Design, green synthesis, molecular docking and anticancer evaluations of diazepam bearing sulfonamide moieties as VEGFR-2 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104350

Novel series of diazepam bearing sulfonamide moieties 5_a-f and 7_a-c were designed, synthesized and evaluated for anticancer activity against HepG2, HCT-116 and MCF-7 cell lines. MCF-7 was the most sensitive cell line to the influence

Full text of DOI:10.1016/j.bioorg.2020.104350

Bioorganic Chemistry 104 (2020) 104350
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, green synthesis, molecular docking and anticancer evaluations of
diazepam bearing sulfonamide moieties as VEGFR-2 inhibitors
,
,d,
Nashwa M. Saleha *, Mohamed S.A. El-Gabyb, Khaled El-Adlc *, Nour E.A. Abd El-Sattare
a Department of Chemistry, Faculty of Science, Al-Azhar University (Girls Branch), PO Box 11754, Cairo, Egypt
b Department of Chemistry, Faculty of Science, Al-Azhar University at Assiut, Assiut 71524, Egypt
c Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884, Cairo, Egypt
d Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, Egypt
e Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel series of diazepam bearing sulfonamide moieties 5a-f and 7a-c were designed, synthesized and evaluated for
anticancer activity against HepG2, HCT-116 and MCF-7 cell lines. MCF-7 was the most sensitive cell line to the
influence of the new derivatives. In particular, compound 5d was found to be the most potent derivative overall
the tested compounds against the three HepG2, HCT116 and MCF-7 cancer cell lines with IC50 = 8.98 ± 0.1,
7.77 ± 0.1 and 6.99 ± 0.1 µM respectively. Compound 5d exhibited higher activity than sorafenib, (IC50 = 9.18
± 0.6, 5.47 ± 0.3 and 7.26 ± 0.3 µM respectively), against HepG2 and MCF-7 but exhibited lower activity against
Benzodiazepines
Sulfonamide
Molecular docking
VEGFR-2 inhibitors
Anticancer agents
HCT116 cancer cell lines respectively. Also, this compound displayed lower activity than doxorubicin, (IC50
=
7.94 ± 0.6, 8.07 ± 0.8 and 6.75 ± 0.4 µM respectively), against HepG2 and MCF-7 but higher activity against
HCT116 cell lines respectively. Compounds 5b, 5c, 5d, 5e, 5f and 7c are respectively, 5.77, 8.58, 9.54, 5.71, 4.68
and 2.31 fold times more toxic in breast cancer cell lines (MCF-7, the most sensitive cells) than in VERO normal
cells. All the synthesized compounds 5a-f and 7a-c were evaluated for their inhibitory activities against VEGFR-2.
Among them, compound 5d was found to be the most potent derivative that inhibited VEGFR-2 at IC50 value of
0.10 ± 0.01 µM, which is equipotent to sorafenib IC50 value (0.10 ± 0.02 µM). Compound 5c exhibited excellent
activity with IC50 value of 0.12 ± 0.01 µM which nearly equipotent to that of sorafenib. Compounds 5b, 5e and 5f
exhibited very good activity with the same IC50 value of 0.14 ± 0.02 µM. Also, compounds 7c and 7b possessed
good VEGFR-2 inhibition with IC50 values of 0.16 ± 0.06 and 0.17 ± 0.06 µM respectively which are more than
the half activity of that of sorafenib. The data obtained from docking studies were highly correlated with that
obtained from the biological screening.
1. Introduction
and products. Moreover, using the preferred safer solvents such as
ethanol and/or acetone and safer reaction conditions (room
Green and sustainable chemistry refers to the design of chemical
products and processes that reduce or eliminate the use and formation of
hazardous chemicals. Therefore, green chemistry practices are intended
to provide economic and environmental benefits to society as a whole
[1]. Chemists can greatly reduce risks to both human health and the
environment by reducing or eliminating hazardous substances usage or
generation associated with a particular synthesis or process.
temperature).
Benzodiazepines (BDZ’s) are privileged bicyclic heteroaromatic
compounds and were considered as an essential class of pharmacologi-
cally active analogues in medicinal and pharmaceutical chemistry
[2–6]. Benzodiazepines are primarily known for their depression in the
central nervous system [7]. In addition to their established anxiolytic
activities, 1,4-benzodiazepines and their analogues demonstrated di-
versity of activity such as antimicrobial [8], in alcohol withdrawal
syndrome [9], sedative [10], hypnotic [11,12], anxiolytic [10,13],
anticonvulsant [14], analgesic [15], anti-inflammatory [16], anti-
In this work, all reactions were carried out at room temperature to
decrease energy usage so increasing energy efficiency. Also, it focused in
designing less hazardous chemical syntheses, designing safer chemicals
* Corresponding authors at: Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884,
Cairo, Egypt (K. El-Adl).
E-mail addresses: drnashwamostafa@azhar.edu.eg (N.M. Saleh), eladlkhaled74@yahoo.com, eladlkhaled74@azhar.edu.eg, khaled.eladl@hu.edu.eg (K. El-Adl).
https://doi.org/10.1016/j.bioorg.2020.104350
Received 19 July 2020; Received in revised form 10 September 2020; Accepted 3 October 2020
Available online 8 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
platelet anti-ulcer [17], antipsychotics [18], anti-proliferative [19] and
anticancer activities [2,7,20–22]. Diazepam consistently inhibited Fatty
acid synthase (FAS) activity, a known anticancer mechanism of flavo-
noids, in addition, it inhibited the release of vascular endothelial growth
factor (VEGF) [23].
tyrosine kinase subtype, is the major regulator of VEGF-driven responses
in endothelial cells and can mediate proliferation, differentiation, and
microvascular permeability. Moreover, it has proven to be a prerequisite
signal transducer in both physiologic and pathologic angiogenesis
[35,36]. VEGFR-2 is over- expressed in several malignancies, including
hepatocellular carcinoma, breast, colorectal, ovarian and thyroid can-
cer, melanoma and medulloblastoma [37–39]. Therefore, VEGFR-2 has
been identified as an excellent remedial target for the production of
novel anticancer agents [40]. Hence, inhibition of VEGFR-2 pathway has
attracted widespread interest as an approach to anti-cancer therapy
[41]. In addition, inhibition of VEGF signaling can also change or
destroy tumor vessels. VEGFR-2 inhibitors, as TKIs, prevent the vital
processes of angiogenesis and lymph angiogenesis. Recently, it was re-
ported that a number of compounds have been confirmed as potent in-
hibitors of VEGFR-2 in vitro or possessed antiangiogenic activity and
achieved clinical success in the treatment of cancer [42]. Due to the
importat role of VEGFR-2 in angiogenesis, this receptor is the most vital
target in anti-angiogenic therapy against cancer. Several potent VEGFR-
2 inhibitors have been developed and approved for treatment of various
cancers, e.g. Pazopanib (IV) [20,43], sunitinib (V) [44,45] and Sorafenib
(Nexavar)®(VI) [46–48] (Fig. 1).
Sulfonamide moieties attached to different heterocyclic compounds
have been reported to inhibit the growth of human tumor cell lines
[24–28]. Ghorab et al. [29] designed (quinoxalin-2-yl)benzene sulfon-
amide derivative I (Fig. 1) with a potent anti-cancer activity against
human liver cancer cell line (Hep G2). In 2014, Shahin et al. [30]
designed a series of new sulfonamide moieties joined to quinoxaline
scaffolds which were biologically evaluated for their inhibitory activity
against VEGFR-2. Also, compound II (Fig. 1) displayed very good anti-
cancer activities against HepG2, HCT-116 and MCF-7 cell lines with IC50
values of 24.5, 12 and 10.23 uM respectively, while compound III
(Fig. 1) exhibited IC50 values of 22.9, 21.9 and 22.9 uM respectively
[31].
Owing to the promising biological activities of benzodiazepines and
sulfonamide derivatives we aimed at incorporation of sulfonamide
moieties into diazepam which may act as potential anticancer molecules
targeting VEGFR-2 enzyme.
Human protein tyrosine kinases (PTKs) play a fundamental role in
human carcinogenesis [32], whereas a cascade of protein kinases
controlled cell cycle progression, cell division and proliferation as a
sequence of events, so PTKs have emerged as promising new cancer
therapy targets [33].
In continuation of our previous efforts in the field of design and
synthesis of new anticancer agents, especially VEGFR-2 inhibitors
[49–56], a new series of diazepam bearing sulfonamide moieties were
designed and synthesized on the basis of the main pharmacophoric
features of VEGFR-2 inhibitors.
The vascular endothelial growth factor (VEGF) is a positive regulator
with its distinct specificity for vascular endothelial cells. The biological
action of VEGF is mediated by three structurally related receptors,
VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 [34]. VEGFR-2, a
A study of the structure–activity relationships (SAR) and binding
pattern of sorafenib and various VEGFR-2 inhibitors revealed that they
shared four main features (as shown in Fig. 2) [57–61]; (i) A flat hetero
aromatic ring system. (ii) A central aryl ring (hydrophobic spacer). (iii)
Fig. 1. Reported VEGFR-2 inhibitors and our derivatives.
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N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
ring (as hydrophobic group), to replace the pyridine and 2,3-dimethyl-
2H-indazole moieties of the reference ligands sorafenib and pazopanib,
respectively. Moreover, phenyldiazene spacer was designed to replace
the central aryl and the methylaminopyrimidine rings of the reference
ligands sorafenib and pazopanib, respectively. Furthermore, the
benzodiazepine moiety occupied the hydrophobic groove formed by
Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916,
Glutamate915, Alanine864 and Leucine838 (Fig. 4).
The diazene and/or sulfonamide linkers interact as H-bond acceptor
–
and as H-bond donor through N N and NH respectively forming H-
–
bonds with the essential amino acid residues Aspartate1044. Also the
terminal (un)substituted hydrophobic tails occupied the hydrophobic
pocket formed by Aspartate1044, Cysteine1043, Isoleucine1042,
Valine897, Leucine887, Lysine866 and Glutamate883. Moreover, the
distal hydrophobic moieties attached to sulfonamide linkers in order to
increase the length of the structure to enable these distal moieties to
occupy new hydrophobic grooves formed by Arginine1025, Histi-
dine1024, Isoleucine1023, Cysteine1022, Lysine1021, Arginine1020,
Leucine1017, Isoleucine890, Histidine889 and Isoleucine886 (Fig. 4).
2. Results and discussion
2.1. Chemistry
At first, the authors expected to get the addition of diazonium salt at
position-3 of diazepam according to the reported procedures [62,63]
and/or at ortho-position of the 5-phenyl ring [64–66] so will obtain
mixture of the corresponding derivatives 3 and/or 4 respectively
(Fig. 5).
But the provided data ensure the addition was occurred at the
preferred para-position (substitution of the activated aromatic ring by
the electrophilic aryldiazonium ion occurs principally at the para posi-
tion. However, if the para position is blocked by a substituent, substi-
tution occurs at the ortho position) [66] due to low temperature,
electronic and steric effects (Fig. 6) and resulted in the corresponding
pure 5a-f derivatives respectively (Scheme 1).
Fig. 2. The basic structural requirements for sorafenib and pazopanib as re-
ported VEGFR-2 inhibitors.
A linker containing a functional group acting as pharmacophore (e.g.
amino or urea) that possesses both H-bond acceptor (HBA) and donor
(HBD) in order to bind with two crucial residues (Glu883 and Asp1044).
(iv) The terminal hydrophobic moiety that occupies the newly created
allosteric hydrophobic pocket via various hydrophobic interactions.
The goal of our work is to synthesize new diazepam bearing sul-
fonamide moieties with the same essential pharmacophoric features of
the reported and clinically used VEGFR-2 inhibitors, in addition, to
being molecularly hybridized in an attempt to get more potent anti-
tumor molecules. The main core of our molecular design rationale was
carried out by ring expansion and bioisosteric modification strategies of
VEGFR-2 inhibitors (sorafenib & pazopanib) at four different positions
(Fig. 3).
The possible reaction mechanism is proposed as described at Fig. 6,
through an electrophilic aromatic substitution. Initially, the acetate
anion catalyst activates the methylene group in the diazepam 2 through
the abstract of a proton to yield the non-isolable intermediate (A) (this
mechanism is very similar to aldol condensation where a carbanion is
generated [67]) which is then stabilized by resonance and delocalization
of the negative charge due to the group the aromatic ring. Finally, the
intermediate (A) then undergo coupling with the beta-nitrogen of a
diazonium ion and subsequent proton transfer to yield the final product
5. The preferred para attack occurs in the reaction of diazepam 2 with
diazonium cation was attributed to the steric effect that inhibited at the
ortho-position [66].
In general, the designed compounds were synthesized and evaluated
for their in vitro anti-proliferative activities against three human tumor
cell lines, namely, hepatocellular carcinoma (HCC) type (HepG2), breast
cancer (Michigan Cancer Foundation-7 (MCF-7)) and human colorectal
carcinoma-116 (HCT-116). The results prompted further examinations
to reach a deep insight into the mechanism of action of the synthesized
compounds. Molecular docking studies were conducted to understand
the expected binding interactions of the target compounds with VEGFR-
2 active sites. Moreover, the most active cytotoxic compounds that
showed promising IC50 values against the three cancer cell lines were
subjected to further investigation for their tyrosine kinase inhibitory
activities against VEGFR-2.
The formation of 7a-c is assumed to proceed through nucleophilic
attack of the amino functional group of compound 5a to the thiocarbonyl
moiety of isothiocyanate followed by intramolecular cyclization via the
addition of NH to carbonyl group to form the non-isolable intermediate
(B) (1,3-diazetidine). The latter intermediate (B) then undergoes a
further stabilization via an intramolecular rearrangement [68] under the
reaction conditions by removal of water molecule to give the unsatu-
rated isothiocyanate 7a-c (Fig. 7).
The synthetic strategy for preparation of the target compounds (5a-f
-
7
a-c) is depicted in Schemes 1 and 2. Synthesis was initiated by addition
of sodium nitrite solution to acidic solution of 4-aminobenzenesulfona-
mide to obtain the corresponding diazonium salt solution, following the
reported procedure [69], which underwent reaction with diazepam 2 in
ethanol in the presence of sodium acetate to obtain the corresponding
derivatives 5a-f respectively (Scheme 1).
1.1. Rationale and structure-based design
Diazepam bearing sulfonamide derivatives have the essential phar-
macophoric features of VEGFR-2 inhibitors (Fig. 3) which include: the
presence of seven membered hetero ring diazepine, fused with benzene
The infrared spectra of compounds 5a-f revealed carbonyl absorption
band at 1685 cmꢀ 1 typical of unsaturated lactams; also, absorption
bands belonging to N N at 1596–1603 cmꢀ 1 and showed absorptions at
–
–
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N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 3. Structural similarities and pharmacophoric features of VEGFR-2 inhibitors and some designed compounds.
3419–3444 cmꢀ 1 for the NH group. In the 1H NMR spectra of 5a-f two
doublets were observed at δ 3.80–3.92 and 4.60 ppm with the same
coupling constant (J = 10.8 Hz) assigned to the methylene protons on C-
3 of diazepam ring, a singlet at δ 3.27–3.39 ppm as assigned to the
methyl protons in addition to the presence of the aromatic protons.
Furthermore, the 13C NMR spectra for these compounds displayed all the
expected signals. The representative 13CNMR spectrum of compound 5b
(DMSO‑d6) revealed the appearance of a signal at 168.96 due to the
used for synthesizing certain organic substances of biological signifi-
cance [70]. Acyl isothiocyanates are bi-functional compounds which
containing a carbonyl group and a thiocyanate group. They are highly
reactive due to the presence of the flow of electrons-withdrawing acyl
group. Thus, the reactivity of acyl isothiocyanates are, determined by
three active centers; the electrophilic carbon atoms of the carbonyl and
thiocarbonyl groups as well as the nucleophilic nitrogen atom, which
enable them to participate in various types of addition and cyclization
reactions. The behavior of benzene sulfonamide 5a towards acyl iso-
thiocyanate was examined. Room temperature reaction of benzene
sulfonamide derivative 5a with acetyl isothiocyanate in dry acetone
gave the acetimidoyl isothiocyanate 7a in quantitative yield, rather than
the expected acyl thioureas 6a. The infrared spectrum of the reaction
product indicated the absence of the NH2 absorption bands and contains
–
presence of C O group and showed three signals at 168.38, 155.73 and
–
–
155.60 which were assigned to the three C N as well as signals at δ
–
154.34, 152.70, 131.18, 130.99, 130.52, 129.84, 129.52, 122.83
122.48, 121.51, 119.98 due to the aromatic carbons and signals at δ
55.91 (CH2) and 34.15 (CH3).
The chemistry of acyl isothiocyanates is very complex, varied and
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N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 4. Superimposition of compound 5d and sorafenib inside the binding pocket of 1YWN.
the characteristic absorption band at 2120 cmꢀ 1 was assigned for the
position and orientation inside the recognized binding site of VEGFR-2
that reveals a large space bounded by a membrane-binding domain,
which serves as entry channel for substrate to the active site (Fig. 8). The
obtained results of the free energy of binding (ΔG) explained that most
of these compounds had good binding affinity toward the receptor and
the computed values reflected the overall trend (Table 1).
– –
N
C S group and three absorption bands at 1865, 1631 and 1601
– –
cmꢀ 1 assignable for C O, C N and N N groups, respectively. The
– –
–
–
– –
1HNMR spectrum (DMSO‑d6) of the reaction product showed absence of
the singlet signal assignable for NH2 while revealed two doublets at δ
3.96 and 4.58 ppm with the same coupling constant (J = 10.8 Hz)
assigned to the methylene protons and two singlet signals at δ 2.09 and
3.35, ppm assigned to the two methyl protons in addition to the presence
of the aromatic protons.
The proposed binding mode of sorafenib revealed affinity value of
ꢀ 95.66 kcal/mol and 4 H-bonds. The urea linker formed one H-bond
with the key amino acid Glutamate883 (2.13 Å) through its NH group
and one H-bond with Aspartate1044 (1.53 Å) through its carbonyl group.
The N-methylpicolinamide moiety was stabilized by formation of 2 H
bonds with Cysteine917 where, the pyridine N atom formed 1H-bond
with the NH of Cysteine917 (2.46 Å) while its NH group formed 1H-
bond with the carbonyl of Cysteine917 (2.20 Å). The N-methyl-
picolinamide moiety occupied the hydrophobic groove formed by
Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916,
Glutamate915, Leucine838 and Alanine864. Moreover, the central phenyl
ring occupied the hydrophobic pocket formed by Cysteine1043,
Leucine1033, Valine914, Valine897 and Lysine866. Furthermore, the
hydrophobic 3-trifluromethyl-4-chlorophenyl moiety attached to the
urea linker occupied the hydrophobic pocket formed by Aspartate1044,
Isoleucine1042, Histidine1024, Leucine1017, Isoleucine890 and
Leucine887 (Fig. 9). The urea linker played an important role in the
binding affinity towards VEGFR-2 enzyme, where it was responsible for
the higher binding affinity of sorafenib. These findings encourage us to
use diazene and sulfonamide linkers hoping to obtain potent VEGFR-2
inhibitors.
In a similar manner, treatment of benzene sulfonamide derivative 5a
with benzoylisothiocyanate and 2-chlorobenzoylisothiocyanate in
acetone at room temperature yielded the corresponding benzimidoyli-
sothiocyanate (7b) and 2-chlorobenzimidoylisothiocyanate (7c) de-
rivatives respectively (Scheme 2).
The structures of (7b,c) were established on their respective analyt-
ical and spectral data. The infrared spectra of compounds 7b,c showed
the disappearance of absorption bands for NH2 and showed the
– –
–
–
appearance of absorption band for C O, C N and N N groups, while
– –
strong absorption band appeared at 2130 cmꢀ 1 for N
C
S group. The
– –
) show–ed t–wo doublets at
1HNMR spectra (DMSO‑d6) of compounds (7b,c
δ 3.81–3.98 and 4.57–4.60 ppm with the same coupling constant (J =
10.8 Hz) which were assigned to the protons of methylene moiety in
addition to the presence of sharp singlet at δ 3.34–3.35 ppm assigned to
the methyl protons as well as aromatic protons. The 13C NMR spectrum
of compound–7b –revealed the presence of a signal at 143.0 due to the
presence of N
C S group and signals at δ 35.25 (CH ), 56.2 (CH ), 151
– –
3 2
–
–
–
(C N), 168 (C O) in addition to the presence of aromatic carbons.
–
The proposed binding mode of compound 5d is virtually the same as
that of sorafenib which revealed higher affinity value of ꢀ 116.78 kcal/
mol and 5H-bonds. The carbonyl group of diazepame scaffold formed
one H-bond with the essential amino acid Cysteine917 through its NH
group with a distance of 1.93 Å. Moreover, the diazene linker formed
2H-bonds with Aspartate1044 (2.06 Å, 2.79 Å) while NH group of sul-
fonamide moiety formed a third H bond with Aspartate1044 (1.75 Å).
2.2. Docking studies
All modeling experiments were carried out using Molsoft software.
Each experiment used VEGFR-2 downloaded from the Brookhaven
Protein Databank (PDB ID 1YWN) [71].
The obtained results indicated that all studied ligands have similar
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N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 5. Addition of the diazonium salt at para-position of the phenyl ring at position-5 of diazepam.
Furthermore, the N atom of the distal pyridine moiety formed one H-
bond with Arginine1025 (2.95 Å). The diazepame scaffold occupied the
hydrophobic groove formed by Leucine1033, Glycine920, Lysine918,
Cysteine917, Phenylalanine916, Glutamate915, Alanine864 and
Leucine838. Moreover, the central phenyldiazene spacer occupied the
hydrophobic pocket formed by Aspartate1044, Cysteine1043,
Leucine1033, Glutamate915, Valine914, Valine897 and Lysine866. The
hydrophobic phenyl tail occupied the hydrophobic pocket formed by
Aspartate1044, Cysteine1043, Isoleucine1042, Valine897, Leucine887,
Lysine866 and Glutamate883. Furthermore, the distal 4,6-dimethylpyri-
dine moiety occupied the hydrophobic groove formed by Argi-
nine1025, Histidine1024, Isoleucine1023, Cysteine1022, Lysine1021,
Arginine1020, Leucine1017, Isoleucine890, Histidine889 and Isoleucine886
(Fig. 10). These interactions of compound 5d may explain its highest
anticancer activity.
a third H bond with Aspartate1044 (2.17 Å). Furthermore, the N atom of
the distal pyridine moiety formed one H-bond with Arginine1025 (2.98
Å). The diazepame scaffold occupied the hydrophobic groove formed by
Leucine1033, Glycine920, Lysine918, Cysteine917, Phenylalanine916,
Glutamate915, Alanine864 and Leucine838. Moreover, the central phe-
nyldiazene spacer occupied the hydrophobic pocket formed by Aspar-
tate1044, Cysteine1043, Leucine1033, Glutamate915, Valine914,
Valine897 and Lysine866. The hydrophobic phenyl tail occupied the
hydrophobic pocket formed by Aspartate1044, Cysteine1043, Isoleu-
cine1042, Valine897, Leucine887, Lysine866 and Glutamate883. Further-
more, the distal 5-methylpyridine moiety occupied the hydrophobic
groove formed by Arginine1025, Histidine1024, Isoleucine1023,
Cysteine1022, Lysine1021, Arginine1020, Leucine1017, Isoleucine890,
Histidine889 and Isoleucine886 (Fig. 11). These interactions of compound
5c may explain its high anticancer activity.
The proposed binding mode of compound 5c is virtually the same as
that of sorafenib and 5d which revealed higher affinity value of
ꢀ 107.58 kcal/mol and 5H-bonds. The carbonyl group of diazepame
scaffold formed one H-bond with the essential amino acid Cysteine917
(1.91 Å). Moreover, the diazene linker formed 2H-bonds with Aspar-
tate1044 (2.27 Å, 2.47 Å) while NH group of sulfonamide moiety formed
The proposed binding mode of compound 5f is virtually the same as
that of sorafenib and 5d which revealed higher affinity value of
ꢀ 101.97 kcal/mol and 5H-bonds. The carbonyl group of diazepame
scaffold formed one H-bond with the essential amino acid Cysteine917
(1.95 Å). Moreover, the diazene linker formed one H-bond with Aspar-
tate1044 (2.47 Å) while NH group of sulfonamide moiety formed a
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N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
[49,72–74]. Sorafenib and doxorubicin were included in the experi-
ments as reference cytotoxic drugs. The results were expressed as growth
inhibitory concentration (IC50) values and summarized in Table 2. From
the obtained results, it was explicated that most of the prepared com-
pounds displayed excellent to modest growth inhibitory activity against
the tested cancer cell lines. In general, investigations of the cytotoxic
activity indicated that MCF-7 was the most sensitive cell line to the in-
fluence of the new derivatives respectively. In particular, compound 5d
was found to be the most potent derivative overall the tested compounds
against the three HepG2, HCT116 and MCF-7 cancer cell lines with IC50
= 8.98 ± 0.1, 7.77 ± 0.1 and 6.99 ± 0.1 µM respectively. Compound 5d
exhibited higher activity than sorafenib, (IC50 = 9.18 ± 0.6, 5.47 ± 0.3
and 7.26 ± 0.3 µM respectively), against HepG2 and MCF-7 but
exhibited lower activity against HCT116 cancer cell lines respectively.
α
β
Also, this compound displayed lower activity than doxorubicin, (IC50
=
7.94 ± 0.6, 8.07 ± 0.8 and 6.75 ± 0.4 µM respectively), against HepG2
and MCF-7 but higher activity against HCT116 cell lines respectively.
With respect to the HepG2 hepatocellular carcinoma cell line, com-
pounds 5c, 5f, 5b and 5e displayed very good anticancer activities with
(IC50 = 11.32 ± 0.3, 11.99 ± 0.3, 14.49 ± 0.3 and 16.11 ± 0.3 µM,
respectively). Compounds 7c and 7b, with IC50 = 21.34 ± 1.6 and 23.11
± 2.2 µM respectively, displayed good cytotoxicity. While compounds 5a
and 7a with (IC50 = 31.76 ± 3.1 and 32.09 ± 2.6 µM) exhibited mod-
erate cytotoxicity.
Cytotoxicity evaluation against colorectal carcinoma (HCT-116) cell
line, discovered that compounds 5f and 5c displayed excellent anti-
cancer activities with (IC50 = 9.33 ± 0.2 and 9.87 ± 0.2 µM, respec-
tively). Compounds 5b, 5e and 7c displayed very good anticancer
activities with (IC50 = 11.39 ± 0.2, 13.23 ± 0.2 and 19.63 ± 1.7 µM,
respectively). Compounds 7b and 7a, with IC50 = 22.21 ± 2.2 and 28.28
± 2.7 µM respectively, displayed good cytotoxicity. While compound 5a
with (IC50 = 37.34 ± 3.2 µM) exhibited moderate cytotoxicity.
Cytotoxicity evaluation against MCF-7 cell line, revealed that com-
pounds 5c, 5b and 5e displayed excellent anticancer activities with (IC50
= 7.24 ± 0.2, 8.65 ± 0.2 and 8.87 ± 0.2 µM, respectively). Compounds
5f and 7c displayed very good anticancer activities with (IC50 = 10.11 ±
0.2 and 17.99 ± 1.6 µM, respectively). Compounds 7b with IC50 = 20.13
± 2.2 µM, displayed good cytotoxicity. While compounds 5a and 7a with
(IC50 = 33.10 ± 3.1 and 30.66 ± 2.6 µM) exhibited moderate
cytotoxicity.
Fig. 6. Proposed mechanism for synthesis of compounds 5a-f.
second H bond with Aspartate1044 (2.23 Å). Furthermore, the distal
quinoxaline moiety was stabilized by formation of two H-bonds with
Arginine1025 (2.17 Å, 2.55 Å). The diazepame scaffold occupied the
hydrophobic groove formed by Leucine1033, Glycine920, Lysine918,
Cysteine917, Phenylalanine916, Glutamate915, Alanine864 and
Leucine838. Moreover, the central phenyldiazene spacer occupied the
hydrophobic pocket formed by Aspartate1044, Cysteine1043,
Leucine1033, Glutamate915, Valine914, Valine897 and Lysine866. The
hydrophobic phenyl tail occupied the hydrophobic pocket formed by
Aspartate1044, Cysteine1043, Isoleucine1042, Valine897, Leucine887,
Lysine866 and Glutamate883. Furthermore, the distal quinoxaline moiety
occupied the hydrophobic groove formed by Arginine1025, Histi-
dine1024, Isoleucine1023, Cysteine1022, Lysine1021, Arginine1020,
Leucine1017, Isoleucine890, Histidine889 and Isoleucine886 (Fig. 12).
These interactions of compound 5f may explain its high anticancer
activity.
Finally, the most potent six derivatives 5b, 5c, 5d, 5e, 5f and 7c were
tested for their cytotoxicity against normal VERO cell lines. The results
revealed that the tested compounds have low toxicity against VERO
normal cells with IC50 values ranging from 41.54 to 66.67 μM. The
From the obtained docking results (Table 1), we concluded that, all
the synthesized compounds exhibited higher affinities towards VEGFR-2
except compound 5a which showed lower affinity and compounds 7a-c
which displayed nearly equipotent affinities in comparing to sorafenib.
The diazene linker occupied the same groove occupied by urea linker of
sorafenib and played the same role which is essential for higher affinity
towards VEGFR-2 enzyme. The distal moieties formed Hydrogen and
hydrophobic bonding interactions and consequently affinities towards
VEGFR-2 enzyme. Elongation of the structure played an important role
in their VEGFR-2 inhibitory activities. The diazene and sulfonamide
linkers formed hydrogen bonding interactions which increased affinity
towards VEGFR-2 enzyme. On the other hand, diazepame scaffold
compensated hydrogen bonding and hydrophobic interactions of N-
methylpicolinamide moiety of sorafenib.
cytotoxicity of the tested compounds against the cancer cell lines was
from 6.99 to 37.34 μM.
Compounds 5b, 5c, 5d, 5e, 5f and 7c are respectively, 5.77, 8.58,
9.54, 5.71, 4.68 and 2.31 fold times more toxic in breast cancer cell lines
(MCF-7, the most sensitive cells) than in VERO normal cells.
2.4. In vitro VEGFR-2 kinase assay
Furthermore, all the synthesized compounds 5a-f and 7a-c were
evaluated for their inhibitory activities against VEGFR-2 by using an
anti-phosphotyrosine antibody with the Alpha Screen system (Perki-
nElmer, USA). The results were reported as a 50% inhibition concen-
tration value (IC50
) calculated from the concentration-inhibition
response curve and summarized in Table 2. Sorafenib was used as pos-
itive control in this assay. The tested compounds displayed high to
medium inhibitory activity with IC50 values ranging from 0.10 ± 0.01 to
0.32 ± 0.06 µM. Among them, compound 5d was found to be the most
potent derivative that inhibited VEGFR-2 at IC50 value of 0.10 ± 0.01
µM, which is equipotent to sorafenib IC50 value (0.10 ± 0.02 µM).
Compound 5c exhibited excellent activity with IC50 value of 0.12 ± 0.01
µM which nearly equipotent to that of sorafenib. Compounds 5b, 5e and
5f exhibited very good activity with the same IC50 value of 0.14 ± 0.02
2.3. In vitro cytotoxic activity
Anti-proliferative activity of the newly synthesized derivatives 5a-f
-
7
a-c was examined against three human tumor cell lines namely, hepa-
tocellular carcinoma (HepG2), colorectal carcinoma (HCT-116) and
breast cancer (MCF-7) using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT) colorimetric assay as described by Mosmann
7

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Scheme 1. Synthetic route for preparation of the target compounds 5a-f.
µM. Also, compounds 7c and 7b possessed good VEGFR-2 inhibition with
IC50 values of 0.16 ± 0.06 and 0.17 ± 0.06 µM respectively which are
more than the half activity of that of sorafenib. On the other hand,
compounds 7a and 5a displayed the moderate VEGFR-2 inhibition with
IC50 values = 0.29 ± 0.06 and 0.32 ± 0.06 µM, respectively.
diazene linkers which interacting as H-bond acceptors and also sulfon-
amide linkers which act as H-bond donor through its NH group. These
linkers interacting with the side chain NH and carboxylate of the
essential amino acid residue Aspartate1044 respectively. Also hydro-
phobic interactions through the attached (un)substituted hydrophobic
distal moieties. The effect of replacement of pyridine and 2,3-dimethyl-
2H-indazole moieties of the reference ligands sorafenib and pazopanib,
respectively by the benzodiazepine moiety of diazepam scaffold of the
synthesized compounds on the antitumor activities also was noticed.
This benzodiazepine moiety occupied the same hydrophobic pocket
2.5. Structure activity relationship (SAR)
The preliminary SAR study has focused on the effect of replacement
of the urea and NH linkers of sorafenib and pazopanib respectively with
8

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 7. Proposed mechanism for the formation of compounds 7a-c.
which occupied by the pyridine moiety of the standard ligand. More-
over, the phenyldiazene spacer was designed to replace the central aryl
and the methylaminopyrimidine rings of the reference ligands sorafenib
and pazopanib, respectively. On the other hand different distal aliphatic,
aromatic and heteroaromatic moieties were introduced to the phenyl
tail of the reference ligand with different lipophilicity and electronic
nature in order to study their effects on the anticancer activity. The
presence of lipophilic distal moieties attached to the phenyl ring through
sulfonamide linkers in order to increase the length of the structure to
enable these distal moieties to form new hydrophobic and H-bonding
interactions with the active site. The data obtained revealed that, the
tested compounds displayed different levels of anticancer activity and
possessed a distinctive pattern of selectivity against the MCF-7 cell lines.
Generally, the spacers, linkers (HBA-HBD), lipophilicity and electronic
nature exhibited an important role in anticancer activity. The presence
of the hydrophobic heteroaromatic distal moieties and the linkers dia-
zene and sulfonamide of compounds 5a-f were found to be responsible
for their highest anticancer activities than the other compounds 7a-c that
containing phenyl and/or methyl ones. From the structure of the syn-
thesized derivatives and the data shown in Table 2 we can divide these
and MCF-7 cell lines. The distal 4,6-dimethylpyrimidine moiety with
two hydrophobic electron donating methyl groups 5d exhibited higher
anticancer activities than the 5-methylpyridine with hydrophobic elec-
tron donating methyl one 5c and the pyrazine one fused to the electron
deficient benzene ring to form quinoxaline moiety 5f against HepG2 and
MCF-7 cell lines. While the distal 4,6-dimethylpyrimidine moiety 5d
exhibited higher anticancer activities than the quinoxaline moiety 5f
and 5-methylpyridine one 5c against HCT116 cell lines respectively.
Moreover, the distal six membered heteroaromatic pyrimidine moieties
5d, 5c and 5b exhibited higher anticancer activities than the five het-
eroaromatic isoxazole one 5e against the three HepG2, HCT116 and
MCF-7 cell lines. Moreover, the distal six membered heteroaromatic
quinoxaline 5f exhibited higher anticancer activities than the five het-
eroaromatic isoxazole one 5e against HepG2 and HCT116 cell lines but
lower activity against MCF-7 cell lines. In the second group 7a-c, the
distal electron deficient phenyl ring substituted with hydrophobic
electron withdrawing chloro group as in compound 7c exhibited higher
activities than that unsubstituted phenyl one 7b and the aliphatic elec-
tron donating methyl one 7a against the three HepG2, HCT116 and MCF-
7 cell lines.
tested compounds into two groups. The first group is compounds 5a-f
,
The data obtained from VEGFR-2 inhibition assay concluded that,
generally the distal heteroaromatic 5b-f displayed higher activity than
the 2-chlorophenyl 7c, phenyl 7b and/or aliphatic methyl one 7a
respectively. Also, these distal moieties attached to sulfonamide linkers
showed higher activities than the sulfonamide group without any distal
moiety 5a. The presence of the distal hydrophobic 4,6-dimethylpyrimi-
dine moiety with two hydrophobic electron donating methyl groups at
positions 4 and 6 as in compound 5d exhibited the highest VEGFR-2
inhibition activity that equipotent to Sorafenib. In addition, 4,6-dime-
thylpyrimidine moiety exhibited higher activity than that mono
substituted with methyl (5-methylpyrimidine) 5c and/or the
the terminal phenyl tail was substituted with different heteroaromatic
moieties linked through sulfonamide linkers as in compound 5b-f, and
the sulfonamide linker was unsubstituted as in compound 5a. Generally,
the substituted sulfonamide linker as in compounds 5b-f displayed
higher activities than the unsubstituted one as in compound 5a against
the three HepG2, HCT116 and MCF-7 cell lines. The presence of distal
4,6-dimethylpyrimidine moiety with two hydrophobic electron
donating methyl groups at positions 4 and 6 as in compound 5d
exhibited higher anticancer activities than the 5-methylpyridine one 5c
and the unsubstituted pyridine one 5b against the three HepG2, HCT116
9

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Scheme 2. Synthetic route for preparation of the target compounds 7a-c.
unsubstituted pyrimidine one 5b respectively. The distal hydrophobic
heteroaromatic pyridine 5b, 5-methylisoxazole 5e and quinoxaline 5f
exhibited the same VEGFR-2 inhibition activity.
amino acid Cysteine917. The distal moieties formed new hydrogen and
hydrophobic bonding interactions and consequently affinities towards
VEGFR-2 enzyme. Elongation of the structure played an important role
in their VEGFR-2 inhibitory activities. The data obtained from the
docking studies were highly correlated with that obtained from the
biological screening. All the tested compounds showed variable anti-
cancer activities. MCF-7 was the most sensitive cell line to the influence
of the new derivatives. In particular, compound 5d was found to be the
most potent derivative overall the tested compounds against the three
HepG2, HCT116 and MCF-7 cancer cell lines with IC50 = 8.98 ± 0.1,
7.77 ± 0.1 and 6.99 ± 0.1 µM respectively. Compound 5d exhibited
higher activity than sorafenib, (IC50 = 9.18 ± 0.6, 5.47 ± 0.3 and 7.26 ±
0.3 µM respectively), against HepG2 and MCF-7 but exhibited lower
activity against HCT116 cancer cell lines respectively. Also, this com-
pound displayed lower activity than doxorubicin, (IC50 = 7.94 ± 0.6,
8.07 ± 0.8 and 6.75 ± 0.4 µM respectively), against HepG2 and MCF-7
but higher activity against HCT116 cell lines respectively. Compounds
3. Conclusion
In summary, nine new diazepam bearing sulfonamide derivatives
have been designed, synthesized and evaluated for their anticancer ac-
tivities against three human tumor cell lines hepatocellular carcinoma
(HepG2), colorectal carcinoma (HCT-116) and breast cancer (MCF-7)
targeting VEGFR-2 enzyme. All the tested compounds showed variable
anticancer activities. The molecular design was performed to investigate
the binding mode of the proposed compounds with VEGFR-2 receptor.
The diazene and/or sulfonamide linkers played the same role of urea
linker of sorafenib which is essential for higher affinity towards VEGFR-
2 enzyme. The diazepam scaffold increased hydrophobic interactions
and form H-bond through its carbonyl group at position-2 with the
10

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 8. Superimposition of some docked compounds inside the binding pocket of 1YWN.
using BRUKER (400 MHz, Varian, UK) using DMSO‑d6 as a solvent and
tetraethylsilane (TMS) as internal standard chemical shifts are expressed
as δ ppm. 13C NMR (100 MHz) spectra, using DMSO‑d6 as a solvent and
TMS (δ) as the internal standard. Analytical data were obtained from
Vario EL III Elemental CHNS analyzer.
Table 1
The calculated free energy of binding (ΔG in Kcal/mole) for the ligands.
Compound
ΔG [kcal molꢀ 1
]
Compound
ΔG [kcal molꢀ 1
]
5a
5b
5c
5d
5e
ꢀ 84.43
5f
ꢀ 101.97
ꢀ 90.48
ꢀ 92.86
ꢀ 95.46
ꢀ 95.66
ꢀ 101.84
ꢀ 107.58
ꢀ 116.78
ꢀ 100.75
7a
7b
4.1.2. General procedure for synthesis of target compounds (5a-f
)
7c
The appropriate 4-Aminobenzenesulfonamide derivative (0.01 mol)
was suspended in water (50 mL). HCl (10 mL, 36.5%) was added drop
wise to this solution with stirring. The mixture was gradually heated up
to 30 ◦C till a clear solution was obtained. The solution was cooled to
0–5 ◦C in an ice bath. A sodium nitrite solution (0.5 g in 5 mL H2O) was
then added over a period of 5 min while stirring to obtain diazonium salt
solution. Diazepam (2.85 gm, 0.01 mol) was dissolved in ethanol in the
presence of sodium acetate and kept at 0–5 ◦C. Subsequently, the dia-
zonium salt solution was added with the occasional slow stirring to the
diazepam solution for 10–15 min. The formed precipitate was filtered,
dried, and washed with ethanol to give the corresponding derivatives 5a-
f respectively.
Sorafenib
5b, 5c, 5d, 5e, 5f and 7c are respectively, 5.77, 8.58, 9.54, 5.71, 4.68 and
2.31 fold times more toxic in breast cancer cell lines (MCF-7, the most
sensitive cells) than in VERO normal cells. All the synthesized com-
pounds 5a-f and 7a-c were evaluated for their inhibitory activities against
VEGFR-2. Among them, compound 5d was found to be the most potent
derivative that inhibited VEGFR-2 at IC50 value of 0.10 ± 0.01 µM,
which is equipotent to sorafenib IC50 value (0.10 ± 0.02 µM). Compound
5c exhibited excellent activity with IC50 value of 0.12 ± 0.01 µM which
nearly equipotent to that of sorafenib. Compounds 5b, 5e and 5f
exhibited very good activity with the same IC50 value of 0.14 ± 0.02 µM.
Also, compounds 7c and 7b possessed good VEGFR-2 inhibition with IC50
values of 0.16 ± 0.06 and 0.17 ± 0.06 µM respectively which are more
than the half activity of that of sorafenib. On the other hand, compounds
7a and 5a displayed the moderate VEGFR-2 inhibition with IC50 values
= 0.29 ± 0.06 and 0.32 ± 0.06 µM, respectively. The obtained results
showed that, our compounds could be useful as a template for future
design, optimization and investigation to produce more potent and se-
lective VEGFR-2 inhibitors with higher anticancer analogs with lower
side effects.
4.1.2.1. 4-([4-{7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-5-yl}phenyl]diazenyl)benzene-(5a). Red
crystals;
m.p.
ꢀ 1
110–112 ◦C; yield 93%; IR (KBr, cm ): 3468, 3396 (NH ),1685(C O),
–
–
2
1
–
–
1597 (N N), 1315, 1156 (SO ); H NMR (DMSO‑d ) δ: 3.30 (s, 3H, N-
2
6
CH3), 3.92 (d, 1H, J = 10.8, CH methylene), 4.59 (d, 1H J = 10.8, CH
methylene), 4.88 (s br, 2H, NH2, D2O exchangeable), 7.25 (d, 1H, J =
2.4, C6H3), 7.49 (d, 2H, J = 7.6, C6H4-SO2), 7.53 (d, 2H, J = 7.6, C6H4-
SO2), 7.58 (s, 2H, NH2, D2O exchangeable), 7.61 (d, 2H, J = 8.3, C6H4-
N2), 7.67 (d, 2H, J = 8.3, C6H4-N2), 7.78 (d, 1H, J = 2.4, C6H3), 7.80 (d,
1H, J = 2.4, C6H3). 13C NMR (DMSO‑d6) δ (ppm): 34.15 (CH3-N), 55.91
(CH2), 119.44 (2), 122.48, 122.83 (2), 128.58 (2), 129.84 (2), 130.52,
4. Experimental
130.99, 131.18, 131.38, 139.62, 141.16, 141.40, 151.19, 152.70,
+
–
–
–
168.38 (C N), 168.69 (C O); MS (m/z): 467.33 (M , 14.11%), 284.09
–
4.1. Chemistry
(66.74%), 218.44 (60.85%), 216.33 (70.17%), 153.56 (100%, base
beak), 133.32 (66.98%); Anal. Calcd for C22H18ClN5O3S (467.93): C,
56.47; H, 3.88; N, 14.97; S, 6.85. Found: C, 56.40; H, 3.70; N, 14.80; S,
6.70.
4.1.1. General
All melting points of the synthesized compounds were recorded on
Büchi melting point apparatus D-545. IR spectra (KBr disks) were
measured on an FTIR plus 460 or Pye Unicam SP-1000 spectropho-
tometer (Pye Unicam, Cambridge, UK). 1H NMR spectra were obtained
11

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 9. Predicted binding mode for sorafenib with 1WYN. H-bonded atoms are indicated by dotted lines.
Fig. 10. Predicted binding mode for 5d with 1WYN.
4.1.2.2. 4-([4-{7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-5-yl}phenyl]-diazenyl)-N-(pyrimidin-2-yl)benzenesulfonamide
(5b). Yellow crystals; m.p. 140–142 ◦C; yield 85%; IR (KBr, cmꢀ 1): 3422
(d, 2H, J = 8.3, C6H4-N2), 7.7 (d, 1H, J = 2.4, C6H3), 7.72 (d, 1H, J = 2.4,
C6H3), 8.53 (s, 1H, NH, D2O exchangeable). 13C NMR (DMSO‑d6) δ
(ppm): 34.15 (CH3-N), 55.91 (CH2), 119.98 (2), 121.51 (2), 122.48,
122.83 (2), 129.52 (2), 129.84 (2), 130.52 (2), 130.99 (2), 131.18 (2),
(NH), 1685 (C O), 1603 (N N), 1314, 1127 (SO2); 1H NMR
–
–
–
–
–
–
–
(DMSO‑d6) δ: 3.27 (s, 3H, N-CH3), 3.80 (d, 1H, J = 10.8, CH methylene),
4.60 (d, 1H, J = 10.8, CH methylene), 7.22–7.44 (dd, 3H, J = 7.4, 2.4,
CH pyrimidine), 7.46 (d, 1H, J = 7.4, C6H3), 7.48 (d, 2H, J = 7.4, C6H4-
SO2), 7.5(d, 2H, J = 7.4, C6H4-SO2), 7.54 (d, 2H, J = 8.3, C6H4-N2), 7.60
152.70, 154.34 (2), 155.60 (C N diazepine), 155.73 (C N pyrimi-
–
+
–
–
–
–
dine), 168.38 (C N pyrimidine), 168.69 (C O); MS (m/z): 546.03 (M ,
12.48%), 509.06 (59.50%), 310.36 (76.40%), 144.12 (78.00%), 119.93
(100%, base beak), 117.33 (81.11%); Anal. Calcd for C26H20ClN7O3S
12

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
Fig. 11. Predicted binding mode for 5c with 1WYN.
Fig. 12. Predicted binding mode for 5f with 1WYN.
(546.0): C, 57.20; H, 3.69; N, 17.96; S, 5.87. Found: C, 57.30; H, 3.50; N,
17.90; S, 5.75.
(dd, J = 7.6, 2H, CH pyrimidine), 7.44 (s, 1H, C6H3), 7.46 (d, 2H, J =
7.8, C6H4-SO2), 7.51 (d, 2H, J = 7.7, C6H4-SO2), 7.56 (d, 2H, J = 8.3,
C6H4-N2), 7.62 (d, 2H, J = 8.3 C6H4-SO2), 7.70 (d, 1H, C6H3), 7.73 (d,
1H, C6H3), 8.5 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6) δ
(ppm): 16.14 (CH3), 34.76 (CH3-N), 55.91 (CH2), 119.98 (2), 122.48 (2),
122.65 (2), 122.83 (2), 129.52 (2), 129.84 (2), 130.52, 130.99 (2),
4.1.2.3. 4-([4-{7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-5-yl}phenyl]diazenyl)-N-(5-methylpyrimidin-2-yl)benzenesulfona-
mide (5c). Orange crystals; m.p. 135–137 ◦C; yield 93%; IR (KBr, cmꢀ 1):
3441 (NH), 1685 (C O), 1603 (N N), 1315, 1166 (SO ); 1H NMR
–
–
–
–
–
–
–
131.18 (2), 153.34 (2), 155.58, 156.48 (C N diazepine), 159.79 (C
N
–
(DMSO‑d6) δ: 2.51 (s, 3H, CH3), 3.28 (s, 3H, N-CH3), 3.812(d, 1H, J =
10.6, CH methylene), 4.60 (d, 1H, J = 10.6, CH methylene), 7.22–7.44
–
–
–
pyrimidine), 168.38 (C N pyrimidine), 168.69 (C O); MS (m/z):
–
560.52 (M+, 18.89%), 506.74 (51.07%), 487.13 (100%, base beak),
13

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
4.1.2.6. 4-([4-{7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-5-yl}phenyl]diazenyl)-N-(quinoxalin-2-yl)benzenesulfonamide
(5f). Orange crystals; m.p. 160–162 ◦C; yield 90%; IR (KBr, cmꢀ 1): 3419
Table 2
In vitro cytotoxic activities of the newly synthesized compounds against HepG2,
HCT-116, MCF-7 and VERO cell lines and VEGFR-2 kinase assay.
(NH), 1685 (C O), 1596 (N N), 1314, 1130 (SO2); 1H NMR
–
–
–
–
Compound
IC50 (µM)a
(DMSO‑d6) δ: 3.35 (s, 3H, N-CH3), 3.80 (d, 1H, J = 10.8 Hz, CH meth-
ylene), 4.60 (d, 1H, J = 10.8 Hz, CH methylene), 6.07 (s, 1H, J = 8.4 Hz,
C6H3), 6.61 (d, 2H, quinoxaline), 7.22 (s, 1H, C6H3), 7.50 (d, 2H, J =
7.4, C6H4-SO2), 7.56 (d, 2H, J = 7.2, C6H4-SO2), 7.62 (d, 2H, J = 8.3,
C6H4-N2), 7.73 (d, 2H, J = 8.3, C6H4-N2), 7.76–7.93 (m, 2H, quinoxa-
line), 8.13 (d, 1H, J = 8.4, C6H3), 8.58 (s 1H, quinoxaline), 11.3 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO‑d6) δ (ppm): 34.76 (CH3-N),
57.14 (CH2), 112.75, 124.25 (2), 124.54, 127.06, 127.57 (2), 128.23,
128.85, 129.14, 129.32 (2), 129.61, 129.90, 130.54, 131.06, 131.14,
131.88 (2), 138.40, 139.09 (2), 139.76, 143.00, 146.75, 153.86, 168.54
HepG2
HCT116
MCF-7
VERO
bNT
VEGFR-2
5a
31.76 ±
3.1
37.34 ±
3.2
33.10 ±
3.1
0.32 ±
0.06
5b
14.49 ±
0.3
11.39 ±
0.2
8.65 ±
0.2
49.88 ±
0.22
0.14 ±
0.02
5c
11.32 ±
0.3
9.87 ± 0.2
7.24 ±
0.2
62.12 ±
0.18
0.12 ±
0.01
5d
8.98 ±
0.1
7.77 ± 0.1
6.99 ±
0.1
66.67 ±
0.21
0.10 ±
0.01
5e
16.11 ±
0.3
13.23 ±
0.2
8.87 ±
0.2
50.61 ±
0.22
0.14 ±
0.02
(CN), 169.65 (C O); MS (m/z): 596.80 (M+, 23.95%), 579.90
–
–
5f
11.99 ±
0.3
9.33 ± 0.2
10.11 ±
0.2
47.31 ±
0.22
0.14 ±
0.02
(92.63%), 392.23 (100%, base beak), 310.68 (68.16%), 207.49
(82.58%), 200.58 (87.11%); Anal. Calcd for: C30H22ClN7O3S (596.06):
C, 60.45; H, 3.72; N, 16.45; S, 5.38. Found: C, 60.40; H, 3.70; N, 16.34;
S, 5.31.
7a
32.09 ±
2.6
28.28 ±
2.7
30.66 ±
2.6
bNT
0.29 ±
0.06
7b
23.11 ±
2.2
22.21 ±
2.2
20.13 ±
2.2
bNT
0.17 ±
0.06
7c
21.34 ±
1.6
19.63 ±
1.7
17.99 ±
1.6
41.54 ±
0.22
0.16 ±
0.06
4.1.3. General procedure for synthesis of target compounds (7a-c
)
Sorafenib
Doxorubicin
9.18 ±
0.6
5.47 ± 0.3
7.26 ±
0.3
bNT
0.10 ±
0.02
A solution of acid chloride (0.005 mol) and ammonium thiocyanate
(0.01 mol) in acetone (15 mL) was heated under reflux for 10 min to
obtain acyl isothiocyanate solution. After the reaction mixture was
cooled to room temperature and the formed precipitate (NH4Cl) was
filtered off. To the filtrate of the freshly prepared solution of acyliso-
thiocyanate, sulfanilamide 5a (0.86 gm, 0.005 mol) was added, and the
mixture was stirred at room temperature for 10 min, the formed pre-
cipitate was filtered and washed with acetone to yield the corresponding
target compounds, 7a-c respectively.
7.94 ±
0.6
8.07 ± 0.8
6.75 ±
0.4
bNT
bNT
a
IC50 values are the mean ± S.D. of three separate experiments.
b
NT: Compounds not tested.
477.16 (43.72%), 439.40 (94.81%), 129.68 (83.22%),; Anal. Calcdfor:
C
27H22ClN7O3S (560.03): C, 57.91; H, 3.96; N, 17.51; S, 5.72. Found: C,
57.90; H, 3.80; N, 17.50; S, 5.40.
4.1.3.1. N-([4-{ (4-[7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e]
[1,4]diazepin-5-yl]phenyl)diazenyl}-phenyl]sulfonyl)acetimidoylisothio-
4.1.2.4. 4-([4-{7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-5-yl}phenyl]diazenyl)-N-(4,6-dimethylpyrimidin-2-yl)benzene-
sulfonamide (5d). Red crystals; m.p. 115–117 ◦C; yield 94%; IR (KBr,
◦C; yield 82%; IR (KBr,
cyanate (7 ). Y–ello–w crystals; m.p. 140–142
–
–
a
cmꢀ 1): 2059 (N
C S), 1693 (C O), 1597 (N N), 1343, 1134 (SO );
– –
2
cmꢀ 1): 3419 (NH), 1685 (C O), 1596 (N N), 1314, 1143 (SO ); 1H
–
–
–
–
1H NMR (DMSO–‑d )–δ: 2.09 (CH ), 3.35 (s, 3H, N-CH ), 3.92 (d, 1H, J =
2
10.8, CH methylene), 4.59 (d, 1H, J = 10.8, CH meth3ylene), 7.26 (s, 1H,
C6H3), 7.55 (d, 2H, J = 7.6, C6H4-SO2), 7.63 (d, 2H, J = 7.6, C6H4-SO2),
7.66 (d, 2H, J = 8.3, C6H4-N2), 7.77 (d, 2H, J = 8.3, C6H4-N2), 7.85 (d,
2H, C6H3). 13C NMR (DMSO‑d6) (ppm) δ: 25.60 (CH3), 34.78 (CH3-N),
57.04 (CH2), 95.75, 95.88, 113.29, 124.37, 124.66, 128.86, 128.89,
129.29, 129.37, 129.66, 129.86, 131.15, 131.96, 138.30, 143.31,
6
3
NMR (DMSO‑d6) δ: 2.24 (s, 6H, 2CH3 pyrimidine), 3.34 (s, 3H, N-CH3
diazepine), 3.81 (d, 1H J = 10.6, CH methylene), 4.60 (d, 1H J = 10.6,
CH methylene), 6.75 (s, 1H, CH pyrimidine), 7.22 (s, 1H, C6H3), 7.45 (d,
2H, J = 7.62, C6H4-SO2), 7.53 (d, 2H, J = 7.0, C6H4-N2), 7.57 (d, 2H, J =
7.0, C6H4-N2),7.63 (d, 3H, J = 7.7, C6H4-SO2), 7.71 (d, 1H, C6H3), 7.73
(d, 1H, J = 2.2, C6H3), 11.97 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6) δ (ppm): 19.40 (2CH3), 34.76 (CH3-N), 57.15 (CH2), 124.31
(2), 128.22 (2), 128.87 (2), 129.32, 129.61 (2), 129.93 (2), 131.07 (2),
–
–
153.64, 157.26 (NCS), 158.89, 168.68, 169.63, 170.35, 170.83 (C O);
MS (m/z): 552.58 (M+, 68.96%), 438.49 (33.35%), 437.68 (58.38%),
131.89, 138.41, 143.04, 143.76, 144.25, 146.75, 153.86, 154.25 (CN
73.47 (55.84%), 71.90 (100%, base beak); Anal. Calcd for:
–
diazepine), 168.53 (2C = N pyrimidine), 169.65 (C O); Anal. Calcd for:
–
C
25H19ClN6O3S (551.06): C, 54.49; H, 3.48; N, 15.25; S, 11.64. Found:
C
28H24ClN7O3S (574.06): C, 58.58; H, 4.21; N, 17.08; S, 5.58 Found: C,
C, 54.40; H, 3.40; N, 15.20; S, 11.60.
58.50; H, 4.20; N, 17.00; S, 5.60.
4.1.3.2. N-([4-{ (4-[7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e]
[1,4]diazepin-5-yl]phenyl)diazenyl} phenyl]sulfonyl)benzimidoylisothio-
4.1.2.5. 4-([4-{7-Chloro-1-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-5-yl}phenyl]diazenyl)-N-(5-methylisoxazol-3-yl)benzenesulfona-
mide (5e). Red crystals; m.p. 130–132 ◦C; yield 92%; IR (KBr, cmꢀ 1):
◦C; yield 83%; IR (KBr,
cyanate (7 ). B–row–n crystals; m.p. 215–217
–
–
b
cmꢀ 1): 2129 (N
C S), 1665 (C O), 1593 (N N), 1311, 1138 (SO );
– – – –
2
3419 (NH), 1685 (C O), 1596 (N N), 1341, 1128 (SO ); 1H NMR
–
–
–
–
(DMSO‑d6) δ: 2.30 (s, 3H, CH3), 3.39 (s, 3H, N-CH3), 3.822(d, 1H, J =
10.8, CH methylene), 4.60 (d, 1H, J = 10.8, CH methylene), 6.16 (d, 2H,
J = 7.6, C6H4-SO2), 6.60 (s, 1H, CH isoxazole), 7.46 (d, 2H, J = 7.6,
C6H4-SO2), 7.51 (s, 1H, C6H3), 7.55 (d, 2H, J = 8.2, C6H4-N2), 7.63 (d,
2H, J = 8.3, C6H4-N2), 7.71 (d, 1H, C6H3), 7.74 (d, 1H, J = 2.4, C6H3),
10.9 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6) δ (ppm):
15.60 (CH3), 34.78 (CH3-N), 57.04 (CH2), 65.38, 95.75, 95.88, 113.09,
124.31, 124.64, 128.26, 128.89, 129.29, 129.37, 129.66, 129.86,
1H NMR (DMSO‑d6) δ: 3.26 (s, 3H, N-CH3), 3.73 (d, 1H, J = 10.8, CH
methylene), 4.55 (d, 1H, J = 10.8, CH methylene), 7.11 (s, 1H, C6H3),
7.43 (d, 2H, J = 7.6, C6H4-SO2), 7.48 (d, 2H, J = 7.6, C6H4-SO2), 7.63 (d,
2H, J = 8.3, C6H4-N2), 7.65 (d, 2H, J = 8.3, C6H4-N2), 7.85 (d, 2H,
C6H3). 13C NMR (DMSO‑d6) (ppm) δ: 34.78 (CH3-N), 57.10 (CH2),
114.39, 117.76, 118.63, 118.80 (3), 124.33, 126.40 (2), 127.84, 128.56
(3), 128.92 (3), 129.02 (3), 129.31 (2), 129.60 (2), 131.13, 131.94,
142.99, 169.73 (2); MS (m/z): 613.06 (M+, 11.67%), 553.70 (40.68%),
490.10 (59.29%), 404.81 (59.07%), 245.20 (56.46%), 61.64 (100%,
base beak); Anal. Calcd for: C30H21ClN6O3S2 (613.11): C, 58.77; H, 3.45;
N, 13.71; S, 10. 46. Found: C, 58.70; H, 3.40; N, 13.70; S, 10. 40.
131.15, 131.96, 138.30, 143.04, 153.72, 157.98, 158.40, 168.68,
+
–
169.63, 170.35, 170.83 (C O); MS (m/z): 549.40 (M , 37.55%), 524.77
–
(70.18%), 465.58 (100%, base beak), 322.20 (48.68%), 272.14
(50.19%); Anal. Calcd for: C26H21ClN6O4S (549.00): C, 56.88; H, 3.86;
N, 15.31; S, 5.84. Found: C, 56.80; H, 3.70; N, 15.30; S, 5.80.
14

N.M. Saleh et al.
Bioorganic Chemistry 104 (2020) 104350
4.1.3.3. 2-Chloro-N-([4-{(4-[7-chloro-1-methyl-2-oxo-2,3-dihydro-1H-
benzo[e][1,4]diazepin-5-yl]phenyl)-diazenyl}phenyl]sulfonyl)benzimidoy-
lisothiocyanate (7c). Orange crystals; m.p. 255–157 ◦C; yield 86%; IR
4.4. In vitro VEGFR-2 kinase assay
The kinase activity of VEGFR-2 was measured by use of an anti-
phosphotyrosine antibody with the Alpha Screen system (PerkinElmer,
USA) according to manufacturer’s instructions [49,72,74]. Enzyme re-
actions were performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl2, 5 mM
(KBr, cmꢀ 1): 2063 (N C = S), 1693 (C O), 1597 (N N), 1152 (SO );
–
–
–
–
–
–
2
1H NMR (DMSO‑d6) δ: 3.34 (s, 3H, N-CH3), 4.01 (d, 1H, J = 10.8, CH
methylene), 4.57 (d, 1H, J = 10.8, CH methylene), 7.27 (s, 1H, C6H3),
7.53, 7.57 (dd, 4H, J = 7.6, C6H4-SO2), 7.62, 7.65 (dd, 4H, J = 8.3, C6H4-
N2), 7.82–7.84 (dd, 2H, C6H3). 13C NMR (DMSO‑d6) (ppm) δ: 35.02
(CH3-N), 52.16 (CH2), 95.75, 95.88, 120.00, 124.80 (2), 127.98 (2),
128.94 (2), 129.21, 132.48 (2), 133.71 (2), 136.73, 142.78 (2), 144.80
MgCl2, 0.01% Tween-20 and 2 mM DTT, containing 10 μM ATP, 0.1 μg/
mL biotinylated poly-GluTyr (4:1) and 0.1 nM of VEGFR-2 (Millipore,
UK). Prior to catalytic initiation with ATP, the tested compounds at final
concentrations ranging from 0 to 300 μg/mL and enzyme were incu-
(NCS), 147.98 (2), 148.94, 157.93, 162.78, 166.73 (2CN), 168.61,
bated for 5 min at room temperature. The reactions were quenched by
the addition of 25 l of 100 mM EDTA, 10 g/mL Alpha Screen strep-
tavidine donor beads and 10 g/mL acceptor beads in 62.5 mM HEPES
+
–
170.90, 179.78 (C O); MS (m/z): 646.65 (M , 14.71%), 636.71
μ
μ
–
(43.17%), 583.15 (50.54%), 427.33 (59.02%), 249.83 (68.63%), 189.67
(100%, base beak); Anal. Calcd for: C30H20Cl2N6O3S2 (647.55): C,
55.56; H, 3.11; N, 12.98; S, 9. 90. Found: C, 55.50; H, 3.10; N, 12.90; S,
9. 90.
μ
pH 7.4, 250 mM NaCl, and 0.1% BSA. Plate was incubated in the dark
overnight and then read by ELISA Reader (PerkinElmer, USA). Wells
containing the substrate and the enzyme without compounds were used
as reaction control. Wells containing biotinylated poly-GluTyr (4:1) and
enzyme without ATP were used as basal control. Percent inhibition was
calculated by the comparison of compounds treated to control in-
cubations. The concentration of the test compound causing 50% inhi-
bition (IC50) was calculated from the concentration–inhibition response
curve (triplicate determinations) and the data were compared with
Sorafenib (Sigma-Aldrich, USA) as standard VEGFR-2 inhibitor.
4.2. Docking studies
In the present work, all the target compounds were subjected to
docking study to explore their binding mode towards VEGFR-2 enzyme.
All modeling experiments were performed using molsoft program,
which provides a unique set of tools for the modeling of protein/ligand
interactions. It predicts how small flexible molecule such as substrates or
drug candidates bind to a protein of known 3D structure represented by
grid interaction potentials (http://www.molsoft.com/icm_pro.html).
Each experiment used the biological target VEGFR-2 downloaded from
the Brookhaven Protein Databank (http://www.rcsb.org/pdb
/explore/explore.do?structureId = 1YWN). In order to qualify the
docking results in terms of accuracy of the predicted binding confor-
mations in comparison with the experimental procedure, the reported
VEGFR-2 inhibitor drug sorafenib was used as reference ligand.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors extend their appreciation and thanking to Dr. Fatma M.
I. A. Shoman, MD in Clinical Pathology, Blood bank specialist, Blood
bank directorate manager, Ministry of Health, Cairo, Egypt for helping
in the pharmacological part.
4.3. In vitro cytotoxic activity
Cancer cells from different cancer cell lines hepatocellular carcinoma
(HepG2), breast cancer (MCF-7) and colorectal carcinoma (HCT-116),
were purchased from American type Cell Culture collection (ATCC,
Manassas, USA) and grown on the appropriate growth medium Roswell
Park Memorial Institute medium (RPMI 1640) supplemented with 100
mg/mL of streptomycin, 100 units/mL of penicillin and 10% of heat-
inactivated fetal bovine serum in a humidified, 5% (v/v) CO2 atmo-
sphere at 37 ◦C Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide (MTT).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104350.
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