Design and Synthesis of DNA-Interactive β-Carboline–Oxindole Hybrids as Cytotoxic and Apoptosis-Inducing Agents

Article abstract of DOI:10.1002/cmdc.201800402

A new series of (E)-3-[(1-aryl-9H-pyrido[3,4-b]indol-3-yl)methylene]indolin-2-one hybrids were synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines, namely, HCT-15, HCT-116, A549, NCI-H460, and MCF-7, including HFL. Among the tested compounds, (E)-1-benzyl-5-bromo-3-{[1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl]methylene}indolin-2-one (10 s) showed potent cytotoxicity against HCT-15 cancer cells with an IC₅₀ value of 1.43±0.26 μm and a GI₅₀ value of 0.89±0.06 μm. Notably, induction of apoptosis by 10 s on the HCT-15 cell line was characterized by using different staining techniques, such as acridine orange/ethidium bromide (AO/EB) and DAPI. Further, to understand the mechanism of anticancer effects, various assays such as annexin V-FITC/PI, DCFDA, and JC-1were performed. The flow cytometric analysis revealed that compound 10 s arrests the HCT-15 cancer cells at the G0/G1 phase of the cell cycle. Additionally, western blot analysis indicated that treatment of 10 s on HCT-15 cancer cells led to decreased expression of anti-apoptotic Bcl-2 and increased protein expression of both pro-apoptotic Bax and caspase-3, -8, and -9, and cleaved PARP with reference to actin. Next, a clonogenic assay revealed the inhibition of colony formation in HCT-15 cancer cells by 10 s in a dose-dependent manner. Moreover, upon testing on normal human lung cells (HFL), the compounds were observed to be safer with a low toxicity profile. In addition, viscosity and molecular-docking studies showed that compound 10 s has typical intercalation with DNA.

Full text of DOI:10.1002/cmdc.201800402

Accepted Article
Title: Design and synthesis of DNA-interactive β-carboline-oxindole
hybrids as cytotoxic and apoptosis inducing agents
Authors: Shankaraiah Nagula, Ramya Tokala, Sowjanya Thatikonda,
Sravani Sana, Usha Sree Vanteddu, and Chandraiah Godugu
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To be cited as: ChemMedChem 10.1002/cmdc.201800402
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800402
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10.1002/cmdc.201800402
ChemMedChem
FULL PAPER
Design and synthesis of DNA-interactive β-carboline-oxindole
hybrids as cytotoxic and apoptosis inducing agents
Ramya Tokala,[a] Sowjanya Thatikonda,[b] Usha Sree Vanteddu,[a] Sravani Sana,[a] Chandraiah
Godugu,[b]* Nagula Shankaraiah[a]*
Abstract: A new series of (E)-3-((1-aryl-9H-pyrido[3,4-b]indol-3-
yl)methylene)indolin-2-one hybrids have been synthesized and
evaluated for their in vitro cytotoxic activity against a panel of
selected human cancer cell lines namely, HCT-15, HCT-116, A549,
NCI-H460, MCF-7 including HFL. Among the tested compounds,
(E)-1-benzyl-5-bromo-3-((1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-
b]indol-3-yl)methylene)indolin-2-one (Compound 10s) showed
potent cytotoxicity against HCT-15 cancer cells with an IC50 value of
1.43 ± 0.26 μM and GI50 value of 0.89 ± 0.06 μM. Notably, induction
of apoptosis by 10s on HCT-15 cell line was characterized by using
different staining techniques such as acridine orange/ethidium
bromide (AO/EB) and DAPI. Further, to understand the mechanism
of anticancer effects various assays such as annexin V-FITC/PI,
DCFDA, and JC-1were performed. The flow cytometric analysis
revealed that compound 10s arrests the HCT-15 cancer cells at the
G0/G1 phase of cell cycle. Additionally, western blot analysis
indicated, the treatment of 10s on HCT-15 cancer cells led to
decreased expression of anti-apoptotic Bcl-2 and increased protein
expression of both pro-apoptotic Bax and caspase-3, 8, 9 and
cleaved PARP with reference to actin. Next, clonogenic assay
revealed the inhibition of colony formation in HCT-15 cancer cells by
10s, in a dose-dependent manner. Moreover, the compounds when
tested on normal human lung cells (HFL) were observed to be safer
with low toxicity profile. In addition, viscosity and molecular docking
studies showed that compound 10s having typical intercalation with
DNA.
ajor target in cancer therapy due to its vital role in cell division
(replication), maintenance (transcription), and new chemical
entities (NCEs) targeting DNA can be remarkable
representatives as potent chemotherapeutics.[3,4] Beyond this,
evading apoptosis stands out as emerging hallmark of cancer[5]
and as a result the compounds inducing apoptosis can evolve as
potential anticancer agents. Considering these facts, the search
towards the development of novel compounds that would target
DNA and induce apoptosis plays an effective role in cancer
therapy.
The β-carboline alkaloids were naturally obtained from seeds
of Peganum harmala (Zygophillaceae) and they contain tricyclic
pyrido[3,4-b] indole ring system.[6] These alkaloids were known
to exert their pharmacological and medicinal properties[7] like
anxiolytic (Abecarnil), anti-alzheimer, anticonvulsant, antifungal,
antimicrobial, antiplasmodial, antiviral, antiplatelet, and
antimutagenic effects along with antitumor activity.[8] This class
of heterocycles was known to exert their activity through variable
mechanisms such as i) interaction with DNA[9], ii) action on
CDK’s,[10-16] and iii) inhibition of topoisomerase I & II.[17] Apart
from these, a mechanism of photocleavage of DNA has also
been reported.[18] Recently, a harmine (A, Figure 1) derivative
CM16 (B, Figure 1) was reported to inhibit translation step of
protein synthesis.[19] 3-Formyl-9H-β-carboline and diversity at
C1-position was reported as an aldo-X bifunctional building
block (AXB3s) by target-oriented synthesis.[20] DNA targeting
anticancer agents show their efficacy by recognition and binding
to DNA through intercalation via G-C base pairs such examples
are Mana-Hox (C, Figure 1), doxorubicin (Hodgkin’s sarcoma)
and dactinomycin (Ewing’s sarcoma).[21]
Introduction
On the other hand, 2-oxindole is a well-known “privileged
heterocyclic scaffold”[22] and displays versatile actions both in
terms of biology and pharmacology. Oxindoles show their potent
anticancer activity through inhibition of diverse kinases like
cyclin-dependent kinase (CDKs) and tyrosine kinases.[23]
Oxindole containing marketed drug sunitinib (D, Figure 1)
shows multi-targeted kinase inhibition and is in clinical use for
the treatment of renal carcinoma and imatinib-resistant
gastrointestinal stromal tumour (GIST).[24] This is the first cancer
drug that has been approved simultaneously for two indications.
Later, investigational new drug candidates such as SU5416 (E,
Figure 1) and SU6668 were also evaluated for their cytotoxic
potential against different tumors. FDA approved sunitinib and
investigational drug candidates like SU5416, SU6668 have been
synthesized based on C3 modification of oxindole moiety.
Moreover, Kamal and co-workers have also reported
imidazopyridine-oxindole conjugates as potential cytotoxic
agents against 50 cancer cell lines.[25a] Hence, the C3
functionalization of oxindole acts as an attractive site for the
development of new potent molecules along with structural
modifications at C5 and N1 positions. [25, 22]
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 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. Despite of the chemotherapeutic agents being
currently available, the tolerance, resistance and the unwanted
toxic effects towards chemotherapy demands for necessity in
the development of new anticancer agents. DNA, till date is a m-
[a]
R. Tokala, U. S. Vanteddu, S. Sana, Dr. N. Shankaraiah
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research (NIPER)
Hyderabad 500 037, India
E-mail: shankar@niperhyd.ac.in
[b]
S. Thatikonda, Dr. C. Godugu
Department of Regulatory Toxicology
National Institute of Pharmaceutical Education and Research (NIPER)
Hyderabad 500 037, India
E-mail: chandra@niperhyd.ac.in
Supporting information for this article is given via a link at the end of the
document.
1
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Results and Discussion
Chemistry
The β-carboline-linked oxindole hybrids 10a–v were synthesized
in convergent approach by utilizing the multifaceted
a
Knoevenagel condensation reaction between variously
substituted oxindoles 3a–d and 1-aryl-9H-pyrido[3,4-b]indole-3-
carbaldehydes 9a–g as depicted in Scheme 1. The 2-oxindoles
3a–d were synthesized according to the previous reports.[22] The
substituted isatins 1a–c were N-alkylated using different
alkylating agents in the presence of base followed by
subsequent Wolff-Kishner reduction with hydrazine hydrate in
reflux conditions to furnish the corresponding 2-oxindoles 3a–d
in overall good yields.[27, 28] Next, another component, 1-aryl-9H-
pyrido[3,4-b]indole-3-carbaldehydes 9a–g were synthesized
29]
according to the reported methods.[9b,
The commercially
available L-tryptophan 4 was esterified using thionyl chloride in
methanol solvent. Thus obtained, L-tryptophan ester 5 was
subjected to Pictet-Spengler reaction with different aldehydes
employing a catalytic amount of TFA. The diastereomers 6a–g
thus obtained was aromatized using KMnO4 followed by
reduction of ester functionality with LiAlH4 provided the
corresponding alcohols 8a–g. Then, the alcohols were
subsequently oxidized into their corresponding aldehydes 9a–g
by activated MnO2. Finally, the desired hybrids 10a–v were
accomplished by employing Knoevenagel condensation reaction
between 9a–g and 3a–d with a catalytic amount of piperidine as
a base. It is important to note that all the final compounds were
obtained and characterized as single isomers and solids by
simple filtration.
Figure 1. Structures of biologically active β-carbolines (A–C), oxindoles (D &
E), and newly designed hybrids 10a–v.
Molecular hybridization is a tool in favor of adjoining two
distinct biologically important chemical entities covalently into
one novel hybrid towards the discovery of desired potent hybrid
molecules.[22] Moreover, hybridization of such two dissimilar
scaffolds with a different mechanism of actions might result in
synergistic effects with improved efficacy and also minimize the
off-target effects. This pharmacophoric alteration presents in the
hybrids with different or dual modes of action possessing high
affinity and selectivity.[26] In this regard, considering the
therapeutic importance of formyl-9H-β-carboline and 2-oxindoles
towards our venture for the synthesis of potent anticancer
agents, herein we have designed hybrids of these two
pharmacophores with structural diversity by employing
Knoevenagel condensation (Figure 2).
One of the representative compounds 10a was confirmed
1
using H NMR by the appearance of a characteristic singlet at δ
7.82 ppm which accounts the proton after condensation reaction.
The two signals at δ 11.92 and 10.55 ppm account for -NH-
protons on oxindole and 9H-indolic proton of β-carboline. All the
remaining protons lie in the range 6.77-9.35 ppm of the aromatic
region. Next, the analysis of 13C NMR depicts the presence of
carbonyl carbon of oxindole at δ 170.3 ppm. All other aromatic
carbons of 10a were in the range of aromatic region δ 79.6-
143.5 ppm. Similarly, the other derivatives 10b–v were well
matched with their respective structures (1H, 13C NMR & FT-IR).
Figure 3. Determination of configuration for representative compound 10b by
NOE analysis.
Figure 2. Molecular hybridization between β-carbolines and 2-oxindoles.
2
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Scheme 1. Design and synthesis of β-carboline linked hybrids 10a–v.
The HRMS (ESI) showed the characteristic protonated [M +
H]+ peak to their corresponding molecular formula. To determine
the obtained isomer, one of the hybrids 10b was subjected to
NOE experiments. The proton NMR obtained was assigned by
gDQFCOSY (see ESI). The presence of NOEs between H5-H6
and H4-H12 suggested that the isomer obtained correspond to
E-configuration (Figure 3).
normal lung fibroblasts (HFL-1) cell lines by employing the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The results from the in vitro anticancer studies revealed
that the compounds exhibited diverse anticancer properties and
are inferred in Table 1. Notably, these compounds exhibited
good to moderate cytotoxicity in the tested cancer cell lines.
From the close analysis of the IC50 values, it was observed that
compound 10s was more significant in inducing cytotoxicity on
all the cancer cells and found to be most potent against HCT-15
colon cancer cell line with IC50 value 1.43 ± 0.26 µM whereas in
A549 lung cancer cell line 50% inhibition was observed at 2.50 ±
0.06 µM. This compound was also found to be active in breast
In vitro cytotoxic activity
The newly synthesized hybrids 10a–v were evaluated for their in
vitro cytotoxic activity against a panel of human cancer cell lines
namely HCT-15, HCT-116, A549, NCI-H460, MCF-7 and a
3
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Table 1. IC50 (µM) values[a] against human cancer cell lines by derivatives10a-v by MTT assay
Compound
HCT-15[b]
> 50
HCT-116[c]
42.11±5.62
A549[d]
NCI-H460[e]
31.21±4.50
MCF-7[f]
HFL-1[g]
-
10a
35.13±4.71
45.41±3.51
10b
4.80±0.24
3.12±0.19
45.80±7.32
> 50
10.32±0.72
9.11±0.21
48.27±8.65
43.60±5.80
37.65±6.38
38.43±6.12
48.11±9.28
31.89±6.21
34.28±0.35
9.39±0.17
36.85±0.48
41.67±9.32
>50
6.78±0.20
9.21±0.51
37.78±3.91
44.12±3.32
48.57±4.12
49.76±1.92
37.21±2.31
39.13±4.43
>50
12.86±0.1
9.10±0.40
37.10±4.91
33.65±4.81
> 50
14.62±0.58
9.19±0.07
33.26±5.54
> 50
27.29±4.20
10c
31.04±5.83
10d
-
10e
-
10f
47.33±4.64
47.12±5.62
35.23±2.81
42.25±4.56
31.25±0.25
2.58±0.02
32.87±0.12
39.57±4.91
35.21±0.21
36.24±0.48
30.12±0.41
38.90±0.05
39.32±5.27
1.43±0.26
48.57±13.27
18.72±0.12
> 50
44.45±3.81
35.64±2.21
35.46±3.62
44.22±2.25
36.84±0.78
12.05±1.27
38.12±0.32
48.18±4.55
38.74±0.45
32.21±0.22
35.85±0.82
31.98±1.28
39.84±5.91
3.36±0.08
48.12±3.53
33.83±2.76
44.65±2.24
46.25±1.43
˗
-
10g
47.21±23.6
33.42±4.53
40.26±5.57
31.98±0.78
8.47±0.03
33.82±0.85
45.78±3.34
34.20±0.12
35.41±0.78
36.71±0.76
32.21±0.87
34.87±3.53
6.71±0.2
-
10h
-
10i
-
10j
-
10k
10.97±0.46
34.21±0.56
41.26±1.56
37.12±0.67
31.97±0.74
37.58±0.46
34.41±0.24
36.46±3.38
2.50±0.06
41.45±3.32
14.46±1.07
40.12±3.64
20.84±3.41
1.24±0.11
34.45±4.21
10l
-
10m
10n
-
-
10o
32.84±0.79
33.51±0.53
36.21±0.72
32.58±3.92
4.18±0.16
47.67±9.48
21.16±0.60
43.48±4.91
31.88±1.16
2.18±0.15
-
10p
-
10q
-
10r
-
10s
49.79±1.17
10t
41.78±1.74
29.76±0.50
37.78±0.71
45.41±3.6
3.57±0.32
-
10u
30.30±6.94
10v
-
Harmine [h]
Sunitinib [h]
37.91±8.02
1.38±0.20
43.61±2.88
˗
[a] 50% Inhibitory concentration after 72 h of drug treatment, [b,c] Human colon cancer, [d,e] Human lung cancer , [f] Human breast cancer, [g] Human normal
lung fibroblasts, [h] Reference compound. All the values are expressed as Mean ± SEM in which each treatment was performed in triplicate wells.
cancer MCF-7 cell line with an IC50 value of 3.36 ± 0.08 µM as
compared to the standard compound harmine. Interestingly, the
compound 10s exhibited the least toxicity on normal HFL-1 cell
line with IC50 value 49.79±1.17 µM. This indicates that the
compound 10s was selective towards the cancer cells rather
than normal cell lines tested as represented in Figure 4. While
the compound 10k also displayed remarkable cytotoxicity with
an IC50 value of 2.58 ± 0.02 µM against the colon cancer cell line
HCT-15. Among the series tested, the compounds 10b and 10c
also showed considerable cytotoxicity in all the cancer cell lines.
It is evident from the IC50 values (Table 1) that C1 position of
the β-carboline displayed better cytotoxic activity with different
phenyl substitutions (10b, 10c, 10k, 10s, and 10u) rather than
the substitutions with heterocyclic entities (10e–10g). C-5
substituted hybrids (X = Cl or Br) were found to be potent than
unsubstituted (X = H) derivatives 10a, 10e, 10j, 10n, and 10t.
Compound 10s, with dimethoxy phenyl at C-1 position of β-
carboline and N-benzylated, a bromine at C-5 position of
oxindole proved to be potent among all the hybrids evaluated.
With respect to promising in vitro cytotoxicity results (Table 1),
the significant activity of newly synthesized hybrids 10a–v could
also be attributed towards C1 modification of β-carboline by
varying different aromatic/heterocyclics in contrast with harmine
which bears a methyl group at this position. These biological
results could suggest a shore up in anticancer studies that may
lead to the discovery of new cytotoxic agents with better activity.
4
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10.1002/cmdc.201800402
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10k and 10u were also efficacious in all the tested cancer cell
lines. The remaining compounds were found to be moderately
active in growth inhibition of cancer cells. In overall comparison
to the standard compound harmine, approximately 20 fold
growth inhibitory effects were observed with the compound 10s
against the HCT-15 cell line (Figure 5). Hence, the compound
10s was further selected for mechanistic studies to disclose the
mechanism of cell growth inhibition at the molecular level in
HCT-15 cell lines. These results may offer a holdup and
demonstrated promising evidence in the treatment of cancer that
may lead to the breakthrough of identifying novel agents in
inhibiting the proliferation of cancer cells.
Figure 4. Cytospecificity of compound 10s towards cancer cells compared to
normal fibroblasts HFL-1. Data was expressed as mean ± S.E.M. (n = 3).
****p<0.0001 versus normal cell line.
Apoptosis detection studies
In vitro growth inhibitory activity
Morphological
microscope
observations
using
phase
contrast
The potent compounds from cytotoxicity studies of 10b, 10c, 10k,
10s, and 10u were further assessed for their growth inhibitory
potential in human cancer cell lines and a normal cell line by
MTT assay. The GI50 (µM) values (concentration required to
inhibit 50% growth of cancer cells) of the tested compounds
were listed in Table 2. It is noticeable from the results that some
of the compounds have shown moderate to potent growth
inhibition against the tested cancer cell lines. Among the series,
compound 10s was found to be the most potent with
considerable cell growth inhibition with GI50 values of 0.89 ± 0.06
µM in HCT-15 cell lines and found to be efficacious than the
parent molecule harmine with GI50 of 18.92 ± 4.55 µM. Also, the
compound 10s was found to be effective towards the lung
(A549) and breast (MCF-7) cancer cells with significant GI50
values of 1.24 ± 0.16, and 1.77 ± 0.17µM, respectively. This
compound effectively discriminated the normal and cancer cells
by showing minimal toxicity towards the normal lung fibroblast
cell line with GI50 value 49.4 ± 0.39 µM, approximately 50 fold
difference was observed to be an on par with HCT-15 colon
cancer cell line. Furthermore, the other derivatives tested among
this series for potential cell growth inhibition, the compounds 10c,
10k, displayed compelling growth inhibitory activity in colon
cancer HCT-15 cells indicating the promising and preferential
specificity towards the colon cancer. In addition, the compounds
The induction of apoptotic bodies’ formation has always been an
ultimate choice in developing anti-cancer therapeutics.[30] To
scrutinize whether the treatment with the most active compound
10s may lead to the induction of apoptosis, HCT-15 cells were
treated with the compound 10s. After 72 h of incubation, images
were captured in phase contrast microscope which depicted the
loss of cell–cell adhesion, cell wall deformation, shrinkage of
cells and resulted in decreased number of viable cells with
increased concentration, whereas these characteristic
morphological features were lacking in control cells as shown in
Figure 6.
Acridine orange/ethidium bromide (AO/EB) staining
Acridine orange/ethidium bromide (AO/EB) staining assay was
carried out to distinguish between live, apoptotic and necrotic
cells. AO can pervade the intact cell membrane and stain the
nuclei green, whereas EB can only stain the nuclei of cells that
have lost membrane integrity. It can be inferred from Figure 7
that the control cells exhibited normal morphology and appeared
green in color. Whereas the compound 10s treated cells clearly
unveiled the morphological changes such as cell shrinkage,
membrane blebbing, chromatin condensation and apoptotic
body formation, dose–dependently implying that the compound
had prompted apoptosis in HCT-15 cells.
60
40
20
DAPI nucleic acid staining
DAPI (4ꞌ,6-diamidino-2-phenylindole) is a fluorescent dye that
binds sturdily to the nucleus and distinguishes the nuclear
damage or chromatin condensation. The DAPI stains the
apoptotic cells where apoptotic nuclei appear bright colored due
to the condensed nucleus which is
a typical apoptotic
****
****
****
****
characteristic. Therefore, to detect nuclear damage or chromatin
condensation induced by the compound 10s in the HCT-15 cell
line, DAPI staining technique was performed in compliance with
previously reported method.[22] From the results, it was noticed
that the nuclear structure of untreated control cells was intact,
whereas with the treatment the compound had induced
apoptosis with the advent of nuclear changes like condensed,
****
0
9
1
0
6
7
-
5
1
-
4
5
6
4
1
1
L
-
F
-
F
T
A
C
H
T
-
H
C
I
M
C
H
C
H
N
Figure 5. Growth inhibitory potential of compound 10s towards cancer cells
compared to normal fibroblasts HFL-1. Data was represented as mean ±
S.E.M. (n=3) ****p<0.0001 versus normal cell line.
5
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horse-shoe shaped, pyknotic or fragmented bright nuclei from
0.25 to 2.5 µM concentrations (Figure 8).
Table 2. In vitro growth inhibitory concentration (GI50 µM) [a] against human cancer cell lines by MTT assay
Compound
10b
HCT-15[b]
1.63±0.19
1.64±0.06
1.62±0.01
0.89±0.06
10.14±2.36
18.92±4.55
0.77±0.05
HCT-116 [c]
7.66±0.7
7.94±0.2
8.52±0.17
2.52±0.38
16.46±1.0
23.80±1.5
ND
A549 [d
NCI-H460 [e]
7.38±1.9
MCF-7 [f]
7.47±0.21
1.66±0.11
6.05±0.20
1.77±0.17
19.0±3.72
20.6±2.47
0.51±0.08
HFL-1 [g]
18.02±1.62
17.80±1.51
26.97±4.34
49.4±0.39
19.8±3.51
28.1±2.24
ND
4.05±0.13
6.53±0.22
8.10±0.20
1.24±0.16
7.08±0.61
10.48±0.17
0.52±0.08
10c
6.51±0.29
3.38±0.21
3.55±0.23
10.52±0.82
35.91±0.86
0.84±0.14
10k
10s
10u
Harmineh
Sunitinib
[a] 50% Growth concentration after 72 h of drug treatment, [b,c] Human colon cancer, [d,e] Human lung cancer, [f] Human breast cancer, [g] Human normal lung
fibroblasts, [h] Reference compound. All the values are expressed as Mean ± SEM in which each treatment was performed in triplicate wells.
Figure 6. Morphological changes observed in colon cancer cells (HCT-15), cells were treated with and without compound 10s 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.
Figure 7. AO/EB staining in colon cancer cell line HCT-15, cells treated with and without compound 10s 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.
6
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Effect on ROS generation
2',7'-dichlorofluorescein (DCF) in the presence of ROS. In our
results, the compound 10s induced the significant generation of
ROS dose–dependently as compared to control cells, which
exhibited a fruitful avenue for further apoptotic studies (Figure 9).
The bar diagram also clearly demonstrated the significant
induction of ROS levels in 10s treated cells compared to
untreated control cells.
The excess ROS generation induces irreversible oxidative
modification of cellular macromolecules which include lipid,
protein or DNA, which disrupt the intracellular redox homeostas-
is and could lead to apoptotic cell death.[31] Hence, to determine
if a pre-treatment with compound 10s could affect the ROS
generation the intracellular ROS levels were determined by
using the fluorescent dye DCFDA, which is readily oxidized to
Figure 8. Nuclear morphology of cancer cells after DAPI staining. HCT-15 cells treated with and without compound 10s 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.
Control
0.5µM
1 µM
2.5 µM
Figure 9. DCFDA staining in colon cancer cell line HCT-15, cells treated with and without compound 10s at various concentrations 0.5, 1 and 2.5 µM for 72 h and
compared with control cells. The images were captured with fluorescence microscope at 200X. The relative DCF fluorescence quantification from the above cells.
7
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Clonogenic assay
Flow-cytometry analysis
The clonogenic assay manifested the effect of compound 10s on
the colony-forming capacity of exponentially growing HCT-15
cancer cells. The results evidenced that the treatment inhibits
colony formation potential in the HCT-15 cell population in a
concentration–dependent manner. After treatment with various
concentrations (0.25, 0.5, 1µM), we observed the formation of
colonies in one week time whereas in treatment ascertained
clear reduction in the number of colonies as shown in Figure 10.
Hence, these results specify the significance of compound 10s
in inhibiting the colony formation potential. The total colonies
were counted by a molecular imaging system (Vilber Fusion Fx
software) and the values were represented as a percent colony
forming ability.
Effect on mitochondrial membrane potential (ΔΨm)
The mitochondria play a fundamental task in initiating the
intrinsic pathway of apoptosis as it is the foremost target of
cellular oxidative stress, which impedes the electron transport
chain, leading to reactive oxygen species (ROS) generation and
[32]
collapse of mitochondrial membrane potential (ΔΨm).
Therefore, to appraise the impact of compound 10s on
mitochondria, the ΔΨM was measured. Treatment of HCT-15
cells with compound 10s caused a noteworthy reduction in the
ΔΨm compared to control cells as shown in Figure 11. The
compound 10s triggered the depolarization of ΔΨm dose-
dependently compared to the control which is illustrating the
fatal effects of the compound on mitochondria, thereby assisting
the later events of apoptosis.
Figure 10. Effect of compound 10s on colony forming ability of exponentially growing colon cancer cells by clonogenic assay. HCT-15 cells were treated with
compound 10s at 0.25, 0.5,1 µM and allowed to form colonies in fresh medium for 7 days. The images were captured and the colonies were counted with vilber
fusion chemdoc imaging system.
Figure 11. Effect on mitochondrial membrane potential. HCT-15 cells were treated with various concentrations of compound 10s for 72 h and JC-1 staining was
performed. The control represents the cells without compound 10s treatment.
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Cell cycle analysis
to 80.61% which gradually subsided with increasing doses. The
G0/G1 phase arrest was distinctive at
concentrations.
1 and 2.5 µM
From the in vitro screening results, it was apparent that the
compound 10s showed significant cytotoxicity against colon
cancer HCT-15 cell lines. Hence, in order to disclose whether
this cytotoxicity may be due to the cell cycle arrest, analysis of
cell cycle was performed. Cells were treated with compound 10s
at concentrations ranging from 0.25 to 2.5 µM for 48 h, and then
the cells were stained with propidium iodide and further
analyzed by using BD FACSVerseTM flow analyzer. The results
from Figure 12 indicated that the HCT-15 untreated control cells
showed 68.71% cells in G0/G1 phase, whereas compound 10s
treatment resulted in elevation in G0/G1 population from 68.7%
Annexin V-FITC/PI assay
Annexin V-FITC/PI dual staining assay was performed using
flow cytometry to quantify the apoptotic cell death. HCT-15 colon
cancer cells were treated with compound 10s at different
concentrations of 0.25, 0.5, 1.0 and 2.5 μM for 72 h. Results
from Figure 13 indicated the percentage of apoptotic cells and
inferred to the significant early and slight late apoptosis induction
(Annexin V positive/PI negative) at both 1.0 and 2.5 μM
concentration in comparison to control.
Control
0.25 µM
0.5 µM
Sub G1:0.45
G0/G1:75.52
S:8.68
Sub G1:0.04
G0/G1:68.71
S:10.45
Sub G1:0.44
G0/G1:75.87
S:6.87
G2/M:15.02
G2/M:20.34
G2/M:16.49
1 µM
2.5 µM
Sub G1:0.58
G0/G1:80.61
S:5.97
Sub G1:0.18
G0/G1:79.82
S:5.53
G2/M:12.84
G2/M:14.08
Figure 12. Effect of 10s on cell cycle progression of colon cancer cells. HCT-15 cells were treated with compound 10s at 0.25, 0.5, 1, 2.5 µM concentration and
cell cycle analysis was performed after 48 h. The analysis of cell cycle distribution was performed by using propidium iodide staining method.
Figure 13. Effect of compound on cell apoptosis as measured by Annexin V-FITC/propidium iodide staining assay. HCT-15 cells were treated with compound 10s
ranging from 0.25 to 2.5 µM concentration for 72 h. Then 10,000 cells from each sample were analyzed by flow cytometry. The percentage of cells positive for
Annexin V-FITC 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.
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Western blotting
eractions of nitrogen on C ring of β-carboline with dG6. Beyond
this, benzylated oxindole moiety also shows strong π-π stacking
interactions with dC5. These interactions also well matched with
co-crystal structure indicating the intercalation into the DNA.
We quested for the molecular mechanisms of apoptosis
induction in colon cancer cells. Hence, we assessed the
regulation of intrinsic apoptosis accompanied by Bcl-2, Bax,
cleaved caspase-3, and 9 proteins, which are intricated in the
mitochondrial apoptosis pathway, thus triggers the caspase-
dependent apoptotic response. Results from the Figure 14
stipulated that compound 10s treatment led to the dose-
dependent elevated expression of Bax and cleaved caspase-3,
9 in HCT-15 cells; hence we also corroborated the effect of
compound 10s on the extrinsic apoptotic pathway. As a result,
we found increased expression of cleaved caspase-8, which is a
key protease, executes apoptosis by cleaving PARP[33] and
consistent clear increase in the expression of PARP along with
caspase-8. Thus the compound 10s treatment prompted the
activation of both intrinsic and extrinsic pathway.[34]
In order to study the stability of the DNA-ligand complex,
Molecular Dynamics (MD) simulations were performed for the
best-scored pose of compound 10s for 20 ns. The images of
10s-DNA complex throughout the 20 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 was
depicted (Figure 2, ESI). From the plot, it can be contemplated
that the complex does not show major deviation from the initial
position over the time period of 20 ns MD simulation. Even the
docked pose of 10s was reasonably stable up to 20 ns MD
simulation. The results obtained from molecular simulation
studies were compatible with in vitro cytotoxicity and relative
viscosity results.
Molecular docking studies
Relative viscosity studies
Most of the β-carboline derivatives were reported to show their
action by interacting into DNA.[9] Therefore, to signify the
interactions of the synthesized ligands towards DNA and to
denote the mode of interaction, molecular modeling studies were
performed on DNA hexamer d(CGATCG) using XP Glide[35]
(Schrödinger, 2017) with default settings. The docked pose of
the compound 10s was depicted in Figure 15, which
demonstrates the intercalation of planar β-carboline moiety
towards GC base pairs of DNA and C1 substituent aligns at right
angles and assists intercalation. The oxindole moiety orients
itself perfectly in between GC base pairs. It is also strongly
assisted by strong π-π stacking interactions of β-carboline
towards dG6, dG8, dC7 residue, along with additional π-cation int-
Relative viscosity experiments furnish the confirmation of
interaction of newly synthesized hybrids with DNA. Changes in
viscosity due to interactions of compounds with DNA can be
measured. Intercalation of a compound into DNA results in the
increase in relative viscosity due to enhancement in the axial
length of DNA helix to naturalize the hybrid. EtBr is a classical
measured DNA intercalator which corresponds to the increase in
viscosity.[36] Covalent binding to DNA ends up in the decrease in
relative viscosity values whereas no; or little change is observed
in case of groove binding.[17] Hoechst 33258 is a well-known
groove binder and does not affect the relative viscosity
(A)
BCL-2
Cleaved caspase 3
Cleaved Caspase
8
BAX
Cleaved PARP
Cleaved Caspase 9
β-Actin
β-Actin
Control
0.5
1
2.5
(µM
)
Control
0.5
1
2.5
(µM)
(B)
.
Figure 14. Effect on apoptotic protein expression in HCT-15 cells. Cells were treated with compound 10s for 48 h and then harvested. Total cell lysates were
prepared and 30 µg proteins was subjected to SDS-PAGE followed by western blot analysis and analysed by chemiluminescence detection. (A) Western blotting
analysis of Bcl-2, Bax, and cleaved caspase-9, 3, 8 and cleaved PARP (B) Bar graphs representing the fold changes in the protein expression compared to
control. Data expressed as mean ± SEM (n=3). **P<0.01 and ***P < 0.001, ***P < 0.0001 significant vs. control. Band intensities were quantified using NIH Image
J software. The western blots shown here are representative of three independent experiments with similar results.
10
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Figure 15. Intercalation of compound 10s into d(CGATCG). a) Side view; b)
Top view 10s showing residual interactions with G-C base pairs of DNA. DNA
is represented as tube and compound 10s is represented in yellow color.
Figure 17. Graphical representation of relative viscosity studies using
compound 10s and 10k with Harmine, Hoechst-33258, and Ethidium Bromide
as reference standards.
Conclusion
In conclusion, a new series of β-carboline-linked oxindole
hybrids have been successfully synthesized and evaluated for
their in vitro cytotoxic potential against a panel of selected
cancer cell lines and a normal human cell line. The results of
prefatory screening using MTT assay suggests that most of the
compounds showed cytotoxic potential at a concentration less
than 50 µM. Interestingly, compound 10s has produced notable
cytotoxicity among all the tested cell lines and found to be potent
on HCT-15 (colon cancer) cell line with an IC50 value of 1.43 ±
0.26 µM. The GI50 values also suggest the compound 10s is
potent in comparison to other compounds with a value of 0.89 ±
0.06 µM. The staining studies using AO/EB, DAPI revealed the
events of apoptosis and elevated generation of reactive oxygen
species (ROS) was indicated by DCFDA staining. Colony
formation in HCT-15 was inhibited by compound 10s in a
concentration-dependent manner. Moreover, the flow cytometric
studies using compound 10s in HCT-15 cell line in a dose-
dependent manner gave the following observations, i) Cells
were arrested at the G0/G1 phase of cell cycle. ii) Annexin V-
FITC/PI studies confirmed the significant early and late
apoptosis. iii) The collapse of mitochondrial membrane potential
(ΔΨM) was evidenced. Further, western-blotting analysis
indicated elevated expression of caspase-3, 8, 9, cleaved PARP,
Bax, and decrease in levels of Bcl-2 which clearly indicate
expression of apoptosis-related proteins. DNA viscosity and
molecular modeling studies indicate the intercalation into DNA
base pairs. Principally, compound 10s showed specificity
Figure 16. Snapshots 10s-DNA complex at 5 ns (A), 10 ns (B), 15 ns (C) and
20 ns (D) timescale.
when bound to DNA.[37] Addition of compounds 10s and 10k
results in the increase in viscosity in comparison to the control in
concentration-dependent manner, thereby accounting for the
DNA intercalation as shown in Figure 17. The graph is plotted
between concentration (compound/CT-DNA) and viscosity
((ŋ/ŋo)1/3 on X and Y axis respectively with harmine, EtBr and
Hoechst 33258 as standards. The viscosity results are
consistent with molecular modeling studies.
towards the cancer cell line in
a cell viability assay in
comparison to normal fibroblasts HFL-1 cells. Overall, these
current studies imply that these new hybrids have the potential
to be developed as lead molecules and suitable structural
modifications may ensue in promising anticancer agents.
11
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37 oC. The formazon product quantity was measured by using a
spectrophotometric microtiter plate reader (Spectra Max, M4 Molecular
devices, USA) at 570 nM wavelength. The day 0 absorbance was
subtracted from the 72 h plates for determining growth inhibition and data
were plotted as a percentage of untreated control.
Experimental Section
Chemistry
General. All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Analytical thin layer
chromatography (TLC) was performed on MERCK precoated silica gel
60-F254 (0.5 mm) aluminum plates. Visualization of the spots on TLC
plates was achieved by UV light. 1H and 13C NMR spectra were recorded
on Bruker 500 MHz by making a solution of samples in the CDCl3 solvent
(or) DMSO using tetramethyl silane (TMS) as the internal standard.
Chemical shifts for 1H and 13C are reported in parts per million (ppm)
downfield from tetra methyl silane. Spin multiplicities are described as s
(singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). Coupling constant (J) values are reported in hertz (Hz).
HRMS were determined with Agilent QTOF mass spectrometer 6540
series instrument. Wherever required, column chromatography was
performed using silica gel (60-120). The reactions wherever anhydrous
conditions required are carried under nitrogen positive pressure using
freshly distilled solvents. All evaporation of solvents was carried out
under reduced pressure using rotary evaporator below 45 oC. Melting
points were determined with an electro thermal digital melting point
apparatus IA9100 and are uncorrected. The names of all the compounds
given in the experimental section were taken from Chem Bio Draw Ultra,
Version 12.0.
Morphological observations. HCT-15 cells were plated in 12 well
culture plates with a cell density of 1x105 cells/mL and allowed to adhere
for overnight.[39] The cells were incubated with the 0.25, 0.5, 1, 2.5 µM
concentrations of the compound 10s. After 72 h treatment, cells were
observed for the morphological changes and photographs were taken
under a phase contrast microscope (Nikon, Inc. Japan).
Acridine orange ethidium bromide (AO/EB) staining. HCT-15 cells
were plated at a concentration of 1x106 cells/ml and treated with different
concentrations of compound 10s and the plates were incubated for 72 h.
10 µl of fluorescent dyes containing Acridine Orange (AO) and Ethidium
Bromide (EB) were added into each well in equal volumes (10 µg/ml)
respectively then the cells were visualized immediately under
fluorescence microscope (Nikon, Inc. Japan) with excitation (488 nM)
and emission (550 nM) at 200x magnification.
DAPI nucleic acid staining. Morphological changes in nucleus were
observed through DAPI staining. After treatment with compound 10s for
72 h, colon cancer cell line HCT-15 cells were washed with PBS and
permeabilized with 0.1% Tween 20 for 10 min followed by staining with 1
µM DAPI. Control and treated cells were observed with fluorescence
microscope with excitation at 359 nM and emission at 461 nM using
DAPI filter at 200x magnification.
General
procedure
for
(E)-3-((1-aryl-9H-pyrido[3,4-b]indol-3-
yl)methylene)indolin-2-one (10a–v). To a mixture of oxindole (3a–d, 1
equiv.), 1-aryl-9H-pyrido[3,4-b]indole-3-carbaldehyde (9a–g, 1.1 equiv.),
in ethanol, catalytic amount of piperdine was added and refluxed at 80 oC
till complete consumption of the starting materials as determined by TLC.
The reaction mixture was cooled, precipitate thus formed was filtered,
washed using ethanol to yield pure (E)-3-((1-aryl-9H-pyrido [3,4-b]indol-
3-yl)methylene)indolin-2-one in moderate to good yields. All the products
were obtained by simple filtration.
DCFDA staining. DCFDA staining was performed to determine the
reactive oxygen species (ROS) levels as per reported method[37] with
slight modifications. For this experiment, HCT-15 cells were plated at
cell density of 3.5
×
105 cells/well into 12-well plates in RPMI
supplemented with 10% FBS. Then the cells were treated with the
compound 10s at various concentrations for 72 h. Then the DCFDA
reagent was added at 10 µM concentration for 15 min and the fluorescent
intensity was captured using fluorescent microscope at 200X
magnification. The relative DCF fluorescence in the cells was measured
by spectramax.
Characterization of the synthesized compounds. A complete list of
compound characterization is available in the supporting information.
Pharmacology
Clonogenic growth inhibition assay. Colon cancer cells HCT-15 were
Cell culture. Cancer cell lines such as colon (HCT-116 and HCT-15),
lung (NCI-H460, A549), breast (MCF-7) were maintained in RPMI media
while normal lung fibroblasts cell line (HFL-1) were maintained in F12k
medium supplemented with 10% fetal bovine serum (FBS) stabilized with
1% antibiotic-antimycotic solution (Sigma) cells were maintained in 5%
CO2 and 98% relative humidity at 37 oC in incubator. When the cells
reached up to 80-90% of confluency, they were sub-cultured using 0.25%
trypsin/1 mM EDTA solution for further passage. The compounds were
dissolved in DMSO to prepare the stock solution of 10 mM. Further
dilutions were made accordingly with respective media to get required
concentration.
passaged in RPMI 1640 supplemented with 10% fetal bovine serum on
o
at 37 C in an atmosphere containing 5% CO2. Exponentially growing
cells were seeded into 12-well culture plates and kept overnight and
treated with the compound 10s. Every 3 days the medium was removed
and replaced with fresh medium. After 7 days of incubation, the colonies
were fixed and stained with 1% crystal violet in methanol for 3 h. The
number of stained colonies were counted under chemdoc imaging
system (Vilber Fusion Fx, France).Colony formation was calculated as a
percentage to untreated control cultures.[40]
Flow cytometric analysis
MTT assay. It is a colorimetric assay that measures the reduction of MTT
(3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide) to
insoluble formazon by mitochondrial succinate dehydrogenase enzyme.
Since reduction of MTT can only happen in metabolically active cells, the
level of activity is the measure of the viability of the cells.[38] Briefly, cells
were seeded in 96-well plates at a density of 1000 to 4000 cells per well
in 100 µl of complete medium and allowed to grow overnight for
attachment onto the wells. Then the cells were treated with various
concentrations of the compounds for a period of 72 h. For cell growth
inhibition analysis after 24 h of cell seeding, before treatment (day 0),
absorbance was measured by adding MTT in few wells. After 72 h
incubation, 100 µl of MTT (0.5 mg/ml) was added and incubated at 37 oC
for 4 h. Then MTT reagent was aspirated and the formazan crystals
formed were dissolved by the addition of 200 µL of DMSO for 20 min. at
Measurement of mitochondrial membrane potential. HCT-15 cells
(1x106 cells/ml) were seeded in 12 well plates and allowed to adhere for
overnight. The cells were incubated with compound 10s at 0.25, 0.5, 1
and 2.5 µM concentrations for 48 h. Cells were collected and washed
with PBS and resuspended in solution of JC-1 (1 µM) and incubated for
30 min in incubator at 37 oC. The cells were washed twice with PBS and
analysed by flow cytometer (BD FACSVerse™, USA).
Cell cycle analysis. Flow cytometric analysis (FACS) was performed to
calculate the distribution of the cell population in various cell cycle
phases. In general the novel compounds exert their cytotoxic or growth
inhibitory effect by arresting the specific checkpoint in cell cycle.[41] Here
HCT-15 cancer cells were incubated with compound 10s at various
12
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10.1002/cmdc.201800402
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concentrations from 0.25 to 2.5 µM for 24 h. Untreated and treated cells
were harvested, washed and fixed overnight in 70% ethanol in PBS at -
20 oC. Fixed cells were pelleted and stained with cell cycle analysis
reagent propidium iodide (50 μg/ml) with RNase A for 20 min at 37 oC in
dark according to the manufacturer instructions and about 10,000 events
were acquired and analyzed on a flow cytometer BD FACSVerseTM (BD
Biosciences, USA).
were estimated based on the ball falling time at 25 oC and densities DNA
solution was prepared in 100 mM Tris-HCl (pH 7.4) and viscosity was
measured while each derivative (5µM) was added to CT-DNA solution
(50 µM). Ethidium bromide, Harmine and Hoechst 33258 at 5 µM
concentration were used as reference standards. Data was represented
1/3
graphically as (ŋ/ŋ0)
vs. the ratio of the concentration of the hybrid to
CT-DNA, where ŋ is the viscosity of CT-DNA in the presence of the
derivative and ŋ0 is the viscosity of CT-DNA solution.
Annexin V assay. It was performed by the method given by Rieger et al.
with slight modifications.[42] Briefly, 1 X 105 cells were seeded in a 12-well
plate and treated with different concentration of compound 10s for 72 h.
Then Untreated and treated cells were harvested and the cells were
processed with Annexin V-FITC and Propidium Iodide (PI) staining
(BioLegend) according to the manufacturer’s instructions. Further, flow
Acknowledgments
The authors are thankful to DoP, Ministry of Chemicals &
Fertilizers, Govt. of India, for the award of NIPER fellowship and
Dr. Gannoju Srinivasulu for his help in the interpretation of NMR
and 2D NMR studies. Dr. N. Shankaraiah gratefully
acknowledges SERB, DST, Govt. of India for research grant
(YSS-2015-001709).
cytometric analysis was performed using
a flow cytometer (BD
FACSVerseTM, USA). Apoptosis and necrosis were analyzed with
quadrant statistics on propidium iodide-negative cells, fluorescein
positive cells and propidium iodide (PI)-positive cells, respectively.
Western blotting. After treatment with various concentrations of
compound 10s in HCT-15 cells for 48 h cells were washed twice in 1X
PBS, then lysed with RIPA buffer which includes protease and
phosphatase inhibitors and incubated on ice for 40 min. Cell lysates
were then centrifuged for 15 min at 13,000 rpm at 4 ºC and the
supernatant was collected. Protein concentration was determined by
bicinchoninic acid method. Equal amounts of protein were separated
using SDS–PAGE and subsequently transferred to nitrocellulose
membrane by using a trans-blot system (Transblot SD: Semidry transfer
cell; Bio-Rad Laboratories) The blots were blocked with 3% BSA in TBS-
T (20 mM Tris– HCl, pH 7.4, 137 mM NaCl, and 0.05 %Tween-20) at
room temperature for 2 h and incubated overnight with the relevant
primary antibody. Immunoreactivity was detected using either an anti-
mouse or anti-rabbit peroxidase-conjugated secondary IgG antibody for 1
hour. To ensure equal loading of protein β-actin was used as an internal
control. Bands were monitored using enhanced chemiluminescence
reagent (Millipore, U.S.A). under chemdoc imaging system (Vilber Fusion
Fx, France).The strength of western blotting bands was determined by
Image J density measurement program.
Keywords: β-Carboline • indoline-2-one • cytotoxicity • DNA
intercalation • flow cytometry
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A series of β-carboline linked oxindole derivatives have been synthesized via knoevenagel condensation. These new compounds
have been evaluated for their in vitro cytotoxic activity and DNA intercalation.
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