Synthesis, vasorelaxant activity and antihypertensive effect of benzo[d]imidazole derivatives

Article abstract of DOI:10.1016/j.bmc.2010.04.027

A series of 1H-benzo[d]imidazole analogues of Pimobendan, substituted at position 5 with either -CF₃ or -NO₂, were synthesized using a short synthetic route. All the nitro derivatives were potent, and exhibited a concentration- and partial endothelium-dependent vasorelaxant effects, with EC₅₀s <5 μM. 2-Methoxy-4-[5-nitro-1H-benzo[d]imidazol-2-yl]phenol (compound 13) was the most potent derivative of the series, showing an EC₅₀ value of 1.81 μM and E_max of 91.7% for ex vivo relaxant response in intact aortic rings, resulting in a 2.5-fold higher activity compared to Pimobendan. The closely related 5-CF₃ analogue (compound 8), was 19 times less potent than 13. The antihypertensive activity of compound 13 was evaluated at doses of 25, 50 and 100 mg kg^-1, using spontaneously hypertensive rats (SHR), showing a statistically significant dose-dependent effect.

Full text of DOI:10.1016/j.bmc.2010.04.027

Bioorganic & Medicinal Chemistry 18 (2010) 3985–3991
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, vasorelaxant activity and antihypertensive effect
of benzo[d]imidazole derivatives
a
a
a
Gabriel Navarrete-Vázquez a, , Sergio Hidalgo-Figueroa , Mariana Torres-Piedra , Jorge Vergara-Galicia ,
*
Julio Cesar Rivera-Leyva a,d, Samuel Estrada-Soto a, , Ismael León-Rivera , Berenice Aguilar-Guardarrama ,
b
b
*
Yolanda Rios-Gómez b, Rafael Villalobos-Molina c, Maximiliano Ibarra-Barajas c
a Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Mor. 62209, Mexico
b Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Mor. 62209, Mexico
c Unidad de Biomedicina, FES Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla, Mex. 54090, Mexico
d Departamento de Farmacia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Gto. 36050, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2010
Revised 7 April 2010
Accepted 8 April 2010
Available online 14 April 2010
A series of 1H-benzo[d]imidazole analogues of Pimobendan, substituted at position 5 with either –CF3 or
–NO2, were synthesized using a short synthetic route. All the nitro derivatives were potent, and exhibited
a concentration- and partial endothelium-dependent vasorelaxant effects, with EC50s <5
4-[5-nitro-1H-benzo[d]imidazol-2-yl]phenol (compound 13) was the most potent derivative of the ser-
ies, showing an EC50 value of 1.81 M and Emax of 91.7% for ex vivo relaxant response in intact aortic
lM. 2-Methoxy-
l
rings, resulting in a 2.5-fold higher activity compared to Pimobendan. The closely related 5-CF3 analogue
(compound 8), was 19 times less potent than 13. The antihypertensive activity of compound 13 was eval-
uated at doses of 25, 50 and 100 mg kgꢀ1, using spontaneously hypertensive rats (SHR), showing a statis-
tically significant dose-dependent effect.
Keywords:
Antihypertensive activity
Benzimidazoles
SHR
Ó 2010 Elsevier Ltd. All rights reserved.
Vasorelaxant effect
1. Introduction
In an effort to identify novel compounds with vasodilatory and
antihypertensive activities, our group initially focused on the mod-
Hypertension is the most common cardiovascular disease,
represents the major risk factor for endothelial dysfunction, meta-
bolic syndrome, renal dysfunction, congestive heart failure, coro-
nary artery disease, and stroke.1 Therapeutic strategies to combat
the consequent damage to the vascular endothelium are generally
aimed at modulating the molecular and biochemical mechanisms
underlying this dysfunction.2 One approach to treat the affected
endothelium involves improving endothelium-dependent vasodila-
tation, which is mediated by augmenting the influence of endothe-
lial protective factors (prostacyclin, nitric oxide, cGMP).
Alternatively, treatment of the damaged endothelium can be at-
tempted by inhibiting the synthesis/release of pathogenic factors
such as angiotensin II and endothelin, as well as prothrombotic fac-
tors, among others.3 Great efforts have been made to obtain novel
antihypertensive agents acting on different targets to control blood
pressure although undesirable side-effects are still encountered.
Therefore, new antihypertensive therapy will need to control hyper-
tension more effectively, with fewer side-effects and neutral impact
on known cardiovascular risk factors.4
estly selective structure of Pimobendan (Fig. 1), a dihydropyridaz-
inone-benzo[d]imidazole derivative that acts as calcium
sensitizer, as well as a partial inhibitor of PDE-3. It is effective in
a
H
O
N
N
N
N
O CH3
H
Pimobendan
Bioisosterism
(F3C-, O2N-)
R2-R4
N
N
H
1-15
* Corresponding authors. Tel./fax: +52 777 3297089.
E-mail addresses: gabriel_navarrete@uaem.mx (G. Navarrete-Vázquez),
enoch@uaem.mx (S. Estrada-Soto).
Figure 1. Drug design of compounds 1–15 through non-classical bioisosteric
approach.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.027

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G. Navarrete-Vázquez et al. / Bioorg. Med. Chem. 18 (2010) 3985–3991
both acute and chronic heart failure and it also causes peripheral
vasodilation.5
tended, it was possible to observe a decrease in the yield due to
the formation of several byproducts. Solid compounds were puri-
fied by recrystallization or by column chromatography, and were
all isolated in satisfactory yields. The chemical structures of the
synthesized compounds were confirmed on the basis of their spec-
tral data (NMR and mass spectra) and the purity was ascertained
by microanalysis.
We recently reported the vasorelaxant effect of six 5-substi-
tuted benzo[d]imidazole derivatives.6 The present study extends
the exploration of the vasorelaxant activity of 5-[nitro/(trifluoro-
methyl)]-2-(alkyloxyphenyl)-1H-benzo[d]imidazoles. As part of
our search for basic information about the structural requirements
for vasodilatory and antihypertensive action, we have designed
and synthesized a series of benzo[d]imidazole derivatives reported
in Table 1, based on the structure of Pimobendan, using non-clas-
sical bioisosteric electron-withdrawing substituents (pyridazinone
/nitro/trifuoromethyl groups). The ex vivo vasorelaxant activity of
these compounds in rat aorta rings pre-contracted with noradren-
aline (NA) is also reported. A selected compound was investigated
for its in vivo antihypertensive activity on spontaneously hyper-
tensive rats (SHR) by tail cuff method.
2.2. In silico PASS screening
We obtained predictive values of the biological activities of
compounds (1–15) by comparing their structures with either the
structures or substructures of more than 46,000 well-known bio-
logically active drugs included in the database of the Prediction
of Activity Spectra for Substances (PASS) software.7 This software re-
ports the predicted activity spectrum of a compound both as prob-
able activity (Pa) and probable inactivity (Pi) with the accuracy of
prediction reported to be as high as 85%.8,9 The results presented
in Table 2 describe two biological activities taken from PASS soft-
ware: vasodilatory and phosphodiesterase inhibition effects. Pa
values estimated for both effects ranged from 0.5 to 0.8. These re-
sults indicated that compounds exhibited chemical structures
similar to known vasoactive drugs, and are likely to reveal this
activity experimentally.
2. Results and discussion
2.1. Chemistry
In this study, 15 benzo[d]imidazole derivatives have been
designed, synthesized and tested as vasorelaxant agents in order
to obtain potential antihypertensive compounds (Table 1). The
reaction sequence shown in Scheme 1 was followed. Compounds
1–9 were prepared from 2-nitro-5-(trifluoromethyl) aniline (16),
followed by catalytic nitroreduction to give 17, and cyclo-conden-
sation with the relevant aromatic aldehyde using conventional
heating. Compounds 10–15 were prepared from 4-nitro-1,2-phen-
ylenediamine (18). Reaction of 18 with the appropriately substi-
tuted benzaldehyde, sodium metabisulfite and dimethoxyethane
(DME) was conducted at 70 °C, under microwave irradiation.
The reaction between 1,2-phenylenediamine 18 and the corre-
sponding aldehyde was carried out in 35–90 s under microwave
irradiation and afforded the corresponding products 10–15 in
acceptable yields. When the microwave irradiation time was ex-
2.3. Vasorelaxant activity
In order to study the vasorelaxant activity of compounds 1–15,
rat thoracic aorta rings with and without endothelium (+E and ꢀE,
respectively) pre-contracted with noradrenaline (NA, 0.1 lM) were
employed. The effects of cumulative concentrations of the com-
pounds were determined. Trifluoromethyl derivatives 1–3, and
5–7, did not show a significant vasorelaxant activity (EC50
>200 M). Compounds 8 and 9 showed low vasorelaxant potency
(EC50s <39 M). However, compound 4 (with a nitro group at-
tached at position 2 of the phenyl ring) showed the best activity
s
l
l
Table 1
EC50 and Emax values for the ex vivo vasorelaxant activity induced by derivatives 1–15, in rat aorta rings pre-contracted with noradrenaline
R1
N
N
R4
H
R2
R3
Compound
R1
R2
R3
R4
Mp (°C)
Ex vivo vasorelaxant effect
With endothelium (+E)
Without endothelium (ꢀE)
M) Emax (%)
91.2 1.18
EC50
(
l
EC50
(
l
M)
Emax (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
–CF3
–CF3
–CF3
–CF3
–CF3
–CF3
–CF3
–CF3
–CF3
–NO2
–NO2
–NO2
–NO2
–NO2
–NO2
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–OH
–OPr
–N(Me)2
–OH
56.0–58.0
369.37 10.2
210.33 11.3
548.5 27.8
3.18 0.30
219.20 14.1
524.49 25.4
550.27 30.1
34.84 5.43
38.53 2.35
4.93 0.30
3.71 0.10
4.89 0.29
1.81 0.08
2.5 0.10
467.75 73.6
574.85 30.3
548.51 77.1
15.03 7.59
219.20 71.6
524.49 19.3
550.27 84.5
140.14 63.2
77.42 9.41
35.1 5.21
15.0 1.12
14.12 1.05
19.49 1.79
301.9 10.2
43.65 2.37
N.T.
75.6 6.31
45.7 15.4
19.8 8.13
85.31 2.63
37.04 10.6
19.0 6.01
30.9 7.53
97.67 3.26
99.6 13.5
60.53 5.58
46.35 7.85
31.69 1.32
55.22 8.85
36.33 6.20
58.91 7.81
N.T.
–OMe
–OEt
–NO2
–H
–H
–H
–H
–H
–H
–OEt
–OiPr
–H
–H
–OMe
204.3–205.5
121.5–122.9
155.8–157.1
263.6–265.4
225.3–226.8
222.1–224.4
214.4–216.6
99.5–102.2
147.1–149.0
128.3–131.2
158.9–163.2
303.4–306.3
169.6–174.1
153.2–155.3
75.14 33.5
90.97 2.30
93.16 3.52
51.15 20.6
51.0 7.33
63.2 4.81
99.55 1.23
101.17 5.83
73.82 5.37
84.82 3.73
80.71 9.41
91.74 2.35
75.0 9.35
90.0 4.56
93.22 5.23
106.3 9.71
N.T.
–OMe
–O–CH2–O–
–H
–H
–H
–OMe
–OMe
–OMe
–H
–H
–H
–OH
–OMe
–OMe
3.23 0.20
4.67 0.83
0.51 1.9
Pimobendan
Carbachol
Nitrendipine
N.A.
0.03 0.003
N.A.
98.90 5.0
N.T.
N.T.: Not tested; N.A.: Not active.

G. Navarrete-Vázquez et al. / Bioorg. Med. Chem. 18 (2010) 3985–3991
3987
F3C
F3C
NH2
NO2
F3C
NH2
NH2
N
O
H
ii
i
R4
R4
+
N
H
R2
R3
R2
R3
16
17
1-9
O2N
O2N
NH2
NH2
N
O
H
iii
R4
+
R4
N
H
R2
R3
R2
R3
18
10-15
Scheme 1. Reagents and conditions: (i) H2, Ni-Raney, EtOH; (ii) Na2S2O5, DMF, reflux; (iii) Na2S2O5, DME,
l
w, 70 °C.
of this series, with an EC50 = 3.18
compounds (10–15) showed fully relaxant effects with EC50
<5 M. Compounds 10 and 12 were as potent as Pimobendan
(EC50 = 4.67 0.83 M). However, compounds 13–15 exhibited an
important vasodilatory effect in a concentration- and partially
endothelium-dependent manner. These compounds were more
potent than Pimobendan. The EC50 values for the relaxant response
of the active compounds were different in rings with a functional
endothelium, and in those without endothelium (Table 1). Com-
pound 13 was the most potent in reducing the NA-induced con-
l
M. All the synthesized nitro-
2.4. Antihypertensive assay
s
l
In order to evaluate the oral antihypertensive activity, we se-
lected compound 13, which showed the highest potency, as well
as good Emax in the rat aortic contraction assay with +E
l
(EC50 = 1.81
lM) and ꢀE (EC50 = 19.49
lM). The in vivo antihyper-
tensive activity of compound 13 was evaluated in spontaneously
hypertensive rats (SHR), by the tail cuff method. Oral dosing (25,
50, and 100 mg kgꢀ1) of 13, led to a robust, sustained and dose-
dependent decrease in systolic blood pressure as shown in Figure
3B. Thus, 50 mg kgꢀ1 of compound 13 caused a moderate descent
in systolic blood pressure, while 100 mg kgꢀ1 provoked a marked
and sustained decrease in systolic blood pressure, which lasted
for more than 6 h (Fig. 3). Interestingly, the antihypertensive effect
tractile response, with EC50 of 1.81 lM and efficacy of 91.74%.
This compound was 2.5 times more active than Pimobendan, but
was not as active as carbachol. However, the Emax values of these
compounds were slighter than the positive control. All the com-
pounds were less potent than nitrendipine in aorta rings ꢀE.
The relaxation effect of benzo[d]imidazole derivatives 10–15 in
endothelium-intact aorta pre-contracted by NA was notably stron-
ger than that in endothelium-denuded aorta (Table 1). In endothe-
lium-denuded (ꢀE) rings, the compounds produced a partial
relaxation with a maximal effect of 60.53 5.58% (vs endothe-
lium-intact group with 91.74 2.35%). The partial endothelium-
dependent relaxation showed by these compounds indicates that
their vasorelaxant effect is through endothelium derived factors
such as cyclooxygenase, endothelium-dependent hyperpolariza-
tion factor (EDHF) or nitric oxide synthase pathways, including
PDE-3 inhibition,10 such as Pimobendan. Further experiments are
in progress in order to determine the possible mode of action of
the most active compound. In order to establish a preliminary
structure–activity relationship, we analyzed the contribution of
substituents of the phenyl ring: bulky oxygenated radical at the
C-2 position (compounds 11: R = –OEt and 12: R = –Oi-Pr) were
non-selective and exhibited less potent vasorelaxation. On the
other hand, the presence of two or even three oxygenated radicals
in the rest of compounds increased the relaxant effect. This sug-
gests that the bioactivity of these compounds depends on the pres-
ence of nitro groups and several small oxygenated radicals
attached at different positions of the phenyl ring. Interestingly,
compound 8 substituted at position 5 with a CF3, and with a pat-
tern of substitution 3-MeO, 4-OH in the phenyl ring, showed less
potent relaxant effect than compound 13, which bears a nitro
group in the same position of the benzo[d]imidazole core. This
small change showed a 19-fold improvement in activity. The same
response was observed with compounds 3 and 11 (–CF3 and –NO2
substituted, respectively), where 11 was 148 times more active
than 3 (Fig. 2). From these results, we can observe that nitro group
is essential for the potent vasodilatory action, suggesting a direct
effect in the mechanism of vasorelaxation.
of compound 13 (50 mg kgꢀ1
)
was similar to captopril
(30 mg kgꢀ1), which is widely used in clinical settings for the treat-
ment of hypertension and some types of congestive heart failure.
The results of the antihypertensive activity study indicate that
compound 13 reduces blood pressure at 50 and 100 mg kgꢀ1; how-
ever, there was no significant effect on heart rate (Fig. 3A). The lack
of activity for a low dose of compound 13 (25 mg kgꢀ1) may be due
to low oral bioavailability, which could be the consequence of
either extensive liver metabolism or limited gastrointestinal
absorption. Furthermore, many other reasons could affect the
in vivo activity of this compound.
The benzo[d]imidazoles prepared in this work are fully compat-
ible with Lipinski’s rule of five (Table 2),11 which should allow for
the development of additional vasorelaxant analogues. Their
advantages include: (i) physical properties known to be compatible
with desirable pharmacokinetic (low molecular weight, favorable
Clog P, favorable hydrogen bond donating and accepting capabili-
ties), (ii) potency and efficacy, with EC50 values at the low micro-
molar level, and (iii) simple synthetic access and thus low
production costs.
Interestingly, a good correlation was found between the cal-
culated log P and the topological polar surface area (TPSA) of
the title compounds. Both parameters were compared in order
to analyze the biological results obtained. The relatively higher
hydrophobicity of the compounds (mi log P >4) and lower TPSA
values might explain the low potency of the ꢀCF3 compounds
1ꢀ9 comparing with those shown by nitroderivatives 10ꢀ15
(Table 2). TPSA is a descriptor that shows good correlation with
passive molecular transport through membranes, and so allows
estimation of transport properties of drugs. These results are
consistent with the activities predicted by PASS: the trifluoro-
methyl derivatives showed lower probabilities of biological ef-
fects than the nitro-analogues.

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G. Navarrete-Vázquez et al. / Bioorg. Med. Chem. 18 (2010) 3985–3991
Table 2
Rule of five properties and predictive values of biological activities calculated with PASS for compounds 1–15
Rule
MW
mi log P No. of H bond acceptors No. of H bond donors TPSA (Å2) Volume (Å3) Violations
PASS probability
Phosphodiesterase
Vasodilatory
effect
inhibition
Compd <500 <5
<10
<5
<140
28.68
<2
Pa
Pi
Pa
Pi
1
2
262
292
306
307
278
270
305
308
306
239
283
297
285
299
329
4.41
4.42
4.80
4.32
3.93
5.35
4.51
3.75
4.30
3.48
3.86
4.28
2.81
3.12
3.30
2
3
3
5
3
3
3
4
4
5
6
6
7
7
8
1
1
1
1
2
1
1
2
1
1
1
1
2
1
1
211.57
237.11
253.91
234.90
219.58
270.72
257.47
245.13
235.50
203.60
245.95
264.54
237.17
254.69
280.24
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0.648
0.570
0.793
0.634
0.663
0.758
0.659
0.698
0.651
0.763
0.846
0.781
0.788
0.792
0.837
0.005
0.005
0.003
0.004
0.010
0.004
0.019
0.007
0.005
0.003
0.003
0.004
0.059
0.003
0.003
0.623
0.725
0.793
0.582
0.568
0.758
0.514
0.521
0.536
0.763
0.846
0.759
0.631
0.683
0.742
0.007
37.91
37.91
74.50
48.91
37.91
31.92
58.14
47.15
74.50
83.74
83.74
103.96
92.97
102.20
0.036
0.003
0.009
0.010
0.004
0.015
0.014
0.013
0.004
0.003
0.004
0.006
0.005
0.004
3
4
5
6
7
8
9
10
11
12
13
14
15
Rule of five parameters were calculated on line at: http://www.molinspiration.com/cgi-bin/properties; PASS probability values were calculated on line at: http://
195.178.207.233/PASS/.
values of biological activities were also investigated using the chem-
istry software server PASS (http://195.178.207.233/PASS/).
3. Conclusion
Compounds 1–15 have been characterized as agents with clear
relaxant effects on isolated rat aorta, although further studies are
needed to determine the mechanisms underlying such activity.
The potent vasorelaxant action of nitro-compounds did not persist
on denuded aortic rings.
4.1.1. General method of synthesis of 5-(trifluoromethyl)-1H-
benzo[d]imidazoles 1–9
A
mixture
of
2-nitro-5-(trifluoromethyl)aniline
(5 g,
0.0242 mol), EtOH (100 mL) and 10% Ni-Raney (500 mg) was
hydrogenated at 25 °C until cessation of H2 uptake. The catalyst
was filtered off on a Whatman paper number 2, washed with EtOH,
and the filtrate concentrated to provide a dark purple-colored li-
quid, which was used immediately in a subsequent step without
purification. A mixture of the last reduction product [4-(trifluoro-
methyl)-1,2-phenylenediamine (0.0313 mol)], 1.01 equiv of appro-
priate aldehyde, and 1.01 equiv of sodium metabisulfite in 10 mL
of DMF was heated under reflux for 3–4.5 h. After cooling, water
(20 mL) was added and the mixture was extracted with EtOAc
(3 ꢁ 15 mL). The organic layer was dried over magnesium sulfate
and removed under vacuum. Purification was done by chromatog-
raphy on silica gel eluting with chloroform and/or recrystallization
from adequate solvent.
The potency of these compounds was dramatically improved by
the addition of a bioisosteric nitro group instead of the trifluoro-
methyl cluster in the benzo[d]imidazole core, and several oxygen-
ated substituents at the phenyl ring. In addition, compound 13 was
orally active, showed an important decrease in blood pressure in a
rat model of hypertension at doses of 50 and 100 mg kgꢀ1. In sum-
mary, we have demonstrated that the 5-nitrobenzo[d]imidazole
structure is a bioisostere of Pimobendan, and it can be a useful tem-
plate for the development of vasorelaxant compounds with better
and higher potency. This core could be a promising lead for future
optimizations as an antihypertensive agent. Further studies on the
mode of action and pharmacology of these compounds and SAR
around the core template will be reported in due course.
4. Experimental
4.1. Chemistry
4.1.1.1. 2-Phenyl-5-(trifluoromethyl)-1H-benzo[d] imidazole
(1). Recrystallized from methanol. Yield 0.62 g (67%) of an white
solid. Mp 56.0–58.0 °C. 1H NMR (200 MHz, CDCl3) d6.89–7.54 (m,
5H, H-20, H-30, H-40, H-50, H-60), 7.70 (d, 1H, H-4, J = 2.0 Hz), 7.94
(dd, 1H, H-6, J = 8.4, J = 2.0 Hz), 8.41 (d, 1H, H-7, J = 8.4 Hz), ppm.
13C NMR (50.28 MHz, DMSO-d6): d 113.9 (C-7), 114.1 (q, C-4,
J = 6.8 Hz), 119.7 (q, C-6, J = 6.8 Hz), 123.8 (q, CF3, J = 271.8 Hz),
126.3 (q, C-5, J = 32.2 Hz), 127.7 (C-30, C-50), 130.0 (C-20, C-60),
131.1 (C-40), 132.6 (C-10), 137.8 (C-3a), 143.0 (C-7a), 152.1 (C-2)
ppm; EIMS: m/z 262 (M+, 100), 243 (25); Anal. Calcd for C14H9N2F3:
C, 64.12; H, 3.46; N, 10.68. Found: C, 64.55; H, 3.77; N, 10.01.
Melting points were determined on an EZ-Melt MPA120 auto-
mated melting point apparatus from Stanford Research Systems
and are uncorrected. Reactions were monitored by TLC on 0.2 mm
precoated Silica Gel 60 F254 plates (E. Merck). Catalytic hydrogena-
tion was carried out in a Parr shaker hydrogenation apparatus.1H
NMR spectra were measured with a Varian EM-390 (300 MHz) spec-
trometer. Chemical shifts are given in ppm relative to tetramethyl-
silane (TMS, d = 0) in DMSO-d6; J values are given in Hz. The
following abbreviations are used: s, singlet; d, doublet; q, quartet;
dd, doubletofdoublet;t, triplet;m, multiplet;brs, broadsignal. Elec-
tron impact (EIMS) and FAB+ mass spectra (mNBA matrix), were re-
corded on a JEOL JMS-SX102A spectrum. Starting material was
commercially available (Aldrich). Carbamoylcholine HCl (carbachol)
and noradrenaline HCl (NA) were purchased from Sigma–Aldrich Co.
All other reagents were analytical grade from local sources. Reac-
tions under microwave irradiation were performed in a CEM Discov-
ery Microwave System apparatus (2450 MHz, 300 W). Predictive
4.1.1.2. 2-(2-Methoxyphenyl)-5-(trifluoromethyl) -1H-benzo[d]
imidazole (2). Recrystallized from ethanol. Yield 0.85 g (65.3%) of
brown solid. Mp 204.3–205.5 °C. 1H NMR (300 MHz, CDCl3) d 4.1 (s,
1H, H–CH3), 7.21–7.26 (m, 1H, H-60), 7.73 (dd, 1H, H-30, J = 8.2,
J = 2.2 Hz), 7.54–7.56 (m, 2H, H-40, H-50), 7.97 (d, 1H, H-4, J =
2.2 Hz), 8.30 (d, 1H, H-7, J = 8.2), 8.62 (dd, 1H, H-6, J = 7.7,
J = 1.9 Hz) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 55.6 (CH3O),
113.7 (C-7), 114.2 (q, C-4, J = 6.7 Hz), 116.4 (C-30), 119.5 (q, C-6,
J = 6.8 Hz), 121.5 (C-10), 122.7 (C-50), 123.8 (q, CF3, J = 271.6 Hz),

G. Navarrete-Vázquez et al. / Bioorg. Med. Chem. 18 (2010) 3985–3991
3989
(61.4%) of yellow solid. Mp 121.5–122.9 °C. 1H NMR (200 MHz,
CDCl3): d 1.56 (t, 3H, CH3), 4.41 (q, 2H, CH2), 7.15 (t, 1H, H-50,
J = 8.2, J = 1.1 Hz), 7.26 (d, 1H, H-30, J = 8.2 Hz), 7.51–7.54 (m, 2H,
H-60, H-40), 7.80 (d, 1H, H-7, J = , 8.2 Hz), 7.98 (d, 1H, H-4,
J = 1.1 Hz), 8.53 (dd, 1H, H-6, J = 1.6, J = 7.7 Hz) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 14.7 (CH3), 62.6 (CH2–O), 113.7 (q, C-4,
J = 6.7 Hz),113.9 (C-7), 115.5 (C-30), 119.7 (q, C-6, J = 6.8 Hz),
121.5 (C-10), 122.7 (C-50), 124.3 (q, CF3, J = 271.8 Hz),125.7 (C-60),
125.9 (q, C-5, J = 32.2 Hz), 132.1 (C-40), 139.9 (C-3a), 145.0 (C-7a),
153.9 (C-2), 154.6 (C-20) ppm; EIMS: m/z 306 (M+, 50), 291 (100);
Anal. Calcd for C16H13F3N2O: C, 62.74; H, 4.28; N, 9.15. Found: C,
62.93; H, 4.55; N, 9.48.
O2N
F3C
N
N
N
H
N
H
148 x
O
O
11: EC50= 3.71 μM
Emax= 84.8 %
3: EC50= 548 μM
Emax= 90.97 %
O2N
F3C
N
N
N
OH
OH
O CH3
N
H
19 x
O CH3
H
13: EC50= 1.81 μM
Emax= 91.7 %
8: EC50= 34.84 μM
4.1.1.4. 2-(2-Nitrophenyl)-5-(trifluoromethyl)-1H-benzo[d]imid
azole (4). Recrystallized from acetonitrile. Yield 0.59 g (61.1%) of
yellow solid. Mp 155.8–157.1 °C. 1H NMR (200 MHz, CDCl3): d
7.15 (t, 1H, H-40, J = 8.2, J = 7.7, J = 1.1 Hz), 7.26 (d, 1H, H-7,
J = 8.2 Hz), 7.5 (d, 1H, H-50, J = 8.2, J = 7.7,), 7.67 (d, 2H, H-60,
J = 8.2), 7.78 (d, 1H, H-4, J = 2.7 Hz), 7.79 (d, 2H, H-6, J = 2.7,
J = 8.2 Hz), 8.0 (d, 1H, H-30, J = 7.7 Hz), ppm. 13C NMR (50.28 MHz,
DMSO-d6): d 111.2 (C-7), 111.4 (q, C-4, J = 6.7 Hz), 119.5 (C-6),
119.7 (q, C-6, J = 6.7 Hz), 123.5 (C-10), 123.2 (q, CF3, J = 271.8 Hz),
125.3 (C-60), 125.6 (q, C-5, J = 32.2 Hz), 126.6 (C-30), 131.9 (C-40),
132.4 (C-50), 140.2 (C-3a), 145.3 (C-7a), 146.4 (C-20), 151.2 (C-2)
ppm; EIMS: m/z 307 (M+, 90), 290 (100); Anal. Calcd for
C14H8F3N3O2: C, 54.73; H, 2.62; N, 13.68. Found: C, 55.03; H,
2.65; N, 13.95.
Emax= 99.5 %
Figure 2. Comparison between potencies and chemical structures of selected
analogues. The improvement of activity is represented under the arrow.
40
CONTROL
13 (25 mg/kg)
13 (50 mg/kg)
13 (100 mg/kg)
CAPTOPRIL (30 mg/kg)
20
0
-20
4.1.1.5. 4-[5-(Trifluoromethyl)-1H-benzo[d] imidazol-2-yl]phe-
nol (5). Recrystallized from methanol–water. Yield 0.90 g (71%)
of beige solid. Mp 300.2–303.4 °C. 1H NMR (300 MHz, CDCl3): d
7.66 (d, 1H, H-7, J = 8.2 Hz), 7.84 (dd, 1H, H-6, J = 8.2, J = 1.6 Hz),
7.85 (s, 1H, H-4, J = 1.6 Hz), 8.01 (dd, 2H, H-20, H-60, J = 8.7,
J = 1.6 Hz), 8.31 (dd, 2H, H-30, H-50, J = 8.7, J = 1.6 Hz) ppm. 13C
-40
*
-60
A
-80
0
1
2
3
4
5
6
NMR (50.28 MHz, DMSO-d6):
d 114.1 (C-7), 113.6 (q, C-4,
TIME (HOURS)
J = 6.1 Hz), 117.8 (C-30,C-50), 119.1 (q, C-6, J = 6.2 Hz), 123.5 (q,
CF3, J = 271.8 Hz), 126.3 (q, C-5, J = 32.0 Hz), 126.5 (C-10), 127.9
(C-20, C-60), 136.9 (C-3a), 143.3 (C-7a), 152.2 (C-2), 161.8 (C-40)
ppm; EIMS: m/z 278 (M+, 100), 249 (33); Anal. Calcd for C14H9
F3N2O: C, 60.44; H, 3.26; N, 10.07. Found: C, 60.15; H, 3.57; N,
10.25.
40
20
CONTROL
13 (25 mg/kg)
13 (50 mg/kg)
13 (100 mg/kg)
CAPTOPRIL (30 mg/kg)
0
*
*
*
*
4.1.1.6. 2-(4-Propoxyphenyl)-5-(trifluoromethyl)-1H-benzo[d]
imidazole (6). Recrystallized from ethanol. Yield 0.60 g (37.1%)
of yellow solid. Mp 225.3–226.8 °C. 1H NMR (200 MHz, CDCl3): d
1.04 (t, 3H, C–CH3), 1.82 (sext, 2H, C–CH2), 4.05 (t, 2H, O–CH2),
7.11 (d, 2H, H-30, H-50, J = 8.7, J = 1.6 Hz), 7.48 (dd, 1H, H-6,
J = 8.2, J = 1.6 Hz), 7.73 (s, 1H, H-4), 7.89 (s, 1H, H-7), 8.31 (d, 2H,
H-20, H-60, J = 8.7, J = 1.6 Hz) ppm. 13C NMR (50.28 MHz, DMSO-
d6): d 10.4 (CH3), 22.2 (CH2), 69.1 (CH2–O), 114.1 (C-7), 113.9 (q,
C-4, J = 6.7 Hz), 116.8 (C-30,C-50), 119.5 (q, C-6, J = 6.7 Hz), 123.4
(q, CF3, J = 271.8 Hz), 124.7 (C-20, C-60), 126.3 (q, C-5, J = 32.2 Hz),
126.4 (C-10), 137.8 (C-3a), 143.1 (C-7a), 152.1 (C-20), 162.1 (C-40)
ppm; EIMS: m/z 320 (M+, 45), 278 (100); Anal. Calcd for
C17H15F3N2O: C, 63.75; H, 4.72; N, 8.75. Found: C, 63.93; H, 4.75;
N, 8.46.
-20
-40
-60
-80
-100
*
*
B
*
*
*
0
1
2
3
4
5
6
TIME (HOURS)
Figure 3. Effect of compound 13 on heart rate (A) and systolic arterial pressure (B)
in conscious SHR at 25, 50, and 100 mg kgꢀ1 po (n = 5). Compound was orally
administrated at time zero. Each point represents mean SEM from five experi-
*
ments. p <0.05, as compared with the control.
4.1.1.7. N,N-Dimethyl-4-[5-(trifluoromethyl)-1H-benzo[d]imida
zol-2-yl]aniline (7). Recrystallized from methanol. Yield 0.51 g
(45.3%) of yellow solid. Mp 222.1–224.4 °C. 1H NMR (200 MHz,
126.0 (C-60), 126.3 (q, C-5, J = 32.2 Hz), 132.5 (C-40), 139.8 (C-3a),
145.1 (C-7a), 154.1 (C-20), 157.2 (C-2) ppm; EIMS: m/z 292 (M+,
100), 273 (33); Anal. Calcd for C15H11F3N2O: C, 61.64; H, 3.79; N,
9.59. Found: C, 60.93; H, 3.75; N, 9.45.
CDCl3):
d
3.04 (s, 6H,
N-(CH3)2), 6.86 (d, 2H, H-30, H-50,
J = 8.7 Hz), 7.45 (d, 1H, H-7, J = 8.2 Hz), 7.69 (dd, 1H, H-6, J = 2.2,
J = 8.2 Hz), 7.85 (d, 1H, H-4, J = 2.2), 8.12 (d, 2H, H-20, H-60,
J = 8.7) ppm. 13C NMR (50.28 MHz, DMSO-d6): d 39.9 (N(CH3)2),
113.3 (C-30,C-50), 113.1 (C-7), 114.1 (q, C-4, J = 6.7 Hz), 119.9 (q,
C-6, J = 6.7 Hz), 127.1 (q, C-5, J = 32.2 Hz), 121.5 (q, CF3,
4.1.1.3.
2-(2-Ethoxyphenyl)-5-(trifluoromethyl)-1H-benzo[d]
imidazole (3). Recrystallized from ethanol–water. Yield 0.73 g

3990
G. Navarrete-Vázquez et al. / Bioorg. Med. Chem. 18 (2010) 3985–3991
J = 271.8 Hz), 127.3 (C-20, C-60), 128.6 (C-10), 137.8 (C-3a), 143.1 (C-
7a), 151.5 (C-40), 152.1 (C-2) ppm; EIMS: m/z 307 (M+, 90), 290
(100); Anal. Calcd for C16H14F3N3: C, 62.95; H, 4.62; N, 13.76.
Found: C, 62.93; H, 4.65; N, 13.85.
2H, H-50, J = 8.2, J = 1.6 Hz), 7.7 (d, 1H, H-4, J = 2.1 Hz), 7.94 (dd, 1H,
H-6, J = 10.2, J = 2.4 Hz), 8.4 (d, 1H, H-7, J = 8.4 Hz), 7.5 (t, 1H, H-30,
J = 8.24, J = 6.8, J = 1.6 Hz) ppm; 13C NMR (50 MHz, DMSO-d6): d
14.76 (CH3), 65.6 (CH2), 114.1 (C-4), 115.4 (C-7), 115.5 (C-30), 116.9
(C-10), 119.4 (C-6), 122.7 (C-50), 125.6 (C-60), 132.1 (C-40), 143.7 (C-
7a), 143.8 (C-3a), 144.9 (C-5), 153.9 (C-2), 154.6 (C-20) ppm; MS
(FAB+): m/z 283 (M+H)+; Anal. Calcd for C15H13N3O3: C, 63.60; H,
4.63; N, 14.83. Found: C, 63.81; H, 4.58; N, 14.95.
4.1.1.8. 2-Methoxy-4-[5-(trifluoromethyl)-1H-benzo[d]imida-
zol-2-yl]phenol (8). Recrystallized from ethanol. Yield 1.02 g
(56.1%) of white solid. Mp 214.4–216.6 °C. 1H NMR (200 MHz,
CDCl3): d 3.91 (s, 3H, CH3O), 6.99 (d, 1H, H-7, J = 8.2 Hz), 7.5 (dd,
1H, H-6, J = 8.2, J = 1.6 Hz), 7.73 (dd, 1H, H-60, J = 8.3, J = 2.2 Hz),
7.81 (d, 1H, H-50, J = 2.2 Hz), 7.87 (d, 1H, H-20, J = 2.2 Hz), 7.9 (d,
1H, H-4, J = 1.6 Hz) ppm. 13C NMR (50.28 MHz, DMSO-d6): d
55.67 (CH3O), 110.24 (C-20), 113.9 (C-7), 114.2 (q, C-4, J = 6.7 Hz),
117.3 (C-50), 119.6 (q, C-6, J = 6.7 Hz), 122.3 (q, CF3, J = 271.8 Hz),
124.7 (C-60), 125.1 (C-10), 125.6 (q, C-5, J = 32.2 Hz), 137.8 (C-3a),
142.9 (C-7a), 146.8 (C-40), 150.2 (C-30), 151.6 (C-2) ppm; EIMS:
m/z 308 (M+, 100), 279 (20); Anal. Calcd for C15H11F3N2O2: C,
58.45; H, 3.60; N, 9.09. Found: C, 58.40; H, 3.75; N, 9.28.
4.1.2.3. 2-(2-Isopropoxyphenyl)-5-nitro-1H-benzo[d]imidazole
(12). Recrystallized from methanol. Yield 6.31 g (90%) of white so-
lid. Mp 158.9–163.2 °C. 1H NMR (200 MHz, DMSO-d6): d 1.36 (d,
6H, (CH3)2), 6.76 (dd, 1H, H-30, J = 7.8, J = 1.2 Hz), 7.13–7.22 (m,
2H, H-4, H-5), 7.63 (d, 1H, H-7, J = 7.7 Hz), 7.72 (dd, 1H, H-60,
J = 7.8, J = 1.8 Hz), 8.02 (dd, 1H, H-6, J = 8.6, J = 1.8 Hz), 8.39 (d,
1H, H-4, J = 1.8 Hz), 10.88 (br s, 1H, N–H) ppm; 13C NMR
(50 MHz, DMSO-d6): d 21.8 (CH3)2, 72.1 (CH), 114.1 (C-4), 114.9
(C-30), 115.5 (C-7), 116.7 (C-10), 118.2 (C-6), 122.7 (C-50), 125.4
(C-60), 131.6 (C-40), 143.7 (C-7a), 143.8 (C-3a), 144.9 (C-5), 154.6
(C-20), 156.0 (C-2) ppm; EIMS: m/z (% rel. int.) 297 (M+, 100); Anal.
Calcd for C16H15N3O3: C, 64.64; H, 5.09; N, 14.13. Found: C, 65.10;
H, 5.12; N, 14.38.
4.1.1.9. 2-(Benzo[d][1,3]dioxol-5-yl)-5-(trifluoro methyl)-1H-
benzo[d]imidazole (9). Recrystallized from methanol. Yield
0.47 g (55.8%) of white solid. Mp 99.5–102.2 °C. 1H NMR
(200 MHz, CDCl3): d 6.11 (s, 2H, O–CH2–O), 7.0 (d, 1H, H-7,
J = 8.2 Hz), 7.49 (dd, 1H, H-6, J = 8.2, J = 2.2 Hz), 7.74 (d, 1H, H-20,
J = 1.6 Hz), 7.79 (dd, 1H, H-50, J = 8.8, J = 1.6 Hz), 7.80 (d, 1H, H-60,
J = 8.8 Hz), 7.89 (d, 1H, H-4, J = 2.2 Hz) ppm. 13C NMR
(50.28 MHz, DMSO-d6): d 101.6 (OCH2O), 106.8 (C-20), 107.4 (C-
50), 113.8 (C-7), 113.9 (q, C-4, J = 6.7 Hz), 119.6 (q, C-6, J = 6.7 Hz),
120.2 (C-60), 123.4 (q, CF3, J = 271.8 Hz), 125.2 (C-10), 125.6 (q, C-
5, J = 32.2 Hz), 137.9 (C-3a), 143.8 (C-7a), 148.8 (C-40), 151.3 (C-
30), 151.3 (C-30), 151.7 (C-2) ppm; EIMS: m/z 306 (M+, 100), 287
(10); Anal. Calcd for C15H9F3N2O2: C, 58.83; H, 2.96; N, 9.15. Found:
C, 58.53; H, 3.05; N, 9.45.
4.1.2.4. 2-Methoxy-4-[5-nitro-1H-benzo[d] imidazol-2-yl]phe-
nol (13). Purified by column chromatography on silica gel, eluted
with CH2Cl2/acetone (85:15). Yield 1.5 g (81%) of yellow solid. Mp
303.4–306.3 °C. 1H NMR (200 MHz, CDCl3): d 3.97 (s, 3H, –OCH3),
6.96 (d, 1H, H-50, J = 8.2 Hz), 7.54–7.67 (m, 2H, H-7, H-60), 7.76
(d, 1H, H-20, J = 1.65 Hz), 8.07 (dd, 1H, H-6, J = 8.8, J = 2.2 Hz),
8.41 (s, 1H, H-4), 9.14 (br s, 2H, N–H, O-H) ppm; 13C NMR
(50 MHz, DMSO-d6): d 55.7 (CH3O), 110.2 (C-20), 114.3 (C-4),
114.9 (C-7), 117.3 (C-5), 119.4 (C-6), 120.5 (C-10), 124.7 (C-60),
141.7 (C-7a), 141.8 (C-3a), 144.9 (C-5), 146.8 (C-40), 150.2 (C-30),
151.6 (C-2) ppm; EIMS: m/z (% rel. int.) 285 (M+, 100); Anal. Calcd
for C14H11N3O4: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.07; H,
3.73; N, 14.55.
4.1.2. General method of synthesis of 1H-benzo[d]imidazoles
10–15
A
mixture of 4-nitro-1,2-phenylenediamine (0.0065 mol),
1.01 equiv of appropriate aldehyde, 1.01 equiv of sodium metabisul-
fite, and DME as solvent, was taken in a 25 mL round bottom flask.
The flask was shaken well and heated under microwave irradiation
system (CEM) fitted with reflux condenser for 35–90 s at 70 °C. After
irradiation, the mixture was poured onto cold water. The precipitate
was collected by filtration, washed with water, dried and recrystal-
lized. In cases where compounds did not precipitate, the mixture
was extracted with EtOAc (3 ꢁ 15 mL). The organic layer was dried
over magnesium sulfate and removed under vacuum. Purification
was done by chromatography on silica gel eluting with chloroform
and recrystallization from adequate solvent.
4.1.2.5. 2-(3,4-Dimethoxyphenyl)-5-nitro-1H-benzo[d]imidaz-
ole (14). Purified by column chromatography on silica gel, eluted
with CH2Cl2/acetone (85:15). Yield 1.6 g (85%) of yellow solid. Mp
1
0
169.6–174.1 °C. H NMR (200 MHz, CDCl3): d 3.82 (s, 3H, C4 –
0
OCH3), 3.88 (s, 3H, C3 –OCH3), 6.74 (d, H-7, J = 8.7 Hz), 7.14 (d, 1H,
H-50, J = 8.2 Hz), 7.51 (dd, 1H, H-60, J = 8.2, J = 1.6 Hz), 7.71 (d, 1H,
H-20, J = 1.6 Hz), 7.88 (dd, 1H, H-6, J = 8.7, J = 2.7 Hz), 7.92 (d, 1H,
H-4, J = 2.7 Hz), 8.66 (s, 1H, N–H) ppm; 13C NMR (50 MHz,
DMSO-d6): d 55.7 (CH3O), 56.0 (CH3O) 108.2 (C-20), 111.4 (C-50),
114.7 (C-4), 115.6 (C-7), 119.4 (C-6), 120.7 (C-60), 121.4 (C-10),
141.5 (C-7a), 142.1 (C-3a), 144.9 (C-5), 151.6 (C-2), 152.1 (C-40),
153.6 (C-30) ppm; MS (FAB+): m/z 300 (M+H)+; Anal. Calcd for
C15H13N3O4: C, 60.20; H, 4.38; N, 14.04. Found: C, 59.97; H, 4.43;
N, 14.25.
4.1.2.1. 5-Nitro-2-phenyl-1H-benzo[d]imidazole (10). Recrys-
tallized from ethanol. Yield 0.97 g (63%) of brown solid. Mp 147.1–
1
149.0 °C. H NMR (200 MHz, DMSO-d6): d 7.46–7.57 (m, 5H, H-20,
H-30, H-40, H-50, H-60) 7.67 (d, 1H, J = 8.5 Hz), 8.12 (dd, 1H, H-6,
J = 2.0, J = 8.5 Hz), 8.21 (d, 1H, H-4, J = 2.0 Hz). 13C NMR (50 MHz,
DMSO-d6): d 114.1 (C-4), 115.6 (C-7), 119.4 (C-6), 127.7 (C-30, C-
50), 128.1 (C-10), 130.1 (C-20, C-60), 131.2 (C-40), 141.7 (C-7a), 141.9
(C-3a), 144.8 (C-5), 152.3 (C-2) ppm; EIMS: m/z (% rel. int.) 239
(M+, 100), 223 (2), 193 (30), 166 (20). Anal. Calcd for C13H9N3O2: C,
65.27; H, 3.79; N, 17.56. Found: C, 65.53; H, 3.85; N, 17.46.
4.1.2.6. 5-Nitro-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imid-
azole (15). Recrystallized from methanol. Yield 1.62 g (76%) of
an orange solid. Mp 153.2–155.3 °C. 1H NMR (200 MHz, CDCl3): d
0
0
0
3.73 (s, 3H, C4 –OCH3), 3.86 (s, 6H, C3 –OCH3, C5 –OCH3), 6.72–
6.75 (m, 1H, H-7, J = 8.7 Hz), 7.38 (s, 2H, H-20, H-60), 7.87 (dd, 1H,
H-6, J = 2.7, J = 8.8 Hz), 7.94 (s, 1H, H-4, J = 2.7), 8.68 (s, 1H, N–H)
ppm; 13C NMR (50 MHz, DMSO-d6): d 56.3 (CH3O)2, 60.6 (CH3O)
102.4 (C-20, C-60), 114.2 (C-4), 115.5 (C-7), 119.2 (C-6), 127.4 (C-
10), 141.4 (C-7a), 142.1 (C-3a), 143.0 (C-40), 144. 9 (C-5), 151.6
(C-2), 152.6 (C-30, C-50) ppm; MS (FAB+): m/z 330 (M+H)+; Anal.
Calcd for C16H15N3O5: C, 58.36; H, 4.59; N, 12.76. Found: C,
58.85; H, 4.77; N, 13.01.
4.1.2.2. 2-(2-Ethoxyphenyl)-5-nitro-1H-benzo[d] imidazole
(11). Recrystallized from methanol. Yield 1.17 g (62%) of beige so-
lid. Mp 128.3–131.2 °C. 1H NMR indicates a mixture of tautomers
(200 MHz, DMSO-d6): d 1.39 (t, 3H, CH3), 4.21 (q, 2H, CH2), 6.8 (d,
1H, H-20, J = 6.9 Hz), 7.26 (dd, 1H, H-60, J = 7.6, J = 1.6 Hz), 7.54 (dd,

G. Navarrete-Vázquez et al. / Bioorg. Med. Chem. 18 (2010) 3985–3991
3991
4.2. Pharmacological assays
the interval of 1 h for 6 h. The decrease in blood pressure (BP) was
calculated and is plotted in Figure 3.
4.2.1. Animals and preparation of aortic rings
Thoracic aorta was removed from healthy male Wistar rats
(250–350 g), maintained under standard laboratory conditions
with free access to food and water. Animals were killed by expo-
sure to ether. All animal procedures were conducted in accordance
with our Federal Regulations for Animal Experimentation and Care
(NOM-062-ZOO-1999, Ministry of Agriculture, México), and were
approved by the Institutional Animal Care and Use Committee.
The aorta was cleaned of adhering connective tissue and cut into
4–5 mm length rings. Endothelium was removed in some rings
by gently rubbing intraluminally with a metallic forceps; in these
preparations, absence of the endothelium was confirmed by a less
4.3. Data analysis
All the results are expressed as the mean of five or six experi-
ments SEM. Concentration- responses curves (CRC) were plotted,
and the experimental data from the CRC were adjusted by the non-
linear curve fitting program (ORIGIN 6.0). EC50 values were calcu-
lated separately for each concentration–response curve by probit
analyses. Maximal relaxations are expressed as Emax. Student’s
t-test was performed to ascertain the significance of the antihyper-
tensive exhibited activity.
than 10% relaxation upon carbachol challenge (1 lM). Then, tissue
Acknowledgments
segments were mounted by stainless steel hooks, under an optimal
tension of 3 g, in 10 mL organ baths containing warmed (37 °C) and
oxygenated (O2/CO2, 19:1) Krebs solution (composition, mM: NaCl,
118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0;
EDTA, 0.026 and glucose, 11.1, pH 7.4). Changes in tension were re-
corded by Grass-FT03 force transducers connected to a MP100 ana-
lyzer, as previously described.12,13
G.N.-V. wishes to thank the postdoctoral fellowship given by
DGAPA-UNAM, and Facultad de Farmacia, UAEM. We are grateful
to Hector Manuel Torres-Gómez and Gudelia León-Méndez for
technical assistance. We thank María Medina Pintor and Victoria
Labastida Galván from Centro de Investigaciones Químicas, UAEM,
for the determination of all mass spectra. Supported in part by
PAPCA (FESI-UNAM), PROMEP-SEP, internal funding from Faculty
of Pharmacy, UAEM and CONACyT 47481. We are in debt to Dr.
Jose Luis Medina Franco and Dr. Austin Yongye, from the Torrey
Pines Institute for Molecular Studies for helpful comments.
4.2.2. Experimental protocol
After equilibration, rings were contracted by NA (0.1
washed every 30 min for 2 h. The absence of endothelium was con-
firmed by the lack of a relaxing response to carbachol (1 M). After
lM) and
l
pre-contraction with NA, the test samples (compounds 1–15, vehi-
cle and positive control) were added to the bath in a volume of
References and notes
100
l
L; then cumulative concentration–response curves were ob-
M). In order to avoid fatigue of
tained for each ring (0.31–100
l
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comparing contraction before and after application of the test
materials. NA, carbachol and nitrendipine were dissolved in dis-
tilled water, whereas compounds 1–15 and Pimobendan in DMSO.
This solvent was determined to have no effect on NA-induced con-
tractions at 1% of concentration. Muscular tone was calculated
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4.2.3. Antihypertensive activity of compound 13
In vivo antihypertensive activity study of compound 13 was
performed on spontaneous hypertensive rats (SHR) by tail cuff
method, using LE 5007 automatic blood pressure computer (Leti-
ca). In each group five rats were taken. Test compound 13 at doses
of 25, 50, and 100 mg kgꢀ1 was administered orally as suspension
in 1% of Tween 20. Measurements (blood pressure and heart rate)
were recorded before and after the treatment of test compound at
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