Design, synthesis and in combo antidiabetic bioevaluation of multitarget phenylpropanoic acids

Article abstract of DOI:10.3390/molecules23020340

We have synthesized a small series of five 3-[4-arylmethoxy)phenyl]propanoic acids employing an easy and short synthetic pathway. The compounds were tested in vitro against a set of four protein targets identified as key elements in diabetes: G protein-coupled receptor 40 (GPR40), aldose reductase (AKR1B1), peroxisome proliferator-activated receptor gama (PPARγ) and solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4). Compound 1 displayed an EC₅₀ value of 0.075 μM against GPR40 and was an AKR1B1 inhibitor, showing IC₅₀ = 7.4 μM. Compounds 2 and 3 act as slightly AKR1B1 inhibitors, potent GPR40 agonists and showed an increase of 2 to 4-times in the mRNA expression of PPARγ, as well as the GLUT-4 levels. Docking studies were conducted in order to explain the polypharmacological mode of action and the interaction binding mode of the most active molecules on these targets, showing several coincidences with co-crystal ligands. Compounds 1–3 were tested in vivo at an explorative 100 mg/kg dose, being 2 and 3 orally actives, reducing glucose levels in a non-insulin-dependent diabetes mice model. Compounds 2 and 3 displayed robust in vitro potency and in vivo efficacy, and could be considered as promising multitarget antidiabetic candidates. This is the first report of a single molecule with these four polypharmacological target action.

Full text of DOI:10.3390/molecules23020340

molecules
Article
Design, Synthesis and in Combo Antidiabetic
Bioevaluation of Multitarget Phenylpropanoic Acids
†
1
1
ID
1
Blanca Colín-Lozano , Samuel Estrada-Soto
, Fabiola Chávez-Silva ,
1
1
ID
2
Abraham Gutiérrez-Hernández , Litzia Cerón-Romero
Julio Cesar Almanza-Pérez
Mario Cappiello 5 , Francesco Balestri , Umberto Mura and Gabriel Navarrete-Vazquez *
, Abraham Giacoman-Martínez ,
2
ID
3
4
4
, Emanuel Hernández-Núñez , Zhilong Wang , Xin Xie ,
ID
5
5
1, ID
1
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico;
clbi_ff@uaem.mx (B.C.-L.); enoch@uaem.mx (S.E.-S.); facasy@gmail.com (F.C.-S.);
ghaa_ff@uaem.mx (A.G.-H.); crlc_ff@uaem.mx (L.C.-R.)
Laboratorio de Farmacología, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana
Iztapalapa, Ciudad de México 09340, Mexico; agmfest@hotmail.com (A.G.-M.);
j.almanza.perez@gmail.com (J.C.A.-P.)
Cátedra CONACyT, Departamento de Recursos del Mar, Centro de Investigación y de Estudios Avanzados
del IPN, Unidad Mérida, Yucatán 97310, Mexico; emanuel.hernandez@cinvestav.mx
CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, endlesslily@hotmail.com (Z.W.);
xxie@simm.ac.cn (X.X.)
Dipartimento di Biologia, Unità di Biochimica, University of Pisa, 56126 Pisa, Italy;
mcappiello@biologia.unipi.it (M.C.); francesco.balestri@unipi.it (F.B.); umberto.mura@unipi.it (U.M.)
Correspondence: gabriel_navarrete@uaem.mx; Tel.: +52-777-329-7089
2
3
4
5
*
†
Taking in part of the Ph.D. Thesis of Blanca Colín-Lozano.
Received: 8 January 2018; Accepted: 3 February 2018; Published: 6 February 2018
Abstract: We have synthesized a small series of five 3-[4-arylmethoxy)phenyl]propanoic acids
employing an easy and short synthetic pathway. The compounds were tested in vitro against
a set of four protein targets identified as key elements in diabetes: G protein-coupled receptor 40
(
GPR40), aldose reductase (AKR1B1), peroxisome proliferator-activated receptor gama (PPAR
solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4). Compound displayed
an EC50 value of 0.075 M against GPR40 and was an AKR1B1 inhibitor, showing IC50 = 7.4 M.
Compounds and act as slightly AKR1B1 inhibitors, potent GPR40 agonists and showed an increase
of 2 to 4-times in the mRNA expression of PPAR , as well as the GLUT-4 levels. Docking studies
γ) and
1
µ
µ
2
3
γ
were conducted in order to explain the polypharmacological mode of action and the interaction
binding mode of the most active molecules on these targets, showing several coincidences with
co-crystal ligands. Compounds
and orally actives, reducing glucose levels in a non-insulin-dependent diabetes mice model.
Compounds and displayed robust in vitro potency and in vivo efficacy, and could be considered
1–3 were tested in vivo at an explorative 100 mg/kg dose, being 2
3
2
3
as promising multitarget antidiabetic candidates. This is the first report of a single molecule with
these four polypharmacological target action.
Keywords: diabetes; GPR40; AKRB1; PPARγ, GLUT-4
1
. Introduction
Type 2 Diabetes Mellitus (T2DM) is a multifactorial metabolic disease that occurs with fasting
blood glucose concentrations above 120 mg/dL (>7 mM), due to the abnormal pancreatic
β-cells and/or
insulin resistance [ ]. Currently, experimental drug discovery for T2DM is focused on developing
1
Molecules 2018, 23, 340; doi:10.3390/molecules23020340
www.mdpi.com/journal/molecules

Molecules 2018, 23, 340
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drugs for insulin sensitizing and/or releasing effect by several mechanisms. One of them is mediated
by the G protein-coupled receptor 40 (GPR40 or FFA1), which is primarily expressed in pancreatic
β
-cells and enteroendocrine cells of the gut [
acids, stimulates insulin secretion only in the presence of high glucose concentration but does not
disturb insulin exocytosis at low glucose levels [ ]. This intriguing mechanism was suggestive in
treating T2DM by small molecules agonists for GPR40, thus playing as novel insulin secretagogues with
little or no risk of hypoglycemia [ ]. An alternative mechanism for the control of glucose levels refers
to the peroxisome proliferator-activated receptors (PPARs) action. PPARs, members of the nuclear
hormone receptor family, occur in three subtypes: PPAR , PPAR , and PPAR ]. Each subtype
regulates tissue-specific target genes acting as lipid sensors and regulators of glucose homeostasis,
such as the solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4) [ ]. Glitazones
function as insulin-sensitizing drugs, via activation of PPAR 10], leading to enforced expression
of target genes involved in glucose-sensing ability of pancreatic -cells in diabetic subjects [11].
2,3]. The GPR40 activation by medium to long chain fatty
4,5
6
α
γ
β/δ [7
8
γ [9,
β
On the other hand, extended hyperglycemia plays a significant key in the development of diabetic
complications: atherosclerosis, blindness, neuropathy and renal failure. The NADPH-dependent
reduction of elevated levels of D-glucose to sorbitol catalyzed by aldose reductase (AKR1B1) is
considered as one of the phenomena leading to the onset of long term diabetic complications [12].
Stressful and damaging cell conditions are produced by several factors such as the osmotic imbalance
due to sorbitol accumulation, the loss of antioxidant power related with NADPH oxidation and
the induction of additional advantageous conditions for protein glycation [13
AKR1B1 represents a potential target of antidiabetic drug action. The purpose of the present work
was to design, synthesize and screen in vitro in vivo and in silico (in combo screens) a small series of
five 3-[4-arylmethoxy)phenyl]propanoic acids to accomplish the treatment of diabetes with a single
molecule with multitarget action: activation of PPAR , GLUT-4 and GPR40 and inhibition of AKR1B1
Figure 1). Multitarget of polypharmacological concept can be defined as the modulation of several
–15]. Inhibition of
,
γ
(
drug targets to achieve a desired therapeutic effect. This multitarget therapy may be an attractive
option for the therapeutic treatment of diabetes that will not only control the glucose concentrations
but also decrease complications linked to hyperglycemic conditions. Compounds
based on the compounds GW9508 [16] and SHF-1 [17], maintaining the typical 4-point pharmacophore
of synthetic GPR40 agonists [ ], that mimic the fatty acid structure of endogenous agonists, such as
1–5 were designed
6
docosahexaenoic acid (DHA, Figure 1): (I) an acidic group (carboxylate or surrogates); (II) a central
p-substituted benzene; (III) a second lipophilic region and (IV) a flexible connector that joins regions
(I) and (IV), and tolerating the structure to assume diverse conformations. The insulin-sensitizer
targets PPAR and GPR40 which are activated by long chain fatty acids and thiazolidine-2,4-diones
γ
are described to function as an integrated two-receptor signal transduction pathway [18] being
sensitive to closely related pharmacophores, as represented by GW9508 and SHF-1 [17]. Then, it is
reasonable to assume that also compounds 1–5 may act as agonists on these targets in a similar
fashion. On the other hand, carboxylic acid derivatives comprise the majority of reported AKR1B1
inhibitors: epalrestat, tolrestat and zopolrestat. The other two chemical groups that function as
inhibitors are: spirohydantoins (Sorbinil) and flavonoids such as quercetin [19]. Based on the relevance
of the phenylpropanoic scaffold (Figure 1) in determining the inhibitory ability of zopolrestat towards
AKR1B1, we expected that also compounds
1–5 may act as inhibitors of the enzyme. As a result of the
multifactorial complexity of chronic diseases, the current therapeutic arsenal and the old-fashioned
“one-molecule, one-target” model seem not so effective [20]. The design of new chemical entities
(
NCE) with two or more balancing bioactivities for the treatment of complex diseases would be very
advantageous. Based on the above considerations, our work was focused on the design, preparation
and evaluation of these novel multitarget molecules to be active against Diabetes. Although compound
1
was previously synthesized [21
this compound and of the accompanying molecules is extended and explored in vivo. A parallel
investigation on the potential antidiabetic action of these compounds: the activation of PPAR and
,22], in the current work the effectiveness as GPR40 agonist of
γ

Molecules 2018, 23, 340
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GLUT-4 and the inhibition of AKR1B1, is also reported for first time. To our knowledge, this is the first
report describing the potential of the simultaneous targeting of these four relevant processes linked to
the diabetic pathology by a single molecule.
DHA
(Endogenous ligand)
SHF-1
(
PPARγ agonist and
GW9508
GPR40 agonist)
Zopolrestat
(AKR1B1 inhibitor)
GLUT4 promoter)
(
Multitarget design
Ar =
1
2
3
4
5
Figure 1. Drug design of multitarget compounds
1–
5, which were designed from pharmacophore
extraction and reorganization from known modulators of four targets: GPR40, PPAR
γ, GLUT-4,
and AKR1B1.
2
. Results and Discussion
2
.1. Chemical Synthesis
Compounds
with malonic acid (
give compound . This compound was hydrogenated under catalytic reduction with 10% Pd/C giving
-(4-hydroxyphenyl)propanoic acid ( ), which was reacted via esterification with thionyl chloride and
1
–5
were prepared starting from 4-hydroxybenzaldehyde (
6), which was condensed
7), following by microwave Knoevenagel reaction and decarboxylation conditions to
8
3
9
ethanol as solvent, obtaining ethyl 3-(4-hydroxyphenyl)propanoate (10). Subsequently, 10 was reacted
with the appropriately substituted methylaryl halide in polar aprotic solvents and potassium carbonate
to give compounds 11
–14. All were treated separately by basic hydrolysis with potassium hydroxide,
to give (Scheme 1). Compound
1
–
4
5
was obtained by direct SN reaction between acid and
9
2
N-1,3-benzothiazol-2-yl-2-chloroacetamide (15). Compounds were recovered with modest yields and
were purified by column chromatography or recrystallization (see experimental part). Their chemical
structures were confirmed by spectroscopic (1D and 2D NMR, see supporting information) and
spectrometric data, and their purity determined by microanalysis.

Molecules 2018, 23, 340
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β-alanine
pyridine,
microwaves
8
(85%)
6
7
Pd/C, H2
EtOH
SOCl2
9
(99%)
1
0 (94%)
1
5
K CO3
K CO MeCN
2 3,
2
Polar aprotic solvent
KOH, Δ
EtOH/H O
2
1
2
3
4
5
: Ar= Biphenyl-3-yl (83%)
: Ar= 1-Naphthyl (51%)
: Ar= 2´-Cyanobiphenyl-4-yl (85%)
: Ar= Quinolyn-2-yl (95%)
: Ar= Benzothiazol-2-ylamino-2-oxoethyl (27%)
1
1
1
1
1: Ar= Biphenyl-3-yl (65%)
2: Ar= 1-Naphthyl (78%)
3: Ar= 2´-Cyanobiphenyl-4-yl (64%)
4: Ar= Quinolyn-2-yl (72%)
Scheme 1. Synthesis of compounds 1–5.
2
.2. In Vitro GPR40 Activity
Compounds
flux assay in GPR40-transfected HEK293 cells [
in Table 1. Linoleic acid (LA), one of the endogenous ligands for GPR40, was designated as positive
control. Compounds were tested in quadruplicates at 100 M. After preliminary screening, compounds
–5 were further tested in a concentration-dependent manner (Table 1).
1–
5
were in vitro tested as GPR40 agonist. This activity was measured with calcium
2
,23]. The preliminary screening results are summarized
µ
1
Table 1. Preliminary screening (Efficacy, maximal response) and GPR40 agonistic activities (Potency,
EC ) of compounds 1–5 in calcium assay for hGPR40/Gα16/HEK293.
5
0
Comp.
Maximal Response % (100 µM)
EC50 (µM) a
1
2
3
4
5
98 ± 10
103 ± 3
105 ± 15
84
0.075 ± 0.02
0.648 ± 0.10
0.797 ± 0.17
≈100
9.9
>100
LA
100
8.30 ± 1.7
a
Means of two experiments.
As we expected, the results indicated that compound
EC50 = 0.075 µM), in accordance with the literature (EC50 = 0.260
1
was the most potent agonist
(
µ
M) [21
,22], followed by compounds
2
and 3, with naphthyl and biphenylcarbonitrile substituents, respectively. All of them showed
potencies in the submicromolar order. Compound
4
, with a quinolynyl substituent, provided
was inactive.
. On the other hand, it is noteworthy
1–3 is orders of magnitude greater than that obtained by endogenous
a noteworthy reduction in agonistic action. Conversely, benzothiazole derivative
Thus, compounds were less active than previously reported
5
2–
5
1
that the potency of compounds
fatty acid ligand LA.

Molecules 2018, 23, 340
5 of 16
2
.3. In Vitro Aldose Reductase (AKR1B1) Inhibition
The in vitro AKR1B1 inhibitory activity of compounds
1–
5
(Table 2) was evaluated by using
a highly purified human recombinant enzyme preparation [24
reference drug.
,25]. Sorbinil was employed as
Table 2. Inhibition of human recombinant AKR1B1 by compounds 1–5.
Comp.
IC50 (µM) a
1
2
3
4
5
8.9 (6.5–12.1)
17.4 (14.3–21.2)
21.0 (12.8–34.3)
31.7 (22.3–44.9)
23.3 (19.0–25.5)
1.2 (0.9–1.5)
Sorbinil
a
95% confidence intervals are reported in brackets.
2
.4. Relative Expression of PPARγ and GLUT-4
Compounds tested were shown interesting AKR1B1 inhibitors in the low micromolar IC50 values.
Once again, compound
series, finding this new activity for this known compound. Compounds
active. Compound was the least active. None of them were more active than sorbinil, a well-known
1, previously identified as GPR40 agonist, was the most active inhibitor of the
2
,
3
and were almost equally
5
4
AKR1B1 inhibitor. As we described previously, AKR1B1 plays a key role in the progress of chronic
diabetic complications, such as retinopathy, nephropathy, neuropathy and also cardiovascular risk.
3
T3-L1 fibroblasts were differentiated to the adipocyte phenotype in order to determine the
action of compounds on PPAR and GLUT-4 expression. Fibroblasts were treated during 24 h with
compounds and pioglitazone (10 M) as reference drug and the mRNA expression level evaluated.
Figure 2 shows that only compounds and enhance significantly the levels of relative expression
for PPAR (around three to four-times) and GLUT-4 mRNA’s in the same way as pioglitazone.
The activation of PPAR could reduce the glucose levels in diabetic patients due to insulin resistance
reduction. Our data also suggest that compounds and induces GLUT-4 expression in the same
γ
1–
5
µ
2
3
γ
γ
2
3
way than pioglitazone does. Several evidences indicate that increased levels of GLUT-4 expression in
skeletal muscle are critical for the regulation of glucose homeostasis [7,17,26].
5
***
2.5
B
A
**
4
3
2
1
0
*
2.0
1.5
1.0
0.5
0.0
***
*
*
Figure 2. (A) Effect of compounds 2 and 3 on expression level of PPARγ B) Effect of compounds
; (
on expression level of GLUT-4. Results are mean
compared with control group.
±
SEM (n = 6)/*** p < 0.001; ** p < 0.01; * p < 0.05

Molecules 2018, 23, 340
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2
.5. Molecular Docking Studies
Based on the in vitro biological assays and the preliminary enzyme inhibition evaluations,
the most active compounds were selected to explain the experimental activities on these relevant
targets. A preliminary molecular docking simulation was performed to assess the presumed binding
mode of
was done using DIA-DB [27], a web server for the prediction of antidiabetic drugs via inverse virtual
screening of the input molecules against a set of 18 protein targets identified as key elements in
diabetes, within which are included PPAR , GPR40 and AKR1B1, among others [28]. Subsequently,
a more specific and refined analysis was carried out for the most active compounds (1–3).
Refined molecular docking reveals that compounds and internalize into the ligand binding site
of PPAR and interact by electrostatic and hydrogen bonds with Ser-289, His-323, His-449 and Tyr-473,
all of them essential for the activation of this receptor. However, compound (the most active in vitro
1–5 into the receptors GPR40, PPARγ and the enzyme AKR1B1. A pilot in silico calculation
1–5
γ
2
3
γ
3
)
showed an additional interaction with Ser-342, characteristic of PPAR
Analyses of a huge number of crystallographic structures of the PPAR
γ
partial agonists (Figure 3).
ligand-binding site bound
χ
to an agonist have revealed that this isotype has two binding modes in a single pocket. These two
binding modes correspond to full and partial agonists [29]. Side effects of glitazones, including weight
gain, edema, congestive heart failure, and the recently reported increased risk of bone fracture are
major undesired effects associated with the use of PPAR
γ full agonists [30]. On the other hand, partial
agonists interact mainly through a hydrogen bond with Ser342. This interaction corresponds to several
carboxylic ligands present in the majority of the PPARγ partial agonists that forms a hydrogen bond
with the Ser342, such as showed by compound 3.
Ser
42
Ile
326
Cys
285
3
Ile
281
Arg
80
Gly
84
2
2
Ile
41
Arg
3
Leu
30
Ser
289
2
88
3
N
His
323
-
O
O
O
Leu
69
Tyr
4
4
73
Met
64
His
49
3
4
Val
39
3
Leu
53
4
(
A)
(B)
Figure 3.
(A) 3D binding model of compounds
1–
3
into the ligand binding site of PPAR
γ
. Compounds
are presented as stick models:
1
(green), (cyan) and
2
3
(magenta), and aminoacids as lines. Dashed
line signifies polar interactions; (B) 2D interaction map of the most active compound 3 and PPARγ.
For GPR40, binding poses depicted in Figure 4 suggest that the in vitro active compounds 1, 2
and
bonds with Tyr-91, Asn-2244, Tyr-2240, all of them showed by well-known GPR40 allosteric agonists
such as TAK-875). On the other hand, the disposition of the biphenyl ring in , which was the
most potent in the in vitro screening, fits into the GPR40 ligand-binding-pocket better than the other
compounds generating interactions with Phe-142 (Figure 4B). The docking score for compound
was the highest ( G = 10.63 kcal/mol), in comparison with compounds and G = 10.31 and
9.96 kcal/mol, respectively).
3 interact through electrostatic bonds with residues of Arg-183 and Arg-2258, and by hydrogen
(
1
π-
π
1
∆
−
2
3
(
∆
−
−

Molecules 2018, 23, 340
7 of 16
(
A)
(B)
Figure 4.
Compounds are presented as stick models:
of the most active compound 1 and GPR40.
(
A
) 3D binding model of compounds
1
–
3
into the allosteric ligand binding site of GPR40.
(cyan) and (magenta). ( ) 2D interaction map
1
(green),
2
3
B
In the case of AKR1B1, solutions of molecular docking into the catalytic site of this enzyme
show that acid moieties of compounds 1, 2 and interact with Tyr-48, His-110 and Trp-111 showed in
several currently inhibitors of this enzyme, such as zopolrestat and tolrestat. Also, the naphthyl ring
3
of
2
conserves an interaction with Trp-111 through
π-
π
stacking (Figure 5). All compounds showed
moderate in vitro inhibition of this enzyme.
(
A)
(B)
Figure 5.
(
A
) 3D binding model of compounds
1
–
3
into the active site of Aldose reductase (AKR1B1).
(cyan) and (magenta); ( ) 2D interaction map
Compounds are presented as stick models:
1
(green),
2
3
B
of the second most active compound 2 and AKR1B1.
2
.6. In Vivo Antidiabetic Effect of Compounds 1–3
Compounds 1–3 were the most active against some of targets identified as key elements in diabetes
in this work, and they were chosen in order to assess their in vivo activity over Streptozotocin (STZ)
and Nicotinamide (NA) diabetic induced mice, a well-known non-insulin-dependent diabetes murine
model [7,17]. Glibenclamide was used as a positive control, in order to ensure that the damage over
β-cells was partial and mice pancreas still produces insulin, that responds to a secretagogue such as
sulfonylurea drug used in this work. The antidiabetic activity of compounds
intragastric route of a single dose of 100 mg/kg (Figure 6).
1–3 were determined via
In the in vivo assay, compound
explain the observed lack of in vivo activity for this compound
1
exhibited potential differences in bioavailability that could
in comparison with the demonstrated
,

Molecules 2018, 23, 340
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activity of
2
and
3. In particular, compounds
2
and
3
showed the best in vivo activity, compared with
control group (Tween 80). Also, compound
2
producing a sustained decrease of blood glucose levels,
close to 80% less, 7 h after compound administration (Figure 6).
This compound showed a statistically significant hypoglycemic effect like glibenclamide during
the time that the experiment lasted. The glucose lowering effect of compound
the first hour of administration ( 20% at 3 h, 50% at 5 h, and 60% at 7 h). Besides, statistically
significant difference is detected between and tween 80 group, and the maximum effect was detected
in the last hours of post-intragastric administration (Figure 6).
3 was increasing after
−
−
−
3
Tween 80
Glibenclamide
1
2
3
80
60
40
20
0
*
*
*
-20
-40
-60
-80
0
1
3
5
7
Time (h)
Figure 6. Effect of a single dose (100 mg/kg) of compounds
1–
3
and glibenclamide (5 mg/kg) in
STZ-NA induced diabetes mice model (intragastric, n = 6). * p < 0.05 versus Tween 80 (10%) group.
2
.7. Off-Target Toxicity Predictions
With the aim of anticipating potential off-targets affinity and toxicity issues of the most active
compounds
1–3, a virtual prediction of safety profiles was accomplished. The toxicity parameters of
1–3, pioglitazone, sorbinil and glibenclamide (in vitro and in vivo antidiabetic reference drugs) were
calculated through the ACD/ToxSuite software, v. 2.95 (Table 3).
Table 3. Toxicity profiles predicted for compounds 1–3, pioglitazone, sorbinil and glibenclamide.
LD50 (mg/kg)
Probability of Inhibition (IC50 or K < 10 µM)
i
Compd
Mouse
Rat
CYP450 Isoform
2D6
hERG
i.p.
p.o.
i.p.
p.o.
3A4
1A2
1
2
3
320
530
580
440
420
980
1400
2000
1990
1900
680
560
460
1200
400
190
500
3800
2700
4000
1100
1600
2400
0.08
0.07
0.07
0.22
0.02
0.20
0.05
0.05
0.02
0.03
0.02
0.08
0.17
0.15
0.04
0.08
0.01
0.01
0.10
0.04
0.13
0.21
0.10
0.60
Pioglitazone
Sorbinil
Glibenclamide
1900
The in silico calculation of inhibition for the three main isoforms of CYP450 for compounds
1–3 were comparable to pioglitazone, sorbinil and glibenclamide at relevant clinical concentrations
(
<10
Besides, several lipophilic compounds are associated with cardiovascular risks due to human
ether-a-go-go related gene (hERG) channel blockade [ 32]. Compounds , contemplated as
polypharmacological molecules, showed low prediction of hERG channel blockage at clinically relevant
µM), showing low probabilities of drug-drug interactions and undesirable adverse effects [31].
7,
1–3

Molecules 2018, 23, 340
9 of 16
concentrations (K < 10
µM), being considered as potentially non-cardiotoxic compounds. In the
i
calculation of acute toxicity, compounds 1–3 demonstrated similar or even high predicted LD50 than
pioglitazone, sorbinil and glibenclamide by two different administration routes, being predicted less
toxic than these common antidiabetic drugs used as reference in this work.
Several examples of polypharmacology have been reported elsewhere [33,34]. Actually, some
multikinase inhibitors used in cancer treatment and all marketed antipsychotics are polypharmacological
drugs, even recently approved compounds. Multitargeting concept explains the efficacy of these drugs by
their modulation of a network of disease relevant targets correcting a pathological imbalance [33].
3
. Materials and Methods
3
.1. Chemistry
All compounds were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Melting points were
recorded using an EZ-Melt MPA120 automated apparatus from Stanford Research Systems (Sunnyvale,
CA, USA). Reactions were monitored by TLC on 0.2 mm precoated silica gel 60 F2 plates (Merck,
54
1
Darmstadt, Germany). H-NMR spectra were determined on a Varian Oxford instrument (Palo Alto,
CA, USA) operating at 600 MHz ( H-NMR) and 150 MHz ( C-NMR) instruments. Chemical shifts are
1
13
given in ppm in DMSO-d and CDCl as deuterated solvents. Mass spectrometry were determined on
6
3
a JMS-700 spectrometer (JEOL, Tokyo, Japan) operating in electronic impact mode.
3
.2. General Procedure for the Synthesis of Compounds 1–4
Ethyl esters 11 14 (0.70 mmol) were treated with a mixture of EtOH/H O (1:1, v/v) and KOH
2 equiv.). The mixture was stirred at reflux for 3.5–5 h. Subsequently, the ethanol was removed under
–
2
(
vacuum and HCl solution (10%, v/v) was added until reach pH 5, to obtain a precipitate which was
filtered and washed with cold water to give a solid. The crude products were recrystallized from
ethanol or methanol affording title compounds.
◦
1
3
(
-[4-(Biphenyl-3-ylmethoxy)phenyl]propanoic acid (
DMSO-d6) : 2.43 (t, 2H, CH ), 2.74 (t, 2H, CH ), 5.13 (s, 2H, CH ), 6.94 (d, 2H, H-3A, H-5A,
J = 7.74 Hz), 7.14 (d, 2H, H-2A, H-6A, J = 8.04 Hz), 7.4 (t, 1H, H-4C), 7.43 (d, 1H, H-4B, J = 7.2 Hz),
.48–7.46 (m, 3H, H-6B, H-3C, H-5C), 7.61 (t, 1H, H-5B, J = 7.5 6Hz), 7.66 (d, 2H, H-2C, H-6C,
J = 8.22 Hz), 7.72 (s, 1H, H-2C) ppm. 13C-NMR (DMSO-d6)
: 30.4 (CH ), 36.9 (CH ), 69.6 (O-CH ),
15.1 (C-3A, C-5A), 126.4 (C-6B), 126.6 (C-4B), 127.1 (C-2C, C-6C), 128 (C-4C), 129.4 (C-3C, C-5C), 129.5
C-5B), 129.7 (C-2A, C-6A), 134 (C-1A), 138.5 (C-1C), 140.4 (C-1B), 140.8 (C-3B), 157.0 (C-4A), 174.7
1). Yield 83%, pearly flakes m.p. 123.7–126 C; H-NMR
δ
2
2
2
7
δ
2
2
2
1
(
(
C=O) ppm. MS/EI: m/z (% int. rel). 353 (M + Na), 178 (M + Na
−
175) 100%. Anal. Calcd. for
C H O : C 79.50, H 6.06; Found C 79.54, H 6.04.
22
20
3
◦
3
-[4-(1-Naphthylmethoxy)phenyl]propanoic acid (
2
). Yield 51%, white powder m.p. 124.8–127.8 C;
1
H-NMR (DMSO-d6) δ: 2.49 (t, 2H, CH ), 2.76 (t, 2H, CH ), 5.50 (s, 2H, CH ), 7.0 (d, 2H, H-3A,
2 2 2
H-5A, J = 8.46 Hz), 7.16 (d, 2H, H-2A, H-6A, J = 8.46 Hz), 7.51 (t, 1H, H-3 , J = 7.56 Hz), 7.58–7.55
m, 2H, H-6 , H-7 ), 7.66 (d, 1H, J = 6.9 Hz), 7.92 (d, 1H, J = 8.52 Hz), 7.97 (d, 1H, J = 7.86 Hz), 8.08
: 30.0 (CH ), 36.1 (CH ), 68.2 (CH ), 115.1 (C-3A, C-5A),
24.3 (C-8 ), 125.8 (C-3 ), 126.4 (C-6 ), 126.9 (C-7 ), 127.03 (C-2 ). 128.9 (C-5 ), 129.0 (C-4 ), 129.7 (C-2A,
C-6A), 131.6 (C-8a), 133.1 (C-1A), 133.6 (C-4a), 133.7 (C-1 ), 157.2 (C-4A, 174.3 (C=O) ppm. MS/EI: m/z
0
0 0
(
(
1
3
d, 1H, J = 7.97 Hz).
δ
:
C-NMR (DMSO-d )
δ
6
2
2
2
0
0
0
0
0
0
0
1
0
+
(
% int. rel.). 306 (M ), 141(M
−
165)100%. Anal. Calcd. for C H O : C 78.41, H 5.92; Found C 78.43,
20 18 3
H 5.91.
0
3
-{4-[(2 -Cyanobiphenyl-4-yl)methoxy]phenyl} propanoic acid (
3
). Yield 85%, pearly flakes m.p. 174–176.9;
1
H-NMR (CDCl3)
δ: 2.66 (t, 2H, CH ), 2.91 (t, 2H, CH ), 5.11 (s, 2H, CH ), 6.93 (d, 2H, H-3A, H-5A,
2
2
2
J = 8.52 Hz), 7.15 (d, 2H, H-2A, H-6A, J = 8.46 Hz), 7.44 (t, 1H, H-4C, J = 7.68 Hz), 7.51 (d, 1H, H-6C,
J = 7.86 Hz), 7.55 (2H, H-3B, H-5B, J = 8.22 Hz), 7.58 (2H, H-2B, H-6B, J = 8.16 Hz), 7.63 (t, 1H, H-5C,
J = 7.62 Hz), 7.76 (d, 1H, H-3C, J = 7.74 Hz), 12.10 (s, 1H, OH) ppm. 13C-NMR (CDCl3)
δ: 29.8 (CH2),

Molecules 2018, 23, 340
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3
5.9 (CH ), 69.6 (CH ), 111.3 (C-2C), 118.6 (CN), 114.9 (C-3A, C-5A), 127.6 (C-4C), 127.7 (C-3B, C-5B),
2 2
1
1
29 (C-2B, C-6B), 129.3 (C-2A, C-6A), 130.0 (C-6C), 132.8 (C-5C), 133.8 (C-3C), 137.7 (C-4B), 137.8 (C-1B),
+
45.1 (C-1C), 157.2 (C-4A), 178.7 (C=O) ppm. MS/EI: m/z (% int. rel). 355 (M ), 192 (M
−
165) 100%.
Anal. Calcd. for C H NO : C 77.29, H 5.36, N 3.92; Found C 77.31, H 5.34, N 3.94.
23
19
3
◦
3
-[4-(Quinolin-2-ylmethoxy)phenyl]propanoic acid (
4
). Yield 95%, yellow crystals m.p. 125.8–128.4 C;
1
H-NMR (DMSO-d6) δ: 2.49 (t, 2H, CH ), 2.74 (t, 2H, CH ), 5.33 (s, 2H, CH ), 6.97 (d, 2H, H-3A, H-5A,
2 2 2
J = 8.82 Hz), 7.15 (d, 2H, H-2A, H-6a, J = 8.34 Hz), 7.62 (t, 1H, H-6 , J = 7.8, J = 7.14 Hz), 7.66 (d, 1H,
H-3 , J = 8.4 Hz), 7.79 (t, 1H, H-7 , J = 6.96, J = 8.34 Hz), 7.99, d, 1H, H-5 , J = 8.16 Hz), 8.02 (d, 1H,
0
0
0
0
0
0
13
H-4 , J = 8.46 Hz), 8.41, d, 1H, H-8 , J = 8.52 Hz), 12.1 (s, 1H, OH) ppm. C-NMR (DMSO-d )
(
1
δ
: 29.9
6
0
0
0
CH ), 35.9 (CH ), 71.2 (CH ), 115.1 (C-3A, C-5A), 119.9 (C-3 ), 127.01 (C-6 ), 128.4 (C-4a), 129.0 (C-7 ),
29.8 (C-2A, C-6A), 130.3 (C-5 ), 133.8 (C-1A), 137.5 (C-4 ), 147.4 (C-8a), 156.9 (C-4A), 158.2 (C-2 ), 174.2
2
2
2
0
0
0
+
(
C=O) ppm. MS/EI: m/z (% int. rel). 307 (M ) 100%, 142 (M
−
165) 75%. Anal. Calcd. for C H NO :
19 17 3
C 74.25, H 5.58, N 4.56; Found C 74.20, H 5.52, N 4.56.
3
.3. Synthesis of 3-{4-[2-(1,3-Benzothiazol-2-ylamino)-2 oxoethoxy]phenyl}propanoic Acid (5)
A stirred mixture of N-1,3-benzothiazol-2-yl-2-chloroacetamide 15 (0.0006 mol), 3-(4-
hydroxyphenyl)propanoic acid (1 equiv.), potassium carbonate (3 equiv.), potassium iodide (10% mol)
in 3 mL of acetonitrile was refluxed. The reaction solvent was dried and the mixture was basified
9
at pH = 3 with HCl 10% (v/v). The solid was filtered and dried to give compound
5
. Yield 27%,
1
low melting point solid; H-NMR (DMSO-d )
δ
: 2.41 (t, CH , H-2), 2.70 (t, CH , H-3), 4.78 (s, CH ,
6
2
2
2
H-4), 7.00 (t, H-5A, J = 7.74 Hz), 7.20 (t, H-6A, J = 7.68 Hz), 7.32 (d, H-2, H-6B, J = 8.64 Hz), 7.45 (s, H-3B,
H-5B), 7.64 (d, H-4A, H-7A, J = 7.80 Hz). 13C-NMR (DMSO-d6)
: 19.24 (C-3), 35.09 (C-2), 65.00 (C-4),
19.47 (C-4B), 120.16 (C-1B), 122.86 (C-4A, C-7A), 125.98 (C-3B, C-5B), 125.99 (C-2B, C-6B), 130.55
δ
1
(
(
C-5A, C-6A), 134.17 (C-7A), 134.29 (C-3A), 136.06 (C-2 A), 157.95 (C-5), 171.56 (C-1); MS-ESI: m/z 357
+
M + H ). Anal. Calcd. for C H N O S: C 60.66, H 4.53, N 7.86; Found C 60.71, H 4.54, N 7.84.
18
16
2
4
3
.4. Synthesis of (2E)-3-(4-Hydroxyphenyl)acrylic Acid (8)
-Hydroxybenzaldehyde (0.25 g, 2.04 mmol), -alanine (0.1 equiv, 0.20 mmol) and malonic acid
(0.21g, 1.1 equiv, 2.06 mmol) were suspended in pyridine (5 mL), following Knoevenagel reaction
4
6
β
7
with subsequent decarboxylation. The flask was shaken well and heated under microwave irradiation
system (Discovery Microwave System apparatus, 2450 MHz, 300 W, CEM, Matthews, NC, USA) for
◦
1
0 min at 70 C. After irradiation, the mixture was verted in cold water and acidified until pH 5.
The precipitate was collected by filtration, washed with water, dried and recrystallized from ethanol.
◦
1
Yield 80%, yellow crystals m.p. 214 C (dec); H-NMR (DMSO-d )
δ
: 6.35 (d, 1H, =CH trans, J = 16 Hz),
6
0
0
0
0
6
8
.86 (d, 2H, H-3 , H-5 , J = 8.4 Hz), 7.55 (d, 2H, H-2 , H-6 , J = 8.4 Hz), 7.58 (d, 1H, =CH trans, J = 16 Hz),
.87 (bs, 2H, OH, COOH) ppm. C-NMR (DMSO-d )
C-2 , C-6 ), 145.3 (C-3), 158.3 (C-4 ), 168.6 (C=O) ppm. MS/EI: m/z (% int. rel). 164 (M ).
13
0
0
0
δ: 115.4 (C-2), 116.2 (C-3 , C-5 ), 126.1 (C-1 ), 130.4
6
0
0
0
+
(
3
.5. Synthesis of 3-(4-Hydroxyphenyl)propanoic Acid (9)
A mixture of acid
8
(0.2 g, 1.20 mmol) and Pd/C 10% (0.02 g) in ethanol (40 mL) was hidrogenated
2
at 60 lb/in in a hydrogenation apparatus for 30 min. After filtration and evaporation of the solvent
uder reduce pressure a yellow oil was obtained. After cooling, a solid compound was obtained which
purificated by recrystallyzation in ethanol. Yield 99%, yellow crystals m.p. 129–131 C; H-NMR
◦
1
0
0
0
(
DMSO-d )
δ
:2.45 (t, 2H, CH ), 2.72 (t, 2H, CH ), 6.65 (d, 2H, H-3 , H-5 , J = 8.01 Hz), 7.05 (d, 2H, H-2 ,
6
2
2
0
13
H-6 , J = 8.02 Hz), 9.54 (bs, 2H, OH, COOH) ppm. C-NMR (DMSO-d )
δ
: 30.1 (CH ), 36.9 (CH ), 115.5
6
2
2
0
0
0
0
0
0
(
1
C-3 , C-5 ), 129.5 (C-2 , C-6 ), 131.8 (C-1 ), 155.5 (C-4 ), 175.1 (C=O) ppm. MS/EI: m/z (% int. rel).
66 (M ).
+

Molecules 2018, 23, 340
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3
.6. Synthesis of Ethyl 3-(4-hydroxyphenyl)propanoate (10)
To a stirred solution of acid (3 g, 18 mmol) in 30 mL of ethanol, under ice-cooling, was added
9
thionyl chloride (1.1 eq, 1.44 mL, 19 mmol) dropwise over 20 min. After stirring the reaction mixture
for 3 h at room temperature, methanol is distilled out and 25 mL of water is added. The separated
ester is extracted with ethyl acetate and washed with 10 mL of saturated sodium bicarbonate solution.
1
Evaporation of the ethyl acetate gave the ester in pure form. Yield 94%, yellow liquid; H-NMR (CDCl )
3
δ
: 1.22 (d, 3H, CH ), 2.59 (t, 2H, CH ), 2.87 (t, 2H, CH ), 4.12, q, 2H, CH ), 6.74 (d, 2H, 2.09, 6.48,
3 2 2 2
13
H-3A-H-5A), 7.03 (d, 2H, 6.54, H-2A, H-6A). C-NMR (CDCl3)
δ: 14.2 (CH ), 30.2 (CH ), 36.3 (CH ),
3 2 2
6
0.6 (CH ), 115.4 (C-3A, C-5A), 129.4 (C-2A, C-6A), 154.3 (C-4A), 173.6 (C=O). MS/EI: m/z (% int. rel).
2
+
1
94 (M ) 49%, 142 (M − 74) 100%.
3
.7. General Method for the Preparation of Compounds 11–14
A solution of ethyl 3-(4-hydroxyphenyl)propanoate 10 (0.5 g, 2.5 mmol) and K CO (0.71 g,
2
3
5
.1 mmol, 2 equiv) in appropriate polar aprotic solvent (5 mL acetonitrile or acetone) was heated
◦
at 40 C for 30 min. Adequately substituted methylaryl halides (1.1 equiv.) were added dropwise.
The mixture was heated to reflux for 8 h. Solvent was evaporated in vacuo. A mixture of ice-water
(
(
10 mL) was added and stirred for 15 min. The emulsion formed was extracted with ethyl acetate
15 mL). The solvent was removed in vacuo to give an oily product, which were purified by
3
×
column chromatography.
1
Ethyl 3-[4-(biphenyl-3-ylmethoxy)phenyl] propanoate (11). Yield 65%, colorless liquid; H-NMR (CDCl )
δ
:
3
0
1
.21 (t, 3H, CH ), 2.58 (t, 2H, CH ), 2.88 (t, 2H, CH ), 4.11 (c, 2H, CH ), 5.08 (s, 2H, CH ), 6.92 (d, 2H
,
3
2
2
2
2
J = 7.8, H-3A, H-5A), 7.11 (d, 2H, J = 8.1, H-2A, H-6A), 7.34 (t, 1H, J = 7.56, H-5B), 7.39 (d, 1H, 7.44,
H-6B), 7.43–7.41 (m, 3H, H-3C, H-4C, H-5C), 7.54 (d, 1H, J = 7.68, H-4B), 7.58, (d, 2H, J = 7.68, H-2C,
H-6C), 7.64 (s, 1H, H-2B) ppm. 13C-NMR (CDCl3)
δ: 14.2 (CH ), 30.1 (CH ), 36.2 (CH ), 60.3 (CH ),
0
3 2 2 2
7
0.1 (CH ), 114.9 (C-3A, C-5A), 126.2 (C-4B), 126.3 (C-2B), 126.7 (C-BB), 127.2 (C-2C, C-6C), 127.4 (C-5B),
2
1
28.9 (C-3C, C-5C), 129 (C-4C), 129.3 (C-2A, C-6A), 133.0 (C-1A), 137.7 (C-1C), 140.9 (C-1B), 141.5
+
(C-3B), 157.2 (C-4A), 172.9 (C=O) ppm. MS/EI: m/z (% int. rel). 360(M ) 31%, 167(M − 193) 100%.
1
Ethyl 3-[4-(1-naphthylmethoxy)phenyl]propanoate (12). Yield 78%, colorless liquid; H-NMR (CDCl )
δ
3
0
:1.23 (t, 3H, CH ), 2.59 (t, 2H, CH ) 2.90 (t, 2H, CH ) 4.12 (c, 2H, CH ), 5.44 (s, 2H, CH ), 6.97 (d, 2H,
3
2
2
2
2
0
7
.14, H-3A, H-5A) 7.14 (d, 2H, J = 7.68, H-2A, H-6A) 7.44 (t, 1H, J = 8.22, J = 8.28, H-3 ) 7.52–7.49 (m, 2H,
0
0
0
0
0
0
H-6 , H-7 ) 7.57 (d, 1H, J = 6.9, H-2 ) 7.83 (d, 1H, J = 8.22, H-4 ) 7.85 (d, 1H, J = 7.5, H-5 ) 8.03 (d, J = 7.86
H-8 ) ppm. C-NMR (CDCl )
,
13
0
δ
: 14.2 (CH ), 30.2 (CH ), 36.2 (CH ), 60.4 (CH ), 68.7 (CH ), 114.9
3
3
2
2
2
2
0
0
0
0
0
0
(
C-3A, C-5A), 123.7 (C-8 ), 125.3 (C-3 ), 125.9 (C-6 ), 126.5 (C-7 ), 128.7 (C-2 ), 128.9 (C-5 ), 129.3 (C-2A,
0
0
C-6A), 131.5 (C-4 ), 132.4 (C-8a), 133.1 (C-4a), 133.7 (C-1 ), 157.4 (C-4a), 172.9 (C=O) ppm. MS/EI: m/z
+
(% int. rel). 334 (M ) 11%, 141(M − 193) 100%.
0
1
Ethyl 3-{4-[(2 -cyanobiphenyl-4-yl)methoxy] phenyl}propanoate (13). Yield 64%, white crystals; H-NMR
0
(
DMSO-d6)
δ
: 1.14 (t, 3H, CH ), 2.56 (t, 2H, CH ) 2.78 (t, 2H, CH ) 4.03, (q, 2H, CH ), 5.16 (s, 2H, CH )
3
2
2
2
2
6
.96 (d, 2H, J = 8.04, H-3A, H-5A) 7.15 (d, 2H, J = 8.04, H-2A, H-6A), 7.64-7.60 (m, 6H, H-2A, H-3A,
13
H-5A, H-6A, H-4C, H-6C), 7.79 (t, 1H, J = 7.56, H-5C) 7.95 (d, 1H, J = 7.68, H-3C) ppm. C-NMR
0
(
DMSO-d6)
δ
:14.5 (CH ), 29.1 (CH ), 35.8 (CH ), 60.2 (CH ), 69.1 (CH ), 110.6 (C-2C), 115 (C-3A, C-5A),
3
2
2
2
2
1
19 (CN), 128.7 (C-4C), 129.2 (C-2B, C-6B), 129.7 (C-2A, C-6A), 130.5 (C-6C), 133.2 (C-1A), 134 (C-5C),
1
34.3 (C-3C), 137.7 (C-4B), 138.3 (C-1B), 144.7 (C-1C), 157.2 (C-4A), 172.5 (C=O) ppm. MS/EI: m/z (%
+
int. rel). 385 (M ) 11%, 192(M − 193) 100%.
Ethyl 3-[4-(quinolin-2-ylmethoxy)phenyl]propanoate (14). Yield 72%, yellow liquid; H-NMR (CDCl ) δ:
1
3
1
.47 (t, 3H, CH ), 2.50 (t, 2H, CH ), 2.75 (t, 2H, CH ), 4.01 (c, 2H, CH ), 5.32 (s, 2H, CH ), 6.96 (d, 2H,
3 2 2 2 2
0
J = 6.66
,
J = 8.67, H-3A, H-5A), 7.13 (d, 2H, J = 8.58, H-2A, H-6A), 7.62, (d, 1H, J = 7.35, H-6 ), 7.65 (d,
0 0 0 0
1H, J = 8.49, H-3 ), 7.79 (dd, 1H, J = 8.28, H-7 ), 7.97 (dd, 1H, J = 8.85, H-5 ), 8.01 (dd, 1H, J = 8.64, H-4 ),
8
0
13
0
2
.39 (d, 1H, 8.55, H-8 ). C-NMR (CDCl3) δ: 14.0 (CH ), 29.4 (CH ), 35.4 (CH ), 59.7 (CH ), 70.7 (CH ),
3 2 2 2

Molecules 2018, 23, 340
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0 0 0 0
14.6 (C-3A, C-5A), 119.5 (C-3 ), 127.1 (C-6 ), 128.1 (C-4 ), 128.5 (C-4a), 129.1 (C-7 ), 129.8 (C-2A, C-6A),
1
1
0
0
0
30.5 (C-5 ), 132.9 (C-1A), 137.9 (C-8 ), 146.9 (C-8a), 158.7 (C-2 ), 172.1 (C=O) ppm. MS/EI: m/z (% int.
+
rel). 335 (M ) 10%.
3
.8. Synthesis of N-1,3-Benzothiazol-2-yl-2-chloroacetamide (15)
A mixture of 2-amino-1,3-benzothiazole (0.100 g), sodium bicarbonate (3 equiv) in acetone (10 mL),
◦
was stirred at 5 C. After 30 min, chloroacetyl chloride (1.5 equiv.) was added dropwise using a drop
funnel apparatus and stirred at room temperature for 8 h. Once the reaction was completed, the solvent
was removed in vacuo, and the white precipitated was washed with cold water. Yield: 90%, m.p.:
◦
1
1
56.5–159.3 C; H-NMR (CDCl )
δ
: 4.32 (s, CH ), 7.36 (t, H-6, J = 8.10 Hz), 7.48 (t, H-5, J = 8.28 Hz),
3
2
+
7
3
3
.84 (q, H-4, H-7, J = 7.92 Hz); MS-EI: m/z 225.75 (M ) 40%, 149.61(M − 77) 100%.
.9. Biological Assays
.9.1. GPR40 Agonistic Activities of Compounds 1–5
4
This bioassay was previously described [
2
]. Briefly: About 4
×
10 h GPR40/HEK293 per well
were seeded in 96-well plate. After culturing for 24 h, cells were loaded with 2
Hanks’ balanced salt solution at 37 C for 40 min. After thorough washing with 50
µmol/L Fluo-4 AM in
◦
µL assay buffer,
25 µL assay buffer containing compound was dispensed into the well, using a FlexStation III microplate
reader and intracellular calcium change was recorded with an excitation wavelength of 485 nm and
emission wavelength of 525 nm.
3
.9.2. Aldose Reductase (AKR1B1) Inhibition Assay
Human recombinant AKR1B1 was purified by electrophoretic homogeneity as previously
◦
described [24]. The enzyme activity was corroborated at 37 C following the decrease of absorbance at
3
40 nm. The assay mixture (final volume of 0.7 mL) contained 0.25 M Na PO buffer (pH 6.8), 4.67 mM
3 4
D,L-glyceraldehyde, 0.11 mM NADPH, 0.38 M (NH ) SO and 0.5 mM EDTA. Compounds
1
–
5
, tested
as AKR1B1 inhibitors were dissolved at appropriate concentrations in a mixture DMSO/methanol
40:60, v/v) and added to the assay mixture containing 5 mU of AKR1B1; the concentrations of DMSO
4
2
4
(
and methanol in all the assays were kept constant at 0.2 and 0.3% (v/v), respectively. IC50 values
was determined by nonlinear regression analysis. For each curve, five different concentrations of
compounds were analyzed [25].
3
.9.3. In Vitro PPARγ and GLUT-4 Assay
Both assays were achieved as described elsewhere [
10 cells per well) were cultured in 6-well plates in Dulbecco’s modified Eagle’s medium.
7
,
17
,
35
–
37]. Briefly: 3T3-L1 fibroblasts
5
(9
×
After 2 days of confluence, the cells were differentiated to the adipocyte phenotype with 0.5 mM
-isobutyl-1-methylxanthine, 0.25 M dexamethasone acetate, and 0.8 M insulin, for 48 h, followed by
insulin alone for other 48 h. To determine the action of compounds on PPAR and GLUT-4 expression,
the cells were treated 24 h. RNA was isolated from cultured cells and two g of total RNA were
3
µ
µ
γ
µ
reverse-transcripted in a thermocycler. The enzyme was inactivated and finally samples were cooled.
Then 1/10 volume of each reverse-transcripted reaction was amplified with SYBR Green master mix
containing 0.5 mM of customized primers for PPAR-
threshold cycles (Ct) were measured in separate tubes and in duplicate. The
in every sample for each gene of interest. Relative changes in the expression level of one specific gene
∆∆Ct) were calculated [37].
γ
and GLUT-4. PCR was conducted and the
∆
Ct values were calculated
(
3
.10. In Vivo Assay
CD1 mice (20–25 g) were housed in groups of six (n = 6) under laboratory conditions. Before
experimentation, all animals were fasted for 16 h with water ad libitum. All animal procedures

Molecules 2018, 23, 340
13 of 16
were conducted in accordance with the Mexican Federal Regulations for Animal Experimentation
and Care (SAGARPA, NOM-062-ZOO-1999), based on US National Institutes of Health Publication
#85-23, revised 1985. Non-insulin-dependent diabetes mice model was induced in overnight fasted
mice by a single i.p. injection of 100 mg/kg STZ, 15 min before the i.p. administration of 40 mg/kg
of NA. Hyperglycaemia was established by the higher plasma glucose concentration, determined
by glucometer after 2 weeks. The animals with glycaemia higher than 200 mg/dL were selected
for the assay [38
(
(
–
41]. The diabetic mice were separated into three groups of six animals each
n = 6). Mice of experimental groups were administered with a suspension of the molecules
100 mg/kg, prepared in 10% (v/v) tween 80). Control group mice were similarly treated with 10%
1–3
tween 80. The hypoglycemic reference drug glibenclamide was used as positive control (5 mg/kg).
The samples of blood were collected from the caudal vein at 0, 1, 3, 5 and 7 h after administration
of each compound. Blood glucose levels were estimated using a commercial glucometer [41–43].
The percentage variation of glycemia for each group was calculated. Values are expressed as
mean ± S.E.M. Statistical significance was estimated by ANOVA.
3
.11. In Silico Docking Studies
The crystal structures of PPARγ, GPR40 and aldose reductase were recovered from the Protein
Data Bank (PDB) with the ID: 1I7I, 4PHU and 1Z3N respectively. Docking calculations were performed
using AutoDock, version 4.2. The program performs several runs (100) in each docking experiment.
Each run provides one predicted binding mode. All water molecules and every co-crystall ligands were
removed from the crystallographic structure and all hydrogen atoms were added. For all ligands and
proteins, Gasteiger charges were assigned and non-polar hydrogen atoms were merged. All torsions
could rotate during docking. The auxiliary program AutoGrid generated the grid maps. Each grid
was centered at the crystallographic coordinates of the crystallographic compound with dimensions of
6
0
×
60
×
60 Å with points separated by 0.375 Å. After molecular docking, all solutions were clustered
into groups with RMSD < 2.0 Å. Discovery Studio version 3.5, Pymol version 1.7.4 and MOE [44] were
used for visualization.
Docking Validation
The docking protocols were validated by re-docking of cocrystal ligands into the active site of
the structure of each protein target: PPAR
γ (Tesaglitazar), GPR40 (TAK-875) and aldose reductase
(
Lidorestat). The RMSD obtained in both cases was less than 2.0 Å in all cases. This value specifies that
the parameters for docking simulations agree in reproducing the orientation and the conformation in
the X-ray crystal coordinates of enzyme and receptors.
4
. Conclusions
Five phenylpropanoic acid derivatives active on different relevant targets involved in the control
of glucose level and in the cell damage linked to hyperglycaemic conditions, are presented as promising
antidiabetic compounds. In fact, compounds to are able with different effectiveness to combine
a significantly increase in the mRNA expression of PPAR , GLUT-4, with a GPR40 agonist action and
a well detectable inhibition of the AKR1B1 activity. Compounds and also revealed their antidiabetic
1
3
γ
2
3
effectiveness in vivo, showing a robust hypoglycemic efficacy similar to glibenclamide. The in vitro
multitargeting profile and the in vivo hypoglycemic efficacy make some of them suitable leads in
developing new chemical entities with polypharmacological ability for treatment of diabetes and its
complications. To our knowledge, this is the first report describing a multiple combined action of
a single molecule facing towards these four relevant targets involved into cellular glucose handling.
Supplementary Materials: Spectra are available online.
Acknowledgments: The work was funded by the Consejo Nacional de Ciencia y Tecnología (CONACyT), grant
No. 253814 (Ciencia Básica 2015) given to Gabriel Navarrete-Vazquez. Blanca Colín-Lozano received a CONACYT

Molecules 2018, 23, 340
14 of 16
fellowship (378373) to complete Doctoral studies. This article is dedicated to all the people, mainly Pharmacists,
who helped in the medication management and classification after the recent Mexico’s earthquake.
Author Contributions: Blanca Colín-Lozano: performed the chemical synthesis of all compounds, did the
acquisition of antidiabetic in vitro and in vivo data, and analyzed the chemical and biological results.
Samuel Estrada-Soto, Fabiola Chavez Silva and Litzia Cerón Romero: Carried out and interpreted the antidiabetic
assays. Abraham Gutierrez-Hernandez: performed and analyzed the in silico refined molecular docking in to
the PPAR
γ
, GPR40 and AKR1B1. Abraham Giacoman-Martinez and Julio Cesar Almanza-Perez: Conducted
and GLUT-4 experiments. Emanuel Hernández-Núñez: performed
and interpreted the expression of PPAR
γ
and interpreted the spectroscopic analysis using nuclear magnetic resonance in 1 and 2 dimensions. He also
wrote the Supplementary Information. Zhilong Wang and Xin Xie: performed and supervised the in vitro GPR40
experiments and edited the manuscript. Mario Capiello, Francesco Balestri and Umberto Mura: performed the
in vitro aldose reductase inhibition assay, interpreted the data for SAR analysis, contributed with reagents and
analysis tools, drafted some parts of the manuscript and did a critical revision. Gabriel Navarrete-Vázquez:
Developed the concept, designed the compounds, analyzed and interpreted data, prepared and wrote
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).