Multi-component synthesis and in vitro and in vivo anticancer activity of novel arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides

Article abstract of DOI:10.1016/j.ejmech.2012.02.004

A three component one-pot protocol has been investigated for the synthesis of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1 from the commercially available materials. The title compounds 1 were also synthesized by a step-wise method and found to b

Full text of DOI:10.1016/j.ejmech.2012.02.004

European Journal of Medicinal Chemistry 50 (2012) 274e279
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Multi-component synthesis and in vitro and in vivo anticancer activity of novel
arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides
,
a
a
a
b
b
E. Rajanarendar a , S. Raju , M. Nagi Reddy , S. Rama Krishna , L. Hari Kiran , A. Ram Narasimha Reddy ,
*
Y. Narasimha Reddy b
a Department of Chemistry, Kakatiya University, Vidyaranyapuri, Warangal, A.P. 506 009, India
b Department of Pharmacology and Toxicology, Kakatiya University, Warangal, A.P. 506 009, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A three component one-pot protocol has been investigated for the synthesis of arylmethylene bis-iso-
xazolo[4,5-b]pyridine-N-oxides 1 from the commercially available materials. The title compounds 1 were
also synthesized by a step-wise method and found to be identical with one-pot synthesis by spectral and
analytical data. The newly synthesized compounds were evaluated for their in vitro anticancer activity
against human cancer cell lines and in vivo anticancer activity on EAC-bearing mice. Compound 1a was
found to be the most active both in in vitro and in vivo cytotoxic studies.
Received 17 August 2011
Received in revised form
2 February 2012
Accepted 2 February 2012
Available online 10 February 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Novel arylmethylene bis-isoxazolo[4,5-b]
pyridine-N-oxides
Multi-component synthesis
In vitro anticancer activity
In vivo anticancer activity
1. Introduction
an important role in anticancer activity [10,11]. Nitric oxide acting
as electron donor or electron acceptor, may participate in the
Multi-component reactions (MCRs) have proved to be remark-
ably successful in generating products in a single synthetic opera-
tion [1,2] and are of increasing importance in organic and medicinal
chemistry [3e5]. In MCRs a high degree of molecular diversity can
be introduced by variation of a single component at a time.
Considering that, rapidity and diversity are key factors in modern
drug discovery, MCR strategies offer significant advantages over
conventional linear-type syntheses, owing to their exceptional
synthetic efficiency [6]. MCRs contribute to the requirements of an
environmentally friendly process by reducing the number of
synthetic steps, energy consumption and waste production. Fused
heterocycles are reported to show a wide variety of applications in
medicinal chemistry [7,8]. Despite several reports on fused
heterocycles, there is a continuing demand for development of new
methods for synthesis of novel fused heterocycles due to their
plethora of medicinal applications [9].
reaction with inorganic molecules, DNA, proteins, consequently
modifies enzymatic and transcriptional factor activities. Nitric
oxide may be responsible for destruction of cellular components
[12e14]. Owing to the potential activity of pyridine-N-oxides as
anti-HIV and antiviral [15e17] agents, and isoxazole nitric oxide
proved to act as anticancer molecule [18], we would like to intro-
duce two isoxazolo[4,5-b] pyridine-N-oxide systems in a single
molecular frame work to study the biological activity. Inspired by
the potential activity of pyridine-N-oxides, and to develop new
synthetic strategies, we performed a multi-component one-pot
synthesis on 3,5-dimethyl-4-nitroisoxazole 5 to synthesize the title
compounds. The present work is an extension of our on-going
efforts towards the development and identification of new mole-
cules with potential biological activity [19e21].
2. Results and discussion
Nitric oxide (NO) is a highly reactive molecule involved in
a number of physiological and pathological processes, which plays
2.1. Chemistry
3,5-Dimethyl-4-nitroisoxazole 5 represents a versatile building
block bearing a number of different functionalities which can be
selectively reacted to generate molecularly diverse products, and
* Corresponding author. Tel.: þ91 870 2456363; fax: þ91 870 2438800.
E-mail address: eligeti_rajan@yahoo.co.in (E. Rajanarendar).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.004

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 274e279
275
O
H3C
N
+
N
O2N
CH3
H3C
NO2
Ar
Ar
O
O
Cyclocondensation
Ar
+
2 Ar
CH3
N
N
O
Ar
O
O
2
N
3
N
+
CH3
O
1
Michael reaction
Knoevenagel
Reaction
H3C
NO2
H3C
NO2
O
H3C
NO2
+
+
Ar
Ar
H
N
N
N
O
CH3
O
CH3
O
5
4
5
6
Fig. 1. A disconnection of compound 1.
we reported many reactions by describing the reactivity of iso-
xazole 5 [22e24]. Our approach to the development of multi-
component one-pot synthesis is based on the creation of building
blocks containing several reactive centers, which can be selectively
reacted. As a part of our endeavour to develop diversity-oriented
synthesis using poly-functional scaffold such as 3,5-dimethyl-4-
nitroisoxazole 5, and products with high pharmacological activity,
we now report a new one-pot, three-component procedure (actu-
ally involving 5 components, MCR-5) which allows the synthesis of
arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides could be
prepared through a multi-component procedure, in which 4 could
be generated first from 5, and 6 by Knoevenagel condensation and
these reacted in situ with 5 through Michael type addition to give 2,
which subsequently undergoes cyclocondensation with 2 mol of
acetophenone 3 to give the title compounds 1.
In a typical experiment, equimolar amounts of 3,5-dimethyl-4-
nitroisoxazole 5, aromatic aldehyde 6, were reacted in the pres-
ence of piperidine in methanol for 1 h at 80 ꢀC with stirring and one
equivalent of 5 was added later and the refluxing continued for
another 1 h at 80 ꢀC, finally 2 equivalents of acetophenone 3 was
added and the reaction was carried out for another 3 h at 80 ꢀC.
After completion of the reaction (monitored by TLC), the solvent
was removed by vacuum distillation and the crude product that
obtained was purified by recrystallization from ethanol.
Compounds 1aej, were isolated in moderate to good yields. The
products were identified as arylmethylene bis-isoxazolo[4,5-b]
pyridine-N-oxides 1 on the basis of elemental analyses and by
spectroscopic data (IR, 1H NMR, 13C NMR and MS) (Scheme 1).
In order to study the scope of this reaction, different substituted
aromatic aldehydes and acetophenones were utilized in this multi-
component synthesis. The desired product was obtained in each
case with moderate to good yield. Finally, the results indicate that
arylmethylene
bis-isoxazolo[4,5-b]pyridine-N-oxides
from
commercially available materials.
We proposed the following retro-synthetic analysis for the
target compounds (Fig. 1). This disconnection looked particularly
attractive due to the commercial availability of 3,5-dimethyl-4-
nitroisoxazole (5), aromatic aldehydes (6) and aromatic ketones
(3) on the market. We reasoned that compound 1 could be ob-
tained, when 2 reacts with 3 by cyclocondensation. Significantly,
compound 2, precursor of 1, could be achieved from 4 and 5 by
Michael type reaction. Finally 4 could arise by Knoevenagel
condensation of 5 and 6, where 5 is the building block in this multi-
component synthesis.
Considering that, tandem synthesis involving sequential
Knoevenagel and Michael processes [25], we anticipated that
O
+
N
H3C
N
H3C
NO2
O
O
Ar
Ar
O
ethanol
N
+
+
Ar
CH3
Ar
H
piperidine
CH3
O
,
5 h.
O
Ar
(1 equiv.)
(2 equiv.)
3
N
(2 equivalents)
5
N
6
+
CH3
O
1
Ar =C6H5, 2-CH3C6H4, 2-CH3OC6H4, 2-ClC6H4, 4-ClC6H4, 4-CH3OC6H4, Ar1 = C6H5, 4-CH3C6H4,
4-CH3OC6H4, 2-OHC6H4 , 4-ClC6H4, 4-NO2C6H4
Scheme 1. Multi-component synthesis of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1.

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E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 274e279
O
H3C
H3C
NO2
NO2
O2N
CH3
H3C
NO2
5
ethanol
piperidine,
Ar
Ar
H
N
N
+
N
N
ethanol
CH3
O
O
O
O
piperidine,
2
4
5
6
Ar
O
ethanol
Ar
CH3
C
piperidine,
(2 moles)
3
O
+
N
H3C
N
O
Ar
Ar
O
N
Ar
N
O
+
CH3
1
Scheme 2. Step-wise synthesis of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1.
our method is compatible with various functional groups present
on benzene ring and the approach proved to be of general appli-
cability. This synthetic strategy permits the introduction of
a diverse array of substituents on to the benzene ring of aldehyde as
well as on ketone.
groups such as methyl and methoxy on the benzene ring present at
6-position (1f, 1g) increased the anticancer activity and has shown
good activity, whereas the introduction of electron attracting
chloro, nitro groups (1i, 1j) on benzene ring decreased the activity
and has shown moderate activity. Compounds 1a and 1h have more
cytotoxic activity. The results suggested that, pyridine-N-oxides
with unsubstituted and hydroxy substituted played a vital role in
the modulation of cytotoxic activity. Among all the compounds
tested 1aej, 1a (bearing unsubstituted benzene) is the most cyto-
toxic in all the three cell lines.
Having obtained the excellent cytotoxic properties of 1a and 1h
in in vitro studies, we evaluated the in vivo antitumor properties of
these compounds in EAC-bearing mice by using liquid tumor model
[30e32]. The effect of the compounds 1a and 1h in two different
doses (5 mg/kg and 10 mg/kg) on body weight, mean survival time,
% increase life span, tumor volume, packed cell volume, tumor cell
count (viable cells) was studied and the results were presented in
Table 2. The compound 1a has significant activity (p < 0.001) in
both doses, decreased the body weight of EAC-bearing mice,
whereas the compound 1h has the significant activity only at
10 mg/kg dose. 1a significantly increased the mean survival time
and deceased the tumor volume, packed cell volume, and viable
tumor cell count in both doses, whereas 1h only in higher dose
The following findings were pivotal to the establishment of
a one-pot procedure. In order to investigate the evidence for the
formation of 1aej, we also carried out a step-wise synthesis. We
reacted 3,5-dimethyl-4-nitroisoxazole 5 (1 mmol) with different
aromatic aldehydes 6 (1 mmol) in alcohol under refluxing condi-
tion on a hot-water bath for 1 h in the presence of catalytic amount
of piperidine, which led to the formation of nitrostyrylisoxazoles 4
by Knoevenagel condensation [26]. Compound 4 (1 mmol) was
later reacted with 5 (1 mmol) in ethanol containing piperidine for
1 h at 80 ꢀC. This reaction afforded Michael type adducts 2 in good
yields [27]. Finally, compound 2 (1 mmol) was reacted with ace-
tophenone 3 (2 mmol) in ethanol containing piperidine for 3 h at
80 ꢀC, which resulted in the formation of title compounds 1aej.
(Scheme 2). The compounds 1aej, obtained in a one-pot multi-
component synthesis and by a step-wise synthesis were found to
be similar from mps, 1H NMR, 13C NMR and mass spectra and by
microanalytical data.
To the best of our knowledge, this report is the first of its kind to
construct arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxide in
a single molecular framework from commercially available mate-
rials in a one-pot synthesis as well as in a step-wise synthesis.
Table 1
Cytotoxic activities of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1aej on
human cancer cell lines [in vitroa (IC50 g/mLb)].
, m
2.2. Anticancer activity
Compound
Ar
Ar1
HeLa
EAC
MCF-7
1a
1b
1c
1d
1e
1f
1g
1h
C6H5
C6H5
C6H5
C6H5
C6H5
28.61
62.40
65.52
79.03
70.74
42.04
47.91
32.83
70.22
80.05
3.84
26.57
65.12
51.85
70.37
60.60
48.42
41.70
36.30
78.35
75.55
4.81
31.87
60.70
57.35
72.06
74.26
44.29
45.96
33.55
79.05
76.22
4.19
2-CH3C6H4
2-CH3OC6H4
2-ClC6H4
4-ClC6H4
C6H5
C6H5
C6H5
C6H5
C6H5
The newly synthesized arylmethylene bis-isoxazolo[4,5-b]pyri-
dine-N-oxides 1aej were evaluated for in vitro anticancer activity
against human cancer cells according to the procedures described
in literature [28,29]. The tumor cell line panel consisted of HeLa,
EAC and MCF-7 cells. Cisplatin (DDP) was used as the reference
drug. The results are presented in Table 1. IC50 values were based on
doseeresponse curves (IC50 value, defined as the concentration
corresponding to 50% growth inhibition). As shown in Table 1, some
of the compounds showed excellent activity against the tumor
cells. All the compounds showed a moderate to good anticancer
activity against three different cell lines and they are not selective
towards any cell lines. While substitution of electron releasing
C6H5
4-CH3C6H4
4-CH3OC6H4
2-OHC6H4
4-ClC6H4
4-NO2C6H4
e
1i
1j
Cisplatin (DDP)
e
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound which
reduced by 50% the optical density of treated cells with respect to untreated cells
using the MTT assay.
b
Data represent the mean values of three independent determinations.

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 274e279
277
Table 2
Anticancer activity of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1a & 1h on EAC-bearing mice.
Parameter
EAC control
Cisplatin (5 mg/kg)
1a (5 mg/kg)
1a (10 mg/kg)
1h (5 mg/kg)
1h (10 mg/kg)
(5 ꢂ 106 cells)
12.20 ꢃ 0.75
13.70 ꢃ 0.03
e
Body weight (g)
3.98 ꢃ 0.43***
29.30 ꢃ 0.36***
113.86***
3.01 ꢃ 0.35***
0.25 ꢃ 0.05***
0.16 ꢃ 0.04***
10.41 ꢃ 0.39*
16.40 ꢃ 0.67**
19.70*
7.61 ꢃ 0.61***
1.25 ꢃ 0.23*
4.05 ꢃ 0.35***
7.70 ꢃ 0.49***
22.10 ꢃ 1.03***
61.31***
4.50 ꢃ 0.39***
0.94 ꢃ 0.11***
3.27 ꢃ 0.43***
11.00 ꢃ 0.27ns
14.80 ꢃ 0.32*
8.02*
8.89 ꢃ 0.35***
18.00 ꢃ 0.63***
31.38**
8.29 ꢃ 0.22**
1.69 ꢃ 0.28ns
4.07 ꢃ 0.44***
Mean survival time (days)
% increase in life span (% ILS)
Tumor volume (mL)
Packed cell volume (mm)
Viable tumor cell count
(ꢂ107 cells/mL)
12.10 ꢃ 0.89
2.75 ꢃ 0.49
6.39 ꢃ 0.51
10.69 ꢃ 0.54ns
2.13 ꢃ 0.33ns
5.21 ꢃ 0.46ns
ns
n ¼ 10 and all values were expressed as mean ꢃ SEM, ***P < 0.001, **P < 0.005, *P < 0.05, non-significant, compared to tumor control.
Table 3
Effect of arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides 1a & 1h on biochemical and haematological parameters in EAC-bearing mice.
Treatment
Parameters
Haemoglobin (mg%)
RBC (million/mm3)
WBC (103 cells/mm3)
Lymphocytes (%)
Neutrophils (%)
Monocytes (%)
Normal
13.10 ꢃ 0.862
5.750 ꢃ 0.655
11.50 ꢃ 1.025***
8.080 ꢃ 0.86*
9.30 ꢃ 1.103**
5.960 ꢃ 0.636ns
7.020 ꢃ 0.797*
4.512 ꢃ 0.084
7.160 ꢃ 0.120
69 ꢃ 1.31
29 ꢃ 1.46
1 ꢃ 0.29
Tumor control
Cisplatin (5 mg/kg)
1a (5 mg/kg)
1a (10 mg/kg)
1h (5 mg/kg)
1h (10 mg/kg)
2.740 ꢃ 0.081
20.60 ꢃ 0.545
23 ꢃ 0.567
73 ꢃ 1.25
2.4 ꢃ 0.12
4.190 ꢃ 0.087***
3.020 ꢃ 0.066**
3.880 ꢃ 0.086**
2.720 ꢃ 0.086ns
3.300 ꢃ 0.158*
9.160 ꢃ 0.231***
18.48 ꢃ 0.355ns
13.92 ꢃ 0.476***
19.90 ꢃ 0.343ns
18.80 ꢃ 0.293**
63 ꢃ 0.782***
31 ꢃ 0.936**
49 ꢃ 0.759**
26 ꢃ 0.583*
40 ꢃ 0.857**
30 ꢃ 1.17***
54 ꢃ 0.85*
39 ꢃ 0.97***
65 ꢃ 0.48*
51 ꢃ 0.84**
1.2 ꢃ 0.16***
2 ꢃ 0.36*
1.6 ꢃ 0.18**
2.3 ꢃ 0.46ns
1.9 ꢃ 0.53*
All the values were expressed as mean ꢃ SEM (n ¼ 10), * p < 0.05, ** p < 0.01, *** p < 0.001 & ns ¼ nonsignificant, compared to tumor control.
(10 mg/kg) increased the mean survival time, decreased the tumor
volume, packed cell volume and viable tumor cell count. On the day
14, the biochemical and haematological parameters, as regard to
haemoglobin level, erythrocytes and leucocytes counts, were
compared with EAC control groups, standard drug Cisplatin treated
groups and the groups injected with the compounds 1a and 1h. As
shown in Table 3, the biochemical and haematological parameters
in the group treated with the compound 1a have been nearly
recovered completely to the normal values. Both derivatives
namely 1a and 1h significantly decreased the ascetic fluid volume
as compared to EAC control. These results could indicate macro-
phage activation and inhibition of vascular permeability, with the
comparison of tumor control. The derivatives 1a and 1h increased
the haemoglobin and RBC levels when compared with the tumor
control, 1a increased these levels more significantly than 1h. The
compounds 1a and 1h decreased the WBC levels when compared
with the tumor control,1a decreased these levels more significantly
than 1h. Treatment with the 1a and 1h brought back the differ-
ential leukocyte count to normal levels. This indicates that the test
drugs possess protective action on haemopoeitic system. These
results suggest that compound 1a is more active in in vivo cytotoxic
studies and by doing simple modification in the structure a potent
anticancer drug can be developed.
mechanism of action, so that, by structureeactivity relationship
a new potent analogue can be generated with the desired anti-
cancer activity with good efficacy.
4. Experimental
All the melting points were determined on a Fisher-Johns
melting point apparatus and are uncorrected. Analytical TLC was
performed on Merck precoated 60 F254 silica gel plates. Visualiza-
tion was done by exposure to iodine vapour. IR spectra (KBr pellet)
were recorded on a Perkin-Elmer BX series FT-IR spectrometer. 1H
NMR spectra were recorded on a Varian Gemini 300 MHz spec-
trometer. 13C NMR spectra were recorded on a Bruker 75 MHz
spectrometer. Chemical shift values are given in ppm (
ramethyl silane as an internal standard. Mass spectral measure-
ments were carried out by EI method on Jeol JMC-300
d) with tet-
a
spectrometer at 70 eV. Elemental analyses were performed on
a Carlo Erba 106 and Perkin-Elmer model 240 analyzers.
4.1. General typical procedure for the multi-component synthesis of
arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides (1aej)
To a stirred solution of 5 (1 mmol) in ethanol (10 mL) were
added piperidine (1 mL) and distilled benzaldehyde 6 (1 mmol).
The reaction mixture was refluxed with stirring at 80 ꢀC for 1 h, and
5 (1 mmol) was added later to the reaction mixture, heating
continued at 80 ꢀC for another 1 h, and finally acetophenone 3
(2 mmol) was further added to it and the reaction was carried out at
80 ꢀC for 3 h. After completion of the reaction (monitored by TLC),
the solvent was removed by vacuum distillation and the crude
product that obtained was purified by recrystallization from
ethanol.
3. Conclusion
In conclusion, we reported the multi-component (MCR-3) one-
pot protocol for the synthesis of arylmethylene bis-isoxazolo[4,5-b]
pyridine-N-oxides, using inexpensive and commercially available
materials with potential medicinal properties. This synthesis
benefits from a simple method of purification, which does not
require chromatography. This ease of purification compliments the
one-pot synthesis, making the technology practical, easy to
perform and facile. The newly synthesized novel arylmethylene bis-
isoxazolo[4,5-b]pyridine-N-oxides 1aej were evaluated for their
anticancer activity in in vitro and in vivo. Compound 1a is proved to
possess remarkable anticancer activity. It can be considered as
future drug candidate for cancer therapy, by simple modification in
the structure and requires further attempts to reveal the exact
4.1.1. 7,70-(Phenylmethylene)bis(3-methyl-6-phenylisoxazolo[4,5-b]
pyridine-N-oxide) (1a)
Yield 65%, mp 211e213 ꢀC; IR (KBr) cmꢁ1: 1380 (¼NþeOꢁ), 1645
(C¼N); 1H NMR (300 MHz, CDCl3)
d: 2.42 (s, 6H, 2CH3), 5.03 (s, 1H,
benzyliceH), 6.81e8.04 (m, 15H, Ar-H), 9.01 (s, 2H, pyridineeH);
13C NMR (75 MHz)
d 11.25, 40.08, 119.68, 120.65, 124.97, 127.31,

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E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 274e279
127.97, 129.08, 129.31, 129.61, 129.71, 130.04, 131.05, 134.21, 144.25,
144.66, 148.55, 152.12, 158.44, 164.69; ESI-MS (m/z): 540 (Mþ);
Anal. Calcd. for C33H24N4O4: C, 73.33; H, 4.44; N, 10.37. Found: C,
73.30; H, 4.47; N, 10.34%.
9.02 (s, 2H, pyridine-H); 13C NMR (75 MHz, CDCl3)
d 11.35, 40.45,
62.75, 120.35, 120.65, 123.08, 124.55, 126.11, 127.55, 128.65, 129.01,
129.55, 130.15, 131.45, 132.55, 134.25, 135.25, 144.01, 145.11, 158.25,
165.20; ESI-MS (m/z): 600(Mþ); Anal. Calcd. for C35H28N4O6: C,
70.00; H, 4.66; N, 9.33. Found: C, 70.02; H, 4.68; N, 9.37%.
4.1.2. 7,70-(2-Methylphenylmethylene)bis(3-methyl-6-phenyli-
soxazolo[4,5-b]pyridine-N-oxide) (1b)
4.1.8. 7,70-(Phenylmethylene)bis(3-methyl-6-(2-hydroxyphenyl)
isoxazolo[4,5-b]pyridine-N-oxide) (1h)
Yield 65%, mp 195e197 ꢀC; IR (KBr) cmꢁ1: 1380 (¼NþeOꢁ), 1640
(C¼N); 1H NMR (300 MHz, CDCl3)
d
: 2.33 (s, 6H, 2CH3), 2.52 (s, 3H,
Yield 70%; mp 216e218 ꢀC; IR (KBr) cmꢁ1: 1365 (¼NþeOꢁ), 1630
CH3), 4.90 (s, 1H, benzyliceH), 7.02e8.03 (m,14H, Ar-H), 9.06 (s, 2H,
(C¼N); 1H NMR (300 MHz, CDCl3)
d: 2.25 (s, 6H, 2CH3), 5.02 (s, 1H,
benzylic-H), 6.92e7.86 (m, 13H, Ar-H), 9.05 (s, 2H, pyridine-H), 9.55
pyridineeH); 13C NMR (75 MHz)
d
11.45, 21.89, 41.05, 120.05,
120.85, 124.99, 127.36, 127.95, 129.22, 129.73, 130.05, 131.00, 132.08,
134.20, 135.26, 144.16, 145.05, 165.12; ESI-MS (m/z): 554 (Mþ); Anal.
Calcd. for C34H26N4O4: C, 73.64; H, 4.69; N,10.10. Found: C, 73.62; H,
4.71; N, 10.13%.
(bs, 2H, OH, D2O exchangeable); 13C NMR (75 MHz, CDCl3)
d 11.20,
41.01, 119.25, 120.20, 125.01, 127.11, 127.80, 129.11, 129.70, 129.80,
130.00, 132.60, 134.10, 144.11, 144.35, 148.11, 152.30, 155.38, 158.01,
164.56; ESI-MS (m/z): 572 (Mþ); Anal. Calcd. for C33H24N4O6: C,
69.23; H, 4.19; N, 9.79. Found: C, 69.27; H, 4.21; N, 9.76%.
4.1.3. 7,70-(2-Methoxyphenylmethylene)bis(3-methyl-6-phenyli-
soxazolo[4,5b]pyridine-N-oxide) (1c)
4.1.9. 7,70-(Phenylmethylene)bis(3-methyl-6-(4-chlorophenyl)
isoxazolo[4,5-b]pyridine-N-oxide) (1i)
Yield 75%; mp 200e202 ꢀC; IR (KBr) cmꢁ1: 1375 (¼NþeOꢁ),
1640 (C¼N)); 1H NMR (300 MHz, CDCl3)
d
: 2.34 (s, 6H, 2CH3),
Yield 60%; mp 240e242 ꢀC; IR (KBr) cmꢁ1: 1370 (¼NþeOꢁ),
3.82 (s, 3H, OCH3), 5.04 (s, 1H, benzylic-H), 6.92e7.51 (m, 14H, Ar-
1650 (C¼N); 1H NMR (300 MHz, CDCl3)
d: 2.30 (s, 6H, 2CH3), 4.97
(s, 1H, benzylic-H), 6.85e7.89 (m, 13H, Ar-H), 9.09 (s, 2H, pyridine-
H), 9.12 (s, 2H, pyridine-H); 13C NMR (75 MHz, CDCl3)
d
11.10,
42.10, 63.21, 120.01, 124.85, 126.54, 126.80, 127.15, 127.85, 129.11,
129.55, 129.85, 130.45, 131.15, 132.35, 134.01, 135.11, 144.28, 145.40,
158.75, 165.15; ESI-MS (m/z): 570 (Mþ). Anal. Calcd. for C34H26N4O5:
C, 71.57; H, 4.56; N, 9.82. Found: C, 71.59; H, 4.53; N, 9.84%.
H); 13C NMR (75 MHz, CDCl3)
d 11.50, 41.11, 118.98, 120.20, 125.20,
126.85, 127.10, 128.65, 129.30, 129.80, 130.15, 131.75, 133.10, 134.75,
143.80, 144.20, 148.33, 152.10, 158.20, 165.45; ESI-MS (m/z): 608
(Mþ); Anal. Calcd. for C33H22N4O4Cl2: C, 65.13; H, 3.61; N, 9.21.
Found: C, 65.10; H, 3.58; N, 9.24%.
4.1.4. 7,70-(2-Chlorophenylmethylene)bis(3-methyl-6-phenyli
soxazolo[4,5-b]pyridine-N-oxide) (1d)
4.1.10. 7,70-(Phenylmethylene)bis(3-methyl-6-(4-nitrophenyl)
isoxazolo[4,5-b]pyridine-N-oxide) (1j)
Yield 65%; mp 235e237 ꢀC; IR (KBr) cmꢁ1: 1360 (¼NþeOꢁ),
1640 (C¼N)); 1H NMR (300 MHz, CDCl3)
d
: 2.23 (s, 6H, 2CH3),
Yield 60%; mp 255e257 ꢀC; IR (KBr) cmꢁ1: 1370 (¼NþeOꢁ),
5.03 (s, 1H, benzylic-H), 6.81e7.52 (m, 14H, Ar-H), 9.14 (s, 2H,
1650 (C¼N); 1H NMR (300 MHz, CDCl3)
d: 2.23 (s, 6H, 2CH3), 5.02
(s, 1H, benzylic-H), 6.92e8.05 (m, 13H, Ar-H), 9.33 (s, 2H, pyridine-
pyridine-H); 13C NMR (75 MHz, CDCl3)
d
12.01, 41.45, 119.25, 120.15,
125.15, 126.95, 127.15, 129.45, 129.95, 130.20, 130.95, 133.45, 134.90,
135.67, 143.96, 144.01, 148.11, 152.25, 158.10, 165.11; ESI-MS (m/z):
574 (Mþ); Anal. Calcd. for C33H23N4O4Cl: C, 68.98; H, 4.00; N, 9.75.
Found: C, 68.95; H, 4.03; N, 9.73%.
H); 13C NMR (75 MHz, CDCl3)
d 11.20, 40.15, 119.60, 120.55, 124.80,
127.15, 127.85, 129.15, 129.55, 129.80, 130.10, 132.27, 134,30, 144.45,
144.75, 148.40, 148.95, 152.10, 158.35, 164.50; ESI-MS (m/z): 630
(Mþ); Anal. Calcd. for C33H22N6O8: C, 62.85; H, 3.49; N, 13.33.
Found: C, 62.88; H, 3.46; N, 13.30%.
4.1.5. 7,70-(4-Chlorophenylmethylene)bis(3-methyl-6-phenyli
soxazolo[4,5-b]pyridine-N-oxide) (1e)
4.2. General procedure for step-wise synthesis of arylmethylene bis-
isoxazolo[4,5-b]pyridine-N-oxides 1
Yield 60%; mp 222e224 ꢀC; IR (KBr) cmꢁ1: 1370 (¼NþeOꢁ),
1650 (C¼N); 1H NMR (300 MHz, CDCl3)
d: 2.32 (s, 6H, 2CH3), 4.93
(s, 1H, benzylic-H), 6.82e7.83 (m, 14H, Ar-H), 9.03 (s, 2H, pyridine-
4.2.1. 3-Methyl-4-nitro-5-styrylisoxazoles (4)
H); 13C NMR (75 MHz, CDCl3)
d
11.95, 42.15, 118.95, 120.75, 124.98,
3,5-Dimethyl-4-nitroisoxazole 5 (1 mmol) and freshly distilled
aromatic aldehyde 6 (1 mmol) were refluxed in ethanol (10 mL) in
the presence of piperidine (0.5 mL) for half an hour. The styr-
ylisoxazole separated while the refluxing is in progress. The reac-
tion mixture was cooled and the product on recrystallization from
ethanol gave lemon yellow crystals.
126.75, 126.85, 127.45, 129.15, 129.75, 130.45, 130.65, 133.15, 134.15,
143.25, 144.25, 148.35, 151.95, 158.35, 165.25; ESI-MS (m/z): 574
(Mþ); Anal. Calcd. for C33H23N4O4Cl: C, 68.98; H, 4.00; N, 9.75.
Found: C, 68.96; H, 4.02; N, 9.78%.
4.1.6. 7,70-(Phenylmethylene)bis(3-methyl-6-(4-methylphenyl)
isoxazolo[4,5-b]pyridine-N-oxide) (1f)
4.2.2. Synthesis of 1,3-bis (3-methyl-4-nitroisoxazol-5-yl)-2-
arylpropanes (2)
Yield 70%; mp 190e192 ꢀC; IR (KBr) cmꢁ1: 1365 (¼NþeOꢁ),1640
(C¼N)); 1H NMR (300 MHz, CDCl3)
d
: 2.34 (s, 6H, 2CH3), 2.52 (s, 6H,
3-Methyl-4-nitro-5-styrylisoxazole 4 (1 mmol) and 3,5-
2CH3), 4.91 (s, 1H, benzylic-H), 6.92e7.93(m, 13H, Ar-H), 9.12(s, 2H,
dimethyl-4-nitroisoxazole 5 (1 mmol), were taken in alcohol
(10 mL) and piperidine (1 mL) was added to it and the contents are
heated at 80 ꢀC with stirring for 1 h. After the completion of the
reaction (monitored by TLC), the solvent was distilled off and the
resulting crude product was recrystallized from alcohol.
pyridine-H); 13C NMR (75 MHz, CDCl3)
d 11.75, 22.15, 41.11, 119.26,
120.75,124.80, 127.26,129.25,129.45,129.66,129.85,130.20,131.65,
134.45,138.40,144.15,144.70,148.32,152.10,158.20,164.30; ESI-MS
(m/z): 568 (Mþ); Anal. Calcd. for C35H28N4O4: C, 73.94; H, 4.92; N,
9.85. Found: C, 73.97; H, 4.95; N, 9.83%.
4.2.3. Arylmethylene bis-isoxazolo[4,5-b]pyridine-N-oxides (1aej)
To a solution of Michael adduct 2 (1 mmol) in ethanol (10 mL),
piperidine (1 mL) and acetophenone 3 (2 mmol) were added and
the contents were refluxed at 80 ꢀC with stirring for 3 h. After
completion of the reaction (monitored by TLC), the solvent was
removed by vacuum distillation and the crude product was purified
4.1.7. 7,70-(Phenylmethylene)bis(3-methyl-6-(4-methoxylphenyl)
isoxazolo[4,5-b]pyridine-N-oxide) (1g)
Yield 72%; mp; 180e182 ꢀC; IR (KBr) cmꢁ1: 1370 (¼NþeOꢁ),
1640 (C¼N); 1H NMR (300 MHz, CDCl3)
d: 2.32 (s, 6H, 2CH3), 3.81
(s, 6H, 2OCH3), 5.02 (s, 1H, benzylic-H), 6.83e8.04(m, 13H, Ar-H),

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 274e279
279
by recrystallization from ethanol. Compounds 1be1j were
prepared by adopting the same procedure as for 1a. The
compounds 1aej obtained in this method were found to be similar
to that of a multi-component one-pot synthesis by mps, 1H NMR,
13C NMR, Mass and elemental analyses.
bearing hosts. The antitumor effects of the extracts were deter-
mined by the change in body weight, mean survival time (MST)
and percentage increased life span (% ILS). The MST of each group
containing five mice was identified by recording the mortality on
a daily basis for 30 days, and the % ILS was calculated using the
following equations. MST ¼ (day of the first death þ day of the last
death)/2; ILS (%) ¼ [(mean survival time of treated group/mean
survival time of control group) e 1] ꢂ 100. The effect of
compounds 1a and 1h was also assessed by the determination of
the body weight, tumor volume, packed cell volume and viable
tumor cell count of EAC-bearing mice by the Trypan blue incor-
poration method.
4.3. Evaluation of anticancer activity
4.3.1. In vitro anticancer activity
The human cell cultures HeLa (cervical), Ehrlich Ascites
Carcinoma (EAC)
& MCF-7 (breast cancer) cell lines were
obtained from National Center for Cancer Cell Sciences (NCCS),
Pune, India. These cell lines were grown in recommended
media supplemented with 10% FBS, 1% L-glutamine and 1%
penicillinestreptomycineamphotericin B in a 5% CO2 humidified
atmosphere at 37 ꢀC. Cells were seeded in 25 cm2 tissue culture
flasks (Tarsons, India), at 250,000 cells/flask in a total volume of
9 mL. When confluent, all the cells were trypsinized (using Trypsin
eEDTA, HiMedia, Mumbai, India), and seeded in 96-well plates
(Tarsons, India). The cell suspension of 1 ꢂ 105 cells/mL was
prepared in complete growth medium. Stock solutions of the
compounds were prepared in DMSO. The stock solutions were
serially diluted with complete growth medium containing 50 mg/
mL of gentamycin to obtain working test solution of required
Acknowledgment
The authors are thankful to Head, Department of Chemistry,
Kakatiya University, Warangal, India for providing necessary facil-
ities and to the Director, Indian Institute of Chemical Technology
(IICT), Hyderabad, India for providing Spectral data.
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