Synthesis and evaluation of the biological activity of N′-[2-oxo-1,2 dihydro-3H-indol-3-ylidene] benzohydrazides as potential anticancer agents

Article abstract of DOI:10.1039/c5ra01528f

New N′-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzohydrazide derivatives were synthesized and evaluated for their cytotoxic properties against murine leukemia, L1210, human leukemia, REH and K562, human T-cell leukemia, CEM and human cervix carcinoma, HeLa

Full text of DOI:10.1039/c5ra01528f

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Katiyar, M. hegde, S. kumar, V. gopalkrishnan, S. A. ramareddy, B. Choudhary, E. De Clercq, D. Schols, K.
Bhatelia, K. Ananthaswamy and S. Raghavan, RSC Adv., 2015, DOI: 10.1039/C5RA01528F.
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Page 1 of 36
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DOI: 10.1039/C15RA01528F
Synthesis and evaluation of the biological activity of N'-[2-oxo-
1,2 dihydro-3H-indol-3-ylidene] benzohydrazides as
potential anticancer agents
Arpit Katiyarac*, Mahesh Hegdeb*, Sujeet Kumara, Vidya Gopalakrishnane, Khyati
D. Bhateliab, Kavya Ananthaswamyb, Sureshbabu A. Ramareddya, Erik De
Clercqd, Bibha Choudharye, Dominique Scholsd, Sathees C. Raghavanb and
Subhas S. Karkia**
aDepartment of Pharmaceutical Chemistry, KLE University's College of
Pharmacy, Bangalore-560 010, India
bDepartment of Biochemistry, Indian Institute of Science, Bangalore-560012,
India
cDepartment of Pharmacy and Medical Science, Singhania University, Pacheri
Bari Jhunjhunu, Rajasthan
dRega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
eInstitute of Bioinformatics and Applied Biotechnology, Electronics City,
Bangalore 560 100, India
*Equal first authors
**Corresponding author: subhasskarki@gmail.com, 080 23325611 (off), 080
23425373 (fax)

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DOI: 10.1039/C25RA01528F
Abstract:
New
N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazide
derivatives were synthesized and evaluated for their cytotoxic properties against
murine leukemia, L1210, human leukemia, REH and K562, human Tꢀcell
leukemia, CEM and human cervix carcinoma, HeLa cells. Among the tested
compounds, the 3,4,5ꢀtrimethoxyꢀN'ꢀ[5ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀ
ylidene]benzoydrꢀazide derivative (5t) emerged as the most potent inhibitor
against all the tumor cell lines evaluated. To investigate the mechanism of action,
5t was further studied by cell cycle analysis, mitochondrial membrane potential
analysis, DNA fragmentation and Annexin VꢀFITC flow cytometric analysis, which
suggested that 5t was able to induce apoptosis at submicromolar range.
Keywords: Antiꢀcancer drugs, Cancer therapeutics, Cytotoxicity, Apoptosis, Cell
death
Running
title:
N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazide
derivativesꢀinduced cell death.

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DOI: 10.1039/C35RA01528F
1. Introduction
Cancer is the most wideꢀspread disease across the world today.
Chemotherapy is one of the main methods used for cancer treatment. The
majority of drugs currently used for the treatment of cancer are cytotoxic,
targeting various pathways in the cell, for example DNA repair pathways [1ꢀ4].
Most of the cytotoxic drugs which are under clinical trial are of potent activity with
sub micromolar range. Therefore, it will be of great importance to design and
develop new compounds with lower IC50 values.
Various compounds having general formula 1 display anticancer activities
[5]. Hydrazide derivatives possess an interesting biological activity such as antiꢀ
platelet [6], analgesic [7, 8], antimicrobial [9], antiꢀinflammatory [10], and
anticancer 2 [11] activities. Various acyclic thiosemicarbazones 3 are lethal to
mice at doses of 25ꢀ100 mg/kg [12]. The isatinꢀbased hydrazones were reported
as antioxidants [13], commercial herbicides and acetohydroxy acid synthase
inhibitors [14], and Bꢀsecretaseꢀ1 (BACE1) inhibitors [15]. The hydrazide (ꢀ
CH=NꢀNHꢀCOꢀ) moiety has an important role as part of antitumor agents 2 [16ꢀ
18].
Previously, we have reported isatinꢀbased thiosemicarbazide derivatives
as cytotoxic agents 4 [19]. The objectives of the present investigation were to
develop analogs of 3 and 4 as potent cytotoxic agents. The derivatives 3 and 4
display cytotoxicity to human CEM Tꢀlymphocytes and their IC50 values were high,
viz, 83,000 and 2,300 nM, respectively. The derivative 4 was more potent,
because of insertion of polar atoms like carbonyl in place of the methylene

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DOI: 10.1039/C45RA01528F
functional group. From this basic design, we inserted an oxygen atom instead of
sulfur as in isatinꢀhydrazones 5 and evaluated their cytotoxicity in human and
murine tumor cell lines (Fig. 1). Moreover the possible underlying mechanism of
action (MOA) has also been investigated.
3
S
R
O
NH
2
S
S
R
NH
NH
2
NH
O
N
NH
NH
NH
N
N
N
F
2
R
N
CH
3
4
1
2
3
CEM IC50= 2300 nM
CEM IC50 = 83000 nM
CH
3
O
O
O
CH
3
NH
O
O
N
H C
H C
3
3
N
H
5t
CEM cells IC50= 660 nM
Figure 1.
2. Experimental
2.1. Chemicals and reagents.
The melting points are uncorrected. The IR spectra were recorded in KBr
1
on a Jasco 430+; the H NMR spectra were recorded in CDCl3 / DMSO on a
Bruker (400 MHz), and J values were reported in hertz (Hz). Mass spectra were
recorded in triple quadrapole LCMSꢀ6410 from Agilent technologies. 5ꢀClꢀisatin

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[20], 5ꢀFꢀisatin [21], 5ꢀCH3ꢀisatin [22], 5ꢀNO2ꢀisatin [23], 4ꢀClꢀphenyl hydrazide
[24], 4ꢀBrꢀphenyl hydrazide [25], 4ꢀNO2ꢀphenyl hydrazide [26], 4ꢀOCH3ꢀphenyl
hydrazide [27] and 3,4,5ꢀtriꢀOCH3ꢀphenyl hydrazide [28] were prepared
according
to
the
literature.
(5b)
4ꢀBromoꢀN'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀ
4ꢀNitroꢀN'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀ
ylidene]benzohydrazide
ylidene]benzohydrazide (5c) and N'ꢀ[5ꢀNitroꢀ2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀ
ylidene]benzohydrazide (5x) were prepared according to the literature [29]. While
compounds N'ꢀ[5ꢀChloroꢀ2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]ꢀ4ꢀmethoxybenzoꢀ
hydrazide (5i) and N'ꢀ[5ꢀFluoroꢀ2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzo
hydrazide (5n) were prepared according to literature [30].
2.2. General Procedure for the synthesis of the N'-[2-oxo-1,2-dihydro-3H-indol-3-
ylidene]benzohydrazide (5a-y)
A mixture of the 1Hꢀindoleꢀ2,3ꢀdione (I, 0.005 mol), aryl hydrazone (II,
0.007 mol) and ethanol (100 ml) was heated under reflux until the reaction was
completed (~4 h). Approximately half of the ethanol was removed in vacuo and
the solution was left overnight at room temperature. The solid which precipitated
was collected, washed with cold ethanol and recrystallized from
ethanol:chloroform (9:1) to give the following compounds.
2.2.1.
4-Chloro-N'-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzohydrazide
(5a). Obtained according to the general procedure for the synthesis of the N'ꢀ[2ꢀ
oxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 75%, MP >300 °C;

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Molecular formula C15H10ClN3O2, Molecular Weight 299.71, IR: 3218, 3163, 3052,
3009, 2918, 2845, 1692, 1670, 1586, 1593, 1539, 1480, 1414, 1379, 1309, 1271,
1
1164. H NMR: 13.85 (1H, s, NH.), 11.41 (1H, s, NH), 7.92ꢀ7.89 (3H, dm, J=8),
7.71 (2H, d, J=8), 7.44ꢀ7.41 (1H, m, ar), 7.27ꢀ7.22 (1H, m, ar), 6.98ꢀ6.94 (1H, m,
ar).
2.2.4. 4-Methoxy-N'-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzohydrazide
(5d). Obtained according to the general procedure for the synthesis of the N'ꢀ[2ꢀ
oxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 74%, MP 268ꢀ270 °C;
Molecular formula C16H13N3O3, Molecular Weight 295.29, IR: 3289, 3165, 3065,
2936, 2833, 1692, 1586, 1523, 1492, 1414, 1330, 1263, 1127. NMR: 13.87 (1H,
s, NH), 11.33 (1H, s, NH), 7.88 (2H, d, J=8), 7.60 (1H, d, J=8), 7.38 (1H, t, J=16),
7.15ꢀ7.09 (3H, dm, J=8), 6.96 (1H, d, J=8).
2.2.5. 3,4,5-Trimethoxy-N'-[2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5e). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 77%, MP 276ꢀ
278 °C; Molecular formula C18H17N3O5, Molecular Weight 355.34, IR: 3268, 3199,
3065, 2942, 2836, 1677, 1589, 1539, 1499, 1465, 1333. NMR: 13.91 (1H, s, NH.),
11.31 (1H, s, NH), 7.62 (1H, d, J=8), 7.40 (1H, t, J=16), 7.19 (2H, s, ar), 7.12 (1H,
t, J=16), 6.98 (1H, d, J=8), 3.87 (6H, s, 2ꢀOCH3), 3.76 (3H, s, OCH3). MS (ESI)
m/z: 354.00 (355.34).

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2.2.6. 4-Chloro-N'-[5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5f). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 74%, MP
>300 °C; Molecular formula C15H9Cl2N3O2, Molecular Weight 334.15, IR: 3231,
3187, 3047, 2917, 2849, 1686, 1593, 1461, 1262, 1147. NMR: 13.81 (1H, s, NH.),
11.49 (1H, s, NH), 7.92 (2H, d, J=8), 7.71 (2H, d, J=8), 7.59 (1H, s, ar), 7.45ꢀ7.43
(1H, m, ar.), 6.99 (1H, d, J=8). MS (ESI) m/z: 332.00 (334.15).
2.2.7. 4-Bromo-N'-[5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5g). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 75%, MP 288ꢀ
290 °C; Molecular formula C15H9BrClN3O2, Molecular Weight 378.60, IR: 3228,
3047, 2918, 2851, 1686, 1592, 1531, 1462, 1308, 1261, 1146. NMR: 13.81 (1H,
s, NH.), 11.48 (1H, s, NH), 7.86ꢀ7.81 (4H, m, ar), 7.59 (1H, s, ar), 7.56 (1H, s, ar),
7.45ꢀ7.43 (1H, m, ar), 6.99 (2H, d, J=8). MS (ESI) m/z: 380.00 (378.60).
2.2.8. N'-[5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-4-nitrobenzo-
hydrazide (5h). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 72%, MP
>300 °C; Molecular formula C15H9ClN4O4, Molecular Weight 344.70, IR: 3247,
3175, 2917, 2849, 1708, 1681, 1601, 1525, 1462, 1352, 1316. NMR: 13.93 (1H,

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s, NH.), 11.57 (1H, s, NH), 8.42 (2H, d, J= 8), 8.14 (3H, dm, J=8), 7.46ꢀ7.43 (1H,
m, ar), 6.99 (1H, d, J=8). MS (ESI) m/z: 343.00 (344.70).
2.2.10. N'-[5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-3,4,5-trimethoxy-
benzohydrazide (5j). Obtained according to the general procedure for the
synthesis of the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield
67%, MP >300 °C; Molecular formula C18H16ClN3O5, Molecular Weight 389.78,
IR: 3265, 3108, 3047, 2942, 2835, 1710, 1661, 1580, 1527, 1492, 1333. NMR:
13.87 (1H, s, NH), 11.42 (1H, s, NH), 7.61 (1H, s, ar), 7.45ꢀ7.42 (1H, m, ar), 7.19
(2H, s, ar), 7.00 (1H, d, J=8), 3.87 (6H, s, 2ꢀOCH3), 3.76 (3H, s, OCH3). MS (ESI)
m/z: 390.10 (389.78).
2.2.11. 4-Chloro-N'-[5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5k). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 75%, MP
>300 °C; Molecular formula C15H9ClFN3O2, Molecular Weight 317.70, IR: 3219,
3055, 2922, 2850, 1715, 1671, 1594, 1540, 1480, 1309, 1271, 1164. NMR: 13.87
(1H, s, NH.), 11.39 (1H, s, NH), 7.92 (2H, d, J=8), 7.71 (2H, d, J=8), 7.43 (1H, s,
ar), 7.27ꢀ7.22 (1H, m, ar), 6.98ꢀ6.95 (1H, m, ar). MS (ESI) m/z: 316.10 (317.70).
2.2.12. 4-Bromo-N'-[5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5l). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 75%, MP
>300 °C; Molecular formula C15H9BrN3O2, Molecular Weight 362.15, IR: 3216,

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3062, 2925, 2850, 1720, 1669, 1590, 1539, 1479, 1390, 1270, 1162. NMR: 13.87
(1H, s, NH.), 11.39 (1H, s, NH), 7.83 (4H, s, ar), 7.44ꢀ7.42 (1H, m, ar), 7.27ꢀ7.22
(1H, m, ar), 6.98ꢀ6.95 (1H, m, ar). MS (ESI) m/z: 362.00 (362.15).
2.2.13. N'-[5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-4-nitrobenzo-
hydrazide (5m). Obtained according to the general procedure for the synthesis
of the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 76%, MP
>300 °C; Molecular formula C15H9FN4O4, Molecular Weight 328.25, IR: 3222,
3059, 2918, 2849, 1674, 1599, 1519, 1479, 1275. NMR: 13.91 (1H, s, NH.),
11.41 (1H, s, NH), 8.44 (2H, d, J=8), 8.14 (2H, d, J=8), 7.45 (1H, s, ar), 7.28ꢀ7.23
(1H, m, ar), 6.98ꢀ6.95 (1H, m, ar). MS (ESI) m/z: 327.10 (328.25).
2.2.15. N'-[5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-3,4,5-trimethoxy-
benzohydrazide (5o). Obtained according to the general procedure for the
synthesis of the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield
71%, MP 228ꢀ230 °C; Molecular formula C18H16FN3O5, Molecular Weight 373.33,
IR: 3229, 3065, 2949, 1674, 1587, 1532, 1482, 1332, 1129. NMR: 13.95 (1H, s,
NH), 11.35 (1H, s, NH), 7.48ꢀ7.45 (1H, m, ar), 7.29ꢀ7.23 (1H, m, ar), 7.22 (2H, s,
ar), 7.01ꢀ6.98 (1H, s, ar), 3.89 (6H, s, 2ꢀOCH3), 3.78 (3H, s, OCH3). MS (ESI)
m/z: 374.33 (373.33).
2.2.16. 4-Chloro-N'-[5-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-

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10
hydrazide (5p). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 75%, MP
>300 °C; Molecular formula C16H12ClN3O2, Molecular Weight 313.73, IR: 3230,
3040, 2919, 2851, 1709, 1671, 1599, 1482, 1378, 1273, 1140. NMR: 13.90 (1H,
s, NH), 11.26 (1H, s, NH), 7.91 (2H, d, J=8), 7.70 (2H, d, J=8), 7.43 (1H, s, ar),
7.22 (1H, d, J=8) 6.86 (1H, d, J=8), 2.31 (3H, s, CH3). MS (ESI) m/z: 314.10
(313.73).
2.2.17. 4-Bromo-N'-[5-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5q). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 77%, MP
>300 °C; Molecular formula C16H12BrN3O2, Molecular Weight 358.18, IR: 3232,
3040, 2919, 2851, 1708, 1671, 1594, 1525, 1481, 1378, 1273. NMR: 13.88 (1H,
s, NH), 11.25 (1H, s, NH), 7.82 (4H, m, ar), 7.42 (1H, s, ar), 7.21 (1H, d, J=8),
6.85 (1H, d, J=8), 2.30 (3H, s, CH3). MS (ESI) m/z: 358.00 (358.18).
2.2.18. N'-[5-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-4-nitrobenzo-
hydrazide (5r). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 74%, MP
>300 °C; Molecular formula C16H12N4O4, Molecular Weight 324.29, IR: 3256,
3113, 2918, 2851, 1702, 1677, 1601, 1523, 1339, 1140. NMR: 14.01 (1H, s, NH.),

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11.31 (1H, s, NH), 8.45 (2H, d, J=8), 8.13 (2H, d, J=8), 7.43 (1H, s, br), 7.23 (1H,
d, J=8), 6.87 (1h, d, J=8), 2.31 (3H, s, CH3). MS (ESI) m/z: 323.10 (324.29).
2.2.19. N'-[5-Methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5s). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 71%, MP
>300 °C; Molecular formula C16H13N3O2, Molecular Weight 279.29, IR: 3232,
3036, 2915, 2848, 1703, 1673, 1594, 1522, 1481, 1382, 1139. NMR: 13.81 (1H,
s, NH), 11.25 (1H, s, NH), 7.84ꢀ7.79 (5H, m, ar), 7.42 (1H, s, ar), 7.21 (1H, d, J=8)
6.86 (1H, d, J=8), 2.30 (3H, s, CH3).
2.2.20. 3,4,5-Trimethoxy-N'-[5-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-
benzohydrazide (5t). Obtained according to the general procedure for the
synthesis of the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield
74%, MP 150 °C; Molecular formula C19H19N3O5, Molecular Weight 369.37, IR:
3502, 3137, 3097, 2919, 1675, 1564, 1357, 1154. NMR: 13.92 (1H, s, NH.),
11.20 (1H, s, NH), 7.45 (1H, s, ar), 7.21ꢀ7.18 (3H, m, ar), 6.87 (1H, d, J=8), 3.86
(6H, s, 2ꢀOCH3), 3.76 (3H, s, OCH3), 2.31 (3H, s, CH3). MS (ESI) m/z: 368.10
(369.37).
2.2.21. 4-Chloro-N'-[5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5u). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 76%, MP

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>300 °C; Molecular formula C15H9ClN4O4, Molecular Weight 344.70, IR: 3176,
3108, 2918, 2849, 1706, 1676, 1601, 1528, 1467. NMR: 13.69 (1H, s, NH.),
12.03 (1H, s, NH), 8.34ꢀ8.29 (2H, m, ar), 7.94 (2H, d, J=8), 7.74 (2H, d, J=8),
7.18 (1H, d, J=8). MS (ESI) m/z: 343.00 (344.70).
2.2.22. 4-Bromo-N'-[5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5v). Obtained according to the general procedure for the synthesis of
the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 71%, MP
>300 °C; Molecular formula C15H9BrN4O4, Molecular Weight 389.16, IR: 3176,
3108, 2845, 1706, 1674, 1593, 1528, 1466, 1340, 1217. NMR: 13.69 (1H, s, NH.),
12.03 (1H, s, NH), 8.34ꢀ8.29 (2H, m, ar), 7.89ꢀ7.84 (4H, m, ar), 7.19 (1H, d, J=8).
MS (ESI) m/z: 388.90 (389.16).
2.2.23. 4-Nitro-N'-[5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzo-
hydrazide (5w). Obtained according to the general procedure for the synthesis
of the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield 75%, MP
>300 °C; Molecular formula C15H9N5O6, Molecular Weight 355.26, IR: 3192, 3108,
2925, 1707, 1681, 1603, 1523, 1343, 1146. NMR: 13.68 (1H, s, NH.), 12.02 (1H,
s, NH), 8.46 (2H, d, J=8), 8.34ꢀ8.31 (2H, m, ar), 8.29 (1H, s, ar), 8.16 (2H, d, J=8),
7.19 (1H, d, J=8). MS (ESI) m/z: 354.10 (355.26).
2.2.25. 3,4,5-Trimethoxy-N'-[5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-

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benzohydrazide (5y). Obtained according to the general procedure for the
synthesis of the N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzohydrazideꢀYield
70%, MP 298ꢀ300 °C; Molecular formula C18H16N4O7, Molecular Weight 400.34,
IR: 3144, 3089, 2949, 2918, 1702, 1662, 1585, 1336, 1132. NMR: 13.70 (1H, s,
NH.), 11.92 (1H, s, NH), 8.32ꢀ8.30 (2H, m, ar), 7.22ꢀ7.17 (3H, m, ar), 3.88 (6H, s,
2ꢀOCH3), 3.77 (3H,s,OCH3). MS (ESI) m/z: 399.10 (400.34).
2.3. Biological evaluations
2.3.1. Cell cultures
Human cell lines K562 (Chronic myelogenous leukemia), REH (Bꢀcell
leukemia) were cultured in RPMI1640 (Sera Lab, UK) containing 10% FBS
(Gibco BRL, USA) along with 100 U of Penicillin G/ml as well as 100 ꢁg of
streptomycin/ml (Sigma–Aldrich, USA) at 37°C in a humidified atmosphere
containing 5% CO2.
2.3.2. Cytostatic assays
The methodology for undertaking the antiproliferative assays has been
published previously [31]. In brief, varying concentrations of the compounds (5ꢀ
fold dilutions) were incubated at 37 °C for 72 h (HeLa and CEM Tꢀlymphocytes)
or 48 h (L1210 cells) in 200 µl 96ꢀwell microtiter plates, and the viable tumor cell
number was counted at the end of the incubation period using a Coulter Counter
(Coulter Electronics, Harpenden Hertz, U.K.).
2.3.3. Trypan blue dye exclusion assay

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The viability of the leukemic cells (REH, K562) were determined by trypan
blue dye exclusion assay after treatment with 5t [32]. Cells were cultured (0.5 ×
105 cells/ml) for 24 h and 5t was added at different concentration (0ꢀ10 µM).
DMSO treated cells were used as vehicle control. Cells were collected at 48 and
72 h after treatment with 5t and viable cells were counted following trypan blue
staining. Each experiment was repeated three independent times and standard
error mean was calculated and plotted with error bars.
2.3.4. MTT assay
The proliferation rate of the cells after treatment with 5t was checked by
using MTT assay [33]. REH, K562 cells (0.5 × 105 cells/ml) were treated with 5t
and MTT assay was carried out after 48 and 72 h incubation. Cells treated with
equal amount of DMSO were used as vehicle control. Experiment was repeated
three independent times each with duplicate reactions and bar diagram was
plotted with error bars.
2.3.5. Cell cycle analysis
Effect of 5t on cell cycle was checked as described previously [34]. Briefly,
REH cells were cultured (0.5 × 105 cells/ml), and after 24 h incubation, 5t was
added (0, 0.1 and 0.5 µM), cells were harvested after 24 and 48 h of treatment,
washed, fixed with 70% ethanol and RNaseꢀA treatment was given overnight ,
finally analyzed in BD FACSVerse™ flow cytometer using propidium iodide stain.
A minimum of 10,000 cells were acquired per sample and histograms were
plotted and analyzed using Flowing software (version2.5). Experiment was
repeated two independent times and each cell cycle phase was indicated in bar
diagram with error bar.

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2.3.6. Measurement of the mitochondrial membrane potential
Mitochondrial transmembrane potential was measured using JCꢀ1 (5, 5’, 6,
6’ꢀ tetrachloroꢀ1, 1, 3, 3’ꢀ tetraethylbenzimidazolcarbocyanamide iodided;
Calbiochem, USA) dye after treatment with 5t. Briefly, after 48 h of treatment with
5t at 0, 0.1 and 0.5 µM concentration, cells were harvested, and incubated in the
media containing 0.5 µM JCꢀ1 for 20 min at 37º C, cells were washed in
phosphate buffered saline and analyzed in (FACS Calibur, BD Biosciences, USA)
flow cytometer. 2, 4ꢀDNP was used as a positive control [35]. Green (low
mitochondrial membrane potential) versus Red (high mitochondrial membrane
potential) ratio of JCꢀ1 florescence was calculated using Flowing software
(version2.5) and represented in a bar diagram with error bar using two
independent experiments.
2.3.7. Annexin V-FITC flow cyometric analysis
Movement of phosphatidylserine to outer membrane upon apoptotic
induction was detected by Annexin VꢀFITC staining (Santacruz, USA) [36]. REH
cells were treated with 5t (0, 0.1 and 0.5 µM) for 48 h and DMSO treated cells
were used as vehicle control, cells were harvested, washed with cold 1x
phosphate buffered saline and reꢀsuspended in binding buffer (HEPESꢀ10 mM
pH 7.4, 144 mM NaCl and 25 mM CaCl2), cells were incubated with Annexin Vꢀ
FITC (200 µg/ml) and propidium iodide (10 µg/ml) for 20 min at room temperature
and analyzed in BD FACSVerse™ flow cytometer. A minimum of 10,000 cells
were acquired per sample and represented in dot plot. Experiment was repeated

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two independent times and bar diagram was plotted for early apoptosis, late
apoptosis and necrosis with error bars.
2.3.8. DNA fragmentation assay
The fragmentation of DNA after treatment with 5t was checked using
agarose gel electrophoresis [37, 38]. Briefly, REH cells were treated with 5t in
different concentration (0, 0.1, 0.5 and 1 µM) for 48 h, DMSO treated cells were
used as a vehicle control. Cells were harvested and total genomic DNA was
isolated using standard protocol, and dissolved in 1X TE, loaded on 2% agarose
gel and run at 50 v for 3 h. DNA Marker was used to check the length of
fragmented DNA ladder.
2.3.9. Detection of intracellular ROS production by flow cytometer
The total intracellular ROS production after treatment with 5t was
determined using cell permeable fluorescent probe 2, 7ꢀdichlorodihydro
fluorescein diacetate (H2DCFDA) in REH cells [39]. Briefly, REH cells were
treated with 0.5 µM of 5t for different time points (5, 15, 30 and 60 min), cells
were harvested , washed with 1x phosphate buffered saline and incubated with
0.5 µM H2DCFDA for 370 C for 15 minutes and the green fluorescence intensity
was analyzed by flow cytometry (FACS Calibur, BD Biosciences, USA). Cells
which are treated with H2O2 were used as positive control. The shift in the peak
after treatment with 5t was compared with control and percentage of ROS
production was determined.
2.3.11. Statistical analysis

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The values were expressed as mean ± SEM for control and experimental
samples and statistical analysis was performed using Oneꢀway ANOVA followed
by the Dunnett test. Significance was mentioned after comparing treated samples
with control. For this analysis, GraphPad software prism 5.1 was used. The
values were considered as statistically significant, if the pꢀvalue was equal to or
less than 0.05.
3. Results and Discussion
3.1. Chemistry
The
synthesis
of
the
N’ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀ
ylidene]benzohydrazide derivatives (5a-y) from 1Hꢀindoleꢀ2,3ꢀdione I and the
benzohydrazide II has been outlined in Scheme 1. The chemical structures of
the newly synthesized 5a-y were established on the basis of analytical and
spectroscopic data. In the IR spectra, the absorption for carbonyl groups and NH
bands were observed in the range of 1720ꢀ1661 cmꢀ1 and 3289ꢀ3165 cmꢀ1,
1
respectively. In the HꢀNMR spectra, the chemical shifts showed for ring NH
between δ = 13.95ꢀ13.66 ppm and for amide NH between 12.02ꢀ11.19 ppm in the
form of singlet. The aromatic protons signals appeared in the range of δ = 8.46ꢀ
6.85 ppm. The signals for CH3 and OCH3 were observed in the range of δ = 2.31ꢀ
2.30 and 3.89ꢀ3.76 ppm, respectively. Furthermore, all compounds were
confirmed by ESIꢀMS and were in agreement with their molecular weight.
3.2. Biological evaluation

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3.2.1. Cytotoxicity in human and murine tumor cell cultures
The evaluation of the compounds in Scheme 1 towards human cervix
carcinoma (HeLa), Tꢀlymphocyte (CEM) and murine leukemia (L1210) cells was
undertaken in order to determine whether the compounds were cytotoxic to
human cancer cells.
The cytotoxicity of the compounds in series 5a-y was determined and
melphalan was used as a standard compound (Table 1). There was no
improvement in cytotoxic activity of compounds 5a-c, 5f-p and 5u-y. Their IC50
values were >250 µM, except for derivative 5g. When hydrogen was substituted
with electronꢀwithdrawing groups like halogen (Cl/F) and nitro (NO2) at the 5th
position of the isatin moiety resulting in the formation of compounds 5f-o and 5u-
y, the cytotoxic activity was poor towards all cell lines. These studies suggest
that electronꢀwithdrawing substitution is not favorable for either the isatin or the
phenyl groups.
However, when the methyl and methoxy groups were substituted on the
isatin and phenyl groups, respectively, resulting in the formation of the
derivatives 5p-t, there was much improvement in cytotoxicity towards all three
cell lines (IC50 33– 0.66 ꢀM) except for compound 5r (IC50 >250 µM). The
structureꢀactivity relationship study suggests that electronꢀdonating groups such
as methyl and methoxy (5d, 5e, 5q, 5s and 5t) are preferred over electronꢀ
withdrawing substituents such as halogens (5f-o) and the nitro (5u-y) group.

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Among the tested compounds, 5t was clearly the more potent in the L1210, CEM
and HeLa tumor cell assays [sub micromolar range].
The data therefore provided ample evidence for pursuing derivative 5t for
further study. As the lead compound 5t showed significant cytotoxicity against
leukemic cells, we further carried out the studies to understand the mechanism of
action in different leukemia cells, namely REH and K562.
3.2.2. Induction of cytotoxicity in REH and K562 cells.
To evaluate the cytotoxicity exerted by 5t, we used the leukemic cell lines,
REH and K562. Bꢀcell leukemia cells (REH) showed a very good response
towards treatment of 5t and we found that the IC50 value was around 0.5 µM
(Fig.1A, B). At the same time, chronic myelogenous leukemia cells (K562)
showed a moderate response to 5t and the IC50 value was found to be ~2 µM
(Fig.1C, D).
3.2.3. Effect of 5t on cell cycle progression
To check whether 5t affects cell cycle progression, REH cells were treated
with 5t at different concentrations (0, 0.1 and 0.5 µM) for 24 and 48 h. The cells
were harvested and analyzed by flow cytometry after staining with propidium
iodide. The results showed no cell cycle arrest at tested concentration and time
point, however, significant accumulation of cells in the SubG1 phase was
observed in a concentration dependent manner (Fig.2A, B).
3.2.4. Effect of 5t on the mitochondrial membrane potential
Mitochondria play a major role in induction of apoptosis [40]. To check the
mitochondrial membrane potential which is very important for maintaining the

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integrity of the cell, we treated REH cells with 5t (100 and 500 nM for 48 h),
stained with the JC1 dye and analyzed the cells by FACS. Interestingly, we
observed disruption of the mitochondrial membrane potential in a concentration
dependent manner, which suggests that 5t induced apoptosis by disruption of the
mitochondrial membrane potential (Fig.3A, B).
3.2.5. Annexin-V-FITC flow cytometry
To check the nature of cell death, annexinꢀVꢀFITC staining was done for
REH cells after treatment with 0.1 and 0.5 µM of 5t at the 48 h timeꢀpoint.
Results showed an increased number of annexinꢀVꢀFITCꢀpositive cells after
treatment with 5t, which clearly suggested the apoptotic mode of cell death (Fig.
4A, B).
3.2.6. DNA fragmentation assay
Fragmentation of DNA by activated endonucleases inside the cell at the
onset of drug treatment is one of the markers for apoptosis [41]. Therefore, we
investigated the possibility of DNA fragmentation in REH cells after 48 h of
treatment with 5t (0, 0.1, 0.5 and 1 µM). Interestingly, the treatment of 5t resulted
in a fragmented DNA ladder formation in a concentrationꢀdependent manner
from 0.5 µM onwards, which suggested possible apoptotic induction upon 5t
treatment (Fig.5A). Both DNA fragmentation and AnnexinꢀVꢀFITC staining
confirmed the induction of apoptosis in REH cells after the treatment with 5t.
3.2.4. Effect of 5t on intracellular ROS production
Generation of reactive oxygen species by inducing cell death or apoptosis
is one of the mechanisms of action of many drugs. To examine the generation of

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ROS, REH cells were treated with 5t (0.5 µM) at different time points such as 5,
15, 30 and 60 minutes. Cells treated with H2O2 served as a positive control. The
results showed no ROS generation by 5t at the selected time points (Fig.5B),
which suggested 5t induced cell death is independent of ROS production.
4. Conclusion
In summary, a series of 25 isatinꢀhydrazone derivatives with various
structural features were synthesized and their cytotoxicity was evaluated against
human cervix carcinoma, Tꢀlymphocyte and murine leukemia cell lines. The
structure activity relationship studies indicated that a methyl group on the isatin
and a 3,4,5ꢀtrimethoxy substituents on the benzene are required for pronounced
cytotoxicity (5s, 5q and 5t). Therefore, derivative 5t was selected for further
studies to understand the mechanism of action in two different leukemic cell
lines, REH and K562. The cell cycle study, analysis of mitochondrial membrane
potential and Annexin VꢀFITC using flow cytometry suggested that 5t was able to
induce apoptosis without arresting the cell cycle. In summary, 5t, compound with
potent anticancer activity could be studied further and developed as an
anticancer agent.
Acknowledgements
We gratefully acknowledge the support for this research effort from All
India Council for Technical Education (AICTE), New Delhi (Ref. No.
8023/BOR/RID/RPSꢀ169/2008ꢀ09) for SSK. The cytotoxic activities were
determined by financial support of the KU Leuven (GOA 10/014). We would also
like to thank the NMR facility, FACS facility, Indian Institute of Science,

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Bangalore for characterization of all compounds. We thank Lizette van
Berckelaer for the cytostatic determinations.
Supplementary material
Supplementary data associated with this article can be found in the online
version.
References
[1]
0. doi:10.1016/j.chembiol.2014.11.013.
[2] M. Srivastava, M. Nambiar, S. Sharma, S.S. Karki, G. Goldsmith, M.
M. Srivastava, S. C. Raghavan. Chem. Biol. 2015, S1074ꢀ5521(14)00422ꢀ
Hegde, S. Kumar, M. Pandey, R. K. Singh, P. Ray, R. Natarajan, M. Kelkar, A.
De, B. Choudhary, S. C. Raghavan, Cell 151 (2012) 1474.
[3]
F. John, J. George, S. V. Vartak, M. Srivastava, P. A. Hassan, V. K. Aswal,
S. S. Karki, S. C. Raghavan. Macromol Biosci. 15 (2015) 521.
doi:10.1002/mabi.201400480.
[4]
X. Chen, S. Zhong, X. Zhu, B. Dziegielewska, T. Ellenberger, G. M.
Wilson, A. D.Jr MacKerell, A. E. Tomkinson. Cancer Res. 68 (2008) 3169.
doi:10.1158/0008ꢀ5472.CANꢀ07ꢀ6636.
[5]
V.E. Anatoly, S.E. Rashel, J.L Margeris, A.Z. Aina, Z.D. Anda, M.K. Iya,
FR 2540868, August 17, 1984 Chem Abstr 1985;102:78567j.

Page 23 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA01528F
23
[6]
A.C. Cunha, J.M. Figueiredo, J.L.M. Tributino, A.L.P. Miranda, H.C.
Castro,R.B. Zingali, C.A.M. Fraga, M.C.B.V. de Souza, V.F. Ferreira, E.J.
Barreiro, Bioorg. Med. Chem. 11 (2003) 2051.
[7]
Fraga, E.J. Barreiro, Farmaco 54 (1999) 747.
[8] P.C. Lima, L.M. Lima, K.C.M. da Silva, P.H.O. Le´da, A.L.P. de Miranda,
C.A.M. Fraga, E.J. Barreiro, Eur. J. Med. Chem. 35 (2000) 187.
[9] C. Loncle, J.M. Brunel, N. Vidal, M. Dherbomez, Y. Letourneux, Eur. J.
L.F.C.C. Leite, M.N. Ramos, J.B.P. da Silva, A.L.P. Miranda, C.A.M.
Med. Chem. 39 (2004) 1067.
[10] A.R. Todeschini, A.L.P. de Miranda, K.C.M. da Silva, S.C. Parrini, E.J.
Barreiro, Eur. J. Med. Chem. 33 (1998) 189.
[11] N. Terzioglu, A. Gu¨rsoy, Eur. J. Med. Chem. 38 (2003) 781.
[12] J.R. Dimmock, D.C. Smith, J.M. Brenner, S.S. Jonnalagadda, M.S.
Sardessai, J.D. Wood, G.E. Bigam, Eur. J. Med. Chem. 21 (1986) 187.
[13] M. Katyal, Y. Dutt, Talanta 22 (1975) 151.
[14] J. Wang, H. Tan, Y. Li, Y. Ma, Z. Li, L.W. Guddat, J. Agric. Food Chem. 59
(2011) 59, 9892.
[15] N.Y. Mok, J. Chadwick, K.A.B. Kellett, N.M. Hooper, A.P. Johnson, C.W.G.
Fishwick, Bioorg. Med. Chem. Lett. 19 (2009) 6770.
[16] P. Vicini, M. Incerti, I. A. Doytchinova, P. La Colla, B. Busonera, R. Loddo,
Eur. J. Med. Chem. 41 (2006) 624.
[17] H. Abadi, A. A. H. Eissa, G. S. Hassan, Chem. Pharm. Bull. 51 (2003) 838.

RSC Advances
Page 24 of 36
View Article Online
DOI: 10.1039/C5RA01528F
24
[18] D. Kumar, N. M. Kumar, S.Ghosh, K. Shah, Bioorg. Med. Chem. Lett. 22
(2012) 212.
[19] S.S. Karki, V.S. Bahaduria, V. Rana, S. Kumar, G.S. Prasanna, U. Das, J.
Balzarini, E. De Clercq, J.R. Dimmock, J. Enz. Inh. & Med. Chem. 24 (2009) 537.
[20] K. Jin, X. Zhang, C. Ma, Y. Xu, Y. Yuan, W. Xu, Bioorg. Med. Chem. 21
(2013) 2663.
[21] A. Natarajan , YunꢀHua Fan , H. Chen , Y. Guo , J. Iyasere , F.
Harbinski, W. J. Christ , H. Aktas , J. A. Halperin, J. Med. Chem. 47 (2004) 1882.
[22] L. HuaꢀQuan, W. DeꢀCai, W. Fei, T. Wei, O. PingꢀKai, Chin. Chem. Lett.
24 (2013) 929.
[23] R.W. Daisley, V.K. Shah, J. Pharm. Sci. 73 (1984) 407.
[24] S.D. Jorge, F. PalaceꢀBerl, A. Masunari, C.A. Cechinel, M. Ishii, K.F.M.
Pasqualoto, L.C. Tavares, Bioorg. Med. Chem. 19 (2011) 5031.
[25] T. Medwick, H.M. Abdou, J. Pharm. Sci. 68 (1979) 301.
[26] N.P. Peet, S. Sunder, J. Het. Chem. 21 (1984) 1807.
[27] N.P. Grimster, S. Connelly, A. Baranczak, J. Dong, L.B. Krasnova, K.B.
Sharpless, E.T. Powers, I.A. Wilson, J.W. Kelly, J. Am. Chem. Soc. 135 (2013)
5656.
[28] K. Sung, L. AnꢀRong, J. Het. Chem. 29 (1992) 1101.
[29] K. Debnath, S. Pathak, A. Pramanik, Tetrahedron Lett. 54 (2013) 4110.
[30] Z. Liang, D. Zhang, J. Ai, L. Chen, H. Wang, X. Kong, M. Zheng, H. Liu, C.
Luo, M. Geng, H. Jiang, K. Chen, Bioorg. Med. Chem. Lett. 21 (2011) 3749

Page 25 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA01528F
25
[31]
P.B. Baraldi, N.M. DelC, M.A. Tabrizo, E.De. Clercq, J. Balzarini, J.
Bermejo, F. Estevez, R. Romagnoli, J. Med. Chem. 47 (2004) 2877.
[32] B.T. Moorthy, S. Ravi, M. Srivastava, K.K. Chiruvella, H. Hemlal, O. Joy,
S.C. Raghavan. Bioorg. Med. Chem. Lett. 20 (2010) 6297.
[33] S. Kumar, V. Gopalakrishnan, M. Hegde, V. Rana, S. S. Dhepe, S. A.
Ramareddy, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, M. Srivastava, S. C.
Raghavan, S. S. Karki, Bioorg. Med. Chem. Lett. 24 (2014) 4682.
[34] S. Sharma, K. Panjamurthy, B. Choudhary, M. Srivastava, M.
Shahabuddin, R. Giri, G.M. Advirao, S.C. Raghavan. Mol. Carcinog. 52 (2013)
413.
[35] S. S. Karki, K. Panjamurthy, S. Kumar, M. Nambiar, S. A. Ramareddy, K.
K. Chiruvella, S. C. Raghavan, Eur. J. Med. Chem. 46 (2011) 2109.
[36] K. K. Chiruvella, K. Panjamurthy, B. Choudhary, O. Joy, S. C. Raghavan,
Int. J. Biomed. Sci. 2010 ;6(3):182ꢀ94.
[37] U. Sahu, H. Sidhar, P. S. Ghate, G. M. Advirao, S. C. Raghavan, R. K. Giri,
PLoS One. 8 (2013) e66430.
[38] S. Kumar, M. Hegde, V. Gopalakrishnan, V. K. Renuka, S. A. Ramareddy,
E.De. Clercq, D. Schols, G. N. Anilkumar, S. C. Raghavan, S. S. Karki, Eur. J.
Med. Chem. 84 (2014) 687.
[39] M. Hegde, S. S. Karki, E. Thomas, S. Kumar, K. Panjamurthy, S. R.
Ranganatha, K. S. Rangappa, B. Choudhary, S. C. Raghavan, PLoS One. 7
(2012) e43632.
[40] C. Wang, R.J. Youle, Annu. Rev. Genet. 43 (2009) 95.

RSC Advances
Page 26 of 36
View Article Online
DOI: 10.1039/C5RA01528F
26
[41] Y. Gavrieli, Y. Sherman, S.A. BenꢀSasson, J. Cell Biol. 119 (1992) 493.
Figure legends
Scheme 1. Synthesis of N’ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀylidene]benzoꢀ
hydrazide derivatives (5aꢀy).
Table 1. Inhibitory effects of derivatives of N'ꢀ[2ꢀoxoꢀ1,2ꢀdihydroꢀ3Hꢀindolꢀ3ꢀ
ylidene]benzohydrazide derivatives on the proliferation of murine leukemia
(L1210), human Tꢀlymphocyte (CEM) and cervix carcinoma (HeLa) cells
Figure 1. Cytotoxic effect of 5t on REH and K562 cells. REH and K562 cells
were seeded (0.5X105 cells/ml) and 5t (0 ꢀ 10 µM) was added after 24 h. Trypan
blue assay (to detect the number of viable cells) and MTT assay (to check
proliferation rate) were performed at 48 and 72 h of treatment. A. Trypan blue
assay for 5t treated REH cells. B. MTT assay for 5t treated REH cells. C. Trypan
blue assay for 5t treated K562 cells. D. MTT assay for 5t treated K562 cells. The
cells treated with equal concentration of DMSO was used as vehicle control and
in all the cases (indicated by ‘C’). Each experiment was performed three
independent times and error bars are indicated.
Figure 2. Cell cycle analysis of 5t treated REH cells. REH cells were seeded
(0.5X105 cells/ml) and were treated with 5t (0, 0.1 and 0.5 µM) after 24 h. Cells
were harvested after 24 and 48 h of treatment, fixed in 70% ethanol, washed and

Page 27 of 36
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DOI: 10.1039/C5RA01528F
27
stained with propidium iodide and analyzed in flow cytometer. A and B.
Histogram representing effect on REH cell cycle by 5t at 24 and 48 h respectively.
C and D. The bar graph representing different phases of cell cycle in REH cells
after treatment with 5t at 24 and 48 h respectively. Error bars were plotted from
two independent experiments.
Figure 3. Measurement of mitochondrial membrane potential. Total
mitochondrial membrane potential after treatment with 5t was measured by using
JCꢀ1 dye. REH cells were treated with 5t (0, 0.1 and 0.5 µM) for 48 h. Cells were
harvested, stained with JCꢀ1 dye at 37º C for 20 min and analyzed in flow
cytometer. 2, 4ꢀDNP treated cells were used as a positive control, negative
control is DMSO treated cells without JCꢀ1 staining and JCꢀ1 stained DMSO
treated cells served as Control. A. Dot plot showing control as well as 5t treated
REH cells. B. Bar diagram showing red (High MMP) and green (Low MMP)
fluorescence ratio of JCꢀ1 with error bars.
Figure 4. Annexin V-FITC flow cytometry. Apoptotic signals from 5t treated
REH cells was detected using Annexin VꢀFITC staining. After 48 h of treatment
with 5t (0, 0.1 and 0.5 µM) cells were harvested, and stained with both Annexin
VꢀFITC and propidium iodide for 20 minutes and analyzed in flow cytometer.
DMSO treated cells were used as vehicle control. A. Dot plot representing control
and 5t treated cells. B. Bar diagram representing early apoptotic, late apoptotic
and necrotic population of control as well as treated cells. Experiment was
repeated minimum two times and error bar was represented.

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28
Figure 5. Detection of DNA fragmentation and ROS generation in 5t treated
REH cells.
A.REH cells were treated with 5t (0, 0.1, 0.5 and 1 µM for 48 h) and cells were
harvested, genomic DNA was isolated and subjected to agarose gel
electrophoresis. DMSO treated cells were used as vehicle control. Samples were
run on a 2% agarose gel (50v for 3 h) along with the DNA marker. ‘C’ represents
the DMSO treated samples, ‘M’ represents the DNA marker lane.
B. REH cells were seeded (0.5X105 cells/ml) and treated with 0.5 µM of 5t for
different time points (5, 15, 30 and 60 min). Cells were harvested and stained
with 0.5 µM H2DCFDA for 15 min at 37o C. ROS production was analyzed using
flow cytometer. Positive control indicates H2O2 treated cells. Cells without
H2DCFDA served as a negative control and DMSO treated cells with H2DCFDA
was served as control. % cells which are positive for green fluorescent of ROS
were measured.

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Scheme 1. Synthesis of N’-[2-oxo-1,2-dihydro-3H-indol-3ylidene]benzohydrazide
derivatives (5a-y)
Compound
5a
R
R1
4-Cl
Compound
5j
R
R1
Compound
5s
R
CH3
R1
H
H
Cl 3,4,5-tri-
OCH3
H
4-Br
F
4-Cl
CH3 3,4,5-tri-
OCH3
5b
5k
5t
H
H
H
4-NO2
4-OCH3
3,4,5-tri-
OCH3
F
F
F
4-Br
4-NO2
H
NO2
NO2
NO2
4-Cl
4-Br
4-NO2
5c
5d
5e
5l
5m
5n
5u
5v
5w
Cl
Cl
Cl
4-Cl
F
3,4,5-tri-
OCH3
4-Cl
NO2
H
5f
5o
5p
5x
5y
4-Br
CH3
CH3
NO2 3,4,5-tri-
OCH3
5g
4-NO2
4-Br
5h
5i
5q
5r
Cl 4-OCH3
CH3 4-NO2