Development of 2-oindolin-3-ylidene-indole-3-carbohydrazide derivatives as novel apoptotic and anti-proliferative agents towards colorectal cancer cells

Article abstract of DOI:10.1080/14756366.2020.1862100

Mitochondrial anti-apoptotic Bcl2 and BclxL proteins, are overexpressed in multiple tumour types, and has been involved in the progression and survival of malignant cells. Therefore, inhibition of such proteins has become a validated and attractive target for anticancer drug discovery. In this manner, the present studies developed a series of novel isatin–indole conjugates (7a-j and 9a-e) as potential anticancer Bcl2 and BclxL inhibitors. The progression of the two examined colorectal cancer cell lines was significantly inhibited by all of the prepared compounds with IC₅₀ ranges132–611 nM compared to IC₅₀ = 4.6 μM for 5FU, against HT-29 and IC₅₀ ranges 37–468 nM compared to IC₅₀ = 1.5 μM for 5FU, against SW-620. Thereafter, compounds 7c and 7g were selected for further investigations. Interestingly, both compounds exhibited selective cytotoxicity against both cell lines with high safety to normal fibroblast (HFF-1). In addition, both compounds 7c and 7g induced apoptosis and inhibited Bcl2 and BclxL expression in a dose-dependent manner. Collectively, the high potency and selective cytotoxicity suggested that conjugates 7c and 7g could be a starting point for further optimisation to develop novel pro-apoptotic and antitumor agents towards colon cancer.

Full text of DOI:10.1080/14756366.2020.1862100

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Development of 2-oindolin-3-ylidene-indole-3-
carbohydrazide derivatives as novel apoptotic and
anti-proliferative agents towards colorectal cancer
cells
Wagdy M. Eldehna , Mahmoud F. Abo-Ashour , Tarfah Al-Warhi , Sara T. Al-
Rashood , Amal Alharbi , Rezk R. Ayyad , Khayal Al-Khayal , Maha Abdulla ,
Hatem A. Abdel-Aziz , Rehan Ahmad & Radwan El-Haggar
To cite this article: Wagdy M. Eldehna , Mahmoud F. Abo-Ashour , Tarfah Al-Warhi , Sara
T. Al-Rashood , Amal Alharbi , Rezk R. Ayyad , Khayal Al-Khayal , Maha Abdulla , Hatem A.
Abdel-Aziz , Rehan Ahmad & Radwan El-Haggar (2021) Development of 2-oindolin-3-ylidene-
indole-3-carbohydrazide derivatives as novel apoptotic and anti-proliferative agents towards
colorectal cancer cells, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 319-328, DOI:
10.1080/14756366.2020.1862100
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 01, 319–328
https://doi.org/10.1080/14756366.2020.1862100
RESEARCH PAPER
Development of 2-oindolin-3-ylidene-indole-3-carbohydrazide derivatives as novel
apoptotic and anti-proliferative agents towards colorectal cancer cells
Wagdy M. Eldehnaa, Mahmoud F. Abo-Ashourb, Tarfah Al-Warhic, Sara T. Al-Rashoodd, Amal Alharbid,
Rezk R. Ayyade, Khayal Al-Khayalf, Maha Abdullaf, Hatem A. Abdel-Azizg, Rehan Ahmadf and Radwan El-Haggarh
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; Department of Pharmaceutical
c
Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Egypt; Department of Chemistry, College of Science, Princess Nourah
d
bint Abdulrahman University, Riyadh, Saudi Arabia; Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
e
f
Riyadh, Saudi Arabia; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt; Colorectal Research
g
Chair, Department of Surgery, King Khalid University Hospital, King Saud University College of Medicine, Riyadh, Saudi Arabia; Department of
Applied Organic Chemistry, National Research Center, Giza, Egypt; Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan
h
University, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 13 October 2020
Revised 18 November 2020
Accepted 1 December 2020
Mitochondrial anti-apoptotic Bcl2 and BclxL proteins, are overexpressed in multiple tumour types, and has
been involved in the progression and survival of malignant cells. Therefore, inhibition of such proteins has
become a validated and attractive target for anticancer drug discovery. In this manner, the present studies
developed a series of novel isatin–indole conjugates (7a-j and 9a-e) as potential anticancer Bcl2 and BclxL
inhibitors. The progression of the two examined colorectal cancer cell lines was significantly inhibited by
all of the prepared compounds with IC50 ranges132–611nM compared to IC50 ¼ 4.6 mM for 5FU, against
HT-29 and IC50 ranges 37–468nM compared to IC50 ¼ 1.5 mM for 5FU, against SW-620. Thereafter, com-
pounds 7c and 7g were selected for further investigations. Interestingly, both compounds exhibited
selective cytotoxicity against both cell lines with high safety to normal fibroblast (HFF-1). In addition, both
compounds 7c and 7g induced apoptosis and inhibited Bcl2 and BclxL expression in a dose-dependent
manner. Collectively, the high potency and selective cytotoxicity suggested that conjugates 7c and 7g
could be a starting point for further optimisation to develop novel pro-apoptotic and antitumor agents
towards colon cancer.
KEYWORDS
Anti-proliferative indoles;
apoptosis; isatin-indole;
Bcl2/BclxL inhibitors;
colorectal cancer;
western blotting
1. Introduction
apoptotic and anti-apoptotic proteins that control the cellular
apoptosis14–16
.
Cancer is a group of diseases that is characterised by uncontrolled
and rapid cell proliferation and differentiation mechanisms with
the potential to invade or spread to other body parts1. Since sev-
eral decades, cancer has been one of the main world health prob-
lems and is still considered a serious leading cause of death
worldwide. Early strategies of cancer treatment were based on the
unspecific induction of cell death mainly targeting the replication
machinery2–5 and/or the DNA synthesis6–9. Therefore, the trad-
itional anticancer drugs were associated with severe adverse
effects due to the unselective toxicity towards the normal cells in
addition to the resistance emerged towards them9. Thus, the
development of effective and safe new antitumour drugs with
increased selectivity towards cancer cells is still an active
search10,11. On the other hand, recent strategies of targeted thera-
pies target specific biomarkers essential for the regulation of can-
cer cells proliferation and/or cell apoptosis such as deregulated,
mutated, or overexpressed proteins12 and thus, selectively affect
cancer cells or their supporting environment with minimum
Apoptosis, a form of programmed cell death takes place in
multicellular organisms, is a series of biochemical events that
result in characteristic cell changes and death17. Apoptosis could
be launched through one of two pathways (intrinsic pathway and
extrinsic pathway). The mitochondria-dependent apoptotic path-
way (intrinsic pathway), one of the main pathways of induction of
the cell apoptosis18,19, is controlled by the Bcl2 proteins family.
The Bcl2 family members have dual functions; some are anti-apop-
totic Bcl2 proteins, such as Bcl2 and BclxL that inhibit apoptosis,
while others are pro-apoptotic Bcl2 proteins such as Bax and Bak
that promote apoptosis14. In this regard, it was reported that
most of cancer cells are characterised by over-expression of
the anti-apoptotic Bcl2 proteins which could lead to apoptosis
prevention as well as drug resistance20,21. Thus, the development
of anti-apoptotic Bcl2 proteins inhibitors has become an import-
ant strategy for introducing potential anti-cancer agents15,16. In
this manner, several heterocyclic scaffolds including isatin22 were
effects on normal cells13. Among these targets are the pro- developed as candidates targeting Bcl2 proteins.
CONTACT Wagdy M. Eldehna
Egypt; Sara T. Al-Rashood
wagdy2000@gmail.com
salrashood@ksu.edu.sa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh,
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

320
W. M. ELDEHNA ET AL.
Figure 1. Chemical structures for some reported isatin-based anticancer conjugates (I-III), and the target conjugates (7a-j and 9a-e).
Isatin (1H-indole-2,3-dione), as a special class in drug design furnish the final compounds 9a-e, respectively. The structure of
and discovery, represents one of the most favourable scaffolds of the synthesised 2-oxindolin-3-ylidene-indole-3-carbohydrazide was
heterocyclic systems which possesses many interesting biological
activities including anti-SARS-CoV-223, antimicrobial24, anticonvul-
sant25 and mainly anticancer26–28. Therefore, isatin nucleus was
broadly used by our group for the development of diverse effect-
ive oxindole-based small molecules (structures I–III29–31, Figure 1)
with anticancer activities that target different enzymatic and cellu-
lar targets such as inhibition of cancer-related carbonic anhydrase
IX isoform32–33, inhibition of different kinases34–35, in addition to
confirmed under the basis of spectral and elemental analyses
which were in full agreement with the proposed structures.
2.2. Biological evaluation
2.2.1. Anti-proliferative activity against HT-29 and SW-620 colorec-
tal cancer cell lines
The anti-proliferative potential of the final compounds (7a-j and
9a-e) was examined against two human colorectal cancer HT-29
and SW-620 cell lines. While, HT-29 is an adenocarcinoma cell line,
SW-620 represents metastatic cancer cell line. These effects were
compared with known anti-cancer drug, 5-Fluorouracil (5FU) com-
monly used in colorectal cancer treatment. All of the compounds
were found to inhibit the cell viability of cancer cells with varied
sub-micro-molar efficacy. The IC50 values ranged from 105 to
611 nM for all compounds were calculated using Graph Pad prism
8 (Table 1).
apoptosis induction in different human cancer cell lines36–37
.
Motivated by the aforementioned findings and in continuation
to our previous work, in the present study, a novel series of isatin/
indole conjugates (7a-j and 9a-e, Figure 1) were designed and
synthesised. The antiproliferative effect of the new compounds
against HT-29 and SW-620 colorectal cancer cell lines were exam-
ined. In addition, the levels of the mitochondria-related anti-apop-
totic proteins Bcl2 and BclxL in HT-29 and SW-620 colorectal
cancer cell lines after incubation with isatin derivatives 7c and 7g
were determined.
Regarding the activity towards HT-29 cell line, all compounds
showed superior activity to the reference drug (5FU) with IC50
ranged from 132-611 nM compared to IC50¼4.6 mM for 5FU.
Compound 7h was the most active against HT-29 cell line with
IC50¼132 nM that is approximately 35-fold more than 5FU. Also,
the results revealed that the N-alkylation of isatin moiety (series
9a-e) resulted in diversified effect on the potency according to
the alkyl group added and/or the substitution on indole moiety.
For the unsubstituted indole (7a, IC50¼408 nM), while N-alkylation
with CH3- group significantly increase the potency (9a,
IC50¼176 nM) and N-alkylation with benzyl- group slightly increase
the potency (9c, IC50¼405 nM), the N-alkylation with propyl
group dramatically decrease the potency (9b, IC50¼525 nM).
Furthermore, N-propylation of both chloro- (7c, IC50¼206 nM) and
bromo- (7d, IC50¼320 nM) derivatives significantly increase the
potency (9d, IC50¼166 nM) and (9e, IC50¼290 nM), respectively.
Similarly, the activity towards SW-620 cell line of all compounds
was more that 5FU with IC50 ranged from 37–468 nM compared
to IC50¼1.5 mM for 5FU. Compound 7f was the most active against
SW-620 cell line with IC50¼37 nM that is approximately 32 folds
2. Results and discussion
2.1. Chemistry
The synthetic strategies deliberate for the development of the
final compounds (7a-j and 9a-e) were illustrated in Schemes 1–2.
In Scheme 1, indole 1 was subjugated to formylation via Vilsmeier
haack reaction to produce 1H-indole-3-carbaldehyde 2, in which
the CHO functionality was oxidised by KMnO4 in acetone to fur-
nish 1H-indole-3-carboxylic acid 3. Then, the acid analogue 3 was
subjected to esterification through refluxing in dry methanol to
get the carboxylate analogue 4, where the ester group reacted
with hydrazine hydrate in methyl alcohol to produce the key
intermediate 1H-indole-3-carbohydrazide 5. Finally, the key inter-
mediate 5 was condensed with different isatin derivatives 6a-j in
glacial acetic acid to give the final targeted compounds 7a-j.
On the other hand, in Scheme 2, three isatins 6a, 6c and 6d
were alkylated with methyl iodide, propyl bromide and benzyl
bromide in DMF with the presence of K2CO3 and catalytic amount
of KI to give the N-substituted isatin derivatives 8a-e, which more than 5FU. However, N-alkylation of isatin moiety resulted in
heated under reflux with the carbohydrazide 5 in acetic acid to reduction of potency for all series except for compounds 9b

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
321
Scheme 1. Synthesis of target 2-oxindolin-3-ylidene-indole-3-carbohydrazide 7a-j; (i) DMF/POCl3/reflux 8 h, (ii) KMnO4/Acetone/Stirring at R.T 12h, (iii) Dry methanol/
H2SO4 (Cat.)/reflux 7 h, (iv) hydrazine hydrate/methanol/reflux 4 h, (v) Glacial acetic acid/reflux (5–7) h.
Scheme 2. Synthesis of target N-substituted 2-oxindolin-3-ylidene-indole-3-carbohydrazide 9a-e; (i) R-Br/DMF/K2CO3/KI (Cat.)/reflux 5 h, (ii) Glacial acetic acid/reflux
(5–7) h.
(IC50¼105 nM) compared to compound 7a (IC50¼145 nM)
Table 1. In vitro anti-proliferative actions of compounds 7a-j and 9a-e towards (Table 1).
HT-29 and SW-620 colorectal cancer cell lines.
Figure 2 illustrated that treatment of HT-29 and SW-620 cell
lines with different concentrations of compounds 7c and 7g
resulted in
a
dose-dependent inhibition of cell viability.
Compound 7c was found to inhibit HT-29 and SW-620 with
IC50¼188 nM and 206 nM, respectively (Figure 2(A,B)), whereas
compound 7g was found to exhibit IC50 of 279 nM against HT-29
and 299 nM against SW-620 cell lines (Figure 2(C,D)), compared to
the IC50 for 5FU that was found to be 1.5 mM against HT-29 and
4.6 mM against SW-620 cell lines.
Furthermore, to investigate the selective cytotoxicity of the
tested compounds, the effect of compounds 7c and 7g was
studied on normal human skin fibroblast (HFF-1). Compounds 7c
and 7g were found to have no/or little effect on cell viability of
HFF-1 (Figure 3(A,B)). This finding indicates that compounds 7c
and 7g inhibited cell viability of cancer cells without affecting nor-
mal fibroblast.
IC50 (nM)a
Comp.
R
R1
R2
HT-29
SW-620
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
F
Cl
H
H
H
H
H
H
H
H
F
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
408
611
206
320
335
142
299
132
140
200
176
525
405
166
290
4600
145
175
188
133
253
37
Br
CH3
OCH3
OCF3
NO2
H
CH3
H
H
H
279
190
468
187
260
105
283
290
414
1500
7j
H
2.2.2. Annexin V-FITC apoptosis assay
9a
9b
9c
9d
9e
5-FU
CH3
propyl
benzyl
propyl
propyl
Induction of apoptosis is mainly considered as an important strat-
egy for the development of new anti-proliferative agents38.
Compounds 7c and 7g were further investigated for their poten-
tial role of apoptosis induction in the colorectal SW-620 cancer
cell line (SW-620 cell line was selected because most of the new
compounds showed significant potency more than HT-29 cell
Cl
Br
aIC50 values are the mean SD of three separate experiments.

322
W. M. ELDEHNA ET AL.
Figure 2. Effect of Compound 7c and 7g on the cell viability. Different concentrations of compound 7c and 7g have been used to study its effect on cell viability by
MTT assay. Results of percent viability vs concentration were plotted and the IC50 was calculated for each cell line using Graph Pad prism 8. (A) HT-29 with compound
7c, (B) SW-620 with compound 7c, (C) HT-29 with compound 7g, and (D) SW-620 with compound 7g.
Figure 3. Effect of Compounds 7c and 7g on HFF-1. HFF-1 fibroblast cells were treated with compound 7c and 7g for 24h and % viability was measured using MTT
assay. (A) HFF-1 with compound 7c and (B) HFF-1 with compound 7g.
line). Exposure of SW-620 cells to compounds 7c and 7g resulted 2.2.3. Impact of compounds 7c and 7g on the anti-apoptotic
in the induction of apoptosis in a dose dependent manner (Figure
4). As shown in the results, compound 7c at concentrations of
250 nM and 500 nM was able to induce approximately 5.2- and
10.66-fold, respectively, total apoptosis increase compared to the
control for SW-620 cell line (Figure 4(A)). Similarly, compound 7g
induced approximately 7.3 and 9.6 fold total apoptosis increase
compared to the control when incubated with SW-620 cell line at
concentration of 250 nM and 500 nM, respectively (Figure 4(B)).
These findings encouraged us to further investigate the effect of
compounds 7c and 7g towards the anti-apoptotic mitochondrial
markers Bcl2 and BclxL.
(Bcl2, and BclxL) markers levels
Bcl2 and BclxL, as anti-apoptotic proteins, are known to be overex-
pressed in diverse tumours causing cancer cell survival and drug
resistance14. Inhibition of these proteins expression resulted in can-
cer cell death and has been exploited as a strategy for anticancer
drug discovery15–16. Treatment of SW-620 with compound 7c
resulted in a dose-dependent inhibition of Bcl2 and BclxL protein
expression (Figure 5(A)). Compound 7g was also found to inhibit
Bcl2 and BclxL expression in SW-620 cells (Figure 5(B)). These find-
ings thus indicate that compound 7c and 7g inhibited cell viability
by inhibiting Bcl2 and BclxL resulting in the apoptosis.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
323
Figure 4. (A) Annexin V apoptosis assay for compound 7c. SW-620 cells were treated with compound 7c (250 and 500 nM) for 24 h. Quadrant was set for the resulted
dots population from the results of the negative auto-fluorescence and the statistical analysis was performed where the significance of data was assessed at p val-
ꢀꢀ
ues < 0.05. p < 0.01;
ꢀꢀꢀ
p < 0.001 control vs treated. (B) Annexin V apoptosis assay for compound 7g. SW-620 cells were treated with compound 7g (250 and
500 nM) for 24 h. Quadrant was set for the resulted dots population from the results of the negative auto-fluorescence and the statistical analysis was performed where
ꢀꢀ
ꢀꢀꢀ
p < 0.001 control vs treated.
the significance of data was assessed at p values < 0.05. p < 0.01;
3. Conclusions
cell lines was significantly inhibited by all of the prepared com-
pounds with IC50 ranges 132–611 nM against HT-29 and IC50
ranges 37–468 nM against SW-620 as compared to IC50¼4.6 and
1.5 mM for 5FU, respectively. For further mechanistic and selective
cytotoxicity studies, compounds 7c and 7g were examined and
In the present work, a series of novel isatin-indole conjugates (7a-
j and 9a-e) was designed and synthesised as potential antiprolifer-
ative agents towards colon cancer cells with promising inhibitory
activity against the anti-apoptotic Bcl2 and BclxL proteins. The cell
growth of two examined colorectal cancer (HT-29 and SW-620) proved to exhibit selective cytotoxicity against both cancer cell

324
W. M. ELDEHNA ET AL.
Figure 5. (A) Effect of compound 7c on anti-apoptotic proteins. SW-620 cancer cells were treated with two concentrations of compound 7c for 24 h. Immunoblotting
was performed with the indicated antibodies. The band densities were measured and the bar charts were created to compare the effect on proteins. The statistical
ꢀꢀꢀ
ꢀꢀ
p < 0.01 control vs treated. (B) Effect of compound 7g on
analysis was performed where the significance of data was assessed at a p values < 0.05.
anti-apoptotic proteins. SW-620 cancer cells were treated with two concentrations of compound 7g for 24 h. Immunoblotting was performed with the indicated anti-
p < 0.001;
bodies. The band densities were measured and the bar charts were created to compare the effect on proteins.
lines with high safety profile to normal fibroblast (HFF-1). In add- 4.1.2. Synthesis of the target compounds 7a-j and 9a-e
To hot stirred solution of 1H-indole-3-carbohydrazide 5 (0.3 g,
1.7 mmole) in 15 ml of glacial acetic acid, an equivalent amount of
appropriate isatin derivatives 6a-j and 8a-e was added. The reac-
tion mixture was heated under reflux for (5–7) h, and then was
cooled to room temperature. The formed precipitate was collected
by filtration, washed with cold water, hexane and recrystallized
from DMF-MeOH mixture to furnish the targeted novel com-
pounds 7a-j and 9a-e, respectively. Representative NMR spectra
charts were provided in the Supplementary data.
ition, towards SW-620 cell line, both candidates inducted apop-
tosis and inhibited anti-apoptotic Bcl2 and BclxL proteins in a
dose dependent manner. Collectively, the high potency and
selective cytotoxicity suggested that conjugates 7c and 7g could
serve as lead compounds for further optimisation to develop
novel antitumor agents and Bcl2/BclxL inhibitors.
4. Experimental
4.1.2.1. N’-(2-Oxoindolin-3-ylidene)-1H-indole-3-carbohydrazide 7a.
4.1. Chemistry
1
Yellow powder; m.p. 259–261 ꢁC; (yield 81%); H NMR d ppm: 6.93
4.1.1. General
(d, 0.3H, Ar-H, J ¼ 8.0 Hz), 6.98 (d, 0.7H, Ar-H, J ¼ 8.0 Hz), 7.07–7.16
(m, 1H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.37 (t, 1H, Ar-H, J ¼ 8.0 Hz),
7.52–7.58 (m, 1H, Ar-H), 7.65 (d, 0.7H, Ar-H, J ¼ 8.0 Hz), 8.13 (d,
0.3H, Ar-H, J ¼ 8.0 Hz), 8.23–8.32 (m, 1.7H, Ar-H), 8.67 (s, 0.3H, Ar-
H), 10.83, 13.45 (s, 1H, NH of isatin), 11.18, 11.30 (s, 1H, hydrazide
NH), 12.01, 12.05 (s, 1H, indole NH); 13 C NMR d ppm: 107.50,
108.17, 110.96, 111.56, 112.56, 112.99, 116.14, 120.75, 120.96,
120.98, 121.73, 121.79, 121.97, 122.21, 123.03, 123.21, 126.19,
The NMR spectra have been recorded by Bruker spectrometer at
400 MHz. 13 C NMR spectra were run at 100 MHz in deuterated
dimethylsulphoxide (DMSO-d6). Chemical shifts (dH) are reported
relative to the solvent (DMSO-d6). Infra-red spectra were recorded
on Schimadzu FT-IR 8400S spectrophotometer. Elemental analyses
have been performed at the Regional Centre for Microbiology and
Biotechnology, Al-Azhar University, Cairo, Egypt.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
325
126.54, 127.75, 131.51, 132.48, 133.62, 136.22, 136.65, 136.78, 4.1.2.7. N’-(2-Oxo-5-(trifluoromethoxy)indolin-3-ylidene)-1H-indole-
3-carbohydrazide 7g. Yellow powder; m.p. 278–280 ꢁC; (yield 72%);
142.41, 143.91 (Aromatic carbons), 162.16, 163.37 (C¼O hydrazide),
165.27, 165.65 (C¼O isatin)); IR (KBr, ꢀ cmꢂ1) 3504, 3280, 3245
(3NH) and 1707, 1654 (2 C¼O); Calcd. Anal. for C17H12N4O2: C,
67.10; H, 3.97; N, 18.41; found C, 66.83; H, 4.57; N, 18.41.
1H NMR d ppm: 7.05 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.23–7.29 (m, 2H, Ar-
H), 7.36 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.55–7.60 (m, 2H, Ar-H), 8.22 (d,
1H, Ar-H, J ¼ 8.0 Hz), 8.31 (s, 1H, Ar-H), 11.46 (s, 1H, NH of isatin),
12.06 (s, 1H, hydrazide NH), 13.39 (s, 1H, Indole NH)); IR (KBr, ꢀ
cmꢂ1) 3647, 3445, 3187 (3NH) and 1710, 1641 (2 C¼O); Calcd.
4.1.2.2. N’-(5-Fluoro-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 7b. Red powder; m.p. >300 ꢁC; (yield 78%); 1H NMR d Anal. for C18H11F3N4O3: C, 55.68; H, 2.86; N, 14.43; found C, 55.53;
ppm: 6.90–9.93 (m, 0.7H, Ar-H), 6.96–7.00 (m, 0.3H, Ar-H), 7.20–7.30
(m, 3H, Ar-H), 7.49–7.58 (m, 1.3H, Ar-H), 8.15 (d, 0.7H, Ar-H,
J ¼ 8.0 Hz), 8.23 (d, 0.3H, Ar-H, J ¼ 8.0 Hz), 8.31 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 8.72 (s, 0.7H, Ar-H), 10.84, 13.41 (s, 1H, NH of isatin),
11.31, 11.35 (s, 1H, hydrazide NH), 12.03 (s, 1H, indole NH); IR (KBr,
ꢀ cmꢂ1) 3659, 3460, 3285 (3NH) and 1712, 1669 (2C¼O); Calcd.
Anal. for C17H11FN4O2: C, 63.35; H, 3.44; N, 17.38; found C, 63.14;
H, 4.01; N, 17.37.
H, 2.91; N, 14.39.
4.1.2.8.
N’-(5-Nitro-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 7h. Yellow powder; m.p. >300 ꢁC; (yield 86%); 1H NMR d
ppm: 7.12 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.22–7.29 (m, 2H, Ar-H), 7.54 (d,
1H, Ar-H, J ¼ 8.0 Hz), 8.20 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.24 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 8.30–8.34 (m, 2H, Ar-H), 11.89 (s, 1H, NH of isatin),
12.11 (s, 1H, hydrazide NH), 13.19 (s, 1H, Indole NH); 13 C NMR d
ppm: 107.64, 111.74, 113.00, 115.84, 120.96, 121.46, 121.92, 122.10,
123.31, 127.21, 132.40, 133.29, 136.71, 143.24, 147.41 (Aromatic
carbons), 163.65 (C¼O hydrazide), 166.06 (C¼O isatin)); IR (KBr, ꢀ
cmꢂ1) 3594, 3426, 3111 (3NH) and 1704, 1671 (2 C¼O); Calcd.
Anal. for C17H11N5O4: C, 58.46; H, 3.17; N, 20.05; found C, 57.91; H,
3.71; N, 20.08.
4.1.2.3. N’-(5-Chloro-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 7c. Yellow powder; m.p. >300 ꢁC; (yield 75%); 1H NMR d
ppm: 6.92 (d, 0.7H, Ar-H, J ¼ 8.4 Hz), 6.97 (d, 0.3H, Ar-H, J ¼ 8.4 Hz),
7.20–7.29 (m, 2H, Ar-H), 7.39–7.43 (m, 1H, Ar-H), 7.51–7.58 (m, 1H,
Ar-H), 7.64 (s, 0.3H, Ar-H), 8.22–8.35 (m, 2H, Ar-H), 8.71 (s, 0.7H, Ar-
H), 10.95, 12.03 (s, 1H, NH of isatin), 11.43 (s, 1H, hydrazide NH),
12.03 (s, 1H, Indole NH); IR (KBr, ꢀ cmꢂ1) 3593, 3429, 3331 (3NH)
and 1720, 1643 (2 C¼O); Calcd. Anal. for C17H11ClN4O2: C, 60.28; H,
3.27; N, 16.54; found C, 59.77; H, 3.88; N, 16.37.
4.1.2.9. N’-(7-Fluoro-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 7i. Yellow powder; m.p. >300 ꢁC; (yield 79%); 1H NMR d
ppm: 7.08–7.17 (m, 1H, Ar-H), 7.21–7.29 (m, 2H, Ar-H), 7.31 (t, 1H,
Ar-H, J ¼ 8.0 Hz), 7.50–7.58 (m, 1H, Ar-H), 8.03 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 8.31 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.68 (s, 1H, Ar-H), 11.29 (s,
1H, NH of isatin), 12.05 (s, 1H, hydrazide NH), 13.43 (s, 1H, Indole
NH); 13 C NMR d ppm: 107.34, 108.02, 112.57, 113.01, 117.04,
118.17, 118.68, 119.11, 119.28,120.96, 121.72, 122.03, 122.56,
122.97, 123.27, 123.70, 123.92, 126.20, 127.76, 129.12, 129.25,
130.80, 131.97, 133.86, 136.21, 136.78, 145.81, 146.21, 148.22,
148.63 (Aromatic carbons), 162.09, 163.17 (C¼O hydrazide),
165.30, 165.44 (C¼O isatin)); IR (KBr, ꢀ cmꢂ1) 3527, 3450, 3286
(3NH) and 1716, 1638 (2 C¼O); Calcd. Anal. for C17H11FN4O2: C,
63.35; H, 3.44; N, 17.38; found C, 63.11; H, 4.08; N, 17.33.
4.1.2.4. N’-(5-Bromo-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
1
drazide 7d. Orange powder; m.p. 247–249 ꢁC; (yield 83%); H NMR
d ppm: 6.91 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.20–7.29 (m, 2H, Ar-H), 7.51
(d, 1H, Ar-H, J ¼ 8.0 Hz), 7.55 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.73 (s, 1H,
Ar-H), 8.23 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.34 (s, 1H, Ar-H), 11.34 (s, 1H,
NH of isatin), 12.04 (s, 1H, hydrazide NH), 13.31 (s, 1H, Indole NH);
13 C NMR d ppm: 107.76, 111.54, 112.97, 113.46, 114.78, 121.03,
122.02, 122.86, 123.24, 126.36, 133.58, 133.92, 136.70, 141.39,
142.39 (Aromatic carbons), 162.99 (C¼O hydrazide), 163.35 (C¼O
isatin)); IR (KBr, ꢀ cmꢂ1) 3621, 3428, 3184 (3NH) and 1710, 1638
(2 C¼O); Calcd. Anal. for C17H11BrN4O2: C, 53.28; H, 2.89; N, 14.62;
found C, 52.79; H, 2.82; N, 14.63.
4.1.2.5. N’-(5-Methyl-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy- 4.1.2.10. N’-(5,7-Dimethyl-2-oxoindolin-3-ylidene)-1H-indole-3-car-
drazide 7e. Red powder; m.p. 201–203 ꢁC; (yield 74%); 1H NMR d
ppm: 1.93, 2.33 (s, 3H, CH3), 6.85 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.16 (d,
1H, Ar-H, J ¼ 8.0 Hz), 7.22–7.29 (m, 2H, Ar-H), 7.45 (s, 1H, Ar-H),
7.55 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.23–8.27 (m, 2H, Ar-H), 11.17 (s, 1H,
NH of isatin), 12.04 (s, 1H, hydrazide NH), 13.44 (s, 1H, Indole NH));
IR (KBr, ꢀ cmꢂ1) 3451, 3322, 3252 (3NH) and 1711, 1660 (2 C¼O);
Calcd. Anal. for C18H14N4O2: C, 67.92; H, 4.43; N, 17.60; found C,
67.25; H, 4.98; N, 17.57.
1
bohydrazide 7j. Red powder; m.p. >300 ꢁC; (yield 74%); H NMR d
ppm: 2.22 (s, 3H, CH3 of position 7 for isatin), 2.30 (s, 3H, CH3 of
position 5 for isatin), 7.00 (s, 1H, Ar-H), 7.15–7.28 (m, 3H, Ar-H),
7.54 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.23–8.34 (m, 2H, Ar-H), 11.20 (s, 1H,
NH of isatin), 12.03 (s, 1H, hydrazide NH), 13.45 (s, 1H, Indole NH);
IR (KBr, ꢀ cmꢂ1) 3528, 3448, 3176 (3NH) and 1711, 1687 (2 C¼O);
Calcd. Anal. for C19H16N4O2: C, 68.66; H, 4.85; N, 16.86; found C,
68.14; H, 5.37; N, 16.68.
4.1.2.6. N’-(5-Methoxy-2-oxoindolin-3-ylidene)-1H-indole-3-carbo-
4.1.2.11. N’-(1-Methyl-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 9a. Yellow powder; m.p. 273–275 ꢁC; (yield 66%); 1H NMR
d ppm: 3.22, 3.26 (s, 3H, N-CH3), 7.10–7.18 (m, 2H, Ar-H), 7.22–7.30
(m, 2H, Ar-H), 7.43–7.49 (m, 1H, Ar-H), 7.53–7.58 (m, 1H, Ar-H), 7.66
(d, 0.3H, Ar-H, J ¼ 8.0 Hz), 8.17 (d, 0.7H, Ar-H, J ¼ 8.0 Hz), 8.24–8.33
(m, 1.3H, Ar-H), 8.67 (s, 0.7H, Ar-H), 11.24, 13.38 (s, 1H, hydrazide
NH), 12.04, 12.07 (s, 1H, Indole NH); 13 C NMR d ppm: 26.65 (N-
CH3), 108.60, 110.78, 113.46, 120.48, 121.10, 121.34, 122.45, 123.69,
124.03, 126.55, 131.90, 132.33, 137.24, 144.14 (Aromatic carbons),
1
hydrazide 7f. Red powder; m.p. 197–199 ꢁC; (yield 80%); H NMR d
ppm: 3.81(s, 3H, OCH3), 6.89 (d, 1H, Ar-H, J ¼ 8.0 Hz), 6.95 (d, 1H,
Ar-H, J ¼ 8.0 Hz), 7.19 (s, 1H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.55 (d,
1H, Ar-H, J ¼ 8.0 Hz), 8.22–8.26 (m, 2H, Ar-H), 11.11 (s, 1H, NH of
isatin), 12.05 (s, 1H, hydrazide NH), 13.52 (s, 1H, Indole NH); 13 C
NMR d ppm: 56.09 (OCH3), 106.08, 108.20, 112.35, 112.99, 117.66,
120.94, 121.51, 121.96, 123.21, 126.12, 131.77, 136.07, 136.79,
155.87 (Aromatic carbons), 162.04 (C¼O hydrazide), 163.51 (C¼O
isatin)); IR (KBr, ꢀ cmꢂ1) 3569, 3432, 3174 (3NH) and 1686, 1655
(2 C¼O); Calcd. Anal. for C18H14N4O3: C, 64.67; H, 4.22; N, 16.76;
found C, 64.09; H, 4.76; N, 16.56.
161.95 (C¼O hydrazide), 162.55 (C¼O isatin)); IR (KBr, ꢀ cmꢂ1
)
3436, 3117 (2NH) and 1774, 1725 (2 C¼O); Calcd. Anal. for

326
W. M. ELDEHNA ET AL.
C18H14N4O2: C, 67.92; H, 4.43; N, 17.60; found C, 67.65; H, 4.97; 4.2. Biological evaluation
N, 17.54.
4.2.1. Cell culture
HT29 and SW620 colorectal cancer cell lines and HFF-1 fibroblast
4.1.2.12. N’-(2-Oxo-1-propylindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 9b. Yellow powder; m.p. 248–250 ꢁC; (yield 69%); 1H NMR
(ATCC, Rockville, USA) were used. HT29 cells were cultured and
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)
(GIBCO, by Thermo Fischer Scientific, NY, USA) supplemented with
10% foetal bovine serum (FBS), 100 units/mL penicillin, and
100 mg streptomycin. SW620 and HFF-1 cells were cultured in
Roswell Park Memorial Institute medium (RPMI-1640) (GIBCO, by
Thermo scientific, NY, USA) supplemented with 10% FBS and 1%
penicillin and streptomycin (Napolitano et al., 2015). All cultures
were incubated at 37 ꢁC and humidified atmosphere of 5% CO2.
d
ppm: 0.92 (t, 3H, –CH2CH3, J ¼ 8.0 Hz), 1.65–1.74 (m, 2H,
–CH2CH3), 3.76 (t, 2H, N-CH2, J ¼ 8.0 Hz), 7.17 (t, 1H, Ar-H,
J ¼ 8.0 Hz), 7.23–7.30 (m, 3H, Ar-H), 7.44 (t, 1H, Ar-H, J ¼ 8.0 Hz),
7.55–7.58 (m, 1H, Ar-H), 7.68 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.23–8.25 (m,
1H, Ar-H), 8.29 (s, 1H, Ar-H), 12.06 (s, 1H, hydrazide NH), 13.40 (s,
1H, Indole NH); 13 C NMR
d ppm: 11.67 (–CH2CH3), 20.95
(–CH2CH3), 41.24 (N-CH2), 108.10, 110.53, 113.00, 120.08, 120.78,
120.94, 122.01, 123.23, 123.45, 126.19, 131.42, 131.84, 134.42,
136.80, 143.00 (Aromatic carbons), 161.51 (C¼O hydrazide), 162.10
(C¼O isatin)); IR (KBr, ꢀ cmꢂ1) 3310, 3151 (2NH) and 1727, 1663
(2 C¼O); Calcd. Anal. for C20H18N4O2: C, 69.35; H, 5.24; N, 16.17;
found C, 69.20; H, 5.73; N, 16.19.
4.2.2. Cell viability assay
The cytotoxicity effect of compounds on the colorectal cancer cell
lines, HT29 and SW620 in addition to the normal human fibro-
blasts was measured by MTT (3–(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA)
as previously described39. Briefly, cells were seeded in 96 well cul-
ture plates at 5000/well for HT29 and 10,000/well for SW620 for
24 h. Cells were then incubated with different compounds from
WAG1 series (1–15) 24 h at 37 ꢁC and humidified 5% CO2 incuba-
tor. Freshly prepared 10 ml of 3–(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT 5 mM) solution were added to
the cells and further incubated for 2 h. Thereafter, 100 ml of
dimethyl sulphoxide (DMSO) were added in each well and the
crystals were dissolved through careful pipetting. In certain experi-
ment, cells were treated different concentration of 5FU for 72 h.
The absorbance of the product was measured at 540 nm using a
SynergyTM 2 multi-mode microplate reader (Biotech, VA, USA). The
experiments were performed in triplicate for each condition.
4.1.2.13. N’-(1-Benzyl-2-oxoindolin-3-ylidene)-1H-indole-3-carbohy-
drazide 9c. Yellow powder; m.p. 274–276 ꢁC; (yield 72%); 1H NMR
d ppm: 5.00 (s, 2H, CH2), 7.06 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.16 (t, 1H,
Ar-H, J ¼ 8.0 Hz), 7.24–7.31 (m, 3H, Ar-H), 7.34–7.43 (m, 5H, Ar-H),
7.57 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.70 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.25 (d,
1H, Ar-H, J ¼ 8.0 Hz), 8.32 (s, 1H, Ar-H), 12.09 (s, 1H, hydrazide NH),
13.35 (s, 1H, Indole NH); 13 C NMR d ppm: 43.03 (CH2), 108.09,
110.81, 113.03, 120.21, 120.86, 120.95, 122.06, 123.28, 123.70,
126.18, 127.83, 128.10, 129.22, 131.33, 131.89, 134.29, 136.20,
136.80, 142.62 (Aromatic carbons), 161.52 (C¼O hydrazide), 162.13
(C¼O isatin); IR (KBr, ꢀ cmꢂ1) 3450, 3210 (2NH) and 1748, 1688
(2 C¼O); Calcd. Anal. for C24H18N4O2: C, 73.08; H, 4.60; N, 14.20;
found C, 72.58; H, 5.14; N, 14.06.
4.1.2.14. N’-(5-Chloro-2-oxo-1-propylindolin-3-ylidene)-1H-indole-3-
carbohydrazide 9d. Yellow powder; m.p. 262–263 ꢁC; (yield 70%);
IR (KBr, ꢀ cmꢂ1) 3450, 3221 (2NH) and 1730, 1660 (2 C¼O); 1H
NMR d ppm: 0.90 (t, 3H, –CH2CH3, J ¼ 8.0 Hz), 1.62–1.71 (m, 2H,
–CH2CH3), 3.72 (t, 2H, N-CH2, J ¼ 8.0 Hz), 7.22–7.28 (m, 3H, Ar-H),
7.45 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.55 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.65 (s,
1H, Ar-H), 8.22 (d, 1H, Ar-H, J ¼ 8.0 Hz), 8.34 (s, 1H, Ar-H), 12.06 (s,
1H, hydrazide NH), 13.26 (s, 1H, Indole NH); 13 C NMR d ppm: 11.61
(–CH2CH3), 20.89 (–CH2CH3), 41.37 (N-CH2), 107.71, 112.06, 112.55,
112.99, 120.35, 120.99, 121.81, 122.06, 123.26, 126.35, 127.70,
130.66, 133.15, 136.73, 141.63 (Aromatic carbons), 161.28 (C¼O
hydrazide), 162.20 (C¼O isatin); IR (KBr, ꢀ cmꢂ1) 3450, 3210 (2NH)
and 1748, 1688 (2 C¼O); Calcd. Anal. for C20H17ClN4O2: C, 63.08; H,
4.50; N, 14.71; found C, 62.89; H, 4.94; N, 14.71.
4.2.3. Measurement of apoptosis by annexin V-FITC/PI assay:
Induction of apoptosis was measured by Dead Cell Apoptosis Kit
with Annexin V FITC and PI, for flow cytometry (Thermofischer sci-
entific, OR, USA) according to the manufacturer’s instruction. As
described previously (39), cells were seeded in a 6-well plate
(3 ꢃ 105 cells per well) and treated with the various compound for
24 h. Both floating and adherent cells were harvested, pooled
together, and incubated with Annexin V-FITC and PI for 15 min on
ice in dark. The cells were analysed by BD FACSCaliburTM cell ana-
lyser (BD Biosciences, CA, USA) at an emission of 530 nm (FL1
channel) and >575 nm (FL3).
4.2.4. Western blot analysis:
All cells were seeded in a 100 mm dish (1 ꢃ 106 cells per dish) in
4.1.2.15. N’-(5-Bromo-2-oxo-1-propylindolin-3-ylidene)-1H-indole-3- 5% CO2 at 37 ꢁC in the appropriate culture medium. The cells with
carbohydrazide 9e. Orange powder; m.p. 255–256 ꢁC; (yield 75%);
1H NMR d ppm: 0.95 (t, 3H, –CH2CH3, J ¼ 8.0 Hz), 1.70–1.79 (m, 2H,
–CH2CH3), 3.78 (t, 2H, N-CH2, J ¼ 8.0 Hz), 6.94 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 7.21–7.30 (m, 2H, Ar-H), 7.51 (d, 1H, Ar-H, J ¼ 8.0 Hz),
7.55 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.75 (s, 1H, Ar-H), 8.24 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 8.35 (s, 1H, Ar-H), 12.10 (s, 1H, hydrazide NH), 13.29 (s,
around 50% confluency were treated with compounds for 24 h. At
the experiment day, cells were washed with 1x PBS, harvested
and lysed in RIPA lysis buffer, combined with protease inhibitors,
(Sigma-Aldrich, St. Louis, MO, USA) as described previously39). The
total protein concentration was evaluated by the colorimetric
Bradford protein assay (BIO-RAD inc, CA, USA) at 595 nm absorb-
ance. Lysates were loaded in equal concentration and separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and then transferred to a nitrocellulose membrane by
semi-dry. Blocking of the membrane was done by 5% non-fat
1H, Indole NH); 13 C NMR
d ppm: 11.62 (–CH2CH3), 20.89
(–CH2CH3), 41.35 (N-CH2), 107.68, 112.51, 112.99, 115.33, 121.00,
122.05, 122.17, 123.04, 123.25, 126.36, 132.37, 132.98, 133.45,
136.72, 142.00 (Aromatic carbons), 161.13 (C¼O hydrazide), 162.22
(C¼O isatin); IR (KBr, ꢀ cmꢂ1) 3450, 3179 (2NH) and 1745, 1688 dried milk for one hour, incubated with the primary antibodies.
(2 C¼O); Calcd. Anal. for C20H17BrN4O2: C, 56.48; H, 4.03; N, 13.17; The primary antibodies used were Bcl2 (cat. no. sc-7382), BclxL
found C, 56.06; H, 4.43; N, 13.16.
(cat. no. sc-8392) and b Actin (cat. no. sc-69879) from (Santa Cruz

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
327
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used were goat anti-mouse IgG-HRP (cat. no. sc-2005) and mouse
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Biotechnology, Inc., Dallas, TX, USA). Detection was done with
Luminol HRP chemiluminescence substrate (cat. no. sc-2048) from
(Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and then visual-
ised by c-digit blot-scanner (LI-COR, Nebraska, USA).
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The statistical analysis by the one-way ANOVA test was performed
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Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The authors extend their appreciation to the Deputyship for 19. Wang B, Xu A. Aryl hydrocarbon receptor pathway partic-
Research & Innovation, "Ministry of Education" in Saudi Arabia for
funding this research work through the project number
IFKSURG-1439–065.
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lating mitochondrial apoptosis. Med Hypotheses 2019;123:
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