Design and synthesis of β-carboline linked aryl sulfonyl piperazine derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing ability

Article abstract of DOI:10.1016/j.bioorg.2020.103983

A series of new β-carboline linked aryl sulfonyl piperazine congeners have been synthesized by coupling various β-carboline acids with substituted aryl sulfonyl piperazines. Evaluation of their anticancer activity against a panel of human cancer cell lines such as colon (HT-29), breast (MDA-MB-231), bone osteosarcoma (MG-63), brain (U87 MG), prostate (PC- 3) and normal monkey kidney (Vero) cell line has been done. Among the series, compound 8ec and 8ed has shown most potent cytotoxicity with an IC₅₀ values of 2.80 ± 0.10 μM and 0.59 ± 0.28 μM respectively against MG-63 cell line and also potent on other cell lines tested. Compounds 8ec and 8ed was found to inhibit Topo II that is confirmed by specific Topo II inhibition assay. DNA binding studies, cell cycle analysis, Annexin V study indicate that these compounds has potential anticancer activity. Molecular docking studies for compound 8ec and 8ed are incorporated to understand the nature of interaction with topoisomerase IIα and dsDNA.

Full text of DOI:10.1016/j.bioorg.2020.103983

Journal Pre-proofs
Design and Synthesis of β-carboline linked aryl sulfonyl piperazine deriva-
tives: DNA topoisomerase II inhibition with DNA binding and apoptosis in-
ducing ability
Kesari Lakshmi Manasa, Sowjanya Thatikonda, Dilep Kumar Sigalapalli,
Arpita Sagar, Gaddam Kiranmai, Arunasree M Kalle, Mallika Alvala,
Chandraiah Godugu, Narayana Nagesh, Bathini Nagendra Babu
PII:
S0045-2068(20)31280-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.103983
YBIOO 103983
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 December 2019
15 April 2020
29 May 2020
Please cite this article as: K. Lakshmi Manasa, S. Thatikonda, D. Kumar Sigalapalli, A. Sagar, G. Kiranmai,
A.M. Kalle, M. Alvala, C. Godugu, N. Nagesh, B. Nagendra Babu, Design and Synthesis of β-carboline linked
aryl sulfonyl piperazine derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing
ability, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103983
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.

Design and Synthesis of β-carboline linked aryl sulfonyl piperazine
derivatives: DNA topoisomerase II inhibition with DNA binding and
apoptosis inducing ability
Kesari Lakshmi Manasa,a,b Sowjanya Thatikonda,c Dilep Kumar Sigalapalli,a,b Arpita Sagar,e
Gaddam Kiranmai,d Arunasree M Kalle,e Mallika Alvala,b Chandraiah Godugu,c* Narayana
Nagesh,d* and Bathini Nagendra Babua*
aFluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad-500007,
India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500037, India
cDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500037, India
dCSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
eDepartment of Animal Biology, School of Life Sciences, University of Hyderabad,
Hyderabad-500 046, India
Abstract
A series of new β-carboline linked aryl sulfonyl piperazine congeners have been synthesized
by coupling various β-carboline acids with substituted aryl sulfonyl piperazines. Evaluation
of their anticancer activity against a panel of human cancer cell lines such as colon (HT-29),
breast (MDA-MB-231), bone osteosarcoma (MG-63), brain (U87 MG), prostate (PC- 3) and
normal monkey kidney (Vero) cell line has been done. Among the series, compound 8ec and
8edhas shown most potent cytotoxicity with an IC50 values of 2.80±0.10 µM and 0.59±0.28
µM respectively against MG-63 cell line and also potent on other cell lines tested.
Compounds 8ec and 8edwas found to inhibit Topo II that is confirmed by specific Topo II
inhibition assay. DNA binding studies, cell cycle analysis, Annexin V study indicate that
these compounds has potential anticancer activity. Molecular docking studies for compound
8ec and 8ed are incorporated to understand the nature of interaction with topoisomerase IIα
and dsDNA.
Keywords: β-carboline; Aryl sulfonyl piperazine; Topoisomerase II inhibition; DNA binding
studies; Cytotoxicity and Molecular docking.

1.
Introduction
In the present scenario, cancer has become a dreadful disease with an increase in the number
of patients [1] and an average, 9.6 million deaths are reported in the year 2018 globally and
were projected to rise over 13.1 million by 2030 [2]. Cancer is characterized by the
uncontrolled proliferation of cells. Most of the chemotherapeutic agents currently in the
market target DNA and are highly recommended as the front-line therapies in several
cancers. However, they have several limitations such as high toxicity, limited efficacy, non-
specificity, poor tolerance, less bioavailability and the development of resistance. Although
several targeted therapies are available for few cancers, till date, DNA is still considered as
the main target in cancer therapy due to its role in replication and transcription [3]. Therefore
there is a need to develop novel compounds targeting the DNA that would induce apoptosis
in cancer cells with high specificity.
The β-carboline alkaloids contain tricyclic pyrido[3,4-b] indole ring system and were
naturally obtained from seeds of Peganum harmala (Zygophillaceae) [4]. These alkaloids are
known to exhibit diverse pharmacological properties such as antialzheimer, antiplatelet,
anxiolytic, anticonvulsant, antiviral, antimicrobial, anticancer and antiplasmodial properties
[5].
N
N
N
H
N
N
N
O
O
N
N
N
H
N
N
H
H
H
N
H2N
Annomontine ( )
E
Harmaline ( )
A
D
Norharman ( )
B
C)
Harman ( )
Harmine (
O
F
amide linker
O
N
O
N
N
H
NH
N
N
N
O
S
R1
O
O
S
S
NH
N
O
aryl
O
sulfonyl
F
F
F
F
N
piperazine
O
H
R
N
-carboline
O

F
G
H
J30 ( )
F
T138067 (
)
Mana-Hox ( )
8aa-fe
Designed Hybrids (
)
Fig. 1. Structures of biologically active β-carbolines (A–F), sulphonamides (G &H) and
newly designed hybrids (8aa-fe).

β-carboline alkaloids are reported to exhibit anticancer activity via multiple
mechanisms i.e, DNA intercalation [6], topoisomerase I and II inhibition [7], kinesin Eg5 [8],
MK-2 [9] and CDK inhibition [10]. Harmine (B), a β-carboline analog, possesses DNA
intercalation property and inhibits topoisomerase I in various cancer cell lines [11]. Recently
Chen et al reported a harmine derivative (CM 16) that inhibits the translational step of protein
synthesis [12]. Mana-Hox (C) is another β-carboline analog that intercalates with DNA via
G-C base pairs [13]. Earlier studies have shown that changes in substitution at C-1 and C-3
positions of β-carboline ring exhibit improved DNA binding ability [14].
Sulphonamide congeners are privileged scaffolds having distinct biological activities
such as antiviral, antihypertensive, antidiabetic, antithyroid and antibacterial activities [15].
Recently T138067 [16] (E, Fig. 1) and ABT-751 [17] are novel sulphonamide derivatives
came into clinical studies. Moreover, piperazine and its derivatives, with well known
heterocyclic scaffold, such as piperazinyl-linked ciprofloxacin dimers [18] are potent
antimicrobial agents but also possess antitumor property. AK301 was able to arrest the
mitosis and induce the cell apoptosis [19].
Pharmacophore hybridization is an attractive tool for covalently adjoining two distinct
biologically important chemical entities into one hybrid molecule [20]. The hybrid molecules
with different mechanism of action, might result in synergistic effect with high affinity and
selectivity [21].Considering the importance of β-carbolines and the biological activity of
sulphonamides, our group has put efforts in developing novel β-carboline derivatives
containing aryl sulfonyl-piperazine scaffold with DNA binding and topoisomerase inhibiting
activities aiming the combined substructures may display a synergistic effect on anticancer
activity. Herein, we report the design and synthesis of the hybrids of these two
pharmacophores with structural diversity by employing simple amidation (Fig. 2).
Furthermore, the synthesized compounds were evaluated for their cytotoxic activity, cell
cycle analysis, DNA binding ability and topoisomerase II inhibitory activity.

O
OH
N
O
N
O
R
N
N
N
H
S
R1
Pharmacophore
Hybridization
-carboline part

O
H
N
N
R
H
8aa-fe)
Designed Hybrids (
N
O
O
-carboline linked aryl sulfonyl piperazine
congeners

S
R1
Aryl sulfonyl piperazine part
Fig. 2. Pharmacophore Hybridization Strategy for the target molecule.
2. Results and discussion
2.1. Chemistry
The β-carboline linked aryl sulfonyl piperazine congeners (8aa‒fe) were synthesized in a
convergent approach by employing the acid amine coupling reaction between substituted β-
carboline acids (5a-f) and substituted phenyl sulfonyl piperazines (7a-e) as depicted in
Scheme 1. The key synthetic precursor L-tryptophan (1) was esterified by using SOCl2 in
methanol to L-tryptophan methyl ester (2), followed by a Pictet-Spengler condensation with
variety of substituted benzaldehydes resulted the corresponding methyl tetrahydro-β-
carboline-3-carboxylates 3a‒f. The diastereomers which are obtained were used directly for
the aromatization, using trichloroisocyanuric acid (TCICA) to afford the methyl-β carboline-
3-carboxylates 4a-f. These were further hydrolized in the presence of aqueous NaOH solution
to get β-carboline acids 5a-f in good yields. Next, a variety of substituted phenyl sulfonyl
piperazine intermediates (7a-e) were synthesized as to the previous synthetic procedures [15].
Compounds 7a-e was synthesized by the reaction of different substituted benzene sulfonyl
chlorides with piperazine in DCM at 0 ºC for 30 min.

O
O
O
COOH
NH2
O
NH2
NH
SOCl2, MeOH,
4 h, rt, 95%
R-CHO, p-TSA,
EtOH, reflux,
12 h, 70-85%
R
N
N
H
N
H
H
1
2
3a-f
O
O
OH
O
TCICA, Et3N,
DMF,
NaOH, MeOH,
H2O,
N
N
R = 4-trifluoromethylphenyl
4-fluorophenyl
4-methoxyphenyl
3,4,5-trimethoxyphenyl
phenyl
0 C-rt, 2 h,
70-95%
reflux, 6 h,
50-70%
R
R
N
H
5a-f
N
H
4a-f
Cl
O
O
H
S
O
S N
O
R1 = 4-methoxy
p-tolyl
Piperazine, DCM
NH
0 C, 30 min,
80-90%
R1
4-chloro
phenyl
4-t-butyl
R1
6a-e
7a-e
H
N
O
N
O
OH
O
O
O
S
N
EDCI, HOBt, DIPEA,
DMF,
N
S
R1
N
N
O
R
rt, 12 h, 80-90%
N
H
5a-f
N
H
R
R1
7a-e
8aa-fe)
Designed Hybrids (
8aa
8ab
8ac
8ad
8ae
8ba
: R = 4-trifluoromethylphenyl, R1 = 4-methoxy
: R = 4-trifluoromethylphenyl, R1 = p-tolyl
: R = 4-trifluoromethylphenyl, R1 = 4-chloro
: R = 4-trifluoromethylphenyl, R1 = phenyl
: R = 4-trifluoromethylphenyl, R1 = 4-t-butyl
: R = 4-fluorophenyl, R1 = 4-methoxy
8da
8db
8dc
8dd
8de
: R = 3,4,5-trimethoxyphenyl, R1 = 4-methoxy
: R = 3,4,5-trimethoxyphenyl, R1 = p-tolyl
: R = 3,4,5-trimethoxyphenyl, R1 = 4-chloro
: R = 3,4,5-trimethoxyphenyl, R1 = phenyl
: R = 3,4,5-trimethoxyphenyl, R1 = 4-t-butyl
: R = phenyl, R1 = 4-methoxy
: R = phenyl, R1 = p-tolyl
8ea
8eb
8ec
8ed
8ee
8bb
8bc
8bd
8be
: R = 4-fluorophenyl, R1 = p-tolyl
: R = 4-fluorophenyl, R1 = 4-chloro
: R = 4-fluorophenyl, R1 = phenyl
: R = 4-fluorophenyl, R1 = 4-t-butyl
: R = 4-methoxyphenyl, R1 = 4-methoxy
: R = 4-methoxyphenyl, R1 = p-tolyl
: R = 4-methoxyphenyl, R1 = 4-chloro
: R = 4-methoxyphenyl, R1 = phenyl
: R = 4-methoxyphenyl, R1 = 4-t-butyl
: R = phenyl,R1 = 4-chloro
: R = phenyl,R1 = phenyl
: R = phenyl, R1 = 4-t-butyl
8ca
8cb
8cc
8cd
8ce
8fa
8fb
8fc
8fd
8fe
: R = H, R1 = 4-methoxy
: R = H, R1 = p-tolyl
: R = H, R1 = 4-chloro
: R = H, R1 = phenyl
: R = H, R1 = 4-t-butyl
Scheme 1. Synthesis of β-carboline linked arylsulfonyl piperazine compounds8aa–fe.
Finally, synthesis of desired β-carboline-linked aryl sulfonyl piperazine congeners
(8aa‒fe) have been achieved by reacting intermediate 5a-f with various substituted phenyl
sulfonyl piperazines (7a-e) in the presence of EDCI and HOBt in DMF and the yields were in

the range of 80-90%. All the structures of these compounds were characterized by 1H NMR,
13C NMR, mass and HRMS spectral data.
1
One of the representative compounds 8cc was confirmed by H NMR by the
appearance of characteristic signal at δ11.73 ppm which accounts for 9H-indolic of β-
carboline and the signal at δ3.91 ppm accounts for methoxy group. All the aromatic protons
of 8ccwere found in the range of δ8.38–7.19 ppm. The 2 signals at δ3.82 and 3.01 ppm
13
accounts for methylinic protons of piperazine. The interpretation of C NMR of compound
8ccdepicts carbonyl signals at δ167.6 ppm. All other aromatic carbons of 8cc appeared in the
1
13
range of δ159.8.5–112.5 ppm and aliphatic methoxy carbon at δ55.4 ppm. The H and C
NMR spectra of all newly designed compounds 8aa–fe were found almost in a similar pattern
and well matched with the respective aromatic and aliphatic signals of their structure. The
HRMS (ESI) of newly designed compounds showed characteristic protonated [M+H]+ peak
to their corresponding molecular formula.
2.2. Evaluation of Biological activity
2.2.1. In vitro cytotoxic activity
All the synthesized compounds were screened initially for the percentage growth inhibition
(GI50 %) on six human cancer cell lines such as PC-3, MDA-MB-231, U87MG, MG-63, HT-
29 and HeLa cell line. Compounds which are able to show the percentage growth inhibition
less than 40% known to be inactive compounds and percentage inhibition more than 50% are
known to be active. The percentage growth inhibition of the tested compounds was listed in
Table 1. It is noticeable from the results that all the tested compounds shown moderate to
potent growth inhibition. Among the series, compounds 8ec and 8ed showed potent growth
inhibition.
Table 1 In vitro growth inhibitory concentration (GI50a %) against human cancer cell lines by
MTT assay.
Compound HT-29b
MDA-MB- MG-63d
231c
U87MGe
PC3f
HeLag
8aa
8ab
8ac
8ad
8ae
8ba
8bb
8bc
8bd
25.64±3.2 21.57±1.4 53.19±1.4
10.97±1.2 3.88±1.1 35.11±1.1
11.07±1.1 28.97±1.5 41.52±1.8
6.41±0.89 8.26±0.89 18.11±0.89 48.92±3.7 42.02±1.2 33.20±1.3
11.02±1.2 15.73±1.1 14.63±1.0
59.84±1.5 57.44±1.5 73.33±1.7
57.61±3.2 25.22±1.25 23.17±1.56
41.92±3.5 51.51±1.5 46.42±1.4
51.71±3.1 25.48±1.6 40.71±1.4
50.23±3.5 48.02±4.5 40.55±1.23
42.51±4.5 50.60±1.59 51.95±1.56
3.46±0.2
8.17±0.3
30.50±1.5 47.77±1.2
38.50±1.6 49.02±1.5
15.87±1.25 30.28±1.23 52.63±1.56 26.43±1.2 52.24±1.56 45.50±1.56
21.94±1.23 23.73±1.45 12.11±1.32 16.28±1.1 51.17±1.25 55.95±1.58

8be
8ca
8cb
8cc
8cd
8ce
8da
8db
8dc
8dd
8de
8ea
8eb
8ec
8ed
8ee
8fa
8fb
8fc
9.84±0.56 19.71±1.2 27.55±1.28 49.20±1.56 37.57±1.45 29.73±1.26
9.76±0.23 6.30±0.56 30.77±0.38 32.64±1.25 23.95±1.22 50.18±1.58
7.00±0.25 15.57±1.2 5.36±0.23
3.53±0.56 23.14±1.2 47.16±1.4
22.17±1.25 47.64±1.45 25.79±1.25
60.92±1.89 27.60±1.22 50.79±1.56
1.07±0.56 22.83±0.25 53.30±1.56 66.53±1.25 54.80±1.56 44.70±1.44
5.30±0.12 38.83±0.38 38.66±1.2 60.00±1.45 51.35±1.89 38.94±1.89
30.43±0.32 32.45±1.32 54.25±0.54 45.05±1.45 52.02±1.56 38.75±1.23
6.02±0.89 10.16±0.89 41.16±1.2
17.64±0.18 37.07±0.56 60.66±1.2
25.84±0.25 49.21±0.89 66.36±1.1
16.74±1.23 49.20±1.89 27.16±1.56
10.92±0.98 40.13±0.45 25.68±1.36
7.10±0.23 53.86±1.56 30.52±1.32
24.23±0.27 9.40±0.96 64.33±0.64 26.61±0.25 6.06±0.89 36.98±1.56
14.46±0.15 37.40±0.36 65.58±0.65 43.43±1.45 58.33±0.58 34.81±1.78
22.61±0.22 78.33±1.7 76.27±0.76 68.61±0.68 71.46±1.75 44.12±1.45
44.97±0.14 70.04±1.2 68.88±1.68 66.35±1.23 77.68±1.89 26.34±1.45
71.25±0.14 55.16±0.89 88.86±1.56 58.23±1.56 47.02±1.23 38.49±1.56
14.76±0.23 13.33±1.2 55.25±1.56 16.43±1.2 87.02±1.89 45.44±1.58
39.84±0.39 28.85±0.28 43.13±1.56 42.74±1.0 53.91±1.23 46.24±1.45
19.97±0.18 25.00±1.25 40.22±1.45 35.00±0.56 35.60±1.56 17.93±1.09
4.05±0.23 18.35±1.23 57.27±1.56 53.74±1.56 8.08±0.56 27.35±0.25
29.71±0.29 28.69±1.25 56.72±1.23 59.84±1.23 40.68±1.89 36.34±0.36
57.20±0.56 18.83±1.2 61.75±1.63 57.61±1.22 37.64±0.38 40.68±0.89
8fd
8fe
aCompound concentration required to decrease cell growth to half that of untreated cells.
c
d
e
bHuman colon cancer, Human breast cancer, Human bone osteosarcoma, Human brain
f
g
cancer, Human prostate cancer, Human cervical cancer. All the values are expressed as %
inhibition in which each treatment was performed in triplicate wells.
The newly synthesized β-carboline linked aryl sulfonyl piperazine congeners (8aa‒fe)
were evaluated for their in vitro cytotoxic activity against a panel of six human cancer cell
lines namely HT-29 (colon cancer), MDA-MB-231 (Breast cancer), MG-63 (bone
osteosarcoma), U87MG ( brain cancer), PC3 (prostate cancer), HeLa (cervical cancer) and
normal monkey kidney (Vero) cell lines by employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The results from in vitro cytotoxic studies
revealed that the compounds 8aa‒fe exhibited diverse cytotoxic properties and are depicted
in Table 2.
Notably, these compounds exhibited good to moderate cytotoxicity in the tested
cancer cell lines. From the close analysis of the IC50 values, it was observed that compound
8ed was more significant in inducing cytotoxicity on all the tested cancer cells and found to
be most potent against MG-63 (bone osteosarcoma) cancer cell line with an IC50 value
0.59±0.28 μM whereas in U87MG cell line the IC50 value was 4.20±0.30 μM. Interestingly,
the compound 8ec, 8ed exhibited least toxicity on normal vero cell line with IC50 value
9.95±0.67 µM and 8.41±0.12 µM respectively. This indicates that the compound 8ed was
selective towards the cancer cells rather than normal cell lines tested as outlined in Table 2.

While the compound 8ec also displayed remarkable cytotoxicity against MG-63, PC-3 and
U87MG with an IC50 value 2.80±0.10, 3.36±0.32 and 3.90±0.20 μM respectively. Among the
series of compounds 8aa, 8cc, 8cd, 8ea and 8eb also showed considerable cytotoxicity in
cancer cell lines. SAR analysis revealed that C-1 position of β-carboline ring displayed better
cytotoxic activity with phenyl substitution (8ed, 8ec, 8ea and 8eb) rather than the
substitutions at various positions in the phenyl ring. Compound 8ed, with phenyl at C-1
position of β-carboline and phenyl sulfonyl piperazine proved to be potent among all the
synthesized compounds. With respect to the promising in vitro cytotoxicity results (Table 2),
the significant activity of newly synthesized hybrids 8aa‒fe could also be attributed towards
C1 modification of β-carboline by H and CH3 group at this position. Hence the most active
compounds (8ec and 8ed) were further explored for their apoptotic induction studies, DNA
topoisomerase II inhibition, DNA interaction studies and molecular docking studies.
a
Table 2 IC50 (μM) of different compounds at 48 h post treatment on the HT-29b, MDA-
MB-231c, MG-63d, U87MGe, PC3f, HeLag and Veroh cells determined by the MTT assay.
Code
HT-29b MDA-MB-
231c
MG-63d
U87MGe
PC3f
HeLag
Veroh
8aa
8ab
8ac
8ad
8ae
8ba
8bb
8bc
8bd
8be
8ca
8cb
8cc
8cd
8ce
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.86±0.25
>10
6.06±0.15 5.06±0.20
3.86±0.20 >10
2.66±0.58
>10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
>10
>10
>10
4.73±0.20
>10
>10
>10
>10
>10
4.43±0.20 >10
4.36±0.11 >10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.30±0.55
4.53±0.20
>10
7.13±0.35
>10
4.83±0.35
5.30±0.10
>10
>10
>10
5.53±0.30
>10
>10
>10
>10
>10
>10
>10
5.23±0.15 4.70±0.10
4.50±0.30 3.36±0.15
5.36±0.25 4.70±0.10
7.33±0.30
>10
8.00±0.17
>10
>10

8da
8db
8dc
8dd
8de
8ea
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
-
-
-
-
-
-
>10
>10
7.5±0.30
8.43±0.49
7.96±0.40
3.93±0.15
>10
5.10±0.10
>10
11.32± >10
0.47
7.30±0.43
8eb
8ec
8ed
13.20± >10
0.52
4.09±0.11
2.80±0.10
0.59±0.28
4.90±0.20 4.20±0.30
3.90±0.20 3.36±0.32
4.20±0.30 >10
>10
>10
>10
-
8.20±0. >10
52
9.95±0
.66
10.10± >10
0.36
8.41±0
.16
8ee
8fa
8fb
8fc
8fd
>10
>10
>10
>10
>10
>10
>10
>10
8.20±0.30
>10
>10
>10
>10
>10
4.47±0.33
>10
>10
>10
>10
>10
>10
-
-
-
-
-
>10
>10
8.06±0.51
9.56±0.20
>10
4.70±0. >10
65
7.36±0.20 >10
8fe
>10
>10
10.30±0.20 8.16±0.35 >10
>10
1.72±0.16
-
-
Doxor 1.38±0. 1.20±0.32
ubicin 04
Harm 4.26±1. 12.26±0.64
2.54±0.20
-
-
1.20±0.14
36.33±0.42
22.44±1.64 16.18±1.02
-
ine
18
a Each data represents mean ± S.D. from three different experiments performed in triplicate.
b HT-29: human colon cancer cell line. c MDA-MB-231: human breast cancer cell line. d MG-
e
f
63: human bone osteosarcoma cell line. U87 MG: human brain cancer cell line. PC3:
human prostate cancer cell line. HeLa: Human cervical cancer cell line. Vero: normal
monkey kidney cell line.
g
h
2.2.2. Topoisomerase II inhibition assay
Topoisomerases are the enzymes that involve in increasing or decreasing the winding of
DNA. They control the topology of the DNA and play a significant role in DNA replication,
transcription and segregation as well as recombination phenomena [22]. Two types of
topoisomerases, Topo I and Topo II are well known. Type I, topoisomerases (Topo I) make a
single nick in DNA strand while type II, topoisomerases (Topo II) make similar nicks on both
the strands of the DNA. Topo II is a nuclear enzyme that influences the DNA topology by
catalyzing the DNA cleavage and by religating the phosphodiester bonds and as a result, the

Topo II initiates multiple recombination/repair pathways that may leads to cell death [23].
The catalytic cycle of Topo II takes place in six different steps like (1) binding of Topo II to
DNA, (2) double-stranded cleavage of the DNA, (3) ATP-dependent DNA strand passage,
(4) religation of the cleaved DNA, (5) ATP hydrolysis and (6) enzyme recycling [24]. Topo
II inhibition studies were carried out by using Topo II Drug Screening Kit (TG 1009,
Topogen, USA). Catenated DNA kinetoplast DNA (kDNA) and topo II enzyme (5 units) was
incubated in presence 10 µM Etoposide for 30 min (lane 3 in Fig. 3), which is considered as a
positive control in the present experiment. These experimental results revealed that
decatenation did not take place (by forming minicircles) and the ternary complex
(kDNA+topo II and Etoposide complex) remained close to the origin. Similar studies when
repeated with catenated kDNA (200–300 ng) in the presence of 10 µM 8ed and 8ec (lane 4
and 5 respectively in Fig. 3) revealed that due to the formation of ternary complex
(kDNA+topo II and 8ec and 8ed) most of the DNA in the presence of 8ec and 8ed
derivatives, remained near the origin and did not enter into the agarose gel. Further it is
noticed that, with 8ec, formation of ternary complex (kDNA+topo II and 8ec) is less
compared to 8ed, indicating lesser topo II inhibition by 8ec compared to 8ed.These results
are supported by the spectroscopy experimental results as well as the Kd values calculated
using the UV-vis abosorption data, which shows that 8ed has better interaction with DNA
compared to 8ec. The results indicate that in the absence of derivatives, kDNA remained in
the catenated form (lane 1) and on addition of topo II, the kDNA has transformed into
decatenated form (lane 2) as depicted in Fig. 3. From these studies it is evident that the two
derivatives considered in the present work (mainly 8ec and 8ed) inhibit topo II activity.
Fig. 3. Topo II inhibition assay. To about 200–300 ng of kDNA, about 5 units of Topo II was
added and incubated at 37oC for 30 min. Lane 1- kDNA alone; Lane 2- kDNA and topo II;
Lane 3- kDNA, topo II and Etoposide; Lane 4- kDNA, topo II and 8ed; Lane 5- kDNA, topo
II and 8ec.

2.2.3. Apoptosis induction studies
2.2.3.1. Cell cycle analysis
In order to validate the results obtained from MTT assay as well as to understand the effect of
8ec and 8ed on cell cycle progression, Cell cycle analysis was performed, wherein MG-63
cells were treated with 8ed (0.5 µM) and 8ec (2 µM) for 24 h and then the cells were stained
with propidium iodide and further analyzed by using BD FACSVerseTM flow analyzer.
There was a significant increase in the population of cells at sub G1 phase was observed with
respect to control cells. The results obtained from Fig. 4 clearly indicated that MG-63
untreated control cells showed 1.05% cells in sub G1 phase, whereas compound 8ed showed
6.95% and 8ec showed 8.02% of cell accumulation in sub G1 phase. These results imply that
active congeners 8ed and 8ec arrest the cell cycle at sub G1 phase thereby induces apoptosis.
Control
8ed (0.5 µM)
Sub G1:1.05
G0/G1:21.21
S:4.46
Sub G1:6.95
G0/G1:11.31
S:5.73
G2/M:14.72
G2/M:23.16
8ec (2 µM)
Doxorubicin (0.5 µM)
Sub G1:33.22
G0/G1:17.72
S:8.08
Sub G1:8.02
G0/G1:16.22
S:5.66
G2/M:9.64
G2/M:15.08
Fig. 4. Effect of 8ed (0.5 µM) and 8ec (2 µM) on cell cycle progression of MG-63 and assay
was performed after 24 h. The analysis of cell cycle distribution was performed by using
propidium iodide staining method.
2.2.3.2. Phase contrast microscopy
To examine whether the treatment with most active congeners 8ed and 8ec may lead to
induction of apoptosis, MG-63 cells were treated with active compounds. After 48 h of
incubation, images were captured in phase contrast microscopy which is depicted in Fig.5 (i)
whereas characteristic morphological features such as shrinkage of cells, membrane blebbing,
detachment from substratum, reduction in number of viable cells was observed in treated

cells compared to the untreated control cells. The same results were observed with
doxorubicin.
2.2.3.3. Acridine orange/ethidium bromide (AO/EB) staining
The morphological changes induced by the active compounds 8ed and 8ec in MG-63 cells
were further studied by using acridine orange/ethidium bromide (AO/EB) staining technique.
AO penetrate through the intact cell membrane and stains the nuclei in green, whereas EB
can stains the nuclei of cells in red that have lost membrane integrity and necrotic cells. It can
be inferred from Fig. 5 (ii) that control cells appeared in green colour due to its normal
morphology. Treated MG-63 cells (8ed and 8ec) demonstrate the morphological changes
such as membrane blebbing, cell shrinkage, chromatin condensation and apoptotic body
formation indicating that the compounds induce apoptosis.
8ec (2 µM)
Doxorubicin (0.5 µM)
Control
8ed (0.5 µM)
Fig. 5. (i) Morphological changes observed in MG-63 cells were treated with and without
compound 8ed (0.5 µM) and 8ec (2µM). After 48 h the images were captured with a phase
contrast microscope (200x magnification, scale bar = 50 µm). (ii) AO/EB staining in MG-63
cells treated with and without compound 8ed (0.5 µM) and 8ec (2µM) for 48 h and compared
with control and standard drug (Doxorubicin).
2.2.3.4. DAPI staining
The characteristic feature of apoptotic cells is condensation of nucleus and it can be observed
by DAPI nuclear staining method [25]. DAPI (4ꞌ,6-diamidino-2-phenylindole) is a blue
fluorescent dye, which can pervade intact membrane of live cells and stains the nucleus in
light blue, wherein the apoptotic cell nuclei appears as bright blue due to chromatin
condensation. MG-63 cells were stained with DAPI followed by the treatment with both the
compounds 8ed (0.5 µM) and 8ec (2 µM), there by cells were observed under fluorescence

microscope for nuclear morphological changes. The results observed from Fig.6 (i) that
control cells were intact with normal morphology, while treated compounds 8ed and 8ec led
to the nuclear changes like horse-shoe shaped, chromatin condensation, pyknotic or
fragmented bright nuclei. From the results, it was evident that these compounds 8ed and 8ec
had induced apoptosis in MG-63 cells.
2.2.3.5. Measurement of reactive oxygen species (ROS)
In order to determine the ability of compound 8ed (0.5 µM) and 8ec (2 µM) on the ROS
generation, the intracellular ROS levels were examined by using DCFDA staining assay [26].
DCFDA (2',7'-dichlorofluorescin diacetate) is a non-fluorescent probe which is readily
oxidized to 2',7'-dichlorofluorescein (DCF) in the presence of ROS. MG-63 cells were treated
with compound 8ed and 8ec at indicated concentrations, significant increase in the green
fluorescence was observed which is comparable with Doxorubicin. These results clearly state
that induction of ROS by the treated compounds is one of the prompting mechanism to
induce apoptosis in MG-63 cells (Fig.6 (ii)).
Control
8ed (0.5 µM)
8ec (2 µM)
Doxorubicin (0.5 µM)
Fig. 6. (i) Nuclear morphology of cancer cells after DAPI staining. MG-63 cells treated with
and without compound 8ed (0.5 µM) and 8ec (2 µM) for 48 h and compared with control and
Doxorubicin. The images were captured with fluorescence microscope (200x magnification,
scale bar = 50 µm). (ii) DCFDA staining in MG-63 cells treated with and without compound
8ed (0.5 µM) and 8ec (2 µM) for 24 h and compared with control and standard drug
(Doxorubicin).

2.2.3.6. Effect on mitochondrial membrane potential (ΔΨm)
Mitochondria play a key role in initiating the intrinsic apoptotic pathway. To further evaluate
the effect of compound on mitochondria, ΔΨm was measured. JC-1 is a lipophilic cationic
dye was used to evaluate ΔΨm, in which polarized mitochondria stains red due to formation
of J-aggregates, while depolarised mitochondria in apoptotic cells stains green because of J-
monomers. MG-63 cells were treated with 8ed (0.5 µM) and 8ec (2 µM) for 48 h and
remarkable increase in the mitochondrial membrane depolarisation was observed as
compared to control cells as shown in the Fig.7. Flow-cytometric analysis of the treated cells
clearly displayed an increase in depolarized cell population (P2) from 1.42% in control to
14.15%, 19.20% for 8ed (0.5 µM) and 8ec (2 µM) respectively. Thus, the treated compounds
clearly induced the loss of mitochondrial membrane potential and suggest the involvement of
mitochondria dependent apoptotic pathway in the mechanism of action.
Control
8ed (0.5 µM)
P1:98.02
P2:1.42
P1:84.63
P2:14.15
Doxorubicin (0.5 µM)
8ec (2 µM)
P1: 79.47
P2: 19.20
P1: 53.29
P2: 46.45
Fig. 7. Effect on mitochondrial membrane potential. MG-63 cells were treated with 8ed and
8ec for 48 h and JC-1 staining was performed. The control represents the cells without
compound.
2.2.3.7. Annexin V-Alexa fluor 488/PI assay
To quantify the percentage of apoptotic cell death induced by the compound 8ed (0.5 µM)
and 8ec (2 µM) on MG-63 cells, Alexa Fluor 488 annexin V/Propidium iodide staining assay
was performed. MG-63 cells were treated with compound 8ed (0.5 µM) and 8ec (2 µM) for
48 h and stained with Annexin V-alexa fluor488 and propidium iodide, and samples were
analysed by flow cytometry. Annexin assay ease the detection of necrotic cells (Q1-UL; AV-

/PI+), live cells (Q2-LL; AV-/PI-), early apoptoticcells (Q3-LR; AV+/PI-), and late apoptotic
cells (Q4- UR;AV+/PI+). Results from Fig. 8 indicate compound 8ed and 8ec induce
apoptosis in comparison to control.
Control
8ed (0.5 µM)
UL
0.02
UL
0.03
LA
1.13
LA
0.05
EA
15.01
EA
0.45
LC
83.83
LC
99.48
Doxorubicin (0.5 µM)
UL
8ec (2 µM)
UL
0.32
LA
6.51
LA
3.68
0.01
EA
32.6
EA
7.79
LC
63.67
LC
85.38
Fig. 8. Effect of compound on cell apoptosis as measured by Annexin V-Alexa fluor
/propidium iodide staining assay. MG-63 cells were treated with compound 8ed (0.5 µM) and
8ec (2 µM) for 48 h. Then 10,000 cells from each sample were analyzed by flow cytometry.
The percentage of cells positive for Annexin V-Alexa fluor 488 and/or Propidium iodide is
represented inside the quadrants.
2.2.4. DNA binding studies
To further ratify the biological activities of these potential congeners (8ec and 8ed) and to
understand the mode of interaction with DNA, spectroscopic studies such as UV-visible,
fluorescence, circular dichroism spectroscopy and viscosity studies were performed.
2.2.4.1. UV-visible Spectroscopic Studies.
In order to find the interaction of 8ec and 8ed with double stranded DNA, UV-visibility
spectroscopy study was carried out. However, both these derivatives have shown maximum
absorption bands at around 208 nm and 206 nm with 8ec and 8ed respectively. But, upon
addition of equal ratio of CT-DNA solution to the compounds, the absorption peak exhibited
gradual hyperchromicity. Further, it is understood that very less hyperchromicity and no shift
in the absorption peak is observed on interaction of CT DNA with each of these derivatives.

Each time after the successive addition of CT-DNA to 8ec and 8ed solution, samples were
equilibrated for 5 min prior to measurement. The data obtained is fitted to Eq.(1), to obtain
the dissociation constant (Kd) taking into account the total amount of 1:1 complex
formed,independently of the derivative binding site(s).
Kd = [CT- DNA] [8ec/8ed compound] / [CT-DNA. 8ec/8ed compound] ------------ (1)
From the absorbance values obtained when 8ec and 8ed compounds interact with CT-DNA,
dissociation constant (Kd) was calculated using eq 1. The Kd values obtained on interaction of
8ec and 8ed with CT- DNA are 37 µM and 42 µM, respectively. Even Kd values obtained
will indicate that 8ed has marginally better binding with CT-DNA compared to 8ec. From the
experimental data, it is understood that 8ecand 8ed compounds are interacting moderately
with CT DNA. But, it is difficult to conclude the nature and mode of binding of compounds
with CT DNA only by UV-visible titration. The UV-visible spectra obtained on interaction of
these derivatives with CT-DNA were shown in Fig.9.
Fig. 9. UV-visible spectra obtained when 8ecand 8ed compounds interact with CT DNA. 25
µM of 8ecand 8ed compounds was taken in 1 cm path length cuvette and equal increments of
CT DNA were added to cuvette. (A) The UV-vis spectra recorded with increasing
concentration of CT DNA added to 25 µM of 8ec. (B) The UV-vis spectra recorded with
increasing concentration of CT DNA added to 25 µM of 8ed.
2.2.4.2. Fluorescence spectroscopy
Fluorescence spectroscopy is another valuable biophysical technique to understand the
binding mode of small molecules with DNA and to study the electronic environment around
the DNA-complex at comparably lower concentrations [27]. The nature of interaction of
compounds with DNA can be monitored at low concentration when they are fluorescent.

However, the emission spectra of compounds 8ec and 8ed shows a prominent peak in the
range from 700-770 nm for 8ec and from 370-670 nm for 8ed. The compounds 8ec and 8ed
are excited at 375 nm and 275 nm respectively. It is noticed that the derivatives fluorescence
emission intensity has reduced gradually upon equal addition of CT DNA. The reduction in
fluorescence intensity of 8ec and 8ed compounds with the addition of CT DNA, may be
either due to the absorption of fluorescence emission energy by the DNA bases which are in
the closely located to conjugate molecules or due to the absorption of emission energy by the
surrounding water molecules. The degree of fluorescence quenching is comparatively high in
case of 8ed than 8ec. The fluorescence spectrum of 8ec and 8ed with CT-DNA was shown in
Fig. 10.
Fig. 10. Fluorescence spectra recorded when 8ec and 8ed compounds interact with CT DNA.
10 µM of 8ec and 8ed compounds was taken in 1 cm path length cuvette and equal
increments of CT DNA (multiples of 0.5 µM) were added to cuvette. The compounds 8ec and
8ed were excited at 375 nm and 275 nm respectively and emission spectrum was recorded.
(A) The fluorescence spectra recorded with increasing concentration of CT DNA added to 25
µM of 8ec. (B) The fluorescence spectra recorded with increasing concentration of CT DNA
added to 25 µM of 8ed.
2.2.4.3. Circular Dichroism Studies
Further, to understand the effect of both 8ec and 8ed compounds on the conformation of CT
DNA, circular dichroism (CD) studies are performed. The CD spectra of CT-DNA alone
exhibited positive and negative bands around 270 nm and 240 nm respectively. On addition
of 10 µM of 8ec and 8ed compounds to CT-DNA (conjugate/DNA, 1:1 ratio), the positive
CD band exhibited hypochromicity, indicating unwinding of CT-DNA on interaction with β-
carboline linked aryl sulfonyl piperazine congeners. Further, on increasing the concentration
of 8ec and 8ed (conjugate /DNA, 2:1 ratio), the positive CD band intensity reduced further
and exhibited slight red shift. These changes can be attributed to absorption flattening and

differential scattering phenomena. The representative CD spectra obtained with 8ec and 8ed
compounds and CT-DNA were shown in Fig. 11.CD studies indicate that β-carboline linked
aryl sulfonyl piperazine compounds interact with DNA and this can exhibit changes in DNA
conformation. The positive CD band hypochromicity is more with 8ed compared to 8ec.Also,
the negative CD band intensities reduced with both the compounds8ec and 8ed indicating
alteration in the DNA helicity. However maximum change was noticed with 8ed. From the
CD data it is evident that β-carboline linked aryl sulfonyl piperazine congeners alter the DNA
helix on its interaction with CT-DNA, which is clear from the changed negative CD band
intensities.
Fig. 11. Circular Dichroism profiles of CT DNA in 100 mM Tris-HCl (pH 7.0) in the absence
or presence of 8ec and 8ed compounds. The conformational changes in CT DNA was
observed when 10.0 µM and 20 µM of 8ec and 8ed compounds interact with 10.0 µM CT
DNA (1:1 and 1:2 ratio of DNA:conjugate) in 1 cm path length cuvette. The spectra were
averaged over 3 scans. (A) The CD spectra recorded when 10.0 µM and 20 µM 8ec interact
with 10.0 µM CT DNA. (B) The CD spectra recorded when 10.0 µM and 20 µM 8ed interact
with 10.0 µM CT DNA.
2.2.4.4. Viscosity studies
From the spectroscopic studies, it was clear that, the active compounds 8ec and 8ed interact
well with CT DNA. Further, to conclude the nature and mode of interaction of 8ec and 8ed
with CT DNA, viscosity studies were also carried out. The relative viscosity of DNA
generally increases when the molecule intercalate with DNA. The enhanced viscosity may be
due to the increase in the axial length of DNA helix on molecule’s intercalation. Hence,
intercalation of a molecule results in an increase in the viscosity of DNA solution [28].
Whereas, reduction in the relative viscosity is typically observed with covalent DNA binding
[29] molecules. Moreover, viscosity does not change or show slight change when molecules
bind to the surface of DNA [30].

In the case of active compounds 8ec and 8ed, the viscosity of DNA solution increased slowly
with the addition of derivatives. But the variation in DNA viscosity on interaction of these
compounds with DNA, is in between ethidium bromide (EtBr) and Hoechst 33342. Among
both the derivatives, slight change in viscosity was observed in case of 8ed compared to 8ec,
while with the ethidium bromide (EtBr), the viscosity of DNA has increased continuously as
EtBr is a well known intercalator. The variation was less with the addition of Hoechst 33342,
due to its binding to the groove. The viscosity change noticed on interaction of different
molecules with CT is shown in Fig. 12. All the spectroscopic results and viscosity study
results indicate that the 8ec and 8ed compounds interact well with DNA and they may bind to
the surface of CT DNA.
Fig. 12. Viscosity studies graphical representation of 8ec (15 µM) and 8ed (15 µM)
compounds with CT-DNA solution (150 µM) at 25 oC, EtBr and Hoechst 33342 were used as
controls.
2.3. Molecular Docking Studies
Molecular docking simulations were performed on biologically most active compounds of
synthesized analogues i.e, β-carboline linked aryl sulfonyl piperazine derivatives (compound
8ed and 8ec) against the ATP binding domain of human topoisomerase IIα (PDB ID: 1ZXM)
[31] using GLIDE docking module of Schrödinger suite 2017-1[32].
The binding mode of compounds8ed and 8ec atthe ATP binding domain of human
topoisomerase IIα was depicted in Fig. 13. Compound 8ed has shown two hydrogen bond
interactions with ATP binding domain residues of human topoisomerase IIα. The β-carboline
NH- group of conjugate8ed has made strong hydrogen bond interaction with side chain of
Asp99 with a distance of 2.79 Å. Carbonyl group of conjugate 8ed has formed hydrogen
bond interaction with side chain of Arg98 with a distance of 2.99 Å. As well, sulfonyl phenyl

moiety has made π-cation interaction with Arg98 and Lys157. In addition, a number of
hydrophobic interactions were observed between conjugate8ed and ATP binding domain
residues i.e Ile33, Tyr34, Pro100, Pro126 and Tyr186 which will enhances the binding of
conjugate 8ed in the ATP binding pocket of human topoisomerase IIα. In case of conjugate
8ec, it has shown strong hydrogen bond interaction with Arg98. The β-carboline NH- and
carbonyl group has established hydrogen bond interaction with back bone and side chain of
Arg98, respectively in a two point contact way with a distance 2.19 Å and 2.97 Å. The β-
carboline moiety also formed π-cation contact with Arg98. Furthermore, numerous
hydrophobic contacts were also noticed between conjugate 8ec and ATP binding domain
residues which promotes the binding of 8ec in theATP binding domain of hTopoIIα.
Fig. 13. View of compounds8ed and 8ec docked in the ATP binding pocket of human
topoisomerase IIα. The red dashed line represents hydrogen bond.

Fig. 14. Binding poses of compounds 8ed and 8ec in the minor groove of
d(CGCGAATTCGCG)2.
Additionally, molecular docking simulation studies were also performed to enumerate
the binding mode of compounds 8ed and 8ec into the DNA duplex d(CGCGAATTCGCG)2
(PDB ID: 1DNH) [33]. Figure 14 illustrate the docking pose of compounds 8ed and 8ec at
the minor groove of duplex DNA. The β-carboline NH- group of compounds 8ed and 8ec has
established strong hydrogen bond interaction with DT8 base pair of DNA with the distance of
3.06 Å and 2.86 Å, respectively. Further, the aromatic ring of the β-carboline moiety of
compounds8ed and 8ec were observed to be involved in π-π stacking interaction with the
base pairs of DNA. All these interactions certainly stabilizes the binding of compounds8ed
and 8ec in the minor groove binding site of DNA duplex d(CGCGAATTCGCG)2.
3.
Conclusion
In conclusion, we have designed and synthesized a new series of β-carboline linked aryl
sulfonyl piperazine compounds, possessing a substituted phenyl at position-1 and aryl
sulfonyl piperazine motif at position-3 of the β-carboline moiety. These active compounds
involving two pharmacophoric features and their cytotoxic property were evaluated against
various human cancer cell lines. Compounds8ec and 8ed showed profound cytotoxic activity
against MG-63 cell line and induced apoptosis. Cell cycle analysis revealed that
compounds8ec and 8ed arrest at sub G1 phase. In addition these compounds inhibit
efficiently topo II enzymatic activity by catalytic inhibition of topo II. The DNA binding
studies states that 8ec and 8ed interact well with DNA and they may bind to the surface of
CT DNA. Further docking studies are well correlated with the spectroscopic, viscosity studies
and topo II inhibition studies. Therefore, all these studies are important to understand the role
of pharmacophores and these hybrids have the potential to be developed as lead
moleculesand suitable structural modifications may ensue in promising anticancer activity.
4. Experimental section
4.1. Materials and methods
All Chemicals and reagents were purchased from the commercial suppliers Alfa Aesar,
Sigma Aldrich and used without further purification. The reaction progress was monitored by
Thin layer chromatography (TLC) was performed using pre-coated silica gel 60 F254
MERCK. TLC plates were visualized and analysed by exposure to UV light or iodine vapors

and aqueous solution of ninhydrin. Column chromatography was performed with Merck flash
silica gel with 60–120 mesh size. Melting points were determined on an Electro thermal
1
melting point apparatus and are uncorrected. Nuclear magnetic resonance spectra for H
NMR were obtained on Avance 300, 400 and 500 MHz and analyzed using Mestrenova
software and the chemical shifts are reported in ppm from tetramethylsilane (0 ppm) or the
solvent resonance as the internal standard (CDCl3 7.26 ppm, DMSO-d6 2.49 ppm) and for 13C
NMR the chemical shifts are reported in ppm from the solvent resonance as the internal
standard (CDCl3 77 ppm, DMSO-d6 39.3 ppm). Spin multiplicities are described as s
(singlet), br s (broad singlet), d (doublet), dd (double doublet), t (triplet), td (triple doublet), q
(quartet), or m (multiplet). Coupling constants are reported in hertz (Hz). Mass spectra were
recorded by electrospray ionization mass spectrometry (ESI-MS). HRMS was performed on a
Varian ESI- QTOF instrument.
4.2.
General synthetic procedure for the preparation of compounds 8aa–fe
To a solution of amine intermediates (7a–e, 1 mmol) and appropriate β-carboline acid (1
mmol) in DMF(5 mL) was added EDCI (1.2 mmol), HOBt (1.2 mmol) and DIPEA (3 mmol)
under nitrogen atmosphere and the reaction mixture was stirred at room temperature for 12 h.
After the complete consumption of starting materials (monitored by TLC), reaction mixture
was poured into ice-cold water (25 mL), extracted by EtOAc (3 × 30 mL) and the combined
organic phases were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The obtained residue was purified by column chromatography using
EtOAc/n-hexane, collected fractions were evaporated in vacuo to afford the titled products
8aa–fe.
4.2.1. (4-((4-Methoxyphenyl)sulfonyl)piperazin-1-yl)(1-(4-(trifluoromethyl)phenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8aa)
White solid:88% yield; mp: 254–256 °C;1H NMR (300 MHz, DMSO-d6) δ: 11.91 (s, 1H),
8.50 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 7.9 Hz, 2H), 7.99 (d, J = 8.0 Hz, 2H), 7.71
(d, J = 8.7 Hz, 2H), 7.63 (dd, J = 16.3, 7.7 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.14 (d, J = 8.7
13
Hz, 2H), 3.83 (s, 7H), 2.98 (s, 4H); C NMR (75 MHz, DMSO-d6) δ: 167.4, 162.8, 142.1,
141.7, 141.5, 138.4, 133.5, 130.1, 129.8, 129.3, 128.8, 126.0, 125.6, 122.5, 122.0, 120.8,
120.1, 115.8, 114.5, 112.5, 111.3, 55.6, 46.3, 41.3; MS (ESI): m/z 595 [M+H]+; HRMS (ESI):
m/z calcd for C30H26F3N4O4S: 595.16214; found: 595.16442 [M+H]+.

4.2.2. (4-Tosylpiperazin-1-yl)(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-
yl)methanone (8ab)
1
Cream solid:85% yield; mp: 275–277 °C; H NMR (300 MHz, DMSO-d6) δ: 11.84 (s, 1H),
8.45 (s, 1H), 8.31–8.23 (m, 1H), 8.15 (d, J = 8.1 Hz, 2H), 7.94 (d, J = 8.2 Hz, 2H), 7.64 (d, J
= 8.2 Hz, 3H), 7.56 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1H), 3.83
13
(s, 4H), 3.01 (s, 4H), 2.39 (s, 3H); C NMR (101 MHz, CDCl3+DMSO-d6) δ: 167.3, 143.6,
142.0, 141.5, 141.4, 138.3, 133.3, 131.7, 130.1, 129.7, 129.2, 128.9, 128.7, 127.5, 125.4,
122.8, 121.8, 120.8, 120.1, 115.7, 112.4, 46.3, 41.3, 20.9; MS (ESI): m/z 579 [M+H]+;
HRMS (ESI): m/z calcd for C30H26F3N4O3S: 579.16722; found: 579.16900 [M+H]+.
4.2.3. (4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)(1-(4-(trifluoromethyl)phenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8ac)
White solid:90% yield;mp: 258–260 °C; 1H NMR (300 MHz, DMSO-d6) δ: 11.91 (s, 1H),
8.51 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 7.8 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H), 7.79
(d, J = 8.5 Hz, 2H), 7.71 (d, J = 7.1 Hz, 3H), 7.63 (d, J = 6.2 Hz, 1H), 7.31 (t, J = 7.0 Hz,
13
1H), 3.82 (s, 4H), 3.01 (s, 4H); C NMR (101 MHz, DMSO-d6) δ: 167.5, 142.2, 141.6,
141.5, 138.5, 138.4, 133.6, 133.4, 130.2, 129.6, 129.6, 129.4, 129.2, 128.9, 125.7, 123.0,
122.1, 120.9, 120.3, 115.9, 112.6, 46.4, 41.4; MS (ESI): m/z 599 [M+H]+; HRMS (ESI): m/z
calcd for C29H23ClF3N4O3S: 599.11260; found: 599.11474 [M+H]+.
4.2.4. (4-(Phenylsulfonyl)piperazin-1-yl)(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-
b]indol-3-yl)methanone (8ad)
1
White solid:84% yield; mp: 195–197 °C; H NMR (300 MHz, DMSO-d6) δ: 11.89 (s, 1H),
8.50 (s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 8.16 (d, J = 7.9 Hz, 2H), 7.99 (d, J = 8.1 Hz, 2H), 7.77
(d, J = 7.5 Hz, 2H), 7.69–7.56 (m, 5H), 7.30 (t, J = 7.3 Hz, 1H), 3.82 (s, 4H), 3.02 (s, 4H);
13C NMR (75 MHz, DMSO-d6) δ: 167.4, 156.3, 142.2, 141.5, 138.4, 134.7, 133.4, 130.1,
129.4, 129.3, 128.9, 127.6, 125.6, 122.0, 120.8, 120.2, 117.8, 115.9, 112.5, 46.4, 41.3; MS
(ESI): m/z 565 [M+H]+; HRMS (ESI): m/z calcd for C29H24F3N4O3S: 565.15157; found:
565.15315 [M+H]+.
4.2.5. (4-((4-(Tert-butyl)phenyl)sulfonyl)piperazin-1-yl)(1-(4-(trifluoromethyl)phenyl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (8ae)

Cream solid:82% yield; mp: 191–193 °C;1H NMR (300 MHz, DMSO-d6) δ: 11.90 (s, 1H),
8.51 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.15 (d, J = 7.9 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H), 7.71
(d, J = 8.2 Hz, 2H), 7.68–7.54 (m, 4H), 7.30 (t, J = 7.0 Hz, 1H), 3.81 (s, 4H), 3.03 (s, 4H),
13
1.27 (s, 9H); C NMR (75 MHz, DMSO-d6) δ: 167.3, 156.3, 142.2, 141.5, 139.5, 138.4,
133.3, 131.8, 130.1, 129.3, 128.8, 127.5, 126.1, 125.6, 122.0, 120.8, 120.2, 115.8, 112.5,
46.4, 34.8, 30.6; MS (ESI): m/z 621 [M+H]+; HRMS (ESI): m/z calcd for C33H32F3N4O3S:
621.21417; found: 621.21719 [M+H]+.
4.2.6. (1-(4-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)(4-((4-
methoxyphenyl)sulfonyl)piperazin-1-yl)methanone (8ba)
Cream solid:90% yield; mp: 186–188°C;1H NMR (300 MHz, DMSO-d6) δ: 11.81 (s, 1H),
8.43 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.00 (dd, J = 8.2, 5.7 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H),
7.65 (s, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 8.8 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 7.16
(d, J = 8.7 Hz, 2H), 3.84 (s, 3H), 3.80 (s, 4H), 2.97 (s, 4H); 13C NMR (75 MHz, DMSO-d6) δ:
167.5, 164.1, 162.8, 142.0, 141.4, 139.1, 134.0, 133.0, 130.7 (d, J = 8.4 Hz), 129.8, 128.7,
126.0, 121.9, 120.9, 120.1, 115.8, 115.5, 115.1, 114.6, 112.5, 55.7, 46.4, 41.3; MS (ESI): m/z
545 [M+H]+; HRMS (ESI): m/z calcd for C29H26FN4O4S: 545.16533; found: 545.16669
[M+H]+.
4.2.7. (1-(4-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)(4-tosylpiperazin-1-
yl)methanone (8bb)
White solid:85% yield; mp: 244–246°C;1H NMR (300 MHz, DMSO-d6) δ: 11.80 (s, 1H),
8.43 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 8.00 (dd, J = 8.7, 5.6 Hz, 2H), 7.65 (dd, J = 8.2, 3.3
Hz, 3H), 7.58 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.5 Hz, 4H), 7.28 (t, J = 7.4 Hz, 1H), 3.80 (s,
4H), 2.98 (s, 4H), 2.39 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ: 167.5, 161.5, 143.9,
142.0, 141.5, 139.2, 134.0, 133.0, 131.7, 130.7 (d, J = 8.3 Hz), 129.9, 129.8, 128.7, 127.6,
121.9, 120.9, 120.1, 115.7 (d, J = 21.6 Hz), 115.1, 112.5, 46.4, 46.0, 21.0; MS (ESI): m/z 529
[M+H]+; HRMS (ESI): m/z calcd for C29H26FN4O3S: 529.17042; found: 529.17126 [M+H]+.
4.2.8. (4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)(1-(4-fluorophenyl)-9H-pyrido[3,4-
b]indol-3-yl)methanone (8bc)
White solid:82% yield; mp: 253–255°C;1H NMR (300 MHz, DMSO-d6) δ: 11.81 (s, 1H),
8.43 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 8.00 (dd, J = 8.7, 5.5 Hz, 2H), 7.77 (s, 1H), 7.73 (d, J =
8.8 Hz, 2H), 7.70–7.63 (m, 2H), 7.58 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.9 Hz, 2H), 7.29 (t, J =

7.0 Hz, 1H), 3.81 (s, 4H), 3.01 (s, 4H); 13C NMR (75 MHz, DMSO-d6) δ: 167.5, 164.1,
142.0, 141.4, 139.1, 138.4, 134.0, 133.6, 133.0, 130.7 (d, J = 8.5 Hz), 129.8, 129.6, 129.5,
128.7, 121.9, 120.9, 120.1, 115.7 (d, J = 21.5 Hz), 115.1, 112.5, 46.3, 41.3; MS (ESI): m/z
549 [M+H]+; HRMS (ESI): m/z calcd for C28H23ClFN4O3S: 549.11579; found: 549.11733
[M+H]+.
4.2.9. (1-(4-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)(4-(phenylsulfonyl)piperazin-1-
yl)methanone (8bd)
White solid: 80% yield; mp: 232–234°C;1H NMR (300 MHz, DMSO-d6) δ: 11.80 (s, 1H),
8.43 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 8.00 (dd, J = 8.8, 5.5 Hz, 2H), 7.80–7.75 (m, 2H), 7.69
(s, 1H), 7.68–7.63 (m, 3H), 7.62 – 7.54 (m, 1H), 7.47 (t, J = 8.9 Hz, 2H), 7.28 (t, J = 7.4 Hz,
1H), 3.81 (s, 4H), 3.01 (s, 4H); 13C NMR (75 MHz, DMSO-d6) δ: 167.5, 164.1, 160.8, 142.0,
141.4, 139.1, 134.6, 134.0, 133.4, 133.0, 130.6 (d, J = 8.4 Hz), 129.6 (d, J = 24.4 Hz), 128.6,
127.5, 121.9, 120.9, 120.1, 115.7 (d, J = 21.5 Hz), 115.1, 112.5, 46.4, 41.3; MS (ESI): m/z
515 [M+H]+; HRMS (ESI): m/z calcd for C28H24FN4O3S: 515.15477; found: 515.15566
[M+H]+.
4.2.10. (4-((4-(Tert-butyl)phenyl)sulfonyl)piperazin-1-yl)(1-(4-fluorophenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8be)
White solid:84% yield; mp: 260–262°C;1H NMR (300 MHz, DMSO-d6) δ: 11.81 (s, 1H),
8.44 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.97 (dd, J = 8.7, 5.5 Hz, 2H), 7.69 (d, J = 1.6 Hz, 4H),
7.65 (d, J = 2.9 Hz, 1H), 7.58 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.9 Hz, 2H), 7.28 (t, J = 7.4 Hz,
1H), 3.81 (s, 4H), 3.01 (s, 4H), 1.28 (s, 9H); 13C NMR (75 MHz, DMSO-d6) δ: 167.5, 164.1,
160.8, 156.3, 142.0, 141.4, 139.1, 134.0, 133.0, 131.7, 130.7 (d, J = 8.4 Hz), 129.8, 128.7,
127.6, 126.3, 121.4 (d, J = 76.6 Hz), 120.1, 115.6 (d, J = 21.5 Hz), 115.1, 112.5, 46.4, 41.4,
34.9, 30.7; MS (ESI): m/z 571 [M+H]+; HRMS (ESI): m/z calcd for C32H32FN4O3S:
571.21737; found: 571.21842 [M+H]+.
4.2.11. (1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)(4-((4-
methoxyphenyl)sulfonyl)piperazin-1-yl)methanone (8ca)
White solid:88% yield; mp: 228–230°C;1H NMR (500 MHz, DMSO-d6) δ: 11.71 (s, 1H),
8.35 (s, 1H), 8.29 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.9 Hz, 2H), 7.66
(d, J = 8.2 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.20–7.13 (m, 4H), 3.90
13
(s, 3H), 3.83 (s, 3H), 3.79 (s, 4H), 2.97 (s, 4H); C NMR (101 MHz, DMSO-d6) δ: 167.7,

162.9, 159.9, 142.0, 141.4, 140.1, 132.9, 130.1, 129.9, 129.8, 129.5, 128.5, 126.0, 121.9,
121.0, 120.0, 114.6, 114.5, 114.3, 112.6, 55.7, 55.4, 46.5, 41.3; MS (ESI): m/z 557 [M+H]+;
HRMS (ESI): m/z calcd for C30H29N4O5S: 557.18532; found: 557.18670 [M+H]+.
4.2.12. (1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)(4-tosylpiperazin-1-
yl)methanone (8cb)
White solid:84% yield; mp: 148–150°C;1H NMR (300 MHz, DMSO-d6) δ: 11.73 (s, 1H),
8.37 (s, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.1 Hz, 3H), 7.57
(t, J = 7.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.27 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.7 Hz, 2H),
3.91 (s, 3H), 3.81 (s, 4H), 2.99 (s, 4H), 2.40 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ: 167.6,
159.8, 143.8, 141.9, 141.4, 140.0, 132.8, 131.7, 130.0, 129.9, 129.8, 129.5, 128.4, 127.6,
121.8, 121.0, 119.9, 114.5, 114.2, 112.6, 55.4, 46.4, 21.0; MS (ESI): m/z 541 [M+H]+;
HRMS (ESI): m/z calcd for C30H29N4O4S: 541.19040; found: 541.19162 [M+H]+.
4.2.13. (4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)(1-(4-methoxyphenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8cc)
White solid:88% yield; mp: 214–216°C;1H NMR (300 MHz, DMSO-d6) δ: 11.73 (s, 1H),
8.38 (s, 1H), 8.30 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.82–7.73 (m, 3H), 7.71–7.63
(m, 2H), 7.57 (t, J = 7.5 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 3.91 (s,
13
3H), 3.82 (s, 4H), 3.01 (s, 4H); C NMR (101 MHz, DMSO-d6) δ: 167.6, 159.8, 141.9,
141.4, 140.0, 138.4, 133.6, 133.1, 132.8, 130.0, 129.8, 129.6, 129.5, 128.4, 121.8, 121.0,
119.9, 114.5, 114.2, 112.5, 55.4, 46.3, 41.3; MS (ESI): m/z 561 [M+H]+; HRMS (ESI): m/z
calcd for C29H26ClN4O4S: 561.13578; found: 561.13720 [M+H]+.
4.2.14. (1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)(4-(phenylsulfonyl)piperazin-
1-yl)methanone (8cd)
1
White solid:82% yield; mp: 225–227°C; H NMR (300 MHz, DMSO-d6) δ: 11.72 (s, 1H),
8.37 (s, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.76 (t, J = 8.5 Hz, 3H), 7.71–
7.64 (m, 3H), 7.58 (dd, J = 14.5, 7.4 Hz, 1H), 7.27 (t, J = 7.3 Hz, 1H), 7.19 (d, J = 8.7 Hz,
2H), 3.91 (s, 3H), 3.81 (s, 4H), 3.02 (s, 4H); 13C NMR (75 MHz, DMSO-d6) δ: 167.6, 164.9,
159.8, 141.3, 140.1, 140.0, 133.4, 132.8, 130.0, 129.7, 129.4, 128.4, 127.5, 121.8, 120.9,
119.9, 114.5, 114.2, 112.5, 109.6, 55.3, 46.3; MS (ESI): m/z 527 [M+H]+; HRMS (ESI): m/z
calcd for C29H27N4O4S: 527.17475; found: 527.17565 [M+H]+.

4.2.15. (4-((4-(Tert-butyl)phenyl)sulfonyl)piperazin-1-yl)(1-(4-methoxyphenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8ce)
1
White solid:90% yield; mp: 145–147°C; H NMR (300 MHz, DMSO-d6) δ: 11.73 (s, 1H),
8.38 (s, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.71–7.63 (m, 5H), 7.57 (t, J
= 7.6 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2H), 3.91 (s, 3H), 3.81 (s, 4H),
13
3.02 (s, 4H), 1.29 (s, 9H); C NMR (101 MHz, DMSO-d6) δ: 167.6, 159.8, 156.4, 141.9,
141.4, 140.1, 132.8, 131.8, 130.0, 129.8, 129.5, 128.5, 127.6, 126.3, 121.8, 121.0, 120.0,
114.6, 114.2, 112.6, 55.4, 46.4, 41.4, 34.9, 30.7; MS (ESI): m/z 583 [M+H]+; HRMS (ESI):
m/z calcd for C33H35N4O4S: 583.23735; found: 583.23908 [M+H]+.
4.2.16. (4-((4-Methoxyphenyl)sulfonyl)piperazin-1-yl)(1-(3,4,5-trimethoxyphenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8da)
Cream colour solid:88% yield; mp: 157–159°C; 1H NMR (300 MHz, DMSO-d6) δ: 11.81 (s,
1H), 8.44 (s, 1H), 8.33 (d, J = 7.2 Hz, 1H), 7.65 (s, 3H), 7.57 (s, 1H), 7.28 (s, 1H), 7.17 (s,
2H), 7.10 (d, J = 27.4 Hz, 2H), 3.91 (s, 7H), 3.84 (s, 6H), 3.79 (s, 3H), 3.00 (s, 4H); 13C NMR
(101 MHz, DMSO-d6) δ: 167.5, 162.9, 153.1, 141.8, 141.4, 140.2, 138.0, 133.1, 133.0, 129.7,
128.6, 126.2, 121.9, 121.0, 120.0, 115.2, 114.6, 112.6, 110.5, 105.8, 60.1, 55.8, 55.7, 46.3,
41.4; MS (ESI): m/z 617 [M+H]+; HRMS (ESI): m/z calcd for C32H33N4O7S: 617.20645;
found: 617.20909 [M+H]+.
4.2.17. (4-Tosylpiperazin-1-yl)(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methanone (8db)
White solid:86% yield; mp: 145–147 °C;1H NMR (300 MHz, DMSO-d6) δ: 11.81 (s, 1H),
8.44 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 7.62 (dd, J = 18.0, 9.1 Hz, 4H), 7.45 (d, J = 8.0 Hz,
2H), 7.34–7.21 (m, 1H), 7.11 (d, J = 35.5 Hz, 2H), 3.91 (s, 7H), 3.79 (s, 6H), 3.01 (s, 4H),
2.40 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ: 167.6, 153.1, 143.9, 141.8, 141.4, 140.2,
138.1, 133.1, 133.0, 131.9, 129.9, 129.6, 128.6, 127.5, 121.9, 121.0, 120.0, 115.3, 112.6,
105.8, 60.1, 55.8, 46.3, 41.4, 21.0; MS (ESI): m/z 601 [M+H]+; HRMS (ESI): m/z calcd for
C32H33N4O6S: 601.21153; found: 601.21387 [M+H]+.
4.2.18. (4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)(1-(3,4,5-trimethoxyphenyl)-9H-
pyrido[3,4-b]indol-3-yl)methanone (8dc)

White solid:84% yield; mp: 180–182°C;1H NMR (300 MHz, DMSO-d6) δ: 11.81 (s, 1H),
8.45 (s, 1H), 8.33 (d, J = 7.8 Hz, 1H), 7.74 (s, 4H), 7.71–7.62 (m, 2H), 7.59 (d, J = 7.5 Hz,
1H), 7.29 (t, J = 7.3 Hz, 1H), 7.18 (s, 1H), 3.87 (d, J = 20.3 Hz, 9H), 3.79 (s, 4H), 3.08 (d, J =
17.8 Hz, 4H); 13C NMR (75 MHz, DMSO-d6) δ: 167.5, 153.0, 141.8, 141.4, 140.1, 138.4,
138.0, 133.9, 133.0, 129.6, 129.3, 128.5, 121.9, 121.0, 120.0, 115.5, 115.2, 112.6, 105.8,
60.1, 55.8, 46.2, 41.4; MS (ESI): m/z 621 [M+H]+; HRMS (ESI): m/z calcd for
C31H30ClN4O6S: 621.15691; found: 621.15944 [M+H]+.
4.2.19. (4-(Phenylsulfonyl)piperazin-1-yl)(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-
b]indol-3-yl)methanone (8dd)
Cream colour solid:85% yield; mp: 162–164°C;1H NMR (300 MHz, DMSO-d6) δ: 11.82 (s,
1H), 8.45 (d, J = 4.5 Hz, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.79–7.71 (m, 3H), 7.70 (d, J = 5.2
Hz, 1H), 7.68–7.61 (m, 3H), 7.57 (dd, J = 9.5, 5.5 Hz, 1H), 7.27 (dd, J = 13.4, 6.4 Hz, 1H),
7.18 (s, 1H), 3.91 (d, J = 4.2 Hz, 9H), 3.78 (s, 4H), 3.05 (d, J = 14.9 Hz, 4H); 13C NMR (101
MHz, DMSO-d6) δ: 167.6, 153.0, 141.7, 141.5, 140.1, 138.0, 134.8, 133.5, 133.2, 133.0,
129.5, 128.5, 127.4, 121.9, 121.0, 119.9, 115.2, 112.6, 110.5, 105.7, 60.1, 55.8, 46.3, 41.4;
MS (ESI): m/z 587 [M+H]+; HRMS (ESI): m/z calcd for C31H31N4O6S: 587.19588; found:
587.19804 [M+H]+.
4.2.20. (4-((4-(Tert-butyl)phenyl)sulfonyl)piperazin-1-yl)(1-(3,4,5-trimethoxyphenyl)-
9H-pyrido[3,4-b]indol-3-yl)methanone (8de)
White solid:86% yield; mp: 188–190°C;1H NMR (300 MHz, DMSO-d6) δ: 11.82 (s, 1H),
8.45 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.66 (s, 4H), 7.59 (dd, J = 14.8, 7.2 Hz, 3H), 7.28 (t, J
= 7.1 Hz, 1H), 7.16 (s, 1H), 3.87 (d, J = 19.9 Hz, 9H), 3.78 (s, 4H), 3.05 (s, 4H), 1.29 (s, 9H);
13C NMR (101 MHz, DMSO-d6) δ: 167.5, 156.4, 153.0, 141.7, 141.4, 140.2, 138.0, 134.0,
133.1, 132.0, 129.6, 128.5, 127.4, 126.3, 121.9, 121.0, 120.0, 115.3, 112.6, 105.8, 60.0, 55.8,
46.3, 41.4, 34.9, 30.7; MS (ESI): m/z 643 [M+H]+; HRMS (ESI): m/z calcd for C35H39N4O6S:
643.25848; found: 643.26157 [M+H]+.
4.2.21. (4-((4-Methoxyphenyl)sulfonyl)piperazin-1-yl)(1-phenyl-9H-pyrido[3,4-b]indol-
3-yl)methanone (8ea)
Brown colour solid:85% yield; mp: >350°C;1H NMR (400 MHz, DMSO-d6) δ: 8.48 (s, 1H),
8.39 (d, J = 7.8 Hz, 1H), 7.67 (s, 2H), 7.65 (d, J = 2.4 Hz, 2H), 7.63 (d, J = 1.7 Hz, 2H), 7.59
(dd, J = 9.2, 3.3 Hz, 3H), 7.37–7.32 (m, 1H), 7.12 (d, J = 8.9 Hz, 2H), 3.83 (s, 3H), 3.75 (d, J
= 19.6 Hz, 4H), 2.95 (s, 4H); 13C NMR (75 MHz, DMSO-d6) δ: 167.3, 162.8, 149.6, 142.7,

141.5, 138.8, 134.3, 129.7, 128.9, 128.5, 128.0, 125.9, 124.6, 121.9, 120.5, 120.3, 114.7,
114.5, 110.6, 55.6, 46.3, 41.3; MS (ESI): m/z 528 [M+H]+; HRMS (ESI): m/z calcd for
C29H27N4O4S: 527.17475; found: 527.17602 [M+H]+.
4.2.22. (1-Phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-tosylpiperazin-1-yl)methanone (8eb)
Brown colour solid:85% yield; mp: >350°C;1H NMR (300 MHz, DMSO-d6) δ: 8.48 (s, 1H),
8.39 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.66 (d, J = 2.7 Hz, 2H), 7.62 (d, J = 2.0 Hz, 2H), 7.59
(d, J = 2.1 Hz, 3H), 7.57 (d, J = 2.5 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.38–7.30 (m, 1H),
3.75 (d, J = 14.0 Hz, 4H), 2.95 (s, 4H), 2.38 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ: 167.4,
156.8, 143.8, 142.7, 141.5, 138.8, 134.3, 131.6, 129.9, 129.7, 128.9, 128.5, 128.0, 127.5,
124.7, 121.9, 120.5, 120.3, 114.7, 110.6, 46.3, 41.3, 21.0; MS (ESI): m/z 512 [M+H]+;
HRMS (ESI): m/z calcd for C29H27N4O3S: 511.17984; found: 511.19204 [M+H]+.
4.2.23. (4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)(1-phenyl-9H-pyrido[3,4-b]indol-3-
yl)methanone (8ec)
Brown colour solid:82% yield; mp: >350°C;1H NMR (300 MHz, DMSO-d6) δ: 8.48 (s, 1H),
8.39 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 8.0 Hz, 3H), 7.69 (d, J = 6.5 Hz, 4H), 7.60 (d, J = 11.4
Hz, 4H), 7.34 (s, 1H), 3.77 (s, 4H), 3.01 (s, 4H); 13C NMR (101 MHz, DMSO-d6) δ: 167.4,
142.7, 141.5, 138.8, 138.3, 134.3, 133.6, 129.8, 129.7, 129.6, 129.4, 129.3, 128.9, 128.5,
128.0, 121.9, 120.5, 120.3, 114.7, 110.6, 46.2, 41.3; MS (ESI): m/z 532 [M+H]+; HRMS
(ESI): m/z calcd for C28H24ClN4O3S: 531.12522; found: 531.12564 [M+H]+.
4.2.24. (1-Phenyl-9H-pyrido[3,4-b]indol-3-yl)(4-(phenylsulfonyl)piperazin-1-
yl)methanone (8ed):
Brown colour solid:80% yield; mp: 224–226°C;1H NMR (500 MHz, DMSO-d6) δ: 8.48 (s,
1H), 8.38 (d, J = 7.8 Hz, 1H), 7.75–7.72 (m, 2H), 7.72–7.68 (m, 2H), 7.67 (d, J = 8.7 Hz,
2H), 7.64 (d, J = 1.4 Hz, 1H), 7.63 (s, 2H), 7.62–7.59 (m, 1H), 7.58 (d, J = 6.7 Hz, 2H), 7.34
(t, J = 7.1 Hz, 1H), 3.76 (d, J = 20.7 Hz, 4H), 2.98 (s, 4H); 13C NMR (101 MHz, DMSO-d6)
δ: 167.4, 142.7, 141.5, 138.8, 134.6, 134.3, 133.4, 129.9, 129.7, 129.4, 128.9, 128.5, 128.0,
127.5, 127.3, 121.9, 120.5, 120.3, 114.7, 110.6, 46.3, 41.3; MS (ESI): m/z 498 [M+H]+;
HRMS (ESI): m/z calcd for C28H25N4O3S: 497.16419; found: 497.16514 [M+H]+.
4.2.25. (4-((4-(Tert-butyl)phenyl)sulfonyl)piperazin-1-yl)(1-phenyl-9H-pyrido[3,4-
b]indol-3-yl)methanone (8ee)