of the carboxylic acid area.
So we kept the structure, then considered the
TAK-875 and the AMG-837 all have biphenyl in the hydrophobic area. Therefore, our design idea was to
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maintain the biphenyl of the hydrophobic area and the phenylalanine structure of the carboxylic acid area
as the mother nucleus, and make modification to the benzene ring, as shown in Figure 1. In addition, many
researches have suggested that ClogP values should be lower in the optimization process. As an effort to
identify novel FFA1 agonists with improved ClogP and molecular weight, we reported the synthesis,
biological evaluation and structure-activity relationship of a series of novel phenylpropanoic acid
derivatives as small molecular FFA1 agonists for the treatment of T2DM.
Figure 1. Structures of representative FFA1 agonists and our strategy to explore novel FFA1 agonists.
2 Methods and materials
2.1 Chemistry
All starting materials, reagents and solvents were obtained from commercial suppliers and used without
further purification unless otherwise indicated. Purifications by column chromatography were performed
on over silica gel (200 to 300 mesh), monitored by thin layer chromatography carried out on GF/UV 254
plates and were visualized by UV light (254 and 365 nm). The NMR (nuclear magnetic resonance) spectra
1
were recorded on a Bruker AVⅢ 400MHz/100MHz spectrometer. (400 MHz for HNMR and 100 MHz
for 13C NMR spectra), The chemical shifts were given in parts per million (ppm) using the 7.26 signal of
1
CDCl3 (1H NMR) and the 77.0 signal of CDCl3 (13C NMR) as internal standards. Data of H NMR were
reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, dd = doublet of doublet, br s = broad singlet), coupling constants, integration and identification.
The LC/MS spectra were recorded on the Waters liquid chromatography-mass spectrometer system (ESI).
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1
TAK-875 was synthesized via previous reported procedures. The physical characteristics, H NMR, 13C
NMR and MS for all intermediates and target compounds, were reported in Supporting Information.
2.2 General methods for preparation of compounds 2a~c
A mixture of 3-Formylphenylboronic acid (5.0 g, 25 mmol), aromatic bromine 1 (25 mmol), Pd(PPh3)4
(1.5 g, 1.25 mmol), ethyl alcohol (25 mL), a solution of sodium carbonate (5.3 g) in water (25 mL) and
methylbenzene (75 mL) ware reacted by stirring at 85oC for 8 h. After completion of the reaction
monitored by TLC, the material was filtered, then the filtrate was diluted with water (10 mL). The aqueous
phase was extracted with ethyl acetate (45mL ×3). The combined organics were washed with brine, dried
over sodium sulfate, and concentrated to afford yellow solid. The solid was purified by column
chromatography.
2.2.1 4'-hydroxy-2',6'-dimethyl-[1,1'-biphenyl]-3-carbaldehyde(2a)
Eluent from PE/ EtOAc =10:1, light yellow solid, 62.5% yield. 1H NMR (400 MHz, DMSO-d6) δ: 10.03
(d, J = 1.4 Hz, 1H, -CH), 7.98 (q, J = 1.7 Hz, 1H, -ArH), 7.92 – 7.84 (m, 1H, -ArH), 7.68 (dt, J = 7.6, 2.0
Hz, 1H, -ArH), 7.50 (t, J = 7.5 Hz, 1H, -ArH), 6.67 (s, 2H, -ArH), 4.89 (s, 1H, -OH), 2.29 (s, 6H, -ArCH3)
2.2.2 4'-hydroxy-2'-methyl-[1,1'-biphenyl]-3-carbaldehyde (2b)
Eluent from PE/ EtOAc =10:1, light yellow solid, 64.5% yield. 1H NMR (400 MH, DMSO-d6) δ : 10.04 (s,
1H, -CH), 8.00 (t, J = 2.2 Hz, 1H, -ArH), 7.89 (dt, J = 7.5, 2.6 Hz, 1H, -ArH), 7.71 (dt, J = 7.3, 2.0 Hz, 1H,
-ArH), 7.50 (t, J = 7.5 Hz, 1H, -ArH), 7.31 (d, J = 7.4 Hz, 1H, -ArH), 6.83 – 6.72 (m, 2H, -ArH), 4.87 (s,
1H, -OH), 2.29 (d, J = 1.0 Hz, 3H, -ArCH3).
2.2.3 2'-fluoro-4'-hydroxy-[1,1'-biphenyl]-3-carbaldehyde (2c)
Eluent from PE/ EtOAc =10:1, light yellow solid, 66.5% yield. 1H NMR (400 MHz, DMSO-d6) δ : 10.06
(s, 1H, -CH), 8.07 (s, 1H, -ArH), 7.87 (dt, J = 7.5, 2.0 Hz, 1H, -ArH), 7.79 (dt, J = 7.3, 2.0 Hz, 1H, -ArH),
7.54 – 7.42 (m, 2H, -ArH), 6.77 (dd, J = 8.9, 2.0 Hz, 1H, -ArH), 6.66 (dd, J = 7.4, 2.1 Hz, 1H, -ArH), 4.95
(s, 1H, -OH).
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2.3 General methods for preparation of compounds 3a~c
A solution of 2 (13.2 mmol) in dry N, N-dimethylformamide (25 mL) was treated with potassium
carbonate (3 g, 13.2 mmol). The contents were treated with toluene-4-sulfonic acid
3-Methanesulfonyl-propyl ester (4.68 g, 15.6 mmol) and then heated to 105°C for overnight. After
completion of the reaction monitored by TLC, the mixture was diluted with sodium hydroxide aqueous
solution (2 mol/L, 200 mL). The aqueous phase was extracted with ethyl acetate (75 mL ×3). The
combined organics were washed with brine (75 mL × 4), dried over sodium sulfate, and concentrated to
afford target compound.
2.3.1 2',6'-dimethyl-4'-(3-(methylsulfonyl) propoxy)-[1,1'-biphenyl]-3-carbaldehyde (3a)
1
White solid, 65.4% yield. H NMR ( 400MHz, CDCl3) δ: 10.05 (s, 1H, -CH), 7.86-7.87 (m, 1H, -ArH),
7.66 (s, 1H, -ArH), 7.59 (t, J = 6.0 Hz, 1H, -ArH), 7.28 (d, J = 7.5 Hz, 1H, -ArH), 6.67 (s, 2H, -ArH), 4.14
(t, J = 8.0 Hz, 2H, -CH2), 3.28 (t, J = 8.0 Hz, 2H, -CH2), 2.98 (s, 3H, -SO2CH3), 2.28-2.38 (m, 2H, -CH2),
1.99 (s, 6H, -ArCH3).
2.32 2'-methyl-4'-(3-(methylsulfonyl) propoxy)-[1,1'-biphenyl]-3-carbaldehyde (3b)
1
Eluent from PE/ EtOAc =10:1, light yellow solid, 64.5% yield. H NMR ( 400MHz, CDCl3) δ: 10.05 (s,
1H, -CH), 7.87 (m, 1H, -ArH), 7.72 (s, 1H, -ArH), 7.60(t, J= 6.0 Hz, 1H, -ArH), 7.42 (d, J = 8.0 Hz 1H,
-ArH), 7.28 (d, J = 7.5 Hz, 1H, -ArH), 6.64 (s, 2H, -ArH), 4.12 (t, J = 8.0 Hz, 2H, -CH2), 3.24 (t, J = 8.0
Hz, 2H, -CH2), 2.94 (s, 3H, -SO2CH3), 2.26(m, 2H, -CH2), 1.99 (s, 3H, -ArCH3).
2.3.3 2'-fluoro-4'-(3-(methylsulfonyl) propoxy)-[1,1'-biphenyl]-3-carbaldehyde (3c)
1
Eluent from PE/ EtOAc =10:1, light yellow solid, 64.5% yield. H NMR (400 MHZ, DMSO-d6) δ: 10.05
(d, J = 1.2 Hz, 1H, -CH), 8.16 – 8.12 (m, 1H, -ArH), 7.81 (m, 2H, -ArH), 7.60 – 7.48 (m, 2H, -ArH), 6.86
(m, 1H, -ArH), 6.75 (m, 1H, -ArH), 4.22 (t, J = 7.4 Hz, 2H, -CH2), 3.39 (t, J = 5.9 Hz, 2H, -CH2), 2.94 (s,
3H, -SO2CH3), 2.27 (m, 2H, -CH2).
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2.4 General methods for preparation of compounds 3d~l
A solution of 2a (6.64 mmol) in dry N,N-dimethylformamide (10 mL) was treated with potassium
carbonate (1.38 g, 9.96 mmol), and the mixture was stirred at 40oC for 0.5 h. The contents were treated
with N-(3-Chloropropyl) morpholine (1.07 g, 6.64 mmol) and then heated to 80oC for overnight. After
completion of the reaction monitored by TLC, the mixture was diluted with water (50 mL). The aqueous
phase was extracted with ethyl acetate (20 mL × 3). The combined organics were washed with brine (20
mL× 4), dried over sodium sulfate, and concentrated to afford white solid 3d (1.6 g, 68.4%). 1H NMR (400
MHZ, DMSO-d6) δ: 10.06 (s, 1H, -CH), 7.90 (d, J = 7.68 Hz, 1H, -ArH), 7.68 (t, J = 7.56 Hz, 2H, -ArH),
7.49(d, J = 7.68 Hz, 1H, -ArH), 6.74 (s, 2H, -ArH), 4.03 (t, J = 6.32 Hz, 2H, -CH2), 3.61-3.57 (m, 4H,
-CH2), 2.43(t, J = 7.12 Hz, 2H, -CH2), 2.40-2.37 (m, 4H, -CH2), 1.95 (s, 6H, -ArCH3), 1.91-1.87 (m, 2H,
-CH2)。
Compounds 3e~3l were prepared in a manner similar to that described for compound 3d.
2.5 General methods for preparation of compounds 4~6
A suspension of 3a~c (18.0 mmol) in acetonitrile (100 mL) and slowly treated with potassium carbonate (5
g, 36.0 mmol), and the mixture was stirred at 40oC. The contents were treated with methyl
3-(4-hydroxyphenyl) propanoate (3.3g, 36.0 mmol) then heated to 80oC for 3h. After completion of the
reaction monitored by TLC, the mixture was diluted with water (100 mL). The aqueous phase was
extracted with ethyl acetate (100 mL× 3). The combined organics were washed with brine (100 mL× 4),
dried over sodium sulfate, and concentrated to afford yellow solid. The yellow solid was added to a mixed
solution (methanol: distilled water: tetrahydrofuran=2:3:1, 50 mL). The contents were treated with sodium
hydroxide solution (0.2M, 2mL) stirring at room temperature for 1h. After completion of the reaction
monitored by TLC, the mixture was diluted with water (20 mL). Subsequently, the pH of the reaction
solution was adjusted to 3-4 with dilute hydrochloric acid (1 M). A solid precipitated, the mixture was
filtered to afford solid.
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2.5.1 3-(4-((2',6'-dimethyl-4'-(3-(methylsulfonyl) propoxy)-[1,1'-biphenyl]-3-yl) methoxy) phenyl)
propanoic acid (4)
Light yellow solid, 63.2% yield. MS (ESI): m/z calcd. for C28H32O6S, [M+H]+ 471, found 497.6 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ: 12.08 (s, 1H, -OH), 7.45-7.41 (m, 1H, -ArH), 7.12-7.06 (m, 3H, -ArH),
7.00 (d, J = 8.4 Hz, 2H, -ArH), 6.90 (s, 2H, -ArH), 6.65 (d, J = 8.4 Hz, 2H, -ArH), 5.13 (s, 2H, -CH2), 4.17
(t, J = 6.4 Hz, 2H, -CH2), 3.32-3.28 (m, 2H, -CH2), 3.04 (s, 3H, -SO2CH3) , 2.76 (t, J = 7.6 Hz, 2H, -CH2),
2.61 (s, 6H, -ArCH3), 2.47 (d, J = 7.6 Hz, 2H, -CH2), 2.21-2.14 (m, 2H, -CH2); 13C NMR (100 MHz,
DMSO-d6) δ : 177.33, 157.32, 146.75, 141.18, 140.53, 137.10, 134.79, 131.19, 129.42, 128.87, 128.70,
127.88, 125.89, 121.24, 113.90, 113.77, 112.93, 65.90, 51.12, 47.20, 32.40, 22.56, 21.1.
2.5.2 3-(4-((2'-methyl-4'-(3-(methylsulfonyl) propoxy)-[1,1'-biphenyl]-3-yl) methoxy) phenyl) propanoic
acid (5)
Light yellow solid, 64.4% yield. MS (ESI): m/z calcd. for C27H30O6S, [M+H]+ 483.1, found 483.6 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ: 12.04 (s, 1H, -OH), 7.35 (t, J = 7.6 Hz, 1H, -ArH), 7.27 (t, J = 8.0 Hz,
2H, -ArH), 7.15 (d, J = 7.2 Hz, 1H, -ArH), 7.09 (d, J = 8.4 Hz, 1H, -ArH), 6.88-6.85 (m, 3H, -ArH), 6.82
(m, 1H, -ArH), 6.50(d, J = 8.4 Hz, 1H, -ArH) , 6.08 (t, J = 6.4 Hz, 1H, -ArH), 5.07 (s, 2H, -CH2), 4.19 (d,
J =6.4 Hz, 2H, -CH2), 3.28 (t, J =6.5 Hz, 2H, -CH2), 3.03(s, 3H, -SO2CH3), 2.62 (t, J =7.6 Hz, 2H, -CH2),
2.33 (t, J =7.6 Hz, 3H, -ArCH3), 2.17-2.12 (m, 4H, -CH2); 13C NMR (100 MHz, DMSO-d6) δ : 181.21,
173.80, 166.79, 164.03, 158.41, 157.86, 155.27, 152.80, 147.28, 142.36, 140.73, 137.21, 136.95, 132.14,
129.12, 127.81, 116.90, 112.87, 112.47, 70.04, 51.14, 32.70, 29.81, 22.32, 19.84.
2.5.3 3-(4-((2'-fluoro-4'-(3-(methylsulfonyl) propoxy)-[1,1'-biphenyl]-3-yl) methoxy) phenyl) propanoic
acid (6)
Light yellow solid, 68.4% yield. MS (ESI): m/z calcd. for C26H27FO6S, [M+H]+ 487.1, found 487.6
[M+H]+. H NMR (400 MHz, DMSO-d6) δ: 12.10 (s, 1H, -OH), 7.58 (s, 1H, -ArH), 7.50-7.45 (m, 4H,
-ArH), 7.15 (d, J = 8.8 Hz, 2H, -ArH), 7.00-6.91 (m, 4H, -ArH), 5.14 (s, 2H, -CH2), 4.17 (t, J = 6.4 Hz, 2H,
-CH2), 3.33-3.28 (m, 2H, -CH2), 3.05 (s, 3H, -SO2CH3) , 2.76 (t, J = 7.6 Hz, 2H, -CH2), 2.48 (d, J = 7.6 Hz,
2H, -CH2), 2.21-2.14 (m, 2H, -CH2); 13C NMR (100 MHz, DMSO-d6) δ : 174.26, 161.32, 157.12, 153.81,
152.09, 138.13, 133.55, 131.68, 129.70, 129.18, 128.16, 127.03, 115.14, 112.96, 111.93, 103.03, 101.26,
93.50, 69.52, 66.71, 50.93, 36.01, 29.98, 22.37.
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2.6 General methods for preparation of compounds 7~18
A mixture of 3-(4-aminophenyl) propionic acid (0.17 g, 1.15 mmol), intermediate 3a (1.15mmol), methyl
alcohol (15 mL) was reacted by stirring at room temperature for 12 h, and then filtered. The solids were
diluted with methyl alcohol (15 mL) and cooled to 0oC. The mixture was slowly treated with sodium
borohydride (0.04 g, 1.15 mmol) and reacted by stirring at room temperature for 0.5 h. The mixture was
extracted with ethyl acetate (50 mL). The organic was washed with brine (25 mL × 2), dried over sodium
sulfate, and concentrated to afford white solid 7 (0.90 g, 56.5%). MS (ESI): m/z calcd. for C28H33NO5S,
[M+H]+ 496.2, found 496.7 [M+H]+. 1H NMR (400 MHz, CDCl3) δ: 7.38 (t, J = 7.6Hz, 1H, -ArH), 7.32 (d,
J = 8.0 Hz, 1H, -ArH), 7.01(s, 1H, -ArH), 7.01 (t, J = 7.2 Hz, 3H, -ArH), 6.63 (s, 2H, -ArH), 6.57 (d, J =
8.4 Hz, 2H, -ArH), 4.35 (s, 2H, -CH2), 4.12(t, J = 6.0 Hz, 2H, -CH2), 3.27 (t, J = 7.6 Hz, 2H, -CH2), 2.97 (s,
3H, -SO2CH3), 2.84 (t, J = 7.6Hz, 2H, -CH2), 2.61 (t, J = 8.0 Hz, 2H, -CH2), 2.38-2.31(m, 2H, -CH2), 1.97
(s, 6H, -ArCH3); 13C NMR (100 MHz, DMSO-d6) δ: 177.33, 157.33, 146.76, 141.18, 140.50, 137.09,
134.79, 131.19, 129.41, 128.85, 128.70, 127.88, 125.89, 121.25, 113.88, 113.75, 112.92, 65.90, 51.10,
47.20, 32.40, 22.57, 21.1.
Compounds 8~18 were prepared in a manner similar to that described for compound 7. Detailed analytical
data is given in Supporting Information.
2.7 In vitro FFA1 agonist activity
CHO cells stably expressing human FFA1 (accession no. NM_005303) were plated at a density of 15 K
cells/well and incubated 16-24 h in 5% CO2 at 37oC. Subsequently, the culture medium in the wells was
removed and washed with 100 mL of Hank's Balanced Salt Solution. Then, cells were incubated in loading
buffer (recording medium containing 2.5 μg/mL fluorescent calcium indicator Fluo 4-AM, 2.5 mmol/L
probenecid and 0.1% fatty acid-free BSA) for 1 h at 37oC. Various concentrations of test compounds or
γ-linolenic acid (Sigma) were added into the cells and the intracellular calcium flux signals after addition
were monitored by FLIPR Tetra system (Molecular Devices) for 90 s. The agonistic activities of test
compounds on human FFA1 were expressed as [(A-B)/(C-B)]* 100 (increase of the intracellular calcium
concentration (A) in the test compounds-treated cells and (B) in vehicle-treated cells, and (C) in 10 μM
γ-linolenic acid-treated cells). All test compounds were measured in three independent experiments. EC50
value of selected compound was obtained with Prism 5 software (GraphPad).
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2.8 Animals and statistical analysis of the data
Male ICR mice (18–22 g) and male C57BL/6 mice (18–22 g) were purchased from Comparative Medicine
Centre of Yangzhou University (Jiangsu, China, SCXK(Su)2017-0007). Mice were acclimatized for 1
week before the experiments. The animal room was maintained under a constant 12 h light/black cycle
with controlled temperature at 23 ±1oC and relative humidity 50 ± 10% throughout the experimental period.
Mice were allowed ad libitum access to standard pellets and water unless otherwise stated, and the vehicle
was oral administrated 0.5% Caboxy Methyl Cellulose aqueous solution for all animal studies. All animal
experimental protocols were approved by the institutional committee at Jiangnan University, conducted
according to the Laboratory Animal Management Regulations in China (SYXK(Su)2017-0052) 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 with the assistance
of GraphPad software (GraphPad InStat version 5.00, San Diego, CA, USA). Unpaired comparisons were
analyzed using the two-tailed Students t-test.
2.8.1 Effect of compounds on OGTT explored in ICR mice.
Ten-week old male ICR mice were fasted 12 h, weighted, bled via the tail vein, and randomized into 8
groups (n = 6). Mice were orally administrated with a single dose of vehicle, TAK-875(50 mg/kg), selected
compounds (50 mg/kg) 60 min before oral glucose load (2 g/kg). Blood samples were collected from the
tail vein immediately before drug administration (-30 min), 0 min (just before glucose load), and at 15, 30,
60 and 120 min after glucose load. The blood glucose levels were measured by using blood glucose test
strips (SanNuo ChangSha, China).
2.8.2 Hypoglycemic effects of compounds explored in type 2 diabetic mice.
Male C57BL/6 mice after 7 days adaptation were fed with high-fat diet (45% calories from fat, from
Mediscience Ltd., Yangzhou, China) ad libitum for further 4 weeks to induce insulin resistance and then
injected intraperitoneally (i.p.) with low dose of STZ (10 mL/kg; 80 mg/kg). The C57BL/6 mice were fed
with high-fat-diet for another 4 weeks, and the development of diabetes was confirmed by measuring
blood glucose levels. The mice with fasting blood glucose level 11 mmol/L or higher were recognized to
be diabetic and were used in the experiment as type 2 diabetic mice model. Type 2 diabetic C57BL/6 mice
were fasted 12 h, weighted, bled via the tail vein, and randomized into 5 groups (n = 6), another group of
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normal fasting C57BL/6 mice was added as negative control. Mice were administrated orally with a single
doses of vehicle, TAK-875 (50 mg/kg), or compounds (50mg/kg) 60 min before oral glucose load (2 g/kg).
Blood samples were collected immediately before drug administration ( -30 min), 0 min (before glucose
challenge), and at 15, 30, 60 and 120 min after glucose load. The blood glucose was measured by blood
glucose test strips (SanNuo ChangSha, China).
3 Results and discussion
3.1 Chemistry
The synthetic routes of target compounds 4-18 are outlined in Scheme 1. The synthesis of this series of
compounds started from reacting commercially available compounds 1 with 3-formylphenylboronic acid to
provide intermediate 2 over Suzuki-Miyaura cross-coupling reaction. Compounds 2 with appropriate
bromides in the presence of potassium carbonate as a base afforded 3. The resultant compounds 3 were
reduced under aldol condensation and further ester hydrolysis to provide the desired compounds 4-18.
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Scheme 1. Reagents and conditions: a) Na2CO3, Pd(PPh3)4, methylbenzene, 3-Formylphenylboronic acid, CH3CH2OH, 85℃,
8h; b) K2CO3, DMF, 85-106℃, overnight; c) K2CO3, CH3CN, 80℃, 3h; NaOH (2M), MeOH:H2O:THF = 2:3:1, reflux;
HCl (1M), reflux, (for compound 4-6); NaBH4 , MeOH , r.t, (for compound 7-18).
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3.2 Biological activity
In order to explore a new series of phenylpropionic acid derivatives, the agonistic activities of the
synthesized compounds with relative lower molecular weight and ClogP values were investigated by a
fluorometric imaging plate reader (FLIPR) assay in Chinese hamster ovary (CHO) cells expressing human
FFA1.The results were shown in Table 1, as well as ligand efficiency (LE), lipophilic ligand efficiency
(LLE) and lipophilic ligand efficiency dependent lipophilic (LELP) of the synthesized compounds 4~18.
We were dedicated to maintaining or improving agonistic potency, while reducing lipophilicity, which is
one of the most important physiochemical properties for druggability, by targeting lower LELP score. First,
the biphenyl structure in the hydrophobic region and the basic nucleus of the phenylpropanoic acid
structure in the carboxylic acid region remained unchanged. A methyl group or fluorine was introduced at
the R1 and R2 positions of the aromatic ring in the hydrophobic region (compound 4~7, Figure 2). The
linking arms of the carboxylic acid region and the hydrophobic region were replaced by oxygen atoms to
nitrogen atoms (compound 7~9). Having made comparisons between compounds 4 (EC50=96nM;
ClogP=4.88) and 7 (EC50=82nM; ClogP=4.14); 5 (EC50=219nM; ClogP=4.68) and 8 (EC50=170nM;
ClogP=3.94); 6 (EC50=396nM; ClogP=4.76) and 9 (EC50=330nM; ClogP=4.02), we found that the
activity of the compounds was improved, and ClogP had obviously declined after replacing the linking
arms of the carboxylic acid region and the hydrophobic region with nitrogen atoms from oxygen atoms.
Therefore, nitrogen atoms were used for the replacement of oxygen atoms in the design of subsequent
compounds. Then, different groups such as weak acid or weak base group were introduced into the tail of
the hydrophobic region to improve the physicochemical properties of the compounds, thus respectively
introducing 1-chloropropane, N-(3-chloropropyl)morpholine and 4-chloromethylpyridine fragment into the
tail R3 at the hydrophobic region. At the same time, the modification of the aromatic ring in the
hydrophobic region continues (compound 10~18, Figure 2). The results showed that when a different side
chain group was introduced into the tail R3 of the hydrophobic region, it also had a great effect on the
activity. When it was a hydrophilic group, it could effectively increase the agonistic activity while reducing
the compound's ClogP. (comparative studies of compounds 7 (ClogP=4.14), 10 (ClogP=6.11), 13
(ClogP=3.21) and 16 (ClogP=5.33)). From the results of Table 1, we had an interesting discovery about
compounds 14 and 15, they were identified as the most potent FFA1 agonists with EC50 values of 79 and
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88 nM, compared with TKA-875(95.1 nM), their ClogP was also the lowest of all compounds, which
might implied a better chance of becoming drug candidates in the future.
Figure 2. Phenylpropanoic acid derivatives progression and Numbering Assignment
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Table1. In vitro FFA1 agonistic activities, Ligand efficiency and lipophilic ligand efficiency of compounds 4-18.
Compound
EC50(nM)
pEC50
ClogPa
LE (kcal/mol)b
LLEc
LELPd
96
219
396
82
7.02
6.66
6.40
7.09
6.77
6.48
6.10
6.46
6.25
6.60
7.10
7.06
6.97
6.76
6.51
7.02
4.88
4.68
4.76
4.14
3.94
4.02
6.11
5.91
6.00
3.21
3.01
3.09
5.33
5.13
5.21
4.70
0.27
0.27
0.26
0.28
0.27
0.26
0.27
0.29
0.28
0.23
0.27
0.27
0.37
0.27
0.26
0.26
2.14
1.98
1.64
2.95
2.83
2.46
-0.01
0.55
0.25
3.39
4.09
3.96
1.64
1.64
1.30
2.05
17.88
17.60
18.61
15.05
14.55
15.54
22.81
20.18
21.19
13.95
11.23
11.63
19.65
18.99
20.04
18.06
4
5
6
7
170
330
786
347
559
251
79
8
9
10
11
12
13
14
88
15
107
173
310
95.1
16
17
18
TAK-875
aClogP was calculated by ChemBioDraw;
bLE=1.36pEC50/Nheavy atoms
cLLE=pEC50-ClogP;
dLELP=ClogP/LE
;
3.3 Oral glucose tolerance test evaluated in the normal mice
In order to screen orally bioavailable compounds, the optimized compounds were selected to evaluate the
glucose-lowering effects by oral glucose tolerance test in normal ICR mice. The time-dependent changes
of glucose levels and the corresponding area under the curve (AUC0–2 h) are shown in Fig.3. All selected
compounds showed good hypoglycemic activity in accordance with in vitro agonistic activity. Among
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these compounds, compounds 7, 14 and 15 made remarkable improvement in glucose to lerance with 17 %,
27 % and 18 % decrease in glucose AUC0–2 h, respectively. Notably, compound 14 revealed a significant
glucose-lowering effect (27%) at the dose of 50 mg/kg, a proximity to the hypoglycemic effect of
TAK-875 (50 mg/kg, 26%).
3.4 Hypoglycemic effects of 15 explored in type 2 diabetic mice
To further assess antihyperglycemic effects of 15 in the diabetic state, the OGTT in STZ-induced type 2
diabetic C57BL/6 mice was conducted. As shown in Fig.3, single oral doses of compound distinctly
reduced the glucose excursion at 50 mg/kg. Compound 15 significantly improved the hyperglycemia state
of diabetic mice as TAK-875. This result proved that these compounds, with a low risk of hypoglycemia,
had great potential for improving the diabetic state.
15
30
Vehicle
Vehicle
14
TAK-875
15
7
TAK-875
25
20
15
10
5
7
14
15
16
16
10
5
*
*
*
*
30%
21%
20%
10%
29%
0
-30
0
0
30
60
90
120
e
5
7
7
4
1
5
1
6
1
l
a
c
i
8
b
-
h
e
K
V
A
Blood Sample Time(min)
T
40
Vehicle
TAK-875
15
60
40
20
0
Vehicle
TAK-875
15
30
20
10
0
*
*
35%
30%
-30
0
30
60
90
120
e
5
7
5
1
l
d
c
i
8
-
c
h
e
K
V
Blood Sample Time(min)
A
T
Figure 3. Effects of selected compounds (50 mg/kg) on glucose excursion in OGTT. a) Blood glucose levels at various times
relative to glucose injection in normal mice; b) Percentage reduction in blood glucose AUC in normal mice; c) Blood
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glucose levels at various times relative to glucose injection in diabetic mice; d) Percentage reduction in blood glucose AUC
in diabetic mice. * P < 0.05 significantly differences from vehicle controls.
4 Conclusions
We found that nitrogen atoms as the linking arms of the carboxylic acid region and the hydrophobic region
can improve the activity of the compounds and decline ClogP obviously. The results showed that when a
hydrophilic group was introduced into the tail R3 of the hydrophobic region, it could effectively increase
the agonistic activity while reducing the compound's ClogP.
In summary, a series of novel phenylpropionic acid derivatives were designed and synthesized to improve
the pharmacokinetic and safety profiles of FFA1 agonists. Their FFA1 agonistic activities were
investigated on the CHO cells expressing human FFA1 by the FLIPR assay. Compounds 7, 14 and 15 were
identified as the most potent FFA1 agonists with EC50 values of 82nM, 79nM and 88nM, compared with
TKA-875 (95.1nM). Additionally, compound 15 showed good hypoglycemic activity both in normal and
type 2 diabetic mice in accordance with in vitro agonistic activity. All these results support further
exploration based on this scaffold and the optimization results will be reported in due course.
ACKNOWLEDGMENTS
The work was supported by the National First-class Discipline Program of Food Science and Technology
(JUFSTR20180101), the National Natural Science Foundation of China (21305051).
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
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Figure Legends
Figure 1. Structures of representative FFA1 agonists and our strategy to explore novel FFA1 agonists.
Figure 2. Phenylpropanoic acid derivatives progression and Numbering Assignment
Figure 3. Effects of selected compounds (50 mg/kg) on glucose excursion in OGTT. a) Blood glucose
levels at various times relative to glucose injection in normal mice; b) Percentage reduction in blood
glucose AUC in normal mice; c) Blood glucose levels at various times relative to glucose injection in
diabetic mice; d) Percentage reduction in blood glucose AUC in diabetic mice. * P < 0.05 significantly
differences from vehicle controls.
Scheme 1. Reagents and conditions: a) Na2CO3, Pd(PPh3)4, methylbenzene, 3-Formylphenylboronic acid,
CH3CH2OH, 85℃, 8h; b) K2CO3, DMF, 85-106℃, over night; c) K2CO3, CH3CN, 80℃, 3h; NaOH
(2M), MeOH:H2O:THF = 2:3:1, reflux; HCl (1M), reflux, (for compound 4-6); NaBH4 , MeOH , r.t, (for
compound 7-18).
Table 1. In vitro FFA1 agonistic activities, Ligand efficiency and lipophilic ligand efficiency of
compounds 4-18.
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Table 1. In vitro FFA1 agonistic activities, Ligand efficiency and lipophilic ligand efficiency of
compounds 4-18.
Compound
EC50(nM)
pEC50
ClogPa
LE (kcal/mol)b
LLEc
LELPd
96
219
396
82
7.02
6.66
6.40
7.09
6.77
6.48
6.10
6.46
6.25
6.60
7.10
7.06
6.97
6.76
6.51
7.02
4.88
4.68
4.76
4.14
3.94
4.02
6.11
5.91
6.00
3.21
3.01
3.09
5.33
5.13
5.21
4.70
0.27
0.27
0.26
0.28
0.27
0.26
0.27
0.29
0.28
0.23
0.27
0.27
0.37
0.27
0.26
0.26
2.14
1.98
1.64
2.95
2.83
2.46
-0.01
0.55
0.25
3.39
4.09
3.96
1.64
1.64
1.30
2.05
17.88
17.60
18.61
15.05
14.55
15.54
22.81
20.18
21.19
13.95
11.23
11.63
19.65
18.99
20.04
18.06
4
5
6
7
170
330
786
347
559
251
79
8
9
10
11
12
13
14
88
15
107
173
310
95.1
16
17
18
TAK-875
aClogP was calculated by ChemBioDraw;
bLE=1.36pEC50/Nheavy atoms
cLLE=pEC50-ClogP;
dLELP=ClogP/LE;
;
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