Design, synthesis and biological evaluation of new β-carboline-bisindole compounds as DNA binding, photocleavage agents and topoisomerase I inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.10.054

A series of new β-carboline-bisindole compounds were designed, synthesized and evaluated for their antiproliferative activity against human cancer cell lines, such as A549 (lung cancer), DU-145 (prostate cancer), HeLa (cervical cancer) and MCF-7 (breast cancer). All the compounds exhibited considerable antiproliferative activity. Among them, compounds 7g and 7r exhibited significant antiproliferative activity against DU-145 cells with IC₅₀ values 1.86 and 1.80 μM respectively. Further, these compounds effectively inhibit DNA topoisomerase I activity and can also cleave the pBR322 plasmid upon irradiation with UV light. In addition, Annexin V-FITC assay suggested that these compounds induced apoptosis in DU- 145 cell line (prostate cancer). To know the binding mode of these compounds with DNA, spectroscopic studies were also carried out. These new compounds were showing a unique mode of binding with DNA, both biophysical studies such as UV–Visible, fluorescence, circular dichroism and molecular docking studies revealed that the β-carboline-bisindole compounds exhibit combilexin type of interaction with DNA.

Full text of DOI:10.1016/j.ejmech.2017.10.054

Accepted Manuscript
Design, synthesis and biological evaluation of new β-carboline-bisindole compounds
as DNA binding, photocleavage agents and topoisomerase I inhibitors
Jeshma Kovvuri, Burri Nagaraju, V. Lakshma Nayak, Ravikumar Akunuri, M.P.
Narasimha Rao, Ayyappan Ajitha, Narayan Nagesh, Ahmed Kamal
PII:
S0223-5234(17)30856-5
10.1016/j.ejmech.2017.10.054
EJMECH 9846
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 September 2017
Revised Date: 16 October 2017
Accepted Date: 17 October 2017
Please cite this article as: J. Kovvuri, B. Nagaraju, V.L. Nayak, R. Akunuri, M.P.N. Rao, A. Ajitha, N.
Nagesh, A. Kamal, Design, synthesis and biological evaluation of new β-carboline-bisindole compounds
as DNA binding, photocleavage agents and topoisomerase I inhibitors, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.054.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of New β-Carboline-Bisindole
Compounds as DNA Binding, Photocleavage Agents and Topoisomerase I
Inhibitors
a,b
a,b
b
d
Jeshma Kovvuri,
Burri Nagaraju, V. Lakshma Nayak, Ravikumar Akunuri, M. P.
b
c
*c
*a,b,d,e
Narasimha Rao, Ayyappan Ajitha, Narayan Nagesh and Ahmed Kamal
Graphical Abstract

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Design, Synthesis and Biological Evaluation of New
β-Carboline-Bisindole
Compounds as DNA Binding, Photocleavage Agents and Topoisomerase I
Inhibitors
a,b
a,b
b
d
Jeshma Kovvuri, Burri Nagaraju, V. Lakshma Nayak, Ravikumar Akunuri, M. P.
b
c
*c
*a,b,d,e
Narasimha Rao, Ayyappan Ajitha, Narayan Nagesh and Ahmed Kamal
a
Academy of Scientific and Innovative Research (AcSIR), New Delhi-110025, India
b
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad-500007, India
c
Centre for Cellular and Molecular Biology, Hyderabad-500007, India
d
National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad-500 037,
India
e
School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi
1
10062, India.
Abstract
A series of new β-carboline-bisindole compounds were designed, synthesized and evaluated for
their antiproliferative activity against human cancer cell lines, such as A549 (lung cancer), DU-
1
45 (prostate cancer), HeLa (cervical cancer) and MCF-7 (breast cancer). All the compounds
exhibited considerable antiproliferative activity. Among them, compounds 7g and 7r exhibited
significant antiproliferative activity against DU-145 cells with IC50 values 1.86 and 1.80 µM
respectively. Further, these compounds effectively inhibit DNA topoisomerase I activity and can
also cleave the pBR322 plasmid upon irradiation with UV light. In addition, Annexin V-FITC

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assay suggested that these compounds induced apoptosis in DU- 145 cell line (prostate cancer).
To know the binding mode of these compounds with DNA, spectroscopic studies were also
carried out. These new compounds were showing a unique mode of binding with DNA, both
biophysical studies such as UV-Visible, fluorescence, circular dichroism and molecular docking
studies revealed that the β-carboline-bisindole compounds exhibit combilexin type of interaction
with DNA.
Keywords: β-Carboline; bis-indole; topoisomerase I; antiproliferative activity; DNA-binding
affinity; Photocleavage.
*
Corresponding authors. E-mail addresses: ahmedkamal@iict.res.in (Ahmed Kamal),
nagesh5112@gmail.com (Narayan Nagesh)
. Introduction
In the present scenario, cancer has become a global problem with an alarming increase in
1
the number of cancer patients. An estimated 8.2 million deaths are reported in 2012 worldwide,
the annual number of new cases due to cancer is projected to rise from 14.1 million in 2012 to
2
1.6 million by 2030 with an increase in the death rate by 60 % [1]. Therefore, enormous efforts
are being made to develop affordable and potent cancer chemotherapeutics. Due to the
complexity of the disease, success rate of finding a potential candidate is low and multiple
biological targets are being pursued to identify a better drug. DNA being the major element
involved in cell division and maintenance has been the most important target to develop
anticancer drugs [2, 3]. The DNA binders are classified into three categories based on their mode
of interaction, such as i) intercalators ii) groove binders iii) and combilexins. Combilexin
molecules exhibit enhanced DNA interaction with a dual mode of binding compared to single
mode [4]. Subsequently, the enzymes associated with the DNA replication and transcription are

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also important targets for inhibiting the cell proliferation. One such enzyme is a topoisomerase I,
which cuts the single strand of DNA double helix and unwinds superhelix of DNA. Studies have
revealed that the topoisomerase I is over expressed in tumour cells in comparison to normal cells,
due to which this has become an attractive tool for developing anticancer agents [5]. Numerous
anticancer drugs containing indolocarbazoles, indenoisoquinolines and camptothecins target
topoisomerases [6]. Considering the proximity of this potential target, medicinal chemists have
put their efforts to design compounds with combilexin binding for a potential interaction, such as
NetAmsa (I) that can target DNA as well as topoisomerases to exhibit improved efficacy with
greater selectivity [7].
Natural products have played a major role in the quest of developing anticancer drugs [8].
One such class of natural products is β-carboline alkaloids that target tumor cell DNA [9]. These
were first isolated from the seeds of peganum harmala and exhibit diverse pharmacological
properties such as antiplasmodial [10], antimutagenic [11], antiplatelet [12, 13] and anticancer
[14]. These compounds have been explored extensively due to their multiple physiological
effects such as (CDK) [15] cyclin-dependent kinase, (IKK) [16] IkappaB kinase, topoisomerase
inhibition [17] and DNA binding ability [18]. Harmine (II) is the common representative
compound of this family with broad spectrum of cytotoxicity against various cancer cell lines
including drug resistant KB sub cells. Due to the planar structure, harmine possesses DNA
intercalating ability and inhibits DNA topoisomerase I. The methoxy group present at C-7
position causes neurotoxicity in animals [19] (Fig. 1). Mana-Hox (III) is another β-carboline
analog that interacts with DNA through intercalation and groove binding, and shows significant
cytotoxic activity against various human cancer cell lines including prostate cancer [20].
Considering the importance of β-carbolines, our group has put efforts in designing β-carboline

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compounds for developing potential DNA binding agents and topoisomerase inhibitors for
antiproliferative activity and the studies have shown that the changes in substitutions at C-1 and
C-3 positions of β-carboline ring exhibited improved DNA binding ability [21].
<
Insert Fig. 1>
On the other hand indoles are the privileged scaffolds with distinct pharmacological
properties [22]. Rebeccamycin (V), an indolocarbazole, it is an antineoplastic antibiotic. The
carbon backbone of rebeccamycin intercalates with DNA and the sugar moiety shows DNA
groove binding along with topoisomerase I inhibition [23]. Another bisindole diindoylymethane
(DIM) (IV), a major component derived from digestion of indole-3-carbinol, has completed the
phase II clinical trials for treating patients with stage I and II prostate cancer [24]. DIM bears
similar apoptosis inducing property as paclitaxel. In view of β-carboline and indole biological
importance a series of β-carboline-bisindole compounds (7a-z, 7aa and 7ab, Fig. 1) were
designed by removing the methoxy group at C-7 and changing various substitutions at C-1
position of β-carboline and introducing substituted bisindoles at the C-3 position for synergistic
effect. The synthesized compounds are evaluated for their DNA binding ability and
topoisomerase I inhibitory potential.
2
2
. Results and discussion
.1 Chemistry
The synthetic strategy for the preparation of β-carboline-bisindole compounds (7a-z, 7aa
and 7ab) is outlined in the Scheme 1. The L-tryptophan methyl ester (2) was prepared by
esterification of L-tryptophan (1) using SOCl and methanol, which was subjected to the Pictet-
2
Spengler condensation with different aldehydes in the presence of catalytic amount of p-TSA to
get the respective methyltetrahydro-β-carboline-3-carboxylates (3a-d). Compounds 3a-d were

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aromatized using trichloroisocyanuric acid to yield methyl-β-carboline-3-carboxylates (4a-d),
followed by reduction of 4a-d with LiAlH to obtain the respective carbinols (5a-d). Carbinols
4
5
a-d were oxidized with DMP to furnish the desired methyl-β-carboline-3-carboxaldehyde (6a-
d). Here the final reaction of various substituted indoles with the synthesized β-carboline
aldehydes (6a-d) in the presence water-ethanol solvent mixture, using Amberlite IR-120H as a
recyclable heterogeneous catalyst under the reflux conditions offered the desired compounds 7a-
1
13
z, 7aa and 7ab in good to excellent yields. All the compounds were confirmed by H, C, ESI-
MS and HRMS, the purity of the active compounds were demonstrated by HPLC.
<
Insert Scheme 1>
2
2
.2. Biological activity
.2.1. Antiproliferative activity
All the newly synthesized β-carboline-bisindole compounds were evaluated for their
antiproliferative activity against human cancer cell lines, such as A549 (lung cancer), DU-145
prostate cancer), HeLa (cervical cancer) and MCF-7 (breast cancer) using MTT assay.
(
Gratifyingly, the series of compounds (7a-z, 7aa & 7ab) exhibited good to moderate cytotoxic
activity ranging from 1.80 to 44.20 µM. The cytotoxicity results were expressed as IC50 values
and the results were summarized in Table 1 compared to positive controls harmine and
doxorubicin. Among the synthesized 28 compounds, 11 of the compounds (7g, 7i, 7j, 7m, 7n,
7
p, 7r, 7t-v, and 7aa) exhibited pronounced cytotoxicity against DU-145 cell line. The most
active compounds of the series were 7g, 7r and 7t with IC values of 1.86, 1.80 to 1.92 µM
5
0
respectively. The cytotoxicity of the β-carboline-bisindole compounds depend on the nature of
1
2
3
substituents on the phenyl ring at C-1 position (R , R and R , Fig. 2) and on indole moiety at C-
4
5
3
position (R and R ).

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<
Insert Fig.2 >
In order to know SAR of the compounds, different substituents on the phenyl and indole
rings were varied. Among all the synthesized compounds 4-Fluoro (7r and 7t-7v) substituent on
the phenyl ring at C-1 position displayed potent antiproliferative activity and then followed by 4-
methyl (7g, 7i and 7j) substituent against DU-145 cells. The number of methoxy substituents on
the phenyl ring had a major influence on antitumor activity. Replacement of 4-methyl group (7g,
7
i and 7aa) on the phenyl ring with more electron rich methoxy group (7m, 7n and 7p)
decreased antiproliferative activity by 1.4 folds in the DU-145 cells and three methoxy
substituents (7a) on the phenyl ring caused a substantial decrease in the antiproliferative activity
by 5.6 folds with respective to 7m compound in the DU-145 cells. We speculate that the electron
deficient substituents enhanced cytotoxic activity in comparable to the electron rich substituents.
In addition, the influence of C-3 substituents on activity was also examined. 5-unsubstituted
indole, electron deficient substituents such as Br, F, CN and NO at C-5 position on indole ring
2
linked to C-3 position of β-carboline enhanced antiproliferative activity, while electron rich
3
OCH group decreased the activity and replacing the hydrogen of indole C-2 position (7d, 7i, 7n
and 7t) with the bulky methyl group (7c, 7h, 7l and 7s) leads to decrease in potency by 1.7 to 8.8
folds in the DU-145 cells, Further, to understand the mode of action, some detailed biological
studies were carried out by taking most active compounds based on MTT assay.
<Insert Table 1>
2
.2.2. DNA topoisomerase I inhibition study
DNA topoisomerase I (topo I) has been identified as the potential target of several
anticancer drugs used clinically today. Since topo I is involved in replication and proliferation
process, over production of topo I was noticed in cancer cells compared to normal cells. In

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general, topo I control the changes in the DNA structure by cutting a single stranded DNA and
re-joining the phosphate backbone during the normal cell cycle. Mechanism of topo I inhibition
is known to occur in two ways, these inhibitors may bind topoisomerase directly or they may
bind to DNA and alter its structure, so that it cannot be recognized by topoisomerases. In the
present study, the potential of the 7g, 7r and 7t compounds towards inhibition of topo I was
studied. A well known quinoline alkaloid known for topo I inhibition namely, camptothecin was
used as a positive control (shown in lane 6 in Fig. 3). Interestingly, 7g, 7r and 7t compounds
along with camptothecin have shown significant topo I inhibition at 20 µM concentration (lane
3
-5, Fig. 3). All the compounds (7g, 7r and 7t) were found to inhibit topo I activity.
Insert Fig 3>
<
2
.2.3. DNA photocleavage
The DNA photocleavage reaction was carried out in the presence of 7g, 7r and 7t
compounds to find their efficiency in cleaving pBR322 supercoiled DNA by generating free
radicals in the system. In general, the photocleavage reactions proceed via the generation of
3
molecular oxygen or hydroxyl radical species. These reactions involve triplet oxygen state ( O
2
)
that could proceed by two major mechanistic pathways. In the first one, the singlet excited
electronic state of the compound through inter system crossing, generate an excited triplet state
of the compound which in turn activate the molecular oxygen in its stable triplet oxygen state
3
1
(
O ) to a more reactive singlet oxygen state ( O ) [25]. In an alternate pathway, the excited state
2 2
molecule could reduce molecular oxygen to generate highly reactive hydroxyl radical. The
ability of these compounds (7g, 7r and 7t) to cleave DNA upon irradiation with UV light of 365
nm wavelength followed by agarose gel electrophoresis depicts that they generate free radicals to
function as potent photocleavage agents and appear to be suitable for photodynamic therapy

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(PDT). In the absence of 7g, 7r and 7t, when the pBR322 plasmid DNA is subjected to
electrophoresis, a dense and relatively fast migration band was noticed in the supercoiled form
Form I). Upon cleaving one strand of supercoiled DNA, it will relax to generate a relaxed
circular form (Form II). The gel electrophoresis separation of pBR322 DNA after the addition of
g, 7r and 7t ligands (20 µM) and upon UV-light irradiation for 1 h was shown in Fig. 4. The
(
7
experimental results indicate that, control (DNA solution alone irradiated) as well as DNA +
compounds (20 µM) incubated in the dark did not show cleavage. However, the DNA samples
irradiated along with 7g and 7r exhibited photocleavage as evidenced by the decrease in the
intensity of Form I. Further, 7g and 7r are considerably active to generate more free radicals that
will effectively cleave the supercoiled DNA. Among 7g and 7r, effect of UV-light irradiation on
supercoiled DNA cleavage was more in case of 7g followed by 7r. Topo I inhibition and UV
cleavage experiments indicate that among 7g, 7r and 7t compounds, 7g and 7r demonstrated
maximum potential. 7t has minimum effect on DNA cleavage in the presence of UV light. Even
cytotoxicity data also indicate that 7g and 7r are more efficient, so we further carried out
experiments only with 7g and 7r.
<
Insert Fig. 4>
2
.2.4. Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combination of
both [26, 27]. The screening results revealed that compounds 7g and 7r showed significant
cytotoxic activity against human prostate cancer cell line (DU-145). A cell cycle analysis study
was performed, wherein DU-145 cells were treated with these compounds (7g and 7r) for 48 h at
1
µM and 2 µM concentrations respectively. The data obtained clearly indicated that these

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compounds show cell cycle arrest at G2/M phase in comparison with the untreated control cells.
G2/M phase cell cycle arrest is a common cellular response to a variety of DNA-damaging
agents [28]. These compounds (7g and 7r) showed 22.04 % and 57.46 % of cell accumulation in
G2/M phase at 1 µM concentration, whereas they exhibited 57.12 % and 62.86 % of cell
accumulation at 2 µM concentration, respectively (Fig. 5 and Table 2).
<
Insert Fig. 5>
<Insert Table 2>
2
.2.5. Apoptosis assay
Induction of apoptosis among DU-145 cell by 7g and 7r compounds were evaluated by
Annexin V FITC/PI (AV/PI) dual staining assay [29] to examine the occurrence of
phosphatidylserine externalization and also to understand whether it is due to physiological
apoptosis or nonspecific necrosis. In this study, DU-145 cells were treated with these compounds
for 48 h at 1 and 2 µM concentrations to examine the apoptotic effect. It was observed that these
compounds showed significant apoptosis against DU-145 cells as shown in Fig. 6. Results
indicated that compounds 7g and 7r showed 12.86 % and 15.04 % at 1 µM concentration,
whereas they exhibited 35.88 % and 43.34 % apoptosis at 2 µM concentrations respectively for
4
8 h (Table 3).
<
Insert Fig. 6>
<Insert Table 3>
2
.2.6. Circular dichroism studies
In order to understand the effect of β-carboline-bisindole compounds on DNA
conformation, circular dichroism studies were performed. The CT-DNA shows positive and
negative CD bands at 275 nm and 245 nm respectively. At an average all the β-carboline-

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bisindole compounds demonstrated around 10-12 % of positive CD band hypochromicity on
interaction with CT DNA. On addition of 10 µM 7g and 7r compounds to CT-DNA (at 1:1
ratio), the positive CD band showed hypochromicity, indicating unwinding of CT-DNA on
interaction with β-carboline-bisindole compounds. Further, on doubling the concentration of β-
carboline-bisindole compounds (at 1:2 ratio), the positive band intensity reduced further and
exhibited slight red shift. Red shift of the positive CD band is an indication of good interaction
between the bound β-carboline-bisindole compounds and CT-DNA. CD studies indicate that β-
carboline-bisindole compounds interact with DNA and they can bring change in DNA
conformation. The extent of positive CD band hypochromicity is more with 7r compared to 7g.
From the CD data it is evident that β-carboline-bisindole compounds alter DNA helix on its
interaction with CT-DNA, which is clear from the changed negative CD band intensities. The
representative CD spectra obtained with 7g and 7r compounds and CT-DNA was shown in Fig.
7
.
<
Insert Fig. 7>
2
.2.7. UV-visible studies
UV-visible studies are useful in understanding the mode of binding of these compounds
with DNA. All the complexes has absorption peak at around 370 nm. On addition of equal
concentration of CT DNA, the absorption band at 370 nm exhibited hypochromicity without
exhibiting any shift. It is known that hypochromicity along with the larger bathochromic shift
(red shift) is an evidence of intercalative mode of binding [30]. The extent of hypochromicity of
the absorption band can be taken as a measure of intercalative binding strength [31]. The
absorption band hypochromicity is usually attributed to the interaction between the electronic
states of the complex and the DNA bases [32]. From the results it is evident that the

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hypochromicity of UV-visible band at 370 nm is comparatively more with 7g compared to 7r.
This shows that the β-carboline-bisindole compounds show very good interaction with double
stranded DNA, may be by intercalative mode. The representative UV-visible spectra obtained on
interaction of compounds with CT-DNA are shown in Fig. 8.
<
Insert Fig. 8>
2
.2.8. Fluorescence titrations
Fluorescence titration is yet another valuable technique for understanding the binding
mode of small molecules with DNA and to study the electronic environment around the DNA-
complex formed with molecules at comparatively lower concentrations [33]. Since the β-
carboline-bisindole compounds are fluorescent, their interaction with DNA can be monitored at
low concentration. The emission spectra of these compounds show prominent peaks in the region
between 395 nm to 470 nm. On addition of equal increments of DNA to the all the compound
solutions, the emission peak intensities decrease gradually. The extent of fluorescence
hypochromicity both at 395 nm and 470 nm is slightly higher in case of 7r compared to 7g.
However, higher fluorescence quenching in case of 7g and 7r is probably due to the stacking of
the compounds to the surface of DNA [34]. Indeed, simple β-carboline alkaloids such as harmine
and harmane are well known DNA intercalators having fully planar nature, whereas the mode of
binding of these β-carboline compounds is slightly different that could be due to their structure
and planarity. The fluorescence spectra were shown in Fig. 9.
<
Insert Fig. 9>
2
.2.9. Viscosity studies:
In order to clearly understand the nature of interaction of these molecules with CT DNA,
viscosity studies were carried out. Spectroscopic studies indicate that they interact well with CT

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DNA, as the relative viscosity of DNA increases when a molecule intercalate with DNA. The
enhancement of viscosity is due to increase in the axial length of DNA helix on molecules
intercalation [35]. Whereas, reduction in the relative viscosity is typically observed due to
covalent DNA binding [36] and on the other hand the viscosity does not change or show very
less change when molecules bind to the surface of the DNA. With the addition of 7g and 7r,
there was a slight enhancement of viscosity in the DNA solution was noticed. Minor positive or
negative changes in DNA solution viscosity were observed when binding occurs in the DNA
grooves. As shown in Fig. 10, on addition of 7g and 7r complexes to CT DNA there is a slight
increase in the viscosity, which is in between the classical intercalators like EtBr [37] and the
groove-binder like Hoechst 33342 [38].
<Insert Fig. 10>
As a control, 5 µM of ethidium bromide (EtBr) and Hoechst 33342 was added to CT-
DNA. The viscosity of DNA on addition of EtBr has increased continuously, on the other hand,
no change was noticed when Hoechst 33342 was added to CT-DNA. Upon plotting the values,
the viscosity graphs with 7g and 7r were seen in between ethedium bromide (a strong
intercalator) and Hoechst (a known groove binder). From these results it is speculated that they
may be exhibited combilexin type of interaction between CT-DNA and 7g/7r molecules. The
1
/3
graph plotted between (ŋ/ŋ ) and complex/CT-DNA was shown in Fig. 10.
o
2
.3. Molecular Docking
The β-carboline derivatives of C-1 and C-3 substitutions are exhibiting efficient DNA
binding abilities and topoisomerase I inhibition. Biophysical studies speculated that the
synthesized β-carboline-bisindole compounds could behave like combilexin-type of molecules.
Hence we chose a pair of crystal structures ( PDB IDs: 1SC7 and 1NAB for DNA-Topo-1

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inhibition and combilexin-type of binding to DNA respectively) to know the structural details of
protein-small molecule complex on interaction with active compounds 7g and 7r within the
constraints of target protein binding site by molecular docking studies.
These compounds fit well into M38 binding pocket present in DNA-Topo 1 complex
(PDB ID: 1SC7), β-carboline ring intercalated with DNA (Fig. 11A) and the C-1 substituents
exhibited π-stacking interactions with dT10, TGP11, dC112, dA113 nitrogen bases and
hydrophobic interactions with side chains of Arg364, Thr718 and Asn722 amino acid residues
(Fig. 11B & 11C). Whereas the C-3 substituents extended out of the groove and showing
hydrophobic interactions with dT10, Ala351, Asn352, Tyr426, Met428 and Lys436. In addition,
the same C-3 substituent present in the compounds 7g and 7r also interact with Ile427 and dT9
respectively.
<Insert Fig. 11>
Biophysical studies and our previous reports also proved that the C-1 and C-3 substituted
β-carboline compounds could behave like combilexin type of molecules [39]. The combilexin
type of interaction of 7g and 7r compounds with DNA is evaluated using the DNA crystal
structure with PDB ID: 1NAB. In this regard, molecular docking of compounds 7g and 7r at the
4
4D binding pocket of DNA (PDB ID: 1NAB) indicates combilexin type of binding (Fig. 12A
and 12B). In which β-carboline ring stacked in between the consecutive DNA base pairs dG12-
dC1 and dC11-dG2, C-1 substituents show good interaction with the DNA helices and interacts
well with dG-2, dA-3. On the other hand, C-3 substituents showing interactions with dA-3, dT-4
and dT-10 nucleotides present in the groove with the help of Sp3 carbon present in bis-indole
motif (Fig. 12C & 12D). In addition, dG-12 present in groove also showing interaction with C-3
Substituent of compound 7g.

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<Insert Fig. 12>
The molecular docking studies revealed that the β-carboline ring is responsible for
intercalation and C-1 substituent especially in 7g come close to dG-2, dA-3 of DNA and show
effective interaction. Further, the C-3 substituent was responsible for interaction with nucleotides
present at the groove in 44D binding pocket of 1NAB. Hence, these compounds have enough
structural features to show the combilexin type of binding even though smaller in molecular size
along with Topoisomerase I Inhibitor properties.
3
. Conclusion
In conclusion, a new series of β-carboline-bisindole compounds of varying substitutions
at C-1 and C-3 positions were synthesized involving two pharmacophores and their
antiproliferative properties were evaluated against various human cancer cell lines. All the
compounds exhibited pronounced antiproliferative activity against prostate cancer cell line.
Among them 7g and 7r compounds were found to possess significant cytotoxicity. Further
assays such as Annexin V-FITC assay revealed that these compounds induce apoptosis. Cell
cycle histograms indicate that these compounds stop the cell cycle at G2/M phase. In addition,
these compounds inhibit topoisomerase I by binding with DNA-topoisomerase complex and can
also cleave the pBR322 plasmid upon irradiation with UV light. DNA binding assays and
molecular docking studies support that these compounds abide by combilexin type interactions
with DNA.
4
. Experimental Section
4
.1. Chemistry
All solvents and reagents were purchased from the commercial sources and were used without
further purification. Reactions were monitored by TLC on silica gel glass plate containing 60

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1
13
GF-254, and visualization was done by UV light and iodine vapor. H and C NMR spectra
were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or InovaVarian-VXR-unity (400,
5
00 MHz) instruments. Chemical shifts were expressed in parts per million (δ in ppm) downfield
1
from TMS expressed as internal standard and coupling constants are expressed in Hz. H NMR
spectral data were reported in the following order: multiplicity (s, singlet; brs, broad singlet; d,
doublet; dd, doublet of doublets; t, triplet; m, multiplet), coupling constants in Hz, and number of
protons. ESI mass spectra were recorded on a Micromass Quattro LC using ESI+ software with
capillary voltage 3.98 kV and an ESI mode positive ion trap detector. High resolution mass
spectra were recorded on a QSTAR XL Hybrid MS-MS mass spectrometer. Melting points were
determined with an electro thermal digital melting point apparatus IA9100 and are uncorrected.
The purity of tested compounds was P98% as determined by HPLC performed on a Shimadzu
LC-20AD apparatus equipped with a SPD-M20A diode array detector and a Shimadzu SIL-
2
0AC auto injector using C18 column (waters 5 µM C18, 4.6 mm 250 mm column). Elution
H
conditions: mobile phase A (85%)- acetonitrile; mobile phase B(15%)-water, P 4.3 adjusted
with 0.1% formic acid + 10 mmol NH OAc. The flow rate was 1.0 mL/min and the injection
4
volume was 5 µL at 25 °C and detection at 254 nm.
4
.1.1. General procedure for the preparation of compounds (3a-d)
To a stirred solution of L-tryptophan (1, 0.1 mol) in methanol (40 ml) was added (0.11
o
o
mol) thionly chloride drop wise at 0 C and the reaction mixture was stirred for 8 h at 27 C then,
excess solvent was removed under vacuum and the crude product was co-distilled with toluene
(2 × 15 mL) to obtain a solid. The resulted solid was extracted with DCM (3 × 40 mL), washed
with saturated NaHCO solution, the combined organic layers were dried over anhydrous
3
Na SO and evaporated under vacuum to obtain white solid tryptophan ester (2). To a solution
2
4

ACCEPTED MANUSCRIPT
mixture of tryptophan ester (0.023 mol) and substituted benzaldehyde (0.023 mol) in toluene (50
o
mL), a catalytic amount of p-TSA was added and the reaction mixture was refluxed at 110 C for
1
2 h. After completion of the reaction, the solvent was removed under the vacuum, the crude
mixture was extracted with ethyl acetate. The organic layers were dried over anhydrous Na SO
2
4
and concentrated under vacuum. The resulted diasteriomeric mixture (3a-d) was directly used for
the next step without any further purification.
4
.1.2. General procedure for the synthesis of methyl-1-phenyl-9H-pyrido[3,4-b]indole-3-
carboxylates (4a-d)
To a stirred solution of compounds 3a-d (0.01 mol) in DMF was added triethylamine
o
(
0.03 mol) and trichloroisocyanuric acid (0.012 mol) solution in DMF drop wise at -20 C. The
o
reaction mixture was slowly warmed to 0 C and stirred for 2 h, after completion of the reaction,
was monitored by TLC, added ice water, filtered the resulted precipitate and dried under the
vacuum to obtain the product (4a-d). These products were directly used for the next step without
any further purification.
4
.1.3. General procedure for the synthesis of (1-phenyl-9H-pyrido[3,4-b]indol-3-yl)methanol
(6a-d)
To the compound 4a-d ( 0.019 mol) taken in dry THF (60 ml) was added LiAlH (0.076
4
o
o
mol) portion wise at -5 to 0 C and continued stirring for 4 h at 27 C, after completion of the
reaction excess of LiAlH was quenched by adding Na SO ·10H O until the bubbling stops,
4
2
4
2
filtered through celite and concentrated under vacuum. To the crude product 5a-d (0.014 mol) in
o
dry CH Cl was added DMP (0.021 mol) and stirred for 2 h at 27 C. After completion of the
2
2
reaction, the reaction mixture was washed with water (2 × 50 ml), the organic layers were dried

ACCEPTED MANUSCRIPT
over Na
2
SO
4
and concentrated under vacuum. The resulted crude products was purified by
column chromatography (ethyl acetate: hexane) to afford the compounds (6a-d) with high purity.
4
.1.3.1. 1-(3,4,5-Trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde (6a)
1
yellow solid: 81 % yield; mp: 220-224 °C; H NMR (300 MHz, CDCl3 + DMSO-d ) δ (ppm):
6
1
7
4
0.21 (s, 1H), 9.2 (bs, 1H), 8.71 (s, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.64 – 7.63 (m, 2H), 7.42 –
+
.39 (m, 1H), 7.13 (s, 2H), 3.92 (s, 9H); MS (ESI): m/z 363 [M + H] .
.1.3.2. 1-(4-Methylphenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde (6b)
1
White solid of 79 % yield; mp: 189-190 °C; H NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.4 (bs,
1
H), 10.25 (s, 1H), 8.69 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.13 (t, J = 8.4Hz, 3H), 7.7 (d, J = 7.7
+
Hz, 1H), 7.6 (t, J = 7.3Hz, 2H), 7.5 (s, 1H), 2.28 (s, 3H); MS (ESI): m/z 287 [M + H] .
4
.1.3.3. 1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde (6c)
1
Pale yellow solid: 82 % yield; mp: 213-217 °C; H NMR (300 MHz, CDCl3 + DMSO-d ) δ
6
(ppm): 10.3 (s, 1H), 8.9 (s, 1H), 8.7 (s, 1H), 8.2 (d, J = 7.94 Hz, 1H), 7.62 (m, 3H), 7.37 ( m,
+
2
H), 7.14 (m, 2H), 3.87 (s, 3H); MS (ESI): m/z 303 [M + H] .
4
.1.3.4. 1-(4-Fluorophenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde (6d)
1
White solid of 85 % yield; mp: 189-190 °C; H NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.42 (bs,
1
H), 10.25 (s, 1H), 8.69 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.13 (t, J = 8.4 Hz, 3H), 7.7 (d, J = 7.7
+
Hz, 1H), 7.6 (t, J = 7.3 Hz, 2H), 7.5 (s, 1H); MS (ESI): m/z 291 [M + H] .
4
.1.4. General Procedure for the synthesis β-carboline-bisindole compounds (7a-7ab)
To a solution of β-carboline aldehyde (1 equiv.) in water-ethanol (3:1) was added
respective substituted indole (2 equiv.) and 2 mol % of catalyst Amberlite IR-120H. The reaction
o
mixture was stirred for 6 h at 80 C, after completion of the reaction, monitored by the TLC and
the appearance of solid is also an indication of the product formation. Then the reaction mixture

ACCEPTED MANUSCRIPT
was cooled to room temperature and filtered the resulted solid. The crude products were purified
by the filter column chromatography to afford the pure respective β-carboline-bisindole
compounds in good to excellent yields.
4
.1.4.1.
3-(bis(5-bromo-1H-indol-3yl)methyl)-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-
b]indole (7a)
1
Brown solid; yield 86 %; mp: 194-196˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.15 (s,
3
6
1
7
3
1
1
H), 10.57 (s, 2H), 7.99 (d, J = 7.8 Hz, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 7.57 (d, J = 12.3 Hz, 3H),
.48 (d, J = 7.6 Hz, 1H), 7.30 – 7.23 (m, 3H), 7.14 (t, J = 8.9 Hz, 3H), 6.92 (s, 2H), 6.16 (s, 1H),
1
3
6
.96 (s, 6H), 3.88 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 152.77, 141.51, 140.55, 137.61,
35.11, 131.68, 128.38, 127.64, 124.81, 123.21, 121.38, 120.94, 120.73, 118.89, 117.40,
12.69,111.97, 111.67, 110.99, 105.54, 60.15, 55.64, 41.92; HRMS (ESI) : calcd for
+
C H N O Br [M+H] 735.0601, found 735.0597.
37
29
4
3
2
4
.1.4.2. 3-(di(1H-indol-3-yl)methyl)-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole (7b)
1
Light brown solid; yield 91 %; mp: 186-188 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.21
3
6
(s, 1H), 10.49 (s, 2H), 7.99 (d, J = 7.8 Hz, 1H), 7.93 (s, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.46 (d, J
=
7.7 Hz, 3H), 7.35 (d, J = 8.1 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 3H), 6.97 (s,
1
3
2
H), 6.87 (t, J = 7.4 Hz, 2H), 6.22 (s, 1H), 3.94 (s, 6H), 3.85 (s, 3H); C NMR (75 MHz,
DMSO-d ) δ 153.03, 136.57, 131.65, 128.03, 126.90, 123.67, 121.50, 120.80, 119.25, 118.15,
6
+
1
5
12.38, 111.40, 105.98, 60.17, 55.88, 42.28; HRMS (ESI) : calcd for C H N O [M+H]
37 31 4 3
79.2391, found 579.2388.

ACCEPTED MANUSCRIPT
4
.1.4.3.
3-(bis(2-methyl-5-nitro-1H-indol-3-yl)methyl)-1-(3,4,5-trimethoxyphenyl)-9H-
pyrido[3,4-b]indole (7c)
Light green solid; yield 85 %; mp: 213-215 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.01
1
3
6
(s, 1H), 10.85 (s, 2H), 7.90 (dd, J = 10.4, 8.3 Hz, 3H), 7.72 (s, 2H), 7.46 (s, 3H), 7.34 (d, J = 9.0
Hz, 2H), 7.05 (s, 2H), 6.89 (s, 1H), 6.40 (s, 1H), 3.86 (d, J = 9.8 Hz, 3H), 3.78 (s, 6H), 2.21 (s,
1
3
6
1
1
6
4
H); C NMR (75 MHz, DMSO-d ) δ 152.04, 151.74, 141.39, 140.59, 140.23, 138.98, 137.40,
6
35.23, 130.92, 126.68, 126.45, 120.12, 119.86, 117.98, 114.71, 114.01, 111.19, 109.16, 104.92,
+
04.55, 59.02, 54.78, 54.35, 40.06, 11.03; HRMS (ESI) : calcd for C39
97.2405, found 697.2403.
33 6 7
H N O [M+H]
.1.4.4. 3-(bis(5-nitro-1H-indol-3-yl)methyl)-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole
7d)
Light yellow solid; yield 81 %; mp: 346-348 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.28
(
1
3
6
(s, 2H), 11.22 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 8.02 – 7.92 (m, 4H), 7.77 (s, 1H), 7.60 (d, J = 8.2
Hz, 1H), 7.47 (t, J = 7.2 Hz, 3H), 7.25 (s, 2H),7.16 (d, J = 8.4 Hz, 3H), 6.37 (s, 1H), 3.95 (s, 6H),
1
3
3
1
1
3 6
.87 (s, 3H); C NMR (75 MHz, CDCl +DMSO-d ) δ 152.80, 150.70, 141.45, 141.14, 140.19,
39.73, 137.61, 133.89, 131.88, 130.23, 127.59, 126.74, 125.93,120.89, 120.75, 120.30, 118.88,
16.43, 116.27,112.02, 111.76, 111.23, 105.52, 60.15, 55.58, 42.16; HRMS (ESI) : calcd for
+
C H N O [M+H] 669.2092, found 669.2099.
37
29
6
7
4
5
.1.4.5. 3,3’-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methelene)bis(1H-indole-
-carbonitrile) (7e)
1
White solid; yield 80 %; mp: 195-197 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 10.97 (s,
3
6
1
7
H), 10.85 (s, 2H), 7.97 (d, J = 7.8 Hz, 1H), 7.83 (s, 1H), 7.78 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H),
.49 (dd, J = 12.9, 8.3 Hz, 4H), 7.32 (d, J = 8.4 Hz, 2H), 7.21 – 7.15 (m, 3H), 6.99 (s, 2H), 6.29

ACCEPTED MANUSCRIPT
13
(
s, 1H), 3.95 (s, 6H), 3.89 (s, 3H); C NMR (75 MHz, CDCl
3
+DMSO-d
6
) δ 152.89, 150.88,
1
1
41.37, 141.21, 138.34, 137.64, 133.95, 131.93, 130.30, 127.61, 126.45, 125.74, 124.91, 123.54,
20.94, 120.69, 118.92, 118.78, 112.06, 111.76, 105.47, 100.65, 60.31, 55.67, 42.19; HRMS
+
(
ESI) : calcd for C H N O [M+H] 629.2296, found 629.2297.
3
9
29
6
3
4
.1.4.6. 3-(di(1H-indol-3-yl)methyl)-1-(p-tolyl)-9H-pyrido[3,4-b]indole (7f)
1
Brown solid; yield 88 %; mp: 323-325˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.24
3
6
(s,1H), 10.62 (s, 2H), δ 8.01 (dd, J = 22.2, 9.9 Hz, 4H), 7.69 (d, J = 3.0 Hz, 1H), 7.61 – 7.58 (m,
2
H), 7.45 (d, J = 7.7 Hz, 2H), 7.37 (dd, J = 12.5, 8.0 Hz, 4H), 7.15 (t, J = 7.1 Hz, 1H), 7.02 (t, J
1
3
=
7.6 Hz, 2H), 6.94 (s, 1H), 6.87 (t, J = 7.5 Hz, 2H), 6.22 (s, 1H), 2.46 (s, 1H); C NMR (75
MHz, CDCl +DMSO-d ) δ 150.92, 141.55, 140.31, 139.86, 137.78, 135.71, 131.98, 130.30,
3
6
1
1
4
29.09, 128.39, 127.69, 126.86, 126.03, 120.96, 120.43, 119.05, 116.83, 116.40, 112.18, 111.64,
+
11.41, 42.56, 21.02; HRMS (ESI) : calcd for C H N [M+H] 503.2230, found 503.2218.
3
5
27
4
.1.4.7. 3-(bis(5-bromo-1H-indol-3-yl)methyl)-1-(p-tolyl)-9H-pyrido[3,4-b]indole (7g)
1
Brown solid; yield 84 %; mp: 186-188 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 10.74 (s,
3
6
1
7
6
1
1
H), 10.37 (s, 2H), 7.96 (d, J = 7.7 Hz, 3H), 7.83 (s, 1H), 7.61 (s, 3H), 7.46 (t, J = 7.6 Hz, 1H),
.38 (d, J = 7.7 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 7.15 (dd, J = 14.4, 7.5 Hz, 3H), 6.91 (s, 2H),
1
3
.13 (s, 1H), 2.45 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 151.98, 141.86, 141.09, 138.23,
6
35.41, 131.81, 130.56, 129.54, 128.76, 128.56, 128.30, 125.28, 123.53, 121.96, 121.60, 121.02,
19.55, 117.70, 113.61, 112.58, 112.05, 111.11, 42.38, 21.19; HRMS (ESI) : calcd for
+
C H N Br [M+H] 659.0440, found 659.0442; HPLC:t 5.63min, purity 97.4%
35
25
4
2
R
.
4
.1.4.8. 3-(bis(2-methyl-5-nitro-1H-indol-3-yl)methyl)-1-(p-tolyl)-9H-pyrido[3,4-b]indole (7h)
1
Yellow solid; yield 81 %; mp: 238-240 ˚C; H NMR (300 MHz, CDCl
3
+DMSO-d
6
) δ 11.24 (s,
2
H), 11.16 (d, J = 7.1 Hz, 1H), 8.08 – 7.95 (m, 2H), 7.85 – 7.80 (m, 6H), 7.61 (t, J = 7.8 Hz,

ACCEPTED MANUSCRIPT
1
1
1
1
H), 7.49 – 7.39 (m, 2H), 7.32 (d, J = 9.5 Hz, 2H), 7.27 (d, J = 8.1 Hz, 1H), 7.16 (t, J = 7.4 Hz,
1
3
H), 6.30 (s, 1H), 2.19 (s, 3H), 2.14 (s, 6H); C NMR (75 MHz, DMSO-d ) δ 149.93, 141.52,
6
39.95, 138.40, 137.60, 135.96, 131.96, 130.02, 129.95, 128.87, 128.26, 127.59, 121.03, 120.90,
18.94, 115.90, 115.14, 114.97, 112.18, 111.96, 110.13, 41.50, 20.89, 12.04; HRMS (ESI) :
+
calcd for C H N O [M+H] 621.2245, found 621.2249.
3
7
29
6
4
4
.1.4.9. 3-(bis(5-nitro-1H-indol-3-yl)methyl)-1-(p-tolyl)-9H-pyrido[3,4-b]indole (7i)
1
Yellow solid; yield 78 %; mp: 242-244 ˚C; H NMR (300 MHz, CDCl
3
+DMSO-d
6
) δ 11.28 (s,
2
H), 11.05 (s, 1H), 8.49 (d, J = 1.8 Hz, 2H), 8.03 – 7.94 (m, 6H), 7.80 (d, J = 5.0 Hz, 1H), 7.60
(d, J = 8.2 Hz, 1H), 7.44 (d, J = 9.0 Hz, 3H), 7.36 (d, J = 7.9 Hz, 2H), 7.23 (s, 1H), 7.17 (t, J =
1
3
7
1
1
.5 Hz, 1H), 6.32 (s, 1H), 2.45 (s, 3H); C NMR (75 MHz, CDCl +DMSO-d ) δ 150.74, 141.37,
3 6
40.13, 139.68, 137.60, 135.53, 131.80, 130.11, 128.91, 128.20, 127.51, 126.67, 125.85, 120.77,
20.24, 118.86, 116.64, 116.21, 112.00, 111.46, 111.22, 42.37, 20.83; HRMS (ESI) : calcd for
+
C H N O [M+H] 593.1932, found 593.1932.
35
25
6
4
4
.1.4.10. 3,3’-((1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)methylene)bis(1H-indole-5-carbonitrile)
7j)
White solid; yield 79 %; mp: 254-256 ˚C; H NMR (300 MHz, DMSO-d
Hz, 3H), 7.96 (dd, J = 19.5, 11.7 Hz, 6H), 7.61 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.3 Hz, 3H), 7.39
(
1
6
) δ 11.15 (d, J = 7.6
1
3
(
d, J = 7.4 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.17 (s, 3H), 6.21 (s, 1H), 2.46 (s, 3H). C NMR
(
75 MHz, CDCl +DMSO-d ) δ 150.94, 141.38, 138.23, 137.59, 131.71, 128.97, 128.15, 127.50,
3
6
1
1
26.35, 125.69, 125.02, 123.26, 120.86, 120.78, 120.53, 118.86, 118.64, 112.26, 112.04, 111.40,
+
00.37, 42.35, 20.85; HRMS (ESI) : calcd for C H N [M+H] 553.2135, found 553.2133.
3
7
25
6

ACCEPTED MANUSCRIPT
4
.1.4.11. 3-(di(1H-indol-3-yl)methyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole (7k)
1
Brown solid; yield 84 %; mp: 212-214˚C; H NMR (300 MHz, DMSO-d ) δ 11.06 (s, 1H), 10.46
6
(s, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 4.1 Hz, 1H), 7.58 (d, J =
8
6
.0 Hz, 1H), 7.45 (d, J = 5.6 Hz, 3H), 7.34 (d, J = 8.0 Hz, 2H), 7.06 (dt, J = 23.4, 7.6 Hz, 5H),
1
3
.88 (dd, J = 15.6, 7.8 Hz, 4H), 6.19 (s, 1H), 3.88 (d, J = 2.4 Hz, 3H); C NMR (75 MHz,
CDCl +DMSO-d ) δ 159.25, 152.48, 141.30, 140.50, 136.38, 131.35, 129.85, 129.51, 127.27,
3
6
1
4
4
26.72, 123.31, 120.84, 120.49, 119.04,118.68, 118.13, 117.83, 113.57, 111.92, 110.96, 54.87,
+
2.53; HRMS (ESI) : calcd for C35
H
27
N
4
O [M+H] 519.2179, found 519.2167.
.1.4.12.
3-(bis(2-methyl-5-nitro-1H-indol-3-yl)methyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-
b]indole (7l)
1
Yellow solid; yield 83 %; mp: 239-241 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.39 (s,
3
6
3
H), 8.02 – 7.94 (m, 3H), 7.90 (s, 1H), 7.82 (d, J = 7.1 Hz, 4H), 7.63 (d, J = 8.2 Hz, 1H), 7.49 (t,
J = 7.6 Hz, 1H), 7.34 (d, J = 9.5 Hz, 2H), 7.19 (t, J = 8.6 Hz, 3H), 6.30 (s, 1H), 3.33 (s, 3H), 2.20
1
3
(
s, 6H); C NMR (75 MHz, DMSO-d
6
) δ 160.59, 150.03, 141.64, 140.25, 139.91, 138.41,
1
1
6
36.07, 134.61, 131.86, 130.37, 130.27, 128.53, 127.89, 127.46, 121.23, 119.17, 115.93, 115.16,
+
14.96, 112.25, 110.33, 63.43, 41.40, 12.04; HRMS (ESI) : calcd for C37
37.2194, found 637.2191.
29 6 5
H N O [M+H]
4
.1.4.13.
3-(bis(5-bromo-1H-indol-3-yl)methyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole
(7m)
1
Brown solid; yield 81 %; mp: 168-170 ˚C; H NMR (300 MHz, CDCl
H), 10.66 (s, 2H), 8.02 (t, J = 9.1 Hz, 2H), 7.86 (s, 2H), 7.60 (d, J = 9.4 Hz, 3H), 7.46 (t, J = 7.4
Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.14 (dd, J = 20.6, 7.9 Hz, 5H), 6.99 (s, 2H), 6.09 (s, 1H), 3.90
3 6
+DMSO-d ) δ 11.05 (s,
1

ACCEPTED MANUSCRIPT
1
3
(
s, 3H); C NMR (75 MHz, CDCl
3
+DMSO-d
6
) δ 159.34, 151.54, 141.38, 135.08, 131.40,
1
29.97, 129.57, 128.35, 127.46, 124.74, 123.11, 121.53, 120.86, 120.76, 118.83, 117.39, 113.70,
+
1
12.83, 112.02, 110.83, 54.90, 42.34; HRMS (ESI) : calcd for C H N OBr [M+H] 675.0390,
35 25 4 2
found 675.0392.
4
.1.4.14. 3-(bis(5-nitro-1H-indol-3-yl)methyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole (7n)
1
Yellow solid; yield 79 %; mp: 299-301 ˚C; H NMR (500 MHz, CDCl
3
) δ 11.29 (s, 2H), 11.06
(s, 1H), 8.50 (d, J = 2.0 Hz, 2H), 8.03 (dd, J = 11.8, 4.9 Hz, 3H), 7.97 – 7.92 (m, 3H), 7.83 (d, J
=
2.0 Hz, 1H), 7.60 (s, 1H), 7.45 (t, J = 7.5 Hz, 3H), 7.24 (s, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.08
1
3
(
d, J = 8.6 Hz, 2H), 6.31 (s, 1H), 3.89 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 159.36, 150.65,
6
1
1
41.35, 140.12, 139.69, 131.64, 130.82, 130.08, 129.59, 129.58, 127.49, 126.66, 125.84, 120.82,
20.23, 118.86, 116.66, 116.23, 113.64, 111.98, 111.20, 54.89, 42.35; HRMS (ESI) : calcd for
+
C H N O [M+H] 609.1881, found 609.1876.
35
25
6
5
4
.1.4.15.
carbonitrile) (7o)
Brown solid; yield 77 %; mp: 238-240 ˚C; H NMR (300 MHz, CDCl
7.6 Hz, 3H), 8.06 (dd, J = 23.1, 8.6 Hz, 6H), 7.62 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 8.3 Hz, 3H),
3,3’-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methelene)bis(1H-indole-5-
1
3
6
+DMSO-d ) δ 11.36 (d, J
=
1
3
7
.32 (d, J = 7.1 Hz, 4H), 7.23 – 7.09 (m, 3H), 6.24 (s, 1H), 3.89 (s, 3H); C NMR (75 MHz,
DMSO-d ) δ 159.49, 151.11, 141.52, 141.04, 138.24, 131.47, 129.61, 126.47, 125.93, 125.26,
6
1
23.33, 121.17, 120.67, 119.07, 118.79, 113.96, 112.56, 112.27, 100.30, 55.09, 42.14; HRMS
+
(
ESI) : calcd for C H N O [M+H] 569.2084, found 569.2077.
3
7
25
6

ACCEPTED MANUSCRIPT
4
.1.4.16.
3-(bis(5-fluoro-1H-indol-3-yl)methyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole
(7p)
1
light brown solid; yield 80 %; mp: 210-212˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.30
3
6
(s, 1H), 10.67 (s, 2H), 8.04 (d, J = 38.6 Hz, 3H), 7.64 (d, J = 25.3 Hz, 2H), 7.45 – 7.37 (m, 6H),
1
3
7
.14 (s, 1H), 6.98 (d, J = 17.7 Hz, 4H), 6.88 (s, 1H), 6.21 (s, 1H), 4.29 (s, 1H); C NMR (75
MHz, CDCl +DMSO-d ) δ 160.59, 160.08, 157.51, (d, J = 234.3 Hz), 148.73, 148.18, 141.27,
3
6
1
1
40.67, 135.35, 129.60, 129.10, 128.79, 127.56, 125.33, 122.14, 120.67, 119.98, 117.18, 115.05,
14.55, 112.00, 111.65, 111.53, 110.05, 105.12, 104.81, 104.32, 55.58, 42.92; HRMS (ESI) :
+
calcd for C35
H
25
N
4
OF
2
[M+H] 555.1991, found 555.1983.
4
.1.4.17. 3-(bis(5-methoxy-1H-indol-3-yl)methyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole
7q)
White solid; yield 85 %; mp: 191-193 ˚C; H NMR (500 MHz, CDCl ) δ 8.39 (s, 1H), 7.94 (d, J
(
1
3
=
7.9 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.82 (s, 1H), 7.47 – 7.41 (m, 2H), 7.17 (dd, J = 10.7, 8.5
Hz, 3H), 7.01 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 2.3 Hz, 1H), 6.79 (s, 1H), 6.77 (s, 1H), 6.76 (s,
1
3
2
1
1
3
H), 6.24 (s, 1H), 3.83 (s, 3H), 3.62 (s, 6H); C NMR (101 MHz, CDCl ) δ 160.01 (s), 153.70,
53.54, 141.75, 140.92, 132.19 , 132.06, 131.46, 130.80, 129.66, 128.25, 127.89, 124.60, 122.24,
22.04, 119.93, 119.27, 114.56, 112.17, 111.92, 111.79, 111.59, 102.27, 55.96, 55.49, 43.24;
+
HRMS (ESI) : calcd for C H N O [M+H] 579.2391, found 579.2380.
37
31
4
3
4
.1.4.18. 3-(di(1H-indol-3-yl)methyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole (7r)
1
Light brown solid; yield 82 %; mp: 195-197˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.06
3
6
(
s, 1H), 10.30 (d, J = 22.1 Hz, 2H), 8.09 (s, 1H), 7.97 (d, J = 7.4 Hz, 1H), 7.91 (s, 1H), 7.76 –
.68 (m, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.46 (t, J = 9.7 Hz, 3H), 7.36 (d, J = 8.1 Hz, 1H), 7.29 (d,
J = 4.8 Hz, 2H), 7.16 (t, J = 7.3 Hz, 1H), 7.05 (dd, J = 13.7, 6.2 Hz, 2H), 6.89 (t, J = 7.1 Hz, 5H),
7

ACCEPTED MANUSCRIPT
1
3
6
1
1
1
4
4
3 6
.28 (s, 1H); C NMR (75 MHz, CDCl +DMSO-d ) δ 161.49 (d, J = 247.1 Hz), 151.88, 140.76,
38.86, 135.74, 130.86, 129.55 (d, J = 7.4 Hz), 128.76, 126.86, 126.03, 125.70, 125.32, 122.70,
21.04, 120.24, 119.88, 118.37, 118.17, 117.36, 117.20, 114.32 (d, J = 21.0 Hz), 111.22, 110.90,
+
10.30, 41.79; HRMS (ESI) : calcd for C H N F [M+H] 507.1979, found 507.1970; HPLC:t
3
4
24
4
R
.87 min, purity 96.9% .
.1.4.19.
3-(bis(2-methyl-5-nitro-1H-indol-3-yl)methyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-
b]indole (7s)
1
Yellow solid; yield 80 %; mp: 241-243 ˚C; H NMR (300 MHz, DMSO-d
6
) δ 10.67 (s, 2H),
1
0.54 (s, 1H), 7.92 (dt, J = 13.0, 7.5 Hz, 5H), 7.83 (s, 1H), 7.75 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H),
.49 (t, J = 7.6 Hz, 1H), 7.42 (s, 1H), 7.30 (d, J = 8.9 Hz, 2H), 7.21 – 7.13 (m, 3H), 6.36 (s, 1H),
7
1
3
2
1
1
.16 (s, 6H); C NMR (75 MHz, CDCl +DMSO-d ) δ 162.20 (d, J = 247.5 Hz), 149.92, 141.45,
3 6
40.46, 139.95, 138.34, 135.84, 134.49, 131.87, 130.15, (d, J = 8.4 Hz), 127.64, 127.41, 120.95,
20.83, 118.95, 115.77, 115.25, 114.81, 144.67, 112.04 (d, J = 21.3 Hz), 109.94, 41.38, 11.94;
+
HRMS (ESI) : calcd for C H N O F [M+H] 625.1994, found 625.1997.
36
26
6
4
4
.1.4.20. 3-(bis(5-nitro-1H-indol-3-yl)methyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole (7t)
1
Light green solid; yield 77 %; mp: 232-234 ˚C; H NMR (300 MHz, CDCl
3
+DMSO-d
6
) δ 11.28
(s, 2H), 11.14 (s, 1H), 8.49 (d, J = 2.1 Hz, 2H), 8.11 (dd, J = 8.7, 5.5 Hz, 2H), 8.03 (d, J = 7.8
Hz, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 2.2 Hz, 1H), 7.80 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H),
1
3
7
.47 (dd, J = 14.7, 8.1 Hz, 3H), 7.22 (dd, J = 11.8, 10.0 Hz, 5H), 6.33 (s, 1H); C NMR (75
MHz, CDCl +DMSO-d ) δ 162.19 (d, J = 247.2 Hz), 150.73, 141.46, 140.08, 139.65, 134.46,
3
6
1
1
31.70, 130.23 (d, J = 8.1 Hz), 127.67, 126.66, 125.78, 120.90, 120.69, 120.09, 118.98, 116.61,
16.18, 115.01 (d, J = 21.3 Hz), 111.97, 111.80, 111.24, 42.29 HRMS (ESI) : calcd for
+
C
34
H
22
N
6
O
4
F [M+H] 579.1681, found 597.1680; HPLC:t
R
5.53min, purity 98.6%
.

ACCEPTED MANUSCRIPT
4
.1.4.21.
carbonitrile) (7u)
Light brown solid; yield 78 %; mp: 198-200 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 10.91
3,3’-((1-(4-fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)methelene)bis(1H-indole-5-
1
3
6
(s, 3H), 8.07 (s, 2H), 8.00 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 5.9 Hz, 2H), 7.59 (s, 2H), 7.47 (d, J =
1
3
8
.5 Hz, 3H), 7.31 (d, J = 7.9 Hz, 4H), 7.19 (s, 1H), 7.04 (s, 2H), 6.24 (s, 1H); C NMR (75
MHz, CDCl +DMSO-d ) δ 162.21 (d, J = 247.8 Hz), 150.88, 141.48, 140.05, 138.26, 134.50,
3
6
1
1
31.68, 130.19 (d, J = 7.9 Hz), 127.69, 126.29, 125.68, 124.98, 123.30, 120.93, 120.71, 120.53,
18.99, 118.47, 115.10 (d, J = 21.4 Hz), 112.23, 111.98, 111.71, 100.39, 42.27; HRMS (ESI) :
+
calcd for C36
H
22
N
6
F [M+H] 557.1884, found 557.1879.
4
.1.4.22. 3-(bis(5-bromo-1H-indol-3-yl)methyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole (7v)
1
Light brown solid; yield 79 %; mp: 194-196 ˚C; H NMR (500 MHz, CDCl ) δ 8.41 (s, 1H), 8.07
3
(s, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.91 (dd, J = 8.5, 5.4 Hz, 2H), 7.83 (s, 1H), 7.63 (s, 1H), 7.52 –
1
3
7
.46 (m, 2H), 7.22 – 7.15 (m, 6H), 6.78 (d, J = 1.6 Hz, 2H), 6.17 (s, 1H); C NMR (75 MHz,
DMSO-d ) δ 162.45 (d, J = 247.03 Hz), 151.96, 141.67, 140.15, 135.39, 134.77, 131.89, 130.53,
6
1
29.50 (d, J = 8.8 Hz), 127.92, 125.04, 123.48, 121.75, 121.18, 121.01, 119.23, 117.63, 115.41
(
[
d, J = 21.45 Hz), 113.12, 112.25, 111.84, 111.19, 42.60; HRMS (ESI) : calcd for C34 Br F
H
22
N
4
2
+
M+H] 663.0190, found 663.0197.
4
.1.4.23. 3-(bis(5-fluoro-1H-indol-3-yl)methyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole (7w)
1
White solid; yield 76 %; mp: 164-166 ˚C; H NMR (400 MHz, CDCl ) δ 8.46 (s, 1H), 8.06 (s,
3
1
7
d
1
H), 7.98 (d, J = 7.8 Hz, 1H), 7.91 (s, 2H), 7.84 (s, 1H), 7.48 (q, J = 8.3 Hz, 2H), 7.21 (s, 3H),
13
.10 (d, J = 9.6 Hz, 2H), 6.89 – 6.84 (m, 4H), 6.18 (s, 1H); C NMR (75 MHz, CDCl +DMSO-
3
6
) δ 156.38 (d, J = 244.7 Hz), 151.97, 141.46, 139.81, 134.74, 133.07, 131.57, 130.27, 127.60,
26.10 (d, J = 7.8 Hz), 120.93, 118.94, 117.88, 115.04 (d, J = 21.2 Hz), 112.00, 111.84, 111.71,

ACCEPTED MANUSCRIPT
+
1
22 4 3
08.88, 108.53, 103.88, 103.57, 42.57; HRMS (ESI) : calcd for C34H N F [M+H] 543.1791,
found 543.1787.
.1.4.24.
7x)
4
3-(bis(5-methoxy-1H-indol-3-yl)methyl)-1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole
(
1
Brown solid; yield 82 %; mp: 178-180 ˚C; H NMR (500 MHz, CDCl ) δ 8.39 (s, 1H), 7.96 (d, J
3
=
7.9 Hz, 1H), 7.89 (dd, J = 15.9, 4.7 Hz, 4H), 7.50 – 7.44 (m, 2H), 7.22 – 7.18 (m, 4H), 6.94 (d,
1
3
J = 2.1 Hz, 2H), 6.80 (d, J = 2.1 Hz, 1H), 6.78 (s, 2H), 6.25 (s, 1H), 3.63 (s, 6H); C NMR (101
MHz, CDCl ) δ 162.94 (d, J = 248.1 Hz), 153.64, 153.55, 140.87, 140.69, 134.80, 132.10,
31.96, 131.05, 130.10 (d, J = 8.2 Hz), 128.40, 127.75, 124.44, 122.01, 120.04, 119.08, 116.01
3
1
(d, J = 21.5 Hz), 112.62, 111.85, 111.68, 111.50, 102.20, 55.87, 43.06; HRMS (ESI) : calcd for
+
C H N O F [M+H] 567.2191, found 567.2183.
36
28
4
2
4
.1.4.25.
3-(bis(5-fluoro-1H-indol-3yl)methyl)-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-
b]indole (7y)
1
White solid; yield 84 %; mp: 190-192 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 11.29 (s,
3
6
1
1
4
H), 10.69 (s, 2H), 7.99 (dd, J = 21.7, 5.5 Hz, 2H), 7.60 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 7.6 Hz,
H), 7.30 (dd, J = 7.6, 4.3 Hz, 2H), 7.25 (s, 2H), 7.17 (d, J = 7.5 Hz, 1H), 7.08 (d, J = 2.8 Hz,
1
3
H), 6.79 (t, J = 8.9 Hz, 2H), 6.10 (s, 1H), 3.93 (s, 6H), 3.84 (s, 3H); C NMR (75 MHz,
CDCl +DMSO-d ) δ 156.25 (d, J = 232.8 Hz), 152.75, 151.79, 141.40, 140.61, 137.56, 133.98,
3
6
1
1
33.00, 131.53, 130.06, 127.46,126.96, 126.84, 125.21, 120.91, 120.76, 118.81, 117.89, 111.50,
08.76, 108.41, 105.53, 103.91, 103.60, 59.96, 55.52, 42.43; HRMS (ESI) : calcd for
+
C H N O F [M+H] 615.2202, found 615.2199.
37
29
4
3 2

ACCEPTED MANUSCRIPT
4
.1.4.26.
3-(bis(5-methoxy-1H-indol-3yl)methyl-1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-
b]indole (7z)
1
brown solid; yield 90 %; mp: 194-196 ˚C; H NMR (300 MHz, CDCl +DMSO-d ) δ 10.78 (s,
3
6
1
7
H), 9.67 (s, 2H), 7.96 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.52 (s, 1H),
.45 (t, J = 7.6 Hz, 1H), 7.26 (s, 1H), 7.22 (d, J = 6.5 Hz, 3H), 7.15 (t, J = 7.4 Hz, 1H), 6.91 (dd,
J = 10.2, 1.8 Hz, 3H), 6.72 (dd, J = 8.7, 2.2 Hz, 2H), 6.20 (s, 1H), 3.90 (d, J = 11.1 Hz, 9H), 3.62
1
3
(
s, 6H); C NMR (75 MHz, CDCl
3 6
+DMSO-d ) δ 152.81, 152.62, 141.25, 140.62, 137.51,
1
1
6
4
34.29, 131.68, 130.20, 127.35, 127.23, 124.18, 121.04, 120.95, 118.73, 118.11, 111.74, 111.31,
+
10.48, 105.50, 101.69, 60.27, 55.62, 55.19, 42.58; HRMS (ESI) : calcd for C39
39.2602, found 639.2606.
35 4 5
H N O [M+H]
.1.4.27. 3-(bis(5-fluoro-1H-indol-3yl)methyl-1-(p-tolyl)-9H-pyrido[3,4-b]indole (7aa)
1
Light brown solid; yield 83 %; mp: 195-197 ˚C; H NMR (400 MHz, CDCl ) δ 8.42 (s, 1H), 7.99
3
–
7
7.95 (m, 2H), 7.81 (s, 2H), 7.48 – 7.42 (m, 2H), 7.32 (d, J = 7.8 Hz, 2H), 7.20 – 7.17 (m, 2H),
1
3
.10 (dd, J = 9.8, 2.2 Hz, 2H), 6.88 – 6.82 (m, 4H), 6.17 (s, 1H), 2.40 (s, 3H); C NMR (75
+DMSO-d ) δ 156.42(d, J = 232.7 Hz), 154.88, 151.83, 141.18, 141.07, 137.50,
35.63, 132.98, 131.68, 130.00, 128.86, 128.12, 127.34, 126.95, 126.82, 125.19, 120.90, 120.81,
MHz, CDCl
3
6
1
1
18.71, 118.06, 111.75, 111.51, 111.39, 111.27, 108.90, 108.55, 104.01, 103.70, 42.59, 20.79;
+
HRMS (ESI) : calcd for C H N F [M+H] 539.2042, found 539.2035.
35
25
4 2
4
.1.4.28. 3-(bis(5-methoxy-1H-indol-3yl)methyl-1-(p-tolyl)-9H-pyrido[3,4-b]indole (7ab)
1
Brown solid; yield 88 %; mp: 192-194 ˚C; H NMR (500 MHz, CDCl ) δ 8.46 (s, 1H), 7.96 (d, J
3
=
7.9 Hz, 1H), 7.89 (s, 1H), 7.83 (d, J = 7.9 Hz, 2H), 7.49 – 7.44 (m, 2H), 7.32 (d, J = 7.6 Hz,
2
1
H), 7.19 (dd, J = 17.9, 8.3 Hz, 3H), 6.95 (s, 2H), 6.80 (d, J = 2.2 Hz, 2H), 6.78 (d, J = 2.4 Hz,
1
3
3
H), 6.27 (s, 1H), 3.64 (s, 6H), 2.17 (s, 3H); C NMR (126 MHz, CDCl ) δ 153.77,153.65,