Rational design, synthesis and QSAR study of vasorelaxant active 3-pyridinecarbonitriles incorporating 1H-benzimidazol-2-yl function

Article abstract of DOI:10.1016/j.ejmech.2013.01.042

A variety of 2-alkoxy-4-aryl-6-(1H-benzimidazol-2-yl)-3- pyridinecarbonitriles 4a-r were prepared via either regioselective reaction of 3-aryl-1-(1H-benzimidazol-2-yl)-2-propen-1-ones 3 with malononitrile or ylidenemalononitriles 6 with 2-acetyl-1H-benzim

Full text of DOI:10.1016/j.ejmech.2013.01.042

European Journal of Medicinal Chemistry 63 (2013) 14e21
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rational design, synthesis and QSAR study of vasorelaxant active
3-pyridinecarbonitriles incorporating 1H-benzimidazol-2-yl function
,
Zeinab M. Nofal a, Aladdin M. Srour a, Wafaa I. El-Eraky b, Dalia O. Saleh b, Adel S. Girgis c
*
a Therapeutical Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
b Pharmacology Department, National Research Centre, Dokki, Cairo 12622, Egypt
c Pesticide Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of 2-alkoxy-4-aryl-6-(1H-benzimidazol-2-yl)-3-pyridinecarbonitriles 4aer were prepared via
either regioselective reaction of 3-aryl-1-(1H-benzimidazol-2-yl)-2-propen-1-ones 3 with malononitrile
or ylidenemalononitriles 6 with 2-acetyl-1H-benzimidazoles 1 in the presence of sodium alkoxide in
the corresponding alcohol. All the synthesized compounds showed significant vasodilation properties
using isolated thoracic aortic rings of rats pre-contracted with norepinephrine hydrochloride standard
technique. Compounds 4d, 4p, 4l, and 4f exhibited remarkable activity compared with prazosin hy-
drochloride, which was used as a reference standard in the present study. QSAR studies revealed
a good predictive and statistically significant 3 descriptor model (r2 ¼ 0.913, ra2djusted ¼ 0:8808,
rp2rediction ¼ 0:7911).
Received 23 November 2012
Received in revised form
20 January 2013
Accepted 24 January 2013
Available online 12 February 2013
Keywords:
3-Pyridinecarbonitrile
1H-Benzimidazol
2-Propen-1-one
Ylidenemalononitrile
Vasodilator
Ó 2013 Elsevier Masson SAS. All rights reserved.
QSAR
1. Introduction
converting enzyme inhibitors [12,13]; angiotensin II receptor
blockers [13,14] and calcium channel blockers [15]; in addition to
Cardiovascular and cerebrovascular disorders are the main
reason for morbidity and death in recent years. These include
coronary heart diseases, hypertension, and peripheral artery dis-
eases in addition to others [1]. Hypertension is the most common
cardiovascular disease that represents the major risk factor for
endothelial dysfunction, metabolic syndrome, renal dysfunction,
congestive heart failure, coronary artery disease, and stroke [2].
Hypertension affects approximately billions of people all around
the world. Hypertension is defined as repeatedly elevated systolic
and/or diastolic blood pressure above 140/90 mm Hg [3]. Consistent
control of blood pressure is of crucial importance and should be
achieved throughout the 24 h dosing interval where, uncontrolled
hypertension is associated with acute end-organ damage [4] as
congestive heart failure [5] or renal failure as in type-2 diabetes
patients [6]. Drugs currently used for hypertension include di-
uretics [7]; drugs that prevent the action of peripheral sympathetic
drugs directly dilating the blood vessels (arterial dilators) [16].
Relaxation of vascular smooth muscles is considered one of the
main strategies for treatment of hypertension [17]. Several agents
have been developed; however they are all associated with side
effects such as fatigue, mood change, sleep disturbances, dry
mouth, blurry vision, impotence etc. [1]. Therefore, there is a con-
tinuous need to explore, search and develop new vasorelaxant
agents with minimal side effects.
One of the most known vasodilatory active heterocycles
are nicotinate esters. Where many nicotinate analogs are well
known as vasodilating active agents such as, micinicate ‘cis-3-
pyridinecarboxylic acid, 2-oxo-1-phenyl-2-[(3,3,5-trimethylcyclo-
hexyl)oxy]ethyl ester’, hepronicate ‘3-pyridinecarboxylic acid, 2-
hexyl-2-[[(3-pyridinylcarbonyl)oxy]methyl]-1,3-propanediyl ester’
and inositol nicotinate ‘myo-inositol hexa-3-pyridinecarboxylate’
[18]. In continuation of our reports directing toward developing
vasorelaxant active agents [19e23], it is intended in the present
work to describe synthesis as well as vasodilation properties of
novel 3-pyridinecarbonitriles incorporating 1H-benzimidazol-2-yl
function. Interest in developing these agents could be attributed
to the fact that, 3-pyridinecarbonitriles are recognized as bio-
isosteric forms of nicotinate analogs (3-pyridinecarboxylates),
activity as
b-adrenergic [8,9] and a-adrenergic [10] blocking
agents; centrally sympathetic a2-adrenoceptors [11]; angiotensin-
* Corresponding author. Tel.: þ20 2 01220447199; fax: þ20 2 33370931.
E-mail addresses: girgisas10@yahoo.com, girgisas10@hotmail.com (A.S. Girgis).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.042

Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
15
where only the cyano group replaces the acid/ester function. This
may develop enhanced pharmacological active agents with higher
potency and fewer side effects. Recent publications describing
vasorelaxant properties of 4,40-[1,2-ethanediylbis(oxy-2,1-phenyl-
ene)]bis(2-alkoxy-6-aryl-3-pyridinecarbonitriles) also support the
present investigation [24]. Bioactivity and applications as pharma-
ceutical active agents and agricultural materials of pyridinecar-
bonitriles also prompted the present study [25,26]. Additionally,
relationships (QSAR) will be also considered in the present study not
only to explore the controlling factors governing the observed
pharmacological properties of the synthesized analogs, but also to
validate the observed activity.
2. Results and discussion
2.1. Chemistry
4-amino-3-pyridinecarbonitriles were reported as PKC
[27e33]. PKC , a novel isoform, is crucial for the activation and
survival of T cells [34e36]. Proof-of-concept studies with mice
where the PKC gene was deleted or ‘knocked out’ validated that the
q inhibitor
q
Reaction of 3-aryl-1-[(1-methyl-1H-benzimidazol)-2-yl]-2-prop-
en-1-ones 3a,b with malononitrile in the presence of sufficient
amountofsodiumalkoxideinthecorrespondingalcoholaffordedonly
one product which structure was assigned to be either 2-alkoxy-4-
aryl-6-[(1-methyl-1H-benzimidazol)-2-yl]-3-pyridinecarbonitriles 4
or their isomeric form 2-alkoxy-6-aryl-4-[(1-methyl-1H-benzimida-
zol)-2-yl]-3-pyridinecarbonitriles 5, based on the observed spectro-
scopic(IR,1H NMR,13CNMR,MS)andelementalanalysis data(Scheme
1). Formation of4aer via reactionof ylidenemalononitriles6aeh with
2-acetyl-1H-benzimidazoles 1a,b in the presence of sufficient amount
of sodium alkoxide in the corresponding alcohol not only adds good
support for the assumed structures but also confirms that the reaction
of 2-propen-1-ones 3 with malononitrile proceeds in a regioselective
q
inhibition of this kinase could have therapeutic utility in diseases
such as multiple sclerosis [37,38], arthritis [39], asthma [40,41],
inflammatory bowel disease [42], and transplant rejection [43,44].
Pyridinecarbonitriles analogs were also reported as fluorescent
materials useful as security markers for treatment of paper [45e47].
Moreover, benzimidazole containing-compounds were reported
to exhibit promising pharmacological properties such as anti-
hypertensive [48] and vasodilation [49] in addition to antitumor
[50e53], anti-inflammatory [54], antibacterial [55,56], and antiviral
[57e59] activity. Additionally, quantitative structureeactivity
O
N
N
O
N
N
(2)
RCHO
KOH, EtOH
CH3
CH3
R
CH3
1b
3
CH2(CN)2
Na, R''OH
R
N
R
CN
N
N
N
N
N
OR''
OR''
CN
R'
R'
4
5
Na, R''OH
O
N
N
RCH=C(CN)2
+
CH3
6
R'
1
1a; R' = H
1b; R' = Me
4a; R = Ph, R', R'' = Me
4b; R = Ph, R' = Me, R'' = Et
4c; R = 4-ClC6H4, R' = H, R'' = Me
4d; R = 4-ClC6H4, R', R'' = Me
4e; R = 4-ClC6H4, R' = Me, R'' = Et
4f; R = 2,4-Cl2C6H3, R', R'' = Me
2a, 3a; R = 4-ClC6H4
2b, 3b; R = 4-H3COC6H4
6a; R = Ph
4g; R = 2,4-Cl2C6H3, R' = Me, R'' = Et
4h; R = 4-FC6H4, R', R'' = Me
6b; R = 4-ClC6H4
6c; R = 2,4-Cl2C6H3
6d; R = 4-FC6H4
6e; R = 4-H3CC6H4
6f; R = 4-H3COC6H4
6g; R = 2-thienyl
6h; R = 5-methyl-2-furanyl
4i; R = 4-FC6H4, R' = Me, R'' = Et
4j; R = 4-H3CC6H4, R', R'' = Me
4k; R = 4-H3CC6H4, R' = Me, R'' = Et
4l; R = 4-H3COC6H4, R', R'' = Me
4m; R = 4-H3COC6H4, R' = Me, R'' = Et
4n; R = 2-thienyl, R', R'' = Me
4o; R = 2-thienyl, R' = Me, R'' = Et
4p; R = 5-methyl-2-furanyl, R' = H, R'' = Me
4q; R = 5-methyl-2-furanyl, R', R'' = Me
4r; R = 5-methyl-2-furanyl, R' = Me, R'' = Et
Scheme 1. Synthetic routes of compounds 4aer.

16
Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
manner via Michael addition (due to active methylene malononitrile
attack at the -carbon of 3 with subsequent cyclization via nucleo-
concentration necessary for 50% reduction of maximal nor-
epinephrine hydrochloride induced contracture ¼ 0.145, 0.202,
0.210, 0.214 mM, respectively), that seem more potent than pra-
zosin hydrochloride (IC50 ¼ 0.487 mM [60]), the used reference
standard in the present study. In order to understand the observed
pharmacological properties and determining the main controlling
factors governing these activities, QSAR study was initiated.
Meanwhile, through the observed vasodilation properties of the
synthesized compounds few SAR rules could be attained. Insertion
b
philic attack of the alkoxide at one of the nitrile groups followed by
dehydration and dehydrogenation to give eventually 4) rather than
Knoevenagel pathway (condensation of the active methylene malo-
nonitrilewith the ketonicresidue of 3withsubsequentcyclizationdue
to alkoxide attack at one of the nitrile groups followed by dehydro-
genation to give 5) (Scheme 2).
IR spectrum of 4a (as a representative example of the synthe-
sized analogs) reveals the presence of a strong stretching nitrile
of
a methoxy function at 2-position of the synthesized 3-
vibration band at
n
¼ 2220 cmꢀ1 while, no assignable band due to
pyridinecarbonitriles afforded more vasorelaxant active agents
than the case when ethoxy group was adopted, as exhibited in all
the tested analogs, except compound 4o. Additionally, 4-
carbonyl stretching vibration was detected. 1H NMR spectrum of 4a
exhibits the characteristic pyridinyl H-5 at
d
¼ 8.24 in addition to
the methoxide signal at
the pyridinyl C-3 and C-5 at
methoxide and nitrile carbons at
Mass spectrum (EI) of 4a reveals the parent ion peak with a con-
siderable relative intensity value. The spectral data (IR, 1H NMR, 13
d
¼ 4.28. 13C NMR spectrum of 4a reveals
chlorophenyl
substitution
at
the
4-position
of
3-
d
¼ 94.4 and 110.2 in addition to the
pyridinecarbonitriles seems the best choice for optimization of
a vasorelaxant active hit as exhibited in compounds 4a, 4d, 4f, 4h,
4j, 4l, 4n and 4q (IC50 ¼ 0.300, 0.145, 0.214, 0.343, 0.295, 0.210,
0.312 and 0.283, respectively) and compounds 4b, 4e, 4g, 4i, 4k,
4m, 4o and 4r (IC50 ¼ 0.364, 0.293, 0.305, 0.362, 0.400, 0.436, 0.249
and 0.336, respectively).
d
¼ 55.2 and 115.3, respectively.
C
NMR, MS) of all the other synthesized analogs exhibit similar ob-
servations to that of compound 4a confirming their established
structures (c.f. Experimental section).
2.3. QSAR study
2.2. Vasodilation properties
This study was performed using Discovery Studio 2.5 software
(Accelrys Inc., San Diego, CA, USA), which permits search for diverse
structural descriptors (connectivity, topological, etc.) capable of
explaining the controlling factors governing bio-activity observa-
tions. A set of 16 compounds (4a,c,eer) was used as a training set
for a QSAR modeling. The remaining 2 synthesized analogs (4b and
4d “mild and high vasodilation active agents, respectively”) were
adopted as an external test subset for validating the QSAR models.
Many molecular descriptors were calculated for each compound
employing calculate molecular properties module. The calculated
descriptors including various simple and valence connectivity
Vasodilation properties of the synthesized 2-alkoxy-4-aryl-6-
(1H-benzimidazol-2-yl)-3-pyridinecarbonitriles 4aer were inves-
tigated using isolated thoracic aortic rings of rats pre-contracted
with norepinephrine hydrochloride standard reported procedure
[20e23] and compared with prazosin hydrochloride (highly se-
lective a1-adrenoceptor antagonist), which was used as a reference
standard. The observed data (Table 1, Figures
1 and 2 of
Supplementary material) reveal that, all the synthesized com-
pounds show significant vasodilation properties. Meanwhile,
compounds 4d, 4p, 4l, and 4f exhibit remarkable activity (IC50
,
O
N
N
R'
R
3
CH2(CN)2
Na, R''OH
-H2O
R
R
CN
N
N
CN
CN
O
N
CN
N
R'
R'
R''OH
-2H
R''OH
-H2O
-2H
R
R
CN
N
N
N
N
OR''
OR''
N
N
CN
R'
R'
5
4
Scheme 2. Schematic diagram explaining the proposed mechanistic pathways toward the synthesized pyridinecarbonitriles.

Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
17
Table 1
2.4. Validation of QSAR
Concentration of compounds necessary to reduce maximal norepinephrine hydro-
chloride induced contracture by 50% (IC50) in rat thoracic aortic rings.
External validation of the determined QSAR model was per-
formed utilizing two of our synthesized analogs exhibiting mild
(4b) and potent (4d) vasodilation activity. The observed activities
and those provided by QSAR study, was presented in Table 3.
Entry Compound
R
R0
R00
Potency
(IC50), mM
1
4a
Ph
Ph
Me Me 0.300
Me Et 0.364
Me 0.286
Me Me 0.145
Me Et 0.293
Me Me 0.214
Me Et 0.305
Me Me 0.343
Me Et 0.362
Me Me 0.295
Me Et 0.400
Me Me 0.210
Me Et 0.436
Me Me 0.312
Me Et 0.249
Me 0.202
2
4b
3
4c
4-ClC6H4
4-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
2,4-Cl2C6H3
4-FC6H4
H
3. Conclusion
4
4d
5
4e
6
4f
2-Alkoxy-4-aryl-6-(1H-benzimidazol-2-yl)-3-pyridinecarbonit-
riles 4aer were successfully prepared via either regioselective re-
action of 3-aryl-1-(1H-benzimidazol-2-yl)-2-propen-1-ones 3 with
malononitrile or ylidenemalononitriles 6 with 2-acetyl-1H-benz-
imidazoles 1 in the presence of sodium alkoxide in the corre-
sponding alcohol. All the synthesized compounds showed
significant vasodilation properties using isolated thoracic aortic
rings of rats pre-contracted with norepinephrine hydrochloride
standard technique. Compounds 4d, 4p, 4l, and 4f exhibited
remarkable activity compared with prazosin hydrochloride, (highly
selective a1-adrenoceptor antagonist), which was used as a refer-
ence standard in the present study. QSAR studies revealed a good
7
4g
8
4h
9
4i
4-FC6H4
10
11
12
13
14
15
16
17
18
19
4j
4k
4l
4m
4n
4o
4p
4q
4-H3CC6H4
4-H3CC6H4
4-H3COC6H4
4-H3COC6H4
2-Thienyl
2-Thienyl
5-Methyl-2-furanyl
H
5-Methyl-2-furanyl Me Me 0.283
5-Methyl-2-furanyl Me Et
4r
0.336
0.487
Prazosin hydrochloride
e
e
e
predictive and statistically significant
3
descriptor model
(r2 ¼ 0.913, ra2djusted ¼ 0:8808, rp2rediction ¼ 0:7911). External vali-
indices, electro-topological state indices, single point quantum-
mechanical descriptors (via the AM1 model) and other molecular
descriptors were considered. Genetic function approximation (GFA)
was employed to search for the best possible QSAR regression
equation capable of correlating the variations in biological activities
of the training set compounds with variations in the generated
descriptors, i.e. multiple linear regression modeling [61]. The pre-
sent equation shows our best-performing QSAR model (Fig. 1 ex-
hibits the corresponding scatter plots of experimental versus
estimated bioactivity values for the training set compounds).
dation of the determined QSAR model utilizing two of our syn-
thesized analogs exhibiting mild (4b) and potent (4d) vasodilation
activity supported the established model. From all the above it
could be concluded that, designing 3-pyridinecarbonitrile analogs
possessing 1H-benzimidazol-2-yl function, seems highly promising
approach toward developing vasodilation active hits.
4. Experimental
Melting points were recorded on a Stuart SMP3 melting point
apparatus. IR spectra (KBr) were recorded on a JASCO 6100 spec-
trophotometer. NMR spectra were recorded on a JEOL AS 500 (1H:
500, 13C: 125 MHz) spectrometer. Mass spectra were recorded on
a Shimadzu GCMS-QP 1000 EX (EI, 70 eV) spectrometer. Com-
pounds 1a,b [62], 3a,b [63], and 6aeh [64] were prepared ac-
cording to the previously reported procedures (Figures 3e73 of
Supplementary material exhibit the spectral features of the syn-
thesized compounds).
IC50 ¼ ꢀ0:203 þ 0:798 Shadow YZfrac þ 0:491 Jurs FPSA 2
ꢀ 0:025 Jurs PPSA 3
Where; Shadow YZfrac, is the area of the molecular shadow in the
YZ plane, Jurs FPSA 2, is the Fractional charged partial surface areas
“set of six descriptors obtained by dividing descriptors 1e6 by the
total molecular solvent-accessible surface area”, and Jurs PPSA 3, is
the atomic charge weighted positive surface area “sum of the
product of solvent-accessible surface area ꢁ partial charge for all
4.1. Synthesis of 2-alkoxy-4-aryl-6-(1H-benzimidazol-2-yl)-3-
pyridinecarbonitriles 4aer
positively charged atoms” (Table
2 exhibits the calculated
descriptor values and the estimated bio-activity value due to the
mentioned model).
The goodness of the model was validated by squared correlation
coefficient (r2) ¼ 0.913, ra2djusted ¼ 0:8808, rp2rediction ¼ 0:7911,
RMS residual error (root mean square) ¼ 0.02253, for N “number of
molecules in the training set” ¼ 16.
4.1.1. Method A
A mixture of equimolar amounts of 3a,b (10 mmol) and malo-
nonitrile in the appropriate alcohol (25 ml) containing sodium
(0.46 g; 20 mmol) was stirred at room temperature (25e30 ꢂC) for
the proper time (TLC control). The separated solid was collected,
washed with water and crystallized from n-butanol affording the
corresponding 4d,e,l,m.
4.1.2. Method B
A mixture of equimolar amounts of 1a,b (10 mmol) and the
corresponding ylidenemalononitrile 6aeh in the appropriate
alcohol (25 ml) containing sodium (0.46 g; 20 mmol) was stirred at
room temperature (25e30 ꢂC) for the proper time (TLC control).
The separated solid was collected, washed with water and crys-
tallized from n-butanol affording the corresponding 4aer.
4.1.2.1. 2-Methoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-phenyl-3-
pyridinecarbonitrile (4a). Reaction time 24 h, colorless crystals, mp
219e221 ꢂC, yield (2.10 g) 62%. IR: nmax/cmꢀ1 2220 (C^N), 1586,
Fig. 1. Predicted versus experimental IC50 values of the training set compounds.

18
Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
Table 2
Estimated activity values of the training set and calculated descriptors governing activity according to the QSAR model.
Entry
Compd.
R
R0
R00
Observed activity (IC50), mM
Estimated activity
Descriptors
Shadow YZfrac
Jurs FPSA 2
Jurs PPSA 3
1
2
3
4
5
6
7
8
4a
4c
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
Ph
4-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
2,4-Cl2C6H3
4-FC6H4
Me
H
Me
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Me
Et
0.300
0.286
0.293
0.214
0.305
0.343
0.362
0.295
0.400
0.210
0.436
0.312
0.249
0.202
0.283
0.336
0.282
0.261
0.335
0.203
0.302
0.353
0.345
0.303
0.408
0.245
0.423
0.326
0.261
0.198
0.261
0.336
0.6879
0.6810
0.7080
0.5902
0.6254
0.6984
0.6365
0.6691
0.7462
0.6051
0.7695
0.6849
0.6780
0.5769
0.6287
0.6765
1.1328
0.9906
1.0187
0.9157
0.9380
1.0920
1.1406
1.1859
1.2119
1.3475
1.3329
1.0194
1.0197
1.2233
1.2296
1.2763
24.7913
24.6475
21.6573
21.8512
20.4677
24.0665
22.9951
24.3749
23.1048
27.6895
25.3009
22.8361
21.3766
25.7432
23.8522
23.2495
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
4-FC6H4
4-H3CC6H4
4-H3CC6H4
4-H3COC6H4
4-H3COC6H4
2-Thienyl
9
10
11
12
13
14
15
16
2-Thienyl
5-Methyl-2-furanyl
5-Methyl-2-furanyl
5-Methyl-2-furanyl
Me
Me
1547 (C]N, C]C). 1H NMR (CDCl3):
OCH3), 7.32e7.82 (m, 9H, arom. H), 8.24 (s, 1H, pyridinyl H-5). 13
NMR (CDCl3): 33.2 (NCH3), 55.2 (OCH3), 94.4 (pyridinyl C-3), 110.2
(pyridinyl C-5), 115.3 (C^N), 117.7, 120.4, 123.3, 124.4, 128.6, 129.0,
130.4, 135.5, 137.4, 142.6, 148.2, 150.5, 156.7, 164.7 (arom. C). MS: m/
z (%) 340 (M, 87), 339 (100). Anal. Calcd. For C21H16N4O (340.39): C,
74.10; H, 4.74; N, 16.46. Found: C, 74.32; H, 4.88; N, 16.52.
d
4.16 (s, 3H, NCH3), 4.28 (s, 3H,
NCH3), 4.35 (s, 3H, OCH3), 7.33e7.84 (m, 8H, arom. H), 8.23 (s, 1H,
pyridinyl H-5). 13C NMR (CDCl3):
33.3 (NCH3), 55.3 (OCH3), 94.4
C
d
d
(pyridinyl C-3), 110.2 (pyridinyl C-5), 115.1 (C^N), 117.5, 120.6,
123.4, 124.5, 129.4, 130.0, 134.0, 136.8, 137.5, 142.7, 148.1, 150.9,
155.5, 164.9 (arom. C). MS: m/z (%) 374 (M, 100), 376 [(M þ 2), 49].
Anal. Calcd. For C21H15ClN4O (374.83): C, 67.29; H, 4.03; N, 14.95.
Found: C, 67.15; H, 3.93; N, 15.12.
4.1.2.2. 2-Ethoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-phenyl-3-
pyridinecarbonitrile (4b). Reaction time 24 h, pale yellow crystals,
mp 228e230 ꢂC, yield (1.95 g) 55%. IR: nmax/cmꢀ1 2220 (C^N),
4.1.2.5. 4-(4-Chlorophenyl)-2-ethoxy-6-(1-methyl-1H-benzimidazol-
2-yl)-3-pyridinecarbonitrile (4e). Reaction time 24 h (Methods A &
B), colorless crystals, mp 243e245 ꢂC, yield (1.94, 2.10 g) 50, 54%
(Methods A & B, respectively). IR: nmax/cmꢀ1 2223 (C^N), 1580,
1584, 1549 (C]N, C]C). 1H NMR (CDCl3):
d 1.55 (t, 3H, OCH2CH3,
J ¼ 6.5 Hz), 4.35 (s, 3H, NCH3), 4.61 (q, 2H, OCH2, J ¼ 6.5 Hz), 7.38e
1546 (C]N, C]C). 1H NMR (CDCl3):
d 1.54 (t, 3H, OCH2CH3,
7.88 (m, 9H, arom. H), 8.32 (s, 1H, pyridinyl H-5). 13C NMR (CDCl3):
J ¼ 6.88 Hz), 4.30 (s, 3H, NCH3), 4.60 (q, 2H, OCH2, J ¼ 6.9 Hz), 7.32e
d
14.6 (OCH2CH3), 33.1 (NCH3), 64.0 (OCH2), 94.7 (pyridinyl C-3),
7.83 (m, 8H, arom. H), 8.20 (s, 1H, pyridinyl H-5). 13C NMR (CDCl3):
110.2 (pyridinyl C-5), 115.3 (C^N), 117.7, 120.5, 123.4, 124.4, 128.6,
129.0, 130.3, 135.7, 137.4, 142.5, 148.4, 150.5, 156.8, 164.5 (arom. C).
MS: m/z (%) 354 (M, 35). Anal. Calcd. For C22H18N4O (354.41): C,
74.56; H, 5.12; N, 15.81. Found: C, 74.75; H, 5.23; N, 15.97.
d 14.6 (OCH2CH3), 33.2 (NCH3), 64.2 (OCH2), 94.7 (pyridinyl C-3),
110.2 (pyridinyl C-5), 115.1 (C^N), 117.4, 120.5, 123.4, 124.5, 129.4,
130.0,134.1, 136.7, 137.4, 142.6,150.8,155.5, 164.5 (arom. C). MS: m/z
(%) 388 (M, 38), 390 [(M þ 2), 13]. Anal. Calcd. For C22H17ClN4O
(388.86): C, 67.95; H, 4.41; N, 14.41. Found: C, 67.86; H, 4.28; N,
14.50.
4.1.2.3. 6-(1H-Benzimidazol-2-yl)-4-(4-chlorophenyl)-2-methoxy-3-
pyridinecarbonitrile (4c). Reaction time 48 h, pale yellow crystals,
mp 273e275 ꢂC, yield (2.10 g) 58%. IR: nmax/cmꢀ1 3343 (NH), 2221
4.1.2.6. 4-(2,4-Dichlorophenyl)-2-methoxy-6-(1-methyl-1H-benzi-
midazol-2-yl)-3-pyridinecarbonitrile (4f). Reaction time 24 h, col-
orless crystals, mp 240e242 ꢂC, yield (2.30 g) 56%. IR: nmax/cmꢀ1
(C^N), 1593, 1545 (C]N, C]C). 1H NMR (DMSO-d6):
d
4.24 (s, 3H,
OCH3), 7.23e7.78 (m, 8H, arom. H), 8.00 (s, 1H, pyridinyl H-5), 13.12
(s, 1H, NH). 13C NMR (DMSO-d6):
55.9 (OCH3), 94.6 (pyridinyl C-3),
d
2226 (C^N), 1582, 1532 (C]N, C]C). 1H NMR (CDCl3):
d
4.19 (s, 3H,
NCH3), 4.34 (s, 3H, OCH3), 7.26e7.82 (m, 7H, arom. H), 8.17 (s, 1H,
pyridinyl H-5). 13C NMR (CDCl3):
33.2 (NCH3), 55.3 (OCH3), 96.6
112.9 (pyridinyl C-5), 115.5 (C^N), 114.8, 120.3, 123.1, 124.8, 129.6,
130.9, 134.7, 135.5, 135.9, 144.5, 149.3, 149.5, 155.5, 165.2 (arom. C).
MS: m/z (%) 360 (M, 18), 362 [(M þ 2), 11]. Anal. Calcd. For
d
(pyridinyl C-3), 110.2 (pyridinyl C-5), 114.2 (C^N), 118.6, 120.6,
123.5, 124.6, 127.7, 130.2, 131.3, 133.3, 136.6, 137.5, 142.7, 148.0,
150.9, 153.8, 164.2 (arom. C). MS: m/z (%) 408 (M, 100), 410 [(M þ 2),
57], 412 [(M þ 4), 13]. Anal. Calcd. For C21H14Cl2N4O (409.28): C,
61.63; H, 3.45; N, 13.69. Found: C, 61.43; H, 3.35; N, 13.87.
C
20H13ClN4O (360.81): C, 66.58; H, 3.63; N, 15.53. Found: C, 66.64;
H, 3.72; N, 15.73.
4.1.2.4. 4-(4-Chlorophenyl)-2-methoxy-6-(1-methyl-1H-benzimida-
zol-2-yl)-3-pyridinecarbonitrile (4d). Reaction time 24 h (Methods
A & B), almost colorless crystals, mp 259e261 ꢂC, yield (2.16,
2.30 g) 58, 61% (Methods A & B, respectively). IR: nmax/cmꢀ1 2220
4.1.2.7. 4-(2,4-Dichlorophenyl)-2-ethoxy-6-(1-methyl-1H-benzimi-
dazol-2-yl)-3-pyridinecarbonitrile (4g). Reaction time 24 h, color-
less crystals, mp 250e251 ꢂC, yield (2.25 g) 53%. IR: nmax/cmꢀ1 2225
(C^N), 1586, 1545 (C]N, C]C). 1H NMR (CDCl3):
d 4.20 (s, 3H,
Table 3
External validation for the established QSAR model utilizing mild (4b) and potent (4d) vasodilatory active agents.
Entry
Compd.
R
R0
R00
Observed activity (IC50), mM
Estimated activity
Descriptors
Shadow YZfrac
Jurs FPSA 2
Jurs PPSA 3
1
2
4b
4d
Ph
4-ClC6H4
Me
Me
Et
Me
0.364
0.145
0.440
0.118
0.79613
0.650737
1.24693
0.87069
24.0706
24.9979

Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
19
(C^N), 1589, 1562 (C]N, C]C). 1H NMR (CDCl3):
d
1.56 (t, 3H,
NCH3), 4.17 (s, 3H, OCH3), 4.32 (s, 3H, OCH3), 7.01e7.84 (m, 8H,
arom. H), 8.23 (s,1H, pyridinyl H-5). 13C NMR (CDCl3):
33.2 (NCH3),
OCH2CH3, J ¼ 6.88 Hz), 4.35 (s, 3H, NCH3), 4.63 (q, 2H, OCH2,
d
J ¼ 6.88 Hz), 7.34e7.84 (m, 7H, arom. H), 8.17 (s, 1H, pyridinyl H-5).
55.2 (OCH3), 55.5 (OCH3), 94.1 (pyridinyl C-3), 110.2 (pyridinyl C-5),
115.6 (C^N), 114.5, 117.6, 120.4, 123.4, 124.4, 127.8, 130.2, 137.4,
142.4, 148.4, 150.2, 156.4, 161.4, 164.9 (arom. C). MS: m/z (%) 370 (M,
100). Anal. Calcd. For C22H18N4O2 (370.41): C, 71.34; H, 4.90; N,
15.13. Found: C, 71.45; H, 5.03; N, 14.97.
13C NMR (CDCl3):
d 14.5 (OCH2CH3), 33.2 (NCH3), 64.2 (OCH2), 96.7
(pyridinyl C-3), 110.2 (pyridinyl C-5), 114.2 (C^N), 118.4, 120.6,
123.4,124.6,127.7,130.2,131.3, 133.3, 133.4,136.6,137.5, 142.7,148.1,
150.9, 153.8, 163.9 (arom. C). MS: m/z (%) 422 (M, 34), 424 [(M þ 2),
22], 426 [(M þ 4), 5]. Anal. Calcd. For C22H16Cl2N4O (423.31): C,
62.42; H, 3.81; N, 13.24. Found: C, 62.53; H, 3.98; N, 13.32.
4.1.2.13. 2-Ethoxy-4-(4-methoxyphenyl)-6-(1-methyl-1H-benzimi-
dazol-2-yl)-3-pyridinecarbonitrile (4m). Reaction time 24
h
4.1.2.8. 4-(4-Fluorophenyl)-2-methoxy-6-(1-methyl-1H-benzimida-
zol-2-yl)-3-pyridinecarbonitrile (4h). Reaction time 24 h, colorless
crystals, mp 254e256 ꢂC, yield (2.25 g) 63%. IR: nmax/cmꢀ1 2222
(Methods A & B), colorless crystals, mp 224e226 ꢂC, yield (1.96,
2.10 g) 51, 55% (Methods A & B, respectively). IR: nmax/cmꢀ1 2220
(C^N), 1585, 1548 (C]N, C]C). 1H NMR (CDCl3):
d 1.54 (t, 3H,
(C^N), 1586, 1549 (C]N, C]C). 1H NMR (CDCl3):
d
4.21 (s, 3H,
NCH3), 4.43 (s, 3H, OCH3), 7.25e8.03 (m, 9H, 8 arom. H þ pyridinyl
H-5). 13C NMR (CDCl3):
33.2 (NCH3), 55.3 (OCH3), 94.5 (pyridinyl
OCH2CH3, J ¼ 6.88 Hz), 3.86 (s, 3H, NCH3), 4.30 (s, 3H, OCH3), 4.59
(q, 2H, OCH2, J ¼ 6.88 Hz), 7.01e7.85 (m, 8H, arom. H), 8.22 (s, 1H,
d
pyridinyl H-5). 13C NMR (CDCl3):
d 14.6 (OCH2CH3), 33.2 (NCH3),
C-3), 110.2 (pyridinyl C-5), 115.1 (C^N), 116.2, 116.4, 117.8, 120.5,
123.5, 124.6, 130.7, 130.8, 131.6, 137.4, 142.4, 148.1, 150.6, 155.7, 164.9
(arom. C). MS: m/z (%) 358 (M, 93), 357 (100). Anal. Calcd. For
55.5 (OCH3), 64.0 (OCH2), 94.3 (pyridinyl C-3), 110.2 (pyridinyl C-5),
115.6 (C^N), 114.5, 117.5, 120.3, 123.4, 124.4, 127.9, 130.2, 137.3,
148.5, 150.1, 156.4, 161.4, 164.6 (arom. C). MS: m/z (%) 384 (M, 44).
Anal. Calcd. For C23H20N4O2 (384.44): C, 71.86; H, 5.24; N, 14.57.
Found: C, 71.92; H, 5.35; N, 14.40.
C
21H15FN4O (358.38): C, 70.38; H, 4.22; N, 15.63. Found: C, 70.47; H,
4.33; N, 15.79.
4.1.2.9. 2-Ethoxy-4-(4-fluorophenyl)-6-(1-methyl-1H-benzimidazol-
2-yl)-3-pyridinecarbonitrile (4i). Reaction time 24 h, almost color-
less crystals, mp 243e245 ꢂC, yield (2.10 g) 56%. IR: nmax/cmꢀ1 2220
4.1.2.14. 2-Methoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-(2-
thienyl)-3-pyridinecarbonitrile (4n). Reaction time 24 h, colorless
crystals, mp 215e217 ꢂC, yield (2.00 g) 58%. IR: nmax/cmꢀ1 2223
(C^N), 1585, 1549 (C]N, C]C). 1H NMR (CDCl3):
d
1.54 (t, 3H,
(C^N), 1582, 1550 (C]N, C]C). 1H NMR (DMSO-d6):
d 4.09 (s, 3H,
OCH2CH3, J ¼ 6.9 Hz), 4.30 (s, 3H, NCH3), 4.61 (q, 2H, OCH2,
NCH3), 4.33 (s, 3H, OCH3), 7.23e7.99 (m, 7H, arom. H), 8.17 (s, 1H,
J ¼ 6.88 Hz), 7.19e7.87 (m, 8H, arom. H), 8.21 (s, 1H, pyridinyl H-5).
pyridinyl H-5). 13C NMR (DMSO-d6 þ TFA):
d 34.1 (NCH3), 55.8
13C NMR (CDCl3):
d
14.6 (OCH2CH3), 33.2 (NCH3), 64.1 (OCH2), 94.6
(OCH3), 94.3 (pyridinyl C-3), 111.9 (pyridinyl C-5), 113.4 (C^N),
114.2, 116.5, 118.8, 127.1, 127.5, 129.1, 131.5, 132.4, 134.5, 136.0, 143.7,
145.2, 148.8, 165.3 (arom. C). MS: m/z (%) 346 (M, 10). Anal. Calcd.
For C19H14N4OS (346.41): C, 65.88; H, 4.07; N,16.17. Found: C, 66.02;
H, 4.13; N, 16.37.
(pyridinyl C-3), 110.2 (pyridinyl C-5), 115.2 (C^N), 116.1, 116.3, 117.5,
120.5, 123.4, 124.5, 130.7, 131.8, 137.4, 142.5, 148.2, 150.7, 155.7,
163.0, 164.5, 165.0 (arom. C). MS: m/z (%) 372 (M, 38). Anal. Calcd.
For C22H17FN4O (372.41): C, 70.96; H, 4.60; N, 15.04. Found: C,
70.80; H, 4.47; N, 14.87.
4.1.2.15. 2-Ethoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-(2-
thienyl)-3-pyridinecarbonitrile (4o). Reaction time 24 h, almost
colorless crystals, mp 234e236 ꢂC, yield (2.20 g) 61%. IR: nmax/cmꢀ1
4.1.2.10. 2-Methoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-(4-
methylphenyl)-3-pyridinecarbonitrile (4j). Reaction time 24 h, col-
orless crystals, mp 211e213 ꢂC, yield (1.95 g) 55%. IR: nmax/cmꢀ1
2219 (C^N), 1581, 1548 (C]N, C]C). 1H NMR (CDCl3):
d 1.54 (t, 3H,
2217 (C^N), 1581, 1545 (C]N, C]C). 1H NMR (CDCl3):
d
2.40 (s, 3H,
ArCH3), 4.11 (s, 3H, NCH3), 4.22 (s, 3H, OCH3), 7.26e7.80 (m, 8H,
arom. H), 8.19 (s, 1H, pyridinyl H-5). 13C NMR (CDCl3):
21.5
OCH2CH3, J ¼ 6.9 Hz), 4.30 (s, 3H, NCH3), 4.59 (q, 2H, OCH2,
J ¼ 6.9 Hz), 7.21e8.02 (m, 7H, arom. H), 8.34 (s, 1H, pyridinyl H-5).
d
13C NMR (CDCl3):
d 14.6 (OCH2CH3), 33.1 (NCH3), 64.1 (OCH2), 92.2
(ArCH3), 33.2 (NCH3), 55.2 (OCH3), 94.2 (pyridinyl C-3), 110.1 (pyr-
idinyl C-5), 115.5 (C^N), 117.5, 120.4, 123.3, 124.3, 128.5, 129.7,
132.6, 137.4, 140.7, 142.6, 148.3, 150.4, 156.6, 164.8 (arom. C). MS: m/
z (%) 354 (M, 100). Anal. Calcd. For C22H18N4O (354.41): C, 74.56; H,
5.12; N, 15.81. Found: C, 74.49; H, 5.08; N, 15.68.
(pyridinyl C-3), 110.2 (pyridinyl C-5), 115.8 (C^N), 116.3, 120.4,
123.4,124.4,128.7, 129.9,130.1,137.2, 137.5, 142.4, 148.2, 150.5,165.0
(arom. C). MS: m/z (%) 360 (M, 44), 362 [(M þ 2), 3]. Anal. Calcd. For
C
20H16N4OS (360.44): C, 66.65; H, 4.47; N, 15.54. Found: C, 66.53; H,
4.38; N, 15.35.
4.1.2.11. 2-Ethoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-(4-
methylphenyl)-3-pyridinecarbonitrile (4k). Reaction time 24 h, col-
orless crystals, mp 226e228 ꢂC, yield (1.95 g) 53%. IR: nmax/cmꢀ1
4.1.2.16. 6-(1H-Benzimidazol-2-yl)-2-methoxy-4-(5-methyl-2-
furanyl)-3-pyridinecarbonitrile (4p). Reaction time 48 h, pale yel-
low crystals, mp 284e286 ꢂC, yield (1.95 g) 59%. IR: nmax/cmꢀ1 3303
(NH), 2222 (C^N), 1603, 1555 (C]N, C]C). 1H NMR (DMSO-d6):
2219 (C^N), 1580, 1546 (C]N, C]C). 1H NMR (CDCl3):
d 1.54 (t, 3H,
OCH2CH3, J ¼ 6.88 Hz), 2.42 (s, 3H, ArCH3), 4.29 (s, 3H, NCH3), 4.59
d
2.34 (s, 3H, ArCH3), 4.12 (s, 3H, OCH3), 6.35e7.69 (m, 6H, arom. H),
8.09 (s, 1H, pyridinyl H-5), 12.90 (s, 1H, NH). 13C NMR (DMSO-d6):
d 14.0 (ArCH3), 55.6 (OCH3), 87.7 (pyridinyl C-3), 109.2 (pyridinyl C-
(q, 2H, OCH2, J ¼ 6.9 Hz), 7.30e7.84 (m, 8H, arom. H), 8.22 (s, 1H,
pyridinyl H-5). 13C NMR (CDCl3):
d
14.6 (OCH2CH3), 21.5 (ArCH3),
33.1 (NCH3), 64.0 (OCH2), 94.4 (pyridinyl C-3), 110.1 (pyridinyl C-5),
115.5 (C^N), 117.4, 120.4, 123.3, 124.3, 128.5, 129.7, 132.8, 137.4,
140.6, 142.6, 148.4, 150.4, 156.7, 164.5 (arom. C). MS: m/z (%) 368 (M,
45). Anal. Calcd. For C23H20N4O (368.44): C, 74.98; H, 5.47; N, 15.21.
Found: C, 75.14; H, 5.54; N, 15.02.
5), 116.0 (C^N), 110.4, 112.9, 116.6, 120.2, 123.0, 124.5, 135.4, 142.7,
144.5, 146.3, 149.4, 156.7, 165.5 (arom. C). MS: m/z (%) 330 (M, 100).
Anal. Calcd. For C19H14N4O2 (330.35): C, 69.08; H, 4.27; N, 16.96.
Found: C, 69.22; H, 4.29; N, 16.89.
4.1.2.17. 2-Methoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-(5-
methyl-2-furanyl)-3-pyridinecarbonitrile (4q). Reaction time 24 h,
colorless crystals, mp 286e288 ꢂC, yield (2.10 g) 61%. IR: nmax/cmꢀ1
4.1.2.12. 2-Methoxy-4-(4-methoxyphenyl)-6-(1-methyl-1H-benzimi-
dazol-2-yl)-3-pyridinecarbonitrile (4l). Reaction time 24
h
(Methods A & B), almost colorless crystals, mp 238e239 ꢂC, yield
(1.81, 2.00 g) 49, 54% (Methods A & B, respectively). IR: nmax/cmꢀ1
2218 (C^N), 1577, 1550 (C]N, C]C). 1H NMR (DMSO-d6):
d 2.25 (s,
3H, ArCH3), 3.98 (s, 3H, NCH3), 4.26 (s, 3H, OCH3), 6.19e7.73 (m, 6H,
arom. H), 8.14 (s, 1H, pyridinyl H-5). MS: m/z (%) 344 (M, 82). Anal.
2217 (C^N), 1585, 1545 (C]N, C]C). 1H NMR (CDCl3):
d 3.86 (s, 3H,

20
Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
Calcd. For C20H16N4O2 (344.38): C, 69.76; H, 4.68; N,16.27. Found: C,
69.84; H, 4.81; N, 16.46.
References
[1] H. Marona, N. Szkaradek, A. Rapacz, B. Filipek, M. Dybata, A. Siwek, M. Cegta,
E. Szneler, Bioorg. Med. Chem. 17 (2009) 1345e1352.
[2] A.E. Kümmerle, J.M. Raimundo, C.M. Leal, G.S. daSilva, T.L. Balliano,
M.A. Pereira, C.A. de Simone, R.T. Sudo, G. Zapata-Sudo, C.A.M. Fraga,
E.J. Barreiro, Eur. J. Med. Chem. 44 (2009) 4004e4009.
[3] J. Vergara-Galicia, R. Ortiz-Andrade, P. Castillo-Espana, M. Ibarra-Barajas,
I. Gallardo-Ortiz, R. Villalobos-Molina, S. Estrada-Soto, Vasc. Pharmacol. 49
(2008) 26e31.
[4] C.J. Vaughan, N. Delanty, Lancet 356 (2000) 411e417.
[5] P. Verdecchia, F. Angeli, C. Cavallini, R. Gattobigio, G. Gentile, J.A. Staessen,
G. Reboldi, Eur. Heart J. 30 (2009) 679e688.
4.1.2.18. 2-Ethoxy-6-(1-methyl-1H-benzimidazol-2-yl)-4-(5-methyl-
2-furanyl)-3-pyridinecarbonitrile (4r). Reaction time 24 h, colorless
crystals, mp 225e227 ꢂC, yield (2.20 g) 61%. IR: nmax/cmꢀ1 2218
(C^N), 1577, 1551 (C]N, C]C). 1H NMR (CDCl3):
d 1.51 (t, 3H,
OCH2CH3, J ¼ 7.25 Hz), 2.43 (s, 3H, ArCH3), 4.27 (s, 3H, NCH3), 4.55
(q, 2H, OCH2, J ¼ 6.85 Hz), 6.21e7.87 (m, 6H, arom. H), 8.46 (s, 1H,
pyridinyl H-5). 13C NMR (CDCl3):
d 14.1, 14.6 (ArCH3, OCH2CH3), 33.2
(NCH3), 63.9 (OCH2), 88.3 (pyridinyl C-3), 109.6 (pyridinyl C-5),
115.9 (C^N), 110.1, 112.5, 116.5, 120.3, 123.4, 124.3, 137.3, 143.2,
146.5, 148.6, 150.2, 156.3, 164.7 (arom. C). MS: m/z (%) 358 (M, 43).
Anal. Calcd. For C21H18N4O2 (358.40): C, 70.38; H, 5.06; N, 15.63.
Found: C, 70.57; H, 5.14; N, 15.69.
[6] B.E. Galan, V. Perkovic, T. Ninomiya, A. Pillai, A. Patel, A. Cass, B. Neal,
N. Poulter, S. Harrap, C. Mogensen, M. Cooper, M. Marre, B. Williams, P. Hamet,
G. Mancia, M. Woodward, P. Glasziou, D.E. Grobbee, S. MacMahon, J. Chalmers,
J. Am. Soc. Nephrol. 20 (2009) 883e892.
[7] M.E. Ernst, M. Moser, N. Engl. J. Med. 361 (2009) 2153e2164.
[8] L.H. Lindholm, B. Carlberg, O. Samuelsson, Lancet 366 (2005) 1545e1553.
[9] L. Kalinowski, L.W. Dobrucki, M. Szczepanska-Konkel, M. Jankowski,
L. Martyniec, S. Angielski, T. Malinski, Circulation 107 (2003) 2747e2752.
[10] A.T. Remaley, Circ. Res. 101 (2007) 116e121.
[11] B. Szabo, Pharmacol. Ther. 93 (2002) 1e35.
4.2. Vasodilation activity screening
[12] R. Donnelly, J. Renin Angiotensin Aldosterone Syst. 8 (2007) 13e22.
[13] H.T. Ong, J. Am. Board Fam. Med. 22 (2009) 686e697.
[14] F.H. Messerli, S. Bangalore, F. Ruschitzka, Eur. Heart J. 30 (2009) 2427e
2430.
[15] M. Pahor, B.M. Psaty, M.H. Alderman, W.B. Applegate, J.D. Williamson,
C. Cavazzini, C.D. Furberg, Lancet 356 (2000) 1949e1954.
[16] D.A. Williams, in: D.A. Williams, T.L. Lemke (Eds.), Foye’s Principles of Me-
dicinal Chemistry, sixth ed., Lippincott Williams & Wilkins, New York, 2008,
pp. 769e796.
The vasodilation activity screening procedures were carried out
according to the standard reported techniques [20e23] by testing
the effects of the synthesized 2-alkoxy-4-aryl-6-(1H-benzimidazol-
2-yl)-3-pyridinecarbonitriles 4aer on isolated thoracic aortic rings
of male Wister rats (250e350 g). After light ether anesthesia, the
rats were sacrificed by cervical dislocation. The aortae were
immediately excised, freed of extraneous tissues and prepared for
isometric tension recording. Aorta was cut into (3e5 mm width)
rings and each ring was placed in a vertical chamber “10 ml jac-
keted automatic multi-chamber organ bath system (Model no.
ML870B6/C, Panlab, Spain)” filled with Krebs solution composed of
(in mM): NaCl, 118.0; KCl, 4.7; NaHCO3, 25.0; CaCl2, 1.8; NaH2PO4,
1.2; MgSO4, 1.2; glucose, 11.0 and oxygenated with carbogen gas
(95% O2/5% CO2) at 37 ꢃ 0.5 ꢂC. Each aortic ring was mounted be-
tween two stainless steel hooks passed through its lumen. The
lower hook was fixed between two plates, while the upper one was
attached to a force displacement transducer (Model no. MLT0201,
Panlab, Spain) connected to an amplifier (PowerLab, AD In-
struments Pty. Ltd.) which is connected to a computer. The Chart for
windows (v 3.4) software was used to record and elaborate data.
Preparations were stabilized under 2 g resting tension during
2 h and then the contracture response to norepinephrine hydro-
chloride (10ꢀ6 M) was measured before and after exposure to
increasing concentrations of the tested synthesized compounds.
The tested compounds were dissolved in dimethylsulfoxide
(DMSO) as stock solution (10 ml of 0.005 M). Control experiments
were performed in the presence of DMSO alone, at the same con-
centrations as those used with the derivatives tested, which dem-
onstrated that the solvent did not affect the contractile response of
isolated aorta. The observed vasodilation activity screening data are
reported (Table 1, Figures 1 and 2 of Supplementary material) and
the potency (IC50, concentration necessary for 50% reduction of
maximal norepinephrine hydrochloride induced contracture) was
determined.
[17] G.S. Stokes, J. Clin. Hypertens. 6 (2004) 192e197.
[18] A. Kleemann, J. Engel, B. Kutscher, D. Reichert, in: Pharmaceutical Substances:
Syntheses Patents Applications, third ed., Thieme, Stuttgart, New York, 1999,
pp. 938, 1007, 1245.
[19] A.S. Girgis, H.M. Hosni, F.F. Barsoum, A.M.M. Amer, I.S. Ahmed-Farag, Boll.
Chim. Farm. 143 (2004) 365e375.
[20] A.S. Girgis, A. Kalmouch, M. Ellithey, Bioorg. Med. Chem. 14 (2006) 8488e
8494.
[21] A.S. Girgis, N. Mishriky, A.M. Farag, W.I. El-Eraky, H. Farag, Eur. J. Med. Chem.
43 (2008) 1818e1827.
[22] A.S. Girgis, N.S.M. Ismail, H. Farag, W.I. El-Eraky, D.O. Saleh, S.R. Tala,
A.R. Katritzky, Eur. J. Med. Chem. 45 (2010) 4229e4238.
[23] A.S. Girgis, N.S.M. Ismail, H. Farag, Eur. J. Med. Chem. 46 (2011) 2397e2407.
[24] F.F. Barsoum, Eur. J. Med. Chem. 45 (2010) 5176e5182.
[25] P. Pierrat, P.C. Gros, Y. Fort, J. Comb. Chem. 7 (2005) 879e886.
[26] S.P. Jose, S. Mohan, Spectrochim. Acta Part A 64 (2006) 240e245.
[27] L.N. Tumey, N. Bhagirath, A. Brennan, N. Brooijmans, J. Lee, X. Yang,
D.H. Boschelli, Bioorg. Med. Chem. 17 (2009) 7933e7948.
[28] J. Shim, C. Eid, J. Lee, E. Liu, D. Chaudhary, D.H. Boschelli, Bioorg. Med. Chem.
Lett. 19 (2009) 6575e6577.
[29] C. Niu, D.H. Boschelli, L.N. Tumey, N. Bhagirath, J. Subrath, J. Shim, Y. Wang,
B. Wu, C. Eid, J. Lee, X. Yang, A. Brennan, D. Chaudhary, Bioorg. Med. Chem.
Lett. 19 (2009) 5829e5832.
[30] A.S. Prashad, D. Wang, J. Subrath, B. Wu, M. Lin, M.-Y. Zhang, N. Kagan, J. Lee,
X. Yang, A. Brennan, D. Chaudhary, X. Xu, L. Leung, J. Wange, D.H. Boschelli,
Bioorg. Med. Chem. Lett. 19 (2009) 5799e5802.
[31] J. Subrath, D. Wang, B. Wu, C. Niu, D.H. Boschelli, J. Lee, X. Yang, A. Brennan,
D. Chaudhary, Bioorg. Med. Chem. Lett. 19 (2009) 5423e5425.
[32] D.H. Boschelli, D. Wanga, A.S. Prashad, J. Subrath, B. Wu, C. Niu, J. Lee, X. Yang,
A. Brennan, D. Chaudhary, Bioorg. Med. Chem. Lett. 19 (2009) 3623e3626.
[33] R.G. Dushin, T. Nittoli, C. Ingalls, D.H. Boschelli, D.C. Cole, A. Wissner, J. Lee,
X. Yang, P. Morgan, A. Brennan, D. Chaudhary, Bioorg. Med. Chem. Lett. 19
(2009) 2461e2463.
[34] G. Baier, D. Telford, L. Giampa, K.M. Coggeshall, G. Baier-Bitterlich, N. Isakov,
A. Altman, J. Biol. Chem. 268 (1993) 4997e5004.
[35] K. Hayashi, A. Altman, Pharmacol. Res. 55 (2007) 537e544.
[36] B.J. Marsland, M. Kopf, Trends Immunol. 29 (2008) 179e185.
[37] S. Salek-Ardakani, T. So, B.S. Halteman, A. Altman, M. Croft, J. Immunol. 175
(2005) 7635e7641.
[38] S.-L. Tan, J. Zhao, C. Bi, X.Y.C. Chen, D.L. Hepburn, J. Wang, J.D. Sedgwick,
S.R. Chintalacharuvu, S. Na, J. Immunol. 176 (2006) 2872e2879.
[39] A.M. Healy, E. Izmailova, M. Fitzgerald, R. Walker, M. Hattersley, M. Silva,
E. Siebert, J. Terkelsen, D. Picarella, M.D. Pickard, B. LeClair, S. Chandra,
B. Jaffee, J. Immunol. 177 (2006) 1886e1893.
Acknowledgment
This work was supported financially by the U.S. e Egypt Science
and Technology Joint Fund under project No. MAN10-007-002.
[40] B.J. Marsland, T.J. Soos, G. Spaeth, D.R. Littman, M. Kopf, J. Exp. Med. 200
(2004) 181e189.
[41] S. Salek-Ardakani, T. So, B.S. Halteman, A. Altman, M. Croft, J. Immunol. 173
(2004) 6440e6447.
[42] K. Nagahama, A. Ogawa, K. Shirane, Y. Shimomura, K. Sugimoto, A. Mizoguchi,
Gastroenterology 134 (2008) 459e469.
Appendix A. Supplementary data
[43] S. Manicassamy, D. Yin, Z. Zhang, L.L. Molinero, M.-L. Alegre, Z. Sun,
J. Immunol. 181 (2008) 513e520.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.042.

Z.M. Nofal et al. / European Journal of Medicinal Chemistry 63 (2013) 14e21
21
[44] L. Wang, Z. Xiang, L.-L. Ma, Z. Chen, X. Gao, Z. Sun, P. Williams, R.S. Chari,
D.-P. Yin, Transplantation 87 (2009) 507e516.
[55] H. Goker, S. Ozden, S. Yildiz, D.W. Boykin, Eur. J. Med. Chem. 40 (2005)
1062e1069.
[45] M. Missori, M. De Spirito, L. Ferrari, S. Selci, A. Gnoli, F.R. Bertani, A.S. Girgis,
H. El-Saied, A.H. Basta, J. Nanopart. Res. 14 (2012) 649.
[46] A.H. Basta, A.S. Girgis, H. El-Saied, M.A. Mohamed, Mater. Lett. 65 (2011)
1713e1718.
[56] M.A. Ismail, R. Brun, T. Wenzler, F.A. Tanious, W.D. Wilson, D.W. Boykin,
Bioorg. Med. Chem. 12 (2004) 5405e5413.
[57] H. Goker, C. Kus, D.W. Boykin, S. Yildiz, N. Altanlar, Bioorg. Med. Chem. 10
(2002) 2589e2596.
[47] A.H. Basta, A.S. Girgis, H. El-Saied, Dyes Pigm. 54 (2002) 1e10.
[48] J.Y. Xu, Y. Zeng, Q. Ran, Z. Wei, Y. Bi, Q.H. He, Q.J. Wang, S. Hu, J. Zhang,
M.Y. Tang, W.Y. Hua, X.M. Wu, Bioorg. Med. Chem. Lett. 17 (2007) 2921e2926.
[49] S.E. Soto, R.V. Molina, F.A. Crespo, J.V. Galicia, H.O. Díaz, M.T. Piedra,
G.N. Vázquez, Life Sci. 79 (2006) 430e435.
[50] S.Y. Hong, K.W. Kwak, C.K. Ryu, S.J. Kang, K.H. Chung, Bioorg. Med. Chem. 16
(2008) 644e649.
[51] M. Boiani, M. Gonzalez, Mini Rev. Med. Chem. 5 (2005) 409e424.
[52] V. Martınez, V. Burgos, J.A. Builla, G. Fernandez, A. Domingo, R.G. Nieto,
F. Gago, I. Manzanares, C. Cuevas, J.J. Vaquero, J. Med. Chem. 47 (2004)
1136e1148.
[58] K. Starcevic, M. Kralj, K. Ester, I. Sabol, M. Grce, K. Pavelic, G.K. Zamola, Bioorg.
Med. Chem. 15 (2007) 4419e4426.
[59] T. Fonseca, B. Gigante, M.M. Marques, L.T. Gilchrist, E.D. Clercq, Bioorg. Med.
Chem. 12 (2004) 103e112.
[60] S.M. Abou-Seri, K. Abouzid, D.A. Abou El Ella, Eur. J. Med. Chem. 46 (2011)
647e658.
[61] A.S. Girgis, J. Stawinski, N.S.M. Ismail, H. Farag, Eur. J. Med. Chem. 47 (2012)
312e322.
[62] G.W.H. Cheeseman, J. Chem. Soc. (1964) 4645e4646.
[63] S.N. Sawhney, D. Vir, A. Gupta, Indian J. Chem. 29 (1990) 1107e1112.
[64] a) R.F. Silver, K.A. Kerr, P.D. Frandsen, S.J. Kelley, H.L. Holmes, Can. J. Chem. 45
(1967) 1001e1006;
[53] S. Demirayak, U.A. Mohsen, A.C. Karaburun, Eur. J. Med. Chem. 37 (2002)
255e260.
[54] K. Taniguchi, S. Shigenaga, T. Ogahara, T. Fujitsu, M. Matsuo, Chem. Pharm.
Bull. 41 (1993) 301e309.
b) M. Mantri, O. De Graaf, J. Van Veldhoven, A. Goeblyoes, J.T. Von Frijtag
Drabbe Kuenzel, T. Mulder-Krieger, R. Link, H. De Vries, M.W. Beukers,
J. Brussee, A.P. Ijzerman, J. Med. Chem. 51 (2008) 4449e4455.