Med Chem Res (2017) 26:929–939 MEDICINAL

DOI 10.1007/s00044-017-1798-9
CHEMISTRY
RESEARCH
ORIGINAL RESEARCH

Design, synthesis and biological evaluation of new 4-(4-substituted-

anilino)quinoline derivatives as anticancer agents
Khaled R. A. Abdellatif1,2 Eman K. A. Abdelall1 Mohamed A. Abdelgawad1,3
● ● ●

Dina M. E. Amin1 Hany A. Omar4,5

●

Received: 7 September 2016 / Accepted: 3 February 2017 / Published online: 23 February 2017
© Springer Science+Business Media New York 2017

Abstract A new series of 4-(4-substituted-anilino)quino- Introduction

line derivatives 6a–f was synthesized from amine deriva-
tives via Gould–Jacobs reaction. All synthesized The quinoline scaffold, a highly versatile drug like template,
compounds were evaluated for their cytotoxic activity is often used for the design of many pharmaceutical active
against two human cancer cell lines; breast carcinoma compounds with different pharmacological activities such
(MCF-7) and non-small cell lung cancer (A549). The tested as antimalarial (Ekoue et al. 2009), antimicrobial (Patel and
compounds showed a broad range of activities (IC50 = Park 2014), anti-inflammatory (El-Feky et al. 2015), hyp-
3.42–23.32 and 5.97–22.01 µM) in comparison with dox- notic (Ferlin et al. 2005), ant tuberculosis (Medapi et al.
orubicin (IC50 = 2.07 and 0.02 µM) and erlotinib (IC50 = 2015), anticancer (Sun et al. 2013), antiviral (Majerz et al.
1.14 and 19.26 µM) for MCF-7 and A549 respectively. The 2011), antischizophrenic (Hamaguchi et al. 2015), anti-
4-(4-chloroanilino)quinoline derivative (6c, IC50 = 3.42 and protozoal (Ma et al. 2009), anticholesterol (Rano et al.
5.97 µM) was the most potent among all compounds against 2009), Anthelmintic (Rossiter et al. 2005), and analgesic
both MCF-7 and A549 cell lines respectively. In addition, (Roma et al. 2008). Quinoline is a privileged anticancer
molecular docking studies were performed and the results scaffold possessing various antiproliferative mechanisms as
were in agreement with the in vitro cytotoxic data. cell cycle arrest, angiogenesis inhibition, apoptosis (Afzal
et al. 2015), DNA repair and tyrosine kinases (TK) inhibitor
Keywords Quinoline Antitumor MCF-7 EGFR
● ● ●
(Solomon and Lee 2011). Epidermal growth factor receptor
(EGFR), one of protein kinases, plays a critical role in
signals transduction (Abdellatif et al. 2014a). Dysregulation
of EGFR causes considerable alteration in different cellular
processes like transcription, proliferation, angiogensis and
inhibition of apoptosis so drugs targeted EGFR are con-
* Khaled R. A. Abdellatif sidered as the main anticancer chemotherapeutics (Hicks
khaled.ahmed@pharm.bsu.edu.eg et al. 2010) especially for breast, ovarian, colon, lung, and
1 prostate cancer at which over expression of EGFR was
Department of Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt established (Amin et al. 2011). Erlotinib (I) is one of 4-
2 anilinoqunazoline derivatives approved by FDA as kinase
Pharmaceutical Sciences Department, Ibn Sina National College
for Medical Studies, Jeddah 21418, Saudi Arabia inhibitor (Arora and Scholar 2005), 6,7-disubstituted-4-
3 anilinoquioline-3-carbonitrile derivatives EKB-567 (II) and
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Al-Jouf University, Sakaka, Al-Jouf, Saudi Arabia HKI-272 (III) are EGFR inhibitors which were selected for
4 further studies in clinical trials as anticancer (Afzal et al.
Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt 2015) were taken as references in our design for new
5 cytotoxic quinoline derivatives (Fig. 1).
Sharjah Institute for Medical Research, Department of
Pharmacology, College of Pharmacy, University of Sharjah, Based on the previous information and in continuation of
Sharjah 27272, United Arab Emirates our previous work for synthesis of new anticancer agents

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930 Med Chem Res (2017) 26:929–939

Fig. 1 Rational design of 4-(4-

substituted-anilino)quinoline
derivatives 6a–f based on
chemical structures of some
anticancer drugs containing
anilinoquinazoline and
anilinoquinoline moieties
(erlotinib I, EKB-569 II and
HKI-272 III) (Arora and
Scholar 2005; Afzal et al. 2015)

(Abdellatif et al. 2013, 2014b; Gouda et al. 2014), we now
describe synthesis, in vitro anticancer evaluation and
molecular docking for a new series of 4-anilinoquinoline
derivatives 6a–f which are structurally related to erlotinib
(I), EKB-567 (II) and HKI-272 (III) with some modifica-
tions; (i) isosteric replacement of quniazoline ring of erlo-
tinib by quinoline ring which bind with adenine portion of
ATP binding site of EGFR protein tyrosine kinase, (ii)
attach different substituents at para position of the aniline
moiety (lipophilic group as methyl or methoxy, 6a, 6b, 6d
and 6e or halogen like Cl, 6c and 6f) to check their effect on
biological activity, (iii) the N of quniazoline ring of erloti-
nib (I) or the cyano group at position 3 of quinoline ring for
both EKB-569 (II) and HKI-272 (III) were replaced by
another electron withdrawing ester group to retain the
charge distribution that would result from such EGFR
interaction and (iv) replacement the large fragment of the
erlotinib, aminoacrylate and alkoxy moieties in positions
6,7 of EKB-569 and HKI-272 quinoline rings by electron
withdrawing groups as COOH or Br.
Material and methods
Experimental
Melting points were determined on a Griffin apparatus and
are uncorrected. Infrared (IR) spectra were recorded on
Shimadzu IR 435 spectrophotometer using KBr discs. 1H
NMR spectra were measured on Varian Gemini 300 MHz or
on Bruker 400 MHz spectrometer in D2O, CDCl3 or
DMSO-d6 with TMS as the internal standard, where J

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Med Chem Res (2017) 26:929–939
(coupling constant) values were estimated in hertz (Hz) and
chemical shifts were recorded in ppm on δ scale. 13C NMR
spectra were carried out on Bruker 400 MHz spectrometer.
Mass spectra were run at 70 ev on Shimadzu QP-2010 plus
spectrometer. Microanalyses were performed for C, H, N
(Micro analytical Center, Cairo University and at the
regional center for Mycology and Biotechnology, Al-Azhar
University) and were within +0.4% of theoretical values.
Progress of the reaction was monitored by TLC using alu-
minum sheets precoated with UV fluorescent silica gel
Merck 60 F254 and were visualized by UV lamp.
General procedure for synthesis of enamine compounds
(3a, b)
A mixture of the respective amine1a or 1b (2.16 g, 10
mmol) with diethylethoxymethylene malonate (2, 2.16 g,
10 mmol) in ethanol (25 mL) was heated under reflux for
5–7 h. The mixture was evaporated under reduced pressure
to give a solid product which crystallized from benzene.
Physical and spectral data of 3a, b are listed below.
3-bromo-4-((3-ethoxy-2-(ethoxycarbonyl)-3-oxoprop-1-en-
1-yl)amino)benzoic acid (3a) White crystals; yield 83%;
m.p. 222–224 °C; IR (KBr/cm−1) 3486 (OH), 3385 (NH),
3090 (C–H aromatic), 2983 (C–H aliphatic), 1692 (C=O),
1627 (C=O); 1H NMR (CDCl3) δ 1.21–1.27 (m, 6H, 2
(CH2CH3)), 4.17 (q, J = 7.2 Hz, 2H, CH2CH3), 4.26 (q, J
= 7.2 Hz, 2H, CH2CH3), 7.72 (d, J = 8.7 Hz, 1H, phenyl H-
6), 7.95 (d, J = 8.7 Hz, 1H, phenyl H-5), 8.14 (s, 1H,
phenyl H-3), 8.54 (d, J = 12.6 Hz, 1H, HC olefenic), 11.21
(d, J = 12.6 Hz, 1H, NH, D2O exchangeable), 13.30 ppm (s,
1H, OH, D2O exchangeable); 13C NMR (CDCl3) δ 14.2,
14.3, 60.6, 60.9, 97.8, 112.9, 114.5, 119.2, 128.8, 130.7,
135.5, 146.1, 149.1, 168.5, 170.2; MS, (relative abundance
þ þ
%) 386 (M  , 9.63 %), 388 (M  +2, 8.87%), 341 (100%);
Anal. Calcd. for C15H16BrNO6 (386): C, 46.65; H, 4.18; N,
3.93. Found: C, 46.75; H, 3.88; N, 4.07.
5-Bromo-2-((3-ethoxy-2-(ethoxycarbonyl)-3-oxoprop-1-en-
1-yl)amino)benzoic acid (3b) Shiny buff crystals; yield
80%; m.p. 198–200 °C; IR (KBr/cm−1) 3465 (OH), 3130
(NH), 3088 (C–H aromatic), 2981 (C–H aliphatic), 1688
(C=O), 1633 (C=O); 1H NMR (CDCl3) δ 1.35–1.43 (m,
6H, 2(CH2CH3)), 4.30 (q, J = 6.8 Hz, 2H, CH2CH3), 4.38
(q, J = 6.8 Hz, 2H, CH2CH3), 7.36 (d, J = 8.4 Hz, 1H,
phenyl H-6), 8.10 (d, J = 8.4 Hz, 1H, phenyl H-5), 8.36 (s,
1H, phenyl H-3), 8.55 (d, J = 12.8 Hz, 1H, HC olefenic),
11.46 (d, J = 12.8 Hz, 1H, NH, D2O exchangeable), 13.25
(s, 1H, OH, D2O exchangeable ); 13C NMR (CDCl3) δ 14.3,
14.3, 60.6, 60.7, 97.6, 106.5, 111.2, 115.6, 116.6, 117.2,
133.8, 135.1, 148.6, 168.5, 170.2; MS, (relative abundance
þ þ
%): 386 (M  , 8.47 %), 388 (M  +2, 7.57%), 115 (100%);
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
931
Anal. Calcd. for C15H16BrNO6 (386): C, 46.65; H, 4.18; N,
3.63. Found: C, 46.88; H, 4.20; N, 3.63.
General procedure for synthesis of quinoline compounds
(4a, b)
A solution of 3a or 3b (0.772 g, 2.0 mmol) in diphenyl ether
(30 mL) was heated at 250–260 °C for 1–2 h .The reaction
mixture was cooled and then diethyl ether (100 mL) was
added. The resulting precipitate was filtered, washed with
petroleum ether followed by methanol, dried and crystal-
lized from ethanol. Physical and spectral data of 4a, b are
listed below.
8-Bromo-3-(ethoxycarbonyl)-4-oxo-1,4-dihydroquinoline-
6-carboxylic acid (4a) Shiny yellow crystals; yield 63%;
m.p. 293–295 °C; IR (KBr/cm−1) 3446 (OH), 3129 (NH),
3090 (C–H aromatic), 2980 (CH aliphatic), 1688 (C=O),
1633 (C=O); 1H NMR (DMSO-d6) δ 1.29 (t, J = 6.9 Hz,
3H, CH2CH3), 4.24 (q, J = 6.9 Hz, 2H, CH2CH3), 8.39 (s,
1H, quinoline H-2), 8.46 (s, 1H, quinoline H-5), 8.70 (s,
1H, quinoline H-7), 11.56 (s, 1H, NH, D2O exchangeable),
13.30 (s, 1H, OH, D2O exchangeable); 13C NMR (DMSO-
d6) δ 14.5, 60.83, 111.7, 112.6, 127.6, 128.2, 136.2, 137.9,
143.2, 146.5, 164.4, 166.1; MS, (relative abundance %):
þ þ
340 (M  , 37.50 %), 342 (M  +2, 35.57%), 341 (100%);
Anal. Calcd. for C13H10BrNO5 (340): C, 45.91; H, 2.96; N,
4.12. Found: C, 45.70; H, 3.00; N, 4.06.
6-Bromo-3-(ethoxycarbonyl)-4-oxo-1,4-dihydroquinoline-
8-carboxylic acid (4b) Shiny light green crystals; yield
60%; m.p. 288–290 °C; IR (KBr/cm−1) 3475 (OH), 3431
(NH), 3076 (C–H aromatic), 2984 (CH aliphatic), 1690
(C=O), 1648 (C=O); 1H NMR (DMSO-d6) δ 1.30 (t, J =
7.2 Hz, 3H, CH2CH3), 4.23 (q, J = 7.2 Hz, 2H, CH2CH3),
8.38 (s, 1H, quinoline H-2), 8.69 (s,1H, quinoline H-5),
8.70 (s, 1H, quinoline H-7), 11.65 (s, 1H, NH, D2O
exchangeable), 13.32 (s,1H, OH, D2O exchangeable); 13C
NMR (DMSO-d6)δ 14.7, 60.1, 111.0, 113.0, 127.67, 127.9,
128.9, 135.2, 143.2, 147.2, 164.4, 165.8, 173.4; MS,
þ þ
(relative abundance %): 340 (M  , 33.66 %), 342 (M  +2,
31.71), 341 (100%); Anal. Calcd. for C13H10BrNO5
(340): C, 45.91; H, 2.96; N, 4.12. Found: C, 45.81; H, 3.02;
N, 3.98.
General procedure for synthesis of 4-chloroquinoline
compounds (5a, b)
A quinoline derivative 4a or 4b (1.02 g, 3.0 mmol) was
added to POCl3 (2.29 g, 15.0 mmol), and the mixture was
heated under reflux until the starting material had dis-
appeared at TLC (8 h). The suspension was then cooled first
to room temperature and then to 0 °C in a water/ice bath.
932
Then, with stirring it was carefully made alkaline with
Na2CO3 aqueous solution, then this filtrate was acidified by
acetic acid (96%) and the resulting precipitate was col-
lected, washed many times with water, dried and crystal-
lized from methanol. Physical and spectral data of 5a, b are
listed below.
8-Bromo-4-chloro-3-(ethoxycarbonyl)quinoline-6-car-
boxylic acid (5a) Canary yellow crystals; yield 73%; m.p.
244–246 °C; IR (KBr/cm−1) 3436 (OH), 3089 (C–H aro-
matic), 2983 (CH aliphatic), 1691 (C=O), 1653 (C=O); 1H
NMR (DMSO-d6 ) δ 1.28 (t, J = 7.2 Hz, 3H, CH2CH3),
4.23 (q, J = 7.2 Hz, 2H, CH2CH3), 8.37 (s, 1H, quinoline
H-7), 8.44 (s, 1H, quinoline H-5), 8.69 (s, 1H, quinoline H-
2), 11.85 (s, 1H, OH, D2O exchangeable); 13C NMR
(DMSO-d6) δ 14.7, 60.1, 111.3, 127.6, 128.2, 128.6, 135.2,
147.5, 164.4, 166.1, 173.4; MS, (relative abundance %):
þ þ
358.5 (M  , 52.35%), 360.5 (M  +2, 14.79%), 57(100%);
Anal. Calcd. for C13H9BrClNO4 (358.5): C, 43.54; H, 2.53;
N, 3.91. Found: C, 43.28; H, 2.79; N, 4.13.
6-Bromo-4-chloro-3-(ethoxycarbonyl)quinoline-8-car-
boxylic acid (5b) Yellow crystals; yield 70%; m.p.
215–217 °C; IR (KBr/cm−1) 3426 (OH), 3076 (C–H aro-
matic) , 2980 (CH aliphatic), 1756 (C=O), 1713 (C=O); 1H
NMR (DMSO-d6) δ 1.29 (t, J = 6.8 Hz, 3H, CH2CH3), 4.24
(q, J = 6.8 Hz, 2H, CH2CH3), 8.38 (s, 1H, quinoline H-5),
8.45 (s,1H, quinoline H-7), 8.69 (s, 1H, quinoline H-2),
11.91 (s, 1H, OH, D2O exchangeable); MS, (relative
þ þ
abundance %), 358.5 (M  , 25.35 %), 360.5 (M  +2,
15.21%), 75 (100%); Anal. Calcd. for C13H9BrClNO4
(358.5): C, 43.54; H, 2.53; N, 3.91. Found: C, 43.64; H,
2.90; N, 3.73.
General procedure for synthesis of 4-substituted
phenylaminoquinoline compounds (6a–f)
A mixture of chloroquinoline derivative 5a or 5b (3.585 g,
10 mmol), the appropriate aromatic amine (10 mmol) and
catalytic amount of sodium iodide (0.2 g) in ethanol (20
mL) was heated under reflux for 2–4 h. Filter, the filtrate
was neutralized to litmus paper with acetic acid (96%). The
formed precipitate was collected by filtration, washed with
water and crystallized from the ethanol.
8-Bromo-3-(ethoxycarbonyl)-4-(p-tolylamino)quinoline-6-
carboxylic acid (6a) Yellow crystals; yield 65%; m.p.
196–198 °C; IR (KBr/cm−1) 3418–3272 (OH, NH), 3127
(C–H aromatic), 2981 (C–H aliphatic), 1705 (C=O); 1H
NMR (CDCl3) δ 1.36 (t, J = 7.2 Hz, 3H, CH2CH3), 2.35 (s,
3H, CH3), 4.48 (q, J = 7.2 Hz, 2H, CH2CH3), 7.17 (d, J =
8.4 Hz, 2H, phenyl H-2,H-6), 7.27 (d, J = 8.4 Hz, 2H,
phenyl H-3,H-5), 8.41 (s, 1H, quinoline H-5), 8.52 (s, 1H,
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Med Chem Res (2017) 26:929–939
quinoline H-7), 8.47 (d, J = 12.9 Hz, quinoline H-2), 11.37
(s, J = 12.9 Hz, 1H, NH ,D2O exchangeable );13C NMR
(CDCl3) δ14.2, 20.9, 61.4, 106.0, 119.2, 123.7, 125.0,
125.7, 129.1, 130.4, 134.1, 135.9, 139.8, 150.1, 153.9,
þ
154.1, 164.5, 168.1; MS, (relative abundance %): 429 (M  ,
þ
6.70 %), 431(M  +2, 6.16%), 322(100%); Anal. Calcd. for
C20H17BrN2O4 (429): C, 55.96; H, 3.99; N, 6.53. Found: C,
55.60; H, 3.70; N, 6.70.
8-Bromo-3-(ethoxycarbonyl)-4-((4-methoxyphenyl)amino)
quinoline-6-carboxylic acid (6b) Yellow crystals; yield
69%; m.p. 182–184 °C; IR (KBr/cm−1) 3421–3272 (OH,
NH), 3090 (C–H aromatic), 2928 (C–H aliphatic), 1716
(C=O); 1H NMR (CDCl3) δ 1.26 (t, J = 7.2 Hz, 3H,
CH2CH3), 3.80 (s, 3H, OCH3), 4.46 (q, J = 7.2 Hz, 2H,
CH2CH3), 6.91 (d, J = 8.8 Hz, 2H, phenyl H-2,H-6), 7.10
(d, J = 8.8 Hz, 2H , phenyl H-3,H-5), 8.43 (s, 1H, quinoline
H-5), 8.53 (s, 1H, quinoline H-7), 9.42 (s, 1H, quinoline H-
2), 10.83 (s, 1H, NH, D2O exchangeable);13C NMR
(CDCl3) δ 14.2, 55.4, 61.4, 105.3, 115.1, 118.6, 124.2,
125.6, 129.0, 134.3, 149.5, 153.9, 154.7, 158.3, 164.1,
þ
168.2; MS, (relative abundance %): 445 (M  12.07 %), 447
þ
(M  +2, 10.82), 278 (100%); Anal. Calcd. for
C20H17BrN2O5 (445): C, 53.95; H, 3.85; N, 6.29. Found: C,
54.06; H, 3.91; N, 6.40.
8-Bromo-4-((4-chlorophenyl)amino)-3-(ethoxycarbonyl)
quinoline-6-carboxylic acid (6c) Yellow crystals; yield
71%; m.p. 200–202 °C ; IR (KBr/cm−1) 3430–3296 (OH,
NH), 3124 (C–H aromatic), 2982 (C–H aliphatic), 1715
(C=O); 1H NMR (DMSO-d6) δ 1.13 (t, J = 7.2 Hz, 3H,
CH2CH3) , 3.95 (q, J = 7.2 Hz, 2H, CH2CH3), 7.12 (d, J =
8.8 Hz, 2H, phenyl H-2,H-6), 7.36 (d, J = 8.8 Hz, 2H,
phenyl H-3,H-5), 8.49 (s, 1H, quinoline H-5), 8.90 (s, 1H,
quinoline H-7), 9.01 (s, 1H, quinoline H-2), 10.03 (s, 1H,
NH,D2O exchangeable); 13C NMR (CDCl3) δ16.7, 64.2,
109.5, 121.6, 127.1, 127.9, 128.0, 131.2, 132.8, 133.8,
136.8, 142.97, 152.5, 155.9, 156.3, 166.9, 170.4; MS,
þ þ
(relative abundance %):449 (M  , 3.61%), 451(M  +2,
6.20%), 322 (100%); Anal. Calcd. for C19H14BrClN2O4
(449): C, 50.75; H, 3.14; N, 6.23. Found: C, 50.55; H, 2.93;
N, 6.01.
6-Bromo-3-(ethoxycarbonyl)-4-(p-tolylamino)quinoline-8-
carboxylic acid (6d) Yellow crystals; yield 62%; m.p.
206–208 °C; IR (KBr/cm−1) 3500–3290 (OH, NH), 3100
(C–H aromatic), 2975 (C–H aliphatic), 1700 (C=O); 1H
NMR (CDCl3) δ 1.39 (t, J = 7.6 Hz, 3H, CH2CH3), 2.78 (s,
3H, CH3), 4.30 (q, J = 7.6 Hz, 2H, CH2CH3), 7.21 (d, J =
8.4 Hz, 2H, phenyl H-2,H-6), 7.53 (d, J = 8.4 Hz, 2H,
phenyl H-3,H-5), 8.09 (s, 1H, quinoline H-5), 8.35 (s, 1H,
quinoline H-7), 8.66 (s, 1H, quinoline H-2), 11.43 (s, 1H,
NH ,D2O exchangeable ); 13C NMR (DMSO-d6) δ 18.7,
Med Chem Res (2017) 26:929–939
20.9, 56.5, 120.5, 122.3, 125.0, 127.0, 128.6, 130.5, 133.7,
134.4, 140.2, 149.2, 152.9, 154.4, 165.9, 169.4; MS,
þ þ
(relative abundance %): 429 (M  , 14.62 %), 431(M  +2,
12.53%), 64 (100%); Anal. Calcd. for C20H17BrN2O4
(429): C, 55.96; H, 3.99; N, 6.53. Found: C, 56.07; H, 4.06;
N, 6.70.
6-Bromo-3-(ethoxycarbonyl)-4-((4-methoxyphenyl)amino)
quinoline-8-carboxylic acid (6e) Buff crystals; yield 69%;
m.p. 203–205 °C; IR (KBr/cm−1) 3440–3208 (OH, NH),
3090 (C–-H aromatic), 2931 (C–H aliphatic), 1710 (C=O);
1
H NMR (DMSO-d6) δ 2.5 (t, J = 7.2 Hz, 3H, CH2CH3),
3.10 (s, 3H, OCH3), 3.76 (q, J = 7.2 Hz, 2H,CH2CH3), 6.91
(d, J = 8.8 Hz, 2H, phenyl H-2,H-6 ), 7.10 (d, J = 8.8 Hz,
2H , phenyl H-3,H-5), 8.33 (s, 1H, quinoline H-5), 8.39 (s,
1H, quinoline H-7), 9.18 (s, 1H, quinoline H-2), 11.36 (s,
1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 22.0,
48.8, 79.4, 110.3, 120.9, 121.0, 123.3, 127.7, 128.0, 128.5,
129.7, 134.32, 141.9, 151.9, 154.1, 165.8, 168.8; MS,
þ þ
(relative abundance %): 445 (M  , 6.37 %), 447(M  +2,
5.77%), 123 (100%); Anal. Calcd. for C20H17BrN2O5
(445): C, 53.95; H, 3.85; N, 6.29. Found: C, 53.70; H, 4.13;
N, 6.14.
6-Bromo-4-((4-chlorophenyl)amino)-3-(ethoxycarbonyl)
quinoline-8-carboxylic acid (6f) Yellow crystals; yield
70%; m.p. 211–213 °C; IR (KBr/cm−1) 3434 (NH), 3075
(C–H aromatic), 2977 (C–H aliphatic), 1678 (C=O); 1 H
NMR (DMSO-d6) δ 2.27 (t, J = 7.2 Hz, 3H, CH2CH3), 2.90
(q, J = 7.2 Hz, 2H, CH2CH3), 6.87 (d, J = 8.8 Hz, 2H,
phenyl H-2,H-6), 7.07 (d, J = 8.8 Hz, 2H, phenyl H-3,H-5),
8.17 (s, 1H, quinoline H-5), 8.23 (s, 1H, quinoline H-7),
8.95 (s, 1H, quinoline H-2), 10.66 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 14.6, 62.0, 108.6,
119.2, 122.2, 123.1, 124.8, 128.1, 128.6, 129.1, 133.9,
140.2, 151.8, 153.2, 164.5, 166.2 ppm; MS, (relative
þ þ
abundance %): 449 (M  , 15.67 %), 451 (M  +2, 18.54%),
64 (100%); Anal. Calcd. for C19H14BrClN2O4 (449): C,
50.75; H, 3.14; N, 6.23. Found: C, 50.93; H, 3.34; N, 5.94.
Biological assay
The two human cancer cell lines used in this study breast
carcinoma (MCF-7) and non-small cell lung cancer (A549)
were purchased from American Type Culture Collection.
Cells were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM: Gibco, USA) supplemented with 10%
fetal bovine serum (Gibco), penicillin/streptomycin
(Gibco).4′,6-Diamidino-2-phenylindole (DAPI) nuclear
staining indicated that these cells were devoid of myco-
plasma contaminations. All incubations were done at 37 °C
in a humidified incubator containing 5% CO2. Cells in
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933
log-phase growth were harvested by trypsinization for use
in various assays.
Measurement of potential cytotoxic activity
Cell viability was assessed using MTT assay (Sigma-
Aldrich, St. Louis, MO, USA) in 6 replicates as described
previously (Omar et al. 2013). In brief, cells were seeded
into 96-well tissue culture plates in DMEM containing 10%
FBS to a final volume of 0.2 mL. The cells were subjected
to different treatments 24 h post seeding. Following the
incubation for 48 h with doxorubicin (positive control), test
drugs or vehicle (DMSO), the media were removed,
replaced by 200 µL DMEM containing 0.5 mg/mL of MTT
and cells were incubated for 2 h. Next, the supernatants
were removed and the precipitated formazan was dissolved
by adding 200 μL of DMSO. Absorbance at 570 nm was
determined using a microplate reader (Model 450 Mior-
oplate Reader; Bio-Rad). Results were calculated by sub-
tracting blank readings. The relation between surviving
fraction and drug concentration is plotted and IC50 [the
concentration required for 50% inhibition of cell viability]
was calculated for each compound.
The results of in vitro cytotoxic activity experiments are
presented in (Table 1).
Docking study
All molecular calculations and docking studies were per-
formed using “molecular operating environment” (MOE)
version 2008.10 release of Chemical Computing Group’s.
The program operated under “Windows XP” operating
system installed on an Intel Pentium IV PC with a 2.8 MHz
processor and 512 RAM.
Preparing for docking
The X-ray crystallographic structure of the Tyrosine Kinase
Domain from EGFR TK complexed with erlotinib was
obtained from the Protein Data Bank (PDBID: 1M17).
Validation of docking procedure
Docking of the co-crysatallized ligand should be carried out
to study the scoring energy (s), root mean square deviation
(RMSD), and amino acid interactions. The root mean
square deviation (RMSD) which is measure of superposing
was 0.50A° for the lead compound. Docking was performed
using London dG force and refinement of the results was
done using force field energy.
Preparing compounds for docking Preparing of synthe-
sized compounds for docking was achieved through their
934 Med Chem Res (2017) 26:929–939

3D structure built by MOE. Certain procedures should be interactions and the hydrogen bond numbers and lengths
done before docking process including: 3D protonation of were summarized in (Table 2).
the structures, running conformational analysis using sys-
temic search, selecting the least energetic conformer, and
applying the same docking protocol used with ligand.
The previous measures were taken and docking for Results and discussion
the synthesized compounds was applied. Amino acid
Chemistry
Table 1 In vitro cytotoxic activity of reference drugs doxorubicin,
erlotinib and 4-(4-substituted-anilino)quinoline derivatives 6a–f The target 4-anlinoquinoline derivatives (6a–f) were syn-
thesized using the reaction sequence illustrated in
R2 Scheme 1. Accordingly, condensation of the bromo deri-
vatives (1a, b) with diethyl ethoxymethylenemalonate
yielded the corresponding enamine derivatives (3a, b) via
Gould–Jacobs reaction. Thermal cyclization of (3a, b) in
HN boiling diphenyl ether yielded the respective quinolone
R1 COOC2H5 derivatives (4a, b) which undergo chlorination by phos-
phorus oxychloride gave 4-chloroquinoline derivatives (5a,
b). Nucleophilic substitution of the active chlorine in (5a, b)
N with the appropriate aromatic amines gave the target 4-
6a-f anlinoquinoline derivatives (6a–f) in moderate yields
R (62–71%).
Compound no. IC50a (µM) Enamine derivatives (3a, b) were confirmed by IR
spectra which showed absorption band at 3486–3130 cm−1
R R1 R2 MCF-7 A549
revealed NH group presence. The 1H NMR spectra dis-
Doxorubicin – – – 2.07 1.14
played two doublet signals, each of one proton intensity at δ
Erlotinib – – – 0.02b 19.26c
8.50–8.54 ppm and 11.21–11.40 ppm with J value = 12.6
6a Br COOH CH3 5.51 7.31 Hz indicating the olefinic CH and NH respectively. These
6b Br COOH OCH3 10.84 11.26 characteristic two doublet signals disappeared in the qui-
6c Br COOH Cl 3.42 5.97 nolone derivatives (4a, b). The IR spectra of the quinoli-
6d COOH Br CH3 18.11 16.32 none derivatives (4a, b) showed sharp peak at 1636–1648
6e COOH Br OCH3 23.32 22.01 cm−1 corresponding to C=O which disappeared in the
6f COOHC Br Cl 14.53 12.32 chloroquinoline compounds (5a, b). Additionally, 13C
a
IC50: concentration of a drug that is required for 50% inhibition of NMR spectra of (4a, b) confirmed the presence of CH3 and
cell viability CH2 carbons at δ 14.2–14.3 ppm and at δ 60.6–60.9 ppm,
b
Data obtained from literature (Lv et al. 2010) also deshielded peak at δ 164.4 ppm of quinolone
c
Data obtained from literature (XQ and TK 2013) C=O which disappeared in the chloroquinoline derivatives

Table 2 Molecular modeling

Compound No. Number of H- Atoms of compound Amino acid residues Binding energy
data for compounds 6a, 6c, 6e
bonds forming H-bonds forming H-bonds(H-bond score(Kcal/mol)
and erlotinib during docking in
length in Ao)
the EGFR active sites
N1 Thr-766 (2.78 Ao) −11.37
Erlotinib 2 N3 Met-769 (2.70 Ao)
6a 2 O (acid) Thr-766 (2.55 Ao) −10.04
C=O (acid) Met-769 (2.44 Ao)
6e 3 C=O (ester) Thr-766 (2.94 Ao)
N (quinoline) Lys-721 (2.47 Ao) −10.78
C=O (acid) Lys-721 (2.19 Ao)
6c 3 O (acid) Thr-766 (2.38 Ao)
C=O (acid) Met-769 (2.82 Ao) −9.82
NH Asp-831 (1.73 Ao)

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Med Chem Res (2017) 26:929–939 935

R respectively confirmed presence of the OCH3 group of 4-

NH2 H COOC2H5 anilinophenyl ring. Finally, the mass spectra of (3a, b, 4a,
+ C C þ
C2H5O COOC2H5 b, 5a, b, and 6a–f) demonstrated molecular ion (M  ) peaks
1
R for all compounds as well as additional peaks with relative
1 a,b 2
abundance equal that of the molecular ion corresponding to
1a, R = Br, R1 = COOH a
1b, R = COOH, R1 = Br M+2 due to presence of bromine.

R COOC2H5
NH
COOC2H5 Biological results
3 a,b
1
R
b
All the target compounds (6a–f) were screened for their
in vitro anticancer activity against human breast cancer cell
O
line (MCF-7) and non-small cell lung cancer cell line
R1 COOC2H5
(A549) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) assay (Omar et al. 2013).
N
The relationship between survival ratio and drug con-
R H
centration was plotted to obtain the survival curve of the
4a,b used cell lines (MCF-7 and A549). The response parameter
calculated was IC50 value (Table 1), which corresponds to
c the compound concentration causing 50% mortality in net
cells. The obtained data revealed that the target compounds
Cl
(6a–f) showed activities against MCF-7 and A549 cell lines
R1 COOC2H5
(IC50 = 3.42–23.32 and 5.97–22.01 µM respectively) in
N
comparison with doxorubicin (IC50 = 2.07 and 0.02 µM
R
respectively) and erlotinib (IC50 = 1.14 and 19.26 µM
5a,b respectively). For both cell lines, the 6-carboxy-8-bromo
analogs (6a, 6b and 6c) exhibited higher potency (IC50 =
d R2
5.51, 10.84, and 3.42 µM respectively for MCF-7 and IC50
= 7.31, 11.26, and 5.97 µM respectively for A549) rather
HN than 6-bromo-8-carboxy analogs (6d, 6e, and 6f) (IC50 =
R1 COOC2H5 18.11, 23.32, and 14.53 µM respectively for MCF-7 and
IC50 = 16.23, 22.01, and 12.32 µM respectively for A549).
N These results revealed that presence of carboxylic group at
6a-f
R position 6 of quinoline ring enhance the activity through
6a, R = Br, R1 = COOH, R2 =CH3; 6b, R = Br, R1 = COOH, R2 =
formation of additional hydrogen bonds with certain amino
OCH3
6c, R = Br, R1 = COOH, R2 = Cl; 6d, R = COOH, R1 = Br, R2 = acids in the active site of EGFR. On the other hand, para
CH3; substitution at aniline moiety with Cl as an electron with-
6e, R = COOH, R1 = Br, R2 = OCH3; 6f, R = COOH, R1= Br, R2 = Cl
drawing group was favored over CH3 or OCH3 substitution
Scheme 1 Synthesis of 4-(4-substituted-anilino)quinoline derivatives since (6c and 6f) were the most active compounds followed
6a–f by CH3 containing derivatives (6a and 6d) then the meth-
oxy derivatives (6b and 6e). Compound (6c) was the most
active derivative with IC50 = 3.42 and 5.97 µM against
(5a, b). The target compounds (6a–f) structure confirmed MCF-7 and A549 cell lines respectively that can be
by the presence of the additional aromatic moieties by explained by the presence of COOH group in position 6 of
presence of two doublet peaks at δ 6.87–7.21 ppm with J quinoline ring, in addition of para substitution of pheny-
value = 8.4–8.8 Hz of phenyl H-2,H-6 and at δ 7.07–7.53 lamino by Cl. Finally, all tested compounds 6a–f had higher
ppm with J value = 8.4–8.8 Hz of phenyl H-3,H-5 in the 1H activity against non-small cell lung cancer cell line (A549)
NMR spectra. Compounds (6a, d) had additional single (IC50 = 5.97–16.23 µM) than erlotinib (IC50 = 19.26 µM)
signal at δ 2.35, 2.78 ppm corresponding to CH3 group at 4- except 6e which was approximately equipotent (IC50 =
anilinophenyl ring in their 1H NMR spectra, while it 19.26 µM) that could be attributed to presence of halogen Br
appeared in 13C NMR spectra at δ 20.9 ppm. 1H NMR and in position 6 of quinoline ring instead of COOH group and
13
C NMR spectra of compounds (6b, e) showed presence of also 4-phenylamino substitution by methoxy group which
a single peak at δ 3.10, 3.80 ppm and at δ 48.8, 55.4 ppm caused decreased activity.

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936 Med Chem Res (2017) 26:929–939

Docking studies the docking reliability. We obtained the X-ray structure of

A molecular modeling study has been performed to under-
stand the protein kinase inhibitor interactions and the struc-
tural features of the ATP active site of EGFR. Also, to
investigate the possible binding conformation for the newly
prepared 4-anlinoquinoline derivatives to ATP binding site of
EGFR, this may give a suggestion about their proposed
mechanism of action. First of all, it is necessary to validate
TK domain of epidermal growth factor receptor (EGFR TK)
in complex with erlotinib which is the ligand from protein
data bank (PDB ID: 1M17). Using MOE version 2008.10 the
ligand erlotinib was docked to the binding site of EGFR TK,
and we selected the lowest energy score docking conformer
as the most probable binding conformation. In addition its
interaction mode revealed that two amino acids are involved
in the interaction; Thr-766 and Met-769 (Fig. 2).

Fig. 2 Binding of erlotinib with EGFR a the proposed binding mode inside the active site of EGFR resulting from docking, the most important
amino acids are shown together with their respective numbers. b 2D interaction

Fig. 3 Binding of 6a with EGFR a the proposed binding mode inside the active site of EGFR resulting from docking, the most important amino
acids are shown together with their respective numbers. b 2D interaction

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Med Chem Res (2017) 26:929–939 937

We then docked the most in vitro cytotoxic active
compounds (6-carboxy-8-bromo analogs, 6a and 6c) and
the least active compound (6-bromo-8-carboxy analog 6e).
The docking results including the energy score associated
with intermolecular interactions obtained upon computa-
tional docking for the three compounds (6a, 6c, and 6e) and
the hydrogen bonding interactions between the amino acid
residues and functional groups of the compounds are
summarized in Table 2.
From the docking results, both compounds (6a and 6c)
showed good binding interactions affinity; compound (6a)
had a Glide score of −10.04 Kcal/mol and exhibited two
hydrogen bonding interactions while the score of compound
6c = −9.82 and formed three hydrogen bonding interac-
tions. Compound (6e) showed −10.78 Kcal/mol score with
three hydrogen bonding interactions in comparison with
erlotinib (I) which had energy score = −11.37 Kcal/mol
and formed two hydrogen bonding interactions. Compound
(6a) formed a hydrogen bond between carbonyl group of
COOH group and Met 769 whiles the other hydrogen bond
with Thr 766 through the remaining O of COOH. These two
hydrogen bonding interactions were observed similarly in
erlotinib (I). Compound (6c) formed the same two hydrogen
bonding interactions with the same amino acid residues in

Fig. 4 Binding of 6c with EGFR a the proposed binding mode inside the active site of EGFR resulting from docking, the most important amino
acids are shown together with their respective numbers. b 2D interaction

Fig. 5 Binding of 6e with EGFR a the proposed binding mode inside the active site of EGFR resulting from docking, the most important amino
acids are shown together with their respective numbers. b 2D interaction

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

938
addition to a third one between NH and Asp 831. The least
in vitro cytotoxic active compound (6e) formed only one
hydrogen bond of 2 hydrogen bonding interactions of the
ligand (carbonyl group of the ester moiety with Thr 766)
and also two other hydrogen bonding interactions not pre-
sent in the ligand binding with Lys 721. These docking
results suggest that compounds 6a and 6c (carboxylic group
in position 6 of quinoline moiety) may exhibit their cyto-
toxicity in a similar manner to erlotinib (Figs. 3–5).
Conclusion
A new series of 4-(4-substituted-anilino)quinoline deriva-
tives (6a–f) was designed and synthesized for evaluation as
anticancer against two human cancer cell lines; breast car-
cinoma (MCF-7) and non-small cell lung cancer (A549).
Structure-activity data and molecular modeling studies
showed that (i) the 6-carboxy-8-bromo analogs (6a, 6b, and
6c) exhibited higher potency than 6-bromo-8-carboxy ana-
logs (6d, 6e, and 6f) which mains presence of carboxylic
group in position 6 of quinoline ring is favored over its
presence in position 8, (ii) substitution at para position of
the aniline moiety with Cl as electron with drawing group is
favored over substitution with CH3 (electron donating
group) and over substitution with OCH3 (electron donating
group) since 6c > 6a > 6b and 6f > 6d > 6e in potency, (iii)
all tested compounds (6a–f) had higher activity against non-
small cell lung cancer cell line (A549) than erlotinib except
(6e) which was approximately equipotent, (iv) molecular
docking for the most in vitro cytotoxic active compounds
(6a and 6c) revealed that these compounds may exhibit their
cytotoxicity in a similar manner to erlotinib and (v) the
previous promising results could help in design of new
potent quinoline derivatives targeting cancer chemotherapy.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no com-
peting interest.
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