Synthesis, crystal structure, cytotoxic and apoptotic activity of 2,4-dichloro-6-methylquinoline on human oral carcinoma cell line

Article abstract of DOI:10.1007/s10870-007-9305-7

The present report describes the synthesis, IR spectra, 3-dimensional structure of the compound 2,4-dichloro-6-methylquinoline and evaluation of its anti-cancer activity using propidium iodide (PI) staining and annexin binding assay techniques. This derivative of quinoline was synthesized from the mixture of p-toluidine and malonic acid and synthesis has been achieved in a one-pot reaction from an aryl amine, malonic acid and phosphorous oxychloride. Crystallographic data reveals that the crystals belong to triclinic crystal system with space group P-1 with the unit cell dimensions of a = 7.14(1) A, b = 11.53(1) A, c = 11.97(1) A and α = 90.18° (10), β = 106.31° (10), γ = 91.07° (10). The in vitro anti-cancer assay indicated that compound has cytotoxic and apoptotic activity on human oral squamous carcinoma (KB) cell line, thus it could be developed as a potent anti-cancer agent. Synthesis, Crystal Structure, Cytotoxic and Apoptotic Activity of 2,4-Dichloro-6-methylquinoline on Human Oral Carcinoma Cell Line R. K. Somvanshia, R. Subashinib, V. Dhanasekaranb, G. Arulprakashb, S. N. Dasc, S. Deya [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10870-007-9305-7

J Chem Crystallogr (2008) 38:381–386
DOI 10.1007/s10870-007-9305-7
ORIGINAL PAPER
Synthesis, Crystal Structure, Cytotoxic and Apoptotic Activity
of 2,4-Dichloro-6-methylquinoline on Human Oral Carcinoma
Cell Line
R. K. Somvanshi Æ R. Subashini Æ V. Dhanasekaran Æ
G. Arulprakash Æ S. N. Das Æ S. Dey
Received: 8 August 2007 / Accepted: 5 November 2007 / Published online: 28 November 2007
Ó Springer Science+Business Media, LLC 2007
Abstract The present report describes the synthesis, IR
spectra, 3-dimensional structure of the compound
Introduction
2
,4-dichloro-6-methylquinoline and evaluation of its anti-
The increase in knowledge of cancer biology has lead to
the development of several new classes of anti-cancer
agents. Quinolines and quinilones are very important in
medicinal chemistry because of their wide occurrence in
several cytotoxic agents [1], natural products [2] and drugs
[3]. These compounds affect mammalian cellular functions
in vitro in several ways; at higher concentrations it inhibits
DNA replication while, at lower concentration it may
affect individual genes [4]. Recent studies have shown their
anti-tumor activity in a variety of human tumor cell either
through arresting the G1, G2/M phase of cell cycle and
cyclin D kinase [5] or by down regulation of Bcl-2 leading
to altered ratio of Bax-Bcl2 that favors apoptosis [6]. It has
been seen that series of quinoline derivatives mainly hal-
ogen derivatives with nitrogen or amino group present in
the aromatic ring exhibit excellent cytotoxic activity [7, 8]
and hence are useful as anti-microbial and/or anticancer
agents.
cancer activity using propidium iodide (PI) staining and
annexin binding assay techniques. This derivative of
quinoline was synthesized from the mixture of p-toluidine
and malonic acid and synthesis has been achieved in a one-
pot reaction from an aryl amine, malonic acid and phos-
phorous oxychloride. Crystallographic data reveals that the
crystals belong to triclinic crystal system with space group
˚
P-1 with the unit cell dimensions of a = 7.14(1) A, b =
˚
˚
1
1
1.53(1) A, c = 11.97(1) A and a = 90.18° (10), b =
06.31° (10), c = 91.07° (10). The in vitro anti-cancer
assay indicated that compound has cytotoxic and apoptotic
activity on human oral squamous carcinoma (KB) cell line,
thus it could be developed as a potent anti-cancer agent.
Keywords Aryl amine Á Malonic acid Á
2
,4-Dichloro-6-methylquinoline Á Crystal structure Á
Flow cytometry Á Apoptosis
One of the major obstacles to effective cancer chemo-
therapy is the existence of multi drug resistance (MDR)
proteins in cancer cells that expel xenobiotics from the
cells via ATP dependent efflux pump [9]. To overcome
MDR, enormous efforts have been made to find an inhib-
itor of the drug efflux pump and various compounds such
as verapamil, cyclosporine, quinodine, tomoxifen, proges-
terone, reserpine, etc have been reported to overcome
MDR in vitro [10] but these have not been successful due
to their dose limiting toxicity. However, being natural
product quinolines derivatives have shown promising use
in MDR resistant tumor cells with lesser side effects. A
clinically approved quinoline derivative drug called
MS209 is used in Japan.
R. K. Somvanshi Á S. Dey (&)
Department of Biophysics, All India Institute of Medical
Sciences, Ansari Nagar, New Delhi 110029, India
e-mail: sharmistha_d@hotmail.com
R. Subashini Á V. Dhanasekaran Á G. Arulprakash
Chemistry Division, School of Science and Humanities,
Vellore Institute of Technology, Vellore 632 014, India
S. N. Das
Department of Biotechnology, All India Institute of Medical
Sciences, New Delhi 110029, India
123

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J Chem Crystallogr (2008) 38:381–386
Although many preparative methods are available in the
temperature (298 K) by slow evaporation. The unit cell
parameters and X-ray intensity data were collected on
CAD4 single crystal diffractometer with Cu Ka radiation.
The unit cell parameters were refined by a least-squares fit
of 25 high angle (25 B h B 40°) reflections. These
reflections were centered individually on the diffractome-
ter. Lorentz and polarization corrections were applied. The
literature [11, 12], it is still challenging to explore new and
simple synthetic methods for quinolines and quinolones;
particularly for the highly functionalized ones [13–15]. The
classical method of quinoline synthesis is Skraup’s proce-
dure [16]. To the best of our knowledge there is no simple
and efficient method for the formation of functionalized
quinolines. In this paper, we report the synthesis, IR, NMR,
X-ray crystal structure of one of new derivative of quino-
line and its anti-cancer activity on human oral squamous
cell carcinoma cell line.
3
crystal dimensions were (0.4 9 0.3 9 0.2 mm ). The
structure was solved with direct methods using the program
SHELXS-97 [17]. The coordinates of non-hydrogen atoms
were refined anisotropically using program SHELXL-97
[
18]. However, the temperature factors for hydrogen atoms
were not refined. The final R-factor for 3,109 observed
reflections [I C 2r(I)] was 0.057. The atomic scattering
factors used in these calculations were those of Cromer and
Mann [19] for non-hydrogen atoms and Steward, Davidson
and Simpson [20] for hydrogen atoms. The final atomic and
positional coordinates are deposited to the Cambridge
Crystallographic Data Centre with the deposition number
CCDC 606999.
Materials and Methods
Synthesis of 2,4-Dichloro-6-methylquinoline (1)
A mixture of p-toludine (2.14 g, 0.02 mole), malonic acid
(
2.08 g, 0.02 mole) and phosphorous oxychloride (50 ml)
was refluxed under dry condition for 15 h in a steam bath.
The solution was cooled to room temperature and poured
over crushed ice carefully and allowed to stand overnight.
The solid settled was filtered to dryness and purified over a
column of silica gel (60–120 mesh; 50 g) eluting with
Petroleum Ether–benzene (4:1) to give 2,4-dichloro-6-
methylquinoline (Fig. 1). The product was re-crystallized
from benzene which melted at 95 °C in 72% yield.
Evaluation of Cytotoxic and Apoptotic Activity
The compound was dissolved in 1 ml of methanol, recon-
stituted in tissue culture media (RPMI 1640) and used in all
the experiments. Control experiments were done with tis-
sue culture media containing similar quantity of vehicle.
Spectral Conformation of 2,4-Dichloro-6-
methylquinoline (1)
Flow Cytometry
In order to confirm the synthesis of the title compound IR
spectra was recorded on Thermo Nicolet Model Avatar 330
FT-IR spectrometer using KBr disc.
Anti-tumor activity of the compound was assessed by flow
cytometry on Human oral squamous cell carcinoma (KB)
cell line (National Cell Science Centre, Pune, India)
established in tissue culture. Tumor cell cytotoxicity was
assayed by Propidium Iodide (PI) staining that intercalates
with DNA of the dead cells.
1
H-NMR was recorded on Varian AMX (400 MHz)
instrument using TMS as an internal reference and CDCl3
as solvent. The mass spectra were recorded on Jeol JMS
3
00 mass spectrometer.
6
Briefly, 2910 tumor cells/ml was cultured in 24 well
tissue culture plates in duplicate at 37 °C in CO incubator
2
with different concentrations (5, 10, 15, 20 and 25 lg/ml)
of the compound for 1 h. The control wells received equal
amount of tissue culture medium. The tumor cells were
harvested after termination of the culture, washed twice
with Phosphate Buffered Saline (PBS pH 7.2) and labeled
with PI for 30 min at room temperature. At least 10,000
events were acquired in flow cytometer (BD LSR II, San
Diego, California, USA) and the data was analyzed using
FACS DIVA software. The results were expressed as
percent cell survival.
Crystal Structure Determination
,4-Dichloro-6-methylquinoline was crystallized from its
2
solution in 80:20 acetone–water mixture at room
Cl
Malonic acid H C
H3C
3
NH2
POCl3
N
1
Cl
Apoptotic activity of the compound was assessed by
annexin binding assay using flow cytometry technique
[21]. Briefly, the cells were treated with compound as
Fig. 1 Reaction scheme
1
23

J Chem Crystallogr (2008) 38:381–386
383
above. After harvesting, cells were washed twice with PBS
Table 1 Crystal data and structure refinement for 2,4-dichloro-6-
methylquinoline
(
pH 7.2) and the cells pellet was treated with annexin V,
FITC at room temperature for 45 min, washed twice with
PBS and re-suspended in 1 ml of 1% paraformaldehyde.
At least 10,000 events were acquired in flow cytometer.
Frequency of apoptotic cells was calculated using FACS
DIVA software and results were expressed as percentage.
All experiments were conducted in triplicates.
Empirical formula
Formula weight
C
10
H
8
Cl
2
N
213.07
Temperature
293(2) K
Radiation type
Cu Ka
˚
1.54178 A
Wavelength
Crystal system, space group
Unit cell dimensions
Triclinic, P-1
˚
a = 7.14 (1) A
˚
b = 11.53 (1) A
˚
c = 11.97 (1) A
Results and Discussion
a = 90.18° (10)
b = 106.31° (10)
c = 91.07° (10)
Synthesis of Compound as a 2,4-Dichloro-6-
methylquinoline
3
˚
Volume
945.44(17) A
3
Z, Calculated density
Absorption coefficient
Crystal size
4, 1.49 mg/m
-
5.736 mm
IR spectra recorded on Thermo Nicolet Model Avatar 330
FT-IR spectrometer using KBr disc showed absorptions at
1
3
-
1
Vmax = 1,650 cm corresponding to (C=N), 1,060 cm
-1
.2 9 .4 9 .2 mm
3.83°–69.88°
-8 B h B 8,
-
1
corresponding to (C–Cl) and 3,010 cm (CH).
h range for data collection
Limiting indices
NMR was obtained in ppm: d 2.5 (s, 3H, C –CH ); d
6
3
-
14 B k B 14,
7
8
.64–7.98 (m, 2H, C –H and C –H); d 8.3 (s, 1H, C –H); d
7
8
5
0 B l B 14
3,463/3,109
82.6%
.9 (s, 1H, C –H).
3
Reflections collected/unique
Completeness to h = 69.88°
Refinement method
Mass spectra recorded a molecular ion peak at m/z 212
M+), 214 (M + 2) and 216 (M + 4) confirmed the com-
(
2
Full-matrix least-squares on F
pound as 2,4-dichloro-6-methylquinoline.
2
Goodness-of-fit on F
1.070
Final R indices [I [ 2r(I)]
R
1
= 0.057, wR
2
= 0.1704
CCDC No
CCDC 606999
Molecular Dimensions and Crystal Packing
The relevant crystal data of the compound, bond length and
bond angles are given in Tables 1 and 2. The structure was
solved by direct methods and refined by full matrix least-
square. The final R factor was 0.057. An ORTEP plot of the
molecule is shown in Fig. 2. The bond distances, N1–C2
and N1–C9, are shorter than the normally expected values
and similar shortening is also observed in quinoline ring
systems of structure like 8-hydroxyquinoline N-oxide [22],
copper 8-hydroxyquinolinate [23] and zinc-hydroxyquino-
linate dehydrate [24].
In-vitro-assay of Anti-cancer Activity
Cytotoxic effect of the compound was tested on human oral
carcinoma (KB) cell line in 1 h culture at 37 °C and the
results are shown in Fig 4. The tumor cells were treated
with different doses (5, 10, 15, 20 and 25 lg/ml) of the
compound and percent cytotoxicity was determined by
Propidium Iodide uptake after termination of the culture.
The compound showed 27% toxicity at 25 lg/ml dose 1 h
of culture period. Time kinetics as determined using 25 lg/
ml dose showed almost 40% cell death (cytotoxicity) at 4 h
treatment (Fig 5). Apoptotic activity of a compound was
assayed using optimum (25 lg/ml cytotoxic dose of the
compound. Almost 60% of the tumor cells showed binding
of annexin V at 4 h treatment period (Fig 6). The results
suggest that 2,4-dichloro-6-methylquinoline is a potent anti
tumor agent and it is able to induce apoptosis in the human
oral carcinoma (KB) cells.
The low value for the bond length [C2a–Cl1a=],
˚ ˚
1
.738(3) A and [C2b–Cl1b=], 1.737(2) A and the exocy-
clic bond angle [N1b–C2b–Cl1b=], 116.47(1)° shows that
the close packed bonding between N1, C2, and Cl1. Tor-
sion angles: [C11–C6–C7–C8=], 179.61° for molecule a
and 179.83° for molecule b and [C11–C6–C5–C10=],
1
79.43° for molecule a and 179.71° for molecule b indi-
cates an extended conformation for the methyl group
attached with the ring. The packing of the compound in
The exact mechanism of anti tumor activity of 2,4-
dichloro-6,methylquinoline is not known. The previous
studies reported that the substitution at C7 position of
unit cell is illustrated in Fig. 3. Hydrogen bond of type
˚
[
C–HÁÁÁCl=], (2.77 A) is observed in the crystal packing for
the molecule [25].
quinoline molecule by aliphatic group, OH or OCH and by
3
123

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J Chem Crystallogr (2008) 38:381–386
˚
Table 2 Bond lengths [A] and angles [°] for 2,4-dichloro-6-
methylquinoline
Table 2 continued
C(5B)–C(10B)–C(9B)
C(5B)–C(10B)–C(4B)
C(9B)–C(10B)–C(4B)
C(7B)–C(8B)–C(9B)
N(1B)–C(2B)–C(3B)
N(1B)–C(2B)–Cl(1B)
C(3B)–C(2B)–Cl(1B)
N(1B)–C(9B)–C(10B)
N(1B)–C(9B)–C(8B)
C(10B)–C(9B)–C(8B)
C(5B)–C(6B)–C(7B)
C(5B)–C(6B)–C(11B)
C(7B)–C(6B)–C(11B)
C(4B)–C(3B)–C(2B)
C(3B)–C(4B)–C(10B)
C(3B)–C(4B)–Cl(2B)
C(10B)–C(4B)–Cl(2B)
C(8B)–C(7B)–C(6B)
C(6B)–C(5B)–C(10B)
119.8(2)
124.7(3)
115.4(2)
120.9(3)
125.9(3)
116.5(2)
117.6(2)
123.4(2)
118.7(2)
117.9(3)
119.2(3)
120.9(3)
119.9(3)
117.1(3)
121.2(2)
119.7(2)
119.1(2)
121.3(3)
120.8(3)
Cl(2A)–C(4A)
1.726(3)
1.738(3)
1.289(4)
1.362(3)
1.409(4)
1.414(4)
1.420(4)
1.367(4)
1.404(4)
1.366(4)
1.505(4)
1.409(4)
1.357(4)
1.398(4)
117.3(2)
125.0(2)
119.2(2)
115.8(2)
121.2(3)
119.0(2)
120.9(3)
120.1(3)
118.9(2)
122.8(2)
118.3(2)
120.8(3)
116.8(3)
121.5(2)
121.2(3)
119.3(2)
119.5(2)
126.2(3)
117.1(2)
116.8(2)
1.720(3)
1.737(3)
1.296(4)
1.363(4)
1.404(4)
1.411(4)
1.423(4)
1.361(5)
1.412(4)
1.501(4)
1.395(4)
1.374(4)
1.394(5)
1.352(4)
116.9(2)
Cl(1A)–C(2A)
N(1A)–C(2A)
N(1A)–C(9A)
C(10A)–C(5A)
C(10A)–C(4A)
C(10A)–C(9A)
C(7A)–C(8A)
C(7A)–C(6A)
C(6A)–C(5A)
C(6A)–C(11A)
C(9A)–C(8A)
C(3A)–C(4A)
C(3A)–C(2A)
C(2A)–N(1A)–C(9A)
C(5A)–C(10A)–C(4A)
C(5A)–C(10A)–C(9A)
C(4A)–C(10A)–C(9A)
C(8A)–C(7A)–C(6A)
C(5A)–C(6A)–C(7A)
C(5A)–C(6A)–C(11A)
C(7A)–C(6A)–C(11A)
N(1A)–C(9A)–C(8A)
N(1A)–C(9A)–C(10A)
C(8A)–C(9A)–C(10A)
C(7A)–C(8A)–C(9A)
C(4A)–C(3A)–C(2A)
C(6A)–C(5A)–C(10A)
C(3A)–C(4A)–C(10A)
C(3A)–C(4A)–Cl(2A)
C(10A)–C(4A)–Cl(2A)
N(1A)–C(2A)–C(3A)
N(1A)–C(2A)–Cl(1A)
C(3A)–C(2A)–Cl(1A)
Cl(2B)–C(4B)
Fig. 2 Perspective view of the molecule 2,4-dichloro-6-methylquin-
oline at 50% ellipsoidal probability
Cl(1B)–C(2B)
N(1B)–C(2B)
N(1B)–C(9B)
C(10B)–C(5B)
the presence of halogens in the ring with Nitrogen inhibits
DNA gyrase and topoisomerase during DNA replication.
Such inhibition leads to the termination of DNA replication
and eventually cell death [8, 26, 27]. Earlier studies show
that 4-quinolones treatment down regulates Bcl-2 expres-
sion in tumor cells consequently leading to an altered ratio
of Bax:Bcl-2. Such alteration triggers apoptotic cell death
in the tumor cells 6. It seems reasonable to presume that
substitution of aliphatic group at C6 position and chlorine
at 2 and 4 positions may provide similar mechanism of
action to the present compound.
C(10B)–C(9B)
C(10B)–C(4B)
C(8B)–C(7B)
C(8B)–C(9B)
C(11B)–C(6B)
C(2B)–C(3B)
C(6B)–C(5B)
C(6B)–C(7B)
C(3B)–C(4B)
C(2B)–N(1B)–C(9B)
1
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J Chem Crystallogr (2008) 38:381–386
385
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
Treated
Untreated Control
0
2
4
6
8
Time (Hrs)
Fig. 5 In-vitro cytotoxicity of the quinoline on KB cell line. Tumor
cell lines were pulsed with 25 lg/ml quinoline for 0, 2, 4, 6 and 8 h,
labeled with PI and acquired in flow cytometer. The result represents
averages of three experiments
6
5
4
0
0
0
Fig. 3 Perspective view of the crystal packing of 2,4-dichloro-6-
methylquinoline
treated
Untreated Control
79
78
77
76
75
74
73
72
71
70
30
20
10
0
0
2
4
6
8
Time (Hrs)
Fig. 6 In-vitro annexin binding assay showing frequency of apopto-
sis in tumor cell lines treated with 25 lg/ml quinoline for different
time intervals. The cells were labeled with annexin V, FITC and
acquired in flow cytometer. The results represent averages of three
experiments
5
10
15
20
25
Dose ( µg/ml)
Fig. 4 In-vitro cytotoxic effect of different doses of quinoline on
human oral carcinoma (KB) cell line. Tumor cell lines were pulsed
with different concentrations of the PI and acquired in flow cytometer.
The result represents averages of three experiments
induces cytotoxicity and apoptosis in mammalian tumor
cell line; hence series of compound with substitution at
position 6 can be synthesized and tested for their anti-
cancer activity.
Conclusions
The compound 2,4-dichloro-6,methylquinoline was syn-
thesized, characterized and evaluated for the anti-cancer
activity on oral carcinoma cell line. This compound con-
tains aliphatic group at C6 position in quinoline molecule
instead of C7 position and chlorine at 2 and 4 positions
which are important for cytotoxic activity of eukaryotic
cells. The cytotoxic activity of the quinolines with substi-
tution at position 6 has been determined successfully for
the first time. It seems that such substitution of aliphatic
group at C6 position as well as halogens at 2 and 4 position
Supplementary Material
CCDC (606999) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free
of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.
html or from the Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge CB2 1EZ, United Kingdom;
Fax: (44) 1223-336-033; e-mail: deposit@cdc.cam.ac.uk
123

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J Chem Crystallogr (2008) 38:381–386
Acknowledgements Authors acknowledge financial support from
the Department of Science and Technology and Council of Scientific
and Industrial Research, Government of India. Author thanks the
service rendered by ‘‘Technology Business Incubator’’ at Vellore
Institute of Technology, Vellore, ‘‘Sophisticated Instrumentation
facility’’ at Indian Institute of Science, Bangalore and ‘‘Central Drug
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