Design, synthesis, biological evaluation and molecular docking studies of novel benzofuran-pyrazole derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2015.08.006

This study deals with design and synthesis of novel benzofuran-pyrazole hybrids as anticancer agents. Eight compounds were chosen by National Cancer Institute (NCI), USA to evaluate their in vitro antiproliferative activity at 10^-5 M in full NC

Full text of DOI:10.1016/j.bioorg.2015.08.006

Accepted Manuscript
Design, synthesis, biological evaluation and molecular docking studies of novel
benzofuran-pyrazole derivatives as anticancer agents
Somaia S. Abd El-Karim, Manal M. Anwar, Neama A. Mohamed, Tamer Nasr,
Samia A. Elseginy
PII:
S0045-2068(15)30015-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.08.006
YBIOO 1838
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 July 2015
25 August 2015
25 August 2015
Please cite this article as: S.S. Abd El-Karim, M.M. Anwar, N.A. Mohamed, T. Nasr, S.A. Elseginy, Design,
synthesis, biological evaluation and molecular docking studies of novel benzofuran-pyrazole derivatives as
anticancer agents, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.08.006
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1
Design, synthesis, biological evaluation and molecular docking
studies of novel benzofuran-pyrazole derivatives as anticancer agents
Somaia S. Abd El-Karima, Manal M. Anwar a,*, Neama A. Mohameda, Tamer
Nasrb, Samia A. Elseginyc,d
aTherapeutical Chemistry Department, National Research Centre, Dokki, Cairo,
12622, Egypt.
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University,
11795 Helwan, Cairo, Egypt
cGreen Chemistry Department, National Research Centre, Cairo, Egypt.
dSchool of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward
VII avenue, Cardiff, CF10 3NB, UK.
* To corresponding author: e-mail: manal.hasan52@live.com (M.M. Anwar)
Tel.: +20 1223956970; fax: + 20 233370931.
________________________________________________________________________
Abstract
This study deals with design and synthesis of novel benzofuran-pyrazole hybrids as
anticancer agents. Eight compounds were chosen by National Cancer Institute (NCI),
USA to evaluate their in vitro antiproliferative activity at 10-5 M in full NCI 60 cell
panel. The preliminary screening of the tested compounds showed promising broad-
spectrum anticancer activity. Compound 4c was further assayed for five dose molar
ranges in full NCI 60 cell panel and exhibited remarkable growth inhibitory activity
pattern against Leukemia CCRF-CEM, MOLT-4, Lung Cancer HOP-92, Colon
Cancer HCC-2998, CNS Cancer SNB-75, Melanoma SK-MEL-2, Ovarian Cancer
IGROV1, Renal Cancer 786-0, RXF 393, Breast Cancer HS 578T and T-47D (GI50:
1.00 – 2.71 µM). Moreover, enzyme assays were carried out to investigate the
possible antiproliferative mechanism of action of compound 4c. The results revealed
that compound 4c has good c-Src inhibitory activity at 10 µM. In Addition, molecular
docking studies showed that 4c could bind to the ATP Src pocket sites. Fulfilling the
Lipinskiís rule of five in addition to its ADME profile and the biological results, all
strongly suggest that 4c is a promising Src kinase inhibitor.
Keywords: Anticancer, Molecular docking, Benzofuran, Pyrazole

2
___________________________________________________________________________
1. Introduction
Cancer still remains a potentially life threatening disease and the number of
cancer related deaths are increasing alarmingly. Literature clearly indicated that more
than 90% of cancer patients die due to chronic tumor metastases. Despite the presence
of a large number of anticancer drugs, no currently available agents can eradicate
cancer cells without harming normal tissues. Thus, the development of newer
chemotherapeutic scaffolds which selectively act on the target without side effects has
become a primary objective of medicinal chemists [1,2].
Literature survey revealed that substituted benzofurans could act as anticancer agents.
It has been reported that phenylbenzofuran-2-carboxylic acid ethyl ester derivative (I)
showed selective and potent cytotoxicity for human fibroblasts of lung tissues (EC50;
40 ng/mL) [3]. In order to get rid of the biologically unstable ester group the 2-
benzoyl-phenylbenzofuran derivative (II) was derived as another lead compound
(EC50; 600 ng/mL) [3]. Similarly, it has been reported that the 2-(2,4-
dimethoxybenzoyl)-phenylbenzofuran derivative III was exhibiting potent cytotoxic
activities against human lung carcinoma cells [4]. Moreover, Ariad Pharmaceuticals
disclosed compound IV which carries benzofuran subunit in its structural molecule
for the treatment of hyper-resorptive bone disorders through the direct inhibition of
the Src protein tyrosine kinase (IC50 = 22 μM) [5].
Further literature survey revealed that various N-substituted pyrazoles have been
implemented as antileukemic, antitumor, antiproliferative, anti-angiogenic, DNA
interacting, proapoptotic, autophagy and antitubulin agents. In addition, these
compounds are capable to exert remarkable anti-cancer effects through inhibition of
different types of enzymes, proteins and receptors which play critical roles in cell
division [6]. Moreover, the compound V bearing N-phenylpyrazole nucleus was
developed by Vertex Pharmaceuticals as an inhibitor of Src kinases and c-Jun N-
terminal kinases (JNKs) [5].
< Insert Fig. 1>
The non-receptor tyrosine kinase c-Src belongs to a family of closely related kinases
and is widely expressed in all cell types. Over the past century, large amounts of data

3
have been generated supporting the role of c-Src as a key messenger in many
important cellular pathways, including those involved in regulating proliferation,
differentiation, survival, motility, adhesion, migration, and angiogenesis [7,8].
Because of its essential role in many intracellular signaling pathways, however,
interrupting c-Src signaling may disrupt oncogenic pathways. Research has revealed
that c-Src is present in a low concentration inactive form in the normal cells, while
increase in its activity can be linked to increased motility/ invasiveness of tumor cells
and tumor progression [9,10]. Although c-Src is rarely found as a mutated oncogene
in human malignancies, elevated expression and activation of Src family kinases
(SFKs) have been implicated in a variety of cancers (including glioblastoma, colon,
lung, breast, and prostate cancers), osteoporosis and stroke-induced vascular
permeability [11-13]. Moreover, beneficial effects of c-Src inhibition in several
pathological models (cell cycle arrest of tumor cell lines, growth inhibition of Src-
transformed fibroblast derived tumors, metastasis inhibition in human tumor models)
has been demonstrated [14,15].
Based on the afore-mentioned findings, and in an attempt to find new potent c-Src
inhibitors as anticancer agents; novel hybrid compounds having benzofuran-pyrazole
backbone have been designed to evaluate their cytotoxic activity against several
tumor cell lines (Fig. 2). This backbone was conjugated with different aromatic or
heteroaromatic rings of documented potent antitumor activity such as isoxazole ring
[16,17] or c-Src inhibiting activity such as pyrimidine heterocycle [18,19] in a trial to
generate a new class exhibiting both enzymatic and cellular c-Src inhibition activities
for maximal anti-tumor effects (Fig. 2). All the newly synthesized compounds were
submitted to the National Cancer Institute (NCI), Bethesda, Maryland, USA, to
evaluate their anticancer activity. Only eight compounds were selected by NCI and
subjected to in vitro anticancer screening against 60 human cancer cell lines.
Furthermore, the inhibitory activity was evaluated against c-Src at Kinexus
Corporation. Molecular docking studies were carried out to explain how these
compounds could act at the molecular level.
< Insert Fig. 2>
2. Results and Discussion
2.1. Chemistry

4
The synthetic pathways adopted for the preparation of the new benzofuran-
pyrazole hybrid derivatives in this study is depicted in (Scheme 1). Using Vilsmeier
Haach reaction, the key starting material 3-(benzofuran-2-yl)-1-phenyl-1H-pyrazole-
4-carbaldehyde (1) was prepared according to the reported method [20]. It is
documented that chalcones are convenient intermediates for the synthesis of five and
six membered-heterocycles [21-23]. The chalcones 2a-d were obtained in good yields
by Claisen-Schmidt condensation of 3-(benzofuran-2-yl)-1-phenyl-1H-pyrazole-4-
carbaldehyde (1) with various substituted ketones namely; p-chloroacetophenone,
cyclohexylmethyl ketone, 2-acetylpyrrole and/or 2-acetylfuran in ethanolic sodium
hydroxide solution. The elemental analyses and spectral data confirmed the molecular
1
structures of the synthesized derivatives. H-NMR spectra of the obtained derivatives
displayed a pair of doublets at δ 6.31-7.10 and 7.60-8.10 ppm, with coupling constant
values (J=15.0-15.5 Hz) due to the trans-olefinic protons. Upon treatment of the
chalcone derivatives 2a-d with hydrazine hydrate in ethanol and/or acetic acid
afforded the corresponding pyrazoline and N-acetylpyrazoline derivatives 3a-d and
4a-d; respectively. The chemical structures of the obtained derivatives were
1
established depending on the elemental and spectral data. For example, H-NMR
spectra of compounds 3a-d exhibited, besides the expected signals of the parent
protons, the two methylene protons of CH2 of the pyrazoline ring as a pair of douplets
at the regions δ 2.91-3.11 and 3.41-3.65 ppm, while its methine proton CH presented
1
as two douplets at the range δ 5.19-5.26 ppm. Regarding H-NMR spectra of 4a-d,
they represented the acetyl protons as a singlet signal at δ 2.19-2.32 ppm, the
methylene protons of the pyrazoline ring as two douplets at δ 2.98-3.20 and 3.86-4.02
ppm and the methine proton as a pair of douplets at δ 5.87-5.98 ppm. Further
cyclocondensation reaction of the chalcone derivatives 2a-d with hydroxyl amine
hydrochloride and thiourea was carried out to obtain the corresponding isoxazole and
thiopyrimidine analogues 5a-d and 6a-d, respectively. The obtained derivatives were
investigated on the basis of elemental analyses and spectral data. For example, their
mass spectra were consistent with their chemical structures (Scheme 1).
<Insert Scheme 1>
2.2.
Pharmacology
2.2.1. Screening of anticancer activity

5
All the newly synthesized compounds were subjected to NCI in vitro
anticancer screening against full 60 human cancer cell lines organized into nine sub-
panels derived from nine different human cancer types: leukemia, lung, colon, CNS,
melanoma, ovarian, renal, prostate and breast cancer cell lines using a single high
dose concentration (10-5 M) under the drug discovery program of the NCI [24]. Eight
compounds were selected on the basis of the degree of structure variations and
computer modelling techniques for evaluation of their antineoplastic activity. The
selected derivatives were 2a, 2c, 2d, 3c, 4c, 4d, 5d and 6d. The screening results of
the promising compounds 2c, 2d, 3c, 4c, 4d, 5d were summarized in (Table 1).
Program generated data associated with the reference standard Staurosprorin
(registered as NSC 618487) may retrieved from the DTP web site
http://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp
<Insert Table 1>
The output of the single dose screening is reported as graphs of mean growth
percent of the treated cells (Figures I-IV, supplementary data). This allows detecting
both growth inhibition values (between 0 and 100) and cytotoxicity values (less than
0). Moreover, the results of single dose screening were analyzed by COMPARE
program. Among the screened compounds, compound 4c, benzofuran nucleus in
conjugation with pyrrole-N-acetylpyrazoline core, exhibited significant growth
inhibition against variety of cell lines. Thus, compound 4c was further evaluated at
five different minimal concentrations (0.01, 0.1, 1, 10 and 100 µM) by NCI team
(Table 2).
Compound 4c displayed cell growth promotion for Leukemia K-562 and SR
(43.16 % and 39.11%; cell growth inhibition: 56.84% and 60.89%; respectively),
Non-small cell lung cancer NCI-H322M and NCI-H460 (59.13% and 19.08%; cell
growth inhibition: 40.87% and 80.92%; respectively), Colon cancer HCT-116, KM12
and SW-620 (27.86% , 58.51% and 59.18%; cell growth inhibition: 72.14%, 41.49
and 40.82%; respectively), CNS cancer SNB-75 and U251 (41.98% and 26.06%; cell
growth inhibition: 58.02% and 73.94%; respectively), Melanoma LOX IMVI and
MDA-MB-435 (27.31% and 49.54; cell growth inhibition: 72.69% and 50.64%;
respectively), MALME-3M (cell growth promotion -2.87%; cytotoxic), Ovarian
cancer OVCAR-4 and OVCAR-8 (43.55% and 55.5%; cell growth inhibition: 56.45%

6
and 44.50%; respectively). In addition to its cytotoxic activity against Melanoma
MALME-3M, it showed cytotoxic activity against the renal cancer 786-0. Also it
displayed cell growth promotion for renal A498, ACHN, CAKI-1, RXF 393, TK-10
and UO-31 in the range (26.36-65.85%) and cell growth promotion for Prostate
cancer PC-3 (42.28%), and Breast cancer MCF7, MDA-MB-231/ATCC, HS 578T,
T-47D in the range (45.95-55.44%).
The replacement of the pyrrole functionality of compound 4c with furan
heterocyclic ring system as compound 4d produced the second active candidate in this
study. Compound 4d showed cell growth promotion for Leukemia MOLT-4,
Melanoma SK-MEL-5, Prostate cancer PC-3, Breast cancer MCF7 and MDA-MB-
468 (21.91%, 26.70%, 58.63%, 58.28% and 22.28%; cell growth inhibition: 87.09%,
73.3%, 41.37%, 41.72%, and 77.72%; respectively). Moreover, the conjugation of the
bio-isosteric isoxazole ring to the furan moiety instead of the pyrazoline heterocycle
as derivative 5d retained the potency against the growth promotion of different types
of the carcinoma cell lines. Compound 5d displayed cell growth promotion for
Leukemia CCRF-CEM, MOLT-4, SR (57.57%, 50.54% and 44.22%; cell growth
inhibition: 42.43%, 49.46 and 55.78%; respectively). Additionally, it showed cell
growth promotion for CNS cancer SNB-75 and Prostate cancer PC-3 (55.60% and
56.34%; cell growth inhibition: 44.40% and 43.66%; respectively). Moreover,
compound 5d showed cell growth promotion for Breast cancer MCF7, MDA-MB-
231/ATCC and T-47D (51.64%, 55.65% and 45.72%; cell growth inhibition: 48.36%,
44.35 and 54.28%; respectively). On the other hand, the sensitivity of the tested
human carcinoma cell lines apparently decreased towards the pyrrole-chalcone 2c, the
furan-chalcone 2d. The results revealed that the pyrrole-chalcone 2c emerged activity
against Leukemia MOLT-4, SR and Breast cancer MCF7, T-47D (cell growth
promotion 69.07, 67.66%, 50.02% and 55.66%, inhibition 30.93, 32.34%, 49.02 and
44.34%; respectively). The furan-chalcone 2d displayed growth inhibition potency
against Leukemia MOLT-4 (cell growth promotion 67.04%, inhibition 32.96%).
Unfortunately, the tested human cell lines exhibited weak response towards the parent
p-chlorophenyl chalcone 2a and the pyrrolo-pyrazoline compound 3c.
2.2.2. In vitro 5 dose full NCI 60 cell panel assay
Compound 4c satisfied pre-determined threshold growth inhibition criteria and
was further selected for NCI full panel five dose assay. All the sixty cell lines,
representing nine tumor subpanels, were incubated at 10-fold dilutions of five

7
different concentrations (0.01, 0.1, 1, 10 and 100 µM). The dose response curve of
nine sub-panel cell lines for compound 4c and the reference drug Staurosprorin were
represented in Figures V, VI (supplementary data).
The results of the tested compound were given by three response parameters
(GI50, TGI and LC50) for each cell line from log concentration vs the percent growth
inhibition curves on nine cancer diseases (Table 2). The GI50 value (growth inhibitory
activity) corresponds to the concentration of the compound causing 50% decrease in
net cell growth, the TGI value (cytostatic activity) is the concentration of the
compound resulting in total growth inhibition and LC50 value (cytotoxic activity) is
the concentration of the compound causing net 50% loss of initial cells at the end of
the incubation period of 48h (Table 2). The mean graph midpoints (MG-MID) were
also calculated for GI50 parameter, giving an average activity parameter over all cell
panels for the tested compound 4c.
< Insert Table 2 >
The results revealed that the compound 4c exhibited promising growth
inhibition potency against the tested tumor cell lines with average GI50 values (MG-
MID) 2.63-7.34 µM, namely, Leukemia CCRF-CEM (GI50: 1.00 µM), MOLT-4 (GI50:
1.00 µM), Non-Small Cell Lung Cancer HOP-92 (GI50: 2.15 µM), Colon Cancer
HCC-2998 (GI50: 2.71 µM), CNS Cancer SNB-75(GI50: 1.79 µM), Melanoma SK-
MEL-2 (GI50: 2.57 µM), SK-MEL-28 (GI50: 2.85 µM), SK-MEL-5 (GI50: 2.80 µM),
Ovarian Cancer IGROV1 (GI50: 1.90 µM), Renal Cancer 786-0 (GI50: 2.54 µM), RXF
393 (GI50: 2.42 µM), Breast Cancer HS 578T (GI50: 1.26 µM) and T-47D (GI50: 2.68
µM) (Table 2).
2.2.3. Enzyme activity inhibition assay
Evaluation of the protein kinase inhibition activity of the most active target
compounds was the main focus of this study. Thus, compound 4c was assayed for its
activity as Src protein kinase inhibitor. The assay was carried out at Kinexus
Corporation, Vancouver, BC, Canada using the radio-labeled 33P-ATP assay format.
The assay conditions were optimized to yield acceptable enzymatic activity with high
signal-to-noise ratio. The results were recorded as the percent change of the activity in
comparison to the control. The intra-assay variability was determined to be less than
10%. Inhibition of the target activity by the compound gives negative value while

8
activation of the target activity gives positive value. Kinexus considers only values of
more than 25% change in activity compared to control to be significant. The results
showed that compound 4c inhibited Src kinase at 10 µM concentration by 59 %
compared to the control. The reference standard Staurosprorin inhibited Src kinase by
83 % at the same concentration. According to this promising result, it was interesting
to find other protein kinase targets for the tested compound. Thus, it was assayed at
10 µM concentration against other seven protein kinases; AURORA A, BRAF,
CDK2/Cyclin A2, ZAP-70, EGFR, FGFR1 and PI3Kγ. Unfortunately, only ZAP-70
was inhibited by 49 % compared to the control and the rest of the protein kinases were
not significantly inhibited (Table 3). The good inhibition activity of compound 4c
against Src and ZAP-70 kinases might explain its high and broad spectrum anti-
proliferative potency against many NCI human cancer cell lines in which elevated
expression of Src family kinases (SFKs) [10-12] and ZAP-70 [25-27] has been
implicated.
< Insert Table 3 >
2.2.4. Docking studies
To further validate our rational design, compound 4c was docked against the
Kinase ATP binding site of Src kinase, using MOE (Molecular Operating
Environment) 2008-10 [28]. The crystal structure of Src kinase in complex with
AP23451 (co-crystalized ligand) was retrieved from the RCSB Protein Data Bank
(PDB; 2BDF) and used as a target for our modelling studies. The native ligand
formed H-bond with the backbone of Met341 and showed hydrophobic interaction
with Leu393 and Leu273. In addition to the conserved amino acids Leu273, Val281,
Ala293, Thr338, Tyr340, Leu393, Gly344 and Ser345 that surrounded the native
ligand [29]. The MOE algorithm was validated by redocking the co-crystalized
ligand. The result of the self-docking showed that the docked pose has the same
binding mode as the native ligand and occupies the same surface area of Src pocket
site (Fig. 3). This result indicates the reliability of MOE algorithm with 2BDF
complex.
< Insert Figure 3 >
The docking results of compound 4c exhibited interesting interactions between
compound 4c and the receptor. It can be identified as following: (i) the NH of the

9
pyrrole ring formed H-bond with Lys343 (2.78Ǻ) and the nitrogen atom of pyrazoline
moiety formed H-bond with Ser345 (3.24Ǻ); one of the conserved amino acids (ii) the
phenyl group of benzofuran moiety was surrounded by Tyr340, Met341 and Ser342
and the pyrazole ring represented closure to Asp348 that might suggest hydrophobic
interactions. The phenyl group of compound 4c showed hydrophobic interaction with
Leu273 and Gly274 (Fig. 4). Moreover, compound 4c showed good fitting within
ATP Src binding site similar to the native ligand (Fig. 5). The good fitting and
interactions of 4c within Src kinase binding site correlate with the enzymatic assay
results.
< Insert Figure 4>
< Insert Figure 5>
Lipinskiís rule of five and ADME profile
We assessed compound 4c using ADME (adsorption, distribution,
metabolism, elimination) method. In particular, we investigated the consistent of
compound 4c to the Lipinskiís rule of five [30]. Briefly, this simple rule is based on
the observation that most orally administered drugs have a molecular weight (MW) of
500 or less, five or fewer hydrogen bond donor sites and 10 or fewer hydrogen bond
acceptor sites. In addition, we calculated the polar surface area (PSA), since it is
another key property that has been linked to the drug bioavailability. Thus, passively
absorbed molecules with a PSA >140 Å are thought to have low oral bioavailability
[31]. Our results showed that compound 4c fulfilled Lipinski rule (MW = 435.48, one
hydrogen bond acceptor and one hydrogen bond donor) (Table 4). In addition, it also
fulfilled the topological descriptors and fingerprints of molecular drug-likeness
structure keys as LogP and Log S.
< Insert Table 4 >
2.2.5. Structure activity relationship (SAR)
The results of the anticancer screening demonstrated the following assumptions
about the structure activity relationship (SAR) (Fig. 6):
< Insert Figure 6 >

10
· The combination of 3-pyrrolo-N-acetylpyrazoline or 3-furano-N-
acetylpyrazoline conjugates with the basic benzofuran-N-phenylpyrazole core
as compound 4c and 4d exhibited the most potent anti-proliferative activity.
· The attachment of the electron withdrawing acetyl group to N-1 of pyrazoline
ring appeared to be essential for potent cancerous inhibitory effect, since the
pyrrolo-pyrazoline candidate 3c represented weak anti-proliferative activity
against most of the tested human cancer cell lines.
· Furthermore, the same high anti-proliferative potency was gained upon
replacement of the N-acetylpyrazoline moiety with the bioisostere isoxazole
ring but retaining the furan heterocycle as compound 5d.
· Apparent decrease in the potency was noticed by the parent chalcones even in
the presence of the pyrrole and furan rings as the derivatives 2a, 2c and 2d.
· The increase in the size of the hetero-ring systems attached to the parent core
as thiopyrimidine rings resulted in weak to moderate anti-proliferative
potency.
· It can be concluded that the highest activity that was obtained by compounds
4c, 4d, 5d could be attributed to the synergistic effects of the parent
benzofuran-N-phenylpyrazole skeleton together with the 3-pyrrolo/furano-N-
acetylpyrazoline or 3-pyrrolo-isoxazole ring systems.
3. Conclusion
In this study, novel benzofuran-pyrazole hybrid derivatives were successfully
synthesized and eight of them were selected by NCI, USA to evaluate their in vitro
anticancer activities against full 60 human cancer cell lines at a single high dose (10-5
M) concentration. The hybrids clubbed with 3-furano-N-acetylpyrazoline and 3-
furano-isoxazole rings 4d, 5d produced remarkable and broad spectrum anticancer
activities. The most potent activity was obtained by the derivative bearing 3-pyrrolo-
N-acetylpyrazoline 4c which was further selected and evaluated by NCI against full
panel of cell lines at five different minimal concentrations. Enzyme assay study
revealed that compound 4c is a good inhibitor of Src and ZAP-70 kinases. Fulfilling
the Lipinskiís rule of five together with its ADME profile and the anticancer results

11
all revealed that compound 4c is a promising scaffold for further modification of more
potent and selective anticancer agents.
4.
4.1. Chemistry
All melting points are uncorrected and were taken in open capillary tubes
Experimental
using Electrothermal apparatus 9100. Elemental microanalyses were carried out at
Microanalytical Unit, Central Services Laboratory, National Research Centre, Dokki,
Cairo, Egypt, using Vario Elementar and were found within ±0.4% of the theoretical
values. Infrared spectra were recorded on FT/IR-4100 Jasco-Japan, Fourier transform,
Infrared spectrometer at cm-1 scale using KBr disc technique at Central Services
1
13
Laboratory, National Research Centre, Dokki, Cairo, Egypt. H-NMR and C-NMR
spectra were determined by using a JEOL AS-500 NMR spectrometer at Central
Services Laboratory, National Research Centre, Dokki, Cairo, Egypt, chemical shifts
are expressed in δ (ppm) downfield from TMS as an internal standard. The mass
spectra were measured with a GC MS-Qp1000EX Shimadzu, Cairo University, Cairo,
Egypt and Finnigan MAT SSQ-7000 mass spectrometer at Central Services
Laboratory, National Research Centre, Dokki, Cairo, Egypt. Follow up of the
reactions and checking the purity of the compounds were made by TLC on silica gel-
precoated aluminium sheets (Type 60, F 254, Merck, Darmstadt, Germany) using
chloroform/methanol (20:1, v/v) and the spots were detected by exposure to UV lamp
at l254 nanometer for few seconds and by iodine vapor. The chemical names given for
the prepared compounds are according to the IUPAC system. 3-(benzofuran-2-yl)-1-
phenyl-1H-pyrazole-4-carbaldehyde (1) was prepared according the reported method
[20].
4.1.1. 3-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(substituted)prop-2-en-1-
one 2a-d
A mixture of carbaldehyde 1 (2.88 g, 0.01 mol) and the appropriate acetyl derivatives
namely; 4-chloroacetophenone, cyclohexylmethyl ketone, 2-acetylpyrrole and/or 2-
acetylfuran (0.01 mol) in alcoholic sodium hydroxide solution (30 mL, 10%) was
stirred for 3h. The reaction mixture was left overnight at room temperature. The
formed precipitate was filtered, washed several times with water, dried and
recrystallized from ethanol to give the target compounds 2a-d respectively.

12
4.1.1.1. 3-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(4-chlorophenyl)prop-
2-en-1-one (2a)
Yield 90%, mp. 154-156oC, pale yellow; IR (KBr, cm-1): 3062 (CH-arom.), 1676
(C=O); 1H NMR (DMSO-d6): d 7.10 (1H, d, J = 15.0 Hz, CH=) 7.22-7.61 (14H, m,
Ar-H), 7.79 (1H, J = 15.0 Hz, CH=), 8.71 (1H, s, pyrazole-H5);13C NMR (DMSO-d6):
d 198.18 (CO), 104.28, 105.94, 111.81, 118.63, 118.95, 119.43, 122.23, 122.46,
125.83, 128.20, 129.24, 129.51, 130.16, 130.37, 130.71, 134.72, 135.70, 138.66, 143,
80, 150.00, 150.48 (arom. C); MS, m/z (%): 424, 426 [M+] (15, 10), 423, 425 [M+-1]
(13, 7), 285 [M+ -C7H4ClO] (100); Anal. For C26H17ClN2O2 (424.88): Calcd. C,
73.50; H, 4.03; N, 6.59; Found: C, 73.23; H, 4.15; N, 6.36.
4.1.1.2. 3-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-cyclohexylprop-2-en-1-
one (2b)
Yield 87%, mp. 176-178oC, yellow powder; IR (KBr, cm-1): 3063 (CH-arom.), 2920,
1
2851 (CH-aliph.), 1640 (C=O); H NMR (DMSO-d6): d 1.31, 1.66, 1.77, 1.90 (10H,
m, 5(CH2)-cyclohexyl protons), 2.69 (1H, m, CH-cyclohexyl proton), 7.02 (1H, d, J =
15.2 Hz, CH=), 7.30-7.86 (10H, Ar-H), 7.97 (1H, d, J = 15.2 Hz, CH=), 9.33 (1H, s,
pyrazole-H5); 13C NMR (DMSO-d6): d 25.76, 26.11, 28.78, 48.95 (cyclohexyl C),
202.34 (CO), 105.73, 111.77, 118.71, 119.35, 122.17, 124.04, 125.52, 125.78, 128.02,
128.49, 129.14, 130.27, 131.83, 139.28, 143.46, 149.76, 154.83 (arom. C); MS, m/z
(%): 396 [M+] (5), 313 [M+-C6H11] (30), 285 [M+-C7H11O] (26), 77 [C6H5] (100);
Anal. For C26H24N2O2 (396.48): Calcd. C, 78.76; H, 6.10; N, 7.07; Found: C, 78.84;
H, 5.91; N, 7.24.
4.1.1.3. 3-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(1H-pyrrol-2-yl)prop-
2-en-1-one (2c)
Yield 88%, mp. 212-214oC, yellow powder; IR (KBr, cm-1): 3237 (NH), 3044 (CH-
1
arom.), 1645 (CO); H NMR (DMSO-d6): 6.31 (1H, d, J = 15.0 Hz, CH=), 7.25-8.10
(14H, m, Ar-H, CH=, pyrrole protons), 9.40 (1H, s, pyrazole-H5), 11.95 (1H, s, NH,
D2O exchangeable); 13C NMR (DMSO-d6): d 178.20 (CO), 105.73, 110.65, 111.79,
117.44, 119.36, 122.14, 124.00, 124.37, 125.66, 126.95, 127.96, 128.54, 129.00,
130.24, 130.56, 133.64, 139.35, 143.38, 149.85, 154.85 (arom. C); MS, m/z (%): 379
[M+] (17), 380 [M++1] (6), 285 [M+-C5H3NO] (100); Anal. For C24H17N3O2 (379.41):
Calcd. C, 75.97; H, 4.52; N, 11.08; Found: C, 75.71; H, 4.88; N, 11.26.

13
4.1.1.4. 3-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(furan-2-yl)prop-2-en-
1-one (2d)
Yield 92%, mp. 144-145oC, yellow powder; IR (KBr, cm-1): 3060 (CH-arom.), 1653
1
(CO); H NMR (DMSO-d6): d 6.67 (1H, d, J = 15.5 Hz, CH=), 7.25-8.10 (14H, m,
Ar-H, CH=, furan protons), 8.69 (1H, s, pyrazole-H5); 13C NMR (DMSO-d6): d
186.50 (CO), 103.60, 103.96, 110.90, 112.25, 112.63, 117.66, 118.56, 124.45, 125.04,
126.88, 128.01, 128.18, 129.47, 139.14, 141.44, 147.22, 148.28, 149.90, 151.94,
151.97, 153.83 (arom. C); MS, m/z (%): 380 [M+] (13), 381 [M++1] (9), 285 [M+-
C5H3O2] (49), 95 [C5H3O2] (100); Anal. For C24H16N2O3 (380.40): Calcd. C, 75.78;
H, 4.24; N, 7.36; Found: C, 75.51; H, 4.50; N, 7.15.
4.1.2. 3-(Benzofuran-2-yl)-4-(3-substituted-4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-
1H-pyrazole 3a-d
A mixture of chalcones 2a-d (0.002 mol) and hydrazine hydrate (0.2 mL, 98%) in
absolute ethanol was refluxed for 3h. The formed precipitate during heating was
filtered, dried and recrystallized from absolute ethanol to give the compounds 3a-d
respectively.
4.1.2.1. 3-(Benzofuran-2-yl)-4-(3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-1-
phenyl-1H-pyrazole (3a)
Yield 56%, mp. 197-199oC, yellow powder; IR (KBr, cm-1): 3233 (NH), 3069 (CH-
arom.), 2969, 2888 (CH-aliph.); 1H NMR (DMSO-d6): d 3.02-3.11 (1H, dd, J = 11.0,
5.7 Hz, pyrazoline-H4), 3.56-3.65 (1H, dd, J = 11.1, 6.8 Hz,pyrazoline-H4ʹ), 5.25-5.31
(1H, dd, J = 12.0, 5.8 Hz, pyrazoline-H5), 5.78 (1H, br.s, NH, D2O exchangeable),
7.09-7.94 (14H, m, Ar-H), 8.62 (1H, s, pyrazole-H5); 13C NMR (DMSO-d6): d 50.04
(pyrazolinyl C-4), 55.70 (pyrazolinyl C-5), 104.83, 111.84, 118.92, 121.84, 123.83,
124.50, 125.28, 127.23, 127.74, 127.84, 128.83, 129.07, 130.17, 132.55, 133.05,
139.71, 141.90, 148.91, 150.08, 154.57 (arom. C); MS, m/z (%): 438, 440 [M+] (14,
5), 77 [C6H5] (100); Anal. For C26H19ClN4O (438.91): Calcd. C, 71.15; H, 4.36; N,
12.77; Found: C, 71.32; H, 4.49; N, 12.53.
4.1.2.2.
3-(Benzofuran-2-yl)-4-(3-cyclohexyl-4,5-dihydro-1H-pyrazol-5-yl)-1-
phenyl-1H-pyrazole (3b)
Yield 98%, mp. 135-137oC, yellow powder; IR (KBr, cm-1): 3128 (NH), 3060 (CH-
arom.), 2925, 2852 (CH-aliph.); 1H NMR (DMSO-d6): d 1.36, 1.70, 1.86 (10H, m,
5(CH2)-cyclohexyl protons), 2.63 (1H, m, CH-cyclohexyl proton), 2.96-3.10 (1H, dd,

14
J = 11.4, 5.1 Hz, pyrazoline-H4), 3.41-3.49 (1H, dd, J = 11.3, 6.1 Hz,pyrazoline-H4ʹ),
5.15-5.21 (1H, dd, J = 11.0, 5.6 Hz, pyrazoline-H5), 7.18-7.99 (10H, m, Ar-H), 8.63
(1H, s, pyrazole-H5), 10.25 (1H, br.s, NH, D2O exchangeable); 13C NMR (DMSO-d6):
d 25.64, 26.07, 30.70, 32.96, 48.97 (cyclohexyl C), 50.57 (pyrazolinyl C-4), 54.75
(pyrazolinyl C-5), 101.82, 104.84, 111.71, 118.85, 118.98, 121.80, 123.76, 127.12,
128.81, 130.16, 139.64, 139.75, 150.17, 154.53 (arom. C); MS, m/z (%): 410 [M+]
(7), 77 [C6H5] (100); Anal. For C26H26N4O (410.51): Calcd. C, 76.07; H, 6.38; N,
13.65; Found: C, 76.34; H, 6.45; N, 13.84.
4.1.2.3. 3-(Benzofuran-2-yl)-4-(4,5-dihydro-3-(1H-pyrrol-2-yl)-1H-pyrazol-5-yl)-1-
phenyl-1H-pyrazole (3c)
Yield 60%, mp. 204-205oC, white powder; IR (KBr, cm-1): 3282, 3212 (2NH), 3056
1
(CH-arom.), 2960, 2856 (CH-aliph.); H NMR (DMSO-d6): d 2.91-3.00 (1H, dd, J =
11.2, 5.0 Hz, pyrazoline-H4), 3.47-3.52 (1H, dd, J = 11.7, 5.9 Hz, pyrazoline-H4ʹ),
5.12-5.16 (1H, dd, J = 12.0, 5.8 Hz, pyrazoline H5), 6.07 (1H, d, J = 6.71 Hz, pyrrole-
H3), 6.28 (1H, m, pyrrole proton), 6.80 (1H, d, J = 6.70 Hz, pyrrole-H5), 7.14-7.93
(10H, m, Ar-H), 8.58 (1H, s, pyrazole-H5), 10.18, 11.19 (2H, 2br.s, 2NH, D2O
13
exchangeable); C NMR (DMSO-d6): d 54.04 (pyrazolinyl C-4), 54.79 (pyrazolinyl
C-5), 104.33, 108.12, 108.84, 109.20, 111.00, 117.99, 118.68, 120.02, 121.00, 121.48,
123.13, 124.65, 125.63, 126.87, 127.50, 128.27, 129.51, 139.18, 141.37, 144.77,
149.63, 154.00 (arom. C); MS, m/z (%): 393 [M+] (85), 392 [M+-1] (39), 77 [C6H5]
(100); Anal. For C24H19N5O (393.44): Calcd. C, 73.27; H, 4.87; N, 17.80; Found: C,
73.55; H, 4.59; N, 17.62.
4.1.2.4.
3-(Benzofuran-2-yl)-4-(3-(furan-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-1-
phenyl-1H-pyrazole (3d)
Yield 52%, mp. 182-183oC, yellow powder; IR (KBr, cm-1): 3229 (NH), 3049 (CH-
arom.), 2934, 2850 (CH-aliph.); 1H NMR (DMSO-d6): d 2.95-3.03 (1H, dd, J = 11.3,
6.1 Hz, pyrazoline-H4), 3.51-3.57 (1H, dd, J = 11.7, 5.8 Hz, pyrazoline-H4ʹ), 5.19-
5.26 (1H, dd, J = 11.9, 6.1 Hz, pyrazoline-H5), 6.56, 6.66 (2H, d, d, J = 6.8 Hz, furan-
H3,5), 7.33-8.00 (11H, m, Ar-H, furan-H4), 8.59 (1H, s, pyrazole-H5), 10.20 (1H, br.s,
13
NH, D2O exchangeable); C NMR (DMSO-d6): d 53.10 (pyrazolinyl C-4), 55.27
(pyrazolinyl C-5), 104.81, 109.86, 111.86, 112.20, 118.92, 121.85, 123.84, 124.35,
125.29, 127.22, 127.84, 130.17, 139.72, 141.86, 142.18, 144.01, 148.92, 150.09,
154.57 (arom. C); MS, m/z (%): 394 [M+] (27), 393 [M+-1] (16), 51 [C4H3] (100);

15
Anal. For C24H18N4O2 (394.43): Calcd. C, 73.08; H, 4.60; N, 14.20; Found: C, 73.31;
H, 4.75; N, 14.43.
4.1.3.
1-(5-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3-substituted-4,5-
dihydropyrazol-1-yl)ethanone 4a-d
A mixture of chalcones 2a-d (0.002 mol) and hydrazine hydrate (0.2 mL, 98%) in
glacial acetic acid (15 mL) was refluxed for 3h. The formed precipitate during heating
was filtered, dried and recrystallized from acetic acid to give the target compounds
4a-d respectively.
4.1.3.1. 1-(5-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(4-chlorophenyl)-
4,5-dihydropyrazol-1-yl)ethanone (4a)
Yield 64%, mp. 160-162oC, creamy powder; IR (KBr, cm-1): 3057 (CH-arom.), 2923,
1
2853 (CH-aliph.), 1662 (C=O); H NMR (DMSO-d6): d 2.23 (3H, s, COCH3), 3.09-
3.16 (1H, m, pyrazoline-H4), 3.98-4.08 (1H, m, pyrazoline-H4ʹ), 5.94-5.98 (1H, m,
pyrazoline-H5), 7.18-7.98 (14H, m, Ar-H), 8.51 (1H, s, pyrazole-H5); 13C NMR
(DMSO-d6): d; 21.26 (COCH3), 42.18 (pyrazolinyl C-4), 52.22 (pyrazolinyl C-5),
168.53 (COCH3), 104.28, 111.77, 118.92, 121.86, 123.87, 124.14, 125.28, 127.26,
128.92, 129.31, 130.07, 135.06, 139.57, 142.34, 150.51, 153.99 (arom. C); MS, m/z
(%): 480, 482 [M+] (13, 5), 437 [M+-COCH3] (8), 77 [C6H5] (100); Anal. For
C28H21ClN4O2 (480.94): Calcd. C, 69.92; H, 4.40; N, 11.65; Found: C, 70.12; H, 4.68;
N, 11.46.
4.1.3.2.
1-(5-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3-cyclohexyl-4,5-
dihydropyrazol-1-yl)ethanone (4b)
Yield 61%, mp. 127-129oC, creamy powder; IR (KBr, cm-1): 3060 (CH-arom.), 2925,
1
2851 (CH-aliph.), 1658 (CO); H NMR (DMSO-d6): d 1.35, 1.64, 1.79 (10H, m,
5(CH2)-cyclohexyl protons), 2.19 (3H, s, COCH3), 2.62 (1H, m, CH-cyclohexyl
proton), 2.98-3.12 (1H, m, pyrazoline-H4), 3.86-4.01 (1H, m, pyrazoline-H4ʹ), 5.87-
5.92 (1H, m, pyrazoline-H5), 7.13-7.94 (10H, m, Ar-H), 8.66 (1H, s, pyrazole-H5); 13C
NMR (DMSO-d6): d; 21.54 (COCH3), 21.96, 25.25, 29.74 (cyclohexyl C), 42.48
(pyrazolinyl C-4), 50.90 (pyrazolinyl C-5), 167.23 (COCH3), 104.01, 104.19, 111.08,
118.12, 118.62, 121.39, 123.19, 124.00, 124.70, 126.11, 126.66, 128.19, 129.49,
139.03, 140.22, 149.42, 154.02, 162.64 (arom. C); MS, m/z (%): 452 [M+] (10), 453
[M++1] (5), 409 [M+-COCH3] (5), 77 [C6H5] (100); Anal. For C28H28N4O2 (452.55):
Calcd. C, 74.31; H, 6.24; N, 12.38; Found: C, 74.42; H, 6.43; N, 12.16.

16
4.1.3.3.
1-(5-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4,5-dihydro-3-(1H-
pyrrol-2-yl)pyrazol-1-yl)ethanone (4c)
Yield 66%, mp. 140-142oC, grey powder; IR (KBr, cm-1): 3222 (NH), 3040 (CH-
1
arom.), 2933, 2852 (CH-aliph.), 1698 (C=O); H NMR (DMSO-d6): d 2.32 (3H, s,
COCH3), 3.13-3.20 (1H, dd, J = 11.2, 5.7 Hz, pyrazoline-H4), 3.88-3.98 (1H, dd, J =
11.7, 5.8 Hz, pyrazoline-H4ʹ), 5.87-5.92 (1H, dd, J = 12.0, 5.6 Hz, pyrazoline-H5),
6.13, 6.95 (2H, d, d, J =7.0 Hz, pyrrole-H3,5), 6.52 (1H, m, pyrrole-H4), 7.27-8.35
(10H, m, Ar-H), 9.62 (1H, s, pyrazole-H5), 11.43 (1H, br.s, NH, D2O exchangeable);
13C NMR (DMSO-d6): d 22.38 (COCH3), 42.50 (pyrazolinyl C-4), 51.69 (pyrazolinyl
C-5), 168.59 (COCH3), 104.58, 109.80, 111.72, 111.84, 112.78, 118.97, 121.86,
122.81, 122.93, 123.89, 124.16, 124.34, 125.29, 127.27, 128.78, 130.07, 139.58,
140.85, 148.75, 150.03, 154.59, 167.70 (arom. C); MS, m/z (%): 434 [M+-1] (14), 433
[M+-2] (12), 93 [C6H7N] (100); Anal. For C26H21N5O2 (435.48): Calcd. C, 71.71; H,
4.86; N, 16.08; Found: C, 71.52; H, 4.61; N, 16.36.
4.1.3.4.
1-(5-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(furan-2-yl)-4,5-
dihydropyrazol-1-yl)ethanone (4d)
Yield 57%, mp. 149-150oC, pale brown powder; IR (KBr, cm-1): 3062 (CH-arom.),
1
2925, 2849 (CH-aliph.), 1659 (CO); 1H NMR (DMSO-d6): d H NMR (DMSO-d6): d
2.31 (3H, s, COCH3), 3.04-3.12 (1H, m, pyrazoline-H4), 3.92-4.02 (1H, m,
pyrazoline- H4ʹ), 5.91-5.95 (1H, m, pyrazoline H5), 6.63, 6.97 (2H, d, d, J =6.9 Hz,
furan-H3,5), 7.29-7.93 (11H, m, Ar-H, furan-H4), 8.42 (1H, s, pyrazole-H5);13C NMR
(DMSO-d6): d 22.50 (COCH3), 42.75 (pyrazolinyl C-4), 52.00 (pyrazolinyl C-5),
168.21 (COCH3), 104.56, 111.79, 112.72, 118.79, 118.94, 121.87, 123.91, 123.98,
125.32, 127.24, 128.76, 130.06, 139.57, 141.00, 146.04, 146.23, 150.00, 154.59
(arom. C); MS, m/z (%): 436 [M+] (47), 435 [M+-1] (34), 393 [M+-COCH3] (36), 60
[C3H8O] (100); Anal. For C26H20N4O3 (436.46): Calcd. C, 71.55; H, 4.62; N, 12.84;
Found: C, 71.31; H, 4.44; N, 12.97.
4.1.4. 3-(Benzofuran-2-yl)-4-(3-substituted)isoxazol-5-yl)-1-phenyl-1H-pyrazole 5a-
d
A mixture of chalcones 2a-d (0.002 mol) and hydroxyl amine hydrochloride (0.14 g,
0.002 mol) in ethanolic sodium hydroxide solution (15 mL, 5%) was refluxed for 10h.
After cooling, the formed precipitate was filtered, washed several times with water,

17
dried and recrystallized from absolute ethanol to give the compounds 5a-d
respectively.
4.1.4.1.
3-(Benzofuran-2-yl)-4-(3-(4-chlorophenyl)isoxazol-5-yl)-1-phenyl-1H-
pyrazole (5a)
Yield 90%, mp. 160-162oC, pale yellow powder; IR (KBr, cm-1): 3063 (CH-arom.);
1H NMR (DMSO-d6): d 7.11-8.02 (15H, m, Ar-H, isoxazole-H4), 8.59 (1H, s,
pyrazole-H5); 13C NMR (DMSO-d6): d 103.66, 104.77, 111.36, 111.61, 118.82,
121.27, 123.68, 124.51, 125.04, 127.05, 127.22, 127.81, 128.53, 129.13, 130.10,
133.64, 135.31, 139.50, 142.51, 143.13, 150.21, 154.33, 155.21 (arom. C); MS, m/z
(%): 437, 439 [M+] (3, 6), 438, 440 [M++1] (5, 2), 77 [C6H5] (100); Anal. For
C26H16ClN3O2 (437.88): Calcd. C, 71.32; H, 3.68; N, 9.60; Found: C, 71.51; H, 3.35;
N, 9.49.
4.1.4.2.
(5b)
3-(Benzofuran-2-yl)-4-(3-cyclohexylisoxazol-5-yl)-1-phenyl-1H-pyrazole
Yield 97%, mp. 221-223oC, pale yellow powder; IR (KBr, cm-1): 3057 (CH-arom.),
1
2922, 2849 (CH-aliph.); H NMR (DMSO-d6): d 1.39-1.42, 1.76-1.81, 1.95, 2.70
(11H, m, cyclohexyl protons), 7.30-8.02 (10H, m, Ar-H, isoxazole-H4), 9.17 (1H, s,
pyrazole-H5); 13C NMR (DMSO-d6): d 26.62, 26.81, 32.16 (cyclohexyl C), 104.86,
111.47, 117.79, 119.19, 120.65, 122.02, 123.74, 123.97, 125.52, 127.27, 127.56,
128.55, 130.10, 139.49, 142.43, 150.42, 154.71, 157.59 (arom. C); MS, m/z (%): 411
[M++2] (61), 326 [M+-C6H11] (8), 77 [C6H5] (100); Anal. For C26H23N3O2 (409.48):
Calcd. C, 76.26; H, 5.66; N, 10.26; Found: C, 76.41; H, 5.42; N, 10.43.
4.1.4.3.
4-(3-(1H-pyrrol-2-yl)isoxazol-5-yl)-3-(benzofuran-2-yl)-1-phenyl-1H-
pyrazole (5c)
Yield 61%, mp. 220-222oC, brown powder; IR (KBr, cm-1): 3234 (NH), 3057 (CH-
1
arom.); H NMR (DMSO-d6): d 6.30 (1H, d, pyrrole proton), 7.19-8.03 (13H, m, Ar-
H, isoxazole-H4, pyrrole protons), 9.40 (1H, s, pyrazole-H5), 11.98 (1H, s, NH, D2O
exchangeable); 13C NMR (DMSO-d6): d 104.35, 106.88, 111.31, 117.53, 120.22,
121.54, 122.34, 123.51, 123.94, 125.08, 126.98, 127.38, 128.41, 129.13, 131.02,
138.89, 141.71, 151.08, 154, 61, 158.82 (arom. C); MS, m/z (%): 392 [M+] (62), 326
[M+-C4H4N] (67), 92 [C6H6N] (100); Anal. For C24H16N4O2 (392.41): Calcd. C,
73.46; H, 4.11; N, 14.28; Found: C, 73.30; H, 4.25; N, 14.09.

18
4.1.4.4. 3-(Benzofuran-2-yl)-4-(3-(furan-2-yl)isoxazol-5-yl)-1-phenyl-1H-pyrazole
(5d)
1
Yield 79%, mp. 119-121oC, white powder; IR (KBr, cm-1): 3060 (CH-arom.); H
NMR (DMSO-d6): d 6.50, 6.72 (2H, d, d, J = 6.8 Hz, furan-H3,5), 7.18-7.96 (12H, m,
Ar-H, furan-H4, isoxazole-H4), 8.67 (1H, s, pyrazole-H5); 13C NMR (DMSO-d6): d
105.21, 106.02, 111.87, 120.95, 121.07, 121.89, 122.48, 122.86, 123.56, 125.41,
126.28, 127.04, 127.92, 128.71, 130.58, 139.04, 140.64, 150.83, 155, 23, 159.03
(arom. C); MS, m/z (%): 393 [M+] (26), 394 [M++1] (9), 77 [C6H5] (100); Anal. For
C24H15N3O3 (393.39): Calcd. C, 73.27; H, 3.84; N, 10.68; Found: C, 73.56; H, 3.61;
N, 10.42.
4.1.5. 6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4- substituted-pyrimidine-
2(1H)-thione 6a-d
A mixture of chalcones 2a-d (0.002 mol) and thiourea (0.16 g, 0.002 mol) in ethanolic
potassium hydroxide solution (15 mL, 5%) was refluxed for 24h. The reaction
mixture was poured onto ice/cold water acidified by hydrochloric acid. The formed
precipitate was filtered, washed several times with water, dried and recrystallized
from absolute ethanol to give the title compounds 6a-d respectively.
4.1.5.1.
6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4-(4-
chlorophenyl)pyrimidine-2(1H)-thione (6a)
Yield 69%, mp. 148-150oC, yellowsh brown powder; IR (KBr, cm-1): 3125 (NH),
1
3057 (CH-arom.), 1252 (CS); H NMR (DMSO-d6): d 5.44 (1H, s, pyrimidine-H5),
7.18-8.04 (14H, m, Ar-H), 8.57 (1H, s, pyrazole-H5), 9.38 (1H, s, NH, D2O
exchangeable); 13C NMR (DMSO-d6): d 96.84 (pyrimidinyl C5), 177.24 (CS), 104.21,
111.51, 118.35, 121.84, 122.59, 123.60, 124.96, 125.49, 127.04, 127.82, 128.83,
129.37, 131.05, 132.46, 133.12, 140.58, 141.86, 148.72, 151.22, 155.63 (arom. C);
MS, m/z (%): 479, 481 [M+-1] (7, 2), 436, 438 [M+-CS] (3, 8), 77 [C6H5] (100); Anal.
For C27H17ClN4OS (480.97): Calcd. C, 67.42; H, 3.56; N, 11.65; S, 6.67; Found: C,
67.28; H, 3.71; N, 11.43; S, 6.40.
4.1.5.2. 6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4-cyclohexylpyrimidine-
2(1H)-thione (6b)
Yield 88%, mp. 228-230oC, pale yellow powder; IR (KBr, cm-1): 3191 (NH), 3078
(CH-arom.), 2924, 2851 (CH-aliph.), 1281 (C=S); 1H NMR (DMSO-d6): d 1.06-1.18,
1.35, 1.60-1.65, 1.96-2.08 (11H, m, cyclohexyl protons), 5.52 (1H, s, pyrimidine-H5),

19
7.26-7.91 (10H, m, Ar-H), 8.35 (1H, s, pyrazole-H5), 9.53 (1H, s, NH, D2O
exchangeable); 13C NMR (DMSO-d6): d 25.98, 26.34, 30.75, 30.93, 47.50
(cyclohexyl C), 95.71 (pyrimidinyl C5), 175.20 (CS), 104.60, 112.00, 119.07, 121.74,
123.77, 125.18, 126.58, 127.40, 128.59, 128.73, 130.23, 139.62, 141.00, 141.25,
150.07, 154.69 (arom. C); MS, m/z (%): 453 [M++1] (25), 454 [M++2] (20), 55
[C4H7] (100); Anal. For C27H24N4OS (452.57): Calcd. C, 71.65; H, 5.35; N, 12.38; S,
7.09; Found: C, 71.51; H, 5.16; N, 12.57; S, 7.22.
4.1.5.3.
6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4-(1H-pyrrol-2-
yl)pyrimidine-2(1H)-thione (6c)
Yield 79%, mp. 138-140oC, yellowish brown powder; IR (KBr, cm-1): 3249, 3126
(2NH), 3059 (CH-arom.), 1254 (CS); 1H NMR (DMSO-d6): d 5.02 (1H, s,
pyrimidine-H5), 7.20-7.91 (13H, m, Ar-H, pyrrole protons), 8.50 (1H, s, pyrazole-H5),
9.20 (1H, s, NH, D2O exchangeable); 13C NMR (DMSO-d6): d 98.54 (pyrimidinyl
C5), 177.87 (CS), 104.23, 110.35, 111.67, 111.91, 112.53, 119.34, 121.28, 122.61,
123.05, 123.78, 124.32, 124.90, 125.49, 126.95, 128.72, 131.10, 140.27, 141.54,
149.25, 151.61, 155.28 (arom. C) ; MS, m/z (%): 435 [M+] (41), 393 [M++1] (35), 80
[C5H6N] (100); Anal. For C25H17N5OS (435.50): Calcd. C, 68.95; H, 3.93; N, 16.08;
S, 7.36; Found: C, 68.74; H, 3.74; N, 16.26; S, 7.54.
4.1.5.4.
6-(3-(Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-4-(furan-2-
yl)pyrimidine-2(1H)-thione (6d)
Yield 71%, mp. 192-194oC, brown powder; IR (KBr, cm-1): 3127 (NH), 3053 (CH-
1
arom.), 1250 (CS); H NMR (DMSO-d6): d 5.51 (1H, s, pyrimidine-H5), 6.53, 6.66
(2H, m, furan protons),7.31-7.93 (11H, m, Ar-H, furan proton), 8.45 (1H, s, pyrazole-
H5), 9.15 (1H, s, NH, D2O exchangeable); 13C NMR (DMSO-d6): d 96.58
(pyrimidinyl C5), 175.66 (CS), 105.21, 111.66, 112.51, 118.82, 119.24, 121.65,
123.72, 124.38, 125.53, 126.94, 128.40, 130.59, 140.02, 141.34, 145.70, 146.08,
150.61, 155.27 (arom. C); MS, m/z (%): 436 [M+] (10), 403 [M+-SH] (12), 77 [C6H5]
(100); Anal. For C25H16N4O2S (436.49): Calcd. C, 68.79; H, 3.69; N, 12.84; S, 7.35;
Found: C, 68.56; H, 3.80; N, 12.62; S, 7.18.
4.2.
Pharmacology
4.2.1. Evaluation of cytotoxic activity against a panel of sixty human cancer cell
lines

20
The selected compounds by NCI were evaluated for their anticancer activity in
a two-stage process. First, these compounds were screened against the full NCI 60
cell lines panel representing leukemia, Non-Small Cell Lung Cancer, melanoma,
colon cancer, CNS cancer, breast cancer, ovarian cancer, renal cancer and prostate
cancer at a single high dose of 10-5 M. Then, the output from the single dose screen is
reported as a mean graph and is available for analysis by the COMPARE program.
Second, compounds exhibiting significant growth inhibition were evaluated against
the 60 cell panel at five different minimal concentrations (0.01, 0.1, 1, 10 and 100
µM).
a. Assay protocol
The human tumor cell lines of the cancer-screening panel are grown in RPMI
1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical
screening experiment, cells are inoculated into 96 well microtiter plates in 100 mL at
plating densities ranging from 5000 to 40,000 cells/well depending on the doubling
time of individual cell lines. After cell inoculation, the microtiter plates are incubated
at 37οC, 5% CO2, 95% air and 100% relative humidity for 24h prior to addition of the
experimental drugs. After 24h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line at the time of drug
addition (Tz). The experimental drugs are solubilized in dimethyl sulfoxide at 400-
fold the desired final maximum test concentration and stored frozen prior to use. At
the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to
twice the desired final maximum test concentration with complete medium containing
50 mg/mL Gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to
provide a total of five drug concentrations plus control. Aliquots of 100 mL of these
different drug dilutions are added to the appropriate microtiter wells already
containing 100 mL of medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37οC, 5%
CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated
by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 ml of
cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at
4οC. The supernatant is discarded, and the plates are washed five times with tap water
and air dried. Sulforhodamine B (SRB) solution (100 mL) at 0.4% (w/v) in 1% acetic
acid is added to each well, and plates are incubated for 10 min at room temperature.

21
After staining, unbound dye is removed by washing five times with 1% acetic acid
and the plates are air dried. Bound stain is subsequently solubilized with 10 mM
trizma base, and the absorbance is read on an automated plate reader at a wavelength
of 515 nm. For suspension cells, the methodology is the same except that the assay is
terminated by fixing settled cells at the bottom of the wells by gently adding 50 ml of
80% TCA (final concentration, 16% TCA).
b. Data analysis
Using the seven absorbance measurements [time zero, (Tz), control growth, (C),
and test growth in the presence of drug at the five concentration levels (Ti)], the
percentage growth is calculated at each of the drug concentrations levels. Percentage
growth inhibition is calculated as: [(Ti-Tz)/(C-Tz)] × 100 for concentrations for which
Ti ≥ Tz and [(Ti-Tz)/Tz] ×100 for concentrations for which Ti < Tz: three dose
response parameters are calculated for each experimental agent. Growth inhibition of
50% (GI50) is calculated from [(Ti-Tz)/(C-Tz)] ×100 = 50, which is the drug
concentration resulting in a 50% reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation. The drug concentration
resulting in total growth inhibition (TGI) is calculated from Ti ¼ Tz. The LC50
(concentration of drug resulting in a 50% reduction in the measured protein at the end
of the drug treatment as compared to that at the beginning) indicating a net loss of
cells following treatment is calculated from [(Ti-Tz)/Tz] ×100 = -50. Values are
calculated for each of these three parameters if the level of activity is reached;
however, if the effect is not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum concentration tested
[32,33]. Results for each compound were reported as a mean graph of the percent
growth of the treated cells when compared to the untreated control cells. There after
obtaining the results for one dose assay, analysis of historical Development
Therapeutics Programme (DTP) was performed and compounds which satisfies
predetermined threshold inhibition criteria is selected for NCI full panel 5 dose assay.
4.2.2. In-vitro enzyme inhibition assay
The profiling evaluation of in-vitro Src kinase inhibition by compound 4c was
carried out in Kinexus Corporation, Vancouver, BC, Canada using radiolabeled 33P-
ATP assay format. The assay condition for the protein kinases was optimized to yield
acceptable enzymatic activity. A radioisotope assay format was used for profiling
evaluation of protein kinase target and all assays are performed in a designated

22
radioactive working area. Protein kinase assays (in singlicate) were performed at
ambient temperature for 20-30 minutes in a final volume of 25 μL according to the
following assay reaction recipe: 5 µL of diluted active protein kinase (~10-50 nM
final concentration in the assay), 5 µL of stock solution of substrate, 5 µL of kinase
assay buffer, 5 µL of compound (50 μM) or 10% DMSO, 5 µL of 33P-ATP (250 µM
stock solution, 0.8 µCi). The assay was initiated by the addition of 33P-ATP and the
reaction mixture incubated at ambient temperature for 20-30 minutes. After the
incubation period, the assay was terminated by spotting 10 µL of the reaction mixture
onto a Multiscreen phosphocellulose P81 plate. The Multiscreen phosphocellulose
P81 plate was washed 3 times for approximately 15 minutes each in a 1% phosphoric
acid solution. The radioactivity on the P81 plate was counted in the presence of
scintillation fluid in a Trilux scintillation counter. Blank control was set up that
included all the assay components except the addition of the appropriate substrate
(replaced with equal volume of assay dilution buffer). The corrected activity for
protein kinase target was determined by removing the blank control value.
4.3.
Docking studies
4.3.1. Target preparation
The crystal structure of Src ATP kinase in complex with AP23451 was
retrieved from the RCSB Protein Data Bank (PDB ID: 2BDF) http://www.rcsb.org
and was used as a target for our modelling studies. All water molecules were deleted,
hydrogen atoms were added and energy of the system was minimized with MOE
2008.10.
4.3.2. Ligand preparation
Compound 4c was prepared with MOE 2008.10 (Molecular Operating
Environment (http: //www.chemcomp.com.). Ligand structure was built with MOE
and the energy was minimized using the MMFF94x force field until RMSD gradient
of 0.05 Kcal mol-1 Ǻ -1 was reached.
4.3.3. Molecular Docking
Co-crystalized ligand and compound 4c were docked with MOE 2008.1027
within the pocket sites of the target kinases. The site of docking was considered using
crystallographic ligand position. The second step of the procedure was Placement that

23
was chosen to be Triangle Matcher. The third step was Rescoring 1 which was set to
be London dG. Refinement step was chosen to be Force field. The last step: Rescoring
2 was selected to be Affinity dG.
Acknowledgments
This work was supported financially by National Research Centre fund under
project No. P 90202. Also, the authors are thankful to the National Cancer Institute
(NCI), Bethesda, Maryland, USA, for performing the anticancer evaluation over the
60-cancer cell line panel.
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Figure captions:

25
Fig. 1: Examples of 2-substituted-benzofurans and N-substituted pyrazoles as Src
protein tyrosine kinase inhibitors
Fig. 2: Design of benzofuran-pyrazole derivatives as Src inhibitors.
Fig. 3: The docked co-crystalized ligand (yellow, stick) showed the same binding
mode of native ligand (green, stick). The protein represented as cartoon (light
magenta). The important amino acids involved in the interaction (grey, stick).
Fig. 4: The interaction of compound 4c (magenta, stick) within Src ATP binding site.
NH of pyrrole moiety formed H-bond with Lys 343 (2.78Å), N of pyrazoline moiety
formed H-bond with Ser345 (3.24Ǻ). Phenyl group of benzofuran moiety was
surrounded on by Tyr340, Met341and Ser342.
Fig. 5: Compound 4c (magenta, stick) and native ligand (green, stick) within Src ATP
showed the same binding mode indicating good fitting of compound 4c within the
pocket site.
Fig. 6: Structure activity relationship (SAR) of benzofuran-pyrazole derivatives as
anticancer agents.
Scheme caption:
Scheme 1: Synthesis of chalcone, pyrazoline, isoxazole and thiopyrimidine
derivatives.
Table captions:
Table 1: Anticancer screening results of eight selected target compounds against sixty
human tumor cell lines at single dose assay (10-5 M concentration); data was
presented as cell growth promotion percentage.
Table 2: NCI in vitro testing results of compound 4c at five different dose levels in
µM.
Table 3: Enzyme inhibition assay of compound 4c against eight different tyrosine
kinases.
Table 4: Calculated Lipinskiís rule of compound 4c within Src ATP Kinase.

26
OMe
OMe
OH
OH
OH
CH3
CH3
CH3
O
O
F
O
O
O
O
O
I
II
III
N
H2N
O
O
S
N
HN
O
HN
N
N
N
N
NH
NH
O
O
IV
V
Fig. 1: Examples of 2-substituted-benzofurans and N-substituted pyrazoles as Src
protein tyrosine kinase inhibitors
2-Carboxy-Benzofuran
Pharmacophore
O
O
O
+
Src Kinase
Inhibition
Anticancer
Activity
N
N
Hyberidization
N
N
N-Phenyl Pyrazole
Pharmacophore
Benzofuran-Pyrazoles
Backbone
Fig. 2: Design of benzofuran-pyrazole derivatives as c-Src inhibitors.

27
Fig. 3: The docked co-crystalized ligand (yellow, stick) showed the same binding
mode of native ligand (green, stick). The protein represented as cartoon (grey). The
important amino acids involved in the interaction (grey, stick).
Fig. 4: The interaction of compound 4c (magenta, stick) within Src ATP binding site.
NH of pyrrole moiety formed H-bond with Lys 343 (2.78Å), N of the pyrazoline
moiety formed H-bond with Ser345 (3.24Ǻ). The phenyl group of benzofuran moiety
was surrounded by Tyr340, Met341and Ser342.

28
Fig. 5: Compound 4c (magenta, stick) and native ligand (green, stick) within Src ATP
showed the same binding mode indicating good fitting of compound 4c within the
pocket site.
Replacement of the pyrazoline ring with isoxazole ring retains the activity.
Replacement of the pyrazoline ring with chalcone moiety decreases the activity.
Increasing the size of the heterocyclic ring decreases the activity.
O
Replacement of the acetyl
group with hydrogen atom
decreases the activity.
2-Pyrrolyl or 2-
furyl give the
highest activity
X
N
O
N
N
N
Fig. 6: Structure activity relationship (SAR) of benzofuran-pyrazole derivatives as
anticancer agents.

29
R
O
N
ii
NH
OHC
N
N
O
N
N
1
3a-d
i
R
O
O
N
N
O
iii
iv
v
R
N
N
4a-d
N
O
N
2a-d
R
O
N
O
N
N
5a-d
N
S
2-6
R
R
NH
a
b
c
4-Cl-C6H4-
Cyclohexyl
2-Pyrrolyl
2-Furyl
d
N
O
N
6a-d
(i) appropriate acetyl derivatives, ethanolic NaOH, stirring, r.t. 3h; (ii) hydrazine hydrate,
EtOH, reflux, 3h; (iii) hydrazine hydrate , glacial acetic acid, reflux, 3h; (iv) hydroxyl amine
hydrochloride, ethanolic NaOH, reflux, 10h, (v) thiourea, ethanolic NaOH, reflux, 24h.
Scheme 1: Synthesis of chalcone, pyrazoline, isoxazole and thiopyrimidine
derivatives.