Towards breast cancer targeting: Synthesis of tetrahydroindolocarbazoles, antibreast cancer evaluation, uPA inhibition, molecular genetic and molecular modelling studies

Article abstract of DOI:10.1016/j.bioorg.2019.103332

A series of some new tetrahydroindolocarbazole derivatives has been synthesized. The structure of the synthesized compounds has been confirmed by different spectroscopic techniques such as IR, NMR, elemental analysis and mass spectrometry. The target compounds were evaluated for their antitumor activity against breast cancer cell line MCF-7, their GI% and their LC₅₀ have been determined. Six of the synthesized compounds exhibited GI% values against MCF-7 cell lines exceeding 70% ranging from 71.9 to 85.0% in addition that compound 11 expressed GI% values of 99.9% and considered the most active derivatives among the synthesized ones. Compound 11 showed a remarkable decrease of u PA level to 3.5 ng/ml compared to DOX. Compound 5, 11 and 15 showed significant decrease in expression of MTAP and CDKN2A, in addition to a remarkable decrease in DNA damage comet assay method. Molecular modeling studies were performed to interpretate the behavior of active ligands as uPA inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.103332

BioorganicChemistry93(2019)103332
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Towards breast cancer targeting: Synthesis of tetrahydroindolocarbazoles,
antibreast cancer evaluation, uPA inhibition, molecular genetic and
molecular modelling studies
⁎
Entesar M. Ahmeda, , Alaadin E. Sarhanb, Dina H. El-Naggarc, Reham R. Khattabd,
⁎
Mohamed El-Naggare, Shahenda M. El-Messeryf, , Ghada S. Hassang, Marwa M. Mounierh,
⁎
Khaled Mahmoudh, Neama I. Alii, Karima F. Mahrousi, Mamdouh M. Alij, Mardia T. El Sayedc,
a Chemistry Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo, Egypt
b Therapeutical Chemistry Department, Pharmaceutical Division, National Research Centre, Dokki- 12311, Egypt
c Department of Applied Organic Chemistry, National Research Centre, 12622 Dokki, Giza, Egypt
d Photochemistry Department, Chemical Industries Research Division, National Research Centre, Dokki 12311, Egypt
e Chemistry Department, Faculty of Sciences, University of Sharjah, Sharjah 27272, United Arab Emirates
f Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt
g Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt
h Pharmacognosy Department, National Research Centre, 12622-Dokki, Egypt
i Cell Biology Department, National Research Centre, 12622-Dokki, Egypt
j Biochemistry Department, National Research Centre, 12622-Dokki, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of some new tetrahydroindolocarbazole derivatives has been synthesized. The structure of the syn-
thesized compounds has been confirmed by different spectroscopic techniques such as IR, NMR, elemental
analysis and mass spectrometry. The target compounds were evaluated for their antitumor activity against breast
cancer cell line MCF-7, their GI% and their LC50 have been determined. Six of the synthesized compounds
exhibited GI% values against MCF-7 cell lines exceeding 70% ranging from 71.9 to 85.0% in addition that
compound 11 expressed GI% values of 99.9% and considered the most active derivatives among the synthesized
ones. Compound 11 showed a remarkable decrease of u PA level to 3.5 ng/ml compared to DOX. Compound 5,
11 and 15 showed significant decrease in expression of MTAP and CDKN2A, in addition to a remarkable de-
crease in DNA damage comet assay method. Molecular modeling studies were performed to interpretate the
behavior of active ligands as uPA inhibitors.
Breast cancer
Tetrahydroindolocarbazoles
Urokinase inhibition
DNA genetics
Molecular modelling studies
1. Introduction
malignant tumour such as colon, liver, breast, lung, stomach and
ovarian cancer, when compared to normal cells also uPA has been used
Urokinase is a type of protease enzyme also known as urokinase
plasminogen activator (uPA), it is a serine type of protease enzyme that
is present in humans and animals. It is mainly present in the blood and
extracellular matrix (ECM) of the tissues and involved in either vascular
diseases or cancer progression [1,2]. Increased level of urokinase ex-
pression with other plasminogen activation system was found to be in
correlation with tumour malignancy via tissue degradation- facilitates
tissue invasion and promotes metastasis as it is implicated in different
physiological and pathological processes including cell migration, an-
giogenesis, inflammation, tumour growth and metastasis. It was also
found that uPA was overexpressed in different types of human
a tumor “marker”. Accordingly, urokinase inhibitors have been sought
to be used as anticancer agents [3,4]. Breast cancer is considered the
second cause of cancer-related deaths in women all over the world.
Multiple drugs have been approved by the US-FDA for treatment of
breast related malignancies. Frequent emergence of resistances leads to
the urgent need for newer moieties to overcome such problems [5].
Recently, amiloride HCl -which has been used for decades as potassium
sparing diuretic- also proved to exhibit significant antitumor and anti-
metastasis activities in biochemical, cellular and animal tumor models.
Several studies have established that amiloride can suppress the me-
tastases formation and the invasive capacity of human breast cancer
⁎ Corresponding authors.
E-mail addresses: entsar.ahmed2016@yahoo.com (E.M. Ahmed), selmessery@gmail.com (S.M. El-Messery), mardia.elsayed2016@gmail.com (M.T. El Sayed).
https://doi.org/10.1016/j.bioorg.2019.103332
Received 7 March 2019; Received in revised form 7 August 2019; Accepted 28 September 2019
Availableonline01October2019
0045-2068/©2019PublishedbyElsevierInc.

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
cell lines of types MDA-MB-231 and MDA-MB-436 in a dose dependant
manner. Through studying its mechanisms of action, it was revealed
that one of the methods to inhibit the breast cancer propagation is
through the inhibition of uPA system by binding reversibly to the en-
zyme's active site, [6]. Furthermore, many researchers correlate be-
tween the level of expression of uPA and the progression of breast
cancer. Previously, the traditional prognosis of breast cancer focused on
age, tumour size, tumour grade and lymph node status etc [7,8], but as
uPA was found to be involved in regulating breast cancer invasion due
to its ability to facilitate (ECM) degradation, cell proliferation, migra-
tion and adhesion, it becomes the first novel tumour prognostic factors
confirmed un highest level of evidence regarding the clinical utility in
breast cancer [9]. Drugs targeting breast cancer with various indole
based derivatives have also been explored [10a,b,11a,b]. Indole pro-
ducts using DNA- established electrochemical biosensors evidenced that
it can meaningfully lessen the advancement of cancer cell lines such as
HOP-92 (lung), A498 (renal) and MDAMB-231/1TCC (breast) [12,13a].
Indoles and Indolocarbazoles have also promising anti-breast cancer
potential [13b], in addition, that was related to DNA expression
[14,15]. That possible role of indole based anticancer agents typically
for the breast related malignancies Fig. 1A and B has put an emphasis
on the synthesis of new derivatives to overcome problems faced by
existing therapeutic agents (see Scheme 1).
mixture. An alternative method was used to synthesize the target
compounds through one step reaction by the use of double amounts of
both indole and the substituted aromatic aldehydes in methanol and in
presence of catalytic amount of concentrated H2SO4. The compounds
produced by both methods were identical according to the spectral data
performed. It was also found that the direct method gave better yield
ranging from 65 to 90%. All compounds (1–20) derived from aldehydes
(1–20) were confirmed by means of analytical and spectral data.
Compounds 1–8 and 18–20 were previously reported and their iden-
tities are equivalent to our reported samples [12,16]. These known
derivatives have been synthesized to completely address the effect of
structure modification whereas the THICZs (9–17) are new derivatives
synthesized and confirmed by the means of 1H- and 13C NMR, ESI-MS
and IR spectra. The 1H NMR spectra of all these compounds showed
singlet signal around δ from 4.5 to 5.5 ppm belonging for 2 protons of
(2CH), the two NH indole protons appeared around δ from 9.94 to
10.4 ppm as a broad signal. The 1H NMR data proved the similarity of
the structure which confirmed additionally by its 13C NMR spectrum
(see Table 1).
2.2. Anticancer evaluation
The antitumor activity of the synthesized compounds has been
performed against MCF-7 breast cancer cell line obtained from Vacsera
(Giza, Egypt). Culturing and subculturing were carried out according to
reported procedure; a negative control composed of DMSO was also
used [17]. Cell % viability was evaluated and described according to
the reported protocols [18,19]. The absorbance was measured con-
suming a microplate multi-well reader at 595 nm and a reference wa-
velength of 690 nm. Cell viability was evaluated rendering to the mi-
tochondrial-supported reduction of yellow MTT (3-(4, 5-
dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide) to ex-
aggerated formazan. The GI% (Growth inhibition percentage) of the
synthesized compounds was illustrated in Table 2. It was observed that
6 of the synthesized compounds exhibited GI% values against MCF-7
cell lines exceeding 70% ranging from 71.9 to 85.0% in addition that
compound 11 expressed GI% values of 99.9% and considered the most
active derivatives among the synthesized ones. LC50 of the most active
compounds were evaluated and illustrated in Table 2. In the same
manner, compounds 11 and 15 exhibited the least LC50 values of 5.7
and 10.8 respectively.
Taking into consideration the above-mentioned facts, and as a part
of our ongoing effort to develop potent indole based anti-breast cancer
agents [13b], in the present work a group of some new tetra-
hydroindolocarbazoles (THICZs) with variety of substituents has been
synthesized and subjected to spectroscopic elucidation using different
techniques such as IR, 1HNMR, 13CNMR, elemental analysis and mass
spectroscopy. They were examined for their in-vitro antibreast cancer
screening, progressive urokinase inhibition, DNA-damage determina-
tion were further evaluated. Computer modelling studies exhausting the
energetic site of uPA as a template for the most active uPA inhibitors
were calculated.
2. Results and discussion
2.1. Chemistry
The target compounds THICZs (1–20) were prepared by the use of
two dissimilar synthetic methodologies according to the reported pro-
cedures [11,12]. The first method involved the preparation of the in-
termediates bis-indolylmethane (BIMs) by condensation of indole and
different substituted aromatic aldehydes followed by reaction of equi-
molar concentration of both BIMs and the same aldehyde used in its
preparation. The reaction proceeded in methanolic solution in presence
of catalytic amount of concentrated H2SO4 added gently to the reaction
2.3. Urokinase inhibition assay
One of the landscapes of a number of distortions counting lung,
prostate, breast, and stomach cancers is the over-expression of the
plasminogen activator urokinase (uPA) [20–22]. In the present work,
Fig. 1. Reported structures of anticancer indoles (A and B) and structures of synthesised derivatives (C).
2

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
Scheme 1. Synthesis of THICZs.
the decrease in the level of (uPA) expression was used to indicate the
activity of the synthesized compounds as antitumor agents. The most
active compounds 11 and 15 were used and tested for their ability to
reduce the uPA protein expression. The levels of expression of uPA
protein articulated in cell extracts of MCF7 cell line were illustrated in
Table 3. The data revealed that, the concentrations of uPA protein were
reduced when the cells were treated with compounds 11 and 15, as
compared with the untreated cancer cells (control) whose value was
Table 1
Structures of the synthesized THICZs.
Compound NO.
R1
R2
R
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
2
Ph
3
p-Br-Ph
4
m-Br-Ph
5
p-N(Me)2-Ph
m-OCH2Ph-Ph
p,m-OH-Ph
6
22.00
3.70 ng/ml, the levels of uPA protein in compounds 11 and
7
15 were decreased by 84% and 50% correspondingly with IC50 identical
8
p-MeO-m-OCH2Ph-Ph
m-MeO-p-OCH2Ph-Ph
p-OCH3-Ph
3.50
0.47 ng/ml for candidate 11 and for compound 15 it was
9
11.00
1.30 ng/ml when compared with regulator cancer cells. Thus
10
11
12
13
14
15
16
17
18
19
20
the decrement in the level of uPA protein manifestation are reliable
with the cytotoxicity outcomes resulting in that the anticancer effect of
tested composites may employ its anticancer activity by hindering uPA
and possess significant anticancer activity comparable to the activity of
the commonly used anticancer drug, doxorubicin [23–25].
p-CN-Ph
2,4-di-NO2-Ph
2,6-di-NO2-Ph
m-OCH3-Ph
P-OH,m-OCH3-Ph
2,5-OH-Ph
p-OCH2Ph-Ph
p-MeO-m-OCH2Ph-Ph
p-MeO-m-OCH2Ph-Ph
m-Cl-Ph
2.4. Molecular genetics
2.4.1. Gene expression analysis
2.4.1.1. Quantitative real time PCR method. The sequences of specific
primer of the genes used are listed in Table 4. The relative
quantification of the target to the reference was determined by using
the 2−ΔΔCT method as follows:
Table 2
The GI% values of the target compounds, LC50 of the most active compounds.
Comp. No
GI%
LC50
Comp. No
GI%
LC50
58.5
10.8
ΔCT(test) = CT(target, test) − CT(reference, test)
ΔCT(calibrator) = CT(target, calibrator) − CT(reference, calibrator)
ΔΔCT = ΔCT(Test) − ΔCT(calibrator)
,
2
29.5
13
73.7
1.3
0.8
,
3
77.7
5.70
63.5
0.9
14
8.30
.
4
15
85.00
13.00
22.70
13.30
36.10
29.90
100.00.
5
71.9
79.8
33.00
53.10
22.60
99.9
33.1
45.6
0.6
1.1
16
As illustrated in Figs. 2 and 3, in comparison to the positive control
group, the transcription levels of MTAP and CDKN2A showed a non-
significant reduction in both 11 and 15 cell lines and a significant de-
crease in cell line with compound 5. Additionally, the expression levels
of MTAP and CDKN2A genes for cell line with compound 5 exhibit a
statistically significant decrease as compared to cell line 11 and a slight
non-significant reduction as compared to cell line 15.
6
17
8
18
9
19
10
11
20
5.7
0.8
Doxorubicin
30.5
2.1
Table 3
2.4.1.2. DNA damage using comet assay. The neutral comet assay for
tumor cell lines was used as described according to the reported
procedures for compounds 5, 11 and 15. For each sample about 100
cells were examined to determine the percentage of cells with DNA
damage that appear like comets based on perceived comet tail length
migration and relative proportion of DNA in the nucleus [26,27].
Behavior of 5, 11 and 15 cell lines has discovered that DNA damage
was amplified meaningfully as associated to the negative control group
accomplishment 19.75, 17 and 16.25 in 11, 15 and 5 compounds,
correspondingly, while, the percentage of the scratched cells in these
three compounds (11, 15 and 5) was markedly decreased with a
The level of expression of uPA protein in the MCF7 cells.
Compound
uPA level (ng/ml)
a
11
3.50
0.47
a
15
11.00
22.00
2.30
1.30
3.70
0.37
DMSO
Dox
a
Data are expressed as mean
S.E. of three separate
experiments.
a
Is significant difference from control untreated at
p < 0.05.
3

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
Table 4
Primers sequence used for RT-qPCR.
Gene
Forward
Reverse
β-actin
MTAP
GAT GCA GAA GGA GAT CAC TGC
CCA CCG CCG TGA AGA TTG GAA
CCA CCG CCG TGA AGA TTG GAA
AGT ACT TGC GCT CAG GAG GA
CCA CCG CCG TGA AGA TTG GAA
AAG TTT CCC GAG GTT TCT CA
CDKN2A
MTAP : Methylthioadenosine Phosphorylase; CDKN2A: Cyclin Dependent Kinase Inhibitor 2A.
Treatment
significant value for samples 15 and 5 and with a non-significant value
for sample 11 tumor cell line as compared to the positive control group.
Hence, the increase of un-fragmented cells values revealed a positive
effect of compounds in comparing with the positive control group and
the decrease of such values indicated that compounds have no effect on
the studied cell lines as compared to the negative control group
(Table 5).
3.00
2.50
2.00
1.50
1.00
0.50
0.00
a
b
ab
b
c
2.5. Molecular modeling study
2.5.1. Docking studies
Conformational analysis of the most active uPA inhibitors have been
performed in addition to Amiloride that was selcted as reference uPA
inhibitor in our modeling study. The least energy conformer for each
compound was obtained by conformational searching in torsional space
using the multiconformer method and is illustrated in Fig. 4a–c. It was
quite interesting to perform a docking study of the best uPA inhibitory
activity synthesized compounds 11 and 15 respectively against
Amiloride which was recognized recently as a lead compound for uPA
inhibitors as anticancer agents [28–30].
Fig. 3. The alterations of CDKN2A gene in tumor cell lines treated. Data are
presented as mean
SEM. a–cMean values within tissue with unlike super-
b and c
script letters were significantly different (a: P < 0.01,
P < 0.05).
Table 5
Visual score of DNA damage in tumor cell lines treated with compounds 5, 11
and 15.
Treatment
No. of cells
Class**
DNA damaged cells %
The results obtained were quite informative. The complex structure
of uPA and amiloride was used as a reference for modeling and docking
(Fig. 5). It showed a tight binding occupying the S1 and S1β sites via
excessive hydrogen bonding formation in addition to salt-bridge inter-
action with Asp189, in addition to important aminoacid in the binding
pocket illustrated in Ser190, Gly219, Val227, Arg 217 and Lys 224, Ser
214 and Tryp 215, the binding energy was estimated by
−12.987635 Kcal/mol [6].
(Mean
SEM)
Analyzed* Comets
0
1
2
9
3
2
Control (-ve)
400
45
94
65
79
68
355 34
11.25
0.75c
a
Control (+ve) 400
306 41 34 19 23.50
0.29
Sample 5
Sample 11
Sample 15
400
400
400
335 38 19
321 42 21 16 19.75
332 41 19 17.00
8
16.25
0.48b
0.49ab
0.41b
8
Docking results revealed that compound 11 has shown high affinity
with a binding energy of −11.3456 Kcal/mol with Ser192 and Gly
A216 via hydrogen bonding type of interaction (64%) and (92%) re-
spectively while Arg217 amino acid residue binds with both aryl part of
indole ring by arene-arene interaction type (Fig. 6). Moreover, Com-
pound 15 binds well with good affinity binding energy of
−12.8776 Kcal/mol with Arg 217 and Leu 222 by an arene-arene in-
teraction while it binds with Gly 216 by a hydrogen binding interaction
through its (31%) (Fig. 7).
* : Number of cells examined per a group,
** : Class 0 = no tail; 1 = tail length < diameter of nucleus; 2 = tail length
between 1X and 2X the diameter of nucleus; and 3 = tail length > 2X the
diameter of nucleus.
and 15 showed their interact action with the importants amino acid
residue in the active site of uPA enzyme such as the cocrystallized li-
gand explaining their activity profile inspite of their dissimilarity in
structure to the reference amiloride under study. A detailed view of
both compounds 15 into the active site, have shown that it occupies a
A deep insight to the interaction of synthesized uPA inhibitors 11
Treatment
3.00
2.50
2.00
1.50
1.00
0.50
0.00
a
a
ab
b
c
Fig. 2. The alterations of MTAP gene in tumor cell lines treated with tested compounds Data are presented as mean
SEM. a–cMean values within tissue with unlike
b and c
superscript letters were significantly different (a: P < 0.01,
P < 0.05).
4

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
Fig. 4. Lowest energy conformers of most active compounds (a) 11 (b) 15 and reference compound (c) Amiloride with balls and cylinders rendering.
deeper location into the cavity which may lead to a higher binding
interaction within the hydrophobic residues in the uPA active site and
hence attribute its higher activity pattern. (Fig. 8).
higher activity of tested compounds. Molecular docking study of the
present work revealed that active uPA inhibitors candidates 11 and 15
bound with similar amino acid residues as amiloride especially men-
tioning Arg 217 and Gly 216 which support our hypothesis that these
compounds could exert their antibreast cancer activity action via uPA
inhibition. Surface mapping has illustrated active candidates under
study showed good binding to uPA by having optimal lipophilicity re-
quired for effective binding. Thus this work presents two active anti-
breast cancer candidates 11 and 15. More studies will be conducted for
further optimization of active derivatives to be drug candidates.
2.5.2. Surface mapping
In a valuable research for reasons behind the activity of compounds
under investigation, hydrophobic surface mapping study was per-
formed. As there is a powerful link between hydrophobicity and ef-
fective binding to uPA [31]. Compounds 11 and 15 clearly showed a
preference for more hydrophobic regions (more greener areas on the
map, Fig. 9a and b that could be attributed to the presence of two indole
ring in addition to phenyl moieties in both compounds causing higher
lipophilic character of the whole molecule even more than the reference
modeling compound amiloride Fig. 9c.
4. Experimental section
The melting points were measured on a Boetius-Mikroheiztisch the
company “VEB weighing, Rapido Radebeul/VEB NAGEMA” measured
and are uncorrected. The carbon, hydrogen and nitrogen content of the
substances were performed on a “CHNS-932” automatic analyzer of the
company “LECO Corporation” in the automatic Micro chemical de-
termined. The halogen content was determined by titration in semi
micro method determined. For the analyzes TLC were with aluminium
foil fluorescent indicator from Merck KGaA (silica gel 60 F254, layer
thickness 0.2 mm) used. Rf -values (run level relative to the solvent
front), The separations were with column chromatography at atmo-
spheric pressure on silica gel 60 (Grain size from 0.063 to 0.200 mm)
from Merck KGaA. The NMR spectra were recorded on a “Gemini 2000”
(400/100 MHz). The ATR spectrawere recorded on an FT-IR spectro-
meter “IFS 28” by “Bruker”, the KBr spectra on an FT-IR Spectrometer
“Spectrum BX” the Company “PerkineElmer” measured. The ESI mass
3. Conclusion
The current research paper was directed towards breast cancer
targeting via the synthesis of some new tetrahydroindolocarbazoles
derivatives (1–20). The results revealed that compounds 11, 15 and 5
possessed the higher cytotoxicity on breast (MCF-7) human tumor cell
line with LC50 5.7, 10.8, 33.1 respectively. By measuring the level of
uPA protein expression for the most active tested compounds 11 and 15
the results indicated that they may exert their anticancer activity by
inhibiting uPA and possess significant anticancer activity comparable to
the activity of the commonly used anticancer drug, doxorubicin. Gene
expression analysis by Quantitative Real Time PCR and DNA damage
assay were performed for the active derivatives, results supported the
Fig. 5. The 3D binding mode and residues involved in the recognition for Amiloride The hydrogen bonding were represented as pink dotted lines.
5

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
Fig. 6. The 3D binding mode and residues involved in the recognition for compound 11. The hydrogen bonding residues at the binding site are tagged in dotted lines.
spectra were recorded on a “Finnigan LCQ Classic” by “thermal
Electron measured” the sample was injected directly.
2H, ArH), 6.96 (d, 4H, J = 7.2 Hz, ArH), 7.17–7.21 (m, 14H, ArH), 7.30
(dd, 2H, J = 2.0, 7.5 Hz, ArH), 10.31 (s, 2H, 2NH), 13C NMR
(100 MHz,acetone‑d6) δ (ppm): 29.2 (CH), 30.6 (CH), 55.5 (OCH3),
56.00 (OCH3), 74.1 (OCH2), 76.1 (OCH2), 109.0–148.1 (40C, Aromatic-
C), EA calcd. C, 80.92, H, 5.61, N, 4.10, found C, 80.91, H, 5.60, N,
4.11, Rf 0.79 (CH2Cl2).
4.1. Chemistry
4.1.1. General procedure for the preparation of THICZs (1 – 20)
0.002 mol of substituted aromatic aldehyde were added to a stirred
methanolic solution of 0.002 mol of indole or its derivative till complete
dissolution. Catalytic amount of concentrated H2SO4 were added while
stirring. Reflux the mixture for additional 1 hr. The completion of the
reaction was monitored by TLC (100% CH2Cl2). The reaction was then
terminated by addition of 50 ml of water followed by neutralization
with NH4OH solution. The product was then extracted twice by ethy-
lacetate, washed with water, and finally dried over anhydrous Na2SO4.
The obtained filterate is concentrated under vacuum and the crude
product was purified via column chromatography on silica gel using
(30% EtAc/hexane) as a solvent to afford the target THICZs derivatives
1–20. The THICZs (1–8 and 18–20) were previously reported and their
identities are equivalent to the synthesized samples, whereas the
THICZs (9–17) new derivatives were confirmed by the means of 1H- and
13C NMR, ESI-MS and IR spectra (see supporting information).
4.1.3. 6,12-bis(4-methoxyphenyl)-5,6,11,12-tetrahydroindolo[3,2-b]
carbazole (10)
Yellow powder, mp. 146–147 °C, yield 56%, 470.56 g/mol,
C
32H26N2O2, ESI-MS: 471.22 [M + H+], IR (ATR,cm−1): 3802 (NH),
2989, 2990 (CH), 1H NMR (400 MHz, DMSO‑d6): δ (ppm): 3.71 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 5.43 (s, 2H, 2CH), 7.32 (t, 4H, J = 7.8 Hz,
ArH), 7.51(d, 2H, J = 8.0 Hz, ArH), 7.44 (d, 2H, J = 8.0 Hz, ArH),
7.65–7.71 (m, 4H, ArH), 7.91 (d, 2H, J = 7.5 Hz, ArH), 8.02–8.11 (m,
2H, ArH), 9.88 (s, 1H, 1NH), 10.14 (s, 1H, 1NH). 13C NMR: (100 MHz,
DMSO‑d6) δ (ppm) = 45.1 (CH), 47.0 (CH), 55.6 (OCH3), 56.7 (OCH3),
111.0–137.3 (28C, Aromatic-C), EA calcd. : C, 81.68, H, 5.57, N, 5.95,
found C, 81.66, H, 5.58, N, 5.93, Rf 0.55 (CH2Cl2).
4.1.4. 4,4′-(5,6,11,12-tetrahydroindolo[3,2-b]carbazole-6,12-diyl)
dibenzonitrile (11)
4.1.2. 6,12-bis(4-(benzyloxy)-3-methoxyphenyl)-5,6,11,12-
tetrahydroindolo[3,2-b]carbazole (9)
Yellow crystals, mp. 138–139 °C, yield 76%, 460.53 g/mol,
C
32H20N4, ESI-MS: 461.11 [M + H+], IR (ATR, cm−1): 3865 (NH),
White powder, Mp 282–284 °C, yield 86%, C46H38N2O4, 682.80 g/
mol, ESI-MS: 683.30 [M + H+], IR (ATR,cm_1): 3368(NH), 1H NMR
(400 MHz, DMSO‑d6): δ (ppm) = 3.83 (s, 6H, 2OCH3), 4.94 (s, 4H,
2OCH2), 5.54 (s, 2H, 2CH), 6.71 (dd, 2H, J = 1.9, 8.6 Hz, ArH), 6.81 (s,
2994, 2999 (CH), 1H NMR (400 MHz, DMSO‑d6): δ (ppm): 5.53 (s, 2H,
2CH), 6.82–6.84 (m, 2H, ArH), 7.12 (t, 4H, J = 7.8 Hz, ArH), 7.41(d,
2H, J = 8.0 Hz, ArH), 7.54 (d, 4H, J = 8.0 Hz, ArH), 7.56–7.57 (m, 4H,
ArH), 10.45 (s, 1H, 1NH), 10.56 (s, 1H, 1NH), 13C NMR: (100 MHz,
Fig. 7. The 3D binding mode and residues involved in the recognition for compound 15. The hydrogen bonding residues at the binding site are tagged in dotted lines.
6

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
Fig. 8. Overelay of compound 15 embedded into the surface mapping pocket Pink: hydrogen bond, blue: mild polar, green: hydrophobic region.
DMSO‑d6) δ (ppm) = 45.1 (CH), 47.0 (CH), 111.0 (CN), 118.7 – 144.3
(29 C, Aromatic-C), EA calcd. : C, 83.46, H, 4.38, N, 12.17, found C,
83.47, H, 4.39, N, 12.16, Rf 0.45 (CH2Cl2).
4.1.7. 6,12-bis(3-methoxyphenyl)-5,6,11,12-tetrahydroindolo[3,2-b]
carbazole (14)
Yellow orange powder, mp. 166–167 °C, yield 75%, 470.56 g/mol,
C32H26N2O2, ESI-MS: 471.15 [M + H+], IR (ATR, cm−1): 3822 (NH),
2988, 2994 (CH), 1H NMR (400 MHz, DMSO‑d6): δ (ppm): 3.72 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 5.46 (s, 2H, 2CH), 6.44 (s, 2H, ArH), 7.34 (t,
4H, J = 7.8 Hz, ArH), 7.42(d, 2H, J = 8.0 Hz, ArH), 7.46 (d, 2H,
J = 8.0 Hz, ArH), 7.57–7.59 (m, 4H, ArH), 7.90 (d, 2H, J = 7.5 Hz,
ArH), 9.98 (s, 1H, 1NH), 10.33 (s, 1H, 1NH), 13C NMR: (100 MHz,
DMSO‑d6) δ (ppm) = 43.1 (CH), 45.0 (CH), 55.7 (OCH3), 58.1 (OCH3),
112.0 – 142.6 (28C, Aromatic-C). EA calcd. : C, 81.68, H, 5.57, N, 5.95,
found C, 81.69, H, 5.59, N, 5.94, Rf 0.75 (CH2Cl2).
4.1.5. 6,12-bis(2,4-dinitrophenyl)-5,6,11,12-tetrahydroindolo[3,2-b]
carbazole (12)
Yellow powder, Mp 232–234 °C, yield 66%, C30H18N6O8, 590.50 g/
mol, ESI-MS: 591.11 [M + H+], IR (ATR, cm−1): 3388(NH), 1H NMR
(400 MHz, DMSO‑d6): δ (ppm) = 5.43 (s, 2H, 2CH), 6.57 (dd, 2H,
J = 1.9, 8.6 Hz, ArH), 6.83 (s, 2H, Ar-H), 7.23 (t, 4H, J = 7.2 Hz, ArH),
7.47–7.49 (m, 4H, ArH), 7.60 (dd, 2H, J 2, 7.5 Hz, ArH), 10.34 (s, 2H,
2NH), 13CNMR (100 MHz,acetone‑d6) δ (ppm): 30.55 (CH), 32.5 (CH),
109.1–150.2 (28C, Aromatic-C), EA calcd. C, 61.02, H, 3.07, N, 14.23,
found C, 61.02, H, 3.06, N, 14.22, Rf 0.29 (CH2Cl2).
4.1.8. 4,4′-(5,6,11,12-tetrahydroindolo[3,2-b]carbazole-6,12-diyl)bis(2-
methoxyphenol) (15)
4.1.6. 6,12-bis(2,6-dinitrophenyl)-5,6,11,12-tetrahydroindolo[3,2-b]
carbazole (13)
Yellow powder, mp. 166–167 °C, yield 75%, 502.56 g/mol,
C
32H26N2O4, ESI-MS: 503.81 [M + H+], IR (ATR, cm−1): 3842 (NH),
Pale yellow powder, Mp 222–224 °C, yield 58%, C30H18N6O8,
590.50 g/mol, ESI-MS: 591.15 [M + H+], IR (ATR, cm−1): 3398(NH),
1H NMR (400 MHz, DMSO‑d6): δ (ppm) = 5.44 (s, 2H, 2CH), 7.21 (d,
4H, J = 7.2 Hz, ArH), 7.45–7.47 (m, 6H, ArH), 7.63 (t, 4H, J = 7.5 Hz,
ArH), 10.51 (s, 2H, 2NH), 13CNMR (100 MHz, acetone‑d6) δ (ppm):
32.51 (CH), 35.4 (CH), 108.1–150.9 (28C, Aromatic-C), EA calcd. C,
61.02, H, 3.07, N, 14.23, found C, 61.03, H, 3.08, N, 14.21, Rf 0.19
(CH2Cl2).
1H NMR (400 MHz, DMSO‑d6): δ (ppm): 3.78 (s, 6H, OCH3), 5.66 (s, 2H,
2CH), 7.40 (t, 4H, J = 7.0 Hz, ArH), 7.45 (d, 2H, J = 9.0 Hz, ArH), 7.46
(d, 2H, J = 8.8 Hz, ArH), 7.52 (s, 2H, ArH), 7.90 (d, 4H, J = 7.0 Hz,
ArH), 10.01 (s, 2H, 2OH), 10.35 (s, 2H, 2NH), 13C NMR: (100 MHz,
DMSO‑d6) δ (ppm) = 44.32 (CH), 45.20 (CH), 56.4 (OCH3), 60.06
(OCH3), 110.0–152.9 (28C, Aromatic-C). EA calcd.: C, 76.48, H, 5.21,
N, 5.57 found C, 76.49, H, 5.20, N, 5.56, Rf 0.45 (CH2Cl2).
Fig. 9. Surface map for (a) the most active compound 11; (b) Compound 15, (c) Amiloride Pink: hydrogen bond, blue: mild polar, green: hydrophobic region.
7

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
4.1.9. 2,2′-(5,6,11,12-tetrahydroindolo[3,2-b]carbazole-6,12-diyl)
dibenzene-1,4-diol (16)
about 10 min or till the optimal blue color density develops. Add 50 μl
of stop solution to each well. The color will change from blue to yellow.
Read the absorbance on a microplate reader at a wavelength of 450 nm
immediately. The concentrations of uPA in the samples were de-
termined based on the uPA standard curve.
Yellow powder, mp. 162–163 °C, yield 75%, 474.51 g/mol,
C
30H22N2O4, ESI-MS: 475.15 [M + H+], IR (ATR, cm−1): 3822 (br. OH
and NH), 2988, 2994 (CH), 1H NMR (400 MHz, DMSO‑d6): δ (ppm):
5.52 (s, 2H, 2CH), 6.46 (s, 2H, ArH), 7.35 (t, 4H, J = 7.4 Hz, ArH), 7.43
(d, 2H, J = 7.8 Hz, ArH), 7.51 (d, 2H, J = 7.5 Hz, ArH), 7.58–7.06 (m,
2H, ArH), 7.78 (d, 2H, J = 8.0 Hz, ArH), 9.88 (brs, 2H, 2OH), 10.12
(brs, 2H, 2OH), 10.26 (s, 2H, 2NH). 13C NMR: (100 MHz, DMSO‑d6)δ
(ppm) = 44.12 (CH), 46.11 (CH), 111.0 – 152.0 (28C, Aromatic-C). EA
calcd.: C, 75.94, H, 4.67, N, 5.90, found C, 75.95, H, 4.68, N, 5.91, Rf
0.49 (CH2Cl2).
4.4. Molecular genetics
4.4.1. Quantitative real time- PCR (qPCR)
Total RNA was isolated from tumor cell lines using the RNeasy Mini
Kit (Qiagen, Hilden, Germany) supplemented with DNaseI (Qiagen)
digestion step, according to the manufacturer’s protocol. Isolated total
RNA was treated with one unit of RQ1 RNAse-free DNAse (Invitrogen,
Germany) to digest DNA residues, re-suspended in DEPC-treated water
and quantified photospectrometrically at 260 nm. Purity of total RNA
was assessed by the 260/280 nm ratio which was between 1.8 and 2.1.
Additionally, integrity was assured with ethidium bromide-stain ana-
lysis of 28 S and 18 S bands by formaldehyde-containing agarose gel
electrophoresis. Aliquots were used immediately for reverse transcrip-
tion (RT), otherwise they were stored at −80 °C. Complete Poly(A)+
RNA isolated from tumor cell lines was reverse transcribed into cDNA in
a total volume of 20 µl using RevertAidTM First Strand cDNA Synthesis
Kit (Fermentas, Germany). An amount of total RNA (5 µg) was used
with a master mix. The master mix was consisted of 50 mM MgCl2, 10×
RT buffer (50 mM KCl; 10 mM Tris-HCl; pH 8.3), 10 mM of each dNTP,
50 µM oligo-dT primer, 20 IU ribonuclease inhibitor (50 kDa re-
combinant enzyme to inhibit RNase activity) and 50 IU MuLV reverse
transcriptase. The mixture of each sample was centrifuged for 30 s at
1000 g and transferred to the thermocycler. The RT reaction was car-
ried out at 25 °C for 10 min, followed by 1 h at 42 °C, and finished with
a denaturation step at 99 °C for 5 min. Afterwards the reaction tubes
containing RT preparations were flash-cooled in an ice chamber until
being used for cDNA amplification through quantitative Real Time-
polymerase chain reaction (qRT-PCR). Step One™ Real-Time PCR
System from Applied Biosystems (Thermo Fisher Scientific, Waltham,
MA USA) was used to determine the tumor cell lines cDNA copy
number. PCR reactions were set up in 25 μl reaction mixtures con-
taining 12.5 μl 1 × SYBR® Premix Ex TaqTM (TaKaRa, Biotech. Co.
Ltd.), 0.5 μl 0.2 μM sense primer, 0.5 μl 0.2 μM antisense primer, 6.5 μl
distilled water, and 5 μl of cDNA template. The reaction program was
allocated to 3 steps. First step was at 95.0 °C for 3 min. Second step
consisted of 40 cycles in which each cycle divided to 3 steps: (a) at
95.0 °C for 15 sec; (b) at 55.0 °C for 30 sec; and (c) at 72.0 °C for 30 sec.
The third step consisted of 71 cycles which started at 60.0 °C and then
increased about 0.5 °C every 10 sec up to 95.0 °C. At the end of each
sqRT-PCR a melting curve analysis was performed at 95.0 °C to check
the quality of the used primers. Each experiment included a distilled
water control. The sequences of specific primer of the genes used are
listed in Table 4. At the end of each qPCR a melting curve analysis was
performed at 95.0 °C to check the quality of the used primers. The re-
lative quantification of the target to the reference was determined by
using the 2−ΔΔCT method as follows:
4.1.10. 6,12-bis(4-(benzyloxy)phenyl)-5,6,11,12-tetrahydroindolo[3,2-b]
carbazole (17)
Light green fine powder, Mp 200–202 °C, yield 85%, C44H34N2O2,
622.75 g/ mol, ESI-MS: 623.26 [M + H+], 621.31 [M−H+], IR (ATR,
cm−1) 3396 (NH), 1H NMR (400 MHz, DMSO‑d6): δ (ppm): 5.03 (s, 4H,
2CH2O), 5.60 (s, 2H, 2CH), 6.82 (t, 4H, J = 7.3 Hz, ArH), 6.99 (d, 4H,
J = 7.9 Hz, ArH), 7.02 (d, 4H, J = 7.2 Hz, ArH), 7.03 (d, 4H,
J = 7.7 Hz, ArH), 7.32 –7.38 (m, 5H, ArH), 7.43–7.49 (m, 5H, ArH),
10.88 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO‑d6): δ (ppm): 36.7 (CH),
40.8 (CH), 69.6 (OCH2), 74.0 (OCH2), 110.4–157.4 (40C, Aromatic-C),
EA calcd. C, 84.86; H, 5.50; N, 4.50, found C, 84.87, H, 5.52, N, 4.49, Rf
0.73 (CH2Cl2).
4.2. Antitumor screening
Human breast carcinoma (MCF-7 cell line) was used in the biolo-
gical assay and they were obtained from Vacsera (Giza, Egypt). Cell
cultures procedures were performed in a sterile area using a laminar air
flow cabinet biosafety class II level. Culture was maintained in DMEM
F12 medium with 1% antibiotic-antimycotic mixture (10,000 U/ml
potassium penicillin, 10,000 μg/ml streptomycin sulfate and 25 μg/ml
amphotericin B), 1% L-glutamine, and supplemented with 10% heat
inactivated fetal bovine serum. Culturing and subculturing were carried
out according to the reported procedure. A negative control composed
of DMSO was also used [17]. Cell viability assay were conducted ac-
cording to reported method [18,19]. Following culturing for 10 days,
the cells were seeded at concentration of 10 × 103 cells per well in a
fresh complete growth medium using 96-well microtiter plastic plates
at 37 °C for 48 h under 5% CO2, in a water jacketed carbon dioxide
incubator. Fresh medium (without serum) was added and cells were
incubated either alone (negative control) or with Samples to give a final
concentration of 100 μg/ml. After 24 h incubation, the medium was
aspirated and then 40 μl MTT salt (2.5 mg/ml) were added to each well
and incubated for further four hours at 37 °C under 5% CO2. To stop the
reaction and dissolve the formed crystals, 200 μl 10% sodium dodecyl
sulphate (SDS) in deionized water was added to each well and in-
cubated overnight at 37 °C. The absorbance was measured using a mi-
croplate multi-well reader at 595 nm and a reference wavelength of
690 nm. Cell viability was assessed according to the mitochondrial-
dependent reduction of yellow MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl tetrazolium bromide) to purple formazan.
ΔCT(test) = CT(target, test) − CT(reference, test)
ΔCT(calibrator) = CT(target, calibrator) − CT(reference, calibrator)
ΔΔCT = ΔCT(Test) − ΔCT(calibrator)
,
,
4.3. Urokinase inhibition assays
.
According to the manufacturer’s instructions, after 24 h from
treatment of the cells with IC50 values of each compound, medium was
collected and centrifuged at 2000 xg for 10 min to remove cellular
debris. Add 50 µl of the cell extract per well and incubate for 2 h. Wells
were washed with 200 µl of wash buffer then add 50 μl of biotinylated
uPA antibody to each well and incubate for 1 h at 25 °C. After washing,
plates were incubated with 50 μl of streptavidin-peroxidase conjugate
per well and incubate for 30 min then wash the microplate as described
above. Add 50 μl of chromogen substrate per well and incubate for
4.4.2. DNA damage using the comet assay
The neutral comet assay for tumor cell lines was used as described
according to the reported protocol [26,27]. After the trypsin treatment
to produce a single cell suspension, approximately 1.5 × 104 cells were
embedded in 0.75% low-gelling-temperature agarose and rapidly pi-
petted onto a pre-coated microscope slide. Samples were lysed for 4 h at
50 °C in 0.5% SDS, 30 mM EDTA, pH 8.0. After rinsing overnight at
room temperature in Tris/borate/EDTA buffer, pH 8.0, samples were
electro-phoresed for 25 min at 0.6 V/cm, and then stained with
8

E.M. Ahmed, et al.
BioorganicChemistry93(2019)103332
propidium iodide. Slides were viewed using a fluorescence microscope
with a CCD camera, and 150 individual comet images were analyzed
from each sample for tail moment, DNA content, and percentage DNA
in tail. For each sample about 100 cells were examined to determine the
percentage of cells with DNA damage that appear like comets. The
nonoverlapping cells were randomly selected and were visually as-
signed a score on an arbitrary scale of 0–3 (i.e., class 0 = no detectable
DNA damage and no tail; class 1 = tail with a length less than the
diameter of the nucleus; class 2 = tail with length between 1 × and
2 × the nuclear diameter; and class 3 = tail longer than 2 × the dia-
meter of the nucleus) based on perceived comet tail length migration
and relative proportion of DNA in the nucleus.
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determination in primary breast cancer tissue for individualized therapy concepts, Clin.
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4.5. Molecular modelling
The coordinates of (PDB ID: 1f51) for docking calculations was
obtained from the Brookhaven Protein Data Bank. Ligands under study
were drawn using MOE 2D sketcher Then energy minimization was
computed Protein was prepared using the MOE 10.9 Water molecules
which are important in aiding the interaction with the receptor were
optimized Lowest energy aligned conformation(s) were identified. This
corrected protein structure was used in the subsequent docking studies.
Moreover addition of Hydrogen atoms at their standard geometry, then
computing the partial charges followed by energy optimization. Scoring
function was conducted by the aid of triangle matcher as placement
method also affinity dG was validated for docking. The retention of 30
conformers with the highest and best score was employed. Amiloride as
areference drug was docked into the active site. The active site has been
defined. All atoms located less than 10.0 A° from any ligand atom were
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‘Molecular Operating Environment’ software (MOE of Chemical
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Declaration of Competing Interest
[22] S. Siicela, J. Barrett, In vitro degradation of radiolabelled, intact basement membrane
mediated by cellular plasminogen activator, Carcinogenesis (Lond.) 3 (1982) 363–369.
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Appendix A. Supplementary material
[25] M.A. Omar, Y.M. Shaker, S.A. Galal, M.M. Ali, S.M. Kerwin, J. Li, H. Tokuda,
R.A. Ramadan, H.I. El Diwani, Synthesis and docking studies of novel antitumor benzi-
midazoles, Bioorg. Med. Chem. 20 (2012) 6989–7001.
Supplementary data to this article can be found online at https://
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