Design, synthesis and ex vivo study of the vasorelaxant activity induced by isosteric derivatives of dihydropyridines (NH-O)

Article abstract of DOI:10.2174/1570180812666151003002501

Background: Hypertension is a complex and multifactorial disorder that is an important cause of morbidity and mortality in all around the world; and the rationalized structural modification of drugs represents an important strategy for to develop new anti

Full text of DOI:10.2174/1570180812666151003002501

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353
Letters in Drug Design & Discovery, 2016, 13, 353-359
ISSN: 1570-1808
eISSN: 1875-628X
Design, Synthesis and ex vivo Study of the Vasorelaxant Activity Induced
by Isosteric Derivatives of Dihydropyridines (NHꢀO)
Impact
Factor:
0.77
Priscila Rendón-Vallejoa, Samuel Estrada-Sotoa,*, Miguel A. Vázquezb,*, Fernando Hernández-
Borjab, Rafael Villalobos-Molinac and Maximiliano Ibarra-Barajasc
BENTHAM
S
CIENCE
aFacultad de Farmacia, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos, 62209, México
bDepartamento de Química, División de Ciencias Naturales y Exactas,
Universidad de Guanajuato, Guanajuato, 36050, México
cUnidad de Biomedicina, Facultad de Estudios Superiores-Iztacala,
Universidad Nacional Autónoma de México, Tlalnepantla, México, 54090,
México
Abstract: Background: Hypertension is a complex and multifactorial disorder that is
an important cause of morbidity and mortality in all around the world; and the rationalized structural modification of
drugs represents an important strategy for to develop new antihypertensive drugs. Objective: To determine the vasorelax-
ant effect of 4H-pyranes and 2-pyridones derivatives designed from nifedipine and milrinone as pharmacophoric scaf-
folds. Methods: The vasorelaxant effect of all designed and synthesized compounds were carried out on the contraction
induced by noradrenaline 0.1 ꢀM in isolated rat aorta rings. Results: Compounds 4a—j, 6a-c and 8a-c showed a concen-
tration–dependent and endothelium-independent relaxation on contraction induced by noradrenaline in isolated rat aorta
rings. Compounds 6a, 8a, 8b, and 8c were the most potent compounds of entire series evaluated; however, were less po-
tent than nifedipine and carbachol used as positive controls. Conclusions: Some compounds were designed and synthe-
sized with significant vasorelaxant effect which can be used for the development of new antihypertensive drugs.
Keywords: 4H-pyrans, 2-pyridones, antihypertensive, drug design, hypertension, vasorelaxant.
1. INTRODUCTION
for about 17 million deaths per year worldwide. Only hyper-
tension provoked at least 45% of deaths from heart disease
and 51% of deaths from stroke. In 2025, it is estimated that
1.56 billion of adults will suffer for this disease [2].
Hypertension is a complex and multifactorial disorder
that is an important cause of morbidity and mortality in the
modern world [1]. The disease is defined as a systolic blood
pressure at or above of 140 mmHg and a diastolic blood
pressure at or above of 90 mmHg. Hypertension is a risk
factor for coronary heart disease and the single most impor-
tant risk factor for stroke [2]. It causes about 50% of ische-
mia and increases the risk of hemorrhagic stroke. Hyperten-
sion can generate atherosclerosis and narrowing of the blood
vessels, damage to the arteries, and can also create weak
places that rupture easily or thin spots that balloon out the
artery wall resulting in an aneurism [2, 3].
NO2
H3COOC
COOCH3
N
H
Fig. (1). Structure of nifedipine.
Around 5-10% of hypertension cases are caused by
pheochromocytoma, renal artery disease or aldosteronism,
types of hypertension which are called secondary, but, for
the remaining 90-95% there is unknown cause of the disease
and is called essential, primary or idiopathic hypertension
[4]. In addition, cardiovascular diseases are responsible
Thus, for the treatment of hypertension there are several
antihypertensive drugs that influence arterial blood pressure
at four effectors sites: the resistance vessels, the capacitance
vessels, the heart, and the kidney [2, 4-6]. Therefore, new
antihypertensive agents with new or known therapeutic tar-
gets are needed to control hypertension more effectively with
less adverse effects [7]. In this context, 1,4-dihydropyridines
(DHPs), a class of calcium antagonists, is widely used in
clinic for the treatment of cardiovascular diseases, such as
hypertension [4]. In this sense, nifedipine (Fig. 1) is the most
DHPs used as a calcium channel blocker in the treatment of
angina pectoris and high blood pressure [8]. Also, nifedipine
has been widely used via oral or sublingual administrations
*Address correspondence to this author at the Facultad de Farmacia,
Universidad Autónoma del Estado de Morelos, P.O. Box: 62209,
Cuernavaca, Morelos, México; Tel/Fax: +52-777-329-7000;
E-mail: enoch@uaem.mx
Departamento de Química, División de Ciencias Naturales y Exactas,
Universidad de Guanajuato, Gto., 36050, México;
E:mail: mvazquez@ugto.mx
1875-628X/16 $58.00+.00
©2016 Bentham Science Publishers

354 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Rendón-Vallejo et al.
O
O
R1
CN
CN
O
NH4OH
R2
R2
CN
R1
R3
O
R3
O
NH2
4a-j
2
1a-e
3a-b
1a, R1= 2-NO2Ph
1b, R1= 3-NO2Ph
1c, R1= 4-NO2Ph
1d, R1= 2,4-NO2Ph
1e, R1= 2,4-ClPh
1f, R1= 2,4-OMePh
3a, R2=OEt, R3=Me
3b, R2-R3=C3H6
Scheme (1). Synthesis of 4H-pyran analogues 4a-j.
O
R
CN
O
CN
NH2
NH4OH
O
NH2NH2
N
CN
R
N
H
O
O
6a-c
1a-c
2
3a
5
1a, R1= 2-NO2Ph
1b, R1= 3-NO2Ph
1c, R1= 4-NO2Ph
Scheme (2). Synthesis of 4H-pyran analogues 6a-c.
O
R
O
R
O
R
H+
[O]
microwave
CN
CN
CN
O
O
O
O
microwave
O
NH2
N
H
O
N
H
1a, R1= 2-NO2Ph
1b, R1= 3-NO2Ph
1c, R1= 4-NO2Ph
8a-c
4a-c
7a-c
Scheme (3). Synthesis of 5-cyano-pyridin-2-ones 8a-c.
MS maXis impact and the samples were analyzed in combi-
nation with methyl stearate as internal standard.
in the management of hypertensive emergencies, severe hy-
pertension associated with chronic renal failure, postopera-
tive hypertension, and pregnancy-induced hypertension [9,
10]. Thus, herein we have synthesized new nifedipine ana-
logues, to establish the pharmacological effect of dihydro-
pyridine isosteric derivatives, which represents an opportu-
nity for the development of new drugs that act on new or
already known therapeutic targets for treating chronic degen-
erative diseases such as hypertension.
Chemical shifts are given in ppm (ꢀ); multiplicities are
indicated by s (singlet), d (doublet), dd (double doublet), t
(triplet), q (quartet), m (multiplet) or bs (broad singlet).
2.2. Chemistry
In Scheme (1), the synthesis of 4H-pyran analogues 4a-j
and 4H-pyranpyrazole analogues is depicted. In Scheme (2),
through a MCR, condensation reactions occur between alde-
hydes, malononitrile and 1,3-dicarbonyl systems to give 6a-
c, by using ammonium hydroxide as the catalyst.
2. MATERIALS AND METHODS
2.1. General Experimental Procedures
2-pyridone analogues 8a-c were synthesized in two steps,
from 4H-pyran opening-closure process in acid medium,
followed by an oxidation reaction, both promoted by micro-
wave radiation as depicted in Scheme (3).
Melting points were determined on an Electrothermal
digital 90100 melting point apparatus and were uncorrected.
The progress of the reaction and the purity of compounds
were monitored by TLC with E. Merck silica gel 60-F254
coated aluminum sheets, in n-hexane/ethyl acetate, and visu-
alized by a 254 nm UV lamp. NMR spectra were recorded,
for solutions in DMSO-d6 with Me4Si as internal standard,
on Varian Mercury (200 MHz), Varian Gemini (300 MHz)
and Bruker UltraShield (500 MHz) instruments. Mass stud-
ies were performed using a spectrometer Bruker ESI-QTOF-
General Procedure for the Synthesis of Compounds 4a-j
A mixture of aldehyde 1 (3.0 mmol), malononitrile 2 (3.0
mmol), ethyl acetoacetate (3a, 3.0 mmol) or 1,3-
cyclohexanedione (3b, 3.0 mmol) and ammonium hydroxide
solution 28% w/w (10 mmol) was placed in a 25 mL round-
bottom flask. The mixture was stirred at room temperature

Design, Synthesis and ex vivo Study of the Vasorelaxant Activity
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5 355
for 30 minutes until the consumption of the substrates. The
reaction was monitored by TLC. The precipitated product
formed was filtered and successively washed with warm
hexane; the residue was purified by recrystallization in etha-
nol to provide pure 4a-j.
133.83, 136.42, 137.14, 138.07, 146.63, 163.47, 163.71,
170.21. HRMS (ESI+ [M+1]+) calculated for [C16H15
Cl2N2O3]+: 353.0460, found 353.0451.
2-amino-4-(2-nitrophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (4f)
Ethyl
6-amino-5-cyano-2-methyl-4-(2-nitrophenyl)-4H-
1
Yield 87% White solid. m.p. 195-196 °C; H NMR (200
pyran-3-carboxylate (4a)
MHz, DMSO-d6) ꢀ 1.90 (m, 2H), 2.18 (m, 2H), 2.58 (t, 2H),
4.93 (s, 1H), 7.18 (s, 2H), 7.40 (dd, J = 7.6, 7.8 Hz, 2H),
7.64 (t, J = 7.8 7.4 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H). ). 13C
NMR (50 MHz, DMSO-d6) ꢀ 19.69, 26.35, 30.09, 35.93,
56.35, 113.26, 119.10, 123.66, 127.81, 130.42, 133.44,
138.98, 148.94, 159.04, 164.71, 196.00. HRMS (ESI+
[M+1]+) calculated for [C16H14N3O4]+: 312.0984, found
312.0978.
Yield 91% Yellow solid. m.p. 182-183 °C; 1H NMR (200
MHz, DMSO-d6) ꢀ 0.91 (t, 3H), 2.33 (s, 3H), 3.90 (q, 2H),
5.01 (s, 1H), 7.09 (s, 2H), 7.43 (m, 2H), 7.70 (t, J = 7.2, 7.4
Hz, 1H), 7.88 (d, J = 7.6 Hz). 13C NMR (50 MHz, DMSO-
d6) ꢀ13.49, 18.33, 32.87, 55.88, 60.31, 106.35, 118.97,
123.76, 128.12, 130.47, 133.79, 139.62, 148.52, 158.31,
158.99, 164.85. HRMS (ESI+, [M+1]+) calculated for
[C16H16N3O5]+ : 330.1090, found 330.1086.
2-amino-4-(3-nitrophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-
Ethyl
6-amino-5-cyano-2-methyl-4-(3-nitrophenyl)-4H-
chromene-3-carbonitrile (4g)
pyran-3-carboxylate (4b)
1
Yield 87% Red solid. m.p. 224-225 °C; H NMR (300
Yield 98% Yellow solid. m.p. 179-181 °C; 1H NMR (200
MHz, DMSO-d6) ꢀ 1.01 (t, 3H), 2.34 (s, 3H), 3.94 (q, 2H),
4.53 (s, 1H), 7.10 (s, 2H), 7.66 (m, 2H), 7.99 (s, 1H), 8.12
(m, 1H). 13C NMR (50 MHz, DMSO-d6) ꢀ 13.67, 18.35,
38.59, 56.37, 60.37, 106.28, 119.41, 121.81, 122.00, 130.20,
134.25, 147.43, 147.82, 157.88, 158.69, 165.13. HRMS (E-
SI+, [M+1]+) calculated for [C16H16N3O5]+: 330.1090, found
330.1083.
MHz, DMSO-d6) ꢀ 1.94 (m, 2H), 2.28 (m, 2H), 2.63 (m,
2H), 4.41 (s, 1H), 7.20 (s, 2H), 7.63 (m, 2H), 7.99 (s, 2H),
8.08 (d, J = 8.1 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) ꢀ
19.74, 26.52, 35.40, 36.24, 57.11, 112.84, 119.44, 121.76,
129.99, 134.22, 147.03, 147.81, 158.65, 165.13, 195.97.
HRMS (ESI+ [M+1]+) calculated for [C16H14N3O4]+:
312.0984, found 312.0980.
2-amino-4-(4-nitrophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (4h)
Ethyl
6-amino-5-cyano-2-methyl-4-(4-nitrophenyl)-4H-
pyran-3-carboxylate (4c)
1
Yield 89% Orange solid. m.p. 178-180°C; 1H NMR (300
MHz, DMSO-d6) ꢀ 1.03 (t, 3H), 2.37 (s, 3H), 3.96 (q, 2H),
4.49 (s, 1H), 7.12 (s, 2H), 7.48 (d, J = 8.7 Hz, 2H), 8.23 (d, J
= 8.7 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) ꢀ 13.63,
18.26, 38.73, 56.22, 60.28, 106.02, 119.22, 123.73, 128.49,
146.38, 152.50, 157.87, 158.55, 165.03. HRMS (ESI+
[M+1]+) calculated for [C16H16N3O5]+ : 330.1090, found
330.1086.
Yield 89% Red solid. m.p. 227-228 °C; H NMR (200
MHz DMSO-d6) ꢀ 1.95 (m, 2H), 2.30 (m, 2H), 2.64 (t, 2H),
4.37 (s, 1H), 7.18 (s, 2H), 7.46 (d, J = 8.8 Hz, 2H), 8.16 (d, J
= 8.8 Hz, 2H). 13C NMR (50 MHz, DMSO-d6) ꢀ 19.73,
26.52, 35.57, 36.21, 56.90, 112.73, 119.36, 123.64, 128.57,
146.24, 152.32, 158.54, 165.13, 195.89. HRMS (ESI+
[M+1]+) calculated for [C16H14N3O4]+: 312.0984, found
312.0983.
2-amino-4-(2,4-dinitrophenyl)-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile (4i)
Ethyl 6-amino-5-cyano-4-(2,4-dinitrophenyl)-2-methyl-4H-
pyran-3-carboxylate (4d)
Yield 81% Brown solid. m.p. 185-187 °C; 1H NMR (200
MHz, DMSO-d6) ꢀ 0.92 (t, 3H), 2.22 (s, 3H), 3.86 (q, 2H),
4.98 (s, 1H,), 7.22 (s, 2H), 7.68 (m, 2H), 8.38 (m, 1H),
8.61(s, 1H). 13C NMR (50 MHz, DMSO-d6) ꢀ 13.52, 18.49,
33.55, 54.34, 60.52, 105.46, 118.55, 119.40, 127.85, 132.31,
146.05, 146.10, 148.14, 159.12, 159.28, 164.56. HRMS (E-
SI+ [M+1]+) calculated for [C16H15N4O7]+: 375.0941, found
375.0935.
Yield 60% Yellow solid. m.p. 205-206 °C; 1H NMR (200
MHz, DMSO-d6) ꢀ 1.95 (m, 2H), 2.20 (m, 2H), 2.61 (t, 2H),
4.99 (s, 1H), 7.38 (s, 2H), 7.74 (d, J = 8.7 Hz, 1H), 8.43 (dd,
J = 8.7, 2.3 Hz, 1H), 8.62 (d, J = 2.3 Hz, 1H). 13C NMR (50
MHz, DMSO-d6) ꢀ 19.65, 26.44, 30.84, 35.81, 55.22,
112.48, 118.82, 119.28, 127.49, 132.42, 145.44, 146.00,
148.75, 159.19, 165.32, 196.13. HRMS (ESI+ [M+1]+) calcu-
lated for [C16H13N4O6]+: 357.0835, found 357.0829.
Ethyl 6-amino-5-cyano-4-(2,4-dichlorophenyl)-2-methyl-
4H-pyran-3-carboxylate (4e)
2-amino-4-(2,3-dimethoxyphenyl)-5-oxo-5,6,7,8-tetrahy-
dro-4H-chromene-3-carbonitrile (4j)
Yield 92% Yellow solid. m.p. 144-145 °C; 1H NMR (500
MHz, DMSO-d6) ꢀ 0.92 (t, J = 7.1 Hz, 3H), 2.27 (s, 3H),
3.86 (dd, J = 7.0, 5.7 Hz, 2H), 6.93 (s, 2H), 4.79 (s, 1H),
7.17 (d, J = 8.4 Hz, 1H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H), 7.48
(d, J = 2.1 Hz, 1H). 13C NMR (125 MHz, DMSO-d6) ꢀ
18.82, 23.41, 40.24, 60.81, 65.42, 110.72, 124.26, 133.21,
Yield 92% Yellow solid. m.p. 216-218 °C; 1H NMR (200
MHz, DMSO-d6) ꢀ 1.95(m, 2H), 2.22 (m, 2H), 2.62 (t, 2H),
3.77 (d, 6H), 4.5 (s, 1H), 6.62 (dd, J = 1.6, 1.8 Hz, 1H), 6.87
(m, 4H). 13C NMR (50 MHz, DMSO-d6) ꢀ 19.89, 26.52,
30.06, 36.40, 57.89, 55.55, 59.99, 111.07, 113.70,

356 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Rendón-Vallejo et al.
119.90,120.43, 123.65, 137.73, 146.26, 152.23, 158.73,
164.64, 195.86. HRMS (ESI+ [M+1]+) calculated for
[C18H19N2O4]+: 327.1345, found 327.1341.
to carry out a recrystallization using a proportion H2O/EtOH
(95/5) to obtain the mixture of the two diastereoisomers.
The obtained solid was collected by vacuum filtration;
the product was allowed to dry and then quantified.
General Procedure for the Synthesis of Compounds 6a-c
A mixture of 1,4,5,6-tetrahydropyridine 7a-c (1.52
mmol), ethanol (3 mL) and DDQ (1.52 mmol) was irradiated
with microwaves until at 100 °C, 39 psi, 10 W for 5 min.
The progress of the reaction was monitored by TLC
(EtOAc/hexane 5:5). The crude product was further purified
by silica gel column chromatography using (hexane/ AcOEt,
1:1) as eluent to afford the pure product 8a-c.
A mixture of aldehyde 1 (3.0 mmol), malononitrile 2 (3.0
mmol), ethyl acetoacetate 3a (3.0 mmol), hydrazine solution
50% 4 (3.0 mmol) and ammonium hydroxide solution 28%
w/w (10% mmol) was placed in a 25 mL round-bottom flask.
The mixture was stirred at room temperature for 30 minutes
until the consumption of the substrates. The reaction was
monitored by TLC. The precipitated product formed was
filtered and successively washed with warm hexane, the
residue was purified by recrystallization in ethanol to pro-
vide pure 6a-c.
Ethyl 5-cyano-2-methyl-4-(2-nitrophenyl)-6-oxo-1,6-dihy-
dropyridine-3-carboxylate (8a)
Yield 91% Brown solid. m.p. 225-226 °C; 1H NMR (500
MHz, DMSO-d6): ꢀ 0.74 (t, J = 7.1 Hz, 3H), 2.56 (s, 3H),
3.82 (q, J = 7.1 Hz, 2H), 7.52 (d, J = 7.6 Hz, 1H), 7.82 (td, J
= 8.0, 0.9 Hz, 1H), 7.93 (t, J = 7.6 Hz, 1H), 8.35 (d, J = 8.3
Hz, 1H), 13.29 (bs,1H). 13C NMR (125 MHz, DMSO-d6): ꢀ
13.13, 19.33, 60.90, 100.78, 109.74, 114.77, 124.71, 129.78,
130.87, 131.80, 134.81, 146.07, 156.18, 158.57, 159.28,
163.62. HRMS (ESI+ [M+1]+) calculated for [C16H14N3O5]+:
328.0933, found 328.0929.
6-amino-3-methyl-4-(2-nitrophenyl)-1,4-dihydropyrano[2,-
3-c]pyrazole-5-carbonitrile (6a)
Yield 83% Yellow solid. m.p. 226-227 °C; 1H NMR (300
MHz, DMSO-d6) ꢀ 1.70 (s, 3H), 5.09 (s, 1H), 7.06 (s, 2H),
7.67 (s, 2H),7.33 (d, J = 7.8 Hz, 1H),7.49 (t, J = 8.4 Hz,
1H),7.67 (t, J = 9.5, 7.2 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H),
12.22(bs,1H). 13C NMR (75 MHz, DMSO-d6) ꢀ 9.49, 31.41,
56.04, 96.39, 120.24, 123.59, 120.32, 131.28, 133.37,
135.72, 137.61, 149.15, 154.97, 161.19. HRMS (ESI+
[M+1]+) calculated for [C14H12N5O3]+: 298.0940, found
298.0938.
Ethyl 5-cyano-2-methyl-4-(3-nitrophenyl)-6-oxo-1,6-dihy-
dropyridine-3-carboxylate (8b)
1
Yield 94% Red solid. m.p. 227-228 °C; H NMR (300
MHz, DMSO-d6) ꢀ 0.74 (t, J = 7.5 Hz, 3H), 2.51 (s, 3H),
3.88 (q, J = 7.2 Hz, 2H), 7.88 (s, 2H), 8.24 (s, 1H), 8.41 (m,
1H), 13.28 (bs,1H). 13C NMR (75 MHz, DMSO-d6) ꢀ 13.12,
18.64, 60.96, 101.39, 111.17, 115.15, 122.10, 124.13,
130.41, 134.07, 137.75, 147.50, 154.79, 157.21, 159.51,
164.29. . HRMS (ESI+ [M+1]+) calculated for [C16H14
N3O5]+: 328.0933, found 328.0929.
6-amino-3-methyl-4-(3-nitrophenyl)-1,4-dihydropyrano[2,-
3-c]pyrazole-5-carbonitrile (6b)
Yield 91% Yellow solid. m.p. 229-230 °C; 1H NMR (200
MHz, DMSO-d6) ꢀ 1.81 (s, 3H), 4.88 (s, 1H), 7.06 (s, 2H),
7.67 (s, 2H), 8.03 (s, 1H), 8.14 (s, 1H), 12.21 (bs, 1H). 13C
NMR (50 MHz, DMSO-d6) ꢀ 9.78, 35.68, 56.18, 96.69,
120.55, 121.88, 122.01, 130.26, 134.41, 135.95, 147.92,
147.92, 154.73, 161.19. HRMS (ESI+ [M+1]+) calculated for
[C14H12N5O3]+: 298.0940, found 298.0983.
Ethyl-5-cyano-2-methyl-4-(4-nitrophenyl)-6-oxo-1,6-dihy-
dropyridine-3-carboxylate (8c)
Yield: 91% Brown solid. m.p. 208-210 °C 1H NMR (200
MHz, DMSO-d6) ꢀ 0.80 (t, 3H, J = 7 Hz), 2.54 (s, 3H), 3.92
(q, 2H, J = 7 Hz), 7.74 (d, 2H, J = 8.4Hz), 8.46 (d, J = 8.4
Hz, 2H), 13.13 (bs, 1H). 13C NMR (50 MHz, DMSO-d6) ꢀ
13.1, 18.7, 61.05, 101.0, 110.9, 115.0, 123.6, 128.9, 142.8,
147.9, 154.9, 157.8, 159.4, 164.2. HRMS (ESI+ [M+1]+)
calculated for [C16H14N3O5]+: 328.0933, found 328.0932.
6-amino-3-methyl-4-(4-nitrophenyl)-1,4-dihydropyrano[2,-
3-c]pyrazole-5-carbonitrile (6c)
Yield 87% Yellow solid. m.p. 230-231°C; 1H NMR (200
MHz, DMSO-d6) ꢀ 2.24 (s, 3H), 5.22 (s, 1H), 7.43 (s, 2H),
7.90 (d, J = 8.2 Hz, 2H), 8.63 (d, J = 8.2 Hz, 2H), 12.59 (bs,
1H). 13C NMR (50 MHz, DMSO-d6) ꢀ 9.72, 35.99, 55.95,
96.47, 120.42, 123.77, 128.76, 135.80, 146.36, 152.01,
154.68, 161.12. HRMS (ESI+ [M+1]+) calculated for
[C14H12N5O3]+: 298.0940, found 298.0938.
2.3. Chemical and Drugs
The synthesis reagents and chemicals were obtained from
Sigma-Aldrich Co. (St. Louis, MO, USA) and used as re-
ceived without any further purification. On the other hand,
pharmacological reagents as carbamylcholine HCl (car-
bachol), noradrenaline HCl (NA) were purchased from
Sigma-Aldrich. All other reagents were analytical grade
from local sources. For ex vivo experiments, the 4H-pyrans
derivatives and 2-pyridones were dissolved in distilled water
and DMSO (1% v/v), while glibenclamide was dissolved in
DMSO and the other reagents were dissolved in distilled
General Procedure for the Synthesis of Compounds 8a-c
In a pressure tube for microwave reactions was placed
4H-pyran 8a-c (1.50 mmol) and 3 mL of EtOH was added.
To the reaction mixture was added concentrated sulfuric acid
(10 mol). The reaction mixture was irradiated with micro-
wave irradiation until a reaction temperature of 100 °C for 5
min, 39 psi, 10 W. The end of the reaction was confirmed by
TLC using a 7:3 (Hex/AcOEt). The reaction was continued

Design, Synthesis and ex vivo Study of the Vasorelaxant Activity
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5 357
water and sonicated just before use. Preliminary experiments
showed that DMSO at 1% (v/v) had no effect on tension
development of isolated aorta.
other nuclei of biological interest; on the basis of this fea-
ture, we took advantage to synthesize 2-pyridones.
Moreover, in previous works were reported the synthesis
of 2-pyridones using microwaves [7] and infrared irradia-
tions (Scheme 3) [12]. By choosing the first energy source,
90 to 92% yields are recorded.
2.4. Experimental Animals
To determine vasorelaxant activity, male Wistar rats
(250ꢀ350 g) were used. Animals were maintained under
standard laboratory conditions with free access to food and
water. All animal procedures were conducted in accordance
with the Mexican Federal Regulations for Animal Experi-
mentation and Care (SAGARPA, NOM-062-ZOO-1999,
México) and approved by the Institutional Animal Care and
Use Committee (Universidad Nacional Autónoma de
México) based on a U.S. National Institutes of Health publi-
cation (No. 85-23, revised 1985).
The first stage reaction is promoted by sulfuric acid (10
mol), yielding a mixture of diastereoisomers, in a ratio
ꢀ80/20 trans/cis. As a mixture, both isomers were oxidized
using DDQ. It was observed that microwave radiation more
efficiently promotes this reaction in comparison with infra-
red radiation, showing shorter times (10 min) and better
yields.
3.2. Vasorelaxant Effect of Compounds
Compounds 4a-j, 6a-c and 8a-c showed a significant
vasorelaxant activity in a concentration-dependent manner
on the contraction induced by noradrenaline (0.1 ꢀM) (Table
1) on aorta rat rings. However, 6a, 8a, 8b and 8c were the
most potent, and revealed an endothelium-independent effect
(Fig. 2). All compounds tested were less potent than positive
controls nifedipine and carbachol.
2.4.1. Vasorelaxant Activity
All animals were sacrificed by cervical dislocation and
the thoracic aorta was removed, cleaned, and cut in about 4-5
mm length rings. In addition, for some aortic rings the endo-
thelium layer was removed by manual procedures. Then,
each piece of tissue was suspended by platinum hooks under
an optimal tension of 3 g in a tissue chamber containing
Krebs solution (composition, mM: NaCl, 118; KCl, 4.7;
CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0;
EDTA, 0.026; glucose, 11.1, pH 7.4) at 37 °C, continuously
gassed with O2/CO2 (95:5). The contractions were recorded
with an isometrical force transducer Grass FT 03 (Astromed,
West Warwick, RI, USA), connected to a MP100 Manager
Biopac System polygraph (Biopac Instruments, Santa Bar-
bara, CA, USA). After the stabilization period, the tissues
were stimulated with noradrenaline (NA, 0.1 ꢀM) during 10
min, and then washed with fresh Krebs solution. This proce-
dure was repeated three times at 30 min intervals before
starting the experiments. The absence or presence of endo-
thelium layer was confirmed by the lack of the relaxant re-
sponse induced by carbachol (1 ꢀM) in the last contraction to
assess viability. Finally, all tissues were contracted with NA
and test samples (pure compounds or positive controls) were
added to the bath in quarter-log cumulative concentrations
(evaluation period). The relaxant effect of the samples was
determined by its ability to induce a maximal vascular con-
traction before and after their addition.
Table 1. Relaxatory effects induced by 4a-j, 6a-c, 8a-c on the
contraction induced by NA (0.1 ꢁM).
With endothelium
(E+)
Without endothelium
(E-)
Contractile agent: NA 0.1 ꢁM
Compound
EC50
(ꢁM)
Efficacy
(%)
EC50
(ꢁM)
Efficacy
(%)
4a
4b
4c
4d
4e
4f
109.5
140
205
ND
115
ND
136
135
ND
ND
94
61 7.92
100 0
ND
140
273
106
ND
185
195
182
133
ND
98
55 0
100 0
100 0
100 0
ND
85 5.17
40 7.57
100 0
76 3.16
51 5.18
70 4.92
70 6.15
38 5.12
40 1.42
90 3.52
50 2.10
44 6.15
100 3.75
82 0
4g
4h
4i
100 0
3. RESULTS AND DISCUSSION
3.1. Synthesis
100 0
100 0
Previously, we reported a procedure to obtain 4H-pyrans
[11], using a multicomponent reaction between aldehydes,
malononitrile and 1,3-dicarbonyl systems. This reaction pro-
ceeds with excellent yields from 80 to 98%.
4j
20 4.59
100 0
6a
6b
6c
8a
8b
8c
ND
ND
30
ND
ND
43
46 4.54
44 7.44
98 4.82
90 7.70
98 1.8
The synthesis of 4H-pyrans derivatives can be performed
using either, direct 1,3-dicarbonyl systems such as ethyl ace-
toacetate and 1,3-cyclohexanodione, or other masked sub-
strates like pyrazolone. It is remarkable that the flexibility of
this procedure let the construction of a wide collection of
4H-pyrans, varying the substitution pattern in the 2 and 3
positions. 4H-pyran core is a scaffold for the formation of
131
28
71
100 0
42

358 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Rendón-Vallejo et al.
a)
0
Vascular tone and blood pressure are determined by the
contractile state of vascular smooth muscle cells within the
blood vessel wall, which is regulated by intracellular calcium
concentration [13]. The contractile activity in smooth muscle
begins by an interaction of calcium-calmodulin complex to
stimulate phosphorylation of myosin light chain, sensitiza-
tion of contractile proteins mediated by calcium is indicated
by RhoA/Rho pathway to inhibit dephosphorylation of the
myosin light chain, and maintain the strength of contraction.
On the other hand, the removal of calcium from the cytosol
and stimulating myosin phosphatase initiate the process of
smooth muscle relaxation. The sarcoplasmic reticulum and
plasma membrane containing Ca2+-Mg2+-ATPase that re-
move calcium from the cytosol, Na+/Ca2+ exchangers are
also found in the plasma membrane promoting a Ca2+ reduc-
tion. During relaxation, receptor- and voltage-operated Ca2+
channels in the plasma membrane are closed resulting in a
reduced Ca2+ entry into the cell [14].
20
40
60
80
Carbachol
Nifedipine
100
6a E+
6a E-
1E-4
1E-3
0.01
0.1
1
10
100
[ ꢀM ]
b)
0
20
In a preliminary SAR analysis, we could observe that
pyridin-2-one scaffold was more active than its isosteric 4H-
pyran analogue. It is important to mention that a hydrogen
bond donor as a part of the heterocyclic ring is necessary in
order to show vasorelaxant effect. Besides, a nitro group
attached at positions ortho and para, is essential for the ac-
tivity (Compounds 8a and 8c). On the other hand, the inclu-
sion of pyrazole fused at 4H-pyran ring enhanced the effect
(Compound 6a). Further experiments are in progress in order
to corroborate this asseveration.
40
60
80
Carbachol
Nifedipine
8a E+
100
8a E-
1E-4
1E-3
0.01
0.1
1
10
100
[ ꢀM ]
c)
In conclusion, all compounds studied showed vasorelax-
ant activity on the contraction induced by NA 0.1 ꢀM in
isolated rat rings, being 6a, 8a, 8b and 8c the most active
compounds obtained and their possible mechanism of action
could be related with calcium channel blockade.
-20
0
20
40
60
80
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
Carbachol
Nifedipine
8b E+
100
8b E-
ACKNOWLEDGEMENTS
1E-4
1E-3
0.01
0.1
1
10
100
This study was financed by a grant from Faculty of
Pharmacy Budgets (FECES 2012, 2013 and 2014), DAIP-
UGto (531/2015) and Fellowship grants (349887 and
482137) for the PhD. studies of P. Rendón-Vallejo and F.
Hernandez-Borja, respectively.
[ꢁM ]
d)
-20
0
We thank the support of National Laboratory of Molecu-
lar Spectroscopy of the Universidad de Guanajuato (CONA-
CyT Grant 260373).
20
40
60
80
100
SUPPLEMENTARY MATERIAL
Carbachol
Nifedipine
8c E+
Available NMR and HRMS spectra of compounds
studied.
8c E-
1E-4
1E-3
0.01
0.1
1
10
100
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Received: July 31, 2015
Revised: September 21, 2015
Accepted: October 02, 2015