Synthesis and characterization of some novel quinoxaline-2, 3-dione derivatives: A preliminary investigation on their activity against a human epithelial carcinoma cell line

Article abstract of DOI:10.2174/157018011794839385

Quinoxaline-2, 3-dione obtained from cyclocondensation reaction of o-phenylene diamine with oxalic acid, was reacted with chlorosulphonic acid under cold condition followed by a reaction with various benzimidazoles to give 2, 3- dioxo-1, 2, 3, 4-tetrahydroquinoxaline-6-sulphonyl benzimidazoles in satisfactory yield. Their structures were confirmed using ¹H NMR, IR and mass analysis. Cytotoxicity of these derivatives were evaluated by growth inhibition of HEp-2 cells in vitro. The preliminary bioassay indicated that these compounds showed moderate cytotoxicity.

Full text of DOI:10.2174/157018011794839385

Letters in Drug Design & Discovery, 2011, 8, 317-320
317
Synthesis and Characterization of some Novel Quinoxaline-2, 3-Dione De-
rivatives: A Preliminary Investigation on their Activity Against a Human
Epithelial Carcinoma Cell Line
,
1
1
,2
1
Selvaraj Jubie* , Rajamanickam Gayathri , Ammayappan Rajam Srividya* , Rajagopal Kalirajan ,
1
1
1
Prabakaran Prabitha , Sundaram Sankar and Kannan Elango
1
2
Department of Pharmaceutical Chemistry; Department of Pharmaceutical Biotechnology, J.S.S. College of Pharmacy,
Off campus - JSS University, Mysore
Received August 05, 2010: Revised January 31, 2011: Accepted February 08, 2011
Abstract: Quinoxaline-2, 3-dione obtained from cyclocondensation reaction of o-phenylene diamine with oxalic acid,
was reacted with chlorosulphonic acid under cold condition followed by a reaction with various benzimidazoles to give 2,
3
- dioxo-1, 2, 3, 4-tetrahydroquinoxaline-6-sulphonyl benzimidazoles in satisfactory yield. Their structures were con-
1
firmed using H NMR, IR and mass analysis. Cytotoxicity of these derivatives were evaluated by growth inhibition of
HEp-2 cells in vitro. The preliminary bioassay indicated that these compounds showed moderate cytotoxicity.
Keywords: Benzimidazole, Cytotoxicity, Quinoxaline-2,3-dione, SRB method, Electrophilic substitution, Human epithelial
carcinoma cell line.
1
. INTRODUCTION
2. MATERIALS AND METHODS
Quinoxaline-2,3-dione derivatives are important classes
All the solvents and materials were reagent grade and pu-
rified as required. Melting points were taken in open glass
capillary using Veego VMP-1 melting point apparatus and
are uncorrected. IR spectra were recorded on Shimadzu FT-
IR spectrometer model using KBr discs. The NMR spec-
of nitrogen containing heterocycles, as they constitute useful
intermediates in organic synthesis [1]. In particular quinox-
aline scaffolds were found as a core unit in a number of bio-
logically active compounds. Quinoxaline including their
fused ring derivatives display diverse pharmacological ac-
tivities such as neuroprotective agents, antifungal, antibacte-
rial, radio protective, anticonvulsant, antimalarial, antican-
cer, potent antithrombotic, analgesic, anti-inflammatory,
antiglaucoma, antiparasite, antituberculosis, hypoglycemic,
antiviral, anti-HIV, anthelmintic activities, anti depressant,
NMDA receptor antagonist and antimalarial activities [1-5].
Also they bear a strong resemblance to well characterized
drugs that act by intercalation. Benzimidazole derivatives
have shown a wide spectrum of pharmacological activities
such as antiviral effects, antagonistic and inhibitory activi-
ties, potential antitumor agents, a treatment for interstitial
cystitis, anthelmintic agents in veterinarian medicine and in
diverse human therapeutic areas such as treatment of ulcers
and antihistaminics [6]. Many examples have been demon-
strated that incorporation of benzimidazoles in structure of
other heterocyclic compounds often resulted in the im-
provement of activities. To develop a convenient pathway in
good agreement with rapid pharmacomodulation, we were
interested in using electophilic substitution. Electrophilic
substitution of aromatic ring of quinoxaline usually occurs at
position 6. Thus we synthesized a vareity of new derivatives
of our lead compound and measured their in-vitro potency
towards a human epithelial carcinoma cell line.
6
trums (DMSO-d ) were recorded on Bruker DRX-300 spec-
trometer with TMS as an internal standard. The mass spectra
were measured on a Shimadzu LCMS 2010A spectrometer.
Elemental analysis was performed on a varion EL III ele-
mental analyzer. Reactions were monitored by thin layer
chromatography (TLC).
2
.1. Synthesis of Quinoxaline-2, 3-dione (2) Under
Thermal Conditions
A powdered mixture of oxalic acid dihydrate (0.01 mole,
1
.26 g) and o-phenylene diamine (0.01 mole, 1.0814 g) was
refluxed using an oil bath for 1.5 hours and cooled. The
product that separated was filtered and washed with water
and crystallized with 5% NaOH/dil HCl to give the colour-
less crystals.
Under Microwave Irradiation
A powdered mixture of oxalic acid dihydrate (0.01 mole,
1
.26 g) and o-phenylene diamine (0.01 mole, 1.0814 g) was
put in an open beaker and 1 ml of water added and mixed
thoroughly. The mixture was irradiated in a catalyst micro-
wave system at an emitted power of 400 W for 3 min. 100
mL of water was added, followed by further irradiation for 1
min to give a clear solution and then left to stand at room
temperature. The product obtained was filtered, washed with
water and crystallized with 5% NaOH/ dil HCl to give col-
0
ourless crystals 2, (88 %), mp >340 C; R
f
(chloro-
*
Address correspondence to these authors at the Department of Pharmaceu-
tical Chemistry, J. S. S. College of Pharmacy, Rocklands, Ooty-643 001,
Tel: 0423-2443393; Fax: 0423-2442937; E-mail: jubiejawahar@gmail.com
form/methanol 9:1) 0.52; IR (KBr): 1708.9 & 1681 (C=O),
1
1
247 (C-N), 3344 (N-H), 854 (Ar C-H), 1593 (C=C); H
Tel: 0423-2443393; Fax: 0423-2442937;
E-mail: pharmarsrividya@jsscpooty.org
NMR (DMSO-d 300 MHZ): ꢀ 11.7 (s, 2H, NH), 7 (m, 4H,
Ar-H); ms (m/z): M+ calculated 162.15, found 161.98.
6
,
1
570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

3
18 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 4
Jubie et al.
2
.2. Synthesis of 2, 3-dioxo-1, 2, 3, 4-tetrahydro-
v/v,10 mL) was added at room temperature with a solution
of potassium dichromate in aqueous acetic acid (5%v/v,10
mL), and the mixture was stirred for 15 min. The separated
product was filtered, washed with water and dried. The dried
product was recrystallized with ethanol. This compund was
quinoxaline-6- sulphonyl Chloride (3)
Quinoxaline -2, 3- dione (0.03 mole, 4.86 g) was added
portion wise to chlorosulphonic acid (0.27 mole, 25mL) at
0
tion solution was added to ice-water and then filtered. The
resulting solid was washed with water and recrystallized
from dry toluene-acetone mixture to give white crystals 3,
o
C. After being stirred 10 h at room temperature, the reac-
o
obtained as pink crystals, (70%), mp 186-190 C, IR (KBr):
1
750 (C=O), 3055.82 (N-H), 2960.69 (C=C), 1425.70 (C-N);
1
H NMR (DMSO-d6,300 MHZ): ꢀ 5.3(d, 1H, NH), 6.5(m,
4H, Ar-H), 2.5 (s, 3H, CH ).
0
3
(
(
5
78%), mp >340 C; R
KBr):1757.2 & 1720 (C=O), 1163.1 (SO
f
(chloroform/methanol 9:1) 0.50; IR
Cl), 1373.3 (SO1 ),
99.8 (Cl), 636.5 (C-S), 835.2 (Ar C-H), 3400 (N-H); H
NMR (DMSO-d6,300 MHZ): ꢀ 11.2 (s, 2H, NH), 7.4-7.8 (m,
2
2
2.4. General procedure for the synthesis of compounds
(4)
4
H, Ar-H); ms (m/z): M+ calculated 260.65, found 261.98
Compound 3 (0.01 mole, 2.6 g) was dissolved in dry di-
methyl formamide (20 mL) followed by addition of substi-
tuted benzimidazoles (0.02 mole) and the resulting mixture
was kept under stirring at room temperature for 10 h. The
resulting mixture was then poured into water (50 mL) to give
a foamy precipitate and recrystallized from hot DMF with
cooling and drop wise addition of water.
2
.3. General Procedure for the Synthesis of Compounds
(
a-d). Under Microwave Irradiation
To O-Phenylene diamine (0.0457 mole) was added with
mono carboxylic acid (0.0457 mole). The mixture was irra-
diated in a catalyst microwave system at an emitted power of
3
50W for 20 min. The resulting mixture was allowed to cool
2.4.1.
2,3-dioxo
-1,2,3,4-tetrahydroquinoxaline-6-
and rendered alkaline by addition of 10% NaOH, as deter-
mined using litmus paper. The precipitate that formed was
filtered and washed with water and recrystallized from hot
water. The reaction was monitored by TLC (Chloroform:
methanol 9:1).
sulphonylbenzimidazole (4a)
This compund was obtained as a white solid, (54%),
o
m.p>360 C, IR (KBr):1716.10 &1678.1 (C=O), 1392.6
(
(
SO
2
), 704.4 (C-S), 854.4 (Ar C-H), 3344.6 (N-H), 1614.4
1
C=N), 1247 (C-N); H NMR (DMSO-d
6
,300 MHZ): ꢀ 11.8
2
.3.1. 1H-benzo[d]imidazole (a)
(s, 2H, NH), 7.05 -7.14 (m, 8H, Ar-H), 3.4 (d, 1H, NH Ben-
zimidazole), 4.4 (m, 1H, CH Benzimidazole); ms (m/z): M+
calculated 344.35, found 345.28. Anal. Calcd. for
C H N O S: C,52.3; H,3.51; N,16.2; O,18.59; S,9.31.
15 12 4 4
Found: C,52.33; H,3.49; N,16.23; O,18.19; S,9.35.
This compund was obtained as a pink crystal, (48%), mp
o
1
1
98-200 C; IR (KBr):3061.83, 3113.21 (Ar C-H), 1458,
1
477.52 (C-N), 1300.07 (tertiary amine), 3061.83 (N-H); H
NMR (DMSO-d6,300 MHZ): ꢀ 4.5 (d, 1H, NH), 7.5 (m, 4H,
Ar-H), 8.2 (s, 1H, CH).
2.4.2. 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulphonyl
(
2-methyl)benzimidazole (4b)
2
.3.2. 2-methyl-1H-benzo[d]-imidazole (b)
This compund was obtained as a white solid, (72%),
m.p.>360 C, IR (KBr):1681 (C=O), 1390.7 (SO2), 638.4 (C-
S), 854.4 (Ar C-H), 3342 (N-H), 1614.4 (C=N), 1247 (C-N);
This compund was obtained as pink crystal, (56%), mp
o
o
1
3
69-174 C, IR (KBr): 1425.44 (C-N), 804 (Ar C-H),
1
053.42 (N-H); H NMR (DMSO-d6,300 MHZ): ꢀ 4.8(d, 1H,
1
H NMR (DMSO-d
m, 8H, Ar-H), 3.4 (d, 1H, NH Benzimidazole), 2.4 (m, 1H,
CH Benzimidazole), 1.5 (d, 3H, CH ); MS (m/z): M+ calcu-
6
,300 MHZ): ꢀ 11.8 (s, 2H, NH), 7.14
NH), 6.5 (m, 4H, Ar-H), 2.3 (d, 3H, CH
3
).
(
2
.3.3. 2-ethyl-1H-benzo[d]-imidazole (c)
3
14 4 4
lated 358.37, found 357.75. Anal. Calcd. for C16H N O S:
C,56.13; H,4.12; N, 16.36;O, 14.02; S,9.37. Found: C,56.08;
H, 4.15; N,16.38;O,14.01; S,9.32.
This compund was obtained as pink crystal, (48%),mp
o
2
13-215 C, IR (KBr): 1425.44 (C-N), 1271.13 (tertiary ami-
1
ne), 3053.42 (N-H); H NMR (DMSO-d
6
,300 MHZ): ꢀ 5.1(d,
), 2.5 (d, 3H,
1
H, NH), 6.3(m, 4H, Ar-H), 2.7 (m, 2H, CH
2
2.4.3.
2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-
CH ).
3
sulphonyl(2-ethyl)benzimidazole(4c)
2
.3.4. 2-propyl-1H-benzo[d]-imidazole (d)
This compund was obtained as a white solid, (72%), m.p
C >360, IR (KBr):1708.9 & 1681 (C=O), 1390.7 (SO ),
2
o
This compund was obtained as pink crystal, (44%),mp
o
638.4 (C-S), 854.4 (Ar C-H), 3342 (N-H), 1614.4 (C=N),
1
1
80-184 C, IR (KBr): 3053.42 (N-H), 2964.69 (C=C),
1
1
1247 (C-N); H NMR (DMSO-d , 300 MHZ): ꢀ 11.7 (s, 2H,
6
421.58 (C-N); H NMR (DMSO-d6,300 MHZ): ꢀ 5.3(d, 1H,
NH quinoxaline), 7 (m, 4H, Ar-H benzimidazole), 7.1 (m,
2 2
NH), 6.5(m, 4H, Ar-H), 2.7 (m, 2H, CH ), 2.1(m, 2H, CH ),
3
H, Ar- H in quinoxaline), 3.3(d, 1H, NH), 2.8 (m, 1H, CH
benzimidazole), 2.4 (m, 2H, CH ), 2.4 (t, 3H, CH ); MS
m/z): M+ calculated 372.4, found 371..75. Anal. Calcd. for
S: C, 54.83; H, 4.33; N, 15.04; O,17.09; S, 8.61.
Found: C, 54.81; H, 4.32; N, 15.0; O, 17.11; S, 8.65.
1
.5 (t, 3H, CH
.3.5. Synthesis of -2- acetyl benzimidazole (e)
O-Phenylene diamine (0.1 mole, 10 g) with lactic acid
0.1 mole, 9 g) was refluxed in a microwave oven at an in-
3
).
2
3
2
(
17 16 4 4
C H N 0
(
tensity of 20% (140 watts) for 7 min. The mixture was
cooled and 10% NaOH was added until to get basic as de-
termined using litmus paper. Then the precipitate was fil-
tered, and the dried product was recrystallized with hot wa-
ter. To the precipitate (10 mmole) in aqueous acetic acid (5%
2
.4.4. 2,3-dioxo-1,2,3, 4-tetrahydroquinoxaline-6-sulphonyl
(
2-propyl) benzimidazole(4d)
This compound was obtained as a white solid, (75.5%),
o
m.p>360 C, IR (KBr): 1718.5 & 1685 (C=O), 1385.7 (SO2)
,

Synthesis and Characterization of Quinoxaline Derivatives
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 4 319
6
1
47.4 (C-S), 832.1 (Ar C-H), 3448 (N-H), 1624.5 (C=N),
247 (C-N); H NMR (DMSO-d , 300 MHZ: ꢀ 11.7 (s, 2H,
6
essential medium supplemented with 2 mmol L-glutamine,
10% Fetal Bovine Serum, Penicillin (100 ꢁg/mL), Strepto-
mycin (100 ꢁg/mL) and Amphotericin B (5 ꢁg/mL) and the
cells were maintained at 37˚C in a humidified atmosphere
1
NH quinoxaline), 7.5 (m, 4H, Ar-H benzimidazole), 6.7 (m,
H, Ar-H quinoxaline), 3.7(d, 1H, NH), 2.8 (m, 1H, CH
benzimidazole), 2.2 (m, 2H, CH ), 2.4 (m, 2H, CH ),1.9 (t,
H, CH ); MS (m/z): M+ calculated 386.42, found 386.15.
Anal. Calcd. for C18 S: C,55.95; H, 4.70; N, 14.50;
3
2
2
2
with 5% CO and subculture twice a week. The total cell
3
3
protein content was determined by sulphoradamine B (SRB)
assay [7]. The monolayer cell culture was trypsinized and the
18 4 4
H N O
5
O,16.56; S, 8.30. Found: C,55.91; H,4.72; N,14.54; O,16.60;
S,8.31.
cell count adjusted to 1.0 x 10 cell/mL using medium
(MEM) containing 10% new born calf serum. To each well
of the 96 well microtitre plate, 0.1ml of diluted cell suspen-
sion (approximately 10,000 cells) was added. After 24 h,
when a partial monolayer was formed, the supernatant was
flicked off, the monolayer was washed once and 100ꢁL of
medium and the culture was exposed to different concentra-
tions of drug in microtitre plates. The plates were then incu-
2
.4.5. 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulphonyl
(
2-acetyl) benzimidazole(4e)
This compund was obtained as a white solid, (68%),
o
m.p>360 C, IR (KBr): 1728.1 & 1690 (C=O), 1387.7 (SO
6
1
2
),
43.2 (C-S), 835.1 (Ar C-H), 3450 (N-H), 1620.5 (C=N),
1
250 (C-N); H NMR (DMSO-d
6
, 300 MHZ): ꢀ 12.1 (s, 2H,
bated at 37°C for 3 days in 5% CO atmosphere, and micro-
2
NH quinoxaline), 7.6 (m, 4H, Ar-H benzimidazole), 6.9 (m,
H, Ar-H quinoxaline), 4.1 (d, 1H, NH), 3 (m, 1H, CH ben-
zimidazole), 1.4 (t, 3H, CH ); MS (m/z): M+ calculated
86.38, found 388.45. Anal. Calcd. for C17 S: C,
2.84; H, 3.65; N,14.50; O, 20.70; S, 8.30. Found: C,52.86;
scopic examination was carried out and observations re-
corded every 24 h. After 72 h, 25ꢁL of 50% trichloro acetic
3
3
acid was added to the wells gently such that it forms a thin
layer over the drug solution to give an overall concentration
of 10%.The plates were incubated at 4°C for one hour. The
culture plates were flicked and washed five times with tap
water to remove traces of medium, drug and serum, and were
then air-dried. The air-dried plates were stained with SRB
for 30 min. The unbound dye was then removed by rapidly
washing four times with 1% acetic acid. The plates were
then air-dried.100ꢁL of 10mM tris base was then added to
3
5
14 4 5
H N O
H, 3.69; N,14.48; O,20.71; S,8.32.
2
.5. Biological Assay
Cell cultures were obtained from National centre for cell
sciences, Puna. HEp-2 cells were grown in Earl’s Minimal
H
N
H
NH2
O
O
ClO2S
N
O
O
COOH
+
COOH
ClSO H
3
NH2
N
H
N
H
o-Phenylene diamine
Oxalic acid
2
,3-dioxo-1,2,34-tetrahydro
Quinoxaline-2,3-dione
quinoxaline-6-sulphonyl chloride
1
2
3
+
NH2
H
N
R
N
+
R-COOH
NH2
o-Phenylene diamine monocarboxylic acid
Benzimidazole
a-e
1
H
N
O2S
N
O
O
HN
R
N
H
2
,3-dioxo-1,2,3,4-tetrahydro
quinoxaline-6-sulfonyl benzimidazole
4a-e
Compound
R
4
4
4
4
4
a
b
c
H
-CH
3
-CH
2
CH
3
d
e
-CH CH
2
2
CH
3
-COCH
3
Scheme 1.

3
20 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 4
Jubie et al.
the wells to solubilize the dye. The plates were shaken vig-
orously for 5 min. The absorbance was measured using mi-
cro plate reader at a wavelength of 540 nm.The percentage
growth inhibition was calculated using the formula below:
In conclusion, this type of quinoxaline compound bears a
strong resemblance to well-characterised drugs that bind to
DNA by intercalation. In general, three major aspects of the
binding of drugs to DNA maybe expected to influence their
biological activity: (i) mode of interaction with the double
helix, (ii) sequence specificity of binding, and (iii) kinetics
of association/dissociation. Compounds 4a-e represents an
attractive model for theoretical and experimental study of
intercalators and their medical applications because of the
diversity in their available forms. Because of the mild reac-
tion condition, good yields, easily accessible starting mate-
rial and straight forward product isolation, we think that the
versatile synthetic approach discussed here in many cases
compares favorably with other existing methods. Moreover,
given the current interest in drug targeting to harmful genes
through development of molecules that recognize specific
DNA sites, we have developed the efficient synthesis of 2,3-
dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulphonyl benzimida-
zole derivatives via electophilic substitution. As a first step
towards studying possible interactions of 4a-e with DNA,
the compounds were screened for cytotoxicity activity
against HEp-2 cells by determination of CTC value by SRB
Mean OD of individual test group X 100
%
Growth inhibition =
Mean OD of control group
3
3
. RESULTS AND DISCUSSION
.1. Synthesis
The o-phenylene diamine 1 reacted with oxalic acid to
give compound 2, quinoxaline-2,3-dione by both conven-
tional and microwave oven methods, The resulting product
was reacted with chlorosulphonic acid to provide compound
3
by the conventional method. In the next step different sub-
stituted benzimidazoles (a-e) were prepared by cycloconden-
sation reaction of o-phenylene diamine with different mono-
carboxylic acids by the microwave oven method. These ben-
zimidazoles were incorporated easily to the compound 3
through the sulphonyl group to give the corresponding com-
pounds, 4a-e by substitution in satisfactory yields at room
temperature. The synthesized compounds were purified by
recrystallization with appropriate solvents. Further the struc-
tures of all the compounds were confirmed by spectral
5
0
assay. The preliminary bioassay indicated that all the com-
pounds showed moderate cytotoxicity against HEp-2 cells
1
analysis such as IR, H NMR and Mass spectra.
ACKNOWLEDGEMENTS
The authors are thankful to Vice Chancellor, JSS Univer-
sity and the Management, JSS Mahavidyapeetha for provid-
ing the facilities to carry out the work.
3
.2. In-Vitro Cytotoxicity Screening
The compounds were screened for cytotoxicity activity
against HEp-2 cells by determination of CTC50 (concentra-
tion of the sample required to kill 50% of the cells) value by
SRB assay. The experiments were each carried out in tripli-
cate and the results are tabulated in Table 1. The compounds
were found to display moderate cytotoxic activities.
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