Synthesis and 2D-QSAR study of active benzofuran-based vasodilators

Article abstract of DOI:10.3390/molecules22111820

A new series of 2-alkyloxy-pyridine-3-carbonitrile-benzofuran hybrids (4a-x) was synthesized. All the new derivatives were examined via the standard technique for their vasodilation activity. Some of the investigated compounds exhibited a remarkable activity, with compounds 4w, 4e, 4r, 4s, 4f and 4g believed to be the most active hits in this study with IC50 values 0.223, 0.253, 0.254, 0.268, 0.267 and 0.275 mM, respectively, compared with amiodarone hydrochloride, the reference standard used (IC50 = 0.300 mM). CODESSA PRO was employed to obtain a statistically significant 2-Dimensional Quantitative Structure Activity Relationship (2D-QSAR) model describing the bioactivity of the newly synthesized analogs (N = 24, n = 4, R² = 0.816, R²cvOO = 0.731, R² cvMO = 0.772, F = 21.103, s² = 6.191, 10..8).

Full text of DOI:10.3390/molecules22111820

molecules
Article
Synthesis and 2D-QSAR Study of Active
Benzofuran-Based Vasodilators
Nagy M. Khalifa 1,2 ID , Aladdin M. Srour 2,*,†, Somaia S. Abd El-Karim 2,†, Dalia O. Saleh 3,† and
Mohamed A. Al-Omar 1
ID
1
Pharmaceutical Chemistry Department, Drug Exploration & Development Chair (DEDC),
College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
nagykhalifa@hotmail.com (N.M.K.); Malomar1@KSU.EDU.SA (M.A.A.-O.)
Therapeutical Chemistry Department, National Research Centre, Dokki, Giza 12622, Egypt;
2
somaia_elkarim@hotmail.com
Pharmacology Department, National Research Centre, Dokki, Giza 12622, Egypt; doabdelfattah@yahoo.com
3
*
†
Correspondence: aladdinsrour@gmail.com; Tel.: +20-2-01229749596
These authors contributed equally to this work.
Received: 18 September 2017; Accepted: 22 October 2017; Published: 26 October 2017
Abstract: A new series of 2-alkyloxy-pyridine-3-carbonitrile-benzofuran hybrids (4a–x) was
synthesized. All the new derivatives were examined via the standard technique for their vasodilation
activity. Some of the investigated compounds exhibited a remarkable activity, with compounds
4w, 4e, 4r, 4s, 4f and 4g believed to be the most active hits in this study with IC50 values 0.223,
0.253, 0.254, 0.268, 0.267 and 0.275 mM, respectively, compared with amiodarone hydrochloride, the
reference standard used (IC50 = 0.300 mM). CODESSA PRO was employed to obtain a statistically
significant 2-Dimensional Quantitative Structure Activity Relationship (2D-QSAR) model describing
the bioactivity of the newly synthesized analogs (N = 24, n = 4, R2 = 0.816, R2cvOO = 0.731,
R2cvMO = 0.772, F = 21.103, s2 = 6.191 × 10−8).
Keywords: benzofuran; vasodilation activity; 2D-QSAR; amiodarone hydrochloride
1. Introduction
Hypertension is the most common cardiovascular and cerebrovascular disorder representing the
major risk factor for endothelial dysfunctions [
to have high blood pressure, which is responsible for half of the mortalities related to stroke and
heart disease [ ]. Under hypertensive conditions, many functional organs can suffer irreparable
lesions [ ]. Essential hypertension is a common trait caused by many factors and it increases the risk
of cardiovascular (heart attacks), cerebrovascular (stroke), peripheral artery, rheumatic heart, congenital
heart, heart failure and renal diseases [ ]. A benzofuran-containing compound, amiodarone,
is one of the most therapeutically important antiarrhythmic drugs for various types of cardiac
dysrhythmias [ 10] (Figure 1). Though the responsible pharmacological mechanisms of amiodarone’s
1,2]. Worldwide, one of every three adults is reported
2,3
4,5
6–8
9
,
antiarrhythmic effects are not settled [11], it has an extreme effect on various ionic currents [12], as well
as sodium, calcium and potassium fluxes. These actions are interrelated in a complex way, but are
of prime importance for its activity. Amiodarone also possesses coronary and peripheral vasodilator
properties [11]—this appears to be mainly due to a release of nitric oxide (NO). Moreover, it expands
the precompressed in vivo human hand veins through the activation of NO synthase and blockade
of α-adrenergic mechanisms as a venodilator [13,14], and amiodarone’s analog KB130015 (Figure 1)
activates the BKCa channels, which relaxes vascular smooth muscle cells. KB130015 is a novel BKCa
activator—its efficacy is based on the subunit composition of the channel complex [15]. Dronedarone
as well as KB130015 (Figure 1) is a noniodinated congener of amiodarone that has been developed and
Molecules 2017, 22, 1820; doi:10.3390/molecules22111820
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1820
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approved by FDA (Food and Drug Administration) to avoid the limiting iodine-associated adverse
effects of the commercially used amiodarone. Additionally, dronedarone displays antiadrenergic
properties, atrial flutter and atrial fibrillation [16]. Consequently, the stimulation of coronary dilation
by dronedarone involving a dual mechanism, putative Ca2+ channels inhibition and stimulation of
NO synthase pathway [16
broad range of chemotherapeutic properties [19
active heterocycles [26 27], also, many nicotinate analogs such as, micinicate, hepronicate and inositol
,
17]. Benzofurans are naturally existing scaffolds [18], associated with a
–
25]. Nicotinate esters are very interesting vasodilatory
,
nicotinate are of significant vasodilating activity [28], (Figure 1).
In the present study, we designed and synthesized some novel hybrids of pyridine-3-carbonitriles
and benzofuran-pyrazole functions, attributed to the fact that, pyridine-3-carbonitriles are interesting
agents in developing new active hits due to the recognition of bioisosterism with the nicotinate
analogs where the acid/ester function is just replaced by a cyano group [28]. Furthermore, it is known
that the benzofuran-pyrazole hybrid is of considerable vasorelaxant interest [29], this may due to
the belief that the aliphatic secondary amine side chain of amiodarone might be responsible for its
vasodilation activity [30,31]. Thus, the insertion of a pyrazole ring system in this scaffold may widen
new pharmacological active hits with higher potency and fewer side effects. In addition, studying the
two-dimensional quantitative structure activity relationships (2D-QSAR) for the newly synthesized
analogs explored the controlling factors governing the observed pharmacological properties as well as
validated of the observed activity of the new chemical entities.
N
N
OH
O
I
I
O
O
O
O
O
O
H
O
N
I
I
S
O
O
O
O
Dronedarone
Amiodarone
KB130015
N
N
O
N
O
O
O
O
O
O
O
N
O
O
N
O
O
O
O
O
O
N
O
O
N
N
O
O
CH3
CH3
O
N
O
N
CH3
Inositol Nicotinate
Micinicate
Hepronicate
Figure 1. Structures of some active vasodilating agents.
2. Results and Discussion
2.1. Chemistry
The assumed synthetic approach to obtain target derivatives is illustrated in (Scheme 1). Treating
2-acetylbenzofuran, the key starting compound in this study, with phenyl hydrazine afforded the
corresponding semihydrazone derivative which undergo formylation via Vilsmeier–Haack reaction
to give 3-(benzofuran-2-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (
1) [32]. The formed carbaldehyde
1
was treated with malononitrile in refluxing ethanol to give 2-((3-(benzofuran-2-yl)-1-phenyl-
1H-pyrazol-4-yl)-methylene)malononitrile (2). Adopting the reported procedure [32], the required
2-alkyloxy-pyridine-3-carbonitrile derivatives 4a were synthesized via the condensation reaction of
–
x
aromatic ketones 3a with ylidenemalononitrile 2 and sodium alkoxide of the corresponding alcohol
–l

Molecules 2017, 22, 1820
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(Scheme 1). As a representative example, the IR spectrum of compound 4a shows a strong stretching
1
vibration band at
signal at
at
= 8.42 ppm. 13C-NMR spectrum of 4a reveals the presence of a methoxide carbon at δ = 54.7 ppm,
pyridinyl C-3, C-5 and nitrile carbon signals appeared at = 93.5, 105.8, and 114.6 ppm, respectively.
Mass spectrum (EI) of 4a reveals the molecular ion peak 482.27 with relative intensity value 30.7%.
ν
= 2213 cm−1 for nitrile group. H-NMR spectrum of 4a exhibits a characteristic
δ
= 4.23 ppm refering to the methoxide group, while the pyridinyl H-5 appeared as singlet peak
δ
δ
The established structures of all new chemical entities 4a
spectral data (IR, 1H-NMR, 13C-NMR and EI-MS).
–x
were certified by their microanalyses and
NC
CN
OHC
(i)
N
N
O
N
O
N
1
2
O
(ii)
N
R
CH3
3
R'
O
R
N
N
O
N
4a-x
3a; R = Ph
4a; R = Ph
4b; R = Ph
4c; R = 4-ClC6H4
4d; R = 4-ClC6H4
4e; R = 4-BrC6H4
4f; R = 4-BrC6H4
4g; R = 4-FC6H4
3b; R = 4-ClC6H4
3c; R = 4-BrC6H4
3d; R = 4-FC6H4
3e; R = 4-H3CC6H4
3f; R = 4-H3COC6H4
3g; R = 1,2,3,4-Tetrahydronaphthalen-6-yl
3h; R = 2-Pyrrolyl
4h; R = 4-FC6H4
3i; R = 2-Furanyl
3j; R = 2-Thienyl
3k; R = 2-Pyridinyl
3l; R = 1-Methyl-1H-benzo[d]imidazol-2-yl
4i; R = 4-H3CC6H4
4j; R = 4-H3CC6H4
4k; R = 4-H3COC6H4
4l; R = 4-H3COC6H4
0
4m; R = 1,2,3,4-Tetrahydronaphthalen-6-yl, R = Me
0
4n; R = 1,2,3,4-Tetrahydronaphthalen-6-yl, R = Et
0
4o; R = 2-Pyrrolyl, R = Me
0
4p; R = 2- Pyrrolyl, R = Et
0
4q; R = 2-Furanyl, R = Me
0
0
4r; R = 2-Furanyl, R = Et
4s; R = 2-Thienyl, R = Me
4t; R = 2-Thienyl, R = Et
4u; R = 2-Pyridinyl, R = Me
0
0
0
4v; R = 2-Pyridinyl, R = Et
0
4w;R = 1-Methyl-1H-benzo[d]imidazol-2-yl, R = Me
0
4x; R = 1-Methyl-1H-benzo[d]imidazol-2-yl, R = Et
Reagents and Conditions: (i) CNCH2CN/Ethanol/r.t./6 h, 75%; (ii) alcohol/Na/r.t./33.3–45.3%.
Scheme 1. Synthetic route of 2-alkyloxy-pyridine-3-carbonitrile derivatives 4a–x.

Molecules 2017, 22, 1820
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2.2. Biological Evaluation
2.2.1. Vasodilation Properties
Vasodilation properties of the synthesized 2-alkyloxy-pyridine-3-carbonitrile derivatives 4a–x
were inspected applying the separated thoracic aortic rings of mice pre-contracted by norepinephrine
hydrochloride according to the standard method [33] using amiodarone hydrochloride as a standard
reference. The observed data (Table 1), (Figures S1 and S2 of Supplementary Materials) reveal that
most the new chemical entities reveal remarkable vasodilation properties. Meanwhile, compounds
4w, 4e, 4r, 4s, 4f and 4g exhibit significant activity (IC50, is the required concentration for 50%
lessening of maximal norepinephrine. HCl induced contracture = 0.223, 0.253, 0.254, 0.268, 0.267
and 0.275 mM, respectively), that seems more potent than the used reference standard in the present
study (IC50 = 0.300 mM). The 2D-QSAR study was initiated to recognize the observed bioactivities
and concluding the most important factors that manage the pharmacological properties. Currently,
throughout the observed vasodilator activities of the new chemical entities, few Structure activity
relationship (SAR) rules could be achieved, the presence of a methoxy group at the 2-position of
3-pyridinecarbonitriles enhances the vasodilation activity more than the ethoxy group, as shown in
all of the tested analogs. Compounds 4k and 4q (IC50 = 0.428, 0.321 mM, respectively) are exceptions.
Benzimidazole ring systems attached to pyridine-C5 seems appropriate for designing vasodilation
active hits (IC50 = 0.223, 0.299 mM) compared with the corresponding substituted phenyl ring systems
or other heterocycles. Thus, the combination of benzimidazole, pyridine and benzofuran has the
potential to be developed into potent vasorelaxant active targets.
Table 1. Vasodilatory activity IC50 (mM) in rat thoracic aortic rings.
0
Entry
Compound
R
R
Potency (IC50), mM
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
41
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
Ph
Ph
4-ClC6H4
4-ClC6H4
4-BrC6H4
4-BrC6H4
4-FC6H4
4-FC6H4
4-H3CC6H4
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
0.281
0.343
0.295
0.397
0.253
0.267
0.275
0.330
0.330
0.452
0.322
0.291
0.286
0.337
0.356
0.400
0.321
0.254
0.268
0.298
0.333
0.370
0.223
0.299
0.300
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
4-H3CC6H4
4-H3COC6H4
4-H3COC6H4
1,2,3,4-Tetrahydronaphthalen-6-yl
1,2,3,4-Tetrahydronaphthalen-6-yl
2-Pyrrolyl
2-Pyrrolyl
2-Furanyl
2-Furanyl
2-Thienyl
2-Thienyl
2-Pyridinyl
2-Pyridinyl
1-Methyl-1H-benzo[d]imidazol-2-yl
1-Methyl-1H-benzo[d]imidazol-2-yl
-
Amiodarone.HCl
-
To validate and understand the observed pharmacological activities and to detect the factors that
control the activities, the 2D-QSAR study was initiated via the CODESSA PRO package. Molecular
descriptors of the 2D-QSAR correlating the chemical structure(s) and property values expressed as
1/IC50 µM are presented in Table 2, arranged on their level of significance (t-criterion). The descriptors
were acquired using the BMLR (Best Multiple Linear Regression) method. The first descriptor

Molecules 2017, 22, 1820
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controlling the BMLR-QSAR model based on its t-criterion value (t = 7.789) is maximum e–e repulsion
for bond C–O which is a semiempirical descriptor. Electron–electron repulsion between two given
atoms is determined by Equation (1) [34].
Eee(AB) =
PµνPλσhµν|λσi
(1)
∑ ∑
µ,ν∈A λ,σ∈B
where, A stands for a given atomic species, B is another atomic species Pµν, Pλσ is density matrix
elements over atomic basis {µνλσ}, hµν|λσi is the electron repulsion integrals on atomic basis {µνλσ}.
The second important descriptor controlling the BMLR-QSAR model (t =
charged partial-negative charged surface area (WNSA1) weighted PNSA (PNSA1
(MOPAC PC) (charge-related descriptor). Surface-weighted charged partial negative-charged surface
−
3.637) is surface-weighted
TMSA/1000)
×
area (WNSA1) is calculated by Equation (2) [34].
PNSA1 · TMSA
WNSA1 =
(2)
1000
where, PNSA1 stands for partial negatively charged molecular surface area, TMSA for total molecular
surface area. The third descriptor controlling BMLR-QSAR model (t = 5.670) is the fractional
−
hydrogen bonding acceptor ability of the molecule FHACA1, which is also a charge-related descriptor
determined by Equation (3) [34].
HACA1
FHACA1 =
(3)
TMSA
where, HACA1 is hydrogen bonding acceptor ability, TMSA is the total molecular surface area.
The fourth descriptor controlling BMLR-QSAR model (t = 6.241) is a semiempirical descriptor,
−
maximum e–n attraction for bond C–N, is determined by Equation (4) [34].
ZB
RiB
Ene(AB) =
Pµνhµ|
|νi
(4)
∑ ∑
B µ,ν∈A
where, A stands for a given atomic species, B is another atomic species Pµν is density matrix
elements over atomic basis {µν}, ZB for charge of atomic nucleus B, RiB for distance between the
ZB
iB
electron and atomic nucleus B, hµ| R |νi for electron–nuclear attraction integrals on atomic basis
{
µν}. The correlation between the observed and predicted vasodilation activities is represented
in Figure 2. The descriptor values for each respective compound are exhibited in Table S1 of
Supplementary Material.
Table 2. Descriptors of the BMLR-QSAR model for the vasodilatory active compounds.
Entry
ID
Coefficient
s
t
Descriptor
−8
N = 24, n= 4, R2 = 0.816, R2cvOO = 0.731, R2cvMO = 0.772, F = 21.103, s2 = 6.191 × 10
1
2
3
4
5
0
1.464
0.0004
0.244
4.751 × 10
1.975 × 10
0.045
6.009
7.789
−3.637
−5.670
−6.241
Intercept
−5
−6
D1
D2
D3
D4
Max. e–e repulsion for bond C–O
WNSA-1 Weighted PNSA (PNSA1*TMSA/1000) (MOPAC PC)
FHACA Fractional HACA (HACA1/TMSA) (MOPAC PC)
Max. e–n attraction for bond C–N
−6
−7.1825 × 10
−0.255
−0.0043
0.001
1/IC50 (µM) = 1.464 + (0.0004 × D1) − [(7.1825 × 10−6) × D2] − (0.255 × D3) − (0.0043 × D3)
The reliability and statistical relevance of the attained BMLR-QSAR model is examined by internal
validation technique, which is an appropriate technique due to the limited data points of the present
study [35–37]. Internal validation is applied by the CODESSA PRO employing both Leave One
Out (LOO), which involves developing a number of models with one example omitted at a time,
and Leave Many Out (LMO) that develops a number of models with many data points omitted
at a time (up to 20% of the total data points). The observed correlations attributed to the internal

Molecules 2017, 22, 1820
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validation techniques are R2cvOO = 0.731, R2cvMO = 0.772. Both of them are significantly correlated
with the R2value of the attained QSAR model (R2 = 0.816). Standard deviation of the regressions
(s2 = 6.191
×
10−8) and Fisher test value (F = 21.103) are also statistical parameters supporting the
QSAR model. The predicted/estimated IC50 values of the new chemical entities according to the
achieved BMLR-QSAR model are displayed in Table 3. The obtained results revealed that the most
potent analogue among all the tested hybrids, compound 4w, shows error value (difference between
estimated and observed IC50 values) =
4a , relative to the standard reference, amiodarone hydrochloride (IC50 = 300
estimated bioproperties matched with their observed potencies (IC50 = 253–397
corresponding to the observed and predicted values respectively, error = 0– 40). Compound 4j is
the only exception with high error value = 83 with (IC50 = 452, 369 M, for observed and predicted
−
0.4. Additionally, the high potent analogues synthesized
M), also exhibited
M, 253–392 M,
–
i
µ
µ
µ
−
µ
bio-data, respectively). This compound is exhibited as an outlier (Figure 2) and shows the lowest
vasodilation properties among all the synthesized hybrids. From all the above, it can be concluded
that the achieved BMLR-QSAR model is statistically significant and also supported by the matched
correlations due to the observed and predicted bio-observations. Success of this study can be attributed
to the homogeneity of chemical structural entities. Additionally, the achieved model can be adopted
for optimizing hits of high potency relative to the standard reference used based on the hybrid design
mentioned in the present study.
Figure 2. BMLR-QSAR model plot of correlations, the yellow dots are representing the observed vs.
predicted vasodilatory active agents for all compounds, while the blue dot is represent an outlier
compound (compound 4j).
Table 3. Observed and estimated/predicated values of the vasodilatory active compounds according
to the BMLR-QSAR model.
0
Entry
Compd.
R
R
Observed IC50, µM Estimated IC50, µM
Error
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
4g
4h
4i
Ph
Ph
Me
Et
Me
Et
Me
Et
Me
Et
281
343
295
397
253
267
275
330
330
452
276
363
272
392
253
307
303
354
299
369
5
−20
23
5
0
−40
−28
−24
31
4-ClC6H4
4-ClC6H4
4-BrC6H4
4-BrC6H4
4-FC6H4
4-FC6H4
4-H3CC6H4
4-H3CC6H4
Me
Et
10
4j
83

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Table 3. Cont.
0
Entry
Compd.
R
R
Observed IC50, µM Estimated IC50, µM
Error
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4k
41
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
4-H3COC6H4
4-H3COC6H4
1,2,3,4-Tetrahydronaphthalen-6-yl
1,2,3,4-Tetrahydronaphthalen-6-yl
2-Pyrrolyl
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
Me
Et
322
291
286
337
268
298
321
254
356
400
333
370
223
299
337
307
265
344
286
311
310
257
328
386
336
369
227
285
−15
−16
21
−7
−18
−13
11
2-Pyrrolyl
2-Furanyl
2-Furanyl
2-Thienyl
2-Thienyl
2-Pyridinyl
2-Pyridinyl
−3
28
14
−3
1
1-Methyl-1H-benzo[d]imidazol-2-yl
1-Methyl-1H-benzo[d]imidazol-2-yl
−4
14
2.2.2. Toxicological Bioassay
The most potent hits in this study, compounds (4a, c, e, f, g, l, m, r, s,t, w and 4x), were tested at
1000 mg kg−1 (mouse body weight), with no toxic symptoms or mortality rates being observed after
24 h post-administrations elucidating the safe behavior of the used doses. Thus, the present study
recommended that the benzofuran-containing compounds may have the potential to be developed
into potent vasodilatory active agents.
3. Experimental Section
3.1. General Information
Melting points were recorded on a Stuart SMP30 melting point apparatus. IR spectra (KBr) were
recorded on a JASCO 6100 spectrophotometer, JASCO, Easton, USA. NMR spectra were recorded on
a JEOL AS 500 (DMSO-d6, 1H: 500 MHz, 13C: 125 MHz) spectrometer, JEOL USA, Inc. (Pleasanton,
CA, USA). Chemical shifts (δH) are reported relative to Tetramethylsilane (TMS) as the internal
standard. All coupling constant (J) values are given in hertz. Chemical shifts (δc) are reported
relative to CDCl3 as internal standards. Mass spectra were recorded on a Shimadzu GCMS-QP
1000 EX (EI, 70 eV) spectrometer, Shimadzu corporation, Kyoto, Japan. Elemental microanalyses were
performed by using a Vario Elemental analyzer, Elementar Analysensysteme GmbH, Langenselbold,
Germany. 2-Acetylbenzofuran [38], 3-(benzofuran-2-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde (
1
) [32
]
and 2-((3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)malononitrile (
1) [32] were prepared
according to the previously reported procedures.
3.1.1. Synthesis of 2-((3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)malononitrile (2)
◦
1 (2.88 g, 10 mmol) was stirred in ethanol at room temperature (25–30 C)
The formed carbaldehyde
for 6 h with malononitrile (0.66 g, 10 mmol) in the presence of few drops of piperidine, the formed
precipitate was filtered, dried and recrystallized from n-butanol, to afford 2.54 g of compound
2
(75% yield).
3.1.2. Synthesis of 2-Alkoxy-4-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-phenylpyridine-3-
carbonitriles (4a–x)
General Procedure
A mixture of equimolar amounts of 2 (3.36 g, 10 mmol) and methyl aryl ketones 3a–l (10 mmol),
in the appropriate alcohol (20 mL) containing sodium (0.46 g, 20 mmol) was stirred at room temperature
(25–30 ◦C) for the proper time controlled by Thin-layer chromatography (TLC). The solid separated
was collected, washed with water and crystallized from n-butanol to afford the title compounds 4a–x.

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4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-phenylpyridine-3-carbonitrile (4a): yield
◦
1
1.86 g (39.7%), m.p. 198–200 C. IR:
(CDCl3): 4.23 (s, 3H), 7.08 (s, 1H), 7.25–7.29 (m, 4H), 7.39–8.04 (m, 6H), 7.09 (d, J = 7.7 Hz, 2H), 8.04
(d, J = 7.7 Hz, 2H), 8.38 (s, 1H), 8.42 (s, 1H). 13C-NMR (CDCl3):
54.7 93.5, 105.7, 105.8, 111.6, 111.7,
ν
max/cm−1 2213 (C
≡
N), 1588, 1543 (C=N, C=C). H-NMR
δ
δ
114.6, 119.8, 120.0, 121.4, 123.3, 125.1, 126.7, 127.4, 127.8, 127.9, 128.4, 128.6, 128.7, 129.0, 129.7, 130.6,
139.3, 154.9, 155.1, 157.8, 165.0. MS: m/z (%) 468.31 (M, 0.47). Anal. for C30H20N4O2 (468.51); Calcd. C,
76.91; H, 4.30; N, 11.96. Found: C, 76.84; H, 4.26; N, 11.87.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-phenylpyridine-3-carbonitrile (4b): yield 1.84 g
◦
(39%), m.p. 186–188 C. IR:
ν
max/cm−1: 2215 (C N), 1590, 1541 (C=N, C=C). 1H-NMR (CDCl3):
δ 1.55
≡
(t, J = 7.6 Hz, 3H), 4.69 (q, J = 7.6 Hz, 2H), 7.05 (s, 1H), 7.20–7.53 (m, 10 H), 7.81–8.10 (m, 4 H), 8.40 (s,
1H), 8.50 (s, 1H). 13C-NMR (CDCl3):
14.8, 63.5, 93.6, 105.8, 111.6, 114.3, 116.1, 118.1, 119.0, 120.0, 121.4,
δ
123.3, 125.1, 127.3, 127.8, 128.6, 128.7, 128.9, 129.7, 132.5, 135.4, 138.5, 140.0, 143.0, 147.3, 148.9, 155.1,
157.7, 164.8. MS: m/z (%) 482.25 (M, 20.34). Anal. for C31H22N4O2 (482.53); Calcd. C, 77.16; H, 4.60; N,
11.61. Found: C, 77.05; H, 4.57; N, 11.72.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(4-chlorophenyl)-2-methoxypyridine-3-carbonitrile (4c):
yield 2.04 g (40.5%), m.p. 259–261 ◦C. IR:
ν
max/cm−1 2214 (C N), 1588, 1542 (C=N, C=C). 1H-NMR
≡
(DMSO-d6):
δ 4.14 (s, 3H), 7.08 (s, 1H), 7.20–7.58 (m, 9H), 7.92 (s, 1H), 8.0 (d, J = 8 Hz, 2H), 8.12 (d,
J = 8 Hz, 2H), 9.1 (s, 1H). MS: m/z (%) 502.11 (M, 0.47). Anal. for C30H19ClN4O2 (502.95); Calcd. C,
71.64; H, 3.81; N, 11.14. Found: C, 71.56; H, 3.78; N, 11.18.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(4-chlorophenyl)-2-ethoxypyridine-3-carbonitrile (4d):
yield 2.27 g (40.4%), m.p. 199–201 ◦C. IR:
ν
max/cm−1 2219 (C N), 1588, 1541 (C=N, C=C). 1H-NMR
≡
(CDCl3):
δ
1.54 (t, J = 6.7 Hz, 3H), 4.67 (q, J = 6.7 Hz, 2H), 7.08 (s, 1H), 7.35–7.53 (m, 10H), 7.81–7.84
14.6, 63.5, 93.8, 105.9, 111.5, 114.1, 116.0, 117.5, 119.9, 121.5,
(m, 4H), 8.43 (s, 1H). 13C-NMR (CDCl3):
δ
123.4, 125.2, 127.9, 128.4, 128.6, 129.0, 129.2, 129.8, 135.8, 136.7, 139.2, 148.9, 149.0, 154.9, 156.4, 164.7.
MS: m/z (%) 516.30, 518.30 (M, M+2, 1.95, 0.68). Anal. for C31H21ClN4O2 (516.98); Calcd. C, 72.02; H,
4.09; N, 10.84. Found: C, 72.14; H, 4.14; N, 10.78.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(4-bromophenyl)-2-methoxypyridine-3-carbonitrile (4e):
yield 2.14 g (39%), m.p. 273–275 ◦C. IR:
ν
max/cm−1 2217 (C N), 1585, 1542 (C=N, C=C). 1H-NMR
≡
(DMSO-d6):
δ 4.14 (s, 3H), 7.09 (s, 1H), 7.20–7.73 (m, 10H), 7.96–7.16 (m, 4H), 9.10 (s, 1H). MS: m/z (%)
546.16 (M, 3.22). Anal. for C30H19BrN4O2 (547.40): Calcd. C, 65.82; H, 3.50; N, 10.24. Found: C, 65.94;
H, 3.38; N, 10.17.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(4-bromophenyl)-2-ethoxypyridine-3-carbonitrile (4f):
yield 1.94 g (34.6%), m.p. 207–209 ◦C. IR:
ν
max/cm−1 2219 (C N), 1588, 1540 (C=N, C=C). 1H-NMR
≡
(CDCl3):
δ
1.54 (t, J = 6.7 Hz, 3H), 4.67 (q, J = 6.7 Hz, 2H), 7.09 (s, 1H), 7.20–7.52 (m, 10H), 7.77–7.81 (m,
14.6, 63.6, 93.9, 105.9, 111.5, 114.1, 115.7, 117.5, 120.0, 121.5, 123.4,
4H), 8.44 (s, 1H). 13C-NMR (CDCl3):
δ
125.2, 127.9, 128.4, 128.8, 129.0, 129.8, 132.2, 136.3, 139.3, 142.4, 147.4, 148.9, 154.9, 156.5, 164.8. MS: m/z
(%) 560.40 (M, 0.24). Anal. for C31H21BrN4O2 (561.43); Calcd. C, 66.32; H, 3.77; N, 9.98. Found: C,
66.54; H, 3.84; N, 9.82.
4-(3-(benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(4-fluorophenyl)-2-methoxypyridine-3-carbonitrile (4g):
yield 1.96 g (40.3%), m.p. 237–239 ◦C. IR:
ν
max/cm−1 2215 (C N), 1590, 1543 (C=N, C=C). 1H-NMR
≡
(CDCl3):
δ 4.22 (s, 3H), 7.07 (s, 1H), 7.25–7.53 (m, 10H), 7.81–7.93 (m, 4H), 8.44 (s, 1H).MS: m/z (%)
486.31 (M, 1.90). Anal. for C30H19FN4O2 (486.5); Calcd. C, 74.06; H, 3.94; N, 11.52. Found: C, 73.95; H,
3.88; N, 11.48.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(4-fluorophenyl)pyridine-3-carbonitrile (4h):
yield 2.12 g (42.4%), m.p. 204–206 ◦C. IR:
ν
max/cm−1 2218 (C N), 1592, 1543 (C=N, C=C). 1H-NMR
≡
(CDCl3):
δ 1.53 (t, J = 7 Hz, 3H), 4.67 (q, J = 7 Hz, 2H), 7.08 (s, 1H), 7.39–7.54 (m, 10 H), 7.81–7.83 (m,

Molecules 2017, 22, 1820
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4H), 8.44 (s, 1H). 13C-NMR (CDCl3):
δ
14.6, 63.6, 93.5, 105.9, 111.5, 115.9, 116.1, 120.0, 121.4, 123.4, 125.1,
127.9, 129.0, 129.3, 129.4, 129.8, 139.3, 142.4, 147.4, 148.9, 154.9, 156.6, 163.3, 164.8, 165.3. MS: m/z (%)
500.36 (M, 2.71). Anal. for C31H21FN4O2 (500.52); Calcd. C, 74.39; H, 4.23; N, 11.19. Found: C, 74.45; H,
4.18; N, 11.14.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-p-tolylpyridine-3-carbonitrile (4i): yield 2.3 g
◦
1
(47.7%), m.p. 258–260 C. IR:
δ
ν
max/cm−1 2218 (C
≡
N), 1588, 1541 (C=N, C=C). H-NMR (CDCl3):
2.39 (s, 3H), 4.22 (s, 3H), 7.06 (s, 1H), 7.22–7.52 (m, 10H), 7.58–7.86 (m, 4H), 8.41 (s, 1H). 13C-NMR
(CDCl3):
δ 21.5, 54.7, 93.1, 105.8, 111.6, 114.2, 115.9, 117.6, 120.1, 121.4, 123.3, 125.1, 127.3, 127.9, 128.4,
129.0, 129.7, 134.5, 139.3, 141.0, 147.2, 148.9, 154.5, 157.9, 165.0. MS: m/z (%) 482.27 (M, 30.7). Anal. for
C31H22N4O2 (482.53): Calcd. C, 77.16; H, 4.60; N, 11.61. Found: C, 77.29; H, 4.42; N, 11.51.
4-(3-(Benzofuran-2-yl)-1-◦phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-p-tolylpyridine-3-carbonitrile (4j): yield 1.92 g
1
(38.7%), m.p. 202–204 C. IR:
δ
4H), 8.41 (s, 1H). 13C-NMR (CDCl3):
ν
max/cm−1 2215 (C
≡
N), 1598, 1543 (C=N, C=C). H-NMR (CDCl3):
1.54 (t, J = 6 Hz, 3H), 2.39 (s, 3H), 4.69 (q, J = 6 Hz, 2H), 7.05 (s, 1H), 7.20–7.56 (m, 10H), 7.81–7.85 (m,
δ
14.7, 21.5, 63.4, 93.2, 105.8, 111.6, 114.0, 116.0, 117.7, 120.0, 121.4,
123.3, 125.1, 127.3, 127.8, 128.4, 129.0, 129.7, 134.6, 139.3, 141.0, 143.0, 147.2, 149.0, 154.9, 157.8, 164.7.
MS: m/z (%) 496.22 (M, 5.52). Anal. for C32H24N4O2 (496.56): Calcd. C, 77.40; H, 4.87; N, 11.28. Found:
C, 77.52; H, 4.76; N, 11.34.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-(4-methoxyphenyl)pyridine-3-carbonitrile (4k):
yield 1.86 g (37.3%), m.p. 256–258 ◦C. IR:
ν
max/cm−1 2213 (C N), 1588, 1543 (C=N, C=C). 1H-NMR
≡
(DMSO-d6):
δ 3.80 (s, 3H), 4.13 (s, 3H), 7.04 (s, 1H), 7.20–7.62 (m, 10H), 7.88–8.20 (m, 4H), 9.08 (s,
1H).MS: m/z (%) 498.38 (M, 100). Anal. for C31H22N4O3 (498.53); Calcd. C, 74.69; H, 4.45; N, 11.24.
Found: C, 74.66; H, 4.38; N, 11.29.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(4-methoxyphenyl)pyridine-3-carbonitrile (4l):
yield 2.14 g (41.7%), m.p. 168–170 ◦C. IR:
ν
max/cm−1 2219 (C N), 1589, 1543 (C=N, C=C). 1H-NMR
≡
(CDCl3):
δ
1H-NMR (CDCl3):
δ
1.54 (t, J = 7 Hz, 3H), 3.83 (s, 3H), 4.67 (q, J = 7 Hz, 2H), 6.90 (s, 1H),
14.7, 55.5, 63.4, 92.6,
7.05 (s, 1H), 7.45–7.52 (m, 9H), 7.81–7.90 (m, 4H), 8.40 (s, 1H). 13C-NMR (CDCl3):
δ
105.8, 111.6, 113.4, 114.3, 117.8, 120, 121.4, 123.3, 125.1, 127.8, 128.4, 128.9, 129, 129.7, 139.3, 142.4, 147.1,
149, 154.9, 157.4, 161.7, 164.7. MS: m/z (%) 512 (M, 22.4). Anal. for C32H24N4O3 (512.56); Calcd. C,
74.99; H, 4.72; N, 10.93. Found: C, 74.91; H, 4.71; N, 10.89.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(1,2,3,4-tetrahydronaphthalen-6-yl)-2-methoxypyridine-3-
carbonitrile (4m): yield 1.78 g (34%), m.p. 255–257 ◦C. IR:
ν
max/cm−1 2221 (C
≡
N), 1591, 1555 (C=N,
1.58 (s, 2H), 1.79 (s, 2H), 2.69 (s, 2H), 2.78 (s, 2H), 4.22 (s, 3H), 7.06 (s, 1H),
7.24–7.58 (m, 10H), 7.81–7.83 (m, 4H), 8.43 (s, 1H). 13C-NMR (CDCl3):
23.1, 29.6, 54.6, 92.8, 105.9,
C=C). 1H-NMR (CDCl3):
δ
δ
111.6, 114.3, 119.9, 121.4, 123.0, 124.5, 125.1, 127.8, 128.1, 128.4, 128.9, 129.7, 129.9, 137.3, 155.0, 158.2,
165.0. Anal. for C34H26N4O2 (522.6); Calcd. C, 78.14; H, 5.01; N, 10.72. Found: C, 73.69; H, 4.14;
N, 15.56.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(1,2,3,4-tetrahydronaphthalen-6-yl)pyridine-3-
◦
carbonitrile (4n): Yield 2.16 g (40.3%), m.p. 213–215 C. IR:
ν
max/cm−1 2213 (C
N), 1585, 1542
≡
(C=N, C=C). 1H-NMR (CDCl3):
(q, J = 7.7 Hz, 2H), 7.05 (s, 1H), 7.12–7.56 (m, 11H), 7.81 (d, J = 8.5 Hz, 2H), 8.42 (s, 1H). 13C-NMR
(CDCl3): 14.7, 23.1, 23.2, 29.6, 29.6 63.4, 92.9, 105.9, 111.6, 114.0, 116.0, 118.0, 120.0, 121.4, 123.3, 124.5,
δ 1.54 (t, J = 7.7 Hz, 3H), 1.78 (s, 4H), 2.69 (s, 2H), 2.78 (s, 2H), 4.68
δ
125.1, 127.8, 128.1, 128.9, 129.7, 129.8, 135.0, 137.8, 140.3, 148.5, 149.5, 155.0, 158.2, 164.8. MS: m/z (%)
536.31 (M, 100). Anal. for C35H28N4O2 (536.62); Calcd. C, 78.34; H, 5.26; N, 10.44. Found: C, 78.52; H,
5.14; N, 10.57.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-(1H-pyrrol-2-yl)pyridine-3-carbonitrile (4o):
yield 1.86 g (40.6%), m.p. 187–189 ◦C. IR:
ν
max/cm−1 2218 (C N), 1587, 1539 (C=N, C=C). 1H-NMR
≡

Molecules 2017, 22, 1820
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(CDCl3):
δ
3.97 (s, 3H), 7.06 (s, 1H), 7.29–7.55 (m, 10H), 7.59–7.82 (m, 2H), 8.45 (s, 1H), 9.05 (s, 1H).
54.5, 102.0, 106.3, 111.6, 115.4, 117.0, 120.0, 121.7, 123.6, 125.4, 128.2, 128.4, 129.8,
13C-NMR (CDCl3):
δ
129.9, 138.9, 139.1, 145.7, 148.9, 155.3, 162.4. MS: m/z (%) 457.25 (M, 1.93). Anal. for C28H19N5O2
(457.48); Calcd. C, 73.51; H, 4.19; N, 15.31. Found: C, 73.69; H, 4.14; N, 15.56.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(1H-pyrrol-2-yl)pyridine-3-carbonitrile (4p):
yield 1.84 g (39%), m.p. 208–210 ◦C. IR:
ν
max/cm−1 2217 (C N), 1588, 1538 (C=N, C=C). 1H-NMR
≡
(CDCl3):
δ
1.53 (t, J = 7.7 Hz, 3H), 4.64 (q, J = 7.7 Hz, 2H), 7.05 (s, 1H), 7.20–7.30 (m, 4H), 7.46–7.54
14.6, 63.8, 93.0, 105.8, 111.6, 112.7, 120.1, 121.4,
(m, 8H), 7.81 (d, 2H), 8.40 (s, 1H). 13C-NMR (CDCl3):
δ
123.3, 125.1, 127.1, 127.9, 128.6, 129.0, 129.7, 130.0, 139.3, 142.4, 143.4, 147.2, 148.8, 152.9, 154.9, 164.7.
MS: m/z (%) 471.37 (M, 100). Anal. for C29H21N5O2 (471.51); Calcd. C, 73.87; H, 4.49; N, 14.85. Found:
C, 73.75; H, 4.43; N, 14.88.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-6-(furan-2-yl)-2-methoxypyridine-3-carbonitrile (4q): yield
1.88 g (41%), m.p. 246–248 ◦C. IR:
δ
ν
max/cm−1 2217 (C N), 1588, 1538 (C=N, C=C). 1H-NMR (CDCl3):
≡
4.21 (s, 3H), 6.54 (s, 1H), 7.05 (s, 1H), 7.20–7.53 (m, 10H), 7.80 (d, 2H), 8.35 (s, 1H). 13C-NMR (CDCl3):
δ
54.7, 93.3, 105.7, 111.6, 112.3, 112.6, 117.4, 120.0, 121.4, 123.2, 125.0, 127.9, 128.4, 129.0, 129.7, 139.3,
142.4, 145.1, 147.6, 148.7, 149.3, 152.5, 154.9, 165.1. MS: m/z (%) 458.21 (M, 100). Anal. for C28H18N4O3
(458.47); Calcd. C, 73.35; H, 3.96; N, 12.22. Found: C, 71.22; H, 4.14; N, 11.49.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(furan-2-yl)pyridine-3-carbonitrile (4r): Yield
◦
2.14 g (45.3%), m.p. 174–176 C. IR:
δ
ν
max/cm−1 2216 (C N), 1590, 1521 (C=N, C=C). 1H-NMR (CDCl3):
≡
1.51 (t, J = 7 Hz, 3H), 4.62 (q, J = 7 Hz, 2H), 6.53 (s, 1H), 7.00 (s, 1H), 7.15–7.30 (m, 3H), 7.39–7.55 (m,
7H), 7.81 (d, 2H), 8.35 (s, 1H). Anal. for C29H20N4O3 (472.49); Calcd. C, 73.72; H, 4.27; N, 11.86. Found:
C, 73.65; H, 4.29; N, 11.83.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-(thiophen-2-yl)pyridine-3-carbonitrile (4s):
yield 1.58 g (33.3%), m.p. 232–234 ◦C. IR:
ν
max/cm−1 2211 (C N), 1629, 1523 (C=N, C=C). 1H-NMR
≡
(CDCl3):
54.8, 92.9, 105.8, 111.6, 112.9, 120.1, 121.4, 123.3, 127.2, 127.9, 128.6, 129.1, 129.7, 130.1, 139.3, 142.4,
δ
4.19 (s, 3H), 7.06 (s, 1H), 7.25–7.52 (m, 11H), 7.81 (d, 2H), 8.40 (s, 1H). 13C-NMR (CDCl3):
δ
143.2, 148.8, 153.0, 154.9, 165.0. MS: m/z (%) 474.18 (M, 100). Anal. for C28H18N4O2S (474.53); Calcd. C,
70.87; H, 3.82; N, 11.81. Found: C, 70.79; H, 3.74; N, 11.74.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(thiophen-2-yl)pyridine-3-carbonitrile (4t): Yield
◦
2.06 g (42.2%), m.p. 202–204 C. IR:
ν
max/cm−1 2217 (C N), 1591, 1546 (C=N, C=C). 1H-NMR (CDCl3):
≡
1H-NMR (CDCl3):
δ
1.52 (t, J = 7 Hz, 3H), 4.63 (q, J = 7 Hz, 2H), 6.35 (s, 1H), 6.65 (s, 1H), 7.26–7.52 (m,
14.6. 63.8,
3H), 7.64–7.83 (m, 7H), 8.25 (d, J = 8 Hz, 2H), 8.36 (s, 1H), 9.65 (s, 1H). 13C-NMR (CDCl3):
δ
93.0, 105.8, 111.6, 112.7, 115.5, 117.5, 120.1, 121.4, 123.4, 125.1, 127.1, 127.9, 128.6, 129.0, 129.7, 130.0,
139.3, 142.4, 143.4, 147.2, 148.8, 153.0, 155.0, 165.0. MS: m/z (%) 488.34 (M, 100). Anal. for C29H20N4O2S
(488.56); Calcd. C, 71.29; H, 4.13; N, 11.47. Found: C, 71.22; H, 4.14; N, 11.49.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-(pyridin-2-yl)pyridine-3-carbonitrile (4u):
Yield 1.85 g (39.4%), m.p. 275–277 ◦C. IR:
ν
max/cm−1 2218 (C N), 1588, 1545 (C=N, C=C). 1H-NMR
≡
(DMSO-d6):
δ 4.18 (s, 3H), 7.09 (s, 1H), 7.25–7.62 (m, 10H), 7.98 (d, J = 6.7 Hz, 2H), 8.26 (s, 1H), 8.66 (s,
1H), 9.15 (s, 1H).MS: m/z (%) 469.39 (M, 100). Anal. for C29H19N5O2 (469.49); Calcd. C, 74.19; H, 4.08;
N, 14.92. Found: C, 74.24; H, 4.15; N, 14.86.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(pyridin-2-yl)pyridine-3-carbonitrile (4v): yield
◦
1.75 g (36.2%), m.p. 204–206 C. IR:
δ
ν
max/cm−1 2219 (C
≡
N), 1589, 1543 (C=N, C=C). 1H-NMR (CDCl3):
1.55 (t, J = 6.7 Hz, 3H), 4.69 (q, J = 6.7 Hz, 2H), 7.03 (s, 1H), 7.19–7.53 (m, 13H), 7.82 (d, J = 8.6 Hz,
2H), 8.27–8.35 (m, 2H), 8.65 (s, 1H). 13C-NMR (CDCl3):
δ
14.6, 63.7, 95.8, 104.9, 105.5, 111.6, 119.8, 120.0,
121.3, 123.1, 124.8, 128.9, 129.7, 137.0, 139.4, 142.5, 142.6, 148.1, 149.6, 154.3, 156.5, 164.6. MS: m/z (%)

Molecules 2017, 22, 1820
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483.25 (M, 100). Anal. for C30H21N5O2 (483.52); Calcd. C, 74.52; H, 4.38; N, 14.48. Found: C, 74.36; H,
4.24; N, 11.56.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-methoxy-6-(1-methyl-1H-benzo[d]imidazole-2-yl)pyridine-
◦
−1
3-carbonitrile (4w): yield 2.05 g (39.2%), m.p. 295–297 C. IR:
ν
max/cm 2214 (C N), 1586, 1538 (C=N,
≡
C=C). 1H-NMR (CDCl3):
δ
4.18 (s, 3H), 4.35 (s, 3H), 7.14 (s, 1H), 7.27–8.01 (m, 13H), 8.26 (s, 1H), 9.16
(s, 1H).MS: m/z (%) 522.1 (M, 20.5). Anal. for C32H22N6O2 (522.56); Calcd. C, 73.55; H, 4.24; N, 16.08.
Found: C, 73.49; H, 4.22; N, 16.13.
4-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-2-ethoxy-6-(1-methyl-1H-benzo[d]-imidazol-2-yl)pyridine-
3-carbonitrile (4x): yield 2.12 g (39.5%), m.p. 247–249 ◦C. IR:
ν
max/cm−1 2218 (C
≡
N), 1587, 1539 (C=N,
1.56 (t, J = 7 Hz, 3H), 4.36 (q, J = 7 Hz, 2H), 4.64 (s, 3H), 7.1 (s, 1H), 7.20–7.53
(m, 11H, Ar–H), 7.77, 7.83 (dd, 2H, J = 7.65, J = 7.65, Ar–H), 8.33 (s, 1H), 8.39 (s, 1H). 13C-NMR (CDCl3):
14.6, 33.2, 64.1, 95.9, 105.4, 110.1, 111.7, 118.7, 120.0, 120.6, 121.4, 123.1, 123.3, 124.4, 124.8, 127.8, 128.9,
C=C). 1H-NMR (CDCl3):
δ
δ
129.7, 137.5, 139.3, 142.5, 142.7, 148.1, 148.3, 148.9, 150.7, 155.0, 164.3. MS: m/z (%) 536.44 (M, 100).
Anal. for C33H24N6O2 (536.58); Calcd. C, 73.87; H, 4.51; N, 15.66. Found: C, 73.91; H, 4.45; N, 15.58.
3.2. Vasodilation Activity Screening
The vasodilation activity screening procedures were carried out according to the standard
reported techniques [35] by testing the effects of the synthesized 2-alkoxy-4-aryl-6-(benzofuran-2-yl)-
3-pyridinecarbonitriles 4a–x on isolated thoracic aortic rings of male Wistar rats (250–350 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 (3–5 mm
width) rings and each ring was placed in a vertical chamber “10 mL jacketed 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
±
0.5 C. Each aortic ring was
mounted between 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 Instruments Pty., Ltd. Victoria,
Australia), 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 hydrochloride (10−6 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 concentrations as those
used with the derivatives tested, which demonstrated that the solvent did not affect the contractile
response of isolated aorta. The observed vasodilation activity screening data are reported and the
potency (IC50, concentration necessary for 50% reduction of maximal norepinephrine hydrochloride
induced contracture) was calculated in three successful replicates and the observed vasodilation
activity data expressed as IC50 has determined mathematically from the dose response curve of
each tested compound (Table 1), (Figures S1 and S2 of Supplementary Materials) and the potency
(IC50, concentration necessary for 50% reduction of maximal norepinephrine hydrochloride-induced
contracture) was determined. This experiment was carried out in according to recommendations
in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH
publication No. 85–23, revised 1996) and under regulations of the Animal Care and Use of National
Research Centre in Egypt.
2D-QSAR Study
The QSAR study was undertaken using comprehensive descriptors for structural and statistical
analysis (CODESSA PRO) software employing the synthesized compounds 4a–x of the present

Molecules 2017, 22, 1820
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study (Table 3). Geometry of the compounds was optimized using molecular mechanics force field
(MM+) followed by the semiempirical AM1 method implemented in the HyperChem 8.0 package.
The structures were fully optimized without fixing any parameters, thus bringing all geometric
variables to their equilibrium values. The energy minimization protocol employed the Polake–Ribiere
conjugated gradient algorithm. Convergence to a local minimum was achieved when the energy
gradient was
for the two semiempirical tools [39
≤
0.01 kcal/mol. The Restricted Hartree–Fock (RHF) method was used in spin-pairing
44]. The resulting output files were exported to CODESSA PRO
–
that includes MOPAC capability for final geometry optimization. CODESSA PRO calculated 797
molecular descriptors including constitutional, topological, geometrical, charge-related, semiempirical,
thermodynamic, molecular-type, atomic-type and bond-type descriptors for the exported 24 bioactive
benzofuran-based hybrids 4a
–
x, which were used in the present study. Different mathematical
transformations of the experimentally observed property/activity (IC50,
µM, which is the concentration
necessary for 50% reduction of maximal norepinephrine hydrochloride induced contracture) of the
training set compounds were utilized for the present QSAR modeling determination including property
(IC50, µM), 1/property, log(property) and 1/log(property) values in searching for the best QSAR
models. Best multilinear regression (BMLR) was utilized, which is a stepwise search for the best
n-parameter regression equations (where, n stands for the number of descriptors used), based on the
highest R2 (squared correlation coefficient), R2cvOO (squared cross-validation “leave one-out, LOO”
coefficient), R2cvMO (squared cross-validation “leave many-out, LMO” coefficient), F (Fisher statistical
significance criteria) values, and s2 (standard deviation). The QSAR models, with up to four descriptor
models describing the bioactivity of the vasodilatory active agents were generated (obeying the thumb
rule of 6:1, which is the ratio between the data points and the number of QSAR descriptor models).
Statistical characteristics of the QSAR models are presented in Table 2. The established QSAR model
is statistically significant. The descriptors are sorted in descending order of the respective values of
the Student’s t-criterion, which is a widely accepted measure of statistical significance of individual
parameters in multiple linear regressions. Figure 2 exhibits the QSAR multilinear model plot of
correlation representing the observed vs. predicted 1/IC50 values for vasodilatory active agents.
The scattered plots are uniformly distributed, covering ranges, Observed 0.00221–0.00448; Predicted
0.00255–0.004411/IC50 units.
3.3. Toxicological Bioassay
Toxicological bioassay of the most promising vasodilatory active compounds (4a
, c, e, f, g, l,
m, r, s, t, w and 4x) was determined using the standard reported method in mice [45]. Albino mice
weighing 25–30 g were divided in 13 groups of 6 mice each. Administrations of the tested compounds
dissolved in saline solution (0.9%) by the aid of few drops of Tween 80 were given intraperitoneally
in 1000 mg kg−1 (mouse body weight). The control group was given saline solution only with few
drops of Tween 80. The toxic symptoms and mortality rates were recorded 24 h post-administration in
each group.
4. Conclusions
The required 2-alkyloxy-pyridine-3-carbonitrile hybrids (4a
via the condensation reaction of aromatic ketones 3a with 2-((3-(benzofuran-2-yl)-1-phenyl-1H-
pyrazol-4-yl)methylene)malononitrile ( ) in the presence of sufficient amount of sodium alkoxide in
–x) were designed and synthesized
-
l
2
the corresponding alcohol. The compounds have evaluated for their vasodilation activity adopting
the standard technique “using isolated thoracic aortic rings of rats precontracted with norepinephrine
hydrochloride”. Some compounds revealed a noteworthy activity, with compounds 4w
4s 4f and 4g believed to be the most active hits in this study. “IC50, concentration necessary for
50% reduction of maximal norepinephrine hydrochloride induced contracture = 223, 253, 254, 268,
267 and 275 M, respectively”, compared with amiodarone hydrochloride, the reference standard
used (IC50 = 300 µM). The CODESSA PRO program was utilized to achieve a statistically significant
, 4e, 4r,
,
µ

Molecules 2017, 22, 1820
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2D-QSAR model describing the bioactivity of the newly synthesized analogues 4a
–
x
, and afforded an
excellent predictive and statistically vital four-crucial 4 descriptor model (R2 = 0.816, R2
= 0.731,
observed
R2pridicted = 0.772). It is obvious that the 2D-QSAR study supported the attained model, so the
applicability of benzofuran-based hybrids incorporating the 3-pyridinecarbonitrile function have
potential to be developed into vasorelaxant active agents.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: The project was financially supported by King Saud University, Vice Deanship of Research
Chairs. The project was also financially supported by National Research Centre, Dokki, Cairo 12622, Egypt,
under project No. 110-10-312.
Author Contributions: The listed authors contributed to this work as described in the following. A.M.S. and
S.S.A.E.-k. carried out the synthetic work, interpreted the results and prepared the manuscript; N.M.K. and
M.A.A.-O. interpreted the results and cooperated in the preparation of the manuscript, and D.O.S. carried and
interpreted the results of the biological activities. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflicts of interest
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).