Developing lipophilic aromatic halogenated fused systems with specific ring orientations, leading to potent anticancer analogs and targeting the c-Src Kinase enzyme

Article abstract of DOI:10.1016/j.molstruc.2019.03.012

This work presents the combined microwave assisted synthesis, spectroscopic analyses, cytotoxic effects, and molecular docking studies of the chromene fused ring systems with various substituents of the terminal halogenated phenyl ring derivatives of 6-me

Full text of DOI:10.1016/j.molstruc.2019.03.012

Accepted Manuscript
Developing lipophilic aromatic halogenated fused systems with specific ring
orientations, leading to potent anticancer analogs and targeting the c-Src Kinase
enzyme
Hany E.A. Ahmed, Mohammed A.A. El-Nassag, Ahmed H. Hassan, Hany M.
Mohamed, Ahmed H. Halawa, Rawda M. Okasha, Saleh Ihmaid, Shimaa Mohamed
Abd El‐Gilil, Essam S.A.E.H. Khattab, Ahmed M. Fouda, Ahmed M. El-Agrody,
Ateyatallah Aljuhani, Tarek H. Afifi
PII:
S0022-2860(19)30264-9
DOI:
https://doi.org/10.1016/j.molstruc.2019.03.012
MOLSTR 26281
Reference:
To appear in: Journal of Molecular Structure
Received Date: 3 September 2018
Revised Date: 1 March 2019
Accepted Date: 5 March 2019
Please cite this article as: H.E.A. Ahmed, M.A.A. El-Nassag, A.H. Hassan, H.M. Mohamed, A.H.
Halawa, R.M. Okasha, S. Ihmaid, S.M. Abd El‐Gilil, E.S.A.E.H. Khattab, A.M. Fouda, A.M. El-Agrody,
A. Aljuhani, T.H. Afifi, Developing lipophilic aromatic halogenated fused systems with specific ring
orientations, leading to potent anticancer analogs and targeting the c-Src Kinase enzyme, Journal of
Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.03.012.
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ACCEPTED MANUSCRIPT
Developing Lipophilic Aromatic Halogenated Fused Systems with Specific
Ring Orientations, Leading to Potent Anticancer Analogs and Targeting the c-
Src Kinase Enzyme
Hany E. A. Ahmed,a,b Mohammed A. A. El-Nassag, c,d Ahmed H. Hassan, c,d Ahmed H.
Halawa,c Rawda M. Okasha,e Saleh Ihmaid,a Shimaa Mohamed Abd El‐Gilil,f Essam S. A.
E. H. Khattab,c Ahmed M. Fouda, g Ahmed M. El-Agrody,c Ateyatallah Aljuhani e and
Tarek H Afifi,e*

ACCEPTED MANUSCRIPT
Developing Lipophilic Aromatic Halogenated Fused Systems with Specific
Ring Orientations, Leading to Potent Anticancer Analogs and Targeting the
c-Src Kinase Enzyme
Hany E. A. Ahmed,a,b Mohammed A. A. El-Nassag,c,d Ahmed H. Hassan,c,d Hany M.
Mohamed,c,d Ahmed H. Halawa,c Rawda M. Okasha,e Saleh Ihmaid,a Shimaa Mohamed
Abd El‐Gilil,f Essam S. A. E. H. Khattab,c Ahmed M. Fouda,g Ahmed M. El-Agrody,c
Ateyatallah Aljuhani e and Tarek H Afifi,e*
a Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah
University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar
University, Cairo 11884, Egypt
c Chemistry Department, Faculty of Science, Al-Azhar University, 11884 Nasr City, Cairo,
Egypt
d Chemistry Department, Faculty of Science, Jazan University, Jazan, Saudi Arabia
e Chemistry Department, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah,
Saudi Arabia, Email: afifith@yahoo.com
f Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar
University, Nasr City, 11754, Cairo, Egypt
g Chemistry Department, Faculty of Science, King Khalid University, Abha, 61413, P.O.
Box Abha 9004, Saudi Arabia
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Abstract: This work presents the combined microwave assisted synthesis, spectroscopic
analyses, cytotoxic effects, and molecular docking studies of the chromene fused ring systems
with various substituents of the terminal halogenated phenyl ring derivatives of 6-methoxy-4H-
benzo[h]chromene (4a-m). The structures of the synthesized compounds were established on the
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13
13
basis of their spectral data, IR, H NMR, C NMR, C NMR-DEPT, and MS data. The target
compounds were evaluated for their antitumor activities against three cancer cell lines: MCF-7,
HCT-116, and HepG-2, in comparison to Vinblastine and Colchicine as reference drugs with the
usage of a MTT colorimetric assay. The most active compounds were subjected to furthur
mechanistic analysis through the assay of the c-Src Kinase inhibition activity. The SAR studies
reported that the substitution at the 4-position of the 2-amino-4H-benzo[h]chromene nucleus
with specific halogen atoms and lipophilic characters increased the ability of the molecule
against the different cell lines. The Molecular Operating Environment (MOE) was used to
evaluate the interactions between the ligand and the c-Src Kinase enzyme. Compound 4m,
having the 3,5-dibromo-2-methoxyphenyl group attached to the 4H-benzo[h]chromene moiety at
the 4-postion, exhibited a promising interaction with the c-Src Kinase, which leads to a strong
inhibition of this enzyme.
Keywords: 4H-Benzo[h]chromenes • Antitumor • Molecular docking • c-Src kinase activity •
SAR
Introduction
Multicomponent reactions (MCRs) are one of the most attractive synthetic strategies to
produce a massive number of biologically active compounds, which can be achieved simply by
varying the reacting components. Furthermore, it is one of the tools that is used to combine
economic aspects with environmental features for the synthesis of heterocyclic compounds [1,2].
Chromene or benzochromene is one of the most advanced medicinal pharmacophores, which
appeared as an important structural component in natural products and generated great attention
because of their interesting pharmacological activities, such as antifungal, [3,4] antimicrobial,
[5,6] anticoagulant, antispasmolytic, estrogenic, [7] anti-HIV, [8] hypolipidemic, [9] herbicidal,
analgesic and anticonvulsant, [10] antioxidant, [11] antitubercular, [12] TNF-α inhibitor, [13]
anticancer, [14] antiviral effects and activities, [15] as well as acting as an inhibitor of diabetes-
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induced vascular dysfunction [16]. Meanwhile, they have also been notorious for the inhibition
of human cancer cell lines with no levels of toxicity against the normal human cells [17-26]. In
recent studies, the curiosity in the c-Src kinase inhibitors as anti-invasive strategies has increased
for the therapy of cancer [27,28].
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1
NH2
O
NH2
CN
2
3
CN
4
R
R
R = Different substitutions
R
R
4
1
CN
2
3
CN
O
NH2
NH2
R1
R = Different substitutions
R1
R
4
1
R
CN
2
3
CN
O
NH2
R = Br, OCH3
NH2
NH2
R1 = Different substitutions
NH2
CN
O
1
R
2
3
CN
R
4
R = OCH3
R1 = Halogens
R1
R1
Fig. (1). Systamatic analysis of different Fused Armatic Systems (FAS).
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Herein, we report that the fused systems of the chromene compounds with different
substituents had a potent anticancer effect [17-22]. The study revealed that the 2,3,4 postions on
the chromene moieties were the most prominent, particularly, with the presence of bromine and
methoxy groups. In continuation with our efforts to identify new candidates that may be valuable
in designing new antitumor agents, we have utilized the three-component synthetic approach to
produce a lipophilic-fused aromatic system with different alignments of halogens, linked with
the methoxy group, and screened their cytotoxicity against a panel of three human cancer cell
lines. Fig. 1 shows the comparison of the previously reported synthezied system with the newly
designed ones [27,28]. The inhibition of the c-Src kinase and the Structural-Activity Relationship
(SAR) of the novel analogs and molecular docking were also examined.
Experimental
Materials and methods
The commercial-grade solvents and reagents were purchased from Sigma–Aldrich (St. Louis,
MO, USA) and used without further purification. Melting points were measured with a Stuart
Scientific (UK) apparatus and are uncorrected. The IR spectra were determined as KBr pellets on
1
a Jasco FT/IR 460 plus spectrophotometer (Jasco, Japan). The H-NMR and 13C-NMR spectra
were recorded, using a Bruker AV 500 MHz spectrometer (Bruker, USA). The 13C-NMR spectra
were obtained, using the distortion-free enhancement by polarization transfer (DEPT) and the
attached proton test (APT). The chemical shifts (δ) are expressed in parts per million (ppm). The
MS data were measured using a Shimadzu GC/MS-QP5050A spectrometer (Shimadzu, Japan).
The elemental analyses were carried out at the Regional Centre for Mycology & Biotechnology
(RCMP), Al-Azhar University, Cairo, Egypt, and the results were within ±0.25 %. The analytical
thin layer chromatography (TLC) on silica gel-precoated F254 Merck plates were used to check
the purity of the compounds.
Chemistry
General procedure for synthesis of 4H-benzo[h]chromene derivatives (4a-m)
A reaction mixture of 4-methoxy-1-naphthol 1 (0.01 mol), different aromatic aldehydes 2a-m
(0.01 mol), malononitrile 3 (0.01 mol), and piperidine (0.5 mL) in ethanol (30 mL) was heated
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under microwave irradiation conditions for 2 min. at 140 °C. After the completion of the
reaction, the reaction mixture was cooled to room temperature, and the precipitated solid was
filtered off, washed with MeOH, and recrystallized from ethanol or ethanol/benzene. The
physical and spectral data of compounds 4a-m are as follows:
2-Amino-6-methoxy-4-(4-fluorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4a)
Prepared as previously described [29].
2-Amino-6-methoxy-4-(2,4-difluorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4b)
ο
Colourless crystals from ethanol; yield 77 %; m.p. 237-238 C; IR (KBr) υ (cm-1): 3467,
3335, 3200 (NH2), 3066, 2977, 2948 (CH), 2198 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.22-
6.47 (m, 8H, aromatic), 7.14 (bs, 2H, NH2 D2Oexchangeable), 5.13 (s, 1H, H-4), 3.82 (s, 3H,
OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 160.72 (C-2), 151.33 (C-6), 137.27 (C-10b), 128.25
(C-10a), 128.15 (C-6a), 127.33 (C-9), 126.35 (C-8), 121.64 (C-7), 120.60 (C-10), 120.26 (C-4a),
116.06 (CN), 102.87 (C-5), 55.70 (CH3), 54.38 (C-3), 35.40 (C-4), 160.25, 159.02, 131.19,
13
124.53, 111.96, 111.79 (aromatic); C NMR-DEPT spectrum at 135оCH, CH3 [positive (up)],
CH2 [negative (down)], revealed the following signals at δ: 131.19 (aromatic ↑), 127.33 (C-9 ↑),
126.35 (C-8 ↑), 121.64 (C-7 ↑), 120.60 (C-10 ↑), 111.96 (aromatic ↑), 111.79 (aromatic ↑),
102.87 (C-5 ↑), 55.70 (CH3 ↑), 35.40 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are
positive (up) and showed δ: 131.19 (aromatic ↑), 127.33 (C-9 ↑), 126.35 (C-8 ↑), 121.64 (C-7 ↑),
120.60 (C-10 ↑), 111.96 (aromatic ↑), 111.79 (aromatic ↑), 102.87 (C-5 ↑), 35.40 (C-4 ↑). In the
DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 131.19 (aromatic ↑),
127.33 (C-9 ↑), 126.35 (C-8 ↑), 121.64 (C-7 ↑), 120.60 (C-10 ↑), 111.96 (aromatic ↑), 111.79
(aromatic ↑), 102.87 (C-5 ↑), 55.70 (CH3 ↑), 35.40 (C-4 ↑); 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.72 (C-2 ↓),
160.25 (aromatic ↓), 159.02 (aromatic ↓), 151.33 (C-6 ↓), 131.19 (aromatic ↑), 137.27 (C-10b ↓),
128.25 (C-10a ↓), 128.15 (C-6a ↓), 127.33 (C-9 ↑), 126.35 (C-8 ↑), 124.53 (aromatic ↓), 121.64
(C-7 ↑), 120.60 (C-10 ↑), 120.26 (C-4a ↓), 116.06 (CN ↓), 111.96 (aromatic ↑), 111.79 (aromatic
↑), 102.87 (C-5 ↑), 55.70 (CH3), 54.38 (C-3 ↓), 35.40 (C-4 ↑); MS m/z (%): 364 (M+, 98.76) with
a base peak at 251 (100); Anal. Calcd for C21H14F2N2O2: C, 69.23; H, 3.87; N, 7.69. Found: C,
69.18; H, 3.83; N, 7.63 %.
2-Amino-6-methoxy-4-(2,6-difluorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4c)
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Colourless crystals from ethanol / benzene; yield 79 %; m.p. 275-276 οC; IR (KBr) υ (cm-1):
1
3477, 3339, 3203 (NH2), 3069, 2979, 2953 (CH), 2199 (CN); H NMR (500 MHz) (DMSO-d6)
δ: 8.21-6.37 (m, 8H, aromatic), 7.17 (bs, 2H, NH2 D2Oexchangeable), 5.32 (s, 1H, H-4), 3.80 (s,
3H, OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 161.58 (C-2), 151.32 (C-6), 137.43 (C-10b),
127.35 (C-9), 126.35 (C-8), 121.64 (C-7), 124.57 (C-10a), 123.45 (C-6a), 120.26 (C-4a), 120.55
(C-10), 114.74 (CN), 102.52 (C-5), 55.72 (CH3), 52.81 (C-3), 30.85 (C-4), 160.87, 159.62,
129.77, 119.69, 112.12, 111.95 (aromatic); 13 C NMR-DEPT spectrum at 135оCH, CH3 [positive
(up)], CH2[negative (down)], revealed the following signals at δ: 129.77 (aromatic ↑), 127.35 (C-
9 ↑), 126.35 (C-8 ↑), 121.64 (C-7 ↑), 120.55 (C-10 ↑), 112.12 (aromatic ↑), 111.95 (aromatic ↑),
102.52 (C-5 ↑), 55.72 (CH3 ↑), 30.85 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are
positive (up) and showed δ: 129.77 (aromatic ↑), 127.35 (C-9 ↑), 126.35 (C-8 ↑), 121.64 (C-7 ↑),
120.55 (C-10 ↑), 112.12 (aromatic ↑), 111.95 (aromatic ↑), 102.52 (C-5 ↑), 30.85 (C-4 ↑). In the
DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 129.77 (aromatic ↑),
127.35 (C-9 ↑), 126.35 (C-8 ↑), 121.64 (C-7 ↑), 120.55 (C-10 ↑), 112.12 (aromatic ↑), 111.95
(aromatic ↑), 102.52 (C-5 ↑), 55.72 (CH3 ↑), 30.85 (C-4 ↑); 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 161.58 (C-2 ↓),
160.87 (aromatic ↓), 159.62 (aromatic ↓), 151.32 (C-6 ↓), 137.43 (C-10b ↓), 129.77 (aromatic ↑),
127.35 (C-9 ↑), 126.35 (C-8 ↑), 121.64 (C-7 ↑), 124.57 (C-10a ↓), 123.45 (C-6a ↓), 120.55 (C-10
↑), 120.26 (C-4a ↓), 119.69 (aromatic ↓), 114.74 (CN ↓), 112.12 (aromatic ↑), 111.95 (aromatic
↑), 102.52 (C-5 ↑), 55.72 (CH3 ↑), 52.81 (C-3 ↓), 30.85 (C-4 ↑); MS m/z (%): 364 (M+, 87.59)
with a base peak at 251 (100); Anal. Calcd for C21H14F2N2O2: C, 69.23; H, 3.87; N, 7.69. Found:
C, 69.17; H, 3.82; N, 7.62 %.
2-Amino-6-methoxy-4-(2-chlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4d)
ο
Pale yellow needles from benzene; yield 86%; m.p. 250-251 C; IR (KBr) υ (cm-1): 3452,
3329, 3202 (NH2), 3045, 2974, 2951 (CH), 2196 (CN); 1H NMR (500 MHz) (DMSO-d6) δ: 8.10-
6.37 (m, 9H, aromatic), 7.15 (bs, 2H, NH2 D2Oexchangeable), 5.33 (s, 1H, H-4), 3.76 (s, 3H,
OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 160.67 (C-2), 151.27 (C-6), 137.11 (C-10b), 128.27
(C-10a), 127.31 (C-9), 126.31 (C-6a), 123.57 (C-8), 121.61 (C-7), 120.63 (C-10), 120.22 (C-4a),
116.44 (CN), 102.44 (C-5), 55.55 (CH3), 54.78 (C-3), 38.60 (C-4), 142.07, 131.89, 130.88,
129.75, 128.97, 124.53 (aromatic); MS m/z (%): 364 (M++2, 17.56), 362 (M+, 53.36) with a base
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peak at 251 (100); Anal. Calcd for C21H14ClN2O2: C, 69.52; H, 4.17; N, 7.72. Found: C, 69.57;
H, 4.22; N, 7.77 %.
2-Amino-6-methoxy-4-(3-chlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4e)
ο
Yellow needles from ethanol; yield 84%; m.p. 220-221 C; IR (KBr) υ (cm-1): 3461, 3323,
1
3199 (NH2), 3049, 2952, 2977 (CH), 2195 (CN); H NMR (500 MHz) (DMSO-d6) δ: 8.23-6.56
(m, 9H, aromatic), 7.17 (bs, 2H, NH2 D2Oexchangeable), 4.90 (s, 1H, H-4), 3.82 (s, 3H, OCH3);
13C NMR (125 MHz) (DMSO-d6) δ: 160.55 (C-2), 151.31 (C-6), 136.95 (C-10b), 127.20 (C-
10a), 126.95 (C-9), 126.32 (C-6a), 123.64 (C-8), 121.66 (C-7), 120.63 (C-10), 120.41 (C-4a),
117.09 (CN), 103.25 (C-5), 55.76 (CH3), 55.76 (C-3), 41.05 (C-4), 147.95, 133.24, 130.66,
127.30, 126.30, 124.50 (aromatic); MS m/z (%): 364 (M++2, 12.58), 362 (M+, 38.61) with a base
peak at 251 (100); Anal. Calcd for C21H14ClN2O2: C, 69.52; H, 4.17; N, 7.72. Found: C, 69.47;
H, 4.11; N, 7.68 %.
2-Amino-6-methoxy-4-(4-chlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4f)
Prepared as previously reported [29].
2-Amino-6-methoxy-4-(2,3-dichlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4g)
ο
Yellow crystals from ethanol/benzene; yield 80 %; m.p. 276-277 C; IR (KBr) υ (cm-1):
1
3467, 3336, 3211 (NH2), 3059, 2983, 2958 (CH), 2188 (CN); H NMR (500 MHz) (DMSO-d6)
δ: 8.23-6.38 (m, 8H, aromatic), 7.19 (bs, 2H, NH2 D2Oexchangeable), 5.45 (s, 1H, H-4), 3.78 (s,
3H, OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 160.69 (C-2), 151.37 (C-6), 137.27 (C-10b),
129.63 (C-10a), 128.27 (C-9), 127.38 (C-8), 124.65 (C-6a), 121.65 (C-7), 120.66 (C-10), 120.07
(C-4a), 115.90 (CN), 102.40 (C-5), 55.66 (CH3), 54.50 (C-3), 39.94 (C-4), 144.61, 132.21,
130.09, 129.37, 128.71, 126.44 (aromatic); 13 C NMR-DEPT spectrum at 135оCH, CH3 [positive
(up)], CH2 [negative (down)], revealed the following signals at δ: 129.37 (aromatic ↑), 128.71
(aromatic ↑), 128.27 (C-9 ↑), 127.38 (C-8 ↑), 126.44 (aromatic ↑), 121.65 (C-7 ↑), 120.66 (C-10
↑), 102.40 (C-5 ↑), 55.66 (CH3 ↑), 39.94 (C-4 ↑). In the DEPT spectrum at 90° only CH signals
are positive (up) and showed δ: 129.37 (aromatic ↑), 128.71 (aromatic ↑), 128.27 (C-9 ↑), 127.38
(C-8 ↑), 126.44 (aromatic ↑), 121.65 (C-7 ↑), 120.66 (C-10 ↑), 102.40 (C-5 ↑), 39.94 (C-4 ↑). In
the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 129.37 (aromatic
↑), 128.71 (aromatic ↑), 128.27 (C-9 ↑), 127.38 (C-8 ↑), 126.44 (aromatic ↑), 121.65 (C-7 ↑),
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120.66 (C-10 ↑), 102.40 (C-5 ↑), 55.66 (CH3 ↑), 39.94 (C-4 ↑); 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.69 (C-2 ↓),
151.37 (C-6 ↓), 144.61 (aromatic ↓), 137.27 (C-10b ↓), 132.21 (aromatic ↓), 130.09 (aromatic ↓),
129.63 (C-10a ↓), 129.37 (aromatic ↑), 128.71 (aromatic ↑), 128.27 (C-9 ↑), 127.38 (C-8 ↑),
126.44 (aromatic ↑), 124.65 (C-6a ↓), 121.65 (C-7 ↑), 120.66 (C-10 ↑), 120.07 (C-4a ↓), 115.90
(CN ↓), 102.40 (C-5 ↑), 55.66 (CH3 ↑), 54.50 (C-3 ↓), 39.94 (C-4 ↑); MS m/z (%): 400 (M++4,
1.18), 398 (M++2, 6.58), 396 (M+, 10.33) with a base peak at 91 (100); Anal. Calcd for
C21H14Cl2N2O2: C, 63.49; H, 3.55; N, 7.05. Found: C, 63.55; H, 3.61; N, 7.10 %.
2-Amino-6-methoxy-4-(2,4-dichlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4h)
ο
Yellow crystals from ethanol; yield 82 %; m.p. 225-226 C; IR (KBr) υ (cm-1): 3468, 3338,
1
3213 (NH2), 3009, 2988, 2959 (CH), 2190 (CN); H NMR (500 MHz) (DMSO-d6) δ: 8.22-6.36
(m, 8H, aromatic), 7.25 (bs, 2H, NH2 D2Oexchangeable), 5.35 (s, 1H, H-4), 3.78 (s, 3H, OCH3);
13C NMR (125 MHz) (DMSO-d6) δ: 160.62 (C-2), 151.33 (C-6), 137.17 (C-10b), 127.37 (C-9),
126.42 (C-8), 124.60 (C-10a), 123.53 (C-6a), 121.64 (C-7), 120.65 (C-10), 120.09 (C-4a),
115.83 (CN), 102.35 (C-5), 55.61 (CH3), 54.32 (C-3), 38.26 (C-4), 141.14, 132.85, 132.38,
13
132.32, 129.13, 128.17 (aromatic); C NMR-DEPT spectrum at 135оCH, CH3 [positive (up)],
CH2 [negative (down)], revealed the following signals at δ: 127.37 (C-9 ↑), 126.42 (C-8 ↑),
121.64 (C-7 ↑), 120.65 (C-10 ↑), 102.35 (C-5 ↑), 55.61 (CH3 ↑), 38.26 (C-4 ↑), 132.32 (aromatic
↑), 129.13 (aromatic ↑), 128.17 (aromatic ↑). In the DEPT spectrum at 90° only CH signals are
positive (up) and showed δ: 127.37 (C-9 ↑), 126.42 (C-8 ↑), 121.64 (C-7 ↑), 120.65 (C-10 ↑),
102.35 (C-5 ↑), 38.26 (C-4 ↑), 132.32 (aromatic ↑), 129.13 (aromatic ↑), 128.17 (aromatic ↑). In
the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 127.37 (C-9 ↑),
126.42 (C-8 ↑), 121.64 (C-7 ↑), 120.65 (C-10 ↑), 102.35 (C-5 ↑), 55.61 (CH3 ↑), 38.26 (C-4 ↑),
132.32 (aromatic ↑), 129.13 (aromatic ↑), 128.17 (aromatic ↑); 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.62 (C-2 ↓),
151.33 (C-6 ↓), 141.14 (aromatic ↓), 137.17 (C-10b ↓), 132.85 (aromatic ↓), 132.38 (aromatic ↓),
132.32 (aromatic ↑), 129.13 (aromatic ↑), 128.17 (aromatic ↑), 127.37 (C-9 ↑), 126.42 (C-8 ↑),
124.60 (C-10a ↓), 123.53 (C-6a ↓), 121.64 (C-7 ↑), 120.65 (C-10 ↑), 120.09 (C-4a ↓), 115.83
(CN ↓), 102.35 (C-5 ↑), 55.61 (CH3 ↑), 54.32 (C-3 ↓), 38.26 (C-4 ↑); MS m/z (%): 400 (M++4,
9.92), 398 (M++2, 64.14) and a base peak at 396 (M+, 100); Anal. Calcd for C21H14Cl2N2O2: C,
63.49; H, 3.55; N, 7.05. Found: C, 63.53; H, 3.59; N, 7.09 %.
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2-Amino-6-methoxy-4-(2,5-dichlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4i)
ο
Yellow crystals from ethanol / benzene; yield 82 %; m.p. 260-261 C; IR (KBr) υ (cm-1):
1
3467, 3323, 3196 (NH2), 3079, 2978, 2947 (CH), 2193 (CN); H NMR (500 MHz) (DMSO-d6)
δ: 8.23-6.37 (m, 8H, aromatic), 7.23 (bs, 2H, NH2 D2Oexchangeable), 5.35 (s, 1H, H-4), 3.77 (s,
3H, OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 160.73 (C-2), 151.37 (C-6), 137.19 (C-10b),
128.26 (C-9), 127.37 (C-8), 124.66 (C-10a), 123.52 (C-6a), 121.64 (C-7), 120.69 (C-10), 120.05
(C-4a), 115.42 (CN), 102.28 (C-5), 55.60 (CH3), 54.09 (C-3), 38.97 (C-4), 143.92, 132.29,
131.68, 130.89, 130.36, 128.87 (aromatic); 13 C NMR-DEPT spectrum at 135оCH, CH3 [positive
(up)], CH2 [negative (down)], revealed the following signals at δ: 131.68 (aromatic ↑), 130.36
(aromatic ↑), 128.87 (aromatic ↑), 128.26 (C-9 ↑), 127.37 (C-8 ↑), 121.64 (C-7 ↑), 120.69 (C-10
↑), 102.28 (C-5 ↑), 55.60 (CH3 ↑), 38.97 (C-4 ↑). In the DEPT spectrum at 90° only CH signals
are positive (up) and showed δ: 131.68 (aromatic ↑), 130.36 (aromatic ↑), 128.87 (aromatic ↑),
128.26 (C-9 ↑), 127.37 (C-8 ↑), 121.64 (C-7 ↑), 120.69 (C-10 ↑), 102.28 (C-5 ↑), 38.97 (C-4 ↑).
In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 131.68 (aromatic
↑), 130.36 (aromatic ↑), 128.87 (aromatic ↑), 128.26 (C-9 ↑), 127.37 (C-8 ↑), 121.64 (C-7 ↑),
120.69 (C-10 ↑), 102.28 (C-5 ↑), 55.60 (CH3 ↑), 38.97 (C-4 ↑); 13CNMR-APT spectrum CH,
CH3 [positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.73 (C-2
↓), 151.37 (C-6 ↓), 137.19 (C-10b ↓), 128.26 (C-9 ↑), 127.37 (C-8 ↑), 124.66 (C-10a ↓), 123.52
(C-6a ↓), 121.64 (C-7 ↑), 120.69 (C-10 ↑), 120.05 (C-4a ↓), 115.42 (CN ↓), 102.28 (C-5 ↑),
55.60 (CH3 ↑), 54.09 (C-3 ↓), 38.97 (C-4 ↑), 143.92 (aromatic ↓), 132.29 (aromatic ↓), 131.68
(aromatic ↑), 130.89 (aromatic ↓), 130.36 (aromatic ↑), 128.87 (aromatic ↑); MS m/z (%): 400
(M++4, 0.66), 398 (M++2, 4.40), 396 (M+, 6.67) with a base peak at 251(100); ; Anal. Calcd for
C21H14Cl2N2O2: C, 63.49; H, 3.55; N, 7.05. Found: C, 63.41; H, 3.50; N, 7.00 %.
2-Amino-6-methoxy-4-(2,6-dichlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4j)
ο
Yellow crystals from benzene; yield 82 %; m.p. 300-301 C; IR (KBr) υ (cm-1): 3479, 3327,
1
3188 (NH2), 3071, 2973, 2957 (CH), 2197 (CN); H NMR (500 MHz) (DMSO-d6) δ: 8.19-6.16
(m, 8H, aromatic), 7.15 (bs, 2H, NH2 D2Oexchangeable), 5.94 (s, 1H, H-4), 3.75 (s, 3H, OCH3);
13C NMR (125 MHz) (DMSO-d6) δ: 160.93 (C-2), 151.24 (C-6), 136.95 (C-10b), 127.35 (C-9),
126.34 (C-8), 124.63 (C-10a), 123.26 (C-6a), 121.62 (C-7), 120.59 (C-10), 119.90 (C-4a),
10

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113.73 (CN), 101.56 (C-5), 55.55 (CH3), 51.94 (C-3), 37.45 (C-4), 137.70, 135.55, 134.87,
13
130.80, 129.92, 128.67 (aromatic); C NMR-DEPT spectrum at 135оCH, CH3 [positive (up)],
CH2 [negative (down)], revealed the following signals at δ: 130.80 (aromatic ↑), 129.92
(aromatic ↑), 128.67 (aromatic ↑), 127.35 (C-9 ↑), 126.34 (C-8 ↑), 121.62 (C-7 ↑), 120.59 (C-10
↑), 101.56 (C-5 ↑), 55.55 (CH3 ↑), 37.45 (C-4 ↑). In the DEPT spectrum at 90° only CH signals
are positive (up) and showed δ: 130.80 (aromatic ↑), 129.92 (aromatic ↑), 128.67 (aromatic ↑),
127.35 (C-9 ↑), 126.34 (C-8 ↑), 121.62 (C-7 ↑), 120.59 (C-10 ↑), 101.56 (C-5 ↑), 37.45 (C-4 ↑).
In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 130.80 (aromatic
↑), 129.92 (aromatic ↑), 128.67 (aromatic ↑), 127.35 (C-9 ↑), 126.34 (C-8 ↑), 121.62 (C-7 ↑),
120.59 (C-10 ↑), 101.56 (C-5 ↑), 55.55 (CH3 ↑), 37.45 (C-4 ↑); 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.93 (C-2 ↓),
151.24 (C-6 ↓), 137.70 (aromatic ↓), 136.95 (C-10b ↓), 135.55 (aromatic ↓), 134.87 (aromatic ↓),
130.80 (aromatic ↑), 129.92 (aromatic ↑), 128.67 (aromatic ↑), 127.35 (C-9 ↑), 126.34 (C-8 ↑),
124.63 (C-10a ↓), 123.26 (C-6a ↓), 121.62 (C-7 ↑), 120.59 (C-10 ↑), 119.90 (C-4a ↓), 113.73
(CN ↓), 101.56 (C-5 ↑), 55.55 (CH3 ↑), 51.94 (C-3 ↓), 37.45 (C-4 ↑); MS m/z (%): 400 (M++4,
0.41), 398 (M++2, 2.56), 396 (M+, 3.81) with a base peak at 251 (100); Anal. Calcd for
C21H14Cl2N2O2: C, 63.49; H, 3.55; N, 7.05. Found: C, 63.42; H, 3.49; N, 7.01 %.
2-Amino-6-methoxy-4-(3,4-dichlorophenyl)-4H-benzo[h]chromene-3-carbonitrile (4k)
ο
Yellow crystals from benzene; yield 79 %; m.p. 230-231 C; IR (KBr) υ (cm-1): 3470, 3331,
1
3196 (NH2), 3089, 2962, 2965 (CH), 2195 (CN); H NMR (500 MHz) (DMSO-d6) δ: 8.22-6.56
(m, 8H, aromatic), 7.20 (bs, 2H, NH2 D2Oexchangeable), 4.94 (s, 1H, H-4), 3.83 (s, 3H, OCH3);
13C NMR (125 MHz) (DMSO-d6) δ: 160.51 (C-2), 151.36 (C-6), 136.94 (C-10b), 127.34 (C-9),
126.42 (C-8), 124.56 (C-10a), 123.62 (C-6a), 121.67 (C-7), 120.66 (C-10), 120.30 (C-4a),
116.62 (CN), 103.16 (C-5), 55.81 (CH3), 55.62 (C-3), 40.48 (C-4), 146.53, 131.15, 131.05,
13
129.57, 129.36, 127.99 (aromatic); C NMR-DEPT spectrum at 135оCH, CH3 [positive (up)],
CH2 [negative (down)], revealed the following signals at δ: 131.05 (aromatic ↑), 129.36(aromatic
↑), 127.99 (aromatic ↑), 127.34 (C-9 ↑), 126.42 (C-8 ↑), 121.67 (C-7 ↑), 120.66 (C-10 ↑), 103.16
(C-5 ↑), 55.81 (CH3 ↑), 40.48 (C-4 ↑). In the DEPT spectrum at 90° only CH signals are positive
(up) and showed δ: 131.05 (aromatic ↑), 129.36(aromatic ↑), 127.99 (aromatic ↑), 127.34 (C-9
↑), 126.42 (C-8 ↑), 121.67 (C-7 ↑), 120.66 (C-10 ↑), 103.16 (C-5 ↑), 40.48 (C-4 ↑). In the DEPT
11

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spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 131.05 (aromatic ↑),
129.36(aromatic ↑), 127.99 (aromatic ↑), 127.34 (C-9 ↑), 126.42 (C-8 ↑), 121.67 (C-7 ↑), 120.66
(C-10 ↑), 103.16 (C-5 ↑), 55.81 (CH3 ↑), 40.48 (C-4 ↑); 13CNMR-APT spectrum CH, CH3
[positive (up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.51 (C-2 ↓),
151.36 (C-6 ↓), 146.53 (aromatic ↓), 136.94 (C-10b ↓), 131.15 (aromatic ↓), 131.05 (aromatic ↑),
129.57 (aromatic ↓), 129.36 (aromatic ↑), 127.99 (aromatic ↑), 127.34 (C-9 ↑), 126.42 (C-8 ↑),
124.56 (C-10a ↓), 123.62 (C-6a ↓), 121.67 (C-7 ↑), 120.66 (C-10 ↑), 120.30 (C-4a ↓), 116.62
(CN ↓), 103.16 (C-5 ↑), 55.81 (CH3 ↑), 55.62 (C-3 ↓), 40.48 (C-4 ↑); MS m/z (%): 400 (M++4,
1.02), 398 (M++2, 6.65), 396 (M+, 9.91) with a base peak at 251 (100); Anal. Calcd for
C21H14Cl2N2O2: C, 63.49; H, 3.55; N, 7.05. Found: C, 63.52; H, 3.59; N, 7.09 %.
2-Amino-6-methoxy-4-(4-bromophenyl)-4H-benzo[h]chromene-3-carbonitrile (4l)
This compound was prepared as previously reported [29].
2-Amino-6-methoxy-4-(3,5-dibromo-2-methoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile
(4m)
ο
Yellow crystals from benzene; yield 77 %; m.p. 245-246 C; IR (KBr) υ (cm-1): 3470, 3331,
1
3196 (NH2), 3089, 2962, 2965 (CH), 2195 (CN); H NMR (500 MHz) (DMSO-d6) δ: 8.23-6.35
(m, 7H, aromatic), 7.21 (bs, 2H, NH2 D2Oexchangeable), 5.13 (s, 1H, H-4), 3.76 (s, 3H, OCH3),
3.72 (s, 3H, OCH3); 13C NMR (125 MHz) (DMSO-d6) δ: 160.78 (C-2), 151.33 (C-6), 137.00 (C-
10b), 127.30 (C-9), 126.28 (C-8), 124.35 (C-10a), 123.63 (C-6a), 121.65 (C-7), 120.61 (C-10),
120.45 (C-4a), 116.26 (CN), 102.58 (C-5), 61.42 (CH3), 55.59 (CH3), 54.69 (C-3), 36.81 (C-4),
13
154.06, 142.31, 134.35, 131.98, 118.19, 116.99 (aromatic);
C NMR-DEPT spectrum at
135оCH, CH3 [positive (up)], CH2 [negative (down)], revealed the following signals at δ: 134.35
(aromatic ↑), 131.98 (aromatic ↑), 127.30 (C-9 ↑), 126.28 (C-8 ↑), 121.65 (C-7 ↑), 120.61 (C-10
↑), 102.58 (C-5 ↑), 61.42 (CH3 ↑), 55.59 (CH3 ↑), 36.81 (C-4 ↑). In the DEPT spectrum at 90°
only CH signals are positive (up) and showed δ: 134.35 (aromatic ↑), 131.98 (aromatic ↑),
127.30 (C-9 ↑), 126.28 (C-8 ↑), 121.65 (C-7 ↑), 120.61 (C-10 ↑), 102.58 (C-5 ↑), 36.81 (C-4 ↑).
In the DEPT spectrum at 45° (CH, CH2 and CH3 positive) revealed signals at δ: 134.35 (aromatic
↑), 131.98 (aromatic ↑), 127.30 (C-9 ↑), 126.28 (C-8 ↑), 121.65 (C-7 ↑), 120.61 (C-10 ↑), 102.58
(C-5 ↑), 61.42 (CH3 ↑), 55.59 (CH3 ↑), 36.81 (C-4 ↑); 13CNMR-APT spectrum CH, CH3 [positive
12

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(up)], CH2, Cq [negative (down)], revealed the following signals at δ: 160.78 (C-2 ↓), 154.06
(aromatic ↓), 151.33 (C-6 ↓), 142.31(aromatic ↓), 137.00 (C-10b ↓), 134.35 (aromatic ↑), 131.98
(aromatic ↑), 127.30 (C-9 ↑), 126.28 (C-8 ↑), 124.35 (C-10a ↓), 123.63 (C-6a ↓), 121.65 (C-7 ↑),
120.61 (C-10 ↑), 120.45 (C-4a ↓), 118.19 (aromatic ↓), 116.99 (aromatic ↓), 116.26 (CN ↓),
102.58 (C-5 ↑), 61.42 (CH3 ↑), 55.59 (CH3 ↑), 54.69 (C-3 ↓), 36.81 (C-4); MS m/z (%): 518
(M++4, 23.10), 516 (M++2, 48.01), 514 (M+, 25.91) with a base peak at 251 (100); Anal. Calcd
for C22H16Br2N2O3: C, 51.19; H, 3.12; N, 5.43. Found: C, 51.24; H, 3.16; N, 5.49 %.
In Vitro Pharmacological Studies
Cell culture
The tumor cell lines of the breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-
116), and hepatocellular carcinoma (HepG-2) were obtained from the American Type Culture
Collection (ATCC, Rockville, MD). The cells were grown on a RPMI-1640 medium,
supplemented with a 10 % inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were
maintained at 37 οC in a humidified atmosphere with 5 % CO2 and were subcultured two to three
times a week.
Cytotoxicity evaluation using viability assay
The tumor cell lines were suspended in a medium at a concentration of 5 x 104 cell/well in
Corning® 96-well tissue culture plates and then were incubated for 24 h. The tested compounds
with concentrations, ranging from 0 to 50 ꢀg/ml, were then added into 96-well plates (six
replicates) to achieve different concentrations for each compound. Six vehicle controls with
media or 0.5 % DMSO were run for each of the 96 well plates. After incubating for 24 h, the
number of viable cells were determined by the MTT test. Briefly, the media was removed from
the 96 well plates and replaced with 100 ꢀl of fresh culture RPMI 1640 medium without phenol
red. Then, 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) was added to
each well, including the untreated controls. The 96-well plates were then incubated at 37 οC and
5 % CO2 for 4 h. An 85-ꢀl aliquot of the media was removed from the wells, and 50 µl of DMSO
ο
was added to each well, mixed thoroughly with the pipette, and incubated at 37 C for 10 min.
Then, the optical density was measured at 590 nm with the microplate reader (Sunrise, TECAN,
Inc, USA) to determine the number of viable cells. The percentage of the viability was calculated
as [1 - (ODt/ODc)] x 100 %, where ODt is the mean optical density of wells treated with the
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tested sample and ODc is the mean optical density of untreated cells. The relation between the
surviving cells and the drug concentration is plotted to get the survival curve of each tumor cell
line after treatment with the specified compound. The 50 % inhibitory concentration (IC50), the
concentration required to cause toxic effects in 50 % of intact cells, was estimated from the
graphic plots of the dose response curve for each conc., using Graph pad Prism software (San
Diego, CA. USA) [30, 31].
c-Src Kinase activity assay
The effect of synthesized compounds on the activity of the c-Src kinase was determined,
using a HT Scan Src Kinase Assay Kit, catalogue number 7776 from Cell Signaling Technology
(Danvers, MA, USA); according to manufacturer's protocol. The streptavidin-coated plates were
purchased from Pierce (Rockford, IL, USA). In brief, the kinase reaction started with the
incubation of the 12.5 ꢀL of the reaction cocktail (0.5 ng/ꢀL of GST-Src kinase in 1.25 mM
DTT) with 12.5 ꢀL of prediluted compounds (dissolved in 1% DMSO) for 5 min at room
temperature. The ATP/substrate (25 ꢀL, 20ꢀM/1.5 ꢀM) cocktail was added to the mixture. The
biotinylated substrate (catalogue number 1366) contains the residues surrounding tyrosine 160
(Tyr 160) of the 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 ꢀL of 50mM EDTA (pH 8.0). The reaction solution (25 ꢀL) was transferred into
96-well streptavidin plates (Pierce, part number 15125), diluted with 75 ꢀL 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 ꢀL of 0.05% Tween-20 in a PBS buffer (PBS/T). After that, a
100 ꢀL of phosphotyrosine antibody (P-Tyr-100) (1:1000 dilution in PBS/T with 1% BSA) was
added to the each well 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 ꢀL of the secondary
anti-mouse IgG antibody, which was HRP conjugated (1:500 dilution in PBS/T with 1 % BSA)
for the 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 ꢀL of the 3,3',5,5'-
tetramethylbenzidinedihydrochloride (TMB) substrate for 5 min. The reaction was stopped by
adding 100 ꢀL /well of the stop solution to each well, mixed well, and read the absorbance a,
using a microplate reader (Molecular devices, spectra Max M2). The IC50 values of the
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compounds were calculated, using ORIGIN 6.0 (origin lab) software. IC50 is the concentration of
the compound that inhibited enzyme activity by 50%. All the experiments were carried out in
triplicate [32].
Molecular docking
The molecular docking simulation was carried out with the program AutoDock 4.0.1 [33].
The three-dimensional (3D) structure of the c-Src kinase was obtained from the Protein Data
Bank (PDB entry no. 2src with resolution 1.5 Å). 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 concentrated on the center of the ligand from the
corresponding crystal structure complexes . Briefly, an initial docking box is calculated from the
coordinates of a bound ligand in the crystal structure, and the box dimensions in x, y, and z are
increased by 10 Å until it reaches the optimum size of the ligand in the active site (25*25 Å).
The Lamarckian genetic algorithm was issued for docking with the following settings: a
maximum number of 2,500,000 energy evaluations, an initial population of 50 randomly placed
individuals, a maximum number of 37,000 generations, a mutation rate of 0.02 –a cross over rate
of 0.80- and an elitism value (number of top individuals that automatically survive) of 1. The
ligand was fully optimized 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 the c-Src active pocket was selected. Hydrogen atoms were added to the structure with the
Molecular Operating Environment (MOE 2012), [34] and atomic partial charges were calculated,
using Auto-Dock Tools. The selected active compounds were docked into the active site of the
microorganisms' targets to predict compound binding modes. For the flexible docking, Auto-
Dock standard parameter settings were applied. High-scoring binding poses were selected on the
basis of visual inspection.
Statistical Analysis
All the statistical calculations were done, using computer programs, Microsoft excel version
10, SPSS (statisticapackage for the social science version 20.00) statisticalprogram at 0.05, 0.01
and 0.001 level of probability [35]. The comparisons of the inhibiting tumor growth between the
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treatment groups or the control were done, using the Student's t test, One way ANOVA and Post
hoc-LSD tests (the least significantdifference) measurement.
Results And Discussions
Chemistry
This work exploits the highly efficient one-pot, three-component synthetic methodology to
generate
a
variety of halogenated 2-amino-4-aryl-6-methoxy-4H-benzo[h]chromene-3-
carbonitriles (4a-m) by the reaction of 4-methoxy-1-naphthol (1) with a mixture of different
aromatic aldehydes (2a-m) and malononitrile (3) in an ethanolic/piperidine solution under
microwave irradiation conditions for 2 min. at 140 °C, presented in Scheme 1. The maximum
power of the microwave irradiation was optimized by repeating the reaction at different watt
powers and time. The microwave radiations at 400 W and a reaction time of 2 min. gave the
highest yield.
The assignment of structure 4 was established on the basis of the spectral data. The IR
spectra of 4b-e, 4g-k, and 4m showed the appearance of the NH2 stretch at υ 3479-3452, 3339-
13
3323, and 3213-3196 cm-1 and the CN stretch band at υ 2199-2188 cm-1. The 1H and C NMR
16

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spectra of 4b-e, 4g-k, and 4m revealed the presence of the 4H signals at δ 5.94-4.90 (s, 1H, H-4)
and 41.05-30.85 ppm (C-4). The 13C NMR-DEPT spectra at 45°, 90°, and 135° and the 13C
NMR-APT spectra of compounds 4b, 4c, 4g-k, and 4m provided additional evidence in
supporting the suggested structures (see experimental part and supplementary materials).
Consequently, the mass spectra of compounds 4a-m gave further evidence for the proposed
structures.
Antitumor assays
The anti-proliferative activity of the newly synthesized compounds 4a-m was examined in
three human cancer cell lines: breast adenocarcinoma (MCF-7), colon carcinoma (HCT-116),
and hepatocellular carcinoma (HepG-2), using the MTT colorimetric assay as described in
literature [30, 31]. The In-vitro cytotoxicity evaluation, using the viability assay, was performed
at the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar University.
Vinblastine and Colchicine were used as references for the cytotoxic compounds under different
concentrations (50, 25, 12.5, 6.25, 3.125, 1.56 and 0 µg/mL). The results were expressed as
growth inhibitory concentration (IC50) values, which represent the required compound
concentrations to produce a 50% inhibition of cell growth after 24 hrs. of incubation in
comparison to the untreated controls as shown in Table 1 and Fig. 2. The obtained data revealed
that some of the target compounds displayed excellent to equipotent or modest growth inhibitory
activity against the three tested cancer cell lines. Investigations of the cytotoxic activity against
MCF-7 indicated that compounds 4e, 4k, 4b, 4h, 4d, 4m, 4i, 4g, 4j, 4a, and 4l (IC50 =1.2-17.7
µg/mL, respectively) were the most sensitive derivatives, evaluated against the standard drugs
Vinblastine and Colchicine. Compounds 4e, 4k, 4b, 4h, 4d, and 4m (IC50 =1.2-5.6 µg/mL,
respectively) were found to be the most potent derivatives of the tested compounds against MCF-
7 as they were 5.1, 4.7, 2.9, 2.0, 1.5, and 1,1 times more active than Vinblastine (IC50 =6.1
µg/mL) while compounds 4e, 4k, 4b, 4h, 4d, 4m, 4i, 4g, 4j, and 4a with (IC50 =10.5-17.4
µg/mL, respectively) were more efficacious than Colchicine (IC50 =17.7 µg/mL), and compound
4l (IC50 =17.7 µg/ml) was equipotent as Colchicine. Furthermore, compounds 4m and 4e (IC50
=1.3 and 1.9 µg/mL) were the most potent derivatives against HCT-116, and they were 2.0 and
1.4 times more active, compared to Vinblastine (IC50 =2.6 µg/mL). Compound 4b (IC50 =2.6
µg/mL) was equipotent as Vinblastine while compounds 4m, 4e, 4b, 4k, 4a, 4d, 4l, 4h, 4c, 4j, 4i,
17

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4g, and 4f (IC50 = 1.3-22.8 µg/mL, respectively) displayed excellent potent antitumor activity
against HCT-116, which was assessed against the standard drug Colchicine (IC50 =42.8 µg/mL).
On the other hand, the cytotoxicity evaluation against the HepG-2 cell line revealed that
compounds 4k, 4b, 4e, and 4h (IC50 = 1.5-2.5 µg/mL) were found to be the most potent tested
derivatives as their values were 3.1, 2.6, 2.1, and 1.8 times the standard Vinblastine (IC50 =4.6
µg/mL), and compounds 4k, 4b, 4e, 4h, 4m, 4l, 4i, 4g, and 4j (IC50 = 1.5-10.1 µg/mL) have
significant potent activity against HepG-2 in comparison to Colchicine (IC50 =10.6 µg/mL).
Finally, the other compounds displayed moderate to low cytotoxicity.
Table 1. SAR of the 4-aryl group and the inhibitory concentration (IC50, in µg/mL) of target
compounds against the three human cancer cell lines in comparison with Vinblastine and
Colchicine as measured with the microculturetetrazolium (MTT) method and c-Src inhibitory
activity (IC50, in µM) in comparison with Staurosporine.a
Cytotoxic activity
IC50 (µg/mL)
c-Src
inhibitory
activity
IC50 (µM)
Selectivity
analysis
LogP
ID
MCF-7
HCT-116
HepG-2
4.33±0.5
4.42±0.6
5.05±0.5
-
4.88±0.5
5.34±0.5
5.34±0.5
5.34±0.5
4a
4c
4l
17.4± 0.04
33.5± 0.02 10.1± 0.12 24.3± 0.2
17.7± 0.3
17.7± 0.12
38.7± 0.18
13.8± 0.3
5± 0.05
11.7±0.09
-
-
-
-
HCT-Selective
Less active
HCT-Selective
-
7.3± 0.05
42.8± 0.2
22.8± 0.2 28.4±0.36 35.5± 0.42
5.9± 0.03
10.6± 0.4
Colchicine
4f
4j
4g
Less active
11.5± 0.14 10.1±0.06
-
-
Less active
11.6± 0.11 18.1± 0.19 10± 0.01
HepG-
Selective
-
4i
10.5± 0.01 16.8± 0.02 6.0± 0.01
-
-
-
Vinblastine
6.1± 0.05
5.6± 0.2
4.1± 0.03
3.0± 0.24
2.1± 0.11
1.3± 0.03
1.2± 0.01
-
2.6± 0.01
1.3± 0.11
4.6± 0.08
5.75±0.7
4.88±0.5
5.34±0.5
4.42±0.6
5.34±0.5
4.88±0.5
-
4m
4d
4h
4b
4k
4e
5.4± 0.09 0.6459±0.66 Non-selective
5.11± 0.03 18.5± 0.7 0.1432±0.50
Bi-selective
Non-selective
-
7.6± 0.08
2.6± 0.28
3.0± 0.08
1.9± 0.02
-
2.5± 0.02 0.420± 0.45
1.8± 0.3 0.1617±0.62
1.5± 0.08 0.3044±0.04
2.2±0.04 0.2459±0.21
Staurosporine
-
0.1069±0.22
IC50 values as the mean values of triplicate wells from at least three experiments and are
reported as the mean ± standard error, SE.
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45
40
35
30
25
20
15
10
5
MCF-7
HCT-116
HepG-2
0
Compounds
Fig. (2). IC50 values expressed in (µg/mL) of halogenated 2-amino-4H-benzo[h]chromene
derivatives 4a-m) against MCF-7, HCT and HepG-2 tumor cells.
(
c-Src inhibitory activity
The most active compounds 4b, 4d, 4e, 4h, 4k, and 4m were selected for further
investigation to test their biological efficacy against the c-Src kinase target. This investigation
could assist us in knowing more details about the action mode of these compounds and how they
work. The desired compounds were compared with Staurosporine, a reference drug. Based on the
calculated IC50 values, all products displayed potent activities, ranged between 0.6459-0.1432
ꢀM. Our results revealed that compound 4d, having a 2-chloro substituent on the aromatic ring,
displayed the best activity (IC50 = 0.1432 ꢀM) while compound 4m, having methoxy and bromo
substituents on the aromatic ring, exhibited the lowest activity with IC50 = 0.6459 ꢀM. However,
the other compounds also showed good activities, ranging between these two values, and are
comparable to the reference drug Staurosporine, whereas compound 4f was inactive with IC50 =
19

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35.5 ꢀM. As a final comparison between the newly designed compounds and the previously
reported designed structures [27, 28], we can conclude that compounds 3-amino-8-bromo-1-(4-
chlorophenyl)-1H-benzo[f]chromene-2-carbonitrile and 2-amino-4-(2 or 3-chloro/2,4-
diflouro/3,4-dichlorophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitrile exhibited higher
c-Src kinase inhibitory activity in evaluation against compounds 2-amino-4-(2-chloro/2,3-
dichlorophenyl)-4H-benzo[h]chromene-3-carbonitrile
and
3-amino-1-(3-chloro/2,3-
dichlorophenyl)-1H-benzo[f]chromene-2-carbonitrile [28], which implies that the grafting of a
lipophilic electron-withdrawing substituent like the bromine atom or an electron-donating
substituent like the methoxy group in the naphthalene moiety is more beneficial than that of the
unsubstituted naphthalene moiety for the c-Src kinase inhibitory activity.
Molecular docking
The c-Src kinase is a non-receptor tyrosine kinase, which performs imperative roles in
controlling the different biological processes, including: cell proliferation, adhesion, migration,
and invasion [36, 37]. The c-Src kinase is over expressed and/or aberrantly activated in a variety
of human tumors, including: breast, colon, pancreatic, bladder, ovarian, and brain carcinomas [
38, 39]. Remarkably, the increased c-Src activity is believed to correlate strongly with metastatic
potential and poor prognosis in breast cancers [38, 40, 41]. Since the c-Src kinase has been
recognized as an attractive molecular target for cancer therapy, the development of the c-Src
inhibitors for cancer treatment is of increasing interest.In this report, the docking protocols were
applied for the chemical scaffold of the target fused systems as a preliminary step for the
identification of its binding mode and behavior in the enzyme pocket, i.e to determine which
derivative is the best binder or inhibitor and is considered a good tool for furthure optimization.
The placement of the scaffold revealed that the tricyclic fused systems tolerates the hydrophobic
Val209 and Ile201 with the aromatic interaction similar to Staurosporine. Additionally, polar
nitrile fragements had the ability to stabilize the system through hydrogen bonding interactions
with Lys222 and presented the importance of its role in the pocket. The active potent
antiproliferative derivative 4b with the substitution of the 2,4-difluro atoms exhibited promising
interactions in the ATP binding pocket of the target enzyme through conserved hydrophobic
processes with the Phe209 and Nitrile-Lys222 hydrogen bonding types. Moreover, the aromatic
hydrophobic interaction of the difluro atoms with Phe333 might be responsible for their activity.
Extra aromatic stacking appears in the pocket region between the terminal aromatic fragments of
20

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the system and the Gly272 residue, making the compound more stable. The second active
derivative 4d that carries the 2-Cl atom on the terminal ring in the system was also docked in the
ATP binding pocket and offered a good interaction, compared to the reference drug. The
hydrogen bonding and hydrophobic interactions with the corresponding amino acid residues
(Phe209, Gly272, Lys222, and Gly204) and the aromatic compound rings with the halogen and
nitrile fragments are prominent and crucial for compound stability. In contrast to the analog 4a
that is monosubstituted, the fluro atom showed less activity in comparison with the others, due to
the loss of interaction with the Phe333 key residue in the pocket. Hence, compound 4a lost most
of its essential interactions and different modes of the binding pocket, which makes the
derivative 4-F inactive. The data are presented in Fig. 3 and Table 2. Meanwhile, it was observed
that the methoxy group is shared strongly in the binding pocket, even with a mixed
hydrophilic/hydrophobic effect, and creates an interaction with the scaffold (Glu267, Val209,
and Ala220) that presents positive activity contribution and stability of the active compounds.
Finally, it is clear that the fused ring system could be a promising scaffold for designing
anticancer agents by targeting the c-Src kinase while focusing on the ring orientations, type, and
postion of the halogens.
Leu
321
Arg
318
Gly
204
Ala
220
Gly
202
Lys
222
NH
Lys
O
203
Phe
333
H
Asp
276
O
N
N
3.81
333..8.88111
3.81
Val
209
H
3.86
333..8.88666
H
3.86
Thr
266
Ile
201
O
N
Glu
267
2.8
222..8.88
2.8
Ser
273
Gly
272
Met
269
Asn
319
Val
249
Ser
331
Tyr
268
(A) Target scaffold
21

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Thr
266
Val
249
Ala
220
Glu
267
Phe
333
Ile
201
Met
269
3.61
333..6.66111
3.61
Leu
321
Val
209
Gly
202
Gly
272
O
4.13
444..1.11333
4.13
H
4.89
444..8.88999
4.89
H
Cl
Ser
331
Lys
222
O
3.22
333..2.22222
3.22
N
NH2
3.64
333..6.66444
3.64
Gly
204
Lys
203
(B) Active analogs
Fig. (3). Binding data of target compounds. (A) 2D depiction map of the docked conformation of
the target scaffold (B) potent active 4d and 4b analogs, in the active site of enzyme revealing the
essential residues and types of interactions.
Table 2. Interaction maps of docked compounds.
Binding
energy (dG,
Kcal/mol)
Cop.
4b
Ar1
Ar2
X
Ar4
CN
Gly272, Met269 Lys209 Phe333 Lys222
Aromatic Hydrophobic
Gly272, Leu321 Lys209 Phe333 Lys222 Lys222, Gly204
Lys222
H-bonding, 2.8
-16.5
-15.4
-12.6
4d
H-bonding, 2.7
Hydrophobic
and 2.9
Leu321
Aromatic Hydrophobic
Leu321 Gly202 Ile201
Hydrophobic
-
4f
The data reported in the table are extracted from the MOE program, showing the corresponding
amino acids residues in the enzyme pocket, aromatic rings of ligands, interaction distances, types
of interaction, and their binding energy to some selected drugs. Conserved amino acids are bold
highlighted.
22

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SAR studies
The partition coefficient (Log P), which is known as the index of lipophilicity and is an
important physicochemical parameter, was measured by the ACD/Labs Log P calculation,
ver.14.02, and is displayed in Table 1. The preliminary SAR study was centered on the effect of
the halogens substituents on the phenyl group at the 4-position of the 4H-benzo[h]chromene
moiety on the antitumor activities of the target compounds. Furthermore, it includes the
relationship between the lipophilicity and the antitumor activity. In a comparison of the
cytotoxicity of the derivatives 4a-m against MCF-7, we discovered that the 4-fluoro-phenyl and
2,6-difluorophenyl analogues 4a and 4c displayed moderate to fair cytotoxic activities (IC50
=17.4 and 33.5 µg/mL), compared to Vinblastine (IC50 =6.1 µg/mL) while the 2,4-fluorophenyl
4b has caused a remarkable enhancement in the antitumor activity (IC50 =2.1 µg/mL) as
disclosed in Table 3. Proposing the incorporation of the fluorine atoms as hydrophobic groups at
the 2,4-positions on the phenyl group in the 4-position of the 4H-benzo[h]chromene moiety may
be beneficial when evaluated against the other positions by increasing the partition coefficient
(Log P), as shown in Tables 1 and 3, respectively.
Table 3. Positive and negative controls and effectiveness of the test compounds against MCF-7
IC50 (µg/mL)
6.1 a
Control / Cpd.
Vinblastine
Cell line
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
MCF-7
F ratio
P value
4e
4k
4b
4h
1.2 d
1.3 f
2.1 e
193.14
0.000 (HS)
3.0 d
4d
4m
Colchicine
4.1 c
5.6 b
17.7 a
1.2 g
4e
4k
4b
4h
4d
4m
4i
4j
4a
4l
1.3 g
2.1 fg
3.0 ef
4.1 de
5.6 d
139.79
0.000 (HS)
10.5 c
13.8 b
17.4 a
17.7 a
23

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Positive control (active compounds) and negative control (standard drugs), same letters in
column do not differ statistically (ANOVA), medium evaluated with LSD test (
α = 0.05) and HS
(highest significant).
In the case of the chlorine series, the 4-chlorophenyl derivative 4f resulted in a large loss of
activity (IC50 =38.7 µg/mL) while changing the position of the chlorine atom from the 4 position
to the 3- or 2-postion (4e and 4d), which has sharply increased their antitumor activity with IC50
values of 1.2 and 4.1 µg/mL, respectively. In contrast, compounds 4k and 4h with two chlorine
atoms at the 3,4- or 2,4- positions, respectively, exhibited a remarkable enhancement in their
antitumor activity against MCF-7 in comparison to the rest of the dichloro substituents, Table 3.
Having a bromine substituent at the 4-position, compound 4l, resulted in a moderate loss in its
activity against MCF-7 while the introduction of two bromine atoms and a methoxy group on the
phenyl ring at the 4-position of compound 4m led to an increase in its activity, which implies
that the bulky group at the 2,3,5-positions is preferred over the bromine atom at the 4-position on
the phenyl group, Tables 1 and 3. Additionally, compounds 4e, 4k, 4b, 4h, 4d, 4m, 4i, 4g, 4j, 4a,
and 4l (IC50 =1.2-17.7 µg/mL, respectively) displayed efficacious cytotoxicity, compared to
Colchicine (IC50 =17.7 µg/mL), indicating that the halogens substituent at certain positions may
be more suitable than the other positions, Table 3.
In the case of HCT-116, the investigation of SAR revealed that compounds 4m and 4e have
the highest significant potent antitumor activity when assessed against Vinblastine, which reveals
that the trisubstituent (more bulky and more hydrophobic groups) is preferred over the
monosubstituent (less bulky and less hydrophobic groups) at the 4-position of the 4H-
benzo[h]chromene moiety, implying that there might be a size limited pocket at the 4-position of
the 4H-benzo[h]chromene moiety. The rest of the compounds 4a-d and 4f-l displayed efficacious
cytotoxicity with IC50 values, ranging from 1.3 to 22.8 µg/mL, in comparison to Colchicine (IC50
=42.8 µg/ml), which intimates that the halogens substituents at certain positions may be more
advantageous over other positions with increasing the value of the partition coefficient (Log P),
Tables 1 and 4.
Furthermore, the HepG-2 cancer cell line was highly sensitive to the 3,4-chloro, 2,4- fluoro,
3-chloro, and 2,4-chloro substituents 4k, 4b, 4e, and 4h (IC50 =1.5-2.5 µg/mL) as compared to
Vinblastine (IC50 =4.6 µg/mL) and Colchicine (IC50 =10.6 µg/mL) while the 3,5-bromo-2-
methoxy, 4-bromo, and 2,5-, 2,3- or 2,6-chloro substituents 4m, 4l, 4i, 4g, and 4j, respectively,
24

ACCEPTED MANUSCRIPT
(IC50 =5.4-10.1 µg/mL) were more potent than Colchicine (IC50 =10.6 µg/ml), which indicates
that the tri/disubstituent is preferred over the monosubstituent as hydrophobic groups at the 4-
position of the 4H-benzo[h]chromene moiety by increasing the partition coefficient (Log P),
presented in Tables 1 and 5, respectively.
Table 4. Positive and negative controls and effectiveness of the test compounds against HCT-116
Control / Cpd. IC50 (µg/ml)
Vinblastine
Cell line
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
HCT-116
F ratio
4.203
P value
2.6 a
1.3 b
4m
4e
Colchicine
0.08 (NS)
1.9 ab
42.8 a
1.3 h
4m
4e
4b
4k
4a
4d
4l
4h
4c
4j
1.9 h
2.6 gh
3.0f gh
5.0 efg
5.1ef
122.36
0.000 (HS)
7.3 de
7.6 de
10.1 d
11.5 d
16.8 c
18.1c
22.8 b
4i
4g
4f
Positive control (active compounds) and negative control (standard drugs), same letters in column do
not differ statistically (ANOVA), medium evaluated with LSD test (α = 0.05) and HS (highest
significant).
Table 5. Positive and negative controls and effectiveness of the test compounds against HepG-2
Control / Cpd. IC50 (µg/ml)
Vinblastine
Cell line
F ratio
P value
4.6 a
1.5 c
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
4k
4b
4e
4h
1.8 bc
2.2 bc
2.5b
18.59
0.0001 (HS)
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Colchicine
10.6 a
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
HepG-2
4k
4b
4e
4h
4m
4l
4i
4g
4j
1.5 e
1.8 de
2.2 cd
2.5 c
224.83
0.000 (HS)
5.4 b
5.9 b
6.0 b
10.0 a
10.1 a
Positive control (active compounds) and negative control (standard drugs), Same letters in
column do not differ statistically (ANOVA), medium evaluated with LSD test (
α = 0.05) and HS
(highest significant).
In general, the order of anticancer activities of the halogenated derivatives 4a-m has widely
varied in accordance to the type of the cell line and the type and position of the halogen atom on
the phenyl ring, attached to the 4-position of the 4H-benzo[h]chromene moiety. The effect of the
halogenated members in the target compounds resulted in a decrease in the activity in the
following order: (3-Cl > 3,4-Cl >2,4-F > 2,4-Cl > 2-Cl > 3,5-Br-2-OMe > 2,5-Cl > 2,3-Cl > 2,6-
Cl > 4-F > 4-Br > 2,6-F > 4-Cl) for the MCF-7, (3,5-Br-2-OMe > 3-Cl > 2,4-F >3,4-Cl > 4-F >2-
Cl > 4-Br > 2,4-Cl > 2,6-F > 2,6-Cl > 2,5-Cl > 2,3-Cl > 4-Cl) for the HCT-116, and (3,4-Cl >
2,4-F> 3-Cl > 2,4-Cl >3,5-Br-2-OMe >4-Br > 2,5-Cl >2,3-Cl > 2,6-Cl > 4-F> 2-Cl > 2,6-F > 4-
Cl) for the HepG-2.
Finally, we can deduce that the substitution pattern on the phenyl moiety is a crucial element
for the antitumor activities. The incorporation of the substituents with the bulky electron-
withdrawing groups as the bromine, chlorine, or fluorine atoms at certain positions on the phenyl
ring has greatly enhanced the activity by increasing the partition coefficient (Log P).
Conclusion
In summary, we have synthesized a variety of halogenated aromatic fused systems, based
upon a previous study of the 2-amino-4-aryl-6-methoxy-4H-benzo[h]chromene-3-carbonitrile
scaffold (4a-m) and evaluated their cytotoxic activities against the three cancer cell lines: MCF-
7, HCT-116, and HepG-2. Most of the prepared compounds displayed relatively potent and
selective cytotoxicity against the three tumor cells. The docking studies have been carried out on
all of the newly synthesized compounds 4a-m in comparison to the reference drugs Vinblastine
26

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and Colchicine in a trial to predict their mode of action as anticancer agents. The most active
compounds were subjected to further investigation against the c-Src kinase, and they were
discovered to be promising candidates as potent c-Src kinase inhibitors. As revealed from the
aforementioned data, the compounds showed significant inhibitory activity, giving rise to the
notion that these derivatives exert their vital role as anticancer agents through the inhibition of
the c-Src enzyme. The Structure Activity Relationships (SARs) concluded that the introduction
of certain halogen substituents on the phenyl ring, attached to the 4-position of the 4H-
benzo[h]chromene moiety, enhanced the antitumor activities of the target compounds by
increasing their partition coefficient (Log P), hydrophobic groups, and lipophilicity.
Acknowledgements
The authors would like to thank chemical computing group for use of the MOE software. The
authors would also like to thank Malak T. Mahmoud for English editing.
Author Contribution Statement
A. M. El-Agrody, R. M. Okasha, Ateyatallah Aljuhani and T. H. Afifi conceived and designed
the experiments; M. A. El-Nassag, A. H. Hassan, A. H. Halawa and A. M. Fouda performed the
experiments and analyzed the data; H. E. Ahmed, S. M. AbdEl-Gilil, E. S. Khattab and S.
Ihmaid achieved the antitumor assay and docking studies. All authors wrote the paper, discussed
the results and commented on the manuscript.
Conflict of Interest
The authors declare no conflicts of interest.
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