Synthesis, molecular docking, dynamic simulation and pharmacological characterization of potent multifunctional agent (dual GPR40-PPARγ agonist) for the treatment of experimental type 2 diabetes

Article abstract of DOI:10.1016/j.ejphar.2021.174244

The current manuscript describes two molecules that were designed against PPARγ and GPR40 receptors. The preparation of the compounds was carried out following a synthetic route of multiple steps. Then, the mRNA expression levels of PPARγ, GLUT4, and GPR4

Full text of DOI:10.1016/j.ejphar.2021.174244

European Journal of Pharmacology 907 (2021) 174244
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
Synthesis, molecular docking, dynamic simulation and pharmacological
characterization of potent multifunctional agent (dual GPR40-PPARγ
agonist) for the treatment of experimental type 2 diabetes
,
*
b
e
,
1
c
Sergio Hidalgo-Figueroaa , Ana Rodríguez-Luevano , Julio C. Almanza-Perez ,
´
´
f
d
´
Abraham Giacoman-Martínez , Rolffy Ortiz-Andrade , Ismael Leon-Rivera ,
g
´
Gabriel Navarrete-Vazquez
a
b
c
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CONACyT, IPICYT/Consorcio de Investigacion, Innovacion y Desarrollo para Las Zonas Aridas, San Luis Potosí, 78216, Mexico
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Posgrado en Biología Molecular, Division de Biología Molecular, Instituto Potosino de Investigacion Científica y Tecnologica (IPICYT), San Luis Potosí, 78216, Mexico
´
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Laboratorio de Farmacología, Depto. Ciencias de La Salud, D.C.B.S, Universidad Autonoma Metropolitana- Iztapalapa, Apdo.-Postal 55-535, Mexico, CP 09340,
CDMx, Mexico
d
´
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Departamento de Farmacobiología, Centro de Investigacion y Estudios Avanzados Del Instituto Politecnico Nacional, Sede Sur, CDMx, Mexico
e
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Area de Farmacología Experimental, Laboratorio de Farmacología, Facultad de Química, Universidad Autonoma de Yucatan, Calle 43 No. 613 X Calle 90, Colonia
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Inalambrica, Merida, Yucatan, 97069, Mexico
f
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Centro de Investigaciones Químicas, IICBA, Universidad Autonoma Del Estado de Morelos, Cuernavaca, Morelos, 62209, Mexico
g
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Facultad de Farmacia, Universidad Autonoma Del Estado de Morelos, Cuernavaca, Morelos, 62209, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
The current manuscript describes two molecules that were designed against PPARγ and GPR40 receptors. The
preparation of the compounds was carried out following a synthetic route of multiple steps. Then, the mRNA
expression levels of PPARγ, GLUT4, and GPR40 induced by compounds were measured and quantified in
adipocyte and β-pancreatic cell cultures. The synthesized compound 1 caused an increase in the 4-fold expression
of mRNA of PPARγ regarding the control and had a similar behavior to the pioglitazone, while compound 2 only
increased 2-fold the expression. Also, the compound 1 increased to 7-fold the GLUT4 expression levels, respect to
the control and twice against the pioglitazone. On the other hand, the 1 increase 3-fold GPR40 expression, and
compound 2 had a minor activity. Besides, 1 and 2 showed a moderated increase on insulin secretion and calcium
mobilization versus the glibenclamide. Based on the molecular docking studies, the first compound had a similar
conformation to co-crystal ligands into the binding site of both receptors. The poses were docked keeping the
most important interactions and maintaining the interaction along the Molecular Dynamics simulation (20 ns).
Finally, compound (1) showed an antihyperglycemic effect at 5 mg/kg, however at higher doses of 25 mg/kg it
controlled blood glucose levels associated with feeding intake and without showing the adverse effects associated
with insulin secretagogues (hypoglycemia). For these reasons, we have concluded that molecule 1 acts as a dual
PPARγ and GPR40 agonist offering a better glycemic control than current treatments.
PPARγ
GPR40
Multifunctional
Antidiabetic
Molecular dynamics
1. Introduction
people affected by diabetes in the world, and it has been estimated that
in 2045 there will be 700 million (IDF, 2020). Type 2 Diabetes (T2D) is
Diabetes Mellitus (DM) is a chronic disease that has been charac-
terized by hyperglycemia, with alterations in the metabolism of carbo-
hydrates, lipids, and proteins (Baynest, 2015). According to the
International Diabetes Federation (IDF), in 2019 there were 463 million
related to an insulin resistance, unbalanced secretion of insulin,
pancreatic β-cell apoptosis, and an increased production of hepatic
glucose (Vieira et al., 2019).
Unfortunately, the main factor in the development of diabetes is
´
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* Corresponding author. CONACyT, Instituto Potosino de Investigacion Científica y Tecnologica/ Consorcio de Investigacion, Innovacion y Desarrollo para las
´
´
Zonas Aridas, Camino a la Presa San Jose 2055, Lomas 4a secc., San Luis Potosí, C.P. 78216, Mexico.
E-mail address: sergio.hidalgo@ipicyt.edu.mx (S. Hidalgo-Figueroa).
1
´
Taken in part from the M. Experimental Biology thesis of A. Rodríguez-Luevano.
https://doi.org/10.1016/j.ejphar.2021.174244
Received 10 February 2021; Received in revised form 5 June 2021; Accepted 7 June 2021
Available online 8 June 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
central obesity, especially visceral adipose tissue (Papaetis et al., 2015).
Also, in adipocytes, there is a high expression of a peroxisome
proliferator-activated receptor-γ (PPARγ); which is a transcription factor
that when it is activated by a ligand, it regulates the carbohydrate
metabolism and decreases the blood lipid levels (Gao et al., 2015;
Hidalgo-Figueroa et al., 2017). Moreover, an augmented expression of
PPARγ in adipose tissue leads to a reduction of insulin resistance by
increasing the expression of the glucose transporter type 4 (GLUT4),
which is responsible for the glucose uptake (Parimala et al., 2015).
Additionally, the G protein-coupled receptor 40 (GPR40) is a novel
and attractive pharmacological target which complements the treatment
through the control of other factors that frequently affect the diabetic
patient, for example insulin deficiency. The novel target stimulates the
secretion of insulin through its natural ligands; the free fatty acids (FFA)
having a high level of expression in pancreatic beta cells (Rodrigues
et al., 2017). This mechanism has a low risk of hypoglycemia because
GPR40 has its effects on the second phase of insulin secretion (Hidal-
go-Figueroa et al., 2017; Liu et al., 2014). However, studies have been
carried out to develop new hybrid molecules based on a phenoxyacetic
acid substructure that has a balanced dual agonism in PPARγ and GPR40
receptors (Darwish et al., 2018; Hidalgo-Figueroa et al., 2017).
For these reasons, we decided to perform the synthesis, pharmaco-
logical evaluation, and in silico studies of two novel molecules with an
antihyperglycemic action. In this study, two phenoxyacetic derivatives
against two important therapeutic targets in the treatment of diabetes:
PPARγ and GPR40 receptors were designed. Also, molecular docking
studies to determine the conformation and occupation into the binding
site of both receptors were performed.
2.1.2. {4-[2-(4-acetylphenoxy)acetamido]phenoxy}acetic acid (2)
A 10 ml solution of ethyl {4-[2-(4 acetylphenoxy)acetamido]phe-
noxy}acetate (4, 0.2 g, 0.5 mmol) and KOH (0.060 g, 1.0 mmol) in H2O/
EtOH (1:3 ratio). The solution was stirred and refluxed until the reaction
was completed (2.5 h). Then, this mixture was poured into water and
acidified with HCl (5%) at pH value of 3; the resulting precipitate (white
solid) was filtered in vacuum and purified using column chromatog-
raphy (mixture of eluents: CH2Cl2–EtOH in 90:10 ratios). Next, the ob-
tained product was dried to give 0.106 g of a white compound. Yield
57%, m. p. 210–212 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ: 2.5 (s, 3H,
–
–
–
–
O
C–CH , H-13), 4.62 (s, 2H, O–CH –C O, H-8), 4.77 (s, 2H, O–CH
3
2
2
–
ꢀ C O, H-11), 6.87 (d, 2H, =CH–CH = , H-2, H-6, J = 9.08 Hz), 7.08 (d,
–
2H, =CH–CH = , H-2′, H-6′, J = 8.94 Hz), 7.51 (d, 2H, =CH–CH = , H-3,
H-5, J = 9.07 Hz), 7.93 (d, 2H, =CH–CH = , H-3′, H-5′, J = 9.01 Hz),
10.0 (s, 1H, O C–N–H, NH-9). 13C NMR (100 MHz, DMSO‑d6) δ: 26.52
–
–
–
–
–
–
–
–
(O C–CH , C-13), 64.7 (O–CH 2 –C O, C-8), 67.1 (O–CH ꢀ C O, C-
11), 114.63(=CH–CH = , C-2, C-6), 114.6 (=CH–CH = , C-2′, C-6′), 121.3
2
′
′
–
–
(=CH–CH = , C-3, C-5), 130.2 (=C–C O, C-4 ) 130.5 (=CH–CH = , C-3 ,
C-5′), 131.8 (=C–N, C-4), 154.1 (=C–O, C-1), 161.7 (=C–O, C-1’), 165.6
–
–
–
–
(O C–OH, C-7), 170.3 (O C–N–H, C-10), 196.4 (CH3 ꢀ C = O, C-12).
HRMSESI 343.3281 (calculated 343.3284).
2.1.3. Ethyl {4-[2-(4-acetamidophenoxy)acetamido]phenoxy}acetate (3)
A 30 ml solution of N-(4-hydroxyphenyl)acetamide (0.1685 g, 1.1
mmol) and K2CO3 (0.2289 g, 1.6 mmol) was stirred in acetonitrile at
room temperature (27 ◦C) for 30 min. Immediately, the compound 5
(ethyl [4-(2-chloroacetamido)phenoxy]acetate, 0.3 g, 0.0001 mol) was
slowly added. The reaction was monitored by thin-layer chromatog-
raphy until the reagents were consumed (17 h). Once the reaction was
completed, the mixture was poured into cold water and it was stirred
during 20 min, and the resulting precipitate was filtered. Finally, the
product that was obtained was dried to give 0.309 g of a white com-
pound. Yield 72%, m. p. 163–165 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ:
1.2 (t, 3H, H-16, J = 8 Hz), 2.0 (s, 3H, H-14), 4.16 (q, 2H, H-15, J = 8
Hz), 4.60 (s, 2H, H-11), 4.70 (s, 2H, H-8), 6.89 (d, 2H, H-2, H-6, J = 5
Hz), 6.93 (d, 2H, H-2′, H-6′, J = 5 Hz), 7.49 (d, 2H, H-3, H-5, J = 5 Hz,
7.55 (d, 2H, H-3′, H-5′, J = 5 Hz), 9.88 (s, 1H, H-12), 9.95 (s, 1H, H-9).
13C NMR (100 MHz, DMSO‑d6) δ: 14.05 (–CH2–CH3, C-16), 23.81
2. Material and methods
2.1. Chemistry
Unaltered and unpurified commercially chemicals and solvents were
used in the following investigation. First, the melting points (m.p.) were
taken into an open glass capillary and uncorrected using the Stuart
SMP10 digital melting point apparatus from Cole-Parmer. Then, their
reactions were examined through a thin-layer chromatography (TLC) on
0.2 mm precoated Silica Gel 60 F254 plates (E. Merck). Next, the 1H and
13C NMR spectra were recorded with a Varian Inova 400 MHz (9.4 T),
the chemical shifts were given in ppm relative to tetramethylsilane
(TMS) in DMSO‑d6. High-Resolution mass spectra were obtained from an
Agilent 6545 LC/Q-TOF. Finally, the GC-MS analysis was performed by
using a Hewlett Packard 6890 A gas chromatograph coupled to a
Hewlett Packard Mass Selective Detector MSD 5970.
–
–
–
(O C–CH, C-14), 60.6 (CH –CH –O, C-15), 65.0 (O–CH –C O, C-8),
–
3
2
2
–
–
67.50 (O–CH –C O, C-11), 114.37 (=CH–CH = , C-2, C-6), 114.76
(=CH–CH = ,2C-2′, C-6′), 120.6 (=CH–CH = , C-3, C-5), 121.3 (=CH–CH
= , C-3′, C-5′), 132.17 (=C–N, C-4), 133.27 (=C–N, C-4′), 153.58 (=C–O,
–
–
C-1), 153.94 (=C–O, C-1’), 166.12 (O C–(CH )N–H, C-10), 167.85
–
3
–
–
(O C–(CH )N–H, C-13), 170.73 (O C–O–CH , C-7). HRMSESI
–
2
2
384.3829 (calculated 384.3830).
2.1.4. Ethyl {4-[2-(4-acetylphenoxy)acetamido]phenoxy}acetate (4)
A 30 ml solution of 1-(4-hydroxyphenyl)ethan-1-one (0.1518 g, 1.1
mmol) and K2CO3 (0.2288 g, 1.6 mmol) in acetonitrile. The previous
mixture was stirred at room temperature (27 ◦C) for 30 min. Immedi-
ately, a compound 5 (ethyl [4-(2-chloroacetamido)phenoxy]acetate,
0.3 g, 0.0001 mol) was slowly added. The reaction was monitored with
the thin-layer chromatography until the reagents were consumed (17 h).
Once the reaction was completed, the mixture was poured into cold
water and stirred for 20 min, the resulting precipitate was filtered.
Finally, the obtained product was dried to give 0.305 g of a white
compound. Yield 74%, m. p. Of 115–117 ◦C. 1H NMR (400 MHz,
2.1.1. {4-[2-(4-acetamidophenoxy)acetamido]phenoxy}acetic acid (1)
A 10 ml solution of ethyl {4-[2-(4-acetamidophenoxy)acetamido]
phenoxy}acetate (3, 0.2 g, 0.5 mmol) and KOH (0.058 g, 1.0 mmol) in
H2O/EtOH (1:3 ratio) was stirred and refluxed until the reaction was
completed (2.5 h). Then, this mixture was poured into water and acid-
ified with HCl (5%) at a pH value of 3; the resulting precipitate (white
solid) was filtered in vacuum and purified by column chromatography
(mixture of eluents: CH2Cl2–EtOH in 90:10 ratio). Eventually, the ob-
tained product was dried to give 0.139 g of a white compound. Yield
74%, m. p. 248–250 ◦C. 1H NMR (400 MHz, DMSO‑d6) δ: 2.0 (s, 3H, H-
14), 4.29 (s, 2H, H-8), 4.70 (s, 2H, H-11), 6.79 (d, 2H, H-2, H-6, J = 7
Hz), 6.91 (d, 2H, H-2′, H-6′, J = 7 Hz), 7.47 (d, 2H, H-3, H-5, J = 7 Hz),
7.49 (d, 2H, H-3′, H-5′, J = 7 Hz), 9.85 (s, 1H, NH-12), 9.95 (s, 1H, NH-
DMSO‑d6) δ: 1.21 (t, 3H, O–CH2–CH3, H-15, J = 8 Hz), 2.51 (s, 3H,
–
O
C–CH , H-13), 4.16 (q, 2H, O–CH –CH , H-14, J = 8 Hz), 4.73 (s, 2H,
3 2 3
–
13
–
–
–
–
–
O–CH –C O, H-11), 4.78 (s, 2H, O–CH –C O, H-8), 6.90 (d, 2H,
9). C NMR (100 MHz, DMSO‑d ) δ: 23.81 (O C–CH , C-14), 65.0
–
=CH–2CH = , H-2, H-6, J = 9.08 Hz), 7.10 (d, 2H, =CH–CH = , H-2′, H-6′,
2
6
3
–
–
–
(O–CH –C O, C-8), 66.85 (O–CH –C O, C-11), 114.37 (=CH–CH = ,
–
2
C-2, C-26), 114.76 (=CH–CH = , C-2′, C-6′), 120.48 (=CH–CH = , C-3, C-
J = 8.94 Hz), 7.52 (d, 2H, =CH–CH = , H-3, H-5, J = 9.07 Hz), 7.95 (d,
′
′
5), 121.23 (=CH–CH = , C-3′, C-5′), 132.11 (=C–N, C-4), 133.17 (=C–N,
–
–
2H, =CH–CH = , H-3 , H-5 , J = 9.01 Hz), 10.1 (s, 1H, O C–N–H, H-
9).13C NMR (100 MHz, DMSO‑d6) δ: 14.30 (O–CH2–CH3, C-15), 26.75
C-4′), 153.58 (=C–O, C-1), 154.94 (=C–O, C-1’), 166.12 (O = C-(CH3)
–
–
–
–
(O C–CH , C-13), 60.9 (O–CH –CH , C-14), 65.1 (O–CH –C O, C-8),
–
N–H, C-13), 167.85 (O C–(CH )N–H, C-10), 170.73 (CH –COOH, C-7).
–
3
2
3
2
2
2
–
67.3 (O–CH –C O, C-11), 114.9 (=CH–CH = , C-2, C-6), 115.1
–
HRMSESI 358.3446 (calculated 358.3448).
2
2

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
(=CH–CH = , C-2′,C-6′), 121.6 (=CH–CH = , C-3, C-5), 130.7 (=CH–CH
= , C-3′, C-5′), 131.8 (=C–N, C-4), 154.1 (=C–O, C-1), 161.5 (=C–O, C-
2.2. In vitro assay
2.2.1. MTT assay
–
–
–
1’), 166.2 (O C–O–H, C-7), 169.3 (O C–N–H, C-10), 196.2
–
–
(CH –C O, C-12). HRMSESI 371.7051 (calculated 371.7054).
MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-
mide] assay (NR, Sigma-Aldrich, MO, USA) was used to evaluate the
cellular viability and cytotoxicity (Loveland et al., 1992) of compounds
1–4. Fibroblasts and RINm5F cells were grown in microplates that had
96 plates (5 × 103 cells per well) in Dulbecco’s modified Eagle’s medium
(DMEM medium) and Roswell Park Memorial Institute medium (RPMI
1640 medium), respectively. Exponential concentrations of title com-
–
3
2.1.5. Ethyl [4-(2-chloroacetamido)phenoxy]acetate (5)
The aniline 6 of the previous reactions (2.5 g, 12.8 mmol) and 2.67
ml of Et3N (19.2 mmol) were dissolved in CH2Cl2 (15 ml). The mixture
was cooled down with an ice bath and 2-chloroacetyl chloride (1.02 ml,
12.9 mmol) were added dropwise to the cooled mixture. After, the ice
bath was removed and the mixture was stirred at room temperature for
12 h. Then, the solvent was removed under a reduced pressure and cold
water (30 ml) was added and the mixture was stirred during another 20
min. The resulting white precipitate was filtered, washed with cold
water (3 × 10 ml) and dried. Yield 2.65 g (76%). m. p. 99.3–100.4 ◦C,
pounds were utilized and preincubated (0.5, 5, 50 and 100 μM) for 24 h
(n = 4), which were measured at 570 nm (Loveland et al., 1992).
2.2.2. 3T3-L1 line cell culture
The 3T3-L1 cells were cultured in 6-well plates at a semi-confluent
density (45,000 cells/well) in Dulbecco’s modified Eagle’s medium
supplemented with 25 mM glucose, 10% fetal bovine serum, 1 mM so-
dium pyruvate, 2 mM glutamine, 0.1 mM non-essential amino acids, and
1% (1000 U/ml) gentamicin, in a 5% CO2 humidified atmosphere at
37 ◦C. After two days of confluence, the cell differentiation was carried
out with 0.5 mM 3-isobutyl-1-methylxanthine, 0.25 mM dexamethasone
acetate, and 80 mM bovine insulin, during 48 h, followed by insulin
alone for another 48 h. The culture medium without insulin was
changed every 2 days during 8 days of differentiation (Garcia-Macedo
et al., 2008; Hidalgo-Figueroa et al., 2017).
C
12H14ClNO4 (271.7). Mass (EI) m/z (% rel. Int.) 271 (80, M+), 198
(20), 184 (30), 168 (10), 91 (10), 77 (159). Anal. Calc. C12H14ClNO4: C,
53.05; H, 5.19; N, 5.16. Found: C, 52.90; H, 5.14; N, 5.26.1H RMN (500
MHz, CDCl3) δ: 1.29 (t, 3H, OCH2CH3), 4.16 (s, 2H, H-2′′), 4.26 (c, 2H,
OCH2CH3), 4.60 (s, 2H, H-2), 6.88 (d, 2H, H-2′, H-6′, J = 9.05 Hz), 7.45
(d, 2H, H-3′, H-5′, J = 8.95 Hz), 8.29 (s, 1H, NH). 13C RMN (125 MHz,
CDCl3) δ: 14.16 (OCH2CH3), 42.85 (C-2′′), 61.45 (OCH2CH3), 65.66 (C-
2), 115.13 (C-2′, C-6′), 121.95 (C-3′, C-5′), 130.75 (C-4′), 155.14 (C-1′),
163.80 (C-1′′), 168.83 (C-1).
2.1.6. Ethyl (4-aminophenoxy)acetate (6)
A suspension of 2.5 g (11.1 mmol) of ethyl (4-nitrophenoxy)acetate
(7) and 0.125 g of Pd/C 10% in 200 ml of ethanol was reduced with
hydrogen at 4 lb/in2 in a Parr hydrogenation reactor (500 ml). After 10
min, the reaction consumed 32 lb/in2. Then, the catalyst was removed
through careful filtration and the filtrate was concentrated under
reduced pressure until there was a presence of a yellow liquid. This
liquid was not purified and it was immediately used for the subsequent
acylation reaction.
2.2.3. RINm5F line cell culture
RINm5F cells were seeded at a density of 80,000 cells/well in 6-well
plates and were maintained in RPMI 1640 supplemented with 100 U/ml
gentamicin, 10% FBS (v/v) (HyClone Laboratories, Logan, UT), 2 mM L-
glutamine and 1 mM sodium pyruvate at 37 C and 5% CO2, and 95% of
humidity (Hidalgo-Figueroa et al., 2017; Jhun et al., 2013; Wu et al.,
2015).
2.2.4. PPARγ and GRP40 mRNA expression, measurement of [Ca2+]i and
measuring the insulin secretion
2.1.7. Ethyl (4-nitrophenoxy)acetate (7)
A 30 ml solution of 4-nitrophenol 8 (1 g, 0.0072 mol) and K2CO3
(1.49 g, 0.0108 mol) in acetone. First, the mixture was stirred for 30
min. Then, 0.83 ml of ethyl bromoacetate 9 (0.0075 mol) was added on
dropwise. The mixture was refluxed and monitored with a thin-layer
chromatography until the raw material was converted (2.5 h). Conse-
quently, the solvent was removed under reduced pressure. Later, cold
water (10 ml) was added and the mixture was stirred for 30 min and the
resulting precipitate was filtered, washed with cold water (3 × 15 ml)
and recrystallized from ethanol obtaining a solid pale yellow. Yield 1.42
g (88%). m. p of 71.5–72.6 ◦C. C10H11NO5 (225.2). Mass (EI) m/z (% int.
Rel.) 225 (100, M+), 152 (90), 122 (25). Anal. Calc. C10H11NO5: C,
53.33; H, 4.92; N, 6.22. Found: C, 52.48; H, 4.89; N, 6.49.1H RMN (500
MHz, CDCl3) δ: 1.3 (t, 3H, OCH2CH3), 4.29 (c, 2H, OCH2CH3), 4.72 (s,
2H, H-2), 6.97 (dd, 2H, H-2′, H-6′, J = 7.1 Hz), 8.21 (d, 2H, H-3′, H-5′, J
= 6.8 Hz). 13C RMN (125 MHz, CDCl3) δ: 14.14 (OCH2CH3), 61.82
(OCH2CH3), 65.45 (C-2), 114.72 (C-2′, C-6′), 125.93 (C-3′, C-5’), 162.66
(C-1).
The objective of this work was to evaluate novel compounds with the
previously published methodologies, without protocol modifications,
equipment, or reagents (Hidalgo-Figueroa et al., 2017), but unlike pre-
ceding molecules, the compounds described in this manuscript were
pre-incubated at 1 μM.
2.3. Antihyperglycemic in vivo assay in normoglycemic rats
2.3.1. Animals
Normoglycemic male Wistar rats (6 weeks old, 150–200 g, body
´
weight) were provided by the Animal house of Universidad Juarez
´
Autonoma de Tabasco (UJAT). These animals were maintained at
standard laboratory conditions; temperature (25 ± 2 ◦C), relative hu-
midity 40–60%, 12 h light/dark cycle, having free access to food and tap
water. The animals were allowed to acclimatize for two weeks before the
experiments started. The experimental protocol was approved by the
´
Institutional Animals Ethics Committee (3/2017) of Universidad Juarez
´
Autonoma de Tabasco, and the animal care was taken as per the
2.1.8. N-(4-hydroxyphenyl)acetamide (acetaminophen)
Guidelines of Mexican Official Norms for Animal Experimentation
(NOM-062-ZOO-1999).
The Acetaminophen was obtained from 10 commercial tablets (500
mg paracetamol, Lab Perrigo. GI) with a 17G102 lot number and expi-
ration date of June 2019, they were powdered in a grinder and the
resulting powder was dissolved in 20 ml of ethanol (99.5% v/v) for 5
min at room temperature. Subsequently, it was filtered under vacuum,
and the solvent was eliminated until a white crystalline solid was ob-
tained (4.7 g, 94%). Acetaminophen was checked by thin-layer chro-
matography and the melting point was measured 169–172 ◦C (reported
m. p. 169–170 ◦C).
2.3.2. Experimental design
The animals were divided into four groups. 1) The Control rats
(group 1) were given 5 ml/kg of distilled water; 2) The Positive control
(group 2) were given 5 mg/kg of glibenclamide (insulin secretagogue
drug); 3) The treatment rats (group 3 and 4) were given 5 and 25 mg/kg
of 1, respectively. The oral glucose tolerance test (OGTT) was performed
in overnight fasted (16 h) for intragastric administration. The rat’s
groups (n = 5) time 0 h was set before any treatments; 30 min later, a
dextrose dose (2 g/kg) was administered, and the blood samples were
3

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
obtained 0, 0.5, 1, 2, and 4 h post carbohydrate ingestion. Plasmatic
glucose levels were measured with a commercial glucometer and
glucose test strips (Accucheck Active, Roche) (Ortiz-Andrade et al.,
2012).
server (http://zarbi.chem.yale.edu/ligpargen/) (Dodda et al., 2017a,
2017b; Jorgensen and Tirado-Rives, 2005). Second, the provided force
field (FF) parameters in ligand (file.prm), the topology (file.rtf), and
molecule structures (file.pdb) were retrieved and the files were used as
input formats of recognition to NAMD. After, each protein-ligand com-
plex was put into a cubic box, Just the GPR40 receptor or complexes
(lig-GPR40) were embedded in the hydrated POPC (1-palmitoyl-2-o-
leoyl-sn-glycero-3-phosphocholine) membrane, which were used to
simulate a lipid bilayer environment around the receptor (dimensions
71 × 69). In addition, the systems were solvated using the water model
of TIP3P and neutralized by adding Clꢀ ions.
2.4. Antihyperglycemic in vivo assay in diabetic rats
2.4.1. Experimental diabetes induction
Experimental Type 2 Diabetes (T2D) was induced in overnight fasted
animals by two intravenous injections of alloxan (Sigma-Aldrich) at
intervals of 48 h (125 mg/kg). Diabetes was confirmed by the elevated
blood glucose levels determined at 72 h and on the 7th day after the
treatment (glucose level above 200 mg/dL was taken into account). Only
the rats that were confirmed with T2D were used in the study (Calzada
et al., 2017).
Then, the energy of these systems was immediately minimized from
2ps to 273.15 K with backbone restraints, followed by a heating protocol
with 288 ps (this protocol was used only with the GPR40 complexed
with bilayer lipid) and/or followed by an equilibration protocol with
100 ps of simulations at 300 K with backbone restraints. Next, 20 ns of
MD simulation were performed at 300 K temperature, with the
isothermal-isobaric (NpT) ensemble and periodic boundary conditions
at a constant pressure of 1 atm without any restraints. During each one
of the MD simulations, the Particle Mesh Ewald (PME) method was used
with updates every 2 fs., consequently the MD simulations were pre-
pared using QwikMD (Ribeiro et al., 2016).
2.5. In silico
2.5.1. GPR40 model preparation
The crystal structure of GPR40 (PDB access code: 5TZR, resolution of
2.2 Å) was prepared using the DockPrep in Chimera 1.14rc (Pettersen
et al., 2004). First, the extra molecules such as ligands, solvent,
non-complexed ions, or additional subunits were deleted. Then, the
incomplete side chains of the structure were repaired using a set of the
Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, 2011) and
all hydrogens and charges were added. After that, the molecule was
saved in a PDB format using it in molecular docking and molecular
dynamics simulations.
Finally, the calculations were conducted using a workstation DELL
Precision 7920 equipped with two Xeon® Gold® 5122 @ 3.60 GHz
processor (Intel, US), 128 Gb of DDR memory and Quadro P4000
(NVIDIA, US) in our institute. Other software packages such as the Vi-
sual Molecular Dynamics (VMD) was employed as an interface,
analyzer, and visualizer (Humphrey et al., 1996).
3. Results
2.5.2. Molecular docking studies
Molecular docking studies in compound 1 were carried out with
PPARγ (2F4B) and GPR40 (5TZR, modified) crystal structures, and the
docking calculations were conducted with the AutoDock Vina (Hum-
phrey et al., 1996). All of the water molecules and also EHA (co-crys-
tallographic ligand from PPARγ) and MK6 (co-crystallographic ligand
from GPR40) were removed from the crystallographic structure. Then,
each one of the hydrogen atoms were added, non-polar hydrogen atoms
were merged and Gasteiger charges were assigned to the all molecules
(ligands and proteins). Next, the torsions from compounds were allowed
to rotate during the docking study. Each grid was centered at the crys-
tallographic coordinates of EHA (center_x = 8.693; center_y = ꢀ 6.961
and center_z = 39.672) and MK6 (center_x = ꢀ 32.741; center_y =
ꢀ 3.538 and center_z = 58.465 of PPARγ and GPR40, respectively. The
grid dimensions were 40 × 40 x 40 points and 58 × 40 x 40 points with a
default spacing of PPARγ and GPR40, respectively. Finally, the
exhaustiveness employed was 64.
3.1. Chemistry
The two phenoxyacetic acid derivatives (1 and 2) were synthesized
through a series of linear transformations, the first reaction was started
from compounds 8 and 9 to give the intermediate ether (7), which was
subjected to the catalytic reduction with H2 and Pd/C to obtain the
aniline product (6) with a totally raw material conversion. Immediately,
an acylation of ethyl (4-aminophenoxy)acetate (6) was carried out with
2-choroacetyl chloride giving the corresponding compound 5 in good
yield. The intermediate 5 has been the most important common moiety
used to synthesize the ester derivative compounds 3 and 4, from phenol
reagents: N-(4-hydroxyphenyl)acetamide (acetaminophen) and 1-(4-
hydroxyphenyl)ethan-1-one, respectively. Then, the Acetaminophen
was isolated from commercial tablets with ethanol. Consequently, the
compound 5 was subjected to SN2 conditions with both phenol moieties
to obtain the required ester intermediates 3 (ethyl {4-[2-(4-acet-
amidophenoxy)acetamido]phenoxy}acetate) and 4 (ethyl {4-[2-(4-ace-
tylphenoxy) acetamido]phenoxy}acetate). Finally, the ester products
were treated under basic conditions with KOH at reflux in EtOH-water to
give the final compounds 1 and 2 in a moderate yield (Scheme 1).
Finally, all compounds and their intermediates were recovered with
appropriate yields (±70%) and characterized by the 1H, 13C-Nuclear
Magnetic Resonance and Mass Spectral Analysis. 1H-NMR spectra of
compounds 1 and 2 showed classic aromatic signals that corresponded
to a benzene system in the range of 6.79–8.21 ppm, the signals of the
amide group were founded in a range of 9.85–10.0 ppm; the methylene
was found at a range of 4.29–4.77 ppm, while the carboxylic group was
assigned by 13C spectra in which the signal was established at
165.6–170.7 ppm.
2.5.3. Molecular docking validation
The molecular docking protocol was validated through a re-docking
of co-crystal ligands (EHA or MK6) into the binding site of both phar-
macological targets. The conditions to reproduce the binding mode of
co-crystallographic ligands were established and after a re-docking, we
found that the root mean squares deviations (RMSD) between the co-
crystal ligand and the re-docked structure were 0.53 Å to PPARγ and
1.6 Å to GPR40, which indicated that the parameters in docking simu-
lations were worthy in reproducing orientation, conformation, and in-
teractions in the original X-ray crystal structure. These conditions
allowed us to obtain good predictions in the compounds of interest.
2.5.4. Molecular dynamics simulations
Molecular dynamics (MD) were performed using the NAnoscale
Molecular Dynamics (NAMD) software package (Phillips et al., 2005)
and as a force field, the standard CHAMM-36 was used. The obtained
complexes of compound 1 with PPARγ and GPR40 were used to perform
the MD simulations. First, the obtained pose of compound 1 to each
receptor was protonated and submitted to the LigParGen web-based
3.2. In vitro assays
3.2.1. PPARγ, GPR40, GLUT-4 expression
Once compounds 1 and 2 have been evaluated with their respective
prodrugs (esters: 3 and 4, respectively) in the MTT functionality test on
4

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
Scheme 1. Synthesis of phenoxyacetic acid derivatives, conditions and reagents: a) K2CO3, acetone, reflux; b) H2 Pd/C 10% EtOH, r. t.; c) d) K2CO3, AcCN, reflux; e)
KOH, EtOH–H2O, reflux.
3T3-L1 and RINm5F culture cells. We found that in the range of 0.5
M–50 M, the molecules were safe to use and did not affect cell func-
3.3. In vivo activity
μ
μ
tionality (Fig. 1). Then, we decided to evaluate the effect in cell cultures
Compound 1 was evaluated in vivo using a rat model for non-insulin-
dependent diabetes mellitus (NIDDM) and an additional group of nor-
moglycemic rats with a complementary assay of oral glucose tolerance
test (OGTT), respectively; glibenclamide was used as a positive control.
The hypoglycemic and antihyperglycemic activity was determined using
5 or 25 mg/kg as a single dose, respectively.
of 3T3L1 and RINm5F at 1
genes.
μM to explore the overexpression of target
Fig. 1 shows the results of tested compounds 1–4 in 3T3-L1 and
RINm5F cells in different concentrations to determine and calculate the
safety concentrations that do not affect the cellular functionality. The
results shown in Fig. 2 support the objective of this research, since
compound 1 shows a high PPARγ expression, almost 3 times the level of
expression concerning to the control (CT) in adipocytes, but showing a
similar effect as pioglitazone (3T3-L1 cell line, Fig. 2A). Nevertheless,
the prodrugs of final compounds (3 and 4) did not show a relevant action
on PPARγ expression, and compound 2 only presented an increase of 2-
fold respect to CT, these results have been shown in Fig. 2A. However,
the increment of PPARγ expression of compound 1 had an impact on
GLUT4 expression levels, which are a very important target expressed
PPARγ activation.
As can be seen in Fig. 3A, compound 1 (dose of 5 mg/kg) did not
induce hypoglycemia in normoglycemic rats compared to saline solu-
tion. However, compound 1 (5 mg/kg) decreased the hyperglycemic
peak on the glucose tolerance test of the same normoglycemic animals,
as well as glibenclamide (Fig. 3C). This indicates that compound 1 is
able to control high blood glucose levels at basal levels but avoiding
hypoglycemia. Afterwards, another OGTT was carried out in diabetic
rats, for instance, rats that have high glucose values and also a dextrose
intake; determining that compound 1 (dose of 5 mg/kg) also lowered the
high glucose concentrations as glibenclamide, decreased to 50% at 7 h
post-administration Fig. 3D). Nevertheless, when the compound was
administered to the same diabetic model but with free feeding and tap
water access, there was a loss effect observed at 5 mg/kg even if at a
higher dose (25 mg/kg) the hyperglycemia were once again controlled
once (Fig. 3B). This indicates that the lower dose is not effective because
the feeding ad libitum increases the glucose level throughout the
experiment, while a dose of 25 mg/kg accomplishes glucose control,
even when there is a free feeding access (ad libitum).
3.2.2. Insulin secretion and [Ca2+]i in RINm5F cells
In order to be able to corroborate that the increment on GPR40
expression led to an insulin release, we evaluated the 1 and 2 com-
pounds in RINm5F cells, in which the insulin release as well as intra-
cellular calcium release were involved in this event. Finally, a moderate
secretion of insulin that supported our hypothesis, which meant that the
designed compounds caused a low insulin release and did not cause
hypoglycemia as glibenclamide (Fig. 2E) through the intracellular cal-
cium quantification was observed.
5

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
Fig. 1. Cell functionality test by reduction of MTT in 3T3-L1 cells and RIN-m5F. The compounds 1–4 are shown in different concentrations tested in 3T3-L1 (A) and
RIN-m5F (B) cells.
3.4. Molecular docking of compound 1
are considered anchoring residues in this receptor since almost all of the
ligands that activate this receptor are anchored to these amino acids.
–
A molecular docking study to explain the outstanding activity
showed by compound 1 on PPARγ and GPR40 structures, which was
achieved to find the possible binding mode into the orthosteric site
(active site) in both receptors was performed. Compound 1 was docked
with a crystallographic structure of PPARγ (pdb code: 2f4b) and GPR40
(pdb code: 5tzr). This molecule showed a good affinity against both
targets with free binding energy of ꢀ 8.2 kcal/mol and ꢀ 8.4 kcal/mol,
Moreover, we found a new interaction between Ser342 and the last C
O
–
of the amidic group from compound 1.
3.5. Molecular dynamics simulation of most active compound (1) into
PPARγ and GPR40
The complexes obtained from molecular docking were used in the
molecular dynamics simulation and it was performed to investigate the
stability and possible variations of interactions between ligand and
targets (GPR40 and PPARγ). The root mean squares deviations (RMSD)
in complex with co-crystal compound, molecule 1, or in absence of li-
gands such as a number of H-bonds are illustrated in Figs. 5 and 6.
Regarding the complex of GPR40 during the progress of molecular
dynamics simulation, the interactions were maintained with Tyr90,
Arg181, and Arg254. However, a new interaction appeared between Leu
137 and nitrogen of the central amidic group of a molecule that
increased the stability and the last overlaying of phenyl group with Phe
142 (Fig. 5A). This stability is correlated with RMSD obtained during
MD (Fig. 5C), which was lower than 1.5 Å after 12 ns of simulation to
compound 1, even lower than the co-crystallized ligand. Also, all the
hydrogen bonds ranged between 3 and 4 during the entire simulation
(Fig. 5B), impacting on the stability of the complex.
respectively (Ki = 0.7944 ± 0.13 μM). This can give us an indication that
the affinity towards both receptors is similar, and could provide a dual
equilibrate effect to this molecule. Fig. 4A–C shows the binding mode of
1 and co-crystal (MK6) into the GP40 receptor with a respective re-
docking pose. We can observe in both figures, the conservation of
three important interactions with Tyr90, Arg181, and Arg254 residues,
where the first scaffold’s portion of the ligands (1 and MK6) is centered
between the transmembrane domains and the tail (acetaminophen and
methylsulfonylpropoxy groups) are in the extra-domain. Therefore, in
both cases, the acid region is primarily recognized and essential for
binding to these therapeutic targets (Li et al., 2019a; Sum et al., 2007).
The most recently published results by other research groups have
demonstrated that FFA1 agonists bearing phenoxyacetic acid scaffold
exerted a greater potential on glucose control than that of TAK-875 (Li
et al., 2019b).
Regarding PPARγ, the results are shown in Fig. 4B–D, which illus-
trate the compound 1 and EHA into the binding site. We can observe in
this representation that in both molecules had interactions at least with
three residues of the binding site (His323, His449 and Tyr473), which
On the other hand, the protein in the absence of ligands shows a
balanced variation in the RMSD throughout the simulation (Fig. 5C), but
in the presence of the co-crystallized ligand, the protein structure is
disturbed, while in the presence of compound 1, the protein structure
6

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
Fig. 2. Effect of compounds 1–4, pioglitazone and glibenclamide on mRNA expression levels of genes of interest: PPARγ (A), GLUT4 (B), GPR40 (C), Percentage of
intracellular Ca2+ concentration (D), insulin release (E) and Images confocal microscopy (Fluo-4 AM) of treated cells. Scale bar indicates the increase in [Ca2+
]
intracellular concentration (F). Results are expressed as relative expression of mRNA (mean ± S.E.M, n = 4) *p < 0.05 compared with control.
7

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European Journal of Pharmacology 907 (2021) 174244
Fig. 3. The effect in percent of the variation of glucose for compound 1 in A) Normoglycemic rats, B) Diabetic rats with free feed access; C) and D) Oral glucose
Tolerance Test (OGTT) in normoglycemic and diabetic rats, respectively.
Fig. 4. Docked pose (carbon atoms colored orange)
of compound 1 and hydrogen bond network is deno-
ted as yellow dashed lines. A) the proposed binding
mode of compound 1 with the best free binding en-
ergy (ꢀ 8.4 kcal/mol) in the binding site of GPR40; B)
the proposed binding mode of compound 1 with the
best free binding energy (ꢀ 8.2 kcal/mol) in the
binding site of PPARγ: C) and D) re-docking pose of
co-crystal ligand (carbon atoms colored pale green)
with the pose of co-crystal structure reference (carbon
atoms colored blue).
varies less, even lower than protein without ligands. The data indicated
that the interaction between compound 1 and GPR40 is possibly a par-
tial activation. Fig. 5D showed that the interactions were maintained by
a co-crystallized ligand along with the simulation with three main res-
idues. Finally, Fig. 5E illustrates the constituted system used for this
simulation.
the hydrogen bond ranged between 2 and 3 (Fig. 6B) impacting on the
stability of the complex; the RMSD obtained during MD (Fig. 6C), which
was less than 1.5 Å during and after 20 ns of simulation to compound 1,
even lower than the co-crystallized ligand. It is worth mentioning that
the co-crystallized ligand undergoes more changes or disturbances than
compound 1 in the first 7 ns of simulation, this indicates that the com-
plex of our compound is more stable than the co-crystallized ligand.
Besides, Fig. 6D illustrated the interactions between the co-crystallized
ligand and receptor that had the mean residues His449 and Tyr473,
which were conserved during MD with system were exposed in Fig. 6E.
Also, the behavior of compound 1 was compared and contrasted to
the PPARy receptor showing that the co-crystallized ligand maintains
polar interactions with the mean residues: His323, His449 and Tyr473
during all simulation (Fig. 6A). Certainly, during the entire process all of
8

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European Journal of Pharmacology 907 (2021) 174244
Fig. 5. Compound 1 into the binding site of GPR40; A) hydrogen bond network during 20 ns of simulation, B) number of H-bonds during all simulation, C) RMSD of
Protein and Ligands, D) final pose of MK6 obtained after of simulation (co-crystal ligand), E) system used for dynamic simulation.
4. Discussion
work (Hidalgo-Figueroa et al., 2017) the modification in the same
portion, which contained the 2-mercaptobenzimidazole moiety (pre-
In this work, the final molecules were analogs sharing important
characteristics, but the last fragment had some differences, for example,
in the case of compound 1 the ending hydrophobic group was acet-
aminophen while in compound 2 this portion was replaced by 4-hydrox-
yacetophenone, which had no amidic group. We reported in previous
incubated at 100 μM) demonstrating an increase PPARγ, GPR40 and
GLUT4 expression, moreover, showing an increase of [Ca2+]i levels
allowing an increment on insulin release, being as active as the positive
control (glibenclamide). However, we have found in the most recent
work that this small modification in the molecule allows us to know and
9

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European Journal of Pharmacology 907 (2021) 174244
Fig. 6. Compound 1 into the binding site of PPARγ; A) hydrogen bond network during 20 ns of simulation, B) number of H-bonds during all simulation, C) RMSD of
Protein and Ligands, D) final pose of ligand EHA obtained after of simulation (co-crystal ligand), E) system used for dynamic simulation.
compare the characteristics that increase the potency and details of the
molecular requirements to improve the pharmacological activity. Also,
this has helped us to be able to discover more effective compounds.
Being able to observe that the newly synthesized compound in this work
(1, containing acetaminophen structure) has greater potency than the
previous molecule since at a lower concentration a similar pharmaco-
logical efficacy has been observed. Indeed, it is an important site to
consider in the future to modify or preserve in the following design of
derivatives.
The increment of PPARγ expression of compound 1 had an impact on
GLUT4 expression levels, which are a very important target expressed
PPARγ activation. The Increment of GLUT4 expression is associated with
glucose uptake and insulin sensibility. Regarding the GPR40 receptor,
compound 1 shows a greater effect than the other compounds (2–4),
10

S. Hidalgo-Figueroa et al.
European Journal of Pharmacology 907 (2021) 174244
´
including higher than glibenclamide (Fig. 2C), which is a novel mech-
anism reported in a previous publication by our scientific group
(Hidalgo-Figueroa et al., 2017).
methodology, reviewing and editing. Ismael Leon-Rivera: chemical
´
characterization and reviewing. Gabriel Navarrete-Vazquez: software
and reviewing.
Correspondingly, we discovered that this release was due to an in-
crease of calcium that affected the mobilization of the vacuoles that
contained insulin, which was mild compared to glibenclamide (Fig. 2D
and F). For this reason, it can be inferred that compound 1 directly
activated the PPARγ receptor and GPR40 receptor.
Declaration of competing interest
The authors declare that there is no conflict of interest regarding the
publication of this paper.
Also, the observed dose-dependent effect (in vivo) suggests that the
dose of 25 mg/kg could be adequate for glycemic control in diabetics
and without any adverse effects such as hypoglycemia, which are
generated by other drugs such as glibenclamide.
Acknowledgements and Funding
This work was supported by the Consejo Nacional de Ciencia y
Tecnología under Grant No. 2001 (APN-2016) and partially supported
by FORDECYT No. 296354 and FORDECYT-PRONACES No. 377882.
Reviewing the literature, we discovered that the PPARγ full agonists
showed undesired side effects associated to these characteristics, which
include weight gain, edema, congestive heart failure, and bone fracture.
Interestingly, we can observe in the molecular study that compound 1
preserved an interaction with Ser342, which is characteristic of partial
agonists (Capelli et al., 2016; Colín-Lozano et al., 2018; Guasch et al.,
2011). As compared to the crystallographic ligands, compound 1 pre-
served numerous important hydrogen bonds with main residues into
both targets. Interestingly, three H-bonds were conserved through car-
boxylic acid in both receptors and these results could explain the
experimental findings.
´
Ana Teresa Rodríguez Luevano acknowledges a Postgrade Scholarship
from CONACYT (No. 636061). The authors acknowledge LANEM (CIQ,
UAEM) for access to analytical equipment, and Lucia Aldana Navarro for
the grammatical review.
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