Synthesis, in vitro cytotoxicity activity against the human cervix carcinoma cell line and in silico computational predictions of new 4-arylamino-3-nitrocoumarin analogues

Article abstract of DOI:10.1016/j.molstruc.2019.127047

A new series of 4-arylamino-3-nitrocoumarin analogues (4–18) have been synthesized and characterized by sophisticated spectroscopic techniques (¹H NMR, ¹³C NMR) and mass spectrometry. All the new synthesized compounds were evaluated for their in vitro cytotoxic activity against the human cervix carcinoma cell line (KB-3-1) using resazurin assay with (+)-griseofulvin as the positive control (IC₅₀ = 19 μM). Among them, thiazolidinylidene derivative 17a that bearing malononitrile unit displayed the best cytotoxic potency with IC₅₀ value of 21 μM. Also, in silico docking simulation studies were conducted on human DNA topoisomerase 1 (Top1) (PDB: 1T8I) to explore and interpret the interaction pattern between the selected compounds and target enzyme as well confirm the acquired cytotoxicity results. In addition to the above, in silico predictions of physicochemical properties, ADME (absorption, distribution, metabolism and excretion) parameters, oral toxicity and indication of toxicity targets were implemented for some title compounds.

Full text of DOI:10.1016/j.molstruc.2019.127047

Journal of Molecular Structure 1200 (2020) 127047
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, in vitro cytotoxicity activity against the human cervix
carcinoma cell line and in silico computational predictions of new 4-
arylamino-3-nitrocoumarin analogues
a
a
,
b
,
*
, Ahmed A. Hassan a , Hesham S. Nassar d,
,
c
a,
Ahmed H. Halawa , Essam M. Eliwa
a,
d
b
,
e
b
b, f
R.A. El-Eisawy , Mohamed Ismail , Marcel Frese , Mohamed Shaaban
Ahmed M. El-Agrody
,
a,
**
a
b
, Ahmed H. Bedair , Norbert Sewald
a
Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City-Cairo, 11884, Egypt
b
c
d
e
f
Organic and Bioorganic Chemistry, Faculty of Chemistry, Bielefeld University, Bielefeld, D-33501, Germany
Chemistry Department, Faculty of Science, Jazan University, Jazan, 82621, Saudi Arabia
Chemistry Department, Faculty of Science and Art, Al-Baha University, Al-Baha, 1988, Saudi Arabia
Microbiology Department, Faculty of Science, Helwan University, Helwan-Cairo, 11795, Egypt
Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, El-Behoos St. 33, Dokki-
Cairo, 12622, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2019
Received in revised form
A new series of 4-arylamino-3-nitrocoumarin analogues (4e18) have been synthesized and character-
1
13
ized by sophisticated spectroscopic techniques ( H NMR, C NMR) and mass spectrometry. All the new
synthesized compounds were evaluated for their in vitro cytotoxic activity against the human cervix
carcinoma cell line (KB-3-1) using resazurin assay with (þ)-griseofulvin as the positive control
5
September 2019
Accepted 7 September 2019
Available online 11 September 2019
(
IC50 ¼ 19
mM). Among them, thiazolidinylidene derivative 17a that bearing malononitrile unit displayed
the best cytotoxic potency with IC50 value of 21
m
M. Also, in silico docking simulation studies were
conducted on human DNA topoisomerase 1 (Top1) (PDB: 1T8I) to explore and interpret the interaction
pattern between the selected compounds and target enzyme as well confirm the acquired cytotoxicity
results. In addition to the above, in silico predictions of physicochemical properties, ADME (absorption,
distribution, metabolism and excretion) parameters, oral toxicity and indication of toxicity targets were
implemented for some title compounds.
Keywords:
4-Arylamino-3-nitrocoumarin
Cervical cancer
Cytotoxic activity
In silico computational predictions
Top1-DNA complex
© 2019 Published by Elsevier B.V.
1. Introduction
(LMICs) [1]. Cervical cancer is caused by sexually gained infection
with certain types of Human papillomavirus (HPV). Approximately
Admittedly, cancer remains one of the most prevalent, knotted
and fatal diseases in the present time. According to the World
Health Organization (WHO) report in 2018, cancer is the second
leading cause of death worldwide after heart disease, cancer
burden rises to 18.1 million new cases and is responsible for an
estimated 9.6 million deaths. About one in six deaths is due to
cancer, so it emerges as a global epidemic. Nearly 70% of deaths
from cancer take place in low- and middle-income countries
all cases of cervical cancer can be attributable to HPV infection,
smoking and HIV infection. Around the world, cervical cancer is the
second most common cancer in women especially living in under
developed regions with an estimated 570 000 new cases in 2018
representing 7.5% of all female cancer deaths. Among them, almost
311000 women died from cervical cancer; more than 85% of these
deaths occurring in LMICs and this number is projected to rise [2].
Thereby, there is an advantage and critically important in finding
new anticancer agents with improved qualifications to gain better
efficacy and selectivity as well low side effects.
*
Corresponding author. Chemistry Department, Faculty of Science, Al-Azhar
Coumarin-containing derivatives are ubiquitous, versatile and
endowed with a broad range of biological with pharmacological
activities including anti-inflammatory [3], antioxidant [4], anti-
fungal [5], antibacterial [6], anticoagulant [7], anticancer [8,9] and
University, Nasr City-Cairo, 11884, Egypt.
** Corresponding author.
E-mail addresses: essameliwa85@azhar.edu.eg (E.M. Eliwa), elagrody_am@
azhar.edu.eg (A.M. El-Agrody).
https://doi.org/10.1016/j.molstruc.2019.127047
0
022-2860/© 2019 Published by Elsevier B.V.

2
A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
anti-HIV [10,11]. Recently, a particular interest in thiazoleebased
compounds has been increased by researches attributable to their
outstanding achievements in the prevention and curing of diseases
of triethylamine (44 mmol) was refluxed for 4 h then cooled. The
resulting solid was collected by filtration, washed with ethanol and
recrystallized from dioxane to give compound 4 as a golden yellow
ꢀ
[
12e16]. Encouraged by the aforementioned findings and in line of
crystals with a yield of 98% and Mp: 210e212 C. NMR and MS data
our continuous research program, we synthesized and character-
ized new coumarin analogues incorporated with thiazole and other
moieties. In addition to, we screened their in vitro cytotoxic activity
against the human cervix carcinoma cell line (KB-3-1) using resa-
zurin assay with (þ)-griseofulvin as a positive control.
for all compounds are presented in supporting information file.
2.2.2. 2-{1-[4-((3-Nitrocoumarin-4-yl)amino)phenyl]ethylidene}
hydrazinecarbothioamide (5)
A mixture of compound 4 (1 mmol) and thiosemicarbazide
(1 mmol) in dioxane (10 mL) containing a catalytic amount of
glacial acetic acid (1 mL) was heated under reflux for 3 h. The
resulting solid on heating was collected by filtration, washed with
hot ethanol and dried to afford compound 5 as a yellow amorphous
Indeed, in silico computational prediction approaches demon-
strate an alternative and foster way for addressing the biological
targets, molecular interaction mechanism of ligands with target
protein and toxicity testing in the pharmaceutical industry and
cosmetics, so these protocols play a crucial role for the progress in
drug discovery and development [17]. In connection with biological
targets, human topoisomerase 1 (Top1) is an enzyme that catalyze
the cleavage and rejoining of a single strand of DNA, releasing the
tension on the DNA twist and enabling DNA replication and/or
mRNA transcription. Interfering with the Top1 would result in in-
hibition of cell replication and consequently cell death, thus it is
recognized as an important target in human cancer tackling ap-
proaches [18,19]. Several naturally occurring and synthesized in-
hibitors classes were discovered and their mode of action has been
characterized as potential antiproliferative agents. They work
through the binding to the enzyme active site after the cleavage of
the DNA backbone, inhibiting the religation step and accumulating
Top1-DNA adduct [20]. Nevertheless, they suffer from low tumor
response rates and dose limiting toxicity, therefore the search of
inhibitors with higher activity is always desired. Accordingly, we
shed light on the in silico computational studies encompassing the
molecular docking of some synthesized 4-arylamino-3-
nitrocoumarin derivatives as potential Top1-DNA inhibitors
compared to the reference camptothecin (EHD) aiming to explore
the binding affinity and key interactions between the synthesized
ligands with target enzyme. Additionally, physicochemical prop-
erties, ADME parameters, acute oral toxicity and toxicity targets
were predicted for some title compounds.
ꢀ
solid with a yield of 87% and Mp: 248e250 C.
2.2.3. 2-Cyano-N'-{1-[4-((3-nitrocoumarin-4-yl)amino)phenyl]
ethylidene}acetohydrazide (6)
Compound 6 was generated as an orange amorphous powder
from 4 in the same fashion that described for compound 5 with a
ꢀ
yield of 98% and Mp: 274e276 C.
2.2.4. 4-{[4-(2-Bromoacetyl)phenyl]amino}-3-nitrocoumarin (7)
To a stirred solution of 4 (0.02 mol) in warm glacial acetic acid
(100 mL), the bromine (0.02 mol) was added dropwise with con-
stant stirring in direct sun light for 5 h, then the reaction mixture
was poured into crushed ice/water (100 g) with stirring. The
separated solid was filtered off, washed with cold water, dried and
recrystallized from dioxane to furnish 7 as a yellow amorphous
ꢀ
solid with a yield of 95% and Mp: 236e238 C.
2.2.5. General procedure for preparation of 4-{[4-(2-substituted-
thiazol-4-yl)phenyl]amino}-3-nitrocoumarin (8a-c)
A mixture of 7 (1 mmol) and thiocarboxamide derivatives
namely: (thiourea, phenyl thiourea, and thioacet-amide) (1 mmol)
in dry dioxane (10 mL) containing TEA (1 mmol) was refluxed for
3 h. The resulting solid on heating was collected by filtration and
washed with hot methanol to afford compounds 8a-c.
4-{[4-(2-Aminothiazol-4-yl)phenyl]amino}-3- nitrocoumarin (8a)
2
. Materials and methods
was obtained as a golden yellow amorphous powder with a yield of
ꢀ
80% and Mp: 338e340 C.
2.1. Materials and instruments
4-{[4-(2-(Phenylamino)thiazol-4-yl)phenyl]amino}-3-
nitrocoumarin (8b) was manifested as a yellow amorphous solid
ꢀ
Melting points were determined on a BÜCHI Melting Point B-
40 apparatus and uncorrected (BÜCHI Germany). The NMR
with a yield of 75% and Mp: 240e242 C.
5
4-{[4-(2-Methylthiazol-4-yl)phenyl]amino}-3-nitrocoumarin (8c)
was collected as an orange amorphous solid with a yield of 82% and
1
13
spectra ( H NMR and C NMR) were measured on Bruker Avance
13
ꢀ
DRX 500 MHz (125 MHz for C NMR) spectrometer (Bruker, USA)
with TMS as an internal standard. Chemical shifts were reported
Mp: 223e225 C.
1
13
relative to residual solvent peaks ([D
6
]DMSO: H: 2.50 ppm, C:
2.2.6. General procedure for preparation of 4-{[4-(2-(2-(4-
substituted-benzylidene)hydrazinyl)thiazol-4-yl)phenyl]amino}-3-
nitrocoumarin (10a,b)
In a similar vein, compounds 10a,b were obtained as above-
mentioned synthetic route for compounds 8a-c by the reaction
between 7 and benzylidene thiosemicarbazides namely: [1-(4-
methoxybenzylidene)-thiosemicarbazide
chlorobenzylidene)thiosemicarbazide].
4-{[4-(2-(2-(4-Methoxybenzylidene)hydrazineyl)thiazol-4-yl)
phenyl]amino}-3-nitrocoumarin (10a) was obtained as a yellow
3
9.5 ppm). ESI mass spectra were recorded using an ion trap mass
spectrometer equipped with a standard ESI/APCI source (Bruker
Daltonik GmbH, Bremen, Germany). Starting materials, reagents
and solvents were obtained from commercial sources and used
without further purification. The purity of the synthesized com-
pounds was investigated by TLC, performed on Merck precoated
silica gel 60 F254 aluminum sheets with solvent mixture of
dichloromethane-methanol (95e5) as eluent. Spots were visual-
ized under UV light at 254 nm. Elemental analyses were carried out
at the Regional Centre for Mycology and Biotechnology (RCMP), Al-
Azhar University, Cairo, Egypt.
or
1-(4-
ꢀ
amorphous solid with a yield of 90% and Mp: 256e258 C.
4-{[4-(2-(2-(4-Chlorobenzylidene)hydrazinyl)thiazol-4-yl)phenyl]
amino}-3-nitrocoumarin (10b) was resulted as a golden yellow
ꢀ
2
2
.2. Synthesis and characterization
amorphous powder with a yield of 72% and Mp: 250e252 C.
4
-{[4-(Quinoxalin-2-yl)phenyl]amino}- 3-nitrocoumarin (11).
.2.1. 4-[(4-Acetylphenyl)amino]-3-nitrocoumarin (4)
The solution of 4-chloro-3-nitrocoumarin (3) (44 mmol) and p-
aminoacetophenone (44 mmol) in ethanol (30 mL) in the presence
Compound 7 (1 mmol) and 1,2-phenylenediamine (1 mmol)
were taken in dry dioxane (10 mL) containing TEA (1 mmol) and
heated for 3 h under reflux. The reaction mixture was cooled to

A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
3
ꢀ
Scheme 1. Synthetic routes of compounds 4e7. Reagents and conditions: (a) HNO
reflux, 4 h; (d) thiosemicarbazide, AcOH, dioxane, reflux, 3 h; (e) cyanoacetohydrazide, AcOH, dioxane, reflux, 3 h; (f) Br
3
/H
2
SO
4
, AcOH, rt, 2 h; (b) POCl
3
, DMF, 10 C then rt, 1 h; (c) p-aminoacetophenone, Et
, AcOH, rt, 5 h.
3
N, EtOH,
2
room temperature and methanol (5 mL) was added. The solid
4-{[4-(Imidazo[1,2-a]pyrimidin-3-yl)phenyl]amino}-3-
precipitate was filtered by suction, washed with hot methanol
nitrocoumarin (13) was accomplished as a reddish brown amor-
phous solid with a yield of 91% and Mp: 243e245 C.
ꢀ
(
10 mL) and dried to afford compound 11 as a yellow amorphous
ꢀ
powder with a yield of 88% and Mp: 243e245 C.
2.2.8. 4-{[4-(2-(1-Methylpiperazin-1-yl)acetyl)phenyl]amino}-3-
nitrocoumain (14)
2.2.7. General procedure for preparation of 12 and 13
By the same token as stated above for 11, compound 14 was
produced from 7 (1 mmol) and 1-methylpiperazine (1 mmol) as a
brown amorphous solid with a yield of 58% and Mp: 233e235 C.
A mixture of 7 (1 mmol) and heterocyclic amines namely: (2-
aminopyridine or 2-aminopyrimidine) (1 mmol) in dry dioxane
10 mL) containing TEA (1 mmol) was refluxed for 2 h. The resulting
ꢀ
(
solid on heating was collected by filtration, washed with hot
methanol and dried to furnish compounds 12 and 13, respectively.
2.2.9. 4-{[4-(2-Thiocyanatoacetyl)phenyl]amino}- 3-nitrocoumarin
(15)
4-{[4-(Imidazo[1,2-a]pyridin-3-yl)phenyl]amino}-3-
nitrocoumarin (12) was demonstrated as a yellow amorphous
powder with a yield of 87% and Mp: 278e280 C.
A mixture of 7 (1 mmol) and potassium thiocyanate (1 mmol) in
dry dioxane (10 mL) was refluxed for 2 h. The resulting solid on
ꢀ
3 3
Scheme 2. Synthetic routes of compounds 8e11. Reagents and conditions: (a) thiourea/phenyl thiourea/thioacetamide, Et N, dioxane, reflux, 3 h; (b) Et N, dioxane, reflux, 3 h; (c)
1
,2-phenylenediamine, Et N, dioxane, reflux, 3 h.
3

4
A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
heating was collected by filtration, washed with hot methanol and
dried to donate compound 15 as a yellow amorphous solid with a
yield of 85% and Mp: 208e210 C.
alignment study for the structures before and after simulation.
ꢀ
2.5. In silico predictions of physicochemical properties and ADME
parameters
2.2.10. General procedure for preparation of 17a and 18a
Malononitrile or ethylcyanoacetate (1 mmol) was added to a
stirred solution of potassium hydroxide (1 mmol) in anhydrous
DMF (10 mL). Phenyl isothiocyanate (1 mmol) was added dropwise.
The physicochemical properties and ADME parameters of
compounds 8a, 10b, 11 and 17a in comparison with omeprazole
drug were predicted using SwissADME web tool as described pre-
viously [23].
The reaction mixture was stirred at room temperature with
a-
bromocarbonyl compound 7 (1 mmol) and left at room tempera-
ture for 6 h, then it was poured into ice/water and acidified with
2.6. In silico acute oral toxicity prediction and indication of toxicity
targets
0
.1 N HCl. The resulting precipitate was filtered off, washed with
water, dried and recrystallized from dioxane to afford compounds
7a and 18a, respectively.
-(4-Hydroxy-4-(4-((3-nitro-2-oxo-2H-chromen-4-yl)amino)
1
The in silico prediction of rodent oral toxicity and indication of
possible toxicity targets for compounds 8a, 10b, 11 and 17a were
implemented utilizing the ProTox web server as demonstrated by
Drwal et al. [24].
2
phenyl)-3-phenylthiazolidin-2-ylidene)malono-nitrile (17a) was
proved as a golden yellow amorphous powder with a yield of 60%
ꢀ
and Mp: 192e194 C.
Eethyl-2-cyano-2-(4-hydroxy-4-(4-((3-nitro-2-oxo-2H-chromen-
3. Results and discussion
4
-yl)amino)phenyl)-3-phenylthiazolidin-2-ylidene)acetate (18a) was
indicated as a pale yellow amorphous powder with a yield of 91%
and Mp: 284e286 C.
3.1. Chemistry
ꢀ
4-Hydroxycoumarin (1) was nitrated under mild condition to
2
.3. In vitro cytotoxicity based upon resazurin assay
give 4-hydroxy-3-nitrocoumarin (2) that was treated with phos-
phorus oxychloride in DMF to form 4-chloro-3-nitrocoumarin (3)
[25]. Reaction of 3 with p-aminoacetophenone afforded 4-[(4-
All the new synthesized compounds were evaluated for their
1
in vitro cytotoxicity activity against the human cervix carcinoma
cell line KB-3-1 (was obtained from the American Type Culture
Collection, ATCC, Rockville, MD) according to our previously re-
ported resazurin assay [21].
acetylphenyl)amino]-3-nitrocoumarin (4). The H NMR spectrum
of 4 shows a singlet signal at
reveals multiplet signals between
to eight aromatic protons present in the molecule. In addition to,
d
10.49 ppm that attributed to NH, also
d
8.38 and 7.32 ppm attributable
1
the acetyl group generated a singlet H chemical shift at
d
2.58 ppm.
13
13
2.4. In silico docking study
The C NMR spectrum of 4 displays a downfield C resonance at
d
d
197.3 ppm for carbonyl acetophenoide and upfield one at
27.1 ppm for methyl carbon. ESI-MS spectrum of 4 exhibits the
Molecular dynamic simulation of some selected coumarin de-
þ
-
rivatives were built and energy minimized using Yet Another Sci-
entific Artificial Reality Application (YASARA) [22]. The structure of
human DNA Topoisomerase I was obtained from protein data bank
molecular ion peak at m/z: 347 [MþNa] and 323 [M-H] .
Condensation of 4 with thiosemicarbazide and cyanoacetohy-
drazide gave the corresponding thiosemi-carbazone 5 and cya-
noacetohydrazone
1
(
PDB code: 1T8I). The camptothecin inhibitor was removed, the
6
derivatives, respectively. The
H NMR
missing hydrogens in the structure were added and the whole
enzyme structure was energy minimized using AMBER14 force
field, 10 Å cutoff and PME for long range electrostatics. Docking
results were analyzed based on the B-factor (binding energy) that
calculated by YASARA and correct positioning of the compounds
within the active site of the enzyme. PyMol was used for the
spectrum of 5 indicate two singlet signal at
attributable to 2NH. The C NMR spectrum of 5 exhibits the
d
10.38 and 10.22 ppm
13
13
absence of a carbonyl acetophenoide signal, while a downfield
resonance at 179.2 ppm for the newly formed C]S group was
appeared. Its ESI-MS spectrum manifests the molecular ion peak at
C
d
þ
þ
-
m/z: 398 [MþH] , 420 [MþNa] and 396 [M-H] . The NMR data of 6
3 3
Scheme 3. Synthetic routes of compounds 12e15. Reagents and conditions: (a) 2-aminopyridine, Et N, dioxane, reflux, 2 h; (b) 2-aminopyrimidine, Et N, dioxane, reflux, 2 h; (c) 1-
methylpiperazine, Et N, dioxane, reflux, 3 h; (d) KSCN, dioxane, reflux, 2 h.
3

A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
5
Scheme 4. Synthetic routes of compounds 17a and 18a.
whilst the signal for methyl unit was disappeared. The 13C NMR
Table 1
In vitro cytotoxicity of the synthesized compounds 4e18 against KB-3-1 cell line.
13
spectrum of 7 displays a C chemical shift at
d 66.8 ppm attribut-
Compound
IC50
(
m
M)a
Compound
IC50
(
m
M)
able to the methylene carbon. (þ)-ESI-MS spectrum of 7 indicates
þ
79
the molecular ion peaks at m/z: 425 [MþNa] (94.92%, Br) and
KB-3-1
KB-3-1
þ
81
4
27 [MþNa] (90.74%, Br), (See supporting information Fig. S13).
4
5
6
7
>100 ± 0.12
e
11
12
13
14
15
17a
18a
43 ± 0.6
e
The behavior of compound 7 towards thiocarboxamide de-
rivatives was investigated. Thus, condensation of 7 with thiourea,
e
e
>100 ± 0.5
44 ± 0.4
>100 ± 0.1
>100 ± 0.2
e
e
phenyl thiourea and thioacetamide afforded the corresponding
1
8
8
8
1
1
a
b
c
0a
0b
>100 ± 0.12
21 ± 0.11
e
thiazolyl-3-nitrocoumarin derivatives 8a-c [26] (Scheme 2). The H
NMR spectra of thiazole derivatives 8a-c show a characteristic
b
resonance of CH-thiazole at
d 7.25 for 8a, 7.41 for 8b and 7.99 ppm
(þ)-Griseofulvin
19.2 ± 0.03
e
66 ± 0.02
DMSOc
for 8c. (þ)-ESI-MS data for 8a-c display the molecular ion peaks at
þ
þ
þ
a
m/z: 381 [MþH] for 8a, 479 [MþNa] for 8b and 402 [MþNa] for
c, in agreement with the proposed empirical formulas. Moreover,
IC50 values expressed in mM as the mean values of triplicate wells from at least
8
three experiments and are reported as the mean ± standard error.
b
Positive control,c negative control, “e” means no obvious inhibitory effect.
reaction of 7 with benzylidene thiosemicarbazides 9a,b [27] affor-
ded the corresponding hydrazinyl thiazole derivatives 10a,b
1
(
Scheme 2). The H NMR spectra reveal the hydrazinyl-NH at
Table 2
d
12.08 and coumarinyl-NH at 10.37 ppm for 10a, also at d 12.29 and
Binding affinity of different coumarin derivatives to Top 1 (PDB: 1T8I) and compared
to the reference camptothecin (EHD).
1
d
0.37 ppm, respectively, for 10b. The CH-thiazole was defined at
13
7.38 for 10a and 7.42 ppm for 10b. The C NMR data of 10a,b
187.6 ppm attributable to thiazolyl
Compound
Binding affinity (Kcal/mol)
No. of H-bonding
display a downfield signal at
d
4
5
8
1
1
8.25
e
2
e
3
1
2
4
C]N that flanked by three heteroatoms. (þ)-ESI-MS spectra for
þ
1
0a,b show the molecular ion peaks at m/z: 536 [MþNa] for 10a
a
0b
7a
8.44
10.97
10.09
9.48
þ
35
þ
37
and 540 [MþNa] (100%, Cl) with 542 [MþNa] (35%, Cl) for
1
0b. Further, reaction of 7 with 1,2-phenylenediamine yielded the
EHD
corresponding quinoxalinyl-3-nitrocoumarin derivative 11. The
formation of 11 is explained by cyclocondensation of 7 with 1,2-
phenylenediamine, followed by subsequent oxidation [26]. The
“
e” means no binding affinity or no H-bonding.
1
H NMR spectrum 11 discloses the absence of 2NH group that
show a 1H resonance at
d 4.29 for methylene protons and the
characteristic for 1,4-dihydroquinoxaline derivative, meanwhile it
1
respective methylene carbon at 25.4 ppm. As well, the carbonyl
amide was demonstrated at 166.4 ppm. Synthesis of 7 was ach-
ieved by the reaction of 4 with bromine in glacial acetic acid
shows
a
H
chemical shift at
d
10.49 ppm attributable to
9.64 ppm that assignable for
d
coumarinyl-NH and another one at
d
CH-quinoxaline. The ESI-MS spectrum of 11 represents the molec-
1
þ
-
(
d
Scheme 1). The 1H NMR spectrum of 7 exhibits H resonance at
ular ion peak at m/z: 433 [MþNa] and 409 [M-H] .
4.78 ppm that being for the newly formed methylene group,
Our investigation was extended to include the behavior of 7

6
A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
Fig. 1. A) esidues (cyan) Predicted binding mode of compound 17a (grey) and B) the reference compound EHD (grey) within the active site of the Top1-DNA complex (PDB: 1T8I),
showing the interaction of the inhibitors with the amino acid rof the enzyme and DNA base pair.
Fig. 2. A) Predicted binding mode of compounds 8a and B) 10b within the active site of Top1 (PDB: 1T8I). Hydrogen bondings are shown in yellow dashed lines. DNA backbone is
shown in orange and Top 1 in slate.
towards another heterocyclic amines to build different fused het-
erocyclic rings. Thus, treatment of compound 7 with different
amines namely: (2-amino-pyridine and 2-aminopyrimidine)
afforded the corresponding derivatives 12, and 13, respectively
NMR spectrum exhibits CN resonance at
d 115.1 ppm. The ESI-MS
spectra of 15 represent the molecular ion peaks at m/z: 404
þ
e
[MþNa] in positive mode and 380 [M-H] in negative mode,
establishing the molecular weight of 15 as 381 Da.
(
Scheme 3). The 1H NMR spectra manifest the absence of a char-
Finally, active methylene reagents such as malononitrile or
ethylcyanoacetate was treated with phenyl isothiocyanate in DMF
containing potassium hydroxide to afford the non-isolable in-
termediates 16a,b that treated directly with 7 to give the corre-
sponding thiazolidinylidene derivatives 17a and 18a, respectively.
The synthetic pathway for thiazole derivatives (17b, 18b) or
thiophene derivatives (17c, 18c), as reported by Fadda et al. [28]
acteristic signal to methylene protons, while display a singlet one
attributable to the newly formed CH-imidazole at 8.82 for 12 and
d
8
.66 ppm for 13. (þ)-ESI-MS spectra for 12 and 13 show the mo-
þ
þ
lecular ion peaks at m/z: 399 [MþH] and 400 [MþH] , respec-
tively. Furthermore, nucleophilic substitution reaction of 7 with 1-
methylpiperazine gave the expected product 1-methylpiperazinyl-
1
3
-nitrocoumain derivative 14 (Scheme 3). The H NMR spectrum of
or Abdelhamid and Al-Shehri [29], respectively, was failed in our
practice (Scheme 4). The structure of 17a was deduced by the
1
1
4 generates the CH
corresponding carbon resonance was detected by C NMR spec-
trum at 51.1, whilst 49.1 ppm was attributed to methyl carbon.
2
-piperazine chemical shift at
d
3.17 ppm. The
H
1
3
NMR spectrum that indicates the thiomethylene diastereotopic
4.12 and 3.82 ppm (See
d
geminal coupling protons (J ¼ 12.4 Hz) at
d
(
4
þ)-ESI-MS spectrum of 14 shows the molecular ion peak at m/z:
supporting information Fig. S45). Also, ESI-MS spectra of 17a
show the molecular ion peak in positive mode at m/z: 546
þ
23 [MþH] . Direct reaction of equimolar amounts from 7 with
2 mechanism yielded the
þ
-
1
potassium thiocyanate according to S
corresponding coumarinyl thiocyanato derivative 15 [26]. Its
N
[MþNa] and 522 [M-H] in negative mode. The H NMR spec-
13
C
trum of 18a exhibits triplet and quartet vicinal protons (J ¼ 7.1 Hz)

A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
7
Fig. 3. A) Superimposed Top1 structure (PDB: 1T8I) before (blue) and after simulation (grey) with docked compound 17a before MD simulation (blue) and after 1 ns simulation
grey); B) an internal view of the active site pocket with the structural deviation of 17a showing the H-bonding (yellow dots) with the Top1 amino acid residues (cyan and green).
(
at
d
4.12 and 1.17 ppm, respectively, attributable to the ester
3.84 and
S fragment. The C NMR spec-
3.2. Cytotoxicity activity evaluation by resazurin assay
group, as well shows geminal protons (J ¼ 12.6 Hz) at
d
13
3
.57 ppm that attributed to CH
2
The in vitro cytotoxicity activity of the new synthesized com-
pounds 4e18 were evaluated against KB-3-1 cell line using
resazurin-based assay [21] with (þ)-griseofulvin as the positive
control. As appeared in Table 1, thiazolidinylidene derivative 17a
that containing malononitrile fragment exhibited the best cytotoxic
trum of 18a displays the corresponding ester carbons at
and 14.8 ppm, respectively.
d 60.5
Table 3
Physicochemical Properties, drug-likeness and lead-likeness parameters of compounds 8a, 10b, 11 and 17a in comparison with omeprazole drug.
Compounds
Predictive models and their
parameters
8a
10b
11
17a
Omeprazole
Physicochemical Properties
MW
380.38 g/mol
517.94 g/mol
410.38 g/mol
523.52 g/mol
345.42 g/
mol
Fraction Csp3
RB
0.00
4
0.00
7
0.00
4
0.07
5
0.29
5
a
H-bond acceptors
H-bond donors
5
2
6
2
6
1
7
2
5
1
b
MR
106.36
155.21 Å
144.45
119.38
147.25
93.70
TPSAc
2
153.58 Å2
113.84 Å2
184.41 Å2
96.31 Å2
Lipophilicity
d
Log Po/w (XLOGP3)
4.11
4.16
7.03
6.48
4.71
5.06
4.93
4.61
2.23
3.61
Log Po/w (WLOGP)
Water Solubility
e
Log S (ESOL)
ꢁ5.10
ꢁ7.57
ꢁ5.71
ꢁ6.29
ꢁ3.52
Qualitative solubility
Moderately soluble
Poorly soluble
Moderately soluble
Poorly soluble
Soluble
Drug-likeness
Lipinski (RO5)
f
Yes; 0 violation
Yes
Yes; 1 violation: MW > 500
Yes; 0 violation
Yes
Yes; 1 violation: MW > 500 Yes;
0
No; 2 violations: MW > 480, Yes
MR > 130
violation
Ghoseg
No; 3 violations: MW > 480,
WLOGP>5.6,
MR > 130
No; 1 violation: TPSA>140
0.55
Veberh
Bioavailability Score
Lead-likeness
No; 1 violation: TPSA>140
0.55
Yes
No; 1 violation: TPSA>140 Yes
0.55
0.55
0.55
i
Rule of three (RO3)
No; 2 violations: MW > 350,
XLOGP3>3.5
No; 2 violations: MW > 350,
XLOGP3>3.5
No; 2 violations: MW > 350,
XLOGP3>3.5
No; 2 violations: MW > 350, Yes
XLOGP3>3.5
a
RB ¼ Rotatable bonds.
b
MR ¼ Molar Refractivity.
c
TPSA ¼ Topological polar surface area.
d
e
f
Log Po/w ¼ The partition coefficient between n-octanol and water.
Log S ¼ The decimal logarithm of the molar solubility in water.
Lipinski (RO5) criteria range are: lipophilicity (Log Po/w) ꢂ 5, MW ꢂ 500, H-bond donors ꢂ 5 and H-bond acceptors ꢂ 10.
g
h
i
Ghose filter criteria range are: Log Po/w in ꢁ0.4 to þ5.6 range, MR from 40 to 130, MW from 180 to 480, No. of atoms from 20 to 70.
2
Veber rule criteria range are: RB ꢂ 10 and TPSA ꢂ140 Å .
RO3 criteria range are: XLOGP3 ꢂ 3.5, MW ꢂ 350, H-bond donors ꢂ3, H-bond acceptors ꢂ3 and RB ꢂ 3 [23,30].

8
A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
Fig. 4. Bioavailability Radar plot of the predicted compounds: A) for 8a; B) for 10b; C) for 11; D) for 17a; E) for omeprazole drug. The pink area shows the optimal range for each
2
properties (Lipophilicity: XLOGP3 between ꢁ0.7 and þ 5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å , solubility: log S not higher than 6,
saturation: fraction of carbons in the sp3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds) [23].
Table 4
Pharmacokinetics parameters of compounds 8a, 10b, 11 and 17a in comparison with omeprazole drug.
Pharmacokinetics parameters
Compounds
8a
10b
11
17a
Omeprazole
GI (HIA) absorptiona
Low
No
No
Low
No
No
Low
No
No
Low
No
No
High
No
Yes
b
BBB permeant
P-gp substratec
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
Yes
Yes
Yes
No
No
Yes
No
No
No
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
CYP3A4 inhibitor
Yes
ꢁ5.70 cm/s
No
ꢁ4.47 cm/s
No
ꢁ5.46 cm/s
Yes
ꢁ5.99 cm/s
Yes
ꢁ6.82 cm/s
d
Log K
p
(skin permeation)
a
GI (HIA) ¼ Human gastrointestinal absorption.
BBB ¼ Blood-brain barrier permeation.
P-gp ¼ Permeability glycoprotein.
b
c
d
Log K
p
¼ The skin permeability coefficient.
potency with IC50 value of 21
(þ)-griseofulvin IC50 ¼ 19.2 M], whereas thiazolyl derivatives 8a
and 10b as well quinoxalinyl analog 11 showed moderate activity
with IC50 values 44, 66 and 43 M, respectively. On the other hand,
derivatives 4, 7, 8b, 8c and 15 indicated lower activity with IC50
values over than 100 M, whilst compounds 5, 6, 10a, 12, 13, 14 and
8a showed no activity toward KB-3-1. In general, the results sug-
gested that compound 17a have potent antiproliferative and cyto-
toxic activity against KB-3-1but limited unfavorable effects on
normal cells.
m
M and very close to the reference
scaffolds possessing different IC50 values to Top1 enzyme as an
effective target for cancer treatment was carried out [22]. As rep-
resented in Table 2, docking results revealed that, the potential
antiproliferative activity of these compounds compared to the
reference camptothecin (EHD) e a known Top1 inhibitor, where
compound 17a that have the highest cytotoxicity activity, showed
high binding affinity of 10.09 kcal/mol to the Top1-DNA complex,
despite the bulky structure of the compound, it can access the
active site, forming important H-bonds with Arg364, Asp533,
Gln633 and 5 -Thio-2 -deoxyguanosine phosphonic acid of the DNA
backbone (TGP11); besides the Pi stacking stabilizing the potential
inhibitor. Coumarin unit of 17a is directed within the active site
between the catalytic 3-phosphotyrosine (Tyr723) and 5-
phosphothiolate (TGP11) of the DNA backbone that might hinder
[
m
m
m
1
0
0
3.3. Docking study
Docking analysis of some selected synthesized coumarin-based

A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
9
Fig. 5. The BOILED-Egg plot of the predicted compounds 8a, 10b, 11 and 17a in comparison with omeprazole drug. The white region is for highly probable HIA (GI) absorption, and
the yellow region (yolk) is for highly probable BBB permeation. The outside grey region stands for molecules are predicted with low absorption and not brain penetration. As well,
the points are colored in blue if predicted as P-gp substrate (PGPþ) and in red if predicted as P-gp non-substrate (PGPꢁ) [23,31].
Table 5
In silico oral toxicity prediction and indication of toxicity targets for compounds 8a, 10b, 11 and 17a.
Compound
Predicted LD50 (mg/kg)
Predicted Toxicity Classa
Average similarity (%)
Prediction accuracy (%)
Toxic fragments
Binding to toxicity targets
8
1
1
1
a
0b
1
300
3
4
4
4
39.77
37.59
41.29
35.87
23
23
54.26
23
Nil
Nil
Nil
Nil
No
No
No
No
1000
1000
1000
7a
a
Toxicity Class ranging from 1 to 6 according to the Global Harmony System (GHS) [24].
the second transesterification required for rejoining DNA backbone.
On the other hand, camptothecin displayed slightly lower binding
affinity (9.48 kcal/mol) than 17a, however four H-bonds with
Asp533, Arg488 and two DNA base pair were formed besides the
base stacking interaction (Fig. 1).
within the active site of the Top1 was observed for compound 5.
In order to further investigate the potentiality of compound 17a
as a Top1 inhibitor, molecular dynamic (MD) simulation was
employed. Interestingly, 17a was more oriented within the active
site pocket of Top 1 that enabled H-bond formation with residues
0 0
Arg362, Arg364, Asp533 and 5 -Thio-2 -deoxyguanosine phos-
Thiazolyl derivative 8a with relatively low IC50 value 44
m
M,
0
0
exhibited lower binding affinity (8.44 kcal/mol) than 17a and EHD,
in spite of it formed three H-bonds with the enzyme and DNA
backbone. Surprisingly, the 4-chlorobenzylidene hydrazinyl thia-
phonic acid (TGP11) and 2 -Deoxyguanosine 5 -monophosphate
(DG12) from the DNA backbone (Fig. 3). In the same manner, MD
simulation was conducted on the reference compound EHD, but no
more deviation was observed after 1 ns of the simulation time with
retention of the same interactions pattern to Top 1-DNA complex
(four H-bonding with Lys532, Asp533, Arg488 and Arg364).
zolyl analog 10b with IC50 value 66 mM, manifested the highest
binding affinity (10.97 kcal/mol), although only one H-bond was
detected with the DNA backbone, but three pi stacking could be
recognized with the DNA backbone that explain the strong binding
and stability of the compound. So, the low IC50 value may be
attributable to low accessibility of 10b through the cell membrane
3.4. Physicochemical properties and ADME parameters
(
Fig. 2). Compound 4 with IC50 > 100 showed low binding energy
To our delight by the findings obtained from cytotoxicity
screening and docking study, we used SwissADME web tool as
described previously [23] to predict the physicochemical properties
(
8.25 kcal/mol) besides it correlated away from the catalytic 3-
phospotyrosine. As the cytotoxicity result, no binding affinity

10
A.H. Halawa et al. / Journal of Molecular Structure 1200 (2020) 127047
and ADME parameters of compounds 8a, 10b, 11 and 17a in com-
parison with omeprazole drug that conforms to Lipinski's rule of
five (RO5). As stated in Table 3, these compounds passed the filter of
Lipinski's rule with zero violation for 8a and 11, whereas com-
pounds 10b and 17a passed with one violation attributable to the
molecular weight that over than 500 g/mol. Consequently, these
compounds have oral bioavailability.
known ligands of that protein target and the ligand mapping to
pharmacophores models (Toxicophores). As tabulated in Table 5,
the evaluated LD50 was ranged from 300 to 1000 mg/kg for the
predicted compounds. The higher the LD50, the lower toxic effect is
the molecule. Our tested molecules were predicted to toxicity
classes 3 or 4 and have not any toxic fragments. Also, all these
compounds are non-binding to toxicity targets.
Interestingly, compound 11 passed the filter of both Ghose and
Veber rules without any violation like omeprazole drug. Besides, all
the predicted compounds as well omeprazole drug displayed the
same bioavailability Score (0.55). For defining lead-likeness, our
predicted compounds share the same violations to the rule of three
4. Conclusions
In summary, a new series of 4-arylamino-3-nitrocoumarin de-
rivatives were synthesized starting from 4-hydroxycoumarin and
characterized by various spectral methods. All the compounds were
subjected for in vitro cytotoxicity activity against KB-3-1 cancer cell
line. Compound 17a exhibited a promising activity in the tested
compounds. The docking and MD simulation study provide an
additional evidence for the potentiality and possible interaction
mechanism of the new coumarin derivatives as antiproliferative
agents targeting Top1-DNA complex and hence as anticancer
agents. As well, in silico prediction of physicochemical properties
and oral toxicity manifested that compound 17a passed the filter of
Lipinski's rule and have low toxic effect, respectively. So, compound
17a would become a valuable lead for additional investigation. Also,
compounds 5 and 6 will be utilized as a starting material for further
modification.
(
RO3) attributable to MW > 350 and XLOGP3>3.5. In that regard,
the Bioavailability Radar planner is displayed for a rapid evaluation
of drug-likeness taken into account six physicochemical in-
spections including lipophilicity, size, polarity, solubility, flexibility
and saturation (Fig. 4) [23]. The pink area indicates the range of
optimal values for each parameter. Obviously, all the predicted
compounds from the first glance showed deviation particularly the
highly unsaturation degree (Fraction Csp3 ¼ 0.0), leading to sub-
optimal physicochemical properties for oral bioavailability, whilst
also omeprazole drug displayed all the properties in the optimal
range (pink area). Moreover, solubility is one pioneer property
affecting the absorption [23] and concerning qualitative water
solubility, all the predicted compounds ranged from poorly to
moderately soluble based on ESOL topological model, but omep-
razole drug was soluble.
Conflicts of interest
With regard to the pharmacokinetic behaviors, the estimation of
our predicted compounds to be substrate or non-substrate of the
permeability glycoprotein (P-gp) and to be inhibitor or non-
inhibitor of the most important cytochromes P450 isoenzymes
The authors declare that they have no conflict of interest.
Acknowledgements
(
CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4), was implemented
by the support vector machine algorithm (SVM) model [23]. As
shown in Table 4, omeprazole drug inhibited the different CYP
isoenzymes, while our evaluated compounds manifested selectivity
for particular isoforms, for instance, compound 8a suppressed four
types among them: CYP1A2, CYP2C19, CYP2C9 and CYP3A4, but
compound 10b only affected CYP2C19. As well, 17a inhibited three
CYP isoforms: CYP2C19, CYP2C9 and CYP3A4. Noteworthy, CYP2D6
isoenzyme was only affected by quinoxalinyl-3-nitrocoumarin de-
rivative 11.
The authors are grateful to the NMR and MS Departments at
Bielefeld University for spectral measurements. We would like to
thank Carmela Michalek for biological activity testing and Marco
Wi
work has been financed by the German Academic Exchange Service
DAAD) with funds from the German Federal Foreign Office in the
bbrock with Anke Nieb for technical assistance. This research
(
frame of the Research Training Network “Novel Cytotoxic Drugs
from Extremophilic Actinomycetes” (Project ID57166072).
Based on BOILED-Egg model (WLOGP vs TPSA) that illustrated in
Fig. 5 [23,31], all the tested compounds demonstrated low GI ab-
sorption, not brain penetration and P-gp non-substrates (PGP-, red
dots), while omeprazole drug was predicted high absorbed, but not
BBB permeant and PGPþ (blue dot). Importantly as well, predicting
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127047.
p
the skin permeability coefficient (K ) of our chemical compounds
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