A new approach to construct a fused 2-ylidene chromene ring: Highly regioselective synthesis of novel chromeno quinoxalines

Article abstract of DOI:10.1039/c2ob25416f

Regioselective construction of a fused 2-ylidene chromene ring was achieved for the first time by using AlCl₃-induced C-C bond formation followed by Pd/C-Cu mediate coupling-cyclization strategy. A number of chromeno[4,3-b]quinoxaline derivatives were prepared by using this strategy. Single crystal X-ray diffraction study of a representative compound e.g. 6-(2,2-dimethylpropylidene)-4-methyl-6H-chromeno[4,3-b]quinoxalin-3-ol confirmed the presence of an exocyclic C-C double bond with Z-geometry. The crystal structure analysis and hydrogen bonding patterns of the same compound along with its structure elaboration via propargylation followed by Sonogashira coupling of the resulting terminal alkyne is presented. A probable mechanism for the formation of 2-ylidene chromene ring is discussed. Some of the compounds synthesized showed anticancer properties when tested in vitro.

Full text of DOI:10.1039/c2ob25416f

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 4774
www.rsc.org/obc
PAPER
A new approach to construct a fused 2-ylidene chromene ring: highly
regioselective synthesis of novel chromeno quinoxalines†
K. Shiva Kumar,a D. Rambabu,a,b Bagineni Prasad,a Mohammad Mujahid,a G. Rama Krishna,c
M. V. Basaveswara Rao,d C. Malla Reddy,c G. R. Vanaja,e Arunasree M. Kallee and Manojit Pal*b
Received 25th February 2012, Accepted 24th April 2012
DOI: 1039.1039/c2ob25416f
Regioselective construction of a fused 2-ylidene chromene ring was achieved for the first time by using
AlCl3-induced C–C bond formation followed by Pd/C–Cu mediate coupling-cyclization strategy.
A number of chromeno[4,3-b]quinoxaline derivatives were prepared by using this strategy. Single crystal
X-ray diffraction study of a representative compound e.g. 6-(2,2-dimethylpropylidene)-4-methyl-6H-
chromeno[4,3-b]quinoxalin-3-ol confirmed the presence of an exocyclic C–C double bond with
Z-geometry. The crystal structure analysis and hydrogen bonding patterns of the same compound along
with its structure elaboration via propargylation followed by Sonogashira coupling of the resulting
terminal alkyne is presented. A probable mechanism for the formation of 2-ylidene chromene ring is
discussed. Some of the compounds synthesized showed anticancer properties when tested in vitro.
anticancer agents.2 Several quinoxalines are also known as antic-
Introduction
ancer drugs3 e.g. NSC 339004 (D, Fig. 1). Thus appropriate
The chromene framework with an exocyclic double bond at C-2
(A, Fig. 1) constitutes a unique class of O-heterocycles and
found to be integral part of progestins B. In addition to their uses
in many therapeutic areas, progestins are known as effective
combination of structural features of A (or B) with a quinoxaline
nucleus in a single template was expected to provide new chemi-
cal space (e.g. C, Fig. 1) to develop novel anticancer agents.3
Since cancer is the second leading cause of death4 worldwide,
the search for new anticancer agents is therefore desirable. We
now report the evaluation of the anticancer properties of novel
small molecules based on C, the synthesis of which has been
carried out using a newly developed strategy.
The only method known for constructing a 2-ylidene chro-
mene ring involves the reaction of a Grignard reagent prepared
from the corresponding benzyl bromide or chloride with a
lactone e.g. 2,2,4-trimethyl-1H-chromeno[3,4-f]quinolin-5(2H)-
one followed by treating the resulting hemi-acetal intermediate
with p-toluenesulfonic acid.1 Alternatively, the lactone can be
reacted with an organo-lithium reagent (prepared via lateral
lithiation of a 2-methyl thiophene derivative with LDA) to give
the hemi-acetal intermediate which under protic acid conditions
provides a 2-ylidene chromene derivative.5 While effective for
the preparation a particular class of compounds, e.g. benzyli-
denes or thienyl methylidenes, these methods lack generality and
also require cumbersome preparation of moisture sensitive
Grignard or organo-lithium reagents. One of the major goals of
the present work was therefore to develop a more straightforward
and robust methodology for the preparation of our target mol-
ecules C. Transition metal mediated synthesis of O-containing
heterocycles via intramolecular addition of an O-nucleophile
across the C–C triple bond has become a powerful tool in
organic chemistry.6 A large variety of structurally diverse com-
pounds has been synthesized by using this strategy and Pd has
Fig. 1 Design of new and potential anticancer agents C.
aInstitute of Life Sciences, University of Hyderabad Campus,
Gachibowli, Hyderabad 500046, India
bDepartment of Chemistry, K. L. University, Vaddeswaram, Guntur 522
502, Andhra Pradesh, India
cDepartment of Chemical Sciences, Indian Institute of Science
Education and Research, Kolkata, West Bengal 741252, India
dDepartment of Chemistry, Krishna University, Machilipatnam-521001,
A. P., India
eDepartment of Animal Sciences, School of Life Sciences,
University of Hyderabad, Hyderabad 500046, India.
E-mail: manojitpal@rediffmail.com
†Electronic supplementary information (ESI) available: Copies of NMR
spectra for all new compounds. CCDC 858005. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI:
10.1039/c2ob25416f
4774 | Org. Biomol. Chem., 2012, 10, 4774–4781
This journal is © The Royal Society of Chemistry 2012

View Article Online
Scheme 2 Preparation of 4-(3-chloroquinoxalin-2-yl)benzene-1,3-diol (3).
Scheme 1 Intramolecular addition of O–H across the C–C triple bond.
Table 1 Effect of reaction conditions on the Pd-catalyzed coupling of
4-(3-chloroquinoxalin-2-yl)benzene-1,3-diol (3a) with phenylacetylene
(4a)a
been the most utilized metal in these catalytic processes.
Recently, the use of Pd/C as an inexpensive, easily separable and
recyclable catalyst has also been explored for the synthesis of
O-heterocycles.7 We envisaged that a phenolic OH present at the
δ-position to the alkynyl moiety of E might undergo an intramo-
lecular cyclization in a regioselective manner under an appropri-
ate reaction conditions (Scheme 1). The challenging issues
however to be addressed here were (i) installation of a phenolic
OH at the δ-position to the alkynyl moiety, (ii) its reactivity
towards transition metal-activated alkynes, (iii) regioselectivity
and (iv) the optimal catalyst system.
Entry
Catalyst
Solvent
Time (h)
%Yieldb
1
2
3
4
5
6
7
8
10% Pd/C-PPh3
10% Pd/C-PPh3
PdCl2(PPh3)2
Pd(OAc)2-PPh3
10% Pd/C-PPh3
10% Pd/C-PPh3
10% Pd/C-PPh3
10% Pd/C-PPh3
Toluene
Toluene
Toluene
Toluene
1,4-Dioxane
CH3CN
EtOH
6
10
8
8
6
7
8
8
82
80
75c
70
78
75
70
72
Results and discussion
The AlCl3 induced C–C bond forming reaction, an effective
strategy for the introduction of a phenol moiety to an N-hetero-
cyclic ring8 was adopted to generate the key starting material.
Thus the chloro compound 3 required for our study, was pre-
pared (Scheme 2) via AlCl3 mediated reaction of 2,3-dichloro-
quinoxaline (1) with phenols (2) in 1,2-dichloroethane (DCE).
To this end, we initially choose to examine the coupling-cycli-
zation of 3a (Z = H) with phenylacetylene (4a) in the presence
of 10% Pd/C-PPh3-CuI7 and Et3N in toluene (Table 1). To our
satisfaction, the reaction proceeded smoothly with high regios-
electivity affording the compound 5a as the only product (entry
1, Table 1). The reaction was carried out for 6 h and a longer
reaction time did not improve the product yield (entry 2,
Table 1). The use of other catalysts, e.g. PdCl2(PPh3)2 and Pd-
(OAc)2-PPh3, afforded the product 5a but in inferior yield
(entries 3–4, Table 1). The absence of CuI did not provide 5a
indicating the need of CuI for the reaction to proceed. The reac-
tion afforded only traces amount of 5a in the absence of Pd-cata-
lyst whereas the use of other solvents, e.g. 1,4-dioxane, CH3CN,
EtOH or MeOH decreased the product yield (entries 5–8,
Table 1). Thus a combination of Pd/C-PPh3-CuI and Et3N in
toluene was found to be optimum for the present coupling-cycli-
zation reaction.
With the optimized reaction conditions in hand, a variety of
terminal alkynes (4) were reacted with chloro compound 3
(Table 2). Both aromatic (entries 1–2, 4 Table 2) and aliphatic
alkynes (entries 3, 5–12 Table 2) participated well in this reac-
tion, affording the desired products in good yields. All the pro-
ducts isolated were characterized by spectral (NMR, IR and MS)
data. Depending on the nature of substituent present a singlet or
triplet was observed in the region δ 6.3–6.5 in the 1H NMR
spectra of 5, indicating the presence of a vinylic proton. Notably,
we were able to isolate the 7-membered ring product i.e.
6-hexyl-4-methylbenzo[b]quinoxalinoxepin-3-ol (5kk) in 10%
yield along with 5k when the reaction of 3b and the alkyne 4k
MeOH
a All reactions were carried out by using 3a (1.0 equiv.), 4a (1.5 equiv.),
Pd-catalyst (0.05 equiv.) or Pd/C (0.035 equiv.), PPh3 (0.35 mmol),
CuI (0.06 equiv.), Et3N (3.0 equiv.) in a solvent (5 mL) at 80 °C under
N2. b Isolated yields. c PPh3 was not used.
was conducted in the presence of PdCl2(PPh3)2 and CuI in
toluene at 80 °C for 8 h (Scheme 3). The presence of a vinylic
proton was indicated by a singlet observed at δ 6.25 in the H
1
NMR spectra of 5kk compared to a triplet at δ 6.31 for 5k.
Nevertheless, the molecular structure of 5f was established
unambiguously by single crystal X-ray diffraction study
(Fig. 2)9a confirming the presence of an exocyclic C–C double
bond with Z-geometry.9b The compound 5f crystallizes in the
monoclinic P21/c space group with one molecule in the asym-
metric unit (Z: 4 Z′: 1) (Fig. 2). The quinoxaline ring of the mol-
ecule in the asymmetric unit is not in coplanar with 2,2-
dimethylpropylidene moiety of the molecule. The torsion angle
of the quinoxaline ring and neopentane moieties are −174.2(2)
(C15–C16–C17–C18). Interestingly, the molecule in the asym-
metric unit form strong hydrogen bonding via O–H⋯N synthon
(N1⋯O2 2.874(2), N1⋯H2 2.06) and are forming the 1D corru-
gated layers as shown in Fig. 3 and 4 along the ac plane. The
free hydrogen of the O–H group interacts with quinoxaline
moiety and forms O–H⋯N intermolecular hydrogen bond and
are stabilized by dimer formation through aromatic C–H⋯π
interactions. These interactions propagate in 2D corrugated type
layered structure in bc plane as shown in Fig. 5.
Further structure elaboration of 5f was carried out via
propargylation followed by Sonogashira coupling of the
resulting terminal alkyne (6) with an iodoarene (7) to give
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 4774–4781 | 4775

View Article Online
Table 2 Pd/C-mediated synthesis of 6-substituted 6H-chromeno[4,3-b]-
quinoxalin-3-ol (5)a
Table 2 (Contd.)
Entry 3; Z =
4; R =
Products (5)
% Yieldb
80
Entry 3; Z =
4; R =
Products (5)
% Yieldb
82
8
3b
3b
3b
4g CMe2OH
4h (CH2)3Me
4i (CH2)4Me
1
3a; H
4a; C6H5
9
78
78
2
3a
3a
4b; C6H4Me-p
80
10
3
4
4c; C3H6CN-p
80
80
11
12
3b
3b
4j (CH2)5Me
80
80
3b; Me 4d; C6H4Me-p
4k (CH2)9Me
5
6
3b
3b
4c
78
80
a All reactions were carried out by using 3 (1.0 equiv.), 4 (1.5 equiv.), 10%
Pd/C (0.035), PPh3 (0.35 equiv.), CuI (0.06 equiv.) and Et3N (3.0 equiv.)
in toluene (5 mL) at 80 °C for 6 h under N2. b Isolated yields.
4e; CMe3
7
3b
4f; SiMe3
82
Scheme 3 Preparation of 7-membered ring product 5kk.
(Z)-6-(2,2-dimethylpropylidene)-4-methyl-3-(3-phenyl prop-2-
ynyloxy)-6H-chromeno[4,3-b]quinoxaline (8) (Scheme 4). Thus
compound 5f was reacted with propargyl bromide in the
4776 | Org. Biomol. Chem., 2012, 10, 4774–4781
This journal is © The Royal Society of Chemistry 2012

View Article Online
Fig. 5 The formation of 2D corrugated layer with channels via
O–H⋯N intermolecular hydrogen bonding and stabilization by aromatic
C–H⋯π interactions.
Fig. 2 ORTEP representation of 5f (thermal ellipsoids are drawn at
50% probability level).
Scheme 4 Structure elaboration of compound 5f.
Fig. 3 The formation of O–H⋯N intermolecular hydrogen bonding in
the ac plane.
Scheme 5 Cu-mediated cyclization of compound 9.
carried out in the presence of Pd(PPh3)2Cl2 and CuI in DMSO to
give the desired product 8 in 50% yield.
Mechanistically, the reaction seems to proceed via Pd/C–Cu
mediated in situ generation of an internal alkyne E (Scheme 1)
which undergoes intramolecular cyclization in the presence of
CuI present in the reaction mixture. To gain further evidence on
intermediacy of alkyne E a representative alkyne i.e. 4-(3-(phenyl-
ethynyl)quinoxalin-2-yl)benzene-1,3-diol (9) prepared via the
reaction of 3a and 4a was treated with CuI in toluene in the pres-
ence of Et3N at 80 °C for 6 h when the cyclized product 5a was
isolated in 67% yield (Scheme 5).
Fig. 4 The formation of corrugated tapes.
presence of K2CO3 in DMF to give the corresponding terminal
alkyne i.e. (Z)-6-(2,2-dimethylpropylidene)-4-methyl-3-(prop-2-
ynyloxy)-6H-chromeno[4,3-b]quinoxaline (6) in 80% yield.
Subsequent coupling of the alkyne 6 with iodobenzene 7 was
The present AlCl3-induced C–C bond formation followed by
Pd/C–Cu mediated coupling-cyclization strategy provided a
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 4774–4781 | 4777

View Article Online
Table 3 The % inhibition of growth of cancer cell lines by compound 5a
K-562 (leukemia) MD-AMB-231 (breast)
Compound (5) 100 μM 10 μM 1 μM 100 μM 10 μM 1 μM
thin layer chromatography (TLC) on silica gel plates (60 F254),
visualizing with ultraviolet light or iodine spray. Flash chromato-
graphy was performed on silica gel (100–200 and
230–400 mesh) using hexene, ethyl acetate, dichloromethane.
1H NMR and 13C NMR spectra were determined in CDCl3,
DMSO-d6, solution by using 400 and 100 MHz spectrometers,
respectively. Proton chemical shifts (δ) are relative to tetra-
methylsilane (TMS, δ = 0.00) as internal standard and expressed
in ppm. Spin multiplicities are given as s (singlet), d (doublet),
t (triplet) and m (multiplet) as well as b (broad). Coupling con-
stants (J) are given in hertz. Infrared spectra were recorded on a
FT-IR spectrometer. Melting points were determined using
melting point apparatus and are uncorrected. MS spectra were
obtained on a mass spectrometer. Chromatographic purity by
HPLC (Agilent 1200 series Chem Station software) was deter-
mined by using area normalization method and the condition
specified in each case: column, mobile phase (range used), flow
rate, detection wavelength, and retention times.
5b
5c
5e
5g
5i
72.3
38.4
37.4
31.0
76.0
55.9
31.5
32.8
34.2
53.3
50.1
25.1
30.3
22.8
45.8
90.4
91.1
90.7
90.5
85.9
58.1
74.9
52.8
75.6
42.3
45.0
63.6
48.0
40.4
41.7
a Data presented are average of three experiments.
number of chromeno[4,3-b]quinoxaline derivatives in good
yields. In compared to other Pd-catalysts the Pd/C catalyst used
here can be easily separated from the product by simple filtration
thereby decreasing the possibility of metal contamination in the
products isolated. Moreover, due to the well known uses of Pd/C
for hydrogenation reaction in industry the present strategy has
potential for scale up synthesis.
General procedure for the preparation of 4-(3-chloroquinox-
alin-2-yl)benzene-1,3-diol (3). A mixture of 2,3-dichloroqui-
noxaline (1, 1.0 equiv.), an appropriate arene (2, 1.0 equiv.) and
AlCl3 (1.1 equiv.) in dichloroethane (5 mL) was stirred at 80 °C
for 30 min under a nitrogen atmosphere. After completion of the
reaction, the mixture was cooled to room temperature, poured
into ice-cold water (15 mL), stirred for 10 min and then extracted
with ethylacetate (3 × 20 mL). The organic layers were collected,
combined, washed with cold water (2 × 20 mL), dried over anhy-
drous Na2SO4 and concentrated under vacuum. The residue
obtained was purified by column chromatography on silica gel
(230–400 mesh) using ethylacetate–hexene to give the desired
product.
Some of the compounds10 (5) synthesized were tested in vitro
against leukemia (K-562) and breast (MD-AMB-231) cancer cell
lines in a MTT assay. Harmine, a member of beta-carboline family
of compounds showed cytotoxicity against HL60 and K562 cell
lines.11 While 5b and 5i showed good activity against leukemia
cells (Table 3), compounds 5b, 5c, 5e, 5g and 5i were found to be
effective against breast cancer and 5c was best among them
(Table 3, entry 2). Notably, IC50 value of Harmine was found to
be 45 and 54 μM when tested against K562 and MDA-MB 231
cell lines in our MTT assay. Overall, this research has led to the
identification of small molecule-based agents that could be useful
for the potential treatment of leukemia or breast cancer. The com-
pounds presented here therefore may have medicinal value.
4-(3-Chloroquinoxalin-2-yl)benzene-1,3-diol
(3a). Yellow
1
solid; mp 176–178 °C; H NMR (400 MHz, DMSO-d6) δ 9.75
(bs, 1H), 9.64 (bs, 1H), 8.10–8.08 (m, 1H), 8.05–8.02 (m, 1H),
7.89–7.85 (m, 2H), 7.15 (d, J = 8.4, 1H), 6.40–6.34 (m, 2H);
13C NMR (100 MHz, DMSO-d6) δ 160.3, 156.9, 153.3, 148.5,
140.7, 140.5, 131.7, 131.2, 130.9, 129.1, 128.1, 116.3, 107.0,
102.9; HPLC: 97.8%, column: Zorbax XDB C-18 150 × 4.6 mm
5μ, mobile phase A: 0.05% formic acid in water, mobile phase
B: CH3CN (gradient) T/%B: 0/20, 2/20, 9/95, 12/95, 15/20,
18/20; flow rate: 1.0 mL min−1; UV 240 nm, retention time
7.8 min; m/z (CI) 273 (M + 1, 100); IR (KBr) vmax 3289, 1623,
Conclusions
In conclusion, we have developed a new strategy that allows
regioselective construction of fused 2-ylidene chromene ring and
thereby rapid access to a library of small molecules based on a
novel structural motif. This is the first example of combining the
AlCl3-induced C–C bond formation with a Pd/C–Cu mediated
coupling-cyclization strategy leading to various chromeno[4,3-b]-
quinoxaline derivatives. Single crystal X-ray diffraction study of a
representative compound confirmed the presence of an exocyclic
C–C double bond with Z-geometry. The crystal structure analysis
and hydrogen bonding patterns of the same compound along with
its structure elaboration via propargylation followed by Sonoga-
shira coupling of the resulting terminal alkyne is presented.
A probable mechanism for the formation of 2-ylidene chromene
ring is discussed. Some of the compounds synthesized showed anti-
cancer properties when tested in vitro. Our study therefore suggests
that the 6-ylidene chromeno quinoxalin framework could be an
attractive template for the identification of novel anticancer agents.
1598 cm−1
.
4-(3-Chloroquinoxalin-2-yl)-2-methylbenzene-1,3-diol (3b).
1
Yellow solid; mp 161–163 °C; H NMR (400 MHz, DMSO-d6)
δ 9.58 (bs, 1H), 9.03 (bs, 1H), 8.10–8.03 (m, 2H), 7.88–7.84
(m, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 2.04
(s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 158.1, 154.7, 153.4,
148.1, 140.54, 140.51, 131.1, 130.9, 129.0, 128.10, 128.08,
116.2, 111.6, 107.0, 9.3; IR (KBr) vmax 3379, 1637, 1688 cm−1
m/z (CI) 287 (M + 1, 100%).
;
Experimental
General procedure for the preparation of chromeno[4,3-b]qui-
noxalin-3-ol (5). A mixture of 4-(3-chloroquinoxalin-2-yl)-
benzene-1,3-diol (3) (1.0 mmol), 10% Pd/C (0.035 mmol), PPh3
(0.35 mmol), CuI (0.06 mmol), and triethylamine (3.0 mmol) in
toluene (5 mL) was stirred at 25–30 °C for 30 min under
Chemistry
General methods. Unless stated otherwise, reactions were per-
formed under nitrogen atmosphere. Reactions were monitored by
4778 | Org. Biomol. Chem., 2012, 10, 4774–4781
This journal is © The Royal Society of Chemistry 2012

View Article Online
nitrogen. A terminal alkyne (4) (1.5 mmol) slowly was added to
this mixture. The mixture was then stirred at room temperature
for 1.0 h and then at 75–80 °C for 6–7 h. After completion of
the reaction the mixture was cooled to room temperature, diluted
with EtOAc (50 mL), and filtered through Celite. The organic
layers were collected, washed with water (3 × 30 mL), dried over
anhydrous Na2SO4, filtered, and concentrated. The crude residue
was purified by column chromatography on silica gel
(230–400 mesh) using hexane–ethyl acetate to afford the desired
product.
(s, 3H), 2.25 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 162.8,
155.0, 146.1, 142.7, 142.5, 141.7, 137.7, 132.0, 131.9, 131.8,
130.8, 129.9 (2C), 129.7 (2C), 129.1, 128.6, 126.8, 112.8,
110.8, 109.0, 102.8, 21.4, 9.3; HPLC: 98.4%, column: Zorbax
XDB C-18 150 × 4.6 mm 5μ, mobile phase A: 0.1% formic acid
in water, mobile phase B: CH3CN (gradient) T/%B: 0/80, 2/80,
9/98, 13/98, 15/80, 18/80; flow rate: 1.0 mL min−1; UV 264 nm,
retention time 9.6 min; IR (KBr) vmax 2923, 1598, 1344 cm−1
m/z (CI) 367 (M + 1, 100%).
;
5-(3-Hydroxy-4-methyl-6H-chromeno[4,3-b]quinoxalin-6-
ylidene)pentanenitrile (5e). Light red solid; mp 146–148 °C;
1H NMR (400 MHz, DMSO-d6) δ 10.31 (bs, 1H), 8.01 (d, J =
8.8 Hz, 1H), 7.95–7.91 (m, 2H), 7.76–7.68 (m, 2H), 6.73 (d, J =
8.4 Hz, 1H), 6.32 (t, J = 7.6 Hz, 1H), 2.62–2.58 (m, 2H),
2.57–2.53 (m, 2H), 2.16 (s, 3H), 1.87–1.85 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 160.4, 153.3, 147.5, 143.0, 142.7,
141.5, 141.1, 130.8, 129.7, 129.0, 128.6, 123.3, 121.0, 111.3,
111.1, 110.5, 110.2, 24.7, 24.1, 16.5, 8.4; HPLC: 97.8%,
column: Zorbax XDB C-18 150 × 4.6 mm 5μ, mobile phase A:
0.01% formic acid in water, mobile phase B: CH3CN (gradient)
T/%B: 0/80, 2/80, 9/98, 12/98, 15/80, 18/80; flow rate: 0.8 mL
min−1; UV 255 nm, retention time 4.9 min; IR (KBr) vmax 3101,
1603, 1343 cm−1; m/z (CI) 344 (M + 1, 100%).
6-Benzylidene-6H-chromeno[4,3-b]quinoxalin-3-ol (5a). Light
red solid; mp 170–172 °C; H NMR (400 MHz, DMSO-d6) δ
1
10.52 (bs, 1H), 8.25–8.23 (m, 1H), 8.04–7.99 (m, 4H),
7.83–7.78 (m, 2H), 7.53–7.49 (m, 2H), 7.40–7.36 (m, 1H),
7.30–7.28 (m, 1H), 6.79–6.78 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 162.8, 154.9, 146.8, 142.6, 141.6, 141.5, 134.6
(2C), 131.0, 130.0 (3C), 129.2, 129.1, 128.6, 128.0, 127.8,
126.8, 112.9, 110.8, 108.8, 102.8; HPLC: 97.1%, column:
Zorbax XDB C-18 150 × 4.6 mm 5μ, mobile phase A: 0.1%
formic acid in water, mobile phase B: CH3CN (gradient) T/%B:
0/80, 2/80, 9/98, 13/98, 15/80, 18/80; flow rate: 1.0 mL min−1
;
UV 229 nm, retention time 6.1 min; IR (KBr) vmax 3439, 2923,
1614 cm−1; m/z (CI) 337 (M − 1, 100%).
4-Methyl-6-(4-methylbenzylidene)-6H-chromeno[4,3-b]quinoxa-
lin-3-ol (5b). Light red solid; mp 231–233 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.44 (bs, 1H), 8.05 (d, J = 8.4 Hz,
1H), 7.95–7.92 (m, 2H), 7.82–7.70 (m, 2H), 7.74–7.67 (m, 2H),
7.63–7.58 (m, 1H), 7.25–7.23 (m, 2H), 7.19 (s, 1H), 6.71–6.68
(m, 1H), 3.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 162.8,
155.0, 146.2, 142.7, 142.6, 141.70, 141.68, 137.7, 131.9, 131.8,
130.8, 130.0, 129.9, 129.7, 129.2, 129.1, 128.6, 126.8, 112.8,
110.8, 109.0, 102.8, 21.4; HPLC: 97.9%, column: Zorbax XDB
C-18 150 × 4.6 mm 5μ, mobile phase A: 0.05% formic acid
in water, mobile phase B: CH3CN (gradient) T/%B: 0/80, 2/80,
9/98, 13/98, 15/80, 18/80; flow rate: 1.0 mL min−1; UV 229 nm,
6-(2,2-Dimethylpropylidene)-4-methyl-6H-chromeno[4,3-b]-
quinoxalin-3-ol (5f). Yellow solid; mp 182–184 °C; 1H NMR
(400 MHz, DMSO-d6) δ 8.20 (d, J = 8.4 Hz, 1H), 7.95–7.91 (m,
2H), 7.63–7.59 (m, 2H), 6.62 (d, J = 8.4 Hz, 1H), 6.48 (s, 1H),
5.34 (bs, 1H), 2.32 (s, 3H), 1.39 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 160.4, 153.5, 145.8, 142.8, 142.6, 141.55, 141.52,
130.7, 129.7, 129.0, 128.6, 123.4, 121.3, 111.4, 110.8, 110.5,
31.9, 30.1 (3C), 9.2; HPLC: 96.3%, column: Zorbax XDB C-18
150 × 4.6 mm 5μ, mobile phase A: 0.1% formic acid in water,
mobile phase B: CH3CN (Isocratic); A : B (10 : 90); flow rate:
1.0 mL min−1; UV 230 nm, retention time 6.0 min; IR (KBr)
v
max 3143, 1603, 1351 cm−1; m/z (CI) 333 (M + 1, 100%).
retention time 7.5 min; IR (KBr) vmax 2923, 1511, 1331 cm−1
m/z (CI) 353 (M + 1, 100%).
;
4-Methyl-6-((trimethylsilyl)methylene)-6H-chromeno[4,3-b]-
1
quinoxalin-3-ol (5g). Yellow solid; mp 194–196 °C; H NMR
(400 MHz, DMSO-d6) δ 10.33 (bs, 1H), 8.00–7.98 (m, 1H),
7.92–7.89 (m, 2H), 7.72–7.66 (m, 2H), 6.70 (d, J = 8.4 Hz, 1H),
6.35 (s, 1H), 2.17 (s, 3H), 0.29 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 160.6, 157.9, 153.5, 143.2, 143.1, 141.9, 140.1,
131.4, 130.0, 129.5, 128.8, 123.6, 111.6, 110.9, 110.7, 106.4,
9.1, 0.001 (3C); IR (KBr) vmax 2943, 1628, 1441 cm−1; m/z (CI)
350 (M + 1, 100%).
5-(3-Hydroxy-6H-chromeno[4,3-b]quinoxalin-6-ylidene)penta-
nenitrile (5c). Light red solid; mp 172–174 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.43 (bs, 1H), 8.13 (d, J = 8.4 Hz,
1H), 7.94–7.90 (m, 2H), 7.75–7.69 (m, 2H), 6.65 (d, J = 8.8 Hz,
1H), 6.50 (s, 1H), 6.28 (t, J = 7.2 Hz, 1H), 2.59–2.52 (m, 4H),
1.82–1.79 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 162.8,
155.6, 147.4, 142.7, 142.6, 141.5, 141.3, 130.9, 129.8, 129.0,
128.7, 126.8, 121.0, 120.4, 112.4, 110.6, 102.7, 24.1, 18.0, 15.9;
HPLC: 96.6%, column: Zorbax XDB C-18 150 × 4.6 mm 5μ,
mobile phase A: 0.01% formic acid in water, mobile phase B:
CH3CN (gradient) T/%B: 0/60, 2/60, 9/98, 12/98, 15/60, 18/60;
flow rate: 0.8 mL min−1; UV 230 nm, retention time 6.2 min;
IR (KBr) vmax 3140, 3140, 1331 cm−1; m/z (CI) 328 (M − 1,
100%).
6-(2-Hydroxy-2-methylpropylidene)-4-methyl-6H-chromeno-
[4,3-b]quinoxalin-3-ol (5h). Light red solid; mp 176–178 °C;
1H NMR (400 MHz, DMSO-d6) δ 10.34 (bs, 1H), 8.04–8.00 (m,
1H), 7.96–7.91 (m, 2H), 7.77–7.68 (m, 2H), 6.74–6.71 (m, 1H),
6.55 (s, 1H), 5.00 (bs, 1H), 2.16 (s, 3H), 1.58 (s, 6H); 13C NMR
(100 MHz, DMSO-d6) δ 160.4, 153.4, 144.0, 142.9, 142.7,
141.5, 141.4, 130.9, 129.8, 129.0, 128.6, 123.4, 121.9, 111.4,
111.0, 110.4, 64.1, 31.5 (2C), 9.0; HPLC: 97.5%, column:
Zorbax XDB C-18 150 × 4.6 mm 5μ, mobile phase A: 0.05%
formic acid in water, mobile phase B: CH3CN (gradient) T/%B:
4-Methyl-6-(4-methylbenzylidene)-6H-chromeno[4,3-b]quinoxa-
lin-3-ol (5d). Light red solid; mp 270–272 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.37 (bs, 1H), 8.05 (d, J = 8.8 Hz,
1H), 7.96–7.91 (m, 2H), 7.83–7.81 (m, 2H), 7.75–7.69 (m, 2H),
7.52–7.45 (m, 2H), 7.25 (s, 1H), 6.77 (d, J = 8.8 Hz, 1H), 2.34
0/40, 2/40, 9/98, 12/98, 15/40, 18/40; flow rate: 0.8 mL min−1
;
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 4774–4781 | 4779

View Article Online
6-hexyl-4-methylbenzo[b]quinoxalinoxepin-3-ol
(5kk). This
UV 230 nm, retention time 8.0 min; IR (KBr) vmax 3230, 1605,
1102 cm−1; m/z (CI) 333 (M − 1, 100%).
compound was isolated in 10% yield along with 5k when the
reaction of 3b and the alkyne 4k was conducted in the presence
of PdCl2(PPh3)2, CuI in toluene at 80 °C for 8 h; yellow solid;
4-Methyl-6-pentylidene-6H-chromeno[4,3-b]quinoxalin-3-ol
(5i). Yellow solid; mp 161–163 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.28 (bs, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.95–7.91
(m, 2H), 7.63–7.59 (m, 2H), 6.62 (d, J = 8.4 Hz, 1H), 6.31 (t,
J = 8.0 Hz, 1H), 2.13 (s, 3H), 2.01–2.03 (m, 2H), 1.54–1.50 (m,
2H), 1.42–1.38 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 159.2, 151.1, 150.1, 143.3, 142.7,
141.6, 129.7, 129.3, 129.2, 128.8, 123.0, 112.8, 112.3, 111.2,
110.7, 110.0, 31.2, 25.1, 22.3, 14.0, 8.1; HPLC: 96.9%, column:
Zorbax XDB C-18 150 × 4.6 mm 5μ, mobile phase A: 0.05%
formic acid in water, mobile phase B: CH3CN (gradient) T/%B:
1
mp 201–203 °C; H NMR (400 MHz, CDCl3) δ 8.05–8.03 (m,
1H), 7.97–7.94 (m, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.67–7.64 (m,
2H), 6.78 (d, J = 8.8 Hz, 1H), 6.25 (s, 1H), 5.67 (bs, 1H), 2.48
(t, J = 7.6 Hz, 2H), 2.30 (s, 3H), 1.76–1.72 (m, 2H), 1.43–1.41
(m, 2H), 1.33–1.32 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 159.9, 157.2, 151.0 (2C), 140.7, 129.74,
129.69, 129.4 (3C), 129.0 (2C), 128.0, 127.9, 116.3, 112.8,
31.6, 29.7, 28.9, 29.7, 22.5, 14.0, 9.1; m/z (CI) 361 (M + 1,
100%).
0/80, 2/80, 9/98, 13/98, 15/80, 18/80; flow rate: 1.0 mL min−1
;
Preparation of 6-(2,2-dimethylpropylidene)-4-methyl-3-(prop-
2-ynyloxy)-6H-chromeno[4,3b]quinoxaline (6). To a mixture of
(Z)-6-(2,2-dimethylpropylidene)-4-methyl-6H-chromeno[4,3-b]-
quinoxalin-3-ol (5f) (0.60 mmol), and K2CO3 (0.90 mmol) in
anhydrous DMF (5 mL) was added propargyl bromide
(0.72 mmol) at room temperature. The resulting mixture was
then stirred at room temperature for 14 h. Upon completion of
the reaction, the reaction mixture was diluted with ice-cold
water. The solid obtained was filtered and washed with water to
afford the desired compound 6 as yellow solid (yield 80%); mp
146–148 °C; 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.8 Hz,
1H), 7.97–7.91 (m, 2H), 7.66–7.59 (m, 2H), 6.83 (d, J = 8.8 Hz,
1H), 6.49 (s, 1H), 4.81 (d, J = 2.4 Hz, 2H), 2.55 (t, J = 2.8 Hz,
1H), 2.32 (s, 3H), 1.40 (s, 9H); 13C NMR (100 MHz, CDCl3)
δ 159.2, 153.4, 145.8, 142.7, 142.6, 142.2, 142.0, 129.7, 129.0,
128.9, 128.6, 123.3, 122.0,114.0, 113.2, 107.1, 78.3, 75.8, 56.3,
31.9, 29.9 (3C), 9.0; IR (KBr) vmax 3231, 2958, 2111, 1608,
1115; m/z (CI) 371 (M + 1, 100%).
UV 255 nm, retention time 9.6 min; IR (KBr) vmax 2925, 1605,
1434 cm−1; m/z (CI) 333 (M + 1, 100%).
6-Hexylidene-4-methyl-6H-chromeno[4,3-b]quinoxalin-3-ol
(5j). Yellow solid; mp 215–217 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.28 (bs, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.90–7.87
(m, 2H), 7.72–7.65 (m, 2H), 6.70–6.68 (m, 1H), 6.32 (t, J = 8.0
Hz, 1H), 2.43–2.39 (m, 2H), 2.12 (s, 3H), 1.54–1.51 (m, 2H),
1.37–1.33 (m, 4H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 160.1, 153.6, 146.7, 143.3, 142.7,
141.6, 129.7, 129.2, 128.7, 123.0, 121.0, 112.8, 112.3, 111.2,
110.9, 110.7, 31.6, 28.7, 25.1, 22.4, 14.1, 8.1; HPLC: 97.8%,
column: Zorbax XDB C-18 150 × 4.6 mm 5μ, mobile phase A:
0.05% formic acid in water, mobile phase B: CH3CN (gradient)
T/%B: 0/90, 2/90, 9/98, 13/98, 15/90, 18/90; flow rate: 1.0 mL
min−1; UV 230 nm, retention time 8.6 min; IR (KBr) vmax 2927,
1603, 1433 cm−1; m/z (CI) 347 (M + 1, 100%).
6-Heptylidene-4-methyl-6H-chromeno[4,3-b]quinoxalin-3-ol
(5k). Yellow solid; mp 201–203 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.27 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.91–7.88
(m, 2H), 7.72–7.68 (m, 2H), 6.70 (d, J = 8.8 Hz, 1H), 6.30 (t,
J = 8.0 Hz, 1H), 2.40 (q, J = 7.2 Hz, 2H), 2.12 (s, 3H),
1.54–1.48 (m, 2H), 1.37–1.33 (m, 2H), 1.28–1.27 (m, 4H), 0.84
(t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 160.4,
153.5, 146.7, 143.1, 142.6, 141.5, 141.4, 130.6, 129.6, 129.0,
128.6, 123.4, 112.7, 111.2, 111.0, 110.5, 31.5, 29.0, 28.9, 25.0,
22.5, 14.4, 8.3; HPLC: 97.1%, column: Zorbax XDB C-18
150 × 4.6 mm 5μ, mobile phase A: 0.05% formic acid in water,
mobile phase B: CH3CN (gradient) T/%B: 0/90, 2/90, 9/98,
13/98, 15/90, 18/90; flow rate: 1.0 mL min−1; UV 230 nm,
Preparation of 6-(2,2-dimethylpropylidene)-4-methyl-3-(3-phe-
nylprop-2-ynyloxy)-6H-chromeno[4,3-b]quinoxaline
(8). A
mixture of iodo benzene (7) (0.26 mmol) in DMSO (5 mL),
Pd(PPh3)2Cl2 (0.006 mmol), CuI (0.01 mmol), and Et3N
(0.39 mmol) were created under a nitrogen atmosphere. The
acetylenic compound 6 (0.26 mmol) was added slowly to the
mixture with stirring. The reaction mixture was allowed to stir at
room temperature for 12 h. Then the reaction mixture was
diluted with water (15 mL) and the product was extracted with
ethyl acetate (3 × 15 mL). The organic layers were collected,
combined, dried over anhydrous Na2SO4, filtered and concen-
trated under vacuum. The residue thus obtained was purified by
column chromatography to afford the desired product as yellow
solid (yield 50%); mp 134–136 °C; 1H NMR (400 MHz,
CDCl3) δ 8.30 (d, J = 8.8 Hz, 1H), 7.97–7.91 (m, 2H),
7.65–7.59 (m, 2H), 7.44 (dd, J = 6.8, 1.6 Hz, 2H), 7.33–7.29
(m, 3H), 6.93 (d, J = 8.8 Hz, 1H), 6.49 (s, 1H), 5.03 (s, 2H),
2.34 (s, 3H), 1.39 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
159.6, 153.5, 145.8, 142.57 (2C), 142.3, 142.0, 131.8, 129.7,
129.04, 129.01, 128.9, 128.8, 128.7, 128.4, 128.3, 123.4,
122.15, 122.11, 114.0, 112.9, 107.4, 87.5, 83.6, 57.2, 31.9, 29.9
(3C), 9.1; IR (KBr) vmax 2924, 2854, 2151, 1600, 1115; m/z (CI)
447 (M + 1, 100%).
retention time 10.6 min; IR (KBr) vmax 2924, 1601, 1326 cm−1
m/z (CI) 361 (M + 1, 100%).
;
4-Methyl-6-nonylidene-6H-chromeno[4,3-b]quinoxalin-3-ol
(5l). Brick red solid; mp 185–187 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.28 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.93–7.90
(m, 2H), 7.73–7.68 (m, 2H), 6.71 (d, J = 8.0 Hz, 1H), 6.31 (t,
J = 8.0 Hz, 1H), 2.45–2.41 (m, 2H), 2.14 (s, 3H), 1.54–1.53 (m,
2H), 1.39–1.25 (m, 14H), 0.82 (t, J = 7.0 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 168.0, 159.8, 159.5, 150.9, 150.0,
140.7, 140.4, 130.1, 129.5, 129.0, 128.6, 128.5, 120.8, 116.3,
112.9, 112.8, 35.4, 31.7, 29.4, 29.3, 29.14, 29.12, 28.8, 26.6,
22.5, 14.3, 9.6; IR (KBr) vmax 2854, 1592, 1338 cm−1; m/z (CI)
417 (M + 1, 100%).
Preparation of 5a from 9. A mixture of 4-(3-(phenylethynyl)-
quinoxalin-2-yl)benzene-1,3-diol (9) (1.0 mmol) (obtained
via the reaction of 3a and 4a in low yield at 45–50 °C),
4780 | Org. Biomol. Chem., 2012, 10, 4774–4781
This journal is © The Royal Society of Chemistry 2012

View Article Online
CuI (0.06 mmol), and Et3N (3.0 mmol) in toluene (5 mL) was
stirred at 80 °C for 6 h. After completion of the reaction the
mixture was cooled to room temperature, diluted with EtOAc
(30 mL), and filtered through Celite. The organic layer was col-
lected, washed with water (3 × 30 mL), dried over anhydrous
Na2SO4, filtered, and concentrated under vacuum. The crude
residue was purified by column chromatography on silica gel
(230–400 mesh) using hexane/ethyl acetate to afford the desired
product 5a.
(5 mg mL−1 in PBS) was added to the fresh medium. After 2 h
incubation at 37 °C, 100 μL of DMSO was added to each well
and plates were agitated for 1 min. Absorbance was read at
570 nm on a multi-well plate reader (Synergy Mx, Biotek Inc.,
USA). Percent inhibition of proliferation was calculated as a frac-
tion of control (without compound).
Acknowledgements
Single crystal X-ray data for compound 5f. Single crystals
suitable for X-ray diffraction of 5f was grown from dimethyl for-
mamide. The crystals were carefully chosen using a stereo zoom
microscope supported by a rotatable polarizing stage. The data
was collected at room temperature on Bruker’s KAPPA APEX II
CCD Duo with graphite monochromated Mo-Kα radiation
(0.71073 Å). The crystals were glued to a thin glass fibre using
FOMBLIN immersion oil and mounted on the diffractometer.
The intensity data were processed using Bruker’s suite of data
processing programs (SAINT), and absorption corrections were
applied using SADABS.12 The crystal structure was solved by
direct methods using SHELXS-97 and the data was refined by
full matrix least-squares refinement on F2 with anisotropic dis-
placement parameters for non-H atoms, using SHELXL-97.13
Crystal data of 5f: Molecular formula = C21H20N2O2, Formula
weight = 332.15, crystal system = monoclinic, space group = P2
(1)/C, a = 19.497(2) Å, b = 5.589(7) Å, c = 16.139(2) Å,
The authors thank Prof. J. Iqbal for encouragement and support.
M. P. thanks DST, new Delhi, India for financial support
(Grant no. SR/S1/OC-53/2009). K. S. thanks UGC, New Delhi,
India for a Dr D. S. Kothari Post doctoral fellowship
[No. F.4-2/2006(BSR)/13-24/2010(BSR)]. B. Prasad thanks
CSIR, New Delhi, India for a Junior Research Fellowship.
Notes and references
1 C. M. Tegley, L. Zhi, K. B. Marschke, M. M. Gottardis, Q. Yang and
T. K. Jones, J. Med. Chem., 1998, 41, 4354.
2 S. Lundgren, Acta Oncol., 1992, 31, 709.
3 H. Gao, E. F. Yamasaki, K. K. Chan, L. L. Shen and R. M. Snapka,
Mol. Pharmacol., 2003, 63, 1382.
4 D.M. Parkin, F. Bray, J. Ferlay and P. Pisani, Ca-Cancer J. Clin., 2005,
55, 74.
5 A. R. Hudson, S. L. Roach, R. I. Higuchi, D. P. Phillips,
R. P. Bissonnette, W. W. Lamph, J. Yen, Y. Li, M. E. Adams,
L. J. Valdez, A. Vassar, C. Cuervo, E. A. Kallel, C. J. Gharbaoui,
D. E. Mais, J. N. Miner, K. B. Marschke, D. Rungta, A. Negro-Vilar and
L. Zhi, J. Med. Chem., 2007, 50, 4699.
6 See for example: (a) B. M. Trost and A. J. Frontier, J. Am. Chem. Soc.,
2000, 122, 11727; (b) T. Yao, X. Zhang and R. C. Larock, J. Am. Chem.
Soc., 2004, 126, 11164; (c) N. G. Kundu, M. Pal, J. S. Mahanty and
M. De, J. Chem. Soc., Perkin Trans. 1, 1997, 2815.
V = 1687.6(4) Å3, T = 296(2) K, Z = 4, Dc = 1.316 Mg m−3
,
μ(Mo-Kα) = 0.09 mm−1, 17 443 reflections measured, 4075
independent reflections, 2670 observed reflections [I > 2.0σ(I)],
R1_obs = 0.040, goodness of fit = 1.042.
7 M. Pal, Synlett, 2009, 2896.
Biological assay
8 (a) M. Pal, V. R. Batchu, K. Parasuraman and K. R. Yeleswarapu, J. Org.
Chem., 2003, 68, 6806; (b) M. Pal, V. R. Batchu, I. Dager, N. K. Swamy
and S. Padakanti, J. Org. Chem., 2005, 70, 2376.
9 (a) Crystallographic data (excluding structure factors) for 5f have been
deposited with the Cambridge Crystallographic Data Centre as sup-
plementary publication numbers CCDC 858005 (b) Based on the results
of single crystal X-ray diffraction study of compound 5f it was assumed
that rest of the compounds represented by 5 possess exocyclic C–C
double bond with Z-geometry.
10 For our earlier effort on identification of novel anticancer agents, see:
(a) N. K. Swamy, L. K. Tatini, J. M. Babu, P. Annamalai and M. Pal,
Chem. Commun., 2007, 1035; (b) C. Rajitha, P. K. Dubey, V. Sunku,
F. J. Piedrafita, V. R. Veeramaneni and M. Pal, Eur. J. Med. Chem., 2011,
46, 4887; (c) N. Mulakayala, D. Rambabu, M. R. Raja, M. Chaitanya,
C. S. Kumar, A. M. Kalle, G. R. Krishna, C. M. Reddy, M. V. B. Rao
and M. Pal, Bioorg. Med. Chem., 2012, 20, 759.
Cell lines and culture conditions. Human metastatic breast
cancer cells, MDA-MB 231 and human chronic myeloid leuke-
mia cells, K562, were procured from National Center for Cell
Sciences, Pune, India. All cells were grown in RPMI-1640 sup-
plemented with 10% heat inactivated fetal bovine serum (FBS),
100 IU mL−1 penicillin, 100 mg mL−1 streptomycin and 2 mM-
glutamine. Cultures were maintained in a humidified atmosphere
with 5% CO2 at 37 °C. The cells were subcultured twice each
week, seeding at a density of about 2 × 103 cells per mL.
MTT assay. Cell viability was determined by (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Cells (5 × 103 cells per well) were seeded to 96-well
culture plate and cultured with or without compounds at 1, 10
and 100 μM concentration for 24 h in a final volume of 200 μL.
After treatment, the medium was removed and 20 μL of MTT
11 F. Jahaniani, S. A. Ebrahimi, N. Rahbar-Roshandel and M. Mahmoudian,
Phytochemistry, 2005, 66, 1581.
12 Bruker SADABS V2008-1, Bruker AXS, Madison, WI, USA, 2008.
13 G. M. Sheldrick, SHELX-97, Program for Crystal Structure Determi-
nation, University of Göttingen, 1997.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 4774–4781 | 4781