Assessment of elementary derivatives of 1,5-benzodiazepine as anticancer agents with synergy potential

Article abstract of DOI:10.1016/j.bioorg.2021.105331

Herein, we designed and synthesized 1,5-benzodiazepines as a lead molecule for anticancer activity and as potent synergistic activity with drug Methotrexate. Working under the framework of green chemistry principles, series of 1,5-benzodiazepine derivativ

Full text of DOI:10.1016/j.bioorg.2021.105331

Journal Pre-proofs
Assessment of elementary derivatives of 1,5-benzodiazepine as anticancer
agents with synergy potential
Sinthiya J. Gawandi, Vidya G Desai, Shrinivas Joshi, Sunil Shingade,
Raghuvir Pissurlekar
PII:
S0045-2068(21)00708-2
DOI:
https://doi.org/10.1016/j.bioorg.2021.105331
YBIOO 105331
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 April 2021
26 August 2021
1 September 2021
Please cite this article as: S.J. Gawandi, V.G. Desai, S. Joshi, S. Shingade, R. Pissurlekar, Assessment of
elementary derivatives of 1,5-benzodiazepine as anticancer agents with synergy potential, Bioorganic Chemistry
(2021), doi: https://doi.org/10.1016/j.bioorg.2021.105331
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Graphical Abstract
Assessment of elementary derivatives of 1,5-benzodiazepine as
anticancer agents with synergy potential
Sinthiya J Gawandi , Vidya G Desai , Shrinivas Joshi , Sunil Shingade and Raghuvir Pissurlekar
———

Assessment of elementary derivatives of 1,5-benzodiazepine as
anticancer agents with synergy potential
Sinthiya J Gawandia , Vidya G Desaia , Shrinivas Joshib , Sunil Shingadec and Raghuvir Pissurlekard
a Department of Chemistry, Dnyanprassarak Mandal’s College & Research Centre, Assagao, Bardez
403507, India.
b Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S.E.T.’s
College of Pharmacy, Sangolli Rayanna Nagar, Dharwad, 580 002, Karnataka, India.
cSSPM’s V P College of Pharmacy, Madkhol, Sawantwadi, Sindhudurg, Maharashtra.
d(Bio) Molecular Simulations Group, Department of Pharmaceutical Chemistry, Goa College of Pharmacy,
Panaji Goa, India.
Dedicated to my Ph.D. mentor Professor Santosh G. Tilve on his 62nd birthday
Abstract:
Herein, we designed and synthesized 1,5-benzodiazepines as a lead molecule for anticancer activity
and as potent synergistic activity with drug Methotrexate. Working under the framework of green
chemistry principles, series of 1,5-benzodiazepine derivatives (3a-3a1) were synthesized using
biocatalyst i.e. thiamine hydrochloride under solvent free neat heat conditions. These compounds
were screened for in vitro anti cancer activity against couple of cancer cell lines (HeLa and HEPG2)
and normal human cell line HEK-293 via MTT assay. The IC50 values for the compounds were in
the range 0.067 to 0.35 µM, better than Paclitaxel and compatible with the drug Methotrexate.
Compound 3x was found to be influential against both the cell lines with IC50 values of 0.067±
0.002 µM against HeLa and 0.087± 0.003 µM against HEPG2 cell line, having activity as
compatible to the standard drug Methotrexate. Bioinformatic analysis showed that these compounds
are good tyrosine kinase inhibitors which was then proved using enzyme inhibition assay. The
studies of apoptosis revealed late apoptotic mode of cell death for the compounds against HEPG2
cancer cell line using flow cytometry method. Synergistic studies of compound 3x and drug
Methotrexate showed that the combination was highly active against cancer HeLa and HEPG2 cell
line with IC50 value 0.046 ± 0.002 µM and 0.057 ± 0.002 µM respectively, which was well
supported by apoptosis pathway. Further the compounds proved its scope as DNA intercalating
agents, as its molecular docking and DNA binding studies revealed that the compounds would fit
well into the DNA strands.
Keywords:
———

1,5-benzodiazepines, thiamine hydrochloride, cytotoxicity, apoptosis, molecular docking.
Corresponding Author:
Tel.: 9822180792; fax: 0832-2268683; e-mail: desai_vidya@ymail.com
1.
Introduction:
Cancer is one of the topmost malignant diseases responsible for millions of demise of people per
year worldwide. Principally, numbers of deaths arise from leukemia, breast, cervical and liver
cancer. There is a continuous struggle for the advancement of new drug therapies for safe and
effective treatment of cancer disease1,2,3. One of the therapies that have proven it has been the drug
combination therapy4,5,6. Combinations of two or more drugs can conquer over toxicity and other
side effects linked with steep shot of single drugs by resisting biological compensation, thereby
treating multifactorial disease. Synergistic drug combination studies have gained a promising
research strategy with the motif of enhancing drug effect; improve drug selectivity by overcoming
unwanted side effects, host immunity and lowering normal cell toxicity current drug therapeutics
by development of novel drug treatment strategies. Administering more than one drug can provide
several benefits that include better efficacy, lower toxicity, and much delay in onset of acquired
drug resistance7,8,9
In cancer drug discovery, Tyrosine kinase is the key enzyme in the sub group of protein kinases,
responsible for phosphate group transfer from ATP to a protein in a cell which is a vital mechanism
in signal transduction and cell regulation10,11,12. Epidermal growth factor receptor (EGFR)-TK is
found to be over expressed in most of the solid tumors. It leads to autophosphorylation of protein,
thus triggering the tumor growth and expansion leading to malignant progression13, 14. The most
common drugs known for inhibition of these enzymes are Gefitinib15, Imatinib16, Erlotinib17, etc.
(Fig 1)
N
H
O
O
N
N
N
N
HN
N
O
N
O
O
O
HN
Cl
F
HN
N
N
O
O
N
N
Imatinib
Erlotinib
Gefitinib
Fig 1: Drugs known as EGFR tyrosine kinase inhibitors
Kinase inhibitors as monotherapy have not been so effective for treating large cases of malignances
and hence synergistic combination is an alternative to look forward to18. Simple compounds have
been synergised with EGFR tyrosine inhibitors in an anticancer synergy19, 20. A potential
combination therapy for the treatment of advanced ovarian cancer has been reported involving FDA-

approved targeted drugs—sunitinib, dasatinib, and everolimus21. In silico modelling methods have
been used to predict synergism of cancer drug combinations22.
The mechanism of action of main anticancer drugs like Cis-platin, Paclitaxel and Methotrexate takes
place by inhibiting DNA synthesis and suppressing RNA transcription 23, stabilization of
microtubule 24 and by inhibition of dihydrofolate reductase25 respectively, can hold a great scope in
combination therapy. (fig 2)
O
O
O
OH
O
OH
OH
O
NH
O
O
O
O
H
O
OH
O
NH2
N
N
OH
O
H
NH3
NH3
Cl
Cl
N
N
O
O
N
N
Pt
H2N
Cis-platin
Paclitaxel
Fig 2. Standard drugs used as anticancer agents
Methotrexate
Amongst these, Methotrexate is one such drug which acts by reducing the amount of
tetrahydrofolate required for the synthesis of purine bases. This action results in death of cancer
cells26. Being also a powerful medication for variety of autoimmue disorders such as psoriasis27 and
rheumatoid arthritis28, it has been commonly used in synergy with variety of drugs that include 5-
Fluorouracil29, Infliximab30, Cytarabine31 etc. Despite efforts, developing an attractive
experimentation on synergism for cancer drug development is indeed a challenging task.
Benzodiazepine a pharmacophoric scaffold containing a ring complex made up of a benzene ring
and a diazepam ring represents a class of psychoactive drugs. Besides, enhancing the effect of
gamma-aminobutyric acid at the GABA receptor resulting in the sedative, hypnotic and anti-
depressant activity, benzodiazepines exhibit wide range other significant biological applications32-
38. Moreover,benzodiazepines have attracted attention of chemist in the field of drugs and
pharmaceuticals39.
The most commonly employed synthetic protocol for 1,5-benzodiazepines includes coupling
42
43
44
diamines with α,β-unsaturated ketone 40, 41, aliphatic ketones , β- diketones , β-ketoesters in
presence of acid or base. Distinct range of catalysts have been employed in the synthesis of 1,5-
benzodiazepines 45-56. Solvent free organic synthesis and transformations are said to be industrially
constructive and green57. In our synthetic strategy towards 1,5-benzodiazepines, we choose
Thiamine hydrochloride also called as vitamin B1, a water soluble vitamin employed as
organocatalyst by virtue of its properties such as non- toxic, metal free, inexpensive, ease of isolation
procedures etc. As a result of its structural appearance it has been considered as a powerful
biocatalyst for various organic transformations by many synthetic organic chemists.58-61 (Fig 3)

a-
Ami
no
Quin
oxali
nes
Fuse
d
phos
Ami
phon
doal
1,4-
Polyhy
pyrid
Cl
ates
NH3
Dibe
dihydr
kyl
dro
ines
nzo
opyrid
xant
Cl
N
quinol
nap
N
S
ines
ines
htho
ls
H3C
hane
s
Ben
OH
H3C
N
zop
Be
nz
yra
ns
oin
Fig 3: Thiamine Hydrochloride (Vitamin B1)
As an extension to our work towards the development of methodologies for the synthesis of heterocyclic
compounds, we hereby report an efficient and ecofriendly method for the synthesis of 1,5-benzodiazepines
from substituted o-phenylenediamine and ketones. A simple model substrate was examined to establish the
viability of the approach and to optimize the reaction conditions in presence of a biocatalyst under solvent
free conditions. In view of the recent developments towards metal free synthesis, we decided to explore
thiamine hydrochloride as catalyst.
2.
Results and Discussion
2.1.
Chemistry
Derivatives of 2-Methyl-2,4-diphenyl-2,3-dihydro-1H-benzo[1,5]diazepine have been synthesized by
condensation of substituted 2,3-diamino benzenes with different ketone substrates in presence of thiamine
hydrochloride as a catalyst under solvent free neat heat conditions at 70-80oC. The synthesis of compounds
3a-3a1 has been carried out as outlined in (scheme 1). To synthesize compounds 3a-3e the condensation was
carried out between o-phenylene diamine and substituted ketones. Similarly, to obtain compound 3f - 3l and
3m - 3s 4-methyl-2-nitro-aniline and 4-chloro-2-nitro-aniline were initially reduced to 4-methyl-1,2-diamino
benzene and 4-chloro-1,2 diamino benzene respectively using Sn/ HCl and were then condensed with ketone
substrates using the same model methodology. The benzodiazepine derivatives 3t-3w were synthesized by
condensation of ketones with 4-bromo-1,2 diamino benzene.
R1
O
5 mol%
Thiamine HCl
N
NH2
NH2
+
2
R2
solvent free
70-80oC
N
R2
R1
H
1
2
3
R1
Scheme 1: Methodology for the synthesis of 1,5-benzodiazepines derivatives
In the initial experiment, optimization was carried out wherein; acetophenone substrate was used as a
representative substrate. The efficiency of the reaction depends on the physical factors such as amount of
catalyst, temperature of the reaction and the type of solvent used. The optimal reaction conditions were
investigated by varying the temperature, catalyst and solvent of the reaction system. Ultimately, 5 mol% of

catalyst under solvent free neat heat reaction condition was witnessed as the most efficient method to carry
out the synthesis. To study the synthetic utility of the employed catalyst and optimized reaction conditions,
the methodology was applied on differently substituted o-phenylenediamine and ketone substrates. All the
substituted aromatic and heterocyclic ketone substrates as well as substituted diamines reacted well to afford
respective derivatives in good yields.
In all the cases, the reaction was monitored using thin layer chromatography. After completion, the reaction
was quenched in water, followed by extraction in ethyl acetate. The crude product obtained was further
isolated using column chromatography. The scope and generality of this method was extended by
scrutinizing electronically divergent substituted ketones bearing various electron withdrawing or electron
donating groups at ortho, meta, or para positions of the aromatic ring. Substitution pattern in target synthesis
plays an important role in drug development. Presence of specific substituents such as halogens, alkyl and
nitro, methoxy can be of great utility towards structure activity relationship studies. The results are
encapsulated in Table 1. Para substituted nitro acetophenone was found to be most reactive for the
condensation to take place with all the different substituted o-phenylenediamines. It was also inferred from
the results that the yields of the product with respect to electron withdrawing substrates were higher in
comparison to that of the electron donating substrates. Likewise, parent o-phenylene diamine showed better
reactivity in terms of yields as compared to those of the substituted diamines
Table 1: List of synthesized 1,5-benzodiazepine derivatives detailing substrate scope for the reaction
Time in
h
Lit M. Pt.
oC
Compound
R1
R2
% yield Expt. M. Pt. oC
3a
3b
3c
3d
3e
3f
H
H
H
1
3
66
66
72
75
68
27
55
55
49
50
52
52
62
66
58
54
57
140-142
140-144
138-144
146-148
156-160
148-150
146-150
156-160
138-142
150-154
134-136
78-80
143-145
145-146
140-142
148-150
158-160
148-150
N.R
4-Br
4-Cl
H
4
4-NO2
3-NO2
H
H
3
H
3.5
4.5
3.5
4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
3g
3h
3i
4-NO2
4-Br
N.R
4-Cl
2.5
4
N.R
3j
3-Br
N.R
3k
3l
3-NO2
4-OMe
H
3
N.R
3
N.R
3m
3n
3o
3p
3q
3.5
3
138-140
162-164
154-156
146-148
138-140
138-142
N.R
4-NO2
4-Br
Cl
Cl
3
N.R
4-Cl
Cl
3.5
3.5
N.R
3-NO2
Cl
N.R

3r
3s
3-Br
4-F
Cl
Cl
Br
Br
Br
Br
3
57
35
62
56
45
57
124-126
138-140
162-164
172-174
122-124
136-138
N.R
N.R
N.R
N.R
N.R
N. R
3.5
2.5
3.5
3.5
4
3t
4-NO2
4-Cl
3u
3v
3w
3,4-OMe
3- NO2
In order to investigate the feasibility of this synthetic methodology, towards differently substituted substrates
and with a perspective of developing a better biologically active molecule, we synthesized scaffolds
containing benzodiazepine. Pyridine being part of many natural products such as vitamins coenzymes is an
interesting molecule with its beneficial drug favoring characteristics which includes its smaller size, basicity,
water solubility, hydrogen bonding capability 62. Naphthalene has also been seen as one of the most privileged
molecule in the drug discovery field and has also been reported to possess anticancer properties63, 64. Thus,
to study the effect of these moieties in combination with benzodiazepine molecule we designed few
molecules and examined the alterations in the biological properties. The synthesis was accomplished through
a condensation reaction between substituted 1,2-diaminobenzenes with 2-acetyl pyridine, 4-acetyl pyridine
and 2- acetyl naphthalene compounds (scheme 2).
HN
N
N
N
H
N
N
Cl
3x
N
H
N
N
N
3a1
3y
N
NH
Cl
3z
Scheme 2: Synthesis of scaffold containing 1,5-benzodiazepines
The newly synthesized compounds have been characterized by 1H NMR, 13C NMR and mass spectroscopy.
1H NMR analysis confirms the formation of 1,5 -benzodiazepine structure. The main distinctive peaks in the

spectra of all the target compounds is the appearance of two doublets in the range of δH at 2.5 to 3.5 ppm
signifying the CH2 protons at C3 of the seven membered benzodiazepine ring. The most deshielded proton
was observed in case of the aromatic hydrogen present on the phenyl ring substituted on C4 of the
benzodiazepine ring. All the aromatic protons appeared as multiplets in the region δH 8.00 to 6.5ppm, thereby
13
marking the formation of 1,5-benzodiazepine derivatives. Along with this, C NMR spectroscopic study
also signifies the benzodiazepine formation. In C13 NMR spectra of all the compounds the characteristic
peak of C4 (C=N) of the benzodiazepine ring appeared in the range of 164.7-167.2 ppm. Signal due to C2
was observed in the range of 72.7-73.4 ppm. Characteristic peak of formation of benzodiazepine ring
signifying C3 (methylene-CH2) carbon appeared at 42.9- 43.4 ppm which was also confirmed in DEPT
analysis. The exact molecular weight was also characterized and confirmed by HRMS studies. Purity of the
compounds was checked using HPLC studies .
2.2.
Biological activity
In-vitro screening
2.2.1.
2.2.1.2. Cytotoxicity assay
In vitro cytotoxicity of the selected compounds 3a, 3k, 3q, 3w, 3y and 3x was determined by the MTT [(3-
(4,5-dimethyl-2- thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide)] assay against a panel of three different
human cancer cell lines namely; HEPG2 (human liver carcinoma), HeLa (human cervical) and HEK-293T
(Human embryonic kidney cells) normal cell line. The concentration required to suppress 50% of the tumor
cells i.e. IC50 values of the screened compounds and the reference drugs, Paclitaxel and Methotrexate are
outlined in Table 2. As noticed, all compounds showed significant antitumor activities with IC50 values in
the range of 0.067-1.65 µM, this indicated that the 1,5-benzodiazepine group can intensify the antitumor
effects. Compound 3x bearing the 2,4-dipyridinyl group, on the benzodiazepine nucleus was found to be the
most potent among all the screened compounds and showed enhanced activity (IC50 values 0.067 µM against
HeLa cell line and 0.087 µM against HepG2 cell line) against both the cancer cell lines . This was followed
by compound 3q with 3’-nitro substituted phenyl group on benzodiazepine ring, which displayed 0.10 ±
0.004 µM against HeLa cell line and 0.16±0.01 µM against HepG2 cancer cell line. All the screened
compounds 3a, 3k, 3q, 3w, 3x and 3y were found to show better activity (0.067-0.21 µM) against HeLa cell
lines compared to one of the reference compound Paclitaxel (IC50 values 0.23 µM). Compound 3x showed
analogous activity with respect to the other reference compound Methotrexate (IC50 values 0.057 and 0.083
µM). Other screened compounds also showed good activity against both the cell lines. It is clear from the
results that almost all the targets exhibit moderate to good cytotoxicity against human liver carcinoma and
cervical cancer cell lines although very weakly toxic against normal human embryonic kidney cells. The cell
viability action was observed in a dose-dependent manner. Impressively, the cell viability decreased with the
increase in concentration of the compounds against the tabbed cancer cell lines. In case of compound 3x the
cell viability dropped off significantly to 65.7%, 48.3%, 38.4%, 34.7%, 29.2%, 24.9% with the
concentrations of 12.5, 25, 50, 100, 200 and 400 μg/mL respectively. figure 4.

Table 2: Evaluation of cytotoxicity against carcinoma cells (IC50 values in µM) of the selected synthesized
compounds
Compound
R
R1
H
HeLaa
HepG2b
0.34±0.02
0.35±0.04
0.16±0.01
0.16±0.02
0.22±0.03
0.087±0.003
0.28±0.03
0.083± 0.002
HEK293c
0.72±0.03
0.39±0.02
1.65±0.03
0.53±0.01
0.61±0.04
0.31±0.02
-
SId-HeLa
5.53
2.60
16.50
4.07
2.90
4.62
-
SI-HEPG2
2.11
1.11
16.5
10.31
2.90
3.56
-
3a
H
0.13 ± 0.008
0.15 ± 0.006
0.10 ± 0.004
0.13 ± 0.007
0.21 ± 0.01
0.067 ± 0.002
0.23± 0.02
3k
3-NO2
CH3
Cl
3q
3-NO2
3w
3y
3-NO2
Br
2-Naphthyl
2-pyridinyl
CH3
CH3
3x
Paclitaxel
Methotrexate
0.057± 0.001
0.44± 0.01
-
-
a: human cervical cancer cell line
b: human liver carcinoma cell line
c: human embryonic kidney cell line
d: selectivity index was calculated as ratio of IC50 value of normal human cell line to that of the cancerous cell line.
IC50 values are obtained as the mean ± SD (µM) from three different experiments.
On the basis of these results structure activity relationship studies were executed by understanding the effect
of substituents on cytotoxicity of the compounds. Compound 3a with no substitution on either of the rings
displayed moderate activity. Replacement of phenyl ring with heterocylic ring i.e. 2-pyridinyl moiety resulted
in highest activity whereas substitution with a non-heterocyclic moiety like naphthalene showed lowest
activity among all the compounds. Considering the substitution on 2 and 3 position on benzodiazepine ring
the order of cytotoxicity can be drawn as follows 2-pyridinyl > 4-NO2-phenyl > H > 2-Naphthyl. Substitution
variation on the phenyl ring of the benzodiazepine ring was also studied. Presence of electron withdrawing
group such as Cl and Br showed good activity compared to the electron donating methyl group. Among the
two halogens, the most electronegative Chlorine group showed better activity as compared to bromine group.
With respect to phenyl ring substitution, the cytotoxicity order is Cl > Br > H > CH3. The selectivity index
(SI) value >2 proved better selectivity and lower cytotoxicity of the synthesized compounds.

B
A
D
C
Figure 4: Dose- dependent effect on cell viability of selected compounds for three different cell lines a: for HeLa cell
line, b: for HepG2 cell line c: for HEK-293 cell line, d: cell viability exhibited by methotrexate for all the three cell
lines.
2.2.2.2. EGFR-Tyrosine kinase inhibition assay
These potent molecules were aimed as EGFR- tyrosine kinase inhibitors. To study the strength of inhibitory
activity and inhibition pathway, the inhibitory concentration IC50 values were determined against EGFR-
tyrosine kinase enzyme using ELISA method. The IC50 values of the synthesized compounds against ranged
from 0.156 to 0.514 µM (Table 3). Interestingly, among all the studied derivatives, compound 3x displayed
prominent inhibition with IC50 value 0.156 ± 0.003 µM. Structural divergences appeared to affect the potency
of these compounds against the screened enzyme.
Table 3: IC50 values of in vitro tyrosine kinase inhibition assay
Tyrosine Kinase
Inhibition
Compound
(IC50 in µM)a
3d
3k
0.514 ± 0.003
0.404 ± 0.002

3y
3x
0.191 ± 0.005
0.156 ± 0.003
0.08 ± 0.04
Erlotinib
a: Data are average of two independent runs
Figure 5: Graphical representation of tyrosine kinase inhibition activity
2.2.2.3. Annexin V- FITC/ Propidium iodide dual staining assay:
To investigate the mechanism underlying the antiproliferative effect of these potent compounds, the mode of
tumor cell death was analyzed by fluorescence activated cell sortor (FACS) caliber on HepG2 liver cancer
cells treated with 20µM of selected compounds for 24 h. The most prevailing technique used to study cell
apoptosis/ necrosis is flow cytometry using propidium iodide (PI) and annexin V-FITC as dyes to spot viable
and dead cells. A negative control was used for the analysis. This assay facilitate the detection of live cells
(Q1-LL; AV-/PI-), early apoptotic cells (Q1-LR; AV+/PI-), late apoptotic cells (Q1-UR; AV+/PI+) and
necrotic cells (Q1-UL; AV-/PI+). As shown in figure 6, flow cytometry analysis revealed that HepG2 cells
treated with compounds 3q, 3w, 3y and 3x showed apoptosis. The percentage of late apoptotic cells induced
by compound 3q, 3w, 3y and 3x was 10.2%, 13.0%, 5.54% and 64.6% respectively.
Control
3q
3w
3y
3x
3x :
Methotrexate

Figure 6. The apoptosis of HepG-2 cells was detected by flow cytometry. HepG-2 cells were pre-treated
without the addition of any samples, the control (C) (I); with a dose of 20 μM of compounds for 24 h. Cells
were stained with Annexin-V and PI. The evaluation of apoptosis is via Annexin V: FITC Apoptosis
Detection Kit per manufacture’s protocol. And the quantitative results were shown in (V). In each scatter
diagrams, the abscissa represents the fluorescence intensity of the cells dyed by Annexin V; and the ordinate
represents the fluorescence intensity of the cells dyed by PI. The percentage of cells positive for Annexin V-
FITC and/or Propidium iodide is represented inside the quadrants. Cells in the upper left quadrant (Q1-UL;
AV-/PI+): necrotic cells; lower left quadrant (Q1-LL; AV-/PI-): live cells; lower right quadrant (Q1-LR;
AV+/PI-): early apoptotic cells and upper right quadrant (Q1-UR; AV+/PI +): late apoptotic cells.
2.2.2.4. Synergistic activity of compound 3x with drug methotrexate:
Synergistic drug combination studies have gained a promising research strategy with the motif of enhancing
drug effect; improve drug selectivity by overcoming unwanted side effects, host immunity and lowering
normal cell toxicity current drug therapeutics by development of novel drug treatment strategies. Synergistic
combinations of two or more drugs can conquer over toxicity and other side effects linked with steep shot of
single drugs by resisting biological compensation, thereby treating multifactorial diseases. Striving to
implement this strategy using our synthesized compound, we combined it with the methotrexate drug which
is widely known as anti cancer agent. Synergistic studies were carried out against HeLa and HEPG2 cancer
cell lines using combination of compound 3x and drug Methotrexate. As can be seen in Table 4, the results
showed very good activity higher than the individual IC50 values against both the tested cancer cell lines. The
combination resulted in improved activity with IC50 values of 0.046 ± 0.002 against HeLa cell line and 0.057
± 0.002 against HEPG2 cell line. Enhancement in activity was also observed in case of flow cytometric
analysis for cell death mode studies. The percentage of late apoptotic cells induced by the combination was
67.6% as shown in figure 6.
Table 4 : Synergistic activity of compound 3x with Methotrexate for cytotoxicity studies
SI-
HEPG2
HeLaa
HEPG2b
HEK293c SId-HeLa
R1
CH3
-
Compound
R
3x
2-pyridinyl
-
0.067 ± 0.002
0.087±0.003
0.31±0.02
-
4.62
3.56
3x
0.046 ± 0.002 0.057 ± 0.002
0.057± 0.001
-
-
-
-
:Methotrexate
Methotrexate
0.083± 0.002 0.44± 0.01
a: human cervical cancer cell line b: human liver carcinoma cell line c: human embryonic kidney cell line
d: selectivity index was calculated as ratio of IC50 value of normal human cell line to that of the cancerous cell line. IC50
values are obtained as the mean ± SD (µM) from three different experiments

2.2.2.
Molecular docking studies
2.2.3.1. As dihydrofolate reductase inhibitors (anticancer agents)
To investigate the mechanism of antitumor activity and detailed intermolecular interactions between the
synthesized compounds, molecular docking studies were performed on the crystal structure of dihydrofolate
reductase complexed with NADPH and Methotrexate using the surflex-dock programme of sybyl-X 2.0
software. All the inhibitors were docked into the active site of enzyme as shown in Figure 7 (A and B). The
docking study revealed that all the compounds have showed very good docking score as dihydrofolate
reductase inhibitors.
As depicted in the figure 8(A and B), compound 3q makes five hydrogen bonding interactions at the active
site of the enzyme (PDB ID: 1DF7), among those three interactions were of oxygen atom of nitro group
present at 3rd position of phenyl ring with hydrogen atoms of ARG60 and ARG32 (O----H-ARG60, 2.20 Å,
2.57Å; O----H-ARG32, 2.14 Å), another oxygen atom of nitro group present at 3rd position of phenyl ring
makes hydrogen bonding interaction with hydrogen atom of ARG32 (O----H-ARG60, 2.14 Å) and nitrogen
atom of nitro group present at 3rd position of phenyl ring makes hydrogen bonding interaction with hydrogen
atom of ARG60 (N----H-ARG60, 2.49 Å).
As depicted in the figure 9(A and B), compound 3w makes six hydrogen bonding interactions at the active
site of the enzyme (PDB ID: 1DF7), among those three interactions were of oxygen atom of nitro group
present at 3rd position of phenyl ring with hydrogen atoms of ARG60 and ARG32 (O----H-ARG60, 2.60 Å,
2.25Å; O----H-ARG32, 2.18 Å), another oxygen atom of nitro group present at 3rd position of phenyl ring
makes hydrogen bonding interactions with hydrogen atoms of ARG32 and TYR100 (O----H-ARG60, 1.71
Å; O----H-TYR100, 2.55 Å) and nitrogen atom of nitro group present at 3rd position of phenyl ring makes
hydrogen bonding interaction with hydrogen atom of ARG60 (N----H-ARG60, 2.50 Å).
The binding interaction of 1DF7 ligand with dihydrofolate reductase active sites shows thirteen bonding
interactions. The synthesized compounds bind to the enzyme in similar manner as that of 1DF7 ligand. The
compounds have same H-bonding interactions with same amino acids ARG60, ARG32 and TYR100 as that
of 1DF7 ligand. Figure 10 (A and B) represents the hydrophobic and hydrophilic amino acids surrounded to
the studied compound 3q and 3w. All the compounds showed consensus score in the range 8.73-1.57,
indicating the summary of all forces of interaction between ligands and the enzyme. These scores and
interactions indicate that molecules preferentially bind to the enzyme in comparison to the reference 1DF7
ligand.

A
B
Figure 7. Docked view of all the compounds at the active site of the enzyme PDB ID: 1DF7
A
B
Figure 8. Docked view of compound 3q at the active site of the enzyme PDB: 1DF7
A
B
Figure 9. Docked view of compound 3w at the active site of the enzyme PDB: 1DF7
B
A
Figure 10. A ) Hydrophobic amino acids surrounded to compounds 3q (green colour) and 3w (cyan colour)
B) Hydrophilic amino acids surrounded to compounds 3q and 3w.

2.2.3.2. As Tyrosine kinase inhibitors
In silico molecular docking study was performed on the compounds with Molegro Virtual Docker (MVD-
2007, 6.0). Figure 11 shows the structure of EGFR-tyrosine kinase complexed with a 4-anilinoquinazoline
inhibitor obtained from Protein Data Bank with the PDB ID: 1m17.
Figure 11: Structure of EGFR-tyrosine kinase domain complexed with 4-anilinoquinazoline inhibitor
(PDB ID: 1m17)
The crystal structure of the target enzyme including forty amino acids from the carboxyl-terminal tail has
been determined to 2.6 A resolution. Unlike any other kinase enzymes, the EGFR family members possess
constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of
phosphorylation, the EGFRK activation loop adopts a conformation similar to that of the phosphorylated
active form of the kinase domain from the insulin receptor. It is observed that the key residues of a dimerized
structure lying between the EGFRK domain and carboxyl terminal substrate docking sites are found in close
contact with the kinase domain. The site at which the known 4-anilinoquinazoline inhibitor binds with the
target protein was selected as the active site. It is lined with amino acid residues such as Leu694, Met769,
Thr830, Asp831, Glu738, Lys721, Cys773; etc.
Hence to identify other residual interactions of the tested compounds, a grid box (include residues within a
15.0 A radius) large enough to accommodate the active site was constructed. Since 4-Anilinoquinazoline is
a known inhibitor, the centre of this site was considered as the centre of search space for docking. Docking
of the synthesized compounds with EGFR tyrosine kinase domain exhibited well conserved hydrogen
bonding with the amino acid residues at the active site. The MolDock scores of the test compounds ranged
from -86.2649 to -116.861 while that of 4-anilinoquinazoline was -127.098.
The molecular docking study revealed that compounds 3n, 3s, 3y and 3x acts as good inhibitors of tyrosine
kinase due to characteristic features. However, compounds 3a-3a’ were found to be active with the target
PDB ID: 1M17. The best docked poses of the compounds are depicted in Figure 12. Compound 3n makes
two hydrogen bond interactions at the active site of the enzyme. The interactions are raised by nitro group
present on phenyl rings with Thr766 and Thr830. Compound 3z and 3x exhibited three hydrogen interactions

of which two are of nitrogen of pyridine rings with Thr766 and Thr830 and the third interaction is of nitrogen
of benzodiazepine ring with Thr766.
Figure 12: Molecular docking data: The compounds docked in best of its conformation into the binding site
of 1M17. a Binding mode of compound 3l forming two H bonds with Thr766 and Thr830;b binding mode
of compound 3n forming one H bonds with Thr766; c binding mode of compound 3s forming two H bonds
with Met769 and Thr766; d & e binding modes of compound 4g and 4h forming two H bonds with Thr766
and Thr830, one H bond with Thr766.
2.2.3.3 Docking studies on DNA intercalation:

A drug is known to act by interaction with macromolecules like DNA and proteins as a target. The studies
of interaction of drug molecule with DNA have been an interesting path towards design and development of
novel highly effective drug molecules. Interaction of drug molecule with DNA leads to a sequence of
operations such as structural modifications, thereby killing the unwanted cells via disrupting its replication,
transcription and repair. To study the interactions of the most active benzodiazepine 3x among the
synthesized derivative and to indicate the site of interaction, primary molecular docking studies were
performed.
2K4L
Ligand
Docking
Score
MMGBSA
bind
3x
-8.318
-60.49
Figure 13: Docked view of compound 3x intercalated into the DNA fragment
The molecule 3x had high docking score of —8.3 kcal/mol. Visual inspection revealed that the ligands get
intercalated in the minor grove of the DNA fragment. N1 atom of benzodiazepine forms hydrogen bond with
Thiamine while N atom of 4-pyridine ring forms hydrogen bond with adenine of the chain B of the DNA
fragment. The 2-pyridine ring exhibits pi-stacked in-between two adenine rings of the DNA chain A.
4.400
4.000
4.400
4.000
3.000
2.000
1.000
3.000
2.000
1.000
0.000
0.000
-0.400
-0.400
200.00
400.00
600.00
800.00
200.00
400.00
600.00
800.00
nm.
nm.
Figure 14: DNA binding studies using UV spectroscopy

2.2.3.3. Analysis of drug scan:
For a molecule to be ‘druglike’, its molecular structure and biological effectiveness must be in a perfect
harmony. There are various methods wherein the druglikeness can be analysed virtually considering the
lipinski’s RO5. In view of this, the toxicity and its feasibility to act like a drug were predicted for the
synthesized compounds using Data Warrior 5.2.1. The toxicity results are presented in the following table.
Based on the property calculation have favorable cLogP which should be ideally between 2.0 and 5.0 with
some outliers (3b, 3c, 3h, 3i, 3j, 3m, 3o, 3p, 3r, 3s, 3u, 3y, 3a’). Compounds that exhibit drug likeliness
based on the prediction are 3a, 3c, 3f, 3l, 3m, 3p, 3v, 3x, 3y, 3z, 3a’. Most of the compounds are devoid of
toxicities except 3l and 3u which are possibly mutagenic while 3a and 3f are probably teratogenic.
Table 5: Toxicity prediction using data warrior 5.2.1
Ligands
Mutagenic
none
Tumorigenic
none
Teratogenic
high
Irritant
none
3a
3f
none
none
high
none
3l
high
none
none
none
3u
high
none
none
none
Table 6: Ligands predicted with druglike properties obeying lipinski’s RO5 with no toxicity parameters
Ligands
Total Mol
Weight
cLogP
H-bond
Acceptors
H-bond
Donors
Total
Surface
Area
Druglikeness
3d
402.41
402.41
416.44
416.44
452.90
436.85
481.31
402.41
328.42
348.84
2.66
2.66
3.00
3.00
3.26
3.26
3.38
2.66
2.95
3.11
8
8
8
8
8
8
8
8
4
4
1
1
1
1
1
1
1
1
1
1
297.48
297.48
309.74
309.74
312.90
312.90
316.11
297.48
259.92
263.08
-3.80
-3.80
-3.80
-3.80
-3.72
-3.72
-5.59
-3.80
1.37
3e
3g
3k
3n
3q
3t
3w
3x
3z
1.43
3.
Conclusion
In conclusion, we have developed an efficient and convenient catalytic method for the synthesis of new
derivatives of 2-Methyl-2,4-diphenyl-2,3-dihydro-1H-benzo[1,5]diazepine under solvent free reaction
conditions. The current method is advantageous due to low environmental impact, clean reaction conditions,
wide substrate scope of the reaction, use of non-toxic, metal free and inexpensive catalyst which makes this
procedure a green alternative to the reported tedious methods for the synthesis of 1,5-benzodiazepines. The
synthesized derivatives were characterized using NMR and Mass spectroscopy. Based on the molecular docking
study, the best docked compounds 3a, 3k, 3q, 3w, 3x and 3y were screened biologically for their anti-tumor
activity studies. Significant anti-proliferative activity was demonstrated by all the assessed compounds against

the HeLa (human cervical cancer), HEPG2 (human liver carcinoma) cancer cell line and HEK-293 (normal
human embryonic kidney) cell line. Compound 3x was found to be the most versatile amongst all the compounds
exhibiting both anti-tumor with the IC50 value of 0.067 ± 0.002 against HeLa cell line supported by apoptosis
inducing mode of cell death. Drug synergism plays a key role in improving efficacy of drug. In order to exploit
the synergistic behavior of the most potent compound 3x, we performed synergistic activity in cytotoxicity
studies of the same with drug Methotrexate. These studies were performed against HepG2 and HeLa cell lines
with IC50 values of 0.046 ± 0.002 and 0.057 ± 0.002 which were better than the values of drug alone. Further
this combination also showed promising results in apoptosis studies. The anticancer mechanistic studies
indicated the compound 3x to be good DNA intercalating agent. This proved that compound 3x has a potency
to be good lead to develop into drug molecule. DNA toxicity assessment prediction studies revealed the drug
likeness of such compounds. Moreover, the present study endeavors an initiative approach for development of
such targets, as good leads for enhancing the research scope for further exploitation of these molecules by
broadening the efficacy of such novel molecules as potential anticancer agents.
4.
Experimental
4.2.
Material methods:
All commercially available solvents and reagents were purchased from SD-fine, Loba Chemie and Avra
synthesis. o-phenylenediamines were used after recrystallization. Melting points were measured using open
capillary tube method. The reactions were monitored by TLC carried out on Macherey – Nagel pre-coated
TLC sheets SIL G/UV254 and visualized under UV light. SDFCL silica gel (200-400 mesh) was used for
flash chromatography (Combi Flash Companion by Teledyne Isco). 1H and 13C NMR spectra were recorded
on Bruker model 400 MHz instrument using CDCl3 solution. Chemical shifts (δ) are expressed in ppm (parts
per million) relative to the residual solvent peaks as an internal standard (1H NMR: CDCl3 at 7.24 ppm, 13C
NMR at 77.00 ppm) and coupling constant (J) are given in hertz. IR spectra were recorded on a Schimadzu
Fourier Transform Infrared Spectrometer using KBr pallets. High resolution mass spectroscopy data were
obtained in electron impact (EI) mode. Intensities are reported as percentages relative to the base peak (I=
100%).
4.3.
General procedure for synthesis:
o-phenylene diamine substrate (1mmol) and substituted ketone derivative (2mmol) were taken in a round
bottom flask. To that 5mol% of thiamine hydrochloride was added and heated on water bath at 70-80oC for
the time indicated in scheme 2. The reaction was monitored periodically till its completion. The reaction
mixture was then quenched in cold water (15 mL), which was further extracted using ethyl acetate (3 x 10
mL) and dried over anhydrous Na2SO4. The crude product obtained was further purified using column
chromatography pet ether- EtOAc (9:1). The isolated pure product was then characterized using IR, NMR
and Mass spectropscopy.
4.4.
Spectral data

2,8-Dimethyl-2,4-bis-(4-nitro-phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepine (3g): Orange solid, mp:
168-170oC; IR (KBr) (νmax , cm-1): 3315, 1597, 1517, 1350. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.72 (s,
a
b
3H, CH3- cyclic ), 2.33 (s, 3H, CH3- Ar) 2.88 (d, 1H, J= 13.2 Hz, CH2 ), 3.08 (d, 1H, J=13.2 Hz, CH2 ),
3.4 (s, 1H, -NH), 6.64 (d, 1H, Ar-H, J= 0.8 Hz), 6.74 (d, 1H, Ar-H, J= 8 Hz), 6.86- 6.89 (dd, 1H, Ar-H,
J=1.8 Hz, J=8 Hz), 7.55-7.11 (m, 8H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 165.6, 155.3, 150.7,
145.6, 136.0, 135.6, 134.6, 133.2, 131.2, 131.0, 129.2, 128.0, 123.3, 118.9, 113.4, 73.2, 43.2 (CH2), 29.2,
21.2; HRMS (ESI-TOF, [M]+) : calcd for C23H20N4O4, 416.15; found 416.1527.
2,4-Bis-(4-bromo-phenyl)-2,8-dimethyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3h): Yellow solid, mp:
1
142-144oC; IR (KBr) (νmax , cm-1): 3323, 1601, 1510, 784. HNMR(400 MHz, CDCl3) (δ, ppm): 1.76 (s,
a
b
3H, CH3- cyclic ), 2.36 (s, 3H, CH3- Ar) 2.90 (d, 1H, J= 13.2 Hz, CH2 ), 3.08 (d, 1H, J=13.2 Hz, CH2 ),
3.4 (s, 1H, -NH), 6.64 (d, 1H, Ar-H, J= 0.8 Hz), 6.74- 7.76 (m, 10H, Ar-H) 13C NMR (75MHz, CDCl3) (δ,
ppm): 165.0, 149.5, 141.5, 137.4, 136.9, 136.8, 132.4, 130.2, 130.0, 129.5, 129.0, 128.9, 125.3, 124.3, 73.1,
43.2(CH2), 29.8, 21.1; Mol. formula : calcd for C23H20N2Br2, 481.99.
2,4-Bis-(4-chloro-phenyl)-2,8-dimethyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3i): Yellow solid, mp:
156-158oC; IR (KBr) (νmax , cm-1): 3061, 1604, 1487, 1328, 831. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.56
a
b
(s, 3H, CH3- cyclic ), 1.85 (s, 3H, CH3- Ar) 3.0 (d, 1H, J= 13.2 Hz, CH2 ), 3.72 (d, 1H, J=13.2 Hz, CH2 ),
13
3.75 (s, 1H, -NH), 6.90 -8.08 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 165.1, 145.9, 140.1,
137.9, 137.0, 136.1, 135.0, 133.2, 131.7, 129.7, 129.5, 129.3, 129.1, 128.8, 128.2, 127.3, 126,9, 121.6, 72.7,
42.9 (CH2), 29.5, 20.5; Mol. formula: Calcd for C23H20N2Cl2, 394.10
2,4-Bis-(3-bromo-phenyl)-2,8-dimethyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3j): Orange solid, mp:
138-140oC; IR (KBr) (νmax , cm-1): 3315, 1597, 1517, 1350, 752. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.74
a
b
(s, 3H, CH3- cyclic ), 2.34 (s, 3H, CH3- Ar) 2.88 (d, 1H, J= 13.2 Hz, CH2 ), 3.07 (d, 1H, J=13.2 Hz, CH2 ),
13
3.43 (s, 1H, -NH), 6.75-7.73 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 164.9, 149.5, 141.5,
137.3, 136.9, 132.4, 130.2, 129.9, 129.4, 128.9, 125.4, 124.32, 122.72, 122.41, 73.3, 43.3 (CH2), 29.8,
21.0; HRMS (ESI-TOF, [M]+) : calcd for C23H20N2Br2, 482.00; found 482.0059.
2,8-Dimethyl-2,4-bis-(3-nitro-phenyl)-2,3-dihydro-1H-benzo[b][1,5]diazepine (3k): Orange solid, mp:
134-136oC; IR (KBr) (νmax , cm-1): 3313, 1598, 1516, 1349. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.72 (s,
a
b
3H, CH3- cyclic ), 2.33 (s, 3H, CH3- Ar), 2.87 (d, 1H, J= 13.2 Hz, CH2 ), 3.08 (d, 1H, J=13.2 Hz, CH2 ),
3.43 (s, 1H, -NH), 6.64 (d, 1H, Ar-H, J= 1.2 Hz), 6.73-7.49(m, 10H, Ar-H) 13C NMR (75MHz, CDCl3) (δ,
ppm): 164.9, 149.5, 141.5, 137.3, 136.9, 136.8, 132.4, 130.2, 130.0, 129.8, 129.0, 129.4, 129.0, 128.9,
125.4, 124.3, 122.7, 122.41, 121.62,73.1, 43.2(CH2), 29.8, 21.8; HRMS (ESI-TOF, [M]+) : Calcd for
C23H20N4O4, 416.15; found 416.1659.

2,4-Bis-(4-methoxy-phenyl)-2,8-dimethyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3l): Yellow solid, mp:
102-104oC; IR (KBr) (νmax , cm-1): 3331, 1589, 1508, 1247. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.65 (s,
a
b
3H, CH3- cyclic ), 2.26 (s, 3H, CH3- Ar), 2.84 (d, 1H, J= 13.2 Hz, CH2 ), 2.98 (d, 1H, J=13.2 Hz, CH2 ),
13
3.42 (s, 1H, -NH), 3.67 (s, 3H, OMe), 3.72 (s, 3H, OMe) 6.59-7.60 (m, 11H, Ar-H) C NMR (75MHz,
Mol. formula : Calcd for C25H26O2N2, 386.20.
8-Chloro-2-methyl-2,4-bis-(4-nitro-phenyl)-2,3-dihydro-1H-benzo[b][1,5]diazepine (3n): Orange solid,
mp: 162-164oC; IR (KBr) (νmax , cm-1): 3383, 1597, 1517, 1348, 856. 1HNMR(400 MHz, CDCl3) (δ, ppm):
a
b
1.77 (s, 3H, CH3- cyclic ), 2.84 (d, 1H, J= 13.76 Hz, CH2 ), 3.02 (d, 1H, J=13.76 Hz, CH2 ), 3.37 (s, 1H, -
13
NH), 6.87-7.58 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 163.8, 153.5, 148.4, 147.0, 138.3,
136.7, 132.7, 131.3, 127.6, 126.7, 123.7, 123.5, 122.0, 120.4, 72.5, 43.2 (CH2), 30.6; HRMS (ESI-TOF,
[M]+) : calcd for C22H17N4O4Cl, 436.098; found 436.1004.
2,4-Bis-(4-bromo-phenyl)-8-chloro-2-methyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3o): Brownish
yellow solid, mp: 154-156oC; IR (KBr) (νmax , cm-1): 3279, 1604, 1589, 1517, 831, 720.1HNMR(400 MHz,
a
b
CDCl3) (δ, ppm): 1.73 (s, 3H, CH3- cyclic ), 2.85 (d, 1H, J= 18 Hz, CH2 ), 3.11 (d, 1H, J=18 Hz, CH2 ),
13
3.52 (s, 1H, -NH), 6.75-7.72 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 166.1, 145.9, 140.8,
138.7, 137.7, 136.1, 131.4, 131.3, 130.1, 129.8, 128.6, 128.5, 128.0, 127.3, 126.8, 125.0, 124.8, 122.4,
121.4, 120.61, 72.7, 43.0 (CH2), 29.6; HRMS (ESI-TOF, [M]+) : Calcd for C22H17N2Br2Cl, 501.95; found
501.9589.
8-Chloro-2,4-bis-(4-chloro-phenyl)-2-methyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3p): Yellow solid,
mp: 146-148oC; IR (KBr) (νmax , cm-1): 3269, 1604, 1591, 1469, 810. 1HNMR(400 MHz, CDCl3) (δ, ppm):
a
b
1.74 (s, 3H, CH3- cyclic ), 2.87 (d, 1H, J= 13.5 Hz, CH2 ), 3.08 (d, 1H, J=13.5 Hz, CH2 ), 3.51 (s, 1H, -
13
NH), 6.73-7.50 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 166.2, 145.4, 141.0, 138.7, 137.6,
136.3, 133.3, 131.5, 130.5, 130.1, 128.5, 128.4, 128.3, 128.0, 127.0, 126.3, 122.5, 121.8, 120.7, 73.7, 43.1
(CH2), 29.7; HRMS (ESI-TOF, [M]+) : Calcd for C22H17Cl3N2, 414.05; found 414.0513.
8-Chloro-2-methyl-2,4-bis-(3-nitro-phenyl)-2,3-dihydro-1H-benzo[b][1,5]diazepine (3q): Orange solid,
mp: 138-140oC; IR (KBr) (νmax , cm-1): 3317, 1605, 1518, 1350. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.97
a
b
(s, 3H, CH3- cyclic ), 2.95 (d, 1H, J= 13.5 Hz, CH2 ), 3.30 (d, 1H, J=13.5 Hz, CH2 ), 3.90 (s, 1H, -NH),
6.92-8.38 (m, 11H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 164.1, 148.7, 148.2, 148.0, 140.4, 138.4,
137.2, 132.5, 132.2, 131.9, 130.5, 129.6, 129.3, 124.6, 122.4, 122.0, 121.6, 120.8, 120.7, 73.3, 43.1 (CH2),
30.3; HRMS (ESI-TOF, [M]+) : Calcd for C23H17N4O4Cl, 436.09; found 436.1004.

2,4-Bis-(3-bromo-phenyl)-8-chloro-2-methyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3r): Yellow solid,
mp: 124-126oC; IR (KBr) (νmax , cm-1): 3277, 1605, 1581, 1487, 821, 715. 1HNMR(400 MHz, CDCl3) (δ,
a
b
ppm): 1.71 (s, 3H, CH3- cyclic ), 2.85 (d, 1H, J= 18 Hz, CH2 ), 3.05 (d, 1H, J=18 Hz, CH2 ), 3.62 (s, 1H, -
13
NH), 6.83-7.65 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 166.1, 149.0, 141.1, 138.7, 137.7,
132.8 131.6, 130.1, 129.0, 128.0, 126.8, 126.5, 125.5, 124.3, 122.8, 122.5, 121.7, 120.7, 73.2, 43.3 (CH2),
30.1; HRMS (ESI-TOF, [M]+) : Calcd for C22H17N2Br2Cl, 501.95; found 501.9589.
8-Chloro-2-methyl-2,4-bis-(4-fluoro-phenyl)-2,3-dihydro-1H-benzo[b][1,5]diazepine (3q): Orange solid,
mp: 138-140oC; IR (KBr) (νmax , cm-1): 3317, 1605, 1518, 1350. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.73
a
b
(s, 3H, CH3- cyclic ), 2.87 (d, 1H, J= 13.5 Hz, CH2 ), 3.09 (d, 1H, J=13.5 Hz, CH2 ), 3.48 (s, 1H, -NH),
6.82-7.52 (m, 11H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 166.5, 142.6, 138.7, 138.2, 136.3, 135.4,
131.2, 129.9, 129.0, 127.4, 127.3, 126.1, 122.6, 121.8, 120.8, 115.2, 115.0, 114.8, 73.1, 43.4 (CH2), 30.2;
HRMS (ESI-TOF, [M]+) : Calcd for C23H17N2F2Cl, 382.10; found 382.1169.
8-Bromo-2-methyl-2,4-bis-(4-nitro-phenyl)-2,3-dihydro-1H-benzo[b][1,5]diazepine (3t): Orange solid,
mp: 162-164oC; IR (KBr) (νmax , cm-1): 3390, 1597, 1517, 1347. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.83
a
b
(s, 3H, CH3- cyclic ), 2.97 (d, 1H, J= 13.32 Hz, CH2 ), 3.35 (d, 1H, J=13.32 Hz, CH2 ), 3.72 (s, 1H, -NH),
7.04-8.06 (m, 11H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 166.6, 142.6, 138.8, 138.2, 136.3, 135.4,
131.2, 129.9, 129.2, 129.1, 127.4, 127.3, 126.1, 122.6, 121.8, 120.8, 115.2, 115.1, 115.0, 73.1, 43.4(CH2),
30.1; HRMS (ESI-TOF, [M]+) : calcd for C22H17N4O4Br, 480.042; found 480.0500.
8-Bromo-2,4-bis-(4-chloro-phenyl)-2-methyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3u): Orange solid,
mp: 172-174oC; IR (KBr) (νmax , cm-1): 3269, 1604, 1469, 831. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.77
a
b
(s, 3H, CH3- cyclic ), 2.84 (d, 1H, J= 13.76 Hz, CH2 ), 3.03 (d, 1H, J=13.76 Hz, CH2 ), 3.37 (s, 1H, -NH),
6.93-7.58 (m, 11H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 166.1, 145.3, 140.9, 138.7, 137.9, 137.5,
136.5, 136.2, 133.2, 131.4, 130.1, 128.5, 128.3, 128.0, 127.0, 126.3, 122.5, 121.8, 120.6, 73.6, 43.1(CH2),
30.0; HRMS (ESI-TOF, [M]+) : calcd for C22H17BrCl2N2, 458.00; found 458.0038.
8-Bromo-2,4-bis-(3,4-dimethoxy-phenyl)-2-methyl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3v): Yellow
solid, mp: 122-124oC; IR (KBr) (νmax , cm-1): 3299, 1589, 1566, 1446, 1252. 1HNMR(400 MHz, CDCl3) (δ,
a
b
ppm): 1.76 (s, 3H, CH3- cyclic ), 2.82 (d, 1H, J= 13.32 Hz, CH2 ), 3.02 (d, 1H, J=13.32 Hz, CH2 ), 3.37 (s,
1H, -NH), 3.65 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.48-6.62 ( m,
9H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 167.2, 153.3, 149.0, 147.6, 147.3, 140.5, 137.3, 130.5,
127.2, 123.0, 121.2, 119.15, 114.6, 114.2, 112.5, 110.0, 109.9, 73.3, 56.1, 43.3(CH2), 26.3; HRMS (ESI-
TOF, [M]+) : Calcd for C26H27Br3N2O4, 510.37; found 510.3710.

8-Chloro-2-methyl-2,4-bis-(3-nitro-phenyl)-2,3-dihydro-1H-benzo[b][1,5]diazepine (3w): Orange solid,
mp: 138-140oC; IR (KBr) (νmax , cm-1): 3319, 1604, 1519, 1348. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.84
a
b
(s, 3H, CH3- cyclic ), 2.94 (d, 1H, J= 13.2 Hz, CH2 ), 3.27 (d, 1H, J=13.2 Hz, CH2 ), 3.91 (s, 1H, -NH),
6.77-8.40 (m, 11H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 164.3, 148.7, 148.0, 140.4, 138.7, 136.5,
132.6, 131.9, 131.3, 130.7, 130.0, 129.6, 129.3, 124.8, 123.0, 122.4, 120.9, 73.9, 43.0(CH2), 30.3; HRMS
(ESI-TOF, [M]+) : Calcd for C23H17N4O4Br, 480.04; found 480.0500.
2,8-Dimethyl-2,4-di-pyridin-2-yl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3x): Orange solid, mp: 112-
114oC; IR (KBr) (νmax , cm-1): 3321, 1620, 1588, 1566, 1471. 1HNMR(400 MHz, CDCl3) (δ, ppm): 1.73 (s,
a
b
3H, CH3- cyclic ), 2.33 (s, 3H, CH3- Ar) 2.87 (d, 1H, J= 12.4 Hz, CH2 ), 3.05 (d, 1H, J=12.4 Hz, CH2 ),
13
3.42 (s, 1H, -NH), 6.64-7.55 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 167.2, 163.4, 163.1,
160.9, 139.4, 129.7, 129.6, 128.6, 127.5, 127.4, 123.5, 115.4, 115.3, 115.2, 115.1, 73.4, 43.0(CH2), 29.7,
HRMS (ESI-TOF, [M]+) : calcd for C21H20N4, 328.18; found 328.1821.
2,8-Dimethyl-2,4-di-naphthalen-2-yl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3y): Yellow solid, mp: 138-
1
140oC; IR (KBr) (νmax , cm-1): 3282, 1612, 1469,1321. HNMR(400 MHz, CDCl3) (δ, ppm): 1.87 (s, 3H,
a
b
CH3- cyclic ), 2.37 (s, 3H, CH3- Ar) 3.15 (d, 1H, J= 13.2 Hz, CH2 ), 3.35 (d, 1H, J=13.2 Hz, CH2 ), 3.61
(s, 1H, -NH), 6.71 (d, 1H, Ar-H, J= 0.8 Hz), 6.80 – 8.10 (m, 17H, Ar-H) 13C NMR (75MHz, CDCl3) (δ,
ppm): 166.7, 145.0, 138.0, 137.7, 137.2, 136.9, 136.4, 135.5, 133.9, 133.8, 133.1, 132.7, 131.6, 128.7,
128.2, 127.6, 127.3, 126.8, 126.7, 126.1, 125.9, 124.3, 124.1, 124.0, 122.6, 73.3, 42.9(CH2), 29.7, 20.6;
HRMS (ESI-TOF, [M]+) : calcd for C31H26N2, 426.21; found 426.2162.
8-Chloro-2-methyl-2,4-di-pyridin-4-yl-2,3-dihydro-1H-benzo[b][1,5]diazepine (3z): Yellow solid, mp:
1
132-134oC; IR (KBr) (νmax , cm-1): 3332, 1618, 1589, 1568, 1476. HNMR(400 MHz, CDCl3) (δ, ppm):
a
b
1.87 (s, 3H, CH3- cyclic ), 3.04 (d, 1H, J= 12.4 Hz, CH2 ), 3.30(d, 1H, J=12.4 Hz, CH2 ), 3.68 (s, 1H, -
13
NH), 6.84-8.45 (m, 11H, Ar-H) C NMR (75MHz, CDCl3) (δ, ppm): 166.3, 155.5, 149.0, 147.3, 145.9,
138.0, 136.0, 133.3, 126.2, 124.8, 123.4, 121.9, 121.0, 118.5, 115.5, 73.4, 43.5(CH2), 26.3; HRMS (ESI-
TOF, [M]+) : Calcd for C20H17N4Cl, 348.11; found 348.1715.
8-Chloro-2-methyl-2,4-di-naphthalen-2-yl-2,3-dihydro-1H-benzo[b][1,5]diazepine: (3a1): Yellow solid,
mp: 168-170oC; IR (KBr) (νmax , cm-1): 3327, 1597, 1574, 1450, 828. 1HNMR(400 MHz, CDCl3) (δ, ppm):
a
b
1.87 (s, 3H, CH3- cyclic ), 3.13 (d, 1H, J= 13.2 Hz, CH2 ), 3.44 (d, 1H, J=13.2 Hz, CH2 ), 3.69 (s, 1H, -
NH), 6.79 – 8.05 (m, 17H, Ar-H) 13C NMR (75MHz, CDCl3) (δ, ppm): 167.8, 144.5, 130.1, 128.4, 128.3,
127.8, 127.7, 127.5, 127.4, 127.0, 126.4, 126.2, 124.2, 124.1, 123.9, 121.8, 73.5, 43.0(CH2), 29.9; HRMS
(ESI-TOF, [M]+) : calcd for C30H23ClN2, 446.15; found 446.1523.

4.5 Tyrosine kinase enzyme inhibition studies
Materials: FBS (Gibco, Invitrogen) Cat No -10270106, Antibiotic – Antimycotic 100X solution
(Thermofisher Scientific)-Cat No-15240062, 96-well plates, Universal Tyrosine Kinase Assay Kit- Cat No
–MK410
Methodology
Cell Treatment:
In a 96-well flat-bottom micro plate, maintained at 37ºC in 95% humidity and 5% CO2 for overnight, the
cells were seeded at a density of approximately 1×105cells/well followed by treatment with different
concentration of test samples. The cells were then incubated for another 24 hours. The phosphate buffer
solution, and 1 ml of extraction buffer was used to wash the cells in well for twice. The cells were then
recovered carefully using cell scraper and were centrifuged at 4ºC for 10 min at 10,000 rpm. The supernatant
was collected and stored for further analysis.
Tyrosine Kinase Assay:
For carrying out the assay, collected supernatant was diluted by 25 times with kinase reacting solution. The
diluted control and the treated sample were added in each well in duplicate. This was followed by addition
of 10 µl of 40 mM ATP-2Na solution into each well and was mixed thoroughly. Treated wells were incubated
for 30 min at 37ºC. after removing the samples, wells were washed thrice with wash buffer. Next, 100µl of
blocking solution was added into each well and incubated for 30 min at 37ºC. The blocking solution was
then discarded and 50 µl of Anti-phosphotyrosine - HRP solution was added into each well and incubated
for 30 min at 37ºC. Once the antibody solution was discared each well was washed 4 times with washing
buffer followed by addition of 100 µl of HRP substrate solution (TMBZ) into each well and incubation
for 30 min at 37ºC . Then 100 µl of
stop solution was added into each well in the same order as HRP
substrate solution and finally absorbance was measured at 450
inhibition was calculated using the below formula
nm with a plate reader. The percent
% Inhibition = 1- (Abs of sample /Abs of control) x 100
The IC50of compounds was calculated using graph Pad Prism Version5.1 by taking a percentage of Inhibition
Tyrosine Kinase Enzyme at six different concentrations of treatment.
4.6
Cytotoxicity assay:
The in-vitro cytotoxicity studies of selective compounds 3a, 3k, 3q, 3w, 3y and 3x was assessed by MTT
assay 65 using three different cell lines namely HeLa, HEPG2 and HEK-293. Cytotoxicity studies were also
evaluated against Paclitaxel and Methotrexate as standard drugs. Firstly, the cell lines were seeded in 96 well
flat-bottom micro plate containing Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% heat
inactivated fetal bovine serum and 1% antibiotic- antimycotic 100X solution. The cells were maintained
overnight at 37oC in 95% humidity & 5% CO2 and were seeded in separate 96 well plates. The compound

concentration of 400, 200, 100, 50, 25, 12.5 µg/mL was treated and cells were incubated for 48 hrs before
addition of MTT solution. The wells were washed with phosphate buffer solution two times followed by
addition of 20 µL of MTT solution and then incubated at 37oC. After 4h, formazan crystals were dissolved
using 100 µL DMSO and absorbance was recorded at 570 nm using microplate reader. The IC50 calculation
was carried out using graph pad prism version 5.1.
4.7
Apoptosis studies using flowcytometry:
Materials: Cell lines – Hep G2 (Liver cancer), DMEM with low glucose (Cat No-11875-093), FBS (Gibco,
Invitrogen) Cat No -10270106, Antibiotic – Antimycotic 100X solution (Thermofisher Scientific)-Cat No-
15240062, AnnexinV-FITC Kit (Beckman Coulter) Cat No –PN IM 3546- 200 Tests, Propedium Idiode
(Sigma-Aldrich) Cat No –P 4170
Methodology:
A 24-well flat bottom micro plate containing cover slips, maintained at 370C in CO2 incubator for overnight
was used for seeding the cells. The cells were treated with 20μg/ml of each sample compound at 24 hrs. After
incubation, cells were then washed with PBS and Centrifuged for 5 minutes at 500 x g at 4°C. Supernatant
was discarded, and the cell pellets were resuspended in ice-cold 1X Binding Buffer to 1 x105 per mL. The
tubes were kept on ice and 1 μL of annexin V-FITC solution and 5 μL PI was added. It was mixed gently
and incubated for 15 minutes in the dark. Followed by this, 400 μL of ice-cold 1X binding buffer was added
and mixed gently. The cell preparations were then analyzed by flowcytometry within 30 minutes.
4.8
Molecular docking:
As DHFR target
4.8.1
Molecular docking was used to clarify the binding mode of the compounds to provide straightforward
information for further structural optimization. The crystal structure of dihydrofolate reductase complexed
with NADPH and Methotrexate (PDB ID 1DF7, 1.7 Å X-ray resolutions) was extracted from the Brookhaven
Protein Database (PDB http://www.rcsb.org/pdb). The proteins were prepared for docking by adding polar
hydrogen atom with Gasteiger-Huckel charges and water molecules were removed. The 3D structure of the
ligands was generated by the SKETCH module implemented in the SYBYL program (Tripos Inc., St. Louis,
USA) and its energy-minimized conformation was obtained with the help of the Tripos force field using
Gasteiger-Huckel charges and molecular docking was performed with Surflex-Dock program that is
interfaced with Sybyl-X 2.0. and other miscellaneous parameters were assigned with the default values given
by the software
4.8.2
As anticancer target
Ligand Preparation
The molecules 3b, 3w and 3x were prepared using LigPrep module in the Schrodinger Suite 2018-4 (Trial
Version). The stereoisomerism of the chiral centers in the molecules synthesized was racemic, hence all the
stereoisomers were generated for the purpose of docking. The molecules were neutral as a results ionization

was not considered. Diverse conformations were generated for all the molecules which were subsequently
used for docking studies.
Docking
The active ligands were docked into the receptor grid generated around the inhibitor bound to the protein
crystal structure. The outer grid 15 Å inx, y, and z extents while the inner grid was 10 Å in extents. To
maximize the probability of forming hydrogen bonds between the residues in the enzyme active site and the
ligand, the side chain hydroxyl groups of the amino acids serine, threonine, and tyrosine were allowed to
rotate. All the calculations were carried with Schrödinger Suite 2018-4 (Trial Edition) running on Linux
Workstation.
DNA binding studies:
Methodology: UV Absorption experiments were performed by maintaining a constant nucleotide
concentration and varying samples concentration (10–2 µg/ml) in buffer. Solutions of calf thymus DNA in
phosphate buffer gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) 1.8–1.9, indicating that the
DNA was pure and sufficiently free of contaminants like the proteins and RNA. The solution of DNA and
desired compounds were kept to equilibrate at 25 °C for 20 min, after which absorption readings were noted.
The data were then fit to following equation (1) to obtain intrinsic binding constant Kb.
[DNA]/ [
-
] = [DNA] / [
f
-
] + 1 / Kb[
f
-
b
f] - - - (1)
∈
∈
∈
∈
∈
∈
a
b
Where, [DNA] = concentration of DNA in base pairs,
∈
∈
∈
a is the extinction coefficient observed for the MLCT absorption band at the given DNA concentration,
f is the extinction coefficient of the complex without DNA
b is the extinction coefficient of the complex when fully bound to DNA.
5.
Acknowledgments
The authors are grateful to the Department of Science and Technology of Goa and UGC- New Delhi, DST-
FIST New Delhi for financial support. We would also like to thank Department of Chemistry, Goa University
for providing 1H NMR and 13C NMR data, Sophisticated analytical instrument facilities, Chandigarh and
Karnatak University, Dharwad, for 1H NMR and 13C NMR data, IIT Ropar and Birla Institute of Technology,
Pilani, Rajasthan, for Mass spectral data.
6.
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