Diverse C-6 substituted 4-methyl-2-(2-, 3- and 4-pyridinyl)quinolines: synthesis, in vitro anticancer evaluation and in silico studies

Article abstract of DOI:10.1007/s00044-016-1775-8

A series of twelve 4-methyl-2-(2-, 3- and 4-pyridinyl)quinolines 7–9 was synthesized using modified Kametani reaction protocol and their in vitro cytotoxicity was tested against human cancer cell lines MCF-7, SKBR-3, PC3, HeLa, comparing with human dermis

Full text of DOI:10.1007/s00044-016-1775-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1775-8
ORIGINAL RESEARCH
Diverse C-6 substituted 4-methyl-2-(2-, 3- and 4-pyridinyl)
quinolines: synthesis, in vitro anticancer evaluation and
in silico studies
Vladimir V. Kouznetsov1 Mayra L. Robles-Castellanos1 Felipe Sojo2,3
●
●
●
Fernando A. Rojas-Ruiz1 Francisco Arvelo2,3
●
Received: 7 June 2016 / Accepted: 30 December 2016
© Springer Science+Business Media New York 2016
Abstract A series of twelve 4-methyl-2-(2-, 3- and 4-pyr-
idinyl)quinolines 7–9 was synthesized using modified
Kametani reaction protocol and their in vitro cytotoxicity
was tested against human cancer cell lines MCF-7, SKBR-
3, PC3, HeLa, comparing with human dermis fibroblast as
non-tumor cells. In general, these molecules displayed
potent anticancer properties, but also demonstrated a narrow
safe margin, as it was observed for doxorubicin. Com-
pounds 8a, 8b, 9a and 9d showed prominent selective
cytotoxicity with higher IC50 values compared with refer-
ence drug doxorubicin in prostate carcinoma, cervical epi-
thelial carcinoma and breast carcinoma (no overexpresses
the HER2/c-erb-2 gene), respectively. The 4-methyl-2-(3-
pyridinyl)quinoline (8a) stands out by its low unspecific
cytotoxicity (IC50 = 476.69 μM) and highly exceptional
selectivity for PC3 cells (IC50 = 4.40 μM) as an interesting
model for antitumor drugs against prostate carcinoma. Its 4-
pyridinyl analog 9a showed superior potency against HeLa
cells (IC50 = 0.016 μM) and an outstanding selectivity (SI
= 4168.1) compared to doxorubicin (IC50 = 3.62 µM, SI =
0.7). Analog 9d revealed potent activity (IC50 = 0.38 µM)
against breast cancer MCF-7. In silico studies are also
reported. ADME profiling, in silico toxicity and drug-score
data of compounds 8a and 9a resulted to be favorable
compared to doxorubicin.
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Keywords Pyridinylquinolines Anticancer properties
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Kametani reaction In silico studies
Introduction
Cancer is one of the foremost causes of death worldwide,
thus several efforts have been developed to treat various
forms of cancer for decades (Jemal et al. 2011; Siegel et al.
2014). Chemotherapeutic drugs are still considered to be the
most essential therapeutic approach for cancer treatment.
However, today drug resistance and adverse side effects are
serious problems. In this sense, designing and developing
new chemotherapeutic drugs for cancer is of great impor-
tance. Quinoline and pyridine are aromatic heterocyclic
nitrogen-containing compounds, whose scaffolds are com-
monly present in a broad number of anticancer drugs.
Therefore, design and synthesis of novel small molecules
based on quinoline and/or pyridine skeletons are active
fields among the anticancer drug development (Solomon
and Lee 2011; Afzal et al. 2015; Arafa et al. 2013; Jun et al.
2014; Karki et al. 2015; Abadia et al. 2009). Moreover, they
have revealed outstanding results through different
mechanism of action such as growth inhibitors by cell cycle
arrest, apoptosis, and inhibition of angiogenesis or cell
migration disruption. Despite the broad spectrum of biolo-
gical and biochemical properties exhibited by quinoline and
pyridine derivatives, few reports focus on designing
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1775-8) contains supplementary material,
which is available to authorized users.
* Vladimir V. Kouznetsov
kouznet@uis.edu.co
vkuznechnik@gmail.com
1
Laboratorio de Química Orgánica y Biomolecular, Universidad
Industrial de Santander, Parque Tecnológico Guatiguara, Km 2 vía
refugio, Piedecuesta, A.A. 681011, Colombia
2
Centro de Biociencias Fundación Instituto de Estudios Avanzados-
IDEA, Caracas, Venezuela
3
Laboratorio de Cultivo de Tejidos y Biología de Tumores,
Instituto de Biología Experimental, Universidad Central de
Venezuela, Código postal 1041, Caracas, Venezuela

Med Chem Res
potential anticancer agents throughout the combination of
these two scaffolds in one single molecule, especially pyr-
idinylquinolines. This is likely due to the difficulty asso-
ciated with their synthesis: described syntheses are plagued
by lengthy multi-step reactions, low yields, hazardous
reagents, and/or the wasteful use of solvents for purification
purposes. Moreover, reported synthetic protocols do not
allow obtaining functionalized pyridylquinolines, and
mainly address to the preparation of 2-(2-pyridinyl)
quinolines (Borel et al. 2015; Mamo et al. 2002; Dick et al.
2012).
Although current pyridylquinolines syntheses have cer-
tain drawbacks, several molecules based on pyr-
idinylquinoline skeleton were evaluated as possible
anticancer agents (Fig. 1). For example, 4-(4-pyridinyl)
quinoline derivative A was identified as an effective PI3K
inhibitor (Solomon and Lee 2011), 6,7-dimethoxy-3-(3-
pyridinyl)quinoline B and 5,7-dimethoxy-3-(4-pyridinyl)
quinoline C emerged as potent inhibitors of platelet-derived
growth factor (PDGFR) (Maguire et al. 1994; Dolle et al.
1994), and 4-anilino-7-(2-pyridinyl)-3-quinolinecarbonitrile
D showed potent activity in both the Src enzyme and cell
assays, and demonstrated in vivo antitumor activity in a
xenograft model (Woynarowski et al. 1994). Other func-
tionalized 2-(4-pyridinyl)quinolines act as topoisomerase-II
To the best of our knowledge there is no systematic
investigation on the preparation and cytotoxic evaluation of
C-6 substituted 4-methylquinolines diversely linked with
three isomeric pyridinyl motifs (H, Fig. 1). Noteworthy, as
they are flat, heteroaromatic molecules like these quinoline
compounds could bind to DNA by intercalating and
stacking between the base pairs of the DNA double helix,
which is an important target in cancer therapy. The planar
structure of DNA intercalating drugs can strongly bind to
DNA resulting in the death of cancer cells. Moreover,
several representative antitumor agents of this class are
traditionally used in clinics, such as topotecan, irinotecan
(derivatives of quinoline alkaloid camptothecin) and dau-
nomycin, and doxorubicin (adriamycin) (anthracycline
antibiotics). Taking into account the above stated and as a
continuation of our previous studies in this field (Kouz-
netsov et al. 2012a, 2012b), we now report the synthesis,
characterization and biological evaluation of new C-6 sub-
stituted 4-methylquinolines linked with α-, β- or γ-pyridine
subunits. Aspects examined include development of a new
and simple protocol for the synthesis of C-6 substituted 4-
.
methyl-2-(pyridinyl)quinolines based on BF3 OEt2-cata-
lyzed Kametani reaction of N-(pyridinyliden)anilines and
2,2-dimethoxypropane (Kouznetsov et al. 2007), evaluation
for their in vitro cytotoxicity against the cell lines MCF-7,
SKBR-3, PC3, HeLa as well as the nonspecific cytotoxicity
against human dermis fibroblast for non-tumor cells, and in
silico studies of the most active molecules tested. The
structure-activity relationships of these quinoline com-
pounds are also discussed.
inhibitor
[2-(4-pyridinyl)quinoline-8-carboxamide
E]
(Zhang et al. 2009) or possess potent in vitro anticancer
activity against human cancer MCF-7, H-460 and SF-268
cell lines without harming normal cells (Vero and THP-1)
[4-methyl-2-(4-pyridinyl)quinolines F and G] (Kouznetsov
et al. 2012a).
On the other hand, the cell cycle-regulated cyclin-
dependent kinases (CDKs), CDK1—CDK6 have been
extensively studied as potential therapeutic targets in cancer
(Mariaule and Belmont 2014; Sánchez-Martínez et al. 2015)
and some quinoline and pyridine molecules can disrupt
CDKs interaction with their cyclin A subunit acting as ATP
competitive inhibitors (Denga et al. 2014; Anderson et al.
2003).
Material and methods
Chemistry
The melting points (uncorrected) were determined on a
Fisher-Johns melting point apparatus. Infrared (FT-IR)
spectra were recorded on a Lumex Infralum FT-02
Fig. 1 Pyridinylquinolines as
anticancer agents

Med Chem Res
spectrometer, νmax in cm−1. Bands are characterized
according to the functional group. H NMR spectra were
acetate to afford the 4-methyl-2-(pyridinyl)quinolines 7–9
(Table 1).
1
recorded on a Bruker Avance-400 (400 MHz) spectrometer.
Data were reported as follows: chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, dd = doublet of doub-
lets, m = multiplet), coupling constants (Hz) and integra-
tion. 13C NMR spectra were recorded on a Bruker Avance-
400 (100 MHz) spectrometer with complete proton decou-
pling. Chemical shifts are reported in ppm (δ) relative to the
4-Methyl-2-(2-pyridinyl)quinoline (7a)
Yield 35%, red oil, Rf = 0.5 (hexane:ethyl acetate, 3:1); IR
(KBr): νmax 3053, 1608, 1594, 827, 748 cm−1; H NMR
1
(CDCl3, 400 MHz), δ = 2.12 (3H, s, 4-Me), 7.23 (1H, dd, J
= 7.3, 5.7 Hz, 5-HPy), 7.78–7.55 (1H, m, 4′-HPy), 7.58 (1H,
t, J = 7.1 Hz, 6-HQu), 7.81 (1H, t, J = 7.1 Hz, 7-HQu), 7.84
(1H, s, 3-HQu), 7.90–7.86 (3H, m, 5-HQu, 8-HQu and 3-
HPy,), 8.45 (1H, d, J = 7.3 Hz, 6-HPy); 13C NMR (CDCl3,
100 MHz), δ = 161.5, 149.1, 136.8, 129.3, 127.6, 126.6,
123.8, 121.9, 117.9, 115.5, 114.3, 113.9, 113.0, 112.1,
22.7; EI-MS, m/z: 220 (M+.); anal. calcd. for C15H12N2: C,
81.79; H, 5.49; N, 12.72. Found: C, 81.55; H, 5.64; N,
12.61.
1
solvent peak (CDCl3, 7.24 ppm for H and 77.23 ppm for
13C). A Hewlett Packard 5890a series II Gas Chromato-
graph interfaced to an HP 5972 mass selective detector with
an HP MS Chemstation Data system was used for MS
identification at 70 eV using a 60 m capillary column coated
with HP-5 [5%-phenyl-poly(dimethyl-siloxane)] (comp.
9a–9d). Additionally, a Bruker X Amazon for analysis by
mass spectrometry with electrospray ionization ion trap
(ESI-MS) was employed (comp. 7a–7d and 8a–8d). Ele-
mental analyses were performed on a Perkin Elmer 2400
Series II analyzer and were within 0.4 of theoretical
values. All work-up and purification procedures were car-
ried out with reagent grade solvents (purchased from
Aldrich and Merck). Thin-layer chromatography was per-
formed using E. Merck silica gel 60 F254 precoated plates
(0.25 mm). Column chromatography was performed using
silica gel 60 (0.063–0.200 mm) 70–230 mesh. Preparation
of N-(pyridinyliden)anilines 3–5 was easily performed
using reaction of p-substituted anilines 1a–d and isomeric
pyridinecarboxyaldehydes 2a–c in dichloromethane in the
presence of Na2SO4 at room temperature following descri-
bed reports (Kouznetsov et al. 2007).
6-Chloro-4-methyl-2-(2-pyridinyl)quinoline (7b)
Yield 45%, white solid, mp 110–111; oC, Rf = 0.4 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3020, 1598, 1520, 753
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.55 (1H, dd, J =
7.3, 1.7 Hz, 6-HPy), 7.62 (1H, td, J = 7.7, 1.8 Hz, 7-HQu),
7.36 (1H, d, J = 7.8 Hz, 8-HQu), 7.20 (1H, s, 5-HQu),
7.10–7.07 (2H, m, 3-HPy, 4-HPy), 6.54–6.40 (2H, m, 5-HPy,
3-HQu), 2.13 (3H, s, 4-Me); 13C NMR (CDCl3, 100 MHz):
δ = 160.7, 149.5, 146.6, 145.4, 136.9, 132.4, 131.9, 130.6,
129.2, 123.8, 122.7, 122.6, 121.9, 115.00, 20.9; ESI-MS,
m/z 275 [M+Na]+; anal. calcd. for C15H11ClN2: C, 70.73;
H, 4.35; N, 11.00. Found: C, 70.54; H, 4.51; N, 10.84.
6-Fluoro-4-methyl-2-(2-pyridinyl)quinoline (7c)
Synthesis of C-6 substituted 4-methyl-2-(2-, 3- and 4-
pyridinyl)quinolines (7–9)
Yield 35%, yellow solid, mp 89–91 °C, Rf = 0.3 (hexane:
ethyl acetate, 3:1); IR (KBr) : νmax 3039, 1531, 1506, 1210
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.56 (1H, dd, J =
.
1
General experimental procedure BF3 OEt2 (20 mmol) was
dropped to a stirred solution of N-(pyridinyliden)anilines 3–
5 (10 mmol) in dichloromethane (5 mL) under nitrogen at
−8 °C for 20 min. Then, 2,2-dimethoxypropane (40 mmol)
dissolved in dichloromethane (4 mL) was very slowly added
to the reaction mixture keeping the inert atmosphere and
cooling. Upon completion of addition, the cold bath was
removed and the temperature was increased to reflux and
maintained for 2–5 h, according to thin layer chromato-
graphy control. The reaction mixture was allowed to cool to
room temperature and treated with saturated NaHCO3
solution to reach neutral pH. Then, the mixture was
extracted with CH2Cl2 (3 × 20 mL). The organic phase was
dried over anhydrous Na2SO4 and the solvent was removed
by distillation under reduced pressure to give crude product,
which was purified by column chromatography on silica
gel, using as eluents solvent mixtures of hexane and ethyl
7.3, 1.7 Hz, 6-HPy), 7.62 (1H, td, J = 7.7, 1.8 Hz, 7-HQu),
7.37 (1H, d, J = 7.8 Hz, 8-HQu), 7.20–7.17 (1H, m, 5-HQu),
6.88–6.80 (2H, m, 3-HPy, 4-HPy), 6.60–6.53 (2H, m, 5-HPy,
3-HQu), 2.14 (3H, s, 4-Me); 13C NMR (CDCl3, 100 MHz):
δ = 160.9 (d, J = 247.9 Hz), 153.8, 154.9, 149.4 (d, J = 5.2
Hz), 143.0, 136.9 (d, J = 9.0 Hz), 128.3, 128.2 (d, J = 9.0
Hz), 122.5, 121.9, 120.0, 119.7 (d, J = 26.1 Hz), 115.9,
107.6 (d, J = 23.2 Hz), 20.8; ESI-MS, m/z 259 [M+Na]+;
anal. calcd for C15H11FN2: C, 75.62; H, 4.65; N, 11.76.
Found: C, 75.56; H, 4.83; N, 11.52.
4-Methyl-2-(2-pyridinyl)-6-(trifluoromethyl)quinoline (7d)
Yield 30%, white solid, mp 118–119 °C, Rf = 0.5 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3037, 1613, 1541, 1110
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.57 (1H, dd, J =

Med Chem Res
Table 1 Physicochemical data
of prepared 2-(pyridinyl)
quinoline derivatives 7–9
Comp.
Formula
Yield (%)
Mp (°C)
MW (g/mol)
ESI-MS (m/z)
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
a
C15H12N2
35
45
35
30
68
75
63
40
69
77
52
41
Red oil
110–111
89–91
220.27
254.71
238.26
288.27
220.27
254.71
238.26
288.27
220.27
254.71
238.26
288.27
221 [M+H]+
275 [M+Na]+
259 [M+Na]+
289 [M+H]+
221 [M+H]+
275 [M+Na]+
239 [M+H]+
289 [M+H]+
220a
C15H11ClN2
C15H11FN2
C16H11F3N2
C15H12N2
118–119
67–69
C15H11ClN2
C15H11FN2
C16H11F3N2
C15H12N2
128–129
102–103
125–127
72–73
C15H11ClN2
C15H11FN2
C16H11F3N2
130–131
104–105
Red oil
254a
238a
288a
Performed using MS, (m/z) (EI, 70 eV)
7.3, 1.7 Hz, 6-HPy), 7.63 (1H, td, J = 7.7, 1.8 Hz, 7-HQu),
7.39–7.34 (3H, m, 5-HQu, 8-HQu and 3-HPy), 7.21–7.17
(1H, m, 5-HPy), 6.65–6.60 (2H, m, 3-HQu, 4-HPy), 2.13 (3H,
m, 4-Me); 13C NMR (CDCl3, 100 MHz): δ = 156.4, 150.7,
149.6, 149.3, 137.1, 131.5, 128.3, 125.5 (2C), 123.6, 122.7,
121.9, 120.4, 119.6, 119.3, 20.9; ESI-MS, m/z 289 [M
+H]+; anal. calcd for C16H11F3N2: C, 66.66; H, 3.85; N,
9.72. Found: 66.75; H, 3.67; N, 9.64.
8-HQu), 7.45 (1H, ddd, J = 8.0, 4.8, 0.8 Hz, 5-HPy), 2.73
(3H, d, J = 0.8 Hz, 4-Me); 13C NMR (CDCl3, 100 MHz): δ
= 154.6, 150.4, 148.8, 146.6, 144.7, 134.9, 134.9, 132.4,
131.9, 130.6, 128.2, 123.8, 122.9, 120.0, 19.1; ESI-MS, m/z
255 [M+H]+; anal. calcd for C15H11ClN2: C, 70.73; H,
4.35; N, 11.00. Found: C, 70.85; H, 4.48; N, 10.79.
6-Fluoro-4-methyl-2-(3-pyridinyl)quinoline (8c)
4-Methyl-2-(3-pyridinyl)quinoline (8a)
Yield 63%, yellow solid, mp 102–103 °C, Rf = 0.4 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3038, 1622, 1550, 1173
1
Yield 68%, yellow solid, mp 67–69 °C, Rf = 0.5 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3039, 1601, 1512, 756,
cm−1; H NMR (CDCl3, 400 MHz): δ = 9.32 (1H, d, J =
1.8 Hz, 2-HPy), 8.70 (1H, dd, J = 4.8, 1.6 Hz, 4-HPy),
8.50–8.44 (1H, m, 6-HPy), 8.16 (1H, dd, J = 9.2, 5.5 Hz, 5-
1
709 cm−1; H NMR (CDCl3, 400 MHz): δ = 9.33 (1H, dd,
J = 2.3, 0.7 Hz, 2-HPy), 8.69 (1H, dd, J = 4.8, 1.7 Hz, 4-
HPy), 8.48 (1H, ddd, J = 8.0, 2.2, 1.8 Hz, 6-HPy), 8.17 (1H,
dd, J = 8.4, 0.6 Hz, 5-HPy), 8.01 (1H, dd, J = 8.4, 0.9 Hz, 5-
H
Qu), 7.73 (1H, s, 3-HQu), 7.60 (1H, dd, J = 9.7, 2.8 Hz, 7-
Qu), 7.51 (1H, dd, J = 9.2, 2.8 Hz, 8-HQu), 7.45 (1H, ddd,
H
J = 8.0, 4.8, 0.6 Hz, 5-HPy), 2.74 (3H, s, 4-Me); 13C NMR
(CDCl3, 100 MHz): δ = 160.4 (d, J = 247.5 Hz), 153.8,
150.3 (d, J = 5.2 Hz), 148.8, 145.3, 144.9, 135.1 (d, J = 9.1
Hz), 132.9, 132.8, 128.3 (d, J = 8.9 Hz), 123.8, 120.0,
119.9 (d, J = 26.1 Hz), 107.6 (d, J = 23.4 Hz), 19.2; ESI-
MS, m/z 239 [M+H]+; anal. calcd for C15H11FN2: C,
75.62; H, 4.65; N, 11.76. Found: C, 75.76; H, 4.89; N,
11.85.
H
Qu), 7.74 (1H, ddd, J = 8.4, 6.9, 1.4 Hz, 8-HQu), 7.70 (1H,
d, J = 0.7 Hz, 3-HQu), 7.57 (1H, ddd, J = 8.2, 6.9, 1.2 Hz,
6-HQu), 7.44 (1H, ddd, J = 7.9, 4.8, 0.8 Hz, 7-HQu), 2.78
(3H, d, J = 0.9 Hz, 4-Me); 13C NMR (CDCl3, 100 MHz): δ
= 154.4, 150.2, 148.9, 148.2, 145.5, 135.3, 135.0, 130.3,
129.8, 129.4, 127.5, 126.6, 123.8, 123.8, 19.2; ESI-MS, m/z
221 [M+H]+; anal. calcd for C15H12N2: C, 81.79; H, 5.49;
N, 12.72. Found: C, 81.61; H, 5.68; N, 12.53.
4-Methyl-2-(3-pyridinyl)-6-(trifluoromethyl)quinoline (8d)
6-Chloro-4-methyl-2-(3-pyridinyl)quinoline (8b)
Yield 40%, white solid, mp 125–126 °C, Rf = 0.5 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3051, 1628, 1603, 1118
Yield 75%, white solid, mp 128–129 °C, Rf = 0.3 (hexane :
ethyl acetate, 3:1); IR (KBr): νmax 3032, 1597, 1545, 783
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 9.32 (1H, d, J =
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 9.32 (1H, dd, J =
1.8 Hz, 2-HPy), 8.70 (1H, dd, J = 4.8, 1.6 Hz, 4-HPy),
8.50–8.44 (1H, m, 6-HPy), 8.16 (1H, d, J = 2.3 Hz, 5-HQu),
7.73 (1H, s, 3-HQu), 7.60 (1H, dd, J = 9.7, 2.8 Hz, 7-HQu),
7.51 (1H, dd, J = 9.2, 2.8 Hz, 8-HQu), 7.45 (1H, dd, J = 4.8,
0.6 Hz, 5-HPy), 2.74 (3H, m, 4-Me); 13C NMR (CDCl3, 100
2.2, 0.6 Hz, 2-HPy), 8.70 (1H, dd, J = 4.8, 1.6 Hz, 4-HPy),
8.46 (1H, ddd, J = 8.0, 2.2, 1.8 Hz, 6-HPy), 8.08 (1H, d, J
= 9.0 Hz, 7-HQu), 7.95 (1H, d, J = 2.3 Hz, 5-HQu), 7.71
(1H, d, J = 0.5 Hz, 3-HQu), 7.66 (1H, dd, J = 9.0, 2.3 Hz,

Med Chem Res
MHz): δ = 156.4, 150.7, 149.3, 148.9, 146.6, 135.2, 134.7,
131.5, 126.6, 125.6, 125.4, 123.8, 121.9, 121.8, 120.4,
19.2; ESI-MS, m/z 289 [M+H]+; anal. calcd for
C16H11F3N2: C, 66.66; H, 3.85; N, 9.72. Found: 66.43; H,
3.95; N, 9.54.
4-Methyl-2-(4-pyridinyl)-6-(trifluoromethyl)quinoline (9d)
Yield 41%, red oil; Rf = 0.5 (hexane: ethyl acetate, 3:1); IR
(KBr): νmax 3029, 1617, 1549, 1110 cm−1 1H NMR
;
(CDCl3, 400 MHz): δ = 8.57 (2H, d, J = 4.6 Hz, 2-HPy, 6-
HPy), 8.48 (2H, d, J = 4.4 Hz, 3-HPy, 5-HPy), 7.81–7.49
(2H, m, 3-HQu, 7-HQu), 7.40–7.31 (2H, m, 5-HQu, 8-HQu),
2.33 (3H, s, 4-Me); 13C NMR (CDCl3, 100 MHz): δ =
150.5, 149.9, 148.2, 141.9, 140.2, 130.7, 126.7, 126.3,
122.1, 121.9, 121.4, 120.2, 114.1, 112.0, 19.9; ESI-MS, m/z
289 [M+H]+; anal. calcd for C16H11F3N2: C, 66.66; H,
3.85; N, 9.72. Found: 66.39; H, 3.76; N, 9.88.
4-Methyl-2-(4-pyridinyl)quinoline (9a)
Yield 69%, white solid, mp 72–73 °C; Rf = 0.5 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3029, 1597, 1541, 833
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.76 (2H, dd, J =
4.5, 1.6 Hz, 2-HPy, 6-HPy), 8.19 (1H, ddd, J = 8.5, 1.2, 0.5
Hz, 5-HQu), 8.04 (2H, dd, J = 4.5, 1.7 Hz, 3-HPy, 5-HPy),
8.03 (1H, m, 6-HQu), 7.75 (1H, ddd, J = 8.4, 6.9, 1.4 Hz, 8-
Biological activity
H
Qu), 7.72 (1H, d, J = 0.8 Hz, 3-H), 7.60 (1H, ddd, J = 8.2,
6.9, 1.3 Hz, 7-HQu), 2.78 (3H, d, J = 0.9 Hz, 4-Me); 13C
NMR (CDCl3, 100 MHz): δ = 154.2, 150.5 (2C), 148.1,
146.9, 145.7, 130.6, 129.9, 127.9, 127.1, 123.8, 121.7 (2C),
119.3, 19.2; EI-MS, m/z: 220 (M+.); anal. calcd for
C15H12N2: C, 81.79; H, 5.49; N, 12.72. Found: C, 81.85; H,
5.58; N, 12.66.
Reagents and compounds
Human tumor cell lines and culture media: PC3 (Prostate
carcinoma), HeLa (Cervical epithelial carcinoma) were
grown in RPMI 1640 medium (Invitrogen) supplemented
with 10% heat inactivated fetal bovine serum, 1% of L-
glutamine, 1% streptomicyn, 100 units/mL penicillin (all
obtained from Sigma Aldrich USA). MCF-7 (breast carci-
noma, no overexpresses the HER2/c-erb-2 gene), SKBR-3
(breast carcinoma, overexpresses the HER2/c-erb-2 gene)
and primary culture of normal human dermis fibroblast used
as control cells were grown in DMEM medium (Invitro-
gen). Cells were grown in a humidified incubator with 5%
CO2 and 95% air at 37 °C until they reached the exponential
growth phase. For treatments exponentially growing cells
were collected, counted, re-suspended in fresh culture
medium and incubated in 96 sterile well plates.
6-Chloro-4-methyl-2-(4-pyridinyl)quinoline (9b)
Yield 77%, white solid, mp 130–131 °C, Rf = 0.3 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3040, 1596, 1539, 784
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.78 (2H, dd, J =
1
4.5, 1.6 Hz, 2-HPy, 6-HPy), 8.12 (1H, d, J = 9.0 Hz, 7-HQu),
8.04 (2H, dd, J = 4.5, 1.6 Hz, 3-HPy, 5-HPy), 7.99 (1H, d, J
= 2.3 Hz, 5-HQu), 7.76 (1H, s, 3-HQu), 7.69 (1H, dd, J =
9.0, 2.3 Hz, 8-HQu), 2.76 (3H, d, J = 0.7 Hz, 4-Me); 13C
NMR (CDCl3, 100 MHz): δ = 154.5, 150.6 (2C), 146.6,
146.5, 144.9, 132.9, 132.2, 130.8, 128.7, 123.0, 121.7 (2C),
120.1, 19.2; EI-MS, m/z: 254 (M+.); anal. calcd for
C15H11ClN2: C, 70.73; H, 4.35; N, 11.00. Found: C, 70.81;
H, 4.23; N, 10.77.
Cytotoxic activity
Cell viability was assessed using the MTT assay, which is
based on the ability of viable cells to metabolically reduce a
yellow tetrazolium salt (MTT; Sigma) to a purple formazan
product. This reaction takes place when mitochondrial
reductases are active (Mosmann 1983). Cells were grown in
96-well plates (5 × 103 cells/well) for 24 h. Cultures were
carried out at 37 °C in a humidified atmosphere with 5%
CO2. Cells were incubated with the synthetics products or
chemotherapeutic drugs in 100 μL of complete culture
medium containing 0, 1, 5, 10, 25, 100 µg/mL of each
compound for 72 h. After incubation, the medium was
removed and the cells were treated with 100 μL 0.4 mg/mL
MTT for 3 h at 37 °C. Subsequently, 100 μL DMSO were
added to the mixture. The solubilized formazan product was
quantified with the help of a microtiter plate reader
TECAN-sunrise at 570 nm. Doxorubicin was used as a
positive control in the assay. In all cases the compounds
were dissolved in DMSO, at the final concentration in the
6-Fluoro-4-methyl-2-(4-pyridinyl)quinoline (9c)
Yield 52%, yellow solid, mp 104–105 °C, Rf = 0.4 (hexane:
ethyl acetate, 3:1); IR (KBr): νmax 3054, 1625, 1543, 1186
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.76 (2H, dd, J =
1
4.5, 1.6 Hz, 2-HPy, 6-HPy), 8.16 (1H, dd, J = 9.2, 5.6 Hz, 5-
H
Qu), 8.02 (2H, dd, J = 4.5, 1.6 Hz, 3-HPy, 5-HPy), 7.72
(1H, s, 3-HQu), 7.58 (7-HQu, dd, J = 9.7, 2.8 Hz, 1H), 7.51
(1H, ddd, J = 9.2, 8.1, 2.8 Hz, 8-HQu), 2.72 (3H, d, J = 0.6
Hz, 4-Me); 13C NMR (CDCl3, 100 MHz): δ = 161.1 (d, J =
246.8 Hz), 153.5, 150.5 (2C), 146.5, 145.2, 145.1 (d, J =
5.3 Hz), 133.1, 133.0 (d, J = 8.8 Hz), 128.8, 128.7, 121.5,
119.9 (d, J = 26.3 Hz), 107.6 (d, J = 23.1 Hz), 19.2; EI-MS,
m/z: 238 (M+.); anal. calcd for C15H11FN2: C, 75.62; H,
4.65; N, 11.76. Found: C, 75.70; H, 4.91; N, 11.54.

Med Chem Res
culture medium was lower than 1%, a concentration that
had neither cytotoxic effect nor caused any interference
with the colorimetric detection method.
(molecules H, Fig. 1) could be potential anticancer agents.
To obtain these small heterocyclic molecules we addressed
the Kametani reaction that involves interaction of Schiff
bases and acetals (Kametani and Kasai 1989). Using simple
addition-elimination reaction of inexpensive and commer-
cially available p-substituted anilines 1a–d and isomeric
pyridinecarboxyaldehydes 2a–c in dichloromethane in the
presence of Na2SO4 at room temperature, N-(pyridinyliden)
anilines 3–5 were easily obtained. Then, the respective
Schiff bases 3–5 (1 mmol) were treated with 2,2-dime-
Selectivity index (SI) and statistical analysis
The selectivity index was calculated as the IC50 (control
cells)/IC50 (tumoral cell line) ratio. A selectivity index >1
indicates that the cytotoxicity on tumorial cells surpassed
the one on healthy non-tumoral cells (Nugroho et al. 2013).
All experiments were performed at least three times. The
results are expressed as mean SD. Anova test were per-
formed. Only post hoc Dunnet test p < 0.01 was considered
to be statistically significant. The dose–response curves
were plotted with the OriginPro ver.8.0 programs, and 50%
growth inhibitory concentrations (IC50) of synthetics pro-
ducts or chemotherapeutic drugs were determined by a non-
linear regression of individual experiments calculated
through computation with GraphPad prism v.5.02 software
program (Intuitive Software for Science, San Diego, CA,
USA).
.
thoxypropane 6 (4 mmol) in the presence of BF3 OEt2 (2
mmol) in dichloromethane to give diverse functionalized 4-
methyl-2-(pyridinyl)quinolines 7–9, which were purified by
column chromatography (silica gel, hexane and ethyl acet-
ate, 3:1) (Scheme 1).
These compounds were prepared in moderate to good
yields, and obtained as stable crystalline substances with
well-defined melting points. Their structures were easily
1
determined by analytical and spectral (IR, H NMR, 13C
NMR, ESI-MS or EI-MS) methods. Some physicochemical
properties are depicted in the Table 1.
Having a 12-membred pyridinylquinoline library in our
hands, we started our pharmacological investigation. Clas-
sifying this library, we divided it in three groups (I, II and
III) depending on chemical nature of pyridine subunits.
Results and discussion
Chemistry
Anticancer properties
Based on our early works (Kouznetsov et al. 2012a, b,
2007), we believed that functionalized lepidines (4-
methylquinolines) with α,- β- and γ-pyridine nucleus
In this investigation, cytotoxic properties of 12 substituted
pyridinylquinolines 7–9 were evaluated in both cancer as
Scheme 1 Two-step synthesis
for 2-(pyridinyl)quinoline
library

Med Chem Res
well as normal cell lines. To study their inhibitory effects on
human tumor cell growth, four types of human malignant
cell lines,—MCF-7, SKBR-3, PC3, HeLa-, were employed.
Cell cytotoxicity was measured using the MTT assay. As
control cells, primary culture of normal human dermal
fibroblasts (NHDF, non-tumor cells) was employed to dis-
cern an unspecific cytotoxicity of prepared pyr-
idinylquinolines. Doxorubicin (DOX), a DNA intercalating
drug commonly used in clinics, was used as a reference
anticancer drug. Biological results are shown in Table 2.
The analysis of the quinoline derivatives of each group
(I, II, and III) and the cytotoxic activities exhibited some
structure-activity relationships. In general, it was noted that
tested quinoline derivatives displayed potent anticancer
properties and demonstrated a narrow safe margin, as
observed for doxorubicin, but with reduced toxic effects.
The most toxic molecule of these groups was 6-flouro-2-(3-
pyridinyl)quinoline derivative (8c) (group II) with IC50
12.34 µM values, while its analog without fluorine atom 8a
can be considered non-toxic molecule against NHDF (IC50
476.69 µM). Interestingly, the compound 8c showed only
moderate anticancer activity against all four human cancer
cells tested in this study, whereas the 2-(3-pyridinyl)qui-
noline compound 8a demonstrated selective activity against
prostate carcinoma cells with IC50 4.40 µM, compared to
doxorubicin (IC50 2.36 µM). Another molecule of this
series, 6-chloro-4-methyl-2-(3-pyridinyl)quinoline (8b)
showed similar activity against PC3 cells (IC50 3.77 µM).
Additionally, this compound exhibited a good inhibitory
effect (IC50 8.79 µM) on breast cancer cell growth
(SKBR-3). However, this 6-chloroquinoline derivative
resulted to be rather toxic against NHDF (IC50 73.61 µM).
Analyzing cytotoxicity data of quinoline molecules from
group III, it was found that 2-(4-pyridinyl)quinoline (9a)
showed a good activity against breast cells SKBR-3 (IC50
10.17 µM), but its more marked inhibitory effect was
observed on cervical epithelial carcinoma cell growth: this
compound displayed a notable activity with IC50 0.016 µM,
227 times more active than doxorubicin (IC50 3.62 µM), for
the inhibition of this cancer cell line. Interestingly, the same
compound 9a demonstrated a poor activity (IC50 64.87 µM)
against breast cancer MCF-7, while its 6-CF3 substituted
analog 9d revealed potent activity (IC50 0.38 µM) against
this type of cancer cells. Noteworthy, the compound 9d was
found to be two times more active than doxorubicin (IC50
0.74 µM).
Regarding quinolines of the group I, to our surprise the
2-(2-pyridinyl)quinoline derivatives 7a–7d did not possess
noticeable cytotoxic properties against all tested cancer cell
lines.
The pyridinylquinolines prepared in this study were
found to be reasonably active (IC50 < 25 µM) against one
(comp. 8a and 9b), two (comp. 7b, 8b, 8d, 9a, and 9c) or
three (comp. 9d) cancer cell lines.
Comparison of cytotoxicity and nonspecific cytotoxicity
of the tested compounds is presented in Table 3. According
to these results, quinoline compounds 7c, 8a, 8b, and 9a
presented a high selectivity for breast cells SKBR-3 (SI >
6.6). The compounds 8a and 8b displayed also a high
selectivity for PC3 cells with SI 108.5 and 19.5 values,
respectively, while the 2-(4-pyridinyl)quinolines 9a and 9d
were more specific for tumor lines MCF-7 and HeLa,
respectively. Noteworthy, the compound 9d showed an
outstanding selectivity (SI = 4168.1).
Table 2 Cytotoxic properties of
the prepared 2-(pyridinyl)
quinolines 7–9
Comp.
Cytotoxicity IC50 (µM)a
Unspecific
cytotoxicity IC50
(µM)
Type MCF-7
SKBR-3
PC3
HeLa
NHDF
7a
7b
7c
I
53.57 1.03
14.64 1.01
125.58 1.10
65.18 1.00
83.76 1.01
54.06 1.33
105.89 1.02
11.93 1.01
64.87 1.09
13.74 1.01
31.48 1.07
0.38 1.74
29.01 1.03
60.66 1.01
28.08 1.04
29.17 1.01
70.14 1.01
8.79 1.04
85.28 1.05
18.21 1.13
10.17 1.04
79.78 1.05
22.08 1.13
8.26 1.02
1.66 0.08
92.48 1.02
14.72 1.06
89.73 1.02
69.21 1.00
4.40 1.74
3.77 1.74
39.07 1.05
48.88 1.01
34.96 1.03
48.41 1,02
61.36 1.03
18.56 1.00
2.36 0.40
92.48 1.10
37.14 1.02
176.15 1.12
57.17 1.00
51.16 1.03
165.01 1.08
68.54 1.00
69.87 1.01
0.016 5.71
47.74 1.02
21.11 1.02
26.43 1.06
3.62 0.12
32.10 1.09
25.09 1.05
361.37 1.08
50.47 1.05
476.69 1.01
73.61 1.08
12.34 1.03
44.82 1.05
66.69 1.01
51.16 1.02
40.67 1.02
23.83 1.01
2.45 0.37
I
I
7d
8a
8b
8c
I
II
II
II
II
III
III
III
III
8d
9a
9b
9c
9d
DOX
a
0.74 0.05
Marked in bold indicated a pronounced anticancer activity

Med Chem Res
Therefore, as they are planar molecules, these simple
quinolines could be interesting models in the development
of new antitumor agents with a possible non-covalent
intercalation mode of action, in which the drug is held
rigidly perpendicular to a DNA helix axis, and as a con-
sequence, could interfere with the action of DNA-binding
enzymes such as DNA topoisomerase complex (Topo I and
Topo II).
Taking into consideration this fact, we addressed to study
some in silico physicochemical parameters of the tested
quinolines including equilibrium binding constant (Keq) of
quinoline molecule to DNA.
values predict a poor bioavailability for reference drug
doxorubicin and a good bioavailability for quinolines tested
in this study.
However, it can be noted that all 12 quinoline molecules
have low hydrogen bonding ability, which could enhance
the biological membranes permeating capacity, but affect
Table 3 Selectivity Index of the prepared 2-(pyridinyl)quinolines 7–9
Comp.
Selectivity index (SI)a
Type
MCF-7
SKBR-3
PC3
0.3
HeLa
0.3
7a
7b
7c
I
0.6
1.7
2.9
0.8
5.7
1.4
0.1
3.8
1.0
3.7
1.3
62.7
3.3
1.1
0.4
12.9
1.7
6.8
8.4
0.1
2.5
6.6
0.6
1.8
2.9
1.5
I
1.7
4.0
0.7
2.1
In silico studies
I
The bioavailability of tested quinoline compounds was
assessed using ADME (absorption, distribution, metabo-
lism, and excretion) prediction methods (van de Water-
beemd and Gifford 2003; Kerns and Di 2008), mainly,
Lipinski’s Rule of Five analysis (Lipinski et al. 1997;
Lipinski 2004). Calculation of Lipinski’s parameters
including total polar surface area (TPSA) descriptor, which
has been also linked to drug bioavailability, were performed
using the Molinspiration Cheminformatics software. Pre-
dicted ADME properties for the studied compounds are
given in Table 4. According to the data reported, all the
tested compounds comply with this rule. Theoretically,
these compounds should present good passive oral
absorption and differences in their bioactivity cannot be
attributed to this property. It is interesting to note that TPSA
7d
8a
8b
8c
I
0.7
0.9
II
II
II
II
III
III
III
III
108.3
19.5
0.3
9.3
0.4
0.2
8d
9a
9b
9c
0.9
0.6
1.9
4168.1
1.1
1.1
0.7
1.9
9d
DOX
1.3
0.9
1.0
0.7
a Marked in bold parameters indicated a pronounced selectivity. SI was
calculated using following equation: SI = IC50 (control cells)/ IC50
(tumoral cell line).
A selectivity index >1 indicates that the
cytotoxicity on tumoral cells surpassed the one on the healthy non-
tumoral cells
Table 4 Molecular properties
related to Lipinski’s parameters
Comp.
Type MW
Log P TPSA
HA
HD ROTB Lipinski’s rule violations
of obtained 2-(pyridinyl)
7a
I
220.27
3.22
3.88
2.39
4.10
3.44
4.10
2.61
4.31
3.37
4.02
2.53
4.24
0.48
<5
25.78
25.78
25.78
25.78
25.78
25.78
25.78
25.78
25.78
25.78
25.78
25.78
206.08
<140b
2
0
0
0
0
0
0
0
0
0
0
0
0
7
1
1
1
2
1
1
1
2
1
1
1
2
5
0
0
0
0
0
0
0
0
0
0
0
0
1
–
quinoline derivatives 7–9a
7b
I
254.71
238.26
288.27
220.27
254.71
238.26
288.27
220.27
254.71
238.26
288.27
543.50
<500
2
7c
I
2
7d
I
2
8a
II
II
II
II
III
III
III
III
–
2
8b
2
8c
2
8d
2
9a
2
9b
2
9c
2
9d
2
DOX
12
<10
Lipinski’s Rule
<5 <10
a
Obtained data are calculated on http://www.molinspiration.com/cgi-bin/properties, accessed September
2015. TPSA (Å2) Topological Polar Surface Area, Log P logarithm of partition coefficient of the molecule in
an octanol/water system, HA hydrogen bond acceptors, HD hydrogen bond donors, ROTB number of
rotatable bonds, DOX Doxorubicin (Adriamycin)
b
Molecules with a TPSA >140 are thought to have low oral bioavailability

Med Chem Res
Table 5 Distribution coefficient and other related parameters at pH7.4
of 2-(pyridinyl)quinolines 7–9a
the water solubility (WS). It is well known that there are
variations on the lipophilicity of a drug with respect to their
ionic states present in biological environments, where pH is
variable (Kerns and Di 2008).
Comp. Type Log D7.4 pKa
Log WS7.4 HIA (%) Log Keq
7a
7b
7c
I
3.62
3.62
3.12
3.63
3.12
3.64
3.01
3.12
3.14
3.66
3.04
3.48
1.12
3.62 −4.24
3.62 −4.24
3.71 −5.19
4.03 −5.51
3.81 −3.35
4.14 −4.09
4.31 −4.82
3.58 −3.57
3.22 −3.33
3.88 −4.08
2.39 −4.85
4.09 −5.25
11.00 −2.67
82.2
82.2
86.7
88.1
84.2
82.1
87.1
84.3
84.1
82.0
87.0
88.4
7.63
7.72
7.53
7.75
7.66
7.75
7.55
7.77
7.65
7.73
7.54
7.76
6.71
In this context, the distribution coefficient (log D) pro-
vides a better approximation of lipophilicity because it
brings more physiologically relevant outcomes (Bhal et al.
2007). Thus, next step was to predict the distribution
coefficient calculated at physiological pH7.4 (Log D7.4),
together with the acid dissociation constant (pKa). Addi-
tionally, data corresponding to the WS prediction at the
same pH values (LogWS7.4) and the percentage of human
intestinal absorption (HIA) were recollected in the Table 5.
Results obtained indicated that the distribution coefficients
calculated at physiological pH7.4 (Log D7.4) were 3.01–3.66
for all tested quinolines, which means that their solubility is
low and their permeability is high. Thus, oral bioavailability
of these quinolines could be moderate to low. However,
their theoretical HIA resulted be good ranged between 82.1
and 88.4%.
I
I
7d
8a
8b
8c
I
II
II
II
II
III
III
III
III
–
8d
9a
9b
9c
9d
DOX
5.00
a Calculated on https://secure.chemsilico.com/pages/menu.php, accessed
September 2015
DOX Doxorubicin (Adriamycin)
The strength of reversible binding to DNA could be
quantified for any molecule by means of its Keq. The Keq
values could be presented as logarithmic-transformed
values Log Keq. Thus, employing the equation developed
by Portugal (Portugal 2009), Log Keq for the tested quino-
lines were calculated (Table 5).
In order to assess the possible pharmacological proper-
ties of these quinoline derivatives, a toxicity profile eva-
luation was performed employing the Osiris software, as it
may point to the presence of some fragments generally
responsible for the mutagenic, tumorigenic, irritant, or
reproductive effects in these molecules. The tested com-
pounds presented a low in silico toxicity risk profile, similar
to doxorubicin. The Osiris program was also used for cal-
culating the drug-scores of all tested molecules (Table 6).
The results showed that compounds 7–9 have slightly
higher drug-score values compared to doxorubicin. The
in silico toxicity profiles and drug-score data of 2-(3-pyr-
idinyl)quinolines 8a, 8b and 9a make these two compounds
promising leads for future development of safe and efficient
anticancer agents.
According to Portugal’s work, it could be stated that
stronger DNA-molecule binding is responsible for a higher
cell growth inhibition. Analyzing the obtained data, we
observed that the calculated Log Keq (7.45–7.76) were
above those values (4.30–6.89) reported by Portugal for
different active anticancer drugs currently used in che-
motherapy. This means that the tested compounds might
have a major affinity for DNA base pairs when compared to
doxorubicin (Log Keq = 6.71). Regarding the most active
compounds of this group, quinolines 8a, b and 9a, d pre-
sented the following Log Keq parameters: 7.66, 7.75, 7.65,
and 7.76 that were very similar to that of less active com-
pounds. Hence, to our regret, the results obtained could not
confirm the main conclusion of Portugal’ work (Portugal
2009). Possibly, the cell growth inhibition process of this
type of heterocycles is not achieved through DNA inter-
calating as doxorubicin does, or the processes of DNA
binding with these simple quinolines are very specific and
complex (Rescifina et al. 2014; Ur Rehman et al. 2015).
However, comparing structural similarities of the most
active compound 9a with imidazo[1,2-a]pyridines that are
known inhibitors of the enzyme CDK2, a target that is
expressed in HeLa cells (Anderson et al. 2003; Denga
et al. 2014), it should be commented that cell growth
inhibition process at least on HeLa cells could be completed
via CDKs inactivation to inhibit uncontrolled cellular
proliferation.
Conclusion
We have developed a simple synthetic protocol for diverse
C-6 substituted 4-methylquinolines linked with three iso-
meric pyridine subunits. Twelve 2-(pyridinyl)quinoline
derivatives were tested for four cancer cell lines (MCF-7,
SKBR-3, PC3, HeLa) as well as a normal cell line (NHDF).
All prepared molecules resulted to be active against all
tested cell lines. Among them, compounds 8a, 8b, 9a and
9d showed prominent selective cytotoxicity possessing
higher IC50 values compared with the ones of the reference
drug doxorubicin in prostate carcinoma, cervical epithelial
carcinoma and breast carcinoma (no overexpresses the
HER2/c-erb-2 gene), respectively. The cytotoxicity

Med Chem Res
Table 6 In silico toxicities and drug-score profiles of 2-(pyridinyl)
quinolines 7–9
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