Full text of DOI:10.1080/14756366.2018.1476503

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Introducing novel potent anticancer agents of 1H-
benzo[f]chromene scaffolds, targeting c-Src kinase
enzyme with MDA-MB-231 cell line anti-invasion
effect
Hany E. A. Ahmed, Mohammed A. A. El-Nassag, Ahmed H. Hassan, Rawda M.
Okasha, Saleh Ihmaid, Ahmed M. Fouda, Tarek H. Afifi, Ateyatallah Aljuhani
& Ahmed M. El-Agrody
To cite this article: Hany E. A. Ahmed, Mohammed A. A. El-Nassag, Ahmed H. Hassan, Rawda
M. Okasha, Saleh Ihmaid, Ahmed M. Fouda, Tarek H. Afifi, Ateyatallah Aljuhani & Ahmed M. El-
Agrody (2018) Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds,
targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect, Journal of Enzyme
Inhibition and Medicinal Chemistry, 33:1, 1074-1088, DOI: 10.1080/14756366.2018.1476503
To link to this article: https://doi.org/10.1080/14756366.2018.1476503
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1074–1088
https://doi.org/10.1080/14756366.2018.1476503
RESEARCH PAPER
Introducing novel potent anticancer agents of 1H-benzo[f]chromene scaffolds,
targeting c-Src kinase enzyme with MDA-MB-231 cell line anti-invasion effect
Hany E. A. Ahmeda,b, Mohammed A. A. El-Nassagc,d, Ahmed H. Hassanc,d, Rawda M. Okashae, Saleh Ihmaida,
Ahmed M. Foudaf, Tarek H. Afifie, Ateyatallah Aljuhanie and Ahmed M. El-Agrodyc
aDepartment of Pharmacy College, Pharmacognosy and Pharmaceutical Chemistry, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia;
c
bPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt; Chemistry Department, Faculty of
d
e
Science, Al-Azhar University, Cairo, Egypt; Chemistry Department, Faculty of Science, Jazan University, Jazan, Saudi Arabia; Chemistry
f
Department, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia; Chemistry Department, Faculty of Science, King
Khalid University, Abha, Saudi Arabia
ABSTRACT
ARTICLE HISTORY
Received 15 February 2018
Revised 8 May 2018
Accepted 9 May 2018
In our effort to develop novel and powerful agents with anti-proliferative activity, two new series of
1H-benzo[f]chromene derivatives, 4a–h and 6a–h, were synthesised using heterocyclocondensation meth-
odologies under microwave irradiation condition. The structures of the target compounds were
established on the basis of their spectral data, IR, 1H NMR, 13 C NMR, 13 C NMR-DEPT/APT, and MS data.
The new compounds have been examined for their anti-proliferative activity against three cancer cell lines,
MCF-7, HCT-116, and HepG-2. Vinblastine and Doxorubicin have been used as positive controls in the
viability assay. The obtained results confirmed that most of the tested molecules revealed strong and
selective cytotoxic activity against the three cancer cell lines. Moreover, these molecules exhibited weak
cytotoxicity on the HFL-1 line, which suggested that they might be ideal anticancer candidates. The
SAR study of the new benzochromene compounds verified that the substituents on the phenyl ring of
1H-benzo[f]chromene nucleus, accompanied with the presence of bromine atom or methoxy group at
the 8-position, increases the ability of these molecules against the different cell lines. Due to their high
anti-proliferative activity, compounds 4c and 6e were selected to be examined their proficiency to inhibit
the invasiveness of the highly sensitive and invasive breast cancer cell line, MDA-MB-231. The anti-invasion
behaviour of these molecules against the highly sensitive, non-oestrogen, and progesterone MDA-MB-231
cell line gave rise to their decreasing metastatic effect compared to the reference drug. Furthermore, this
report explores the apoptotic mechanistic pathway of the cytotoxicity of the target compounds and
reveals that most of these compounds enhance the Caspase 3/7 activity that could be considered as
potential anticancer agents.
KEYWORDS
Microwave synthesis;
1H-benzo[f]chromenes;
antitumour activity; SAR
study; Caspase 3/7
1. Introduction
inducers16,17. It has also been cited in literature that the 4-aryl-4H-
chromenes have been developed as future anticancer agents with
potent apoptosis induction through cell-based mediated Caspases
3/7 activation16,17. Fallah et al. also reported the synthesis of 4-aryl-
4H-chromenes with mild selective c-Src kinase anticancer activity24.
Indeed, benzochromene is a successful choice in the treatment of
several human diseases. For instance, 2-amino-4-(3-nitrophenyl)-4H-
benzo[h]chromene-3-carbonitrile (E) is a potent anti-proliferative
agent for a variety of cell types and acts as an inhibitor for mitosis
Chromene and benzochromene molecules are notorious for exhib-
iting a wide range of biological and pharmacological activities
such as antimicrobial1–5, antileishmanial6,7, antioxidant8,9, vascular-
disrupting10, oestrogenic, anticoagulant and antispasmolytic11,
blood platelet antiaggregating12, analgesic13, and hypolipidemic
effects14. In addition, the current interest in chromenes and benzo-
chromenes arises from their applications in the treatment of
human diseases15–21. Furthermore, chromene derivatives have
emerged as promising and desirable scaffolds in the development
of potent antitumour agents22,23. For example, CrolibulinTM (A) is
currently in phase I/II of clinical trials for the treatment of
and microtubules25,26
.
Meanwhile, 2-amino-5-oxo-4-phenyl-4,5-
dihydropyrano[3,2-c]chromene-3-carbonitrile (F) serves as a precursor
for the blood anticoagulant warfarin19, and 4-substituted-2-
(N-succinimido)-4H-benzo[h]chromene-3-carbonitriles (G) displays
anti-rheumatic activity20 as shown in Figure 1.
advanced solid tumours15
,
while 2-amino-4-(3-bromo-4,5-
dimethoxyphenyl)-7-(dimethylamino)-4H-chromene-3-carbonitrile (B)
has been known as a tubulin inhibitor21, and ethyl 2-amino-6-
bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (C)
and 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-
In our efforts to be a part of the development process of novel
cytotoxic agents, we have recently focussed on utilising the
1H-benzo[f]chromene scaffold with methoxy substituent at the
9-position to create various potent and high anti-proliferative
carbonitrile (D) act as inhibitors of Bcl-2 protein and as apoptosis compounds such as 3-amino-1-(4-chlorophenyl)-9-methoxy-1H-
CONTACT Hany E. A. Ahmed heahmad@taibahu.edu.sa Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University,
Al-Madinah Al-Munawarah, Saudi Arabia
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1075
OMe
OMe
MeO
Br
MeO
Br
CN
CN
Me2N
O
NH2
H2N
O
NH2
NH2
B
A
MX58151
Tublin inhibitor
EPC2407
Treatment of solid tumor
EtO2C
CN
CN
EtO2C
CN
CO2Et
Br
Br
CN
O
NH2
O
NH2
C
D
O
NH2
HA14-1
inhibitor of Bcl-protein
Anticancer
and Bcl-2 inhibitor
2-amino-4H-chromene-3-carbonitrile
O
O
O
NH2
O
N
CN
NH2
O
O
CN
CN
O
F
NO2
E
G
Blood anticoagulant
LY290181
A potent antiproliferative agent
Anti-rheumatic activity
O
NH2
CN
4H-benzo[h]chromene
O
NH2
CN
O
NH2
CN
R = Br and -OCH
3
R
O
Cl
Cl
H
8-Postion substituted 1H-benzo[f ]chromene
9-Postion substituted 1H-benzo[f ]chromene
Target compounds
Figure 1. Reference drugs bearing chromene (red highlighted) or 1H-benzo[f]chromene (blue highlighted) with different biological activities.
benzo[f]chromene-2-carbonitrile27 (H). Since it was revealed that to chromene scaffold which seems to play a crucial role in the
the position and the type of the substituent have a great effect cytotoxic activity.
Herein, we introduced the microwave irradiation synthesis of
the 3-amino-1H-benzo[f]chromene-2-carbonitrile derivatives. The
target compounds were examined for their anti-proliferative activ-
ity towards three cancer cell lines, MCF-7, HCT-116, and HepG-2
on the cytotoxic activity of the desired compounds, the main goal
of the present study is to synthesise a new family of substituted
1H-benzo[f]chromene at the 8-postion with electron-withdrawing
and electron-donating groups. The rational design of the new
compounds was based on the following considerations: (i) the 1H- and the viability assay was evaluated against two standard drugs,
benzo[f]chromene scaffold itself, (ii) the presence of the effective Vinblastine and Doxorubicin. The Structure-Activity Relationships
8-postion substitution, and (iii) the type of the aryl groups linked of these molecules elucidated the effect of the substituents at the

1076
H. E. A. AHMED ET AL.
C-1 and C-8 positions of the 1H-benzo[f]chromene moiety on their 13C NMR d: 159.52 (C-3), 147.07 (C-4a), 132.15 (C-10a), 130.28 (C-7),
cytotoxic activity.
129.90 (C-9), 128.76 (C-6), 128.28 (C-6a), 125.89 (C-10), 120.21
(C-10b), 118.16 (CN), 118.15 (C-5), 115.82 (C-8), 57.65 (C-2), 37.05
(C-1), 161.79, 141.68, 128.87, 128.83, 115.56, 115.39 (aromatic); 13C
NMR-DEPT spectrum at 135ꢀCH, CH3 [positive (up)], CH2 [negative
(down)], revealed the following signals at d: 130.28 (C-7 "), 129.90
(C-9 "), 128.87 (aromatic "), 128.83 (aromatic "), 128.76 (C-6 "),
125.89 (C-10 "), 118.15 (C-5 "), 115.56 (aromatic "), 115.39
(aromatic "), 37.05 (C-1 "). In the DEPT spectrum at 90ꢀ only CH
signals are positive (up) and showed d: 130.28 (C-7 "), 129.90 (C-9
"), 128.87 (aromatic "), 128.83 (aromatic "), 128.76 (C-6 "), 125.89
(C-10 "), 118.15 (C-5 "), 115.56 (aromatic "), 115.39 (aromatic "),
37.05 (C-1 "). In the DEPT spectrum at 45ꢀ (CH, CH2 and CH3 posi-
tive) revealed signals at d: 130.28 (C-7 "), 129.90 (C-9 "), 128.87
(aromatic "), 128.83 (aromatic "), 128.76 (C-6 "), 125.89 (C-10 "),
118.15 (C-5 "), 115.56 (aromatic "), 115.39 (aromatic "), 37.05 (C-1
"); 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [nega-
tive (down)], revealed the following signals at d: 161.79 (aromatic
#), 159.52 (C-3 #), 147.07 (C-4a #), 141.69 (aromatic #), 132.15
(C-10a #), 130.28 (C-7 "), 129.90 (C-9 "), 128.87 (aromatic "), 128.83
(aromatic "), 128.76 (C-6 "), 128.28 (C-6a #), 125.89 (C-10 "), 120.21
(C-10b #), 118.16 (CN #), 118.15 (C-5 "), 115.82 (C-8 #), 115.56 (aro-
matic "), 115.39 (aromatic "), 57.65 (C-2 #), 37.05 (C-1 "); m/z (%):
396 (Mþþ2, 10.91), 394 (Mþ, 34.12) with a base peak at 271 (100);
Anal. Calcd for C20H12BrFN2O: C, 60.78; H, 3.06; N, 7.09. Found: C,
60.90; H, 3.17; N, 7.20%.
2. Experimental section
2.1. Materials and equipments
All chemicals were purchased from Sigma-Aldrich Chemical Co.
(Sigma-Aldrich Corp., St. Louis, MO). All melting points were meas-
ured with a Stuart Scientific Co. Ltd apparatus are uncorrected.
The IR spectra were recorded on a KBr disc on a Jasco FT/IR 460
plus spectrophotometer. The 1H NMR (500 MHz) and 13C NMR
(125 MHz) spectra were measured on BRUKER AV 500 MHz spec-
trometer in DMSO-d6 as a solvent, using tetramethylsilane (TMS)
as an internal standard, and chemical shifts were expressed as d
(ppm). The 13C NMR spectra were obtained using distortion less
enhancement by polarisation transfer (DEPT), where the signals of
CH and CH3 carbon atoms appear normal (up), and the signals of
carbon atoms in CH2 environments appear negative (down). The
13C N MR spectra were also obtained using attached proton test
(APT), with this technique, the signals of CH and CH3 carbon
atoms appear normal (up) and the signal of CH2 and Cq environ-
ments appears negative (down). The Microwave apparatus used is
Milestone Sr1, Microsynth. The mass spectra were determined on
a Shimadzu GC/MS-QP5050A spectrometer. Elemental analysis was
carried out at the Regional Centre for Mycology and
Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt, and the
results were within 0.25%. Reaction courses and product mixtures
were routinely monitored by thin layer chromatography (TLC) on
silica gel precoated F254 Merck plates.
2.2.3. 3-Amino-8-bromo-1-(4-chlorophenyl)-1H-benzo[f]chromene-
2-carbonitrile 4c
Yellow crystals from ethanol; yield 86%; m.p. 260–261 ꢀC (Lit. m. p.
265 ꢀC28); IR (KBr) t (cmꢁ1): 3449, 3322, 3200 (NH2), 2200 (CN); 1H
NMR d: 8.23–7.19 (m, 9H, aromatic), 7. 10 (bs, 2H, NH2), 5.37 (s, 1H,
H-1); 13C NMR d: 159.55 (C-3), 147.11 (C-4a), 132.14 (C-10a), 130.30
(C-7), 129.96 (C-9), 128.79 (C-6), 128.73 (C-6a), 125.85 (C-10), 120.15
(C-10b), 118.20 (CN), 118.15 (C-5), 115.49 (C-8), 57.29 (C-2), 37.15
(C-1), 144.38, 131.28, 128.97 (aromatic); m/z (%): 414 (Mþþ4, 3.11),
412 (Mþþ2, 8.65), 410 (Mþ, 6.31) with a base peak at 299 (100);
Anal. Calcd for C20H12 BrClN2O: C, 58.35; H, 2.94; N, 6.80. Found: C,
58.23; H, 2.83; N, 6.69%.
2.2. General procedure for synthesis of 1H-benzo[f]chromenes
4a–h and 6a–h
A reaction mixture of 6-bromo-2-naphthol 1 or 6-methoxy-2-naph-
thol 5 (0.01 mol), different aromatic aldehydes 2a–h (0.01 mol),
malononitrile 3 (0.01 mol) and piperidine (0.5 ml) in ethanol (30 ml)
was heated under Microwave irradiation conditions for 2 min at
140 ꢀC. After completion of the reaction, the reaction mixture was
cooled to room temperature and the precipitated solid was
filtered off, washed with methanol, and was recrystallised from
ethanol or ethanol/benzene. The physical and spectral data of
compounds 4a–h and 6a–h are as follows:
2.2.4. 3-Amino-8-bromo-1-(4-bromophenyl)-1H-benzo[f]chromene-
2-carbonitrile 4d
Colourless crystals from ethanol; yield 88%; m.p. 250–251 ꢀC (Lit.
m. p. 250 ꢀC29);IR (KBr) t (cmꢁ1): 3447, 3321, 3205 (NH2), 2201
2.2.1. 3-Amino-8-bromo-1-phenyl-1H-benzo[f]chromene-2-carbonitrile 4a
Colourless crystals from ethanol; yield 89%; m.p. 245–246 ꢀC (Lit. (CN); 1H NMR d: 8.23–7.12 (m, 9H, aromatic), 7.09 (bs, 2H, NH2),
m. p. 240 ꢀC28); IR (KBr) t (cmꢁ1): 3477, 3329, 3213 (NH2), 2204 5.37 (s, 1H, H-1); 13C NMR d: 159.54 (C-3), 147.11 (C-4a), 132.14
(CN); 1H NMR d: 8.21–7.15 (m, 10H, aromatic), 7.04 (bs, 2H, NH2, (C-10a), 130.31 (C-7), 129.97 (C-9), 128.98 (C-6), 128.73 (C-6a),
D2O exchangeable), 5.32 (s, 1H, H-1); 13C NMR d: 159.53 (C-3), 125.85 (C-10), 120.13 (C-10b), 118.21 (CN), 118.14 (C-5), 115.42
147.12 (C-4a), 132.12 (C-10a), 130.24 (C-7), 129.85 (C-9), 128.73 (C-8), 57.21 (C-2), 37.21 (C-1), 144.80, 131.65, 129.16, 119.79
(C-6), 128.51 (C-6a), 126.67 (C-10), 125.92 (C-10b), 120.31 (C-5), (aromatic); m/z (%): 458 (Mþþ4, 31.91), 456 (Mþþ2, 63.81), 454
118.12 (CN), 116.01 (C-8), 57.78 (C-2), 37.89 (C-1), 145.42, (Mþ, 32.12) with a base peak at 272 (100); Anal. Calcd for
128.84,128.75, 126.92 (aromatic); m/z (%): 378 (Mþþ2, 18.99), 376
C20H12Br2N2O: C, 52.66; H, 2.65; N, 6.14. Found: C, 52.52; H, 2.53;
(Mþ, 57.92) with a base peak at 299 (100); Anal. Calcd for N, 6.02%.
C20H13BrN2O: C, 63.68; H, 3.47; N, 7.43. Found: 63.79; H, 3.59;
N, 7.55%.
2.2.5. 3-Amino-8-bromo-1-(4-methylphenyl)-1H-benzo[f]chromene-
2-carbonitrile 4e
Colourless needles from ethanol; yield 87%; m.p. 215–216 ꢀC (Lit.
m. p. 215 ꢀC29); IR (KBr) t (cmꢁ1): 3480, 3369, 3217 (NH2), 2181
2.2.2. 3-Amino-8-bromo-1-(4-fluorophenyl)-1H-benzo[f]chromene-
2-carbonitrile 4b
Colourless crystals from ethanol; yield 88%; m.p. 242–243 ꢀC; IR (CN); 1H NMR d: 8.22–7.04 (m, 9H, aromatic), 7.00 (bs, 2H, NH2,
(KBr) t (cmꢁ1): 3457, 3337, 3201 (NH2), 2203 (CN); 1H NMR d: D2O exchangeable), 5.26 (s, 1H, H-1), 2.21 (s, 3H, CH3); 13C NMR d:
8.22–7.08 (m, 9H, aromatic), 7.04 (bs, 2H, NH2), 5.37 (s, 1H, H-1); 159.42 (C-3), 147.04 (C-4a), 132.12 (C-10a), 130.21 (C-7), 129.81

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1077
(C-9), 128.86 (C-6a), 128.67 (C-6), 125.99 (C-10), 120.32 (C-10b), 128.28 (C-6 "), 125.32 (C-10 "), 117.98 (C-5 "), 105.79 (aromatic "),
118.11 (C-5), 118.07 (CN), 116.14 (C-8), 57.89 (C-2), 37.51 (C-1), 98.68 (aromatic "), 31.26 (C-1 "). In the DEPT spectrum at 45ꢀ (CH,
20.51 (CH3), 142.53, 135.78, 129.26, 126.82 (aromatic); 13C NMR- CH2, and CH3 positive) revealed signals at d: 130.20 (C-7 "), 129.86
DEPT spectrum at 135ꢀCH, CH3 [positive (up)], CH2 [negative (C-9 "), 129.21 (aromatic "), 128.28 (C-6 "), 125.32 (C-10 "), 117.98
(down)], revealed the following signals at d: 130.21 (C-7 "), 129.81 (C-5 "), 105.79 (aromatic "), 98.68 (aromatic "), 55.96 (CH3 "), 55.08
(C-9 "), 129.26 (aromatic "), 128.67 (C-6 "), 126.82 (aromatic "), (CH3 "), 31.26 (C-1 "); 13CNMR-APT spectrum CH, CH3 [positive
125.99 (C-10 "), 118.11 (C-5 "), 37.51 (C-1 "), 20.51 (CH3 "). In the (up)], CH2, Cq [negative (down)], revealed the following signals at
DEPT spectrum at 90ꢀ only CH signals are positive (up) and d: 159.88 (C-3 #), 159.25 (aromatic #), 156.61 (aromatic #), 147.35
showed d: 130.21 (C-7 "), 129.81 (C-9 "), 129.26 (aromatic "), (C-4a #), 131.95 (C-10a #), 130.20 (C-7 "), 129.86 (C-9 "), 129.21
128.67 (C-6 "), 126.82 (aromatic "), 125.99 (C-10 "), 118.11 (C-5 "), (aromatic "), 128.97 (C-6a #), 128.28 (C-6 "), 125.73 (aromatic #),
37.51 (C-1 "). In the DEPT spectrum at 45ꢀ (CH, CH2, and CH3 posi- 125.32 (C-10 "), 120.34 (C-10b #), 117.98 (C-5 "), 117.93 (CN #),
tive) revealed signals at d: 130.21 (C-7 "), 129.81 (C-9 "), 129.26 116.40 (C-8 #), 105.79 (aromatic "), 98.68 (aromatic "), 57.09 (C-2 #),
(aromatic "), 128.67 (C-6 "), 126.82 (aromatic "), 125.99 (C-10 "), 55.96 (CH3 "), 55.08 (CH3 "), 31.26 (C-1 "); MS m/z (%): 438 (Mþþ2,
118.11 (C-5 "), 37.51 (C-1 "), 20.51 (CH3 ");13CNMR-APT spectrum 10.03) 436 (Mþ, 10.33) with a base peak at 63 (100); Anal. Calcd for
CH, CH3 [positive (up)], CH2, Cq [negative (down)], revealed the C22H17BrN2O3: C, 60.43; H, 3.92; N, 6.41. Found: C, 60.55; H, 4.03;
following signals at d: 159.42 (C-3 #), 147.04 (C-4a #), 142.53 N, 6.55%.
(aromatic #), 135.78 (aromatic #), 132.12 (C-10a #), 130.21 (C-7 "),
129.81 (C-9 "), 129.26 (aromatic "), 128.86 (C-6a #), 128.67 (C-6 "),
2.2.8. 3-Amino-8-bromo-1-(3,4-dimethoxyphenyl)-1H-benzo[f]chromene-
126.82 (aromatic "), 125.99 (C-10 "), 120.32 (C-10b #), 118.11
2-carbonitrile 4h
(C-5 "), 118.07 (CN #), 116.14 (C-8 #), 57.89 (C-2 #), 37.51 (C-1"),
20.51 (CH3 "); m/z (%): 392 (Mþþ2, 29.87), 390 (Mþ, 30.02) with a
Yellow crystals from ethanol/benzene; yield 79%; m.p. 200–201 ꢀC;
IR (KBr) t (cmꢁ1): 3409, 3327, 3205 (NH2), 2197 (CN); 1H NMR d:
base peak at 271 (100); Anal. Calcd for C21H15BrN2O: C, 64.46; H,
8.22–6.55 (m, 8H, aromatic), 6.98 (bs, 2H, NH2, D2O exchangeable),
5.26 (s, 1H, H-1), 3.68 (s, 3H, OCH3), 3.67 (s, 3H, OCH3); 13C NMR d:
159.40 (C-3), 147.00 (C-4a), 132.11 (C-10a), 130.19 (C-7), 129.80 (C-
9), 128.97 (C-6a), 128.65 (C-6), 126.09 (C-10), 120.40 (C-10b), 118.95
(C-5), 118.08 (CN), 116.17 (C-8), 57.98 (C-2), 55.48 (CH3), 55.43
3.86; N, 7.16. Found: C, 64.35; H, 3.76; N, 7.06%.
2.2.6. 3-Amino-8-bromo-1-(4-methoxyphenyl)-1H-benzo[f]chromene-
2-carbonitrile 4f
Colourless needles from ethanol; yield 84%; m.p. 234–235 ꢀC (Lit. (CH3), 37.43 (C-1), 148.65, 147.51, 138.08, 118.09, 112.16, 111.04
13
m. p. 235 ꢀC28); IR (KBr) t(cmꢁ1): 3431, 3319, 3199 (NH2), 2196 (aromatic); C NMR-DEPT spectrum at 135ꢀCH, CH3 [positive (up)],
(CN); 1H NMR d: 8.21–6.81 (m, 9H, aromatic), 6.99 (bs, 2H, NH2, CH2 [negative (down)], revealed the following signals at d: 130.19
D2O exchangeable), 5.26 (s, 1H, H-1), 3.67 (s, 3H, OCH3);13C NMR d: (C-7 "), 129.80 (C-9 "), 128.65 (C-6 "), 126.09 (C-10 "), 118.95 (C-5
159.38 (C-3),146.99 (C-4a), 132.13 (C-10a), 130.21 (C-7), 129.78 "), 118.09 (aromatic "), 112.16 (aromatic "), 111.04 (aromatic "),
(C-9), 128.85 (C-6a), 127.99 (C-6), 126.00 (C-10), 120.38 (C-10b), 55.48 (CH3 "), 55.43 (CH3 "), 37.43 (C-1 "). In the DEPT spectrum at
118.12 (C-5), 118.06 (CN), 116.29 (C-8), 58.07 (C-2), 54.95 (CH3), 90ꢀ only CH signals are positive (up) and showed d: 130.19 (C-7 "),
37.10 (C-1), 157.88, 137.62, 128.62, 128.28, 114.07 (aromatic); 129.80 (C-9 "), 128.65 (C-6 "), 126.09 (C-10 "), 118.95 (C-5 "),
13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq [negative 118.09 (aromatic "), 112.16 (aromatic "), 111.04 (aromatic "), 37.43
(down)], revealed the following signals at d: 159.38 (C-3 #), (C-1 "). In the DEPT spectrum at 45ꢀ (CH, CH2, and CH3 positive)
157.88 (aromatic #), 146.99 (C-4a #), 137.62 (aromatic #), 132.13 revealed signals at d: 130.19 (C-7 "), 129.80 (C-9 "), 128.65 (C-6 "),
(C-10a #), 130.21 (C-7 "), 129.78 (C-9 "), 128.86 (C-6a #), 128.62 126.09 (C-10 "), 118.95 (C-5 "), 118.09 (aromatic "), 112.16 (aro-
(aromatic "), 128.28 (aromatic "), 127.99 (C-6 "), 126.00 (C-10 "), matic "), 111.04 (aromatic "), 55.48 (CH3 "), 55.43 (CH3 "), 37.43
120.38 (C-10b #), 118.12 (C-5 "), 118.06 (CN #), 116.29 (C-8 #), (C-1 "); 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2,
114.73 (aromatic "), 114.07 (aromatic "), 58.09 (C-2 #), 54.96 (CH3 "), Cq[negative (down)], revealed the following signals at d: 159.40
37.10 (C-1"); m/z (%): 408 (Mþþ2, 9.89), 406 (Mþ, 10.12) with a base (C-3 #), 148.65 (aromatic #), 147.51 (aromatic #), 147.00 (C-4a #),
peak at 271 (100); Anal. Calcd.for C21H15BrN2O2: C, 61.93; H, 3.71; N, 138.08 (aromatic #), 132.11 (C-10a #), 130.19 (C-7 "), 129.80 (C-9 "),
6.88. Found: C, 61.81; H, 3.60; N, 6.77%.
128.97 (C-6a #), 128.65 (C-6 "), 126.09 (C-10 "), 120.40 (C-10b #),
118.95 (C-5 "), 118.09 (aromatic "), 118.08 (CN #), 116.17 (C-8 #),
112.16 (aromatic "), 111.04 (aromatic "), 57.98 (C-2 #), 55.48 (CH3 "),
55.43 (CH3 "), 37.43 (C-1 "); MS m/z (%):438 (Mþþ2, 38.99) 436 (Mþ,
41.38) with a base peak at 79 (100); Anal. Calcd for C22H17BrN2O3: C,
60.43; H, 3.92; N, 6.41. Found: C, 60.28; H, 3.77; N, 6.78%.
2.2.7. 3-Amino-8-bromo-1-(2,4-dimethoxyphenyl)-1H-benzo[f]
chromene-2-carbonitrile 4g
Colourless crystals from ethanol/benzene; yield 83%; m.p.
220–221 ꢀC; IR (KBr) t (cmꢁ1): 3410, 3327, 3202 (NH2), 2198 (CN);
1H NMR d: 8.19–6.37 (m, 8H, aromatic), 6.90 (bs, 2H, NH2, D2O
exchangeable), 5.47 (s, 1H, H-1), 3.85 (s, 3H, OCH3), 3.69 (s, 3H,
OCH3); 13C NMR d: 159.88 (C-3), 147.35 (C-4a), 131.95 (C-10a),
2.2.9. 3-Amino-1-phenyl-8-methoxy-1H-benzo[f]chromene-2-
carbonitrile 6a
130.20 (C-7), 129.86 (C-9), 128.97 (C-6a), 128.28 (C-6), 125.32 (C-10), Colourless crystals from ethanol; yield 84%; m.p. 237–238 ꢀC (Lit.
120.34 (C-10b), 117.98 (C-5), 117.93 (CN), 116.40 (C-8), 57.09 (C-2), m. p. 246 ꢀC30); IR (KBr) t (cmꢁ1): 3475, 3327, 3197 (NH2), 2191
55.96 (CH3), 55.08 (CH3), 31.26 (C-1), 159.25, 156.61, 125.73, 129.21, (CN); 1H NMR d: 7.85–7.09 (m, 10Hs, aromatic), 6.97 (bs, 2H, NH2),
13
105.79, 98.68 (aromatic); C NMR-DEPT spectrum at 135ꢀCH, CH3 5.27 (s, 1H, H-1), 3.82 (s, 3H, OCH3); 13C NMR d: 159.80 (C-3),
[positive (up)], CH2 [negative (down)], revealed the following sig- 156.42 (C-8), 145.76 (C-4a), 132.18 (C-6a), 128.65 (C-10a), 126.92
nals at d: 130.20 (C-7 "), 129.86 (C-9 "), 129.21 (aromatic "), 128.28 (C-6), 126.53 (C-10), 120.55 (C-10b), 119.03 (C-9), 117.08 (C-7),
(C-6 "), 125.32 (C-10 "), 117.98 (C-5 "), 105.79 (aromatic "), 98.68 115.85 (CN), 107.24 (C-5), 57.77 (C-2), 55.18 (CH3), 38.15 (C-1),
(aromatic "), 55.96 (CH3 "), 55.08 (CH3 "), 31.26 (C-1 "). In the 145.33, 128.29, 128.17, 125.11 (aromatic); m/z (%): 328 (Mþ, 16.52)
DEPT spectrum at 90ꢀ only CH signals are positive (up) and with a base peak at 251 (100); Anal. Calcd for C21H16N2O2: C,
showed d: 130.20 (C-7 "), 129.86 (C-9 "), 129.21 (aromatic "), 76.81; H, 4.91; N, 8.53. Found: C, 76.94; H, 5.03; N, 8.69%.

1078
H. E. A. AHMED ET AL.
2.2.10. 3-Amino-1-(4-flourophenyl)-8-methoxy-1H-benzo[f]chromene- NMR d: 7.83–6.81 (m, 9H, aromatic), 6.91 (bs, 2H, NH2), 5.21 (s, 1H,
H-1), 3.82 (s, 3H, OCH3), 3.67 (s, 3H, OCH3); 13C NMR) d: 159.65
2-carbonitrile 6b
Pale yellow needles from ethanol; yield 81%; m.p. 256–257 ꢀC; IR
(KBr) t (cmꢁ1): 3467, 3324, 3215 (NH2), 2201 (CN); 1H NMR d:
7.85–7.07 (m, 9H, aromatic), 6.95 (bs, 2H, NH2), 5.32 (s, 1H, H-1),
3.83 (s, 3H, OCH3); 13C NMR d: 159.78 (C-3), 156.45 (C-8), 145.27
(C-4a), 132.22 (C-6a), 128.73 (C-10a), 126.29 (C-6), 125.06 (C-10),
(C-3), 156.39 (C-8), 145.20 (C-4a), 132.17 (C-6a), 127.96 (C-6), 125.15
(C-10), 125.12 (C-10a), 120.62 (C-10b), 118.95 (C-9), 117.09 (C-7),
116.13 (CN), 107.21 (C-5), 58.06 (C-2), 55.18 (CH3), 54.95 (CH3),
37.35 (C-1), 157.80, 137.95, 128.04, 114.00 (aromatic); 13C NMR-
DEPT spectrum at 135ꢀCH, CH3 [positive (up)], CH2 [negative
120.43 (C-10b), 119.07 (C-9), 117.11 (C-7), 115.66 (CN), 107.24 (C-5), (down)], revealed the following signals at d: 128.04 (aromatic "),
57.64 (C-2), 55.19 (CH3), 37.30 (C-1), 161.73, 142.01, 128.79, 115.47,
127.96 (C-6 "), 125.15 (C-10 "), 118.95 (C-9 "), 117.09 (C-7 "),
115.30 (aromatic); m/z (%): 346 (Mþ, 10.03) with a base peak at
114.00 (aromatic "), 107.21 (C-5 "), 55.18 (CH3 "), 54.95 (CH3 "),
37.35 (C-1 "). In the DEPT spectrum at 90ꢀ only CH signals are
251 (100); Anal. Calcd for C21H15FN2O2: C, 72.82; H, 4.37; N, 8.09.
Found: C, 72.92; H, 4.50; N, 8.22%.
positive (up) and showed d: 128.04 (aromatic "), 127.96 (C-6 "),
125.15 (C-10 "), 118.95 (C-9 "), 117.09 (C-7 "), 114.00 (aromatic "),
107.21 (C-5 "), 37.35 (C-1 "). In the DEPT spectrum at 45ꢀ (CH,
2.2.11. 3-Amino-1-(4-chlorophenyl)-8-methoxy-1H-benzo[f]chromene-
CH2, and CH3 positive) revealed signals at d: 128.04 (aromatic "),
127.96 (C-6 "), 125.15 (C-10 "), 118.95 (C-9 "), 117.09 (C-7 "),
2-carbonitrile 6c
Pale yellow crystals from ethanol; yield 89%; m.p. 247–248 ꢀC; IR
114.00 (aromatic "), 107.21 (C-5 "), 55.18 (CH3 "), 54.95 (CH3 "),
(KBr) t (cmꢁ1): 3461, 3323, 3206 (NH2), 2201 (CN); 1H NMR d: 37.35 (C-1 "); 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2,
7.86–7.10 (m, 9H, aromatic), 7.01 (bs, 2H, NH2), 5.33 (s, 1H, H-1), Cq [negative (down)], revealed the following signals at d: 159.65
3.83 (s, 3H, OCH3); 13C NMR d: 159.81 (C-3), 156.47 (C-8), 145.29
(C-3#), 157.80 (aromatic #), 156.39 (C-8 #), 145.20 (C-4a #), 137.95
(C-4a), 132.21 (C-6a), 128.65 (C-10a), 128.39 (C-6), 125.03 (C-10),
(aromatic #), 132.17 (C-6a #), 128.04 (aromatic "), 127.96 (C-6 "),
120.37 (C-10b), 119.12 (C-9), 117.11 (C-7), 115.33 (CN), 107.31 (C-5),
125.15 (C-10 "), 125.12 (C-10a #), 120.62 (C-10b #), 118.95 (C-9 "),
57.26 (C-2), 55.20 (CH3), 37.39 (C-1), 144.73, 131.11, 128.77, 124.98
117.09 (C-7 "), 116.13 (CN #), 114.00 (aromatic "), 107.21 (C-5 "),
(aromatic); MS m/z (%): 364 (Mþþ2, 3.39), 362 (Mþ, 11.44) with a
base peak at 111 (100); Anal. Calcd for C21H15ClN2O2: C, 69.52; H,
4.17; N, 7.72. Found: C, 69.61; H, 4.27; N, 7.84%.
58.06 (C-2 #), 55.18 (CH3 "), 54.95 (CH3 "), 37.35 (C-1 "); m/z (%):
358 (Mþ, 13.92) with a base peak at 251 (100); Anal. Calcd for
C22H18N2O3: C, 73.73; H, 5.06; N, 7.82. Found: C, 73.61; H, 4.06;
N, 7.70%.
2.2.12. 3-Amino-1-(4-bromophenyl)-8-methoxy-1H-benzo[f]chromene-
2-carbonitrile 6d
2.2.15.
3-Amino-1-(2,4-dimethoxyphenyl)-8-methoxy-1H-benzo[f]
Pale yellow crystals from ethanol; yield 88%; m.p. 261–262 ꢀC; IR
(KBr) t (cmꢁ1): 3457, 3321, 3192 (NH2), 2201 (CN); 1H NMR d:
7.86–7.10 (m, 9H, aromatic), 7.02 (bs, 2H, NH2), 5.32 (s, 1H, H-1),
3.83 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO-d6) d: 159.80 (C-3),
156.47 (C-8), 145.29 (C-4a), 132.21 (C-6a), 128.40 (C-10a), 128.29
(C-6), 125.02 (C-10), 120.37 (C-10b), 119.13 (C-9), 117.10 (C-7),
115.25 (CN), 107.32 (C-5), 57.20 (C-2), 55.20 (CH3), 37.48 (C-1),
145.14, 131.57, 129.16, 119.61 (aromatic); MS m/z (%): 408 (Mþþ2,
1.30), 406 (Mþ, 1.46) with a base peak at 75 (100); Anal. Calcd for
C21H15BrN2O2: C, 61.93; H, 3.71; N, 6.88. Found: C, 61.81; H, 3.59;
N, 6.75%.
chromene-2-carbonitrile 6g
Colourless crystals from ethanol/benzene; yield 85%; m.p.
210–211 ꢀC; IR (KBr) t (cmꢁ1): 3414, 3331, 3203 (NH2), 2196 (CN);
1H NMR d: 7.80–6.36 (m, 8H, aromatic), 6.81 (bs, 2H, NH2), 5.46 (s,
1H, H-1), 3.88 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.69 (s, 3H, OCH3);
13C NMR d: 159.10 (C-3), 156.35 (C-8), 145.59 (C-4a), 131.98 (C-6a),
128.29 (C-10a), 127.72 (C-6), 125.25 (C-10b), 124.46 (C-10), 119.03
(C-9), 116.97 (C-7), 116.27 (CN), 107.25 (C-5), 57.20 (C-2), 55.99
(CH3), 55.17 (CH3), 55.07 (CH3), 31.12 (C-1), 160.12, 156.50, 129.12,
120.54, 105.82, 98.59 (aromatic); 13C NMR-DEPT spectrum at
135ꢀCH, CH3 [positive (up)], CH2 [negative (down)], revealed the
following signals at d: 129.12 (aromatic "), 127.72 (C-6 "), 124.46
(C-10 "), 119.03 (C-9 "), 116.97 (C-7 "), 107.25 (C-5 "), 105.82 (aro-
matic "), 98.59 (aromatic "), 55.99 (CH3 "), 55.17 (CH3 "), 55.07
(CH3 "), 31.12 (C-1 "). In the DEPT spectrum at 90ꢀ only CH signals
are positive (up) and showed d: 129.12 (aromatic "), 127.72 (C-6 "),
124.46 (C-10 "), 119.03 (C-9 "), 116.97 (C-7 "), 107.25 (C-5 "),
105.82 (aromatic "), 98.59 (aromatic "), 31.12 (C-1 "). In the DEPT
spectrum at 45ꢀ (CH, CH2, and CH3 positive) revealed signals at d:
129.12 (aromatic "), 127.72 (C-6 "), 124.46 (C-10 "), 119.03 (C-9 "),
116.97 (C-7 "), 107.25 (C-5 "), 105.82 (aromatic "), 98.59
2.2.13. 3-Amino-8-methoxy-1-(4-methylphenyl)-1H-benzo[f]chromene-
2-carbonitrile 6e
Colourless crystals from ethanol; yield 87%; m.p. 232–233 ꢀC (Lit.
m. p. 232 ꢀC30); IR (KBr) t (cmꢁ1): 3476, 3330, 3208 (NH2), 2203
(CN); 1H NMR d: 7.84–7.01 (m, 9H, aromatic), 6.92 (bs, 2H, NH2),
5.21 (s, 1H, H-1), 3.82 (s, 3H, OCH3), 2.21 (s, 3H, CH3); 13C NMR d:
159.70 (C-3), 156.40 (C-8), 145.26 (C-4a), 132.16 (C-6a), 128.29
(C-10a), 128.08 (C-6), 125.13 (C-10), 120.57 (C-10b), 118.97 (C-9),
117.07 (C-7), 115.96 (CN), 107.21 (C-5), 57.89 (C-2), 55.18 (CH3), (aromatic "), 55.99 (CH3 "), 55.17 (CH3 "), 55.07 (CH3 "), 31.12
(C-1 "); 13CNMR-APT spectrum CH, CH3 [positive (up)], CH2, Cq
37.79 (C-1), 20.51 (CH3), 142.85, 135.60, 129.18, 126.82 (aromatic);
m/z (%): 342 (Mþ, 82.13) with a base peak at 241 (100); Anal.
[negative (down)], revealed the following signals at d: 160.12 (aro-
Calcd for C22H18N2O2: C, 77.17; H, 5.30; N, 8.18. Found: C, 77.01; H,
5.27; N, 8.08%.
matic #), 159.10 (C-3 #), 156.50 (aromatic #), 156.35 (C-8 #), 145.59
(C-4a #), 131.98 (C-6a #), 129.12 (aromatic "), 128.29 (C-10a #),
127.72 (C-6 "), 125.25 (C-10b #), 124.46 (C-10 "), 120.54 (aromatic #),
119.03 (C-9 "), 116.97 (C-7 "), 116.27 (CN #), 107.25 (C-5 "), 105.82
2.2.14. 3-Amino-8-methoxy-1-(4-methoxyphenyl)-1H-benzo[f]chromene-
(aromatic "), 98.59 (aromatic "), 57.20 (C-2 #), 55.99 (CH3 "), 55.17
(CH3 "), 55.07 (CH3 "), 31.12 (C-1 "); MS m/z (%): 388 (Mþ, 84.51)
2-carbonitrile 6f
Yellow needles from ethanol; yield 85%; m.p. 222–223 ꢀC (Lit. m. p. with a base peak at 208 (100); Anal. Calcd for C23H20N2O4: C, 71.12;
220 ꢀC30); IR (KBr) t (cmꢁ1): 3447, 3331, 3213 (NH2), 2200 (CN); 1H H, 5.19; N, 7.21. Found: C, 71.00; H, 5.05; N, 7.09%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1079
Table 1. Cytotoxic activity of the target compounds against MCF-7, HCT-116,
2.2.16.
3-Amino-1-(3,4-dimethoxyphenyl)-8-methoxy-1H-benzo[f]
and HepG-2 cell lines.
chromene-2-carbonitrile 6h
MeO
Br
Ar
Colourless crystals from ethanol/benzene; yield 81%; m.p.
200–201 ꢀC; IR (KBr) t (cmꢁ1): 3431, 3337, 3219 (NH2), 2185 (CN);
1H NMR d: 7.83–6.57 (m, 8H, aromatic), 6.92 (bs, 2H, NH2), 5.22 (s,
1H, H-1), 3.82 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 3.66 (s, 3H, OCH3);
13C NMR d: 159.69 (C-3), 156.40 (C-8), 145.24 (C-4a), 132.15 (C-6a),
128.28 (C-10a), 128.07 (C-6), 125.24 (C-10), 120.67 (C-10b), 118.94
(C-9), 117.05 (C-7), 115.99 (CN), 107.19 (C-5), 58.00 (C-2), 55.45
(CH3), 55.42 (CH3), 55.16 (CH3), 37.72 (C-1), 148.63, 147.43, 138.43,
Ar
CN
CN
O
NH2
O
NH2
4a–h
6a–h
IC50 (mg/mL)a
Compound
Ar
MCF-7
HCT-116
HepG-2
HFL-1
13
112.11, 111.04 (aromatic); C NMR-DEPT spectrum at 135ꢀCH, CH3
4a
4b
4c
4d
4e
4f
4g
4h
6a
6b
6c
6d
6e
6f
C6H5
4-FC6H4
4-ClC6H4
0.9 0.29
5.2 0.03
0.5 0.23
2.5 0.14
2.5 0.18
1.2 0.97
1.0 0.12
0.7 0.11
0.5 0.35
0.7 0.25
0.7 0.5
0.8 0.12
1.1 0.12 15.2 0.10
1.8 0.2
0.9 0.14 22.4 0.21
4.4 0.05
2.8 0.17
11.0 0.02 12.9 0.13
11.2 0.18
0.5 0.01
[positive (up)], CH2 [negative (down)], revealed the following sig-
nals at d: 128.07 (C-6 "), 125.24 (C-10 "), 118.96 (aromatic "),
118.94 (C-9 "), 117.05 (C-7 "), 112.11 (aromatic "), 111.04 (aromatic
"), 107.19 (C-5 "), 55.45 (CH3 "), 55.42 (CH3 "), 55.16 (CH3 "), 37.72
(C-1 "). In the DEPT spectrum at 90ꢀ only CH signals are positive
(up) and showed d: 128.07 (C-6 "), 125.24 (C-10 "), 118.96 (aro-
matic "), 118.94 (C-9 "), 117.05 (C-7 "), 112.11 (aromatic "), 111.04
(aromatic "), 107.19 (C-5 "), 37.72 (C-1 "). In the DEPT spectrum at
45ꢀ (CH, CH2, and CH3 positive) revealed signals at d: 128.07 (C-6
"), 125.24 (C-10 "), 118.94 (C-9 "), 118.96 (aromatic "), 117.05 (C-7
"), 112.11 (aromatic "), 111.04 (aromatic "), 107.19 (C-5 "), 55.45
(CH3 "), 55.42 (CH3 "), 55.16 (CH3 "), 37.72 (C-1 "); 13CNMR-APT
spectrum CH, CH3 [positive (up)], CH2, Cq [negative (down)],
revealed the following signals at d: 159.69 (C-3 #), 156.40 (C-8 #),
148.63 (aromatic #), 147.43 (aromatic #), 145.24 (C-4a #), 138.43
(aromatic #), 132.15 (C-6a #), 128.28 (C-10a #), 128.07 (C-6 "),
125.24 (C-10 "), 120.67 (C-10b #), 118.94 (C-9 "), 117.05 (C-7 "),
115.99 (CN #), 112.11 (aromatic "), 111.04 (aromatic "), 107.19 (C-5
"), 58.00 (C-2 #), 55.45 (CH3 "), 55.42 (CH3 "), 55.16 (CH3 "), 37.72
(C-1 "); MS m/z (%): 388 (Mþ, 16.00) with a base peak at 251 (100);
Anal. Calcd for C23H20N2O4: C, 71.12; H, 5.19; N, 7.21. Found: C,
70.99; H, 5.02; N, 7.07%.
4-BrC6H4
4-MeC6H4
4-MeOC6H4
2,4-MeOC6H3
3,4-MeOC6H3 0.66 0.04
C6H5
4-FC6H4
4-ClC6H4
4-BrC6H4
4-MeC6H4
4-MeOC6H4
3.8 0.36 44.9 0.14
3.6 0.01
3.7 0.15
4.1 0.16
2.1 0.23
4.9 0.01
2.5 0.97
0.9 0.58
0.8 0.06
3.8 0.14
2.7 0.2
4.4 0.05
3.1 0.29
2.0 0.25
0.3 0.05
0.6 0.08
0.3 0.08
0.7 0.13 35.5 0.22
0.6 0.21 29.7 0.17
29.3 0.12
6g
6h
2,4-MeOC6H3 16.0 0.92 10.9 0.5
3,4-MeOC6H3 41.1 0.25 13.9 0.3
9.0 0.3
30.7 0.5
Vinblastine
Doxorubicin
–
–
6.1 0.03
0.4 0.01
2.6 0.08
0.5 0.015
4.6 0.01
0.9 0.04
aIC50 values expressed in mg/mL as the mean values of triplicate wells from at
least three experiments and are reported as the mean standard error.
2.3. Biological screening
2.3.1. Cell culture and cytotoxicity evaluation using viability assay
Compounds 4a–h and 6a–h were initially evaluated for in vitro
antitumour activity against three different human cell lines: MCF-7,
HCT-116, and HepG-2, and only the representative active cytotoxic
compounds were further evaluated against the normal HFL-1 cell
line. The in vitro cytotoxicity evaluation was performed at the
Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar
University under different concentrations (50, 25, 12.5, 6.25, 3.125,
1.56, and 0 mg/mL); Vinblastine and Doxorubicin are used as refer-
ence cytotoxic compounds. The measurements of cell growth and
the in vitro cytotoxicity evaluation were determined using viability
assay as described in literature31–33 and the result was cited in
Table 1 and Figure 2.
2.3.2. Cell invasion assay
The cell invasion assay was performed by Reaction Biology
Corporation, One Great Valley Parkway, PA. All compounds were
dissolved in DMSO and tested for their ability to cell invasion
assay. Compounds 4c, 6e, and reference compound bosutinib
were purchased from Sellechem (Houston, TX). Corning Bio Coat
Figure 2. Heatmap of the distribution of IC50 values with colour codes of target
compounds against the panel of three cancer cell lines.
Fluoro Blok Insert System-96-well plates were purchased from bovine serum (FBS), 100 mg/mL of penicillin, and 100 mg/mL of
Corning (Corning Incorporated, Tewksbury, MA). Calcein AM label- streptomycin. Cultures were maintained at 37 ꢀC in a humidified
ling reagent was purchased from Molecular Probes (Eugene, atmosphere of 5% CO2 and 95% air-following incubation; medium
Oregon). MDA-MB-231 cell line was purchased from American was carefully removed from the apical chambers. The insert
Type Culture Collection (Manassas, VA). MDA-MB-231 cells system was transferred into a second 96-square well plate contain-
were grown in DMEM medium supplemented with 10% foetal ing 200 ml/well of 4 mg/mL Calcein AM in HBSS. The plates were

1080
H. E. A. AHMED ET AL.
incubated for 1 h at 37 ꢀC, 5% CO2. Fluorescence of invaded cells 2.4. Molecular docking
was read at wavelengths of 492/535 nm (Ex/Em) from bottom
Molecular docking simulation was carried out with the program
reading using Envision 2104 Multilabel Reader (PerkinElmer, Santa
Clara, CA). The IC50 curves were plotted and IC50 values were
calculated using the Graph Pad Prism 4 program for windows
(Graph Pad software, San Diego California. www.graphpad.com)
based on a sigmoidal dose-response equation.
AutoDock 4.0.134 (GNU General Public License). The three-dimen-
sional (3D) structure of c-Src kinase was obtained from Protein
Data Bank (PDB entry no. 2src). The ligand and solvent molecules
were removed from the crystal structure to obtain the docking
grid and the active site was defined using AutoGrid. The grid box
was centred on the centre of the ligand from the corresponding
crystal structure complexes. The Lamarckian genetic algorithm
issued for docking with the following settings: a maximum num-
2.3.3. Caspase 3/7 activity assay
The Caspase-Glo 3/7 activity assay was performed by Reaction ber of 2,500,000 energy evaluations, an initial population of 50
randomly placed individuals, a maximum number of 37,000 gener-
ations, a mutation rate of 0.02, across over rate of 0.80, and an
elitism value (number of top individuals that automatically survive)
of 1. The ligand was fully optimised inside the binding site during
the docking simulations. Finally, the conformation with the lowest
predicted binding free energy of the most occurring binding
modes in c-Src active pocket was selected. Hydrogen atoms were
added to the structure with the Molecular Operating Environment
(MOE 2012)35 and atomic partial charges were calculated using
Auto-Dock Tools. Selected active compounds were docked into
the active site of microorganisms' targets to predict compound
binding modes. For flexible docking, Auto-Dock standard param-
eter settings were applied. High-scoring binding poses were
selected on the basis of visual inspection.
Biology Corporation, One Great Valley Parkway, PA. Examined
compounds 4c, 4e, 4h, 6d, and 6e were dissolved in DMSO as
20 mM stock and tested for their ability to Caspase-Glo 3/7 activity
assay. The reference compound staurosporine was purchased from
Sigma-Aldrich (Saint Louis, MO) and dissolved in DMSO in 10 mM
stock. Caspase-Glo 3/7 activity assay reagent was purchased from
Promega (Madison, WI). MDA-MB-231 cell line was provided by
ATCC. MDA-MB-231 cell line culture conditions: DMEM with 10%
FBS. 100 mg/mL penicillin and 100 mg/mL streptomycin were added
to the culture media. Cultures were maintained at 37 ꢀC in a
humidified atmosphere of 5% CO2 and 95% air.
2.3.4. c-Src kinase assay
The effect of the synthesised compounds on the activity of c-Src
kinase was determined by HTScan Src Kinase Assay Kit, catalogue
number 7776 from Cell Signaling Technology (Danvers, MA);
according to the manufacturer’s protocol. Streptavidin-coated
plates were purchased from Pierce (Rockford, IL). In brief, the kin-
ase reaction was started with the incubation of the 12.5 mL of the
reaction cocktail (0.5 ng/mL of GST-Src kinase in 1.25 mM DTT) with
12.5 mL of prediluted compounds (dissolved in 1% DMSO) for
5 min at room temperature. ATP/substrate (25 mL, 20 mM/1.5 uM)
cocktail was added to the mixture. The biotinylated substrate
(catalogue number 1366) contains the residues surrounding tyro-
sine 160 (Tyr160) of signal transduction protein and has a
sequence of EGIYDVP. The reaction mixture was incubated for
30 min at room temperature. The kinase reaction was stopped
with the addition of 50 mL of 50 mM EDTA (pH 8.0). The reaction
solution (25 mL) was transferred into 96-well streptavidin plates
(Pierce, part number 15125), diluted with 75 mL double distilled
water, and incubated at room temperature for 60 min. At the end
of the incubation, the wells were washed three times with 200 mL
of 0.05% Tween-20 in PBS buffer (PBS/T). After that to each well
was added 100 mL of phosphotyrosine antibody (P-Tyr-100) (1:1000
dilution in PBS/T with 1% BSA) and the wells were incubated for
another 60 min. After washing three times with 0.05% Tween-20 in
PBS/T, the wells were incubated with 100 mL secondary anti-mouse
IgG antibody, which was HRP-conjugated (1:500 dilution in PBS/T
with 1% BSA) for next 30 min at room temperature. The wells
were washed five times with 0.05% Tween-20 in PBS and then
were incubated with 100 mL of 3,30,5,50-tetramethylbenzidine
dihydrochloride substrate for 5 min. The reaction was stopped by
adding 100 mL/well of stop solution to each well and mixed well
and read the absorbance at 450 nm using a microplate reader
(Molecular devices, spectra Max M2). IC50 values of the compounds
were calculated using ORIGIN 6.0 (Origin Lab Corporation,
Northampton, MA) software. IC50 is the concentration of the com-
pound that inhibited enzyme activity by 50%. All the experiments
2.5. Statistical analysis
All statistical calculations were done using computer programs,
Microsoft excel version 10, SPSS version 20.00 (SPSS Inc., IBM in
2009, Chicago, IL) statistical program at 0.05, 0.01, and 0.001 level
of probability36
. Comparisons of inhibiting tumour growth
between treatments groups or the control were done using
Student's t test, one-way ANOVA, and post hoc the least significant
difference tests measurement.
3. Results and discussion
3.1. Chemistry
Benzochromene molecules have been synthesised using the syn-
thetic strategy illustrated in Schemes 1 and 2. In a one-pot three
component heterocyclocondensation process, 3-amino-1-aryl-8-
bromo-1H-benzo[f]chromene-2-carbonitrile 4a–h were obtained via
the reaction of 6-bromo-2-naphthol 1 with a mixture of appropri-
ate aromatic aldehydes 2a–h and malononitrile 3 in ethanol/
piperidine solution under microwave irradiation conditions for
2 min at 140 ꢀC.
The IR spectra of compounds 4a–h revealed the presence of
three peaks at t 3477–3409, 3369–3321, 3217–3199 cmꢁ1, and one
peak at t 2204–2181 cmꢁ1 which are assigned to the amino and
cyano groups, respectively. In the meantime, the 1H NMR spectra
of compounds 4a–h displayed a singlet signal of the 1H protons
in the range of d 5.47–5.26 ppm and a broad signal of the amino
group in the range of d 7.10–6.90 ppm. In addition, compounds
4a and 4e–h revealed the presence of D2O as a replacement to
the NH2 proton at d 7.04–6.90 ppm. Furthermore, the 1H NMR
spectra of compounds 4e and 4f–h exhibited signals of the
methyl and methoxy groups at d 2.21 and 3.85–3.67 ppm, respect-
ively. On the other hand, the 13C NMR spectra of compounds
4a–h displayed one signal of the chiral carbon resonating at d
37.89–31.26 ppm, and compounds 4f–h exhibited signals of the
methyl and methoxy groups at d 20.51 and 55.96–54.96 ppm,
were carried out in triplicate24
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1081
9
Br
10
Ar
8
10a
10b
1
c
OH
+
CN
7
2
3
MW / 400 W / 2 min.
EtOH / pip.
e
d
f
6a
6
+
CH2(CN)2
b
Ar
-CH
O
Oa NH2
Br
4a
4a–h4
5
1
2a–h
3
a; Ar = C6H5
b; Ar = 4-FC6H4
c; Ar = 4-ClC6H4
d; Ar = 4-BrC6H4
e; Ar = 4-MeC6H4
f; Ar = 4-MeOC6H4
g; Ar = 2,4-MeOC6H3
h; Ar = 3,4-MeOC6H3
Scheme 1. Synthesis of 3-amino-1-aryl-6-bromo-1H-benzo[f]chromene-3-carbonitriles (4a–h).
9
MeO
10
Ar
8
10a
10b
1
c
OH
CN
7
2
3
MW / 400 W / 2 min.
e
d
f
6a
+
+
CH2(CN)2
Ar-
CHO
b
Oa NH2
EtOH / pip.
6
MeO
4a
5
6a–h4
5
2a–h
3
a; Ar = C6H5
b; Ar = 4-FC6H4
c; Ar = 4-ClC6H4
d; Ar = 4-BrC6H4
e; Ar = 4-MeC6H4
f; Ar = 4-MeOC6H4
g; Ar = 2,4-MeOC6H3
h; Ar = 3,4-MeOC6H3
Scheme 2. Synthesis of 3-amino-1-aryl-8-methoxy-1H-benzo[f]chromene-3-carbonitriles (6a–h).
13
respectively. Also, the C NMR-DEPT spectra at 45ꢀ, 90ꢀ, and 3.2. Antitumour assay
135 ꢀC as well as the 13C NMR-APT spectra of compounds 4b,
4e–h provided additional evidence to support the proposed struc-
tures (See supplemental data).
The in vitro cytotoxic evaluation of the prepared compounds 4a–h
and 6a–h was performed against MCF-7 (breast cancer), HCT-116
(human colon cancer), HepG-2 (liver cancer), and HFL-1(normal
fibroblast lung) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) colorimetric assay as described in litera-
ture31–33. Doxorubicin, one of the most effective anticancer agents,
and the cancer drug Vinblastine was included in the experiment
as positive controls. The results were expressed as growth inhibi-
tory concentration (IC50) values, which represent the compounds’
concentrations required to produce a 50% inhibition of the cell
growth after 24 h of incubation, compared to the untreated con-
trols as shown in Table 1 and Figure 2.
According to the obtained results, it was obvious that most of
the synthesised compounds displayed excellent anti-proliferative
activity against the tested cancer cell lines. Investigations of the
cytotoxic activity against the three cancer cell lines, MCF-7, HCT-
116, and HepG-2, indicated that the HepG-2 was the most sensi-
tive cell line to the influence of the target compounds. In the
meantime, the treatment of the MCF-7 cancer cell lines was very
successful using the first series compounds, 4a–h which displayed
IC50 values in the range of 0.5–5.2 mg/mL. The same effect was
obtained using compounds 6a–f of the second series which exhib-
ited IC50 values between 0.3 and 4.4 mg/mL, Table 1. These values
were found to be more potent and efficacious against MCF-7 can-
cer cell lines than Vinblastine (IC50 ¼ 6.1 mg/mL) while compound
6e (IC50 ¼ 0.3 mg/mL) was found to be the most potent derivative
against MCF-7 as compared to Doxorubicin (IC50 ¼ 0.4 mg/mL);
compounds 4c and 6f (IC50¼ 0.5 and 0.6 mg/mL) are almost
equipotent as Doxorubicin and emerged as the most potent coun-
terpart against MCF-7 in this study. Concerning the cytotoxic activ-
ity against HCT-116, compounds 4a–e of the first series with IC50
values of 0.5–1.2 mg/mL and compounds 6a, 6c–e with IC50 in the
The same methodology has been employed to prepare the
second series of benzochromene molecules; compounds 6a–h,
Scheme 2 illustrated the interaction of 6-methoxy-2-naphthol 5
with a mixture of appropriate aromatic aldehydes, 2a–h, and
malononitrile 3 to afford the corresponding 3-amino-1-aryl-8-
methoxy-1H-benzo[f]chromene-2-carbonitriles, 6a–h. The max-
imum power of the microwave irradiation was optimised by
repeating the reaction at different watt powers and periods of
time. The optimum condition was obtained by using the micro-
wave irradiations at 400 W and reaction time 2 min, which gave
the highest yield of the desired compounds. It is also important to
note that the 1-position of compounds 4a–h and 6a–h is a chiral
centre and all the reactions were controlled using the
TLC technique.
The IR spectra of the target compounds 6a–h confirmed the
presence of the characteristics of the NH2 absorption bands
around t 3475–3414, 3337–3321, 3219–3192 cmꢁ1 and the CN
absorption bands around t 2203–2185 cmꢁ1. The 1H NMR spectra
of 6a–h showed singlet signals of 1H, NH2, and methyl or methoxy
groups protons at
d 5.46–5.21, 7.02–6.81 and 2.21, and
3.88–3.66 ppm, respectively. Furthermore, the 13C NMR spectra of
compounds 6a–h showed signals resonating in the range of d
38.15–31.12 ppm which are attributed to the chiral carbon, while
the carbons of the methyl and methoxy groups appeared in the
range of d 20.51 and 55.45–54.95 ppm, respectively. Besides, the
13C NMR-DEPT spectra at 45ꢀ, 90ꢀ, and 135 ꢀC as well as the 13C
NMR-APT spectra of compounds 6f–h provided additional evi-
dence in supporting the proposed structures (See supplemen-
tal data).

1082
H. E. A. AHMED ET AL.
range of 0.8–2.5 mg/mL displayed significant growth inhibitory
activity in comparison to Vinblastine (IC50 ¼ 2.6 mg/mL). In particu-
lar, compound 4d (IC50 ¼ 0.5 mg/mL) was equipotent as
Doxorubicin (IC50 ¼ 0.5 mg/mL). The same effect has also been
achieved using compounds 4c, 4e, 6d, and 6e with IC50 between
0.7 and 0.9 mg/mL. On the other hand, the cytotoxic evaluation
against HepG-2 cell lines revealed that the second series elicited
considerable anti-proliferative activity than the first series. For
instance, seven members of the first series 4a–g with IC50 values
in the range of 0.5–4.4 mg/mL and four members of the second
series 6b–e with IC50 values between 0.3 and 0.7 mg/mL were
more potent and efficient than Vinblastine (IC50 ¼ 4.6 mg/mL) while
compounds 6b–e, 4a, and 4b with IC50 ranging from 0.3 to
0.8 mg/mL displayed significant growth inhibitory activity in com-
parison to Doxorubicin (IC50 ¼ 0.9 mg/mL). Compound 4e
(IC50 ¼ 0.9 mg/mL) was equipotent as Doxorubicin. In addition, the
potent compounds 4c, 4e, 4h, 6d, and 6e were evaluated for their
cytotoxic effect against normal fibroblast cell line. The obtained
data revealed that all compounds displayed weak cytotoxicity
(IC50 ¼ 13–35 mg/mL) compared to their anticancer effect, proving
the selectivity behaviour. Finally, the remaining compounds exhib-
ited equipotent or moderate to fair cytotoxic activities against the
three tumour cell types compared to Vinblastine and Doxorubicin,
and the highly active ones could be considered as selective anti-
cancer agents.
Cell Invasion Assy (MDA-MB-231 cell line)
200
Bosutinib (M)
4C (M)
150
100
50
6E (M)
0
−14 −12 −10
−8
−6
−4
−2
Log (M)
Figure 3. The effect of selected benzochromene derivatives on the invasion of
MDA-MB-231 cell line.
3.5. c-Src kinase assay
As a biological step for the discovery of their anti-proliferative
mechanism, further c-Src kinase inhibitory activity of the potent
compounds 4c, 4e, 4h, 6d, and 6e were performed, Table 4. It
is clearly presented that their inhibitory activity is potent and
the IC50 values ranged between 9 and 329 nM, which mostly
exhibited higher activity than the reference drug. For instance,
compound 4e, with the 4-methylphenyl substitution, exhibited
higher c-Src kinase inhibitory activity (IC50 ¼ 9 nM) than the rest.
Similarly, compound 6e, with the 4-methylphenyl substitution,
was the most effective compound in the 8-methoxy series with
an IC50 value of 9 nM. Additionally, 3,4-dimethoxyphenyl and 4-
bromophenyl substituted compounds 4 h and 6d were among
the moderately effective inhibitors in both the 8-bromo and the
8-methoxy series of compounds with IC50 ranging from 214 to
329 nM. Lastly, the substitution of the aryl terminal moiety with
the electron-donating group (methyl group) in compounds 4e
and 6e led to increase in inhibitory activity, suggesting that
substitution at position 4 of the phenyl ring is detrimental on
the c-Src kinase inhibition.
3.3. Cell invasion assay
Tumour invasion is a common feature of triple-negative breast
cancers. Triple-negative breast cancers (12–24% of breast can-
cers)37,38 which are characterised by the absence of ER, progester-
one receptors, and HER-2 expression, resulted in high morbidity
and mortality owing to their rapid growth rate, invasive potential,
and frequently acquired treatment resistance. The invasion process
has become an important prerequisite for cancer progression and
metastasis. Therefore, therapeutic strategies for preventing or sup-
pressing cancer invasion and metastasis can significantly improve
the survival of triple-negative breast cancer patients. The two
most active compounds 4c and 6e were examined for their ability
to inhibit the invasiveness of the highly sensitive and invasive
breast cancer cell line, MDA-MB-231. A bosutinib drug was used as
a control ant-invasive agent. As shown in Figure 3, 4c and 6e
derivatives have markedly suppressed the invasion of the MDA-
MB-231 cell up to the highest percentage. As shown in Table 2,
they showed comparable potent ant-invasion activity with IC50 of
10.5 and 16.5 mM, respectively. The obtained data might confirm
that the cytotoxicity of novel molecules led to the death of the
cancer cells and stopped the metastatic processes.
3.6. SAR studies
The preliminary SAR study has focussed on the effect of the pres-
ence of the phenyl ring on the antitumour activities of the syn-
thesised compounds. The influence of replacing the H atom at the
4-position in the phenyl ring of the 1H-benzo[f]chromene moiety
with the electron-withdrawing (F, Cl, Br) or electron-donating (Me,
MeO) groups, and the presence of a bromine atom (electron-with-
drawing) or methoxy group (electron-donating) at the 8-position
has also been discussed. In a comparison of the cytotoxic activities
of the two series 4a–h and 6a–h against MCF-7 breast cancer, we
found that the highest growth inhibitory effect of the first series
4a–h was associated with the presence of 4-chlorophenyl 4c, 3,4-
dimethoxyphenyl 4h, unsubstituted phenyl 4a, 4-bromophenyl 4d,
4-methylphenyl 4e, 2,4-dimethoxyphenyl 4g, 4-methoxyphenyl 4f,
and 4-fluorophenyl 4b, respectively. As previously mentioned,
these molecules showed IC50 values in the range of 0.5–5.2 mg/mL
against MCF-7, which are considered excellent activities relative to
Vinblastine (IC50 ¼ 6.1 mg/mL), Table 5. On the other hand, the
inhibitory effect of the second series 6a–h was more potent and
3.4. Caspase 3/7 activity assay
Caspases are ATP-dependent enzymes that promote apoptosis.
Caspases are vital biochemical features of apoptotic signalling. The
apoptotic response of MDA-MB-231 cells to potent anti-prolifera-
tive compounds was examined via quantifying the activity of cas-
pases 3/7. To determine whether the cell growth inhibition in
response to the benzochromene derivatives was mediated by
apoptosis and was caspase dependent, MDA-MB-231 cells were
treated with 10 different concentrations of the target compounds efficacious than Vinblastine (IC50 ¼ 6.1 mg/mL). The IC50 values of
for 24 h. As shown in Figure 4, caspase-3/7 activity was activated these molecules were between 0.3 and 4.4 mg/mL against MCF-7,
in a dose-dependent manner. The target benzochromene com- and their effect was as follows: 4-methylphenyl 6e, 4-methoxy-
pounds significantly increased caspase-3/7 activity to a 10-fold at phenyl 6f, 4-bromophenyl 6d, un-substituted phenyl 6a, 4-chloro-
a certain concentration and time point, Table 3 and Figure 4.
phenyl 6c, and 4-fluorophenyl 6b, respectively, Table 5. According

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1083
Figure 4. Apoptosis induction by caspase-3/7 activity assay detection in MDA-MB-231 cell line after compound treatment against reference drug.
Table 2. IC50 values for two most potent
ant-invasion compounds in MDA-MB-231
breast cancer cells.
Table 5. Positive and negative controls and effectiveness of the test compounds
against MCF-7.
Control/Cpd.
IC50 (lg/mL)
Cell line
F ratio
p Value
Compound ID
IC50 (M)
Vinblastine
6.1 0.03
0.5 0.23
0.66 0.04
0.9 0.29
2.5 0.14
2.5 0.18
3.6 0.01
3.8 0.36
5.2 0.03
0.3 0.05
0.6 0.08
2.0 0.25
2.7 0.2
4c
6e
1.08 ꢂ 10ꢁ5
4c
4h
4a
4d
4e
4g
4f
4b
6e
6f
6d
6a
6c
6b
1.65 ꢂ 10ꢁ5
Table 3. Caspase-Glo 3/7 activity assay.
MCF-7
221.234
0.000 (HS)
Compound ID
MDA-MB-231 cell line, IC50 (M)
4c
4e
4h
6d
2.26 ꢂ 10ꢁ5
1.95 ꢂ 10ꢁ5
3.15 ꢂ 10ꢁ5
2.56 ꢂ 10ꢁ5
3.89 ꢂ 10ꢁ5
6.85 ꢂ 10ꢁ8
6e
Staurosporine
3.1 0.29
4.4 0.05
Positive control (active compounds) and negative control (standard drugs).
All statistical calculations were done as the mean values of triplicate.
HS: Highest significantly at p values <0.05.
Table 4. Inhibition of c-Src kinase activity
data as IC50 values (nM) of selected
1H-benzo[f]-chromene derivatives.
Compound ID
IC50 (nM)
Table 6. Positive and negative controls and effectiveness of the test compounds
against HCT-116.
4c
4e
4h
6d
89
9
214
329
9
Control/Cpd.
IC50 (lg/mL)
Cell line
F ratio
p Value
Vinblastine
2.6 0.08
0.5 0.35
0.7 0.11
0.7 0.25
1.0 0.12
1.2 0.97
0.8 0.06
0.9 0.58
2.1 0.23
2.5 0.97
6e
4d
4c
4e
4b
4a
6e
6d
6a
6c
Staurosporine
112
to the obtained data and regarding the first series, it was
found that the replacement of the H atom by the chlorine atom
(electron-withdrawing group) at the 4-position in the phenyl ring
of the 1H-benzo[f]chromene moiety was significant for its activity
against the MCF-7 compared to the dimethoxy (electron-donating
group) at 3,4-positions on the same ring with bromine atom at
the 8-position. However, the introduction of a methyl or methoxy
group at 4-position (electron-donating group) on the phenyl ring,
at position 1 of the second series is more beneficial than an elec-
tron-withdrawing substituent like Br, Cl, or F atoms, taking into
consideration that the second series 6a–h (more electron-donating
group) exhibited significant growth inhibitory activity against
MCF-7 which is more effective than the first series 4a–h (more
electron-withdrawing group). In general, the SAR image of the
novel potent derivatives indicates that the presence of different
pharmacophoric functionalities with small hydrophobic moieties as
halogens or slightly hydrophobic fragments as methoxy play an
important role in the cytotoxic activity.
HCT-116
57.243
0.000 (HS)
Positive control (active compounds) and negative control (standard drugs).
All statistical calculations were done as the mean values of triplicate.
HS: Highest significantly at p value <0.05.
Investigations of the cytotoxic activity against HCT-116, human
colon cancer, indicated that compounds 4a–e of the first series
with IC50 ¼ 0.5–1.2 mg/mL and compounds 6a,c–e of the second ser-
ies with IC50 ¼ 0.8–2.5 mg/mL displayed a good inhibitory activity
compared to Vinblastine (IC50 ¼ 2.6 mg/mL), Table 6. These results are
intimate that the grafting of a lipophilic electron-withdrawing sub-
stituent (halogen atoms) is more beneficial than an electron-donating
substituent (methyl or unsubstituted phenyl group) for the activity of
the bromine atom or the methoxy group at the 8-position.

1084
H. E. A. AHMED ET AL.
Table 7. Positive and negative controls and effectiveness of the test compounds
against HepG-2.
Moreover, the first series 4a–h (electron-withdrawing group) revealed
significant growth inhibitory activity against HCT-116 in comparison
to the second series 6a–h (electron-donating group).
Regarding the activity against the HepG-2 cell line (liver cancer),
the order of the antitumour activity of the first series was found to
be 4a> 4b> 4e> 4c> 4d> 4g> 4f (IC50 ¼ 0.7–4.4 mg/mL) and for
the second series 6c> 6b> 6e> 6d, with IC50 ¼ 0.3–0.7 mg/mL,
Control/Cpd.
IC50 (lg/mL)
Cell line
HepG-2
F ratio
p Value
Vinblastine
4.6 0.18
0.7 0.06
0.8 0.06
0.9 0.06
1.1 0.08
1.8 0.12
2.8 0.11
4.4 0.09
0.3 0.02
0.5 0.03
0.6 0.01
0.7 0.04
0.9 0.05
0.7 0.08
0.8 0.03
0.9 0.06
0.3 0.08
0.5 0.06
0.6 0.04
0.7 0.02
270.518
0.000 (HS)
4a
4b
4e
4c
Table 7. These results suggested that the un-substituted phenyl 4d
4g
4f
6c
6b
6e
6d
(electron-donating group) of the first series is preferred as an antitu-
mor agent in comparison to the substituted phenyl (electron-with-
drawing group) with bromine atom at the 8-position; however, the
electron-withdrawing substituent is preferred compared to electron-
donating substituent for the second series with methoxy group
at the 8-position, keeping in mind that the second series, 6a–h
(electron-withdrawing and electron-donating groups), is more
effective towards the HepG-2 than the first series, 4a–h (electron-
donating and electron-withdrawing groups).
On the other hand, the rest of the compounds showed almost
equipotent or moderate to fair cytotoxic activities against the
three tumour cell lines compared to Vinblastine. While compound
6e (IC50 ¼ 0.3 mg/mL) was found to be the most potent derivative
against MCF-7 compared to Doxorubicin (IC50 ¼ 0.4 mg/mL) as
shown in Table 8. Compound 4c (IC50 ¼ 0.5 mg/mL) was almost as
equipotent as Doxorubicin, and compound 4d (IC50 ¼ 0.5 mg/mL)
was equipotent as Doxorubicin against HCT-116. Furthermore,
compounds 6c, 6b, 6e, 4a, and 4b with IC50 ¼ 0.3–0.8 mg/mL dis-
played significant growth inhibitory activity against HepG-2 in
comparison to Doxorubicin (IC50 ¼ 0.9 mg/mL) as shown in Table 7,
and compound 4e (IC50 ¼ 0.9 mg/mL) was equipotent as
Doxorubicin indicating that the electron-withdrawing substituent
is preferred for antitumour activity compared to the electron-
donating substituent.
Doxorubicin
HepG-2
8.210
0.000 (HS)
4a
4b
4e
6c
6b
6e
6d
Positive control (active compounds) and negative control (standard drugs).
All statistical calculations were done as the mean values of triplicate.
HS: Highest significantly at p value <0.05.
Table 8. Positive and negative controls and effectiveness of the test compound
against MCF-7.
Control/Cpd.
IC50 (lg/mL)
Cell line
MCF-7
T test
p Value
Doxorubicin
6e
0.4 0.01
0.3 0.05
1.225
0.288 (NS)
Positive control (active compounds) and negative control (standard drugs).
All statistical calculations were done as the mean values of triplicate.
NS: not significant at p values >0.05.
Finally, we can deduce that the substitution pattern of the phenyl
group at the 1-position of the 1H-benzo[f]chromene moiety played a
vital role in its antitumour activity. The incorporation of halogen
atoms (electron-withdrawing group) has also greatly enhanced the
activity of the desired compounds in comparison to the electron-
donating groups as methyl or methoxy groups with bromine atom or
methoxy group at the 8-position. Overall, the growth inhibitory activ-
ity of the second series exceeded the first one.
a donor with all nitrile fragments of the structures. However, in
some cases, the interaction was supported with extra hydrogen
bonds with the carbonyl group of Glu 339 as an acceptor through
the NH2 fragment. Moreover, Asp 404 and Lys 295 exhibited
additional hydrogen bonds through the OCH3 substituents. In add-
ition, a hydrophobic interaction was found between the naphtha-
lene substructure of compounds within the hydrophobic pockets
in the active site, Figure 5 and Table 9. Finally, the hydrogen-
bonding donor-acceptor motif which was proposed as a crucial
key for the kinase inhibitory activity was accomplished by the
active compounds fragments with the corresponding Met 341 or
Glu 399 residues45. This behaviour towards c-Src kinase might con-
firm the cytotoxic effect of the studied compounds is mediated
through the inhibition of such promising target.
3.7. Molecular docking
c-Src kinase is a non-receptor tyrosine kinase which plays essential
roles in controlling a myriad of fundamental cellular processes,
including cell proliferation, adhesion, migration, and invasion39,40
.
c-Src kinase is overexpressed and/or is aberrantly activated in a
variety of human tumours, including breast, colon, pancreatic,
bladder, ovarian, and brain carcinomas41,42. Notably, increased c-
Src activity is believed to correlate strongly with the metastatic
potential and the poor prognosis in breast cancers41,43,44. Thus, c-
Src kinase has been recognised as an attractive molecular target
for cancer therapy, and the development of c-Src inhibitors for
cancer treatment is an increasing interest. In this report, we
docked the potent active anti-proliferative derivatives 4c, 4e, 4h,
6d, and 6e in the ATP-binding pocket of the target enzyme.
The placement was performed based upon the position of the co-
crystallised ligand ANP. A root mean square deviation (rmsd) of
0.29 and binding energies data were resulted, suggesting a high
similarity of interaction to the referenced ligand. Investigation of
3.8. Prediction of drug-likeness and ADME properties
In a way to design a drug with a good oral bioavailability profile,
certain parameters are considered to play an important role46,47
.
About one-third of the invented drugs fail during development
processes due to their unreasonable pharmacokinetic profiles48.
Therefore, a computational study for the prediction of the ADME
properties of the synthesised molecules was performed by the cal-
culation of lipophilicity, topological polar surface area (TPSA),
absorption (% ABS), and simple molecular descriptors used by
Lipinski in formulating his “rule of five”49. Calculations were per-
formed using the Molinspiration online property calculation tool-
kit50. Table 10 represents
a calculated percentage of the
the docking results reported different binding modes of the target absorption (% ABS), TPSA, and Lipinski parameters of the com-
compounds. These compounds consistently showed stable pounds. A percentage of absorption (% ABS) was estimated using
hydrogen-bonding interaction with the NH of Met 341 residue as the equation: % ABS ¼109ꢁ(0.345 ꢂ TPSA), according to Zhao
a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1085
Figure 5. Binding data of target compounds. (A) 3D and 2D depiction of the docked conformation of the selected active compounds (A) 4c, (B) 4h, and (C) 6e in the
active site of enzyme revealing the essential residues and types of interactions.
et al.51 polar surface area, together with lipophilicity, is an essen- calculated percentages of absorption for compounds are ranged
tial property of a molecule of transportation across biological between 88.6% and 79.1%. The number of the hydrogen bond
membranes. Extremely high TPSA values give rise to poor bioavail- donors was constant for all of the compounds, and the number of
ability and absorption of a drug. According to the above criterions, hydrogen bond acceptors varied from 1 to 4. The investigation of

1086
H. E. A. AHMED ET AL.
Table 9. Description of the docking data of selected target compounds.
Cop.
Fragment
Target residues (distance, Å)
Met341 (2.64), H2O-Ser342 (2.65)
Glu339 (1.80)
Interaction
Binding energy (dG)
4c
CN
NH2
Cl
H-bonding
H-bonding
Hydrophobic
Hydrophobic
H-bonding
H-bonding
H-bonding
Hydrophobic
H-bonding
H-bonding
H-bonding
Hydrophobic
Hydrophobic
216.5
Leu273
Br-Naphthalene
Val281, Asp404, Leu273, Lys295
Met341 (3.15)
Glu339 (1.84)
Lys295, H2O-Asp346, H2O-Ser345, H2O-Ala390 (3.64) (3.53) (2.29)
Asp404, Lys295
Met341 (2.92)
H2O-Ser342 (2.55)
Lys295, H2O-Asn391, Asp404 (2.51) (2.57)
Asn391, Asp404
Val281, Leu273
4h
6e
CN
NH2
215.4
215.6
MeO
Br-Naphthalene
CN
NH2
MeO
Me
MeO-Naphthalene
The data reported in the table are extracted from MOE program showing the corresponding amino acids residues in enzyme pocket, corre-
sponding fragments of ligands, interaction distances, types of interaction, and their binding energy to some selected drugs. Conserved amino
acids are bold highlighted.
essential for the activity than the electron-withdrawing substituent
like a bromine atom. Caspase 3/7 intrinsic apoptotic assay has
been used to explore the demise of the three cell lines upon their
treatment with the target compounds. Meanwhile, the interaction
behaviour of selected active benzochromene derivatives was
examined computationally via their docking simulation within the
c-Src kinase target, which resulted in a good interaction profile
compared to the reference ligand. This behaviour classifies
these molecules as potential c-Src kinase candidates with potent
cytotoxic activity.
Table 10. Predicted ADME, Lipinski parameters, and molecular properties of the
synthesised compounds.
Cpd.
M. wt.
LogS
n-ON
n-OHNH
TPSA
LogP(o/w)
%ABS
4a
4b
4c
4d
4e
4f
4g
4h
6a
6b
6c
6d
6e
6f
377
395
412
456
407
391
437
437
328
346
363
407
342
358
388
388
ꢁ7.3
ꢁ7.6
ꢁ8
1
1
1
1
2
1
3
3
2
2
2
2
2
3
4
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
59
59
59
59
68
59
78
78
68
68
68
68
68
78
87
87
4.8
4.9
5.4
5.6
4.7
5.1
4.7
4.5
3.9
4.1
4.5
4.7
4.2
3.9
3.9
3.6
88.6
88.6
88.6
88.6
85.4
88.6
82.3
82.3
85.4
85.4
85.4
85.4
85.4
82.3
79.1
79.1
ꢁ8.3
ꢁ7.3
ꢁ7.7
ꢁ7.4
ꢁ7.4
ꢁ6.2
ꢁ6.5
ꢁ6.9
ꢁ7.3
ꢁ6.7
ꢁ6.3
ꢁ6.3
ꢁ6.3
Acknowledgements
The authors would like to thank chemical computing group for
use of the MOE software. The Deanship of Scientific Research at
Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia was
acknowledged.
6g
6h
% ABS: Percentage of absorption; TPSA: topological polar surface area; n-ON: number
of hydrogen bond acceptors; n-OHNH: number of hydrogen bond donors; logS: solu-
bility coefficient; Log P: partition coefficient. Calculations were performed using
Molinspiration online property calculation toolkit (http://www.molinspiration.com).
Disclosure statement
No potential conflict of interest was reported by the authors.
the Lipinski profile of the synthesised compounds indicated that
most of the synthesised derivatives are considered to be drug-like
candidates for the novel cytotoxic agents with a slight increase in
the log p values for two bromobenzochromene compounds.
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