Pharmacological characterization of the cardiovascular effect of Nibethione: ex vivo, in vivo and in silico studies

Research Paper

Pharmacological characterization of the cardiovascular

effect of Nibethione: ex vivo, in vivo and in silico studies

a

b

, Sergio Hidalgo-Figueroa^c

,

ꢀ

Amanda Sanchez-Recillas

, Gabriel Navarrete-Vazquez

d

, Rolffy Ortiz-Andrade^a, Maximiliano Ibarra-Barajas^e

,

ꢀ

Marcos Bonilla-Hernandez

a

f

ꢀ

ꢀ~

ꢀ

and Juan Carlos Sanchez-Salgado

Vıctor Yanez-Perez

a

b

ꢀ

c

ꢀ

Laboratorio de Farmacologıa, Facultad de Quımica, Universidad Autonoma de Yucatan, Merida, Mexico, Laboratorio de Quımica Farmaceutica,

ꢀ

Facultad de Farmacia, Universidad Autonoma del Estado de Morelos, Cuernavaca, Mexico, CONACyT, IPICYT/Consorcio de Investigacion,

d

ꢀ

Innovacion y Desarrollo para las Zonas Aridas, San Luis Potosı, Mexico, Unidad de Materiales, Centro de Investigacion Cientıﬁca de Yucatan,

e

ꢀ

Merida, Mexico, Unidad de Investigacion en Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Autonoma de Mexico,

f

ꢀ

Tlalnepantla de Baz, Mexico and Instituto de Inmunologıa y Enfermedades Complejas SA de CV, Ciudad de Mexico, Mexico

Keywords

Abstract

antihypertensive effect; calcium channel;

mechanism of action; molecular docking;

Nibethione; vasorelaxant activity

Objective This work describes the vasorelaxant and antihypertensive effects and

the mechanism of action on vascular smooth muscle cells of Nibethione, a synthetic

thiazolidinedione derivative. Additionally, evidence of its cytotoxicity is assessed.

Methods Nibethione (NB) was synthesized, and its vasorelaxant effect and

mechanism of action were assessed through ex vivo experiments. Molecular dock-

ing studies were used to predict the mode of interaction with L-type Ca²⁺chan-

nel, and in vivo antihypertensive activity was assayed on spontaneously

hypertensive rats (SHR). The cytotoxicity potential was evaluated in porcine aor-

tic endothelial cells (PAECs) from primary explants.

Correspondence

ꢀ

Amanda Sanchez-Recillas, Laboratorio de

ꢀ

Farmacologıa, Facultad de Quımica,

ꢀ

Universidad Autonoma de Yucatan, Calle 43

ꢀ

No. 613 x Calle 90, Colonia Inalambrica,

ꢀ

C.P. 97069 Merida, Yucatan, Mexico.

E-mail: amanda.sanchez@correo.uady.mx

Key ﬁndings Nibethione vasorelaxant effect was efﬁcient on KCl (80 m^M) and

NE-contraction. This effect was deleteriously modiﬁed in the presence of potas-

sium channel block drugs, while the maximal contraction induced with NE was

signiﬁcantly decreased by NB; the CaCl₂-induced contraction was abolished

entirely. In vivo experiments showed that NB decreased diastolic blood pressure

in 20.3 % after its administration on SHR. The molecular docking showed that

NB blocks L-type Ca²⁺channel, and in vitro tests showed that NB did not pro-

duce cytotoxic activity on PAECs (IC₅₀>1000 µM).

Received February 6, 2020

Accepted April 25, 2020

doi: 10.1111/jphp.13295

Conclusions Nibethione showed in vivo antihypertensive and ex vivo vasorelax-

ant effects with implication of voltage-dependent L-type Ca²⁺channel blocking,

and this may contribute to the research of novel antihypertensive drugs.

complications.^[2,3]In this context, drug discovery and

pharmacological research of new antihypertensive agents

Introduction

Cardiovascular diseases (CVD) comprise cerebrovascular

disease, coronary heart disease and hypertension. Almost

17.9 million people die due to CVD every year worldwide.

This number accounts for 31% of global deaths in low- and

middle-income countries. Hypertension is the most preva-

lent CVD, and it is associated with high morbidity and

mortality.^[1]Current hypertension therapies reduce mor-

bidity and mortality in the affected population. However,

long-term use of this medication is associated with severe

adverse events that could induce drug intolerance, poor

therapy compliance and high economic burden due to

remain an ongoing challenge in medical sciences.

2-Thioxo-1,3-thiazolidin-4-one and 2,4-thiazolidinedione

derivatives are ﬁve-membered heterocyclic molecules that

have emerged as central scaffold for design and synthesis of

new drug with therapeutic potential chronic non-degenera-

tive diseases.^[4]Besides, they are both a privileged class of

molecules due to possess a large number of biological

effects, such as anticonvulsant, antibacterial, antiviral,

antidiabetic and cardiovascular beneﬁts among others.^[5–9]

Bhandari et al. evaluated thiazolidine derivatives in

electrocardiographic, antiarrhythmic, vasorelaxant and

1

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Cardiovascular evaluation of Nibethione

Amanda Sanchez-Recillas et al.

antihypertensive activity as well as for in vitro nitric oxide

(NO) releasing ability and found that some of these hetero-

cycles are very potent.^[10]Thereby, the study of 2-thioxo-

1,3-thiazolidin-4-one and 2,4-thiazolidinedione derivatives

occupies a prominent place in the science of chemistry

mainly because of their biological importance. Thus, the

aim of current work was synthesizing Nibethione, a

2-thioxo-1,3-thiazolidin-4-one derivative and pharmaco-

logically characterize its potential as an antihypertensive

agent by ex vivo, in vivo and in silico assays.

light at 254 nm. The uncorrected melting point was deter-

mined by the melting point apparatus EZ-Melt MPA12

(Stanford Research Systems). NMR experiments were per-

formed in a 400 MHz-Varian Inova 400 device (Varian

Inc., Palo Alto, CA, USA). Chemical shifts (d_Hand d_C) and

coupling constants (J) values are given in ppm and Hz,

respectively. TMS (d_H= 0, d_C= 0) in CDCl₃was used as

standard reference for NMR experiments. The following

abbreviations represent signal multiplicities: s (singlet), d

(doublet), t (triplet), q (quartet) and m (multiplet). Mass

spectroscopy was performed in a Jeol JMS-700 MStation

(JEOL, Ltd., Peabody, MA, USA).

Material and Methods

Chemicals and supplies

Synthesis of (Z)-5-(4-nitrobenzylidene)-2-thioxoth-

iazolidin-4-one (Nibethione)

Most chemical reagents (dimethylsulfoxide), raw material

(4-nitrobenzaldehyde, 98%) and pharmacological agents

(2-aminopyridine, carbamylcholine chloride, calcium chlo-

ride, captopril, glibenclamide, nifedipine, norepinephrine

L-bitartrate hydrate, pentobarbital, tetraethylammonium

chloride and potassium chloride) were purchased from a

Sigma-Aldrich’s local provider (St. Louis, MO, USA).

Reagents for Krebs solution were acquired from Fermont

(Monterrey, NL, Mexico).

Nibethione was prepared in a one-step reaction, beginning

from nitrobenzaldehyde (0.5 g, 3.30 mmol), which was

condensed with 2-thioxo-1,3-thiazolidin-4-one (1.5 Equiv),

following Knoevenagel reaction conditions: benzoic acid,

piperide, toluene as solvent under reﬂux^[8](Figure 1).

Pharmacological evaluation

Cell culture reagents and supplies were purchased from

different providers; Dulbecco’s modiﬁed Eagle’s medium

(DMEM) and penicillin-streptomycin solution (1009, Ref.

PSL01) were purchased from Caisson Laboratories (Smith-

ﬁeld, UT, USA); Gibco’s fetal bovine serum (FBS) was pur-

chased from Invitrogen (Waltham, MA, USA; Cat. No.

16000044); sodium bicarbonate was purchased from Sigma-

Aldrich; Cell Titer-Blue cell viability assay was acquired from

Promega (Madison, WI, USA; Cat. No. G8081); Trypsin-

EDTA solution (0.25% Trypsin, 0.53 m^MEDTA; ATCC-

30-2101) was purchased from ATTC (Manassas, VA, USA).

Animals

Adult Wistar rats were obtained from Unidad de Pro-

duccioꢀn, Cuidado y Experimentacioꢀn Animal from Univer-

sidad Juꢀarez Autoꢀnoma de Tabasco (Villahermosa,

Tabasco, Mꢀexico). Spontaneously hypertensive rats (SHR)

and Wistar–Kyoto (WKY) rats were obtained from Insti-

ꢀ

tuto de Fisiologıa Celular at Universidad Nacional

Autoꢀnoma de Mꢀexico (Mexico City, Mexico). All animals

were male with bodyweight ranged 250–300 g.

Animals were housed under standard laboratory condi-

tions (humidity, temperature and light/dark cycle) and

were ad libitum-fed with rodent diet and tap water. All pro-

cedures were conducted following Mexican regulations for

animal experimentation and care from SAGARPA (NOM-

062-ZOO-1999, Mꢀexico).^[11]This protocol was revised and

approved by Internal Committee for the Care and Use of

Laboratory Animals of Universidad Juꢀarez Autoꢀnoma de

Chemistry

Procedures and devices

All chemical reactions were monitored by thin-layer chro-

matography (1.5 9 3.0 cm) and were visualized by UV

O

H

HN

S

N

O

S

HN

S

N

O

S

4-Nitrobenzaldehyde

Nibethione

benzoic acid (cat), piperidine (cat)

toluene, reflux

Figure 1 Scheme of synthesis of (Z)-5-(4-nitrobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (Nibethione).

2

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Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

Tabasco with assigned code CICUAL-001. Also, experimen-

tal procedures met the US Guide for Care and Use of Labo-

ratory Animals (National Academy Sciences, No. 85-23,

revised 1996).^[12]Ex vivo experiments were performed with

six animals per group and euthanized by cervical disloca-

tion technique after deep anaesthesia with pentobarbital

(65 mg/kg, i.p). In vivo experiments were performed with

ﬁve animals per group.

Determination of the vasorelaxant mechanism of

action of Nibethione

To elucidate the vasorelaxant mechanism of action of

Nibethione, the following experiments were performed

after tissue sensitization:

(a)Adrenergic pathway involvement. For establishing an

antagonism of the adrenergic receptor or disruption of the

norepinephrine (NE) pathway, the following procedures

were performed on endothelium-denuded aortic rings. A

cumulative NE-induced contraction CRC (0.1–10 l^M) was

established as a positive control (control-CRC). In another

experiment, aortic rings were pre-incubated with

Nibethione (30, 20, 14 or 9 l^M) for 15 min. Then, the CRC

to NE-induced contraction was performed to compare the

NE-induced contraction in the absence and presence of

Nibethione.

(b)K⁺channel involvement. To identify the role of K⁺chan-

nels in the Nibethione-induced vasorelaxation effect,

endothelium-denuded aortic rings were pre-incubated with

tetraethylammonium chloride (a large-conductance K_Ca

channel blocker (10 lM)), glibenclamide (an ATP-sensitive

potassium channel blocker (10 l^M)) or 2-aminopyridine (a

voltage-gated potassium channel blocker (100 l^M)) during

15 min prior to the contraction with norepinephrine

(1 l^M). The CRC of Nibethione (1.14–376 lM) was

obtained as previously described. The highest vasorelaxant

effect of Nibethione was compared in the absence and pres-

ence of channel blockers.

(c)Ca²⁺channel involvement. For identifying the role of

Ca²⁺channels in the Nibethione-induced vasorelaxant

effect, endothelium-denuded aortic rings were contracted

by depolarization with a high potassium solution (KCl

(80 m^M)), and the CRC of Nibethione (1.14–376 lM) was

built as described previously. In this test, nifedipine (a cal-

cium channel blocker, (0.11 n^M–10 lM)) was used as the

positive control.

(d)Ca²⁺inﬂux involvement. To identify whether the inhibi-

tion of extracellular Ca²⁺inﬂux was responsible for the

Nibethione-induced vasorelaxation. Endothelium-denuded

aortic rings were washed with Ca²⁺-free Krebs–Henseleit

Solution containing high potassium concentration, after

sensitization and were stabilized for 15 min. Then, a CRC

for the CaCl₂-induced contraction (0.113 lM–10 mM) was

built in the absence of Nibethione, and this experiment was

considered as control. After the tissue was washed with

Ca²⁺-free Krebs–Henseleit Solution, 20 min later

Nibethione (EC₅₀= 20 lM) or nifedipine (positive control,

(10 l^M)) was incubated for 15 min, and CRC of CaCl₂was

built again. The maximal contractile effect induced by

CaCl₂was compared in the absence and presence of test

samples.

Ex vivo experiments

General procedure

Following euthanasia, thoracic aorta was dissected and

cleaned of adjacent and connective material, then cut into

rings of 3 mm in length. In addition, the endothelial layer

of some aortic rings was gently removed by manual rubbing

process. Then, aortic rings were ﬁxed to the bottom of the

chambers using stainless-steel hooks at a tension of 3 g.

Tissues were allowed to stabilize for 10 min in incubation

chambers at 37°C containing Krebs–Henseleit Solution

constantly bubbled with an O₂: CO₂(95:5%) mixture. Ten-

sion variations were measured using force transducers

(Astro Med, West Warwick, RI, USA) connected to an MP-

150 analyzer (BIOPAC 4.1 Instruments) as described previ-

ously by Estrada-Soto.^[13]

The vasorelaxant activity of Nibethione on con-

traction induced by norepinephrine

By using a standard protocol, the vasorelaxant activity

was measured.^[14]After a period of stabilization, the aor-

tic rings were submitted to sensitization period, using

norepinephrine (NE; 0.1 l^M); tissue stimulation was car-

ried out during a period of 15 min; then, they were

washed with Krebs–Henseleit Solution, allowing stabiliz-

ing for 15 min; this whole process was carried out in

triplicate. The presence of a functional endothelium was

assessed by testing the ability of acetylcholine/carbachol

to reduce the phenylephrine-induced contraction by a

value ≥50%. After sensitization, tissues were stabilized

for 20 min, then were contracted with norepinephrine

(0.1 l^M) and Nibethione (1.14–376 lM), vehicle (DMSO;

1% ﬁnal concentration) and positive controls (carbachol

for endothelium-intact aortic rings: E+ or nifedipine for

endothelium-denuded aortic rings: Eꢀ, (0.11–10 l^M))

were added in cumulative concentrations (Concentra-

tion–Response Curves (CRC)). The vasorelaxant effect of

test samples was determined by measuring its capacity

to reduce the maximal vascular contraction induced by

norepinephrine and comparing tissue tension before and

after sample addition.

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Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

ꢁ

groups with RMSD of 2.0 A. The reproducibility of inter-

action mode between LCC and Br-dihydropyridine was

used for internal validation of the homology model due to

its conformation, and two binding sites in the molecular

structure of CavAb (PDB-ID: 5KLG) were previously

reported.^[21]The results of molecular docking for Br-di-

hydropyridine in site 1 and site 2 are shown in Figure 2b

and 2c, respectively. MOE 2018.01^[22]and Pymol 1.7.4.

were employed for adequate visualization.

In silico evaluation of L-type Ca²⁺Channel

Molecular modelling of the Human L-type Ca²⁺

Channel Model (LCCM)

The amino acid sequence of human voltage-dependent

L-type Ca²⁺Channel (VLCC) was acquired from National

Center for Biotechnology Information,^[15]with accession

number Q13936 and aligned as described in the Results

and Discussion sections. PSI-BLAST server was used

against the Protein Data Bank (PDB) database for the com-

parative analysis and similar sequence search.

Cytotoxicity activity

The query sequence had a high sequence identity with

two sequences of the structures with the PD-ID: 5GJV

(69%), 3JBR (69%). The crystallographic structure of

ꢁ

5GJV (Resolution of 3.6 A) showed 83% of query cover,

ꢁ

while 3JBR (Resolution of 4.2 A) only 77%. For this rea-

Cell culture

Porcine Aortic Endothelial Cells (PAECs) were obtained

from primary culture. Cells were cultured in T-25 polystyr-

ene ﬂasks with DMEM/High glucose, phenol red-free,

2 m^ML-glutamine supplemented with 10% inactivated

FBS, antibiotics (100 U/ml of penicillin and 100 µg/ml of

streptomycin) and grown at 37 °C in a humidiﬁed atmo-

sphere of 5% CO₂in air. Cultured cells (80–90% conﬂu-

ence monolayer) were trypsinized and seeded in 96-well

plate at a density of 5 9 10³cells/well. PAECs were used at

passages 4–6 for all experiments.

son, the sequence 5GJV was used as a template and it was

aligned with the query sequence, and Figure 2a shows a

sequence alignment between template crystal structure

and query sequence. Then, the heterotetrameric pore

assembly was built in MODELLER v9.18^[16–19]according

to the structure of the mammalian voltage-gated calcium

channel Cav1.1 (PDBID: 5GJV) as a structural template.

One hundred 3D-dimensional models were constructed.

The best model was selected based on DOPE score, and

the geometry was optimized to avoid some steric clashes.

For this purpose, the MMFF94 force ﬁeld was applied.

Although the heterotetrameric pore modelled were com-

pared with the template of the crystallographic structure

(5GJV) were obtained analogous residues into the two

binding pockets. All representations of molecular dock-

ing results were carried out using PyMOL (Molecular

Graphics System, v1.2, Schro€dringer).

Resazurin reduction assay for cytotoxicity

assessment

In vitro cytotoxicity of Nibethione was tested against

PAECs through the measure of cell viability in prolifera-

tion using resazurin reduction assay.^[23]Cells were seeded

and incubated for 24 h at 37°C with 5 % CO₂. Then cells

were treated with increasing concentrations of test sample

(125, 250, 500, 750 and 1000 µ^M). After 24 h of exposi-

tion, 20 µl of CellTiter-Blue reagent (1 : 5 v/v) was added

and absorbance measured at 570 and 600 nm in 96-well

plates. Multifunctional microplate reader Cytation 3

(Biotek Instruments, Inc., Winooski, VT, USA), Gen 5

Microplate Reader and Imager Software (Biotek Instru-

ments, Inc.,Winooski, VT, USA) were used for this step.

The percentage of cells showing cytotoxicity relative to

the control group (Ctrl) was then determined. A higher

percentage of reduction of Resazurin reﬂects a more sig-

niﬁcant cell proliferation. The concentration leading to

50 % inhibition of viability (IC₅₀) was calculated by

regression analysis (per cent survival versus log

concentration). The criteria used to categorize cytotoxic-

ity of Nibethione against PAECs were based on U.S.

National Cancer Institute (NCI) and were as follows: IC₅₀

≤ 20 lg/ml = highly cytotoxic, IC₅₀ranged between 21

and 200 lg/ml = moderately cytotoxic, IC₅₀ranged

between 201 and 500 lg/ml = weakly cytotoxic and

IC₅₀> 501 lg/ml = no cytotoxicity.^[24]

Molecular docking simulations

Docking of Br-dihydropyridine (UK-59811) and

Nibethione was performed with AutoDock 4.2 software

package.^[20]Polar hydrogens were added for LCC model

by using the hydrogen module in AutoDock Tools (ADT).

Later, Gasteiger charges were given for all ligands and

receptors. The semi-rigid molecular docking performed

an automated dock of the ligand with user-speciﬁed dihe-

dral ﬂexibility within a rigid binding site of a protein; it

was carried out using the Lamarckian Genetic algorithm,

applying the standard parameters. The two grids were

centred at the geometric centre of the cavity of the pore

with dimensions of 50 9 50 9 50 points (site 1), while

the second had dimensions of 40 9 40 9 40 points cov-

ering site 2, both binding pockets with points separated

ꢁ

by 0.375 A. The program performed 100 runs. After

molecular docking, all solutions were clustered into

4

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Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

(a)

(b)

(c)

Figure 2 (a) 5GJV sequence alignment between template crystal structure and query sequence (b) proposed binding of the Br-dihydropyridine

into the site 1 and (c) into the site 2 for the LCC receptor.

5

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Cardiovascular evaluation of Nibethione

Amanda Sanchez-Recillas et al.

(30% mol) in the presence of 10 ml of toluene. The reaction

was heated under reﬂux with continuous elimination of

water formed during the reaction using a Dean-Stark appa-

ratus for 3–4 h. The reaction mixture was cooled, and the

yellow solid was ﬁltered off and dried to afford Nibethione

compound. The crude solid was puriﬁed by conventional

procedures.^[28]The chemical structure of Nibethione was

conﬁrmed using spectroscopic and spectrometric data

(NMR and mass spectra): yellow solid, melting point: 265–

266°C, yield: 88 %; ¹H-NMR (400 MHz, DMSO-d6) d: 7.76

(2H, d, H-2, H-6), 7.93 (1H, s, H-7), 8.26 (2H, d, H-3, H-5),

12.08 (1H, s, NH) ppm; ¹³C-NMR (100 MHz, DMSO-d6)

d: 124.2 (C-3, C-5), 127.9 (C-1), 129.0 (C-7), 130.9 (C-2, C-

6), 139.3 (C-5⁰), 147.3 (C-4), 162.3 (C = S), 167.3 (C = O)

ppm; MS/FAB⁺: m/z 267 (M + H)⁺.

The antihypertensive effect of Nibethione

The antihypertensive effect of Nibethione was assessed in

spontaneously hypertensive rats (SHR) and Wistar–Kyoto

(WKY) rats were used as the control group.^[25]Animals were

allotted into three groups (ﬁve animals each): Control rats

(group 1), treated rats (group 2) and positive control (group

3). Group 1 was administrated with a single intragastric dose

of puriﬁed water (5 ml/kg), group 2 was administrated with

Nibethione (187.76 µmol/Kg), and group 3 was adminis-

trated with Captopril (138.06 µmol/Kg). Blood pressure

(systolic and diastolic) and heart rate values were recorded

before (T0) and after the administration test sample at 1, 3, 5

and 7 h (T1–T7) by a tail-cuff method using a LE 5001 auto-

matic blood pressure computer (PanLab, S.L.U; Harvard

Apparatus, Barcelona, Spain). Percentage of decrease in

heart rate (HR), systolic blood pressure (SBP) and diastolic

blood pressure (DBP) were calculated.

Vasorelaxant effect

The vasorelaxant effect of Nibethione (1.14–376 lM) was

evaluated on the NE-induced contraction on aorta rings

isolated from rats. Figure 3 shows the vasorelaxant effect

induced by Nibethione, which was efﬁcient (Maximum

Effect: E_max= 101.54 %) on intact-endothelium aortic

rings (E+) compared with carbachol (E_max= 74.20 %), a

muscarinic agonist (the positive control). As we observed,

Nibethione induced an endothelium-independent vasore-

laxation (Eꢀ) effect that completely abolished contraction

induced by norepinephrine (E_max= 103.46 %). Nifedipine

a calcium channel blocker was used as E-positive control

(E_max= 99.8 %). Table 1 shows the pharmacodynamics

parameters obtained from the concentration–response

Data analysis

The plotted values are mean of the percentage of six experi-

ments for isolated rat aortic ring assay (n = 6), three inde-

pendents experiments for in vitro cytotoxicity assay (n = 3)

considerating the mean of three replicates each experiment,

and ﬁve experiments for in vivo experiments (n = 5) ꢁ

standard deviations. Concentration–response curves were

plotted and ﬁtted whit Origin Lab version 8.0 software

(Microcal). Pharmacodynamics parameters such as maxi-

mum effect (E_max) and half-effective concentration (EC₅₀)

were obtained ﬁtting the Hill equation to dose–response

curves with Origin Lab software.

Half-inhibitory concentration (IC₅₀) for cytotoxicity

assay was calculated using regression analysis (survival % vs

log concentration). Criteria used to categorize cytotoxicity

were based on the U.S. National Cancer Institute criteria as

follow: IC₅₀≤ 20 lg/ml as highly cytotoxic, IC₅₀21–

200 lg/ml as mildly cytotoxic, IC₅₀201–500 lg/ml as

weakly cytotoxic and IC₅₀>501 lg/ml as non-cytotoxic.^[24]

Statistical signiﬁcance was calculated by one-way analysis

of variance (ANOVA) and post hoc Tukey’s tests. All values

with p < 0.05 were considered statistically signiﬁcant

(*).^[26,27]

Results

Chemistry

The synthesis of a (Z)-5-(4-nitrobenzylidene)-2-thioxothia-

zolidin-4-one (Nibethione) was prepared in a one-step reac-

tion. The nitrobenzaldehyde (0.5 g, 3.30 mmol) was

condensed with 2-thioxo-1,3-thiazolidinone (1.5 equiv.),

benzoic acid (0.12 g, 0.99 mmol, 30% mol) and piperidine

Figure 3 Concentration–response curves of vasorelaxant effect of

Nibethione on aortic rings precontracted with norepinephrine (0.1 µM).

All results are expressed as the mean ꢁ SEM of six experiments. No sta-

tistically signiﬁcant difference between Nibethione groups E+ and

Eꢀ (ANOVA: E_max: F = 0.02, P = 0.8790; CE₅₀: F: 0.08, P = 0.7901).

6

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Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

Table 1 Pharmacological parameters of the vasorelaxant effect of Nibethione and positive controls used

Nibethione

Control

Vasorelaxant effect

E_max(%)

EC₅₀(µM)

E_max(%)

EC₅₀(µM)

Aorta rings with endothelium (E+)

101.54 ꢁ 1.78

103.46 ꢁ 2.28

19.07 ꢁ 2.67

20.6 ꢁ 2.23

Carbachol

Nifedipine

74.2 ꢁ 2.75

99.8 ꢁ 4.38

0.47 ꢁ 0.05

0.03 ꢁ 0.02

Aorta rings without endothelium (Eꢀ)

E

_max, maximum effect (Efﬁcacy); EC₅₀, half-effective concentration (Potency), no statistically signiﬁcant difference between Nibethione groups E+

and Eꢀ (E_max; ANOVA: F = 0.02, P = 0.8790; CE₅₀; ANOVA: F: 0.08, P = 0.7901).

curve obtained from Nibethione and positive controls.

CE₅₀values of Nibethione groups E+ and Eꢀ did not show

statistically signiﬁcant difference by one-way ANOVA:

E_max: F = 0.02, P = 0.8790; CE₅₀: F: 0.08, P = 0.7901.

mechanism induced by Nibethione, we investigated the

molecular interaction of this compound on L-type calcium

channel, a large-conductance voltage-operated calcium

channel widely expressed on vascular smooth muscle cells.

Determination of the vasorelaxant

mechanism of action of Nibethione

In silico evaluation of L-type Ca²⁺Channel

(LCC)

Since Nibethione showed an endothelium-independent

vasorelaxant following mechanistic, experiments were focused

on studying the vascular smooth muscle-related molecular

pathways involved, such as the adrenergic pathway, as well as

K+ or Ca²⁺channel involvement.^[29,30]The Figure 4a showed

how Nibethione abolished NE-induced contraction in a con-

centration-dependent fashion (NB at (10 µ^M): E_max= 7.0 ꢁ

0.39 g; (14 µ^M): E_max= 4.0 ꢁ 0.5 g (ANOVA: F = 21.07,

p = 0.0013); (20 µ^M): E_max= 3.5 ꢁ 0.3 g (F = 50.86,

p = 0.0001); and (30 µ^M): E_max= 3.0 ꢁ 0.3 g (F = 51.66,

p = 0.0001)) compared with the control (NE-contraction

curve without Nibethione; E_max= 7.15 ꢁ 0.33 g). On the

other hand, Figure 4b showed that the concentration–

response curve of Nibethione was shifted right in aortic rings

pre-incubated with each K⁺channel blocker proved (gliben-

clamide CE₅₀:110 µM (ANOVA: F = 11.25, P = 0.0153), TEA

CE₅₀: 109 µM (F = 9.99, P = 0.0195) and 2-aminopyridine

CE₅₀: 128 µM (F = 14.60, P = 0.0087)) in relation to the con-

trol (Nibethione in the absence of blockers, CE₅₀: 20.6 µM).

This evidence suggested that the mechanism of action of

Nibethione is through an ion ﬂux modulation or ionic chan-

nel regulation. To conﬁrm either ionic channel or ion ﬂux

involvement, the vasorelaxant effect of test sample was chal-

lenged on aorta rings contracted with depolarizing high-

concentration KCl solution (80 m^M), which produces Ca²⁺

entry through voltage-operated Ca²⁺channels.

Molecular interaction

The building of voltage-operated L-type Ca²⁺channel

model allowed us to hypothesize some overall characteris-

tics for a molecular binding mechanism of dihydropyridine

and Nibethione (used as control). We could identify com-

mon residues involved in binding sites. To analyze protein–

ligand interaction and binding aspects, we performed

molecular docking studies on this template.

Two binging pockets were identiﬁed, and the molecular

docking was performed into these sites, amino acid resi-

dues: (I-S6) S393, V396, L397, N398, V340, L401; (II-S6)

L745, F748; (III-S6) T1048, I1049, I1052, V1053; (IV-S6)

L1378, V1381, M1382, L1384, V1385, L1387 and L1388

comprised a hydrophobic pocket (site 1) (Figure 5a)

whereas a second site was comprised of following residues:

(II-S6) S723, F724, M727, L728, C730, I731, I734; (I-S6)

F349, A350, M353 and L354 where the Nibethione and Br-

dihydropyridine also they bind (Figure 5b).

Cytotoxicity activity

Cell viability of PAECs was not modiﬁed for different con-

centrations of sample test: 125 µ^M(130.30 ꢁ 9.08%),

250 µ^M(131.89 ꢁ 11.89%), 500 µM (105 ꢁ 6.28%),

750 µM (105 ꢁ 7.15%) or 1000 µM (112 ꢁ 9.08%) after

24 h. Calculated IC₅₀was greater than 1000 µM suggesting

no cytotoxicity under the US National Cancer Institute

criteria.^[31]

Figure 4c showed the efﬁcient vasorelaxant effect of

Nibethione (E_max= 87%) on depolarization by KCl solu-

tion (80 mm). Besides Ca²⁺inﬂux involvement was evalu-

ated when a CaCl₂solution was added to the bath in a

Ca²⁺-free incubation medium. Figure 4d shows CaCl₂-in-

duced contraction curve in the presence of Nibethione

(EC₅₀= 20 µM). In this case, the vascular contraction was

completely abolished (ANOVA: F = 124.7, P = 0.0001),

similarly as nifedipine control. Once we identiﬁed an active

involvement of calcium channel on vasorelaxant

The antihypertensive effect of Nibethione

The in vivo acute antihypertensive activity was evaluated in

spontaneously hypertensive rats (SHR) by the non-invasive

tail-cuff method. Figure 6 shows the acute effect of

Nibethione on arterial pressure. Intragastric dosing of

7

ꢀ

Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

(a)

(b)

(c)

(d)

Figure 4 (a) Inhibitory effect of Nibethione on NE-contraction in aortic rings, ANOVA: (10 µM; F = 0.02, P = 0.8890), (14 µM; F = 21.07,

P = 0.0013); (20 µ^M; F = 50.86, P = 0.0001); and (30 µM; F = 51.66, P = 0.0001), (b) effect of potassium channel blockers (Glibenclamide: GB

(ANOVA: F = 11.25, P = 0.0153), tetraethyl ammonium: TEA (F = 9.99, P = 0.0195) and 2-aminopyridine: 2-AP (F = 14.60, P = 0.0087) on

Nibethione-induced vasorelaxant effect in aortic rings precontracted with norepinephrine (0.1 µ^M), (c) vasorelaxant effect of Nibethione on the KCl

(80 m^M)-induced contraction and (d) inhibitory effect of Nibethione (20 µM) on the CaCl₂contraction (ANOVA: F = 124.7, P = 0.0001). All experi-

ments were realized in endothelium-denuded aortic rings, and the results are presented as the mean ꢁ SEM of six experiments; *P < 0.05 repre-

sents a signiﬁcant difference compared with control.

187 µmol/kg led to a moderate decrease in diastolic blood

pressure (DBP) at one hour (20.3%) (ANOVA: F = 16.07,

P = 0.0102) and three hours (14.19%) (F = 7.96,

P = 0.037) postadministration (Figure 6a). The systolic

blood pressure (SBP) and heart rate (HR) were not signiﬁ-

cantly modiﬁed respect to control rats (WKY) (Figure 6b,

F = 1.74, P = 0.2447 and 6(c) F = 0.01, P = 0.9083).

uterus, penis, gut and bladder.^[30]Vascular SMCs play a

pivotal role in the physiology of vessels contraction and

relaxation. Thus, impaired contractibility may cause or

contribute to cardiovascular diseases, such as hypertension,

vascular stroke and heart failure.^[32]Nibethione (Nitroben-

zylydenethiazolidinethione) is a chemical structure that is

present in a wide range of molecules, and it is easy to

obtain, which is being prepared in a one-step reaction using

the Knoevenagel condensation. Indeed, this core (2-thioxo-

1,3-thiazolidin-4-one) is considered as a sulfur analog of

thiazolidinedione, which is present into pioglitazone or

rosiglitazone structures.^[33]Regarding the mentioned

Discussion

Smooth muscle cells (SMCs) comprise functional layers in

many contractile tissues such as blood vessels, airways,

8

ꢀ

Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

(a)

(b)

SITE 2

SITE 1

Figure 5 Bidimensional and Tridimensional image proposed to binding of the Nibethione (a) into the site 1 and (b) the site 2 for the L-type Ca²⁺

channel receptor.

moiety, it has been reported that thiazolidinedione deriva-

tives have emerged as potent antidiabetic agents, which act

in several pharmacological targets for the treatment of

diabetes and could be attributed antihypertensive

properties.^[34,35]

factor (EDHF) and vasorelaxant prostacyclin (PGI₂), which

stimulate the synthesis of cyclic nucleotides.^[38]Vasorelax-

ant effect of Nibethione was independent of endothelium

presence; for this reason, EDRF involvement was promptly

discarded.^[39]

Results exposed in Figure 3 showed that Nibethione pro-

duces a signiﬁcantly vasorelaxant effect in aortic rings.

Based on this, its underlying vasorelaxant mechanism of

action was determined through ex vivo functional experi-

ments. The SMCs account in the wall of blood vessels

(VSMC) represents a central point in the modulation of

vasoconstriction processes and subsequently hypertension

initiation. VSMC contraction begins when agonists as

angiotensin II, norepinephrine or endothelin bind to recep-

tors coupled to a heterotrimeric G_q/11protein (PG_q/11),

which actives the phospholipase C (PLC). This enzyme

hydrolyses the membrane lipids as phosphatidylinositol

4,5-bisphosphate (PIP₂) which catalyses the formation of

two secondary messengers: diacylglycerol (DAG) and inosi-

tol triphosphate (IP₃). The IP₃binds to receptors in the sar-

coplasmic reticulum (RS) resulting in the Ca²⁺release into

the cytosol, whereas DAG activates protein kinase C (PKC),

which phosphorylates speciﬁc target proteins. PKC has

contraction-promoting effects through phosphorylation of

L-type Ca²⁺channels.^[36]This contraction is regulated by

increasing intracellular calcium concentration and myosin

and actin contractile proteins activation.^[30,36,37]VSMC

relaxation occurs either removal of contractile stimulus or

directed action of a substance that stimulates inhibition of

the contractile mechanism. Regardless, the relaxation pro-

cess needs a decreased intracellular Ca²⁺concentration and

increased MLC phosphatase activity.^[30]In addition, vascu-

lar endothelium contributes to relaxation through release

endothelium-derivative relaxant factors (EDRF), such as

nitric oxide (NO), endothelium-derivative hyperpolarizing

Once endothelium involvement was discarded, we decide

to explore a well-known VSM mechanism, such as

a₁-adrenoceptors antagonism, potassium channel opening,

and Ca²⁺channel blockade or Ca²⁺–CaM complex activity

inhibition.^[40–43]Interestingly, Nibethione at 14, 20 and

30 µ^Mcaused a dose-dependent depletion of NE-induced

contraction (Figure 4a). The curve was non-parallel shifted

with suppression of maximum effect in a concentration-

dependent manner. So, this picture is characteristic of

non-competitive antagonism of adrenergic receptor or

disruption of the norepinephrine mechanism.^[36]This

evidence suggested that Nibethione could be involved in a

mechanism as inactivation of contractile intracellular pro-

teins, the blockade of calcium inﬂux, or the opening of

potassium channels.^[30,36,37]It is well known that a variety

of ion channels, pumps, transporters and exchangers are

important in regulating the ionic balance and smooth mus-

cle membrane potential. These proteins regulate vascular

tone, especially modulating the vascular resistance, where

systemic blood pressure is regulated.^[44]Large-conductance

calcium-activated potassium channels (BK_Ca) and ATP-

sensitive potassium channels (K_ATP) signiﬁcantly con-

tribute to the regulation since these channels are associated

with an open–close probability of voltage-gated Ca²⁺chan-

nels.^[45]Conversely, K⁺channels opening results in charge

switching, membrane hyperpolarization, and consequently

vasorelaxation.^[45]

The vasorelaxant effect of Nibethione was signiﬁcantly

modiﬁed in presence of glibenclamide, 2-aminopyridine,

and tetraethylammonium (Figure 4b), suggesting the

9

ꢀ

Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

(a)

(b)

µmol/kg)

(c)

µmol/kg)

Figure 6 Maximal decrease in (a) diastolic blood pressure, (b) systolic blood pressure and (c) heart rate (BPM) elicited by a single intragastric

administration of 187.76 µmol/kg of Nibethione in conscious SHR rats. Results are expressed as the means ꢁ SEM of ﬁve rats per group. *Indi-

cates signiﬁcance respect to WKY (control) with P < 0.05 (ANOVA: F = 16.07, P = 0.0102 (T1), F = 7.96 P = 0.037 (T2)).

participation of K_Ca, K_ATPor Kv channels. K-channels

opening leads to the closure of Ca²⁺channels, and thus

to the decrease of intracellular Ca²⁺and VSMC relax-

ation.^[46]To corroborate this hypothesis, we decided to

explore the vasorelaxant effect related to Ca²⁺channel

blockade.

(Figure 4d). This possibility took relevance when we

observed that Nibethione could bind active sites of dihy-

dropyridine simultaneously. On the other hand, the antihy-

pertensive experiment in SHR only showed an acute

decrease in diastolic blood pressure (DBP) at 1 and 3 h

after administration.

High-concentration KCl solution is a typical strategy for

depolarizing cell membrane by an electrochemical force,

which results in the opening of voltage-dependent L-type

Ca²⁺channels (VLCC).^[46,47]

The DBP is the pressure in the arteries when the heart

rests between beats, it refers to the function of ﬁlling the

heart with blood between muscle contractions, and it

describes that portion of the cardiac cycle opposite to con-

traction. Its elevation represents an increase of systemic

vascular resistance (SVR), which refers to the resistance

that the vascular system offers to blood ﬂow. When blood

vessels constrict (vasoconstriction), this leads to an increase

in SVR, contrary to when blood vessels dilate (vasorelax-

ation), this leads to a decrease in SVR. If Nibethione pro-

duced vasorelaxant effect due to calcium channel blockage

Nibethione showed an efﬁcient vasorelaxation in KCl-

induced contraction similar as nifedipine (a calcium

channel blocker; Figure 4c); this behaviour suggests VLCC-

blockade, thus leading to a decrease of intracellular Ca²⁺

repolarization, and VSMC relaxation.^[47]Additionally, the

CaCl₂-induced contraction curve was entirely inhibited by

Nibethione, supporting a direct involvement of VLCC

10

ꢀ

Amanda Sanchez-Recillas et al.

Cardiovascular evaluation of Nibethione

in VSMC; this activity could decrease peripheral vascular

resistance explaining DBP decrease.^[48]

para el tratamiento experimental de la diabetes tipo 2’

(APN-2001), and CONACyT #253814 (CB-2015, G.N.V).

A. Sꢀanchez-Recillas, J.C. Sꢀanchez-Salgado, S. Hidalgo-Fig-

ueroa, G. Navarrete-Vꢀazquez and R. Ortiz-Andrade are

With the purpose of identifying the potential vascular

toxicity of Nibethione, its cytotoxic activity was measured

in porcine aortic endothelial cells (PAEC). Even though the

effect of Nibethione was endothelium-independent, the

presence of voltage-dependent calcium channels in

endothelial cells has been described.^[49]Results obtained

from cytotoxicity assay in PAEC of Nibethione did not

show changes in cell viability, and its IC₅₀was >1000 µM;

this value suggests the absence of cytotoxicity. Nevertheless,

it is necessary to perform acute and chronic studies of toxi-

city in rodents to discard its possible systemic toxicity.

In conclusion, this investigation suggests that Nibethione

is a potential lead able to induce a vasorelaxant effect with

participation of blockade of voltage-dependent L-type Ca²⁺

channels, and the ﬁndings presented here will have implica-

tions for the research of antihypertensive drugs.

ꢀ

members of ‘Red Mexicana de Investigacioꢀn Preclınica y

Desarrollo Farmacꢀeutico’.

Acknowledgements

The authors thank IPICYT/CIIDZA and CONACyT for

funding this research project (APN-2001). Sꢀanchez-Recillas

ꢀ

A and Ortiz-Andrade R thank the Facultad de Quımica-

UADY for providing facilities for this research. Sꢀanchez-

Recillas A. thanks the CONACyT postdoctoral fellowship

#298553.

Author’s contribution

Amanda Sꢀanchez-Recillas performed major experiments

and analysed data. Gabriel Navarrete-Vꢀazquez carried out

the design, synthesis and chemical characterization of

Nibethione compound. Sergio Hidalgo-Figueroa designed

in silico experiments, analysed, and interpreted data. Mar-

cos Bonilla-Hernꢀandez participated in toxicity assays.

Rolffy Ortiz-Andrade and Maximiliano Ibarra-Barajas

participated in antihypertensive experiment. Vꢀıctor

Yꢀan~ez-Pꢀerez carried out the ex vivo experiments and sta-

tistical analysis. Juan Carlos Sꢀanchez-Salgado contributed

to the grammar review and ﬁnal manuscript preparation.

All authors contributed to drafted and revised the manu-

script.

Declarations

Conﬂict of interest

The Author(s) declare(s) that they have no conﬂicts of

interest to disclose.

Funding

This work was supported by IPICYT/CIIDZA and CONA-

~

CyT under the project: ‘Diseno molecular y sıntesis de

compuestos multifuncionales (PPAR alfa/gamma-GPR40)

ꢀ

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13

Article Doi

Pharmacological characterization of the cardiovascular effect of Nibethione: ex vivo, in vivo and in silico studies

DOI: 10.1111/jphp.13295

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