Synthesis and biological evaluation of 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one structural derivatives as anti-cancer and apoptosis inducing agents

Article abstract of DOI:10.1016/j.ejmech.2015.12.007

A series of thirteen 5H-dibenzo [b,e][1,4]diazepin-11(10H)-one structural derivatives has been synthesized and evaluated for anti-proliferative activity against five human cancer cell lines. Compound 9a exhibited potent tumour growth inhibition in all cel

Full text of DOI:10.1016/j.ejmech.2015.12.007

Accepted Manuscript
Synthesis and biological evaluation of 5,10-Dihydro-11H-dibenzo[b,e]
[1,4]diazepin-11-one structural derivatives as anti-cancer and apoptosis inducing
agents
Chintakunta Praveen Kumar, T. Srinivasa Reddy, Prathama S. Mainkar, Vipul Bansal,
Ravi Shukla, Srivari Chandrasekhar, Helmut M. Hügel
PII:
S0223-5234(15)30391-3
10.1016/j.ejmech.2015.12.007
EJMECH 8247
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 August 2015
Revised Date: 19 November 2015
Accepted Date: 2 December 2015
Please cite this article as: C.P. Kumar, T.S. Reddy, P.S. Mainkar, V. Bansal, R. Shukla, S.
Chandrasekhar, H.M. Hügel, Synthesis and biological evaluation of 5,10-Dihydro-11H-dibenzo[b,e]
[1,4]diazepin-11-one structural derivatives as anti-cancer and apoptosis inducing agents, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.007.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of 5,10-Dihydro-11H-dibenzo[b,e][1,4]diazepin-11-
one structural derivatives as anti-cancer and apoptosis inducing agents
Chintakunta Praveen Kumara,b,c, T. Srinivasa Reddya,d,e,f, Prathama S. Mainkarc, Vipul
Bansala,d,e, Ravi Shuklaa,d,e,f, Srivari Chandrasekharc*, Helmut M. Hügela*
aSchool of Applied Sciences, RMIT University, Melbourne 3001, Australia
b
IICT-RMIT Research Centre SIR-Indian Institute of Chemical Technology, Hyderabad 500
007, India
cDivision of Natural Products Chemistry, CSIR-IICT, Hyderabad 500 007, India
dHealth Innovations Research Institute, RMIT University, Melbourne 3083, Australia
eIan Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of
Applied Sciences, RMIT University, Melbourne 3000, Australia
fCentre for Advanced Materials and Industrial Chemistry, RMIT University, Melbourne
3000, Australia
*Corresponding authors:
Associate Prof. Helmut M. Hügel
School of Applied Sciences, RMIT University
GPO Box 2476, Melbourne 3001 VIC Australia
E-mail: helmut.hugel@rmit.edu.au
Tel:
+61 3 9925 2626
Dr. S. Chandrasekhar
Director
CSIR-Indian Institute of Chemical Technology,
Tarnaka, Hyderabad, India-500 007
E-mail: srivaric@iict.res.in
Fax: +91-40-27160512

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Abstract
A series of thirteen 5H-dibenzo [b,e][1,4]diazepin-11(10H)-one structural derivatives has
been synthesized and evaluated for anti-proliferative activity against five human cancer cell
lines. Compound 9a exhibited potent tumour growth inhibition in all cell lines with IC50
values in the range of 0.71-7.29 µM. Experiments on lung (A549) and breast (MDAMB-231)
cancer cell lines to investigate the mechanisms of growth inhibition and apoptosis inducing
effects of 9a showed that it arrested both cancer cell lines in the G2/M phase of cell cycle in a
dose dependent manner. Hoechst staining analysis revealed that 9a inhibited tumour cell
proliferation through apoptosis induction. Additionally, the mitochondrial membrane
potential (∆Ψm) was affected and the levels of reactive oxygen species (ROS) were raised.
The simple synthetic preparation and their biological properties make these
dibenzodiazepinone-triazole scaffolds promising new entities for the development of cancer
therapeutics.
Key words
Dibenzodiazepinones; triazoles; anti-cancer activity; apoptosis
1.0 Introduction
Cancer is the leading global health burden that at some time will directly or indirectly affect
the lives of most people. Local cancer treatments, such as surgical and radiation therapies are
not always viable due to the position of the tumour in the body. Furthermore, these methods
are often unsuccessful in completely removing tumours. Chemotherapy is a systemic cancer
treatment and chemotherapeutic drugs interfere with the cell cycle and thus cell division,
angiogenesis or induce tumour cell apoptosis by several signalling pathways. However, due

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to the high cancer mortality rate, development of drug resistance and undesirable side effects
there is an urgent need to design and synthesise new anti-cancer drugs.
The dibenzodiazepinone [5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one]1(Figure1)
scaffold is known to exhibit wide range of biological activities including neuroleptic [1], anti-
inflammatory [2], anti-depressant [3], anti-microbial [4], anti-hypertensive [5] and anti-viral
[6] efficacies. Moreover, dibenzodiazepinone research led to the discovery of drug leads such
as Chk1 kinase inhibitors 2 (Figure 1) [7] and the histone deacetylase HDAC [8] inhibitor 3.
Diazepinomicin 4 is an unusual farnesylated-dibenzodiazepinone secondary metabolite
isolated [3, 9] from Micromonospora and contains dibenzodiazepinone skeleton that is
unprecedented among natural products and its biosynthesis [10] has been described recently.
The anti-tumour, anti-bacterial and anti-inflammatory activities of 4 have also been reported
[11]. Compound 4 (Figure 1) induces apoptosis by selectively targeting the peripheral
benzodiazepine receptor (PBR) [12].
<Figure 1>
On the other hand, many triazole containing compounds exhibit extensive range of
pharmacological activities including potent anticancer properties [13]. For instance,
cefatrizine, tazobactam and carboxyamidotriazole are some of the triazole containing
compounds which are in clinical or preclinical studies [14, 15]. Among the triazoles, 1,2,3-
triazoles are capable of interacting with the biological targets through H-bonding and are
stable to metabolic degradation [16]. Based on these observations and inspired by the
promising anticancer activities of dibezodiazepinones and 1,2,3-triazoles, we designed a
series of dibezodiazepinone-triazole hybrid compounds with a view to produce promising
anticancer agents.

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The diverse therapeutic application of dibenzodiazepine derivatives and related compounds in
medicinal chemistry has stimulated considerable synthesis interest in the construction of this
tricyclic ring privileged scaffold. Eight chemical retro synthetic approaches are presented in
Figure 2. In 1985, a one-step synthesis delivered in a modest 30% yield the dibenzodiazepine
scaffold [17, 18] by refluxing 2-chlorobenzoic acid and o-phenylendiamine in the presence of
copper as catalyst and chlorobenzene as solvent that is denoted as route I. The reaction
product from 2-iodoaniline and a substituted1-fluoro-2-nitrobenzene was then employed in a
palladium mediated intramolecular carbonylation reaction [19] to provide the 1,4-
benzodiazepine product identified as route II. The ring closing reductive lactamization
methodology [20] using the dissolving-metal condition of iron and acetic acid was another
synthesis approach [route III]. The amine ring closure path gave an efficient synthesis of 1,4-
benzodiazepines [21] via the double amination of ortho-substituted aryl bromides is shown as
route IV. The one step preparation of dibenzodiazepines [22] and related derivatives
illustrated as route V was accomplished by the palladium-catalyzed reaction between alkyl 2-
(2-chlorophenylamino)benzoate and ammonia. The utilization of POCl3 enabled substituted
ethyl 2-(phenylamino)phenylcarbamate to participate in an intramolecular lactamization via
the electrophilic aromatic substitution to furnish route VI to the desired 1,4-benzodiazepine
[23]. The synthesis of the target compound was also achieved through a high temperature,
Cs2CO3 driven, double nucleophilic aromatic substitution reaction [24] of anthranilamide
with 2,5-dichloronitrobenzene depicted as route VII. Recently, route VIII, the 1,4-
benzodiazepine core [25] structure was assembled from the substituted N-methoxy-2-
(methylphenylamino)benzamide through an intramolecular PIDA-mediated oxidative C-N
bond synthesis in 82% yield in this cyclisation step.
<Figure 2>

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2.0 Results and Discussion
2.1 Chemistry
We utilized a slightly modified one step dibenzodiazepine synthesis [route I in Figure 2]. Our
dibenzodiazepinone-triazole analogues were prepared following the reaction sequence
depicted in Scheme 1. Initially, ethyl-2-iodobenzoate 5 was reacted with o-phenylenediamine
o
6 in presence of 10 mol% CuI in a pressure tube at 100 C to give the dibenzodiazepinone 1
in 50% yield [26]. Propargylation of 1 was first carried out with propargyl bromide and NaH
in anhydrous THF solvent at ice-bath temperature. But, these conditions did not afford the
required compound 7 (Scheme 1). When, the reaction was carried out with n-BuLi at -20 oC
in freshly distilled THF, the desired N-propargylated compound was obtained in 72% yield.
Decreasing the reaction temperature to -78 oC did not improve the reaction yield.
<Scheme 1>
1
13
The formation of product 7 was evident from H NMR and C NMR spectroscopy. Then, 7
was subjected to click reaction conditions to construct the 1,2,3-triazole ring. Various benzyl
azides 8a-k used in the reaction were prepared from respective aldehydes following literature
procedures [27]. Azides underwent 3+2 cycloaddition (CuAAC) with 7 in presence of
CuSO4·5H2Oand sodium ascorbate in tBuOH/H2O (1:1) to give triazole-products 9a-k
(Scheme1). Formation of triazole ring was determined by the characteristic singlet in the
1
aromatic region in H, by peak at around 120 ppm in 13C NMR, and from mass spectral
analysis.

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2.2 Biological studies
2.2.1 In vitro cytotoxic activity
All the synthesised dibenzodiazepinone-triazole derivatives were screened for their
anticancer activity against five different human tumor cell lines including prostate (PC3),
lung (A549), brain (U87MG), and breast (MCF7 and MDA-MB-231) using MTT assay and
the results are illustrated in Table.1. The results from Table 1 indicated that most of the
synthesised compounds (7, 9a-j) except 9k displayed moderate to potent growth inhibition
against the tested cancer cells and selective potency against MDA-MB-231 cells. Compounds
9a, 9c-9d and 9f-9i displayed remarkable growth inhibition on MDA-MB-231 cells which are
superior to the standard 5-Fluorouracil. Moreover, 9a, 9c and 9f displayed significant broad
spectrum growth inhibition against all the tested cancer cells with IC50 values in the range of
0.71-8.23 µM. Further, 9c displayed selective potency growth inhibition of PC-3 (IC50-
0.47µM) and MCF-7 (IC50-0.65µM) cell lines which are superior to the standard drug 5-
Flurouracil. Interestingly, N-propargylated compound 7 that doesn’t contain triazole ring also
exhibited considerable anti-tumour activity against PC-3 tumour cells (IC50- 0.98 µM) and
breast cancer cells MCF-7 (IC50-7.63µM). Substituents at meta-position of the ring C seemed
to have significant effect on activity for example, as both 3-NO2 (9c) and 3-F (9f) substituted
compounds exhibited significant growth inhibition activities. However, the unsubstituted
compound (9a) on the C ring also displayed substantial growth inhibition. On the other hand,
para-substituted compounds displayed mixed results. For instance, 4-fluoro (9d), 4-
trifluoromethoxy (9k), and 4-nitro (9b) substituted compounds are ineffective and 4-chloro
(9g) scaffold showed remarkable cytotoxic activity.
<Table 1>

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2.2.2 Colony formation inhibition assay
Anti-proliferative activity of the most active compound in the series 9a was further confirmed
by clonogenic cell survival assay on A549 and MDA-MB-231 cells. This assay reflects the
capability of a single cell to rise into a colony and it is also considered as the standard for
determining long term cell viability because they reflect all modes of cell death or arrest [28].
As shown in Figure 3 treatment of A549 and MDA-MB-231 cells with 9a resulted in
inhibition of colony formation in a dose dependent manner. The formation of colonies was
completely inhibited by the compound 9a at 10 µM concentration in both the treated cells.
This indicates the ability of 9a on dose dependent inhibition of colony formation ability and
proliferation of A549 and MDA-MB-231 cells.
<Figure 3>
2.2.3 In vitro cell migration assay
Migratory and invasive activities of tumor cells are the important characteristics of metastatic
cancers. Therefore, it was of interest to investigate the effect of compound 9a on tumor cell
migration using wound healing assay [29]. Artificial wounds were created in the confluent
monolayer A549 and MDA-MB-231 cells using sterile 200 µL pipette tip and were treated
with the different concentrations of compound 9a. Images were taken immediately (0 h) and
24 and 48 h after treatment. As depicted in Figure 4 treatment with 9a resulted in suppression
of A549 and MDA-MB-231 cell migration to the wounded area in a concentration dependent
manner in comparison to the control.
<Figure 4>

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2.2.4 Cell cycle analysis
We next analysed the effect of compound 9a on cell cycle distribution using flow cytometry.
A549 and MDA-MB-231 tumor cells were treated with different concentrations (0.5, 1and 2
µM) of 9a for 48 h; cells were fixed in ethanol and stained with propidium iodide. Results
from the Figure 5 revealed that the proportion of A549 and MDA-MB-231 cells in G2/M
phase gradually increased respectively from 31% (control) to 51% (2 µM) and 20% (control)
to 63 % (2 µM). Similarly, the population in G0/G1 phase declined from 49% to 34% and
73% to 33%, respectively after treatment with the compound. This indicates that 9a treatment
resulted in the arrest of A549 andMDA-MB-231 cells in G2/M phase of cell cycle.
<Figure 5>
2.2.5 Apoptosis detection studies
Apoptosis is a programmed cell death which plays an important role in the maintenance of
tissue homeostasis and cell survival. Disruption or inappropriate regulation of apoptotic
processes results in several diseases including cancer [30]. Thus, inducing apoptosis in cancer
cells has emerged as an attractive strategy in cancer therapy [31].
2.2.5.1Hoechst staining
Chromatin condensation, nuclear shrinking and DNA fragmentation are the typical
characteristics of an apoptotic cell. Therefore, apoptosis inducing effect of compound 9a was
investigated using the Hoechst staining (H 33242) assay. A549 and MDA-MB-231 cells
treated with different concentrations of 9a for 48 h were stained with Hoechst. As evident
from Figure 6, control, A549 and MDA-MB-231 cells treated with DMSO showed
uniformly dispersed chromatin, whereas compound 9a treated cells displayed typical
apoptotic characteristics such as condensation of nuclei (arrowheads indicates apoptotic
nuclei).These finding demonstrates that 9a could induce apoptosis in A549 and MDA-MB-
231 cells.

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<Figure 6>
2.2.5.2 Effect of compounds on mitochondrial membrane potential (∆Ψm)
Mitochondrial membrane potential regulates the mitochondrial permeability and plays an
important role in triggering apoptosis [32]. In order to further investigate the apoptosis-
inducing effect of compound 9a, changes in the mitochondrial membrane potential were
measured using rhodamine-123 staining. Mitochondria with the intact ∆Ψm can hold the Rh-
123 probe and exhibits strong green fluorescence. Loss of ∆Ψm results in decrease in green
fluorescence due to the lack of Rhodamine-123 retention [33]. A549 and MDA-MB-231 cells
were treated with 9a for 48 h, and then stained with Rhodamine-123. The results from Figure
7 indicated that, treatment of 9a induced 20-35% decrease in ∆Ψm in MDA-MB-231 cancer
cells and 15-30% in A549 cells in concentration dependant manner in compared to respective
control cells, which indicates that 9a induces apoptosis in A549 and MDA-MB-231 cells
through change in ∆Ψm.
<Figure 7>
2.2.5.3 Measurement of reactive oxygen species (ROS) levels
Decline in mitochondrial membrane potential (∆Ψm) and increase in the level of intracellular
ROS are closely related in the course of apoptosis. Moreover, generation of ROS is
considered as one of the key mediators of apoptotic signalling and many if not most of the
anti-tumour agents exhibit their cytotoxic effects through the generation of ROS [34,
35].Thus to investigate whether ROS accumulation was involved in compound 9a induced
apoptosis, A549 and MDA-MB-231 cells were treated with different concentrations of
compound 9afor 48 h and incubated with carboxy-DCFDA to inspect 9a mediated production
of ROS. As shown in Figure 8, there is a gradual increase in green carboxy-DCF fluorescence
(correlates with ROS concentration) with increasing the concentration of compound 9a
treated A549 (1.8-2.5 times) and MDA-MB-231(2.09-2.98 times) (Figure 8) cells and it is

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more prominent with MDA-MB-231 cells. These results clearly indicate that 9a could induce
the ROS generation.
<Figure 8>
2.2.5.4 AnnexinV-FITC/Propidium iodide dual staining assay
To quantify the percentage of apoptosis induced by the compound 9a, Annexin V-FITC-PI
dual staining assay has been carried out which facilitates 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 9, the percentage of total apoptotic
cells (early and late apoptotic cells) was increased to 32.4 % (MDA-MB-231) and 34.9%
(A549) after treatment with compound 9a (2 µM),in comparison to the control MDA-MB-
231 (9.8%) and A549 (6.5%) cells respectively. The percentage of early and late apoptotic
cells were markedly increased with increase in concentration of the compound 9a.Therefore,
these results confirmed that the compound 9a treated MDAMB-231 and A549 cells undergo
apoptosis in a dose dependent manner.
<Figure 9>
3.0 Conclusion
In an effort to discover novel anti-cancer agents, a series (9a-k) of dibenzodiazipinone-
triazole derivatives were prepared, characterized and evaluated against five human tumour
cell lines. All the synthesized compounds exhibited moderate to good activity against five
cancer cell lines. In particular, compound 9a exhibited potent in vitro anti-cancer activity
among the tested compounds with the IC50 values in the range of 0.71 to 7.29 µM. It is
evident from the cell migration and clonogenic experiments that, migration and colony
formation of the A549 and MDA-MB-231 cells was significantly affected by compound 9a.
Flow cytometry analysis revealed that 9a arrested both these cells in G2/M phase. Further

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apoptosis studies conducted on A549 and MDA-MB-231 cells revealed that compound 9a
effectively induces apoptosis through drop in mitochondrial membrane potential (∆Ψm) and
increases the ROS generation in A549 and MDA-MB-231. Overall, these results suggest that
compound 9a has the potential to be developed as lead and their further structural
modification may produce promising anticancer agents.
4.0 Experimental section
4.1 Chemistry
All reagents and solvents were of commercial grade purchased and used without further
purification. Melting points were recorded after column chromatography of compound on
Electro thermal melting point apparatus and were uncorrected. 1H NMR,19F NMR (300 MHz)
13
and C NMR (75 MHz) spectra were recorded in CDCl3 or CD3SOCD3 solvents on Bruker
Avance 300 MHz spectrometer at ambient temperature. Chemical shifts (δ) are reported in
ppm downfield from internal TMS standard. Signal multiplicities are represented by s
(singlet), d (doublet), t (triplet), dd (double doublet), m (multiplet) and br s (broad singlet).
HRMS results were run on the AB Sciex QSTAR ELITE mass spectrometer using an electron
spray ionization ESI source. IR spectra were recorded on Perkin-Elmer Infrared spectrometer
and samples were scanned in grounded KBr powder. Routine monitoring of reaction was
performed by TLC (Thin layer chromatography) on aluminium silica plates obtained from E-
Merck. For TLC visualisation UV light and iodine were used. All the column
chromatographic separations were done by using silica gel (Acme’s, 60-120 mesh).
Evaporation of solvents was performed at reduced pressure on a rotary evaporator.
4.1.1 5,10-Dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one (1)
To a dried pressure tube was added o-phenylenediamine (0.3 g, 2.7 mmol), ethyl-2-
iodobenzoate (1.5 g, 5.5 mmol) under nitrogen followed by ethylene glycol (6 mL), 10 mol %
CuI (52 mg, 0.27 mmol) and K3PO4 tribasic (1.17 g, 5.5 mmol). Pressure tube was closed

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o
tightly and heated at 100 C for 4 h. After completion of the reaction (monitored by TLC)
reaction mixture was cooled to room temperature and filtered over a pad of silica with
CH2Cl2 to remove CuI and base residues. Then solvent was removed on rotary evaporator
and the residue was purified on silica column chromatography using 25-30% EtOAc in
hexane as solvent to give 1 Yield 50% (0.29 g)
o
1
mp: 251-253 C; IR (υmax):3320, 3025, 1603, 1579, 1471, 1394, 1157, 748 cm-1; H NMR
(300 MHz, DMSO-d6): δ 8.63 (s, 1H, Amide NH), 6.60 (s, 1H, Amine NH), 6.46 (dd, J = 7.9,
1.4 Hz, 1H, ArH), 6.19 – 6.08 (m, 1H, ArH), 5.87 – 5.63 (m, 6H, ArH);13CNMR (75 MHz,
DMSO): δ 168.1, 150.4, 140.0, 133.2, 131.9, 129.6, 124.6, 123.0, 122.7, 121.2, 120.9, 119.8,
119.0 ;HRMS (ESI) m/z calculated for [M+H]+ [C13H11N2O] 211.0866, found 211.0860.
4.1.2 10-(Prop-2-yn-1-yl)-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one (7)
H
N
N
O
7
In a two neck round bottom flask compound 2 (0.1 g, 0.47 mmol) was dissolved in freshly
distilled dry THF (5 mL), under inert conditions. To that was added 1.5 mL of 1.6 M n-BuLi
o
(5 equiv) at -20 C. After 5 min 80% propargyl bromide in toluene (0.054 mL, 0.52 mmol)
was added to the reaction and the mixture stirred for 1 h at the same temperature. Reaction
was monitored by TLC after consumption of the starting material reaction was quenched with
saturated NH4Cl solution and partitioned between H2O and EtOAc. Organic layers were
separated, combined, dried over anhydrous Na2SO4 and concentrated on rotary evaporator
o
below 46 C. Product was purified on silica column chromatography to yield propargylated
scaffold 7.
Yield: 72 % (80 mg); mp: 165-170 oC ; IR (υmax): 3321, 3292, 2923, 1623, 1546, 1454, 1160
cm-1; 1H NMR (300 MHz, CDCl3): δ 7.94 (dd, J = 7.8, 1.3 Hz, 1H, ArH), 7.72 – 7.57 (m, 1H,

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ArH), 7.39 – 7.25 (m, 1H, ArH), 7.20 – 6.90 (m, 4H, ArH), 6.83 (d, J = 8.0 Hz, 1H, ArH),
5.53 (s, 1H, NH), 4.68 (d, J = 2.4 Hz, 2H, propargylic-CH2), 2.39 (t, J = 2.4 Hz, 1H, triple
bond H); 13CNMR (75 MHz, CDCl3): δ 168.1, 150.6, 143.1, 134.3, 133.1, 132.8, 126.2,
124.4, 124.2, 122.9, 122.6, 120.3, 118.6, 80.1, 72.0, 40.3 ; HRMS (ESI): m/z calculated for
[M+H]+ [C16H13N2O] is 249.1022 found 249.1015
4.1.3 General procedure for the click reaction:
To the propargylated compound 7 (50 mg, 0.20 mmol) and azide 8a-k (0.60 mmol) in 3 mL
of 1:1 t-BuOH/H2O was added 100 µL saturated CuSO4·5H2Osolution followed by catalytic
amount of Na-ascorbate at ambient temperature. Reaction was allowed to stir for 5-6 h,
solvent was removed and product was extracted with CH2Cl2. Dichloromethane layer was
dried over anhydrous sodium sulphate, solvent was evaporated in vacuo and residue was
purified using silica column chromatography to obtain 9a-k.
4.1.3.1 10-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9a)
o
Yield: 82% (50 mg); mp: 128-133 C; IR (υmax): 3301, 3069, 2936, 2123, 1631, 1480, 1320,
1160, 765 cm-1;1H NMR (300 MHz, CDCl3) δ: 7.77 – 7.65 (m, 2H), 7.61 (dd, J = 7.5, 1.6
Hz, 1H, ArH), 7.35 – 7.10 (m, 6H, ArH), 7.05 – 6.85 (m, 3H, ArH), 6.80 (dd, J = 7.4, 3.8 Hz,
1H, ArH), 6.71 (d, J = 7.9 Hz, 1H, ArH), 5.42 (s, 3H, benzyl- CH2&-NH), 5.13 (d, J = 12.5
Hz, 2H, triazole-CH2);13CNMR (75 MHz, CDCl3)δ:168.6, 150.8, 143.4, 134.7, 134.7, 132.6,
129.0, 128.6, 127.9, 126.1, 125.0, 124.6, 124.2, 122.6, 120.2, 118.6, 54.1, 47.1; HRMS (ESI):
m/z calculated for [M+H]+ [C23H20N5O] 382.1662 found 382.1663.

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4.1.3.2 10-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9b)
o
Yield: 61% (31 mg); mp: 159-164 C; IR (υmax): 3132, 3075, 1628, 1522, 1474, 1344, 1049,
767 cm-1 ;1H NMR (300 MHz, CDCl3) δ: 8.13 (d, J = 8.7 Hz, 2H, ArH ), 7.79 (s, 1H, triazole
H), 7.68 (d, J = 8.1 Hz, 1H, ArH), 7.60 (d, J= 7.6 Hz, 1H)7.32-7.16 (m, 3H, ArH), 7.06-6.88
(m, 3H, ArH), 6.81 (d, J = 7.5 Hz, 1H, ArH), 6.72 (d, J = 8.0 Hz, 1H, ArH), 5.54 (s, 2H,
benzylic-CH2), 5.33 (brs, 1H, -NH), 5.13 (s, 2H, triazole-CH2); 13CNMR (75 MHz, CDCl3) δ:
168.7, 150.7, 148.0, 145.9, 143.3, 141.7, 134.7, 132.7, 132.5, 128.5, 126.2, 124.9, 124.8,
124.7, 124.2, 124.1, 122.7, 120.2, 118.6, 53.0, 47.1; HRMS (ESI): m/z calculated for [M+H]+
[C23H19N6O3] 427.1513, found 427.1504.
4.1.3.3 10-((1-(3-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9c)
Yield: 68 % (35 mg); mp: 124-130 ºC; IR (υmax): 3314, 3012, 2102, 1634, 1528, 1477, 1350,
758 cm-1;1H NMR (300 MHz, CDCl3) δ: 8.26-8.17 (m, 1H), 8.14 (s, 1H), 7.89 (s, 1H), 7.80
(dd, J = 12.2, 8.2 Hz, 1H, ArH), 7.67 (d, J = 8.8 Hz, 1H, ArH), 7.62 – 7.52 (m, 2H, ArH),
7.31 (m, 1H, ArH), 7.14-6.86 (m, 4H, ArH), 6.81 (d, J = 7.9 Hz, 1H, ArH), 5.63 (s, 2H,
benzylic-CH2), 5.45 (s, 1H, -NH), 5.24 (s, 2H, triazole-CH2); 13C NMR (75 MHz, CDCl3)
δ:168.7, 150.7, 143.4, 136.8, 134.6, 133.8, 132.7, 132.5, 130.2, 126.2, 124.9, 124.7, 124.1,

ACCEPTED MANUSCRIPT
123.6, 122.8, 122.7, 120.3, 118.6, 53.1, 47.0; HRMS (ESI): m/z calculated for [M+H]+
[C23H19N6O3] 427.1513, found 427.1503.
4.1.3.4 10-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9d)
Yield: 70% (43 mg); mp: 109-114 ºC; IR (υmax): 3305, 3129, 3069, 1628, 1513, 1474, 1154,
764 cm-1;1H NMR (300 MHz, CDCl3) δ: 7.70 (s, 1H, triazole H), 7.68 (dd, J = 1.6, 7.8 Hz,
1H, ArH), 7.62 ( dd, J = 1.8, 7.8 Hz, 1H, ArH), 7.25-7.12 (m, 3H, ArH), 7.05-6.89 (m, 5H,
ArH), 6.80 (dd, J = 1.9, 7.4 Hz, 1H, ArH), 6.72 (d, J = 7.9 Hz, 1H, ArH) 5.40 (s, 2H,
benzylic-CH2), 5.31 (brs, 1H, NH), 5.10 (s, 2H, triazole-CH2); 13CNMR (75 MHz, CDCl3) δ:
168.6, 150.7, 145.6, 143.3, 134.7, 132.6, 132.6, 130.6, 130.5, 129.9, 129.8, 126.1, 124.9,
124.7, 124.4, 124.2, 122.7, 120.1, 118.5, 116.2, 115.9, 53.3, 47.1; 19F NMR (300 MHz,
CDCl3) δ: -112.7-112.9 (m);HRMS (ESI): m/z calculated for [M+H]+ [C23H19FN5O]
400.1568 found 400.1567.
4.1.3.5 10-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9e)
Yield: 73 % (35 mg); mp: 168-173 oC; IR (υmax): 3326, 3151, 3057, 1613, 1486, 1226, 1042,
758 cm-1;1H NMR (300 MHz, CDCl3) δ:7.87 (s, 1H, triazole H), 7.80 (dd, J = 7.8, 1.3 Hz,
1H, ArH), 7.70 (dd, J= 7.4, 1.6 Hz, 1H, ArH), 7.46-6.97 (m, 8H, ArH), 6.90 (d, J = 7.3 Hz,

ACCEPTED MANUSCRIPT
1H, ArH), 6.82 (d, J = 7.9 Hz, 1H, ArH), 5.59 (s, 2H, benzylic-CH2), 5.45 (s, 1H, -NH), 5.22
(s, 2H, triazole-CH2); 13CNMR (75 MHz, CDCl3) δ: 168.6, 162.1, 158.8, 150.7, 143.3, 134.7,
132.6 (split), 130.8 (split), 130.7, 130.3, 126.1, 125.0, 124.7, 124.2 (split), 122.7, 122.0,
19
121.9, 120.2, 118.5, 115.9 115.6, 47.6 (split), 47.1; F NMR (300 MHz, CDCl3) δ -117.8-
118.0 (m); HRMS (ESI): m/z calculated for [M+H]+ [C23H19FN5O] 400.1568 found
400.1563.
4.1.3.6 10-((1-(3-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9f)
Yield: 70% (33 mg); mp: 143-148 ºC ; IR (υmax): 3312, 3051, 1622, 1471, 1120, 766 cm-1; 1H
NMR (300 MHz, CDCl3) δ: 7.73 (s, 1H, triazole H), 7.69 (dd, J = 7.8, 1.4 Hz, 1H, ArH), 7.60
(dd, J= 7.6, 1.7 Hz, 1H, ArH), 7.31-7.15 (m, 3H, ArH), 7.07-6.87 (m, 6H, ArH), 6.81 (dd, J =
12.6, 5.3 Hz, 2H, ArH), 6.72 (d, J = 7.9 Hz, 1H, ArH), 5.42 (s, 2H), 5.14 (s, 2H, triazole-
13
CH2); CNMR (75 MHz, CDCl3) δ:168.6, 164.6, 161.3, 150.7, 143.4, 137.0, 134.6, 132.6,
130.7, 130.6, 126.2, 124.9, 124.7, 124.2, 123.5, 122.7, 120.2, 118.6, 115.8, 115.5, 115.0,
19
114.7, 53.5, 47.0; F NMR (300 MHz, CDCl3) δ: -111.6 (td, J= 8.9, 5.9 Hz); HRMS (ESI):
m/z calculated for [M+H]+ [C23H19FN5O] 400.1568 found 400.1569.
4.1.3.7 10-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9g)

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o
Yield: 75% (50 mg); mp: 135-140 C; IR (υmax): 3307, 3135, 1624, 1479, 1395, 1157, 1052,
1
766 cm-1; H NMR (300 MHz, CDCl3) δ: 7.71 (s, 1H, triazole H), 7.68 (dd, J = 1.7, 8.0 Hz,
1H, ArH), 7.60 (dd, J = 2.0, 7.4 Hz, 1H, ArH), 7.28-7.16 (m, 4H, ArH), 7.12-7.06 (m, 2H,
ArH), 7.04-6.88 (m, 3H, ArH), 6.79 (dd, J = 1.8, 7.1 Hz, 1H, ArH), 6.71 (d, J = 7.9 Hz, 1H,
13
ArH) 5.39 (s, 2H, benzylic-CH2), 5.34 (brs, 1H, NH), 5.11 (s, 2H, triazole-CH2); CNMR
(75 MHz, CDCl3) δ: 168.6, 150.7, 145.6, 143.3, 134.7, 134.6, 133.2, 132.6, 132.6, 129.3,
129.2, 126.1, 124.9, 124.7, 124.5, 124.1, 122.7, 120.2, 118.5, 53.6, 47.2; HRMS (ESI): m/z
calculated for [M+H]+ [C23H19ClN5O] 416.1273, found 416.1236.
4.1.3.8 10-((1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-
11H-dibenzo[b,e][1,4]diazepin-11-one (9h)
o
Yield: 90% (80 mg); mp: 122-128 C; IR (υmax): 3311, 3132, 3946, 1631, 1504, 1462, 1238,
1126, 761 cm-1;1H NMR (300 MHz, CDCl3) δ: 7.76 (s, 1H, triazole H ), 7.68 (dd, J = 7.8, 1.4
Hz, 1H, ArH), 7.61 (dd, J= 7.5, 1.7 Hz, 1H, ArH), 7.27-7.17 (m, 2H, ArH), 7.06-6.89 (m, 3H,
ArH), 6.85-6.76 (m, 1H, ArH), 6.73 (d, J = 7.9 Hz, 1H, ArH), 6.41 (s, 2H, ArH), 5.36 (s, 2H,
13
benzylic-CH2), 5.14 (s, 2H, triazole-CH2), 3.79 – 3.72 (m, 9H, -OCH3); CNMR (75 MHz,
CDCl3) δ: 168.6, 153.6, 150.7, 143.3, 138.2, 134.7, 132.7, 132.5, 130.1, 126.2, 124.9, 124.7,
124.6, 124.1, 122.7, 120.2, 118.6, 105.2, 60.8, 56.2, 54.5, 47.1; HRMS (ESI): m/z calculated
for [M+H]+ [C26H26O4N5] 472.1979 found 472.1961.
4.1.3.9 10-((1-(2,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-dihydro-11H-
dibenzo[b,e][1,4]diazepin-11-one (9i)

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o
Yield: 68% (50 mg); mp: 111-115 C; IR (υmax): 3259, 3220, 2948, 1640, 1504, 1226, 1036,
770 cm-1; 1H NMR (300 MHz, CDCl3) δ: 7.84 (s, 1H, triazole H), 7.76 (ddd, J = 1.6, 7.7, 13.9
Hz, 2H, ArH), 7.34-7.27 (m, 1H, ArH), 7.14-6.97 (m, 3H, ArH), 6.92-6.83 (m, 3H, ArH),
6.80 (d, J= 7.9 Hz, 1H, ArH), 6.72 (d, J = 7.6 Hz, 1H, ArH), 6.75-6.71 (m, 1H, ArH), 5.51 (s,
2H, benzylic-CH2), 5.37 (brs, 1H, NH), 5.20 (s, 2H, triazole-CH2), 3.83 (s, 3H,-OCH3), 3.74
(s, 3H, -OCH3); 13CNMR (75 MHz, CDCl3) δ: 168.5, 153.6, 151.2, 150.7, 144.9, 143.3,
134.8, 132.6, 132.5, 126.0, 125.1, 124.8, 124.7, 124.3, 124.0, 122.6, 120.1, 118.5, 115.8,
114.7, 111.7, 55.9, 55.7, 49.0, 47.1; HRMS (ESI): m/z calculated for [M+H]+ [C25H24O3N5]
442.1874 found 442.1876.
4.1.3.10 10-((1-(Benzo[d][1,3]dioxol-4-ylmethyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-
dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one (9j)
Yield: 80% (41 mg); mp: 118-123 oC; IR (υmax): 3328, 2922, 1670, 1451, 1035, 772 cm-1; 1H
NMR (300 MHz, CDCl3) δ:7.86-7.75 (m, 2H, ArH& triazole-H), 7.71 (d, J = 7.5 Hz, 1H,
ArH), 7.36-7.24 (m, 1H, ArH), 7.17- 6.97 (m, 3H, ArH), 6.94-6.70 (m, 5H, ArH), 5.99 (s,
2H, -O-CH2-O-), 5.42 (s, 2H, benzylic-CH2), 5.38 (brs, 1H, -NH), 5.20 (s, 2H, triazole-CH2);
13CNMR (75 MHz, CDCl3) δ: 168.6, 150.7, 148.2, 147.9, 145.4, 143.3, 134.7, 132.6, 132.6,
128.3, 126.1, 125.0, 124.7, 124.2, 122.7, 121.9, 120.1, 118.5, 108.5, 108.5, 101.3, 54.0, 47.1;
HRMS (ESI): m/z calculated for [M+H]+ [C24H20O3N5] 426.1566 found 426.1563.

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4.1.3.11 10-((1-(2-(Trifluoromethoxy)benzyl)-1H-1,2,3-triazol-4-yl)methyl)-5,10-
dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one (9k)
o
Yield: 80% (45 mg); mp:130-135 C; IR (υmax): 3305, 2927, 2958, 1628, 1477, 1253, 1048,
1
761 cm-1; H NMR (300 MHz, CDCl3) δ 7.76 (s, 1H, triazole-H), 7.71 (dd, J = 7.8, 1.4 Hz,
1H, ArH), 7.66-7.56 (m, 1H, ArH), 7.33 (m, 1H, ArH), 7.29-7.16 (m, 4H), 7.13-6.90 (m, 4H,
ArH), 6.85-6.77 (m, 1H, ArH), 6.72 (d, J = 7.9 Hz, 1H, ArH), 5.55 (s, 2H, benzylic-CH2),
5.28 (s, 1H, -NH), 5.15 (s, 2H, triazole-CH2);13C NMR (75 MHz, CDCl3) δ:168.5, 150.6,
146.8, 143.3, 134.7, 132.6, 130.2, 130.1, 127.4, 126.1, 125.0, 124.7, 124.2, 122.7, 120.5,
120.1, 118.5, 48.4, 47.0; 19F NMR (300 MHz, CDCl3) δ -57.21 (d, J = 1.5 Hz).; HRMS
(ESI): m/z calculated for [M+H]+ [C24H19F3 N5O2] 466.1491 found 466.1504.
4.3 Biology
4.3.1 Cell culture
PC-3, A549, U87MG, MCF-7 and MDA-MB-231 cells were purchased from ATCC
(Manassas, VA). The PC-3, A549 and MDA-MB-231 cells were grown in RPMI 1640
medium (GIBCO-Invitrogen, NY) with 10% fetal bovine serum (FBS) and supplemented
with penicillin G (100 µg/mL), glutamine (2 mmol/L), and streptomycin (100 µg/mL) at 37
oC under 5% CO2. U87MG and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin/ streptomycin at
o
37 C under 5% CO2. The medium was changed every two days. After reaching 80-90%
confluence, cells were treated with 0.25% trypsin-EDTA for further passage. For all the
experiments cells were used at passages 4-10.

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4.3.2 MTT assay
MTT cell proliferation assay was conducted on PC-3, A549, U87MG, MCF-7 and MDA-
MB-231 cancer cell lines to test the compounds 9a-k, 1 and7. Cells were seeded at a density
of 3000-5000 cells per well in a 96 well plate depending on the doubling time. Seeded plates
o
were allowed for 24 h at 37 C and 5% CO2 in incubator. After 24 h, the culture media was
removed; 100µL of test compounds dissolved in DMSO and culture media was added to the
cells in each well. Wells in which test compound was not added (containing only culture
medium and DMSO) were used as the control. Then, the compound treated cells were
incubated for 72 h maintaining the same conditions. 72 h later, culture medium was removed
and 100 µL of MTT (Thiazoyl blue tertrazolium bromide) solution (5 mg/mL in culture
media) was added to each well including control wells and further incubated for 4 h. When
the purple precipitate was visible in wells, media containing MTT was aspirated from the
wells and to each well 100 µL of DMSO was added to dissolve the Formazan product. Plate
covers were removed and absorbance in each well was measured in microtiter plate reader at
570 nm and a reference wavelength of 630 nm. The absorbance readings at 630 nm were
subtracted from the 570 nm readings and the results were adjusted by dividing the average by
the DMSO control to adjust for any toxicity that may have occurred in this control treatment
set. The percentage inhibition was calculated as 100-[(Mean OD of treated cell ×100)/Mean
OD of vehicle treated cells (DMSO)]. The IC50 Values were calculated using Probit Software.
All the tests were repeated in at least three independent experiments.
4.3.3 Colony formation inhibition assay
A549 and MDA-MB-231 cells at the exponential phase were plated at a single cell density
(500 cells/well) in two separate 6 well plates and allowed to adhere for 24 h in the incubator.
Then, cells were incubated with culture medium containing compound 9a in 10, 1and 0.1 µM
concentrations. After 24 h, the medium was replaced with fresh culture medium and cells

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were incubated for further 14 days. The cells were washed with PBS, fixed with 4 % para
formaldehyde and stained with 0.5% methylene blue in PBS for 30 min. Then, excess dye
was rinsed off with distilled water several times. Plates were photographed with a digital
camera.
4.3.4 Cell cycle analysis
A549 and MDA-MB-231 cells were seeded in 6 well plates at a density of 1x106 cells/well
and after 24 h; cells were treated with compound 9a in three different concentrations 0.5,
1and 2µM. Cells treated with DMSO were used as control. 48 h after treatment, both floating
and trypsinised cells were harvested and washed with PBS. Cells were fixed with 70%
o
ethanol for 30 mins at 4 C. Then, cells were centrifuged (1200 rpm for 5 min), the
supernatant was discarded and the pellet was washed with PBS and stained the cells with
propidium iodide staining buffer (PI (200 µg), 0.1% (v/v) Triton X-100, 2 mg DNAse-free
RNAse A (Sigma) in 10 mL PBS)) for 15 min at ambient temperature in absence of light.
Later, samples were analysed for propidium iodide-DNA fluorescence from 10000 events by
flow cytometry using BD-C6 accuri flow cytometer.
4.3.5 In vitro Migration Assay
The wound healing assay has been used to examine the cell migration. In two separate 6 well
plates A549 cells and MDA-MB-231 cells (5×105 cells per well) were cultured as confluent
monolayers for 24 h. Then, using 200µL sterile pipette tip the monolayers were gently
scraped one time in the middle from one end to the other end to create a denuded zone of
constant width. The wounded mono layers were twice washed with phosphate saline buffer
(PBS) to remove non-adherent cells. Then, cells were treated with compound 9a in different
concentrations (0.5, 1 and 2µM). Cell migration across the inflicted wounds were
photographed under the phase contrast microscope (Nikon) in 0 , 24 and 48 h time intervals
in three or more randomly selected fields.

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4.3.6 Hoechst staining
A549/MDA-MB-231 cells (25×104 cells/well) at their growth phase were seeded and allowed
for 24 h to adhere. Test compound 9a in three different concentrations 0.5, 1 and 2µM in
o
culture medium was added to the cells and incubated for 48 h at 37 C. Then, culture
medium was removed from the wells, washed with PBS and stained with Hoechst 33242
(5µg/mL) for half an hour at room temperature. Excess dye was removed by PBS washing
twice after which, morphological changes in the cells were examined with fluorescence
microscope using 350 nm excitation and 460 nm emission (Nikon, magnification 10X).
4.3.7 Measurement of Mitochondrial membrane Potential (MMP)
In a 6 well plate A549/ MDA-MB-231MDA-MB-231 cells were cultured at a density of
5×105cells/mL and incubated for overnight. Then the test compound at 0.5, 1 and 2µM
concentrations was added to the wells. 24 h after the treatment, adherent cells were collected
by trypsinisation, washed with PBS (500µL) and treated with the 400µL/well of Rhodamine-
123 solution in PBS (2µM) in the absence of light. After 30 min of incubation at room
temperature, cells were twice washed with PBS and resuspended in PBS. The samples were
then analysed for fluorescence using spectrofluorometer.
4.3.8 Measurement of reactive Oxygen Species (ROS) levels
A549 cells were seeded in a 6 well plate at a density of 5×105cells/mL and allowed to grow
overnight. The cells were incubated with the 0.5, 1 and 2µM concentration of compound 9a
for 24 h. After which the medium was removed from the wells, culture medium containing
DCFDA (10µM) was added to the wells in the dark and incubated for half an hour at ambient
temperature. Cells were washed with PBS, collected and resuspended in PBS. Intensity of the
formed 2', 7'-dichlorofluorescein as a result of Carboxy-DCFDA hydrolysis, was analysed in
spectrofluorometer at an excitation and emission wavelength of 488 and 525 nm respectively.

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Qualitative cellular fluorescence images were captured by Nikon ECLIPSETE2000-S
fluorescence microscope.
4.3.9. AnnexinV-FITC/Propidium iodide
The Annexin V-FITC/propidium iodide dual staining assay has been carried out to quantify
the percentage of apoptotic cells. Briefly, A549 and MDA-MB-231 cells (1×106/mL per well)
were plated in six-well plates and allowed to grow for 24. Cells were treated with increasing
concentrations of compound 9a (0.5, 1 and 2 µM) for 24 h and were collected by
trypsinisation. The collected cells were washed twice with ice-cold PBS, then incubated with
200 µL1×binding buffer containing 5 µL Annexin V-FITC, and then in 300 µL1×binding
buffer containing 5 µL Propidium iodide (PI) for 5 min at room temperature in the dark. After
15 min incubation, cells were analysed using BD-c6 accuri flow-cytometer.
Acknowledgements
C.P.K thanks RMIT and IICT-RMIT research centre for the research fellowship. R.S.
acknowledges the support of Maxwell Eagle Cancer Endowment award for this research.

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