Synthesis, antitumor activity, enzyme assay, DNA binding and molecular docking of Bis-Schiff bases of pyrazoles

Article abstract of DOI:10.1007/s13738-020-02004-y

A novel series of Bis-Schiff bases of pyrazoles 9–24 were synthesized by the direct condensation of 5-aminopyrazoles 4a–d with dialdehydes 8a–d in ethanol. The newly synthesized Bis-Schiff bases of pyrazoles 9–24 were characterized and confirmed by analyt

Full text of DOI:10.1007/s13738-020-02004-y

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02004-y
ORIGINAL PAPER
Synthesis, antitumor activity, enzyme assay, DNA binding
and molecular docking of Bis‑Schiꢀ bases of pyrazoles
1
1
1
1
2
Nesrin M. Morsy · Ashraf S. Hassan · Taghrid S. Hafez · Mohamed R. H. Mahran · Inass A. Sadawe ·
Abdul M. Gbaj2
Received: 5 May 2020 / Accepted: 11 July 2020
©
Iranian Chemical Society 2020
Abstract
A novel series of Bis-Schiꢀ bases of pyrazoles 9–24 were synthesized by the direct condensation of 5-aminopyrazoles
4
a–d with dialdehydes 8a–d in ethanol. The newly synthesized Bis-Schiꢀ bases of pyrazoles 9–24 were characterized and
conꢁrmed by analytical and spectroscopic data. Some selected Bis-Schiꢀ bases were investigated for their in vitro anti-
proliferation activity toward three human carcinoma cell lines {HepG2 (liver), MCF-7 (breast) and RPE-1 (normal retina
pigmented epithelium)} using MTT assay. The result in vitro showed that the compound 23 was found to be the active
candidate against HepG2 and MCF-7 cells, while compound 16 was found to be the most potent derivative against RPE-1
cells. All the Bis-Schiꢀ bases of pyrazoles 9–24 were evaluated for their screening on thymidine phosphorylase and DNA
binding energy. The DNA binding energy showed that the compound 12 shows the lowest IC compared to other series of
5
0
compounds and is the nearest one to the IC of the standard taxol. The molecular docking of the new Bis-Schiꢀ base 9 was
5
0
carried out and showed good binding energies (−4.45, −4.95, −2.62, −3.83 and −5.03 kcal/mol with 1bna, 102d, 1k2j,
2
gvr and 2des double-strand DNA targets, respectively) when compared to standard doxorubicin. This study is an introduc-
tion to promising compounds.
Keywords Bis-Schiꢀ bases · Pyrazole · Dialdehyde · Antitumor activity · DNA binding energy · Molecular docking
Introduction
interactions. Currently, it is too important to be careful
about the design and synthesizing of more eꢀective and
safer human therapeutic agents to treat and overcome tumor
growth.
A cancer disease is a malignant disease due to the uncon-
trolled growth of abnormal cells. Chemotherapy is a major
treatment for cancer disease. Damaging of DNA protein
constituents is a principle target to destroy cell’s growth
Schiꢀ bases exhibited wide range of medicinal applica-
tions with its azomethine (imine) functional group since this
linkage is an essential feature for bioactivity. Also, Schiꢀ
bases containing heterocyclic derivatives were assigned to
have diꢀerent pharmacological activities like compound
A, 1-cyclopropyl-6-fluoro-7-(4-(((Z)-5-fluoro-3-(4-((E)-
4-hydroxybenzylideneamino)phenylimino)-2-oxoindolin-
1-yl)methyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-
[
1]. DNA in cancer cells could be selectively damaged, as a
result of interactions with anticancer agents; consequently,
blocking of cells division leads to cells death [2, 3]. The
molecules that interact with DNA are generally bound to
DNA through non-covalent bonds by three main mecha-
nisms: groove binding, intercalation or static electronic
3
-carboxylic acid, which is a promising antimicrobial agent
4]. Compound B, 3-(3,5-dichlorobenzylideneamino)-
-methylquinazolin-4(3H)-one, was found to be the
most potent anthelmintic agent [5]. Compound C,
-(2-hydroxybenzylideneamino)-2-phenylquinazolin-4(3H)-
[
*
Ashraf S. Hassan
2
ashraf_salmoon@yahoo.com
https://www.scopus.com/authid/detail.uri?authorId=55318442700
3
1
2
Organometallic and Organometalloid Chemistry
Department, National Research Centre, Cairo, Dokki 12622,
Egypt
one, showed better antiviral activity [6], and compounds,
N-(2,4-dichlorobenzylidene)-4-phenyl-5-(1H-1,2,4-tria-
zol-1-yl)thiazol-2-amine (D), N-(3-nitrobenzylidene)-4-
phenyl-5-(1H-1,2,4-triazol-1-yl)thiazol-2-amine (E) and
Department of Medicinal Chemistry, Faculty of Pharmacy,
University of Tripoli, Tripoli, Libya
Vol.:(0
1
123456789)
3

Journal of the Iranian Chemical Society
N-(2,4-dinitrobenzylidene)-4-phenyl-5-(1H-1,2,4-triazol-
evaluated their antitumor activity, enzyme assay, DNA bind-
ing capability and molecular docking study.
1
-yl)thiazol-2-amine (F), were found to be good antitumor
candidates [7].
Pyrazole is an important unit to build biologically active
moieties for detection and expansion of new drugs used par-
ticularly in cancer treatment. Moreover, pyrazole moiety has
important biological activities as cytotoxic, antiviral, anti-
microbial and antitumor [8–11].
Results and discussion
Chemistry
Schiff bases of pyrazole exhibited biological activi-
ties; compounds, 4-((3-phenyl-1H-pyrazol-4-yl)
methyleneamino)-4H-1,2,4-triazole-3-thiol (G) and 5-ethyl-
A series of 5-aminopyrazoles 4a–d was prepared as
described in the literature (Scheme 1) [36, 37].
Also, dibromoalkyl 5a, b {dibromomethane (5a) and
1,2-dibromoethane (5b)} were reacted with salicylaldehyde
(6) or 4-hydroxybenzaldehyde (7) to yield dialdehydes 8a,
b {2,2′-methylenebis(oxy)dibenzaldehyde (8a) [38] and
2,2′-(ethane-1,2-diylbis(oxy))dibenzaldehyde (8b) [39]} or
8c, d {4,4′-methylenebis(oxy)dibenzaldehyde (8c) [40] and
4,4′-(ethane-1,2-diylbis(oxy))dibenzaldehyde (8d) [40]},
respectively (Scheme 2).
4
4
-((3-(4-ꢂuorophenyl)-1H-pyrazol-4-yl)methyleneamino)-
H-1,2,4-triazole-3-thiol (H), have exhibited signiꢁcant
biological activities against Staphylococcus aureus [12],
and compound I, 5-((5-chloro-1-(4-ꢂuorophenyl)-3-methyl-
1
H-pyrazol-4-yl)methyleneamino)-1-(4-(triꢂuoromethyl)
phenyl)-1H-pyrazole-4-carbonitrile, showed the most potent
anti-tobacco mosaic virus (TMV) [13]. Bis-Schiꢀ bases also
exhibited antibacterial, antifungal and antiviral activities
The synthesized target compounds, Bis-Schiꢀ bases of
pyrazoles 9–16 and 17–24 were shown in Scheme 3 and
were synthesized by direct condensation of 5-aminopyra-
zoles 4a–d with dialdehydes 8a, b and 8c, d, respectively.
(Scheme 3, Table 1).
[
14] (Fig. 1).
From the above facts and in continuation to our research
program for developing more promising molecules [15–35],
we synthesized Bis-Schiff bases of pyrazoles 9–24 and
Fig. 1 Structures of the biological activities of Schiꢀ bases A–I
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Journal of the Iranian Chemical Society
Scheme 1 Synthesis of compounds 4a–d, reagents and conditions: (1) Ar=Ph–NCS or 4–CH O–C H –NCS/KOH/EtOH. (2) CH I/EtOH/r.t.
3
6
4
3
(
3) NH NH /TEA/EtOH/reꢂux 4 h
2 2
Scheme 2 Synthesis of dialde-
hydes 8a, b and 8c, d
Scheme 3 Synthesis of Bis-Schiꢀ bases 9–24
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Journal of the Iranian Chemical Society
Table 1 Bis-Schiꢀ bases 9–24
RPE-1 (human normal retina pigmented epithelium) using
MTT assay [41–44]. The percentage and activities of the
intact cells were measured and compared to the control
Compounds
n
R
Ar
Ph
9
1
1
1
1
2
2
2
2
1
1
1
1
2
2
2
2
H
Ph
(
Table 2, Fig. 2). The activities of the Bis-Schiꢀ bases of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4–CH O–C H
4–CH O–C H
3 6
4–CH O–C H
Ph
4–CH O–C H
4–CH O–C H
4–CH O–C H
3 6
Ph
4–CH O–C H
4–CH O–C H
4–CH O–C H
3 6
Ph
4–CH O–C H
4–CH O–C H
4–CH O–C H
3 6
3 6
4
4
4
pyrazoles against the three human cells were compared with
®
4–CH –C H
4–Cl–C H
H
Ph
4–CH –C H
4–Cl–C H
H
Ph
4–CH –C H
4–Cl–C H
H
Ph
4–CH –C H
4–Cl–C H
3
6
4
4
4
4
that of Doxorubicin .
6
4
3 6
From Table 1 and Fig. 2, we can deduce that, among the
derivatives, compound 23 was found to be the active candi-
date against HepG2 and MCF-7 cells, while compound 16
was found to be the most potent derivative against RPE-1
cells.
3
6
4
4
4
3
6
3 6
6
4
3
6
4
4
4
Thymidine phosphorylase
3
6
3 6
6
4
Thymidine phosphorylase enzyme TP has a great focusing
as a cancer goal as it plays a function in tumor angiogenesis,
and is observed at higher levels in the plasma of patients
having cancer and in solid tumors compared to healthy tis-
sues. Inhibitors of TP inhibit angiogenesis and metastasis as
well as promoting apoptosis. Inhibitors of TP also potentiate
the actions of anticancer.
3
6
4
4
4
3
6
3 6
6
4
The structures of the Bis-Schiꢀ bases of pyrazoles 9–16
No compound had any inhibition on thymidine phos-
phorylase enzyme as the compounds were not designed for
this target.
and 17–24 were established and conꢁrmed on the basis of
their elemental analysis and spectral data. For example,
structure 24, 5,5′-(4,4′-ethylenebis(oxy)bis(benzylideneam
ino))bis(3-(4-methoxyphenylamino)-N-(4-chlorophenyl)-
DNA binding properties
1
H-pyrazole-4-carboxamide), was supported by its
mass (949.84), which agrees with its molecular formula
The ꢂuorescence intensity of DNA-bound EB at 612 nm
decreased outstandingly with the increase in these com-
pounds concentrations. This decrease perhaps is attributed
to the quenching of some EB molecules that were perme-
ated from DNA into the substituted solution by the products.
The summarized IC was indicated in the attached table
−
1
C H Cl N O ; its IR (KBr) ν /cm spectrum showed
5
0
42
2
10
6
max
3
426, 3272 for NH function and 1656 for C=O group; its
1
H NMR (DMSO-d , δ ppm) spectrum showed two signals
6
at 4.50 (4H) and 8.66 (2H) corresponding to two CH and
2
1
two –N=CH– groups, respectively. Also, the H NMR spec-
5
0
trum showed the following: 3.72 (6H, s, 2OCH ), 6.90 (4H,
(Table 3).
3
d, J=8.1 Hz, ArH), 7.18–7.26 (6H, m, ArH), 7.41 (4H, d,
J=8.6 Hz, ArH), 7.69 (4H, d, J=8.6 Hz, ArH), 7.88 (2H, d,
J=8.6 Hz, ArH), 8.04 (4H, d, J=8.2 Hz, ArH), 8.96 (2H, s,
Compound 12 shows the lowest IC compared to other
5
0
series of compounds and is the nearest one to the IC of the
5
0
standard taxol.
1
3
2
NH), 9.88 (2H, s, 2NH), 12.63 (2H, s, 2NH). Its C NMR
spectrum (DMSO-d , δ ppm) was characterized by signals
Molecular docking analysis
6
at 66.71 and 156.52 assigned to CH and –N=CH– groups,
2
1
3
respectively. Moreover, the C NMR showed 55.24 (2C,
Table 4 shows the binding energies of our compounds and
DNAs fragments obtained by the molecular docking strat-
egy. The study of our compounds with ꢁve B-DNA frag-
ments was achieved using AutoDock 4.2 to investigate the
way of interaction forces between our compounds and DNA.
The synthesized compounds and DNA were kept as ꢂexible
molecules and were docked into ꢁve forms of rigid B-DNA
fragments to obtain the preferential binding site to the series
of compounds on B-DNAs. The molecular docking results
are shown in Table 4. The modeling studies showed that
there are van der Waals, hydrogen bonding and electrostatic
interactions between the compounds and DNAs. The sum of
the ꢁrst two interactions and desolvation free energy is much
2
1
1
OCH ), 93.51 (2C , pyrazole), 114.41, 115.47, 120.57,
3 4
28.92, 129.88, 131.42, 133.24, 137.50, 151.86 (34C, Ar),
53.10 (2C , pyrazole), 155.27 (2C , pyrazole), 162.33 (2C,
5
3
Ar), 162.83 (2C=O).
Biological evaluation
Cytotoxicity activity
Selected eight Bis-Schiꢀ bases of pyrazoles were examined
in vitro for their cytotoxicity against HepG2 (human liver
carcinoma), MCF-7 (human breast adenocarcinoma) and
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Journal of the Iranian Chemical Society
Table 2 The cytotoxicity IC (µM) values of selected eight Bis-Schiꢀ bases of pyrazoles using MTT assay against three human cancer cell lines
5
0
types (HepG2, MCF-7 and RPE-1)
Compounds
n
R
Ar
IC50 (µM)
HepG2
MCF-7
RPE-1
1
1
1
1
1
2
2
2
1
2
5
6
9
0
3
4
1
1
2
2
1
1
2
2
–
4–CH –C H
4–CH O–C H
111.6
108.4
100.5
88.4
90.8
89.8
84.2
86.2
25.3
125.1
136.7
125.3
107.9
117.9
106.5
99.4
143.7
148.5
143.1
127.7
132.2
137.1
138.2
137.2
19.1
3
6
4
4
4
4
3
6
4
4
4
4
4
4
4
4
4–Cl–C H
4–CH O–C H
6
4
3 6
4–CH –C H
4–CH O–C H
3 6
3
6
4–Cl–C H
4–CH O–C H
6
4
3 6
4–CH –C H
4–CH O–C H
3 6
3
6
4–Cl–C H
4–CH O–C H
6
4
3 6
4–CH –C H
4–CH O–C H
3 6
3
6
4–Cl–C H
4–CH O–C H
100.7
20.9
6
4
3
6
Doxorubicin
–
–
Fig. 2 Antitumor activity of the selected eight Bis-Schiꢀ bases of pyrazoles against HepG2, MCF-7 and RPE-1 cell lines using MTT assay
greater than the last interaction since the contributions of the
three top energies together are larger than the electrostatic
energy [45, 46].
Since these sequence eꢀects lie outside the principal binding
sites for these ligands, they may reꢂect changes in the local
DNA structure and/or dynamics. This is similar to those seen
in protein–DNA interactions [47, 48].
Some of the binding energies obtained by performing
molecular docking simulation of the compounds with the
Molecular docking is used for virtual screening of our
compounds employing binding aꢃnity and the best orienta-
tion possible with respect to the target DNA to illustrate the
DNA (9–24) compounds interactions; compound 9 as an
example from the series was chosen and done. DNA inter-
actions with compound 9 are shown in Fig. 3. Compound
9 showed good binding energies (− 4.45, − 4.95, − 2.62,
ꢁ
ve DNA fragments did not lie in the predeꢁned range
of − 5 kcal/mol to − 15 kcal/mol (Table 4). The obtained
binding energy results demonstrate that the aꢃnity of com-
pounds for their “preferred” sites is modulated by the local
DNA sequence. In some cases, this eꢀect is relatively small,
while in other cases, as in 9, 13 and 19 the eꢀect is good.
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Journal of the Iranian Chemical Society
Table 3 DNA binding (IC (µM)) of Bis-Schiꢀ bases of pyrazoles
with three B-DNA fragments were performed using Auto-
Dock 4.2 to investigate the binding mode of Bis-Schiꢀ
bases with B-DNA and to obtain information about inter-
action forces between synthesized compounds and DNA.
The synthesized compounds and DNA were kept as ꢂex-
ible molecules and were docked into seven forms of rigid
B-DNA fragments to obtain the preferential binding site to
synthesized compounds on B-DNAs. The modeling studies
showed that there are van der Waals, hydrogen bonding
and electrostatic interactions between compound 9 ligand
and DNAs. This fact is clear in the modeling studies of
compound 9 ligand and is consistent with the literature
5
0
9
–24
Compounds
IC50 (µM)
Compounds
IC50 (µM)
9
55.3± 3.32
60.3± 2.32
72.4± 3.32
52.3± 1.42
55.3± 2.12
62.2± 2.12
73.3± 2.22
53.2± 2.12
63.3± 2.12
18
70.2± 2.22
72.2± 1.32
55.3± 1.32
72.4± 2.32
79.3± 1.92
80.4± 3.42
60.3± 2.61
35.0± 1.12
10
11
12
13
14
15
16
17
19
20
21
22
23
24
Taxol
[
49, 50].
All Bis-Schiꢀ bases of pyrazole 9–24 were bound to the
minor groove of the three DNA fragments. This binding
often has cytotoxic activity because they interfere with
the binding of proteins necessary for DNA replication
and transcription. In the literature, compounds that bind
to the minor groove of DNA have proven to be very use-
ful as antitumor agents because they selectively kill rap-
idly dividing cells [49, 51]. This has encouraged eꢀorts
to design molecules that bind at designated sites in the
minor groove. It is contemplation that groove binders
with increased selectivity will produce a greater biologi-
cal response for a given dose and consequently have fewer
−
3.83 and − 5.03 kcal/mol with 1bna, 102d, 1k2j, 2gvr
and 2des double-strand DNA targets, respectively) when
compared to standard doxorubicin as mentioned in Table 4.
Double-helical structure of 1bna, 102d, 1k2j, 2gvr and 2des
hydrogen bounded to the compound 9 at the minor groove
is shown in Fig. 3.
The results showed that the binding energies of the
Bis-Schiꢀ bases of pyrazole 9–24 and DNAs fragments
obtained by the molecular docking strategy. In this study,
molecular dockings of the Bis-Schiꢀ bases of pyrazole
Table 4 Various energies in the binding process of Schiꢀ bases of pyrazole 9–24 with DNAs obtained from molecular docking. The unit of all
energies (ΔG) is kcal/mol
Compounds 1bna
102d
1k2j
2gvr
2des
DNA (5′-D(*CP* DNA (5′-D(*C 5′-D(*CP*GP*TP*AP*CP*G)-3′ 5′-D(*CP*GP*C DNA
GP*CP*GP*AP* P*GP*CP*AP
AP*TP*TP*CP* *AP*AP*TP*
P*GP*AP*AP*T (5′-D(*CP*GP*TP*AP*CP*G)-3′)
P*TP*CP*GP*C
GP*CP*G)-3′)
TP*TP*GP*C
P*G)-3′
P*G)-3′)
ΔG
Binding energy
Binding energy Binding energy
Binding energy
Binding energy
Doxorubicin −7.64
−8.41
−4.95
−4.13
−1.7
−0.99
−4.31
−2.8
−0.07
−2.74
−4.35
−2.52
−1.98
−1.81
−3.62
−2.14
−1.25
−2.92
−6.32
−2.62
+4.11
+3.23
+18.66
−2.93
+13.09
+3.38
+95.28
−4.42
+1.43
−1.93
+6.11
−2.99
−0.88
+5.1
−7.64
−3.83
−3.14
−0.35
+1.27
−2.79
−1.76
+0.23
−0.25
−4.15
−0.84
−1.67
−2.94
−2.76
−2.38
+0.18
−1.1
−4.85
−5.03
−2.39
−0.49
+2.2
−0.35
−1.84
+0.53
+2.08
+1.05
−1.52
−0.18
+1.62
+1.21
−1.88
+1.53
+2.03
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
−4.45
−3.28
−3.61
−0.47
−4.13
−2.55
−0.52
−2.18
−3.59
−1.82
−2.26
−2.04
−3.81
−1.38
−2.41
+1.04
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
+4.11
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Journal of the Iranian Chemical Society
toxic and side eꢀects than non-selective groove binders
52].
[
Conclusion
In summary, we have synthesized a novel series of Bis-
Schiꢀ bases of pyrazoles 9–16 and 17–24 by the direct
condensation of 5-aminopyrazoles 4a–d with dialdehydes
8
a, b and 8c, d, respectively, in reꢂuxing ethanol with
very high yields for evaluation of their antitumor activities
against three human carcinoma cell lines (HepG2, MCF-7
and RPE-1) using MTT assay. Most of Bis-Schiꢀ bases
of pyrazoles displayed moderate antitumor activity. The
molecular docking study of the Bis-Schiꢀ base of pyrazole
with B-DNA was performed. There are three modes of
interaction. The modeling studies showed that the contri-
bution of the sum of van der Waals and hydrogen bonding
interaction is much greater than that of the electrostatic
interaction. Compound 9 showed good binding energies
when compared to the standard doxorubicin due to the
sum of three interactions between a ligand and DNAs.
Accordingly, this class of Bis-Schiꢀ bases of pyrazoles
9
–24 could be considered as useful templates for future
development in derivatizations or modiꢁcations to obtain
more potent and selective antitumor agents.
Experimental
Chemistry
All melting points were measured on a Gallenkamp
1
melting point apparatus and are uncorrected. H NMR
1
3
(
400 MHz) and C NMR (100 MHz) spectra were
recorded on a Varian spectrometer using DMSO-d as
6
solvent and TMS as an internal standard. Chemical shifts
are reported in ppm. Coupling constants (J) are expressed
in Hz. Mass spectra were recorded on a Varian MAT 112
spectrometer at 70 eV. Elemental analyses were performed
at the Microanalytical Center, Cairo University, Egypt.
Progress of the reactions was monitored by thin-layer
chromatography (TLC) using aluminum sheets coated with
silica gel F (Merck), viewing under a short-wavelength
2
54
UV lamp eꢀected detection. All evaporations were carried
out under reduced pressure at 40 °C.
Fig. 3 Double-helical structure of 1bna, 102d, 1k2j, 2gvr and 2des
hydrogen bounded to the compound 9 at the minor groove
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Journal of the Iranian Chemical Society
Synthesis
5,5′‑(2,2′‑Methylenebis(oxy)bis(benzylideneamino))bis(3
‑(phenylamino)‑1H‑pyrazole‑4‑carboxamide) (9) Yellow
of 5‑amino‑3‑(arylamino)‑1H‑pyrazole‑4‑carboxamides
−
1
4a–d
crystals, m.p. 270 °C, yield (82%). IR (KBr) ν /cm
max
1
3
370 (NH), 1653 (C=O). H NMR (DMSO-d , 400 MHz,
6
Compounds of this series were prepared according to the
literature procedure:
δ ppm) 6.18 (2H, s, CH ), 6.87 (2H, t, ArH), 7.20 (2H, t,
2
ArH), 7.28 (4H, t, ArH), 7.36–7.39 (4H, m, ArH), 7.48
(
4H, s, 2NH ), 7.57 (2H, d, J=8.3 Hz, ArH), 7.63 (2H, d,
2
5
-Amino-3-anilino-lH-pyrazole-4-carboxamide (4a)
m.p. 178–180 °C [36].
-Amino-3-(4-methoxyphenylamino)-N-phenyl-1H-
pyrazole-4-carboxamide (4b) m.p. 175–177 °C [37].
-Amino-3-(4-methoxyphenylamino)-N-(4-methylphe-
nyl)-1H-pyrazole-4-carboxamide (4c) m.p. 198–200 °C
37].
-Amino-3-(4-methoxyphenylamino)-N-(4-chlorophe-
nyl)-1H-pyrazole-4-carboxamide (4d) m.p. 190–192 °C
37].
J=6.8 Hz, ArH), 8.07 (2H, d, J=7.0 Hz, ArH), 9.11 (2H,
1
3
s, 2-N=CH-), 9.25 (2H, s, 2NH), 12.73 (2H, s, 2NH).
C
5
NMR (DMSO-d , 75 MHz, δ ppm) 90.57 (1C, CH ), 93.49
6
2
(2C, C -pyrazole), 115.44, 116.14, 120.14, 122.78, 123.67,
4
5
127.78, 128.83, 129.16, 134.78, 141.07 (22C, Ar), 153.71
(4C, C and C , pyrazole), 156.90 (2C, 2–N=CH–), 162.19
5
3
[
(2C, Ar), 166.35 (2C=O). Anal. Calcd. (%) for C H N O
35 30 10 4
5
(654.68): C, 64.21; H, 4.62; N, 21.39. Found: C, 64.30; H,
4.55; N, 21.30%.
[
5
,5′‑(2,2′‑Methylenebis(oxy)bis(benzylideneamino))bis(3‑(
Synthesis of dialdehydes 8a–d
4‑methoxyphenylamino)‑N‑phenyl‑1H‑pyrazole‑4‑carboxa‑
mide) (10) Yellow crystals, m.p. 246 °C, yield (80%). IR
−
1
1
To a solution of salicylaldehyde (6) or 4-hydroxybenzalde-
hyde (7) (0.2 mol) and K CO (0.2 mol) in DMF (100 ml),
(KBr) ν /cm 3427, 3301 (NH), 1657 (C=O). H NMR
max
(DMSO-d , 400 MHz, δ ppm) 3.74 (6H, s, 2OCH ), 6.20
2
3
6
3
dibromoalkyl derivatives 5a, b (0.1 mol) {dibromomethane
(2H, s, CH ), 6.91 (4H, d, J = 8.4 Hz, ArH), 7.03 (2H, t,
2
(
5a) or 1,2-dibromoethane (5b)} were added dropwise. The
ArH), 7.29–7.40 (10H, m, ArH), 7.57–7.69 (8H, m, ArH),
reaction mixture was allowed to stir for 10 h at 150–155 °C
and then 5 h at room temperature. After the addition was
completed, then 200 ml distilled water was added and was
placed in refrigerator. Then, after 1 h the solid product was
8.16 (2H, d, J=7.5 Hz, ArH), 8.69 (2H, s, 2–N=CH–), 9.33
1
3
(2H, s, 2NH), 9.92 (2H, s, 2NH), 12.27 (2H, s, 2NH).
C
NMR (DMSO-d , 100 MHz, δ ppm) 55.28 (2C, 2OCH ),
6
3
91.16 (3C, 1CH and 2C -pyrazole), 114.49, 115.94, 118.98,
2
4
ꢁ
ltered oꢀ, washed with ethanol, dried and ꢁnally recrystal-
123.27, 124.30, 127.02, 129.01, 134.82, 138.51, 149.88
lized from DMF/H O to aꢀord the corresponding dialde-
(34C, Ar), 152.85 (2C , pyrazole), 154.61 (2C , pyrazole),
2
5
3
hydes 8a–d:
157.12 (2C, 2–N=CH–), 161.15 (2C, Ar), 162.71 (2C=O).
Anal. Calcd. (%) for C H N O (866.92): C, 67.89; H,
4
9
42 10
6
2
,2′-Methylenebis(oxy)dibenzaldehyde (8a) m.p. 132 °C
4.88; N, 16.16. Found: C, 68.00; H, 4.80; N, 16.20%.
[
38].
2
1
4
,2′-(Ethane-1,2-diylbis(oxy))dibenzaldehyde (8b) m.p.
22 °C [39].
5,5′‑(2,2′‑Methylenebis(oxy)bis(benzylideneamino))bis(3
‑(4‑methoxyphenylamino)‑N‑(4‑methylphenyl)‑1H‑pyra‑
zole‑4‑carboxamide) (11) Yellow crystals, m.p. 268 °C,
,4′-Methylenebis(oxy)dibenzaldehyde (8c) m.p. 215–216
−
1
[
40].
yield (82%). IR (KBr) ν /cm 3427, 3285 (NH), 1651
max
1
4
,4′-(Ethane-1,2-diylbis(oxy))dibenzaldehyde (8d) m.p.
(C=O). H NMR (DMSO-d , 400 MHz, δ ppm) 2.22 (6H,
6
2
10–212 [40].
s, 2CH ), 3.73 (6H, s, 2OCH ), 6.19 (2H, s, CH ), 6.91 (4H,
3
3
2
d, J = 8.1 Hz, ArH), 7.09 (4H, d, J = 7.9 Hz, ArH), 7.26–
Synthesis of Bis‑Schiꢀ bases 9–24
7.68 (14H, m, ArH), 8.11 (2H, d, J = 7.2 Hz, ArH), 8.70
(
2H, s, 2–N=CH–), 9.28 (2H, s, 2NH), 9.82 (2H, s, 2NH),
1
3
A reaction mixture of compounds 4a–d (0.02 mol) and dial-
dehydes 8a–d (0.01 mol) {namely 2,2′-methylenebis(oxy)
dibenzaldehyde (8a), 2,2′-(ethane-1,2-diylbis(oxy))diben-
zaldehyde (8b), 4,4′-methylenebis(oxy)dibenzaldehyde (8c)
and 4,4′-(ethane-1,2-diylbis(oxy))dibenzaldehyde (8d)} with
a catalytic amount of glacial acetic acid (1 ml) in absolute
ethanol (25 ml) was reꢂuxed for 1 h and then left to cool.
The solid product was ꢁltered oꢀ, dried and ꢁnally recrys-
tallized from ethanol to aꢀord the corresponding Bis-Schiꢀ
bases 9–24.
12.69 (2H, s, 2NH). C NMR (DMSO-d , 75 MHz, δ ppm)
6
20.37 (2C, 2CH ), 55.20 (2C, 2OCH ), 90.64 (2C, CH ),
3
3
2
91.09 (2C, C -pyrazole), 114.39, 115.96, 117.55, 119.76,
4
122.66, 123.20, 124.30, 127.85, 129.31, 132.05, 134.81,
135.92, 136.30 (31C, Ar), 154.13 (4C, C and C pyrazole),
5
3
155.58 (2C, Ar), 157.02 (2C, 2–N=CH–), 162.48 (2C, Ar),
165.16 (2C=O). Anal. Calcd. (%) for C H N O (894.97):
51
46 10
6
C, 68.44; H, 5.18; N, 15.65. Found: C, 68.50; H, 5.10; N,
15.70%.
1
3

Journal of the Iranian Chemical Society
5
,5′‑(2,2′‑Methylenebis(oxy)bis(benzylideneamino))bis(3
115.46, 121.63, 123.51, 126.77, 129.39, 132.05, 134.78,
‑
(4‑methoxyphenylamino)‑N‑(4‑chlorophenyl)‑1H‑pyra‑
136.08, 151.18 (34C, Ar), 153.35 (2C , pyrazole), 155.47
5
zole‑4‑carboxamide) (12) Yellow crystals, m.p. 262 °C,
(2C , pyrazole), 156.66 (2C, 2–N=CH–), 159.49 (2C, Ar),
3
−
1
yield (80%). IR (KBr) ν /cm 3436 (NH), 1655 (C=O).
162.60 (2C=O). Anal. Calcd. (%) for C H N O (909.00):
max
52 48 10
6
1
H NMR (DMSO-d , 400 MHz, δ ppm) 3.73 (6H, s,
C, 68.71; H, 5.32; N, 15.41. Found: C, 68.80; H, 5.25; N,
6
2
7
OCH ), 6.19 (2H, s, CH ), 6.89 (4H, d, J=8.9 Hz, ArH),
15.47%.
3
2
.25–7.37 (10H, m, ArH), 7.56–7.68 (8H, m, ArH), 8.09
(
(
2H, d, J = 6.7 Hz, ArH), 8.61 (2H, s, 2–N=CH–), 9.25
5,5′‑(2,2′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑
(4‑methoxyphenylamino)‑N‑(4‑chlorophenyl)‑1H‑pyra‑
zole‑4‑carboxamide) (16) Orange crystals, m.p. 270 °C,
2H, s, 2NH), 9.94 (2H, s, 2NH), 12.30 (2H, s, 2NH). Anal.
Calcd. (%) for C H Cl N O (935.81): C, 62.89; H, 4.31;
4
9
40
2
10
6
−
1
N, 14.97. Found: C, 62.80; H, 4.35; N, 15.00%.
yield (75%). IR (KBr) ν /cm 3429, 3277 (NH), 1657
max
1
(
C=O). H NMR (DMSO-d , 400 MHz, δ ppm): 3.69 (6H, s,
6
5
,5′‑(2,2′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑(
2OCH ), 4.60 (4H, s, 2CH ), 6.79 (4H, d, J=8.7 Hz, ArH),
3 2
phenylamino)‑1H‑pyrazole‑4‑carboxamide) (13) Yellow
7.17–7.35 (12H, m, ArH), 7.57–7.64 (6H, m, ArH), 8.10
(2H, d, J=7.3 Hz, ArH), 8.59 (2H, s, 2–N=CH–), 9.32 (2H,
1
crystals, m.p. 258 °C, yield (79%). H NMR (DMSO-d ,
6
1
3
4
7
00 MHz, δ ppm) 4.62 (4H, s, 2CH ), 6.84 (2H, t, ArH),
s, 2NH), 9.98 (2H, s, 2NH), 12.42 (2H, s, 2NH). C NMR
(DMSO-d , 100 MHz, δ ppm) 55.17 (2C, 2OCH ), 67.97
2
.08 (2H, t, ArH), 7.26 (6H, t, ArH), 7.33 (4H, d, J=7.8 Hz,
6
3
ArH), 7.45 (4H, d, 2NH ), 7.57 (2H, t, ArH), 8.02 (2H, d,
(2C, 2CH ), 92.86 (2C , pyrazole), 114.29, 118.15, 120.41,
2
2 4
J=7.3 Hz, ArH), 9.08 (2H, s, 2 N=CH-), 9.19 (2H, s, 2NH),
121.72, 123.58, 124.99, 126.48, 126.82, 128.74, 132.92,
13
1
6
1
2.12 (2H, s, 2NH). C NMR (DMSO-d , 100 MHz, δ ppm)
134.77, 137.50, 148.11 (34C, Ar), 152.54 (2C , pyrazole),
6
5
8.26 (2C, 2CH ), 93.86 (2C , pyrazole), 114.28, 116.85,
157.11 (2C , pyrazole), 159.58 (2C, 2–N=CH–), 162.09 (2C,
2
4
3
21.90, 123.79, 127.90, 129.51, 130.10, 135.14, 141.68
Ar), 162.67 (2C=O). Anal. Calcd. (%) for C H Cl N O
5
0
42
2
10
6
(
22C, Ar), 152.17 (2C , pyrazole), 154.06 (2C , pyrazole),
(949.84): C, 63.23; H, 4.46; N, 14.75. Found: C, 63.30; H,
5
3
1
59.77 (2C, 2–N=CH–), 163.96 (2C, Ar), 166.76 (2C=O).
4.39; N, 14.80%.
Anal. Calcd. (%) for C H N O (668.70): C, 64.66; H,
3
6
32 10
4
4
.82; N, 20.95. Found: C, 64.60; H, 4.90; N, 21.00%.
5,5′‑(4,4′‑Methylenebis(oxy)bis(benzylideneamino))bis(3‑
(
phenylamino)‑1H‑pyrazole‑4‑carboxamide) (17) Yellow
1
5
,5′‑(2,2′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑(4
crystals, m.p. > 300 °C, yield (73%). H NMR (DMSO-d ,
6
‑
methoxyphenylamino)‑N‑phenyl‑1H‑pyrazole‑4‑carbox‑
400 MHz) δ 6.09 (2H, s, CH ), 6.84 (2H, t, ArH), 7.30–7.48
2
amide) (14) Orange crystals, m.p. 256 °C, yield (83%).
(16H, m, ArH+2NH ), 7.85–7.99 (4H, m, ArH), 8.88 (2H,
2
−
1
1
IR (KBr) ν /cm 3281 (NH), 1655 (C=O). H NMR
s, 2N=CH–), 9.12 (2H, s, 2NH), 12.65 (2H, s, 2NH). Anal.
max
(
(
DMSO-d , 400 MHz, δ ppm) 3.70 (6H, s, 2OCH ), 4.61
Calcd. (%) for C H N O (654.68): C, 64.21; H, 4.62; N,
6
3
35 30 10
4
4H, s, 2CH ), 6.84 (4H, d, J = 8.9 Hz, ArH), 7.05 (2H, t,
21.39. Found: C, 64.10; H, 4.70; N, 21.45%.
2
ArH), 7.20 (2H, t, ArH), 7.31–7.36 (10H, m, ArH), 7.61 (6H,
d, J=7.8 Hz, ArH), 8.15 (2H, d, J=7.7 Hz, ArH), 8.65 (2H,
s, 2–N=CH–), 9.36 (2H, s, 2NH), 9.98 (2H, s, 2NH), 12.83
5,5′‑(4,4′‑Methylenebis(oxy)bis(benzylideneamino))bis(3‑(
4‑methoxyphenylamino)‑N‑phenyl‑1H‑pyrazole‑4‑carboxa‑
1
(
2H, s, 2NH). Anal. Calcd. (%) for C H N O (880.95):
mide) (18) Orange crystals, m.p. 250 °C, yield (76%). H
5
0
44 10
6
C, 68.17; H, 5.03; N, 15.90. Found: C, 68.20; H, 5.00; N,
NMR (DMSO-d , 400 MHz, δ ppm) 3.73 (6H, s, 2OCH ),
6
3
1
5.85%.
6.14 (2H, s, CH ), 6.91 (4H, m, ArH), 7.08 (2H, t, ArH),
2
7
.35–7.41 (12H, m, ArH), 7.68 (4H, d, J = 7.8 Hz, ArH),
5
,5′‑(2,2′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑
8.08 (4H, d, J=8.1 Hz, ArH), 8.72 (2H, s, 2–N=CH–), 9.04
(2H, s, 2NH), 9.85 (2H, s, 2NH), 12.48 (2H, s, 2NH). Anal.
Calcd. (%) for C H N O (866.92): C, 67.89; H, 4.88; N,
(
4‑methoxyphenylamino)‑N‑(4‑methylphenyl)‑1H‑pyra‑
zole‑4‑carboxamide) (11) Yellow crystals, m.p. > 300 °C,
4
9
42 10
6
−
1
yield (75%). IR (KBr) ν /cm 3426, 3277 (NH), 1653
16.16. Found: C, 67.80; H, 4.95; N, 16.10%.
max
1
(
C=O). H NMR (DMSO-d , 400 MHz, δ ppm) 2.25 (6H,
6
s, 2CH ), 3.70 (6H, s, 2OCH ), 4.61 (4H, s, 2CH ), 6.84
5,5′‑(4,4′‑Methylenebis(oxy)bis(benzylideneamino))bis(3
‑(4‑methoxyphenylamino)‑N‑(4‑methylphenyl)‑1H‑pyra‑
3
3
2
(
4H, d, J=8.7 Hz, ArH), 7.12 (4H, d, J=8.0 Hz, ArH), 7.19
(
2H, t, ArH), 7.34 (6H, d, J = 8.1 Hz, ArH), 7.49 (4H, d,
zole‑4‑carboxamide) (19) Yellow crystals, m.p. 266 °C,
1
J=8.1 Hz, ArH), 7.62 (2H, t, ArH), 8.12 (2H, d, J=7.4 Hz,
ArH), 8.67 (2H, s, 2–N=CH–), 9.33 (2H, s, 2NH), 9.90
yield (81%). H NMR (DMSO-d , 400 MHz, δ ppm): 2.27
6
(6H, s, 2CH ), 3.73 (6H, s, 2OCH ), 6.15 (2H, s, CH ),
3
3
2
1
3
(
2H, s, 2NH), 12.29 (2H, s, 2NH). C NMR (DMSO-d ,
6.91 (4H, s, ArH), 7.15 (4H, d, J=8.1 Hz, ArH), 7.32–7.57
(12H, m, ArH), 8.07 (4H, d, J = 8.1 Hz, ArH), 8.73 (2H,
s, 2–N=CH–), 8.93 (2H, s, 2NH), 9.91 (2H, s, 2NH), 12.47
6
1
6
00 MHz, δ ppm) 20.42 (2C, 2CH ), 55.21 (2C, 2OCH ),
3 3
7.84 (2C, 2CH ), 89.81 (2C , pyrazole), 113.78, 114.40,
2
4
1
3

Journal of the Iranian Chemical Society
(
2H, s, 2NH). Anal. Calcd. (%) for C H N O (894.97):
5,5′‑(4,4′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑
(4‑methoxyphenylamino)‑N‑(4‑methylphenyl)‑1H‑pyra‑
5
1
46 10
6
C, 68.44; H, 5.18; N, 15.65. Found: C, 68.35; H, 5.24; N,
1
5.71%.
zole‑4‑carboxamide) (23) Yellow crystals, m.p. > 300 °C,
1
yield (68%). H NMR (DMSO-d , 400 MHz, δ ppm): 2.27
6
5
‑
,5′‑(4,4′‑Methylenebis(oxy)bis(benzylideneamino))bis(3
(4‑methoxyphenylamino)‑N‑(4‑chlorophenyl)‑1H‑pyra‑
(6H, s, 2CH ), 3.72 (6H, s, 2OCH ), 4.52 (4H, s, 2CH ), 6.90
3
3
2
(4H, d, J = 6.6 Hz, ArH), 7.16–7.28 (12H, m, ArH), 7.55
(4H, d, J=7.8 Hz, ArH), 8.04 (4H, d, J=6.4 Hz, ArH), 8.72
(2H, s, 2–N=CH–), 8.93 (2H, s, 2NH), 9.88 (2H, s, 2NH),
12.60 (2H, s, 2NH). Anal. Calcd. (%) for C H N O
zole‑4‑carboxamide) (20) Yellow crystals, m.p. 262 °C,
−
1
yield (80%). IR (KBr) ν /cm 3432, 3270 (NH), 1654
max
1
(
C=O). H NMR (DMSO-d , 400 MHz, δ ppm): 3.73 (6H,
6
52 48 10
6
s, 2OCH ), 6.14 (2H, s, CH ), 6.90 (4H, d, J=8.6 Hz, ArH),
(909.00): C, 68.71; H, 5.32; N, 15.41. Found: C, 68.62; H,
5.40; N, 15.35%.
3
2
7
8
.31–7.42 (12H, m, ArH), 7.70 (4H, d, J = 8.8 Hz, ArH),
.07 (4H, d, J=8.6 Hz, ArH), 8.66 (2H, s, 2–N=CH–), 8.99
1
3
(
2H, s, 2NH), 9.99 (2H, s, 2NH), 12.64 (2H, s, 2NH).
C
5,5′‑(4,4′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑
(4‑methoxyphenylamino)‑N‑(4‑chlorophenyl)‑1H‑pyra‑
zole‑4‑carboxamide) (24) Yellow crystals, m.p. 282 °C,
NMR (DMSO-d , 100 MHz, δ ppm): 55.72 (2C, 2OCH ),
6
3
8
1
1
9.74 (3C, 1CH and 2C -pyrazole), 114.97, 116.84, 117.29,
2 4
−
1
18.33, 121.24, 121.83, 128.90, 129.40, 131.88, 132.28,
yield (74%). IR (KBr) ν /cm 3426, 3272 (NH), 1656
max
1
38.42, 149.34 (34C, Ar), 153.84 (2C , pyrazole), 155.07
(C=O). H NMR (DMSO-d , 400 MHz, δ ppm): 3.72 (6H, s,
5
6
(
2C , pyrazole), 158.30 (2C, 2–N=CH–), 160.47 (2C, Ar),
2OCH ), 4.50 (4H, s, 2CH ), 6.90 (4H, d, J=8.1 Hz, ArH),
3
3
2
1
62.74 (2C=O). Anal. Calcd. (%) for C H Cl N O
6
7.18–7.26 (6H, m, ArH), 7.41 (4H, d, J=8.6 Hz, ArH), 7.69
(4H, d, J=8.6 Hz, ArH), 7.88 (2H, d, J=8.6 Hz, ArH), 8.04
(4H, d, J=8.2 Hz, ArH), 8.66 (2H, s, 2–N=CH–), 8.96 (2H,
4
9
40
2
10
(
935.81): C, 62.89; H, 4.31; N, 14.97. Found: C, 62.95; H,
4
.26; N, 14.92%.
1
3
s, 2NH), 9.88 (2H, s, 2NH), 12.63 (2H, s, 2NH). C NMR
(DMSO-d , 100 MHz, δ ppm): 55.24 (2C, 2OCH ), 66.71
5
,5′‑(4,4′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑(
6
3
phenylamino)‑1H‑pyrazole‑4‑carboxamide) (21) Yellow
(2C, 2CH ), 93.51 (2C , pyrazole), 114.41, 115.47, 120.57,
2 4
1
crystals, m.p. > 300 °C, yield (77%). H NMR (DMSO-d ,
128.92, 129.88, 131.42, 133.24, 137.50, 151.86 (34C, Ar),
153.10 (2C , pyrazole), 155.27 (2C , pyrazole), 156.52 (2C,
6
4
7
00 MHz, δ ppm): 4.43 (4H, s, 2CH ), 6.86 (2H, t, ArH),
2
5
3
.20 (4H, d, J=8.1 Hz, ArH), 2.27 (4H, t, ArH), 7.34, 7.39
2–N=CH–), 162.33 (2C, Ar), 162.83 (2C=O). Anal. Calcd.
(%) for C H Cl N O (949.84): C, 63.23; H, 4.46; N,
(
4H, 2s, 2NH ), 7.49 (4H, d, J=7.7 Hz, ArH), 7.97 (4H, d,
2
50 42
2
10
6
J=7.8 Hz, ArH), 8.88 (2H, s, 2N=CH–), 9.12 (2H, s, 2NH),
2.21 (2H, s, 2NH). Anal. Calcd. (%) for C H N O
14.75. Found: C, 63.14; H, 4.51; N, 14.69%.
1
3
6
32 10
4
(
668.70): C, 64.66; H, 4.82; O, 9.57. Found: C, 64.72; H,
Biological evaluation
4
.76; O, 9.62%.
In vitro cytotoxicity activity
5
,5′‑(4,4′‑Ethylenebis(oxy)bis(benzylideneamino))bis(3‑(4‑
methoxyphenylamino)‑N‑phenyl‑1H‑pyrazole‑4‑carboxam‑
ide) (22) Yellow crystals, m.p. > 300 °C, yield (80%). IR
Cell culture of HepG2 (human liver carcinoma), RPE-1
(human normal retina pigmented epithelium) and MCF-7
(human breast adenocarcinoma) cell lines was purchased
from the American Type Culture Collection (Rockville, MD)
and maintained in DMEM medium which was supplemented
with 10% heat-inactivated fetal bovine serum (FBS), 100
U/ml penicillin and 100 U/ml streptomycin. The cells were
−
1
1
(
(
(
KBr) ν /cm 3427, 3268 (NH), 1652 (C=O). H NMR
max
DMSO-d , 400 MHz, δ ppm): 3.74 (6H, s, 2OCH ), 4.50
6
3
4H, s, 2CH ), 6.90 (4H, d, J = 8.4 Hz, ArH), 7.08 (2H, t,
2
ArH), 7.20 (2H, d, J=8.6 Hz, ArH), 7.28 (4H, d, J=8.3 Hz,
ArH), 7.37 (4H, t, ArH), 7.67 (4H, d, J=8.2 Hz, ArH), 7.89
(
2H, d, J = 8.6 Hz, ArH), 8.05 (4H, d, J = 8.3 Hz, ArH),
grown at 37 °C in a humidiꢁed atmosphere of 5% CO .
2
8
2
.71 (2H, s, 2–N=CH–), 8.98 (2H, s, 2NH), 9.98 (2H, s,
13
NH), 12.61 (2H, s, 2NH). C NMR (DMSO-d , 100 MHz,
MTT cytotoxicity assay
6
δ ppm): 55.29 (2C, 2OCH ), 66.78 (2C, 2CH ), 90.21 (2C ,
3
2
4
pyrazole), 115.08, 117.60, 121.44, 123.37, 127.44, 129.16,
The cytotoxicity activities against HepG2, RPE-1 and
MCF-7 human cell lines were estimated using the
3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay, which is based on the cleavage of
the tetrazolium salt by mitochondrial dehydrogenases in
viable cells [41–44]. Cells were dispensed in a 96-well ster-
1
1
2
29.91, 131.89, 132.78, 135.64, 138.53, 150.79 (34C, Ar),
53.52 (2C , pyrazole), 155.39 (2C , pyrazole), 156.33 (2C,
5
3
–N=CH–), 162.51 (2C, Ar), 162.98 (2C=O). Anal. Calcd.
(
%) for C H N O (880.95): C, 68.17; H, 5.03; N, 15.90.
50 44 10 6
Found: C, 68.10; H, 5.09; N, 15.95%.
4
ile microplate (5× 10 cells/well) and incubated at 37 °C
with series of diꢀerent concentrations, in DMSO, of each
1
3

Journal of the Iranian Chemical Society
®
tested compound or Doxorubicin (positive control) for
8 h in a serum-free medium prior to the MTT assay. After
using ꢂuorescence emission spectra. All experiments were
conducted in Tris buꢀer (0.01 M Tris, 0.1 M NaCl, at pH
7.4). Glass-distilled deionized water and analytical-grade
reagents were used throughout experiments. pH values of
solutions were measured with a calibrated Jenway pH meter
model 3510 (Staꢀordshire, UK). All buꢀer solutions were
ꢁltered through Millipore ꢁlters (Millipore, UK) of 0.45 mm
pore diameter.
4
incubation, media were carefully removed, and 40 µL of
MTT (2.5 mg/mL) was added to each well and then incu-
bated for an additional 4 h. The purple formazan dye crystals
were solubilized by the addition of 200 µL of DMSO. The
®
absorbance was measured at 570 nm using a SpectraMax
®
Paradigm Multi-Mode microplate reader. The relative cell
viability was expressed as the mean percentage of viable
cells compared to the untreated control cells. All experi-
ments were conducted in triplicate and repeated in three dif-
ferent days. All the values were represented as mean± SD.
IC s were determined by probit analysis using SPSS soft-
Fluorescence spectra and DNA binding studies
Fluorescence emission and excitation spectra were measured
using a Jasco FP-6200 spectroꢂuorometer (Tokyo, Japan)
using ꢂuorescence four-sided quartz cuvettes of 1.00 cm path
length. The automatic shutter-on function was used to mini-
mize photo-bleaching of the sample. The selected excitation
wavelength for ethidium bromide was 480 nm. The emission
spectrum was corrected for background ꢂuorescence of the
buꢀer. The ethidium bromide (EB) ꢂuorescence displace-
ment experiment was performed by sequential addition of
aliquots of 1790 μl Tris buꢀer, 10 μl EB (ꢁnal concentration
of 72 μM), 100 μl G-DNA from stock solutions (1.5 mg/
ml) and ꢁnally 10 μl of the compounds, ꢁnal concentration
of 30 μM. Emission spectra were recorded for each system
using excitation wavelengths of maximum ꢂuorescence
intensity determined for the systems to be 480 nm using a
slit width of 5 nm to examine alterations in emission spectra
resulting from the complex construction of both systems. On
construction of the full systems, the system was allowed to
equilibrate for 30 min at room a temperature and emission
spectra (500–730 nm) were recorded to monitor changes
in EB intensity. Ethidium bromide displacements IC was
5
0
ware program (SPSS Inc., Chicago, IL).
Absorbance spectra
Absorbance spectra were measured on a Jenway UV–vis-
ible spectrophotometer, model 6505 (London, UK) using
quartz cells of 1.00 cm path length. The UV–Vis absorbance
spectra were recorded in the 200–500 nm range and spectral
bandwidth of 3.0 nm. For the ꢁnal spectrum of each solu-
tion analyzed, baseline subtraction of the buꢀer solution was
performed. Genomic DNA was used in a concentration of
7
5 μg/ml. DNA was extracted from peripheral lymphocytes
of anticoagulated blood (EDTA) samples by proteinase K
digestion and phenol/chloroform extraction [53]. The purity
was determined by measuring the absorbance at 260/280 nm
indicating that the sample is free from protein contamination
[
53]. The concentration was assayed spectrophotometrically
−
1
−1
using 6600 M cm as a molar extinction coeꢃcient at
60 nm.
5
0
2
determined, in which the IC values are the concentration of
50
the tested substances required to decrease the ꢂuorescence of
Screening on thymidine phosphorylase
the ethidium bromide–DNA complex by 50% [54].
Enzyme rate spectral scans, studies of stoichiometric inhi-
bition and absorbance readings at ꢁxed wavelengths were
conducted using a Jenway UV–visible spectrophotometer,
model 6505 (London, UK) using quartz cells of 1.00 cm
path length. T. Assays used thymidine as a substrate. The
assay mixture contained (in 1 ml) 20 mM thymidine, 0.1 M
potassium phosphate (pH 7.4) and limiting amounts of
human TP (ca.0.00177 units per assay). The reaction was
initiated by addition of enzyme, and the change in absorb-
ance was monitored at 265 nm at 25 °C. Reaction enzyme
kinetics data were analyzed using Graꢁt version 3 software
Binding energy
The molecular docking simulation method is primarily vali-
dated on the basis of the obtained binding energy. The prede-
ꢁ
ned range of binding energy is supposed to be in the range
between −5 and −15 kcal/mol to productively validate the
molecular docking process.
Molecular docking study
MGL (Molecular Graphics Laboratory) tools 1.5.4 with
AutoDock 4 and AutoGrid 4.0 were used to set up and exert
blind docking calculations between our derivatives and DNA
sequences. DNA sequences:
(
Erithacus software).
DNA binding properties
To study how competently the synthesized compounds inter-
act with G-DNA, we investigated their DNA binding ability
DNA (5′-D(*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP
*CP*G)-3′) (PDB ID: 1bna),
1
3

Journal of the Iranian Chemical Society
DNA (5′-D(*CP*GP*CP*AP*AP*AP*TP*TP*TP*GP*
CP*G)-3′) (PDB ID: 102d),
6. K.S. Kumar, S. Ganguly, R. Veerasamy, E.D. Clercq, Eur. J. Med.
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7
.
X. Zhou, L. Shao, Z. Jin, J.-B. Liu, H. Dai, J.-X. Fang, Heteroat.
DNA (5′-D(*CP*GP*TP*AP*CP*G)-3′) (PDB ID:
Chem. 18, 55–59 (2007)
1
k2j),
8. A.S. Hassan, T.S. Hafez, M.M. Ali, T.K. Khatab, Res. J. Pharm.
DNA (5′-D(*CP*GP*CP*GP*AP*AP*TP*TP
CP*GP*CP*G)-3′) (PDB ID: 2gvr) and DNA
5′-D(*CP*GP*TP*AP*CP*G)-3′) (PDB ID: 2des), were
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G. Ouyang, Z. Chen, X.-J. Cai, B.-A. Song, P.S. Bhadury, S. Yang,
*
L.-H. Jin, W. Xue, D.-Y. Hu, S. Zeng, Bioorg. Med. Chem. 16,
(
9
699–9707 (2008)
obtained from the Protein Data Bank and were used for
the docking studies. Bis-Schiꢀ bases of pyrazoles 9–24
structures were drawn and optimized using ChemDraw
Ultra (version 8.0, Cambridge soft Com., USA). Chem3D
Ultra was used to convert 2D into 3D structures, and the
energy was minimized using the semiempirical AM1
method which is based on the neglect of differential
diatomic overlap (NDDO) integral approximation. The
molecular dockings of Bis-Schiꢀ bases of pyrazole 9–
10. E.M. Sharshira, N.M.M. Hamada, Molecules 17, 4962–4971
(2012)
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1. S. Bondock, S. Adel, H.A. Etman, F.A. Badria, Eur. J. Med.
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4. A. Jarrahpour, D. Khalili, E.D. Clercq, C. Salmi, J.M. Brunel,
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2
4 with B-DNAs (B: right-handed double-helix DNA)
15. A.S. Hassan, A.A. Askar, A.M. Naglah, A.A. Almehizia, A.
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were accomplished by AutoDock 4.2 software from the
Scripps Research Institute (TSRI) (http://autodock.scrip
ps.edu/). Firstly, the polar hydrogen atoms were added
into B-DNA molecules. Then, the partial atomic charges
of the B-DNA and Bis-Schiꢀ bases of pyrazole 9–24 were
calculated using Kollman methods [55]. In the process
of molecular docking, the grid maps of dimensions (62
Å×62 Å×62 Å) with a grid-point spacing of 0.376 Å and
the grid boxes centered. The number of genetic algorithm
runs and the number of evaluations were set to 100. All
other parameters were default settings. Cluster analysis
was performed on docking results by using a root mean
square (RMS) tolerance of 2.0 Å, dependent on the bind-
ing free energy. Lastly, the dominating conꢁguration of
the binding complex of our compounds and B-DNA frag-
ments with minimum binding energy can be determined.
1
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1
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8. A.M. Naglah, A.A. Askar, A.S. Hassan, T.K. Khatab, M.A. Al-
Omar, M.A. Bhat, Molecules 25, 1431 (2020)
9. A.S. Hassan, A.A. Askar, E.S. Nossier, A.M. Naglah, G.O.
Moustafa, M.A. Al-Omar, Molecules 24, 3130 (2019)
20. T.K. Khatab, A.S. Hassan, T.S. Hafez, Bull. Chem. Soc. Ethiop.
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3, 135–142 (2019)
2
1. A.S. Hassan, G.O. Moustafa, A.A. Askar, A.M. Naglah, M.A.
Al-Omar, Synth. Commun. 48, 2761–2772 (2018)
22. A.A. Magd-El-Din, H.A. Mousa, A.A. Labib, A.S. Hassan, A.S.
Abd El-All, M.M. Ali, A.A. El-Rashedy, A.H. El-Desoky, Z.
Naturforsch. C 73, 465–478 (2018)
2
3. H.R. Elgiushy, S.F. Hammad, A.S. Hassan, N. Aboutaleb, K.A.M.
Abouzid, J. Adv. Pharm. Res. 4, 221–237 (2018). https://doi.
org/10.21608/aprh.2018.2839.1049
24. A.S. Hassan, T.S. Hafez, J. Appl. Pharm. Sci. 8, 156–165 (2018)
2
2
2
5. M. El-Naggar, A.S. Hassan, H.M. Awad, M.F. Mady, Molecules
2
3, 1249 (2018)
6. A.S. Hassan, H.M. Awad, A.A. Magd-El-Din, T.S. Hafez, Med.
Chem. Res. 27, 915–927 (2018)
Acknowledgements Authors thank National Research Centre for the
ꢁ
nancial support through scientiꢁc project number 12010103.
7. A.S. Hassan, G.O. Moustafa, H.M. Awad, Synth. Commun. 47,
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963–1972 (2017)
Compliance with ethical standards
28. A.S. Hassan, D.M. Masoud, F.M. Sroor, A.A. Askar, Med. Chem.
Res. 26, 2909–2919 (2017)
2
9. A.S. Abd El-All, A.S. Hassan, S.A. Osman, H.A.A. Yosef, W.H.
Abdel-Hady, M.A. El-Hashash, S.R. Atta-Allah, M.M. Ali, A.A.
El-Rashedy, Acta Pol. Pharm. 73, 79–92 (2016)
Conflict of interest The authors declare that they have no conꢂict of
interest.
3
0. A.S. Hassan, T.S. Hafez, S.A. Osman, M.M. Ali, Turk. J. Chem.
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