Synthesis and in vitro cytotoxicity evaluation of β-carboline-linked 2,4-thiazolidinedione hybrids: potential DNA intercalation and apoptosis-inducing studies

Article abstract of DOI:10.1039/c8nj03248c

A series of new β-carboline-thiazolidinedione hybrids was synthesized and assessed for in vitro cytotoxicity potential against selected human cancer cell lines, namely, PC-3, A549, MG-63, HCT-15, MDA-MB-231, A431, and PANC-1 along with a normal human cell line (L-132). Among this new series, compound 19e was found to exhibit promising cytotoxic effects against triple negative breast cancer cell line (MDA-MB-231) with IC₅₀ value of 0.97 ± 0.13 μM. Hence, further mechanistic studies of the apoptosis-inducing effect of 19e were conducted on the MDA-MB-23 cell line. Moreover, characteristic apoptotic features such as membrane blebbing, chromatin condensation, and apoptotic body formation were observed with the effect of 19e on MDA-MB-231 cells using AO/EB and DAPI staining. The Annexin V-Alexa Flour 488/PI assay confirmed significant early apoptosis induction. Notably, DCFDA assay indicated that 19e induced ROS generation. Moreover, mitochondrial membrane potential collapse was observed through JC-1 staining by 19e. Furthermore, cell cycle analysis revealed that 19e arrested cells in the sub G1 phase. In addition, clonogenic and wound healing assays indicated inhibition of colony formation and cell migration by 19e in a dose-dependent manner. Next, molecular modelling and DNA binding affinity studies such as relative viscosity, circular dichroism and UV-visible spectroscopy denoted classic intercalation of 19e with CT-DNA with binding constant of 1 × 10⁵ M^?1.

Full text of DOI:10.1039/c8nj03248c

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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Synthesis and in vitro cytotoxicity evaluation of β-carboline-linked
2,4-thiazolidinedione hybrids: Potential DNA intercalation and
apoptosis inducing studies
Received 00th January 20xx,
Accepted 00th January 20xx
Ramya Tokala,a Sowjanya Thatikonda,b Sravani Sana,a Phanindranath Regur,c Chandraiah
Godugu,b* Nagula Shankaraiaha*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of new β-carboline-thiazolidinedione hybrids has been synthesized and assessed for their in vitro cytotoxicity
potential against selected human cancer cell lines namely PC-3, A549, MG-63, HCT-15, MDA-MB-231, A431, and PANC-1
along with a normal human cell line (L-132). Among this new series, compound 19e was found to exhibit promising
cytotoxic effect against triple negative breast cancer cell line (MDA-MB-231) with an IC50 value of 0.97 ± 0.13 μM. Hence,
further mechanistic studies of apoptosis inducing effect by 19e were studied on MDA-MB-23 cell line. Gratifyingly, the
characteristic apoptotic features such as membrane blebbing, chromatin condensation, and apoptotic body formation
were observed with the effect of 19e on MDA-MB-231 cells using AO/EB and DAPI staining studies. Annexin V-Alexa Flour
488/PI assay confirmed significant early apoptosis induction. Notably, DCFDA assay indicated that 19e inducing ROS
generation. Moreover, mitochondrial membrane potential collapse was also observed through JC-1 staining by 19e.
Further, cell cycle analysis revealed that 19e arrested the cells at sub G1 phase. In addition, clonogenic and wound healing
assays indicated the inhibition of colony formation and cell migration by 19e in a dose-dependent manner. Next,
molecular modelling and DNA binding affinity studies such as relative viscosity, circular dichroism and UV-visibile
spectroscopy denoted the classic intercalation of 19e with CT-DNA with binding contant of
1 ×
105 M-1.
apoptosis. The improper regulation of apoptotic pathway has
been indicated in various diseases including cancer [5].
Besides, DNA till date remains as a main target in cancer
treatment due to its fundamental role in cell division
(replication) and maintenance (transcription) [6]. Therefore,
the search towards the development of novel compounds that
would induce apoptosis and target DNA will be fruitful strategy
in cancer therapy.
Introduction
Cancer is growing as the leading cause of mortality and
accounts for the major morbidity worldwide [1]. According to
World Health Organisation (WHO), cancer is liable for 8.8
million deaths in the year 2015 and was predicted to rise over
13.1 million by 2030 [2]. It is characterized by the growth of
cells beyond their boundaries and invasion of tissues making it
dreadful of all the diseases. In spite of the enormous efforts to
implement novel chemotherapeutic strategies to treat
different types of cancer, this disease still remains as one of
the major concerns globally [3]. Despite, many
chemotherapeutic agents are currently available, the
tolerance, resistance [4] and the unwanted toxic effects
towards chemotherapy, shows the necessity in development
of new and efficient anticancer agents in drug discovery.
β-Carbolines having a planar tricyclic pyrido[3,4-b] indole
ring system and are isolated from seeds of Peganum harmala;
Family: Zygophillaceae [7]. These are widely distributed in
nature marking their presence in plants, insects including
marine organisms and are well-known for their multifarious
biological and pharmacological properties such as anxiolytic
(Abecarnil), antiparkinson, anti-alzheimer, anticonvulsant,
antifungal,
antimicrobial,
antiplasmodial,
antiviral,
antithrombotic, and antimutagenic effects including with
antitumor activity [8]. The best known natural products of this
The critical cellular process which plays an essential role in
the maintenance of tissue growth and homeostasis is
class include harmine (A), norharman (B), and harmaline (C,
Figure 1). These molecules are known to inhibit cancer cell
proliferation and leading to apoptosis [9] through the multiple
mechanisms of action such as inhibition of DNA-topoisomerase
I & II [10], CDK’s [11], DYRK1A [12], MK-2 [13], PLKs [14] and
interaction with DNA (by intercalation with DNA or by binding
through minor groove) [15]. Recent reports indicate that these
compounds also act by inhibition of translation of protein
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synthesis and by photo cleavage of DNA [16]. Due to the have designed and synthesized a series of new hybrids
presence of aromatic planar tricyclic nucleus, β-carbolines can containing β-carboline bearing 2,4-thiazolidinedione moieties
possibly intercalate through stacking between base pairs of having antiproliferative properties. Herein, we have adjoined
DNA [15a, b]. In addition, some of the well-known anticancer the structural features of β-carboline to the head-part and
drugs that act through DNA intercalation under clinical use for substituted benzyl, phenacyl variations at the tail-part of the
the treatment of different types of cancers are doxorubicin 2,4-thiazolidinedione and evaluated for their in vitro
(Hodgkin’s sarcoma) and dactinomycin (Ewing’s sarcoma) [17]. cytotoxicity profile on selected human cancer cell lines and
On the other hand, in modern medicinal chemistry also compared their cytotoxicity profile on normal human cell line.
thiazolidinedione templates has acquired
a status of
“privileged heterocyclic scaffold” due to its wide range of
medicinal properties [18]. These derivatives are well-known
for the blockbuster drugs, a whole class in hypoglycaemic
agents known as “glitazones” (pioglitazone and its analogues)
[19]. This scaffold also possess a superior affinity for various
bio-targets
namely
enzyme-aldose
signal regulated
reductase,
kinase),
Raf/MEK/ERK(extracellular
(phosphoinositide 3-kinase) PI3K/Akt signaling pathways,
peroxisome proliferator-activated receptor-γ (PPAR-γ) [20].
Besides, this structural fragment has shown a broad spectrum
of activity including antimicrobial, antileukemic, wound healing
Figure 2. The design of hybrid bearing β-carboline-thiazolidinedione as anticancer
agents.
and anti-inflammatory, etc. GSK1059615 (
D, Figure 1) [20c]
and AS605240 , Figure 1) [20d] are thiazolidinedione
(E
derivatives are identified to exert their anticancer effect
through inhibition of PI3K-α and PI3K-γ, respectively. In
addition, the thiazolidinediones were also reported to show
their interaction with DNA thereby causing cytotoxicity [21a].
Owing to their diversity in pharmacological actions and affinity
to varied bio-targets, thiazolidinedione derivatives has
attained enormous interest in recent years and established
their promising role in oncology [18, 21]. A broad spectrum of
anticancer activity was also observed when thiazolidinedione-
Results and discussion
Chemistry
The β-carboline-linked 2,4-thiazolidinedione hybrids 19a–aa
were synthesized in a convergent approach by utilizing the
multifaceted Knoevenagel condensation reaction between 1-
aryl-9H-pyrido[3,4-b]indole-3-carbaldehydes 6a
substituted 2,4-thiazolidinediones 13 16a , and 18a
depicted in Scheme 3. The key precursor’s 6a (Head-part)
–c
and various
9,
,
–
d
–d
as
–
c
linked to benzimidazole motif (F, Figure 1) [18b].
Figure 1. Structures of some bioactive β-carbolines (A-C) and thiazolidinediones
D-F).
(
In recent scenario of drug discovery, the combination of
two divergent chemical units/pharmacophores with two (or
more than two) structural domains having similar/different
biological properties is often considered for the exploration of
novel and potentially active scaffolds [22]. In continuation of
our earlier efforts in design and synthesis of new chemical
entities (NCEs) as significant anticancer agents [23], herein we
Scheme 1. Synthesis of 1-aryl-9H-pyrido[3,4-b]indole-3-carbaldehydes 6a–c.
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were synthesized according to the reported methods [15b,
24]. The commercially available L-tryptophan
using thionyl chloride in methanol. Thus obtained, L- synthesized according to the earlier reported procedures [18b,
tryptophan ester was subjected to Pictet-Spengler reaction 21a]. The reaction of chloroacetic acid ( ) with thiourea (
with different aldehydes employing a catalytic amount of TFA. afforded thiazolidinedione ( , TZD) which was then converted
The diastereomers 3a thus obtained was aromatized using into potassium salt of TZD 10. Subsequently, the morpholine
KMnO4 affords 4a followed by the reduction of ester 11) was reacted with chloroacetyl chloride (12) to obtain
functionality with LiAlH4 provided the corresponding alcohols intermediate 13, and then it was reacted with 10 to afford 14
5a . Then, these alcohols were subsequently oxidized into Similarly, different benzyl bromides 15a and phenacyl
their corresponding aldehydes 6a by activated MnO2 as bromides 17a were reacted with potassium salt of 10 to
shown in Scheme 1 obtain different N-substituted thiazolidinediones 14 16a
and 18a respectively (Scheme 2) in good yields. Thus, the N-
Next,
a
variety of N-substituted 2,4-thiazolidinedione
1
was esterified intermediates (Tail-part)
9, 14, 16a–d, and 18a–c were
2
7
8)
9
–c
–
c
(
.
–
c
–d
–c
–d
.
,
–d,
–
c
substitutions of thiazolidinedione with a variety of benzyl,
phenacyl and a heterocyclic amine (morpholine) moiety were
introduced with different functionalities and established the
scope of diversity.
Finally, the title compounds 19a
employing Knoevenagel condensation reaction between key
precursor 6a and different substituted thiazolidinediones
14 16a , and 18a in EtOH with a catalytic amount of
–aa were accomplished by
–c
9,
,
–
d
–d
piperidine as a base and the yields were in the range of 70–
90% (Scheme 3). It is important to note that all the final
compounds were obtained and characterized as single isomers
as solids by simple filtration. All the derivatives synthesized
1
were characterized carefully by using H, 13C NMR, FT-IR and
HRMS. One of the representative compounds 19a was
confirmed by 1H NMR by the appearance of characteristic
singlet at δ 7.95 ppm which accounts for the proton after
condensation reaction. The 2 signals at δ 12.28 and 11.90 ppm
accounts for –NH– protons of 9H-indolic of β-carboline and 3H-
proton on thiazolidine-2,4-dione respectively. All other
aromatic protons of 19a were found in the range of δ 8.59–
7.26 ppm and 3 protons appeared as singlet at 3.91 ppm is a
Scheme
2
.
Synthesis of thiazolidinedione
9 and N-
substituted thiazolidinediones 14
,
16a–d and 18a–d
.
Scheme 3. Synthesis of β-carboline-linked thiazolidinedione derivatives
19a aa
–
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characteristic peak accounting for methoxy group. The spectra of all other derivatives 19b
Journal Name
–
aa were found almost in a
interpretation of 13C NMR of compound 19a depicts 2 carbonyl similar pattern and well matched with the respective aromatic
signals at δ 173.2 and 168.7 ppm. All other aromatic carbons and aliphatic signals of their structure. The HRMS (ESI) showed
of 19a appeared in the range of δ 160.5–113.5 ppm and the characteristic protonated [M
+
H] peak to their
formula.
aliphatic methoxy carbon at δ 55.8 ppm FT-IR spectrum of 19a corresponding
showed the –NH– stretching at 3221 cm-1. The 1H and 13C NMR
molecular
Table 1. In vitro cytotoxicity data of β-carboline linked thiazolidinedione hybrids 19a–aa
MDA-MB-
231e
Compound
PC-3a
A549b
MG-63c
HCT-15d
A431f
PANC-1g
L132h
19a
19b
> 50
38.20±2.38
36.28±4.23
37.22±2.56
35.51±0.31
6.66 ± 0.96
32.57±2.10
37.27±0.85
34.23±3.33
38.25±5.22
38.62±1.23
23.28±0.33
22.40±2.04
38.27±2.34
12.16±0.03
23.34±1.51
> 50
41.66±2.66
32.56±3.22
35.25±4.34
38.25±0.29
1.40 ± 0.11
34.27±0.47
35.27±3.22
38.79±2.37
35.14±0.62
37.28±3.25
17.38±3.34
20.60±0.87
35.98±2.58
26.15±2.34
21.84±0.51
47.21±0.14
46.28±0.85
> 50
> 50
37.28±2.35
35.28±0.25
36.25±1.24
37.28±0.35
0.97 ± 0.13
31.25±1.27
32.99±6.88
35.88±3.89
37.12±0.27
36.99±1.89
12.89±1.45
2.05±0.37
39.58±0.65
9.24±0.75
19.51±2.2
49.91±0.47
38.21±2.31
49.89±0.98
48.94±2.64
47.28±0.98
40.21±0.68
45.78±0.25
35.89±2.35
47.65±0.54
14.48±0.56
48.89±0.65
46.25±0.31
1.18±0.06
14.26±0.67
>100
39.25±5.28
33.61±2.33
36.15±3.78
42.27±0.25
4.51 ± 0.42
38.25±0.13
31.32±0.56
38.20±0.88
39.22±0.87
40.24±0.57
5.55±0.26
20.13±.1.2
42.42±0.14
6.86±0.66
10.74±1.62
> 50
42.85±2.88
31.75±2.34
38.25±1.58
39.27±0.54
5.08 ± 0.48
34.99±0.47
32.66±2.59
41.25±2.22
41.44±0.17
37.21±2.11
10.37±0.70
3.58±0.15
38.66±0.85
4.99±0.34
28.93±1.01
46.28±0.97
47.21±2.85
47.28±0.74
49.99±1.54
46.21±2.65
42.56±2.95
48.54±1.65
48.22±0.58
46.85±1.32
13.16±3.9
42.75±0.61
44.65±0.75
0.74±0.13
10.80±0.74
ND
-
34.26±2.66
39.25±3.66
37.53±0.12
1.34 ± 0.24
30.23±2.11
42.58±0.57
37.68±0.25
35.89±1.99
35.21±0.87
09.96±1.5
17.85±1.57
36.21±0.23
26.12±1.23
25.6±0.66
42.51±0.58
> 50
45.11±2.95
> 50
-
19c
-
19d
40.23±0.65
1.80 ± 0.15
36.80±0.84
39.21±2.33
45.22±0.28
32.66±1.37
41.20±0.79
8.43±3.17
2.81±0.19
39.87±0.77
9.81±1.75
10.30±0.77
48.22±0.41
> 50
-
19e
3.33 ± 0.93
19f
-
19g
-
19h
-
19i
-
19j
-
19k
5.94±0.50
19l
9.99±1.94
19m
19n
-
21.99±2.41
19o
41.36±4.96
19p
-
19q
45.21±0.78
> 50
41.20±2.35
> 50
-
19r
46.21±2.34
> 50
42.31±1.54
> 50
-
19s
46.25±1.56
> 50
> 50
> 50
-
19t
48.75±2.48
> 50
42.23±2.58
45.21±1.23
42.28±2.58
36.28±0.68
> 50
45.28±2.54
48.28±0.56
43.58±0.87
45.84±0.54
42.10±0.89
32.12±2.4
47.25±0.52
45.20±2.01
2.38±0.28
24.41±0.44
ND
> 50
-
19u
46.22±2.21
>50
47.22±0.87
> 50
-
19v
41.28±3.22
34.59±2.58
39.54±0.87
22.93±0.69
40.23±2.22
> 50
-
19w
19x
39.57±0.24
> 50
> 50
-
45.98±2.35
19.16±2.8
46.28±0.54
42.98±0.89
1.48±0.08
22.84±0.54
ND
-
19y
23.80±1.65
> 50
19.82±4.07
45.89±0.89
44.02±2.00
2.75±0.20
32.33±0.41
>100
28.98±2.6
19z
-
-
19aa
Doxi
Harminei
Pioglitazonei
47.63±0.85
1.90±0.36
18.14±0.96
>100
1.20±0.15
20.55±1.64
>100
3.80±0.02
24.65±2.48
ND
aProstate cancer cells, bLung cancer cells, cOsteosarcoma cells, dColon cancer cells, eBreast cancer cells, fSkin cancer cells, gPancreatic cancer cells, hHuman
pulmonary epithelial cells, iReference compound, ND: Not determined.
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ARTICLE
Apoptosis studies
Morphological observations using phase contrast microscope.
The induction of apoptotic bodies’ formation has always been
a vital choice in developing anticancer therapeutics [25]. To
examine whether the compound could cause loss of cell
viability and induce apoptosis, MDA-MB-231 cancer cells were
treated with the active compound 19e. Loss of distinctive
morphological features such as cell-cell adhesion was observed
from phase contrast microscopic images Figure 3 captured
after an incubation period of 72 h. Further, cell wall
deformation, shrinkage of cells, reduction in number of viable
cells was also observed in treatment in comparison to control.
In vitro cytotoxicity evaluation. The new β-carboline-
thiazolidinedione hybrids 19a–aa were evaluated for their in
vitro cytotoxic activity at 50 µM against selected human cancer
cell lines namely PC-3, A549, MG-63, HCT-15, MDA-MB-231,
A431 and PANC-1 as well as on the normal cell line L-132 by
employing
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The results from
the in vitro cytotoxic activity of the compounds 19a–aa along
with standard harmine were represented in Table 1.
Notably, these compounds exhibited good to moderate
cytotoxicity on the tested cancer cell lines. From the close
analysis of the IC50 values, it was observed that compound 19e
was more significant in inducing cytotoxicity on all the cancer
cells and found to be most potent against MDA-MB-231 breast
cancer cell line with an IC50 value 0.97 ± 0.13 µM whereas in
PC-3 prostate cancer cell line 50% inhibition was observed at
1.34 ± 0.24 µM. This compound was also found to be active in
bone (MG-63) and colon cancer (HCT-15) cell line with IC50
values of 1.40 ± 0.11 µM and 1.80 ± 0.15 µM, respectively.
Further, 19e was found to be effective on epidermoid skin
cancer cell line (A-431) and pancreatic cancer cell line (PANC-1)
with IC50 values of 4.51 ± 0.42 and 5.08 ± 0.48 µM respectively.
Moreover, 19e was also showed to be active on lung cancer
cell line A549 with IC50 value 6.66 ± 0.96 µM, whereas, in
normal lung cells 3.33 ± 0.93 µM 50% inhibitory concentration
was observed respectively. This denotes 19e was found to be
efficacious when compared to the reference natural harmine
in all the tested human cancer cell lines. While the compound
19l also displayed remarkable cytotoxicity with IC50 values of
2.05 ± 0.37 µM, 2.81 ± 0.19 µM and 3.58 ± 0.15 µM against the
breast cancer MDA-MB-231, colon cancer HCT-15 and
pancreatic cancer PANC-1 cell lines, respectively. Besides, 19n
was found active on PANC-1 with IC50 value of 4.99 ± 0.34 µM
and on A-431with IC50 value of 6.86 ± 0.66 µM. Among the
series tested, the compound 19k also showed considerable
cytotoxicity on A431 cancer cell lines.
Figure 3. Morphological changes observed in breast cancer cells (MDA-MB-231),
cells were treated with and without compound 19e at concentrations 0.25, 0.5,1,
2.5 µM. After 72 h the images were captured with a phase contrast microscope at
200X magnification. Scale Bar in the image indicates 50 μm.
Acridine orange/ethidium bromide (AO/EB) staining.
Acridine orange/ethidium bromide (AO/EB) fluorescence
staining assay differentiates between live, apoptotic and
necrotic cells [26]. AO can infuse the intact cell membrane and
It is evident from the IC50 values that C1 position of the β-
carboline displayed better cytotoxic activity when substituted
with benzaldehydes bearing an electron-withdrawing group (R
=
Cl) at para position 19e
,
19k followed by simple
19n 19o, and 19y
benzaldehyde (R = H) at para position 19l
,
,
rather than the benzaldehyde containing an electron-donating
group at 4th position (R = OMe). Based on in vitro cytotoxicity
results, regarding the substitutions at nitrogen of
thiazolidinedione, compound 19e was proved to be the best
among the series tested to possess a heterocyclic secondary
Figure 4. AO/EB staining in breast cancer cells (MDA-MB-231), cells treated with
and without compound 19e at various concentrations 0.25, 0.5, 1 and 2.5 µM for
2 h and compared with control. The images were captured with fluorescence
microscope at 200X. Red scale Bar indicates 50 μm.
amine (morpholine) at N on TZD. N-benzylated derivatives 19k
19l 19n, and 19o were found to be potent when compared to
phenacyl substituted derivatives 19y and free NH 19a 19b
,
,
,
,
and 19c containing derivatives. These biological results could
shed light in anticancer studies which may lead to the
discovery of new cytotoxic agents with better activity.
stain the nuclei green, whereas EB can only permeates the
nuclei of cells that have lost membrane integrity and stains
them red. It can be observed from Figure 4 that the control
cells exhibited normal morphology and appeared green in
color. The morphological changes such as cell shrinkage,
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membrane blebbing, chromatin condensation and apoptotic
body formation can be observed in compound 19e treated
MDA-MB-231 cells in a dose-dependent manner indicating the
compound had induced apoptosis.
DAPI nucleic acid staining. The classic feature of apoptotic
cells is condensation of nucleus and it can be observed by DAPI
staining. DAPI (4ꞌ,6-diamidino-2-phenylindole) is a fluorescent
dye that distinguishes the nuclear damage or chromatin
condensation by firmly binding to the A-T rich regions of DNA
[18b]. DAPI infuses into dead cells efficiently rather than the
live cells and stains the apoptotic nuclei to appear bright.
Hence, to detect nuclear damage or chromatin condensation
induced in MDA-MB-231 by the compound 19e, DAPI staining
study was performed in compliance with previously reported Figure 6. DCFDA staining in breast cancer cell line MDA-MB-231. Cells were
method [23]. It can be observed from Figure 5 that the control treated with and without compound 19e at various concentrations 0.5, 1 and 2.5
cells were intact with normal morphology, while the µM for 24 h and compared with control cells. The images were captured with
compound 19e treatment led to the nuclear changes like fluorescence microscope at 200X. Red scale Bar indicates 50 μm.
condensed, pyknotic or fragmented bright nuclei. From the Clonogenic growth inhibition assay. Clonogenic cell growth
results, it was evident that the compound 19e had induced inhibition assay reflect the ability of single cell to grow in to
apoptosis in dose dependent manner
.
colony [23d, 28]. The potential disruption of colony forming
ability in breast cancer cells by 19e was evaluated by
clonogenic assay. After treatment with various concentrations
(0.25, 0.5, 1 µM), we observed the formation of colonies in
control cells in one week time whereas in treatment at 0.5 and
1 µM apparent reduction in the number of colonies was
observed as shown in Figure 7. Thus, indicating the dose-
dependent inhibition of colony formation and proliferation of
compound 19e in MDA-MB-231 cells. The total colonies were
counted by molecular imaging system Vilber Fusion Fx
software and the values were represented as a percent colony
forming ability.
Figure 5. Effect of 19e on nuclear morphology of cancer cells after DAPI staining.
Breast cancer (MDA-MB-231) cells treated with and without compound 19e at
various concentrations 0.25, 0.5, 1 and 2.5 µM for 72 h and compared with
control. The images were captured with fluorescence microscope at 200X. Red
scale Bar in the image indicates 50 μm.
Measurement of ROS levels. ROS levels were measured as the
excess generation prompts irreversible oxidative modification
of cellular macromolecules which include lipid, protein or DNA.
It also disrupts the intracellular redox homeostasis by oxidative
damage to the mitochondrial potential and permeability and
thereby, causes apoptosis [27]. Therefore, to determine the
ability of compound 19e on the ROS generation, the Figure 7. Effect of compound 19e on disruption of colony forming ability of breast
cancer cells by clonogenic assay. MDA-MB-231 cells were treated with compound
intracellular ROS levels were determined by using the DCFDA
staining assay. We investigated the effect of 19e at different 19e at 0.25, 0.5, and 1 µM and control (untreated cells) are allowed to form
concentrations of 0.5, 1, 2.5 and 5 μM on MDA-MB-231 cells colonies for 7 days. The images were captured and the colonies were counted
using DCFDA which is readily oxidized to 2',7'- with vilber fusion chemdoc imaging system
dichlorofluorescein (DCF) in the presence of ROS. We observed In vitro cell migration/wound healing assay. Metastasis is spread
significant increase in green fluorescence proportionally with of cancer cells from the place of formation to another part of body
increase in the concentration of 19e treatment as compared to which involves migration of cancer cells [23]. MDA-MB-231 is
the control cells in Figure 6. These results clearly indicate the aggressive and highly invasive cancer cell line [29] and in order to
potential of 19e in inducing ROS generation and thereby it establish the effect of compound 19e on cell migration, wound
could be one of the mechanisms in prompting apoptosis in healing assay was performed. This assay is based on the
MDA-MB-231 cancer cells.
observation that, upon formation of a new artificial wound, so
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Figure 8. Effect of compound 19e on migration potential of MDA-MB-231 cells. Cells were exposed to different concentrations (0.5, 1, 2.5, 5μM) of 19e and scratched
with sterile 200 μL pipette tip and compared with control (untreated cells). The images were captured at 0 h and 24 h using phase contrast microscope.
called “scratch” on a confluent cell monolayer, the cells on the
edge of the wound will progress toward the opening to close
the wound until new cell-cell interactions are established.
The treatment of cells with anticancer molecules restricts the
migration of cells to close the wound [21a]. Therefore, to test
the effect of compounds on cell migration, wounds were
created on MDA-MB-231 cell's monolayer by using a 200 μL
sterile pipette tip and cells are treated with 0.5, 1, 2.5, 5 μM
concentrations of compound 19e and cells were allowed to
migrate into the wounded area. The images of cells to fill-up
the wounds were captured at 0 h and 24 h after the treatment
with the compounds by using phase contrast microscopy. It
can be inferred from Figure 8 that the compound 19e
effectively suppressed the migration of cells into the wounded
area, however almost complete healing was observed in Figure 9. Effect on mitochondrial membrane potential. MDA-MB-231 cells were
control cells after 24 h.
treated with various concentrations (0.25, 0.5, 1, 2.5 µM) of compound 19e for
Flow-cytometry analysis
72 h and JC-1 staining was performed. The control represents the cells without
Effect on mitochondrial membrane potential
(ΔΨM)
.
By compound 19e treatment. P1 represents formation of J-aggregates in healthy
monitoring the changes in mitochondria membrane potential, mitochondria, whereas P2 represents depolarised mitochondria in cells due to
mitochondrial function can be evaluated [23]. The the presence of J-monomers.
mitochondria play an essential role in initiating the intrinsic depolarisation of ΔΨM which is indicating the cytotoxic effect
pathway of apoptosis as it is the primary target of cellular associated with mitochondria.
oxidative stress, which hamper the electron transport chain, Cell cycle analysis. The cytotoxic compounds exhibit their
leading to reactive oxygen species (ROS) formation and growth inhibitory action by blocking the cell cycle at a specific
collapse of ΔΨM. Enhanced generation of ROS was evidenced checkpoint [23]. From the in vitro screening results, it was
with compound 19e by DCFDA staining technique. To further noticeable that the compound 19e showed noteworthy
evaluate the impact of compound 19e on mitochondria, the cytotoxicity against breast cancer cell line MDA-MB-231.
ΔΨM was measured. The lipophilic cationic dye JC-1 was used
Therefore, to unveil whether this cytotoxicity may be due to
for evaluating ΔΨM, in which normal polarized mitochondria the cell cycle arrest, analysis of cell cycle was performed. Cells
stains red due to formation of J-aggregates and green color were treated with compound 19e at concentrations ranging
was observed for depolarized mitochondria of apoptotic cells from 0.25 to 2.5 µM for 24 h, and then the cells were stained
due to formation of J-monomers. MDA-MB-231 cells were with propidium iodide and further analyzed by using BD
treated with different concentrations (0.25, 0.5, 1, 2.5 μM) of
FACSVerseTM flow analyzer. The results from Figure 10
compound 19e and remarkable increase in the mitochondrial
indicated that the MDA-MB-231 untreated control cells
membrane depolarisation was observed as compared to showed 4.03% cells in sub G1 phase, whereas compound 19e
control cells as shown in the Figure 9. Flow-cytometric analysis treatment resulted in increase in subG1 population from
of the treated cells clearly displayed an increase in depolarised 7.09% to 19.66%. From the results of cell cycle analysis, it was
cell population (P2) in a concentration dependent manner observed that compound 19e induces blockade at sub G1
from 11.69% in control to 92.23% respectively at 2.5 μM phase, also indicates apoptotic cell death.
respectively. Thus the compound 19e triggered the
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DNA binding affinity studies
These studies will indicate the affinity between the DNA and
the synthetic ligand. It will also be useful in understanding the
molecule’s nature of interaction with a macromolecule namely
DNA. Interaction with DNA results in alterations in processes
of cell division and maintenance which leads in cytotoxicity.
Relative viscosity study. Relative viscosity experiment
indicates the interaction pattern of newly synthesized
derivatives with DNA. The measurement of changes in
viscosity of DNA provides the idea of binding of the
compounds to DNA. Decrease in relative viscosity is observed
if compounds exhibit covalent binding with DNA, whereas a
sharp rise in values is observed when a compound exhibits
intercalation into DNA owing to the enhancement in the axial
length of DNA helix to adapt the hybrid. Ethidium Bromide
(EtBr) is a classical DNA intercalator which corresponds to the
increase in viscosity [30]. Groove binding indicates little or no
change in terms of relative viscosity. Hoechst 33258 is a well-
known groove binder and does not affect the relative viscosity
when bound to DNA [31]. Addition of compounds 19e and 19n
resulted in the increase in viscosity in comparison to the
control DNA in concentration-dependent manner, thereby
Figure 10. Effect of 19e on cell cycle phase distribution and arrest of triple
negative breast cancer cells. MDA-MB-231 cells were treated with compound 19e
at 0.25, 0.5, 1, 2.5 µM concentration and cell cycle analysis was performed after
24 h along with control (untreated cells). The analysis of cell cycle distribution
was performed by using propidium iodide staining method.
Annexin V-FITC/PI assay. Annexin V-FITC/PI dual staining assay
was performed to quantify the apoptotic cell death caused by
compound 19e on MDA-MB-231 cells by using flow cytometry
accounting for the DNA intercalation as shown in Figure 12
.
The graph is plotted between concentration (compound/CT-
DNA) and viscosity ((ŋ/ŋo)1/3 on X and Y axis respectively with
doxorubicin, harmine, EtBr and Hoechst 33258 as standards.
The viscosity results are compatible with molecular modelling
studies.
3
2
1
0
Ethidium
bromide
Dox
Harmine
19e
Figure 11. Effect of compound 19e on cell apoptosis induction of triple negative
breast cancer cells was measured by Annexin V-Alexa Flour 488/propidium iodide
staining assay. MDA-MB-231 cells were treated with compound 19e ranging from
0.25 to 1.0 µM concentration and control (untreated cells) for 72 h. Then 10,000
cells from each sample were analyzed by flow cytometry. The percentage of cells
positive for Annexin V-Alexa Flour 488 and/or Propidium iodide is represented
inside the quadrants. Cells in the upper left quadrant (Q1-UL; AV-/PI+): necrotic
cells; lower left quadrant (Q2-LL; AV-/PI-): live cells; lower right quadrant (Q3-LR;
AV+/PI-): early apoptotic cells and upper right quadrant (Q4- UR; AV+/PI +): late
apoptotic cells.
19n
Hoechst
33258
0
0.02
0.04
0.06
0.08
0.1
[Compound/CTꢀDNA]
Figure 12. Graphical representation of relative viscosity studies using compounds
19e and 19n with Harmine, Hoechst-33258, and Ethidium Bromide as reference
standards. Ethidium bromide and Dox are potent intercalators. The natural
product harmine also shows intercalation where as Hoechst 33258 is a minor
groove binder.
[23d]. Annexin assay facilitates the detection of necrotic cells
(Q1-UL; AV-/PI+), live cells (Q2-LL; AV-/PI-), early apoptotic
cells (Q3-LR; AV+/PI-), and late apoptotic cells (Q1- UR;
AV+/PI+). MDA-MB-231 breast cancer cells were treated with
compound 19e at different concentrations of 0.25, 0.5, and 1
μM for 72 h. Results from Figure 11 indicated the percentage
of apoptotic cells and inferred to the significant early (AV+/PI-
), and slight late apoptosis (AV+/PI+) which indicates
compound 19e induces apoptosis dose dependently in
comparison to control.
Circular dichroism (CD). Circular dichroism studies are carried
out to find the interaction of DNA with ligand (19e) and its
effect on DNA conformation. DNA is
a
biological
macromolecule, which is responsible for having cell
proliferation and several other biological functions. Hence,
interaction and changes in the DNA topology will give enough
indication on the nature of DNA-complex interactions and
conformational changes on derivatives binding to DNA. The CD
spectrum of CT-DNA exhibited a positive band at 275 nm and a
negative band at 245 nm. These indicate π–π base stacking
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and right-hand helicity, the characteristic profile of B form Molecular modelling studies. The β-carboline derivatives
DNA. In the present study, addition of compound 19e to CT possess a flat, planar structure which generally shows their
DNA, the positive CD indicated continuous hyperchromicity interactions by intercalating into DNA [9]. Thus, to denote the
even at 1:1 and 1:2 stoichiometry of DNA:compound. This interactions of the synthesized ligands towards DNA and to
suggests that stabilization of the DNA structure on compound indicate the mode of interaction, molecular modeling studies
interaction [32]. The intensities of negative band has increased were performed on DNA octamer d(GAAGCTTC)2 [35] using XP
on the negative scale, indicating ligands effect on DNA helicity Glide (Schrödinger, 2017) [35] with default settings. The best
during interaction. The CD spectra of the complex on docked pose of the compound 19e indicates the intercalation
interaction with CT DNA are shown in Figure 13
.
of planar β-carboline moiety towards GC base pairs of DNA
and is represented in Figure 15. The morpholine ring orients
into the active site through the acyl linker attached to the
nitrogen of thiazolidinedione. The docked position is best
supported by strong π-π stacking interactions by all the rings
of β-carboline towards dG4, dG12, dC5 residues. Further, the
hydrogen-bonding interactions have been observed between
oxygen of the carbonyl and oxygen of morpholine in the tail
part with the residue dG12. The ring structure has also been
stabilized by hydrophobic interactions in the active pocket.
Figure 13. Circular dichroism studies of compound 19e at different ratio
with CT-DNA.
UV-Visible Spectral study. The present study indicates the
topological changes in CT-DNA on interaction with the ligand.
The UV-Vis spectral studies conducted with CT-DNA and ligand
exhibited a prominent absorption bands at 390 nm. When the
CT-DNA is added to ligand solution (19e), the absorption band
of
ligand
exhibited
hypochromicity.
Continuous
hypochromicity of ligand absorption band on continuous
addition of DNA, suggests that the ligand may be intercalating
with CT DNA [33].
Figure 15. Intercalation of compound 19e into d(GAAGCTTC)2. Side view of 19e
showing residual interaction with G-C base pairs of DNA. DNA is represented as
blue ribbon and compound 19e is represented in yellow color with surface.
In order to study the stability of the DNA-ligand complex,
Molecular Dynamics (MD) simulations were performed for the
best-scored pose of compound 19e with DNA for 5 ns. The
images of 19e-DNA complex during the 5 ns MD simulation
have shown in Figure 16. The plot of root-mean-square
deviation (RMSD) of the backbone atoms as a function of time
Figure 14. UV-Visible spectra representing the intercalation of compound 19e
with CT-DNA
The results obtained from UV-visible spectral data manifests that
compound 19e interact with DNA and possibly through
intercalation between the two stacks of DNA . The UV-vis spectra
obtained with ligand 19e is shown in Figure 14. The binding
constant for the ligand-CT-DNA is calculated using online software
Bindfit [34]. The binding constant was found to be 1.40 × 105 M-1.
was depicted (Figure 2, ESI). It is inferred from the plot that
during the time period of 5 ns MD simulation, the complex
does not show much deviation as of the initial position over
the time. Even the docked pose of 19e was rationally stable up
to 5 ns MD simulation. The results obtained from molecular
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simulation studies were consistent with in vitro cytotoxicity manner. Moreover, cell cycle arrest is observed at sub G1
and DNA affinity results.
phase of cell cycle as analyzed by using flow cytometry. In
addition, molecular modelling studies and DNA affinity studies
(relative viscosity studies, circular dichroism and UV-visible
spectroscopic studies) supports intercalation of this β-
carboline-linked thiazolidinedione hybrids into DNA. Overall,
these new series of hybrids detailed in the current work
comprise the potential to be developed into novel cancer
therapeutics through suitable structural modifications.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
We are thankful to DoP, Ministry of Chemicals & Fertilizers,
Govt. of India, New Delhi, for the award of NIPER fellowship.
Dr N. Shankaraiah gratefully acknowledges SERB, DST, Govt. of
India for research grant (YSS-2015-001709).
References
1. (a) https://www.cancer.gov/about-
cancer/understanding/statistics;
(b)http://www.who.int/mediacentre/factsheets/fs297/en/
2. R. Siegel, K. D. Miller and A. Jemal CA, Cancer. J.Clin., 2017, 67,
7.
3. K. R. Senwar, T. S. Reddy, D. Thummuri P. Sharma, V. G. M.
Naidu, G. Srinivasulu and N. Shankaraiah, Eur. J. Med. Chem.,
2016, 118, 34.
Figure 16. Snapshots 19e-DNA complex at 1 ns (A), 2 ns (B), 4 ns (C) and 5 ns (D)
4. S. Raguz and E. Yague, Br. J. Cancer, 2008, 99, 387.
5. D. Hanahan and R. A. Weinberg, Cell, 2011, 5, 646.
6. (a) L. H. Hurley, Nat. Rev. Cancer., 2002, 2, 188; (b) J. Sheng, J.
Gan and Z. Huang, Med. Res. Rev., 2013, 33, 1119.
timescale in a molecular dynamics study.
Conclusion
7. (a) R. A. Abrimovitch and I. D. Spencer,Adv. Heterocycl. Chem.,
1964, 3, 79; (b) M. Moloudizargari, P. Mikaili, S. Aghajanshajeri,
M. H. Asghari and J. Shayegh, Pharmacogn. Rev., 2002, 14, 199.
8. (a) R. Cao, W. Peng, Z. Wang and A. Xu, Curr. Med. Chem., 2007,
14, 479; (b) W. Polanski, H. Reichmann and G. Gille, Expert. Rev.
Neurother., 2011, 11, 845; (c) Y. Rook, K. U. Schmidtke, F.
Gaube, D. Schepmann, B. Wunsch, J. Heilmann, J. Lehmann and
T. Winckler, J. Med. Chem., 2010, 53, 3611; (d) R. B. Volk and F.
H. Furkert, Microbiol. Res., 2006, 161, 180; (e) H. J. Shin, H. S.
Lee and D. S. Lee, J. Microbiol. Biotechnol., 2010, 20, 501; (f) K.
V. Rao, N. Kasanah, S. Wahyuono, B. L. Tekwani, R. F. Schinazi
and M. T. Hamann, J. Nat. Prod., 2004, 67, 1314; (g) K. L.
Rinehart Jr., J. Kobayashi, G. C. Harbour, J. Gilmore, M. Mascal,
T. G. Holt, L. S. Shield and F. Lafargue, J. Am. Chem. Soc., 1987,
109, 3378; (h) N. Shankaraiah, C. Jadala, S. Nekkanti, K. R.
Senwar, N. Nagesh, S. Shrivastava, V. G. M. Naidu, M. Sathish
and A. Kamal, Bioorg. Chem., 2016, 64, 42; (i) R. Frederick, C.
Bruyere, C. Vancraeynest, J. Reniers, C. Meinguet, L. Pochet, A.
Backlund, B. Masereel, R. Kiss and J. Wouters, J. Med. Chem.,
2012, 55, 6489.
9. Q. Xu, X. Chen, J. Feng, J. Miao, J. Liu, F. Liu, B. Niu, J. Cai, C.
Huang, Y. Zhang and Y. Ling, Sci. Rep., 2016, 6, 36238.
10. (a) A. Kamal, V. Srinivasulu, V. L. Nayak, M. Sathish, N.
Shankaraiah, C. Bagul, N. V. S. Reddy, N. Rangaraj and N.
Nagesh, ChemMedChem., 2014, 9, 2084; (b) J. Jiménez, L.
Riverón-Negrete, F. Abdullaev, J. Espinosa-Aguirre and R.
Rodríguez-Arnaiz, Exp. Toxicol. Pathol., 2008, 60, 381; (c) A.
Kamal, M. Sathish, V. L. Nayak, V. Srinivasulu, B. Kavitha, Y.
Tangella, D. Thummuri, C. Bagul, N. Shankaraiah and N. Nagesh,
Bioorg. Med. Chem., 2015, 23, 5511.
In conclusion, a new series of β-carboline-thiazolidinedione
hybrids were synthesized in good yields and characterized by
IR, 1H, 13C and HRMS spectral data. These hybrids were
obtained as single isomers by simple filtration. Preliminary in
vitro screening was performed to evaluate their in vitro
cytotoxicity against PC-3, A549, MG-63, HCT-15, MDA-MB-231,
A431, PANC-1 human cancer cell lines and a normal cell line L-
132. Notably, the hybrid 19e with a chloro atom at para
position of phenyl on β-carboline (head-part) adjoined to
thiazolidinedione which contain morpholine (tail-part) ring was
found to be the most potent hybrid. This displayed broad
spectrum of antiproliferative activity, specifically on MDA-MB-
231, PC-3, MG-63 and HCT-15 with IC50 values of 0.97±0.13
μM, 1.34±0.24 μM, 1.40±0.11 μM, 1.80±0.15 μM respectively.
The compound 19e treated MDA-MB-231 cells displayed
inhibition of in vitro cell migration assay, and clonogenic assay.
The staining studies including AO/EB and DAPI evidenced the
characteristic features of apoptosis dose dependently and are
supported by Annexin V-Alexa Flour 488/Propidium iodide
assay. Further, induction of reactive oxygen species (ROS) by
DCFDA staining assay and analysis of depolarisation of
mitochondrial membrane potential by JC-1 staining study
revealed the collapse of mitochondrial membrane of MDA-MB
231 cells by compound 19e in concentration dependent
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11. Y. Li, F. Liang, W. Jiang, F. Yu F, R. Cao, Q. Ma, X. Dai, J. Jiang, Y.
Wang and S. Si, Cancer. Biol. Ther., 2007, 6, 1193; (b) P. A.
Barsanti, W. Wang, Z. Ni , D. Duhl, N. Brammeier and E. Martin,
Bioorg. Med. Chem. Lett., 2010, 20, 157.
12. (a) N. Göckler, G. Jofre, C. Papadopoulos, U. Soppa, F. J. Tejedor
and W. Becker, FEBS J.,2009, 276, 6324; (b) A. Seifert, L. A.
Allan, and P. R. Clarke, FEBS J., 2008, 275, 6268.
Med. Chem., 2017, 136, 511; (c) P. Sharma, T. S. Reddy, D.
Thummuri, K. R. Senwar, N. P. Kumar, V. G. M. Naidu, S. K.
Bhargava and N. Shankaraiah, Eur. J. Med. Chem., 2016, 124,
608; (d) K. Metwally, H. Pratsinis and D. Kletsas, Eur. J. Med.
Chem., 2017, 133, 340; (e) A. Vivek, K. M. Debarshi and K. B.
Sanjay, Eur. J. Med. Chem., 2014, 87, 814.
22. C. Viegas-Junior, A. Danuello,V. S. Bolzani, E. J. Barreiro, C. A.
Fraga, Curr. Med. Chem., 2007, 14, 1829.
13. J. L. Trujillo, M. J. Meyers, D. R. Anderson, S. Hegde, M. W.
Mahoney, W. F. Vernier, I. P. Buchler, K. K. Wu, S. Yang, S. J. 23. (a) P. Sharma, D. Thummuri, T. S. Reddy, K. R. Senwar, V. G. M.
Hartmann and D. B. Reitz, Bioorg. Med. Chem. Lett., 2007, 17,
4657.
Naidu, G. Srinivasulu, S. K. Bhargava and N. Shankaraiah, Eur. J.
Med. Chem., 2016, 122, 584; (b) N. P. Kumar, S. Thatikonda, R.
Tokala S. S. Kumari, U. J. Lakshmi, C. Godugu, N. Shankaraiah,
and A. Kamal Bioorg. Med. Chem., 2018, 17, 32454; (c) N.
Shankaraiah, P. Sharma, S Pedapati, S. Nekkanti, V. Srinivasulu,
N. P. Kumar and A. Kamal, Lett. Drug. Des. Discov., 2016, 13,
335; (d) R. Tokala, S. Bale, I. P. Janrao, A Vennela, N. P. Kumar,
K. R. Senwar, C. Godudu and N. Shankaraiah, Bioorg. Med.
Chem. Lett., 2018, 28, 1919.
14. (a) J. Zhang, Y. Li, L. Guo, R. Cao, P. Zhao, W. Jiang, Q. Ma, H. Yi,
Z. Li, J. Jiang, J. Wu, Y. Wang, and S. Si, Cancer. Biol. Ther., 2009,
8, 2374; (b) X. Li, B. Bai, L. Liu, P. Ma, L. Kong, J. Yan, J. Zhang, Z.
Ye, H. Zhou, B Mao, H. Zhu and Y. Li, Cell. Death. Discov., 2015,
1, 15033.
15. (a) J. Wu, M. Zhao, K. Qian, K. H. Lee, S. Morris-Natschke and S.
Peng, Eur. J. Med. Chem., 2009, 44, 4153; (b) A. Kamal, M. P. N.
Rao, P. Swapna, V. Srinivasulu, C. Bagul, A. B. Shaik, K. Mullagiri, 24. N. Devi, G. Kaur, D. Singh, S. Mor, V. P. R. K. Putta, S. Polina, C.
J. Kovvuri, V. S. Reddy, K. Vidyasagar, and N. Nagesh, Org. C. Malakar and V. Singh, New. J. Chem., 2016, 41, 1082.
Biomol. Chem., 2014, 12, 2370; (c) Z. Chen, R. Cao, L. Yu, B. Shi, 25. S. Elmore, Toxicol. Pathol., 2007, 35, 495.
J. Sun, L. Guo, Q. Ma, W. Yi, X. Song and H Song, Eur. J. Med. 26. N. P. Kumar, P. Sharma, S. S. Kumari, U. R. Brahma, S. Nekkanti,
Chem., 2010, 45, 4740; (d) N. Shankaraiah, K. P. Siraj, S.
N. Shankaraiah and A. Kamal, Eur. J. Med. Chem., 2017, 140,
Nekkanti, V. Srinivasulu, P. Sharma, K. R. Senwar, M. Sathish, M.
128.
V. P. S. Vishnuvardhan, S. Ramakrishna, C. Jadala, N. Nagesh, 27. M. L. Circu and T. Y. Aw, Free. Radic. Biol. Med., 2010, 48, 749.
and A. Kamal, Bioorg. Chem., 2015, 59, 130; (e) Z. Chen, R. Cao, 28. P. Sharma, K. R. Senwar, M. K. Jeengar, T. S. Reddy, V. G. M.
B. Shi, W. Yi, L. Yu, H. Song, Z. Ren, and W. Peng, Bioorg. Med.
Chem. lett., 2010, 20, 3876; (f) J. M. Giulietti, P. M. Tate, A. Cai,
Naidu, A. Kamal and N. Shankaraiah, Eur. J. Med. Chem., 2015,
104, 11.
B. Cho and S. P. Mulcahy, Bioorg. Med. Chem Lett., 2016, 26, 29. L. A. Gordon, K. T. Mulligan, H. Maxwell-Jones, M. Adams, R. A.
4705; (g) S. Nekkanti, R. Tokala, N. Shankaraiah, Curr. Med.
Chem., 2017, 24, 2887.
16. (a) A. Carvalho, J. Chu, C. Meinguet, R. Kiss, G. Vandenbussche,
Walker, J. L. Jones, Int. J. Cancer., 2003, 106, 8.
30. S. Nekkanti, V. Pooladanda, M. Veldandi, R. Tokala, C. Godugu,
and N. Shankaraiah, ChemistrySelect., 2017, 2, 7210.
B. Masereel, J. Wouters, A. Kornienko, J. Pelletier, V. Mathieu, 31. S. Nekkanti, K. Veeramani, S.S. Kumari, R. Tokala and N.
Eur. J. Pharmacol., 2017, 805, 25; (b) J. Kovvuri, B. Nagaraju, V. Shankaraiah, RSC. Adv., 2016, 6, 103556.
L. Nayak, R. Akunuri, M. P. N. Rao, A. Ajitha, N. Nagesh and A. 32. L. Wang, Y. Wen, J. Liu, J. Zhou, C. Li and C. Wei, Org. Biomol.
Kamal, Eur. J. Med. Chem., 2018, 143, 1563. Chem., 2011, 9, 2648.
17. (a) O. Tacar, P. Sriamornsak and C. R. Dass, J. Pharm. 33. J. K. Barton, J.J. Dennenberg and J. B. Chaires, Biochemistry.,
Pharmacol.,2013, 65, 157; (b) N. Jaffe, D. Paed, D. Traggis, S.
Salian and J. R. Cassady, Cancer., 1976, 38, 1925.
18. (a) A. Vivek, K. M. Debarshi and K. B. Sanjay, Eur. J. Med. Chem.,
2014, 87, 814; (b) L. Nagarapu, B. Yadagiri, R Bantu, C. G. Kumar,
1993, 32, 2573.
34. K. Yadav, P. N. R. Meka, S. Sadhu, S. D. Guggilapu, J. Kovvuri, A.
Kamal, R. Srinivas, P. Devyani, B. N. Babu and N. Nagesh,
Biochemistry., 2017, 56, 4392.
S. Pombala and J. Nanubolu, Eur. J. Med. Chem., 2014, 71, 91; 35. M. Shinomiya, W. Chu, R. G. Carlson, R. F. Weaver and F.
(c) P. Sharma, T. S. Reddy, N. P. Kumar, K. R Senwar, S. K. Takusagawa, Biochemistry., 1995, 34, 8481.
Bhargava, and N. Shankaraiah, Eur. J. Med. Chem., 2017, 138, 36. Schrödinger suite 2017-1; Schrödinger, LLC: New York, 2017.
234.
19. A. K. M. Iqbal, A. Y. Khan, M. B. Kalashetti, N. S. Belavagi, Y. D.
Gong and I. A. M. Khazi, Eur. J. Med. Chem., 2012, 53, 308.
20. a) K. Liu, W. Rao, H. Parikh, Q. Li, T. L. Guo, S. Grant, G. E.
Kellogg and S. Zhang, Eur. J. Med. Chem., 2012, 47, 125; (b) W.
Motomura, S. Tanno, N. Takahashi, M. Nagamine, M. Fukuda, Y.
Kohgo and Okumura, Biochem. Biophys. Res. Commun., 2005,
332, 89; (c) S. D. Knight, N. D. Adams, J. L. Burgess, A. M.
Chaudhari, M. G. Darcy, C. A. Donatelli, J. I .Luengo, K. A.
Newlander, C. A. Parrish, K. H. Ridgers, M. A. Sarpong, S. J.
Schmidt, G. S. V. Aller, J. D. Carson, M. A . Diamond, P. A. Elkins,
C. M. Gardiner, E. Garver, S. A. Gilbert, R. R. Gontarek, J. R.
Jackson, K. L. Kershner, L. Luo, K. Raha, C. S. Sherk, C. M. Sung,
D. Sutton, P. J. Tummino, R. J. Wegrzyn, K. R. Auger and D.
Dhanak, ACS. Med. Chem. Lett., 2010, 1, 39; (d) X. Wei, J. Han, Z.
Z. Chen, B. W. Qi, G. C. Wang, Y. H. Ma, H. Zheng, Y. F. Luo, Y. Q.
Wei and L. J. Chen, Biochem. Biophys. Res. Commun., 2010, 397,
311; (e) J. J. Mansure, R. Nassim and W. Kassouf, Cancer. Biol.
Ther., 2009, 8, 6.
21. (a) L. S. Vijaya, P. Anil, G. Rajitha, A. J. Rao, P. A. Crooks and B.
Rajitha, J Chem Biol., 2016, 9, 97; (b) A. L. Elizabeth, M. V. d. A.
Sinara, V. C. S. Renata, F. d. O. Jamerson, A. d. C. A. Cezar, M. F.
d. S. Rosali, G. d. R. P. Maria, d. R. P. Ivan, O. d. M. Ricardo, B. d.
C. J. Luiz, J. B. d. M. R. Moacyr and d. C. A. d. L. Maria, Eur.J.
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DOI: 10.1039/C8NJ03248C
Graphical Abstract
Synthesis and in vitro cytotoxicity evaluation of β-carboline-linked 2,4-
thiazolidinedione hybrids: Potential DNA intercalation and apoptosis
inducing studies
Ramya Tokala,a Sowjanya Thatikonda,b Sravani Sana,a Phanindranath Regur,c Chandraiah
Godugu,b* Nagula Shankaraiaha*
b
aDepartment of Medicinal Chemistry & Department of Regulatory Toxicology, National
Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
cCSIR-Centre for Cellular and Molecular Biology, Hyderabad-500007, India.
Corresponding authors: Dr Nagula Shankaraiah, E-mail.: shankar@niperhyd.ac.in; and Dr.
Chandraiah Godugu, E-mail.: chandra.niperhyd@gov.in
A series of β-carboline-linked 2,4-thiazolidinedione hybrids were synthesized and studied for their DNA affinity
and cytotoxicity. The most potent compound was 19e with IC50 of 0.97 ± 0.13 µM