Design, synthesis, in vitro antimicrobial and anticancer activity of novel methylenebis-isoxazolo[4,5-b]azepines derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.02.013

A series of novel methylene bis-isoxazolo[4,5-b]azepines have been synthesized by reaction of 3,5-dimethyl-4-nitroisoxazole 6 with an appropriate methylene bis-chalcones 7 to obtain various Michael adducts 8a-i, which on treatment with SnCl₂-Me

Full text of DOI:10.1016/j.ejmech.2012.02.013

European Journal of Medicinal Chemistry 50 (2012) 344e349
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, in vitro antimicrobial and anticancer activity of novel
methylenebis-isoxazolo[4,5-b]azepines derivatives
,
a
a
a
b
c
E. Rajanarendar a , M. Nagi Reddy , S. Rama Krishna , K. Govardhan Reddy , Y.N. Reddy , M.V. Rajam
*
a Department of Chemistry, Kakatiya University, Warangal 506 009, A.P., India
b Department of Pharmacology and Toxicology, Kakatiya University,Warangal 506009, India
c Department of Genetics, University of Delhi, South Campus, New Delhi 110021, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of novel methylene bis-isoxazolo[4,5-b]azepines have been synthesized by reaction of
Received 8 July 2011
Received in revised form
6 February 2012
Accepted 6 February 2012
Available online 13 February 2012
3,5-dimethyl-4-nitroisoxazole 6 with an appropriate methylene bis-chalcones 7 to obtain various
Michael adducts 8aei, which on treatment with SnCl2eMeOH underwent reductive cyclization to afford
the title compounds 9aei. Structure of these compounds were established on the basis of IR, 1H NMR, 13
C
NMR and mass spectral data. The title compounds 9aei were evaluated for their in vitro antimicrobial
and anticancer activities. Compounds 9h and 9i exhibited potent antimicrobial and anticancer activities
as that of standard drugs.
Keywords:
Methylene bis-isoxazolo[4,5-b]azepine
Michael addition
Ó 2012 Elsevier Masson SAS. All rights reserved.
Reductive cyclization
Antimicrobial and anticancer activities
1. Introduction
such as isoxazolo[4,5-c]azepine (1) is found to contain many phar-
macological applications [6,7], where as dibenzoisoxazolo[2,3-a]
Cancer is a disease of striking significance in the world today. It
represents the second leading cause of human mortality after
cardiovascular diseases [1,2]. The identification of novel structures
that can act as more effective and reliable anticancer agents is still
a major challenge to medicinal chemistry researchers [3]. Despite of
the important advances achieved over recent decades in the
research and development of various cancerostatic drugs, current
antitumour chemotherapy still suffers from major limitations, such
as lack of selectivity of conventional chemotherapeutic agents for
cancer tissues, bringing about unwanted side effects, and the
acquisition by cancer cells of multiple-drug resistance [4].
The current scenario highlights the need for the discovery and
development of new lead compounds of simple structure, exhibiting
optimal in vivo antitumour potency and new mechanisms of action
[5]. The search for new drugs which can selectively target the
tumour cells is today’s goal of cancer therapy and is a never ending
process, till the goal is reached. We are currently engaged in
a program aimed at synthesizing novel heterocyclic compounds that
inhibit the growth of cancer cells. Azepines have been subject of
much investigation, since they are a class of totally synthetic phar-
macological agents with diverse action (Fig. 1). Azepine derivatives
azepine (2) is a 5-HT2A/2C receptor antagonist [8]. Isoxazolo[4,5-b]
azepine (3), and isoxazolo[5,4-b]azepine (4) are active against Gram-
positive and Gram-negative bacteria and also exhibited cytotoxicity
[9e11]. The bis-azepine 5 is found to possess in vitro anticancer
activity and DNA binding affinity [12]. Based on biological activity of
isoxazoloazepines, it seemed that introduction of two iso-
xazoloazepine rings in a single molecular frame work may enhance
the pharmacological activity of these compounds. Inspired by the
anticancer activity of bis-azepine 5, we are interested to synthesize
analogous of bis-azepine and to study the in vitro antimicrobial and
anticancer activity. As a sequel to our project on searching for new
biologically active molecules possessing isoxazole moiety [13e17],
we herein, report the synthesis, antimicrobial and anticancer activity
of novel methylene bis-isoxazolo-[4,5-b]azepines.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 9 was accomplished by synthetic
sequence show in Scheme 1. The preparation of methylene bis-
chalcones (7) was carried out by the condensation of 5,5’-methy-
lene bis-salicylaldehyde with substituted acetophenones [18,19]. The
synthesis of methylene bis-[3-methyl-4-nitro-5-(1-phenyl-3-(2-
* 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.2012.02.013

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 344e349
345
R
( )
N
R
n
R
R
O
Ar
N
N
N
OR
H C
N
R
N
Ar
O
O
R
isoxazolo[4,5-b]azepine
isoxazolo[4,5-c]azepine
dibenzoisoxazolo[2,3-a]azepine
(1)
(3)
(2)
HO
OH
OH
OH
H C
HO
HO
O
O
N
N
OH
N
)
n
N
O
O
(
)
n
(
OH
isoxazolo[5,4-b]azepine
bis-azepine
(4)
(5)
Fig. 1. Structures of biologically active azepines as rational compounds designtemplate.
O
C
O
C
NO2
H3C
CH CH
OH
HC
HO
Ar
HC
Ar
N
+
O
CH3
7
6
i)
Ar
Ar
O
NO2
O
NO2
CH3
N
H3C
CH2
CH
CH2
CH
N
CH2
CH2
O
O
OH
HO
8
ii)
Ar
Ar
N
CH3
N
H3C
N
N
O
O
HO
OH
9
7, 8, 9 : Ar = a) C6H5 ; b) 4- Br C6H4 ; c) 4-NO2 C6H4; d) 4-Cl C6H4; e) 4-OCH3 C6H4;
f) 4-CH3 C6H4; g) 2-OH C6H4; h) 2- furyl; i) 2-Thienyl
Scheme 1. Synthesis of novel methylene bis-isoxazolo[4,5-b] azepines (9aei).

346
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 344e349
hydroxyphenyl)-1-keto-4-n-butyl)isoxazoles 8 were achieved by
reaction of methylene bis-chalcones (7) with 3,5- dimethyl-4-
nitroisoxazole (6) [20] (2 mmol) in the presence of piperidine in
alcohol under refluxing conditions by the Michael addition. Michael
adducts 8 were further converted in to methylene bis-(7,8-dihydro-
6H-3-methyl-5-phenyl-7-(2-hydroxyphenyl)-isoxazolo[4,5-b]aze-
pines 9 by reductive cyclization on treatment with SnCl2-MeOH.
Structure of the compounds 8 and 9 were confirmed by IR, 1H NMR,
13C NMR and Mass spectral studies, and by elemental analyses.
Nine new derivatives of both Michael adducts 8and methylene
bis isoxazolo- [4,5-b]azepine derivatives 9 were reported. The IR
spectral of 8aei showed absorption bands around 1550e1570 cmꢀ1
for nitro group and 1680e1698 cmꢀ1 for carbonyl group. Similarly
IR spectra of compounds 9aei have showed absence of absorption
bands at 1550e1570 cmꢀ1 and 1680e1698 cmꢀ1 and exhibited
a sharp band at 1650e1654 cmꢀ1 due to C]N stretching vibration
confirming reductive cyclization. In particular, it must be pointed
and 9d exhibited good activity towards Gram-negative and Gram-
positive bacteria, where as compounds 9e, 9f, and 9g exhibited
moderate activity towards Gram-negative and Gram-positive
bacteria. Compound 9a exhibited least activity because it has no
substituents on benzene ring. Most of the newly synthesized
compounds exhibited good activity against fungi as compared with
the reference drug Nystatin. Compounds 9h and 9i showed excel-
lent antifungal activity and they inhibited the growth of fungal
organisms to a remarkable extent with low MIC as that of standard
drug. The enhanced activity of 9h and 9i may be due to the pres-
ence of thiophene and furan groups as substituents on bis-
isoxazoloazepine moiety. The activity is tested at concentration of
100 mg/disk. The minimum inhibitory concentrations (MIC) of the
compounds are presented in Table 2 were in accordance with the
results obtained in the primary screening.
2.3. Anticancer activity
out that 1H NMR of compounds 8aei showed two multiplets at
d at
3.41 and 4.13 due to methylene protons attached to isoxazole and
carbonyl group respectively. A characteristic peak as a multiplet at
The newly synthesized compounds 9aei were also evaluated for
their in vitro anticancer activity against two breast cancer cell lines,
MCF-7 (ER-positive), MDA-MB-231 (ER-positive), and a human
kidney cancer epithelial cell line HEK-293 according to MTT assay
method [23] by using Cisplatin (DDP) as reference drug.
The results of in vitro anticancer activity reveal that, some of the
compounds exhibited excellent activity against tumour cells. The
compounds bearing benzene or substituted benzene rings at 5-
position showed moderate to good anticancer activity against two
breast cell lines, MCF-7 (ER-positive) and MDA-MB-231 (ER-posi-
tive) and a normal human kidney epithelial cell line HEK-293 and
not selective towards any particular cell line. While substitution of
d
3.73 indicated the presence of methine proton. Title compounds
9aei have shown three multiplets at 3.32, 4.05 and 3.84 due to
two methylene groups and methine proton respectively. The 13C
NMR spectra of 8aej exhibited a prominent signal at 196.20
d
d
assignable to carbonyl carbon. The reductive cyclization of 8,
leading to the formation of title compounds 9aej was supported by
13C NMR spectra of the compounds, which showed a peak at
d
164.50 due to CN carbon, with the disappearance of carbonyl
carbon peak at 196.20 present in 8aej. Data from the elemental
d
analyses and electron impact mass spectra further confirmed the
assigned structures of 8a and 9a by exhibiting the molecular ion
peaks [M]þ at m/z 744 and 648 respectively.
the electron attracting groups such as chloro (9d) IC50 of 30
mM,
bromo (9b) IC50 of 34 M, and nitro (9c) IC50 of 36 M, groups on
m
m
the benzene ring exhibited moderate activity against MCF-7 cancer
cell lines, whereas the introduction of electron releasing groups
such as, methyl (9f), methoxy (9e) and hydroxy (9g) groups on the
benzene decreased the anticancer activity, and has shown
moderate activity. Compound 9a did not show significant activity in
all the tested cell lines. Replacement of benzene by furan or thio-
phene rings ( 9h and 9i), in the molecule enhanced their anticancer
activity remarkably. It is interesting to find that compounds 9h and
9i exhibited anticancer activity against MCF-7, MDA-MB-231 and
HEK-293 cancer cell line with the IC50 values of 12,10, 30 and 10, 18,
2.2. Antimicrobial activity
In vitro antimicrobial screening of the newly synthesized
compounds 9aei were evaluated against two Gram-positive bacteria
viz; Bacillus subtilis and Streptococcus lactis, two Gram-negative
bacteria viz; Escherichia coli and Pseudomonas aeuroginosa, and two
fungal strains viz; Aspergillus flavus and Trichoderma viridae. The
in vitro antimicrobial activity of the tested compounds (Table 1) were
evaluated by agar diffusion method [21,22]. Nalidixic acid and
Nystatin were used as standard drugs for comparison.
10 mM, respectively. These results suggest that, bis-isoxazolo [4,5-b]
The results of in vitro antimicrobial screening reveals that,
compounds 9i and 9h exhibited good activity towards Gram-
positive bacteria, and exhibited excellent activity towards Gram-
negative bacteria as compared with the reference drug Nalidixic
acid. This may be due to the presence of thiophene and furan rings
as substituents on bis-isoxazoloazepine system. Compounds 9b, 9c
azepines, bearing furan and thiophene rings played a vital role in
the modulation of cytotoxicity. Among all the tested compounds
(9aei), it is interesting to note that compounds 9h and 9i (bearing
furan and thiophene rings) are most cytotoxic towards all the three
cancer cell lines in comparison with that of standard drug Cisplatin.
The results are illustrated in Table 2.
Table 1
The MIC (minimum inhibitory concentration) as a criterian of antimicrobial activity
of the methylene bis-isoxaozolo[4,5-b]azepines (9aei) in mg/mL.
Table 2
In vitro anticancer activity of methylene bis-isoxazolo[4,5-b]azepines (9aei).
IC50(m
M)a
Compds.
Gram þve bacteria Gram eve bacteria
Fungi
Compds.
B. subtilis B. lactis E. coli P. aeuroginosa A. flavus T. viridae
MCF-7
MDA-MB-231
HEK-293
9a
9b
9c
9d
9e
9f
9g
9h
9i
35
30
30
28
35
35
38
20
19
40
25
26
25
30
32
35
22
20
25
e
40
27
28
28
35
35
36
16
14
28
e
35
30
28
30
32
40
38
14
12
30
e
40
30
30
32
35
35
38
20
15
e
40
32
30
30
40
38
40
16
14
e
9a
9b
9c
9d
9e
9f
9g
9h
80.56 ꢁ 0.43
34.21 ꢁ 0.36
36.34 ꢁ 0.05
30.42 ꢁ 0.51
40.38 ꢁ 0.04
42.26 ꢁ 0.03
44.32 ꢁ 0.63
12.45 ꢁ 0.05
10.20 ꢁ 0.04
12.25 ꢁ 0.35
82.61 ꢁ 0.61
51.23 ꢁ 0.33
53.42 ꢁ 0.27
52.48 ꢁ 0.11
58.08 ꢁ 0.32
61.05 ꢁ 0.61
63.42 ꢁ 0.29
10.32 ꢁ 0.43
18.25 ꢁ 0.36
24.16 ꢁ 0.67
85.43 ꢁ 0.43
54.50 ꢁ 0.23
55.61 ꢁ 0.39
53.30 ꢁ 0.43
57.40 ꢁ 0.54
63.38 ꢁ 0.49
65.52 ꢁ 1.03
30.23 ꢁ 0.44
10.26 ꢁ 0.11
40.40 ꢁ 0.35
9i
Nalidixic acid 30
Nystatin
Cisplatin (DDP)
e
22
20
a
Values are mean ꢁ SEM of three independent experiments.

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 344e349
347
3. Conclusion
Calcd. For C41H34N4O10Br2: C, 54.66; H, 3.77; N, 6.22. Found: C,
54.62; H, 3.81; N, 6.20%.
In conclusion, we report the synthesis of novel methylene bis-
isoxazolo[4,5-b] azepines, from 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 this synthetic technology practical, easy to perform
and facile. The newly synthesized novel methylene bis-isoxazolo
[4,5-b]azepines 9aei were evaluated for in vitro antimicrobial and
anticancer activity. Compounds 9h and 9i are proved to possess
remarkable antimicrobial and anticancer activity. By simple modi-
fication in the structure, a new potent analogue can be generated
with the desired antimicrobial and anticancer activity with good
efficacy and further attempts are necessary to reveal
structureeactivity relationship.
4.1.3. Methylenebis-[3-methyl-4-nitro-5-(1-(4-nitrophenyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8c)
Yield 86%, mp 158e160 ꢂC. IR (KBr) cmꢀ1: 1660, 1560, 3254. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.42 (s, 6H, 2CH3), 3.54 (m, 4H,
2CH2eCH), 3.73 (m, 2H, 2CH), 3.84 (s, 2H, CH2), 4.15 (m, 4H, 2CH2
CO), 6.91e7.82 (m,14H, Ar-H),11.22 (bs, 2H, OH, D2O exchangeable).
EIMS (EI): m/z 834 (Mþ). Anal. Calcd. For C41H34N6O14: C, 58.99; H,
4.07; N, 10.07. Found: C, 58.95; H, 4.02; N, 10.09%.
4.1.4. Methylenebis-[3-methyl-4-nitro-5-(1-(4-chlorophenyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8d)
Yield 88%, mp 167e169 ꢂC. IR (KBr) cmꢀ1: 1698, 1565, 3258. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.22 (s, 6H, 2CH3), 3.32 (m, 4H,
2CH2eCH), 3.64 (m, 2H, 2CH), 3.71 (s, 2H, CH2), 4.22 (m, 4H, 2CH2
CO), 6.94e7.93 (m,14H, Ar-H),11.12 (bs, 2H, OH, D2O exchangeable).
EIMS: m/z 812 (Mþ). Anal. Calcd. For C41H34N4O10Cl2: C, 60.59; H,
4.18; N, 6.89. Found: C, 60.64; H, 4.21; N, 6.91%.
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 exposing to iodine vapour. IR spectra (KBr pellet)
were recorded on a PerkineElmer 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
4.1.5. Methylenebis-[3-methyl-4-nitro-5-(1-(4-methoxyphenyl-3-
(2-hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8e)
Yield 84%, mp 172e174 ꢂC. IR (KBr) cmꢀ1: 1682, 1550, 3252. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.33 (s, 6H, 2CH3), 3.32 (m, 4H,
2CH2eCH), 3.71 (m, 2H, 2CH), 3.82 (s, 6H, 2OCH3), 3.94 (s, 2H, CH2),
4.15 (m, 4H, 2 CH2CO), 6.71e7.73 (m,14H, Ar-H),11.22 (bs, 2H, OH, D2O
exchangeable).EIMS: m/z 804 (Mþ). Anal. Calcd. For C43H40N4O12: C,
64.17; H, 4.97; N, 6.96. Found: C, 64.19; H, 4.95; N, 6.94%.
spectrometer. Chemical shift values are given in ppm (d) with tet-
ramethyl silane as internal standard. Mass Spectral measurements
were carried out by EI method on a Jeol JMC-300 spectrometer at
70 eV. Elemental analyses were performed on a Carlo Erba 106
andPerkin-Elmer model 240 analysers.
4.1.6. Methylenebis-[3-methyl-4-nitro-5-(1-(4-methylphenyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8f)
4.1. General procedure for the synthesis of methylene bis-[3-
methyl-4-nitro-5-(1-phenyl- 3-(2-hydroxyphenyl)-1-keto-4-n-
butyl)isoxazoles (8aei)
Yield 84%, mp 153e155 ꢂC. IR (KBr) cmꢀ1: 1680, 1555, 3250. 1H
NMR (300 MHz, CDCl3) d ppm: 2.30 (s, 6H, 2CH3), 2.52 (s, 6H, 2CH3),
3.36 (m, 4H, 2CH2eCH), 3.70 (m, 2H, 2CH), 3.90 (s, 2H, CH2), 4.10 (m,
4H, 2CH2 CO), 6.76e7.72 (m, 14H, Ar-H), 11.20 (bs, 2H, OH, D2O
exchangeable). EIMS: m/z 772 (Mþ). Anal. Calcd. For C43H40N4O10: C,
66.83; H, 5.18; N, 7.25. Found: C, 66.88; H, 5.14; N, 7.20%.
A mixture of 3,5-dimethyl-4-nitroisoxazoloe 6 (2 mmol) and
methylene-bis-chalcone 7 (1 mmol) in ethanol (20 mL) were
refluxed in the presence of 2e3 drops of piperidine for 4 h. After
completion of the reaction (monitored by TLC), the reaction
mixture on cooling gave the solid, which was filtered and washed
with cold ethanol. Recrystallization from ethanol afforded pure
Michael adducts 8.
4.1.7. Methylene bis-[3-methyl-4-nitro-5-(1-(2-hydroxyphenyl-3-
(2-hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8g)
Yield 82%, mp 161e163 ꢂC. IR (KBr) cmꢀ1: 1681, 1570, 3257. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.24 (s, 6H, 2CH3), 3.42 (m, 4H,
2CH2eCH), 3.71 (m, 2H, 2CH), 3.82 (s, 2H, CH2), 4.13 (m, 4H, 2CH2
CO), 6.84e7.85 (m, 14H, Ar-H), 10.52 (bs, 2H, OH, D2O exchange-
able), 11.13 (bs, 2H, OH, D2O exchangeable). EIMS: m/z 776 (Mþ).
Anal. Calcd. For C41H36N4O12: C, 63.40; H, 4.63; N, 7.21. Found: C,
63.42; H, 4.66; N, 7.18%.
4.1.1. Methylene bis-[3-methyl-4-nitro-5-(1-phenyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8a)
Yield 80%, mp 170e172 ꢂC. IR (KBr) cmꢀ1: 1690, 1550, 3200. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.42 (s, 6H, 2 CH3), 3.41 (m, 4H,
2CH2eCH), 3.73 (m, 2H, 2CH), 3.84 (s, 2H, CH2), 4.13 (m, 4H, 2CH2
CO), 6.80e8.03 (m, 16H, Ar-H), 11.04 (bs, 2H, OH, D2O exchange-
4.1.8. Methylenebis-[3-methyl-4-nitro-5-(1-(2-furyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8h)
able). 13C NMR(75 MHz, CDCl3):
d 9.80 28.60, 34.80, 40.20, 44.06,
100.30, 120.45, 125.52, 126.60, 127.90, 128.82, 132.40, 133.90,
138.65, 139.32, 154.08, 156.36, 158.90, 196.20. EIMS: m/z 744 (Mþ).
Anal. Calcd. For C41H36N4O10: C, 66.12; H, 4.83; N, 7.52. Found: C,
66.08; H, 4.81; N, 7.55%.
Yield 86%, mp 189e191 ꢂC. IR (KBr) cmꢀ1: 1680, 1570, 3258. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.21 (s, 6H, 2CH3), 3.42 (m, 4H,
2CH2eCH), 3.64 (m, 2H, 2CH), 3.83 (s, 2H, CH2), 4.12 (m, 4H, 2CH2
CO), 6.42e8.51 (m, 12H, Ar-H & Furan-H), 11.35(bs, 2H, OH, D2O
exchangeable). EIMS: m/z 724 (Mþ). Anal. Calcd. For C37H32N4O12: C,
61.32; H, 4.41; N, 7.73. Found: C, 61.36; H, 4.44; N, 7.70%.
4.1.2. Methylenebis-[3-methyl-4-nitro-5-(1-(4-bromophenyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8b)
Yield 85%, mp 163e166 ꢂC. IR (KBr) cmꢀ1: 1685, 1570, 3250. 1H
4.1.9. Methylenebis-[3-methyl-4-nitro-5-(1-(2-thienyl-3-(2-
hydroxyphenyl)-1-keto-4-n-butyl)isoxazole (8i)
NMR (300 MHz, CDCl3)
d ppm: 2.33 (s, 6H, 2CH3), 3.42 (m, 4H,
2CH2eCH), 3.61 (m, 2H, 2CH), 3.94 (s, 2H, CH2), 4.02 (m, 4H, 2CH2
CO), 6.92e7.93 (m, 14H, Ar-H), 11.03 (bs, 2H, OH, D2O exchange-
Yield 87%, mp 184-184 ꢂC. IR (KBr) cmꢀ1: 1685, 1560, 3251. 1H
NMR (300 MHz, CDCl3)
d ppm: 2.13(s, 6H, 2CH3), 3.62 (m, 4H,
able). 13C NMR(75 MHz, CDCl3):
d
9.85 28.52, 34.20, 40.50, 44.26,
2CH2eCH), 3.72 (m, 2H, 2CH2eCH), 3.93 (s, 2H, CH2), 4.24 (m, 4H,
2CH2 CO), 6.35e8.44 (m, 12H, Ar-H & Thiophene-H), 11.21 (bs, 4H,
OH, D2O exchangeable). EIMS: m/z 756 (Mþ), Anal. Calcd. For
100.08, 120.15,125.32, 126.40, 127.50,128.70, 132.65, 133.40, 138.28,
139.18, 154.02, 156.16, 158.45, 196.10. EIMS: m/z 900 (Mþ). Anal.

348
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 344e349
C37H32N4O10S2: C, 58.73; H, 4.23; N, 7.40. Found: C, 58.76; H, 4.26;
N, 7.36%.
exchangeable). EIMS: m/z708 (Mþ). Anal. Calcd. For C43H40N4O6 : C,
72.88; H, 5.64; N, 7.90. Found: C, 72.85; H, 5.65; N, 7.93%.
4.2.6. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(4-
4.2. General procedure for the synthesis of methylene bis-(7,8-
dihydro-6H-3-methyl-5-phenyl-7-(2-hydroxyphenyl)-isoxazolo
[4,5-b]azepines (9aei)
methylphenyl)-7-(2-hydroxy phenyl)-isoxazolo[4,5-b]azepine (9f)
Yield 88%, mp 183e1985 ꢂC. IR (KBr) cmꢀ1: 1655, 3270. 1H NMR
(300 MHz, CDCl3) d ppm: 2.30 (s, 6H, 2CH3), 2.50 (s, 6H, 2CH3), 3.24
(m, 4H, 2 CH2eCH), 3.60 (m, 2H, 2CH), 3.96 (s, 2H, CH2), 4.08 (m, 4H,
2CH2 CN), 6.70e7.90 (m, 14H, Ar-H), 10.46 (bs, 2H, OH, D2O
exchangeable). EIMS: m/z676 (Mþ). Anal. Calcd. For C43H40N4O4: C,
76.33; H, 5.91; N, 8.28. Found: C, 76.29; H, 5.95; N, 8.32%.
The Michael adducts 8 (1 mmol) and SnCl2.2H2O (3 mmol) were
dissolved in 20 mL of methanol and refluxed for 4 h. After
completion of the reaction (monitored by TLC), solvent was
removed in vacuum. The solid mass was decomposed with cold
water and the reaction solution was carefully adjusted to pH 8 with
10% NaHCO3 solution and then extracted with ethyl acetate
(2 ꢃ 20 mL). The combined organic layers were dried over Na2SO4
and evaporated under vacuum and purified by recrystallization
from ethanol to gave pure product 9.
4.2.7. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(2-
hydroxyphenyl)-7-(2-hydroxy phenyl)-isoxazolo[4,5-b]azepine (9g)
Yield 85%, mp 196e197 ꢂC. IR (KBr) cmꢀ1: 1651, 3271. 1HNMR
(300 MHz, CDCl3) d ppm: 2.02 (s, 6H, 2CH3), 3.10 (m, 4H, 2CH2eCH),
3.71 (m, 2H, 2CH), 3.83 (s, 2H, CH2), 4.05 (m, 4H, 2CH2 CN), 6.76e7.92
(m,14H, Ar-H),10.41 (bs, 2H, OH, D2O exchangeable),10.82 (bs, 2H, OH,
D2O exchangeable). EIMS: m/z 680 (Mþ). Anal. Calcd. For C41H36N4O6:
C, 72.35; H, 5.29; N, 8.23. Found: C, 72.30; H, 5.32; N, 8.26%.
4.2.1. Methylenebis-(7,8-dihydro-6H-3-methyl-5-phenyl-7-(2-
hydroxyphenyl)-isoxazolo[4,5-b]azepine (9a)
Yield 84%, mp 220e222 ꢂC. IR (KBr) cmꢀ1: 1652, 3273. 1H NMR
(300 MHz, CDCl3)
3.84(m, 2H, 2CH), 3.91 (s, 2H, CH2), 4.05(m, 4H, 2CH2 CN),
6.80e8.02 (m, 16H, Ar-H), 10.51 (bs, 2H, OH, D2O exchangeable). 13
NMR(75 MHz, CDCl3): 9.76, 27.20, 32.82, 41.27, 43.26, 102.20,
d
ppm: 2.20 (s, 6H, 2CH3), 3.32 (m, 4H, 2CH2eCH),
4.2.8. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(2-furyl)-7-(2-
hydroxyphenyl)-isoxazolo[4,5-b]azepine (9h)
C
Yield 89%, mp 215e217 ꢂC. IR (KBr) cmꢀ1: 1653, 3277. 1H NMR
d
(300 MHz, CDCl3) d ppm: 2.30 (s, 6H, 2CH3), 3.42 (m, 4H, 2CH2eCH),
121.45,124.62,125.80,126.40,128.22,133.60,134.40,136.25,138.62,
155.28, 158.16, 160.30, 164.50. EIMS: m/z 648 (Mþ). Anal. Calcd. For
C41H36N4O4: C, 75.92; H, 5.55; N, 8.64. Found: C, 75.95; H, 5.51; N,
8.66%.
3.84 (m, 2H, 2CH), 3.91 (s, 2H, CH2), 4.22 (m, 4H, 2CH2 CN),
6.50e8.14 (m, 12H, Ar-H & Furan-H), 10.72 (bs, 2H, OH, D2O
exchangeable). EIMS: m/z 628 (Mþ). Anal. Calcd. For C37H32N4O6: C,
70.70; H, 5.09; N, 8.91. Found: C, 70.73; H, 5.06; N, 8.94%.
4.2.2. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(4-
4.2.9. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(2-thienyl)-7-(2-
hydroxyphenyl)-isoxazolo[4,5-b]azepine (9i)
bromophenyl)-7-(2-hydroxy phenyl-isoxazolo[4,5-b]azepine (9b)
Yield 88%, mp 195e197 ꢂC. IR (KBr) cmꢀ1: 1650, 3270 1H NMR
Yield 85%, mp 205e208 ꢂC. IR (KBr) cmꢀ1: 1650, 3275. 1H NMR
(300 MHz, CDCl3)
d
ppm: 2.30(s, 6H, 2 CH3), 3.43 (m, 4H, 2CH2eCH),
(300 MHz, CDCl3) d ppm: 2.10 (s, 6H, 2CH3), 3.32 (m, 4H, 2CH2eCH),
3.62 (m, 2H, 2CH), 3.81 (s, 2H, CH2), 4.22 (m, 4H, 2CH2 CN), 6.90e8.10
3.74 (m, 2H, 2CH), 3.81 (s, 2H, CH2), 4.12 (m, 4H, 2CH2 CN),
6.64e7.96 (m, 12H, Ar-H & Thiophene-H), 10.62 (bs, 2H, OH, D2O
exchangeable). EIMS: m/z 660 (Mþ), Anal. Calcd. For C37H32N4O4S2:
C, 67.27; H, 4.84; N, 8.48. Found: C, 67.23; H, 4.88; N, 8.44%.
(m,14H, Ar-H),10.41 (bs, 2H, OH, D2O exchangeable).13CNMR(75MHz,
CDCl3): d 9.70, 28.10, 32.42, 41.20, 43.42,101.30,121.25, 124.32, 125.43,
126.30, 128.60, 133.40, 134.20, 136.67, 138.42, 155.08, 158.24, 160.65,
164.30. EIMS: m/z 804 (Mþ). Anal. Calcd. For C41H34N4O4Br2 : C, 61.19;
H, 4.22; N, 6.96. Found: C, 61.22; H, 4.25; N, 6.93%.
4.3. Evaluation of antimicrobial activity
4.2.3. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(4-nitrophenyl)-
7-(2-hydroxy phenyl)-isoxazolo[4,5-b]azepine (9c)
The bacterial isolates representing Gram-negative and Gram-
positive bacteria were recovered on Nutrient and MacConkey
agar. The two fungal isolates A. flavus and T. viridae were isolated on
Sabouraud dextrose agar (oxoid). They are isolated from clinical
samples and identified to the species level according to different
API systems (biomerilux). The selected compounds were tested
in vitro using the agar disk diffusion method taking Nalidixic acid
[21] and Nystatin [22] as reference drugs for bacteria and fungi,
respectively. The antimicrobial potentialities of the tested
compounds were estimated by placing pre-sterilized filter paper
Yield 87%, mp 210e213 ꢂC.IR (KBr) cmꢀ1: 1653, 3271. 1H NMR
(300 MHz, CDCl3) d ppm: 2.10 (s, 6H, 2CH3), 3.42 (m, 4H, 2CH2eCH),
3.71 (m, 2H, 2CH), 3.84 (s, 2H, CH2), 4.03 (m, 4H, 2CH2 CN),
6.72e7.90 (m, 14H, Ar-H), 10.61 (bs, 2H, OH, D2O exchangeable).
EIMS: m/z 738 (Mþ). Anal. Calcd. For C41H34N6O8: C, 66.66; H, 4.60;
N, 11.38. Found: C, 66.62; H, 4.62; N, 11.41%.
4.2.4. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(4-
chlorophenyl)-7-(2-hydroxy phenyl)-isoxazolo[4,5-b]azepine (9d)
disks (5 mm in diameter) impregnated with 100 mg/disk using
Yield 86%, mp 199e201 ꢂC. IR (KBr) cmꢀ1: 1654, 3270. 1HNMR
dimethylsulfoxide (DMSO) as solvent which showed no inhibition
zones. The inhibition zones of the tested compounds were
measured after 24e28 h incubation at 37ꢂ C for bacteria and at 28ꢂ
C after 5 days for fungi. The minimal inhibitory concentration (MIC)
determination method of the biologically active compounds
(Table 1) was applied using different concentrations per disk
against Gram-negative and Gram-positive bacteria, and fungi.
(300 MHz, CDCl3) d ppm: 2.04 (s, 6H, 2 CH3), 3.1(m, 4H, 2CH2eCH),
3.62(m, 2H, 2CH), 3.75(s, 2H, CH2), 4.12 (m, 4H, 2CH2 CN),
6.84e8.02 (m, 14H, Ar-H), 10.52 (bs, 2H, OH, D2O exchangeable).
EIMS: m/z 716 (Mþ). Anal. Calcd. For C41H34N4O4Cl2: C, 68.71; H,
4.74; N, 7.82. Found: C, 68.68; H, 4.73; N, 7.82%.
4.2.5. Methylenebis-(7,8-dihydro-6H-3-methyl-5-(4-
methoxyphenyl)-7-(2-hydroxy phenyl)-isoxazolo[4,5-b]azepine (9e)
4.4. Evaluation of anticancer activity
Yield 89%, mp 189e191 ꢂC. IR (KBr) cmꢀ1: 1650, 3271. 1H NMR
(300 MHz, CDCl3)
d
ppm: 2.34 (s, 6H, 2CH3), 3.20 (m, 4H, 2CH2eCH),
Cancer cell lines (1 ꢃ 104 cells/well) were seeded in 200
ml
3.62 (m, 2H, 2CH), 3.83 (s, 6H, 2OCH3), 3.91 (s, 2H, CH2), 4.13 (m, 4H,
2 CH2 CN), 6.72e7.95 (m, 14H, Ar-H), 10.42 (bs, 2H, OH, D2O
DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h
at 37 ꢂC in

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 50 (2012) 344e349
349
a
humidified atmosphere containing 5% CO2 incubator. The
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The authors are thankful to the Head, Department of Chemistry,
Kakatiya University, Warangal for providing the facilities, the
Director, Indian Institute of Chemical Technology, Hyderabad for
recording 1H NMR, 13C NMR, and Mass Spectral data. Financial
assistance from the UGC SAP (phase-1)-DRS programme, New Delhi,
India, is greatly acknowledged. We are grateful to Prof. J.R. Falck,
Departments of Biochemistry and Pharmacology, University of Texas,
Southwestern Medical Center, Texas, USA for helpful discussions.