Pyridine-catalyzed synthesis of quinoxalines as anticancer and anti-tubercular agents

Article abstract of DOI:10.1007/s00044-016-1558-2

Quinoxaline derivatized with coumarin viz., 3a–f and with sydnones viz., 7a–0 were synthesized using pyridine as catalyst. Among the coumarin derivatives, 3a and 3b have been screened for anticancer activity against 60 human cancer cell lines at NIH (USA)

Full text of DOI:10.1007/s00044-016-1558-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1558-2
ORIGINAL RESEARCH
Pyridine-catalyzed synthesis of quinoxalines as anticancer
and anti-tubercular agents
Atulkumar A. Kamble1 Ravindra R. Kamble1
•
•
Mahadev N. Kumbar1 Gireesh Tegginamath1
•
Received: 9 March 2015 / Accepted: 14 March 2016
Ó Springer Science+Business Media New York 2016
Abstract Quinoxaline derivatized with coumarin viz.,
3a–f and with sydnones viz., 7a–0 were synthesized using
pyridine as catalyst. Among the coumarin derivatives, 3a
and 3b have been screened for anticancer activity against
60 human cancer cell lines at NIH (USA). Compound 3a
has shown 55.75 % GI against Melanoma (MALME-M)
tumor cell line. Further, the sydnone derivatives 7d–i in-
hibited the Mycobacterium tuberculae H37RV.
positive bacteria and they are active against various trans-
plantable tumors (Sandra, 2004). Coumarins belong to fla-
vonoid class of plant secondary metabolites, which have
been found to exhibit variety of biological activities asso-
ciated with low toxicity and have raised considerable interest
because of their potential beneficial effects on human health
(Cooke et al., 1997; Patel et al., 2010). The cytotoxic
activities of the coumarin derivatives are reported against
several human tumor cell lines (Zhiwei et al., 2013).
Keywords Pyridine Á Coumarin Á Sydnone Á Anticancer Á
Mesoionic compounds are non-benzenoid heterocycles
containing different combination of heteroatoms. Sydnones
constitute an important class of mesoionic compounds
which have been extensively studied due not only to their
structural features and chemical properties but also their
biological properties. Also, sydnone derivatives have
shown biological activities like anti-inflammatory, anti-
convulsant, antitumor, diuretic, antimicrobial anti-tuber-
cular (Chandrasekhar and Nanjan, 2012) etc. Sydnone has
been used extensively as synthon in organic synthesis (Taj
et al., 2014; Bansode and Kamble, 2012).
Anti-tubercular
Introduction
In the vast heterocyclic structural space, quinoxaline is the
most widely distributed heterocyclic ring system found in
many drugs and occupies a position of major importance due
to its therapeutic and pharmacological activities (Fig. 1)
such as antimalarial (Chandra Shekhar et al., 2014),
antibacterial, antifungal (Dalia, 2013), anti-inflammatory
(Abu-Hashem et al., 2010), anti-tubercular (Rao et al., 2009;
Esther et al., 2008) and anticancer (Noolvi et al., 2011;
Mohamed et al., 2015) etc. Quinoxaline nucleus is also found
in many antibiotics such as echinomycin, levomycin and
actinoleutin which are known to inhibit the growth of gram-
Cancer, a diverse group of diseases is characterized by
uncontrolled growth of abnormal cells. It is a major cause
of death worldwide followed by heart disease. Presently
there are many research groups working on the discovery
of potent, safe and selective anticancer drug. Tuberculosis
(TB) is a highly infectious disease caused by Mycobac-
terium tuberculosis. This disease is caused due to dense
population, poor nutrition, and poor sanitation found in all
developing countries. It is estimated that every year 2
million deaths occur because of TB, which after infection
occurs in two stages. The first is an asymptomatic state that
can persist for many years in the host called latent TB. The
second stage requires only a weakened immune response to
get activated; then, the bacteria begin replicating and
causes characteristic symptoms such as cough, chest pain,
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1558-2) contains supplementary
material, which is available to authorized users.
& Ravindra R. Kamble
kamchem9@gmail.com
1
Department of Chemistry, Karnatak University Dharwad,
Pavate Nagar, Dharwad 580003, Karnataka, India
123

Med Chem Res
Fig. 1 Chemical structures of
some quinoxalines used as
therapeutic agents
S
CO2H
H
O
N
Br
H
N
N
N
H
N
N
N
N
N
N
H
NH
O
N
CO2H
Quinacillin
(antibiotic)
Brimonidine
(Ocular hypertension)
Varenicline
(Nicotine addiction)
fatigue and unexplained weight loss. If left untreated, the
disease eventually culminates into death (Musiliyu et al.,
2011; Hadda et al., 2007; Puratchikody et al., 2011).
Various heterocycles viz., Biginelli derivatives (Teggi-
namath et al., 2013), b-aminoketones (Dorababu et al.,
2014), 1,3,4-thiadiazole, imidazo[2,1-b][1,3,4]thiadiazoles
(Gireesh et al., 2013) have been introduced at position-4 of
sydnone. For the first time, we herein report the sydnone
having quinoxaline appended at this position to explore
their biological activities.
Therefore, pyridine (10 mol%) was used as catalyst in
all of the conversions viz., formation of either title com-
pounds 3a–f from compound 1a–b and arylenediamines
2a–c or for the formation of 7a–o from the compounds 6a–
e and 2a–c (Schemes 1 and 2). The efficiency of the cat-
alyst may be observed in terms of yield provided in the
characterization of each title compounds.
The proposed mechanism for the formation of final
compounds is given in Scheme 3. This depicts the role
of pyridine in the formation of title compounds. The
initial attack of pyridine on active methylene carbon
gave pyridinium bromide salt I. This induces the attack
of lone pair of electrons on nitrogen of arylenediamine
on the more active methylene carbon attached to qua-
ternary nitrogen of pyridine to form an intermediate II.
The intermediate II undergoes ring closure due to the
enhancement of electrophilicity of carbonyl group by
pyridinium cation as shown in the scheme to form
intermediate III. This intermediate then undergoes
dehydrogenation followed by oxidation to form the title
compounds 3a–f and 7a–o (the driving force for dehy-
dration followed by oxidation is attaining aromaticity by
forming quinoxaline).
Hence, in search for new small molecules, quinoxaline
derivatives incorporated with coumarin 3a–f and sydnone
7a–o moiety, respectively, have been synthesized via
substituted 3-(2-bromoacetyl)-2H-chromen-2-one 1a–
b and substituted 3-bromo-1-sydnon-4-yl-ethanone 6a–e as
starting material in order to explore in vitro anticancer
activity against 60 human cancer cell lines and anti-tu-
bercular activity, respectively.
Results and discussions
Reaction of 3-(2-bromoacetyl)-2H-chromen-2-one 1a–b or
3-bromo-1-sydnon-4-yl-ethanone 4a–e with arylenediami-
nes 2a–e was optimized for the suitable catalyst as follows:
Initially, the reaction between bromoacetyl group of
compound 1a and compound 6a with arylenediamine 2a–
b was tried in the presence of various catalysts viz,
Zn(OTf)3, CAS, ZnBr2, AlCl3, ZnCl2, CAN, LiCl2, MgBr2
and Pyridine in THF at room temperature. Among all of
these, Pyridine showed the most efficient catalytic activity
in terms of both time and yield as shown in Table 1.
The structures of synthesized compounds 3a–f and 7a–
I
o were confirmed by IR, H, 13C NMR, MS and elemental
analyses. The compounds have shown strong adsorption
band for carbonyl of coumarin and sydnone rings at
1713–1723 and 1753–1772 cm-1, respectively. The 1H
NMR spectral analyses of all the compounds exhibited a
singlet in the range 9.59–9.88 ppm for one proton on C2
carbon of quinoxaline ring. The aromatic protons in all
compounds appeared as multiplets in the range
6.82–7.78 ppm. Compounds 3c, 3f, 7c, 7f, 7i–j, 7k–l, 7o
have exhibited a singlet for methyl protons attached to
phenyl ring in the range 2.29–2.38 ppm. In case of 13C
NMR spectral analysis, all the compounds exhibited the
signals in consistent with number of magnetically non-
equivalent carbon atoms. Also the mass spectra of all the
compounds showed molecular ion peaks at their respective
molecular masses.
N
X
O
2b, 2a
Br
Ar
Ar
N
Catalyst
3b, 7a
1a, 6a
Ph
N
1a, 3b; Ar =
6a, 7a; Ar =
N
O
O
O
O
X = H
X = Cl
123

Med Chem Res
Table 1 Optimization of various catalysts for the synthesis of compounds 3b and 7a
Entry
No.
Catalyst
Reaction time
(h) for 3b
Yield
(%)
Reaction time
(h) for 7a
Yield
(%)
1
2
–
20
22
67
79
76
80
35
84
67
82
94
12
25
66
73
80
84
65
79
72
81
92
MgBr2
LiCl2
CAN
5.5
22
16
3
21
4
8.0
10
9.0
8.5
23
5
ZnCl2
AlCl3
ZnBr2
CAS
6
20
7
18
20
8
6.5
6.0
2.0
7.5
5.0
1.0
9
Zn(OTf)3
Pyridine
10
N
R'
O
R
H2N
H2N
R'
Br
R
THF/Pyridine
N
RT, Stirr
O
O
O
O
3a-f
1a-b
2a-c
Scheme 1 Synthesis of 3-(quinoxalin-2-yl)-2H-chromen-2-one derivatives 3a–f. 1a; R = H, 1b; R = Br, 2a; R0 = H, 2b; R0 = Cl, 2c;
R0 = CH3, 3a; R = H, R0 = H, 3b; R = H, R0 = Cl, 3c; R = H, R0 = CH3, 3d; R = Br, R0 = H, 3e; R = Br, R0 = Cl, 3f; R = Br, R0 = CH3
lines at NCI under DTP drug discovery program. Results from
the single dose concentration are reported as a graph of mean
growth percent of the treated cells. This allows detecting both
growth inhibition values (between 0 and 100) and cytotoxicity
values (less than 0). The results of single dose screening were
analyzed by COMPARE program. Compounds 3a (NSC:
778920/1) and 3b (NSC: 778921/1) have been screened for
single high-dose (10-5 M) concentration on all the 60 human
cancer cell lines organized into nine sub-panels derived from
the nine different human cancer types: leukemia, non-small
cell lung, colon, CNS, melanoma, ovarian, renal, prostate,
breast cancer cell lines. The following are the percentage
growth inhibition (GI%) of the treated cells at (10-5 M)
concentration with the compound 3a 3-(Quinoxaline-2-yl)-
2H-chromen-2-one (Table 2): Leukemia MOLT-4 (GI%
11.62), non-small cell lung cancer NCI-H23 (GI% 12.02),
colon cancer HCT-116 (GI% 6.49), CNS cancer U251 (GI%
5.26), Melanoma MALME-3M (GI% 55.75), ovarian cancer
NCI/ADR-RES (GI% 5.87), renal cancer UO-31 (GI%
15.72), prostate cancer DU-145 (GI % 1.53), breast cancer
MDA-MB-468 (GI % 16.75). Similarly, compound 3b has
shown moderate activity against different cancer cell lines
(Table 3).
Biological evaluation
It is interesting to note that synthetic studies on quinoxa-
lines have been focused on C-2 substituted analogs (Radhia
et al., 2014), which have proven to be capable of over-
coming drug resistance against various diseases. Therefore,
available drug results and the structures of various
quinoxalines suggest structural pharmacophore like Fig. 2.
In view of this, we have synthesized the quinoxalines
containing another potent moiety on C2 position and sub-
stituted the same for anti-cancer as well as anti-tubercular
activity.
Anticancer activity assay
The anticancer activity assay was performed at National
Institute of Health, Bethesda, USA, under the drug discovery
program of National Cancer Institute (NCI). All the synthe-
sized compounds 3a–f have been registered on its website.
Out of these the compounds 3a and 3b have been selected for
anticancer activity. The synthesized molecules viz, 3a and 3b
were tested for in vitro anticancer activity in a single dose
(10-5 M) concentration against full 60 human cancer cell
123

Med Chem Res
O
O
Br
R"
R"
H
R"
CH3
N
N
N
Br2
CH3COOH,
N
N
N
O
O
O
CHCl3
O
O
O
P2O5
6a-e
4a-e
5a-e
THF/Pyridine
RT, Stirr
2a-c
R'
N
N
R''
N
N
O
O
7a-o
Scheme 2 Synthesis of 2-(3-arylsydnon-4-yl)quinoxalines 7a–o. 6a;
R00 = –C6H5, 6b; R00 = 4-Cl–C6H4, 6c; R00 = 4-Br–C6H4, 6d;
R00 = 4-CH3–C6H4, 6e; R00 = 4-NO2–C6H4, 7a; R00 = –C6H5,
R0 = H, 7b; R00 = –C6H5, R0 = Cl, 7c; R00 = –C6H5, R0 = CH3,
7d; R00 = 4-Cl–C6H4, R0 = H, 7e; R00 = 4-Cl–C6H4, R0 = Cl, 7f;
R00 = 4-Cl–C6H4, R0 = CH3, 7g; R00 = 4-Br–C6H4, R0 = H, 7h;
R00 = 4-Br–C6H4, R0 = Cl, 7i; R00 = 4-Br–C6H4, R0 = CH3, 7j;
R00 = 4-CH3–C6H4, R0 = H, 7k; R00 = 4-CH3–C6H4, R0 = Cl, 7l;
R00 = 4-CH3–C6H4, R0 = CH3, 7m; R00 = 4-NO2–C6H4, R0 = H, 7n;
R00 = 4-NO2–C6H4, R0 = Cl, 7o; R00 = 4-NO2–C6H4, R0 = CH3
H2N
H2N
O
Br
H
N
R
O
Br
N
Br
N
H
N
R
O
H2N
R
I
II
N
Br
H
-H2O
[O]
HN
HN
N
NH
NH
N
HO
R
R
R
III
IV
3a-f, 7a-o
Scheme 3 Plausible mechanism for the formation of quinoxaline derivatives indicating the catalytic role of pyridine. R = coumarin for 3a–f, N-
arylsydnone for 7a–o
Anti-tubercular activity
reference drug i.e., Pyrazinamide. The remaining com-
pounds have shown moderate to weak inhibition.
The compounds 7a–o were screened for their activity against
standard strain of Mycobacterium tuberculosis H37Rv.
The results of anti-tubercular activity of the test samples are
depicted in Table 4. It was observed that the halogen sub-
stituted phenyl ring on sydnone derivatives viz., 7d–i and
methyl substitution on the phenyl ring of sydnones 7j–l have
shown excellent activitywhich was found to be more than the
Conclusions
In conclusion, some novel quinoxalines were designed and
prepared by simple, mild and expeditious synthetic protocol
to form 3-(quinoxalin-2-yl)-2H-chromen-2-ones 3a–f and
123

Med Chem Res
7.40–7.35 (1H, t, J = 15.0 Hz, Ar–H), 7.43 (1H, m, Ar–
H), 7.46 (1H, m, Ar–H), 7.66–7.61 (1H, t, J = 15.0 Hz,
Ar–H), 7.74–7.72 (1H, t, J = 15.0 Hz, Ar–H), 7.82 (1H,
m, Ar–H), 8.16–8.13 (1H, d, J = 9.0 Hz, Ar–H), 8.86 (1H,
s, Coumarin C4–H), 9.82 (1H, s, Quinoxaline C3–H); 13C
NMR (d, ppm, 400 MHz, CDCl3); 116.67 (Coumarin C8),
119.36 (Coumarin C10), 124.07 (Coumarin C6), 124.89
(Coumarin C5), 129.13 (Coumarin C7), 129.34 (Coumarin
C3), 129.36 (Quinoxaline C5), 130.27 (Quinoxaline C8),
130.30 (Quinoxaline C6), 132.94 (Quinoxaline C7), 142.16
(Quinoxaline C5a), 142.21 (Quinoxaline C8a), 144.55
(Quinoxaline C3), 145.44 (Coumarin C4), 147.34 (Cou-
marin C9), 154.44 (Quinoxaline C2), 160.12 (Coumarin
C = O); Anal. Calcd. For C17H10N2O2: C, 74.44; H, 3.67;
N, 10.21. Found C, 74.56; H, 3.78; N, 10.34 %. MS (EI,
70 eV) m/z: 273.70 [M?1], 245.15, 214.42, 189.19.
N
N
X
Subsꢀtuted phenyl ring
Heterocycle
Fig. 2 SAR of quinoxaline drugs
2-(3-phenylsydnon-4-yl)quinoxalines 7a–o using pyridine
as catalyst with excellent yields. According to the prelimi-
nary SAR of quinoxalines, compound 3-(quinoxalin-2-yl)-
2H-chromen-2-one 3a has shown 55.75 % GI against Mel-
anoma MALME-3M tumor cell line. Similarly, compounds
with substituents like bromo, chloro and methyl group at
para position of phenyl ring present at 3 position of sydnone
nucleus (7d–i and 7j–l) enhanced their activity and were
found to be more active against Mycobacterium tuberculae
H37Rv than reference drug Pyrazinamide.
3-(6-Chloroquinoxalin-2-yl)-2H-chromen-2-one (3b) Yield:
95 %. m.p: 185–187 °C; IR (KBr) cm-1: 1720 (C=O),
1
1607 (C–O). H NMR (CDCl3): d ppm 7.28 (1H, m, Ar–
H), 7.42–7.36 (1H, t, J = 18.0 Hz, Ar–H), 7.45 (1H, m,
Ar–H), 7.48 (1H, m, Ar–H), 7.67–7.63 (1H, t,
J = 12.0 Hz, Ar–H), 7.75–7.72 (1H, t, J = 9.0 Hz, Ar–H),
8.15–8.13 (1H, d, J = 6.0 Hz, Ar–H), 8.87 (1H, s, Cou-
marin C4–H), 9.85 (1H, s, Quinoxaline C3–H); 13C NMR
(d, ppm, 400 MHz, CDCl3): 117.68 (Coumarin C8), 122.42
(Coumarin C10), 124.13 (Coumarin C6), 124.87 (Coumarin
C5), 129.28 (Coumarin C7), 129.37 (Coumarin C3), 129.38
(Quinoxaline C5), 130.29 (Quinoxaline C8), 130.54
(Quinoxaline C6), 135.92 (Quinoxaline C7), 142.19
(Quinoxaline C5a), 143.26 (Quinoxaline C8a), 144.13
(Quinoxaline C3), 146.40 (Coumarin C4), 147.42 (Cou-
marin C9), 155.38 (Quinoxaline C2), 161.23 (Coumarin
C = O); Anal. Calcd. For C17H9N2ClO2: C, 66.14; H, 2.94;
N, 9.07. Found: C, 66.29; H, 3.06; N, 9.21 %. MS (EI,
70 eV) m/z: 310.68 [M?2], 308.50 [M?1], 282.34, 280.24,
251.51, 249.16, 226.43, 224.42.
Experimental protocols
Melting points were recorded in an open capillaries and are
uncorrected. The purity of the compounds was checked by
Aluchrosep Silica Gel 60/UV 254 TLC on silica gel coated
plates in ethylacetate and hexane (7:3) as an eluent. IR
spectra were recorded in a KBr disk matrix using an
IMPACT-410 Nicolet (USA). 1H and 13CNMR spectra were
recorded on Bruker Avance 300-MHz FT NMR spectrom-
eters with CDCl3 as solvent and tetramethylsilane (TMS) as
an internal standard. The mass spectra were recorded on
Shimadzu GC–MS instrument operating at 70 eV. Com-
pounds 1a–b, 4a–e were prepared according to the reported
methods (Kulkarni et al., 1981; Fun et al., 2012).
General procedure for the preparation of title
compounds 3a–f and 7a–o
3-(6-Methylquinoxalin-2-yl)-2H-chromen-2-one (3c) Yield:
96 %. m.p: 190–192 °C; IR (KBr) cm-1: 1719 (C=O),
1609 (C–O). H NMR (CDCl3): d ppm 2.38 (3H, s, CH3),
1
A mixture of 6-substituted-3-(2-bromoacetyl)-2H-chromen-
2-ones 1a–b or substituted 3-bromo-1-(3-arylsydnon-4-yl)e-
thanones 6a–e (0.1 M), pyridine (10 % M) and substituted
1,2-diaminobenzene 2a–c (0.1 M) were taken in THF (5 ml)
and stirred for 1 h at RT. The progress of reaction was mon-
itored by TLC using hexane and ethyl acetate (7:3) as solvent.
After the completion of the reaction, the reaction mass was
quenched using ice water. The crude product formed was
filtered, washed, dried and purified by column chromatogra-
phy using ethyl acetate and hexane (1:9) as solvent.
7.32 (1H, m, Ar–H), 7.44–7.38 (1H, t, J = 18.0 Hz, Ar–
H), 7.48 (1H, m, Ar–H), 7.53 (1H, m, Ar–H), 7.62–7.59
(1H, t, J = 9.0 Hz, Ar–H), 7.77–7.73 (1H, t, J = 12.0 Hz,
Ar–H), 8.13–8.10 (1H, d, J = 9.0 Hz, Ar–H), 8.91 (1H, s,
Coumarin C4–H), 9.84 (1H, s, Quinoxaline C3–H); 13C
NMR (d, ppm, 400 MHz, CDCl3): 25.16 (CH3), 117.76
(Coumarin C8), 120.45 (Coumarin C10), 124.21 (Coumarin
C6), 124.78 (Coumarin C5), 129.23 (Coumarin C7), 129.45
(Coumarin C3), 129.54 (Quinoxaline C5), 130.29
(Quinoxaline C8), 130.39 (Quinoxaline C6), 133.06
(Quinoxaline C7), 142.22 (Quinoxaline C5a), 142.28
(Quinoxaline C8a), 144.78 (Quinoxaline C3), 145.81
3-(Quinoxalin-2-yl)-2H-chromen-2-one (3a) Yield: 97 %.
m.p: 198–200 °C; IR (KBr) cm-1: 1718 (C=O), 1609 (C–
O). 1H NMR (CDCl3): d ppm 7.26 (1H, m, Ar–H),
123

Med Chem Res
Table 2 Growth Percent and Growth Inhibition (GI %) in single
dose assay (10-5
778920/1)
Table 2 continued
M concentration) for Compound 3a (NSC:
Panel/Cell line
Growth
Percent
Growth Inhibition
(GI %)
Panel/Cell line
Growth
Percent
Growth Inhibition
(GI %)
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
97.56
94.13
2.44
5.87
Leukemia
CCRF-CEM
HL-60(TB)
K-562
108.26
-8.26
92.42
117.28
93.34
7.58
-17.28
6.66
99.74
102.48
97.84
0.26
-2.48
2.16
A498
MOLT-4
RPMI-8226
SR
88.38
11.62
-5.13
10.72
ACHN
105.13
89.28
CAKL-1
106.92
93.48
-6.92
6.52
SN12C
Non-small cell lung cancer
A549/ATCC
HOP-62
TK-10
118.40
84.28
-18.40
15.72
93.18
97.75
95.48
98.48
87.98
96.34
104.65
6.82
2.25
UO-31
Prostate cancer
PC-3
HOP-92
4.52
99.64
98.47
0.36
1.53
NCI-H226
NCI-H23
1.52
DU-145
12.02
3.66
Breast cancer
MCF7
NCI-H322M
NCI-H460
Colon cancer
COLO-205
HCC-2998
HCT-116
72.17
97.55
27.83
2.45
-4.65
MDA-MB-231/ATCC
HS 578T
BT-549
100.12
107.49
92.38
-0.12
-7.49
7.62
110.78
91.40
93.51
104.84
97.19
96.62
106.79
-10.78
8.6
T-47D
6.49
MDA-MB-468
83.25
16.75
HCT-15
-4.84
2.81
HT29
(Coumarin C4), 147.56 (Coumarin C9), 154.47 (Quinoxa-
line C2), 161.32 (Coumarin C=O); Anal. Calcd. For
C18H12N2O2: C, 74.99; H, 4.20; N, 9.72. Found: C, 75.12;
H, 4.34; N, 9.88 %. MS (EI, 70 eV) m/z: 288.10 [M?1],
260.34, 229.23, 204.76.
KM12
3.38
SW-620
-6.79
CNS cancer
SF-268
98.13
98.71
1.87
1.29
SF-295
SF-539
110.19
94.85
-10.19
5.15
6-Bromo-3-(quinoxalin-2-yl)-2H-chromen-2-one (3d) Yield
93 %. m.p: 176–178 °C; IR (KBr) cm-1: 1723 (C=O),
1618 (C–O). H NMR (CDCl3): d ppm 7.29 (1H, m, Ar–
SNB-19
1
SNB-75
114.15
94.74
-14.15
5.26
U251
H), 7.45–7.39 (1H, t, J = 18.0 Hz, Ar–H), 7.41 (1H, m,
Ar–H), 7.49 (1H, m, Ar–H), 7.61–7.56 (1H, t,
J = 15.0 Hz, Ar–H), 7.71–7.68 (1H, t, J = 9.0 Hz, Ar–H),
8.12–8.08 (1H, d, J = 12.0 Hz, Ar–H), 8.81 (1H, s, Cou-
marin C4–H), 9.74 (1H, s, Quinoxaline C3–H); 13C NMR
(d, ppm, 400 MHz, CDCl3): 115.62 (Coumarin C8), 120.43
(Coumarin C10), 124.23 (Coumarin C6), 124.82 (Coumarin
C5), 129.13 (Coumarin C7), 129.47 (Coumarin C3), 129.54
(Quinoxaline C5), 130.24 (Quinoxaline C8), 131.18
(Quinoxaline C6), 132.10 (Quinoxaline C7), 142.25
(Quinoxaline C5a), 143.19 (Quinoxaline C8a), 144.21
(Quinoxaline C3), 146.37 (Coumarin C4), 147.47 (Cou-
marin C9), 155.41 (Quinoxaline C2), 160.74 (Coumarin
C=O); Anal. Calcd. For C17H9N2BrO2: C, 57.81; H, 2.57;
N, 7.93. Found: C, 57.95; H, 2.69; N, 8.07 %. MS (EI,
70 eV) m/z: 354.52 [M?2], 352.75 [M?1], 326.13, 324.21,
295.21, 293.46, 270.89, 268.12.
Melanoma
LOX IMVI
MALME-3M
M14
100.78
44.25
108.65
114.18
97.24
110.58
96.36
99.77
97.70
-0.78
55.75
-8.65
-14.18
2.76
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Overian cancer
IGROV1
-10.58
3.64
0.23
2.3
103.58
108.02
109.84
110.22
-3.58
-8.02
-9.84
-10.22
OVCAR-3
OVCAR-4
OVCAR-5
123

Med Chem Res
Table 3 Growth Percent and Growth Inhibition (GI %) in single
dose assay (10-5 M concentration) for Compound 3b (NSC:
778921/1)
Table 3 continued
Panel/Cell line
Growth
Percent
Growth Inhibition
(GI %)
Panel/Cell line
Growth
Percent
Growth Inhibition
(GI %)
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
91.80
8.2
102.70
-2.70
Leukemia
CCRF-CEM
HL-60(TB)
K-562
89.29
121.60
87.25
74.70
95.54
72.22
10.71
-21.6
12.75
25.3
106.75
107.69
98.71
-6.75
-7.69
1.29
A498
ACHN
MOLT-4
CAKL-1
100.56
106.87
127.36
75.53
-0.56
-6.87
-27.36
24.47
RPMI-8226
SR
4.46
SN12C
27.78
TK-10
Non-small cell lung cancer
A549/ATCC
HOP-62
UO-31
93.99
103.09
92.71
94.55
87.13
89.03
109.71
6.01
-3.09
7.29
Prostate cancer
PC-3
96.83
3.17
HOP-92
DU-145
106.03
-6.03
NCI-H226
NCI-H23
5.45
Breast cancer
MCF7
12.87
10.97
-9.71
68.41
101.55
108.45
107.25
79.54
31.59
-1.55
-8.45
-7.25
20.46
11.14
NCI-H322M
NCI-H460
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
MDA-MB-231/ATCC
HS 578T
BT-549
109.93
104.43
100.94
99.44
-9.93
-4.43
-0.94
-0.56
-0.38
5.38
T-47D
MDA-MB-468
88.86
HT29
100.38
94.62
KM12
SW-620
107.59
-7.59
Table 4 Anti-tubercular activity of the synthesized compounds
(strain H37Rva)
CNS cancer
SF-268
100.45
102.93
104.07
101.73
102.06
95.53
-0.45
-2.93
-4.07
-1.73
-2.06
4.47
Entry
No.
MIC (lg/ml)
SF-295
SF-539
7a
7b
7c
7d
7e
7f
7.70
10.90
8.40
2.30
3.50
2.80
2.60
3.50
3.20
6.28
6.12
6.30
24.0
27.0
30.0
7.50
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
91.21
77.32
103.39
97.34
85.65
105.60
94.03
93.79
94.81
8.79
22.68
-3.39
2.66
7g
7h
7i
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Overian cancer
IGROV1
14.35
-5.60
5.97
7j
7k
7l
6.21
7m
7n
7o
Std
5.19
94.31
92.86
99.96
112.37
90.21
5.69
7.14
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
Standard streptomycin 7.5 lg/ml, pyrazinamide 10 lg/ml. All com-
pounds tested at concentration of 5, 10, 25 lg/ml. The active com-
pounds are marked in bold letters
0.04
-12.37
9.79
a
ATCC number
123

Med Chem Res
2-(6-Bromo-2H-chromen-3-yl)-6-chloroquinoxaline (3e) Yield:
94 %. m.p: 181–183 °C; IR (KBr) cm-1: 1713 (C=O),
1621 (C–O). H NMR (CDCl3): d ppm 7.19 (1H, m, Ar–
(Quinoxaline C3), 141.15 (Ar–C), 142.64 (Quinoxaline
C2), 166.49 (Sydnone C=O); Anal. Calcd. For
C16H10N4O2: C, 66.20; H, 3.47; N, 19.30. Found: C, 66.32;
H, 3.58; N, 19.41 %. MS (EI, 70 eV) m/z: 290.02 [M?1],
247.04, 232.02, 203.99.
1
H), 7.35–7.29 (1H, t, J = 18.0 Hz, Ar–H), 7.46 (1H, m,
Ar–H), 7.66–7.61 (1H, t, J = 15.0 Hz, Ar–H), 7.74–7.72
(1H, t, J = 15.0 Hz, Ar–H), 8.23 (1H, d, J = 9.0 Hz, Ar–
H), 8.82 (1H, s, Coumarin C4–H), 9.85 (1H, s, Quinoxaline
C3–H); 13C NMR (d, ppm, 400 MHz, CDCl3): 117.32
(Coumarin C8), 119.83 (Coumarin C10), 124.17 (Coumarin
C6), 124.82 (Coumarin C5), 129.26 (Coumarin C7), 129.41
(Coumarin C3), 129.49 (Quinoxaline C5), 130.34
(Quinoxaline C8), 130.38 (Quinoxaline C6), 132.91
(Quinoxaline C7), 142.24 (Quinoxaline C5a), 142.32
(Quinoxaline C8a), 144.67 (Quinoxaline C3), 145.76
(Coumarin C4), 147.99 (Coumarin C9), 154.78 (Quinoxalin
C2), 161.34 (Coumarin C=O); Anal. Calcd. For C17H10-
N2BrClO2: C, 54.65; H, 2.70; N, 7.50. Found: C, 54.77; H,
2.82; N, 7.63 %. MS (EI, 70 eV) m/z: 376.16 [M?4],
374.23 [M?2], 372.01 [M?1], 348.54, 346.32, 344.54,
317.36, 315.28, 313.75, 292.84, 290.49, 288.84.
6-Chloro-2-(3-phenylsydnon-4-yl)quinoxaline
(7b) Yield:
95 %. m.p: 118–120 °C; IR (KBr) cm-1: 1768 (C=O),
1
1662 (C=N). H NMR (CDCl3): d ppm 6.55–6.42 (5H, m,
Ar–H), 6.92–6.86 (2H, t, J = 18.0 Hz, Ar–H), 7.29 (1H,
m, Ar–H), 7.66–7.47 (1H, m, Ar–H), 9.61 (1H, s, C3-
quinoxaline ring) ppm; 13C NMR (d, ppm, 400 MHz,
CDCl3): 124.94 (Sydnone C4), 126.12 (Ar–C), 128.97 (Ar–
C), 129.32 (Ar–C), 129.35 (Quinoxaline C6), 129.68
(Quinoxaline C8), 130.23 (Quinoxaline C5), 130.52 (Ar–
C), 131.90 (Ar–C), 133.16 (Quinoxaline C7), 135.89
(Quinoxaline C5a), 140.41 (Quinoxaline C8a), 141.13
(Quinoxaline C3), 141.75 (Ar–C), 142.69 (Quinoxaline
C2), 166.64 (Sydnone C=O); Anal. Calcd. For C16H9N4-
ClO2: C, 59.18; H, 2.79; N, 17.25. Found: C, 59.30; H,
2.92; N, 17.36 %. MS (EI, 70 eV) m/z: 326.22 [M?2],
324.14 [M?1], 283.12, 281.23, 268.54, 266.08, 239.57,
237.14.
6-Bromo-3-(6-methylquinoxalin-2-yl)-2H-chromen-2-one (3f)
Yield: 90 %. m.p: 186–188 °C; IR (KBr) cm-1: 1723
(C=O), 1603 (C–O). 1H NMR (CDCl3): d ppm 2.36 (3H, s,
CH3), 7.37–7.31 (1H, t, J = 18.0 Hz, Ar–H), 7.39 (1H, m,
Ar–H), 7.41 (1H, m, Ar–H), 7.67–7.61 (1H, t,
J = 18.0 Hz, Ar–H), 7.71–7.68 (1H, t, J = 6.0 Hz, Ar–H),
7.85 (1H, m, Ar–H), 8.11–8.07 (1H, d, J = 12.0 Hz, Ar–
H), 8.82 (1H, s, Coumarin C4–H), 9.87 (1H, s, Quinoxaline
C3–H); 13C NMR (d, ppm, 400 MHz, CDCl3): 24.68
(CH3), 117.98 (Coumarin C8), 121.47 (Coumarin C10),
124.54 (Coumarin C6), 124.87 (Coumarin C5), 129.33
(Coumarin C7), 129.48 (Coumarin C3), 129.76 (Quinoxa-
line C5), 130.32 (Quinoxaline C8), 130.36 (Quinoxaline
C6), 133.18 (Quinoxaline C7), 142.32 (Quinoxaline C5a),
142.57 (Quinoxaline C8a), 144.73 (Quinoxaline C3), 146.13
(Coumarin C4), 147.76 (Coumarin C9), 155.45 (Quinoxa-
line C2), 161.83 (Coumarin C=O); Anal. Calcd. For C18-
H11N2BrO2: C, 58.88; H, 3.02; N, 7.63. Found: C, 59.01;
H, 3.15; N, 7.76 %. MS (EI, 70 eV) m/z: 368.38 [M?2],
366.21 [M?1], 340.25, 338.54, 309.34, 307, 284.56,
282.87.
6-Methyl-2-(3-phenylsydnon-4-yl) quinoxaline (7c) Yield:
92 %. m.p.: 121–123 °C; IR (KBr) cm-1: 1760 (C=O),
1
1658 (C=N). H NMR (CDCl3): d ppm 2.32 (3H, s, CH3),
6.58–6.44 (5H, m, Ar–H), 6.91–6.85 (2H, t, J = 18.0 Hz,
Ar–H), 7.20 (1H, m, Ar–H), 9.68 (1H, s, C3-quinoxaline
ring) ppm; 13C NMR (d, ppm, 400 MHz, CDCl3): 26.29
(CH3), 125.32 (Sydnone C4), 126.74 (Ar–C), 129.06 (Ar–
C), 129.35 (Ar–C), 129.41 (Quinoxaline C6), 129.75
(Quinoxaline C8), 130.29 (Quinoxaline C5), 130.57 (Ar–
C), 133.27 (Ar–C), 133.83 (Quinoxaline C7), 136.19
(Quinoxaline C5a), 140.78 (Quinoxaline C8a), 141.24
(Quinoxaline C3), 141.81 (Ar–C), 142.96 (Quinoxaline
C2), 166.70 (Sydnone C=O); Anal. Calcd. For
C17H12N4O2: C, 67.10; H, 3.97; N, 18.41. Found: C, 67.21;
H, 4.09; N, 18.53 %. MS (EI, 70 eV) m/z: 304.21 [M?1],
261.57, 246.34, 217.08.
2-(3-Chlorophenylsydnone-4-yl)quinoxaline
(7d) Yield:
96 %. m.p: 122–124 °C; IR (KBr) cm-1: 1759 (C=O),
1
1651 (C=N). H NMR (CDCl3): d ppm 6.82–6.70 (5H, m,
2-(3-Phenylsydnon-4-yl)quinoxaline (7a) Yield: 93 %.
m.p: 115–117 °C; IR (KBr) cm-1: 1765 (C=O), 1667
Ar–H), 6.98–6.92 (2H, t, J = 18.0 Hz, Ar–H), 7.18 (1H,
m, Ar–H), 9.66 (1H, s, C3-quinoxaline ring) ppm; 13C
NMR (d, ppm, 400 MHz, CDCl3): 124.99 (Sydnone C4),
126.43 (Ar–C), 128.97 (Ar–C), 129.34 (Ar–C), 129.48
(Quinoxaline C6), 129.86 (Quinoxaline C8), 130.34
(Quinoxaline C5), 130.90 (Ar–C), 131.92 (Ar–c), 133.29
(Quinoxaline C7), 136.78 (Quinoxaline C5a), 140.57
(Quinoxaline C8a), 141.11 (Quinoxaline C3), 141.78 (Ar–
C), 142.73 (Quinoxaline C2), 166.67 (Sydnone C=O);
Anal. Calcd. For C16H19N4ClO2: C, 59.18; H, 2.79; N,
1
(C=N). H NMR (CDCl3): d ppm 6.57–6.45 (5H, m, Ar–
H), 6.94–6.89 (2H, t, J = 15.0 Hz, Ar–H), 7.26 (1H, m,
Ar–H), 7.68–7.48 (1H, m, Ar–H), 9.64 (1H, s, C3-
quinoxaline ring) ppm; 13C NMR (d, ppm, 400 MHz,
CDCl3): 124.85 (Sydnone C4), 125.50 (Ar–C), 128.83 (Ar–
C), 129.28 (Ar–C), 129.31 (Quinoxaline C6), 129.42
(Quinoxaline C8), 130.04 (Quinoxaline C5), 130.41 (Ar–
C), 131.85 (Ar–C), 132.35 (Quinoxaline C7), 135.80
(Quinoxaline C5a), 140.26 (Quinoxaline C8a), 141.06
123

Med Chem Res
17.25. Found: C, 59.30; H, 2.91; N, 17.36 %. MS (EI,
70 eV) m/z: 326.45 [M?2], 324.23 [M?1], 283.48, 281.18,
268.34, 266.37, 239.42, 237.16.
[M?2], 368.10 [M?1], 327.12, 325.24, 312.84, 310.18,
283.06, 281.36.
2-(3-4-Bromophenylsydnon-4-yl)-6-chloroquinoxaline (7h)
Yield: 88 %. m.p: 119–121 °C; IR (KBr) cm-1: 1767
6-Chloro-2-(3-4-chlorophenylsydnon-4-yl)quinoxaline (7e)
Yield: 91 %. m.p: 124–126 °C; IR (KBr) cm-1: 1757
1
(C=O), 1669 (C=N). H NMR (CDCl3): d ppm 6.68–6.52
1
(C=O), 1669 (C=N). H NMR (CDCl3): d ppm 6.62–6.51
(5H, m, Ar–H), 6.89–6.83 (2H, t, J = 18.0 Hz, Ar–H),
9.56 (1H, s, C3-quinoxaline ring) ppm; 13C NMR (d, ppm,
400 MHz, CDCl3): 125.06 (Sydnone C4), 126.48 (Ar–C),
128.97 (Ar–C), 129.23 (Ar–C), 129.38 (Quinoxaline C6),
129.56 (Quinoxaline C8), 130.24 (Quinoxaline C5), 130.68
(Ar–C), 131.90 (Ar–C), 133.12 (Quinoxaline C7), 135.97
(Quinoxaline C5a), 140.37 (Quinoxaline C8a), 141.16
(Quinoxaline C3), 141.24 (Ar–C), 142.79 (Quinoxaline
C2), 166.66 (Sydnone C=O); Anal. Calcd. For C16H8N4-
BrClO2: C, 47.61; H, 2.00; N, 13.88. Found: C, 47.73; H,
2.12; N, 14.00 %. MS (EI, 70 eV) m/z: 407.41 [M?4],
405.24 [M?2], 403.75 [M?1], 364.21, 362.43, 360.56,
349.74, 347.45, 345.72, 320.05, 318.06, 316.48.
(5H, m, Ar–H), 6.98–6.95 (2H, t, J = 9.0 Hz, Ar–H), 9.60
(1H, s, C3-quinoxaline ring) ppm; 13C NMR (d, ppm,
400 MHz, CDCl3): 124.94 (Sydnone C4), 126.34 (Ar–C),
128.97 (Ar–C), 129.30 (Ar–C), 129.37 (Quinoxaline C6),
129.86 (Quinoxaline C8), 130.12 (Quinoxaline C5), 130.89
(Ar–C), 132.32 (Ar–C), 132.79 (Quinoxaline C7), 136.80
(Quinoxaline C5a), 140.84 (Quinoxaline C8a), 141.17
(Quinoxaline C3), 141.98 (Ar–C), 143.54, (Quinoxaline
C2) 167.11 (Sydnobe C=O); Anal. Calcd. For C16H8N4-
Cl2O2: C, 53.50; H, 2.25; N, 15.60. Found: C, 53.61; H,
2.37; N, 15.72 %. MS (EI, 70 eV) m/z: 362.20 [M?4],
360.14 [M?2], 358.05 [M?1], 319.24, 317.34, 315.12,
304.54, 302.01, 300.08, 275.66, 273.89, 271.46.
2-(3-4-Bromophenylsydnon-4-yl)-6-methylquinoxaline (7i)
Yield: 91 %. m.p: 123–125 °C; IR (KBr) cm-1: 1760
(C=O), 1659 (C=N). 1H NMR (CDCl3): d ppm 2.30 (3H, s,
CH3), 6.68–6.56 (5H, m, Ar–H), 6.99–6.93 (2H, t,
J = 18.0 Hz, Ar–H), 9.51 (1H, s, C3-quinoxaline ring) ppm;
13C NMR (d, ppm, 400 MHz, CDCl3): 24.38 (CH3), 124.87
(Sydnone C4), 125.59 (Ar–C), 128.92 (Ar–C), 129.30 (Ar–
C), 129.36 (Quinoxaline C6), 129.56 (Quinoxaline C8),
130.19 (Quinoxaline C5), 130.58 (Ar–C), 131.92 (Ar–C),
132.56 (Quinoxaline C7), 135.89 (Quinoxaline C5a), 140.34
(Quinoxaline C8a), 141.11 (Quinoxaline C3), 141.19 (Ar–C),
142.75 (Quinoxaline C2), 166.59 (Sydnone C=O); Anal.
Calcd. For C17H11N4BrO2: C, 53.28; H, 2.89; N, 14.62.
Found: C, 53.39; H, 3.01; N, 14.74 %. MS (EI, 70 eV) m/z:
385.23 [M?2], 383.10 [M?1], 342.04, 340.85, 327.61,
325.09, 298.32, 296.34.
2-(3-4-Chlorophenylsydnon-4-yl)-6-methylquinoxaline (7f)
Yield: 96 %. m.p: 111–113 °C; IR (KBr) cm-1: 1769
(C=O), 1660 (C=N). 1H NMR (CDCl3): d ppm 2.34 (3H, s,
CH3), 6.68–6.56 (5H, m, Ar–H), 7.01–6.96 (2H, t,
J = 15.0 Hz, Ar–H), 9.58 (1H, s, C3-quinoxaline ring)
ppm; 13C NMR (d, ppm, 400 MHz, CDCl3): 25.53 (CH3),
124.87 (Sydnone C4), 126.54 (Ar–C), 128.86 (Ar–C),
129.30 (Ar–C), 129.33 (Quinoxaline C6), 129.54
(Quinoxaline C8), 130.04 (Quinoxaline C5), 131.17 (Ar–
C), 131.65 (Ar–C), 133.42 (Quinoxaline C7), 136.75
(Quinoxaline C5a), 140.44 (Quinoxaline C8a), 141.13
(Quinoxaline C3), 141.59 (Ar–C), 142.97 (Quinoxaline
C2), 166.85 (Sydnone C=O); Anal. Calcd. For C17H11N4-
ClO2: C, 60.28; H, 3.27; N, 16.54. Found: C, 60.40; H,
3.39; N, 16.65 %. MS (EI, 70 eV) m/z: 340.25 [M?2],
338.06 [M?1], 297.56, 295.13, 282.38, 280.19, 253.72,
251.27.
2-(3-p-Tolylsydnon-4-yl)quinoxaline (7j) Yield: 93 %.
m.p: 124–126 °C; IR (KBr) cm-1: 1772 (C=O), 1665
(C=N). 1H NMR (CDCl3): d ppm 2.31 (3H, s, –CH3),
6.51–6.39 (5H, m, Ar–H), 6.92–6.88 (2H, t, J = 12.0 Hz,
Ar–H), 7.65–7.45 (1H, m, Ar–H), 9.58 (1H, s, C3-
quinoxaline ring) ppm; 13C NMR (d, ppm, 400 MHz,
CDCl3): 25.12 (CH3), 124.89 (Sydnone C4), 126.23 (Ar–
C), 128.97 (Ar–C), 129.43 (Ar–C), 129.56 (Quinoxaline
C6), 129.67 (Quinoxaline C8), 130.11 (Quinoxaline C5),
130.52 (Ar–C), 131.89 (Ar–C), 132.45 (Quinoxaline C7),
136.97 (Quinoxaline C5a), 140.58 (Quinoxaline C8a),
141.19 (Quinoxaline C3), 141.26 (Ar–C), 142.76
(Quinoxaline C2), 166.75 (Sydnone C=O); Anal. Calcd.
For C17H12N4O2: C, 67.10; H, 3.97; N, 18.41. Found: C,
67.21; H, 4.08; N, 18.53 %. MS (EI, 70 eV) m/z: 304.11
[M?1], 261.59, 246.85, 217.46.
2-(3-4-Bromophenylsydnon-4-yl)quinoxaline (7g) Yield:
89 %. m.p: 120–122 °C; IR (KBr) cm-1: 1753 (C=O),
1
1656 (C=N). H NMR (CDCl3): d ppm 6.69–6.57 (5H, m,
Ar–H), 6.97–6.94 (2H, t, J = 9.0 Hz, Ar–H), 7.29 (1H, m,
Ar–H), 9.55 (1H, s, C3-quinoxaline ring) ppm; 13C NMR
(d, ppm, 400 MHz, CDCl3): 124.62 (Sydnone C4), 126.47
(Ar–C), 128.98 (Ar–C), 129.56 (Ar–C), 129.63 (Quinoxa-
line C6), 129.89 (Quinoxaline C8), 130.12 (Quinoxaline
C5), 130.57 (Ar–C), 131.76 (Ar–C), 133.04 (Quinoxaline
C7), 136.72 (Quinoxaline C5a), 140.70 (Quinoxaline C8a),
141.43 (Quinoxaline C3), 141.57 (Ar–C), 143.36
(Quinoxaline C2), 166.80 (Sydnone C=O); Anal. Calcd.
For C16H9N4BrO2: C, 52.05; H, 2.46; N, 15.18 %. Found:
C, 52.16; H, 2.57; N, 15.31. MS (EI, 70 eV) m/z: 370.27
123

Med Chem Res
6-Chloro-2-(3-p-tolylsydnon-4-yl)quinoxaline (7k) Yield:
97 %. m.p: 113–115 °C; IR (KBr) cm-1: 1755 (C=O),
1657 (C=N). H NMR (CDCl3): d ppm 2.30 (3H, s, CH3),
ppm 6.67–6.56 (5H, m, Ar–H), 6.96–6.92 (2H, t,
J = 12.0 Hz, Ar–H), 9.57 (1H, s, C3-quinoxaline ring)
ppm; 13C NMR (d, ppm, 400 MHz, CDCl3): 124.96
(Sydnone C4), 126.76 (Ar–C), 128.98 (Ar–C), 129.30 (Ar–
C), 129.38 (Quinoxaline C6), 129.69 (Quinoxaline C8),
130.14 (Quinoxaline C5), 130.76 (Ar–C), 131.90 (Ar–C),
133.25 (Quinoxaline C7), 135.91 (Quinoxaline C5a), 141.17
(Quinoxaline C8a), 141.19 (Quinoxaline C3), 142.34 (Ar–
C), 143.74 (Quinoxaline C2), 166.63 (Sydnone C=O);
Anal. Calcd. For C16H8N5ClO4: C, 51.98; H, 2.18; N,
18.94. Found: C, 52.10; H, 2.31; N, 19.07 %. MS (EI,
70 eV) m/z: 371.24 [M?2], 369.36 [M?1], 328.06, 326.24,
313.09, 311.56, 284.02, 282.79.
1
6.62–6.50 (5H, m, Ar–H), 6.89–6.83 (2H, t, J = 18.0 Hz,
Ar–H), 9.64 (1H, s, C3-quinoxaline ring) ppm; 13C NMR
(d, ppm, 400 MHz, CDCl3): 25.46 (CH3), 124.97 (Sydnone
C4), 126.34 (Ar–C), 128.89 (Ar–C), 129.37 (Ar–C), 129.42
(Quinoxaline C6), 129.65 (Quinoxaline C8), 130.11
(Quinoxaline C5), 130.43 (Ar–C), 131.89 (Ar–C), 132.44
(Quinoxaline C7), 135.94 (Quinoxaline C5a), 140.65
(Quinoxaline C8a), 141.23 (Quinoxaline C3), 141.47 (Ar–
C), 142.78 (Quinoxaline C2), 166.69 (Sydnone C=O);
Anal. Calcd. For C17H11N4ClO2: C, 60.28; H, 3.27; N,
16.54. Found: C, 60.40; H, 3.38; N, 16.65 %. MS (EI,
70 eV) m/z: 340.30 [M?2], 338.24 [M?1], 297.06, 295.18,
282.24, 280.45, 253.67, 251.96.
6-Methyl-2-(3-4-nitrophenylsydnon-4-yl)quinoxaline (7o)
Yield: 96 %. m.p: 132–134 °C; IR (KBr) cm-1: 1768
1
(C=O), 1654 (C=N), 1536 (–N–O); H NMR (CDCl3): d
6-Methyl-2-(3-p-tolylsydnon-4-yl)quinoxaline (7l) Yield:
94 %. m.p: 129–131 °C; IR (KBr) cm-1: 1767 (C=O),
1658 (C=N). H NMR (CDCl3): d ppm 2.29 (3H, s, CH3),
ppm 2.30 (3H, s, –CH3), 6.68–6.57 (5H, m, Ar–H), 7.28
(1H, m, Ar–H), 7.65–7.49 (1H, m, Ar–H), 9.67 (1H, s, C3-
quinoxaline ring) ppm; 13C NMR (d, ppm, 400 MHz,
CDCl3): 25.82 (CH3), 124.95 (Sydnone C4), 126.42 (Ar–
C), 128.90 (Ar–C), 129.67 (Ar–C), 129.72 (Quinoxaline
C6), 129.89 (Quinoxaline C8), 130.23 (Quinoxaline C5),
130.54 (Ar–C), 131.97 (Ar–C), 133.38 (Quinoxaline C7),
137.79 (Quinoxaline C5a), 140.43 (Quinoxaline C8a),
141.17 (Quinoxaline C3), 141.56 (Ar–C), 142.87
(Quinoxaline C2), 166.89 (Sydnone C=O); Anal. Calcd.
For C17H11N5O4: C, 58.45; H, 3.17; N, 20.05. Found: C,
58.57; H, 3.30; N, 20.17 %. MS (EI, 70 eV) m/z: 349.10
[M?1], 306.54, 291.38, 262.24.
1
2.34 (3H, s, –CH3), 6.61–6.52 (5H, m, Ar–H), 7.23 (1H, m,
Ar–H), 7.61-7.42 (1H, m, Ar–H), 9.54 (1H, s, C3-quinox-
aline ring) ppm; 13C NMR (d, ppm, 400 MHz, CDCl3):
25.43 (CH3), 24.51 (CH3), 124.87 (Sydnone C4), 125.60
(Ar–C), 128.93 (Ar–C), 129.38 (Ar–C), 129.42 (Quinoxa-
line C6), 129.53 (Quinoxaline C8), 130.12 (Quinoxaline
C5), 130.55 (Ar–C), 131.90 (Ar–C), 132.46 (Quinoxaline
C7), 135.89 (Quinoxaline C5a), 140.34 (Quinoxaline C8a),
141.13 (Quinoxaline C3), 141.22 (Ar–C), 142.59
(Quinoxaline C2), 166.87 (Sydnone C=O); Anal. Calcd.
For C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found: C,
68.04; H, 4.54; N, 17.73 %. MS (EI, 70 eV) m/z: 318.24
[M?1], 275.47, 260.27, 231.75.
Methodology of in vitro anticancer screening
The human tumor cell lines of cancer screening panel were
grown in RPMI 1640 medium containing 5 % fetal bovine
serum and 2 mM L-glutamine. For a typical screening
experiment, cells were inoculated into 96-well microtiter
plates in 100 lL at plating densities ranging from 5000 to
40,000 cells/well depending on the doubling time of indi-
vidual cell lines. After cell inoculation, the microtiter plates
were incubated at 37 °C, 5 % CO2, 95 % air and 100 %
relative humidity for 24 h prior to the addition of samples 3a
and 3b. After 24 h, two plates of each cell line were fixed
in situ with TCA, to represent a measurement of the cell
population for each cell line at the time of sample addition
(Tz). Experimental drugs (compounds 3a and 3b) were sol-
ubilized in DMSO at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate was thawed
and diluted to twice the desired final maximum test con-
centration with complete medium containing 50 lg/ml
gentamicin. Additional four, 10-fold or ‘ log serial dilutions
were made to provide a total of five drug concentrations plus
2-(3-4-Nitrophenyl sydnon-4-yl)quinoxaline (7m) Yield:
83 %. m.p: 135–137 °C; IR (KBr) cm-1: 1758 (C=O),
1659 (C=N), 1539 (–N–O). 1H NMR (CDCl3): d ppm
6.68–6.56 (5H, m, Ar–H), 6.98–6.95 (2H, t, J = 9.0 Hz,
Ar–H), 7.69–7.51 (1H, m, Ar–H), 9.62 (1H, s, C3-
quinoxaline ring) ppm; 13C NMR (d, ppm, 400 MHz,
CDCl3): 124.95 (Sydnone C4), 126.59 (Ar–C), 128.92 (Ar–
C), 129.34 (Ar–C), 129.39 (Quinoxaline C6), 129.76
(Quinoxaline C8), 130.74 (Quinoxaline C5), 130.86 (Ar–
C), 131.98 (Ar–C), 133.42 (Quinoxaline C7), 138.78
(Quinoxaline C5a), 141.15 (Quinoxaline C8a), 141.23
(Quinoxaline C3), 141.98 (Ar–C), 143.55 (Quinoxaline
C2), 166.94 (Sydnone C=O); Anal. Calcd. For C16H9N5O4:
C, 57.32; H, 2.71; N, 20.89. Found: C, 57.44; H, 2.83; N,
231.02 %. MS (EI, 70 eV) m/z: 335.20 [M?1], 292.41,
277.62, 248.18.
6-Chloro-2-(3-4-nitrophenylsydnon-4-yl)quinoxaline (7n)
Yield: 90 %. m.p: 124–126 °C; IR (KBr) cm-1: 1760
1
(C=O), 1656 (C=N), 1541 (–N–O). H NMR (CDCl3): d
123

Med Chem Res
control. Aliquots of 100 ll of these different drug dilutions
were added to the appropriate microtiter wells already con-
taining 100 ll of medium, resulting in the required final drug
concentrations. Following drug addition, the plates were
incubated for an additional 48 h at 37 °C, 5 % CO2, 95 %
air, and 100 % relative humidity. For adherent cells, the
assay was terminated by the addition of cold TCA. Cells
were fixed in situ by the gentle addition of 50 ll of cold 50 %
(w/v) TCA (final concentration, 10 % TCA) and incubated
for 60 min at 4 °C. The supernatant was discarded, and the
plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 ll) at 0.4 % (w/v) in
1 % acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. After staining,
unbound dye was removed by washing five times with 1 %
acetic acid and the plates were air dried. The bound stain was
subsequently solubilized with 10 mM Trizma base, and the
absorbance was read on an automated plate reader at a
wavelength of 515 nm. For suspension cells, the methodol-
ogy was the same except that the assay was terminated by
fixing settled cells at the bottom of the wells by gently adding
50 ll of 80 % TCA (final concentration, 16 % TCA). Using
the seven absorbance measurements [time zero, (Tz), control
growth, (C), and test growth in the presence of drug at five
concentration levels (Ti)], the percentage growth was cal-
culated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as:
tuberculae H37Rv (Sahm and Washington, 1991). Antibiotic
standards used were streptomycin and Pyrazinamide. The
procedure followed for anti-tubercular activity involved the
use of Middle-brook 7H-9 broth and standard strain of M.
tuberculosis H37Rv. The basal medium was prepared
according to manufacturer’s instructions (Hi-Media) and
sterilized by autoclaving. Then broth (4.5 ml) was poured
into each one of the sterile bottles. To this, ADC (0.5 ml)
supplement was added. The supplement consists of catalase,
dextrose and BSA fraction v. Then a stock solution was
prepared (10 lg/ml). From this appropriate amount of
solution was transferred to media bottles to achieve final
concentrations of 25, 50,100 lg/ml. Finally, 10 ll suspen-
sion of M. tuberculosis H37Rv strain (1 lakh organisms/ml
adjusted by McFarmland’s turbidity standard) was trans-
ferred to each of the tubesand incubated at 37 °C. Along with
this, one growth control without compound and drug controls
were also set up. The bottles were inspected for growth twice
a week for a period of 3 weeks. The appearance of turbidity
indicated the growth and infers the resistance to the com-
pound. The growth was confirmed by making a smear from
each bottle and performing a ZN strain.
Acknowledgments The authors acknowledge the NCI, NIH,
Bethesda, USA for selecting samples for in vitro anticancer analyses
under DTP. The authors are grateful to NGH College of Dental sci-
ences, Belgaum, Karnataka, India for anti-tubercular activity. The
authors wish to thank the University Scientific Instrumentation Centre
(USIC), Karnataka University, Dharwad, NMR Research Centre,
Indian Institute of Science (IISc), Bengaluru, India for carrying out
the spectral analyses. The authors (AAK and RRK) are also thankful
to UGC, New Delhi for providing financial assistant under UGC-UPE
thrust area ‘‘Antitumor activity: An Integrated Approach’’. Vide F.
No. 14-3/2012 (NS/PE).
½ðTi À TzÞ=ðC À Tzފ  100 for concentrations for which Ti [ = ¼ Tz
:
½ðTi À TzÞ=TzŠ  100 for concentrations for which Ti\Tz:
Three dose response parameters were calculated for each
experimental agent. Growth inhibition of 50 % (GI50) was
calculated from [(Ti-Tz)/(C-Tz)] 9 100 = 50, which was
the drug concentration resulting in a 50 % reduction in the
net protein increase (as measured by SRB staining) in control
cells during the drug incubation. The drug concentration
resulting in total growth inhibition (TGI) was calculated
from Ti = Tz. The LC50 (concentration of drug resulting in
a 50 % reduction in the measured protein at the end of the
drug treatment as compared to that at the beginning)
indicating a net loss of cells following treatment was
calculated from [(Ti-Tz)/Tz] 9 100 = -50. Values were
calculated for each of these three parameters if the level of
activity was reached. However, if the effect was not reached
or was exceeded, the value for that parameter was expressed
as greater or less than the maximum or minimum
concentration tested (Kattimani et al., 2013; http://dtp.nic.
nih.gov/docs/compare.html).
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