Synthesis, antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives

Article abstract of DOI:10.1016/j.bmc.2019.02.002

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.

Full text of DOI:10.1016/j.bmc.2019.02.002

Accepted Manuscript
Synthesis, antiproliferative and apoptosis induction potential activities of novel
Bis(indolyl)hydrazide-hydrazone derivatives
Reddymasu Sreenivasulu, Kotthireddy Thirumal Reddy, Pombala Sujitha, C.
Ganesh Kumar, Rudraraju Ramesh Raju
PII:
S0968-0896(18)31799-1
https://doi.org/10.1016/j.bmc.2019.02.002
BMC 14738
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 October 2018
29 January 2019
1 February 2019
Please cite this article as: Sreenivasulu, R., Reddy, K.T., Sujitha, P., Kumar, C.G., Raju, R.R., Synthesis,
antiproliferative and apoptosis induction potential activities of novel Bis(indolyl)hydrazide-hydrazone derivatives,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.002
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Synthesis, antiproliferative and apoptosis induction potential activities of
novel Bis(indolyl)hydrazide-hydrazone derivatives
a
a
b
b#
Reddymasu Sreenivasulu, Kotthireddy Thirumal Reddy, Pombala Sujitha, C. Ganesh Kumar,
a
and Rudraraju Ramesh Raju *
1
Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar – 522 510, Andhra
Pradesh, India.
2
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Uppal Road, Tarnaka, Hyderabad 500007, Telangana, India.
Abstract:
In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth
inhibition and apoptosis induction characteristics are being strongly screened for their cancer
chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl
bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to
evaluate for their anti- proliferative and apoptosis induction potential against cervical (HeLa),
breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293
cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye
exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further,
mechanistic studies were conducted on compound 14a to understand the biochemical
mechanisms and functional interactions with various signaling pathways triggered in HeLa and
MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the
participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related
kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic
proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen
species. In downstream signaling pathway, activated p53 protein interacted with MAPK
pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases
resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38
on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of
specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125)
prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP

kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing
and were dependent on p53 activity. Based on the results, compound 14a was identified as a
promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo
experiments on anti-proliferative activity.
Keywords: Bis(indole)hydrazide-hydrazone, anti-proliferative, apoptosis, MTT assay,
mechanistic studies.
*
#
Corresponding author Email: rrraju1@gmail.com
Co- Corresponding author Email: cgkumar5@gmail.com
1
. Introduction
Heterocyclic moieties play a vital role in the synthesis of potent drug molecules. Most of the
heterocyclic systems with two or more hetero atoms act as anticancer agents in cancer
chemotherapy and showed better anticancer results towards different human tumor cancer cell
1
-10
lines.
Indole moiety compounds are considered as most ubiquitous heterocyclic privileged
scaffold and these compounds act as variety of potent therapeutic agents with different
1
1
12,13
14-19
pharmacological activities such as antioxidant, anti-HIV,
anticancer
and anti-
2
0
rheumatoidal . Indole moiety attached to different heterocyclic compounds containing various
functional groups have lead to the enchanting array of potent bioactive natural products and also
2
1-24
25
a variety of most active pharmaceutical ingredients.
type indole built simple small molecules act as potent active anticancer targets and block the cell
cycle transition predominantly at G /M phase followed by destabilization of microtubules.
Indolyl glyoxalyl amide (D-24851)
2
Similarly, the pharmaceutical ingredient with -CO-NH-N=CH- (hydrazide-hydrazones)
2
6-29
30
31
showed different biological activities including anti-tumor,
anti-malarial, anti-convulsant,
3
2
33
34
35
anti-inflammatory, antimicrobial, antileishmanial, anti-tubercular .
Further improvements lead to the synthesis of oxindole hydrazides with IC50 values
3
6
ranging between 0.19-0.97 µM against potent inhibitors of tubulin polymerization. 3-
3
7
phenyl-5-sulfon-amidoindole-2-carboxylic acid hydrazide derivatives were known to act as
anti- depressant agents at 100 mg/kg dose. The hydrazide derivatives of 5-chloro-3-methyl-
3
8
indole-2-carboxylic acid benzylidene function as powerful apoptotic inducers against T47D

(breast cancer cell line), with IC50 value of 0.2 µM by obstructing tubulin polymerization in
3
9
G2/M phase. 3,4,5-trimethoxy benzohydrazides showed their antitumor activities against
human prostate cancer cell line (PC3). Aryl hydrazone derivatives of 5-butyl-2-(4-
4
0
methoxyphenyl)indole-3-carbaldehydes showed potent growth inhibitory activity against breast
cancer cell lines with IC50 values ranging between 19-115 nM followed by cell cycle arrest in
G
2
/M phase resulting in apoptosis.
A library of bis(indolyl)ketohydrazide-hydrazones were screened for their anticancer
activities against HCT-116, MCF-7, JURKAT and MDA-MB-231 cancer cell lines. The most
active compound, 4-chloro benzyl derivative showed caspase-dependent apoptosis in cells. This
compound also arrested cell cycle in G2/M phase by restricting tubulin polymerization with IC50
4
1
42
value of 0.6 µM. Bis(indolyl)hydrazide-hydrazone derivatives were screened for their anti-
cancer activity with IC50 values ranging between 1.0 to >100 µM against six human cancer cell
43
lines. N’-((5-Methoxy-1H-indol-3-yl)methylene)-1H-indole-2-carbohydrazide was efficient and
most selective agent with a incredible ability to encourage apoptosis and mitotic block in A549
(lung adenocarcinoma) cell line by concentrating the cellular microtubule system through tubulin
binding.
μ
structures of potent anticancer
compounds are shown in Figure 1.

Figure 1: Structures of potent anticancer bisindole compounds.
Previously, a series of novel indole-indazolyl hydrazide-hydrazone derivatives were
synthesized and screened for their anti-tumor activities against HeLa, A-549, MDA-MB-231,
and MCF-7 cancer cell lines and showed IC50 values varied from 1.93 to 29.58 µM,
6
45
respectively. Further, bis(indolyl) triketo diazo compounds were also synthesized and screened
for anticancer activities against HeLa, A-549, MDA-MB-231, and MCF-7 human cancer cell
lines which showed IC50 values varied from 1.3 to 44.5 µM, respectively. The remarkable
significant activity of these indole-indazolyl hydrazide-hydrazones motivated us to synthesize N-
methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone derivatives. These compounds were
subjected to cytotoxicity against cervical (HeLa), breast (MCF-7, MDA-MB-231) and lung

(A549) cancer cell lines. The cytotoxicity of these compounds were also checked against human
embryonic kidney (HEK293) cells.
2
2
. Results and discussion
.1 Chemistry
3
Vilsmeir-Haack reaction of Indole and 5–bromo indoles 7a-b with POCl and DMF gave
46,47
indole-3-carboxaldehydes 8a-b in good yields.
These indole carboxaldehydes were converted
4
8
to corresponding indole carboxylic acid derivatives 9a-b with KMnO
This acid derivatives were refluxed with conc. H SO and methanol mixture over 6 to 7 hours
period then gave the ester compounds 10a-b. N-methyl indolyl derivatives were obtained from
ester compounds by refluxing with dimethyl carbonate and K CO in DMF solvent over 4 hours
4
and acetone solvent.
2
4
4
9
2
3
5
0
time period. Finally, Indolyl carbohydrazide derivatives (12a-b) were achieved by refluxing the
N-protected ester with hydrazine hydrate and ethanol solvent over 8 – 10 hours time period (12a-
b) in good yields.
CHO
KMnO /Acetone
COOH
CH OH, Conc.H SO
4
R
R
R
i) POCl ,DMF
3
4
3
2
4
- 5 h,rt
6 h, Reflux
ii) Aqu.NaOH, reflux
N
H
N
H
N
H
8
a-b
7
a-b
9 a-b
9
9
a, R=H
b, R=Br
7
a, R=H
8a, R=H
b, R=Br
8
7b, R=Br
O
O
O
NH
OCH3
OCH3
NH2
R
R
R
NH NH H O/Ethanol
DMC, DMF
2
2 2
Reflux, 8h
K CO ,Reflux
2
3
N
N
N
H
0 a-b
CH3
CH3
1
11 a-b
12 a-b
1
1
2a, R=H
2b, R=Br
1
0a, R=H
11a, R=H
11b, R=Br
10b, R=Br
Scheme 1: Synthesis of N-methyl indole-3-carbohydrazide derivatives
Finally, the reaction of N-methyl indolyl -3-carbohydrazides 12a-b with different indole-
o
3
-carboxaldehydes 13a-13c in the presence of glac. CH
3
COOH at 90 C over a period of 6 h to
afford N-methyl and N,N-dimethyl bis(indolyl) hydrazide-hydrazones (14a-14d) in good yields.

O
O
NH
H
O
R
N
1
NH2
R
1
R
NH
R
Glac. AcOH
+
o
9
0 c, 6h, heating
N
N
N
2
N
CH3
R
2
1
3a-13c
R
1
2a-12b
H C
14a-14d
3
Scheme 2: Synthesis of bis(indolyl) hydrazide-hydrazone derivatives
1
2
1
2
Where, 12a, R=H
2b, R=Br
Where, 13a, R =H and R =CH
3
Where, 14a, R=Br, R =H and R =CH
3
1
2
1
2
1
13b, R =H and R =H
14b, R=H, R =H and R =CH
3
1
2
1
2
1
3c, R =OCH
3
and R =H
14c, R=Br, R =H and R =H
1
2
1
3
4d, R=Br, R =OCH and R =H
2
.2 Biological Evaluation
.2.1 In vitro anticancer activity
Four compounds were screened for cytotoxicity against four different human cancer cell lines
Such as Cervical cancer cell lines (HeLa), human breast adenocarcinoma cells (MDA-MB-231
MCF-7), Human alveolar adenocarcinoma cells (A549) and normal human embryonic kidney
2
&
cells (HEK-293) and shown in table 1. IC50 value is defined as the concentration at which 50%
of cell lines shall be destroyed and represented in µM. The obtained values varied from 0.793-
3
1.26 µM.
Compound 14a was found to be the most potent analog especially on A549 with IC50
value of 0.793 µM and it may be identified as drug target in cancer chemotherapy. The removing
of N-methyl from the compound 14a gave 14c which showed selective activity on MDA-MB-
2
1
31 (IC50 = 14.25 µM). Compound 14b exhibit moderate cytotoxicity on MDA-MB-231 (IC50
=
8.91 µM) and lung cancer cell lines A549 (IC50 = 18.96 µM). While the compound 14d
exhibited moderate cytotoxicity against HeLa and A549 with IC50 values of 14.25 µM and 12.85

µM respectively. The test compound 14a showed potent selective cytotoxicity against A549 with
IC50 = 0.793 µM, then it is selected for complete further mechanic studies.
Table 1: Cytotoxicity of synthesized derivatives 14a-14d
Test
IC50 (µM)
MCF-7
compound
HeLa
MDA-MB-
A549
HEK293
2
31
1
1
4(a)
4(b)
–
–
-
–
–
0.793 ± 0.29
-
-
18.91 ± 0.57 31.26 ± 0.89 18.96 ± 0.72
1
1
4(c)
4(d)
14.25 ± 0.41
-
-
-
-
-
14.25 ± 0.45 22.74 ± 0.28
12.85 ± 0.39
Doxorubicin 0.36 ± 0.14 0.47 ± 0.4 0.98 ± 0.14 0.89 ± 0.26
-
“
-“ shows no activity.
2
.2.2 Evaluation of in vitro anticancer activity of compound 14a on different cancer cell
lines
The anticancer activity of compound 14a on different cancer cell lines is shown in Table 1. The
sensitive cancer cell lines to the test compound were MCF-7 (breast), HeLa (cervical) and SW-
6
80 (colon) with IC50 values nearer to standard drug, “doxorubicin”. In addition, MDA-MB-231
(breast cancer) and A549 (lung cancer) also showed high sensitivity to the test compound. The
test compound also showed considerable anticancer activity towards head and neck squamous
cell carcinomas (FaDu, SCC-25 and Detroit-562). As shown in Figure 2, compound 14a exerted
dose-dependent inhibition in all tested human tumor cell lines. It is noteworthy to mention that
against HeLa, MCF-7 and SW-680 cell lines, maximum inhibitory activity was achieved.
However, on MDA-MB-231 and A549 cell lines significant decline in cell growth was observed.
In case of HNSCC (head and neck squamous cell carcinoma) cell lines such as FaDu and SCC-
2
5 cell lines, the test compound showed considerable inhibitory activity and cell viability was
reduced in a dose-dependent manner. It is noteworthy to mention that in normal HEK-293 cell
line at higher concentration of 200 μM, 95% of total cells were viable indicating the non-toxic

nature of this compound on the normal cell line. On comparison of inhibition data, it was clearly
established that a considerable reduction in cell viability was appeared in HeLa, MCF-7, SW-
6
80, MDA-MB-231, A549 and the entire set of HSNCC cells; however, it was more significant
against HeLa, MCF-7 and SW-680 cell lines. Since the test compound 14a showed potent
selective cytotoxicity against HeLa and MCF-7 with IC50 values of 1.69 and 1.19 µM, it was
selected for further mechanistic studies.
Table 2. Cytotoxicity of compound 14a against different cancer cell lines
Cell line
IC50 values (in μM )
Compound 14a
Doxorubicin
1.09 ± 0.11
3.1 ± 0.59
0.23 ± 0.01
1.2 ± 0.9
HeLa
1.69 ± 0.05
5.49 ± 0.35
1.19 ± 0.07
2.77 ± 0.74
5.49 ± 0.35
9.64 ± 0.09
10.13 ± 0.23
22.69 ± 1.44
>200
MDA-MB-231
MCF-7
SW-680
A549
2.11 ± 0.09
0.68 ± 0.14
2.24 ± 0.11
1.26 ± 0.09
>200
FaDu
SCC-25
Detroit-562
HEK-293 (Normal)

1
1
20
00
HeLa
MDA-MB-231
SW-680
MCF-7
A549
FaDu
SCC-25
HEK-293
Detroit-562
8
6
4
2
0
0
0
0
0
1
10
20
50
100
200
Concentration (in μM)
Figure 2: Dose dependent cell growth inhibition (%) of compound 14a on different cancer
cell lines
2
.2.3 Annexin V/PI staining for cell apoptosis
Annexin V/propidium iodide (PI) co-staining assay was performed to decide the way of
cell death encouraged by compound 14a. This assay distinguish between necrotic cell death and
apoptotic. HeLa and MCF-7 cell lines were reacted with compound 14a (1 μM) for 8, 12 and 16
h, then double stained with FITC-conjugated annexin V and propidium iodide (PI), and it was
analyzed by flow cytometry (Figure 3). After the completion of 12 h reaction with compound
1
4a, a little amount of cell lines (~9%) had either inhibited or were in late stage of the apoptosis
(Annexin V positive, PI positive), and ~24% of cancer cells were appeared in early stage of the
apoptosis (Annexin V positive, PI negative). After 16 h of treatment to compound 14a, ~76% of
cells were appeared at the stage of dead/late apoptotic quadrant, ~26% of cells were appeared in
the early stage apoptosis, and the final ~8% of the cell lines were viable. The experimental results
were the average of three independent experiments treated with compound 14a incubated at time intervals
of 8h, 12h and 16h. At 12 h of incubation, the data 9% refers to the percentage of cells that are Positive
for both Annexin V and Propidium Iodide and 24% of cells were Annexin V positive and Propidium
iodide negative suggesting the early stage of the apoptosis. After 16h of incubation the percentage of cells
in late apoptosis were 76% suggesting that the cell death induced by compound 14a is due to apoptosis
and not by necrosis. Thus, a remarkable quantity of cell lines progress with the Annexin V

positive/PI-negative quadrant, mightily suggesting that the observed anticancer activity in MCF-
and HeLa cells is through apoptosis
7
Figure 3: Effect of compound 14a treatment for 8 to 16 h induced apoptotic cell death in (a)
MCF-7 and (b) HeLa cancer cell lines evaluated based on Annexin V apoptosis assay
2
.2.4 Cell cycle analysis
Treatment of HeLa and MCF-7 cells with compound 14a caused no important changes in
the levels of cell lines at each phase of the cell cycle over 16 hours time period (Figure 4). The
data showed that compound 14a did not produce cell cycle arrest in these cancer cell lines. The
inability of compound 14a to induce cell cycle arrest suggests that topoisomerase obstruction
can not be the first mechanism of action for compound 14a, since small molecules functioning as
topoisomerase inhibitors were known to arrest the cells in definite phases of cell cycle.

G2/M
S
HeLa
MCF-7
G1
0
20
40
60
80
100
120
Cell Population (%)
Figure 4. Effect of compound 14a on cell cycle arrest. MCF-7 and HeLa cell lines were exposed
to compound 14a (1 μM) for 16 h. Values are represented as the percentage of the cell
population in G1, S and G1/M phases of cell cycle. P<0.05, significantly different from the
vehicle group.
2
.2.5 ROS production
To determine if ROS production is induced in response to compound 14a treatment and it
is achieved the cause of demise of reacted cancer cell lines, MCF-7 and HeLa cells were reacted
with 14a and produced ROS levels were observed with DCF(dichlorofluorescein diacetate),
which is a non-fluorescent dye that treated with peroxides to afford fluorescent
dichlorofluorescein. Flow cytometry was used to quantify the dye in live cells with the level of
intracellular fluorescence. t-butyl peroxide was used as positive control for these experiments
and it create the peroxides in cancer cells. As compared to t-BHP, cells treated with compound
1
4a at 5 μM concentration produced significant amount of ROS in 4 h, as indicated by DCF
staining (Figure 5) in both HeLa cells (29.9%) and MCF-7 (36.8%). After 8 h of incubation,
ROS production in both HeLa and MCF-7 cells increased to 58.9% and 48.27%, respectively.
However, when compound 14a treated cells were pretreated with NAC, a known ROS inhibitor;
a significant reduction in the ROS production in both HeLa and MCF-7 cells was observed. This
clearly indicates that there is a rapid formation of ROS upon compound 14a treatment which

suggests that it directly produced by compound 14a treatment and its not a byproduct of cell
death.
1
1
20
00
A
4
h
8h
8
6
4
2
0
0
0
0
0
1
5
10
NAC +
t-BHP
Compound
Compound concentration (μM)
(5 μM)
1
20
00
B
4
h
8h
1
8
6
4
2
0
0
0
0
0
1
5
10
NAC +
Compound
5 μM)
t-BHP
Compound concentration (μM)
(
Figure 5. Effect of compound 14a on ROS production. (A) MCF-7 and (B) HeLa cell lines when
exposed to compound 14a (1 μM) for 3 h. ROS production was monitored using DCFH-DA
assay. P<0.05, significantly different from the vehicle group.

2
.2.6 NO production
The experimental analysis showed that in both HeLa and MCF-7 cancer cells, the
compound 14a induced NO production in a dose-dependent way. Through the primary drug
target interactions, compound 14a at 5 μM produced significant levels of NO production after 8
h of treatment, as indicated by Griess reagent analysis (Figure 6). In case of MCF-7 cells, the NO
levels produced by treatment with compound 14a at concentrations 1 and 5 μM were 32.1% and
4
8.9%, respectively. Similar effect was found in HeLa cells where the observed NO levels were
2
9.8% and 42.4% at 1 and 5 μM, respectively. The results demonstrated that through primary
drug target interactions, compound 14a caused oxidative stress response in both HeLa and MCF-
cells, thereby induced production of NO, a secondary messenger molecule which is responsible
for the downstream signaling process leading to apoptotic cell death.
7
1
00
MCF-7
HeLa
8
6
4
2
0
0
0
0
0
1
5
Concentration (μM)
10
Normal cells
Figure 6: Effect of compound 14a on NO production. MCF-7 and HeLa cells were exposed to
compound 14a at concentrations of 1, 5 and 10 μM for 8 h. NO production was monitored using
Griess reagent assay. P<0.05, significantly different from the vehicle group.

2
.2.7 Effect on PKA, ABL, and PTP1B enzymes
The enzymes used in the study such as tyrosine kinase ABL (ABL), cAMP-dependent
protein kinase A (PKA) and protein-tyrosine phosphatase 1B (PTP1B) plays a key role in
anticancer drug discovery. PTP1B is a positive factor in cancer phenotypes; PKA and ABL are
the protein kinases where the proteins are mostly mutated in malignant tumors.
Compound 14a moderately inhibited the activity of PKA and ABL and exerted moderate
effect on the phosphorylation of these twice protein kinases (Figure 7). However, a dose-
dependent inhibitory effect of PKA activity was appeared. At a concentration of 50 μM,
compound 14a inhibited 45% of the measured PKA activity. Similarly, compound 14a was
found to restrict PTP1B in a dose-dependent way (Figure 7). The PTP1B activity was inhibited
by 48% at 50 μM concentration of compound 14a. The results demonstrated that compound 14a
is a cytotoxic compound, which exibit its anti-proliferative effects through various mechanisms
that add the generation of oxidative species such as ROS and NO and inhibition of protein kinase
activity.
1
20
9
6
3
0
0
0
0
PKA
ABL
PTP1B
0
50
100
150
200
250
Concentration (μM)

Figure 7: The effect of compound 14a on the enzymatic activity of cAMP-dependent protein
kinase A (PKA), protein-tyrosine phosphatase 1B (PTP1B) and tyrosine kinase ABL (ABL).
Enzyme activity was monitored in two separate experiments and results are noted as % of control
activity in the absence of compound 14a. Each point represents the average of two parallel run
means.
2
.2.8 Mitochondrial membrane potential in HeLa and MCF-7 cell lines
The mechanism of action of anticancer activity of compound 14a on HeLa and MCF-7
cell lines was studied by evaluating caspase dependent and caspase independent apoptotic
pathways. Mitochondria plays an important characteristic role in both extrinsic and intrinsic
apoptosis pathways and hence the effect of compound 14a on mitochondrial membrane potential
was measured. Compound 14a affected the mitochondrial membrane potential and related
proteins. HeLa and MCF-7 cell lines were exposed to compound 14a at a concentration range of
5
µM for the mentioned times (1 h, 2 h, 4 h and 8 h) and were analyzed by flow cytometry
(Figures 8A and 8B). Results showed a band shift situation observed after incubation over 1 h
and 2 h in both HeLa and MCF-7 cells, respectively; these phenomena were changed
significantly over 4 h time period. These results indicate that compound 14a affected the
mitochondrial membrane potential.

Figure 8: Effect of compound 14a on the mitochondrial membrane potential in (A) HeLa and
B) MCF-7 cell lines. The cells were treated with 10 µM rhodamine 123 and incubated at 37
C for 30 min in the presence of 5 µM of compound 14a and then quantified by flow
(
°
cytometric analysis at 1 h, 2 h, 4 h and 8 h with rhodamine 123. The horizontal axis shows the
relative fluorescence intensity, and the vertical axis indicates the cell counts or cell number.
The green curve indicates the control. The blue curve indicates 14a treated cells. Loss of
mitochondrial membrane potential denotes the shifting from green curve to the blue curve.
Data are expressed from at least three separate determinations.

2
.2.9 Caspase activation in HeLa and MCF-7 cells
In both extrinsic and intrinsic apoptotic pathways, the activation of caspase pathway
engage an important role. The activation of caspases – 3 play a crucial role among the caspases
in the induction of apoptotic cell death occurred in apoptosis pathway. Hence, the role of
compound 14a in the activation of caspase-3 was studied in both MCF-7 and HeLa cell lines.
The results demonstrated that in both MCF-7 and HeLa cell lines, caspase-3 was not activated by
compound 14a (Figures 9 and 10). In addition, the positive control - paclitaxel (3 µM) activated
caspase-3 in both HeLa and MCF-7 cell lines.
Figure 9: Effect of compound 14a on caspase-3 activation in HeLa cell line. HeLa cells were
treated with compound 14a at concentrations 1, 3 and 5 µM for 24 h and caspase-3 activation
was detected using Western blot analysis. Paclitaxel at 3 µM was used as a positive control.

Figure 10: Effect of compound 14a on caspase-3 activation in MCF-7 cell lines. MCF-7 cells
were treated with compound 14a at concentrations 1, 3 and 5 µM for 24 h and caspase-3
activation was detected using Western blot analysis. Paclitaxel at 3 µM was used as a positive
control.
In addition to caspase-3, the activation and cleavage of caspase -6, -7, -8, and -9 was not
observed upon the treatment with 14a in both MCF-7 and HeLa cell lines, even at 5 µM
concentration for 24 h (Figure 11 and 12). The results demonstrate that compound 14a induced
apoptotic cell death in both MCF-7 and HeLa cell lines which is independent of caspase
activation.
Figure 11. Effect of compound 14a on caspase-6, 7, 8 and 9 activation in HeLa cell lines.
HeLa cells were treated with compound 14a at concentrations 1, 3 and 5 µM for 24 h. The
proteins were separated and activation of caspase-6, 7, 8 and 9 were evaluated using Western
blot analysis.

Figure 12. Effect of compound 14a on caspase-6, 7, 8 and 9 activation in MCF-7 cell line.
MCF-7 cells were treated with compound 14a at concentrations 1, 3 and 5 µM for 24 h. The
proteins were separated and activation of caspase-6, 7, 8 and 9 were evaluated using Western
blot analysis
2
.2.10 Expression of pro-apoptotic and anti-apoptotic proteins in HeLa and MCF-7 cells
The effect of compound 14a on Bcl-2 family protein expression which was known to
play an important crucial role in the mitochondria, including the study of mitochondrial
membrane potential mediation. The results demonstrated that in both HeLa and MCF-7 cells,
compound 14a showed no effect on the regulation of Mcl-1 long form (Mcl-1L) protein, which
moderate inhibition of cell apoptosis (anti-apoptotic). However, compound 14a induced the
expression of the Mcl-1 short form (Mcl-1s) protein (pro-apoptotic), which encourages the
apoptotic cell death (Figure 13 and 14).
Furthermore, in both MCF-7 and HeLa cell lines, there was no change in the expression
levels of Bcl-2 and Bid proteins (Figure 13 and 14). The results demonstrated that in both HeLa
and MCF-7 cells, Bak and Mcl-1 played an crucial role in compound 14a induced caspase
independent apoptotic cell death.

Figure 13: Effect of compound 14a on mitochondrial apoptotic or anti-apoptotic protein
levels in HeLa cells. In HeLa cells, compound 14a induced upregulation of Mcl-1s and Bak
(
pro-apoptotic) proteins whereas Bcl-2 and Bid protein levels were not affected by compound
4a treatment. Cells were treated with 5 µM of compound 14a for 24 h and proteins were
detected by Western blotting. Data are expressed from at least three separate determinations.
1

Figure 14: Effect of compound 14a on mitochondrial apoptotic or anti-apoptotic protein
levels in MCF-7 cells. In MCF-7 cells, compound 14a significantly induced the activation of
Bak and also upregulated Mcl-1s expression. On the other hand, Bcl-2 and Bid protein
expressions remained unchanged upon treatment with compound 14a. Cells were treated with
5
µM of compound 14a for 24 h and proteins were detected by Western blotting. Data are
expressed from at least three separate determinations.
2
.2.11 Oxidative stress induction
Reactive oxygen species is an important biomarker of apoptosis. The increase in
intracellular ROS eventually induces apoptosis and cell cycle arrest. In this study, the effect of
compound 14a on ROS induction at 12 h and 24 h in MCF-7 and HeLa cells was evaluated using
H2DCFDA dye and the histograms are shown in Figures 15 and 16. It is clearly evident that in
cells treated with compound 14a (blackpanel), ROS concentration increased in a time dependent
way as compared to the untreated control (pink panel). From the histograms, it was observed that
in case of HeLa cells treated with compound 14a, ROS were induced at 12 h as compared to
untreated cells (Figure 15). However, in case of compound 14a treated MCF-7 cells, ROS
induction was pronounced at 24 h as compared to untreated cells (Figure 16).

Figure 15: Representative histograms of treated and untreated HeLa cells stained with CM-
H2DCFDA dye. The pink histogram depicts stained untreated control cells while black shows
the cells treated with 5 µM of compound 14a at 12 and 24ꢀh, respectively.
Figure 16: Representative histograms of treated and untreated MCF-7 cells stained with CM-
H2DCFDA dye. The pink histogram depicts stained untreated control cells while black shows
the cells treated with 5 µM of 14a at 12 and 24ꢀh, respectively.

2
.2.12 Acetylation of p53
The effect of compound 14a on the induction and acetylation status of p53 levels in both
MCF-7 and HeLa cell lines during cell apoptosis was investigated using ELISA assay. The
results are expressed as control index and are shown in Figure 17. From the results it is clearly
evident that, compound 14a could induce p53 acetylation in MCF-7 and HeLa cells and also
significant increase in total p53 protein levels were observed in a time-dependent way. After 24 h
of the reaction with compound 14a in HeLa cells (Figure 17), the acetylated p53 levels were
upregulated as compared to control cells (DMSO treated). Similar increase in acetylated p53
levels was observed in MCF-7 cells after 24 h of treatment (Figure 18). In case of total p53
levels, a significant upsurge in both HeLa and MCF-7 cancer cell lines was observed with
increasing time i.e., after 12 h of treatment. In control untreated cells, a negligible effect was
observed on total p53 levels in both cell lines at time intervals studied (Figure17 and 18).
Figure 17: The ELISA assay of total and acetylated p53 protein levels expressed in Hela cells
based on control index. Cells were treated 5 µM of compound 14a for 0, 12, 24 and 48 h.
Values are the means ± SD of triplicate experiments. *P<0.001 vs. all other groups in
different times. Total P53 in HeLa cells without compound 14a treatment and with compound
1
4a treatment at time periods 12, 24 and 48 h. No significant difference was shown in total
and acetylated p53 content in other groups.

Figure 18: The ELISA assay of total and acetylated p53 protein levels expressed in MCF-7
cells based on control index. Cells were treated 5µM of compound 14a for 0, 12, 24 and 48 h.
Values are the means ± SD of triplicate experiments. *P<0.001 vs. all other groups in
different times. Total P53 in HeLa cells without compound 14a treatment and with compound
1
4a treatment at time periods 12, 24 and 48 h. No significant difference was shown in total
and acetylated p53 content in other groups.
2
.2.13 Activation of MAPK in HeLa and MCF-7 cells
The role of MAPK activation in compound 14a induced MCF-7 and HeLa cells was elucidated
in vitro through the protein phosphorylation assessment. Compared with positive control, 5 µM
IC50 value of the compound 14a efficiently induced through the activation of MAPK in cervical
(HeLa) cell lines (Figure 19) and indicated with enhanced p-P38/total P38 (Pꢁ=ꢁ0.0168; Figure
1
9A), p-ERK1/2/total ERK1/2 (Pꢁ=ꢁ0.0002; Figure 19B), and p-JNK/total JNK ratio (Pꢁ=ꢁ0.0034;
Figure 19C). However, MAPK activation was totally restricted by pre-treatment with the P38
kinase inhibitor SB2035809 (Pꢁ=ꢁ0.0187; Figure 19A), ERK1/2 inhibitor PD98059 (Pꢁ=ꢁ0.0003;
Figure 19B) and JNK inhibitor SP600125 (Pꢁ=ꢁ0.0022; Figure 19C). Similarly, MCF-7 cells upon
exposure to compound 14a (5 µM) significantly induced MAPK activation (Figure 20), as
evidenced by enhanced levels of p-P38/total P38 (Pꢁ=ꢁ0.0075; Figure 20A), p-ERK1/2/total
ERK1/2 (P<0.0001; Figure 20B), and p-JNK/total JNK ratio (Pꢁ=ꢁ0.0022; Figure 20C).

Furthermore, this MAPK activation was significantly attenuated by pretreatment with the P38
kinase inhibitor SB2035809 (Pꢁ=ꢁ0.0187; Figure 20A), ERK1/2 inhibitor PD98059 (Pꢁ=ꢁ0.0003;
Figure 20B) and JNK inhibitor SP600125 (Pꢁ=ꢁ0.0019; Figure 20C). MAPK is required for
maintenance of the malignant state, but that short-term activation of MAPK may direct cells to
apoptosis. Phosphorylation of p53 determines the biological activity of the protein. At several
serine residues, including serine 6 and serine 15, phosphorylation has been shown to be relevant
to DNA damage and apoptosis. Compound 14a showed activation and nuclear translocation of
MAPK (extracellular signal-regulated kinase 1/2) and cellular abundance of the oncogene
suppressor protein p53, phosphorylation of p53, and abundance of c-fos, c-jun, and p21. Thus,
compound 14a acts via a ERK1/2-MAPK kinase-MAPK signal transduction pathway to increase
p53 expression, phosphorylation of p53, and p53-dependent apoptosis in MCF-7 and Hela cell
lines.

Figure 19: Effect of compound 14a on activation of MAPK pathway in HeLa cells. (A)
Phosphorylation of P38, (B) ERK1/2, and (C) JNK was determined by cell-based ELISA and
expressed as the ratio of the phosphorylated to the total form of each protein. nꢁ=ꢁ3 and
#
*
P<0.0001 vs. control and P<0.0001 vs. compound 14a in the presence of SB2035809,
PD98055, or SP600125, respectively.

Figure 20: Effect of compound 14a on activation of MAPK pathway in MCF-7 cells. (A)
Phosphorylation of P38, (B) ERK1/2, and (C) JNK was determined by cell-based ELISA and
expressed as the ratio of the phosphorylated to the total form of each protein. nꢁ=ꢁ3 and
#
*
P<0.0001 vs. control and P<0.0001 vs. compound 14a in the presence of SB2035809,
PD98055, or SP600125, respectively.

3
Experimental section
3
.1 General
All chemicals, salts, reagents and solvents were purchased from AVRA laboratories,
Hyderabad, India; Aldrich (USA) and Alfa Aesar (USA). Reaction progress were observed by
1
13
Thin Layer Chromatography plates. NMR ( H & C) spectral data were obtained from 400 MHz
Gemini Varian-VXR-unity instrument. Chemical shifts (δ) values are mentioned in ppm. Mass
ESI spectra were taken from Micromass, Quattro LC using ESI+ software with capillary voltage
3
.98 kV and ESI mode positive ion trap detector. Melting points were taken from an
electrothermal melting point apparatus.
3
.2 Synthesis of bis(indolyl) hydrazide-hydrazone derivatives 14(a-d)
1
.86 mmol 1-methyl-indolyl-3-carbohydrazides 12a or 12b were soluble in 3 mL Glac.
CH
3
COOH. To this 1.86 mM indole-3-aldehydes were added and allowed for heating at 90 °C
solution.
over 6 h time period. Later the reaction mixture was neutralized by a cold NaHCO
3
Further it was filtered and purified by recrystallization technique from ethanol then gave yellow
color solid.
3
.2.1 5-bromo-1-methyl-N'-[(E)-(1-methyl-1H-indol-3-yl)methylidene]-1H-indole-3-carbohydra
o
zide 14(a) Yield: 90%; m.p.: 233-235 C; IR (KBr): 3439, 2916, 1627, 1523, 1462, 1370, 1285,
-1 1
8
46, 744 cm ; H NMR (DMSO-d
6
, 400 MHz):  3.83 (s, 3H), 3.91 (s, 3H), 7.28-7.20 (m, 2H),
7
.41 (s, 1H), 7.55-7.50 (m, 2H), 7.84 (s, 1H), 8.46-8.16 (m, 4H), 11.10 (brs, NH) ppm; HRMS
+
+
4
m/z calculated for C20H18ON Br: 409.06585 (M+H) ; found m/z:409.06546 (M+H) .
3
.2.2 1-methyl-N'-[(E)-(1-methyl-1H-indol-3-yl)methylidene]-1H-indole-3-carbohydrazide 14(b)
o
m.p.: 248-250 C; Yield: 86%; IR (KBr): 3216, 2912, 1625, 1547, 1463, 1382, 1237, 831, 738
-
1 1
cm ; H NMR (DMSO-d
6
, 400 MHz):  3.88 (s, 6H), 7.26 (s, 4H), 7.52 (s, 2H), 7.78 (s, 1H),
: 331.15534
8
.46-8.23 (m, 4H), 11.06 (brs, NH) ppm; HRMS m/z calculated for C20H19ON
4
+
+
(
M+H) ; found m/z: 331.15482 (M+H) .

3
.2.3 5-bromo-N'-[(E)-(1H-indol-3-yl)methylidene]-1-methyl-1H-indole-3-carbohydrazide 14(c)
o
Yield: 87%; m.p.: 278-280 C; IR (KBr): 3377, 3148, 2982, 1745, 1673, 1544, 1460, 1379, 1242,
-1 1
8
74, 789 cm ; H NMR (DMSO-d
6
, 400 MHz):  3.89 (s, 3H), 7.23-7.14 (m, 2H), 7.55-7.38 (m,
3
H), 7.80 (s, 1H), 8.54-8.22 (m, 4H), 11.26 (brs, NH), 11.63 (brs, NH) ppm; HRMS m/z
+
+
4
calculated for C19H16ON Br: 395.05020 (M+H) ; found m/z: 395.05038 (M+H) .
3
.2.4 5-bromo-N'-[(E)-(5-methoxy-1H-indol-3-yl)methylidene]-1-methyl-1H-indole-3-carbohyd
o
razide 14(d) Yield: 92%; m.p.: 249-251 C; IR (KBr): 3368, 3146, 2986, 1686, 1544, 1383, 843,
-1 1
7
52 cm ; H NMR (DMSO-d
6
, 400 MHz):  3.84 (s, 6H), 6.79 (s, 1H), 7.30 (m, 3H), 7.51 (s,
1
H), 7.84-8.07 (m, 2H), 8.41-8.50 (m, 2H), 11.01 (brs, NH), 11.14 (brs, NH) ppm; HRMS m/z
+
+
18 2 4
calculated for C20H O N Br: 425.06076 (M+H) ; found m/z: 425.06008 (M+H) .
3
.3 Cell lines and treatment
Cancer cell lines including Human cervical cancer (HeLa; ATCC No. CCL-2), Human
breast adenocarcinoma (MDA-MB-231; ATCC No. HTB-26), Human breast adenocarcinoma
MCF-7; ATCC No HTB-22), Human alveolar adenocarcinoma (A549; ATCC No. CCL-185),
(
Human colon adenocarcinoma (SW-680; ATCC No. CCL-227), along with head and neck
squamous cell carcinoma (HNSCC) cell lines such as human pharynx squamous cell carcinoma
(FaDu; ATCC No. HTB-43), human pharyngeal carcinoma (Detroit-562; ATCC No. CCL-138)
and human tongue squamous cell carcinoma (SCC-25; ATCC No. CRL-1628) and normal
human embryonic kidney (HEK-293; ATCC No. CRL-1573) cells were used in the present
study. Cells were preserved in DMEM medium containing 100 mg/ml streptomycin and 100
units/ml penicillin and supplemented with heat-inactivated 10% FBS (fetal bovine serum). All
2
cultured cells were kept in a humidified atmosphere at 37°C with 5% CO . Doxorubicin (Sigma-
Aldrich, Schnelldorf, Germany) was taken as a target positive (cytotoxic) control. DMSO
concentration was very less that was not greater than 0.1% in overall experiments.
3
.4 MTT assay
Cells were cultured until a confluence of 80%, trypsinized and were sent to a falcon tube
followed by centrifugation at 3000 rpm over 5 minutes before they were diluted in growth
medium and seeded into 96 well microtitre plate. Number of cells suspended in 100 μL growth