Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.08.031

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45 nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05 nM and 13.84 nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.

Full text of DOI:10.1016/j.bmc.2018.08.031

Accepted Manuscript
Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conju-
gates as potential cytotoxic and DNA topoisomerase I inhibitors
Manda Sathish, Sabanis Chetan Dushantrao, Shalini Nekkanti, Ramya Tokala,
Soujanya Thatikonda, Yellaiah Tangella, Gunda Srinivas, Shirisha
Cherukommu, Namballa Hari Krishna, Nagula Shankaraiah, Narayana Nagesh,
Ahmed Kamal
PII:
S0968-0896(18)30996-9
https://doi.org/10.1016/j.bmc.2018.08.031
BMC 14518
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 May 2018
22 August 2018
25 August 2018
Please cite this article as: Sathish, M., Chetan Dushantrao, S., Nekkanti, S., Tokala, R., Thatikonda, S., Tangella,
Y., Srinivas, G., Cherukommu, S., Hari Krishna, N., Shankaraiah, N., Nagesh, N., Kamal, A., Synthesis of DNA
interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I
inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.031
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Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline
conjugates as potential cytotoxic and DNA topoisomerase I inhibitors
Manda Sathish,a Sabanis Chetan Dushantrao,b Shalini Nekkanti,b Ramya Tokala,b Soujanya
Thatikonda,c Yellaiah Tangella,a Gunda Srinivas,d Shirisha Cherukommu,d Namballa Hari
Krishna,b Nagula Shankaraiah*,b Narayana Nagesh*,d Ahmed Kamal*a,e
aMedicinal Chemistry & Biotechnology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
bDepartment of Medicinal Chemistry, cDepartment of Regulatory Toxicology, National
Institute of Pharmaceutical Education and Research (NIPER), Hyderabad-500 037, India
dCSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
eSchool of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi,
110 062, India
Abstract: A series of new C3-trans-cinnamide linked β-carboline conjugates has been
synthesized by coupling between various β-carboline amines and substituted cinnamic acids.
Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines
such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer)
and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly
designed conjugates are considerably active against all the tested cancer cell lines with IC50
values 13‒45 nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells
(14.05 nM and 13.84 nM respectively) and also even potent on other cell lines tested. Further,
detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I
inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates
are DNA interactive topoisomerase I inhibitors.
Keywords: β-Carbolines; trans-cinnamides; DNA intercalation; topoisomerase I; anticancer
activity.
Corresponding authors: Dr. Nagula Shankaraiah, E-mail: shankar@niperhyd.ac.in;
shankarnbs@gmail.com; Dr. Narayana Nagesh, nagesh@ccmb.res.in, Dr. Ahmed Kamal,
ahmedkamal@iict.res.in
1

1. Introduction
Due to the well-known medical need for new cancer drugs and the resulting commercial
prospects, the academia/pharmaceutical industry continues to rigorously pursue the discovery of
oncology therapeutics.1 Over the past several years, the major drug discovery trends include the
expansion of the cancer-drug candidate pipelines, the advancement of the clinical-trials, and the
increase of accessible clinical data. Advances in understanding of disease pathophysiology and
the development of rational technologies are producing drug candidates with superior therapeutic
and safety profiles. Despite such advances, cancer drug development remains challenging rapid
drug resistance, and ineffective tumor penetration of drugs.2 Hence, there is a need for the
development of more effective and safer cancer drugs.
In the recent years, cinnamoyl compounds particularly, trans-cinnamic acid derivatives from
natural and synthetic sources have gained much attention due to their wide range of biological
activities such as antitumor,3 antioxidant4 and antimicrobial activities.4b,5 The cinnamic acid
derivatives such as cinnamates,6 cinnamides,7 cinnamoyl hydrazides,8 etc are reported to be most
potent than their parent cinnamic acid.8 Among these, trans-cinnamides are an important class of
organic molecules possessing a wide variety of biological and medicinal properties such as
antitubercular,9 antidiabetic,10 anti-inflammatory,11 anti-neoplastic,3 anti-fungal12 and anti-
allergic13 profile. For example, a simple cinnamide like 2‐methyl cinnamide (A, Fig. 1) isolated
from Streptomyces species has shown potent antimetastatic property.14 Also, cinnamides serve in
the field of agrochemistry as fungicidal, avian repellent and insecticidal agents.15 Another
example duocarmycin A,16 a natural antitumor antibiotic which was extracted from cultures of
Streptomyces species which could bind to the DNA minor groove by forming N3‐adenine
covalent bond. Nagamura et al.17 have developed cinnamoyl-duocarmycin A derivatives (B, Fig.
1) and assayed for their antitumor activity against HeLa S3 cell line. Recently, cinnamoyl group
with hydroxamic acid has gained great importance in the development of new histone
deacetylase (HDAC) inhibitors. The HDAC is a Zinc dependent hydrolase which play an
important role in oncogenesis.18 Numerous cinnamoyl hydroxamic acids such as LBH589 (C,
Fig. 1), LAQ 824, PXD 101, etc are under in clinical studies as non selective inhibitors of
HDACs.19,20
2

Fig. 1. Representative bio-active cinnamides (A‒C), β-carbolines (D and E) and newly
synthesized C3-trans-cinnamide linked β-carboline conjugates 8a‒x.
On the other hand, the planar tricyclic β-carbolines are originally isolated from Peganum
harmala (Zygophillaceae, Syrian Rue) which have shown broad spectrum of biological
properties including anticancer activity. The well-known molecules from this class of β-carboline
alkaloids are harman, harmine, norharman and harmaline. Basically, β-carboline derivatives
show their anticancer property through multi kinds of mechanisms of action, such as DNA minor
groove,21 DNA interacting (intercalation with DNA or external binding to DNA),22 inhibition of
MK-2,23 DNA topo I and II,24 kinesin Eg525 and CDK.26 However, interaction with DNA and
inhibition of DNA topo I and II have been studied widely and received much importance in the
area of cancer chemotherapy. Henceforth, some of the anticancer drugs are currently in use for
the treatment of many cancers via these type of mechanism of actions.27
The easy construction of cinnamides possessing various potential biological activities is the
main reason for the design and development of new hybrid molecules in the field of
pharmaceutical sciences. Moreover, many observations revealed that the enhancement of activity
of the cinnamide derivatives is significantly potent when compared to their parent natural
molecules. Similarly, β-carboline alkaloids are also well-known class of natural products with
3

numerous biological properties including anticancer activity. In addition, many synthetic β-
carboline derivatives (D and E, Fig. 1) display improved anticancer activity compared with their
natural substrates.28 The available structure activity relationship (SAR) study of β-carbolines
revealed that basically the activity of molecules depends on nature of substituents at position-1, -
3 and -9, particularly an aryl substitution at C1-position and a side chain at C3-position is greatly
affect the activity. Based on the pharmacophoric properties of both trans-cinnamides as well as
β-carbolines, a synthetic design has been made to develop an array of new series of molecules as
potent anticancer agents (Fig. 2). In this context, we have designed and synthesized a new series
of C3-trans-cinnamide linked β-carboline conjugates 8a‒x and evaluated for their in vitro
cytotoxicity, DNA binding affinity, DNA topo I inhibition and molecular docking studies.
Fig. 2. Synthetic strategy of the target hybrid molecules.
2. Results and Discussion
2.1. Chemistry
The C3-trans-cinnamide linked β-carboline conjugates were synthesized as shown in
Scheme 1. The L-tryptophan methyl ester (2) was obtained by esterification of L-tryptophan (1)
by using SOCl2. Next, Pictet-Spengler condensation of amino ester 2 with substituted
benzaldehydes in the presence of acid catalyst gave the corresponding methyl tetrahydro-β-
carboline-3-carboxylates 3a‒c which were further utilized without any purification for the next
aromatization reaction by employing trichloroisocyanuric acid (TCICA) to afford the methyl-β-
carboline-3-carboxylates 4a–c. Subsequently, the reduction of 4a–c with LiBH4 afforded the
related alcohols 5a‒c respectively, which was taken-up for further azidation with DPPA and
DBU to provide the corresponding azides 6a‒c in moderate to good yields. Later, Staudinger
reduction with azides 6a‒c in to the amines was achieved by using TPP in acetonitrile and water
to afford β-carboline-3-methyl amines 7a‒c. Finally, the desired C3-trans-cinnamide linked β-
4

carboline conjugates 8a‒x were achieved in moderate to excellent yields by the coupling of 7a‒c
with substituted trans-cinnamic acids29 and compounds 8a-e, 8h and 8w were also shown minor
rotamers in minor quantity in 1H NMR (see ESI). The representative β-carboline-3-methyl amine
7c was coupled with crotonic acid (9) and 3-chloro-propionyl chloride (11) to yield C3-trans-
acrylamide linked β-carboline conjugates 10, 12 in good yields. Further, the best performed 3-
hydroxy cinnamic acid (13) was coupled with allylamine (14) to yield 15 as shown in Scheme 2.
Scheme 1. Synthesis of a library of C3-trans-cinnamide linked β-carboline conjugates 8a–x.
5

Scheme 2. Synthesis of acrylamide conjugates 10, 12, and 15.
2.2. Evaluation of Biological Activity
2.2.1. Cytotoxicity
The C3-trans-cinnamide linked β-carboline conjugates 8a‒x were evaluated for their in vitro
cytotoxicity against selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast
cancer), B16 (melanoma), HeLa (cervical cancer) and normal cell line NIH3T3 (mouse
embryonic fibroblast cancer) using MTT assay.30 The MTT assay results were expressed as half
maximal inhibitory concentration (IC50) values and the cytotoxicity data of 8a‒x are shown in
Table 1. The newly synthesized conjugates 8a‒x are potentially active against the tested cell
lines (IC50 = 13‒45 nM) and compared with doxorubicin as control. However, all the derivatives
are very potent than the reference doxorubicin (>500 nM) and these new conjugates were
particularly most active against MCF-7 cells (13 nM to 20 nM) when compared with other cell
lines tested. In the present study, a member of the anthracycline family namely, doxorubicin has
shown higher half maximal inhibitory concentration (IC50) values may be due to the formation of
metabolites generated by carbonyl reduction that is less toxic to the cells.31 The conjugates 8v
and 8x were most active against MCF-7 cells (14.05 nM and 13.84 nM respectively) including
other cell lines tested. Although these new conjugates activity, the nature of substitutions on
6

position-1 and -3 slightly affected on this cytotoxicity. The most active conjugates 8q, 8v and 8x
were having 4-methoxy phenyl group at position-1 and 3,4,5-trimethoxy or 4-chloro or 3-
hydroxy on cinnamide part at position-3. Moreover, all the conjugates with 4-methoxyphenyl
group at position-1 (8q‒x) as well as 3,4,5-trimethoxy on cinnamide at position-3 (8a and 8i) are
considerably active than the other conjugates with IC50 values <24.0 nM. Based on these
observations, the 4-methoxy phenyl group at position-1 and 3,4,5-trimethoxy on cinnamide at
position-3 may be crucial for the in vitro cytotoxicity. However, it was observed that the
acrylamide containing conjugates 10, 12, 15 were found to be less active when compared to the
β-carboline-cinnamide conjugates as represented in Table 1. Hence, the most active conjugates
8v and 8x were further explored for their apoptosis induction assay, DNA binding affinity, DNA
topoisomerase I inhibition and molecular docking studies.
7

Table 1. In vitro cytotoxicity (IC50 values in nM)a of C3-trans-cinnamide linked β-carboline
conjugates 8a‒x and 10, 12, 15 on selected human cancer cell lines
Compound
A549b
MCF-7c
16.23±1.5
17.02±1.2
16.46±1.8
16.54±0.6
17.54±0.3
18.34±0.8
17.93±0.7
19.23±0.6
16.54±1.1
17.52±1.4
16.37±0.8
19.65±1.1
17.23±0.6
17.53±0.9
17.55±0.7
16.83±0.5
13.93±0.4
16.43±0.5
18.23±0.7
17.23±1.2
16.46±0.7
14.05±0.9
17.23±0.5
13.84±0.4
10.83±0.26
>50
B16d
HeLae
NIH3T3f
35.98±1.3
38.98±1.1
30.77±2.1
35.98±2.3
39.65±1.8
43.65±1.1
36.86±1.9
35.66±2.2
38.87±2.5
39.23±1.9
42.98±1.1
36.29±1.7
38.97±1.9
35.62±2.1
43.08±1.3
38.16±1.5
39.26±1.7
37.45±1.4
36.87±1.6
37.54±1.3
34.44±1.6
38.31±1.3
39.87±1.6
37.16±1.1
2.67±0.32
>50
8a
17.92±0.6
18.02±0.8
16.33±0.3
21.03±1.2
22.23±1.1
19.56±1.6
22.42±1.1
23.43±0.8
21.34±0.9
20.43±0.7
18.74±0.7
21.57±1.2
18.77±1.4
18.85±1.7
18.26±1.4
19.50±1.5
19.69±0.6
18.43±0.7
20.17±1.3
19.97±1.1
18.94±1.2
18.89±1.1
21.23±1.5
18.86±1.4
3.36±0.19
>50
19.76±0.6
18.93±0.4
16.84±0.8
18.23±0.3
19.85±0.7
18.23±1.3
19.45±1.1
24.93±0.5
17.23±0.4
16.87±0.7
17.25±0.3
24.08±0.7
19.23±1.4
18.69±1.1
19.04±0.9
21.83±0.8
17.35±0.4
22.98±0.5
21.87±0.3
23.87±0.2
22.14±0.4
16.64±0.5
19.76±0.5
16.31±0.3
-
22.98±0.7
24.87±0.4
24.32±0.6
23.56±0.5
21.87±0.2
19.23±0.6
23.85±0.4
26.98±0.5
23.48±0.3
25.87±0.2
19.98±0.5
23.82±0.3
25.09 ±0.2
26.83±0.4
25.98±0.3
23.65±0.1
22.89±0.2
21.65±0.4
22.76±0.6
24.65±0.4
25.37±0.3
21.55±0.6
24.87±0.2
22.32±0.3
5.41±0.62
>50
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
10g
12g
-
15g
>50
>50
-
>50
>50
Doxorubicin
740±0.4
580±0.2
670±0.7
780±0.5
1720±0.8
a50% Inhibitory concentration after 48 h of drug treatment and the values are average of three individual experiments.
bLung cancer.
cBreast cancer.
dMelanoma.
eCervical cancer.
fMouse embryonic fibroblast cell line.
gValues in μM.
8

2.2.2. Cell cycle analysis
The MTT results indicate that among the compounds tested, 8v and 8x exhibited lowest IC50
(14.05 nM and 13.84 nM respectively) in breast cancer cell lines (MCF-7). In order to
understand the unique potential of these conjugates at their respective IC50 and to shed more light
on various aspects responsible for the cytotoxic effect of 8v and 8x, we examined their influence
on the cell cycle progression in MCF-7 cells as shown in Fig. 3 and the results are displayed in
Table 2. After 48 h of treatment, hybrids 8v and 8x caused a small increase in the percentage of
population of cells in G2/M phase compared to the control (33.65 % to 39.25 % and 37.42 % in
case of 8v and 8x respectively), indicating the onset of apoptosis in MCF-7 cells on treatment
with 8v and 8x conjugates.32
Fig. 3. Cell cycle analysis: Cell cycle studies indicate the effect of 8v and 8x on cell cycle arrest.
(A) MCF-7 cells without 8v and 8x hybrids treatment (B) MCF-7 cells treated with 0.5µM 8v
hybrid (C) MCF-7 cells treated with 1µM 8v. (D) MCF-7 cells treated with 0.5µM 8x hybrid (E)
MCF-7 cells treated with 1µM 8x. The analysis of cell cycle distribution was performed by using
propidium iodide staining method. With the increase in the concentration of 8v and 8x hybrids
from 0 µM to 1 µM, the cell cycle arrest was noticed at G2/M level indicating the onset of
apoptosis among MCF-7 cells.
9

Table 2. Data obtained from cell cycle study, indicating the percentage of cells distributed in
different phases of cell cycle before (control) and after the addition of 8v and 8x (each 0.5 µM
and 1.0 µM)
S.No.
1.
Conjugate
Control
sub G1
3.04
5.40
2.97
4.61
4.32
G1
S
G2/M
33.65
33.10
37.42
32.46
39.25
45.98
42.32
42.38
46.11
40.12
15.64
17.60
16.72
16.81
16.29
2.
8v (0.5 µM)
8v (1.0 µM)
8x (0.5 µM)
8x (1.0 µM)
3.
4.
5.
2.3. Apoptosis induction studies
2.3.1. Annexin V-FITC
Since the 8v and 8x treated cancer cells have accumulated in G2/M phase of cell cycle, the
effect of conjugates 8v and 8x on induction of apoptosis was further studied by using Annexin-V
FITC/PI (AV/PI) dual staining assay. It is also useful to examine the occurrence of
phosphatidylserine externalization as well as to understand whether it is due to physiological
apoptosis or nonspecific necrosis.33 Phosphatidylserine is a label for cell death-switches from the
inner cell membrane to the outer cell membrane during the cascade of apoptosis.34 Annexin-V is
a cellular protein of the annexin group which selectively binds to phosphatidylserine. In this
study, MCF-7 cells were treated with 8v and 8x for 48 h at 0.5 and 1.0 µM concentrations and
found that these molecules displayed significant apoptosis inducers as evidenced in Fig. 4. The
cells without ligand treatment are considered as control (A, Fig. 4). The results indicated that 8v
and 8x showed 7.46 % and 9.12 % of apoptosis at 0.5 µM concentration. Further on enhancing
the concentration to 1.0 µM, 8v and 8x showed 12.25 % and 13.15 % of apoptosis. However, it
is also suggest that hybrid 8x is a better inducer of apoptosis than 8v. The details of apoptosis
assay were provided in Table 3.
10

Fig. 4. Flow cytometry analysis is performed to determine the percentage of apoptosis induced
among MCF-7 cells before and after 8v and 8x hybrids treatment. The study was done with
Annexin V-FITC/propidium iodide (PI) staining. Annexin V-FITC vs Propidium Iodide plots
(dot plots) obtained from the gated cells show the population of cells corresponding to viable or
non-apoptotic (Annexin V– - PI–), early (Annexin V+ -PI–), and late (Annexin V+ -PI+) apoptotic
cells. In this assay, A) Control; B) 8v (0.5 µM); C) 8v (1.0 µM); D) 8x (0.5 µM); E) 8x (1.0
µM).
Table 3. Data obtained from dot plots indicating the percentage of viable and apoptotic cells
present, before and after treatment of 8v and 8x
Percentage of apoptotic cells
S. No.
Conjugate
Percentage of Viable Cells (LL)
UR
LR
1.
2.
3.
4.
5.
Control
96.18 %
91.24 %
81.27 %
84.85 %
77.29 %
0.06 %
0.81 %
2.50 %
2.43 %
3.69 %
0.09 %
6.65 %
9.75%
6.69 %
9.46 %
8v (0.5 µM)
8v (1.0 µM)
8x (0.5 µM)
8x (1.0 µM)
11

2.3.2. Hoechst staining
Apoptosis is one of the main pathways that lead to cell death in which the fragmented nuclei
with chromatin condensation are known as the common characteristics.35 This study provides the
visual evidence and physical appearance of MCF-7 cells before and after the treatment of these
conjugates. In this study, the cellular DNA was stained with Hoechst 33342 and the effect of 8v
and 8x (each 1.0 µM) on MCF-7 cells were shown in Fig. 5. It is evident from Figure 5 that more
number of wrinkled cells and DNA fragmented cells were observed in case of both 8v and 8x
treatment, indicating that these new conjugates efficiently induced apoptosis in MCF-7 cells.
However, more number of DNA fragmented cells and wrinkled cells were observed in case of
conjugate 8x when compared to 8v, suggesting that the 8x is a potent apoptosis inducing agent.
There is no apoptotic cells observed in control (Fig. 5).
Fig. 5. Hoechst staining assay: This assay was carried out to understand the role of 8x and 8v
hybrids on the cell viability. A) Control (untreated MCF-7 cells); B) MCF-7 cells treated with 8v
(1.0 µM); C) MCF-7 cells treated with 8x (1.0 µM).
2.3.3. Cell viability Assay
Further to confirm the viability of MCF-7 cells, the cell viability assay was also performed.
The cancerous (MCF-7) and non-cancerous (NIH 3T3) cells were treated with 8v and 8x (each
1.0 µM) for 24 h. The amounts of dead cells (which are labeled with propidium iodide staining
and appearing as red florescent spots) were observed in case of 8x treated with MCF7 cells
12

compared with 8v. However, the control sample (normal cells, NIH 3T3) which was either
treated with 8v or 8x and the red florescent spots were not observed much, moreover the blue
fluorescent spots (Hoechst 3342 staining) dominated. These observations suggested the
cytotoxicity of conjugates 8v and 8x against cancer cells (Fig. 6) and also observed that 8x is
more potent than 8v.
Fig. 6. Cell viability Assay: This assay is carried out understand the effect of 8v and 8x
molecules on MCF-7 cells. a) Control (Untreated MCF-7 cells); b) MCF-7 cells treated with 8v
(1.0 µM); c) MCF-7 cells treated with 8x (1.0 µM).
2.4. DNA binding studies
To further confirm the biological behavior of these active conjugates 8v and 8x and to
understand the mode of interaction with DNA, spectroscopic studies such as UV-Visible
spectroscopy, florescence spectroscopy and circular dichroism spectroscopy were carried out.
2.4.1. UV-visible studies
In order to find the nature of binding mode of 8v and 8x with DNA, UV-visibility
spectroscopy study was carried out. However, both these derivatives have shown maximum
absorption bands at around 287 and 369 nm whereas up on addition of equal ratio of CT-DNA
solution, the conjugate 8v absorption peak exhibited gradual hypochromicity with a slight red
13

shift (about 6 nm). While no shift was observed in case of conjugate 8x. It is evident that the
hypochromicity along with bathochromic shift (red shift) is considered as intercalative mode of
binding.36,37. From experimental data, the 8v and 8x derivatives with CT-DNA binding constants
were found to be in the range of ≈ 3 x 105 M-1 in case of 8v and ≈ 1 x 103 M-1 in case of 8x. But,
it is impossible to conclude the nature of binding mode by only UV-visible titration and the
representative UV-visible spectra obtained on interaction of conjugates with CT-DNA was
shown in Fig. 7a.
Fig. 7. Spectroscopic study of the interaction of 8v and 8x with double stranded DNA. a) The
representative UV-visible spectra of 8v and 8x with CT-DNA; b) The representative
fluorescence spectra of 8v and 8x with CT-DNA; c) The representative CD spectra of 8v and 8x
with CT-DNA.
2.4.2. Fluorescence spectroscopy
The nature of interaction of conjugates with DNA can be monitored at low concentration
when they are fluorescent.38 However, the emission spectra of conjugates 8v and 8x show a
prominent peak in the range from 400 nm to 600 nm. The fluorescence emission intensity has
reduced gradually upon equal addition of CT-DNA solution to the conjugates 8v and 8x
solutions. The reduction in fluorescence intensity of 8v and 8x molecules with the addition of
14

CT-DNA, may be due to the absorption of fluorescence emission energy by the DNA bases
which are in the closely located to conjugate molecules. The degree of fluorescence quenching is
comparatively high in case of 8x than 8v. The representative fluorescence spectrum of 8v and 8x
with CT-DNA was shown in Fig. 7b.
2.4.3. Circular dichroism studies
Further, to understand the effect of both 8v and 8x molecules with DNA conformation
performed circular dichroism (CD) studies. The CD spectra of CT-DNA alone exhibited positive
and negative bands at 275 nm and 245 nm respectively. On addition of conjugates 8v and 8x (10
μM) to CT-DNA (at 1:1 ratio), the positive CD band showed hypochromicity, which indicated
unwinding of CT-DNA and this may be hint of interaction with 8v and 8x molecules.39 Further,
on doubling the conjugate concentration (at 1:2 ratio of CT DNA:conjugate), the hypochromicity
of positive band further increased. Hence, CD studies indicate that 8v and 8x conjugates interact
with DNA and bring little changes in DNA conformation. It was observed that, the extent of
change in DNA conformation is more with 8x compared to 8v. Also, the negative CD band
intensities reduced with both 8v and 8x conjugates, however maximum change was noticed with
8x. These observed results emphasize the role of these new molecules in considerably interacting
with the DNA helix and the CD spectra obtained was shown in Fig. 7c.
2.4.4. Viscosity studies
According to the spectroscopic studies, the active conjugates 8v and 8x interact well with
CT-DNA. Further, to conclude the interacting nature of 8v and 8x with CT-DNA, viscosity
studies were also carried out. Intercalation of a molecule results in an increase of the viscosity of
DNA solutions,40 whereas reduction in the relative viscosity is typically observed with covalent
DNA binding molecules.41 Moreover, viscosity does not change or show slight change when
molecules bind to the surface of DNA.42 In the case of active conjugates 8v and 8x, the viscosity
of DNA solution increased with the addition of 8v and 8x molecules. Among both the
conjugates, maximum change in viscosity was observed in case of 8x compared to 8v, while with
the ethidium bromide (EtBr), the viscosity of DNA has increased continuously. This is due to
strong intercalation of EtBr molecules with CT-DNA and the graph plotted between (ŋ/ŋo)1/3 and
conjugate/CT-DNA was shown in Fig. 8. All the spectroscopic results and viscosity study results
15

indicate that the conjugates 8v and 8x interacted well with DNA and the interaction may be
through intercalation between the two stacks of CT-DNA.
Fig. 8. The viscosity studies of EtBr, 8v and 8x with CT-DNA. The effect of 8v and 8x
molecules on the DNA viscosity was studied using Ostwald viscometer.
2.5. Topo I inhibition assay
The topoisomerase-I (topo-I) binds to double stranded supercoiled DNA and cuts the single
stranded portion. This will release the super helical tension in DNA transforming it into relaxed
form. Recently, DNA topo-I inhibitors gained much importance in cancer chemotherapy because
the small molecules which inhibit DNA topo-I activity will leave the strand breaks in DNA that
leads to damage of genome integrity. Since the abundance of topo-I usually high in cancer cells
compared to normal cells, DNA topo-I has been identified as the potential target for the several
anticancer drugs used in clinical studies. The topo-I inhibitors stabilize and accumulate the
DNA-topoisomerase cleavage complexes and inhibit the DNA relegation step, thus resulting
cleavage of DNA strand.43 In the present study, the effect of conjugates 8v and 8x towards
inhibition of DNA topo-I was studied. As discussed above, the 8v and 8x show good interaction
with DNA, they may bind to DNA topoisomerase-I, cleavage complex and interfere with its
religation step.
Cao et al. have recently reported that camptothecin inhibit DNA topo-I at 100 µM
concentration where as simple β-carboline harmine derivatives inhibit DNA topo-I at 150 µM.44
Interestingly, in the present study 8v and 8x has lower IC50 values (approximately about 14 nM)
and considerably high interaction with DNA. Hence, these conjugates were studied for DNA
16

topo-I inhibition assay using pBR 322 plasmid DNA. The conjugates 8v and 8x have shown
significant DNA topo-I inhibition at 10 µM concentration (Fig. 9). The topo-I has completely
converted the super coiled pBR 322 plasmid DNA to open circular form in the absence of 8v and
8x (lane B, Fig. 9). Although, both the conjugates inhibiting DNA topo-I effectively, the 8x is
superior to 8v as shown in Fig. 9 (Lane C-D). Based on these characteristic properties such as
DNA intercalation and DNA topo-I inhibition, they could considered as potential candidates for
anticancer therapy.
Fig. 9. The DNA topo I studies of 8v and 8x using pBR 322 plasmid DNA. Topo I inhibition by
8v and 8x was studied after treating pBR322 plasmid DNA with 1 µM and 10 µM concentration
of each molecule.
2.6. Molecular docking studies
Molecular docking studies were performed to obtain further insight into the binding mode of
the conjugates 8v and 8x with the DNA topo-I ternary complex. The docking poses showed that
both these conjugates intercalate with DNA at the topo-I binding site of the ternary complex. The
docking poses for 8v and 8x show the β-carboline ring stacked with TGP11, the C3-substituent
was oriented towards cleaved DNA strand and the C1-substitution lying in the minor groove
(Fig. 10). In 8x, the hydroxyl group of the phenyl in the cinnamide part forms hydrogen-bonding
with both Asp440 and Lys751. Additionally, they hold hydrophobic interactions with the amino
acid residues around the binding site: Arg364, Glu365, Lys425, Asp533, Ile535 and Asn722.
These studies indicate that the synthesized conjugates 8v and 8x are capable of binding
efficiently in the active site of the DNA topo-I ternary complex.
17

Fig. 10. Binding poses of A) 8v and B) 8x in the active site of DNA topo-I.
Additionally, docking studies were performed to obtain a better insight into the binding
mode of these conjugates to the DNA. Docking studies showed that all the conjugates bind to the
DNA through intercalation. The docking poses of 8v and 8x show that the β-carboline ring
stacked between the G–C base pairs of the DNA intercalation site, while the cinnamide part at
the C3-substitution extended towards the minor groove and forms side wise π–π stacking with
the base pairs in the minor groove (Fig. 11). The phenyl ring at C1 forms π–π stacking while the
oxygen in the amide forms hydrogen-bonding with the dG12 residue. Further, in the case of 8x,
the hydroxyl group of the phenyl in the cinnamide part forms hydrogen-bonding with the side
chain of dC13. Docking studies on DNA topo-I and CT-DNA showed that these conjugates have
the potential to bind to the DNA topo-I and also intercalate with DNA.
Fig. 11. Binding poses of A) 8v and B) 8x in the intercalation mode with DNA.
18

3. Conclusion
In conclusion, we have synthesized a series of new C3-trans-cinnamide linked β-carboline
conjugates possessing a substituted phenyl at the position-1 and a trans-cinnamide moiety at
position-3 of the β-carboline moiety. These conjugates have exhibited promising cytotoxicity as
well as significant DNA intercalation, among which, two conjugates 8v and 8x were found to be
most active. In addition, the role of these active conjugates as DNA topo-I inhibitors was also
studied. Further, docking and DNA binding studies of these conjugates confirmed that DNA
topo-I inhibition was mainly due to the formation of a ternary complex with DNA and topo-I.
Consequently, all these studies are important to understand the role of different pharmacophores
conjugated to the naturally occurring β-carboline ring system that possess bioactivities such as
cytotoxicity, apoptosis induction and inhibition of DNA topo-I which play a vital role in cancer
chemotherapy.
4. Experimental Section
4.1. Materials and methods
Chemical reagents were purchased from Sigma-Aldrich and used without further purification.
All the solvents were of commercial grade and were purified prior to use when necessary. 1H and
13C NMR experiments were performed on Avance (300 MHz, 400 MHz and 500 MHz)
spectrometers. Chemical shifts are measured relative to an internal standard tetramethylilane
(TM) set to δ 0 ppm and are reported in parts per million (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). TLC analyses were
performed with silica gel plates (0.25 mm, E. Merck, 60 F254) using iodine, KMnO4, and a UV
lamp for visualization. Specific rotations were measured on a Perkin-Elmer 341MC polarimeter.
Mass spectra were recorded by electron spray ionization mass spectrometry (ESI-MS). HRMS
was performed on a Varian QFT-ESI instrument. Melting points were determined on an
electrothermal melting point apparatus and are uncorrected. IR spectra were measured on Bruker
FT-IR Equinox 55 and Bruker TENSOR 27 instruments.
19

4.2. General reaction procedure for the preparation of compounds 8a‒x
To a solution of amine intermediates (7a‒c,1 mmol) and appropriate trans-cinnamic acid (1.1
mmol) in dry DMF (10 mL) was added HBTU (1.2 mmol) and TEA (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 x 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 20% EtOAc/n-hexane,
collected fractions were evaporated in vacuo to afford the desired products 8a‒x.
4.2.1. (E)-N-((1-(3-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (8a)
Yellow solid, 149 mg (85% yield); mp: 148‒150 oC; 1H NMR (300 MHz, CDCl3 + DMSO‒d6) δ
(ppm): 10.90 (s, 1H), 8.15‒8.08 (m, 1H), 8.02‒7.77 (m, 4H), 7.65‒7.43 (m, 4H), 7.29‒7.16 (m,
2H), 6.80 (s, 2H), 6.64 (d, J = 15.6 Hz, 1H), 4.88 (d, J = 5.4 Hz, 2H), 3.89 (s, 6H), 3.84 (s, 3H);
13C NMR (125 MHz, CDCl3 + DMSO‒d6) δ (ppm): 165.66, 163.84, 161.90, 153.49, 147.47,
142.07, 140.99, 140.00, 139.51, 139.11, 132.55, 131.09, 130.90, 128.71, 124.92, 122.06, 121.93,
121.15, 119.97, 115.79, 112.86, 112.23, 107.96, 105.37, 60.52, 56.29, 45.27; ESI-MS: m/z
HRMS calculated for C30 H27O4N8F 512.19618 [M+H]+, found 512.19801.
4.2.2. (E)-3-(3,4-Dichlorophenyl)-N-((1-(3-fluorophenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8b)
o
1
Yellow solid, 118 mg (70% yield); mp: 255‒258 C; H NMR (500 MHz, DMSO‒d6) δ (ppm):
11.56 (s, 0.7H), 8.76 (s, 1H), 8.26 (d, J = 7.6 Hz, 0.7H), 8.10 (s, 0.7H), 7.94‒7.81 (m, 3H),
7.70‒7.62 (m, 3H), 7.61‒7.53 (m, 2H), 7.49 (d, J = 15.8 Hz, 1H), 7.40‒7.34 (m, 1H), 7.25 (t, J =
7.0 Hz, 1H), 6.89 (d, J = 15.7 Hz, 1H), 4.72 (s, 2H), peaks at 11.70, 8.43 and 8.17 are due to the
30% minor rotamer; 13C NMR (125 MHz, DMSO‒d6) δ (ppm): 141.0, 140.9, 136.7, 136.4,
133.0, 132.5, 131.1, 132.0, 131.5, 130.9, 129.9, 128.8, 127.7, 125.0, 124.9, 124.3, 122.4, 122.1,
121.7, 121.1, 120.0, 115.6, 115.4, 114.5, 112.9, 112.4, 45.2; ESI-MS: m/z HRMS calculated for
C27H19FN3O2Cl2 490.08687 [M+H ]+, found 490.08731.
20

4.2.3. (E)-3-(4-Fluorophenyl)-N-((1-(3-fluorophenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8c)
o
1
Pale yellow solid, 109 mg (73% yield); mp: 258‒264 C; H NMR (500 MHz, DMSO‒d6) δ
(ppm): 11.56 (s, 0.7H), 8.74 (s, 1H), 8.27 (d, J = 7.7 Hz, 0.7H), 8.10 (s, .07H), 7.94‒7.88 (m,
1H), 7.87‒7.82 (m, 1H), 7.69‒7.62 (m, 4H), 7.58‒7.49 (m, 2H), 7.39‒7.34 (m, 1H), 7.29‒7.23
(m, 3H), 6.77 (d, J = 15.7 Hz, 1H), 4.72 (d, J = 4.8 Hz, 2H), peaks at 11.70, 8.44 and 8.16 are
due to 30% minor rotamer; 13C NMR (125 MHz, DMSO‒d6) δ (ppm): 165.4, 164.1, 163.8,
162.1, 161.9, 147.4, 142.0, 140.0, 138.1, 131.1, 130.9, 130.1, 130.2, 128.8, 124.9, 122.6, 122.1,
121.8, 115.6, 114.4, 112.9, 112.7, 112.4, 45.2; ESI-MS: m/z HRMS calculated for C27H20ON8F2
440.15474 [M+H]+, found 440.15690.
4.2.4. (E)-3-(3,5-Difluorophenyl)-N-((1-(3-fluorophenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8d)
Yellow solid, 117 mg (75% yield); mp: 215‒220 oC; 1H NMR (300 MHz, DMSO−d6 + CDCl3) δ
(ppm): 10.95 (s, 0.7H), 8.30 (t, J = 5.3 Hz, 1H), 8.14−8.05 (m, 1H), 7.99 (s, 1H), 7.96−7.75 (m,
m, 3H), 7.65−7.43 (m, 3H), 7.28−7.15 (m, 2H), 7.06 (d, J = 6.4 Hz, 2H), 6.84−6.75 (m, 2H),
13
4.87 (d, J = 5.4 Hz, 2H), peak at 11.15 is due to the 30% minor rotamer; C NMR (75 MHz,
DMSO−d6 + CDCl3) δ (ppm): 164.5, 163.8, 163.6, 160.4, 145.4, 141, 139.3, 137.8, 136.6, 131.8,
130.1, 129.4, 127.5, 123.7, 120.6, 120.3, 120, 118.8, 114.8, 114.5, 114.2, 111.5, 109.3, 103.8,
103.5, 103.1, 44.5; ESI-MS: m/z HRMS calculated for C27 H19ON8F3 458.14559 [M+H]+, found
458.14747.
4.2.5. (E)-3-(2,5-Dimethoxyphenyl)-N-((1-(3-fluorophenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8e)
Yellow solid, 130 mg (79% yield); mp: 228‒230 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.87 (s, 0.8H), 8.10 (d, J = 7.7 Hz, 1H), 800‒7.78 (m, 5H), 7.65‒7.42 (m, 3H),
7.28‒7.15 (m, 2H), 7.07 (s, 1H), 6.86 (s, 2H), 6.75 (d, J = 15.7 Hz, 1H), 4.88 (d, J = 5.4 Hz, 2H),
13
3.83 (s, 3H), 3.78 (s, 3H), peaks at 11.08 and 8.06 are due to the 20% minor rotamer; C NMR
(75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 164.8, 163.0, 152, 150.9, 145.9, 145.5, 140.5,
138,133.2, 131.2, 129.5, 129.0, 128.9, 126.8, 123.1, 121.3, 120.0, 119.7, 118.2, 114.3, 113.9,
113.6, 111.6, 111.1, 110.6, 54.7, 54.2, 43.9; ESI-MS: m/z HRMS calculated for C29H25O3N8F
482.18536 [M+H]+, found 482.18504.
21

4.2.6. (E)-3-(4-Chlorophenyl)-N-((1-(3-fluorophenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8f)
1
Yellow solid, 117 mg (75% yield); mp: 248‒250 oC; H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 11.02 (s, 1H), 8.33‒8.20 (m, 1H), 8.14‒8.06 (m, 1H), 8.02‒7.94 (m, 1H), 7.93‒7.77 (m,
2H), 7.66‒7.45 (m, 6H), 7.34 (d, J = 8.3 Hz, 2H), 7.27‒7.15 (m, 2H), 6.74 (d, J =15.6 Hz, 1H),
4.86 (d, J = 5.2 Hz, 2H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 163.7, 159.4, 145.2,
140.2, 139.2, 136.2, 132.7, 132.2, 130.8, 129.1, 128.6, 127.4, 127.2, 126.5, 122.6, 121.2, 119.7,
119.3, 113.9, 113.4, 113.6, 112.2, 110.8, 110.2, 43.5; ESI-MS: m/z HRMS calculated for
C27H20ON3ClF 456.12541 [M+H]+, found 456.12734.
4.2.7. (E)-N-((1-(3-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (8g)
Yellow solid, 109 mg (68% yield); mp: 205‒210oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.80 (s, 1H), 8.19‒8.04 (m, 1H), 8.03‒7.93 (m, 1H), 7.92‒7.73 (m, 3H), 7.65‒7.48 (m,
5H), 7.31‒7.14 (m, 2H), 7.08‒6.98 (m, 1H), 6.91‒6.83 (m, 1H), 6.52 (d, J = 15.6 Hz, 1H), 4.86
(s, 2H), 4.70 (s, 1H), 3.90 (s, 3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 165.3,
147.2, 146.7, 145.7, 140.7, 139, 131.5, 129.8, 129.6, 129.3, 129.2, 127.1, 125.7, 123.3, 123.1,
120.8, 120.2, 118.4, 117.6, 114.5, 114.2, 113.9, 111.5, 111.3, 110.8, 109.3, 54.7, 44.1; ESI-MS:
m/z HRMS calculated for C30H27O4N8F 468.16518 [M+H]+, found 468.16801.
4.2.8. (E)-N-((1-(3-Fluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)-3-(3-
hydroxyphenyl)acrylamide (8h)
o
Yellow solid, 97 mg (65% yield); mp: 142‒148 C;1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 11.01 (s, 1H), 8.29‒8.19 (m, 1H), 8.16‒8.05 (m, 1H), 8.01‒7.94 (m, 1H), 7.92‒7.77 (m,
2H), 7.66‒7.48 (m, 3H), 7.29‒7.13 (m, 4H), 7.07‒6.96 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H), 6.69
13
(d, J = 15.6 Hz, 1H), 4.86 (d, J = 5.0 Hz, 2H), 4.69 (s, 1H); C NMR (75 MHz, DMSO‒d6 +
CDCl3) δ (ppm): 165.2, 156.9, 145.7, 142.8, 140.9, 139.1, 135.4, 131.7, 130, 129.7, 128.7, 127.3,
123.5, 120.4, 119.9, 118.6, 118.1, 115.9, 114.7, 114.4, 114.1, 113.3, 112.7, 111.4, 111.1, 111.7,
44.3; ESI-MS: m/z HRMS calculated for C27H21N3O2F 438.15969 [M+H]+, found 438.16123.
22

4.2.9. (E)-3-(3,4,5-Trimethoxyphenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-
b]indol-3-yl)methyl)acrylamide (8i)
Yellow solid, 131 mg (82% yield); 165‒170 oC; 1H NMR (500 MHz, CDCl3) δ (ppm): 8.54 (bs,
1H), 8.16 (d, J = 7.7 Hz, 1H), 7.98 (s, 1H), 7.63‒7.50 (m, 3H), 7.32 (t, J = 7.1 Hz, 1H), 7.14 (s,
2H), 6.94 (s, 1H), 6.74 (s, 2H), 6.44 (d, J = 15.5 Hz, 1H), 4.92 (d, J = 5.1 Hz, 2H), 3.96 (s, 6H),
3.95 (s, 3H), 3.88 (s, 6H), 3.87 (s, 3H), peaks at 8.04, 7.48, 4.02 and 4.00 are due to 10% minor
13
rotamer; C NMR (125 MHz, DMSO‒d6 + CDCl3) δ (ppm): 164.9, 160.1, 152.1, 145.6, 140.8,
138.8, 131.6, 129.8, 129.3, 129.2, 127.3, 127.2, 123.4, 120.3, 120.2, 120.2, 118.5, 114.3, 114.0,
112.6, 111.4, 111.0, 103.9, 59.6, 55.0, 44.2; ESI-MS: m/z HRMS calculated for C33H34O7N3
584.23803 [M+H]+, found 584.23913.
4.2.10. (E)-3-(3,4-Dichlorophenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8j)
1
Yellow solid, 130 mg (84% yield); 165‒170oC; H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 11.0 (s, 1H), 8.17‒8.0 (m, 2H), 7.96 (s, 1H), 7.65‒7.57 (m, 3H), 7.55‒7.44 (m, 4H),
7.41‒7.36 (m, 1H), 7.27‒7.19 (t, J = 7.9 Hz, 1H), 6.76 (d, J = 15.6 Hz, 1H), 4.89 (d, J = 5.4 Hz,
2H), 4.01 (s, 6H), 3.92 (m, 3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 164.1,
152.0, 145.1, 140.6, 136.9, 138.1, 136.0, 135.7, 134.4, 133.4, 132.9, 131.3, 129.5, 129.3, 127.9,
126.8, 125.8, 122.9, 120.1, 119.9, 118.2, 111.2, 110.0, 104.8, 59.4, 54.9, 44.0; ESI-MS: m/z
HRMS calculated for C30H26O4N3Cl2 562.12828 [M+H]+, found 562.12949.
4.2.11. (E)-3-(4-Fluorophenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8k)
Yellow solid, 123 mg (88% yield); mp: 214‒218 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.99 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 8.01 (t, J = 7.3 Hz, 1H), 7.97 (s, 1H), 7.67‒7.45
(m, 6H), 7.28‒7.20 (m, 2H), 7.07 (t, J = 8.6 Hz, 2H), 6.65 (d, J = 15.6 Hz, 1H), 4.89 (d, J = 5.4
13
Hz, 2H), 4.00 (s, 6H), 3.92 (s, 3H); C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 165.3,
164.1, 160.8, 152.7, 145.5, 141.3, 141.1, 138.7, 137.5, 133.4, 132.0, 130.7, 130.0, 128.7, 127.5,
120.7, 118.8, 114.9, 111.7, 110.8, 105.2, 120.5, 60.0, 55.5, 44.5; ESI-MS: m/z HRMS calculated
for C30H27O4N8F 512.19610 [M+H]+, found 512.19801.
23

4.2.12. (E)-3-(3,5-Difluorophenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8l)
Yellow solid, 113 mg (78% yield); mp: 228‒230 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.68 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.96 (s, 1H), 7.80 (t, J =4.9 Hz, 1H), 7.64‒7.48
(m, 3H), 7.40 (s, 2H), 7.25 (t, J = 6.6 Hz, 1H), 7.04 (d, J = 6.4 Hz, 2H), 6.84‒6.75 (m, 1H), 6.68
13
(d, J = 15.6 Hz, 1H), 4.91 (d, J = 5.4 Hz, 2H), 4.00 (s, 6H), 3.94 (s, 3H); C NMR (125 MHz,
DMSO‒d6 + CDCl3) δ (ppm): 165.3, 164.1, 162.0, 146.1, 141.8, 138.7, 138.2, 137.5, 134.2,
132.7, 130.6, 128.1, 124.6, 121.4, 121.2, 119.5, 112.4, 110.4, 110.2, 105.9, 104.3, 60.8, 56.2,
45.3, 40.2; ESI-MS: m/z HRMS calculated for C30H26O4N8F2 530.18649 [M+H]+, found
530.18859.
4.2.13. (E)-3-(2,5-Dimethoxyphenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-
3-yl)methyl)acrylamide (8m)
1
Yellow solid, 123 mg (81% yield); mp: 148‒150 oC; H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.66 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.98 (s, 1H), 7.90 (d, J = 15.8 Hz, 1H), 7.63‒7.42
(m, 3H), 7.29‒7.22 (m, 3H), 7.05 (s, 1H), 6.86 (s, 2H), 6.69 (d, J = 15.8 Hz, 1H), 4.90 (d, J = 5.2
Hz, 2H), 4.00 (s, 6H), 3.93 (3H), 3.83 (s, 3H), 3.78 (s, 3H); 13C NMR (75 MHz, DMSO‒d6 +
CDCl3) δ (ppm): 165.5, 158.5, 152.4, 151.4, 145.3, 141.0, 140.6, 137.2, 134.1, 133.0, 131.7,
129.8, 127.3, 123.6, 121.6, 120.5, 120.2, 118.6, 114.8, 114.4, 112.3, 111.5, 110.5, 105, 59.8,
55.2, 54.7, 44.1; ESI-MS: m/z HRMS calculated for C32H32O6N3 554.22685 [M+H]+, found
554.22856.
4.2.14. (E)-3-(4-Chlorophenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8n)
Yellow solid, 116 mg (80% yield); mp:148‒150 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 11.00 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 8.08‒8.01 (m, 1H), 7.97 (s, 1H), 7.66‒7.57 (m,
2H), 7.56‒7.45 (m, 3H), 7.34 (d, J = 8.5 Hz, 2H), 7.30‒7.20 (m, 3H), 6.71 (d, J = 15.8 Hz, 1H),
4.89 (d, J = 5.2 Hz, 2H), 4.00 (s, 6H), 3.92 (s, 3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 164.9, 152.4, 145.2, 141.0, 140.7, 137.5, 137.2, 137.0, 133.8, 133.0 132.8, 131.7, 129.7,
128.0, 127.3, 121.5, 120.5, 120.2, 118.6, 111.5, 110.5, 105.0, , 59.7, 55.2, 44.2; ESI-MS: m/z
HRMS calculated for C30H27O4N3Cl 528.16718 [M+H]+, found 528.16846.
24

4.2.15. (E)-3-(4-Hydroxy-3-methoxyphenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-
b]indol-3-yl)methyl)acrylamide (8o)
Yellow solid, 109 mg (74% yield); mp: 175‒178 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.53 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.96 (s, 1H), 7.65‒7.48 (m, 4H), 7.37 (s, 1H),
7.35 (m, 4H), 7.07‒6.99 (m, 2H), 6.87 (d, J = 8.6 Hz, 1H), 6.45 (d, J = 15.4 Hz, 1H), 4.90 (s,
2H), 4.00 (s, 6H), 3.93 (s, 3H), 3.89 (s, 3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm):
166.6, 153.5, 148.5, 146.3, 142.0, 140.3, 138.2, 133.8, 133.8, 132.6, 128.3, 126.8, 122.0, 121.5,
121.1, 119.6, 118.6, 115.7, 112.5, 111.5, 110.5, 106.1, 60.7, 56.2, 55.9; ESI-MS: m/z HRMS
calculated for C31H29O6N3 540.20564 [M+H]+, found 540.21173.
4.2.16. (E)-3-(3-Hydroxyphenyl)-N-((1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8p)
o
Yellow solid, 98 mg (70% yield); mp: 175‒178 C; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.79 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.96 (s, 1H), 7.71‒7.58 (m, 3H), 7.57‒7.40 (m,
4H), 7.30‒7.14 (m, 4H), 7.11‒6.96 (m, 2H), 6.84 (d, J = 9.4 Hz, 1H), 6.61 (d, J = 15.6 Hz, 1H),
4.90 (d, J = 5.2 Hz, 2H), 4.71 (s, 1H), 4.00 (s, 6H), 3.92 (s, 3H); 13C NMR (75 MHz, DMSO‒d6
+ CDCl3) δ (ppm): 165.0, 156.6, 152.2, 146, 145.4, 140.7, 138.9, 135.2, 133.1, 132.6, 131.5,
126.9, 129.4, 120.5, 120.3, 120.1, 118.3, 117.8, 115.7, 114.5, 113.1, 111.3, 110.1, 104.8, 59.5,
55.0, 44.1; ESI-MS: HRMS calculated for C30H28O5N3 [M+H]+ 510.20051, found 510.20235.
4.2.17. (E)-N-((1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (8q)
o
Yellow solid, 138 mg (80% yield); mp: 148‒150 C;1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.22 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.92 (s, 1H), 7.62‒7.49
(m, 4H), 7.25 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.7 Hz, 2H), 6.76 (s, 2H), 6.55 (d, J = 15.4 Hz,
1H), 4.89 (d, J = 5.1 Hz, 2H), 3.90 (s, 3H), 3.88 (s, 6H), 3.86 (s, 3H); 13C NMR (75 MHz,
DMSO‒d6 + CDCl3) δ (ppm): 165.2, 159.2, 152.4, 145.2, 140.8, 140.0, 139.2, 138.3, 131.7,
130.0, 129.8, 129.2, 127.4, 124.9, 120.6, 120.4, 120.3, 118.7, 113.4, 111.6, 110.4, 104.1, 55.9,
55.2, 54.6, 44.4; ESI-MS: m/z HRMS calculated for C31H30O5N3 524.21800 [M + H]+, found
524.21613.
25

4.2.18. (E)-3-(3,4-Dichlorophenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8r)
Yellow solid, 137 mg (83% yield); mp: 188‒190 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.01 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 8.02‒7.71 (m, 4H), 7.63‒7.48 (m, 4H), 7.47‒7.25
(m, 3H), 7.11 (d, J = 8.4 Hz, 2H), 6.61 (d, J = 15.6 Hz, 1H), 4.88 (d, J = 4.9 Hz, 2H), 3.90 (s,
3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 164.4, 159.1, 145.2, 141.3, 140.9, 136.4,
134.7, 133.3, 131.9, 131.8, 130.3, 129.9, 129.7, 128.3, 127.3, 126.2, 123.0, 120.6, 120.5, 118.7,
113.4, 111.5, 110.4, 110.1, 54.6, 44.6; ESI-MS: m/z HRMS calculated for C28H22O2N3Cl2
502.10836 [M+H]+, found 502.10691.
4.2.19. (E)-3-(4-Fluorophenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8s)
Yellow solid, 126 mg (85% yield); mp: 145‒148 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.84 (s, 1H), 8.19 (t, J = 5.0 Hz, 1H), 8.11 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 8.5 Hz, 2H),
7.94 (s, 1H), 7.64‒7.60 (m 2H), 7.58‒7.47 (m, 3H), 7.23 (t, J = 7.5 Hz, 1H), 7.15‒7.03 (m, 4H),
6.67 (d, J = 15.6 Hz, 1H), 4.85 (d, J = 5.4 Hz, 2H), 3.92 (s, 3H); 13C NMR (75 MHz, DMSO‒d6
+ CDCl3) δ (ppm): 163.8, 159.5, 158.1, 144.9, 140.0, 139.7, 136.3, 130.5, 129.8, 129.3, 128.5,
128.2, 127.8, 126.2, 120.3, 119.6, 119.3, 117.6, 113.9, 112.3, 110.8, 109.1, 53.6, 43.5; ESI-MS:
m/z HRMS calculated for C28H28FO2N3 451.16961 [M+H]+, found 452.17471.
4.2.20. (E)-3-(3,5-Difluorophenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8t)
Yellow solid, 136 mg (88% yield); mp: 230‒232 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.92 (s, 1H), 8.43 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.93 (s,
1H), 7.62 (d, J = 8.3 Hz, 1H), 7.50 (t, J = 6.6 Hz, 2H), 7.23 (t, J = 7.5 Hz, 1H), 7.15‒7.06 (m,
4H), 6.81 (d, J = 15.4 Hz, 2H), 4.84 (d, J = 5.4 Hz, 2H), 3.91 (s, 3H); 13C NMR (75 MHz,
DMSO‒d6 + CDCl3) δ (ppm): 164.8, 158.8, 145.1, 140.7, 136.2, 131.5, 130.1, 129.3, 128.9,
126.9, 123.7, 120.3, 120.2, 118.4, 113.1, 111.3, 110.0, 109.4, 109.2, 109.1, 103.2, 54.3, 44.3;
ESI-MS: m/z HRMS calculated for C28H22O2N8F2 470.16746 [M+H]+, found 470.16570.
26

4.2.21. (E)-3-(2,5-Dimethoxyphenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8u)
Yellow solid, 146 mg (90% yield); mp: 130‒135 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.58 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.96 (s, 1H), 7.90 (d, J =
15.8 Hz, 1H), 7.76 (t, J = 5.3, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.53 (t. J = 6.9 Hz, 1H), 7.26 (t, J =
7.5 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 6.86 (s, 2H), 6.71 (d, J = 15.8 Hz, 1H), 4.87
13
(d, J = 5.4 Hz, 2H), 3.91 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H); C NMR (75 MHz, DMSO‒d6 +
CDCl3) δ (ppm): 166.1, 159.6, 152.8, 151.9, 144.9, 141.4, 140.8, 134.9, 131.9, 130.4, 129.5,
129.4, 127.9, 124.0, 121.6, 121.0, 120.6, 119.2, 115.3, 113.8, 112.9, 111.8, 111.9, 111.0, 55.5,
55.1, 54.8, 44.3; ESI-MS: m/z HRMS calculated for C30H28O4N3 494.20743 [M+H]+, found
494.20549.
4.2.22. (E)-3-(4-Chlorophenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8v)
Yellow solid, 127 mg (83% yield); mp: 140‒145 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 11.18 (s, 1H), 8.60 (t, J = 5.4 Hz, 1H), 8.11 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 8.7 Hz, 2H),
7.92 (d, J = 3.2 Hz, 2H), 7.63 (d, J = 8.3 Hz, 1H), 7.57 (m, 3H), 7.36 (d, J = 8.5 Hz, 2H), 7.21 (t,
J = 7.4 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 15.8 Hz, 1H), 4.80 (d, J = 5.6 Hz, 2H),
3.90 (s, 3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 164.9, 159.4, 146.2, 141.3,
141.1, 137.5, 133.9, 133.5, 131.8, 130.6, 129.8, 129.5, 128.7, 128.5, 127.5, 122.6, 120.9, 120.7,
118.9, 113.6, 112.1, 110.4, 54.9, 4.8; ESI-MS: m/z HRMS calculated for C28H23O2N3 Cl
468.14733 [M+H]+, found 468.14598.
4.2.23. (E)-3-(4-Hydroxy-3-methoxyphenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-
b]indol-3-yl)methyl)acrylamide (8w)
Yellow solid, 121 mg (77% yield); mp: 215‒220 oC; 1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.53 (s, 1H), 8.09 (d, J = 7.5 Hz, 1H), 8.00 (d, J = 8.7 Hz, 2H), 7.94‒7.87 (m, 2H),
7.74‒7.45 (m, 4H), 7.28‒7.17 (m, 1H), 7.21 (d, J = 8.5 Hz, 2H), 7.06‒6.98 (m, 1H), 6.86 (d, J =
7.9 Hz, 1H), 6.50 (d, J = 15.6 Hz, 1H), 4.86 (d, J = 4.9 Hz, 2H), 4.65 (s, 1H), 3.90 (s, 3H), 3.89
13
(s, 3H); C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 165.7, 159.2, 147.5, 146.9, 145.5,
140.9, 139.6, 131.8, 130.4, 129.7, 129.2, 128.9, 127.3, 126.2, 121.3, 120.7, 120.6, 118.7, 117.8,
27

113.5, 111.6, 110.4, 109.6, 55.1, 54.6, 44.5; ESI-MS: m/z HRMS calculated for C29H26O4N3
480.19178 [M+H]+, found 480.19010.
4.2.24. (E)-3-(3-Hydroxyphenyl)-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-
yl)methyl)acrylamide (8x)
Yellow solid, 103 mg (70% yield); mp: 165‒170oC;1H NMR (300 MHz, DMSO‒d6 + CDCl3) δ
(ppm): 10.69 (s, 1H), 9.12 (bs, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.04‒7.92 (m, 4H), 7.64‒7.48 (m,
5H), 7.28‒7.07 (m, 3H), 7.02‒6.97 (m, 2H), 6.83 (d, J = 9.4 Hz, 1H), 6.64 (d, J = 15.5 Hz, 1H),
4.86 (d, J = 5.1 Hz, 2H), 3.90 (s, 3H); 13C NMR (75 MHz, DMSO‒d6 + CDCl3) δ (ppm): 165.2,
158.9, 156.7, 145.2, 140.9, 140.5, 139.1, 135.4, 131.5, 129.8, 129.6, 129.0, 128.7, 127.1, 120.5,
120.4, 120.2, 118.5, 118.0, 115.9, 113.3, 113.1, 111.4, 110.2, 54.4, 44.2; ESI-MS: m/z HRMS
calculated for C28H23O3N3 450.18122 [M+H]+, found 450.17918.
4.2.25. (E)-N-((1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)but-2-enamide (10)
1
Faint yellow solid, 80 mg (80% yield); mp: 190‒200 oC; H NMR (500 MHz, DMSO) δ (ppm):
11.40 (s, 1H), 8.50 (t, J = 5.5 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.99 (d, J = 5.6 Hz, 2H), 7.94 (s,
1H), 7.63 (d, J = 8.1 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.24 (t, J = 7.4 Hz, 1H), 7.18 (d, J = 8.4
Hz, 2H), 6.70 (dd, J = 13.5, 6.7 Hz, 1H), 6.08 (dd, J = 15.3, 1.5 Hz, 1H), 4.63 (d, J = 5.8 Hz,
2H), 3.88 (s, 3H), 1.86 (t, d = 10.0 Hz, 3H); 13C NMR (125 MHz, DMSO) δ (ppm): 165.4, 160.1,
147.5, 141.9, 141.5, 138.2, 132.2, 131.2, 130.3, 130.2, 128.4, 126.5, 121.9, 121.3, 119.8, 114.5,
112.8, 111.0, 55.8, 55.3, 45.0, 17.8; ESI-MS: m/z HRMS calculated for C23H21O2N3 372.1712
[M+H]+, found 372.1724.
4.2.26. 3-Chloro-N-((1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl)propanamide
1
(12) Faint yellow solid, 77 mg (77% yield); mp: 180‒185 oC; H NMR (500 MHz, DMSO) δ
(ppm): 11.41 (s, 1H), 8.66 (t, J = 5.8 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.09 – 7.99 (m, 2H), 7.97
(s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.28 – 7.22 (m, 1H), 7.20 – 7.10 (m, 2H),
4.60 (d, J = 5.8 Hz, 2H), 3.91 (t, J = 6.3 Hz, 2H), 3.88 (s, 3H), 2.77 (t, J = 8.5 Hz, 2H); 13C NMR
(125 MHz, DMSO) δ (ppm): 169.5, 160.1, 147.3, 141.9, 141.5, 132.2, 131.2, 130.4, 130.2,
128.5, 121.7, 121.3, 119.8, 114.5, 112.9, 110.7, 55.8, 45.1, 41.8, 38.9; ESI-MS: m/z HRMS
calculated for C22H20O2N3Cl 394.1322 [M+H]+, found 394.1325.
4.2.27. (E)-N-allyl-3-(3-hydroxyphenyl)acrylamide (15) Cream solid, 84 mg (84% yield); mp:
150‒155 oC; 1H NMR (500 MHz, DMSO) δ (ppm) 9.57 (s, 1H), 8.26 (t, J = 5.6 Hz, 1H), 7.34 (d,
J = 15.8 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.6 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.78
28

(dd, J = 8.1, 1.7 Hz, 1H), 6.59 (d, J = 15.8 Hz, 1H), 5.86 (ddt, J = 17.1, 10.4, 5.3 Hz, 1H), 5.18
(dq, J = 17.2, 1.7 Hz, 1H), 5.09 (ddd, J = 10.3, 3.1, 1.5 Hz, 1H), 3.88 – 3.78 (m, 2H); 13C NMR
(126 MHz, DMSO) δ (ppm): 165.2, 158.1, 139.4, 136.6, 135.7, 130.3, 122.3, 119.1, 117.1,
115.7, 114.1, 41.5; ESI-MS: m/z HRMS calculated for C12H13O2N 204.1025 [M+H]+, found
204.1029.
4.3. Materials and methods of biological studies
4.3.1. MTT assay
The anticancer activities of the complexes were determined using MTT assay.45,46 Cancer cells
(DU-145, MCF-7 HeLa and A549) were used in this assay. 1 x 104 cells/well were seeded in 200
µl Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS in each well of
o
96-well microculture plates and incubated for 24 h at 37 C in a CO2 incubator. All the
derivatives diluted to the desired concentrations (500 nM, 1 µM, 5 µM,10 µM, 25 µM, 50 µM,
75 µM, 100 µM, 150 µM) in culture medium, were added to the wells with respective vehicle
control. Doxorubicin treated cells, in the same concentration range were used as control. After 48
h of incubation, 10 µl MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5
mg/ml) was added to each well and the plates were further incubated for 4 h. Then the
supernatant from each well was carefully removed, formazan crystals were dissolved in 100 µl of
DMSO and absorbance at 570 nm wavelengths was recorded at a wavelength of 540 nm using an
EL x 800 microplate reader (BioTek, USA).
4.3.2. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells through
the cell cycle phases. MCF-7 cells were incubated with hybrids 8v and 8x at 0.5 and 1 µM
concentrations (at their IC50 concentration) for 48 h whereas, doxorubicin was used as a
reference. Untreated and treated cells were harvested, washed with PBS, fixed in cold ethanol
(70%) and stained with propidium iodide (Sigma Aldrich). Cell cycle was performed by flow
cytometry (Becton Dickinson FACS Caliber; Szumilak et al., 2010).47
4.3.3. Confocal studies
Cells were seeded at a density of 10,000 cells over 18-mm cover slips and incubated for 24 h.
After incubation, cells were treated with the conjugates 8v and 8x at 1 µM concentration for 48
h. Hoechst 33258 (Sigma Aldrich) was added to the cells at a concentration of 0.5 mg/mL and
29