Development of novel benzofuran-isatin conjugates as potential antiproliferative agents with apoptosis inducing mechanism in Colon cancer

Article abstract of DOI:10.1080/14756366.2021.1944127

In the current work, a new set of carbohydrazide linked benzofuran-isatin conjugates (5a–e and 7a–i) was designed and synthesised. The anticancer activity for compounds (5b–d, 7a, 7b, 7d and 7g) was measured against NCI-55 human cancer cell lines. Compound 5d was the most efficient, and thus subjected to the five-dose screen where it showed excellent broad activity against almost all tested cancer subpanels. Furthermore, all conjugates (5a–e and 7a–i) showed a good anti-proliferative activity towards colorectal cancer SW-620 and HT-29 cell lines, with an excellent inhibitory effect for compounds 5a and 5d (IC₅₀ = 8.7 and 9.4 μM (5a), and 6.5 and 9.8 μM for (5d), respectively). Both compounds displayed selective cytotoxicity with good safety profile. In addition, both compounds provoked apoptosis in a dose dependent manner in SW-620 cells. Also, they significantly inhibited the anti-apoptotic Bcl2 protein expression and increased the cleaved PARP level that resulted in SW-620 cells apoptosis.

Full text of DOI:10.1080/14756366.2021.1944127

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Development of novel benzofuran-isatin
conjugates as potential antiproliferative agents
with apoptosis inducing mechanism in Colon
cancer
Wagdy M. Eldehna, Rofaida Salem, Zainab M. Elsayed, Tarfah Al-Warhi,
Hamada R. Knany, Rezk R. Ayyad, Thamer Bin Traiki, Maha-Hamadien
Abdulla, Rehan Ahmad, Hatem A. Abdel-Aziz & Radwan El-Haggar
To cite this article: Wagdy M. Eldehna, Rofaida Salem, Zainab M. Elsayed, Tarfah Al-Warhi,
Hamada R. Knany, Rezk R. Ayyad, Thamer Bin Traiki, Maha-Hamadien Abdulla, Rehan
Ahmad, Hatem A. Abdel-Aziz & Radwan El-Haggar (2021) Development of novel benzofuran-
isatin conjugates as potential antiproliferative agents with apoptosis inducing mechanism in
Colon cancer, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 1424-1435, DOI:
10.1080/14756366.2021.1944127
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1424–1435
https://doi.org/10.1080/14756366.2021.1944127
RESEARCH PAPER
Development of novel benzofuran-isatin conjugates as potential antiproliferative
agents with apoptosis inducing mechanism in Colon cancer
Wagdy M. Eldehnaa, Rofaida Salema, Zainab M. Elsayedb, Tarfah Al-Warhic, Hamada R. Knanyd, Rezk R. Ayyade,
Thamer Bin Traikif, Maha-Hamadien Abdullaf, Rehan Ahmadf, Hatem A. Abdel-Azizg and Radwan El-Haggarh
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; Scientific Research and Innovation
c
Support Unit, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; Department of Chemistry, College of Science, Princess Nourah
d
bint Abdulrahman University, Riyadh, Saudi Arabia; Department of Pharmacognosy, College of Pharmacy, Mansoura University, Mansoura,
e
f
Egypt; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt; Colorectal Research Chair, Department
g
of Surgery, King Khalid University Hospital, King Saud University College of Medicine, Riyadh, Saudi Arabia; Department of Applied Organic
Chemistry, National Research Center, Dokki, Giza, Egypt; Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University,
h
Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 14 April 2021
Accepted 11 June 2021
In the current work, a new set of carbohydrazide linked benzofuran-isatin conjugates (5a–e and 7a–i) was
designed and synthesised. The anticancer activity for compounds (5b–d, 7a, 7b, 7d and 7g) was meas-
ured against NCI-55 human cancer cell lines. Compound 5d was the most efficient, and thus subjected to
the five-dose screen where it showed excellent broad activity against almost all tested cancer subpanels.
Furthermore, all conjugates (5a–e and 7a–i) showed a good anti-proliferative activity towards colorectal
KEYWORDS
Benzofuran hydrazide;
Isatin; Cleaved PARP; Bcl2
inhibitors; Colon cancer
cancer SW-620 and HT-29 cell lines, with an excellent inhibitory effect for compounds 5a and 5d (IC50
¼
8.7 and 9.4 mM (5a), and 6.5 and 9.8 mM for (5d), respectively). Both compounds displayed selective cyto-
toxicity with good safety profile. In addition, both compounds provoked apoptosis in a dose dependent
manner in SW-620 cells. Also, they significantly inhibited the anti-apoptotic Bcl2 protein expression and
increased the cleaved PARP level that resulted in SW-620 cells apoptosis.
GRAPHICAL ABSTRACT
1. Introduction
strategies towards cancer cells has received more attention and
still ongoing active search11,12
.
Cancer, a large family of diseases, is characterised by fast and
uncontrolled cell division and differentiation mechanisms and has
the potential to spread to or invade other body parts1. For several
decades, cancer is considered one of the major world public
health problems, and it remains a serious reason of the death of
human beings all over the world2. Despite the presence of a var-
iety of cancer treatment strategies, the majority of which induces
non-selective cell death by targeting the DNA synthesis3–6 and/or
the replication machinery7–10. These early strategies are accompa-
nied by severe side effects due to the unspecific cytotoxicity
towards the cancer cells in addition to the resistance developed
against them4. Therefore, the development of safe and effective
Recent strategies for anticancer development are to target spe-
cific biomarkers required for cancer cells division and/or induction
of cell apoptosis such as deregulated, mutated, or over expressed
proteins13 and thus, affect cancer cells selectively with minimum
influences on normal cells14. Among these targets are the anti-
apoptotic protein Bcl2 and Poly ADP-ribose polymerase (PARP). In
this regard, several reports stated that numerous of cancer cells
are characterised by anti-apoptotic proteins (Bcl2) over-expression,
which could lead to prevention of cell apoptosis as well as devel-
opment of drug resistance15,16. On the other hand, PARP is a fam-
ily of proteins involved in numerous cellular functions such as
novel anticancer agents with increased selective treatment DNA repair and genomic stability17–19 and also, PARP was
CONTACT Wagdy M. Eldehna
Kafrelsheikh, Egypt; Hatem A. Abdel-Aziz
wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, P.O. Box 33516,
hatem_741@yahoo.com Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, Egypt
Supplemental data for this article can be accessed here.
ß 2021 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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1425
Figure 1. Structures of some reported isatin-bearing conjugates (I–V), as well as structures for target benzofuran-isatin conjugates (5a–e and 7a–i).
reported to activate programmed cell death, through cleavage (compound III)53, isatin-indole (compound IV)54 and isatin-quinazoline
into PAR (Poly ADP-ribose), which motivates mitochondria to pro- (compound V)55 conjugates were reported to possess promising anti-
duce apoptosis inducing factors20. Thus, the development of com-
cancer activities (Figure 1).
pounds that inhibit the antiapoptotic Bcl2 proteins and/or
potentiate the cleavage of PARP could be a promising approach
to identify new anticancer therapies.
Encouraged by the aforementioned findings and considering the
need to develop safe and effective novel anticancer agents, a new
attempt to study the significance of utilisation of heterocycles hybrid-
isation approach to furnish efficient anti-proliferative activity was
reported herein. A novel series of benzofuran-isatin conjugates (5a–e
and 7a–i, Figure 1) linked by a carbohydrazide group, was designed
and synthesised. The new compounds were screened for their poten-
tial anticancer activity following NCI, USA protocol against fifty-five dif-
ferent cell lines under nine different cancer panels. In addition, the
cytotoxic effect of these conjugates against SW-620 and HT-29 colorec-
tal cancer cell lines was investigated and their ability to induce cell
apoptosis was examined. Furthermore, the level of the mitochondrial
antiapoptotic protein Bcl2 and the level of cleaved PARP in both SW-
620 and HT-29 colorectal cancer cell lines were also determined.
Heterocyclic compounds in particular oxygen containing het-
erocycles represent an important class of compounds possessing
interesting pharmacological and biological activities21–23
.
Benzofuran nucleus, as a key functional scaffold, represents a basic
structure in a diversity of biologically active synthetic and natural
products24–26, with broad range of desirable activities including;
anti-Alzheimer’s27, antibacterial28, anti-tubercular29, antioxidant30,
anti-inflammatory31, as well as antitumor activities32. Benzofuran
derivatives exert their antiproliferative activity with diversified
mechanisms such as inhibition of tubulin polymerisation33,34, HIF-
135, Aurora B kinase36 and VEGFR-2 activity37. Furthermore, some
benzofurans mediate their antiproliferative activity via apoptosis
induction in various human cancer cell lines38–40. In addition, ben-
zofuran-based conjugates were largely studied and were found to
exert significant anticancer activity, such as conjugation of benzo-
2. Results and discussion
2.1. Chemistry
furan with pyrazole41, indole42 and others43,44
.
The adopted synthetic strategy to develop the target N-unsubsti-
tuted 3-methyl-N0-(oxoindolin-3-ylidene)benzofuran-2-carbohydra-
zide derivatives 5a–e was outlined in Scheme 1.
On the other hand, isatin is identified as a privileged nucleus that
included in many pharmacologically active small molecules, such as
antiviral45, antimicrobial46, anticonvulsant47, CNS-acting48, as well as
anticancer49,50 agents. Over the last few years, hybridisation of isatin
nucleus with different heterocycles has been reported as a successful
approach to develop efficient antitumor agents towards different
Key starting ester 3-methylbenzofuran-2-carboxylate 2, was
prepared in 85% yield through cyclisation of 1–(2-hydroxypheny-
l)ethan-1-one 1 and ethyl bromoacetate in anhydrous acetonitrile
with the presence of potassium carbonate. Thereafter, heating of
cancer types through diverse enzymatic and cellular mechanisms49,50
.
To name just a few, isatin-phthalazine (compound I)51, isatin-thia- ester derivative 2 with hydrazine hydrate in methanol afforded
zolo[3,2-a]benzimidazole (compound II)52, isatin-thiazolidinone the corresponding key intermediate 3-methylbenzofuran-2-

1426
W. M. ELDEHNA ET AL.
Scheme 1. Synthesis of target conjugates 5a–e; (i) Anhydrous CH3CN/potassium carbonate/reflux 8 h, (ii) Hydrazine hydrate/methanol/reflux 4 h, (iii) Ethanol/drops gla-
cial acetic acid (Cat.)/reflux 3–6 h.
Scheme 2. Synthesis of target benzofurans 7a–i; (i) (R-Br or Ar-Br)/Acetonitrile/KI (Cat.)/potassium carbonate/reflux 3 h, (ii) Ethanol absolute/drops glacial acetic acid
(Cat.)/reflux 3–6 h.
carbohydrazide 3. Finally, carbohydrazide 3 was condensed with addition, 1H NMR spectra of N-benzyl bearing derivatives 7d–f, 7h
and 7i displayed the characteristic singlet signal of the benzylic pro-
tons at d 4.98–5.07 ppm, while spectra for hybrids 7a, 7b and 7g
revealed the presence of the aliphatic protons corresponding to the
N-substituents in these derivatives at d (3.28 ppm), (0.93, 1.66 and
3.76 ppm) and (0.97, 1.69 and 3.80 ppm), respectively.
different indoline-2,3-dione derivatives 4a–e, through heating
under reflux temperature in absolute ethyl alcohol and few drops
of acetic acid, to give the desired benzofuran-based compounds
5a–e, respectively, in 72–89%yield.
On the other hand, Scheme 2 illustrated the synthetic pathway
utilised to synthesise N-substituted 3-methyl-N0-(oxoindolin-3-yli-
dene)benzofuran-2-carbohydrazide derivatives 7a–i. In this
scheme, alkylation of indoline-2,3-diones 4a and 4c was accom-
plished via heating with different alkyl bromide or benzyl bromide
derivatives in anhydrous acetonitrile to produce N-substituted
indoline-2,3-dione derivatives 6a–i. Then indoline-2,3-diones 6a–i
were condensed with the key intermediate carbohydrazide 3 pro-
ducing target benzofurans 7a–i, respectively, in 75–87% yield.
Structures of the newly prepared benzofuran-based derivatives
5a–e and 7a–i were verified based on spectral and elemental analy-
On the other hand, 13 C NMR spectra for the novel compounds
5a–e and 7a–i showed one signal belonging to the carbon of CH3 of
benzofuran ring at d 8.12–9.49 ppm, also, they showed two signals
belonging to the carbon of C ¼ O functionalities for both the hydra-
zide linker and isatin moiety at range d (161.15–163.62) and
(164.08–167.02) ppm, respectively. In addition, the existence of ben-
zylic carbon in N-benzyl bearing derivatives 7d–f, 7h and 7i was con-
firmed by a signal at d 42.14–46.03 ppm, whereas, the carbons of
propyl moiety in compounds 7b and 7g appeared as signals at range
d (11.68–13.00), (20.92–22.98) and (41.30–48.74) ppm.
1
ses. H NMR spectra of 5a–e and 7a–i revealed the presence of two
singlet peaks for the protons of C-3 CH3 of benzofuran ring and NH
of the hydrazide linker at range d (2.52–2.72) and (11.37–14.10) ppm,
respectively. Moreover, structure of compounds 5a–e was confirmed
via presence of another singlet D2O exchangeable signal attributable
2.2. Biological evaluation
2.2.1. Nci screening of anticancer activity
In the present investigation, the chemical structures for the novel
to the proton of NH for isatin moieties at d 10.91–11.98 ppm. In benzofuran-isatin
conjugates
were
presented
to
the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1427
Table 1. In vitro Anticancer screening results of compounds 5b–d,7a, 7b, 7d
and 7g against fifty-five human tumour cell lines with single dose assay (10ꢀ5 M
concentration). Data was provided as cell growth inhibition percentage.
Compounda
Subpanel / tumour
cell lines
5b
5c
5d
7a
7b
7d
7g
Leukaemia
CCRF-CEM
MOLT-4
HL-60(TB)
K-562
–
–
–
–
–
–
–
–
42.92
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
49.14
46.13
57.50
56.52
21.46
18.04
17.62
–
10.94
11.45
–
SR
RPMI-8226
Non-small cell lung cancer
EKVX
NA
NT
NT
NT
29.63 32.13
62.73 25.07 32.35 30.23 18.10
A549/ATCC
HOP-92
HOP-62
NCI-H322M
NCI-H23
NCI-H522
NCI-H460
Colon cancer
HCC-2998
COLO 205
SW-620
–
–
–
–
–
27.92
26.54
41.41
31.80
33.05
123.59
143.99
124.09
69.41
89.35
45.72
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.64
18.06 20.82
11.87 17.83
15.06 13.19
10.32 21.72
15.60 24.98
Figure 2. Mean % growth inhibition of compounds 5b–d,7a, 7b, 7d and 7g
against NCI-55 cancer cell line panel.
10.36 10.33
–
42.60
122.83
Developmental Therapeutics Program at the National Cancer
Institute (NCI), USA. Seven conjugates (5b–d,7a, 7b, 7d and 7g)
were selected, according to NCI’s-DTP selection guidelines56, for
evaluating their potential in vitro anticancer activity against a
panel of fifty-five human cancer cell lines representing nine
tumour panels according to the NCI, Bethesda, Drug Evaluation
–
–
–
–
–
–
–
–
–
56.46
77.24
64.85 30.57
128.55
50.91
72.67
56.20
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.57
32.03
–
11.21
HCT-116
HCT-15
HT-29
KM12
–
–
–
–
–
–
–
–
–
Branch protocol57,58
.
20.46
CNS cancer
SF-539
–
–
–
–
–
57.88
11.66
20.30
42.58
31.48
62.58
42.33
94.95 11.84 11.86
97.95
57.29
–
–
–
–
–
–
–
–
–
–
–
–
–
SF-268
SF-295
U251
SNB-19
2.2.1.1. Preliminary single high dose screening at 10 lM concen-
tration. Firstly, the seven selected conjugates (5b–d,7a, 7b, 7d
and 7g) were screened at a dose of 10 lM for their antiprolifera-
tive activity against a panel of fifty-five cancer cell lines. The mean
percent growth inhibition values (GI%) for conjugates 5b–d,7a,
7b, 7d and 7g against NCI-55 cancer cell lines were displayed in
(Figure 2, Table 1). The primary assay data analysis revealed that
the new benzofuran-isatin hybrids showed weak to moderate
inhibitory activity some of the subpanel cancer cell lines except
for compound 5d that possessed excellent activity against nearly
all the cancer cell lines. Although compound 5b, 7a, 7b, 7d and
7g proved inactive against most of the subpanels cancer cell lines
with mean GI% ¼ 0.75%, 0.09%, 5.18%, 6.01%, and ꢀ0.8%,
respectively, they showed selective moderate anticancer activity
against certain cell lines such as Ovarian-IGROV1, Non-small cell
lung-EKVX, Renal-UO-31 and Breast/MCF7 cancer cell lines with
GI% range 17–53% (Table 1).
–
–
–
–
11.60
Melanoma
MALME-3M
LOX IMVI
MDA-MB-435
M14
UACC-257
UACC-62
SK-MEL-2
SK-MEL-28
Ovarian cancer
NCI/ADR-RES
IGROV1
OVCAR-3
OVCAR-8
OVCAR-4
OVCAR-5
Renal cancer
786-0
12.43 64.93
129.46
83.71
61.04
71.32
49.98
65.68
29.13
84.16
–
–
–
–
–
–
–
–
–
–
–
–
–
–
16.04
–
–
–
–
33.82
–
25.40
–
13.84
–
–
–
–
–
–
–
–
–
–
10.91 30.94
–
–
16.86 20.56
–
–
–
–
–
–
–
29.33
69.51
–
10.33 10.19
–
33.72 44.59
93.67 32.80 42.55 33.59 22.38
165.51
62.23
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
22.46
31.04 15,285
–
52.33
–
34.70
166.85
–
–
–
–
In particular, compound 5d was the most efficient anti-prolif-
erative agent and exhibited excellent activity against almost all
subpanel cancer cell lines with mean growth inhibitory activity of
87.33%. Remarkably, compound 5d exerted excellent growth
inhibition properties against Non-small cell lung cancer (NCI-H23),
CNS cancer (SF-295, U251), Melanoma (LOX IMVI, SK-MEL-28),
Ovarian cancer (IGROV1), Prostate cancer (DU-145) and Breast can-
cer (MDA-MB-468) cell lines with GI% of 89.35, 94.95, 97.95, 83.71,
84.16, 93.67, 82.38 and 80.10%, respectively (Table 1). In addition,
conjugate 5d showed good potency with GI% equals or greater
than 60% towards Non-small cell lung cancer (EKVX), Colon cancer
(COLO 205, HT-29 and SW-620), Melanoma (M14, MDA-MB-435
and UACC-62), Ovarian cancer (OVCAR-8), CNS cancer (SF-539),
Renal cancer (SN12C), Breast cancer (MCF7, MDA-MB-231/ATCC
and HS 578 T) and Prostate cancer (PC-3) cell lines with GI% of
62.73, 77.24, 72.67, 64.85, 71.32, 61.04, 65.68, 62.23, 62.58, 65.13,
77.14, 76.19, 74.50and 78.23% respectivly (Table 1).
CAKI-1
ACHN
SN12C
RXF 393
UO-31
TK-10
Prostate cancer
PC-3
19.50 26.59
151.66 19.44 11.31 18.98 14.14
–
–
–
35.48
29.81
11.08
150.93
65.13
136.15
155.33
193.95
–
–
–
–
–
11.60 13.30
–
–
–
–
–
–
–
26.72 43.42
34.27 33.64 21.86
–
–
–
–
–
–
–
13.75
17.00
78.23
82.38
–
–
10.82 15.73
–
–
DU-145
Breast cancer
MCF7
–
–
19.04 26.66
22.06
14.64 29.88
77.14 28.25 25.04 16.66 13.20
BT-549
–
29.88
76.19
74.50
80.10
–
–
–
–
–
–
–
–
–
–
–
MDA-MB-231/ATCC
HS 578 T
MDA-MB-468
T-47D
Mean inhibition, %
Sensitive cell lines no.
28.99 18.32
–
–
–
–
–
46.27
–
42.74
–
–
139.16 21.80 23.76 33.31
0.75 21.99
40
87.33 0.09 5.18 6.01 –0.8
54 19 20
9
7
6
aOnly GI % higher than 10% are shown. NT: not tested.

1428
W. M. ELDEHNA ET AL.
Table 2. NCI in vitro screening results (GI50, TGI, and LC50 (lM) of 5d (NSC: D- Table 3. Median growth inhibitory concentrationsa (GI50, mM) of in-vitro cancer
819833/1) in the five-dose test.
Subpanel /tumour cell lines
cell lines subpanel for compound 5d.
Compound 5d
TGI(mM)
Compound 5d
Selectivity index
GI50(mM)
LC50(mM) Subpanel /tumour cell lines
MG-MID
Leukaemia
CCRF-CEM
HL60(TB)
K-562
MOLT-4
SR
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
non-small cell lung cancer
Colon Cancer
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
2.80
1.92
2.60
3.18
2.29
1.98
2.83
2.51
0.89
1.30
0.96
0.78
1.09
1.26
0.88
NT
>100
NT
NT
NT
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Breast Cancer
NT
NT
NT
Full panel MG-MIDb
aMedian value assessed according to the results obtained from NCI’s screening.
bGI50 (mM) full panel mean-graph midpoint (MG-MID) ¼ the average sensitivity
for all cell lines towards the examined compound.
2.94
1.92
1.84
2.19
1.86
NT
>100
4.02
3.98
NT
4.43
NT
>100
8.44
NT
>100
>100
>100
NT
It is worthy to mention that 5d exhibited a lethal cytotoxic
impact with GI% >100 against Non-small cell lung cancer (HOP-
62, A549/ATCC, HOP-92 and NCI-H460), Colon cancer (HCT-116),
Melanoma (MALME-3M), Ovarian cancer (OVCAR-4 and OVCAR-3),
Renal cancer (CAKI-1, 786–0, RXF 393, ACHN, TK-10 and UO-31)
and Breast cancer (T-47D) cells with GI% values equals 124.09,
123.59, 143.99, 122.83, 128.55, 129.46, 152.85, 165.51, 151.66,
166.85, 136.15, 150.93, 193.95, 155.33 and 139.16%, respectively
(Table 1).
NT
6.07
NT
>100
>100
NT
NT
1.92
NT
NT
NT
NT
>100
>100
NT
>100
NT
>100
>100
>100
>100
NT
>100
>100
>100
>100
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
On the other hand, compound 5c showed moderate to good
activity against some cell lines with mean GI% ¼ 21.99%. The best
results of compound 5c was against cancer cell lines Non-small
cell lung-HOP-62 (GI% ¼ 41.41%), Non-small cell lung-NCI-H460
(GI% ¼ 42.60%), Renal-UO-31 (GI% ¼ 43.42%), Ovarian-IGROV1
(GI% ¼ 44.59%), Breast-HS-578T (GI% ¼ 46.27%), CNS-SF-539 (GI%
¼ 57.88%) and Melanoma-MALME-3M (GI% ¼ 64.93%) (Table 1).
5.18
2.03
1.66
3.45
1.25
2.03
56.8
4.13
3.16
16.2
2.75
4.26
>100
NT
NT
>100
6.04
NT
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
3.16
1.81
NT
NT
2.56
NT
NT
NT
5.19
>100
3.79
>100
>100
6.62
NT
NT
>100
NT
>100
>100
54.7
2.2.1.2. In vitro 5 dose full NCI-55 cell panel screening.. The pre-
liminary screening results showed that conjugate 5d (NSC: D-
819833/1) was the most potent compound in the present study,
and displayed effectiveness towards various cell lines represent
numerous tumour subpanels (Figure 2). Accordingly, 5d was pro-
moted to the five-dose (0.01–100 mM) screening assay.
Accordingly, three main response parameters (GI50, TGI and LC50)
towards each of the examined cancer cell line were calculated for
hybrid 5d and displayed in Table 2. Where, GI50 values represents
molar concentration which produces 50% inhibitory effect in the
net cell growth; TGI (cytostatic activity) is the molar concentration
with total growth inhibition and LC50 is the cytotoxicity parameter
that reflects the molar concentration that results in 50% net cell
death. In addition, the mean graph midpoints (MG-MID), repre-
senting the GI50 average for the individual subpanels as well as
the full panel cell lines were calculated giving an average potency
parameter for the examined compound 5d, (Table 3).
Furthermore, by dividing the full panel MID by their individual
subpanel MID, the selectivity index of compound 5d was calcu-
lated and was used to measure the selectivity of 5d towards dif-
ferent cancer cell subpanels.
NT
>100
>100
>100
>100
>100
2.10
1.84
NT
NT
NT
NT
NT
>100
NT
3.83
>100
NT
NT
>100
>100
>100
NT
NT
3.22
2.47
1.82
1.99
1.63
1.77
1.56
1.79
2.82
2.32
NT
3.84
4.04
NT
3.24
3.70
>100
4.02
NT
NT
NT
NT
NT
NT
>100
NT
NT
NT
NT
>100
NT
>100
>100
DU-145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578 T
BT-549
Results displayed in Table 2, revealed that conjugate 5d exhib-
ited powerful anti-proliferative activity at a single-digit micromolar
level towards all the examined human cancer cell subpanels with
GI50 values range: 1.25 ꢀ 6.07 mM, except for Melanoma HL60(TB)
cell line (more than100 mM). Moreover, regarding the cytostatic
activity, hybrid 5d exhibited excellent cytostatic activity with TGI
values range 2.75–7.72 mM against numerous cell lines including
NSCLC (HOP-62, NCI-H23 and HOP-92), CNS Cancer (SF-295,
NT
>100
5.10
7.72
33.7
NT
>100
>100
>100
>100
>100
>100
2.07
2.41
5.38
1.70
2.60
T-47D
MDA-MB-468
6.52
NT: not tested.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1429
Figure 3. Effect of benzofuran–isatin conjugates (5a–e and 7a–i) on the cell viability. (A) SW-620 with hybrids 5a–e and 7a–i, (B) HT-29 with hybrids 5a–e and 7a–i.
SF-539, SNB-75 and U251), Melanoma (MALME-3Mand SK-MEL-2), 7e, 7h and 7i) showed >50% inhibition of HT-29 cancer cells via-
Ovarian Cancer (OVCAR-8), Renal Cancer (786–0, A498, CAKI-1, RXF
393 andTK-10) and Breast Cancer (MDA-MB-231/ATCC, HS 578 T
and MDA-MB-468). On the other hand, while, compound 5d
showed weak to moderate cytostatic activity towards CNS Cancer
(SF-268 and SNB-19), and Breast Cancer (BT-549) with TGI ¼ 56.8,
16.2 and 33.7 mM, respectively, it proved to have no cytostatic
impact (TGI >100 mM) against entire Leukaemia, Colon cancer and
Prostate Cancer and the remaining examined cancer cell lines
(Table 2). Furthermore, compound 5d as revealed by the results
could be considered as a non-lethal agent that exhibited LC50 val-
ues more than 100 mM for the all of cancer cell lines herein exam-
ined, except for three cancer cell lines; Non-Small Cell Lung
Cancer (HOP-62), CNS Cancer (SNB-75) and Melanoma (MASK-MEL-
2) which possessed a lethal effect of IC50 ¼ 8.44, 6.04 and 54.7,
respectively (Table 2).
On the other hand, as shown in Table 3, all tested subpanels
were sensitive to compound 5d with MG-MID spinning between
1.92 and 3.18 mM and the most susceptible subpanels were Colon
Cancer and Renal Cancer that exhibited MG-MID ¼ 1.92 and
1.98 mM, respectively. Furthermore, it is well known that com-
pounds with selectivity index between 3 and 6 are considered to
be of a moderate selectivity, ratios more than six indicated high
selectivity towards the corresponding cell line, while compounds
not meeting either of these values are considered as non-select-
ive59. Therefore, as displayed in the Table 3, the calculated select-
ivity index for compound 5d ranged from 0.78 to 1.30 indicated
that conjugate 5d has non-selective, broad spectrum antiprolifera-
tive activity against all tested subpanels cancer cells.
bility (Figure 3).
The results revealed that compounds 5a and 5d exhibited
promising cytotoxic activity for both cell lines. For this reason,
compounds 5a and 5d were pursued for further studies. Starting
with determination of IC50s and cytotoxic selectivity studies. Serial
concentrations of compounds 5a and 5d were used to examine
their impact on cell viability using MTT protocol. Results of con-
centration vs percent viability were charted, and the IC50 was cal-
culated for SW-620 and HT-29 cell lines using Graph Pad prism 8
(Figure 4). Compound 5a was found to have IC50 ¼ 9.4 mM and
8.7 mM against SW-620 and HT-29 cell lines, respectively. In add-
ition, the IC50 for compound 5d equals 9.8 mM and 6.5 mM against
SW-620 and HT-29 cell lines, respectively, compared to IC50 of
Irinotecan, a reference drug, which was found to be 1.0 mM
against SW-620 cell line and 6.18 mM against HT-29 cell line
(Figure 4).
Furthermore, selective cytotoxicity of compounds 5a and 5d
was studied on human skin fibroblast (HFF-1) normal cells. Both
conjugates were found to possess a little effect on fibroblast nor-
mal cell viability (Figure 5). These results revealed that compounds
5a and 5d possessed a selective cytotoxicity against SW-620 and
HT-29 cancer cell lines with non-significant effect on normal fibro-
blast cells.
2.2.2. Annexin V-FITC/propidium iodide apoptosis assay
Further investigation for compounds 5a and 5d concerning their
potential role of apoptosis induction, using Annexin V-FITC/PI dou-
ble staining assay61, was performed to evaluate their impact on
both early and late apoptosis percentages in SW-620 cancer cell
lines (Figure 6). The assay findings showed that compounds 5a
and 5d resulted in a dose dependent induction of apoptosis for
SW-620 cancer cells. As shown, compound 5a induced approxi-
mately 1.7-folds and 3.8-folds total increase in apoptosis at con-
centration of 5 mM and 10 mM, respectively, in comparison to the
control untreated SW-620 cell line (Figure 6(A)).
Similarly, compound 5d, at concentration of 5 mM and 10 mM
approximately induced 2.9-folds and 3.8-folds total increase in
apoptosis, respectively, when incubated with SW-620 cell line,
compared to the untreated cells (Figure 6(B)). Encouraged by
these results compounds 5a and 5d were further investigated for
their effect on the anti-apoptotic mitochondrial protein Bcl2 and
their effect on the level of cleaved PARP in SW-620 colorectal can-
2.2.2. In vitro anti-cancer activity against SW-620 and HT-29colo-
rectal cancer cell lines
In the present investigation a new set of benzofuran–isatin
hybrids (5a–e and 7a–i) was synthesised to be evaluated for their
potential anticancer activity towards two human colorectal cancer
cell lines, SW-620 and HT-29. The anticancer activity of the new
conjugates was assessed using MTT assay60, and the results were
shown in Figure 3. The most active compound in the NCI assay
(5d), in addition to another one from untested compounds by NCI
(5a), were selected to explore their activity. Both, SW-620 and HT-
29 cells were treated with 10 mM of each compound for 24 h and
the percent cell viability was calculated using MTT assay.
Regarding impact of the target conjugates towards SW-620 cancer
cells viability, compound 5d exhibited about 52% inhibition,
whereas, compound 5a showed 46% inhibition. On the other
hand, the results showed that seven compounds (5a, 5d, 7b, 7c, cer cell line.

1430
W. M. ELDEHNA ET AL.
Figure 4. IC50 of Compound 5a and 5d. (A) SW-620 with compound 5a, (B) HT-29 with 5a, (C) SW-620 with compound 5d, and (D) HT-29 with 5d.
Figure 5. Impact of 5a and 5d on normal HFF-1 fibroblast cells, upon incubation for 24 h. (A) compound 5a and (B) compound 5d.
2.2.3. Effect of compounds 5a and 5d on the anti-apoptotic
markers Bcl2 and the level of cleaved PARP
Similarly, compound 5d was found to follow the same pattern
with significant inhibition of the anti-apoptotic Bcl2 protein
expression and significant increase in the level of cleaved PARP in
SW-620 cancer cells (Figure 7(B)). These findings indicated that
both compounds 5a and 5d inhibited SW-620 cells viability by
deregulating apoptosis-related proteins (anti-apoptotic Bcl2 and
cleaved PARP) resulting in the induction of apoptosis.
To further examine the possible mechanism of apoptosis, the
effect of compounds 5a and 5d on certain apoptosis-related pro-
teins was studied. Bcl2 protein as a critical component of the
mitochondrial apoptotic pathway is reported to be overexpressed
in numerous tumours causing survival of cancer cell62. In addition,
it was reported that caspase activation during apoptosis leads to
proteolytic cleavage of several cellular substrates participating in
DNA reparation including [poly (ADP-ribose) polymerase]63.
Therefore, the impact of compounds 5a and 5d on the anti-apop-
totic protein Bcl2 and the level of cleaved PARP was examined
3. Conclusions
In summary, a novel series of benzofuran-isatin conjugates linked
by a carbohydrazide group, (5a–e and 7a–i) was designed and
(Figure 7). The results showed that, Western blot analysis of the synthesised. Seven compounds (5b–d and 7a,b,d,g) were selected
extracts prepared from SW-620 cells incubated with compound 5a according to NCI’s DTP selection guidelines for the assessment of
(5 lM and 10 lM) for 24 h, resulted in a dose dependent inhibition their antitumor activity against NCI-55 human cancer cell lines. All
of Bcl2 protein expression and significant increase in the level of compounds proved effective against diverse cell lines among
cleaved PARP (Figure 7(A)).
which compound 5d was promoted to the five-dose screen and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1431
Figure 6. (A) AnnexinV/PI apoptosis assay for compound 5a. Tow concentrations (5 and 10 mM) of compound 5a, in addition untreated plate as a control were used
to test the apoptotic effect by using Annexin V/PI in SW-620 cell line. Cells were treated with the compound 5a for 24h. (B) AnnexinV/PI apoptosis assay for com-
pound 5d. Tow concentrations (5 and 10mM) of compound 5d, in addition untreated plate as a control were used to test the apoptotic effect by using Annexin V/PI
in SW-620 cell line. Cells were treated with the compound 5d for 24 h.
showed good to excellent growth inhibitory activity against against two human colorectal cancer cell lines, SW-620 and HT-29,
almost all subpanel cancer cell lines. In addition, the novel conju- with excellent inhibitory activity for compounds 5a and 5d that
gates (5a–e and 7a–i) showed good anti-proliferative activity showed IC50 ¼ 8.7 mM and 9.4 mM for 5a and IC50 ¼ 6.5 mM and

1432
W. M. ELDEHNA ET AL.
Figure 7. (A) Effect of hybrid 5a on anti-apoptotic Bcl2 protein and the level of cleaved PARP. Statistical analysis was performed where the significance of data was
ꢁꢁꢁ ꢁꢁ
assessed at a p values < 0.05.
Statistical analysis was performed where the significance of data was assessed at a p values < 0.05.
p < 0.001;
p < 0.01 control vs treated. (B) Effect of hybrid 5d on anti-apoptotic Bcl2 protein and the level of cleaved PARP.
ꢁꢁꢁ ꢁꢁ
p < 0.001;
p < 0.01 control vs treated.
9.8 mM for 5d against SW-620 and HT-29 cell lines, respectively,
and proved to have selective cytotoxicity with increased safety
profile to fibroblast (HFF-1) normal cells. Further mechanistic stud-
ies revealed that both compounds 5a and 5d were able to induce
apoptosis in a dose dependent manner with an approximately
1.7–3.8 folds and 2.9–3.8 folds total increase in apoptosis for com-
pounds 5a and 5d, respectively, compared to the control
untreated SW-620 cell line. Furthermore, both conjugates signifi-
cantly inhibited the expression of the anti-apoptotic Bcl2 protein
and increased the level of the cleaved PARP and resulted in SW-
620 cells apoptosis. Collectively, the significant potency and high
selective cytotoxicity of this series specially compounds 5a and 5d
4. Experimental
4.1. Chemistry
4.1.1. General
Solvents of HPLC grade have been used and purchased from
Thermo Fisher. Follow up of reactions has been performed utilis-
ing precoated TLC F254 Merck plates. Schimadzu FT-IR spectrom-
eter has been used for functional groups analysis for the
synthesised derivatives. NMR spectrometric analyses have been
conducted using Bruker-Avance 400 NMR spectrometer (100 MHz
for 13CNMR and 400 MHz for 1H NMR). Chemical shifts have been
recorded in ppm. Multiplicities have been reported with their 1st
suggested that these conjugates might serve as starting point for order J coupling constants (Hz) for doublets (d); Stuart apparatus
additional optimisation to develop potential anticancer agents has been used to determine the melting points. FLASH 2000
and apoptotic inducers.
CHNS/O analyser has been adopted to perform the elemental

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1433
analysis.
reported previously.
Compounds
364,
and
6a–i65,66
have
been
8. Nitiss JL. DNA topoisomerases in cancer chemotherapy:
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4.1.2. Synthesis of target derivatives 5a–e and 7a–i
To stirred hot solution of 3-methylbenzofuran-2-carbohydrazide 3
(0.25 g, 1.3 mmol) in 13 ml of absolute EtOH with catalytic drops
of ethanoic acid, equivalent amount of appropriate indoline-2,3-
dione compounds 4a–e or 6a–i has been added. The reaction
mixture has been then refluxed for (3–6) h. The produced precipi-
tate, after cooling, was collected by filtration, washed with water
then recrystallized from glacial acetic acid to produce target deriv-
atives 5a–e and 7a–i, respectively in a good yield (70–87%).
Full characterisation (NMR, IR, and elemental analysis) data for
target compounds (5a–e and 7a–i) have been presented in the
Supporting Materials.
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All in vitro biological assays in this study; NCI anticancer screen-
ing67,68, MTT cell viability assay54, Annexin V-FITC/PI assay54 and
Western blot analysis54 were performed as reported earlier. All
experimental
procedures
were
provided
in
the
Supporting materials.
Disclosure statement
17. Bai P. Biology of Poly(ADP-Ribose) polymerases: the facto-
tums of cell maintenance. Mol Cell 2015;58:947–58.
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No potential conflict of interest was reported by the author(s).
Funding
This research was funded by the Deanship of Scientific Research
at Princess Nourah bint Abdulrahman University through the Fast-
track Research Funding Program.
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mediates poly(ADP-ribose) (PAR) polymer-induced cell death.
Proc Natl Acad Sci USA 2006;103:18314–9.,
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tures: applications in drug discovery. Comb Chem High
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