Marine-inspired bis-indoles possessing antiproliferative activity against breast cancer; design, synthesis, and biological evaluation

Article abstract of DOI:10.3390/md18040190

Diverse indoles and bis-indoles extracted from marine sources have been identified as promising anticancer leads. Herein, we designed and synthesized novel bis-indole series 7a-f and 9a-h as Topsentin and Nortopsentin analogs. Our design is based on replacing the heterocyclic spacer in the natural leads by a more flexible hydrazide linker while sparing the two peripheral indole rings. All the synthesized bis-indoles were examined for their antiproliferative action against human breast cancer (MCF-7 and MDA-MB-231) cell lines. The most potent congeners 7e and 9a against MCF-7 cells (IC₅₀ = 0.44 ± 0.01 and 1.28 ± 0.04 μM, respectively) induced apoptosis in MCF-7 cells (23.7-, and 16.8-fold increase in the total apoptosis percentage) as evident by the externalization of plasma membrane phosphatidylserine detected by Annexin V-FITC/PI assay. This evidence was supported by the Bax/Bcl-2 ratio augmentation (18.65- and 11.1-fold compared to control) with a concomitant increase in the level of caspase-3 (11.7- and 9.5-fold) and p53 (15.4- and 11.75-fold). Both compounds arrested the cell cycle mainly in the G2/M phase. Furthermore, 7e and 9a displayed good selectivity toward tumor cells (S.I. = 38.7 and 18.3), upon testing of their cytotoxicity toward non-tumorigenic breast MCF-10A cells. Finally, compounds 7a, 7b, 7d, 7e, and 9a were examined for their plausible CDK2 inhibitory action. The obtained results (% inhibition range: 16%-58%) unveiled incompetence of the target bis-indoles to inhibit CDK2 significantly. Collectively, these results suggested that herein reported bis-indoles are good lead compounds for further optimization and development as potential efficient anti-breast cancer drugs.

Full text of DOI:10.3390/md18040190

marine drugs
Article
Marine-Inspired Bis-indoles Possessing
Antiproliferative Activity against Breast Cancer;
Design, Synthesis, and Biological Evaluation
Wagdy M. Eldehna 1 , Ghada S. Hassan 2 , Sara T. Al-Rashood *, Hamad M. Alkahtani ,
3,
3
Abdulrahman A. Almehizia 3 and Ghada H. Al-Ansary 4,5
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh
3
3516, Egypt; wagdy2000@gmail.com
2
3
4
5
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt;
Ghadak25@yahoo.com
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; haalkahtani@ksu.edu.sa (H.M.A.); mehizia@ksu.edu.sa (A.A.A.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo P.O. Box 11566,
Egypt; ghada.mohamed@pharma.asu.edu.eg
Department of Pharmaceutical Chemistry, Pharmacy Program, Batterejee Medical College,
Jeddah P.O. Box 6231, Saudi Arabia
*
Correspondence: salrashood@ksu.edu.sa; Tel.: +966-555-295-929
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 6 March 2020; Accepted: 29 March 2020; Published: 2 April 2020
Abstract: Diverse indoles and bis-indoles extracted from marine sources have been identified as
promising anticancer leads. Herein, we designed and synthesized novel bis-indole series 7a and
as Topsentin and Nortopsentin analogs. Our design is based on replacing the heterocyclic spacer
–f
9a–h
in the natural leads by a more flexible hydrazide linker while sparing the two peripheral indole rings.
All the synthesized bis-indoles were examined for their antiproliferative action against human breast
cancer (MCF-7 and MDA-MB-231) cell lines. The most potent congeners 7e and 9a against MCF-7
cells (IC50 = 0.44
±
0.01 and 1.28
±
0.04 µM, respectively) induced apoptosis in MCF-7 cells (23.7-,
and 16.8-fold increase in the total apoptosis percentage) as evident by the externalization of plasma
membrane phosphatidylserine detected by Annexin V-FITC/PI assay. This evidence was supported
by the Bax/Bcl-2 ratio augmentation (18.65- and 11.1-fold compared to control) with a concomitant
increase in the level of caspase-3 (11.7- and 9.5-fold) and p53 (15.4- and 11.75-fold). Both compounds
arrested the cell cycle mainly in the G2/M phase. Furthermore, 7e and 9a displayed good selectivity
toward tumor cells (S.I. = 38.7 and 18.3), upon testing of their cytotoxicity toward non-tumorigenic
breast MCF-10A cells. Finally, compounds 7a, 7b, 7d, 7e, and 9a were examined for their plausible
CDK2 inhibitory action. The obtained results (% inhibition range: 16%–58%) unveiled incompetence
of the target bis-indoles to inhibit CDK2 significantly. Collectively, these results suggested that herein
reported bis-indoles are good lead compounds for further optimization and development as potential
efficient anti-breast cancer drugs.
Keywords: marine-inspired; breast cancer; bis-indoles; synthesis; apoptosis
1
. Introduction
Drug discovery from marine sources is a prehistoric praxis. Recently, the identification and
development of novel molecules based on natural heterocyclic scaffold have been an area of growing
focus. Surveying the literature reveals that the anticancer activity of diverse bis-indole compounds
Mar. Drugs 2020, 18, 190; doi:10.3390/md18040190
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2020, 18, 190
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extracted from marine sources including plants, fungi, algae, and marine mollusks was broadly
discussed in diverse manuscripts [1,2].
Diverse substituted indole and bis-indole derivatives extracted from marine sources have been
shown to exhibit significant antiproliferative activity [3,4]. Recently, Edwards et al. [5] conducted a
study on purified 6-bromoisatin (Figure 1) extracted from the Australian marine mollusk Dicathais
orbita known for its antineoplastic activity. The study revealed that 6-bromoisatin markedly reduced
the proliferation and concomitantly induced apoptosis in human colon cancer cell lines HT29 and
Caco2 cells [5]. A latter study was conducted on the same isatin derivative by Esmaeelian et al. [6]
which supported the efficacy of 6-bromoisatin at a concentration of 0.05 mg/g to induce apoptosis in
colorectal cancer cells leading ultimately to inhibition of cancer proliferation [6].
Figure 1. Indole and bis-indole marine products that have reported anticancer activity.
Moreover, many bis-indole derivatives isolated from marine sources manifest anticancer activity.
Asterriquinone (Figure 1), isolated from Aspergillus fungi, possesses symmetrical bis-indole moieties
separated by quinone spacer and showed in vivo activity against Ehrlich carcinoma, ascites hepatoma
AH13, and mouse P388 leukemia [
deepwater sponges showed modest cytotoxic activity, Figure 1 [
from the Mediterranean sponge Topsentia genitrix, exhibited antitumor and antiviral activities [11
7
]. In addition, Dragmacidins A–C, isolated from a large number of
10]. Topsentins (Figure 1), extracted
12].
8
–
,
Nortopsentins A–C (Figure 1), that feature imidazole ring spacer, were isolated from Spongosorites
ruetzleri and showed in vitro cytotoxicity against P388 cells [13,14].
As the reservoir of living organisms is inevitably limited, it became an urgent necessity for
medicinal chemists to synthesize biologically active natural molecules and their derivatives to meet
the expanding need of such medicinal agents.
Inspired by the aforementioned discoveries and in connection with our research work concerning
the development of effective anti-breast cancer agents [15–18], we were endeavored to design novel
indole derivatives that promisingly possess antiproliferative activity. Perceiving the significance
of these facts and based on our growing research interest in marine natural products, we were
persuaded to tackle this study to design and synthesize marine-inspired bis-indole derivatives that
have potential in vitro antitumor activity against breast cancer. Herein, we designed and synthesized
three novel bis-indole sets 7a
based on replacing the rigid heterocyclic spacer in the natural products by a more flexible hydrazide
linker while sparing the two peripheral indole rings to furnish the first set of target compounds 7a
Figure 2). Thereafter, the oxindole moiety was decorated with different N-alkyl (allyl, n-propyl,
iso-butyl; compounds 9a ) and N-benzyl (compounds 9d ) substituents to fulfill further elaboration
–f, 9a–h, and 11 as Topsentin and Nortopsentin analogs. Our design was
–f
(
–
c
–h

Mar. Drugs 2020, 18, 190
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for the target bis-indoles and to probe a worthy structure-activity relationship (SAR). Furthermore,
a bioisosteric replacement approach was adopted to replace the oxindole ring with carbocyclic tetralin
ring (compound 11), to explore the significance of the bis-indole scaffold, Figure 2.
Figure 2. Structure-based design of target bis-indole derivatives (7a–f and 9a–h), and 11.
In this study, all of the synthesized compounds 7, 9 and 11 were evaluated for their antiproliferative
activity against MCF-7 cells and MDA-MB-231 cancer cell lines. Three of the most potent compounds
induced apoptosis in MCF-7 cells as evidenced by the externalization of plasma membrane
phosphatidylserine detected by Annexin V-FITC/PI dual staining assay. This evidence was supported
by the Bax/Bcl-2 ratio augmentation with a concomitant increase in the level of caspase-3 and p53.
Moreover, scrutinizing the results of cell cycle analysis unraveled that these compounds arrest the cell
cycle in the G0/G1 phase.
2
. Results
2
.1. Chemistry
The synthetic pathways proposed to obtain the target bis-indole derivatives (7a
were depicted in Schemes 1 and 2. In Scheme 1, the Fischer esterification procedure was applied to
-indoleacetic acid 3 [19] to afford methyl 1H-indole-2-carboxylate 4, which subsequently undergone
–f and 9a–h), and
1
3
1
hydrazinolysis through reaction with 99% hydrazine hydrate in ethyl alcohol under reflux temperature
to furnish key intermediate 1H-indole-2-carbohydrazide 5 in 82% yield.
Thereafter, 1H-indole-2-carbohydrazide
5
was condensed with different N-unsubstituted
, or 1-tetralone 10 in absolute
1
H-indole-2,3-diones 6a , N-substituted 1H-indole-2,3-diones 8a–h
–
f
ethyl alcohol with catalytic drops of acetic acid to produce target indole derivatives 7a
–f
,
9a
–h, and 11,
respectively (Schemes 1 and 2).
Postulated structures of the herein reported indole derivatives 7a–f, 9a–h, and 11 are in full
agreement with the spectral and elemental analyses data.

Mar. Drugs 2020, 18, 190
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Scheme 1. Synthesis of target bis-indole derivatives 7a
–
f
and 9a
–h; Reagents and conditions: (i) (a)
◦
◦
KOH / heating at 250 C 18 h, (b) H O, cooling to 10 C, HCl; (ii) MeOH/H SO (catalytic)/reflux 8 h;
2
2
4
(
iii) 99% NH NH .H O/EtOH/reflux 3 h; (iv) EtOH/AcOH (catalytic)/reflux 2 h.
2 2 2
Scheme 2. Synthesis of target compound 11; Reagents and conditions: (i) EtOH/AcOH (catalytic)/reflux
h.
2
2
.2. Biological Evaluation
2
.2.1. Antiproliferative Activity against Breast Cancer MCF-7 and MDA-MB-231
The biological evaluation journey started by exploring the antiproliferative activity of the pursed
indole derivatives (7a
–f, 9a–h, and 11) against breast cancer cell line; MCF-7 and triple-negative
breast cancer cell line; MDA-MB-231, adopting procedures of the sulforhodamine B colorimetric (SRB)

Mar. Drugs 2020, 18, 190
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assay [20]. Staurosporine was utilized as the reference drug for its well-known broad anticancer activity
against diverse tumors.
All of the tested indole derivatives exhibited gradual cellular log kill with IC50 values ranging
from 0.44
of antiproliferative activity against MDA-MB-231 cell line with IC50 values ranging from 0.34
7.30 M, aside from compound 11 which showed very weak antiproliferative activity against MCF-7
cell line (IC50 = 84.70 M) and no antiproliferative activity against MDA-MB-231 cell line (IC50 > 100 M)
in a proof of concept of the importance of oxindole moiety for boosting the antiproliferative activity.
Scrutinizing the IC50 values of series 7a against MCF-7 cell line revealed that grafting a halide
µM to 47.1
µM against breast cancer cell line; MCF-7, while they exerted a much wider range
µM up to
7
µ
µ
µ
–f
atom on the isatin moiety interestingly influences the antiproliferative activity, where the activity
significantly decreased by increasing the size of the halide detected by the IC50 values of the floro
(
5a), chloro (5b), and bromo (5c) derivatives (IC50 = 1.53
This suggested that a floro substitution on the isatin group is advantageous for the antiproliferative
activity where compound 5a (IC50 = 1.53 M) is 4.45 times more potent than the reference drug
IC50 = 6.81 µM), Table 1.
µM, 8.87 µM, and 36.19 µM, respectively).
µ
(
Table 1. In vitro antiproliferative activity of 7a
–
f,
9a–h
, and 11 against breast MCF-7 and MDA-MB-231
cancer cell lines.
IC50 (µM) a
Cpd.
R
R1
R2
MCF-7
MDA-MB-231
7
a
F
Cl
Br
CH3
NO2
CH3
H
H
H
H
H
H
H
H
H
H
CH3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1.53 ± 0.02
8.87 ± 0.43
36.19 ± 2.78
10.95 ± 0.81
0.44 ± 0.01
9.04 ± 0.32
2.88 ± 0.08
36.57 ± 1.81
0.34 ± 0.02
1.32 ± 0.03
77.30 ± 6.21
18.24 ± 0.62
51.27 ± 3.59
7
b
7
c
7
d
7
7
e
f
b
NA
9
a
−CH CH=CH
1.28 ± 0.04
28.24 ± 1.53
47.10 ± 3.65
1.51 ± 0.03
10.43 ± 0.81
8.72 ± 0.39
2.76 ± 0.14
20.89 ± 0.04
84.70 ± 4.02
6.81 ± 0.22
2
2
9
b
−CH CH CH
2
2
3
b
9
c
−CH CH(CH )
NA
2
3 2
9
d
−CH C H
4.14 ± 0.19
17.66 ± 0.55
25.41 ± 1.56
2.85 ± 0.07
2.29 ± 0.09
2
6
5
9
e
−CH C H -4-F
2
6
4
9
f
H
−CH C H -4-CN
2
6
4
9
g
Br
Br
-
−CH C H
2
6
5
9
h
−CH C H -4F
2
6
4
b
1
1
-
-
NA
Staurosporine
-
10.29 ± 0.72
a
b
IC50 values are the mean ± S.D. of three separate experiments. NA: Compounds having IC50 value > 100 µM.
Furthermore, the influence of grafting electron-donating and electron-withdrawing groups
within the oxindole moiety on the antiproliferative activity of the MCF-7 cell line was closely
investigated. Interestingly, substitution with the electron-donating (CH ) group, compound 5d
,
3
decreased the activity in comparison to Staurosporine (IC50 = 10.95
an electron-withdrawing nitro group on the oxindole moiety, compound 5e, markedly enhanced the
antiproliferative potency (IC50 = 0.44 M), which is, fortunately, 15.5-times the potency of Staurosporine.
Noteworthy, decoration of the oxindole moiety with 5,7-dimethyl substitution, compound 7f, resulted
in the abolishment of the growth inhibitory action toward MCF-7 cells (IC50 > 100 M), Table 1.
µM vs. 6.81 µM), whereas, grafting
µ
µ
Conclusively, grafting a fluorine atom or a nitro group on C-5 of the oxindole moiety significantly
boosts the activity against MCF-7 cells with a more pronounced effect for the nitro group.
As part of our work, we extended our investigation to explore the effect of substituting the nitrogen
atom of the oxindole moiety by different alkyl and benzyl moieties in series
unraveled that substitution of the nitrogen atom with an allyl group in 9a significantly increased the
activity 5.3-times compared to the reference drug (IC50 = 1.28 M vs. 6.81 M, respectively). Conversely,
9. Exploring the IC50 values
µ
µ

Mar. Drugs 2020, 18, 190
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N-substitution with propyl group in 9b (IC50 = 28.24
markedly dwindled the growth inhibitory activity toward MCF-7 cell line.
µ
M) and isobutyl group in 9c (IC50 = 47.1
µ
M)
M)
Alternatively, substitution of the nitrogen atom with a benzyl moiety in 9d (IC50 = 1.51
µ
significantly increased the activity 4.5-times in comparison to Staurosporine. Conversely, utilizing
substituted benzyl groups in 9e (4-F-benzyl) and 9f (4-cyanobenzyl) was not advantageous for the
activity as their IC50 values were less than the reference drug (IC50 = 10.43 µM and 8.72 µM, respectively).
Moreover, two compounds (9g and 9h) were synthesized as analogues of compounds 9d and 9e, where
the oxindole moiety was further substituted with a bromo group in the 5-position. Investigation of the
IC50 values of 9g and 9h (IC50 = 2.76 and 20.89 µM, respectively) clearly depicts a deterioration of the
activity to the half as compared to 9d and 9e. This suggested that bromination of oxindole ring is not
advantageous for the antiproliferative activity, an observation that is in accordance with the structure
activity relationship extracted from series 7 (compound 7c, IC50 = 36.19 µM).
Triple negative breast cancer (TNBC) is a stubborn type of cancer resistant to many
chemotherapeutic agents, thus it represents a powerful challenge for medicinal chemists. Accordingly,
we evaluated the potential antiproliferative activity for our compounds against TNBC cell line;
MDA-MB-231 (Table 1). Analyzing the IC50 values of series 7a
–
f
and 9a–h reveals very interesting
results as many of the synthesized derivatives (7a
potencies compared to Staurosporine (IC50 = 9.04 M, 2.88
.29 M and 10.29
a chloro (7b) group on the oxindole ring results in activity enhancement (IC50 = 9.04
respectively), while grafting of a bromo group (7c) markedly decreased the activity (IC50 = 36.57
in comparison to Staurosporine (IC50 = 10.29 M). Moreover, substitution with a methyl group (7d
,
7b
,
7d
µ
,
7f
,
9d 9g and 9h) exhibited superior
,
µ
M, 0.34
µM, 1.32
µ
M, 4.14
µ
M, 2.85
µ
M,
M,
2
µ
µ
M, respectively). The IC50 values of series
7
unraveled that grafting a fluoro (7a) or
µM and 2.88
µ
µM)
µ
)
and a nitro group (7e) interestingly resulted in boosting the activity by 30.3- and 7.8-times, respectively.
The effect of the N-substituent of the oxindole ring was further investigated in series 9. Substitution of
the nitrogen atom with an allyl group (9a) did not result in enhancement of the antiproliferative activity
compared to Staurosporine (IC50 = 18.24
isobutyl even worsens the case producing much less potent derivatives (9b) (IC50 = 51.27
which failed to produce any marked cytotoxic effect up to 100 M. These results are in accordance with
the observed results for series where the addition of a larger or branched alkyl group proved to be
µM vs. 10.29
µM). Extending the substituent to propyl or
µ
M) and (9c
)
µ
7
detrimental to the antiproliferative action against MDA-MB-231 cell line (Table 1).
In addition, the impact of substitution of the nitrogen atom by un/substituted benzyl moieties was
explored, revealing that unsubstituted benzyl moiety (9d) is advantageous for activity as it enhanced
the activity by 2.5-times while utilizing 4-F-benzyl group (9e) or 4-CN-benzyl group (9f) resulted
in a marked decrease of the activity compared to compound 9d (IC50 = 17.66
µM and 25.41 µM vs.
4
.14 M). The antiproliferative activity for the 5-bromo substituted analogs (9g and 9h) against the
µ
MDA-MB-231 cell line was compared to that of 9d and 9e. The results revealed that, in contrast to
the results observed for the antiproliferative activity against MCF-7 cell line, the bromo substitution
of the oxindole ring was advantageous for the activity as compound 9g (IC50 = 2.85
µM) proved to
be 1.5-times more potent than its unsubstituted bioisostere 9d, also, compound 9h (IC50 = 2.29
proved even to be 7.7-times more potent than 9e counterpart.
µM)
2
.2.2. In Vitro Cytotoxic Activity against Non-Tumorigenic Human Breast Cell Line
To investigate the selectivity and safety profile for the here reported bis-indoles toward the
normal cells, compounds that displayed good activity towards MCF-7 and/or MDA-MB-231 cells were
examined for their cytotoxic activity against non-tumorigenic human breast epithelial (MCF-10A) cell
line (Table 2).

Mar. Drugs 2020, 18, 190
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Table 2. Cytotoxic activity toward non-tumorigenic human breast MCF-10A cell line, and selectivity
index (MCF-10A/MCF-7).
IC50 (µM)
MCF-10A
Selectivity Index
Comp.
MCF-7
MCF-10A/MCF-7
7
a
14.06
39.54
54.92
17.06
23.47
19.12
48.39
42.01
17.16
26.09
1.53
8.87
10.95
0.44
1.28
1.51
10.43
8.72
2.76
9.2
4.5
5.0
38.7
18.3
12.7
4.7
4.8
6.2
1.2
7
7
b
d
7
9
e
a
9
d
9
9
e
f
9
g
9
h
20.89
The examined bis-indoles exerted non-significant or modest cytotoxic action against
non-tumorigenic MCF-10A cells with IC50 range: 14.06–54.92 M, respectively. Bis-indoles 7e and 9a
µ
showed excellent selectivity indexes (SIs) equal to 38.7 and 18.3, respectively, whereas the remaining
compounds, except 9h, displayed good SIs spanning in the range 4.5–12.7 (Table 2).
2
.2.3. Cell Cycle Analysis
Anticancer agents exert their cytotoxic action by aborting cellular proliferation at certain
checkpoints. These checkpoints are distinguishable phases in the cell cycle, whose suppression
results in termination of the cell proliferation. To deeply comprehend the antiproliferative activity of
our tested compounds, the most active two compounds (7e and 9a) toward MCF-7 cells were further
investigated for their effect on the different phases of the cell cycle in MCF-7 cell line. MCF-7 cells
were treated with IC50 concentrations of the two compounds and their effect on the cell population in
different cell phases was recorded and displayed in Table 3. Interestingly, exposure of MCF-7 cells to
7
e
and 9a resulted in marked augmentation in the proportion of cells in the G2/M phase by 3- and
2
.21-fold, and in the Sub-G1 phase by 18.77- and 13.42-fold, respectively, in comparison to the control.
This clearly indicates that the target bis-indoles arrested the cell cycle proliferation of MCF-7 cells in
the G2/M phase.
Table 3. Effect of compounds 7e and 9a on the phases of the cell cycle of MCF-7 cells.
Comp.
%G0-G1
%S
%G2/M
%Sub-G1
7
9
e
a
31.66
43.82
57.26
25.44
24.91
28.59
42.9
31.27
14.15
33.61
24.02
1.79
Control
2
.2.4. Effect of 5e, 5f, and 8a on the Level of the Apoptotic Markers (Bax, Bcl-2, caspase-3, and p53)
Synchronization of the mitochondrial pathway is headed by the Bcl-2 family of proteins. These
proteins are classified into two groups: anti-apoptotic proteins exemplified by Bcl-2 protein and
the counteracting pro-apoptotic proteins including Bax protein [21]. As induction of the apoptotic
machinery is one of the most useful strategies in cancer therapy [22,23], we investigated the effect of
our compounds to boost the pro-apoptotic protein; Bax and reduce the anti-apoptotic protein; Bcl-2 in
an attempt to explore the underlying mechanism for their cytotoxic activity (Table 1). As 7e and 9a
proved to be the most active compounds, their effect on the level of Bax and Bcl-2 was investigated.
Fortunately, both compounds markedly boosted the level of Bax by 8.3- and 6.4-fold, respectively
(
Table 4, Figure 3). Conformingly, they decreased the level of Bcl-2 by 2.25- and 1.74-fold, respectively.
A more indicative parameter is the Bax/Bcl-2 ratio [24], which proved to be augmented by 7e and 9a

Mar. Drugs 2020, 18, 190
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18.65- and 11.1-fold, respectively (Table 4, Figure 3). This further emphasizes that target bis-indoles
trigger apoptosis by significantly boosting the Bax/Bcl-2 ratio.
Table 4. Effect of bis-indoles 7e and 9a on the expression levels of Bcl-2 and Bax in MCF-7 cancer cells.
Bax
Bcl-2
Compound
Bax/Bcl-2
(
pg/mg of Total Protein) (ng/mg of Total Protein)
7
9
e
a
318.0 ± 10.5
243.6 ± 12.4
38.3 ± 2.2
2.07 ± 0.14
2.67 ± 0.16
4.65 ± 0.23
153.6
91.2
8.2
Control
2
1
1
0
5
0
5
0
5
f
8a
5f
8a
5f
8a
5f
p53
8a
Bax
Bax/Bcl-2 Caspase-3
Figure 3. The numbers of fold increase in Bax/Bcl-2 ratio and expression levels of Bax, caspase-3, and
p53 in MCF-7 cancer cells upon treatment with compounds 7e and 9a in comparison to the control.
Moreover, their effect on the level of caspase-3; the executioner caspase and p53 was evaluated in
the MCF-7 cell line. Results revealed that 7e and 9a up-regulated the level of caspase-3 by 11.7- and
9
.5-fold, respectively as compared to the control (Table 5). In addition, they augmented the level of p53
by 15.4- and 11.75-fold, respectively in comparison to the control (Table 5, Figure 3).
Table 5. Effect of compounds 7e and 9a on the expression levels of active caspase-3 and p53 in MCF-7
cancer cells.
Compound
Caspase-3 (pg/mg)
p53 (pg/mg)
7
9
e
a
409.2 ± 17.2
331.0 ± 12.5
35.92 ± 1.8
631.8 ± 35.8
482.3 ± 27.4
41.26 ± 2.7
Control
2
.2.5. Annexin V-FITC Apoptosis Assay
Annexin V-based flow cytometry analysis is a useful tool to investigate whether cell death is
pertaining to physiological apoptosis or nonspecific necrosis. Evaluation of the apoptotic effect of
bis-indoles 7e and 9a was carried out using AnxV-FITC/DAPI dual staining assay (Figure 4).
Treatment of MCF-7 cells with IC50 concentration of 7e and 9a exerted marked increase in the
AnxV-FITC apoptotic cells percentage in both early (from 1.03% to 9.56% and 7.01%, respectively) and
late apoptosis (from 0.29% to 21.76% and 15.20%, respectively) phases, Table 6. This corresponds to an
increase in the total apoptosis percentage by 23.7-, and 16.8-fold, respectively compared to the control.
This proved that the antiproliferative activity of here reported bis-indoles is due to physiological
apoptosis, not nonspecific necrosis.

Mar. Drugs 2020, 18, 190
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Control
7e
9a
Figure 4. Influence of bis-indoles 7e and 9a on the percentage of annexin V-FITC-positive staining
in MCF-7 cells. (Lower right: early apoptotic; upper right: late apoptotic; lower left: viable; upper
left: necrotic).
Table 6. Distribution of apoptotic cells in the AnnexinV-FITC/PI dual staining assay in MCF-7 cells
after treatment with bis-indoles 7e and 9a.
Early Apoptosis
Late Apoptosis
(Upper Right %)
Total
(L.R % + U.R %)
Compound
(Lower Right %)
7
9
e
a
9.56
7.01
1.03
21.76
15.20
0.29
31.32
22.21
1.32
Control
2
.2.6. CDK2 Inhibitory Activity
The efficient cell cycle disturbance influence of the herein reported bis-indoles (Table 7) prompted
a more examination for their plausible inhibitory action toward the cell cycle regulator CDK2 protein
kinase, in an attempt to gain further mechanistic insights for their promising growth inhibitory effect.
The potent antiproliferative agents 7a
at a single dose of 10 µM, (Table 7).
, 7b, 7d, 7e, and 9a were examined for their % inhibition of CDK2
Table 7. Inhibitory effect of bis-indoles 7a, 7b, 7d, 7e, and 9a against CDK2 kinase activity at a single
dose of 10 µM.
Compound
% Enzyme Inhibitory Activity
7
a
41
41
58
16
23
99
7
7
b
d
7
9
e
a
Staurosporine
As displayed in Table 7, the examined bis-indoles exerted moderate to weak CDK2 inhibition
with a % inhibition range of 16%–58%. Compound 7d displayed the best % inhibition against CDK2
equals 58, whereas, both 7a and 7b showed % inhibition equals 44, Table 7.
These results unveiled incompetence of the target bis-indoles to inhibit CDK2 significantly,
highlighting that the cell growth inhibitory and cell cycle arrest capabilities of the target bis-indoles
toward the examined human breast cancer cell lines is attributable to another target rather than CDK.
Accordingly, further optimization for the herein reported bis-indoles including many mechanistic
investigations are in progress and will be reported upon in the future.

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3
3
3
. Experimental
.1. Chemistry
.1.1. General
Melting points were measured with a Stuart melting point apparatus and were uncorrected.
Infrared spectra were recorded on Schimadzu FT-IR 8400S spectrophotometer. The NMR spectra were
1
13
obtained on JEOL ECA-500 II spectrophotometer (500 MHz H and 125 MHz C NMR), in deuterated
dimethylsulfoxide (DMSO-d ). Chemical shifts ( H) are reported relative to TMS as the internal
standard. All coupling constant (J) values are given in hertz. Chemical shifts ( C) are reported relative
to DMSO-d as internal standards. Elemental analyses were carried out at the Regional Center for
δ
6
δ
6
Microbiology and Biotechnology, Al-Azhar University. Compounds methyl 1H-indole-2-carboxylate
and 1H-indole-2-carbohydrazide 5 were prepared as reported earlier [25].
4
3
.1.2. General Procedure for Synthesis of the Target Bis-indoles (7a–f and 9a–h), and 11
To a hot stirred solution of key intermediate 1H-indole-2-carbohydrazide (0.18 gm, 1 mmoL) in
absolute ethyl alcohol (7 mL) and glacial acetic acid (catalytic amount), the appropriate N-unsubstituted
H-indole-2,3-dione 6a , N-substituted 1H-indole-2,3-dione 8a , or 1-tetralone 10 (1 mmoL) was
5
1
–
f
–h
added. The resulting mixture was refluxed for 2 hours, and then the formed solid was filtered off
while hot, washed with cold isopropyl alcohol, dried and recrystallized from DMF to afford target
bis-indoles (7a–f and 9a–h), and 11, respectively.
0
N -(5-Fluoro-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (7a)
◦
1
Red powder, m.p. 281–283 C; (yield 70%), IR: 3327, 3270 (NH) and 1694 (C=O); H NMR δ ppm:
4
7
.19 (brs, 2H, CH2-C=O), 6.85–6.88 (m, 1H, Ar-H), 6.95 (m, 1H, Ar-H), 7.05 (t, 1H, Ar-H, J = 7.5 Hz),
.19 (t, 1H, Ar-H, J = 7.5 Hz), 7.32 (m, 2H, Ar-H), 7.58 (d, 1H, Ar-H, J = 7.5 Hz), 8.14 (s, 1H, Ar-H), 10.82
(
s, 1H, NH of isatin, D O exchangeable), 10.96 (s, 1H, NH of hydrazide, D O exchangeable), 11.23 (s,
2
2
13
1
1
1
H, NH of indol, D O exchangeable); C NMR
δ
ppm: 28.26 (
C
H -C=O), 107.26, 107.89, 111.33, 111.42,
2
2
13.06, 113.26, 115.58, 115.65, 18.52, 118.63, 121.07, 124.56, 127.28, 136.02, 138.54, 139.95, 156.56, 158.44,
+
62.57 (C=O of isatin), 164.81 (C=O of hydrazide); MS m/z [%]: 356.15 [M , 100], 157.03 [11.35], 130.12
[
25.10]; Anal. Calcd. for C H FN O : C, 64.28; H, 3.90; N, 16.66; found C, 63.93; H, 3.94; N, 16.73.
18 13 4 2
0
N -(5-Chloro-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (7b)
◦
1
Orange powder, m.p. 292–294 C; (yield 74%), IR: 3450, 3338 (NH) and 1694 (C=O); H NMR
ppm: 4.11 (brs, 2H, CH2-C=O), 6.87 (d, 1H, Ar-H, J = 8.5 Hz), 6.96-6.99 (m, 1H, Ar-H), 7.05 (t, 1H,
δ
Ar-H, J = 7.5 Hz), 7.31-7.35 (m, 2H, Ar-H), 7.38 (d, 1H, Ar-H, J = 8.0 Hz), 7.57 (d, 1H, Ar-H, J = 8.0 Hz),
.33 (s, 1H, Ar-H), 10.92 (s, 1H, NH of isatin, D O exchangeable), 11.20 (s, 1H, NH of hydrazide, D O
8
2
2
exchangeable), 11.33 (s, 1H, NH of indol, D O exchangeable); Anal. Calcd. for C H ClN O : C,
2
18 13
4
2
6
1.28; H, 3.71; N, 15.88; found C, 60.95; H, 3.66; N, 15.97.
0
N -(5-Bromo-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (7c)
◦
Orange powder, m.p. > 300 C; (yield 85%), IR: 3365, 3219, 3182 (NH) and 1726, 1692 (C=O);
1H NMR
7
δ
ppm: 4.11 (brs, 2H, CH2-C=O), 6.83 (d, 1H, Ar-H, J = 8.5 Hz), 6.96–6.99 (m, 1H, Ar-H),
.04 (t, 1H, Ar-H, J = 7.5 Hz), 7.31-7.35 (m, 2H, Ar-H), 7.51 (d, 1H, Ar-H, J = 8.0 Hz), 7.57 (d, 1H,
Ar-H, J = 8.0 Hz), 8.43 (s, 1H, Ar-H), 10.93 (s, 1H, NH of isatin, D O exchangeable), 10.96 (s, 1H, NH
2
of hydrazide, D O exchangeable), 11.33 (s, 1H, NH of indol, D O exchangeable); Anal. Calcd. for
2
2
C H BrN O : C, 54.43; H, 3.30; N, 14.10; found C, 54.85; H, 3.28; N, 14.02.
18
13
4
2

Mar. Drugs 2020, 18, 190
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0
2
-(1H-Indol-3-yl)-N -(5-methyl-2-oxoindolin-3-ylidene)acetohydrazide (7d)
◦
1
Red powder, m.p. > 300 C; (yield 78%), IR: 3363, 3309, 3191 (NH) and 1691 (C=O); H NMR
δ
ppm: 2.24, 2.50 (2s, 3H, CH ), 3.87, 4.18 (2s, 2H, CH2-C=O), 6.76-6.81 (m, 1H, Ar-H), 6.97 (t, 1H,
3
Ar-H, J = 7.5 Hz), 7.07-7.14 (m, 2H, Ar-H), 7.30–7.59 (m, 4H, Ar-H), 10.95 (s, 1H, NH of isatin, D O
2
exchangeable), 11.08, 11.12 (2s, 1H, NH of hydrazide, D O exchangeable), 12.51, 12.96 (2s, 1H, NH of
2
13
indol, D O exchangeable); C NMR
δ
ppm: 18.57, 20.53, 18.14, 32.32, 106.38, 107.03, 110.83, 111.42,
2
1
1
11.52, 118.45, 118.67, 119.87, 120.92, 121.04, 121.29, 124.32, 124.81, 126.99, 127.27, 131.61, 131.79, 140.02,
62.55, 168.32, 173.32; MS m/z [%]: 332.11 [M , 80.08], 157.24 [40.03], 130.19 [100]; Anal. Calcd. for
+
C H N O : C, 68.66; H, 4.85; N, 16.86; found C, 68.83; H, 4.80; N, 16.97.
19
16
4
2
0
2
-(1H-Indol-3-yl)-N -(5-nitro-2-oxoindolin-3-ylidene)acetohydrazide (7e)
◦
1
Yellow powder, m.p. 289–291 C; (yield 80%), IR: 3399, 3147 (NH) and 1728, 1686 (C=O); H NMR
ppm: 4.13 (brs, 2H, CH2-C=O), 6.95-7.00 (m, 1H, Ar-H), 7.04–7.08 (m, 2H, Ar-H), 7.33–7.35 (m, 2H,
δ
Ar-H), 7.58 (d, 1H, Ar-H, J = 8.0 Hz), 8.19-8.28 (m, 1H, Ar-H), 9.04 (s, 1H, Ar-H), 10.96 (s, 1H, NH of
isatin, D O exchangeable), 11.50 (s, 1H, NH of hydrazide, D O exchangeable), 11.81 (s, 1H, NH of
2
2
13
indol, D O exchangeable); C NMR
δ
ppm: 18.57 (
C
H -C=O), 111.31, 111.40, 111.49, 115.08, 115.62,
2
2
118.51, 118.63, 120.70, 121.03, 121.48, 127.21, 136.00, 136.12, 141.99, 142.75, 147.42, 149.13, 162.76 (C=O
of isatin), 165.04 (C=O of hydrazide); Anal. Calcd. for C H N O : C, 59.50; H, 3.61; N, 19.28; found
18
13
5
4
C, 59.67; H, 3.57; N, 9.34.
0
N -(5,7-Dimethyl-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (7f)
◦
1
Brown powder, m.p. 294–296 C; (yield 73%), IR: 3432, 3267, 3180 (NH) and 1725, 1692 (C=O); H
NMR
δ ppm: 2.13 (s, 3H, CH of isatin), 2.20 (s, 3H, CH of isatin), 4.14 (brs, 2H, CH2-C=O), 6.96–6.99 (m,
3 3
2
H, Ar-H), 7.07 (brs, 1H, Ar-H), 7.35 (br s, 2H, Ar-H), 7.58 (d, 1H, Ar-H, J = 8.0 Hz), 7.84 (s, 1H, Ar-H),
1
0.71 (s, 1H, NH of isatin, D O exchangeable), 10.98 (s, 1H, NH of hydrazide, D O exchangeable), 11.06
2
2
13
(s, 1H, NH of indol, D O exchangeable); C NMR
δ
ppm: 15.80, 15.99, 20.34, 107.21, 111.40, 111.48,
2
115.01, 118.32, 118.59, 119.59, 121.05, 121.27, 124.67, 127.28, 130.55, 131.55, 134.05, 136.05, 139.85, 162.20;
Anal. Calcd. for C H N O : C, 69.35; H, 5.24; N, 16.17; found C, 69.46; H, 5.20; N, 16.11.
20
18
4
2
0
N -(1-Allyl-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9a)
◦
1
Orange powder, m.p. 218–220 C; (yield 75%), IR: 3354, 3238 (NH) and 1704 (C=O); H NMR
δ
ppm: 4.12 (brs, 2H, CH2-C=O), 4.36 (s, 2H, N-CH ), 5.13-5.16 (m, 2H, CH2=CH-), 5.82-5.87 (m, 1H,
2
N-CH -C
H
), 6.96-7.09 (m, 4H, Ar-H), 7.34 (s, 2H, Ar-H), 7.39 (t, 1H, Ar-H, J = 8.0 Hz), 7.58 (d, 1H,
2
Ar-H, J = 8.0 Hz), 8.16 (s, 1H, Ar-H), 10.99 (s, 1H, NH of hydrazide, D O exchangeable), 11.02 (s, 1H,
2
13
NH of indol, D O exchangeable); C NMR
δ
ppm: 29.84 (
CH -C=O), 41.48 (N-CH ), 107.22, 109.84,
2 2
2
1
11.45, 114.73, 116.93, 118.64, 121.15, 122.18, 124.62, 125.60, 131.80, 132.27, 136.06, 143.75, 163.09; MS m/z
+
[
%]: 358.23 [M , 41.07], 157.15 [34.19], 130.26 [100]; Anal. Calcd. for C H N O : C, 70.38; H, 5.06; N,
21 18 4 2
1
5.63; found C, 70.21; H, 5.13; N, 15.75.
0
2
-(1H-Indol-3-yl)-N -(2-oxo-1-propylindolin-3-ylidene)acetohydrazide (9b)
◦
1
Orange powder m.p. 207–208 C; (yield 77%), IR: 3317, 3282 (NH) and 1705 (C=O); H NMR
δ ppm:
0
6
.85 (brs, 3H, CH -CH3), 1.59 (brs, 2H, CH2-CH ), 3.68 (t, 2H, N-CH2), 3.89, 4.20 (2s, 2H, CH2-C=O),
.96 (t, 1H, Ar-H, J = 7.5 Hz), 7.07–7.19 (m, 2H, Ar-H), 7.30–7.43 (m, 4H, Ar-H), 7.58 (d, 1H, Ar-H,
2
3
J = 7.5 Hz), 7.69 (s, 1H, Ar-H), 10.95, 11.09 (2s, 1H, NH of hydrazide, D O exchangeable), 12.44, 12.91
2
(
2s, 1H, NH of indol, D O exchangeable); Anal. Calcd. for C H N O : C, 69.98; H, 5.59; N, 15.55;
2 21 20 4 2
found C, 70.12; H, 5.62; N, 15.43.

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0
N -(1-(sec-Butyl)-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9c)
◦
1
Orange powder, m.p. 199–201 C; (yield 68%), IR: 3451, 3294 (NH) and 1707 (C=O); H NMR
δ
ppm: 0.87 (d, 6H, -CH(CH3)2, J = 2.5 Hz), 2.03 (brs, 1H, -C
J = 2.5 Hz), 4.13 (brs, 2H, CH2-C=O), 6.97–7.07 (m, 3H, Ar-H), 7.12 (d, 1H, Ar-H, J = 8.5 Hz), 7.34 (brs,
H, Ar-H), 7.39 (t, 1H, Ar-H, J = 8.5 Hz), 7.59 (d, 1H, Ar-H, J = 7.5 Hz), 8.14 (brs, 1H, Ar-H), 10.98 (s,
H(CH ) ), 3.52 (d, 2H, N-CH2-CH(CH3)2,
3 2
2
13
1H, NH of hydrazide, D O exchangeable), 11.18 (s, 1H, NH of indol, D O exchangeable); C NMR
2
2
δ
ppm: 19.91 (-CH(
C
H ) ), 26.62 (-
C
H(CH ) ), 46.58 (N-CH ), 107.23, 109.74, 111.44, 114.60, 118.66,
3
2
3 2
2
+
1
21.12, 122.01, 124.61, 125.58, 127.24, 132.31, 132.45, 136.06, 144.38, 163.55; MS m/z [%]: 374.23 [M , 100],
57.10 [18.76], 130.38 [59.05]; Anal. Calcd. for C H N O : C, 70.57; H, 5.92; N, 14.96; found C, 70.69;
1
22
22
4
2
H, 5.87; N, 15.02.
0
N -(1-Benzyl-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9d)
◦
1
Red powder, m.p. 223–225 C; (yield 76%), IR: 3308, 3245 (NH) and 1706 (C=O); H NMR
δ
ppm:
4
.15 (brs, 2H, CH2-C=O), 4.96 (s, 2H, benzylic protons), 6.98 (d, 3H, Ar-H, J = 7.5 Hz), 7.06 (t, 1H, Ar-H,
J = 7.5 Hz), 7.25-7.35 (m, 8H, Ar-H), 7.60 (d, 1H, Ar-H, J = 8.5 Hz), 8.17 (brs, 1H, Ar-H), 10.99 (s, 1H,
13
NH of hydrazide, D O exchangeable), 11.23 (s, 1H, NH of indol, D O exchangeable); C NMR
δ ppm:
2
2
4
2.64, 107.22, 109.86, 111.45, 114.82, 118.66, 121.14, 122.32, 124.64, 125.68, 127.23, 127.51, 128.72, 132.23,
+
1
36.08, 136.18, 143.60, 163.54; MS m/z [%]: 408.25 [M , 32.23], 157.11 [100], 130.05 [48.95]; Anal. Calcd.
for C H N O : C, 73.51; H, 4.94; N, 13.72; found C, 73.63; H, 4.91; N, 13.63.
25
20
4
2
0
N -(1-(4-Fluorobenzyl)-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9e)
◦
1
Red powder, m.p. 227–229 C; (yield 72%), IR: 3353, 3115 (NH) and 1723 (C=O); H NMR
δ
ppm:
4
.15 (brs, 2H, CH2-C=O), 4.94 (s, 2H, benzylic protons), 6.95–7.02 (m, 3H, Ar-H), 7.06 (t, 1H, Ar-H,
J = 8.0 Hz), 7.13 (t, 2H, Ar-H, J = 8.0 Hz), 7.34–7.37 (m, 5H, Ar-H), 7.59 (d, 1H, Ar-H, J = 7.5 Hz), 8.16
brs, 1H, Ar-H), 10.99 (s, 1H, NH of hydrazide, D O exchangeable), 11.23 (s, 1H, NH of indol, D O
(
2
2
exchangeable); Anal. Calcd. for C H FN O (344.38): C, 70.41; H, 4.49; N, 13.14; found C, 70.73; H,
25
19
4
2
4
.46; N, 13.11.
0
N -(1-(4-Cyanobenzyl)-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9f)
◦
Yellow powder, m.p. 233–234 C; (yield 80%), IR: 3290, 3247 (NH), 2230 (C
≡
N) and 1704 (C=O);
1H NMR
7
δ
ppm: 4.14 (brs, 2H, CH2-C=O), 4.95 (s, 2H, benzylic protons), 6.99 (d, 2H, Ar-H, J = 7.5 Hz),
.06 (t, 2H, Ar-H, J = 7.5 Hz), 7.34 (t, 3H, Ar-H, J = 7.5 Hz), 7.52 (t, 1H, Ar-H, J = 7.5 Hz), 7.60 (d, 1H,
Ar-H, J = 7.5 Hz), 7.64 (d, 1H, Ar-H, J = 7.5 Hz), 7.73 (d, 1H, Ar-H, J = 7.5 Hz), 7.85 (s, 1H, Ar-H),
.14 (brs, 1H, Ar-H), 11.00 (s, 1H, NH of hydrazide, D O exchangeable), 11.24 (s, 1H, NH of indol,
8
2
13
D O exchangeable); C NMR
δ ppm: 42.01 (CH ), 107.21, 109.68, 111.46, 111.62, 115.02, 118.63, 122.46,
2
2
1
23.28, 124.64, 129.90, 129.98, 130.91, 131.40, 132.10, 137.96, 142.32, 143.32, 160.79 (C=O of isatin), 163.67
(
C=O of hydrazide); Anal. Calcd. for C H N O : C, 72.04; H, 4.42; N, 16.16; found C, 71.82; H, 4.48;
26
19
5
2
N, 16.28.
0
N -(1-Benzyl-5-bromo-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9g)
◦
1
Orange powder, m.p. 210–211 C; (yield 82%), IR: 3367, 3282 (NH) and 1739, 1669 (C=O); H NMR
ppm: 4.14 (brs, 2H, CH2-C=O), 4.96 (s, 2H, benzylic protons), 6.93 (d, 1H, Ar-H, J = 8.0 Hz), 6.96 (t, 1H,
δ
Ar-H, J = 7.5 Hz), 7.05 (t, 1H, Ar-H, J = 7.5 Hz), 7.25–7.36 (m, 7H, Ar-H), 7.53 (d, 1H, Ar-H, J = 8.0 Hz),
.59 (d, 1H, Ar-H, J = 8.0 Hz), 8.47 (brs, 1H, Ar-H), 10.97 (s, 1H, NH of hydrazide, D O exchangeable),
7
2
13
1
1
1
1.48 (s, 1H, NH of indol, D O exchangeable); C NMR δ ppm: 42.71 (CH ), 107.21, 111.44, 111.62,
2 2
14.35, 116.46, 118.53, 118.63, 121.06, 124.54, 127.19, 127.55, 128.03, 1289.74, 134.24, 135.89, 136.01, 142.65,
63.26; Anal. Calcd. for C H BrN O : C, 61.61; H, 3.93; N, 11.50; found C, 61.79; H, 3.88; N, 11.57.
25
19
4
2

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0
N -(5-Bromo-1-(4-fluorobenzyl)-2-oxoindolin-3-ylidene)-2-(1H-indol-3-yl)acetohydrazide (9h)
◦
1
Brown powder, m.p. 183–185 C; (yield 80%), IR: 3423, 3343 (NH) and 1730 (C=O); H NMR
δ
ppm: 4.13 (s, 2H, CH2-C=O), 4.95 (s, 2H, benzylic protons), 6.97 (d, 2H, Ar-H, J = 8.5 Hz), 7.05 (t, 1H,
Ar-H, J = 8.0 Hz), 7.13 (t, 2H, Ar-H, J = 8.5 Hz), 7.33–7.37 (m, 4H, Ar-H), 7.55 (d, 1H, Ar-H, J = 8.0 Hz),
7
.58 (d, 1H, Ar-H, J = 7.5 Hz), 8.45 (s, 1H, Ar-H), 10.97 (s, 1H, NH of hydrazide, D O exchangeable),
2
1
1
1.49 (s, 1H, NH of indol, D O exchangeable); Anal. Calcd. for C H BrFN O : C, 59.42; H, 3.59; N,
1.09; found C, 59.58; H, 3.55; N, 11.16.
2
25 18
4
2
0
N -(3,4-Dihydronaphthalen-1(2H)-ylidene)-2-(1H-indol-3-yl)acetohydrazide (11)
◦
1
White crystals, m.p. 244–245 C; (yield 84%), IR: 3315, 3246 (NH) and 1692 (C=O); H NMR
δ
ppm: 1.81 (d, 2H, CH , J = 6.0 Hz), 2.61 (m, 2H, CH ), 2.74 (d, 2H, CH , J = 5.6 Hz), 3.78, 4.13 (2s, 2H,
2
2
2
C
(
H2-C=O), 6.95–7.09 (m, 2H, Ar-H), 7.19–7.27 (m, 4H, Ar-H), 7.34 (t, 1H, Ar-H, J = 7.6 Hz), 7.57, 7.62
2d, 1H, Ar-H, J = 8.0, 8.0 Hz), 8.00, 8.10 (2d, 1H, Ar-H, J = 7.2, 7.6 Hz), 10.41, 10.43 (s, 1H, NH of
hydrazide, D O exchangeable), 10.86, 10.90 (s, 1H, NH of indol, D O exchangeable); Anal. Calcd. for
2
2
C H N O: C, 75.69; H, 6.03; N, 13.24; found C, 75.86; H, 5.99; N, 13.32.
20
19
3
3
.2. Biological Evaluation
The detailed experimental procedures adopted in the different biological assays for target
bis-indoles (7a–f and 9a–h); 11 were supplied in the Supplementary Materials.
3
.2.1. Cytotoxic Activity against Human Breast Cancer and Non-Tumorigenic Cell Lines
The two examined human breast cancer cell lines (MCF-7 and Breast MDA-MB-231), and
non-tumorigenic human breast epithelial cell line (MCF-10A) have been obtained from the American
Type Culture Collection (ATCC). Assessment of cytotoxicity for target indole derivatives has been
performed following the SRB colorimetric assay procedures [20], as reported earlier [26].
3
.2.2. Cell Cycle Analysis
The influence of bis-indoles 7e and 9a on cell cycle progression was examined in breast cancer
MCF-7 cells, after 24 h of treatment, through DNA flow cytometric assay by the use of BD FACS
Caliber flow cytometer, as described previously [27]. The cell cycle distributions were calculated using
CellQuest software (Becton Dickinson).
3
.2.3. ELISA Immunoassay
Effects of treatment of breast cancer MCF-7 cells with bis-indoles 7e and 9a on the expression
levels of the pro-apoptotic markers (Bax, caspase-3, and p53) in addition to the anti-apoptotic protein
Bcl-2 marker was assessed by the use of ELISA colorimetric kits as per the manufacturer’s instructions,
as described earlier [28].
3
.2.4. Annexin V-FITC/PI Apoptosis Assay
The apoptotic action of bis-indoles 7e and 9a was further explored through the investigation of their
effect on the phosphatidylserine externalization in breast cancer MCF-7 cells, using Annexin-V-FITC
Apoptosis Detection Kit according to manufacturer’s protocol, as reported earlier [27].
3
.2.5. CDK2 Kinase Inhibitory Activity
The in vitro CDK2 kinase inhibition assay was carried out by Reaction Biology Corp. (Reaction
Biology Corp., Chester, PA, USA) Kinase HotSpotSM service (http://www.reactionbiology.com).

Mar. Drugs 2020, 18, 190
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4
. Conclusions
In summary, the adopted approach of replacing the rigid heterocyclic spacer in the marine natural
products Topsentin and Nortopsentin by the flexible hydrazide linker resulted in the discovery of
promising marine-inspired bis-indole scaffold with good in vitro antitumor activities toward breast
cancer cell lines. All the synthesized bis-indoles (7a
their antiproliferative action against human breast cancer (MCF-7 and MDA-MB-231) cell lines. All the
examined bis-indoles 7a and 9a displayed excellent to low antiproliferative activities against
MCF-7 and MDA-MB-231 cells with IC50 values in ranges 0.44–47.1 and 0.34–77.30 M, respectively.
Bioisosteric replacement of the oxindole ring with the carbocyclic tetralin ring (compound 11) resulted
in a dramatic worsening of effectiveness against the examined cancer cell lines (IC50 = 84.70 4.02
and > 100 M, respectively) in comparison to bis-indoles , which pointed out the importance of
oxindole moiety for the antiproliferative activity. The most potent congeners 7e and 9a against MCF-7
cells (IC50 = 0.44 0.01 and 1.28 0.04 M, respectively) induced apoptosis in MCF-7 cells (23.7-,
–f and 9a–h), and indole 11 were characterized for
–
f
–h
µ
±
µ
7
±
±
µ
and 16.8-fold increase in the total apoptosis percentage) as evident by the externalization of plasma
membrane phosphatidylserine detected by AnnexinV-FITC/PI assay. This evidence was supported by
the Bax/Bcl-2 ratio augmentation (18.65- and 11.1-fold compared to control) with a concomitant increase
in the level of caspase-3 (11.7- and 9.5-fold) and p53 (15.4- and 11.75-fold). Moreover, scrutinizing
results of the cell cycle analysis unraveled that both compounds arrest the cell cycle mainly in the G2/M
phase. On the other hand, 7e and 9a displayed good selectivity toward tumor cells (S.I. = 38.7 and 18.3),
upon testing of their cytotoxicity toward non-tumorigenic breast MCF-10A cells. Finally, compounds
7
a
,
7b, 7d, 7e, and 9a were examined for their plausible CDK2 inhibitory action. The obtained results
(% inhibition range: 16%–58%) unveiled incompetence of the target bis-indoles to inhibit CDK2
significantly. Collectively, these results suggested that herein reported bis-indoles are good lead
compounds for further optimization and development as potential efficient anti-breast cancer drugs.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/4/190/s1.
Author Contributions: For research articles with several authors, a short paragraph specifying their individual
contributions must be provided. The following statements should be used “Conceptualization, W.M.E., G.S.H.
and G.H.A.-A.; methodology, W.M.E., S.T.A.-R. and G.H.A.-A.; software, H.M.A.; validation, S.T.A.-R. and A.A.A.;
formal analysis, W.M.E., H.M.A. and G.H.A.-A.; investigation, G.S.H.; resources, S.T.A.-R. and H.M.A.; data
curation, A.A.A. and G.H.A.-A.; writing—original draft preparation, W.M.E. and G.H.A.-A.; writing—review
and editing, W.M.E. and G.S.H.; visualization, G.S.H. and H.M.A.; supervision, W.M.E. and G.S.H.; project
administration, W.M.E.; funding acquisition, S.T.A.-R.” Please turn to the CRediT taxonomy for the term
explanation. Authorship must be limited to those who have contributed substantially to the work reported.
All authors have read and agreed to the published version of the manuscript.
Funding: The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at
King Saud University for its funding of this research through the Research Group Project no. RG-1439–65.
Conflicts of Interest: The authors declare no conflict of interest.
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