Synthesis and biological evaluation of new bisindole-imidazopyridine hybrids as apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2019.03.061

A series of diindolylmethanes (5a-t) were designed, synthesized, and examined for their cytotoxicity against four human cancer cell lines like prostate (DU-145), lung (A549), breast (MCF-7) and cervical cancer (HeLa). These results revealed that among all

Full text of DOI:10.1016/j.bioorg.2019.03.061

Accepted Manuscript
Synthesis and biological evaluation of new bisindole-imidazopyridine hybrids
as apoptosis inducers
Satish Sunkari, Srinivasa Reddy Bonam, A.V.Subba Rao, Sd Riyaz, V. Lakshma
Nayak, Halmuthur Mahabalarao Sampath Kumar, Ahmed Kamal, Bathini
Nagendra Babu
PII:
S0045-2068(18)30617-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.061
YBIOO 2887
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 June 2018
19 February 2019
21 March 2019
Please cite this article as: S. Sunkari, S. Reddy Bonam, A.V.Subba Rao, S. Riyaz, V. Lakshma Nayak, H.
Mahabalarao Sampath Kumar, A. Kamal, B. Nagendra Babu, Synthesis and biological evaluation of new bisindole-
imidazopyridine hybrids as apoptosis inducers, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.03.061
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Synthesis and biological evaluation of new bisindole-
imidazopyridine hybrids as apoptosis inducers
Satish Sunkaria,c, Srinivasa Reddy Bonamb,c†, A.V.Subba Raof, Sd Riyazd, V. Lakshma Nayaka,
Halmuthur Mahabalarao Sampath Kumarb,c, Ahmed Kamal a,c,e *, Bathini Nagendra Babua,c *
aCentre for Semio Chemicals, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad,
500007, India
bVaccine Immunology Laboratory, Natural Product Chemistry Division, CSIR-Indian Institute of
Chemical Technology (IICT), Hyderabad, 500007, India
cAcademy of Scientific and Innovative Research, New Delhi, 110 025, India
d Department of chemistry, Jawaharlal Nehru Technological University, Hyderabad-500085
†Present address: CNRS, Biotechnology and cell signaling, University of Strasbourg, 67000
Strasbourg, France /Laboratory of excellence Medalis, France.
eSchool of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi,
110062, India
fPresent address: Department of Pharmacology and Molecular Sciences, Johns Hopkins
University, School of Medicine, 725 North Wolfe St., Baltimore, Maryland 21205, United States
Keywords: Bisindole-imidazopyridine, heterocyclic, ROS, cytotoxicity, apoptosis
Corresponding author: Dr. Bathini Nagendra Babu and Dr. Ahmed Kamal
Email: bathini@iict.res.in, ahmedkamal.iict@gov.in

Abstract
A series of diindolylmethanes (5a-t) were designed, synthesized, and examined for their
cytotoxicity against four human cancer cell lines like prostate (DU-145), lung (A549), breast
(MCF-7) and cervical cancer (HeLa). These results revealed that among all the hybrids, two (5k
and 5r) were identified and exhibited significant cytotoxic effect against A549 cancer cells with
IC50 values of 1.65±0.3 and 1.80±0.8 µM respectively. To investigate the reasons for the
cytotoxic activity, the conventional biological assays were carried out with 5k and 5r on the
A549 cancer cells. Both hybrids led to the arrest of A549 cell lines at the G2/M phase of the cell
cycle and strongly induced apoptosis. Further the apoptotic effects of 5k and 5r were confirmed
by ROS, annexin-V FITC, and mitochondrial membrane potential. Moreover, structure−activity
relationships were elucidated with various substitutions on these hybrids.
Introduction
Indoles and their derivatives are well known as an important class of heterocyclic
compounds in many distinct areas of biology and medicinal chemistry, probably one of the most
important frameworks in drug discovery.1,2 Bisindole nucleus is present in many natural products
isolated from marine sources with broad spectrum of biological activities.3 Interestingly the
bisindoles are active metabolites of indole containing natural products responsible for their
anticancer activity. Indole‐3‐carbinol (I3C, 1) is an ingredient of cruciferous vegetables known
for suppressing the proliferation and induces apoptosis in various cancer cells.4,5 In many
early‐phase clinical trial studies indicated that I3C works like a prodrug, which forms active
metabolites in the stomach, 6,7 which are the acid condensation products of I3C usually referred
to as oligomer, that exerts in vivo antitumor effects. Among the 20 oligomers namely
diindolylmethane (DIM, 2), is the major oligomer that reduces prostate tumor growth in mice.8
various anticancer bisindole derivatives of DIM have been developed by different research
groups across the globe. Further investigations showed that diindolylmethane is a strong
mitochondrial Hþ-aTPase inhibitor and stimulates mitochondrial reactive oxygen species (ROS)
production.9 Thus DIM could be considered an important class of anticancer chemotherapeutic
lead that may provide a number of medicinally useful compounds.
On the other hand, nitrogen-bridgehead fused heterocycles containing an imidazole ring
are common structural components in several pharmacologically important molecules that

display a wide range of activities for diverse number of targets. One of the most widely used
heterocyclic systems from this research group is the imidazopyridines,10 which shows a broad
spectrum of biological activities such as inhibitors of aromatase, estrogen production
suppressors,11 positive inotropic agents,12 platelet aggregation inhibitors and thromboxane
synthetase inhibitors.13 Imidazopyridines exhibit cytotoxic activity through different molecular
mechanisms such as vascular endothelial growth factor (VEGF)-receptor KDR (kinase insert
domain receptor) inhibition and the induction of apoptosis.14,15 Recently, we have reported that
the synthesis of imidazopyridine-oxindole (3)16 and imidazopyridine-benzimidazole derivatives
(4)17 as potential anticancer agents.
As a part of our continuous research to develop potent anticancer agents and considering
the diverse chemotherapeutic potential of bisindole and imidazopyridine scaffolds, we have
planned to explore the potential beneficial effects of incorporating both the scaffolds in a single
structure. Thus, a new hybrid has been designed by combining both these active scaffolds as
shown in Figure 1. The present work describes the synthesis of a series of
bisindole‐imidazopyridine hybrids (5a-t) and their evaluation for cytotoxic activity apart from
their effect of apoptotic cell death.

Figure 1 Lead structure of biologically active antitumor agents. (1, Carbinol), (2,
Dimethylindole), and imidazopyridine containing scaffolds (3, 4, and 5a-t).
Results and discussions
Chemistry
Synthesis of the bisindole-imidazopyridine hybrids (5a-t) was accomplished as described
in Scheme 1. The intermediates N1-(2-pyridylmethyl)-substituted benzamides (8a-e) were
prepared by the reaction of different substituted benzoyl chlorides (7a-e) and 2-
pyridylmethanamine (6) in the presence of triethylamine. The cyclization of N1-(2-
pyridylmethyl)-substituted benzamides (8a-e) in POCl3 gave phenyl substituted imidazo[1,5-
a]pyridines (9a-e) in good yields, which were subjected to Vilsimier-formylation with POCl3 in
dry DMF to give 3-(substituted phenyl)imidazo[1,5-a]pyridine-1-carbaldehyde (10a-e). The
reaction between (10a-e) and substituted indoles (11a-d) in ethanol using sulfamic acid afforded
thebisindole-imidazopyridine hybrids (5a-t) in excellent yields.

Reagents and Conditions , , C to rt, 3 h; (b) POCl3, reflux, 3 h; (c) DMF,
POCl3, C, 3 h; (d) EtOH, Cat. Sulfamic acid, rt, 8 h.
Result and Discussion
Biology
Cytotoxic activity
All the synthesized bisindole-imidazopyridine hybrids (5a-t) were evaluated for their
antiproliferative activity against four human tumor cell lines: DU-145 (prostate cancer), A549
(lung cancer), MCF (breast cancer), and HeLa (cervical cancer) employing MTT assay.18 DIM
was used as the reference standard and the results obtained are shown in Table 1. Interestingly
but not surprisingly, all the compounds (5a-t) showed more pronounced activity against A549

cells compared to other cancer cell lines tested. These conjugates displayed cytotoxic activity
comparable to the standard DIM.
Table 1 Cytotoxicity of bisindole-imidazopyridine hybrids (5a-t) against a panel of human
cancer cell lines.
Compound
DU-145b
A549c
MCF-7d
HeLae
22.4±1.8
9.8±0.6
5.1±1.2
3.7±0.89
5.5±0.04
6.9±0.2
22.2±1.1
9.7±0.06
19.7±1.4
16.8±0.1
12.8±0.5
15.5±0.04
14.7±0.5
16.5±0.9
12.4±1.07
16.8±0.9
2.1±0.5
16.7±1.8
8.8±0.3
5a
5b
5c
8.9±0.7
15.2±1.3
11.6±1.3
11.8±0.02
11.4±6.7
11.2±1.4
23.7±1.7
11.7±0.05
15.3±0.3
6.7±1.5
12.9±0.2
10.0±0.3
13.0±1.3
11.8±0.14
13.5±0.7
9.4±0.7
5d
5e
10.5±0.4
10.8±0.6
9.5±0.7
5f
5g
5h
5i
11.7±1.1
11.9±0.5
11.5±0.2
1.65±0.3
10.9±0.1
16.6±0.6
3.9±0.2
11.4±0.6
4.7±0.8
5j
5k
5l
9.3±0.7
15.7±1.2
18.9±0.8
12.7±0.4
11.6±0.3
11.9±0.5
10.2±1.1
2.3±0.2
10.9±0.07
13.9±0.6
13.0±3.2
12.6±1.5
13.0±0.5
11.1±0.5
7.2±2.0
13.8±0.4
9.5±0.3
5m
5n
5o
5p
5q
5r
10.8±0.8
10.6±0.8
15.1±0.6
7.9±1.6
2.0±0.2
3.2±0.4
11.6±0.9
1.80±0.8
12.3±0.2
13.2±1.5
4.9±0.08
17.8±0.4
37.9±1.2
7.8±0.12
15.5±4.2
16.3±0.7
5.3±1.88
13.2±0.7
17.2±0.01
10.8±0.18
5s
5t
DIM
a50% Inhibitory concentration after 48 h of drug treatment; bhuman prostate cancer; chuman lung
cancer; dhuman breast cancer; ehuman cervical cancer. Results are represented as mean ± SD of
three independent experiments, each performed in triplicates.

Structure-activity Relationship
The structure-activity relationship (SAR) is established Based on cytotoxic results against four
different cell lines (Table 1). The effect of various groups on the imidazopyridine ring as well as
indole ring were exemplified by preparing analogs possessing both electron donating as well as
electron withdrawing substituents viz. methoxy, dimethoxy, bromo, cyano, fluoro, and
trifluoromethyl groups. It was observed that hybrids with either unsubstituted or substituted
imdiazopyridine (5a-5t) showed weaker potency against all the cancer cell lines expect few
hybrids such as 5b, 5k, 5n, 5o, 5p, and 5r which are active against A549 cell line. Methoxy
substituent on the imidazopyridine ring (5e-h) showed significant loss in potency against all the
cell lines, comparably better activity was observed with unsubstituted hybrids (5a-d) against
A549 cell lines. Introduction of an additional methoxy group on the imidazopyridine ring (5i-l)
also resulted in no improvement in the potency, except 5k which dramatically increased the
potency in all the cell lines especially in A549 by 5k with IC50 v lue with 1.65 μM. Moreover,
introduction of strong electron withdrawing substituent like trifluoromethyl (5m-5p) and fluoro
(5q-5t) on the imidazopyridine ring also did not improve the potency against all the cancer cell
lines except A549 cell line where compounds 5n, 5o, 5p and 5r showed significant cytotoxicity
with values ranging from 1.8-3.9 μM. However, for MCF-7 cell line, fluoro substituent increased
the cytotoxicity activities of the hybrids.
In addition, we further examined the effect of various groups such as methoxy, bromo
and cyano on the indole ring at the 5-position. Unsubstituted indole ring containing hybrids (5a,
5e, 5i, 5m, and 5q) showed decreased activity against all the cell lines evaluated. Electron-
releasing methoxy group on the indole ring (5b, 5f, 5j, 5n, and 5r) substantially altered the
potency. Particularly, hybrids 5b, 5n, and 5r resulted in a 4 to 6-fold increase in antiproferative
activity against A549 cell lines. Among which, 5r is the potent most hybrid with IC50 values
ranging from 1.65-6.7 μM g inst all the cell lines. Electron-withdrawing substituents such as
bromo and cyano have no substantial effect on the cytotoxicity, where bromo substituted hybrids
(5c, 5g, 5k, 5o, and 5s) were comparably more potent than the corresponding cyano
substituentuted hybrids (5d, 5h, 5l, 5p, and 5t) in A549 cell lines. Among all the synthesized
hybrids, 5k and 5r were the potent most compounds against all the cell lines, particularly against
A549 cell lines.

Based on the results obtained from the cytotoxicity assay, the most cytotoxic conjugates
(5k and 5r) were taken up for the detailed biological studies, such as cell cycle analysis, reactive
oxygen species (ROS) generation, apoptosis determination, and measurement of mitochondrial
membrane potential.
Cell cycle analysis
Many cytotoxic compounds exert their effect by blocking cell cycle progression or by
inducing apoptosis or combined effect of both.20,21 In order to understand the mode of action of
these potential conjugates such as (5k and 5r), we examined their effects on cell cycle
progression by flow cytometry analysis on A549 cancer cells. In this study, A549 cells were
treated with mentioned conjugates at concentrations of 1.5 and 3 µM for 24 h. The data obtained
clearly indicates that DIM, conjugates 5k and 5r are altering the cell cycle progression
profoundly. The compounds 5k at 1.5 µM and 3 µM shows 18.11% and 21.43% in G2/M phase.
Where in 5r has shown 18.58% and 23.91% in G2/M phase arrest at 1.5 µM and 3 µM
respectively. Whereas DIM showed 24.27% arrest in G2/M at 5 µM concentration. Moreover,
percentage distribution of cells in various phases of the cell cycle is indicated by the bar graph
(Figure 2).

Figure 2 Cell cycle analysis. A549 cells were treated without or with DIM, 5k, and 5r at
indicated concentrations for 24 h. DNA content in each phase was measured by propidium iodide
(PI) staining. Data was represented as histograms (a) and bar graphs (b). Error bars are indicative
of mean ± SD of two independent experiments.
Measurement of reactive oxygen species (ROS)

Various categories of chemotherapeutic drugs, either natural or synthetic induce ROS
signals, which is an important strategy in drug development studies. Accumulation of ROS in the
A549 cells treated with DIM, 5k, and 5r fo 24 h w s estim te b DC D . 2’, 7’–
dichlorofluorescindiacetate a non-fluorescent substance and is converted to 2’, 7’–
dichlorofluorescein (DCF) inside the cells through oxidation by ROS. Upon treatment with 5k
and 5r at indicated concentrations increased ROS production was observed in a dose dependent
manner (Figure 3). However, this is not comparable to DIM.

Figure 3 Determination of ROS generation in A549. Fluorescent images are obtained from the
cells treated without or with the DIM, 5k, and 5r for 24 h and then observed the production of
ROS by DCFDA (a). Bar graph represents the mean fluorescence intensity of corrected total cell
fluorescence (CTCF) (b). Error bars are indicative of mean ± SD of two independent
experiments, each performed in triplicates.
Determination of apoptosis by using Annexin V-FITC/PI
Quantification of apoptosis induced by DIM, 5k, and 5r was determined by using
annexin V-FITC/PI staining. This assay provides the extent of live, early apoptotic, late
apoptotic, and necrotic cells as shown in Figure 4. The percentage of early apoptotic cells in the
5k and 5r increased at 1.5 µM (26.13% and 37.93%) to 3 µM (28.05% and 48.77%), whereas
DIM has shown 24.56% at 5 µM. Therefore, these results prove that the conjugates 5k and 5r
induce apoptosis in a dose dependent manner.

Figure 4 Compounds induce apoptosis in A549 cells. Cells were treated without or with DIM,
5k, and 5r for 24 h. Cells were labelled with Annexin V FITC, PI and analyzed by flow
cytometry. Percentage of cells (a) in each phase is represented in the bar graph (b). Error bars are
indicative of mean ± SD of two independent experiments.

Measurement of mitochondria membrane potential (∆Ψm)
Mitochon i memb ne potenti l ∆Ψm w s ete mine b using JC-1 dye. Intact
mitochondrion with energized membrane potential shows red aggregates in presence of JC-1
dye. Potent apoptotic inducers manifest green fluorescence with JC-1 dye by losing the
membrane potential as de-energized. Cells treated with DIM, 5k, and 5r showed the increased
green fluorescence as compared to untreated cells (Figure 5).

Figure 5 Compounds induce mitochondrial membrane potential in A549 cells. a). Cells were
treated without or with DIM, 5k, and 5r for 24 h and observed the changes in mitochondrial
membrane potential by flow cytometry. b). Bar graph represents the percentage distribution of J-
aggregates and J-monomer cells in all groups. Error bars are indicative of mean ± SD of two
independent experiments.
Conclusion
A series of diindolylmethanes (5a-t) were designed, synthesized using sulfhamic acid and
evaluated for their cytotoxicity against three human cancer cell lines like prostate (DU-145), lung
(A549), breast (MCF-7) and cervical cancer (HeLa) with IC50 values ranging from 1.6 to 22.4
µM. Among them, two (5k and 5r) were identified and exhibited significant cytotoxic effect
against A549 cancer cells with IC50 values of 1.65±0.3 and 1.80±0.8 µM respectively. To
investigate the reasons for the cytotoxic activity, the conventional biological assays were carried
out with 5k and 5r on the A549 cancer cells. Both hybrids led to the arrest of A549 cell lines at
the G2/M phase of the cell cycle and strongly induced. Further the apoptotic effects of 5k and 5r
were confirmed by ROS, annexin-V FITC, and mitochondrial membrane potential. Moreover,
structure−activity relationships were elucidated with various substitutions on these hybrids.
Detailed biological studies like ROS, annexin-V FITC and mitochondrial membrane potential
demonstrate that these hybrids induce apoptotic cell death. These results suggest that such
hybrids have the potential to be developed as new template for the potential anticancer agents.
Acknowledgements
The authors thank CSIR-India and UGC, New Delhi, India for the award of fellowships and the
author RS thank CSIR-HRDG (13(8906-A)/2017-pool). We also thanks funding received from
the p oject, entitle ‘ ffo ble C nce he peutics C ’ un e XIIth five-year plan.
Manuscript Communication Number: IICT/Pubs./2018/180
Material and methods
Chemistry

All reagent, starting materials, and solvents were purchased from commercial sources and used
1
without further purification. H and 13C NMR spectra were recorded with 300, 400, and 500
MHz spectrometer in CDCl3, DMSO-d6 solutions with tetramethylsilane (TMS) as an internal
standard. High-resolution mass spectra (ESIHRMS) were obtained by using ESI-QTOF mass
spectrometer. All reactions were monitored by thin-layer chromatography (TLC) using precoated
silica gel 60 F254 Merck and components were visualized by exposure to UV light. Column
chromatography was performed using silica gel (60-120 mesh). Melting points were determined
on an electro thermal melting point apparatus and are uncorrected.
General procedure for the synthesis of N1-(2-pyridylmethyl) substituted benzamides (8a-e)
Method A: To a stirred solution of 2-pyridylmethanamine 6 (1 mmol) in dry THF was added
triethylamine (3.0 mmol) followed by substituted benzoylchlorides (7a-e, 1.1 mmol) at 0 °C. The
reaction mixture was stirred for 3 h and the reaction was monitored by TLC. After the
completion of the reaction, THF was removed under vacuum to get the crude products. This was
further purified by column chromatography (EtOAc-Hexane) to obtain the pure compounds (8a-
e).
N1-(2-Pyridylmethyl) benzamide (8a): This compound was prepared according to the method
A, employing benzoylchlorides 7a (1 mmol) to obtain the pure product 8a. Yield: 76%; 1H NMR
(300 MHz, CDCl3 δ 8.53 , J = 4.7 Hz, 1H), 7.88 (d, J = 6.4 Hz, 2H), 7.67 (t, J = 7.7 Hz, 2H),
7.47–7.39 (m, 3H), 7.32 (d, J = 7.9 Hz, 1H), 7.20 (t, J = 5.0 Hz, 1H), 4.73 (d, J = 4.7 Hz, 2H);
MS (ESI): m/z 213 [M + H]+.
N1-(2-Pyridylmethyl)-4-methoxybenzamide (8b): This compound was prepared according to
the method A, employing 4-methoxy benzoylchloride 7b (1 mmol) to obtain the pure product 8b.
1
Yield: 82%; H NMR (300 MHz, CDCl3 δ 8.56 , J = 4.8 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H),
7.68 (t, J = 7.8 Hz, 1H), 7.47 (brs, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 5.8 Hz, 1H), 6.93 (d,
J = 8.7 Hz, 2H), 4.75 (d, J = 4.8 Hz, 2H), 3.85 (s, 3H); MS (ESI): m/z 243 [M + H]+.
N1-(2-Pyridylmethyl)-3,4-dimethoxybenzamide (8c): Compound was prepared according to
the method A, employing 3,4-dimethoxy benzoylchloride 7c (1 mmol) to obtain the pure product
1
8c Yield 78%, H NMR (300 MHz, CDCl3 δ 8.52 , J = 4.5 Hz, 1H), 7.70-7.62 (m, 2H), 7.45
(d, J = 2.2 Hz, 1H), 7.39-7.31 (m, 2H), 7.20 (dd, J = 6.0, 5.2 Hz, 1H), 6.82 (d, J = 8.3 Hz, 1H),
4.70 (d, J = 5.2 Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H); MS (ESI): m/z 273 [M + H]+.

N-(Pyridin-2-ylmethyl)-4-(trifluoromethyl)benzamide (8d): This compound was prepared
according to the method A,employing 4-trifluoromethyl benzoylchloride 7c (1 mmol) toobtain
1
the pure product 8c. Yield: 77%; H NMR (300 MHz,CDCl3 δ 8.51 , J = 5.2 Hz, 1H), 8.15
(brs, 1H), 7.96 (d, J = 7.5Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 7.5 Hz, 1H), 7.19(q, J =
5.2, 1.5 Hz, 1H), 4.71 (d, J = 4.5 Hz, 2H); MS (ESI): m/z281 [M + H]+.
N1-(2-Pyridylmethyl)-4-fluorobenzamide (8e): This compound was prepared according to the
1
method A,employing 4-fluoro benzoylchloride Yield 75%, H NMR (300 MHz, CDCl3 δ 8.55
(d, J= 4.6 Hz, 1H), 8.00 (brs,1H), 7.72 (d, J = 8.1 Hz, 2H), 7.53 (t, J = 6.2, 6.0 Hz, 1H), 7.40 (d,
J = 8.1 Hz, 2H), 7.25 (m, 1H), 7.2 (dd, J = 6.2,5.6 Hz, 1H), 4.71 (d, J = 5.0 Hz, 2H); MS (ESI):
m/z 231 [M + H]+.
General procedure for the synthesis of substitutedphenylimidazo[1,5-a]pyridine (9a–e)
Method B: To N1-(2-pyridylmethyl)-substituted benzamides (8a-e) (1 mmol), 4 mL of POCl3
was added and refluxed for 3 h. This was poured into cold water and neutralized with NaHCO3
solution. This water layer was extracted three times with ethylacetate. The combined organic
phases were dried over anhydrous Na2SO4 and evaporated under vacuum. Thus, the residue
obtained was purified by column chromatography using ethylacetate and hexane as solvent
system to afford the pure compounds (9a-e).
3-Phenylimidazo[1,5-a]pyridine (9a): This compound was prepared according to the method B.
1
Yield: 78%; H NMR (300 MHz, CDCl3 δ 8.24 , J = 7.5 Hz, 1H), 7.76 (d, J = 6.7 Hz, 2H),
7.50-7.36 (m, 5H), 6.67 (dd, J = 9.0, 6.7 Hz, 1H), 6.51 (t, J = 6.7 Hz, 1H); MS (ESI): m/z 195
[M + H]+.
3-(4-Methoxyphenyl)imidazo[1,5-a]pyridine (9b): This compound was prepared according to
the method B. Yield: 80%; 1H NMR (300 MHz, CDCl3 δ 8.34 , J = 8.7 Hz, 1H), 8.32 (d, J =
6.8 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.32-7.17 (m, 1H), 7.07 (d, J = 8.7 Hz, 2H), 6.98 (s, 1H),
6.88 (t, J = 6.8 Hz, 1H), 3.90 (s, 3H); MS (ESI): m/z 225 [M + H]+.
1
3-(3,4-Dimethoxyphenyl)imidazo [1,5-a]pyridine (9c): Yield 76%, H NMR (500 MHz,
CDCl3 δ 8.18 , J = 7.2 Hz, 1H), 7.47 (s, 1H), 7.40 (d, J = 9.3 Hz, 1H), 7.30 (s, 1H), 7.27 (d, J
= 8.3 Hz, 1H), 6.93 (d, J = 7.2 Hz, 1H), 6.64 (dd, J = 7.2, 6.2 Hz, 1H), 6.49 (t, J = 7.2, 6.2 Hz,
1H), 3.90 (s, 3H), 3.87 (s, 3H); MS (ESI): m/z 255 [M+H]+.

3-(4-(Trifluoromethyl)phenyl)imidazo[1,5-a]pyridine (9d): This compound was prepared
according to the method B. Yield: 78%; 1H NMR (300 MHz, CDCl3 δ 8.28 , J = 7.5 Hz, 1H),
7.91 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.54 (s, 1H), 7.47 (d, J = 9.0 Hz, 1H), 6.73 (q,
J = 6.7, 2.2 Hz, 1H), 6.59 (t, J = 6.0 Hz, 1H); MS (ESI): m/z 263 [M + H]+.
1
3-(4-Fluorophenyl)imidazo [1,5-a]pyridine (9e): Yield 85%, H NMR (300 MHz, CDCl3 δ
8.16 (d, J = 7.5 Hz, 1H), 7.75 (dd, J = 9.0, 5.2 Hz, 2H), 7.49 (s, 1H), 7.47 (d, J = 9.0 Hz, 1H),
7.26-7.17 (m, 2H), 6.69 (dd, J = 9.0, 6.7 Hz, 1H), 6.53 (t, J = 6.7, 6.0 Hz, 1H); MS (ESI): m/z
213 [M + H]+.
General procedure for the synthesis of 3-(substituted phenyl)- imidazo[1,5-a]pyridine-1-
carbaldehydes (10a-e) Method C: To an ice-water cooled solution of substitutedphenyl
imidazo[1,5-a]pyridines (9a-c) (1 mmol) in DMF(1.4 mmol) was added drop wise POCl3 (1.4
mmol)with stirring,and then reaction mixture was heated to 100 °C for 2 h. After the completion
of the reaction, the reaction mixture waspoured into ice water and quenched with ammonium
hydroxidesolution. The aqueous solution was extracted with ethylacetate and the resultant
organic layer was dried over anhydroussodium sulphate and evaporated under vacuum. Thus,the
residue obtained was purified by column chromatographyusing ethylacetate and hexane as
solvent system to afford purecompounds (10a-e).
3-Phenylimidazo[1,5-a]pyridine-1-carbaldehyde (10a): Thiscompound was prepared
1
according to the method C. Yield:80%; H NMR (300 MHz, CDCl3 δ 1 .1 s, 1 , 8.39 , J
=8.3 Hz, 2H), 7.77 (d, J = 6.6 Hz, 2H), 7.58–7.46 (m, 3H), 7.22(dd, J = 9.4, 6.7 Hz, 1H), 6.86 (t,
J = 6.7 Hz, 1H); MS (ESI): m/z223 [M + H]+.
3-(4-Methoxyphenyl)imidazo[1,5-a]pyridine-1-carbaldehyde (10b): This compound was
1
prepared according to the methodC. Yield: 76%; H NMR (400 MHz, CDCl3 δ 1 .16 s, 1 ,
8.34(d, J = 8.7 Hz, 2H), 8.31 (d, J = 6.8 Hz, 1H), 7.26-7.23 (dd, J =8.7 Hz, 2H), 7.07 (d, J = 8.7
Hz, 2H), 6.88 (t, J = 7.8 Hz, 1H),3.90 (s, 3H); MS (ESI): m/z 253 [M + H]+.
3-(3,4-dimethoxyphenyl)imidazo[1,5-a]pyridine-1-carbaldehyde (10c): This compound was
1
prepared according to the method C. Yield 82%, H NMR (500 MHz, CDCl3 δ 1 .12 s, 1 ,
8.35 (d, J = 5.6 Hz, 2H), 7.30 (s, 1H), 7.25 (s, 1H), 7.21 (t, J = 7.2, 6.4 Hz, 1H), 6.96 (d, J = 8.0
Hz, 1H), 6.84 (t, J = 7.2, 6.2 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H); MS (ESI): m/z 283 [M + H]+.

3-(4-(Trifluoromethyl)phenyl)imidazo[1,5-a]pyridine-1-carbaldehyde (10d): This compound
1
was prepared according tothe method C. Yield: 80%; H NMR (300 MHz, CDCl3 δ 1 .16 s,
1H), 8.42–8.38 (m, 2H), 7.95 (d, J = 8.3 Hz, 2H), 7.83 (d, J =8.3 Hz, 2H), 7.34-7.29 (q, J = 6.0,
3.0 Hz, 1H), 6.97 (t, J =7.5 Hz, 1H); MS (ESI): m/z 291 [M + H]+.
3-(4-(Fluoro)phenyl)imidazo[1,5-a]pyridine-1-carbaldehyde (10e): This compound was
1
prepared according to the method C. Yield: 80%; H NMR (400 MHz, CDCl3 δ 1 .17 s, 1 ,
8.37 (dt, J = 9.1, 1.1 Hz, 1H), 8.31 (dt, J = 7.1, 1.0 Hz, 1H), 7.82-7.77 (m, 2H), 7.33-7.24 (m,
3H), 6.96-6.89 (m, 1H); MS (ESI): m/z 241 [M + H]+.
General procedure for the synthesis of compounds (5a-t) Method D: To a solution
appropriate aldehyde (10a-e) in ethanol (10 mL), appropriate indole substituents (11a-e) (2.5
mmol) and catalytic amount of sulfamic acid dissolved in H2O (5 mL) were sequentially added,
and then the reaction mixture was refluxed for 8 h. After the completion of reaction, solvent was
removed by vacuum to obtain crude product, which was extracted with ethyl acetate. The
combined organic extracts were dried over anhydrous Na2SO4 and concentrated to obtain crude
products, which were further purified by column chromatography using ethylacetate and hexane
as solvent system to afford pure compounds (5a-t). The purities of isolated products were
determined by HPLC column Symmetry [R]-C18, 5µM, 4.6nm×254nm, 75% AcCN, 25%
NH4OAc, PH-4.2 adjusted with formic acid.
Di(1H-indol-3-yl)methyl)-3-phenylimidazo [1,5-a]pyridine (5a):This compound was prepared
according to the method D. Yield: 93%; ; Rf = 0.64 (ethyl acetate/n-hexane, 1:1); Melting point:
275-277 0C; 1H NMR (300 MHz, CDCl3+DMSO-d6 δ 1 .19 s, 1 , 8.2 -8.08 (m, 1H), 7.71 (d,
J = 7.3 Hz, 2H), 7.47-7.30 (m, 5H), 7.27 (d, J = 8.1 Hz, 2H), 7.22-7.14 (m, 1H), 6.96 (t, J = 7.7
13
Hz, 2H), 6.89 (s, 2H), 6.80 (t, J = 7.4 Hz, 2H), 6.47-6.38 (m, 2H), 6.20 (s, 1H); C NMR (75
MHz, CDCl3+DMSO-d6 δ135.64, 134.9 , 134.2 , 129.23, 127.74, 127.14, 126.69, 126.43,
125.85, 122.52, 120.07, 119.86, 118.21, 117.68, 117.21, 116.51, 116.41, 111.89, 110.36, 32.64;
HRMS (ESI) Calcd for C30H23N4[M+H]+ 439.1917, found 439.1912. Purity by HPLC UV (254
nm) ESI-MS: 97.30%.
1-(Bis(5-methoxy-1H-indol-3-yl)methyl)-3-phenylimidazo[1,5-a]pyridine
(5b):
This
compound was prepared according to the method D. Yield: 92%; Rf = 0.42 (ethyl acetate/n-

hexane, 1:1); Melting point: 280-282; 1H NMR (400 MHz, DMSO-d6 δ 1 .59 s, 2 , 8.39 , J
= 7.2 Hz, 1H), 7.86-7.81 (m, 2H), 7.71 (d, J = 9.1 Hz, 1H), 7.53 (t, J = 7.3 Hz, 2H), 7.42 (t, J =
7.4 Hz, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 7.18 (d, J = 2.3 Hz, 2H), 7.04 (d, J = 2.4 Hz, 2H), 6.72-
6.68 (s, 1H), 6.67 (d, J = 2.4 Hz, 1H), 6.65 (d, J = 2.4 Hz, 1H), 6.62 (td, J = 7.3 1.2 Hz, 1H), 6.17
(s, 1H), 3.61 (s, 6H); 13C NMR (100 MHz, DMSO-d6 δ 152.47, 135.51, 135.39, 131.5 , 13 .23,
128.85, 128.01, 127.27, 127.12, 127.01, 123.98, 121.55, 118.31, 117.68, 117.11, 113.15, 111.77,
110.40, 101.56, 55.11, 32.28;HRMS (ESI) Calcd forC32H27N4O2[M+H]+ 499.2134, found
499.2202. Purity by HPLC UV (254 nm) ESI-MS: 96.22%.
1-(Bis(5-bromo-1H-indol-3-yl)methyl)-3-phenylimidazo[1,5-a]pyridine (5c):This compound
was prepared according to the method D. Yield: 86%; Rf = 0.74 (ethyl acetate/n-hexane, 1:1);
Melting point: 273–275;1H NMR (300 MHz, CDCl3+DMSO-d6 δ 1 .77 s, 2 , 8.26 , J = 6.7
Hz, 1H), 8.04 – 7.95 (m, 1H), 7.78 (d, J = 7.7 Hz, 2H), 7.65 (s, 1H), 7.47 (t, J = 7.7 Hz, 3H),
7.36 (t, J = 7.4 Hz, 1H), 7.26-7.18 (m, 2H), 7.14-7.01 (m, 4H), 6.66-6.48 (m, 2H), 6.14 (s,
1H);`13C NMR (75 MHz, CDCl3+DMSO-d6 δ 136.16, 134.35, 133.73, 132.85, 128.71, 127.2 ,
126.87, 126.56, 126.05, 125.78, 123.30, 121.77, 120.24, 119.75, 116.64, 116.23, 115.43, 111.57,
111.49, 109.51, 31.21; HRMS (ESI) Calcd for C30H21N4Br2[M+H]+ 595.0033,found 594.9945.
Purity by HPLC UV (254 nm) ESI-MS: 87.07%.
3,3'-((3-Phenylimidazo[1,5-a]pyridin-1-yl)methylene)bis(1H-indole-5-carbonitrile) (5d):
This compound was prepared according to the method D. Yield: 84%; Rf = 0.25 (ethyl acetate/n-
hexane, 1:1); Melting point: 278-280;1H NMR (300 MHz, CDCl3+DMSO-d6 δ 11.41 s, 2 ,
8.39 (d, J = 7.1 Hz, 1H), 8.39 (s, 2H), 7.90 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 7.4 Hz, 2H), 7.59-
7.54 (m, 4H), 7.53-7.43 (m, 3H), 7.36 (d, J = 8.2 Hz, 2H), 6.80 (t, J = 6.6 Hz, 1H), 6.67 (t, J =
13
6.6 Hz, 1H), 6.46 (s, 1H); C NMR (125 MHz, DMSO-d6 δ 138. 3, 135.86, 128.91, 127.49,
127.42, 127.18, 126.25, 126.09, 125.97, 125.20, 125.14, 123.43, 121.72, 121.57, 120.88, 118.37,
117.93, 117.87, 113.43, 112.78, 100.05, 30.97; HRMS (ESI) Calcd for C32H21N6[M+H]+
489.1828, found 489.1874. Purity by HPLC UV (254 nm) ESI-MS: 92.27%.
1-(Di(1H-indol-3-yl)methyl)-3-phenylimidazo [1,5-a]pyridine (5e): This compound was
prepared according to the method D. Yield: 90%; Rf = 0.35 (ethyl acetate/n-hexane, 3:2);
1
Melting point: 281-283; H NMR (300 MHz, CDCl3+ DMSO-d6 δ 1 .73 s, 2 , 8.27 , J =
7.3 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.22-

13
7.10 (m, 5H), 7.04-6.93 (m, 5H), 6.93-6.84 (m, 1H), 6.49 (s, 1H), 3.91 (s, 3H); C NMR (75
MHz, CDCl3+DMSO-d6 δ 159.31, 136.56, 135.75, 134.93, 130.02, 129.15, 126.84, 123.45,
122.72, 121.40, 120.88, 119.27, 118.43, 118.22, 117.58, 117.45, 114.40, 112.99, 111.46, 55.31,
33.01;HRMS (ESI) Calcd for C31H25N4O [M+H]+ 469.2028, found 469.2024. Purity by HPLC
UV (254 nm) ESI-MS: 86.27%.
1-(Bis(5-methoxy-1H-indol-3-yl)methyl)-3-(4-methoxyphenyl)imidazo[1,5-a]pyridine (5f):
This compound was prepared according to the method D. Yield: 94%; Rf = 0.26 (ethyl acetate/n-
1
hexane, 3:2); Melting point: 253-255; H NMR (300 MHz, CDCl3+DMSO-d6 δ 9.67 s, 2 ,
8.02 (dd, J = 12.5, 7.9 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.12-7.05 (m,
1H), 6.93 (d, J = 8.7 Hz, 2H), 6.83 (dd, J = 11.8, 1.9 Hz, 4H), 6.63 (dd, J = 8.7, 2.3 Hz, 2H), 6.39
– 6.31 (m, 2H), 6.10 (s, 1H), 3.77 (s, 3H), 3.57 (s, 6H); 13C NMR (100 MHz, DMSO-d6 δ
159.19, 152.61, 135.60, 135.14, 131.63, 128.97, 127.15, 126.78, 124.10, 122.79, 121.50, 118.40,
117.41, 117.28, 114.43, 113.01, 111.93, 110.54, 101.68, 55.30, 55.25, 32.47; HRMS (ESI) Calcd
for C33H29O3N4[M+H]+ 529.2234, found 529.2232. Purity by HPLC UV (254 nm) ESI-MS:
97.81%.
1-(Bis(5-bromo-1H-indol-3-yl)methyl)-3-(4-methoxyphenyl)imidazo[1,5-a]pyridine
(5g):
This compound was prepared according to the method D. Yield: 87%; Rf = 0.41 (ethyl acetate/n-
1
hexane, 3:2); Melting point: 253-255; H NMR (300 MHz, CDCl3+DMSO-d6 δ 1 .44 s, 1 ,
8.15 (d, J = 6.2 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 1.5 Hz, 1H), 7.48 (d, J = 1.7 Hz,
1H), 7.33 (s, 1H), 7.18 (d, J = 1.8 Hz, 1H), 7.15 (d, J = 1.8 Hz, 1H), 7.12-7.05 (m, 3H), 6.99 (s,
2H), 6.70-6.60 (m, 2H), 6.27 (s, 1H), 3.88 (s, 3H); 13C NMR (75 MHz, CDCl3+DMSO-d6 δ
159.44, 143.28, 134.36, 134.00, 128.82, 127.10, 125.84, 124.17, 122.73, 120.29, 118.49, 117.38,
114.47, 113.80, 113.71, 113.48, 112.38, 112.26, 110.48, 54.27, 31.30;HRMS (ESI) Calcd for
C31H23ON4Br2[M+H]+ 625.0195, found 625.0052. Purity by HPLC UV (254 nm) ESI-MS:
96.85%.
3,3'-((3-(4-Methoxyphenyl)imidazo[1,5-a]pyridin-1-yl)methylene)bis(1H-indole-5-
carbonitrile) (5h): This compound was prepared according to the method D. Yield: 86%; Rf =
0.20 (ethyl acetate/n-hexane, 3:2); Melting point: 284-286; 1H NMR (300 MHz, CDCl3+DMSO-
d6 δ 1 .69 s, 2 , 8.18-8.12 (m, 1H), 7.83 (s, 2H), 7.70 (d, , J = 8.6 Hz, 2H), 7.43 (s, 1H), 7.40
(s, 1H), 7.31 (d, J = 1.5 Hz, 1H), 7.29 (d, J = 1.3 Hz, 1H), 7.16-7.10 (m, 1H), 7.09-7.04 (m, 2H),

7.03 (d, J = 8.6 Hz, 2H), 6.55-6.45 (m, 2H), 6.26 (s, 1H), 3.86 (s, 1H);13C NMR (75 MHz,
CDCl3+DMSO-d6 δ159.35, 138.32, 136.17, 133.24, 129.09, 126.90, 126.33, 125.88, 125.04,
123.47, 122.47, 121.28, 120.82, 118.19, 117.85, 114.23, 112.90, 112.65, 100.42, 55.16, 32.23;
HRMS (ESI) Calcd forC33H23ON6[M+H]+ 519.19279, found 519.19295. Purity by HPLC UV
(254 nm) ESI-MS: 92.20%.
1-(Di(1H-indol-3-yl)methyl)-3-(3,4-dimethoxyphenyl)imidazo[1,5-a]pyridine
(5i):
This
compound was prepared according to the method D. Yield: 94%; Rf = 0.35 (ethyl acetate/n-
hexane, 3:2); Melting point: 203-205; 1H NMR (300 MHz, DMSO-d6 δ 1 .75 s, 2 , 8.36-8.29
(m, 1H), 7.59-7.49 (m, 4H), 7.36-7.28 (m, 4H), 7.15.7.10 (m, 2H), 7.20 (d, J = 8.6 Hz, 2H), 7.00
(t, J = 7.7 Hz, 2H), 6.86 (t, J = 7.7 Hz, 2H), 6.66-6.53 (m, 2H), 6.22 (s, 1H), 3.85-3.79 (m, 6H);
13C NMR (100 MHz, DMSO-d6 δ 148.93, 148.83, 136.43, 135.71, 134.98, 126.76, 123.38,
122.87, 121.67, 120.79, 120.07, 119.40, 119.29, 118.33, 117.57, 117.52, 115.01, 113.02, 111.89,
111.99, 111.38, 55.65, 55.56, 32.69; HRMS Calcd for C32H27N4O2[M+H]+ 499. 2134, found
499.2131. Purity by HPLC UV (254 nm) ESI-MS: 98.45%.
1-(Bis(5-methoxy-1H-indol-3-yl)methyl)-3-(3,4-dimethoxyphenyl)imidazo[1,5-a]pyridine
(5j): This compound was prepared according to the method D. Yield: 96%; Rf = 0.16 (ethyl
1
acetate/n-hexane, 3:2); Melting point: 255-257; H NMR (300 MHz, CDCl3+DMSO-d6 δ 9.8
(s, 2H), 8.21-8.11 (m, 1H), 7.34-7.28 (m, 2H), 7.25 (s, 1H), 7.22 (s, 1H), 7.20-7.12 (m, 1H), 7.03
– 6.98 (m, 1H), 6.95-6.89 (m, 5H), 6.72 (dd, J = 8.7, 2.1 Hz, 2H), 6.47-6.39 (m, 1H), 6.21 (s,
1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.66 (s, 6H);13C NMR (75 MHz, CDCl3+DMSO-d6 δ 151.37,
147.65, 147.61, 146.50, 145.41, 134.35, 133.41, 130.45, 125.75, 125.63, 122.68, 121.60, 119.86,
118.59, 117.15, 115.65, 111.28, 110.42, 110.23, 110.15, 109.18, 100.11, 99.99, 54.29, 53.90,
32.26; HRMS(ESI) Calcd for C34H31O4N4[M+H]+ 559.2345, found 559.2299. Purity by HPLC
UV (254 nm) ESI-MS: 97.52%.
1-(Bis(5-bromo-1H-indol-3-yl)methyl)-3-(3,4-dimethoxyphenyl)imidazo[1,5-a]pyridine
(5k): This compound was prepared according to the method D. Yield: 92%; Rf = 0.42 (ethyl
acetate/n-hexane, 3:2); Melting point: 213-215; 1H NMR (300 MHz, CDCl3+DMSO-d6 δ 1 .55
(s, 2H), 8.23-8.17 (m, 1H), 7.62-7.57 (m, 2H), 7.35-7.32 (m, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.23-
7.18 (m, 2H), 7.16-7.10 (m, 2H), 7.05-7.01 (m, 1H), 7.00-6.95 (m, 2H), 6.55-6.48 (m, 2H), 6.17
13
(s, 1H), 3.96-3.91 (m, 6H); C NMR (75 MHz, CDCl3+DMSO-d6 δ 147.62, 147. 1, 133.87,

126.96, 126.50, 123.44, 121.95, 121.23, 120.23, 119.96, 119.53, 118.64, 116.71, 116.25, 115.36,
111.80, 111.55, 110.58, 110.12, 109.64, 110.19, 54.36, 54.28, 31.56;HRMS(ESI) Calcd for
C32H25Br2N4O2[M+H]+ 655.0.344, found 655.0330. Purity by HPLC UV (254 nm) ESI-MS:
98.42%.
3,3'-((3-(3,4-Dimethoxyphenyl)imidazo[1,5-a]pyridin-1-yl)methylene)bis(1H-indole-5-
carbonitrile) (5l): This compound was prepared according to the method D. Yield: 90%; Rf =
0.13 (ethyl acetate/n-hexane, 3:2); Melting point: 219-221; 1H NMR (300 MHz, CDCl3+DMSO-
d6 δ 1 .91 s, 2 , 8.19 m, 1 , 7.91-7.77 (m, 2H), 7.42 (d, J = 7.4 Hz, 1H), 7.29 (s, 1H), 7.18
(d, J = 7.5 Hz, 1H), 7.10 (s, 2H), 7.01 (d, J = 7.2 Hz, 1H), 6.57-6.44 (m, 2H), 6.25 (s, 1H), 3.92
(s, 6H); 13C NMR (75 MHz, CDCl3+DMSO-d6 δ 148.14, 147.29, 137.56, 137.50, 135.33,
126.16, 125.39, 124.82, 124.15, 122.65, 120.38, 119.91, 119.87, 119.06, 119.92, 114.24, 111.97,
111.54, 110.68, 110.30, 99.81, 99.71, 54.86, 54.79, 32.15; MS(ESI): m/z 548 [M+H]+. Purity by
HPLC UV (254 nm) ESI-MS: 91.13%.
1-(Di(1H-indol-3-yl)methyl)-3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]pyridine
(5m):
This compound was prepared according to the method D. Yield: 88%; Rf = 0.75 (ethyl acetate/n-
hexane, 1:1); Melting point: 263-265; 1H NMR (400 MHz, DMSO-d6 δ 1 .74 s, 1 , 8.45 , J
= 6.6 Hz, 1H), 8.03 (d, J = 7.9 Hz, 2H), 7.85 (d, J = 7.8 Hz, 2H), 7.62 (d, J = 8.9 Hz, 1H), 7.47
(d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.12 (d, J = 1.8 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H),
6.85 (t, J = 7.5 Hz, 1H), 6.77-6.66 (m, 1H), 6.25 (s, 1H);13C NMR (75 MHz, CDCl3+DMSO-d6)
δ 136.21, 135.58, 134. 1, 133.48, 128.51, 127.27, 126.42, 125.13, 125. 9, 124.46, 123. 3,
120.78, 120.53, 120.41, 118.87, 118.52, 117.86, 117.44, 117.05, 113.00, 110.74, 33.47;
MS(ESI): m/z 507 [M+H]+. Purity by HPLC UV (254 nm) ESI-MS: 98.43%.
1-(Bis(5-methoxy-1H-indol-3-yl)methyl)-3-(4-(trifluoromethyl)phenyl)imidazo[1,5-
a]pyridine (5n): This compound was prepared according to the method D. Yield: 90%; Rf =
1
0.60 (ethyl acetate/n-hexane, 1:1); Melting point: 213–215; H NMR (500 MHz, DMSO-d6 δ
10.60 (s, 2H), 8.52 (d, J = 7.3 Hz, 1H), 8.09 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.78
(d, J = 9.1 Hz, 1H), 7.20 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 2.2 Hz, 2H), 7.03 (d, J = 2.4 Hz, 3H),
6.80-6.76 (m, 1H), 6.71 (t, J = 7.3 Hz, 1H), 6.66 (dd, J = 8.6, 2.4 Hz, 2H), 6.20 (s, 1H), 3.61 (s,
13
6H); C NMR (75 MHz, DMSO-d6 δ 152.57, 136.44, 134. 6, 133.93, 131.57, 128.85, 128. 8,
127.90, 127.53, 127.04, 126.02, 125.80, 125.75, 124.08, 122.41, 121.89, 118.57, 118.43, 116.95,

113.84, 111.87, 110.50, 101.57, 55.18, 32.34; HRMS (ESI) Calcd forC33H26F3N4O2[M+H]+
567.2008, found 567.2009. Purity by HPLC UV (254 nm) ESI-MS: 92.97%.
1-(Bis(5-bromo-1H-indol-3-yl)methyl)-3-(4-(trifluoromethyl)phenyl)imidazo[1,5-a]pyridine
(5o): This compound was prepared according to the method D. Yield: 86%; Rf = 0.82 (ethyl
acetate/n-hexane, 1:1); Melting point: 271-273; 1H NMR (400 MHz, DMSO-d6 δ 11. 1 s, 2 ,
8.53 (d, J = 7.3 Hz, 1H), 8.10 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 9.1 Hz, 1H), 7.87 (d, J = 8.3 Hz,
2H), 7.82 (d, J = 1.8 Hz, 2H), 7.32 (d, J = 2.2 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 7.12 (dd, J =
8.6, 1.9 Hz, 1H), 6.86-6.81 (m, 1H), 6.77-6.72 (m, 1H), 6.32 (s, 1H); 13C NMR (75 MHz,
DMSO-d6 δ135.47, 135. , 134.1 , 133.93, 128.31, 127.99, 127.63, 125.94, 125.74, 125.69,
124.97, 123.13, 122.35, 121.89, 121.74, 120.96, 118.84, 118.15, 116.95, 113.92, 113.35, 110.69,
31.41; HRMS (ESI) Calcd for C31H20Br2F3N4[M+H]+ 663.0007, found 663.00088. Purity by
HPLC UV (254 nm) ESI-MS: 96.37%.
3,3'-((3-(4-(Trifluoromethyl)phenyl)imidazo[1,5-a]pyridin-1-yl)methylene)bis(1H-indole-5-
carbonitrile) (5p): This compound was prepared according to the method D. Yield: 84%; Rf =
0.50 (ethyl acetate/n-hexane, 1:1); Melting point: 277-279;1H NMR (300 MHz, DMSO-d6 δ
11.42 (s, 2H), 8.53 (d, J = 7.1 Hz, 1H), 8.17 (s, 2H), 8.10 (d, J = 7.7 Hz, 2H), 7.96 (d, J = 8.8 Hz,
2H), 7.87 (d, J = 7.9 Hz, 2H), 7.60-7.45 (m, 3H), 7.37 (d, J = 8.2 Hz, 2H), 6.88 (t, J = 6.6 Hz,
13
1H), 6.75 (t, J = 6.6 Hz, 1H), 6.49 (s, 1H); C NMR (100 MHz, DMSO-d6 δ 148.73, 148.27,
138.02, 134.90, 134.39, 133.90, 128.86, 128.02, 127.74, 126.23, 126.09, 125.76, 125.72, 125.65,
125.06, 123.47, 122.96, 122.00, 120.87, 119.15, 118.24, 117.98, 114.00, 112.77, 100.08,
30.89;MS (ESI): m/z 557 [M+H]+. Purity by HPLC UV (254 nm) ESI-MS: 98.33%.
1-(Di(1H-indol-3-yl)methyl)-3-(4-fluorophenyl)imidazo[1,5-a]pyridine (5q): This compound
was prepared according to the method D. Yield: 88%; Rf = 0.66 (ethyl acetate/n-hexane, 1:1);
1
Melting point: 281-283; H NMR (300 MHz, CDCl3+DMSO-d6 δ 1 .2 s, 2 , 7.68-7.58 (m,
1H), 7.44-7.32 (m, 3H), 7.44-7.13 (m, 4H), 6.96 (t, J = 7.3 Hz, 2H), 6.89 (d, J = 1.5 Hz, 2H),
6.81 (t, J = 7.6 Hz, 2H), 6.52-6.42 (m, 2H), 6.22 (s, 1H);13C NMR (75 MHz, CDCl3+DMSO-d6)
δ 159.46, 156.18, 134.94, 133.97, 13 .38, 129.35, 129.19, 125.75, 125.16, 123.26, 121.79,
120.43, 120.36, 119.21, 117.51, 116.56, 116.27, 115.80, 114.61, 114.32, 111.16, 109.78, 31.60;
HRMS (ESI) calcd forC30H22N4F [M+H]+457.1823, found 457.1807. Purity by HPLC UV (254
nm) ESI-MS: 97.51%.

1-Bis(5-methoxy-1H-indol-3-yl)methyl)-3-(4-fluorophenyl)imidazo[1,5-a]pyridine (5r): This
compound was prepared according to the method D. Yield: 90%; Rf = 0.50 (ethyl acetate/n-
1
hexane, 1:1); Melting point: 305-307; H NMR (300 MHz,CDCl3+DMSO-d6 δ 1 . 4 s, 2 ,
8.11-8.04 (m, 1H), 7.75-7.62 (m, 3H), 7.21-7.08 (m, 4H), 6.88 (d, J = 1.9 Hz, 2H), 6.80 (d, J =
13
2.2 Hz, 2H) 6.61 (dd, J = 8.8, 2.4 Hz, 2H), 6.45-6.40 (m, 2H), 6.08 (s, 1H), 3.56 (s, 6H); C
NMR (75 MHz, CDCl3+DMSO-d6 δ 162.75, 159.47, 151.83, 134.14, 133.94, 13 .88, 128.7 ,
128.59, 126.42, 126.15, 125.59, 125.55, 123.19, 119.91, 117.66, 116.40, 116.04, 114.89, 114.61,
112.01, 110.90, 109.78, 100.36, 54.42, 32.77; HRMS (ESI) calcd forC32H26FN4O2
[M+H]+517.2040, found 517.2040. Purity by HPLC UV (254 nm) ESI-MS: 96.47%.
1-(Bis(5-bromo-1H-indol-3-yl)methyl)-3-(4-fluorophenyl)imidazo[1,5-a]pyridine (5s): This
compound was prepared according to the method D. Yield: 86%; Rf = 0.73 (ethyl acetate/n-
1
hexane, 1:1); Melting point: 283-285; H NMR (300 MHz, DMSO-d6 δ 11. s, 2 , 8.3 s,
1H), 7.90-7.82 (m, 2H), 7.76 (s, 2H), 7.70 (t, J = 7.0 Hz, 1H), 7.61-7.54 (m, 1H), 7.46-7.37 (m,
2H), 7.33-7.27 (m, 3H), 7.11 (d, J = 8.5 Hz, 2H), 6.82-6.77 (m, 1H), 6.68 (t, J = 6.5 Hz, 1H),
6.30 (s, 1H);` 13C NMR (100 MHz, DMSO-d6 δ 16 .53, 158. 6, 135. , 134.85, 131.84, 131.11,
130.90, 128.33, 127.15, 124.96, 123.12, 122.25, 122.19, 121.71, 118.29, 117.79, 117.09, 116.39,
116.18, 113.33, 113.03, 110.70, 30.62; HRMS (ESI) calcd forC30H20Br2FN4[M+H]+613.0039,
found 612.9979. Purity by HPLC UV (254 nm) ESI-MS: 95.29%.
3,3'-((3-(4-Fluorophenyl)imidazo[1,5-a]pyridin-1-yl)methylene)bis(1H-indole-5-
carbonitrile) (5t): This compound was prepared according to the method D. Yield: 85%; Rf =
0.40 (ethyl acetate/n-hexane, 1:1); Melting point: 294-296; 1H NMR (300 MHz, CDCl3+DMSO-
d6 δ 11.15 s, 2 , 8.23 , J = 7.0 Hz, 1H), 7.92 (s, 2H), 7.82 (dd, J = 8.7, 5.4 Hz, 2H), 7.54-
13
7.40 (m, 4H), 7.34-7.20 (m, 5H), 6.68-6.55 (m, 2H), 6.31 (s, 1H); C NMR (75 MHz, DMSO-
d6 δ 163.75, 16 .48, 138.34, 135.34, 134.12, 13 . 6, 129.95, 127.42, 126.77, 126.51, 126.31,
125.36, 123.83, 121.78, 121.21, 118.79, 118.61, 118.11, 116.36, 116.07, 113.82, 113.10, 100.33,
31.35; HRMS (ESI) Calcd for C32H20N6F [M+H]+507.17280, found 507.17187. Purity by HPLC
UV (254 nm) ESI-MS: 97.95%.
Biology
Cytotoxic activity

The cytotoxic activity of the compounds was determined by using MTT assay.22 Cells
were seeded in 100 µL DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37 ºC in a CO2 incubator. After incubation, cells
were treated with test compounds of desired concentrations for 48 h. After 48 h incubation, 10
µL MTT (3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) (5 mg/mL) was
added to each well and the plates were further incubated for 4 h. Then the supernatant from each
well was carefully removed, formazon crystals were dissolved in 100 µL of DMSO and
absorbance at 570 nm wavelength was recorded.
Cell cycle analysis
Effect of 5k and 5r on DNA content by cell cycle progression was assessed using
A549cells. Cells (2×105/well) were incubated in a 6-well plate with incomplete media (serum
23,24
free) for 24 h, to bring all cells in a synchronized state (Margit Rosner, 2013).
After 24 h,
incomplete media was replaced with complete media without or with DIM, 5k, and 5r at
indicated concentrations and further incubated for 24 h. The cells were washed thrice with PBS,
harvested, fixed in ice cold PBS in 70% ethanol, and stored at -20 ºC for 30 min. After fixation,
these cells were incubated with RNase A (0.1 mg/mL) at 37 ºC for 30 min, stained with PI (50
µg/mL) for 30 min on ice in dark, and then measured for DNA content using BD FACSVerse
flow cytometer (Becton Dickinson, USA).
Measurement of reactive oxygen species (ROS)
ROS production from A549 cells treated with DIM, 5k, and 5r conjugates was measured
b using 2’,7’–dichlorofluorescin diacetate (DCFDA) dye and the method was followed by
Reddy, T. Srinivasa, et al. 2015.25,26 A549 cells were seeded in a 6-well plate at a density of
2×105 cells/well and incubated overnight. After incubation, media was replaced with fresh
medium containing DIM, 5k and 5r conjugates at indicated concentrations for 24 h. After 24 h
incubation, medium was replaced with fresh medium containing DCFDA (20 mM) and
incubated in dark for 30 min at room temperature. Cells were washed with PBS twice and
fluorescence intensity was measured by fluorescence microscope (Olympus-1X71SIF-3), capture
7 pro software.
Determination of apoptosis by Annexin V-FITC/PI stain

Induction of apoptosis was determined by using the Annexin V-FITC apoptosis detection
kit (Sigma Aldrich cat. No: APOAF) (Deng, Sanming, et al. 2012).27 Briefly, cells were seeded
in 6-well plate at a density of 1×105 cells/well and allowed to attach overnight, followed by
treatment with DIM, 5k, and 5r at 1 and 2 µM concentrations for 24 h. After 24 h, media was
discarded, and cells were washed twice with PBS. Gently trypsinize and resuspend the cells in 1x
binding buffer at a concentration of 1×106 cells/mL. Incub te the cell suspension with 5 μL
Annexin V- I C n 1 μL of PI fo 1 min t oom tempe tu e n p otect f om light.
Analysis was carried out by flow cytometry (FACSVerse, Becton-Dickinson, USA).
Measurement of mitochondria membrane potential (∆Ψm)
Mitochon i l memb ne potenti l w s me su e b cco ing to the m nuf ctu e ’s
protocol BDTM Mitoscreen kit. Briefly, 2×105 cells were seeded in to the 6-well plates and
incubated for overnight. Cell culture medium was replaced with fresh medium containing DIM,
5k, and 5r at indicated concentrations for 24 h. After incubation, cells were trypsinized and
collected into 15 mL tubes and centrifuged at 400g for 5 min at room temperature. To the cell
pellet 0.5 mL of freshly prepared JC-1 working solution was added and incubated for 15 min at
37 ºC in CO2 incubator. After incubation, cells were washed twice with 1x assay buffer at room
temperature. Cells were resuspended in 0.5 mL of 1x assay buffer and analyzed by flow
cytometry (Reers, Martin, et al. 1995)28.
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Date:20th Feb 2019
Dear Professor Dr. Timothy Bugg
Please find the attached revised m nusc ipt entitle “Synthesis and biological evaluation of
new bisindole-imidazopyridine hybrids as apoptosis inducers” fo the consi e tion of
publication in your esteemed journal “Bioorganic Chemistry”
The above manuscript has the following significant features:
 In the present studies, we have synthesized imidazo[1,5-a]pyridine-bisindole
conjugates(5a-t)were design, synthesized and evaluated for their cytotoxic activity
against four human cancer cell lines.
 Among the synthesized conjugates 5k and 5rexhibited good activity and possessing IC50
values <2 µM against Lung cancer cell line.
 Conjugates 5k have shown cytotoxic activity with IC50 value1.6µM againt Lung cancer
cell line.
 These conjugates arrest cell cycle at G2/M phase.
Based on the reviewers comments, we have modified the entire manuscript and all the
reservations are carefully addressed. Therefore, it is considered that “Bioorganic
Chemistry”with its wide readership will be the suitable journal to publish this research work.
Looking forward to hearing from you.
With kind regards,
Dr Bathini Nagendra Babu
Dept. of Fluoro-Agrochemicals
CSIR-IICT, Hyderabad
Tel: +91-40-27191898;
E-mail: bathini@iict.res.in; bniict@gmail.com