Design, synthesis, anticancer, antimicrobial activities and molecular docking studies of novel quinoline bearing dihydropyridines

Article abstract of DOI:10.1016/j.jphotobiol.2016.10.009

A new series of eight quinoline bearing dihydropyridine derivatives (A1–A8) were synthesized in high yield and in short reaction time by a four component reaction of 2-chloro-3-fomyl quinoline, malononitrile, arylamines and dimethyl acetylenedicarboxylate in the presence of a catalytic amount of triethylamine. The compounds were fully characterized by IR, NMR and GC–MS. These compounds were screened for potential biological activity in an A549 lung cancer cell line and were also evaluated for their antibacterial activities against Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 whilst their molecular docking properties in an enzymatic system were also determined. Compounds A2, A3, A4 and A8 showed anti-proliferative activity; with A4 having the highest toxicity at 250 μg/mL and A8 has high toxicity at 125, 250 and 500 μg/mL, respectively. Antibacterial results indicated that A4 have significant activity against tested microorganisms at the minimum inhibitory concentration (MIC) values of 32 μg/mL against Pseudomonas aeruginosa and Escherichia coli, and 16 μg/mL against Staphylococcus aureus. Docking of A1 with human mdm2 indicated the lowest binding energy (? 6.111 Kcal/mol) thereby showing strong affinity of the ligand molecule with the receptor which has been stabilized by strong hydrogen bond interactions in the binding pocket. This confirms that A1 is a better inhibitor for E3 ubiquitin-protein ligase mdm2.

Full text of DOI:10.1016/j.jphotobiol.2016.10.009

Design, synthesis, anticancer, antimicrobial activities and molecular docking
studies of novel quinoline bearing dihydropyridines
S’busiso Mfan’vele Nkosi, Krishnan Anand, Srinivasan Anandakumar,
Sanil Singh, Anil Amichund Chuturgoon, Robert Moonsamy Gengan
PII:
DOI:
Reference:
S1011-1344(16)30751-5
doi:10.1016/j.jphotobiol.2016.10.009
JPB 10608
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Received date:
Revised date:
Accepted date:
1 September 2016
3 October 2016
11 October 2016
Please cite this article as: S’busiso Mfan’vele Nkosi, Krishnan Anand, Srinivasan Anan-
dakumar, Sanil Singh, Anil Amichund Chuturgoon, Robert Moonsamy Gengan, Design,
synthesis, anticancer, antimicrobial activities and molecular docking studies of novel
quinoline bearing dihydropyridines, (2016), doi:10.1016/j.jphotobiol.2016.10.009
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ACCEPTED MANUSCRIPT
Design, synthesis, anticancer, antimicrobial activities and molecular
docking studies of novel quinoline bearing dihydropyridines
S’busiso Mfan’vele Nkosi1, Krishnan Anand1, Srinivasan Anandakumar2, Sanil Singh3, Anil
Amichund Chuturgoon4, Robert Moonsamy Gengan1*
1Department of Chemistry, Faculty of Applied Science, Durban University of Technology,
Durban 4001, South Africa
2Department of Bioinformatics, School of life Sciences, Bharathiyar University, Coimbatore
641046, India
3Department of Biomedical Resource Centre, University of KwaZulu-Natal, Westville,
Durban 4001, South Africa.
4Discipline of Medical Biochemistry, School of Laboratory Medicine and Medical Sciences,
University of KwaZulu-Natal, Durban 4001, South Africa.
* Corresponding authors. E-mail address: genganrm@dut.ac.za

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Abstract
A new series of eight quinoline bearing dihydropyridine derivatives (A1-A8) were
synthesized in high yield and in short reaction time by a four component reaction of 2-chloro-
3-fomyl quinoline, malononitrile, arylamines and dimethyl acetylenedicarboxylate in the
presence of a catalytic amount of triethylamine. The compounds were fully characterised by
IR, NMR and GC-MS. These compounds were screened for potential biological activity in an
A549 lung cancer cell line and were also evaluated for their antibacterial activities against
Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Staphylococcus
aureus ATCC 29213 whilst their molecular docking properties in an enzymatic system were
also determined. Compounds A2, A3, A4 and A8 showed anti-proliferative activity; with A4
having the highest toxicity at 250 µg/mL and A8 has high toxicity at 125, 250 and 500
µg/mL, respectively. Antibacterial results indicated that A4 have significant activity against
tested microorganisms at the minimum inhibitory concentration (MIC) values of 32 µg/mL
against Pseudomonas aeruginosa and Escherichia coli, and 16 µg/mL against Staphylococcus
aureus. Docking of A1 with Human mdm2 indicated the lowest binding energy (-6.111
Kcal/mol) thereby showing strong affinity of the ligand molecule with the receptor which has
been stabilized by strong hydrogen bond interactions in the binding pocket. This confirms
that A1 is a better inhibitor for E3 ubiquitin-protein ligase mdm2.
Keywords: 2-chloro-3-formylquinoline; dihydropyridines; molecular docking; anti-cancer;
human mdm2

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Introduction
Quinolines and their derivatives represent an important class of nitrogen-containing
heterocycles as they are useful dyes and intermediates in organic synthesis (Heravi et al.,
2007). In recent years, much attention has been focused on their synthesis as they possess
useful biological activities such as anti-malarial (Golden et al., 2015), anti-inflammatory (Liu
et al., 2015), bactericidal (Maddela et al., 2014), fungicidal (Nadaraj et al., 2012) and anti-
cancer (Wu et al, 2003). Because of their important role in the pharmaceutical field (Litvinov
et al., 2004), the profiles of quinolines and their pharmaceutical properties are well
documented (Chipeleni et al., 2007; Kumar et al., 2009).
On the other hand, many naturally occurring and synthetic compounds bearing
pyridine scaffold possess interesting biological properties (Temple et al., 1992) and they are
well known for their versatile biological activities like antimicrobial (Mahmoud et al., 2007),
antitubercular (Ashrafali et al., 2010; Lourenco et al., 2007), anticancer (Kawase et al., 2002)
and antitumor activity (Safak et al., 2007). The polysubstituted pyridine represent molecular
framework that serves as a platform for development of pharmaceutical agents. Thus, the
synthetic of highly functionalized pyridine derivatives has become an active area of research
(Anabha et al., 2007; Fletcher et al., 2006; Sridhar et al., 2009; Shinde et al., 2010).
Encouraged by quinolines and pyridines potent clinical applications and in continuation of
our previous investigations on biopotent heterocycles (Gengan et al., 2010, 2015) in this
study, our efforts focused on the design and synthesis of more biological potent heterocyclic
systems via combination of both therapeutically active moieties of quinoline and pyridine in a
single scaffold.
Tandem multi-component reactions, in which multiple reactions are simulated in one
synthetic operation, have been used comprehensively to form carbon-carbon bonds (Taylor et
al., 2005). Such reactions offer a wide range of possibilities for the efficient formation of
highly complex molecules in a single operation. Multi-component reactions (MCRs) are
reactions using more than two starting materials that form a product which contains the key
parts of all of the starting materials (Sinha et al., 2013) in a few in-situ reaction steps.
Compared with conventional organic reactions, MCRs are more profitable and requires
minimum energy to complete the reaction. Several MCRs are known which include the Ugi 4
component condensation of amine, ketone, carboxylic acid and isocyanide (Ugi et al., 1962).

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The Strecker reaction, documented in 1850, was the first MCR described (Degussa et al.,
2003). Recently, there has been a tremendous development in three and four-component
reactions and huge attempts have been made are continuously being made to discover and
amplify new MCRs. Therefore, the development of new synthetic approaches using MCRs
remains an active research area of study (Shestopalov et al., 2002). The literature reveals that
quinoline bearing dihydropyridine derivatives in Figure 1 have received much interest in the
field of chemistry owing to their association with a variety of biological activities; (Yan et
al., 2010; Nirmal et al, 2009; Ladani et al, 2011)
Cancer, a worldwide epidemic and one of the leading causes of death in developed
countries, is responsible for 20-25% of all deaths (Bepler et al., 2007, El-Hussein et al.,
2016). Lung cancer in humans is common and often fatal (Mehrotra et al., 2010). Quinolines
play a significant role in the development of new anti-cancer agents; their derivatives have
shown excellent results through different mechanism of action such as growth inhibitors by
cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and
modulation of nuclear receptor responsiveness (Firestone et al., 2009).
A number of developments have advanced drug discovery (Shoichet et al., 2002,
Singh et. al., 2013). Increasingly, as the structures of more potential compounds are
becoming available, molecular docking is progressively being studied for pharmacologically
or biologically active chemical compounds (Clauben et al., 2001). Docking is a method
which predicts the preferred orientation of one molecule to a moment when they bound to
each other to form a stable complex. Docking it is a key tool in structural molecular biology
and computer-assisted drug design. The goal of ligand-protein docking is to predict the
predominant binding mode of a ligand with a protein of known three-dimensional structure
(Kharb et al., 2011).
Pathogenic bacteria are becoming resistance to anti-microbial treatment because of
acquired resistance genes in the DNA of the micro-organism. Therefore, the antibiotic
resistance problem demands continuous discovery and development of new antibacterial
agents that could be used for the effective treatment of infectious diseases (Gandhi and Khan
et. al., 2016). Realizing the medicinal importance of quinoline and pyridine based
compounds; we undertook an investigation to synthesize novel quinoline bearing
dihydropyridine derivatives and study their application in biological systems. The scope of
this study was to synthesize novel quinoline bearing dihydropyridine type molecules from
simple and cost effective starting materials, hence, purify the compounds by chromatographic

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techniques and characterize them by spectroscopy. Furthermore, the study seeks to also,
assess and compare their potential as anti-cancer drugs in A549 lung cancer cell lines,
evaluate their molecular modeling properties and anti-microbial activities.
Materials and Methods
Chemistry
All reagents and solvents such as aniline, m-toluidine, o-anisidine, fluoroaniline,
chloroaniline, petroleum ether, ethanol, ethyl acetate and triethylamine were purchased from
Lasec S.A, Aldrich, Fluka and Merck. These reagents and solvents were used without further
1
purification. Melting points were recorded on Stuart Digital melting point apparatus. The H
13
19
and C NMR, F NMR, COSY, NOESY, HSQC and HMBC spectra were recorded either
on Bruker (600 MHz) or Bruker (400 MHz) spectrophotometers in CDCl3 or DMSO using
tetramethylsilane (TMS) as an internal reference. The chemical shifts were quoted in parts
per million (ppm). The IR spectra were recorded on Perkin Elmer 537 spectrophotometer
and Shimadzu-8201 FT instrument, using ATR. Lung cancer cells (A549) were purchased
from Highveld Biologicals (Johannesburg, SA). Cell culture reagents were purchased from
Whitehead Scientific (Johannesburg, SA). Fresh Nutrient Agar, Oxoid LTD was purchased
from Hamsphire, England and Fresh Mueller Hinton Broth from Sigma-Aldrich, (SA) all
other reagents and consumables were purchased from Merck (SA), unless otherwise stated.
Synthesis of a starting compound 2-chloro-3-formyl quinoline
Dry DMF (3 mmol) was cooled to 0ºC in a flask equipped with a drying tube and then
POCl3 (12 mmol) was added drop-wise with stirring. To this solution, acetanilide (1 mmol)
was added in small portions and after 25-30 minutes the reaction mixture was heated for 24
hours on a boiling water bath. The reaction mixture was poured into ice water and stirred for
30 minutes. The work-up was performed with aqueous NaOH to form a precipitate, to
hydrolyse the imine salt and remove any acid formed. The solid was filtered, dried and
purified from ethyl acetate to give 2-chloro-3-formyl quinoline in high yield (90 %).
General procedure for synthesis of quinoline bearing dihydropyridine derivatives (A1-
A8)
In a round bottom flask, a mixture of 2-chloro-3-formyl quinoline (2.0 mmol,
0.3831g), malononitrile (2.0 mmol, 0.1324g) and triethylamine (1.0 ml) in 20 mL ethanol

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was stirred at room for thirty minutes. Then a solution of arylamines (2.0 mmol) and
dimethyl acetylenedicarboxylate (2.0 mmol, 0.284g) in 25.0mL ethanol was added to it. The
solution was stirred at room temperature for ten hours. The resultant precipitate was collected
by filtration and washed with cold ethanol to give the pure product and purified by column
chromatography (50:50 Petroleum ether: Ethyl acetate).
N
O
O
Cl
CN
H3CO
H3CO
N
NH2
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-phenyl-1,4-dihydropyridine-2,3-
dicarboxylate (A1): Light yellow solid, 87%, m.p. 275-2760C, 1H NMR (600MHz, CDCl3) δ:
8.14 (s, 1H, ArH), 8.02 (d, J= 8.4Hz, 1H, ArH), 7.84 (d, J= 8.1Hz, 1H, ArH), 7.71 (t, J=
7.3Hz, 2H, ArH), 7.55 (t, J= 7.6Hz ,1H, ArH), 7.52-7.51 (m, 2H, ArH), 7.39-7.38 (m, 2H,
ArH), 5.36 (s, 1H, CH), 4.13 (s, 2H, NH2), 3.51 (s, 3H, OCH3), 3.44 (s, 3H, OCH3); 13C
NMR (600MHz, CDCl3) δ: 165.28, 163.20, 150.22, 149.88, 146.93, 142.99, 138.59, 136.14,
134.89, 130.81, 130.47, 130.37, 130.09, 128.34, 127.62, 127.55, 127.11, 119.74, 103.55,
61.22, 52.66, 52.12, 36.94; (IR) υ/cm-1: 3486, 3356, 2171, 1748, 1713, 1647, 1524, 1488,
1448, 1324, 1296, 1247, 1105, 1024, 992, 841, 835, 778, 762, 669, 667, 550. MS (m/z): 475.
Anal Calculated for C25H19ClN4O4: C 63.23; H 4.03; Cl 7.47; N 11.80; O 13.48: Found, C
63.87; H 4.05; Cl 7.50; N 11.88; O 13.81.
N
O
O
Cl
CN
H3CO
H3CO
N
NH2
OCH3
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-(2-methoxyphenyl)-1,4-
1
dihydropyridine-2,3-dicarboxylate (A2): Yellow solid, 40%, m.p. 263-2640C, H NMR
(600MHz, CDCl3) δ: 8.39 (s, 1H, ArH), 8.01 (d, J= 8.4Hz, 1H, ArH), 7.84 (d, J= 8.1Hz, 1H,

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ArH), 7.80 (d, J= 8.1Hz, 1H, ArH) 7.69 (t, J= 7.6Hz, 1H, ArH), 7.53 (d, J= 7.1Hz, 1H. ArH),
7.49 (d, J= 7.8Hz, 1H, ArH), 7.30 (d, J= 7.3Hz, 1H, ArH), 7.06-7.02 (m, 1H, ArH), 5.36 (s,
1H, CH), 4.17 (s, 2H, NH2), 3.50 (s, 3H, OCH3), 3.49 (s, 3H, OCH3), 3.46 (s, 3H, OCH3); 13C
NMR (600MHz, CDCl3) δ: 165.39, 163.33, 157.37, 150.89, 149.91, 146.91, 138.66, 132.57,
130.38, 128.53, 128.31, 127.56, 127.31, 127.03, 121.14, 121.04, 119.99, 112.7, 112.63,
61.80, 52.66, 52.54, 52,10, 36.36; (IR) υ/cm-1: 3437, 3315, 3217, 2185, 1716, 1746, 1665,
1577, 1496, 1421, 1354, 1329, 1222, 1117, 933, 932, 859, 817, 782, 755, 624; MS (m/z): 504.
Anal Calculated for C26H21ClN4O5: C 61.85; H 4.19; Cl 7.02; N 11.10; O 15.84: Found, C
61.87; H 4.25; Cl 7.10; N 11.08; O 15.81.
N
O
O
Cl
CN
H3CO
H3CO
N
NH2
CH3
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-m-tolyl-1,4-dihydropyridine-2,3-
dicarboxylate (A3): Yellow solid, 73%, m.p. 237-2380C, 1H NMR (600MHz, CDCl3) δ: 8.12
(s, 1H, ArH), 8.00 (d, J= 9.7Hz, 1H, ArH), 7.84 (d, J= 8.1Hz, 1H, ArH), 7.69 (t, J= 10.1Hz,
1H, ArH), 7.53 (t, J= 11.2Hz, 1H, ArH), 7.36 (t, J= 9.9Hz, 1H, ArH), 7.29 (d, J= 7.6Hz, 1H,
ArH), 7.23 (s, 1H, ArH), 7.16 (d, J= 6.4Hz, 1H, ArH), 5.33 (s, 1H, CH), 4.23 (s, 2H, NH2),
13
3.50 (s, 3H, OCH3), 3.44 (s, 3H, OCH3), 2.39 (s, 3H, CH3). C NMR (600MHz, CDCl3) δ:
165.31, 163.20, 150.41, 149.87, 146.89, 143.06, 140.45, 138.53, 136.47, 134.74, 131.51,
130.70, 130.42, 129.79, 128.30, 127.67, 127.55, 127.15, 127.08, 119.91, 60.83, 52.61, 52.07,
36.73, 21.23; (IR) υ/cm-1: 3386, 3318, 2180, 1702, 1647, 1565, 1488, 1420, 1358, 1328,
1253, 1224, 1136, 1114, 1056, 1019, 968, 928, 786, 763, 708, 686; MS (m/z) 487. Anal
Calculated for C26H21ClN4O4: C 63.87; H 4.33; Cl 7.25; N 11.46; O 13.09: Found, C 63.20;
H 4.31; Cl 7.11; N 11.36; O 13.23.

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N
O
O
Cl
CN
H3CO
H3CO
N
NH2
Cl
Dimethyl-6-amino-1-(4-chlorophenyl)-4-(2-chloroquinolin-3-yl)-5-cyano-1,4-
1
dihydropyridine-2,3-dicarboxylate (A4): Light yellow solid, 79%, m.p. 287-2890C, H NMR
(600MHz, DMSO) δ: 8.38 (s, 1H, ArH), 8.17 (d, J= 8.1Hz, 1H, ArH), 7.98 (d, J= 8.5Hz, 1H,
ArH), 7.84 (t, J= 6.9Hz, 1H, ArH), 7.70 (t, J= 7.9Hz, 1H, ArH), 7.60 (d, J= 8.8Hz, 2H, ArH),
7.49 (d, J= 11.2Hz, 2H, ArH), 5.86 (s, 2H, NH2), 5.24 (s, 1H, CH), 3.45(s, 3H, OCH3),
3.43(s, 3H, OCH3).13C NMR (600MHz, DMSO) δ: 165.21, 163.13, 151.42, 149.57, 146.54,
143.31, 139.11, 137.73, 135.37, 134.41, 132.98, 131.37, 130.17, 128.52, 128.01, 127.92,
127.89, 127.87, 120.77, 103.14, 58.54, 52.43, 52.01, 39.60; (IR) υ/cm-1: 3428, 3218, 2174,
1745, 1703, 1650, 1623, 1593, 1571, 1489, 1354, 1257, 1219, 1107, 1090, 1018, 850, 825,
780, 741, 723; MS (m/z) 510. Anal Calculated for C25H18Cl2N4O4: C 58.95; H 3.56; Cl 13.92;
N 11.00; O 12.56: Found, C 63.19; H 3.55; Cl 7.15; N 11.39; O 13.22.
N
O
O
Cl
CN
H3CO
H3CO
N
NH2
F
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-(2-fluorophenyl)-1,4-
1
dihydropyridine-2,3-dicarboxylate (A5): White solid, 64%, m.p. 247-2480C, H NMR
(600MHz, DMSO) δ: 8.33 (s, 1H, ArH), 7.84 (d, J= 7.3Hz, 2H, ArH), 7.67 (d, J= 7.1Hz, 2H,
ArH), 7.59 (m, 2H, ArH), 7.37-7.33 (m, 2H, ArH), 5.99 (s, 2H, NH2), 5.22 (s, 1H, CH), 3.46
13
(s, 6H, OCH3). C NMR (600MHz, DMSO) δ: 165.59, 165.02, 164.10, 163.04, 160.57,
158.91, 151.33, 146.57, 143.21, 133.50, 133.44, 131.46, 129.07, 128.89, 128.66, 128.40,

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128.07, 120.59, 79.62, 53.61, 53.15, 49.07, 40.53, 39.57, 29.44; (IR) υ/cm-1: 3376, 3282,
2178, 1747, 1712, 1654, 1571, 1488, 1422, 1360, 1329, 1258, 1231, 1171, 1032, 967, 922,
872, 851, 761, 648, 644.
N
O
O
Cl
CN
H3CO
H3CO
N
NH2
F
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-(3-fluorophenyl)-1,4-
1
dihydropyridine-2,3-dicarboxylate (A6): White solid, 31%, m.p. 269-2710C, H NMR
(600MHz, DMSO) δ: 8.40 (s, 1H, ArH), 8.19 (d, J= 8.4Hz, 1H, ArH), 7.99 (d, J= 8.5Hz, 1H,
ArH), 7.84 (t, J= 7.0Hz, 1H, ArH), 7.70 (t, J= 6.9Hz, 1H, ArH), 7.61-7.56 (m, 1H, ArH),
7.42 (m, 1H, ArH), 7.31 (d, J= 7.2Hz, 1H, ArH), 5.86 (s, 2H, NH2), 5.25 (s, 1H, CH), 3.45
(m, 3H, OCH3), 3.40 (m, 3H, OCH3), 13C NMR (600MHz, DMSO) δ: 165.75, 163.09,
161.65, 151.60, 149.57, 145.55, 143.15, 139.12, 137.87, 136.84, 131.61, 131.56, 131.36,
128.55, 128.00, 127.74, 120.74, 121.91, 118.74, 117.86, 58.75, 52.96, 52.09, 37.10; (IR)
υ/cm-1: 3449, 3303, 2177, 1746, 1715, 1652, 1596, 1570, 1487, 1421, 1329, 1305, 1251,
1194, 1136, 1106, 1033, 966, 924, 819, 763, 706, 899. 19F NMR (600MHz, DMSO) δ:
111.23; MS (m/z) 494. Anal Calculated for C25H18ClFN4O4: C 60.92; H 3.68; Cl 7.19; F 3.85;
N 11.37; O 12.98: Found, C 60.90; H 3.68; Cl 7.11; F 3.84; N 11.36; O 12.99.
N
O
O
Cl
CN
H3CO
H3CO
N
F
NH2
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-(4-fluorophenyl)-1,4-
dihydropyridine-2,3-dicarboxylate (A7): White solid, 60%, m.p. 253-2540C, H NMR
1

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(600MHz, DMSO) δ: 8.38 (s, 1H, ArH), 8,17 (d, J= 8.0Hz, 1H, ArH), 7.96 (d, J= 7.6Hz, 1H,
ArH), 7.82 (t, J= 7.3Hz, 1H, ArH), 7.66 (t, J= 7.2Hz, 1H, ArH), 7.54-7.52 (m, 2H, ArH),
7.38-7.34 (m, 2H, ArH), 5.83 (s, 2H, NH2), 5.23 (s, 1H, CH), 3.43 (s, 3H, OCH3) 3.38 (s, 3H,
OCH3); 13C NMR (600MHz, DMSO) δ: 150.99, 149.9, 149.14, 145.99, 143.03, 142.97,
138.60, 137.44, 137.34, 133.38, 133.37, 133.34, 133.33, 133.26, 113.1.1, 130.06, 128.00,
127.95, 127.38, 120.37, 116.32, 61.65, 57.83, 57.32, 36.77; (IR) υ/cm-1: 3309, 3269, 2171,
1745, 1702, 1622, 1506, 1569, 1329, 1298, 1247, 1213, 1153, 1106, 1027, 1002, 938, 918,
840, 778, 768, 766, 741, 728, 615. 19F NMR (600MHz, DMSO) δ: 110.91; MS (m/z) 494.
Anal Calculated for C25H18ClFN4O4: C 60.92; H 3.68; Cl 7.19; F 3.85; N 11.37; O 12.98:
Found, C 60.90; H 3.68; Cl 7.11; F 3.84; N 11.38; O 12.97.
N
O
O
Cl
CN
H3CO
H3CO
N
NH2
Cl
Cl
Dimethyl-6-amino-4-(2-chloroquinolin-3-yl)-5-cyano-1-(3,4-dichlorophenyl)-1,4-
1
dihydropyridine-2,3-dicarboxylate (A8): Light yellow solid, 72%, m.p. 280-2820C, H NMR
(600MHz, DMSO) δ: 8.45 (s, 1H, ArH), 8.20 (d, J= 8.1Hz, 1H, ArH), 7.99 (d, J= 8.4Hz, 1H,
ArH), 7.85 (d, J= 7.7Hz, 1H, ArH), 7.84 (t, J= 7.7Hz, 1H, ArH), 7.70 (t, J= 7.6Hz, 1H, ArH),
7.48 (d, J= 2.2Hz, 1H, ArH), 7.47 (d, J= 2.3Hz, 1H, ArH), 6.02 (s, 2H, NH2), 5.25 (s, 1H,
CH), 3.43 (s, 3H, OCH3), 3.39 (s, 3H, OCH3); 13C NMR (600MHz, DMSO) δ: 165.20,
163.10, 151.14, 149.58, 146.54, 142.88, 139.38, 137.78, 135.36, 133.79, 133.53, 132.33,
131.79, 131.55, 131.34, 130.17, 128.54, 127.96, 127.84, 120.73, 79.65, 53.09, 53.00, 39.60;
(IR) υ/cm-1: 3439, 3302, 2179, 1742, 1702, 1647, 1573, 1506, 1423, 1366, 1324, 1254, 1214,
1108, 1032, 932, 758; MS (m/z) 544. Anal Calculated for C25H17Cl3N4O4: C 55.22; H 3.15;
Cl 19.56; N 10.30; O 11.77: Found, C 65.19; H 3.16; Cl 19.15; N 10.39; O 11.22.
Anticancer study

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Maintenance of A549 Cells in Culture
The best environment for growing cells in vitro should be matched as close as
possible to the natural physiological conditions. The essential requirements are an
environment of optimum temperature, pH, gas phases, growth substrate and media containing
necessary nutrients. The optimal temperature is provided by the use of a humidified incubator
supplied with 5% CO2. The gas phases supplied to the culture includes oxygen which is
maintained at atmospheric pressure, and CO2 to ensure that the bicarbonate and CO2 tension
is in equilibrium. A549 cells were cultured (370C, 5% CO2) to 90% confluency in 25mL
flasks in complete culture media (CCM) [Eagle’s minimum essential medium, 10% foetal
calf serum, 1% L-Glutamine and 1% penstrepfungizone]. The culture medium is by far the
most important single factor in culturing cells. The extracellular medium must meet the
essential requirements (nutritional, hormonal and stromal factors) for survival and growth.
Trypsinisation
In order to sub-culture and plate cells for the various experimental assays, the process
of trypsinisation was used to detach cells once 90% confluency was reached. The process of
trypsinisation involved the critical step of rinsing the cells with 3 mL aliquots of warm 0.1M
PBS and incubating the cells with 1 mL of trypsin-EDTA (Lonza) for 1 minute. The cells
were monitored using an inverted light microscope (Olympus IXSI; 20x magnification) and
once rounded, the trypsin was discarded and CCM was added to the flask of cells. The flask
was agitated to detached cells and the cell suspension was then enumerated by dye exclusion
using a haemocyto meter. Trypan blue (0.4%) was utilized in a dye exclusion procedure for
cell counting. The principle of dye exclusion using trypan blue is based on compromised cell
membranes in dead/damaged cells which readily allow entry of the dye into the cells and are
stained blue whereas viable cells remain unstained.
Cell antiproliferation assay
The effect of newly synthesised compounds in A549 cells was measured using a
methyl tetrazolium dye reduction assay, the [3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] (MTT) assay. This assay measures cell antiproliferative assay in vitro.
This technique is particularly useful for cells that are metabolically active based on their
redox potential and capacity of dehydrogenase enzymes to convert yellow water-soluble salt
into a purple water-insoluble formazan product. The insoluble crystals are then dissolved in
dimethyl sulfoxide (DMSO) and the absorbance is read on a spectrophotometer. The amount

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of formazan produced is directly proportional to cell number thus allowing for the
determination of cell viability and proliferation (Supino et al., 1995).
A549 cells (15,000/well) were incubated for 24 hours with a range of concentrations
of each compound in triplicate in a micro-titre plate together with an untreated control (cells
incubated with CCM only). Each experiment was conducted twice on separate occasions. The
data results from the first set matched the repeated experiment. The cells were then incubated
(370C, 5% CO2) with the MTT substrate (5 mg/mL in PBS) for 4 hours. Thereafter all
supernatants were aspirated, and DMSO (100 µl/well) was added to the wells. Finally the
optical density was measured at 570 nm and a reference wavelength of 690 nm with an
ELISA plate reader (Bio-Tek µQuant).
Antibacterial study
Preparation of Media and Nutrient Broth
Fresh Nutrient Agar and Fresh Mueller Hinton Broth were prepared according to the
manufacturer’s instructions. The cultures of Staphylococcus aureus, Escheriachia coli and
Pseudomonas aeuginosa were maintained on nutrient agar slopes at 4°C and sub cultured on
to blood agar plates for 24 hours before use.
Microplate Alamar Blue Assay (MABA)
An amount of 0.2g of Resazurin powder was dissolved in 10 mL autoclaved distilled
water. The dye solution was vortexed vigorously; the solution was immediately covered with
aluminium foil to keep away from light as it is light sensitive.
The in vitro antibacterial activities of the synthesized compounds were studied by
MABA using 96-wells microplates. Bacterial cultures were diluted in broth to turbidity
comparable to that of a 0.5 McFarland turbidity standard and 20 μL of each bacterial dilution
was distributed in all 96 wells of microplate including positive control (containing standard
antibiotic) and growth control (containing culture broth without testing materials). Then 20
μL of each concentration of the synthesized compounds were added to two neighbour wells
except for positive and growth control wells.
After adding Alamar Blue (20 μL) to all of 96 wells, the total volume in each well
reached 200 μL. The final concentrations of the tested compounds were 256, 128, 64, 32, 16,
8, 4, 2, 1 μg/mL etc. After incubation, results were recorded as MIC (minimum concentration
of each synthesized compound which completely inhibited growth of microorganism). The

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stock solutions of the synthesized compounds were prepared by dissolving in the minimum
volume of DMSO.
Molecular Docking
The crystal structure of human mdm2 (PDB ID: 3VZV), were retrieved from the
Protein Data Bank After selected the protein structure, protein preparation wizard of
Schrodinger suite was used to prepare protein structure. All the water molecules were
removed from the protein structure. Metal was treated and hydrogen atoms were added. All
atom force field (OPLS-2005) charges and atom types were assigned. Protein structure
energy was minimized. The chemical compounds structures were not available in pubchem
database. Therefore, we used chem sketch to draw the compound structure. All the ligands
were prepared for molecular docking studies using ligprep version 2.3 (Ligprep et al., 2009).
The ligand structure energy was minimized; partial atomic charges were computed using the
OPLS-2005 force field by using Schrödinger suite.
The prepared protein structure was followed up by the grid-fashion kinetic docking
which commonly holds several physical parameters and finds greater significance in
prescribing the ligand interaction with the receptor. Grid files generation for protein was
accomplished with “Receptor Grid Generation” panel of Glide. The grid box was generated
by assigning a common constituency point. From there an actually cubic grid box was
extended to touch the bounty of 20Å in size. In other words, the allocated size for grid
generation of protein-ligand docking was 20Å since our approach was site-specific not
generalized. Having observed that co-crystal ligand binding pattern with protein is already
well documented in recent studies, we attempted to crop the grid box to focus on the centroid
of human mdm2 refined structure and box coordinate X, Y, Z were set at (X=18.341215Å,
Y=-3.662453Å, Z=0.929674Å). The foremost process in the hit identification pipeline,
docking is performed using Glide of Schrodinger (Glide et al., 2009).
Results and Discussion
The focus of this research was to synthesize novel compounds, especially compounds
that contain functional groups with potential biological activities. Since heterocycles
containing quinoline are reported to improve the biological activity of organic compounds,
our aim was to synthesize novel quinolone bearing dihydropyridine derivatives. Also, we
decided to use MCR, therefore, our first objective was to synthesize a formyl quinoline

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derivative using Vilsmeier-Haack reaction (Srivastava et al., 2005) which could be used as
one of the substrates for the one pot MCR.
Scheme 1: Synthesis of quinoline bearing dihydropyridines
N
O
NH2
R
CHO
CN
CN
O
O
Cl
Et3N
OCH3
OCH3
+
+
+
CN
H3CO
H3CO
EtOH
N
Cl
O
N
NH2
R = m-CH3, o-OCH3, p-Cl, m,p-Cl, o-F, m-F, p-F
R
Table 1: Synthesis of quinoline bearing dihydropyridines derivatives
Mp (°C)
275~276
263~264
237~238
287~288
247~248
269~271
253~254
280~282
Entry
Compound
Ar
% Yield
87
1
2
3
4
5
6
7
8
A1
A2
A3
A4
A5
A6
A7
A8
C6H4
o-OCH3C6H4
m-CH3-C6H4
p-ClC6H4
o-FC6H4
m-FC6H4
p-FC6H4
m,p-ClC6H4
40
73
79
64
31
60
72
The identity of all the compounds (A1- A8) were confirmed by analysing the spectral
1
13
data obtained from IR, H NMR, C NMR and GCMS. All the spectra were well resolved.
We selected A1 as the template and characterised the structure fully with the aid of 2D NMR
techniques especially HSQC, HMBC, COSY and NOESY which led to the unambiguous
assigning of all protons and carbons. We used the characterization of A1 as a template to
elucidate the other 7 derivatives. The IR spectra of A1 showed the C=O stretching at 1713

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cm-1 and 1748 cm-1, the C-N stretching at 2171 cm-1, the C-Cl stretching at 669-778 cm-1
whilst the NH stretching at 3486 cm-1 and 3356 cm-1 was not well resolved.
1
The H NMR spectrum of A1 showed singlets at δ 3.44 and δ 3.51 for two acetoxy
groups (H-9', H-10'). The singlets at δ 4.13 and δ 5.35 were assigned to the amino (NH2) and
aliphatic proton (H-4') on the dihydropyridine ring, respectively. The peaks at δ 8.14-7.38
were attributed to aromatic protons. The chemical shifts, spin multiplicities and coupling
constants (in Hertz) were assigned as: δ 8.14 (H-4, s); δ 7.84 (d, H-6, 3.2); δ 7.71 (t, H-7,
7.6); δ 7.64 (t, H-8, 7.4); δ 8.02 (d, H-9, 8.4); δ 7. 39-7.36 (m, H-2", H-4", H-6"); δ 7.54-7.51
(m, H-3"-H-5"). The COSY spectrum, presented in Figure S4, shows H-9 has a strong
coupling with H-8 but a weaker coupling with H-7. The corresponding carbon resonances of
H-7, H-8 and H-9 appeared at δ 130.17, 130.87 and 130.47, respectively and the absence of
correlation between the carbons to the amino protons confirm the assignment of the NH2,
according to the HSQC spectrum, presented in Figure S6. The NOESY spectrum, presented
in Figure S7, shows the coupling of H-9 to H-8; H-6 is coupled to H-7 and H-8 whilst H-7 is
coupled to H-6 and H-8. Furthermore, the COSY spectrum shows a strong coupling between
H-6 and H-7 and between H-7 and H-8 whilst a weak coupling is observed between H-7 and
H-9. Also H-4 lacked any coupling signals. Both H-4 and H-6 shows the HMBC correlations
to C-2, C-4' and C-5, presented in Figure S5, at δ 150.99, 120.33 and 134.90, respectively.
4
5
The J correlations at H-4' to C-5 and H-4' to C-8, J correlation at H-6 to C-4' were also
observed. The H-6" proton resonance shows a COSY correlation to H-5" and was confirmed
by HSQC correlations between C-6" and C-5" occurring at δ 128.00 and 127.49, respectively.
Figure 2 shows the HMBC correlation for A1.
The 13C NMR spectrum of A1 is presented in Figure S8. The peaks at δ 51.90 and δ
52.34 were assigned to the two acetoxy carbons (C-9', C-10'); the peak at δ 58.06 was
assigned to carbon of CN (C-3') and the peak at δ 39.03 was assigned to the C-H (C-4') on the
dihydropyridine ring. The peaks at δ 162.64 are assigned to the two C=O (C-7', C-8'); the
peak at δ 164.73 was assigned to the C-NH2 (C-2') whilst the peaks at δ 150.99-120.33 were
13
attributed to aromatic carbons. The C NMR spectra of all compounds were appropriate to
their formulas. Mass spectra of the compounds showed m/z in accordance with their
formulas. The mass spectrum of A1 is presented in Figure S9, it shows the m/z value for the
molecular ion corresponding to the proposed molecular formula at 475.
The cancer study was used to investigate the cytotoxicity of synthesized quinoline
derivatives (A1-A8) in the A549 lung cancer cell line. These compounds differed in
substituents like OCH3, CH3, F and Cl attached to the benzene ring. Herein we decided to

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investigate the effect of these compounds based on the different functional groups to study
their anticancer potential. At this stage of our investigation, we were unable to source out a
suitable standard containing the dihydropyridine nucleus and hence we selected A1 as a
reference point. Our hypothesis was that the presence of functional groups present in the
phenyl part will influence cytotoxicity. The parent compound A1 (with no substituent in the
benzene ring) is not cytotoxic but rather increases metabolic activity as all concentrations
tested show greater activity than control. The compound A2 (with an electron donating
group, OCH3 substituent in the benzene ring), shows a dose dependent toxicity due to its
substituent as the results gives evidence that the compound has potential as an anti-cancer
drug. The compound A3 (with an electron donating group, CH3 substituent in the benzene
ring) shows high toxicity due to its CH3 substituent but only at higher concentrations; has
potential as an anti-cancer drug. The compound A4 (with the chloro group, which withdraws
electrons by induction, but donates electrons by resonance in the benzene ring) showed a
dose dependent toxicity with the highest toxicity at 250µM. The compound had potential as
an anti-cancer drug. A5, A6 and A7 (with the fluoro group in the benzene ring) were not
cytotoxic. A8 (with the two chloro groups, which withdrew electrons by induction, but
donated electrons by resonance in the benzene ring) showed a dose dependent toxicity with
the highest toxicity at 125, 250 and 500 µM. The compound had potential as an anti-cancer
drug.
All Compounds (A1-A8) were evaluated for antibacterial activity against two Gram-
negative bacteria, E. coli and P. aeruginosa and one Gram-positive bacterium, S. aureus.
Standard antibiotics, ciprofloxacin and nalidixic acid were used as positive controls and
DMSO used as negative control. DMSO had no effect on the bacteria in the concentrations.
The antimicrobial activity results, Table 3 revealed that the synthesized compounds showed
moderate to good activity towards the mentioned panel of bacteria strains. It is interesting to
note that compound with substituted anilines in the para position (A4, A7 and A8) showed
very good activity against all strains. These compounds were found more potent than both
standards, ciprofloxacin (MIC = 50 μM) and nalidixic acid (MIC = 50 μM) towards S. aureus
and P. aeruginosa. Compound A1 (MIC = 128 μM) exhibited comparable activity as the
standard nalidixic acid (MIC = 128 μM) against P. aeruginosa but showed no inhibitory
effect against S. aureus E. coli. Compounds A2 and A3 showed poor activity towards all
strains compared with the standard antibiotics.

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Table 3: Antibacterial activities of (A1-A8): Minimum Inhibitory Concentration
Pseudomonas
aeruginosa
Staphylococcus
aureus (µM)
Escherichia coli
Compound
(µM)
(µM)
A1
A2
A3
A4
A5
A6
A7
A8
-
128
256
256
32
256
256
256
32
256
256
16
128
-
256
256
32
128
-
32
8
32
16
32
50
128
16
Nalidixic
Acid
50
-
25
-
25
-
Ciprofloxacin
DMSO
The binding mode of A1 within the active site of the human mdm2 was analyzed and
the suitable shape of the compound helped it to bind tightly with the active site of the human
mdm2. There was one hydrogen bond interaction formed between the side chains of the
hydrophobic residue of TYR 104. The bond lengths between A1 into the active site of the
human mdm2 were observed (2.15Å). Docking of A1 with Human mdm2 indicated lowest
binding energy, glide score -6.111 Kcal/mol and glide energy -62.406 Kcal/mol thereby
showing strong affinity of the ligand molecule with the receptor which had been stabilized by
strong hydrogen bond interactions in the binding pocket. Furthermore, the following residues
were mainly involved in hydrophobic interaction LEU27, VAL28, MET50, LEU54, PHE55,
ILE61, MET62, TYR67, VAL75, VAL93, and ILE99. Table 3 shows the docking results of
all of the compounds (A1-A8), glide score (Kcal/mol), glide energy ( Kcal/mol), interacting
residues, distance between the protein and ligand (Å), hydrogen bond donor and hydrogen

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bond acceptor. Figure 11a, 11b, and 11c shows hydrogen bond formation, hydrogen bond
formation and bond length and the binding mode of A1 compound with E3 ubiquitin-protein
ligase mdm2.
Table 3: Glide extra-precision (XP) for (A1- A8) with human mdm2 by use of Schrodinger
Glide
Score
Glide
energy
No. of H
bonds
Interacting
Residues
Hydrogen bond
donor
Hydrogen bond
acceptor
Title
Distance (Å)
-6.111
-2.616
-4.391
-62.406
-27.659
-35.217
1
-
Tyr 104
-
2.15
-
Ligand: (H)
A: TYR 104: (O)
A1
A2
A3
-
-
1
Thr 26
2.04
A: THR 26: (H)
Ligand: (O)
A4
A5
-3.963
-4.995
-16.574
-44.146
1
1
Thr 26
Thr 26
1.97
2.05
A: THR 26: (H)
Ligand: (O)
Ligand: (O)
A: THR 26:
(H)HG1
A6
A7
-4.667
-1.089
-42.032
-19.121
1
4
Thr 26
Arg 29
2.04
A: THR 26:
(H)HG1
Ligand: (O)
Ligand: (N)
1.93
2.25
A: ARG29:
(H)HH21
Ligand: (N)
Ligand: (H)
Ligand: (O)
B: LYS 51:
(H)HZ1
Glu 25
2.25
2.46
GLY 25
A: GLY 25:
(O)OE1
A: GLY 25:
(H)H1
A8
-3.497
-49.899
1
Thr 26
2.15
A: THR 26:
(H)HG1
Ligand: (O)
Conclusion
All Compounds containing quinoline and dihydropyridine nucleus in a single
molecular frame work were efficiently synthesized and fully characterized. In vitro
anticancer and antibacterial studies were carried out; molecular docking was also
investigated. The biological studies revealed that these compounds are selective in their
action. The anti-cancer assay indicated that compounds A2, A3, A4 and A8 have good potential as an
anticancer drug. The results obtained from the antibacterial assay showed that compounds A4, A7 and
A8 have good activity compared to that of standard drugs, whereas A2 and A3 showed poor
activity against the tested bacterial strains. Compound A1 showed no inhibitory effect against S.
aureus E. coli. Docking of A1 with Human mdm2 indicated the lowest binding energy thereby

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showing strong affinity of the ligand molecule with the receptor which has been stabilized by strong
hydrogen bond interactions in the binding pocket. This confirms that A1 is a better inhibitor for
mdm2. Hence compound A2-A8 showed an interesting correlation between docking scores
and experimental binding data.
Acknowledgement
We are grateful to the National Research Foundation (NRF) and Durban University of
Technology (DUT) for funding this project.

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List of figures
Figure 2 Selected HMBC correlations for A1
Figure 3 The cytotoxic effects of A1 in the A549 lung cancer cell line
Figure 4. The cytotoxic effects of A2 in the A549 lung cancer cell line
Figure 5. The cytotoxic effects of A3 in the A549 lung cancer cell line
Figure 6. The cytotoxic effects of A4 in the A549 lung cancer cell line
Figure 7. The cytotoxic effects of A5 in the A549 lung cancer cell line
Figure 8. The cytotoxic effects of A6 in the A549 lung cancer cell line
Figure 9. The cytotoxic effects of A7 in the A549 lung cancer cell line
Figure 10. The cytotoxic effects of A8 in the A549 lung cancer cell line
Figure 11a, 11b & 11c. Molecular docking result of A1 with human mdm2

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Figure 1: Dihydropyridine-based bioactive compounds

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Figure 2. Selected HMBC correlations for A1
H
8
H
H
H
N
7
6
9
10
1
5
4
2
O
7'
Cl
3
H
CN
9'
H3CO
5
'
4'
3'
H3CO
10'
1'
N
6'
8'
2'
NH2
H
H
O
1''
6''
2''
3''
5''
H
H
4''
H

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Figure 3. The cytotoxic effects of A1 in the A549 lung cancer cell line

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Figure 4. The cytotoxic effects of A2 in the A549 lung cancer cell line

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Figure 5. The cytotoxic effects of A3 in the A549 lung cancer cell line