Novel diindoloazepinone derivatives as DNA minor groove binding agents with selective topoisomerase I inhibition: Design, synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1016/j.bioorg.2020.103629

We present here-in the molecular design and chemical synthesis of a novel series of diindoloazepinone derivatives as DNA minor groove binding agents with selective topoisomerase I inhibition. The in vitro cytotoxicity of the synthesized compounds was evaluated against four human cancer cell lines including DU143, HEPG2, RKO and A549 in addition to non-cancerous immortalized human embryonic kidney cells (HEK-293). Compound 11 showed significant cytotoxicity against all the four human cancer cell lines with IC₅₀ values ranging from 4.2 to 6.59 μM. 11 was also found to display 13-fold selective cytotoxicity towards A549 cancerous cells compared to the non-cancerous cell lines (HEK-293). The decatenation, DNA relaxation and intercalation assays revealed that the investigational compounds 10 and 11 act as highly selective inhibitors of Topo-I with DNA minor groove binding ability which was also supported by the results obtained from circular dichroism (CD), UV-visible spectroscopy and viscosity studies. Apoptosis induced by the lead 11 was observed using morphological observations, AO/EB and DAPI staining procedures. Further, dose-dependent increase in the depolarization of mitochondrial membrane was also observed through JC-1 staining. Annexin V-FITC/PI assay confirmed that 11 induced early apoptosis. Additionally, cell cycle analysis indicated that the cells were arrested at sub-G1 phase. Gratifyingly, in silico studies demonstrated promising interactions of 11 with the DNA and Topo I, thus supporting their potential DNA minor groove binding property with relatively selective Topo I inhibition compared to Topo II.

Full text of DOI:10.1016/j.bioorg.2020.103629

BioorganicChemistry99(2020)103629
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel diindoloazepinone derivatives as DNA minor groove binding agents
with selective topoisomerase I inhibition: Design, synthesis, biological
evaluation and docking studies
Manasa Kadagathura, G. Parimala Devib, Preeti Grewalc, Dilep Kumar Sigalapallia,
⁎
⁎
Priyanka N. Makhala, Uttam Chand Banerjeec, , Nagendra Babu Bathinib,
,
⁎
Neelima D. Tangellamudia,
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, India
b Department of Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
c Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Punjab 160062, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
We present here-in the molecular design and chemical synthesis of a novel series of diindoloazepinone deri-
vatives as DNA minor groove binding agents with selective topoisomerase I inhibition. The in vitro cytotoxicity of
the synthesized compounds was evaluated against four human cancer cell lines including DU143, HEPG2, RKO
and A549 in addition to non-cancerous immortalized human embryonic kidney cells (HEK-293). Compound 11
showed significant cytotoxicity against all the four human cancer cell lines with IC50 values ranging from 4.2 to
6.59 μM. 11 was also found to display 13-fold selective cytotoxicity towards A549 cancerous cells compared to
the non-cancerous cell lines (HEK-293). The decatenation, DNA relaxation and intercalation assays revealed that
the investigational compounds 10 and 11 act as highly selective inhibitors of Topo-I with DNA minor groove
binding ability which was also supported by the results obtained from circular dichroism (CD), UV-visible
spectroscopy and viscosity studies. Apoptosis induced by the lead 11 was observed using morphological ob-
servations, AO/EB and DAPI staining procedures. Further, dose-dependent increase in the depolarization of
mitochondrial membrane was also observed through JC-1 staining. Annexin V-FITC/PI assay confirmed that 11
induced early apoptosis. Additionally, cell cycle analysis indicated that the cells were arrested at sub-G1 phase.
Gratifyingly, in silico studies demonstrated promising interactions of 11 with the DNA and Topo I, thus sup-
porting their potential DNA minor groove binding property with relatively selective Topo I inhibition compared
to Topo II.
Indoloazepinone
Diindoloazepinone
Anticancer activity
DNA minor groove binding
Selective topoisomerase I inhibitor
Molecular Docking
1. Introduction
replication and transcription progression [2]. This fact makes them
important targets for several chemotherapeutic agents. Topoisomerase I
One of the primary objectives of a medicinal chemist is the dis-
covery of new, efficient and safer chemotherapeutic agents. In the re-
cent past, interest has been particularly drawn to that group of agents
that target the human DNA [1]. The major step in DNA replication is
DNA relaxation (releasing entanglement), which is performed by DNA
topoisomerases I and II [2–4]. Topoisomerase II (Topo II) introduces
splits into the double-strand DNA segments and transports another DNA
helix through the cut segments, thus, untangling the DNA duplexes.
Topoisomerase I (Topo I) is another nuclear enzyme that mediates the
relaxation of both positively and negatively supercoiled DNA via re-
versible DNA single-strand cleavage and religation which is essential for
inhibitors prevent the religation step by stabilizing the transient to-
poisomerase I–DNA covalent complex.
The cellular processes in cancer cells are more susceptible to DNA-
interactive drugs than their normal counterparts. Thus, DNA has been
considered as potential target for the development of anticancer mod-
alities [5]. The minor groove of double helical B-DNA is a site of non-
covalent interactions that are highly specific to AT-rich sequence, thus
making the binding of nonintercalators (termed as minor-groove bin-
ders, MGBs) to minor groove a novel chemotherapeutic approach.
MGBs contain several fused aromatic rings arranged in a crescent shape
rendering very similar dimensions as that of base pairs thereby
⁎ Corresponding authors.
E-mail address: neelima@niperhyd.ac.in (N.D. Tangellamudi).
https://doi.org/10.1016/j.bioorg.2020.103629
Received 1 November 2019; Received in revised form 18 January 2020; Accepted 26 January 2020
Availableonline28January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Fig. 1. Anticancer agents containing indole and heteroaromatic-fused azepinone moiety.
complementing the shape of the groove [6]. Moreover, MGBs also
showcase hydrophobic interactions in addition to the common ionic
contacts [7].
effects that the ring fusion concepts offer, we were prompted to fuse
indole and azepinone (aldisine) in a single heteroaromatic framework
to afford conformationally constrained diheterocyclic azepinone ana-
logues with potential antiproliferative effect. With this rationale, the
designed analogues were synthesized and evaluated for their in vitro
cytotoxicity, DNA binding and topoisomerase inhibition studies to ob-
tain highly promising results.
N-containing heterocyclic rings generally offer a polarized character
which helps in increasing the stability of the molecule-receptor complex
by establishing optimum interaction with the receptor and thus, pro-
duce anticancer effects [8]. In particular, the structural diversity of
biologically active indoles [9] (A-G, Fig. 1) and seven-membered rings
makes them important structural components in many pharmaceutical
agents [8,10–15]
2. Results and discussion
Azepines and azepinones have recently gained a considerable
amount of attention and interest because of their continuing pharma-
cological significance. Many natural products and reported scaffolds
with anticancer property were found to normally contain tetra-
hydroazepinone pharmacophore fused with a heterocycle, such as in-
dole, pyrrole or benzofuran, as their main component (HeP, Fig. 1)
[16–26].
2.1. Chemistry
A library of diindoloazepinone analogues comprising 25 compounds
were synthesized. The synthesis of the desired intermediate, in-
doloazepinone 5, was achieved by the method outlined in Scheme 1, as
per the reported method [29]. EDC.HCl along with DMAP was used for
the coupling of indole-2-carboxylic acid 1 with β-alanine ethyl ester 2
to give the intermediate 3 in high yield of 90%. Basic hydrolysis yielded
the cyclisation precursor 4. Further, cyclodehydration was carried out
in the presence of phosphorous pentoxide in methanesulfonic acid to
give indoloazepinone 5 in 60% yield. N-alkylated indoloazepinones 7a-
b were prepared according to a reported protocol [26] by treating 5
with the respective benzylbromides 6a-b in the presence of potassium
carbonate as base, which was confirmed by the disappearance of indolic
NeH peak in 1H NMR at 12.47 ppm with amidic NeH seen at 8.90 ppm,
whereas, N,N-dialkylated indoloazepinones 8a-b were obtained from
the reaction between indoloazepinone 5 and benzylbromides 6a-b by
the employment of a stronger base – sodium hydride. The N,N-dialky-
lation of indoloazepinone could be confirmed by the disappearance of
Tailor-made design of the molecules by fusion of two or more het-
erocyclic rings in a single molecular frame renders better pharmaco-
kinetic properties and improved molecular interactions between the
drug and its cellular target. [8,27]. Syntheses of such hybrid molecules
by amalgamation of two molecular entities are fascinating to work upon
for developing modified scaffolds with unusual and greatly improved
properties in the area of medical science [28]. In addition, the in-
troduction of backbone rigidity and/or the conformational control of
molecular scaffolds through cyclization [24] have been proven to have
a beneficial effect on pharmacological efficiency. In view of the po-
tential antiproliferative effect of indole and indole-fused azepinones,
and recognizing the conformation control and enhanced synergistic
2

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Scheme 1. Reagents and conditions: (a) EDCI.HCl (2 equiv), DMAP (2.2 equiv), DMF, rt, N2, 20 h; (b) NaOH (3 equiv), EtOH, rt, 30 min; (c) P2O5 (1.2 equiv),
MeSO3H (1 equiv), 85–110 °C, 2 h; (d) K2CO3 (3 equiv), DMF, 0 °C to reflux, 8 h; (e) NaH (2 equiv), DMF, 0 °C to rt, 12 h.
both indolic (12.47 ppm) and amidic NeH peaks (8.90 ppm) from 1H
NMR (experimental section).
noncancerous cells, the cytotoxicity of 10, 11 and 13 was also eval-
uated on HEK-293 non-cancerous immortalized human embryonic
kidney cells. We found that the tested compounds displayed 13-fold
selectivity to A549 cancerous cell lines over HEK-293 non-cancerous
cell lines.
Scheme 2 shows the preparation of diindoloazepinone carried out
by Fischer indole synthesis [30]. Employing this well-known method,
the analogues (10–34) were prepared by reacting the substituted and
unsubstituted indoloazepinones with appropriate phenylhydrazines 9
in ethanol: glacial acetic acid (1:1). Cyclization and aromatization of
the intermediate hydrazones with loss of ammonia, upon addition of
concentrated HCl, provided the desired diindoloazepinone products.
Compound 10 containing unsubstituted indolic and amidic nitro-
gens, showed an IC50 of 8.9 μM on HEPG2 cell line and ≤15 μM on
other tested cell lines. Methyl substitution at the C4 position on the
newly formed indole ring, yielded a more potent analogue 11 with a 4-
fold and 2-fold increase in the activity as compared to the unsubstituted
10 in A549 and HEPG2 cell lines, respectively. Fluoro (12) and chloro
(13) substitutions were acceptable modifications which showed nearly
comparable activities as that of 11. Compound 14 which flaunted a
bromo substitution resulted in slightly diminished activities in all the
tested cell lines. N-alkylated analogues with electron withdrawing
groups (18–20, 24 and 26) on the newly formed indole ring gave better
activities, whereas decreased potency was witnessed as an effect of
electron donating groups as can be seen in 17, 21 and 23. The im-
portance of the free amidic NeH was emphasized by the negligible
activity of the N,N-dialkylated analogues 28–31, 33 and 34.
Nevertheless, compound 32 did preserve its cytotoxic activity with
IC50 < 20 μM in all the tested cell lines. It is noteworthy to emphasize
on the importance of fluoro-substitution on the newly formed indole
ring (at R3, as seen in 12, 18, 24 and 30), which showed good activity
in A549 cell line (ranging from 5.23 to 12.5 μM), irrespective of the
substitution on nitrogen atoms. The structure activity relationship is
summarized in Fig. 2.
2.2. Biological evaluation
2.2.1. Cytotoxic assays
The cytotoxicity of the synthesized library of compounds was
evaluated in vitro against DU143, HEPG2, RKO, A549 and HEK-293
with doxorubicin as a positive control. As a screening assay, the cyto-
toxicity was expressed in terms of IC50 values (concentration of the drug
causing 50% inhibition of the cell growth) by taking into account the
upshots of the MTT tetrazolium reduction assay. Results shown in
Table 1 indicate that the diindoloazepinone derivatives displayed good
cytotoxicity with their IC50 values in the range of 4.2–89.6 μM.
Compounds 10–13, 16, 18, 19, 24 and 26 showed good activity
with their IC50 values < 10 μM for, at least, one of the cell lines tested,
while the 11 exhibited high level of cytotoxicity against all the cell lines
screened initially which encouraged us to choose 11 for further in-
vestigation of mechanistic studies. To determine if our newly synthe-
sized compounds have differential cytotoxic effects on cancerous and
3

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Scheme 2. Reagents and conditions: (a) CH3COOH:EtOH (1:1), (b) HCl reflux, 10 h.
Based on the MTT assay results, further detailed mechanistic studies
on cell growth inhibition viz., DNA binding studies, topoisomerase in-
hibition, apoptotic studies, flow cytometric analysis and molecular
docking studies were investigated on the most active derivatives.
envisioned mechanism of action. Additionally, the effect of the active
compound 11 on the viscosity of DNA solution was also studied to as-
certain its mode of binding with DNA.
2.2.2.1. Decatenation assay. When topoisomerase acts on catenated
circular DNA - kDNA, it catalyzes the release of catenanes and
various decatenated forms such as supercoiled and relaxed forms as
major products along with the minor products such as open circular,
nicked open circular, linear forms. Inhibition of the human
topoisomerase IIα will decrease or completely inhibit the decatenated
product formation, as a result kDNA will be higher in wells. We used
etoposide, a well-known topoisomerase inhibitor, as standard in this
procedure. Upon treatment with the investigational compounds, the
presence of kDNA in the wells was not observed, indicating that the
investigational compounds do not interfere with Topo-IIα enzyme (A,
Fig. 3). Fig. 3B depicts the results obtained upon quantification of the
2.2.2. Topoisomerase inhibition and DNA binding studies
Since, the primary goal of the synthesized novel diindoloazepinone
analogues was to serve as possible DNA binding drugs, it is speculated
that the investigated compounds have some effect on Topo I and/or II
enzyme inhibition which may possibly be through DNA interaction. In
the present study, in vitro decatenation assay, relaxation assay, topoi-
somerase IIα-mediated relaxation and DNA cleavage assay, topoi-
somerase I-mediated relaxation assay, and DNA binding studies such as
DNA intercalation assay, circular dichroism and UV-visible spectro-
scopy were performed on the active compounds 10, 11 and 13 to
evaluate whether their antiproliferative effect was a consequence of the
4

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Table 1
In vitro cytotoxicity (IC50 values in µM)a data for the synthesized compounds.
Code
R1
R2
R3
DU143
HEPG2
RKO
A549
HEK-293
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Doxb
–
H
H
13.6
6.59
4.77
4.73
38.9
11.29
10.27
49.28
34.11
14.7
14.3
30.69
25.6
53.5
27.9
36.05
6.3
0.23
0.35
0.89
0.25
1.25
0.89
0.78
1.89
0.78
1.2
8.9
0.56
0.89
12.5
5.63
6.3
0.56
0.56
0.145
1.02
0.15
0.14
0.56
0.56
0.45
0.89
1.4
15.00
4.2
0.31
65.3
1.23
–
H
CH3
F
4.89
12.9
4.8
0.02
53.6
0.56
–
H
0.78
0.45
0.58
0.23
0.23
0.45
1.23
0.89
0.78
5.23
5.23
15.63
14.56
9.6
0.12
0.56
0.89
0.12
–
–
H
Cl
22.1
19.16
43.94
32.09
35.3
18.1
6.8
89.6
–
1.58
–
H
Br
18.0
16.6
19.5
34.5
21.7
4.4
–
CH3
H
CH3
H
–
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
Cl
Cl
–
0.23
–
H
CH3
F
20.5
6.2
0.23
0.125
0.56
0.56
1.56
0.145
0.12
0.56
0.89
0.23
0.89
0.56
0.12
0.89
1.23
0.45
0.56
1.25
0.056
–
H
–
H
Cl
15.2
15.6
65.3
10.2
56.3
8.5
–
H
Br
0.56
0.23
0.25
0.56
1.23
0.78
0.89
0.89
0.56
0.78
0.56
1.23
1.45
1.235
1.36
0.023
21.2
43.67
21.2
27.06
14.98
17.08
18.5
24.5
35.38
46.69
30.06
31.2
12.5
30.34
27.5
2.45
27.1
33.16
26
–
CH3
H
CH3
H
0.89
0.12
0.56
0.45
0.589
0.23
1.23
1.58
0.25
1.25
0.56
0.89
1.45
0.012
0.23
1.78
0.156
–
–
H
CH3
F
0.55
30
–
H
0.12
0.56
23.64
33
–
H
Cl
38.9
22.5
12.3
86.3
89.6
12.5
24.2
12.63
15.63
36.9
1.23
–
H
Br
0.52
32
–
CH3
H
CH3
H
21.2
33.68
36.9
67.4
25
0.78
16.3
23.9
37.8
46.5
22.2
15.6
28.9
31.5
1.37
–
0.89
–
H
CH3
F
1.89
0.45
–
H
–
H
Cl
1.02
–
CH3
H
CH3
CH3
F
11.4
31.5
25.2
1.23
0.22
–
0.25
–
H
1.58
–
–
–
0.012
9
1.1
The bold indicate compounds with IC50 values < 10 μM.
a
50% inhibitory concentration determined by taking the mean value of n = 3 independent MTT assay experiments after 48 h of compound treatment using a non-
linear regression analysis. DU143 - prostate cancer cells, HEPG2 - liver carcinoma cells, RKO - colon carcinoma cells, A549 - lung cancer cells, HEK-293 - human
embryonic kidney cells.
b
Dox - doxorubicin.
witnessed that, in the case of etoposide, supercoiled form was observed
as major product, while 10, 11 and 13 majorly showed the relaxed form
of DNA as the major product, thus indicating the investigated
compounds are not Topo IIα inhibitors (Fig. 4).
2.2.2.3. Topo IIα-mediated DNA cleavage assay. In order to corroborate
the catalytic inhibitory behaviour of our compounds, Topo II cleavage
complex assay was performed, which mainly helps in monitoring the
ability of compounds to induce the linearization of plasmid DNA in the
presence of Topo IIα. Fig. 5 shows that, etoposide, which is a known
Topo II poison, strongly induced DNA cleavage thus resulting in the
formation of band corresponding to linear form of DNA. Whereas, our
Fig. 2. SAR analysis of diindoloazepinone derivatives.
investigational compounds do not show the formation of linear band,
indicating that the tested compounds have no capacity to poison Topo
IIα. The results obtained from the decatenation, relaxation and
cleavage complex assays confirm that the compounds have no action
on Topo IIα (neither as catalytic inhibitors nor as poisons).
percent inhibition of decatenation by the investigational compounds in
comparison with etoposide. Additionally, densitometric analysis using
supercoiled (SC) and relaxed form (Rel.) as decatenation products for
relative decatenation was performed (C, Fig. 3). Relative decatenation
is the total amount of products formed with 100 ng of DNA. It was
found that the products obtained were higher than the standard drug -
etoposide, which ultimately support the output obtained from the
percentage inhibition of the decatenation assay. The results, thus,
indicate the absence of enzyme inhibition. In order to confirm this
result, we further performed the relaxation assay for human
topoisomerase IIα.
2.2.2.4. Topo I-mediated DNA relaxation assay. Topo I is a known
potential target for several clinically used anticancer drugs owing to
its involvement in the replication and proliferation process as
confirmed by its major expression in actively replicating cells as
compared to normal cells. Under normal circumstances, Topo
I
controls the changes in the DNA topology by cutting single stranded
DNA and re-joining the phosphate backbone. Topo I inhibitors act by
two mechanisms, either they bind to topoisomerase directly or they
may bind to DNA thereby altering its structure, thus, masking it from
being recognized by the enzyme. With the confirmation that the
investigational compounds do not act as Topo IIα inhibitors or
poisons, Topo I relaxation assay was performed taking camptothecin
(Topo I inhibitor) as reference standard. When these compounds were
incubated with Topo I, we found out that two of our compounds 10 and
11 showed supercoiled form along with some nicked open circular
(NOC) form in agarose gel as compared to camptothecin. This clearly
2.2.2.2. Topo IIα-mediated DNA relaxation assay. Topoisomerase II
(Topo II) makes nicks in both the strands of the DNA and modifies
the topology of the DNA resulting in the initiation of many repair
pathways by religating the phosphodiester bonds [31]. Topoisomerase
releases the entanglement of the supercoiled DNA and as a result, a
large number of relaxed form of DNA were observed on the agarose gel,
which migrated more slowly [3,32]. Results of our experiment were
compared with etoposide (Topo II inhibitor) as shown in Fig. 4. We
5

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Fig. 3. A. Agarose gel image for the decatenation assay: kDNA (100 ng) was used substrate. kDNA was treated with human topoisomerase IIα along with either
etoposide (100 μM) or compounds (100 μM) and decatenation products formed were observed and quantified. 1st lane: kDNA; 2nd lane: kDNA + h Topo IIα 3rd lane:
kDNA + h Topo IIα + etoposide (100 μM); lane 4–6: kDNA + h Topo IIα + compounds (100 μM), B. Quantification of kDNA left for percent inhibition of
decatenation by investigational compounds. C. Quantification of relative decatenation of kDNA by investigational compounds as compared to etoposide. E: etoposide,
NOC: nicked open circular DNA, Lin.: linear form, SC: supercoiled form, Rel.: relaxed form.
Fig. 4. Relaxation assay for h Topo IIα: pUC19 was used as substrate and in-
cubated with either etoposide (100 μM) or compounds (100 μM) without
ethidium bromide. Relaxed DNA as product was observed for relaxation. E:
etoposide, NOC: nicked open circular DNA, SC: supercoiled form.
Fig. 6. Relaxation assay for Topo I: Negatively supercoiled DNA pBR322 was
used as substrate which was incubated with 100 μM camptothecin or com-
pounds without ethidium bromide. Relaxed DNA as product was observed for
relaxation. C: camptothecin, NOC: nicked open circular DNA, SC: supercoiled
form.
shows that among the three compounds investigated, 10 and 11 are
good and relatively selective inhibitors of Topo I compared to Topo IIα
(Fig. 6). Compound 11, which showed high level of cytotoxicity against
all the cell lines screened, also showed high potency against Topo I.
However, compound 10, with relatively low cytotoxicity displayed
similar potency against Topo I inhibition. Thus, we cannot exclude the
possibility that other pathways might also be targets for this series of
compounds, with Topo I inhibition playing a fundamental role in
cytotoxicity. We proceeded with DNA binding studies to examine if the
investigational compounds bind at the minor groove of the DNA in
addition to their interaction with Topo I by trapping the Topo I-DNA
cleavage complex by interacting at the cleavage site, which is the
mechanism of action of most interfacial poisons, such as camptothecin
[2].
Fig. 5. Stabilization of cleavage complex assay: After treatment with h Topo
IIα, negatively supercoiled plasmid was incubated with either 100 μM etopo-
side or investigational compounds. The agarose gel was run in excess of ethi-
dium bromide to check the formation of linear band. In contrast to etoposide, a
linear band was not observed in any of the tested compounds. E: etoposide, SC:
supercoiled form.
2.2.2.5. DNA intercalation assay. Compounds with linear geometry
tend to intercalate the DNA and either distort the DNA structure or
change its basic structure, thereby, changing the degree of supercoiling
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M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
DNA bands because of the weak electrostatic interactions involved (Fig.
S1, supporting information). Looking at the Y-axis values after
incubation with different concentrations of investigational compounds
10, 11 and 13, it was found that the CD spectra for CTDNA showed
minor changes at 275 nm and 245 nm wavelength. It can be due to
minor groove binding or due to the interference in the CD spectra in
milieu of CTDNA.
2.2.2.7. UV-Visible spectroscopy. In UV-visible spectrophotometer,
CTDNA has a characteristic spectrum of the B-form DNA with a λmax
at 260 nm. With an increasing concentration of the investigated
compound, a change in the absorbance profile is observed indicating
DNA binding property (either DNA-intercalating or minor/major
groove binding) [34]. When 10, 11 and 13 were treated with CTDNA
at various concentrations,
a weak hypochromicity (decrease in
absorbance) without bathochromic shift was observed, which is
consistent with minor groove binding, indicating the compounds have
DNA groove binding capability and thus impede the incoming
wavelength causing changes in the absorption spectrum [34,35]
(Fig. 9). The minor groove binding mode of the most active
compound 11, was further confirmed by viscosity studies [35] (Fig.
S2, supporting information).
Fig. 7. Intercalation assay: Supercoiled DNA (SC DNA) pUC19 was used as
substrate and incubated with either compounds (100 μM) or ethidium bromide
(100 μM). SC: supercoiled form, EtBr: ethidium bromide, Lin.: linear form, Rel.:
relaxed form.
2.2.3. Apoptotic studies
2.2.3.1. Morphological observations. Apoptosis is typically characterized
by cell death-related biochemical and morphological changes. In this
study, to investigate whether the cell death was induced by apoptosis,
A549 cells were treated with various concentrations (0, 1, 2.5, 5 and
10 µM) of our most active compound 11. After 48 h of incubation,
images were captured in phase contrast microscopy which are depicted
in Fig. 10A, wherein, characteristic morphological features like the
shrinkage of cells, deformation of cell wall, detachment from
substratum, reduction in the number of viable cells were observed in
a dose-dependent manner as compared to the control, clearly indicating
apoptosis.
or mass of the plasmid DNA that could be detected by observing the
retardation in migration during electrophoresis. Negatively supercoiled
form pUC19 was used as substrate in this assay. It was found that in the
presence of ethidium bromide (EtBr), a strong DNA intercalator, there
was retardation in the DNA migration during gel electrophoresis
(Fig. 7). The investigational compounds, however, have displayed less
pronounced retardation in comparison with EtBr, thus indicating that
the tested compounds may not be DNA intercalators, but are possible
DNA groove binding agents [3,32a,d]. From the decatenation,
relaxation and DNA intercalation assays, it is revealed that the
investigational compounds (10, 11 and 13) inhibit Topo-I mostly by
binding at the interface of Top1cc-DNA complexes by interacting with
the minor-groove of the DNA, hence can be considered as good
candidates for development as anticancer molecules.
2.2.3.2. Acridine Orange/Ethidium bromide (AO/EB) staining. Dual
acridine orange/ethidium bromide (AO/EB) fluorescent staining
which is visualized under a fluorescent microscope, can be used to
identify apoptosis-associated changes in cell membrane during
apoptosis. It is also effective in precisely distinguishing cells in
different stages of apoptosis. AO penetrates normal and early
apoptotic cells with intact membranes, fluorescing green when bound
to DNA, while EB only enters cells with damaged membranes, such as
late apoptotic cells and dead cells, and binds to concentrated DNA
fragments or apoptotic bodies thus emitting orange-red fluorescence
[36]. It can be construed from Fig. 10B that control cells maintained
their normal morphology and appeared green in colour and on the
contrary, the cells treated with our compound 11 showed green
fluorescence as that of control but with morphological changes like
2.2.2.6. Circular dichroism. The primary observation of these novel
synthesized compounds as DNA binding agents from the preceding
studies, has been further validated for their ability to bring about
changes in DNA conformation by employing circular dichroism (CD) as
a tool (Fig. 8) [33]. The CD spectrum of CTDNA, displays positive cotton
effect, with a peak at 275 nm and a trough at 245 nm due to base
stacking and helicity of B-form of DNA, respectively. Intercalators
disrupt both the bands while MGBs do not show major changes in the
Fig. 8. CD spectra of CTDNA with various concentrations of compounds 10 (A), 11 (B) and 13 (C), where a: Compound/DNA = 1/10 ratio, b: 1/6, c: 1/4, d: 1/3 and
CTDNA: CTDNA in Tris-HCl buffer (pH-7.4).
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BioorganicChemistry99(2020)103629
Fig. 9. UV-Vis Spectra with CTDNA (100 μg) with various concentration of investigational compounds 10 (A), 11 (B) and 13 (C).
cell shrinkage, membrane blebbing, condensation of chromatin and
apoptotic body formation indicating that the compound induces dose-
dependent apoptosis in A549 cells.
compound 11 on mitochondria, A549 cells were initially treated with
compound 11 at concentrations 1, 2.5, 5 and 10 µM and were further
stained with a lipophilic cationic dye JC-1, in which normal polarized
mitochondria stains red due to formation of J-aggregates and green
color was observed for depolarized mitochondria of apoptotic cells due
to formation of J-monomers.
2.2.3.3. DAPI nucleic acid staining. 4′-6-Diamidino-2-phenylindole
(DAPI), a fluorescent dye, pervades intact membrane of live cells and
stains the nucleus light blue, whereas, the apoptotic cell nuclei appears
as bright blue due to chromatin condensation. To further examine the
nuclear morphological changes in A549 cells, DAPI staining was
Flow-cytometric analysis of the treated cells clearly showed re-
markable increase in the mitochondrial membrane depolarisation as
compared to the control cells, as a result, increase in depolarised cell
population (P2) in a concentration dependent manner (Fig. 11) from
4.44% in control to 47.81% at 5 μM. This dose-dependent increase in
depolarised cell population is represented as bar graph (Fig. S3, sup-
porting information). This assay further suggested the involvement of
mitochondria-dependent apoptotic pathway in the mechanism of action
of the lead compound 11.
performed after 48
h of treatment with compound 11. In this
investigation, it was noticed that the control cells were intact with
normal morphology, while those treated with compound 11 at
concentrations 1, 2.5, 5 and 10 µM displayed nuclear changes like
horse-shoe shaped, irreversible chromatin condensation and
fragmented bright nuclei. From the results, it was evident that the
compound 11 had induced apoptosis in A549 cells (Fig. 10C).
2.2.4.2. Cell cycle analysis. Cell cycle arrest displayed by the cytotoxic
compounds can be analysed by cell cycle analysis. Flow cytometric
analysis of cell distribution was performed on the A549 human lung
cancer cells treated with compound 11 for 48 h and further stained with
propidium iodide dye that is generally excluded from viable cells [38].
According to the analysis results given below, it was evident that
compound 11 treatment induces apoptosis cell death, which was
corroborated by the appearance of the characteristic sub-diploid DNA
content peak (sub-G1). Fig. 12 shows the relative percentages of A549
cells in each phase of the cell cycle following the treatment. We noticed
2.2.4. Flow cytometric analysis
2.2.4.1. Effect of mitochondrial membrane potential (Δψm). Changes in
the mitochondrial membrane potential (Δψm) as
a result of
mitochondrial dysfunction have been originally postulated to be an
obligate event in the intrinsic apoptotic signalling pathway [37]. As a
consequent outcome of apoptosis, opening of the mitochondrial
permeability transition pore is observed which induces depolarization
of the transmembrane potential (Δψm), release of apoptogenic factors
and loss of oxidative phosphorylation. To evaluate the effect of
Fig. 10. A) Morphological changes observed in A549 cells treated with the compound 11 at concentrations of 0, 1, 2.5, 5 and 10 µM. After 48 h, the images were
captured with a phase contrast microscope at 200X magnification. B) AO/EB staining in A549 cells treated with, 1, 2.5, 5 and 10 µM concentrations of compound 11
for 48 h and compared with control. The images were captured with fluorescence microscope at magnification of 200X. C) DAPI staining images displaying the
nuclear morphology of A549 cells treated with compound 11 at concentrations 0, 1, 2.5, 5 and 10 µM for 48 h. The images were captured with fluorescence
microscope at magnification of 200X.
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M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Fig. 11. Effect of compound 11 on mitochondrial membrane potential (Δψm). A549 cells were treated with the lead compound 11 for 48 h and JC-1 staining was
performed on these treated cells along with the control (untreated cells). P1 represents formation of J-aggregates in healthy mitochondria, whereas P2 represents
depolarised mitochondria in cells due to the presence of J-monomers.
that the untreated control cell population showed 2.47% cells in sub-G1
phase, whereas compound 11 treatment resulted in an increase in sub-
G1 population from 8.68 to 22.07% with increasing concentrations
from 0.5 to 5 μM, thus, demonstrating rise in apoptotic cells. The dose-
dependent blockade of sub-G1 phase is also represented as bar diagram
in Fig. S4, supporting information.
2.2.4.3. Annexin V-FITC/PI assay. The quantification of apoptotic cell
death can be carried out by performing annexin V-FITC/PI assay by
using flow cytometry. One of the most commonly used cytofluorometric
stains is fluorochrome‐labeled annexin V, which binds to the negatively
charged phosphatidylserine (PS) exposed on the outer cell membrane, a
hallmark of apoptosis, thereby giving fluorescence. Propidium iodide
Fig. 12. Effect of compound 11 on cell cycle phase distribution of A549 cells examined by performing cell cycle analysis assay using propidium iodide staining
method. The assay was carried out after 48 h of treatment with compound 11 at various concentrations of 0.5, 1, 2.5 and 5 μM along with control (untreated cells).
9

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Fig. 13. Effect of compound on cell apoptosis as measured by annexin V/propidium iodide staining assay. A549 cells were treated with different concentrations of
compound 11 ranging from 0.5 to 5 μM for 48 h. Then 10,000 cells from each treated sample and control were harvested and labelled with annexin V-FITC and PI.
The percentage of cells in each stage of cell apoptosis were quantified by flow cytometry and is represented inside four quadrants, i.e. upper left quadrant shows the
necrotic cells; bottom left quadrant represents live cells, while the bottom right quadrant represents early apoptotic cells and the upper right quadrant indicates late-
apoptotic cells.
(PI),
a
widely used non-specific, phenanthrene-containing DNA
molecules, in silico studies were carried out using three-dimensional
crystal structure of AT-rich A-tract DNA dodecamer d(CGCAAATTT
GCG) complexed with minor-groove binding drug: Hoechst 33258. The
co-crystal, Hoechst, when re-docked showed hydrogen bonding with
dT7A and dT8A (A, Fig. 15), thus validating our docking protocol (Fig.
S7, supplementary information). Similarly, compound 11 also aligned
itself in the minor groove like the co-crystal and displayed hydrogen
bonds with dT9A and dG10A (B, Fig. 15). From the docking results, it
was observed that the compound 11 gets excellently aligned along the
minor groove, akin to Hoechst 33,258 (C, Fig. 15), This indicates the
possible binding capability of our compound at the interface of Topo I
and DNA thus, supporting the results obtained from the in vitro biolo-
gical assays.
intercalating agent, is excluded by the plasma membrane of living
cells. Thus, cells which stained positive for annexin V-FITC/PI represent
those in the late apoptotic stage that have lost membrane integrity [39].
Cells in different stages, such as, necrotic cells (Q1-UL; AV-/PI + ), live
cells (Q2-LL; AV-/PI-), early apoptotic cells (Q3-LR; AV+/PI-), and late
apoptotic cells (Q4- UR; AV+/PI + ) can be detected using annexin
assay. A549 cells upon treatment with compound 11 at varied
concentrations (1, 2.5, 5 and 10 µM) have shown increased binding
to the annexin V-FITC/PI indicating dose-dependent cellular apoptosis.
The percentage of early apoptotic cells (AV+/PI-) showed and dose-
dependent increase after treatment with different concentrations of
compound 11 for 48 h, in comparison to the control cells (Fig. 13).
Graphical representation of the results is depicted in Fig. S5 (supporting
information).
3. Conclusion
2.3. Molecular docking
In conclusion, a new series of diindoloazepinone analogues were
designed, synthesized and further evaluated for their in vitro cytotoxic
potential against different cancer cell lines and also a non-cancerous
immortalized human embryonic kidney cells. The results of this pre-
liminary cytotoxicity screening using the MTT assay suggested that
most of the compounds showed good activity and the most potent one,
11, displayed a broad spectrum of activity against all the tested cancer
cell lines specifically in A549 (lung cancer cells) with IC50 value of
4.2 μM. Interestingly, it showed a 13-fold selectivity upon the non-
cancerous immortalized human embryonic kidney cells. Further de-
tailed biological studies including cell cycle analysis, DNA binding
studies, Topo I inhibition, circular dichroism, UV-visible spectroscopy,
viscosity studies, apoptotic studies and flow cytometric analysis deliv-
ered promising results. The flow cytometric analysis revealed that these
derivatives caused cell cycle arrest at sub-G1 phase. Furthermore, it
Topotecan, a Topo I poison, was re-docked into the Topo I active site
(PDB: 1K4T) to validate the docking protocol (Fig. S6, supplementary
information), which showed hydrogen bonding with Arg364, Lys532
and Asp533 (A, Fig. 14). After the validation of the docking protocol,
we have performed molecular docking studies on compound 11 which
showed hydrogen bond with Gln633 and Ala635 along with hydro-
phobic interactions with Ile535 and π-cation interaction with Arg364
(B, Fig. 14). It can be seen that both topotecan and compound 11 bind
at the site surrounded by amino acid residues like Asp533, Thr718 and
Arg364 which suggested that our compound 11 also lodged at the ac-
tive site of Topo I in a comparable manner as that of the co-crystal.
In order to examine and understand the DNA-binding property of
the most active molecule 11 amongst the library of synthesized
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M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
Fig. 14. A and B represent the binding pose, hydrogen bonds (yellow dotted lines) and hydrophobic interactions of topotecan (co-crystal) and compound 11 with
Topo I (PDB code: 1K4T). The co-crystal and compound 11 are indicated as yellow and turquoise ball and stick models, respectively. Hydrogen bonding is indicated
by yellow dotted line.
per million (ppm) downfield from tetramethylsilane. Spin multiplicities
are described as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet) and m (multiplet). Coupling constant (J) values are reported
in hertz (Hz). HRMS was performed with an Agilent QTOF mass spec-
trometer 6540 series instrument. Melting points were determined using
an electrothermal digital melting point apparatus digital melting point
apparatus IA9100. Infrared spectra (IR) were recorded on a Perkin
Elmer 1600 series FTIR spectrometer. The names of all the compounds
given in the experimental section were taken from ChemBioDraw Ultra,
Version 14.0.
4.2. Chemistry
4.2.1. 3-(1H-Indole-2-carboxamido)propanoic acid 4
Fig. 15. A and B represent minor groove binding of co-crystal, Hoechst 33258,
To a solution of β-alanine ethyl ester hydrochloride 2 (1 equiv) in
and compound 11 into d(CGCAAATTTGCG) (DNA) (PDB code: 264D), respec-
DMF (50 mL) under nitrogen atmosphere was added indole-2-car-
tively. DNA is represented as a turquoise ribbon, while the co-crystal is in-
boxylic acid 1 (31 mmol), followed by anhydrous DMAP (2.2 equiv)
dicated in yellow and compound 11 is represented as red colored ball and stick
and finally EDCI.HCl (2 equiv) at 0 °C. The reaction was allowed to stir
models. C shows the superimposition of Hoechst (orange) and compound 11
at 0 °C for 4 h and continued at room temperature till the completion of
the reaction (20 h). To the reaction mixture, was added crushed ice and
stirred continuously to obtain solid. The solid intermediate was filtered,
exhaustively washed with water and the solid was dried under vacuum
to obtain the desired ester intermediate 3 as white solid (90%). This
ester was dissolved in EtOH and NaOH (3 equiv) was added and the
reaction mixture was allowed to stir for 30 min at rt. After the com-
pletion of the reaction, the solvent was evaporated under vacuum and
water was added. To this, 1 N HCl was added slowly until acidic and the
precipitate was filtered and dried to obtain the acid 4 (92%).
(mustard yellow) along the minor groove of DNA. The yellow dotted lines in-
dicate hydrogen bonding.
forms a stable complex with efficient binding to the minor groove of
DNA, they can effectively and, most importantly, selectively inhibit
Topo I without having an effect on Topo IIα. Molecular docking studies
also showed that the interactions of 11 matched well with the co-
crystal, Hoechst, which is indicative of minor-groove binding into DNA.
It also showed good interactions with Topo I, thus, implying its possible
mechanism of action as interfacial Topo 1 inhibition via minor groove
binding.
4.2.2. 3,4-Dihydro-2H,10H-azepino[3,4-b]indole-1,5-dione 5
In methanesulfonic acid (1 equiv) was dissolved phosphorus pent-
oxide (1.2 equiv) and heated to 80 °C. Once homogenous, carboxylic
acid 5 (0.76 mmol) was added, the reaction stirred and monitored by
TLC until complete. The reaction was quenched with ice and ex-
haustively extracted with ethyl acetate. The combined organic extrac-
tions were dried (Na2SO4) and concentrated under vacuum to afford the
desired intermediate 5 as a solid (60%)
4. Experimental section
4.1. General procedures
Reagents and solvents were purchased from commercial sources. All
the reactions were monitored routinely by thin layer chromatography
(TLC) performed on MERCK silica gel 60-F254 (0.5 mm) pre-coated
aluminium plates and visualized using ultraviolet light or by treatment
with iodine or anisaldehyde dip. Column chromatography was per-
formed by using silica gel (60–120). Evaporation of the solvents was
performed under reduced pressure by using a rotary evaporator below
45 °C. 1H and 13C NMR spectra were recorded with a Bruker 500 MHz
instrument in CDCl3 or [d6] DMSO solvent with tetramethylsilane as the
internal standard. Chemical shifts for 1H and 13C are reported in parts
4.2.3. 10-Benzyl-3,4-dihydroazepino[3,4-b]indole-1,5(2H,10H)-dione 7a
To a solution of indoloazepinone 5 (2.33 mmol) in DMF (5 mL)
maintained at 0 °C was added K2CO3 (3 equiv) was stirred for 15 min.
To this, benzylbromide 6a (1 equiv) was added dropwise and the re-
action mixture was refluxed for 8 h. After the completion of the reaction
(TLC), the reaction mixture was cooled to rt and was poured onto
crushed ice. Further extraction with EtOAc and concentration of the
11

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
dried organic layers followed by purification with column chromato-
graphy yielded the desired N-alkylated indoloazepinone 7a as pale
yellow solid (92%); MP: 166–168 °C. IR (neat): νmax 3191, 3058, 1645,
cooling to rt, the reaction mixture was poured into a 5% sodium acetate
solution (15 mL). The mixture was extracted with EtOAc and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography, eluted with ethyl acetate:hexane (30:70) to afford the
desired product 10. Yield 54%, yellow solid; MP: 115–117 °C. IR (neat):
νmax 3381, 3221, 1631, 1487, 1451, 1315, 1259, 742, 686, 442,
410 cm−1; 1H NMR (500 MHz, CDCl3) δ 9.81 (s, 1H), 8.79 (s, 1H), 8.04
(d, J = 8.0 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H),
7.45 (d, J = 8.3 Hz, 1H), 7.38 (t, J = 7.3 Hz, 1H), 7.28 (m, 1H),
7.25–7.18 (m, 2H), 6.41 (s, 1H), 4.44 (d, J = 4.9 Hz, 2H). 13C NMR
(125 MHz, CDCl3): δ165.0, 137.5, 137.0, 136.4, 132.8, 125.9, 125.9,
123.8, 122.3, 121.6, 120.8, 120.6, 117.8, 112.7, 111.8, 111.4, 109.6,
36.5; HRMS (ESI): m/z calcd for C18H13N3O 288.1131; found 288.1139
1504, 1449, 1449, 1377, 1313, 1253, 1199, 749, 728, 692, 543 cm−1
;
1H NMR (500 MHz, DMSO) δ 8.90 (t, J = 6.0 Hz, 1H), 8.30 (d,
J = 7.4 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.37–7.31 (m, 2H),
7.31–7.29 (m, 1H), 7.28 (d, J = 7.5 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H),
7.13 (d, J = 7.1 Hz, 2H), 5.91 (s, 2H), 3.43 (dd, J = 10.6, 5.7 Hz, 2H),
2.84 (dd, J = 6.6, 3.8 Hz, 2H). 13C NMR (125 MHz, DMSO) δ 196.5,
162.9, 138.3, 138.0, 134.6, 129.0, 127.7, 127.0, 125.8, 125.3, 123.8,
123.3, 116.1, 112.1, 48.2, 45.5, 37.1. HRMS (ESI): m/z calcd for
C
19H16N2O2 305.1285; found 305.1289 [M+H]+
.
4.2.4. 10-(3-Chlorobenzyl)-3,4-dihydroazepino[3,4-b]indole-
1,5(2H,10H)-dione 7b
[M+H]+
.
According to the procedure described for the synthesis of 7a, 7b was
prepared from 5 and 6b in 89% yield as pale yellow solid; MP:
177–179 °C. IR (neat): νmax 3183, 1661, 1644, 1599, 1578, 1505, 1461,
4.2.8. 10-Methyl-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']diindol-6-
one 11
Starting from 5, compound 11 was obtained as golden yellow solid
using a similar procedure as 10. Yield 63%; MP: 142–145 °C; IR (neat):
νmax 3267, 2922, 2862, 1747, 1624, 1489, 1334, 1307, 743, 441,
405 cm−1; 1H NMR (500 MHz, CDCl3) δ 9.47 (s, 1H), 8.57 (s, 1H), 8.05
(d, J = 7.8 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.44–7.41 (m, 1H), 7.40
(s, 1H), 7.37 (s, 1H), 7.33–7.27 (m, 1H), 7.09–7.04 (m, 1H), 6.21 (s,
1H), 4.45 (d, J = 5.1 Hz, 2H), 2.49 (s, 3H);13C NMR (125 MHz, CDCl3):
δ 165.1, 140.4, 136.5, 135.3, 132.9, 129.9, 126.1, 125.9, 123.9, 123.8,
121.5, 120.9, 120.7, 117.5, 112.7, 111.1, 109.2, 36.5, 21.6; HRMS
1428, 1381, 1260, 800, 745, 773, 680, 561 cm−1
;
1H NMR (500 MHz,
CDCl3) δ 8.52–8.50 (m, 1H), 7.40–7.36 (m, 2H), 7.35–7.33 (m, 1H),
7.21 (s, 1H), 7.18 (d, J = 7.9 Hz, 1H), 7.12 (s, 1H), 6.97 (d, J = 6.5 Hz,
1H), 6.91 (s, 1H), 5.84 (s, 2H), 3.58 (dd, J = 10.9, 6.1 Hz, 2H), 2.98–
2.96 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 195.5, 163.6, 139.4, 138.3,
134.7, 132.4, 130.0, 127.8, 126.7, 126.3, 125.5, 124.7, 124.1, 117.3,
110.7, 48.3, 45.1, 37.7. HRMS (ESI): m/z calcd for C19H15ClN2O2
339.0895; found 339.0898 [M+H]+
.
(ESI): m/z calcd for C18H13N3O 302.1288; found 300.1286 [M+H]+
.
4.2.5. 2,10-Dibenzyl-3,4-dihydroazepino[3,4-b]indole-1,5(2H,10H)-dione
8a
4.2.9. 10-Fluoro-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']diindol-6-
To a solution of indoloazepinone 5 (2.33 mmol) in DMF (5 mL)
maintained at 0 °C under nitrogen atmosphere was added NaH (2 equiv)
was stirred for 30 min. To this benzylbromide 6a (2 equiv) was added
dropwise and the reaction mixture was allowed to stir at rt for 12 h.
After the completion of the reaction (TLC), the reaction mixture was
poured onto crushed ice and acidified with 1 N HCl and then extracted
with EtOAc followed by purification with column chromatography
yielding the N,N-dialkylated indoloazepinone 8a as semi-solid in 90%
yield. IR (neat): νmax 1739, 1641, 1505, 1429, 1392, 1355, 1246, 1151,
one 12
Starting from 5, compound 12 was obtained as golden yellow solid
using a similar procedure as 10. Yield 47%; MP: 150–153 °C. IR (neat):
νmax 3404, 3216, 2970, 1743, 1611, 1484,1452, 1256, 1156, 934, 792,
747, 548, 437, 405 cm−1; 1H NMR (500 MHz, CDCl3 + DMSO) δ 11.72
(s, 1H), 11.39 (s, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.86 (s, 1H), 7.82 (d,
J = 8.3 Hz, 1H), 7.77–7.67 (m, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.47–7.40
(m, 1H), 7.09 (dt, J = 9.2, 2.3 Hz, 1H), 4.56 (d, J = 5.0 Hz, 2H). 13C
NMR (125 MHz, CDCl3) δ 164.5, 160.30 (d, J = 203.80 Hz), 136.3,
134.3, 133.5, 129.1, 125.5 (d, J = 9.56 Hz), 124.5, 123.1, 121.2,
120.1, 112.3, 112.1 (d, J = 9.33 Hz), 110.5, 108.9, 108.8, 108.5, 101.6
(d, J = 23.52 Hz), 35.5; HRMS (ESI): m/z calcd for C18H12FN3O
741, 698, 537 cm−1
;
1H NMR (500 MHz, CDCl3) δ 8.42 (dd, J = 6.6,
2.1 Hz, 1H), 7.43 (dd, J = 6.9, 1.9 Hz, 1H), 7.40–7.31 (m, 2H),
7.30–7.27 (m, 6H), 7.11 (dd, J = 6.2, 2.7 Hz, 4H), 5.90 (s, 2H), 4.81 (s,
2H), 3.60 (s, 2H), 2.79 (dd, J = 5.6, 4.7 Hz, 2H). 13C NMR (125 MHz,
CDCl3) δ 195.8, 161.6, 138.4, 137.7, 136.6, 134.1, 128.9, 128.8, 128.0,
127.8, 127.6, 126.7, 125.8, 125.1, 123.8, 123.5, 116.3, 110.6, 50.3,
48.6, 44.1, 43.6. HRMS (ESI): m/z calcd for C26H22N2O2 395.1754;
306.1037; found 306.1044 [M+H]+
.
4.2.10. 10-Chloro-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']diindol-6-
one 13
found 395.1754 [M+H]+
.
Starting from 5, compound 13 was obtained as pale yellow solid
using a similar procedure as 10. Yield 44%; MP: 133–135 °C. IR (neat):
4.2.6. 2,10-Bis(3-chlorobenzyl)-3,4-dihydroazepino[3,4-b]indole-
1,5(2H,10H)-dione 8b
νmax 3013, 2894, 1634, 1529, 1313, 1151, 1033, 442, 407 cm−1
;
1H
NMR (500 MHz, CDCl3 + DMSO) δ 11.35 (s, 1H), 11.18 (s, 1H), 8.21
(d, J = 7.3 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.48 (d,
J = 8.2 Hz, 1H), 7.41–7.32 (m, 2H), 7.30–7.20 (m, 1H), 7.07 (d,
J = 7.9 Hz, 1H), 4.36 (s, 2H). 13C NMR (125 MHz, CDCl3 + DMSO) δ
164.2, 136.3, 135.3, 134.1, 129.6, 126.3, 124.4, 124.3, 123.0, 121.2,
120.4, 120.0, 116.2, 112.6, 112.4, 110.0, 108.5, 35.1; HRMS (ESI): m/z
According to the procedure described for the synthesis of 8a, 8b was
prepared from 5 and 6b in 86% yield as semi-solid. IR (neat): νmax
1751, 1573, 1475, 1455, 1428, 1394, 1272, 1095, 992, 873, 741, 562,
550 cm−1
;
1H NMR (500 MHz, CDCl3) δ 8.43 (d, J = 7.3 Hz, 1H),
7.42–7.33 (m, 3H), 7.31–7.26 (m, 2H), 7.26–7.19 (m, 4H), 7.13 (s, 1H),
7.01 (dd, J = 15.0, 7.3 Hz, 2H), 5.85 (s, 2H), 4.79 (s, 3H), 3.66 (s, 2H),
2.84 (dd, J = 5.7, 4.6 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 195.4,
163.6, 139.4, 138.3, 134.7, 132.4, 130.0, 127.8, 126.7, 126.3, 125.5,
124.7, 124.1, 124.1, 117.3, 110.7, 48.3, 45.1, 37.7, 29.7. HRMS (ESI):
calcd for C18H12ClN3O 322.0742; found 322.0644 [M+H]+
.
4.2.11. 10-Bromo-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']diindol-6-
one 14
m/z calcd for C26H20Cl2N2O2 463.0975; found 463.0969 [M+H]+
4.2.7. 5,7,8,13-Tetrahydro-6H-azepino[3,4-b:5,6-b']diindol-6-one 10
.
Starting from 5, compound 14 was obtained as pale brown solid
using a similar procedure as 10. Yield 42%; MP: 124–127 °C. IR (neat):
νmax 3267, 2926, 2839, 1631, 1486, 1450, 1333, 1014, 744, 445,
A mixture of indoloazepinone 5 (0.5 mmol) and arylhydrazine hy-
drochloride (1.5 equiv) was refluxed in glacial acetic acid (2 mL) and
ethanol (2 mL). After stirring at 70 °C for 1 h, the reaction mixture was
cooled to rt. Concentrated HCl (0.1–0.5 mL) was added and stirring is
continued at 70 °C for 7 h. Reaction was monitored by TLC. After
407 cm−1
;
1H NMR (500 MHz, CDCl3 + DMSO) δ 11.72 (s, 1H), 11.42
(s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.79 (s, 3H), 7.71 (d, J = 1.4 Hz, 1H),
7.61 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.39–7.32 (m, 1H),
7.28–7.21 (m, 1H), 7.19 (dd, J = 8.6, 1.7 Hz, 1H), 4.33 (d, J = 4.6 Hz,
2H). 13C NMR (125 MHz, CDCl3 + DMSO) δ 169.6, 141.6, 140.9,
12

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
139.2, 134.8, 132.3, 129.7, 128.3, 128.3, 126.5, 125.4, 124.6, 118.3,
δ 164.2, 158.4 (d, J = 235.81 Hz), 139.0, 138.0, 134.6, 133.5, 129.4,
128.6, 127.3, 126.6, 125.8, 122.9, 121.6, 120.8, 112.8, 112.2 (d,
J = 9.54 Hz), 111.9 (d, J = 4.38 Hz), 111.5, 110.5, 110.3, 102.9 (d,
J = 24.04 Hz), 48.5, 36.0; HRMS (ESI): m/z calcd for C25H18FN3O
396.1507; found [M+H] + 396.1518.
117.7, 117.3, 115.4, 113.6, 40.5; HRMS (ESI): m/z calcd for
C
18H12BrN3O 366.0237; found 366.0242 [M+H] + .
4.2.12. 10,12-Dimethyl-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']
diindol-6-one 15
Starting from 5, compound 15 was obtained as greenish yellow solid
using a similar procedure as 10. Yield 75%; MP: 130–133 °C. IR (neat):
νmax 3438, 3259, 3156, 2913, 1737, 1480, 1442, 1308, 1172, 840, 740,
577, 565, 513. 442 cm−1; 1H NMR (500 MHz, CDCl3 + DMSO) δ 11.11
(s, 1H), 10.19 (s, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.55–7.38 (m, 1H), 7.25
(t, J = 7.7 Hz, 1H), 7.21–7.14 (m, 2H), 6.72 (s, 1H), 4.26 (d,
J = 5.2 Hz, 2H), 2.81 (s, 3H), 2.34 (s, 3H). 13C NMR (125 MHz,
CDCl3 + DMSO) δ 170.0, 141.6, 140.1, 138.0, 134.0, 133.8, 130.7,
129.7, 128.8, 128.5, 127.1, 125.9, 125.1, 119.5, 117.4, 116.5, 114.8,
40.9, 26.3, 21.9; HRMS (ESI): m/z calcd for C20H17N3O 316.1444;
4.2.16. 5-Benzyl-10-chloro-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']
diindol-6-one 19
Starting from 7a, compound 19 was obtained as pale yellow solid using
a similar procedure as 16. Yield 44%; MP: 115–118 °C. IR (neat): νmax 3339,
2944, 2832, 1637, 1583, 1479, 1449, 1332, 1025, 790, 407 cm−1; 1H NMR
(500 MHz, CDCl3) δ 8.83 (s, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.56 (d,
J = 1.7 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 4.3 Hz, 1H), 7.38
(d, J = 8.1 Hz, 1H), 7.30 (d, J = 7.0 Hz, 1H), 7.28 (s, 1H), 7.25 (s, 1H),
7.22 (d, J = 7.1 Hz, 1H), 7.18 (dd, J = 8.5, 1.8 Hz, 1H), 7.13 (d,
J = 7.0 Hz, 2H), 6.38 (s, 1H), 5.91 (s, 2H), 4.23 (d, J = 6.1 Hz, 2H). 13
C
found 316.1454 [M+H]+
.
NMR (125 MHz, DMSO) δ 163.7, 138.8, 138.5, 136.0, 134.1, 131.2, 129.0,
127.1, 127.0, 126.7, 125.7, 123.0, 122.0, 121.5, 117.7, 117.5, 113.7, 112.0,
111.9, 47.8, 35.1; HRMS (ESI): m/z calcd for C25H18ClN3O 434.1036; found
434.1042 [M + Na]+.
4.2.13. 5-Benzyl-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']diindol-6-
one 16
A mixture of N-alkylated indoloazepinone 7a (0.5 mmol) and ar-
ylhydrazine hydrochloride 6 (1.5 equiv) was refluxed in glacial acetic
acid (2 mL) and ethanol (2 mL). After stirring at 70 °C for 1 h, the
reaction mixture was cooled to rt. Concentrated HCl (0.1–0.5 mL) was
added and stirring is continued at 70 °C for 7 h. Reaction was monitored
by TLC. After cooling to rt, the reaction mixture was poured into a 5%
sodium acetate solution (15 mL). The mixture was extracted with
EtOAc and concentrated in vacuo. The residue was purified by silica gel
column chromatography, eluted with ethyl acetate:hexane (15:85) to
afford the titled product 16 as orange solid. Yield 51%; MP:
268–270 °C. IR (neat): νmax 3188, 3029, 1738, 1633, 1451, 1326, 735,
4.2.17. 5-Benzyl-10-bromo-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']
diindol-6-one 20
Starting from 7a, compound 20 was obtained as cream solid using a
similar procedure as 16. Yield 47%; MP: 212–215 °C. IR (neat): νmax
3452, 3162, 3025, 2919, 1639, 1520, 1449, 1329, 1296, 1199, 1031,
893, 793, 739, 726, 479, 434, 407 cm−1
;
1H NMR (500 MHz, CDCl3) δ
11.71 (s, 1H), 8.65 (d, J = 6.6 Hz, 1H), 8.13 (s, 1H), 7.96 (d,
J = 9.9 Hz, 26H), 7.88–7.82 (m, 2H), 7.85 (s, 1H), 7.67 (d, J = 4.2 Hz,
2H), 7.64 (d, J = 6.6 Hz, 2H), 7.58 (d, J = 2.4 Hz, 2H), 6.35 (s, 2H),
4.67– 4.52 (m, 2H). 13C NMR (125 MHz, DMSO) δ 163.7, 138.8, 138.5,
136.2, 134.0, 131.2, 129.0, 127.6, 127.4 127.1, 125.7, 124.0, 123.0,
122.0, 121.5, 120.5, 114.2, 112.5, 111.8, 111.7, 47.9, 35.1; HRMS
694, 509, 470, 407 cm−1
;
1H NMR (500 MHz, DMSO) δ 11.65 (s, 1H),
8.41 (s, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.63 (t, J = 7.5 Hz, 2H), 7.53 (d,
J = 7.9 Hz, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.31–7.28 (m, 4H),
7.24–7.21 (m, 1H), 7.18 (d, J = 7.3 Hz, 2H), 7.17–7.14 (m, 1H), 7.10
(t, J = 7.4 Hz, 1H), 5.93 (s, 2H), 4.17 (d, J = 5.9 Hz, 2H). 13C NMR
(125 MHz, DMSO) δ 163.8, 139.0, 138.5, 137.6, 132.5, 130.8, 129.0,
127.6, 127.1, 125.6, 125.6, 123.1, 122.1, 121.7, 121.3, 119.8, 118.1,
112.4, 112.3, 112.3, 111.8, 47.8, 35.2; HRMS (ESI): m/z calcd for
(ESI): m/z calcd for
C25H18BrN3O 494.0270; found 494.0276
[M + K]+
.
4.2.18. 5-Benzyl-10,12-dimethyl-5,7,8,13-tetrahydro-6H-azepino[3,4-
b:5,6-b']diindol-6-one 21
Starting from 7a, compound 21 was obtained as buff yellow solid
using a similar procedure as 16. Yield 81%; MP: 220–223 °C. IR (neat):
νmax 3229, 3161, 3002, 2912, 1634, 1474, 1441, 1322, 1203, 934, 843,
791, 726, 693, 590, 486, 421 cm−1; 1H NMR (500 MHz, CDCl3) δ 8.42
(s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.42–7.36
(m, 2H), 7.33–7.28 (m, 2H), 7.22 (d, J = 7.3 Hz, 1H), 7.15 (d,
J = 7.3 Hz, 3H), 6.90 (s, 1H), 6.21 (s, 1H), 5.93 (s, 2H), 4.26 (d,
J = 6.0 Hz, 2H), 2.58 (s, 3H), 2.46 (s, 3H). 13C NMR (125 MHz, CDCl3)
δ 164.2, 139.0, 138.1, 134.9, 132.7, 130.1, 128.6, 127.3, 126.7, 125.7,
125.3, 124.7, 123.2, 123.2, 121.5, 120.8, 120.3, 115.3, 112.2, 111.5,
108.8, 48.5, 36.2, 21.5, 16.8; HRMS (ESI): m/z calcd for C27H23N3O
C
25H20N3O 378.1601; found 378.1634 [M+H]+
.
4.2.14. 5-Benzyl-10-methyl-5,7,8,13-tetrahydro-6H-azepinoq[3,4-b:5,6-
b']diindol-6-one 17
Starting from 7a, compound 17 was obtained as mustard yellow
solid using a similar procedure as 16. Yield 60%; MP: 193–194 °C. IR
(neat): νmax 3278, 3028, 2914, 1645, 1452, 1331, 1306, 1195, 1029,
800, 744, 700, 580, 450 cm−1
;
1H NMR (500 MHz, DMSO) δ 8.74 (s,
1H), 8.05 (d, J = 7.9 Hz, 1H), 7.40 (s, 1H), 7.38 (s, 2H), 7.35–7.30 (m,
2H), 7.25 (s, 1H), 7.23 (s, 1H), 7.22–7.20 (m, 1H), 7.12 (d, J = 6.9 Hz,
2H), 7.05 (d, J = 8.2 Hz, 1H), 6.38 (s, 1H), 5.89 (s, 2H), 4.22 (d,
J = 5.9 Hz, 2H), 2.48 (s, 3H). 13C NMR (125 MHz, DMSO) δ 161.8,
136.7, 136.4, 133.9, 130.4, 128.4, 126.7, 126.2, 125.3, 124.9, 123.6,
123.3, 121.1, 121.0, 120.1, 119.0, 115.5, 110.6, 109.8, 109.6, 109.4,
45.7, 33.1, 19.6; HRMS (ESI): m/z calcd for C26H21N3O 392.1757;
406.1914; found 406.1927 [M+H]+
.
4.2.19. 5-(3-Chlorobenzyl)-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']
diindol-6-one 22
Starting from 7b, compound 22 was obtained as pale brown solid
using a similar procedure as 16. Yield 53%, pale brown solid; MP:
216–219 °C. IR (neat): νmax 3174, 3008, 1626, 1599, 1474, 1439, 1328,
1207, 934, 779, 738, 679, 565, 463 cm−1; 1H NMR (500 MHz, CDCl3) δ
8.74 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.51 (d,
J = 7.8 Hz, 1H), 7.40 (d, J = 5.7 Hz, 1H), 7.33–7.29 (m, 1H), 7.23 (dd,
J = 2.7, 1.3 Hz, 1H), 7.21 (s, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.20–7.18
(m, 1H), 7.16 (s, 1H), 7.04–7.01 (m, 1H), 6.32 (t, J = 5.5 Hz, 1H), 5.87
(s, 2H), 4.31 (d, J = 6.1 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 164.1,
140.2, 138.9, 137.1, 134.6, 132.7, 129.9, 129.2, 127.6, 126.9, 126.0,
125.5, 124.9, 123.2, 122.4, 121.8, 121.0, 120.6, 117.9, 113.1, 112.0,
111.5, 111.3, 48.1, 36.1; HRMS (ESI): m/z calcd for C27H23N3O
found 392.1764 [M+H]+
.
4.2.15. 5-Benzyl-10-fluoro-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']
diindol-6-one 18
Starting from 7a, compound 18 was obtained as pale yellow solid
using a similar procedure as 16. Yield 48%; MP: 136–139 °C. IR (neat):
νmax 3277, 3030, 1740, 1643, 1628, 1451, 1331, 1304, 1164, 930, 799,
742, 700, 529, 470, 436 cm−1
;
1H NMR (500 MHz, CDCl3) δ 8.94 (s,
1H), 8.03 (d, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.7, 4.3 Hz, 1H), 7.37–7.33
(m, 2H), 7.25 (s, 1H), 7.21 (dd, J = 10.7, 3.2 Hz, 3H), 7.10 (d,
J = 6.7 Hz, 3H), 6.97 (dt, J = 9.0, 2.2 Hz, 1H), 6.36 (t, J = 5.0 Hz,
1H), 5.88 (s, 2H), 4.18 (d, J = 6.0 Hz, 2H). 13C NMR (125 MHz, CDCl3)
412.1211; found 412.1218 [M+H]+
.
13

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
4.2.20. 5-(3-Chlorobenzyl)-10-methyl-5,7,8,13-tetrahydro-6H-azepino
[3,4-b:5,6-b']diindol-6-one 23
3241, 3156, 3013, 2914, 1634, 1598, 1473, 1441, 1372, 1321, 1202,
1065, 935, 844, 778, 737, 590 487, 420 cm−1
;
1H NMR (500 MHz,
Starting from 7b, compound 23 was obtained as mustard yellow
solid using a similar procedure as 16. Yield 66%; MP: 145–147 °C. IR
(neat): νmax 3282, 1703, 1622, 1598, 1474, 1445, 1330, 1307, 1198,
741, 680, 438 cm−1; 1H NMR (500 MHz, CDCl3) δ 8.62 (s, 1H), 8.06 (d,
J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.39–7.36 (m, 2H), 7.32–7.28 (m, 2H),
7.19 (dd, J = 4.7, 0.9 Hz, 2H), 7.16 (s, 1H), 7.10–7.05 (m, 1H),
7.05–6.99 (m, 1H), 6.31 (s, 1H), 5.86 (s, 2H), 4.28 (d, J = 6.1 Hz, 2H),
2.49 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 164.3, 140.2, 139.0, 135.5,
134.5, 132.7, 129.9, 129.8, 129.7, 127.5, 126.8, 126.5, 126.0, 125.7,
124.9, 124.0, 123.2, 121.7, 121.0, 117.6, 111.6, 111.2, 111.1, 48.1,
36.1, 21.5; HRMS (ESI): m/z calcd for C26H20ClN3O 426.1368; found
CDCl3) δ 8.45 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.43–7.37
(m, 1H), 7.34–7.29 (m, 1H), 7.22 (s, 1H), 7.19 (s, 1H), 7.19 (d,
J = 1.4 Hz, 1H), 7.16 (s, 1H), 7.06–7.00 (m, 1H), 6.90 (s, 1H),
6.30–6.13 (m, 1H), 5.87 (s, 2H), 4.28 (d, J = 5.8 Hz, 2H), 2.57 (s, 3H),
2.46 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 164.1, 140.2, 138.9, 135.0,
134.5, 132.6, 130.1, 129.9, 129.0, 127.5, 126.9, 125.9, 125.3, 124.9,
124.8, 123.2, 121.7, 121.0, 120.3, 115.3, 113.4, 112.2, 111.3, 48.0,
36.2, 21.5, 16.7; HRMS (ESI): m/z calcd for C27H22ClN3O 478.1088;
found 478.1100 [M + K]+
.
426.1378 [M+H]+
.
4.2.25. 5,7-Dibenzyl-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-b']
diindol-6-one 28
A mixture of N,N-dialkylated indoloazepinone 8a (0.5 mmol) and
arylhydrazine hydrochloride 9 (1.5 equiv) was refluxed in glacial acetic
acid (2 mL) and ethanol (2 mL). After stirring at 70 °C for 1 h, the
reaction mixture was cooled to rt. Concentrated HCl (0.1–0.5 mL) was
added and stirring is continued at 70 °C for 7 h. Reaction was monitored
by TLC. After cooling to rt, the reaction mixture was poured into a 5%
sodium acetate solution (15 mL). The mixture was extracted with
EtOAc and concentrated in vacuo. The residue was purified by silica gel
column chromatography, eluted with ethyl acetate:hexane (10:90) to
afford the desired product 28 as mustard yellow solid. Yield 65%; MP:
112–115 °C. IR (neat): νmax 3267, 3029, 1734, 1594, 1482, 1450, 1326,
4.2.21. 5-(3-Chlorobenzyl)-10-fluoro-5,7,8,13-tetrahydro-6H-azepino
[3,4-b:5,6-b']diindol-6-one 24
Starting from 7b, compound 24 was obtained as mustard yellow
solid using a similar procedure as 16. Yield 55%; MP: 132–135 °C. IR
(neat): νmax 3281, 3069, 2946, 1625, 1598, 1475, 1448, 1331, 1200,
1164, 930, 741, 680, 531, 485, 433 cm−1; 1H NMR (500 MHz, CDCl3) δ
8.75 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 4.3 Hz, 1H), 7.41 (d,
J = 3.9 Hz, 1H), 7.39 (d, J = 0.8 Hz, 1H), 7.33–7.28 (m, 2H), 7.23 (d,
J = 2.6 Hz, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.20–7.19 (m, J = 2.3,
1.2 Hz, 1H), 7.14 (s, 1H), 7.04–6.95 (m, J = 11.5, 7.8, 2.1 Hz, 3H),
6.27 (s, 1H), 5.87 (s, 2H), 4.26 (d, J = 6.2 Hz, 2H). 13C NMR (125 MHz,
DMSO) δ 163.7, 157.8 (d, J = 231.99 Hz), 141.5, 138.4, 134.3, 134.3,
133.6, 130.9, 130.9, 127.8, 127.1, 125.8, 123.1, 122.1, 121.6, 113.2 (d,
J = 9.75 Hz), 112.5, 112.1, 111.7, 109.7 (d, J = 26.56 Hz), 103.1,
102.9, 47.5, 35.2; HRMS (ESI): m/z calcd for C25H17ClFN3O 468.0681;
1190, 735, 695, 511, 407 cm−1
;
1H NMR (500 MHz, CDCl3) δ 9.01 (s,
1H), 8.02 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.37 (t,
J = 7.9 Hz, 3H), 7.27 (s, 3H), 7.25 (s, 3H), 7.22 (d, J = 7.2 Hz, 1H),
7.16 (s, 1H), 7.14–7.10 (m, 5H), 5.89 (s, 2H), 4.71 (s, 2H), 4.10 (s, 2H).
13C NMR (125 MHz, CDCl3) δ 162.1, 138.9, 138.3, 137.1, 136.9, 133.2,
131.0, 128.7, 128.6, 127.8, 127.4, 127.4, 126.9, 125.9, 125.4, 122.9,
122.1, 121.3, 120.8, 120.3, 117.7, 112.3, 111.4, 111.3, 111.0, 50.9,
48.4, 41.6; HRMS (ESI): m/z calcd for C32H25N3O 506.1629; found
found 468.0687 [M + K]+
.
4.2.22. 10-Chloro-5-(3-chlorobenzyl)-5,7,8,13-tetrahydro-6H-azepino
[3,4-b:5,6-b']diindol-6-one 25
506.1642 [M + K]+
.
Starting from 7b, compound 25 was obtained as brwon solid using a
similar procedure as 16. Yield 47%; MP: 135–138 °C. IR (neat): νmax
3258, 1630, 1598, 1474, 1330, 1296, 1199, 1093, 741, 679, 520,
4.2.26. 5,7-Dibenzyl-10-methyl-5,7,8,13-tetrahydro-6H-azepino[3,4-
b:5,6-b']diindol-6-one 29
466 cm−1
;
1H NMR (500 MHz, DMSO) δ 11.84 (s, 1H), 8.44 (s, 1H),
8.21 (d, J = 7.9 Hz, 1H), 7.73 (d, J = 2.1 Hz, 1H), 7.63 (d, J = 8.5 Hz,
1H), 7.52 (d, J = 8.5 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.34–7.32 (m,
1H), 7.31 (d, J = 2.3 Hz, 1H), 7.30–7.28 (m, 1H), 7.18–7.08 (m, 2H),
5.91 (s, 2H), 4.18 (d, J = 5.9 Hz, 2H). 13C NMR (125 MHz, DMSO) δ
163.7, 141.5, 138.4, 136.0, 134.1, 133.6, 131.1, 130.9, 127.6, 127.1,
126.7, 125.9, 125.8, 124.6, 123.1, 122.1, 121.7, 121.6, 117.5, 113.7,
112.0, 111.9, 111.7, 47.5, 35.1; HRMS (ESI): m/z calcd for
Starting from 8a, compound 29 was obtained as light orange solid
using a similar procedure as 28. Yield 69%; MP: 210–213 °C. IR (neat):
νmax 3233, 3026, 2886, 1628, 1589, 1475, 1449, 1335, 1200, 789, 740,
726, 583, 504, 484, 407 cm−1
;
1H NMR (500 MHz, CDCl3) δ 8.90 (s,
1H), 8.01 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.37–7.32 (m,
2H), 7.31–7.26 (m, 5H), 7.25–7.22 (m, 2H), 7.13–7.09 (m, 5H), 7.04
(dd, J = 8.3, 1.2 Hz, 1H), 5.88 (s, 2H), 4.72 (s, 2H), 4.08 (s, 2H), 2.45
(s, 3H). 13C NMR (125 MHz, CDCl3) δ 162.2, 138.8, 138.3, 137.1,
135.4, 133.3, 130.8, 130.7, 129.5, 128.7, 128.6, 127.9, 127.3, 126.8,
126.1, 125.3, 123.6, 122.9, 121.2, 120.9, 117.4, 112.7, 112.6, 111.1,
110.9, 50.9, 48.3, 41.7, 21.6; HRMS (ESI): m/z calcd for C33H28N3O
C
25H17Cl2N3O 484.0386; found 484.0394 [M + K]+
.
4.2.23. 10-Bromo-5-(3-chlorobenzyl)-5,7,8,13-tetrahydro-6H-azepino
[3,4-b:5,6-b']diindol-6-one 26
482.2232; found 482.2388 [M+H]+
.
Starting from 7b, compound 26 was obtained as orange solid using
a similar procedure as 16. Yield 49%; MP: 145–148 °C. IR (neat): νmax
3258, 3065, 1632, 1597, 1475, 1446, 1328, 1296, 1200, 1067, 742,
679, 516 cm−1
;
1H NMR (500 MHz, CDCl3 + DMSO) δ 11.86 (s, 1H),
4.2.27. 5,7-Dibenzyl-10-fluoro-5,7,8,13-tetrahydro-6H-azepino[3,4-b:5,6-
b']diindol-6-one 30
8.44 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 7.88 (s, 1H), 7.63 (d, J = 8.3 Hz,
1H), 7.48 (d, J = 8.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.37–7.30 (m,
4H), 7.25 (s, 1H), 7.12 (d, J = 7.0 Hz, 1H), 5.91 (s, 2H), 4.19 (d,
J = 5.3 Hz, 2H). 13C NMR (125 MHz, DMSO) δ 163.7, 141.5, 138.4,
136.3, 133.9, 133.6, 131.1, 130.9, 127.6, 127.4, 127.1, 125.9, 125.8,
124.1, 123.1, 122.1, 121.7, 120.6, 114.2, 112.6, 112.5, 111.9, 111.8,
111.7, 47.5, 35.1; HRMS (ESI): m/z calcd for C25H17BrClN3O 529.9860;
Starting from 8a, compound 30 was obtained as mustard yellow
solid using a similar procedure as 28. Yield 51%; MP: 212–214 °C. IR
(neat): νmax 3233, 3026, 2886, 1628, 1589, 1475, 1449, 1335, 1200,
789, 740, 726, 583, 504, 484, 407 cm−1
;
1H NMR (500 MHz, CDCl3) δ
9.12 (s, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.45–7.35 (m, 4H), 7.27 (s, 5H),
7.10 (s, 5H), 6.98–6.91 (m, 2H), 5.88 (s, 2H), 4.70 (s, 2H), 4.03 (s, 2H).
13C NMR (125 MHz, CDCl3) δ 162.0, 158.3 (d, J = 235.63 Hz), 138.8,
138.1, 136.8, 134.9, 133.4, 131.1, 128.9, 128.7, 128.7, 127.9, 127.5,
127.4, 126.8, 126.6, 125.5, 123.8, 123.6, 122.7, 121.4, 120.8, 112.1.
112.0, 111.9, 111.3 (d, J = 4.81 Hz), 111.0, 110.6, 110.3, 110.0, 102.8
(d, J = 24.67 Hz), 50.9, 48.4, 41.5; HRMS (ESI): m/z calcd for
found 529.9879 [M + K]+
.
4.2.24. 5-(3-Chlorobenzyl)-10,12-dimethyl-5,7,8,13-tetrahydro-6H-
azepino[3,4-b:5,6-b']diindol-6-one 27
Starting from 7b, compound 27 was obtained as yellow solid using a
similar procedure as 16. Yield 66%; MP: 244–246 °C. IR (neat): νmax
C
27H22ClN3O 592.0761; found 592.0773 [M + K]+
.
14

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
4.2.28. 5,7-Dibenzyl-10-chloro-5,7,8,13-tetrahydro-6H-azepino[3,4-
b:5,6-b']diindol-6-one 31
4.3. Biological assay methods
4.3.1. Cell culture
Starting from 8a, compound 31 was obtained as pale yellow solid
using a similar procedure as 28. Yield 51%, pale yellow solid; MP:
218–220 °C. IR (neat): νmax 3161, 3028, 2895, 1632, 1594, 1586, 1449,
Cells were obtained from National Centre for Cell Sciences (NCCS),
Pune, India and stocks were maintained in the laboratory. Prostate
cancer cells (DU143), Liver carcinoma cells (HEPG2), Colon carcinoma
cells (RKO) and Lung cancer cells (A549) and human embryonic kidney
cells (HEK) were grown in tissue culture flasks in DMEM (Dulbecco
Modified Eagle Medium), Sigma or RPMI (Roswell Park Memorial
Institute medium) supplemented with 10% fetal bovine serum with 1X
stabilized antibiotic–antimycotic solution (Sigma) in a CO2 incubator at
37 °C with 5% CO2 and 90% relative humidity.
1334, 1298, 1200, 1058, 899, 789, 738, 725, 699, 527, 434, 407 cm−1
;
1H NMR (500 MHz, DMSO) δ 11.92 (s, 1H), 8.21 (d, J = 8.1 Hz, 1H),
7.75 (s, 1H), 7.73 (d, J = 5.7 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.44 (t,
J = 7.7 Hz, 1H), 7.36–7.28 (m, J = 11.7, 6.0 Hz, 4H), 7.23 (t,
J = 6.1 Hz, 3H), 7.19 (d, J = 8.3 Hz, 2H), 7.13 (t, J = 7.7 Hz, 3H),
5.93 (s, 2H), 4.76 (s, 2H), 4.45–4.18 (m, 2H). 13C NMR (125 MHz,
CDCl3) δ 162.0, 138.8, 138.1, 136.7, 135.5, 134.4, 131.3, 130.3, 128.7,
128.7, 128.0, 127.6, 127.4, 126.8, 125.6, 124.7, 122.8, 121.5, 120.8,
120.6, 120.4, 120.3, 113.6, 112.7, 111.1, 110.7, 50.9, 48.4, 41.4;
HRMS (ESI): m/z calcd for C32H25ClN3O 502.1686; found 502.1689 [M
4.3.2. Evaluation of cytotoxicity with MTT assay
The cytotoxicity of these azepino diindolone derivatives were de-
termined using MTT assay. Cell lines (DU143, HEPG2, RKO, A549 AND
HEK-293) were used in this assay. 1X104 cells/well were seeded in
200 μL Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS in each well of 96-well microculture plates and incubated
for 24 h at 37 °C in a CO2 incubator. All the derivatives diluted to the
desired concentrations (500 nM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM,
75 μM, 100 μM and 150 μM) in culture medium, were added to the
wells with respective vehicle as control. Doxorubicin treated cells, in
the same concentration range were used as standards. After 48 h of
incubation period, 10 μL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazoliumbromide) (5 mg/mL) was added to each well and the
plates were incubated for 4 h. The supernatant was then carefully re-
moved from each well plate and the formed formazon crystals were
dissolved in 100 μL of DMSO and the absorbance at 570 nm wavelength
was recorded using an ELx800 microplate reader (BioTek, USA). Assay
was repeated thrice and mean values were considered.
+H]+
.
4.2.29. 5,7-Dibenzyl-10,12-dimethyl-5,7,8,13-tetrahydro-6H-azepino[3,4-
b:5,6-b']diindol-6-one 32
Starting from 8a, compound 32 was obtained as mustard yellow
solid using a similar procedure as 28. Yield 64%; MP: 130–133 °C. IR
(neat): νmax 3279, 3029, 1746, 1634, 1603, 1452, 1331, 1192, 1030,
799, 740, 699, 442, 407 cm−1
;
1H NMR (500 MHz, DMSO) δ 11.35 (s,
1H), 8.21 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.43–7.38 (m,
1H), 7.32–7.27 (m, 4H), 7.25 (d, J = 6.1 Hz, 1H), 7.24–7.22 (m, 2H),
7.21 (s, 1H), 7.17 (dd, J = 7.3, 1.8 Hz, 2H), 7.14–7.10 (m, 2H), 6.77 (s,
1H), 5.91 (s, 2H), 4.73 (s, 2H), 4.26 (s, 2H), 2.55 (s, 3H), 2.36 (s, 3H).
13C NMR (125 MHz, DMSO) δ 161.9, 138.9, 138.6, 138.1, 135.6, 133.0,
131.1, 129.0, 128.8, 128.6, 128.0, 127.6, 127.4, 127.1, 126.1, 125.6,
124.4, 123.1, 122.9, 121.2, 121.0, 115.5, 112.7, 111.7, 111.4, 51.1,
47.8, 42.5, 21.7, 17.6; HRMS (ESI): m/z calcd for C34H29N3O 496.2389;
found 496.2392 [M+H]+
.
4.4. Topoisomerase inhibition and DNA binding studies
4.2.30. 5,7-Bis(3-chlorobenzyl)-10-methyl-5,7,8,13-tetrahydro-6H-
azepino[3,4-b:5,6-b']diindol-6-one 33
Topoisomerase assay was performed using the assay kit that is
provided by TopoGEN Inc. The assay kit contains human topoisomerase
I and IIα enzymes (quantity provided in units), substrate DNA, buffer,
proteinase K, SDS and a positive control drug etoposide. Etoposide and
camptothecin are provided as lyophilized powders which we need to be
reconstituted using DMSO as solvent to form a final concentration of
10 mM. The enzymes need to be preserved at −80 °C.
Starting from 8b, compound 33 was obtained as light brown solid
using a similar procedure as 28. Yield 58%; MP: 181–183 °C. IR (neat):
νmax 3244, 2924, 2854, 1585, 1472, 1454, 1333, 1201, 1123, 794, 772,
744, 690, 681, 434 cm−1
;
1H NMR (500 MHz, CDCl3) δ 8.64 (s, 1H),
8.04 (d, J = 8.0 Hz, 1H), 7.47–7.43 (m, 1H), 7.43–7.40 (m, 1H), 7.39
(d, J = 4.5 Hz, 1H), 7.37 (s, 1H), 7.32–7.28 (m, 1H), 7.23 (d,
J = 1.7 Hz, 1H), 7.22 (s, 1H), 7.21–7.20 (m, 1H), 7.19–7.17 (m, 1H),
7.16–7.12 (m, 1H), 7.11 (s, 1H), 7.06 (d, J = 1.0 Hz, 1H), 7.05–7.02
(m, 1H), 7.01 (s, 1H), 5.87 (s, 2H), 4.76 (s, 2H), 4.21 (s, 2H), 2.46 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 162.0, 140.3, 139.3, 138.8, 138.8,
135.4, 134.6, 134.1, 133.1, 130.5, 130.0, 129.9, 129.8, 128.1, 127.6,
127.6, 126.9, 126.0, 125.9, 125.7, 125.0, 123.9, 123.1, 123.0, 121.6,
120.9, 117.4, 113.6, 112.7, 110.0, 110.0, 110.9, 50.7, 48.0, 41.9, 21.5;
HRMS (ESI): m/z calcd for C32H22Cl3N3O 592.0761; found 592.0773 [M
4.4.1. Decatenation assay
Kinetoplastid DNA (kDNA) (100 ng), buffer (freshly prepared by
mixing component A & B), investigational compounds (100 µM) or
etoposide (100 µM), and human topoisomerase IIα was added in re-
action mixture. Double distilled water was added to make the volume
upto 20 µL. The two components of buffer are provided separately that
we need to mix immediately before the reaction and thus makes final
buffer concentration 5X. Buffer A contained 0.5 M Trish-HCl (pH 8),
1.5 M sodium chloride, 5 mM dithiothreitol, 300 µg/mL bovine serum
albumin and buffer B contains 20 mM ATP in water. The reaction
mixture was incubated for 30 min at 37 °C. After reaction was complete
we stopped the reaction by adding proteinase K (500 µM/mL) and SDS
(10%). Incubation for 37 °C for 30 min will complete the digestion. The
samples was mixed with 10X loading dye. Resolution of the DNA
component was done on 0.8% agarose gel containing 0.5 µg/mL ethi-
dium bromide. Tris-acetate-EDTA buffer (pH 7.4) was employed for the
resolution. Visualization and quantification will be performed on Gel
DocTM EZ imager (BIO-RAD) and image Lab (BIO-RAD) respectively.
+H]+
.
4.2.31. 5,7-Bis(3-chlorobenzyl)-10-fluoro-5,7,8,13-tetrahydro-6H-azepino
[3,4-b:5,6-b']diindol-6-one 34
Starting from 8b, compound 34 was obtained as brown solid using a
similar procedure as 28. Yield 50%; MP: 210–213 °C. IR (neat): νmax
3325, 2943, 2832, 1598, 1474, 1452, 1335, 1201, 1079, 1025, 742,
681, 433, 410 cm−1; 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.03 (d,
J = 8.0 Hz, 1H), 7.47–7.44 (m, 1H), 7.44–7.41 (m, 2H), 7.24 (s, 1H),
7.22 (s, 1H), 7.21 (d, J = 4.3 Hz, 2H), 7.19 (s, 1H), 7.17 (s, 1H), 7.05 (s,
1H), 7.03 (d, J = 7.6 Hz, 1H), 6.99–6.97 (m, 1H), 6.97–6.93 (m, 2H),
5.87 (s, 2H), 4.76 (s, 2H), 4.19 (s, 2H); 13C NMR (125 MHz, CDCl3) δ
165.6, 161.4 (d, J = 345.60 Hz), 140.1, 139.0, 138.7, 137.0, 134.7,
133.0, 130.3, 130.0, 130.0, 128.0, 127.8, 127.7, 126.9, 126.0, 125.0,
121.8, 120.7, 120.4, 120.2 (d, J = 2.19 Hz), 111.1, 110.51 (d,
J = 24.21 Hz), 110.4, 50.7, 48.0, 41.9; HRMS (ESI): m/z calcd for
4.4.2. Topoisomerase IIα-Mediated DNA relaxation assay
Supercoiled plasmid DNA (pRYG) was used as substrate. In reaction
mixture 200 ng of substrate supercoiled plasmid DNA (pRYG), 5X
freshly prepared buffer (premixing component A & B), followed by
investigational compounds (100 µM) or standard drug (100 µM) and
C
32H22Cl3N3O 592.0761; found 592.0773 [M+H]+
.
15

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
lastly human topoisomerase IIα was added. Volume of reaction mixture
was adjusted upto 20 μL by adding double distilled water. The final
reaction mixture was incubated at 37 °C for 30 min. After completion
we stop the reaction by adding proteinase K (500 µM/mL) and SDS
(10%) and incubation at 45 °C for 30 min. Electrophoresis will be
carried out without ethidium bromide on 1% agarose gel in TAE buffer.
Staining of gel with ethidium bromide (0.5 µg/mL) and destaining with
water will be carried out. Visualization and quantification will be
performed on Gel DocTM EZ imager (BIO-RAD) and image Lab (BIO-
RAD), respectively.
4.5. Morphological observations
A549 cells were seeded at density of 1 × 105 cells/mL in a 6 well
plates and were treated with compound 11 at various concentrations (0,
1, 2.5, 5, 10 µM). After 48 h of treatment, morphological changes in the
cells were observed and images were captured under a phase contrast
microscope (Nikon, Inc. Japan).
4.5.1. Acridine Orange/Ethidium bromide (AO/EB) staining
A549 cells were plated at a concentration of 1x106 cells/mL were
treated with compound 11 at concentration of 0, 1, 2.5, 5, 10 µM. Plates
were incubated for 48 h at 37 °C. 10 μL each from 1 mg/mL stock
solution of fluorescent dyes containing acridine orange (AO) ethidium
bromide (EB) were added into each well in equal volumes (10 µg/mL),
respectively. Cells were visualized under fluorescence microscope
(Nikon, Inc. Japan) with excitation (488 nM) and emission (550 nM) at
200X magnification.
4.4.3. Topoisomerase IIα-Mediated DNA cleavage assay
Topo IIα cleavage assay was performed as per the reported protocol
[40]. Reaction mixtures (20 μL each) containing 40 mM Tris, pH 7.5,
100 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA,
30 μg/mL bovine serum albumin, 50 ng of 32P-labeled DNA (or 20 μg/
mL of unlabeled dimeric pRR322 DNA), 20 ng of calf thymus DNA to-
poisomerase II (or 70 ng of enzyme if unlabeled pBR322 DNA is used),
and compounds were incubated at 37 °C for 30 min. The reactions were
terminated by the addition of 2 μL of 5% SDS. Unless indicated, reaction
mixtures were treated with 150 μg/mL proteinase K for another h at
37 °C.
4.5.2. DAPI nucleic acid staining
Nuclear morphological changes were observed by DAPI staining.
After treatment with compound 11 (1, 2.5, 5 and 10 µM) for 48 h, A549
cells were washed with PBS and permeabilized with 0.1% Tween 20 for
10 min followed by staining with DAPI. Control and treated cells were
observed with fluorescence microscope with excitation at 359 nM and
emission at 461 nM using DAPI filter at 200X magnification.
4.4.4. Topoisomerase I-mediated relaxation assay
Supercoiled DNA pBR322 was used as a substrate in the topoi-
somerase I-mediated relaxation assay. In 200 ng DNA pBR322 10X
buffer (100 mM Tris-HCl (pH-7.9), 10 mM EDTA, 1.5 M sodium
chloride, 1% BSA, 1 mM spermidine, 50% glycerol), 100 µM in-
vestigational compounds or camptothecin and topoisomerase I was
added. The reaction mixture was then incubated at 37 °C for 30 min.
Proteinase K (500 µM/mL) and SDS (10%) was added to stop the re-
action and further incubation at 50 °C for 20 min will give the digested
products. Final DNA products can be resolved on agarose gel (1%).
Digested DNA fragments were photographed and analysed by Gel
DocTM EZ imager (BIO-RAD).
4.6. Flow cytometric analysis
4.6.1. Effect on mitochondrial membrane potential (Δψm)
6 well plates were seeded with A549 cells (1 × 106 cells/mL) and
allowed to adhere for overnight. The cells were incubated with 1.25,
2.5 and 5 µM concentrations of the compound 11 for 48 h. Cells were
collected and washed with PBS followed by re-suspension in solution
JC-1 (2.5 µg/mL) and incubated for 45 min in incubator at 37 °C. The
stained cells were washed twice with PBS and cells were trypsinized,
centrifuged and analysed by flow cytometer (BD FACSVerseTM, USA).
4.6.2. Cell cycle analysis
4.4.5. DNA intercalation assay
Based on the MTT assay, it was clear that compounds 11 exhibited
cytotoxicity at 4.2 μM concentration in A549 cells. Flow cytometry
analysis (FACS) was performed to evaluate the distribution of the cells
under different cell cycle phases. A549 cancer cells were incubated with
1, 2.5, 5 and 10 µM concentrations of the investigated compound 11 for
48 h. Untreated (control) and treated cells were harvested, washed with
PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide
(PI) (Sigma Aldrich). Cell cycle was performed by flow cytometry (BD
FACSVerseTM, USA).
DNA intercalation assay was carried out using negatively super-
coiled DNA (pUC19) as a substrate. Each reaction mixture contains
200 ng DNA, 100 µM investigational compounds or 1 µM ethidium
bromide and double distilled water to make the reaction volume upto
20 µL. After incubation at 37 °C for 30 min, resolution was performed at
1% agarose gel in TAE buffer without ethidium bromide. After the
staining and destaining of the gel, the gel was visualized under Gel
DocTM EZ imager (BIO-RAD) to observe the retardation.
4.6.3. Annexin V-FITC/PI assay
4.4.6. Circular dichroism
A549 cells (1x106 cells/mL) were seeded in six-well plates and al-
lowed to grow overnight. The medium was then replaced with complete
medium containing compound 11 at 1, 2.5, 5 and 10 µM concentra-
tions. After 48 h of treatment, cells from the supernatant and adherent
monolayer cells were harvested by trypsinization, washed with PBS at
3000 rpm. Then the cells were processed with Annexin V-assay kit
(FITC Annexin V Apoptosis Detection Kit, BD PharmingenTM) ac-
cording to the instructions given by the manufacturer. Further, flow
Stock solution of calf thymus DNA (CTDNA) was prepared by dis-
solving 6 mg/mL in 10 mM Tris-HCl buffer (pH-7.4) and was stored in
4 °C. It was stirred occasionally for 24 h to get homogeneous mixture.
The investigational compounds of various concentrations with com-
pound/DNA ratio 1/10, 1/6, 1/4, 1/3 were incubated at 25 °C for 1 h.
CD spectra of the complexes were recorded on JASCO-J-815CD
Spectrophotometer. Spectra were measured at far-UV (200–320 nm),
with band width of 1 nm, path length 0.1 cm at scanning speed
100 nm/min. Three scans were accumulated and averaged. Spectrum of
buffer was subtracted from CTDNA and CTDNA-compound complex.
cytometric analysis was performed using
FACSVerseTM, USA).
a flow cytometer (BD
4.7. Molecular docking
4.4.7. UV-Visible spectroscopy
UV-Visible spectra were recorded in LabIndia spectrophotometer
with quartz cuvette of having path length 1 nm. Various concentrations
of investigational compounds were incubated with CTDNA (50 μg/mL)
at 25 °C for 1 h.
Molecular docking studies were performed by using Maestro, ver-
sion 11.1 of Schrödinger suite 2017-1 [41]. The protein structure of
human DNA topoisomerase I (70 kda) in complex with the poison to-
potecan along with covalent complex with a 22 base pair DNA duplex
16

M. Kadagathur, et al.
BioorganicChemistry99(2020)103629
(PDB code: 1K4T, resolution 2.1 Å) and the three-dimensional crystal
structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) com-
plexed with minor-groove-binding drug Hoechst 33,258 (PDB code:
264D, resolution 2.44 Å) and was obtained from the RCSB PDB. It was
prepared using the Protein Preparation Wizard, wherein the protein
was hydrogenated and those protons which are necessary to define the
correct ionization and tautomerization of protein were also included at
pH 7.0. The missing or incomplete residues were repaired by employing
the Prime module. OPLS-2005 force field was used to attenuate steric
clashes that may exist in the structures under study. The energy mini-
mization was done until the energy was found to converge or the Root
Mean Square Deviation (RMSD) reached a maximum cut off of 0.30 Å.
Water molecules beyond 5 Å from hetero groups were deleted.
Molecular docking was done keeping the grid box of size 12 Å from
the centroid to ensure complete coverage of the active site. To start
with, the active site was defined by using the co-crystals of the re-
spective proteins, followed by the validation of docking protocol which
was achieved by re-docking the bound-ligand at the active site of
human DNA topoisomerase I and DNA. With the validated docking
protocol, the investigated compound is docked at the active sites of
human DNA topoisomerase I and DNA and the results were compared
with their respective co-crystallized ligands.
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Authors are thankful to NIPER Hyderabad for facilities. MK, PG,
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