Facile synthesis of 1,2,3-triazole-fused indolo- and pyrrolo[1,4]diazepines, DNA-binding and evaluation of their anticancer activity

Article abstract of DOI:10.1016/j.bioorg.2019.103306

A facile synthetic strategy has been developed for the generation of structurally diverse N-fused heterocycles. The formation of fused 1,2,3-triazole indolo and pyrrolodiazepines proceeds through an initial Knoevenagel condensation followed by intramolecu

Full text of DOI:10.1016/j.bioorg.2019.103306

Journal Pre-proofs
Facile synthesis of 1,2,3-triazole-fused Indolo- and Pyrrolo[1,4]diazepines,
DNA-binding and evaluation of their anticancer activity
Jitendra Gour, Srikanth Gatadi, Venkatesh Pooladanda, Shaik Mahammad
Ghouse, Satyaveni Malasala, Y.V. Madhavi, Chandraiah Godugu, Srinivas
Nanduri
PII:
S0045-2068(19)31025-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.103306
YBIOO 103306
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
27 June 2019
17 September 2019
19 September 2019
Please cite this article as: J. Gour, S. Gatadi, V. Pooladanda, S. Mahammad Ghouse, S. Malasala, Y.V. Madhavi,
C. Godugu, S. Nanduri, Facile synthesis of 1,2,3-triazole-fused Indolo- and Pyrrolo[1,4]diazepines, DNA-binding
and evaluation of their anticancer activity, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103306
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Facile
synthesis
of
1,2,3-triazole-fused
Indolo-
and
Pyrrolo[1,4]diazepines, DNA-binding and evaluation of their
anticancer activity
Jitendra Gour,a Srikanth Gatadi,a Venkatesh Pooladanda,b Shaik Mahammad Ghouse,a
Satyaveni Malasala,a Y.V. Madhavi,a Chandraiah Godugu,b and Srinivas Nanduri*a
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India
bDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500 037, India
Abstract: A facile synthetic strategy has been developed for the generation of structurally
diverse N-fused heterocycles. The formation of fused 1,2,3-triazole indolo and
pyrrolodiazepines proceeds through an initial Knoevenagel condensation followed by
intramolecular azide–alkyne cycloaddition reaction at room temperature without recourse to
the traditional Cu(I)-catalyzed azide−alkyne cycloadditions. The synthesized compounds
were evaluated for their in vitro anti-cancer activity against the NCI 60 cell line panel.
Among the tested compounds, 3a and 3h were found to exhibit potent inhibitory activity
against many of the cell lines. Cell cycle analysis indicated that the compounds inhibit the
cell cycle at sub G1 phase. The DNA- nano drop method, viscosity experiment and docking
studies suggested these compounds possess DNA binding affinity.

Keywords: Fused polycyclics; Cytotoxicity; DNA-binding; MDA-MB-468 cancer cells
Corresponding author.Dr.Srinivas Nanduri, Tel: +91-40-23073740; Fax: +91-40-
23073751; E-mail: nandurisrini92@gmail.com

Introduction
Cancer is a major public health concern, responsible for an estimated 9.6 million deaths in
2018 globally [1]. The World Health Organization (WHO) reported that about 1 in 6 deaths
globally are due to cancer. Therefore, development of new heterocyclic compounds with
potent anti-cancer properties continues to be the major area of research [2].
Chemotherapeutic agents targeting DNA, such as groove binders, DNA alkylating agents, or
intercalators are found to be the most effective when used in combination with drugs that
have different mechanisms of action [3]. DNA-intercalators consisting of polycyclic planar
moieties could insert between adjacent base pairs of a DNA molecule without disturbing the
overall stacking pattern [4]. However, the selectivity could be improved by introduction of
specific substituents on the intercalative aromatic core.
Indole and pyrrole fused polycyclics [5] represent some of the most versatile and
privileged scaffolds [6] occupying important position in medicinal and synthetic
organic chemistry owing to their unique structural features and promising
pharmacological properties [7]. Particularly, indole and pyrrole fused seven-member
diazepine class of compounds are known to possess interesting biological properties
[8]. For example, the diazepino-indole derivatives are reported to exhibit melanin
concentrating hormone receptor 1 activity [9]. Lixivaptan [10], a pyrrolo-diazepine is
in clinical development for cardiovascular diseases (Phase III). Structurally related
polycyclic fused indoles are known to possess antitumor, estrogen receptor
modulation, tubulin polymerization inhibitory and kinase inhibitory activities [11]. On
the other hand, 1,2,3-triazoles particularly condensed with heterocycles are known to
exhibit a broad range of biological activities including anticancer, anti-HIV, anti-
inflammatory
and
antibacterial
activities
[12].
Benzo[e]pyrrolo[1,2-
a][1,2,3]triazolo[5,1-c][1,4]diaze-pin-8(4H)-one is known to exhibit protease

inhibitory activity, (+)-JQ-1 [13] and I-BET762 [14] are potent inhibitors of
bromodomain and extra-C terminal domain (BET) proteins. Benzo-pyrido-triazolo-
diazepines [15] are used as effective anti-cancer compounds. Pyrrolo[2,1-
c][1,4]benzodiazepines (PBDs) exert their cytotoxic and antitumor activity through
covalent binding to DNA [16,17] (figure 1). To broaden the scope of this class of
compounds, we designed a series of new1,2,3-triazole-fused indolo- and
pyrrolo[1,4]diazepines, where the triazole ring is fused to diazepine moiety. The
presence of a larger aromatic system can lead to a higher affinity for the DNA
molecule and consequently to a greater growth inhibitory potency. Additionally, 1,2,3-
triazole frameworks possess striking connections in biological systems because of
their remarkable metabolic stability and ability to form hydrogen bonding to
biomolecular targets [18]. Doxorubicin and Dactinomycin drugs are under clinical use
for the treatment of different types of cancers which act through DNA intercalation
[18c].
<Insert Figure 1 here>
A perusal of literature indicated limited number of reports on synthesis and
biological profile of indole and pyrrole fused diazepines [19]. In 2008, Fujii and Ohno
described the synthesis of indole 1,2-fused benzo-1,4-diazepines via copper-catalyzed
domino three component cyclization from N-mesyl-2-ethynylanilines [20]. Hajela et
al. reported a tandem C-2 functionalization followed by intramolecular azide-alkyne
1,3-dipolar cycloaddition reaction to access pyrrolotriazolodiazepines [19b]. In 2015,
Balci and co-workers pioneered the use of gold-catalyzed and NaH-supported
intramolecular cyclization of N-propargyl indoles with pyrazole and pyrrole attached
to indole at C2 position to generate pyrazolodiazepinoindole, pyrazolopyrazinoindole
and pyrrolopyrazinoindole (scheme 1a) [21]. Kurth et al. developed an Indium (III)-

catalyzed one-pot MCR for the synthesis of imidazotriazolobenzodiazepines (Scheme
1b) [22]. Sun and co-workers demonstrated a palladium-catalyzed synthesis of 1,2-
fused-indole diazepines via [5+2] annulation of O-indolo anilines with alkynes [19a].
<Insert Scheme 1 here>
Among the various approaches, copper and Ru-catalyzed intramolecular azide-
alkyne cascade reactions have proven to be versatile method for the construction of
diverse 1,2,3-triazole fused N-heterocycles. However, copper(I) and Ru possess
undesirable side effects even in low concentrations [23]. Thus, the there exists a need
for developing metal free and easily accessible methods. But the metal free reaction
conditions normally require high temperatures (>100 ̊C) or use some other methods
like microwave or photo catalysis [24]. Hence, a convenient and novel strategy for an
efficient construction of these frameworks is greatly desirable. As part of our
continued interest toward the design and synthesis of fused polycyclic indole and
pyrrole derivatives [25], we aimed at direct synthesis of polycyclic heteroaromatic
compounds 3 via reaction of 1-(prop-2-yn-1-yl)-1H-indole-2-carbaldehyde with
different substituted phenacylazides under basic conditions in a one pot operation
(Scheme 1c). To the best of our knowledge, 1,2,3-Triazole-fused indolo and pyrrlo(1,4
diazepine) have not been reported so far. We report herein the details of the synthesis,
biological studies, DNA binding properties, and molecular modelling of the new
compounds.
Results and Discussion
Chemistry
N-propargyl indole and pyrrole derivatives are useful precursors for intramolecular
cyclizations. The initial reaction was conducted with 1-(prop-2-yn-1-yl)-1H-indole-2-

carbaldehyde 1a (0.5 mmol) and 2-azido-1-phenylethan-1-one 2a (0.6 mmol) in
presence of Cs2CO3 (1.0 mmol) in ethanol at room temperature for 24 hours. The
desired product (4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(phenyl)
methanone 3a was obtained in low, but isolable, 15% yield (Table 1, entry 1). When
the same reaction was repeated using KOtBu as base, the anticipated, 1,2,3-triazole-
fused indolo-[1,4]diazepine product 3a did not form even after 48 hours (Table 1,
entry 2). For further improvement, we turned our attention to investigate different
inorganic and organic bases. No significant improvement in the formation of the
product was observed when K2CO3, KOH and NaOH were used (Table 1, entries
3−6). 3a was obtained in 62% yield when the reaction was carried out in ethanol as
solvent and TEA as a base (Table 1, entry 7). When the solvent was changed to
methanol, the product was obtained in comparable yields (65%; Table 1, entry 8).
However, when DMF was used as solvent 3a was obtained in low yield (38%; Table
1, entry 9). When the base was changed to DBU and methanol as solvent, the yield
further decreased. (Table 1, entry 10). When piperidine was used as base (1.0 mmol),
the reaction completed in 28 hours and 3a was obtained in nearly quantitative yield
79% (Table 1, entry 11). Increase of the reaction time did not improve the yield (Table
1, entry 12). When the concentration of piperidine is reduced to 0.5 mmol, the yield
was found to be the best 81% (Table 1, entry 13). Further reduction in the
concentration of piperidine (0.4 mmol) resulted in loss of yield (Table 1, entry 14).
When the reaction was conducted at higher temperatures (70 °C) the yield was found
to be less (42%, entry 15, Table 1). Furthermore, when the reaction was conducted in
water (PEG 2000), only trace amount of the desired product was detected. The above
studies led to the following optimized protocol: 2a (0.6 mmol) was added to a solution
of 1a (0.5 mmol) in methanol (5.0 ml) as solvent in presence of piperidine (0.5 mmol)

as base at room temperature. The reaction was stirred for 28 h yielding the desired
1,2,3-triazole-fused indolo-[1,4]diazepine 3a in 81% isolable yield.
<Insert Table 1 here>
Subsequent to the optimisation of the reaction conditions, we examined the
substrate scope of this metal-free synthetic protocol by employing various N-propargyl
aldehydes 1 and various substituted phenacylazides 2. The results obtained from this
study (Table 2) suggested the generality of this synthetic method. It was observed that
both electron withdrawing and electron-donating substituents on phenacylazide were
well tolerated and afforded the fused triazolo-indolo-diazepines. While, the substituted
phenacylazide 2 containing electron-donating and neutral groups (R1, e.g. methoxy,
methyl, amine, hydroxy and acetamide) proceeded smoothly to provide the desired
products in good to excellent yields 67-91% (3a, 3c, 3d, 3f and 3h), the electron-
withdrawing substituents such as halide functionalities (e.g. F, Cl) exhibited lower
efficiency (3e, 3k, 69 and 71% yields). Much to our satisfaction, phenacylazide with
4-Br substitution yielded the triazoloindolodiazepine 3b in excellent yield. Further,
substrates with steric hindrance (R1 = 3-acetamido and 4-phenyl) were well tolerated
and afforded the products 3f and 3l in 81% and 91% yields respectively.
Phenacylazide with NO2 group at para position gave moderate yield and 3g was
obtained in 60% yield. Reaction of 1-(prop-2-yn-1-yl)-1H-indole-2-carbaldehyde 1
with 4-(2-azidoacetyl) benzonitrile 2 failed to give the corresponding 1,2,3-triazole-
fused indolodiazepine 3x under the same reaction conditions. However, when 3c (R1=
OMe,) was compared with 3g (R1= NO2), a strong electron donating effect of the
methoxy groups is observed. Consequently, the yield was reduced from 88% to 60%.
Furthermore, the C3 substituted indole (R= methyl) gave the corresponding products

(3i and 3j) but the yield was lower than equivalent entries using N-propargyl indole-2-
carboxaldehyde.
<Insert Table 2 here>
Further exploration of the scope of the reaction was carried out by replacing indole with
pyrrole. The pyrrole fused diazepine tricyclics were obtained in moderate to high yields (58-
82%) (Table 2, 3m-w). Various substituted phenacylazides with both electron-donating
groups (such as CH3, OMe, acetamido) and electron-withdrawing groups (such as Cl, Br, F,
NO2) were found to be viable substrates for this reaction, while 3-NO2 substituted product 3t
was afforded in moderate yield (58%). Increase in the reaction time did not improve the yield
even after 35 h. Electron-rich and neutral substrates proceeded in good yields (62-82%). On
the other hand, a slight decrease in the yield was observed when methyl substituents were
present at the para position of phenacylazide (3v, 60%). Furthermore, sterically hindered
substrates such as (R1= 3-acetamido, 4-phenyl) phenacylazide were also found to be suitable
for this transformation 3n and 3u (82% and 80% respectively). It should be pointed out that
azide-alkyne cycloaddition proceeded without any metal-catalyst at room temperature.
Further, when piperidine catalyzed cyclization reaction was explored by replacing indole with
imidazole, the reaction did not progress (3y).
To demonstrate the synthetic utility of the developed method, a gram-scale reaction
was carried out under optimal conditions and the desired product 3l (2.1 g) was obtained in
78% yield. (Scheme S3, Supporting Information). The structures of the synthesized
1
compounds were confirmed by H NMR, 13C NMR and HRMS (ESI) spectroscopic
techniques and the structure of the compound 3k was confirmed by single crystal X-ray
crystallographic analysis (figure 2). The model compound 3a could be easily confirmed by
1H NMR spectrum, the CH2 proton appeared at 5.4 ppm as a singlet and all other aromatic
13
proton found in the range of 6.9–7.9 ppm. The analysis of C NMR spectrum of compound

3a showed the one carbonyl signals at 189.0 ppm. The aromatic carbons of 3a appeared in the
range of 138.5–109.3 ppm and aliphatic carbon signal at 37.2 ppm. The 1H and 13C spectra of
other derivatives 3b-w were found to be almost in similar pattern like 3a for their respective
aliphatic and aromatic peaks. The HRMS (ESI) of all the compounds (3a-w) showed the
characteristic [ M + H]+ molecular ion peaks to their respective molecular formula.
<Insert Figure 2 here>
The plausible reaction mechanism for the piperidinecatalyzed tandem
Knoevenagel condensation and azide–alkyne 1,3-dipolar cycloaddition reaction is
shown in Scheme 2 [27].
<Insert Scheme 2 here>
Biological activity
In vitro cytotoxic activity
The synthesized compounds were submitted to National Cancer Institute (NCI), USA for
evaluation of their in vitro anticancer activity. The compounds were subjected to preliminary
screening at a single dose (single dose study; 10 µM) against the NCI 60 cell line panel
comprising nine human cancer cell types, including, leukemia, non-small cell lung, colon,
CNS, melanoma, ovarian, renal, prostate and breast cancer. The compounds were added at a
concentration of 10 µM and the cells were incubated for 48 h. End point determinations were
made with a protein binding dye, Sulforhodamine B [26]. A perusal of the screening results
indicated that the compounds 3b, 3f and 3u are less active against most of the cancer cell
lines. Compounds 3a and 3h exhibited broad spectrum potent inhibitory activity against all
nine subpanels of cancer cell lines tested (Table 3). Among all, compound 3a exhibited
maximum growth inhibition (~94.02%) in MDA-MB-468 cell line compared to other cells.
We have tested the cytotoxicity of compound 3a in normal human keratinocytes (HaCaT

cells), where we observed that compound 3a showed 35.23% growth inhibition. Hence,
molecular mechanistic studies of compound 3a were performed on MDA-MB-468 cells.
<Insert Table3 here>
Colony forming assay
Antiproliferative activity of compound 3a was further confirmed by colony forming assay or
clonogenic assay. Clonogenic assay is used as an in vitro cancer cell survival assay to
evaluate the reproductive integrity of cancer cells in a dose-dependent manner. Results from
figure 3 showed that compound 3a effectively inhibited the clonogenic growth at 0.5 μM. At
2.5 μM concentration, breast cancer cell (MDA-MB-468) colony formation was completely
inhibited by compound 3a.
<Insert Figure 3 here>
Cell cycle analysis
Most of the chemotherapeutic compounds exert their growth inhibitory effect by arresting the
cell cycle at a specific checkpoint. In vitro screening (NCI 60 cell line panel study, table3)
revealed that the compound 3a showed significant activity against MDA-MB-468 cells.
Therefore, we examined the effect of the compound 3a on cell cycle by using flow
cytometry. MDA-MB-468 cells were treated with various concentrations of compound 3a for
24 h, and stained with propidium iodide, which were analysed by using flow cytometry. The
results from figure 4 indicated that the untreated cells exposed to DMSO showed 4.12% cells
in sub G1 phase, whereas compound 3a treatment resulted in dose dependent accumulation of
cells in sub G1 phase. For instance 6.82, 19.33 and 30.04% of cells in sub G1 phase after
treatment with 0.5, 1 and 2.5 μM concentrations of compound 3a. These results demonstrated
that treatment of MDA-MB-468 cells with compound 3a resulted in sub G1 phase cell cycle
arrest.
<Insert Figure 4 here>

Annexin V binding assay
The apoptosis inducing effect of compound 3a on MDA-MB-468 cancer cells was further
investigated using annexin V-Alexa flour 488/propidium iodide staining assay. MDA-MB-
468 cells were treated with 0.5, 1 and 2.5 μM concentration of compound 3a for 24 h and
stained with Annexin V-Alexa flour 488 and propidium iodide. As shown in figure 5, the
compound 3a increased the percentage of early apoptotic cells (from 0.02% (ctrl) to 2.66%
(0.5 μM), 7.08% (1.0 μM) and 66.70% (2.5 μM) respectively) which indicated that 3a
induced apoptosis of MDA-MB-468 cancer cells in a dose dependent manner.
<Insert Figure 5 here>
Mitochondrial dysfunction
Mitochondria are the principal organelles related to energy metabolism and ATP synthesis,
and by monitoring the changes in mitochondria membrane potential, mitochondrial function
can be examined. Cancer cells exhibit many adaptive responses to drugs, including the
changes in mitochondrial function. Mitochondrial dysfunction is involved in apoptotic cell
death. The loss of mitochondrial membrane potential (MMP, ΔΨm) is a hallmark of
mitochondrial dysfunction. The cationic dye JC-1 was used as the MMP-sensitive probe in
which normal polarized mitochondria stains red due to formation of J-aggregates and green
colour was observed for depolarized mitochondria of apoptotic cells due to formation of J-
monomers. As shown in figure 6, MDA-MB-468 cancer cells displayed a distinct red to
green colour shift in the presence of different concentrations of 3a, indicating the loss of
MMP compared with the untreated group. The representative JC-1 green signals recorded by
flow cytometry are displayed in figure 6. These results indicated that mitochondria-mediated
pathways participated in the apoptosis of MDA-MB-468 cancer cells caused by compound
3a.

<Insert Figure 6 here>
DNA-nanodrop method
DNA-nanodrop experiment indicates the interaction pattern of newly synthesized derivatives
with DNA. The quantity and quality of small amount of DNA obtained were determined by
Nanodrop UV–vis spectrophotometers. The measurement of changes in absorbance of DNA
samples provides the idea of binding of the compounds to DNA. Sharp rise in values is
observed when a compound exhibits intercalation into DNA owing to the enhancement in the
axial length of DNA helix to adapt the hybrid. Ethidium Bromide (EtBr) is a classical DNA
intercalator which corresponds to the increase in absorbance. Groove binding indicates little
or no change in terms of absorbance. Compound 3a with DNA resulted in increase in
absorbance in comparison to the control DNA, thereby accounting for the DNA intercalation
as shown in (figure 7A and 7B). The graph is plotted between wavelength and absorbance on
X and Y axis respectively with doxorubicin, EtBr, compound 3a and control DNA.
Relative viscosity study
Additionally, we performed relative viscosity studies to determine the DNA intercalation,
where we observed that compound 3a showed more viscosity compared to EtBr and DOX.
But the minor groove binder Hoechst-33258 showed more viscosity in comparison with
compound 3a. compounds 3a showed more viscosity compared to the control DNA in
concentration-dependent manner, thereby accounting for the DNA intercalation as shown in
(figure 7C and 7D). The graph is plotted between concentration (compound/CTDNA) and
viscosity (ŋ/ŋo)1/3 on X and Y axis respectively with EtBr, doxorubicin, and Hoechst 33258 as
standards. The viscosity results are well-suited with DNA-nanodrop method and molecular
docking studies.
<Insert Figure 7 here>

Molecular docking
The biophysical studies have already shown that these compounds possess DNA intercalation
properties. Therefore, docking studies were performed to obtain a better insight into the
binding mode of the active compounds 3a to the DNA ternary complex. The coordinates of
the protein were obtained from the Protein Data Bank (PDB ID: 1NAB) [28a]. Molecular
docking simulations were carried out by using Schrödinger Suite 2017-1 with default settings
into DNA duplex and the results were analyzed on the basis of the GLIDE docking score and
molecular recognition interactions. All the 3D figures were obtained using Schrödinger Suite
2017-1 [28b].
The docking pose of 3a shows that the 1,2,3-triazole fused diazepine ring lies in the central
part of the DNA intercalation cavity and is stacked between the dC5, dG6 of chain A and
dC7, dG8 and dA9 of chain B, while benzoyl substitution of 3a interacting with dG-B8
nucleotides present in the groove (figure 8). The docked compound 3a was stabilized
electronically by π–π stacking interactions of the DNA base pairs. The framework has also
been stabilized by hydrophobic interactions in the active pocket. Docking studies suggested
that 1,2,3-triazole-fused frameworkshave the potential as DNA interacting agents.
<Insert Figure 8 here>
Conclusions
In summary, a facile and efficient method has been developed for the synthesis of fused
1,2,3-triazole indolo- and pyrrolodiazepine derivatives in one pot process. The reaction
proceeds via a tandem pathway of an initial Knoevenagel condensation followed by azide–
alkyne 1,3-dipolar cycloaddition. The present protocol is compatible with a large variety of
substrates, easy to work up, and provides medicinally important polycyclic fused molecules
with good to high yields. The synthesized compounds were evaluated for their cytotoxic
potential against a panel of 60 cancer cell lines at NCI. The compounds 3a and 3h exhibited

potent activity in the preliminary screening. Colony formation assay have shown that at 2.5
μM concentration, breast cancer cell (MDA-MB-468) colony formation was completely
inhibited. Further biological studies on compound 3a, cell cycle arrest at sub G1 phase,
apoptosis, mitochondrial dysfunction, DNA-nanodrop experiment, viscosity experiment and
molecular docking studies suggested that these compounds possess DNA interacting affinity.
Encouragingly, the compound 3a also exhibited low cytotoxicity on Human keratinocytes,
HaCaT.
Experimental Section
General Information.1H and 13C NMR spectra were recorded in CDCl3 or DMSO–d6
on a 500 MHz and 125 MHz spectrometer respectively, using tetramethylsilane as the
internal standard. Spin multiplicities were described as s (singlet), d (doublet), dd
(double douplet), t (triplet), and m (multiplet). Coupling constant (J) values were
expressed in hertz (Hz). High-resolution mass spectra (HRMS) were recorded on ESI-
QTOF mass spectrometer. All the melting points were recorded on micromelting point
apparatus and are uncorrected. Thin layer chromatography (TLC) was performed on
MERCK precoated silica gel 60F-254 (0.5 mm) aluminum plates. TLC spot
visualization was achieved under UV light. Column chromatography was performed
using silica gel 100-200. All starting materials commercially available were used.
General procedures for the synthesis 1,2,3-Triazole-Fused Indolo- and
Pyrrlo[1,4] diazepine Frameworks (3a-x).
To a stirred solution of 1 (0.5 mmol) and 2 (0.6 mmol) in methanol (5 mL) was
added 0.50 mmol of piperidine and the resulting reaction mixture was stirred at room
temperature. After completion of the reaction (28-30 h), methanol was evaporated in a
vacuum and the residue extracted with EtOAc/ Water (3 × 15 mL). The combined
organic layers were dried over anhydrous Na2SO4 and concentrated at reduced

pressure. The crude reaction mixture was purified by column chromatography on silica
gel (100–200 mesh) using ethyl acetate/hexane as an eluent in increasing polarity to
yield desired 1,2,3-Triazole-Fused indolo and pyrrlo (1,4 diazepine) frameworks (3a-
w).
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(phenyl)methanone
(3a): Yield: 81% (132 mg); yellow solid; m.p. 220–221 oC; 1H NMR (500 MHz, CDCl3) δ
7.87 – 7.79 (m, 2H), 7.76 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.62 – 7.56 (m, 1H), 7.47 (t, J
= 7.8 Hz, 3H), 7.38 (m, 1H), 7.22 (s, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.91 (s, 1H), 5.43 (s,
13
2H). C NMR (125 MHz, CDCl3) δ 189.0, 138.3, 136.4, 133.6, 133.2, 132.7, 131.2,
129.9, 129.5, 128.8, 128.7, 125.2, 122.1, 121.3, 118.6, 111.1, 109.3, 37.2; HRMS (ESI):
calcd for C20H15N4O [M + H]+ 327.1246, found: 327.1248.
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(4-
bromophenyl)methanone (3b): Yield: 82% (198 mg); yellow solid; m.p. 187–189 oC
; 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 8.7 Hz, 1H), 7.70 – 7. 65(m, 3H), 7.62 (d,
J = 8.6 Hz, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.43 – 7.37 (m, 1H), 7.25 (s, 1H), 7.22 –
7.17 (m, 1H), 6.95 (s, 1H), 5.44 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 187.9, 138.5,
135.3, 133.1, 132.6, 132.1, 131.3, 130.8, 129.4, 128.7, 128.3, 125.4, 122.2, 121.4,
118.8, 111.5, 109.3, 37.2; HRMS (ESI): calcd for C20H14BrN4O [M + H]+ 405.0351,
407.0331, found: 405.0361, 407.0339.
4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(4-
methoxyphenyl)methanone (3c): Yield: 88% (187 mg); pale-yellow solid; m.p. 178–
180 oC; 1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 8.7 Hz, 2H), 7.78 (s, 1H), 7.65 (d,
J = 7.9 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.20 – 7.14(m, 2H),
6.96 (d, J = 8.8 Hz, 2H), 6.89 (s, 1H), 5.43(s, 2H), 3.89 (s, 3H). 13C NMR (125 MHz,

CDCl3) δ 187.6, 164.1, 138.2, 133.3, 132.8, 132.1, 131.2, 130.1, 129.0, 128.3, 125.0,
122.0, 121.2, 117.5, 114.1, 110.5, 109.2, 55.6, 37.2; HRMS (ESI): calcd for
C21H17N4O2 [M + H]+ 357.1352, found: 357.1357.
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(p-tolyl)methanone
o
1
(3d): Yield: 78% (132 mg); pale-yellow; m.p. 234–236 C; H NMR (500 MHz,
CDCl3) δ 7.79 – 7.76 (m, 3H), 7.68 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.43
– 7.39 (m, 1H), 7.32 – 7.28 (m, 2H), 7.23 (s, 1H), 7.21 (t, J = 7.3 Hz, 1H), 6.93 (s,
1H), 5.46 (s, 2H), 2.47 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 188.6, 144.7, 138.3,
133.7, 133.3, 132.8, 131.1, 130.0, 129.7, 129.5, 128.3, 125.1, 122.1, 121.3, 118.2,
110.8, 109.2, 37.2, 21.8; HRMS (ESI): calcd for C21H17N4O [M + H]+ 341.1402,
found: 341.1409.
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(4-
chlorophenyl)methanone (3e): Yield: 69% (148 mg); brown solid; m.p. 185–187 oC;
1H NMR (500 MHz, CDCl3) δ 7.82 – 7.72 (m, 3H), 7.66 (d, J = 8.0 Hz, 1H), 7.49 –
7.43 (m, 3H), 7.41 – 7.36 (m, 1H), 7.24 (s, 1H), 7.18 (t, J = 7.3 Hz, 1H), 6.94 (s, 1H),
5.43 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 187.7, 140.1, 138.4, 134.8, 133.1, 132.6,
131.3, 130.8, 129.4, 129.2, 128.3, 125.4, 122.2, 121.4, 118.8, 111.5, 109.3, 37.2;
HRMS (ESI): calcd for C20H14ClN4O [M + H]+ 361.0856, found: 361.0852.
N-(3-(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indole-12-
carbonyl)phenyl)acetamide (3f): Yield: 81% (184 mg); yellow solid; m.p. 221–223
oC; 1H NMR (500 MHz, CDCl3) δ 8.11 (s, 1H), 8.03 (s, 1H), 7.74 (s, 1H), 7.67 – 7.60
(m, 2H), 7.59 – 7.55 (m, 1H), 7.45 (d, J = 5.0 Hz, 1H), 7.40 – 7.32 (m, 2H), 7.27 (s,
1H), 7.21 – 7.14 (m, 1H), 6.94 (s, 1H), 5.47 (s, 2H), 2.11 (s, 3H). 13C NMR (125 MHz,
CDCl3) δ 188.7, 169.1, 138.7, 138.4, 136.8, 133.6, 132.6, 131.2, 129.6, 129.4, 128.2,

125.3, 124.6, 124.3, 122.2, 121.3, 120.4, 119.3, 111.4, 109.3, 37.2, 24.5; HRMS (ESI):
calcd for C22H17N5NaO2 [M + H]+ 406.1280, found: 406.1275.
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(4-
o
nitrophenyl)methanone (3g): Yield: 60% (132 mg); orange solid; m.p. 204–206 C;
1H NMR (500 MHz, DMSO-d6) δ 8.31 (d, J = 8.6 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H),
7.96 (s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.72 – 7.67 (m, 2H), 7.39 (t, J = 10 Hz, 1H),
7.23 (s, 1H), 7.16 (t, J = 10 Hz, 1H), 5.88 (s, 2H). 13C NMR (125 MHz, DMSO-d6) δ
187.9, 150.3, 142.3, 139.1, 135.0, 133.4, 132.1, 130.6, 128.9, 128.1, 125.4, 124.4,
122.3, 121.4, 120.8, 112.4, 111.1, 36.8; HRMS (ESI): calcd for C20H14N5O3 [M + H]+
372.1097, found: 372.1105.
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(4-
hydroxyphenyl)methanone (3h): Yield: 82% (140 mg); yellow solid; m.p. 227–229
oC; 1H NMR (500 MHz, DMSO-d6) δ 10.57 (s, 1H), 7.95 (s, 1H), 7.83 (d, J = 8.4 Hz,
1H), 7.75 – 7.69 (m, 2H), 7.66 (d, J = 7.9 Hz, 1H), 7.40 – 7.32 (m, 2H), 7.13 (t, J = 7.5
Hz, 1H), 7.08 (s, 1H), 6.90 – 6.84 (m, 2H), 5.81 (s, 2H). 13C NMR (125 MHz, DMSO-
d6) δ 187.4, 163.2, 138.5, 134.8, 133.6, 132.5, 131.9, 130.3, 128.1, 127.7, 124.7,
121.9, 121.1, 117.3, 116.1, 110.9, 110.3, 36.8; HRMS (ESI): calcd for C20H15N4O2 [M
+ H]+ 343.1195, found: 343.1192.
(4-hydroxyphenyl)(10-methyl-4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-
o
a]indol-12-yl)methanone (3i): Yield: 71% (125 mg); yellow solid; m.p. 177–179 C;
1H NMR (500 MHz, DMSO-d6) δ 10.56 (s, 1H), 7.93 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H),
7.72 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 7.9 Hz, 1H), 7.38 – 7.32 (m, 2H), 7.13 (t, J = 7.5
13
Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 5.73 (s, 2H), 2.40 (s, 3H). C NMR (125 MHz,
DMSO-d6) δ 187.5, 163.1, 137.9, 135.0, 132.5, 131.7, 130.3, 129.7, 128.3, 127.9,

125.1, 120.6, 120.3, 118.4, 116.0, 116.0, 110.6, 36.8, 9.2; HRMS (ESI): calcd for
C21H17N4O2 [M + H]+ 357.1352, found: 357.1349.
(4-bromophenyl)(10-methyl-4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-
a]indol-12-yl)methanone (3j): Yield: 68% (172 mg); yellow solid; m.p. 186–188 oC;
1H NMR (500 MHz, CDCl3) δ 7.77 (s, 1H), 7.69 – 7.59 (m, 5H), 7.46 – 7.38 (m, 2H),
7.36 (s, 1H), 7.19 (t, J = 7.3 Hz, 1H), 5.38 (s, 2H), 2.44 (s, 3H). 13C NMR (125 MHz,
CDCl3) δ 188.0, 138.0, 135.6, 133.4, 132.1, 131.1, 130.7, 129.7, 128.5, 128.5, 128.4,
125.8, 120.9, 120.8, 120.5, 117.7, 109.1, 37.3, 9.3; HRMS (ESI): calcd for
C21H16BrN4O [M + H]+ 419.0507, 421.0487, found: 419.0515, 421.0493.
(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-yl)(4-
fluorophenyl)methanone (3k): Yield: 71% (122 mg); yellow solid; m.p. 203–205 oC;
1H NMR (500 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.93 – 7.88 (m, 2H), 7.84 (d, J = 8.4
Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.39 – 7.33 (m, 3H), 7.17 – 7.12 (m,
2H), 5.86 (s, 2H). 13C NMR (125MHz, DMSO-d6) δ 187.7, 165.5 (d, JC-F = 252.8 Hz),
138.8, 134.9, 133.5, 133.5, 132.6 (d, JC-F = 15.5 Hz), 132.0, 129.5, 128.1, 125.0,
122.1, 121.3, 119.1, 116.5 (d, JC-F = 22.1 Hz), 111.3, 111.0, 36.8; HRMS (ESI): calcd
for C20H14FN4O [M + H]+ 345.1152, found: 357.1147.
[1,1'-biphenyl]-4-yl(4H-[1,2,3]triazolo[5',1':3,4][1,4]diazepino[1,7-a]indol-12-
o
1
yl)methanone (3l): Yield: 91% (218 mg); yellow solid; m.p. 247–249 C; H NMR
(500 MHz, DMSO-d6) δ 7.96 (d, J = 7.9 Hz, 1H), 7.93 – 7.89 (m, 2H), 7.87 – 7.81 (m,
3H), 7.78 – 7.74 (m, 2H), 7.68 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.54 – 7.49 (m, 2H),
7.47 – 7.42 (m, 1H), 7.40 – 7.35 (m, 1H), 7.17 – 7.12 (m, 2H), 5.88 (s, 2H). 13C NMR
(125 MHz, DMSO-d6) δ 188.6, 145.4, 139.2, 138.8, 135.6, 134.9, 133.5, 132.0, 130.3,
129.8, 129.6, 129.0, 128.1, 127.5, 125.0, 122.1, 121.2, 118.9, 111.2, 111.0, 36.8;
HRMS (ESI): calcd for C26H19N4O [M + H]+ 403.1559, found: 403.1562.

phenyl(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-yl)methanone
(3m):
Yield: 79% (132 mg); yellow solid; m.p. 185–187 oC; 1H NMR (500 MHz, CDCl3) δ 7.79 (d,
J = 7.4 Hz, 2H), 7.67 (s, 1H), 7.61 – 7.54 (m, 1H), 7.50 – 7.43 (m, 2H), 7.16 (s, 1H), 6.94 (s,
1H), 6.57 (dd, J = 23.1, 3.4 Hz, 1H), 6.30 (d, J = 13.2 Hz, 1H), 5.29 (s, 2H). 13C NMR (125
MHz, CDCl3) δ 188.0, 135.7, 132.8, 132.0, 130.9, 130.7, 128.2, 127.0, 127.0, 126.9, 120.0,
119.0, 111.7, 41.5; HRMS (ESI): calcd for C16H13N4O [M + H]+ 277.1089, found: 277.1094.
N-(3-(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepine-10-
carbonyl)phenyl)acetamide (3n): Yield: 82% (162 mg); yellow solid; m.p. 178–180
oC; 1H NMR (500 MHz, CDCl3) δ 8.25 (s, 1H), 7.93 (s, 1H), 7.64 (s, 1H), 7.59 (d, J =
8.1 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.20 (s, 1H), 6.93 (s,
1H), 6.61 (d, J = 2.7 Hz, 1H), 6.33 – 6.27 (m, 1H), 5.28 (s, 2H), 2.08 (s, 3H). 13C
NMR (125 MHz, CDCl3) δ 188.9, 169.0, 138.6, 137.2, 133.3, 130.8, 129.3, 127.0,
127.0, 124.5, 124.1, 120.4, 120.3, 119.0, 111.6, 41.5, 24.5; HRMS (ESI): calcd for
C18H16N5O2 [M + H]+ 334.1304, found: 334.1301.
(3-aminophenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
o
1
yl)methanone (3o): Yield: 82% (162 mg); pale-yellow solid; m.p. 172–174 C; H
NMR (500 MHz, CDCl3) δ 7.69 (s, 1H), 7.29 (s, 1H), 7.23 (s, 1H), 7.15 (s, 1H), 7.10
(d, J = 6.2 Hz, 1H), 6.97 – 6.87 (m, 2H), 6.61 (s, 1H), 6.34 (s, 1H), 5.27 (s, 2H). 13C
NMR (125 MHz, CDCl3) δ 194.2, 142.4, 138.2, 135.6, 135.5, 134.1, 132.1, 131.8,
131.6, 124.8, 124.6, 123.9, 123.1, 120.3, 116.0, 45.9; HRMS (ESI): calcd for
C16H14N5O [M + H]+ 292.1198, found: 334.1204.
(4-chlorophenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
o
1
yl)methanone (3p): Yield: 63% (117 mg); brown solid; m.p. 182–184 C; H NMR
(500 MHz, CDCl3) δ 7.76 – 7.65 (m, 3H), 7.47 – 7.38 (m, 2H), 7.16 (s, 1H), 6.95 (s,
1H), 6.69 – 6.58 (m, 1H), 6.35 – 6.33 (m, 1H), 5.28 (s, 2H). 13C NMR (125 MHz,

CDCl3) δ 187.8, 139.6, 135.2, 132.8, 130.9, 130.6, 129.0, 127.0, 126.9, 119.9, 119.0,
111.7, 41.5; HRMS (ESI): calcd for C16H1lClN4NaO [M + Na]+ 333.0519, found:
333.0516.
(4-methoxyphenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
o
1
yl)methanone (3q): Yield: 82% (150 mg); yellow solid; m.p. 168–170 C; H NMR
(500 MHz, CDCl3) δ 7.82 – 7.75 (m, 2H), 7.65 (s, 1H), 7.06 (s, 1H), 6.94 – 6.89 (m,
3H), 6.55 (dd, J = 3.8, 1.4 Hz, 1H), 6.29 (dd, J = 3.8, 2.7 Hz, 1H), 5.24 (s, 2H), 3.86
13
(s, 3H). C NMR (125 MHz, CDCl3) δ 187.8, 163.8, 132.9, 131.9, 130.8, 129.3,
127.5, 127.0, 126.3, 118.7, 117.9, 114.0, 111.3, 55.6, 41.5; HRMS (ESI): calcd for
C17H15N4O2 [M + H]+ 307.1195, found: 307.1210.
(4-bromophenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
o
1
yl)methanone (3r): Yield: 70% (148 mg); yellow solid; m.p. 167–169 C; H NMR
(500 MHz, CDCl3) δ 7.68 (s, 1H), 7.61 (dd, J = 19.7, 8.6 Hz, 4H), 7.16 (s, 1H), 6.94
(s, 1H), 6.62 (dd, J = 3.8, 1.3 Hz, 1H), 6.37 – 6.31 (m, 1H), 5.27 (s, 2H). 13C NMR
(125 MHz, CDCl3) δ 187.9, 135.7, 132.7, 132.0, 130.9, 130.7, 128.2, 127.0, 126.9,
119.8, 118.9, 111.7, 41.5; HRMS (ESI): calcd for C16H12BrN4O [M + H]+ 355.0194,
357.0174, found: 355.0198, 357.0181.
(4-hydroxyphenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
yl)methanone (3s): Yield: 76% (132 mg); grey solid; m.p. 271–273 oC; 1H NMR (500
MHz, DMSO-d6) δ 10.48 (s, 1H), 7.85 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.22 (m, 2H),
6.83 (d, J = 8.7 Hz, 2H), 6.68 (dd, J = 3.8, 1.5 Hz, 1H), 6.28 (dd, J = 3.7, 2.6 Hz, 1H),
13
5.57 (s, 2H). C NMR (125 MHz, DMSO-d6) δ 187.5, 162.7, 134.3, 132.3, 131.4,
128.1, 127.6, 127.4, 127.1, 118.6, 118.1, 115.9, 111.0, 40.6; HRMS (ESI): calcd for
C16H13N4O2 [M + H]+ 293.1039, found: 293.1043.

(3-nitrophenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
o
1
yl)methanone (3t): Yield: 58% (112 mg); yellow solid; m.p. 199–201 C; H NMR
(500 MHz, CDCl3) δ 8.46 – 8.37 (m, 2H), 8.06 (d, J = 6.7 Hz, 1H), 7.76 – 7.61 (m,
2H), 7.32 (s, 1H), 7.01 (s, 1H), 6.70 (s, 1H), 6.33 (s, 1H), 5.34 (s, 2H). 13C NMR (125
MHz, CDCl3) δ 186.5, 148.1, 138.7, 134.5, 132.6, 131.2, 130.0, 127.8, 127.1, 126.0,
123.7, 120.9, 120.0, 112.1, 41.6; HRMS (ESI): calcd for C16H12N5O3 [M + H]+
322.0940, found: 322.0949.
[1,1'-biphenyl]-4-yl(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-
o
1
yl)methanone (3u): Yield: 80% (168 mg); pale-yellow solid; m.p. 192–194 C; H
NMR (500 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.85 – 7.81 (m, 2H), 7.79 (d, J = 8.5 Hz,
2H), 7.77 – 7.72 (m, 2H), 7.53 – (m, 2H), 7.43 (t, J = 7.3 Hz, 1H), 7.40 (s, 1H), 7.30 –
7.27 (m, 1H), 6.76 (dd, J = 3.8, 1.5 Hz, 1H), 6.32 (dd, J = 3.8, 2.6 Hz, 1H), 5.64 (s,
13
2H). C NMR (125 MHz, DMSO-d6) δ 188.6, 144.9, 139.3, 136.1, 134.4, 131.6,
130.1, 129.6, 128.9, 128.3, 127.5, 127.3, 127.2, 126.9, 120.1, 119.1, 111.3, 40.6;
HRMS (ESI): calcd for C22H17N4O [M + H]+ 353.1402, found: 343.1398.
(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-yl)(p-tolyl)methanone
o
1
(3v): Yield: 60% (105 mg); brown solid; m.p.181–183 C; H NMR (500 MHz,
CDCl3) δ 7.65 – 7.58 (m, 3H), 7.18 (m, 2H), 7.05 (s, 1H), 6.87 – 6.82 (m, 1H), 6.51
(dd, J = 3.8, 1.4 Hz, 1H), 6.25 (dd, J = 3.8, 2.7 Hz, 1H), 5.19 (s, 2H), 2.35 (s, 3H). 13C
NMR (125 MHz, CDCl3) δ 189.0, 144.3, 134.2, 133.0, 130.9, 129.7, 129.5, 127.7,
127.2, 126.6, 119.5, 118.3, 111.6, 41.6, 21.9; HRMS (ESI): calcd for C17H15N4O [M +
H]+ 291.1246, found: 291.1229.
(4-fluorophenyl)(4H-pyrrolo[1,2-d][1,2,3]triazolo[1,5-a][1,4]diazepin-10-yl)methanone
(3w): Yield: 66% (116 mg); yellow solid; m.p. 165–167 oC; 1H NMR (500 MHz, DMSO-d6)
δ 7.85 (s, 1H), 7.83 – 7.79 (m, 2H), 7.37 (s, 1H), 7.33 – 7.29 (m, 2H), 7.29 – 7.26 (m, 1H),

13
6.75 (dd, J = 3.8, 1.5 Hz, 1H), 6.31 (dd, J = 3.7, 2.6 Hz, 1H), 5.61 (s, 2H). C NMR (125
MHz, DMSO-d6) δ 187.7, 165.2 (d, JC-F = 251.9 Hz), 134.4, 134.0 (d, JC-F = 2.8 Hz), 132.3
(d, JC-F = 9.4 Hz), 131.5, 128.4, 127.2, 126.6, 120.3, 119.2, 116.3, (d, JC-F = 22.1 Hz), 111.3,
40.6; HRMS (ESI): calcd for C16H12FN4O [M + H]+ 295.0995, found: 295.0989.
Biology
In vitro cytotoxic (NCI-screening)
The synthesized compounds were evaluated to National Cancer Institute (NCI), USA for
evaluation of their in vitro anticancer activity. The compounds were subjected to preliminary
screening at a single dose (single dose study; 10 µM) for their growth inhibition effects
against the NCI 60 cell line panel comprising nine human cancer cell types, including,
leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast
cancer. The compounds were added at a concentration of 10 µM and the cells were incubated
for 48 h. Compounds were submitted to Developmental Therapeutics Program (DTP) at
National Cancer Institute (NCI) for anti-cancer on single dose basis.
Determination of growth inhibition on normal cell (Human keratinocytes, HaCaT)
Cells (5000 cells per well) were seeded in 96-well plate and allow to grow for overnight.
After attaching the cells and before going to treatment (day 0), the absorbance was taken by
adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (0.5 mg/ml)
reagent to a few wells. After 48 h of incubation, MTT was added, and the cells were
incubated for 4 h at 37 °C in CO2 incubator. Then the cells were washed with and PBS and
formazan crystals were dissolved in DMSO and the absorbance was taken at 570 nm using
multimode plate reader (Spectra Max, M4 Molecular devices, USA). The day 0 absorbance
was subtracted from the 48 h reading to determine the percentage growth inhibition.

Colony formation assay
MDA-MB-468 cancer cells were seeded into 12-well culture plate at a single cell density of
250 cells/well. After cells adhered to the culture well plate, cells were treated with different
concentrations of 3a (0.5, 1 and 2.5 µM) for 24 hrs. Post incubation, media was discarded and
the cells were maintained in fresh medium, cells were washed with PBS and incubated for
o
next 12 days at 37 C. After 12 days, the so called colonies were stained with 1% crystal
violet for 3 h and washed with PBS. Then, the Plates were photographed and colonies were
counted using Vilber Fusion Fx software (Vilber Lourmat, France). The results were
represented as total colony number vs. concentration.
Cell cycle analysis
To perform cell cycle analysis assay, MDA-MB-468 cancer cells (1 X 106 cells/well) in 6
well plate were treated with 0.5, 1 and 2.5 μM of compound 3a for 24 hrs. cells were
o
harvested by trypsinisation, washed with PBS and fixed with ethanol (70 %) at 4 C for 30
min. After fixation, cells were again washed with PBS (400 µL) and stained with propidium
iodide staining buffer for 30 min in dark at room temperature. Then, the samples were
analyzed for propidium iodide fluorescence by flow cytometry using BD Accuri C6 flow-
cytometer.
AnnexinV/Propidium iodide staining assay
MDA-MB-468 cells were plated at a density of 5 x 103 cells in a 12-well culture plates and
allowed to grow for 24 h. Then cells were treated with 3a at concentration of 0.5, 1 and 2.5
µM. After 48 hrs, cells were collected by trypsinisation. The collected cells were rinsed with
ice-cold PBS (two times), then incubated with 200 µL 1X binding buffer containing 5 µL
Annexin V-FITC, and then in 300 µL 1X binding buffer containing 5 µL Propidium iodide

(PI) in the dark for 5 min at room temperature and incubate for 15 min. After incubation,
cells were analysed for percentage of apoptosis using BD-C6 accuri flow-cytometer.
Assay of mitochondrial membrane potential (Δψm)
The mitochondrial- specific cationic dye JC-1 (Invitrogen, USA), which undergoes potential-
dependent accumulation mitochondria, was used to detect mitochondrial membrane potential.
Briefly, MDA-MB-468 cells were plated at density of 2×105 cells/well in a 12-well plate.
Then cells were treated with 3a at concentration of 0.5, 1 and 2.5 µM for 48 hrs. After 48 hrs
cells were incubated with 2 μM JC-1 in the dark for 30 min at room temperature. After
incubation, cells were observed by flow cytometry for the quantitative analysis of Δψm. At
least 10,000 events per sample were recorded.
DNA-nanodrop method
DNA intercalation is determined by nanodrop spectrophotometric analysis. The intercalating
agents decrease the absorption and increase the wavelengths [29]. In our experiment, we
incubated the 50 μM of calf thymus DNA (Sigma-Aldrich, USA) with compound 3a,
Ethidium bromide (EtBr) and Doxorubicin (DOX) for 10 min at 1 μM concentration. Later,
the TAE buffer used as blank and aborbance and concentrations were determined by
NanoDrop™ 2000/2000c Spectrophotometer (Thermo fisher scientific, USA).
DNA binding affinity studies
Relative studies were determined by Ostwald viscometer. The titrations were conducted for
3a, DOX, EtBr and Hoechst-33258 at 1.0 μM while and they are added to CT-DNA solution
(50 μM) and exposed to viscometer. Here, the DNA solution was prepared in 100 mM Tris-
HCl (pH 7.0). Data were represented as (ŋ/ŋo)1/3 versus the ratio of the concentration of the

compound 3a, DOX, EtBr and Hoechst-33258 to CT-DNA, where ŋ is the viscosity of CT-
DNA in the presence of intercalating agents and ŋo is the viscosity of CT-DNA alone.
Molecular modelling
Protein preparation and Grid generation
The 3D coordinates of the disaccharide anthracycline with DNA hexamer d(CGATCG)
was obtained from Protein Data Bank (PDB ID code: PDB 1NAB). The PDB protein-ligand
structures were processed with the Protein Preparation Wizard in the Schrödinger suite. The
protein structure integrity was checked and adjusted, and missing residues and loop segments
near the active site were added using Prime. A 3D box was generated around each ligand to
enclose the entire vicinity of active site. The receptor grid was prepared with the help of
OPLS_2005 force field. The grid center was set to be the centroid of the co-crystallized
ligand, and the cubic grid had a size of 20 Å.
Ligand preparation
3D structures were generated by Schrödinger suite. Schrödinger’s LigPrep program was
used to generate different conformations of ligands.
Molecular docking
Molecular docking studies were performed by using a GLIDE docking module of
Schrödinger suite 2017-1. For the validation of docking protocol, the cocrystalized ligand
(disaccharide anthracycline) was subjected to re-docking into DNA (PDB code: 1NAB) using
GLIDE, then the prepared ligands were docked into the generated receptor grids using Glide
SP docking precision. The results were analyzed on the basis of the GLIDE docking score
and molecular recognition interactions. The 3D images were generated using Schrödinger
Suite 2017-1[28].

Statistical analysis
Biological assays results were expressed as mean ± SD, and n refers to the number of sample
repeats. The statistical differences between the means were determined by one-way ANOVA
then Tukey’s multiple comparison tests with Prism software (version 6.01; GraphPad, USA).
p< 0.05 was considered to be statistically significant.
Acknowledgments
We are grateful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New
Delhi, for the award of NIPER fellowship. We are thankful to Dr. Mohammed Nayel,
Developmental Therapeutics Program (DTP), National Cancer Institute (NCI),
Chemotherapeutic Agents Repository, Fisher BioServices, Rockville, MD, USA, and
the team for in-vitro anticancer screening of the compounds. We thank N. Jagadeesh
Babu, Centre for X-ray Crystallography, IICT, Hyderabad for carrying out X-ray
Crystallography studies.
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