L-Proline mediated synthesis of quinoxalines; Evaluation of cytotoxic and antimicrobial activity

Article abstract of DOI:10.1039/c4ra08615e

A simple, greener and highly efficient method for the synthesis of functionalized quinoxalines has been developed employing l-proline as a catalyst in water. To the best of our knowledge this transformation is achieved for the first time using an organic catalyst. A small library of quinoxaline-sulphonamide conjugates have been synthesized using this protocol. The newly synthesized conjugates have been tested for their cytotoxicity and antimicrobial activity against several bacterial strains including one fungal strain. The majority of the compounds have exhibited significant cytotoxicity as well as antimicrobial activity. Compounds 5a, 5b and 5d were found to be promising with respect to both cytotoxicity and antimicrobial activity. This journal is

Full text of DOI:10.1039/c4ra08615e

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L-Proline mediated synthesis of quinoxalines;
evaluation of cytotoxic and antimicrobial activity†
Cite this: RSC Adv., 2014, 4, 46369
*
Ahmed Kamal, Korrapati Suresh Babu, Shaikh Faazil, S. M. Ali Hussaini
and Anver Basha Shaik
A simple, greener and highly efficient method for the synthesis of functionalized quinoxalines has been
developed employing L-proline as a catalyst in water. To the best of our knowledge this transformation is
achieved for the first time using an organic catalyst. A small library of quinoxaline–sulphonamide
conjugates have been synthesized using this protocol. The newly synthesized conjugates have been
tested for their cytotoxicity and antimicrobial activity against several bacterial strains including one fungal
strain. The majority of the compounds have exhibited significant cytotoxicity as well as antimicrobial
activity. Compounds 5a, 5b and 5d were found to be promising with respect to both cytotoxicity and
antimicrobial activity.
Received 13th August 2014
Accepted 17th September 2014
DOI: 10.1039/c4ra08615e
www.rsc.org/advances
Owing to extensive applications of quinoxalines several
synthetic methods for their preparation both in solution11 as
well as in solid-phase12 have been developed. Among them
Introduction
Quinoxalines represent as important class of biologically active
compounds that are known to possess antibacterial,1 anti-
cancer,2 and antiviral activities.3 This scaffold is present in
several anticancer agents such as chloroquinoxaline sulfon-
amide (1), XK469 (2) and NCG555879-01 (3)4 and in some
natural products like izumiphenazine C (4)5 (Fig. 1). It is also a
part of various antibiotics such as levomycin, echinomycin and
actinoleutin, which are known to inhibit the growth of Gram
positive bacteria.6 In addition, quinoxaline derivatives nd
application in cavitands,7 efficient electroluminescent mate-
rials,8 organic semiconductors,9 dyes,10 etc.
the condensation of 1,2-diamines with 1,2-diketone13 and
oxidative cyclization of a-hydroxy ketones with 1,2-
diamines14 under various conditions are widely used. Recent
reports indicate the use of several catalysts such as MnO2,15a
Ru/C in the presence of b-CD,15b manganese oxide octahedral
molecular sieves (OMS-2),15c HgI2,15d RuCl2(PPh3)3-TEMPO,15e
KF/Al2O3,15f and Au-NPs15g for onepot synthesis of quinoxa-
line from a-hydroxy ketones. However, they oen suffer from
one or more disadvantages such as long reaction time, use of
hazardous organic solvents, unsatisfactory product yields
and harsh reaction conditions.
On the other hand, organic catalysis is an emerging area of
organic synthesis wherein small molecules are used to cata-
lyze organic transformations. L-Proline is a versatile catalyst
reported to catalyze many important organic reactions and
asymmetric transformations such as aldol,16 Mannich,17
Michael,18 and Diels–Alder reaction.19 It is also excellent
promoter for the copper-catalysed coupling reactions.20 It's
ease of handling, experimental simplicity, cost effectiveness
and excellent solubility in water and organic solvents are
some reasons for its extensive use in the development of
synthetic methods. Further, use of water as a solvent is one of
the greener way of organic synthesis. Therefore in order to
overcome the disadvantages of previous methods and
considering the advantages of L-proline as a catalyst and
ecofriendly nature of water as a solvent we have developed a
new method to synthesize quinoxalines. We herein report a
green, simple and practical method for the synthesis of
quinoxaline derivatives from hydroxy ketones and 1,2-
diamines catalyzed by L-proline.
Fig. 1 Biologically important quinoxalines.
Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India. E-mail: ahmedkamal@iict.res.in
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra08615e
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Table 1 Condensation of benzoin and 1,2-diaminobenzene in water
at different catalyst (L-proline) concentrations
Results and discussion
Chemistry
Entry
Catalyst (mol%)
Time (h)
Yielda (%)
In the beginning, a systematic study was carried out for the
catalytic evaluation of L-proline towards the synthesis of qui-
noxalines. Initially a blank reaction was performed using
benzoin and 1,2-diaminobenzene in water without any catalyst
at room temperature and the completion of the reaction was
monitored by TLC. It was observed that the reaction did not
proceed even until 24 hours. Whereas the same reaction was
executed in the presence of catalytic amounts of L-proline in
water at room temperature and traces of the product were found
(less than 5%). Later, this reaction was carried out under
reuxing conditions and the desired transformation was
observed furnishing the product in very good yield (Scheme 1).
Aer obtaining the desired product, the amount of catalyst and
the time required for the completion of reaction were evaluated.
The reaction was performed using 5, 10, 20 and 30 mol% of the
catalyst and was monitored for 12–16 hours. It was observed
that 20 mol% of the catalyst loading provided maximum yield
(87%) in 12 hours. While 5 and 10 mol% of the catalyst afforded
64% and 72% of the product even aer reuxing the reaction for
16 hours. An additional increase of the catalyst loading to 30%
did not improve the yield. On the contrary, the reaction slows
down on adding more than 20 mol% of the catalyst (Table 1).
With the optimized conditions in hand, the reaction was
performed with different set of substituents to explore the scope
and generality of the present protocol. The quinoxaline deriva-
tives were synthesized using two hydroxy ketones namely
benzoin (2a) and furoin (2b) with varying 1,2-diamines. The
diamines used possessed both ring activating as well as deac-
tivating substituents and the results of these observations are
summarized in Table 2. From the results it can be concluded
that the electronic factors of 1,2-diamine inuences the prog-
ress of the reaction. Electron donating substituents such as
methyl (entry 3 and 8) provided excellent yields of the corre-
sponding products. In presence of weak ring deactivating
groups such as uoro and dichloro (entry 2, 5, 7 and 10) the
reaction progressed smoothly and the product was obtained in
good yields. This trend was also observed in the absence of
substituents on the diamine moiety. However, in case of ring
deactivating groups such as nitro (entry 4 and 9) the reaction
was slower and the yields were also comparable very lower.
1
2
3
4
5
6
7
8
9
—
5
5
10
10
20
20
30
30
24
12
16
12
16
12
16
12
16
Nil
62
64
71
72
87
87
82
86
a
Isolated yield.
ion resulting from the condensation of L-proline and a-hydroxy
ketone. The iminium ion gets converted to an alkene by reor-
ganisation of proton from a-position. The alkene formed is
electron rich and readily adds on oxygen from atmospheric air
to form a diradical. Further rearrangement of a-hydroxyl group
generates a-keto peroxide. The peroxy oxygen is then ionized
with the help of lone pair of proline and leaves to form diketo
imine which is hydrolysed to form 1,2-diketobenzene. The
condensation of 1,2-diketobenzene and 1,2-diaminobenzene
results in the formation of functionalized quinoxalines.
Controlled experiments
In order to determine whether the reaction is proceeding
through the mechanism anticipated, a series of experiments
have been performed. Firstly, the reaction was performed using
other secondary amines such as piperidine, pyrrolidine and
morpholine as a catalyst. The reaction was found to proceed
under the similar reaction conditions. However, the reaction
times were longer and the yields are lower compared to the
proline catalyzed reaction. Similarly, another reaction involving
heating of benzoin with proline in water without the addition of
1,2-diamine has been carried out in presence of air. Formation
of benzil, the oxidized product of benzoin was observed under
these conditions. Since the mechanism depicts the formation of
H2O2, it was of interest to test its effect on the reaction. In this
context, a reaction was carried out using benzoin, 1,2-diamine,
proline and H2O2 under inert conditions (in presence of
nitrogen). Surprisingly, the reaction did not proceed how ever
on removal of nitrogen gas the reaction proceeded to comple-
tion. This indicates the necessity of air which serves as a source
of oxygen (Table 3).
Since quinoxaline structure nds many applications in
pharmaceutical research, it was of interest to develop a library
of quinoxaline conjugates. In continuation to our research to
develop newer anticancer21 and antimicrobial agents,22 the
protocol developed was applied to synthesize a set of quinoxa-
line derivatives (5a–i). The synthetic route to access these
derivatives is outlined in Scheme 2. As shown, the quinoxaline
derivative 3i obtained from the reaction of furoin with nitro-1,2-
diamine was hydrogenated in presence of Pd/C to yield amino
derivative (4).23 This amino derivative was then reacted with
Plausible mechanism
The plausible mechanism for the formation of quinoxaline ring
from 1,2-diaminobenzene and a-hydroxy ketone is shown in
Fig. 2. The reaction proceeds with the formation of an iminium
Scheme 1 L-Proline catalyzed synthesis of quinoxalines.
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Table 2 L-Proline mediated synthesis of quinoxalines from hydroxy ketone with 1,2-diamines
Entry
1,2-Diamine (1)
Hydroxyketone (2)
Product (3a–h)
Time (h)
12
Yielda (%)
1
87
2
3
4
5
12
12
14
13
83
88
65
85
6
7
8
12
12
12
85
82
86
9
15
67
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Table 2 (Contd.)
Entry
1,2-Diamine (1)
Hydroxyketone (2)
Product (3a–h)
Time (h)
Yielda (%)
10
13
82
a
Isolated yield.
In order to determine the effect of substituent on the cyto-
toxicity the quinoxalin–sulphonamide conjugates have been
synthesized with various substitutions on the phenyl ring
attached to sulphonamide (Scheme 2). The compounds 5a and
5b possessing ring activating substituents such as methoxy
group on phenyl ring have signicantly inhibited the growth of
HeLa and A549 cells with IC50 of 6.7; 5.0 mM and 8.4; 7.4 mM
respectively. While the same compounds have shown moderate
growth inhibition on the DU145 cell line (IC50: 5a ¼ 12.3 and 5b
¼ 11.1 mM). In comparison, 5c and 5e functionalized with ring
deactivating substituents such as nitro and triuoromethyl on
phenyl ring exhibited lower cytotoxicity with IC50 values of 26.8
and 13.2 mM respectively against HeLa cells. Based on these
observations it is clear that substitution at C4 position on the
phenyl ring plays an important role with respect to the activity.
The presence of electropositive groups on the phenyl ring
signicantly enhances the cytotoxicity than electronegative
groups such as halogen and nitro. However, there is an insig-
nicant effect on the activity by the substitution at C3 position
of the phenyl ring.
Antimicrobial activity. The synthesized quinoxalin–sulpho-
namide conjugates 5a–i were also tested for their activity
against various Gram positive bacteria like Bacillus subtilis
MTCC121, Staphylococcus aureus MTCC 96, staphylococcus MLS-
16 MTCC2940, Micrococcus luteus MTCC2470 and Gram nega-
tive bacteria like Escherichia coli MTCC739, Pseudomonas aeru-
ginosa MTCC2453, Klebsiella planticola MTCC530 taking
chloramphenicol as a positive control, and the results are
summarized in Table 5. As evident from the results, most of the
Fig. 2 Plausible mechanism of quinoxaline formation.
substituted sulphonyl chlorides in pyridine to yield nal
conjugates 5a–i. These derivatives were later evaluated for their
cytotoxic as well as antimicrobial activities.
Biology
Antiproliferative activity. These quinoxalin–sulphonamide
conjugates 5a–i were investigated for their antiproliferative
activity against three human cancer cell lines namely Hela
(cervical cancer), DU145 (prostate cancer) and A549 (non small
cell lung cancer). Nocodazole was employed as reference stan-
dard and the IC50 values are summarized in Table 4. All the
compounds (5a–i) have exhibited moderate to good cytotoxicity
with the IC50 values ranging between 5.0 and 38.9 mM. Notably,
the compounds 5a and 5b have shown signicant activity on all
the cell lines examined.
Table 3 Controlled experiments to determine mechanism of L-proline catalyzed synthesis of quinoxalines
S. no.
Catalyst (mol%)
In presence of
Time (h)
Yielda (%)
1
2
3
4
5
6
Piperidine (20 mol%)
Pyrrolidine (20 mol%)
Morpholine (20 mol%)
L-Proline (20 mol%)
L-Proline (20 mol%) + H2O2
L-Proline (20 mol%) + H2O2
Air
Air
Air
Air
Nitrogen
Air
16
16
16
12
12
12
82
80
72
87
No reaction
87
a
Isolated yield.
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Compounds 5b, 5c and 5d were found to be most effective from
the series displaying MIC 20 mg mLꢀ1, however the compounds
have not superseded the standard nistatin (MIC 12 mg mLꢀ1).
Scheme 2 Reagents and condition: (i) H2, Pd/C, MeOH, rt, 6 h (ii) aryl
sulfonyl chloride, pyridine, rt, 2 h.
Conclusion
In conclusion, we have developed a simple, efficient and eco-
friendly method for the synthesis of quinoxalines from 1,2-
diamines and a-hydroxy ketones using cost-effective and readily
available catalyst L-proline. To the best of our knowledge this
transformation has not been reported with an organic catalyst.
The advantages of this method over previous reports include its
simplicity of operation, cleaner reactions, higher yields, shorter
reaction times and use of inexpensive catalyst. The mild reac-
tion condition makes this methodology an alternative proce-
dure to the conventional acid or base-catalyzed processes for the
synthesis of quinoxalines and has practical applicability.
Further, using this protocol a short library of quinoxaline–sul-
phonamide conjugates have been developed. The conjugates
were found to be cytotoxic and effectively inhibit the growth of
many bacterial strains.
Table 4 Anticancer activity of the synthesized compounds against
various cancer cell lines expressed in mM
IC50a (mM)
Compound
R1
R2
HeLab
DU145c
A549d
5a
5b
5c
5d
5e
5f
5g
5h
5i
OMe
OMe
NO2
F
CF3
Cl
Br
H
H
—
H
OMe
H
H
H
Cl
H
Cl
NO2
—
6.7 ꢁ 1.0
5.0 ꢁ 1.1
12.3 ꢁ 1.1
11.1 ꢁ 0.1
38.9 ꢁ 1.6
26.5 ꢁ 1.6
16.7 ꢁ 0.3
32.6 ꢁ 0.4
23.6 ꢁ 0.6
21.4 ꢁ 0.8
15.8 ꢁ 1.1
1.2 ꢁ 0.7
8.4 ꢁ 0.6
7.4 ꢁ 1.0
26.8 ꢁ 1.4
14.1 ꢁ 1.7
13.2 ꢁ 0.4
15.3 ꢁ 0.3
27.9 ꢁ 0.7
19.2 ꢁ 1.2
18.1 ꢁ 1.0
0.9 ꢁ 0.9
25.7 ꢁ 1.2
33.6 ꢁ 1.5
16.0 ꢁ 0.6
20.7 ꢁ 0.9
15.8 ꢁ 0.9
16.0 ꢁ 0.4
32.1 ꢁ 1.6
1.3 ꢁ 0.7
Noc
a
IC50: the amount of the compounds concentration required to reduce
b
the cells to half of its original number. HeLa: human cervical cancer
cell line. DU145: human prostate cancer cell line. A549: non small
cell lung cancer cell line; Noc: nocodazole.
c
d
Experimental
General remarks
conjugates have shown activity against all the strains tested.
Interestingly, the compounds have shown selectivity towards
Gram positive bacteria. Some of the conjugates 5b, 5c and 5d
Melting points were determined with an electrothermal melting
point apparatus and are uncorrected. Infrared (IR) spectra were
recorded on Perkin-Elmer model 683 or 1310 spectrometers
with sodium chloride optics. 1H NMR spectra were recorded on
an Avance 300 MHz spectrometer (Bruker, Fallanden, Switzer-
land) and 13C NMR spectra were recorded on a UNITY 300 MHz
(Varian, Switzerland). Chemical shis (d) are reported in ppm,
downeld from internal TMS standard. Mass spectra were
recorded using a quadruple ion trap mass spectrometer
(Thermo Finnign, San Jose, CA, USA) equipped with an electro
spray source.
have exhibited excellent antibacterial activity (MIC 10 mg mLꢀ1
)
comparable to and sometimes even better than the standard
against the pathogens B. subtilis, S. MLS and M. luteus. However,
other conjugates have displayed moderate activity against both
Gram positive and Gram negative bacteria. Furthermore, all the
conjugates were tested against a fungal strain, Candida albicans
(MTCC3017). All the compounds have shown moderate activity
against it with MIC values ranging between 20 and 80 mg mLꢀ1
.
Table 5 Antimicrobial activity of the compounds 5a–i and values are expressed in MIC (mg mLꢀ1 a
)
Compound
R1
R2
B sa
S ca
M la
S aa
E cb
P ab
K pb
C ac
5a
5b
5c
5d
5e
5f
5g
5h
OMe
OMe
NO2
F
CF3
Cl
Br
H
H
H
OMe
H
H
H
Cl
H
Cl
NO2
40
10
10
10
40
25
10
25
40
20
—
10
10
10
16
40
50
50
25
40
15
—
40
10
10
10
40
20
20
20
40
10
—
40
25
15
25
40
30
40
25
50
12
—
80
30
25
40
>100
80
40
80
>100
14
80
40
30
40
>100
80
40
40
>100
20
80
30
25
40
>100
40
80
80
>100
25
40
20
20
20
80
30
25
30
80
—
12
5i
Chloramphenicol
Nistatin
—
—
—
a
a ¼ gram positive bacteria; b ¼ gram negative bacteria; c ¼ fungal strain; B sa ¼ Bacillus subtilis MTCC121; S ca ¼ Staphylococcus MLS-16
MTCC2940; M la ¼ Micrococcus luteus MTCC2470; S aa ¼ Staphylococcus aureus MTCC 96; E cb ¼ Escherichia coli MTCC739; P ab ¼ Pseudomonas
aeruginosa MTCC2453; K pb ¼ Klebsiella planticola MTCC530; C ac ¼ Candida albicans MTCC3017.
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Representative experimental procedure for the synthesis
quinoxalines (3a–j)
6-Fluoro-2,3-di(furan-2-yl)quinoxaline (3g)
Brown solid; Mp: 106–108 ꢂC; 1H NMR (300 MHz CDCl3): d 6.56–
In a 50 mL round bottom ask 1,2-diamine (1 mmol) and a- 6.58 (m, 2H), 6.66 (dd, J ¼ 3.02, 9.80 Hz, 2H), 7.49–7.56 (m, 1H),
hydroxy ketone (1 mmol) were taken in water (5 mL). Catalytic 7.62–7.65 (m, 2H), 7.75 (dd, J ¼ 3.02 & 9.06 Hz, 1H), 8.10–8.15
amount (20 mol%) of L-proline was added and the reaction (m, 1H); 13C NMR (75 MHz CDCl3): d 111.23, 111.61, 112.01,
mixture was reuxed for 12 hours. The progress of the reaction 112.27, 112.76, 119.92, 123.64, 130.33, 136.74, 140.46, 140.95,
was monitored by TLC. Aer completion of the reaction, the 142.25, 143.66, 148.74, 149.52; ESI-MS: m/z ¼ 281 (M + H)+.
mixture was cooled to room temperature. The precipitated solid
was collected by ltration, washed with water and recrystallized
using methanol.
2,3-Di(furan-2-yl)-6-methylquinoxaline (3h)
Brown solid; Mp: 122–124 ꢂC; 1H NMR (300 MHz CDCl3): d 6.61
(d, J ¼ 16.42 Hz, 4H), 7.59 (t, J ¼ 8.87 Hz, 3H), 7.89 (s, 1H), 7.99
(d, J ¼ 8.49 Hz, 1H); 13C NMR (75 MHz CDCl3): d 21.26, 111.31,
111.95, 112.20, 127.20, 127.87, 132.14, 138.33, 139.96, 140.45,
141.07, 141.82, 143.32, 143.45, 150.20; ESI-MS: m/z ¼ 277 (M +
H)+.
2,3-Diphenylquinoxaline (3a)
White solid; Mp: 126–127 ꢂC; 1H NMR (300 MHz CDCl3): d 7.31–
7.4 (m, 6H), 7.52–7.57 (m, 4H), 7.76–7.82 (m, 2H), 8.17–8.22 (m,
2H); 13C NMR (75 MHz CDCl3): d 127.50, 128.13, 128.40, 129.14,
129.35, 138.33, 140.38, 152.57; ESI-MS: m/z ¼ 283 (M + H)+.
6-Fluoro-2,3-diphenylquinoxaline (3b)
2,3-Di(furan-2-yl)-6-nitroquinoxaline (3i)
Brown solid; Mp: 100–102 ꢂC; 1H NMR (500 MHz CDCl3): d 7.32–
7.40 (m, 6H), 7.49–7.52 (m, 4H), 7.53–7.57 (m, 1H), 7.81 (dd, J ¼
2.74 & 9.30 Hz, 1H), 8.16–8.19 (m, 1H); 13C NMR (75 MHz
CDCl3): d 112.52, 112.78, 120.20, 120.51, 128.32, 128.90, 129.08,
129.80, 129.86, 131.20, 131.34, 138.44, 138.70, 138.82, 141.88,
142.05, 152.84, 154.24, 161.19, 164.54; ESI-MS m/z ¼ 301 (M +
H)+.
Brown solid; Mp: 152–154 ꢂC; 1H NMR (300 MHz CDCl3): d 6.60–
6.63 (m, 2H), 6.87 (dd, J ¼ 3.58 & 16.80 Hz, 2H), 7.67 (dd, J ¼ 0.94
& 5.09 Hz, 2H), 8.23 (d, J ¼ 9.253 Hz, 1H), 8.50 (dd, J ¼ 2.45 &
9.253 Hz, 1H), 9.01 (d, J ¼ 2.45 Hz, 1H); 13C NMR (75 MHz
CDCl3): d 111.28, 111.40, 113.44, 114.28, 122.39, 123.93, 129.37,
137.92, 141.78, 142.93, 143.45, 143.95, 144.41, 146.64, 148.89,
148.98; ESI-MS: m/z ¼ 308 (M + H)+.
6-Methyl-2,3-diphenylquinoxaline (3c)
Brown solid; Mp: 120–122 ꢂC; 1H NMR (300 MHz CDCl3): d 2.59
(s, 3H), 7.32 (d, J ¼ 6.79 Hz, 6H), 7.49 (d, J ¼ 6.79 Hz, 4H), 7.59
(dd, J ¼ 1.51, 8.68 Hz, 1H), 7.90 (s, 1H), 8.03 (d, J ¼ 8.49 Hz, 1H);
13C NMR (75 MHz CDCl3): d 20.78, 126.79, 127.04, 127.52,
127.58, 128.76, 131.18, 138.07, 138.45, 139.27, 140.02, 151.20,
151.95; ESI-MS: m/z ¼ 297 (M + H)+.
6,7-Dichloro-2,3-di(furan-2-yl)quinoxaline (3j)
Brown solid; Mp: 135–137 ꢂC; 1H NMR (300 MHz CDCl3): d 6.58–
6.59 (m, 2H), 6.73 (d, J ¼ 3.50 Hz, 2H), 7.63 (d, J ¼ 1.06 Hz, 2H),
8.24 (s, 2H); 13C NMR (75 MHz CDCl3): d 111.46, 113.26, 128.73,
133.72, 138.42, 142.49, 143.96, 149.50; ESI-MS: m/z ¼ 330 (M +
H)+.
6-Nitro-2,3-diphenylquinoxaline (3d)
Brown solid; Mp: 140–142 ꢂC; 1H NMR (300 MHz CDCl3): d 7.34–
7.46 (m, 6H), 7.54–7.58 (m, 4H), 8.28 (d, J ¼ 9.25 Hz, 1H), 8.54
(dd, J ¼ 2.45 & 9.25 Hz, 1H), 9.09 (d, J ¼ 2.45 Hz, 1H); 13C NMR
(75 MHz CDCl3): d 123.28, 125.60, 128.43, 129.60, 129.74,
129.78, 129.86, 130.72, 137.97, 138.04, 139.94, 143.55, 147.84,
155.66, 156.29; ESI-MS: m/z ¼ 328 (M + H)+.
2,3-Di(furan-2-yl)quinoxalin-6-amine (4)
6-Nitro-2,3-di(furan-2-yl)quinoxaline in methanol (20 mL) was
hydrogenated in the presence of 5% Pd/C at room temperature
for 3 h. Aer completion of the reaction, the mixture was ltered
over celite. The ltrate was collected and concentrated.
6,7-Dichloro-2,3-diphenylquinoxaline (3e)
General procedure for the synthesis of quinoxalin-6-amine
sulphonamides
Brown solid; Mp: 141–143 ꢂC; 1H NMR (300 MHz CDCl3): d 7.31–
7.39 (m, 6H), 7.48–7.52 (m, 4H), 8.29 (s, 2H); 13C NMR (75 MHz
CDCl3): d 127.63, 128.62, 129.05, 133.44, 137.61, 139.17, 153.77;
ESI-MS: m/z ¼ 351 (M + H)+.
To a round bottom ask containing pyridine (5 mL) 2,3-
di(furan-2-yl)quinoxalin-6-amine (4.1 mmol) and corresponding
benzene sulphonyl chlorides (1.5 mmol) were added. The
reaction mixture was stirred for 3 hours and the progress of the
reaction was monitored by TLC. Aer completion of the reac-
2,3-Di(furan-2-yl)quinoxaline (3f)
Brown solid; Mp: 134–136 ꢂC; 1H NMR (300 MHz CDCl3): d 6.56– tion, aqueous CuSO4$5H2O solution was added and the product
6.58 (m, 2H), 6.65 (dd, J ¼ 0.56 & 3.58 Hz, 2H), 7.63 (dd, J ¼ 0.56 was extracted using ethyl acetate (3 ꢃ 20 mL). The combined
& 1.70 Hz, 2H), 7.74–7.78 (m, 2H), 8.11–8.17 (m, 2H); 13C NMR organic layer was dried over anh. Na2SO4 and concentrated on
(75 MHz CDCl3): d 111.85, 112.93, 129.02, 130.33, 138.46, rotavapor. The crude product was puried by column chroma-
140.51, 142.55, 144.15; m/z ¼ 263 (M + H)+.
tography using hexane and ethyl acetate (7 : 3) as eluent.
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1H);13C NMR (75 MHz DMSO d6): d 110.42, 110.50, 110.98,
111.58, 114.24, 122.78, 124.97, 127.13, 128.43, 129.89, 131.46,
135.51, 135.96, 137.79, 138.07, 139.33, 139.67, 141.24, 142.53,
142.84, 149.00, 149.09; IR (KBr) (nmax/cmꢀ1): n ¼ 3247, 2924,
1619, 1491, 1327, 1157, 1068, 1022, 919 cmꢀ1; ESI-MS: m/z 486
(M + H)+: HRMS (ESI) m/z for C22H14O4N3Cl2S calculated m/z:
486.00766, found m/z: 486.00459.
N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-
methoxybenzenesulfonamide (5a)
1
ꢂ
Yellow solid; Mp: 230–232 C; H NMR (300 MHz DMSO d6): d
3.78 (s, 3H), 6.56 (s, 2H), 6.61 (d, J ¼ 2.64, 1H), 6.88 (dd, J ¼ 2.07
& 8.87 Hz, 2H), 7.43 (s, 1H), 7.59–7.65 (m, 3H), 7.83–7.87 (m,
3H), 7.93 (dd, J ¼ 2.64 & 8.87 Hz, 1H), 10.43 (brs, 1H); 13C NMR
(75 MHz DMSO d6): d 54.11, 110.31, 110.48, 111.28, 111.52,
112.53, 112.80, 113.57, 122.62, 127.42, 127.69, 128.26, 129.43,
129.69, 135.74, 138.53, 139.47, 142.19, 142.45, 148.94, 149.20;
ESI-MS: m/z 448 (M + H)+: HRMS (ESI) m/z for C23H18O5N3S
calculated m/z: 448.09617, found m/z: 448.09330.
N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-(triuoromethyl)
benzenesulfonamide (5f)
1
ꢂ
Yellow solid; Mp: 255–257 C; H NMR (300 MHz DMSO d6): d
6.56–6.75 (m, 4H), 7.60–7.81 (m, 5H), 7.85–8.01 (m, 2H), 8.05–
8.25 (m, 2H), 10.86 (brs, 1H); 13C NMR (75 MHz DMSO d6): d
28.52, 111.01, 111.09, 111.70, 112.24, 115.12, 122.91, 122.95,
123.51, 128.51, 128.55, 128.98, 129.16, 129.41, 136.64, 138.43,
139.84, 140.02, 140.36, 141.93, 143.05, 143.31, 149.56; IR (KBr)
(nmax/cmꢀ1): n ¼ 3247, 2924, 1619, 1535, 1488, 1347, 1250, 1160,
919 cmꢀ1; ESI-MS: m/z 486 (M + H)+: HRMS (ESI) m/z for
N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-3,4-
dimethoxybenzenesulfonamide (5b)
1
ꢂ
Yellow solid; Mp: 245–247 C; H NMR (300 MHz DMSO d6): d
3.84 (s, 6H), 6.57–6.61 (m, 4H), 6.85–6.88 (m, 1H), 7.40 (s, 1H),
7.49–7.51 (m, 1H), 7.60–7.65 (m, 3H), 7.84 (s, 1H), 7.89–7.92 (m,
1H), 10.57 (brs, 1H); 13C NMR (75 MHz DMSO d6): d 55.06,
108.60, 109.56, 110.95, 111.47, 112.03, 114.41, 120.06, 123.46,
128.70, 130.09, 136.41, 139.26, 140.10, 142.92, 143.19, 147.90,
151.56; IR (KBr) (nmax/cmꢀ1): n ¼ 3446, 2934, 1620, 1508, 1156,
1138, 1018, 913 cmꢀ1; ESI-MS: m/z 478 (M + H)+: HRMS (ESI) m/z
for C24H20O6N3S calculated m/z: 478.10673, found m/z:
478.10325.
C
23H15O4N3F3S calculated m/z: 486.07299, found m/z:
486.06939.
4-Bromo-N-(2,3-di(furan-2-yl)quinoxalin-6-yl)
benzenesulfonamide (5g)
1
ꢂ
Yellow solid; Mp: 220–222 C; H NMR (300 MHz DMSO d6): d
6.56–6.62 (m, 4H), 7.57–7.66 (m, 5H), 7.76–7.84 (m, 3H), 7.90–
7.94 (m, 1H), 10.81 (brs, 1H); 13C NMR (75 MHz DMSO d6): d
111.68, 110.75, 111.24, 111.82, 114.20, 123.04, 126.29, 127.36,
128.57, 131.02, 136.15, 137.41, 138.40, 139.68, 139.85, 141.50,
142.70, 142.98, 149.27, 149.34; IR (KBr) (nmax/cmꢀ1): n ¼ 3086,
2853, 1618, 1571, 1527, 1489, 1343, 1218, 1087, 914 cmꢀ1; ESI-
MS: m/z 495 (M + H)+: HRMS (ESI) m/z for C22H15O4N3BrS
calculated m/z: 495.99612, found m/z: 495.99265.
N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-
nitrobenzenesulfonamide (5c)
1
ꢂ
Yellow solid; Mp: 260–262 C; H NMR (300 MHz DMSO d6): d
6.56–6.63 (m, 3H), 7.61–7.65 (m, 3H), 7.70–7.78 (m, 1H), 7.81–
7.86 (m, 2H), 7.89–7.94 (m, 1H), 8.19–8.23 (m, 1H), 8.34–8.39
(m, 1H), 8.70–8.72 (m, 1H), 11.10 (brs, 1H); 13C NMR (75 MHz
DMSO d6): d 110.77, 111.36, 111.92, 114.91, 120.71, 123.19,
126.14, 128.72, 129.63, 131.40, 137.85, 139.60, 140.20, 140.56,
142.77, 143.05, 143.30, 146.79, 149.20; IR (KBr) (nmax/cmꢀ1): n ¼
3246, 3095, 1619, 1571, 1527, 1353, 1165, 1123, 912 cmꢀ1; ESI-
MS: m/z 463 (M + H)+: HRMS (ESI) m/z for C22H15O6N4S calcu-
lated m/z: 463.07068, found m/z: 463.06729.
3-Chloro-N-(2,3-di(furan-2-yl)quinoxalin-6-yl)
benzenesulfonamide (5h)
1
ꢂ
Yellow solid; Mp 216–218 C; H NMR (300 MHz DMSO d6): d
6.57–6.63 (m, 4H), 7.42–7.54 (m, 2H), 7.63 (d, J ¼ 7.93, 3H), 7.78–
7.83 (m, 2H), 7.89–7.95 (m, 2H), 10.91 (s, 1H); 13C NMR (75 MHz
DMSO d6): d 110.53, 110.61, 111.09, 111.69, 114.13, 122.86,
123.82, 125.25, 128.44, 129.36, 131.58, 133.24, 135.99, 138.12,
139.47, 139.71, 139.95, 141.34, 142.59, 142.90, 149.09, 149.17; IR
(KBr) (nmax/cmꢀ1): n ¼ 3241, 2977, 1735, 1618, 1420, 1324, 1250,
1019, 1162, 911 cmꢀ1; ESI-MS: m/z 452 (M + H)+: HRMS (ESI) m/z
for C22H15O4N3ClS calculated m/z: 452.04663, found m/z:
452.04381.
N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-
uorobenzenesulfonamide (5d)
1
ꢂ
Yellow solid; Mp: 235–237 C; H NMR (300 MHz DMSO d6): d
6.58–6.61 (m, 4H), 7.10–7.16 (m, 2H), 7.58–7.63 (m, 3H), 7.86–
7.94 (m, 4H), 10.73 (s, 1H); 13C NMR (75 MHz DMSO d6): d
110.43, 110.52, 110.90, 111.52, 113.57, 114.74, 115.03, 122.69,
128.24, 128.34, 134.13, 135.74, 138.26, 139.35, 139.45, 141.15,
142.50, 144.81, 149.05; IR (KBr) (nmax/cmꢀ1): n ¼ 3251, 2924,
1620, 1491, 1325, 1229, 1088, 1018, 912 cmꢀ1; ESI-MS: m/z 436
(M + H)+: HRMS (ESI) m/z for C22H15O4N3FS calculated m/z:
436.07618, found m/z: 436.07273.
N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-3-
nitrobenzenesulfonamide (5i)
1
ꢂ
Yellow solid; Mp 260–262 C; H NMR (300 MHz DMSO d6): d
6.57–6.61 (m, 4H), 7.61–7.72 (m, 4H), 7.89 (s, 1H), 7.97 (d, J ¼
9.06 Hz, 1H), 8.23 (d, J ¼ 7.36 Hz, 1H), 8.37 (d, J ¼ 8.12 Hz, 1H),
8.78 (s, 1H), 11.01 (brs, 1H); 13C NMR (75 MHz DMSO d6): d
3,4-Dichloro-N-(2,3-di(furan-2-yl)quinoxalin-6-yl)
benzenesulfonamide (5e)
1
ꢂ
Yellow solid; Mp: 236–238 C; H NMR (300 MHz DMSO d6): d 110.16, 110.24, 110.68, 111.29, 113.92, 119.84, 122.47, 125.83,
6.63 (d, J ¼ 9.90 Hz, 4H), 7.65 (t, J ¼ 7.33 Hz, 4H), 7.78 (t, J ¼ 7.33 128.22, 129.50, 130.84, 135.54, 137.30, 138.83, 139.30, 140.83,
Hz, 2H), 7.96 (d, J ¼ 8.80 Hz, 1H), 8.01 (s, 1H), 11.01 (brs, 142.48, 142.82, 146.20, 148.58, 148.67; IR (KBr) (nmax/cmꢀ1): n ¼
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3249, 1618, 1569, 1526, 1468, 1352, 1228, 1164, 1081, 911 cmꢀ1
;
prepared sterile Mueller Hilton agar plates.26 Subsequently
ESI-MS: 463 [M + H]+: HR-MS (ESI) m/z for C22H15O6N4S calcu- these plates were incubated at 35 C for 24 h for zone of inhi-
ꢂ
lated m/z: 463.07068, found m/z: 463.06717.
bition, if any, around the disks. The lowest concentration
(higher dilution) of the test compound required to arrest the
growth of tested strains was dened as minimum inhibitory
concentrations (MICs). 20 mL of DMSO was loaded on sterile
disc and placed on the culture. Aer incubation for 24 h,
observed no zone of inhibition around the disc, indicated that
DMSO has no inhibitory effect on the tested strains and which
was used as a negative control. Chloramphenicol was employed
as the reference standard. The method followed for antifungal
bioassay was similar to that followed for antibacterial assay
where the medium was potato dextrose agar 39 g Lꢀ1. The
equivalent amount of solvent (DMSO) did not exhibit any
activity in the assay. The treated compounds and the controls
were kept in an incubator at 28 ꢁ 2 ꢂC for 48 h. All the
compounds were tested for minimum inhibitory concentration
(MIC). Nistatin was employed as positive control.
Materials and methods
Cell culture and reagents
All the cell lines used in this study were obtained from the
American Type Culture Collection (ATCC). DU145 (human
prostate carcinoma epithelial) cells have cultured in Eagle's
minimal essential medium (MEM) containing nonessential
amino acids, 1 mM sodium pyruvate, and 10% FBS. HeLa
(human epithelial cervical cancer) and A549 (human lung
carcinoma epithelial) were grown in Dulbecco's modied
Eagle's medium (DMEM) containing nonessential amino acids
and 10% FBS. All the cells maintained under humidied
atmosphere of 5% CO2 at 37 ꢂC. Cells were trypsinized when sub
conuent from T75 asks per 90 mm dishes and seeded on to
96 well test plates at a concentration of 1 ꢃ 104 cells per mL in
complete medium, treated with compounds at desired
concentrations and harvested as required.24
Acknowledgements
The authors (KSB, SMAH, AB and SF) are thankful to CSIR and
UGC, New Delhi for nancial support and XIIth Five Year plan
project “Affordable Cancer Therapeutics (ACT)”
MTT assay
Cell proliferation and viability was determined by 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay. The pale yellow colored tetrazolium salt (MTT) reduces to
a dark blue water-insoluble formazan by metabolically active
cells and which is measured quantitatively aer soluble in
DMSO. The absorbance of the soluble formazan is directly
proportional to the number of viable cells. Cells were seeded at
a density of 1 ꢃ 104 cells in 200 mL of medium per well of 96-well
plate. The 96-well microtiter plates were incubated for 24 h prior
to addition of the experimental compounds. Cells were treated
with vehicle alone (0.4% DMSO) or compounds (drugs were
dissolved in DMSO previously) at different concentrations (1, 10
and 25 mM) of test compounds for 48 hours. The assay was
completed with the addition of MTT (5%, 10 mL) and incubated
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