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E. Rajanarendar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 149–153
R1
( )
R2
N
R3
n
R2
O
N
Ar
N
N
OR
H3C
R3
N
N
Ar
O
O
R4
isoxazolo[4,5-c]azepine
dibenzoisoxazolo[2,3-a]azepine
isoxazolo[4,5-b]azepine
(1)
(2)
(3)
HO
OH
OH
OH
H3C
HO
HO
O
O
N
N
OH
N
O
O
N
( )
( )
n
n
OH
isoxazolo[5,4-b]azepine
bis-azepine
(4)
(5)
Figure 1. Structures of biologically active azepines as rational compounds design template.
cancer) cell lines, were obtained from National Centre for Cell
Sciences (NCCS), Pune, India. These cell lines were grown in recom-
mended media supplemented with 10% FBS, 1% L-glutamine and 1%
of 5 mg/kg and 10 mg/kg were administered each day to the trea-
ted groups, and the standard drug Cisplatin at a dose of 5 mg/kg
was administered to each animal from the positive control group.
The pharmacological treatment lasted for 9 days. Fourteen days
after the treatment, five mice from each group were killed for
the study of antitumor activity. The rest of the animal groups were
kept to check the mean survival time of EAC tumor bearing hosts.
The antitumor effects of the extracts were determined 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 equa-
tions MST = (day of the first death + day of the last death)/2; and
ILS (%) = [(mean survival time of treated group/mean survival time
of control group) ꢁ 1] ꢀ 100. The effect of compounds 10i and 10j
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 incorporation method.
penicillin–streptomycin amphotericin B in a 5% CO2 humidified
atmosphere at 37 °C. Cells were seeded in 25 cm2 tissue culture
flasks (Tarsons, India) at 25,0000 cells/flask in a total volume of
9 mL. When confluent, all the cells were trypsinized (using
Trypsin–EDTA, HiMedia, Mumbai, India), and seeded in 96-well
plates (Tarsons, India). The cell suspension of 1 ꢀ 105 cells/mL
was prepared in complete growth medium. Stock solutions of the
compounds 10a–j 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 concentrations (having <1% DMSO). The 100
suspension was added to each well of the 96-well plates. The test
materials in complete growth medium (100 L) were added after
lL of cell
l
24 h incubation to the wells containing cell suspension. After
48 h of treatment with different concentrations of test compounds,
the cells were incubated with MTT (2.5 mg/mL) for 2 h. The med-
The cytotoxic activity of the newly synthesized phenylmethyl-
ene bis-ixoazolo[4,5-b] azepines 10a–j was evaluated for in vitro
anticancer activity against human cancer cell lines Hela, EAC
and MCF-7 according to MTT assay method18,19 by using Cisplatin
(DDP) as a reference drug. The results are presented in Table 1.
IC50 values were based on dose-response curves (IC50 values, de-
fined as the concentration corresponding to 50% growth inhibi-
tion). From Table 1, it is clear that some of the compounds
showed excellent activity against tumor cells. The compounds
bearing benzene or substituted benzene rings at 5-position
showed moderate to good anticancer activity against three differ-
ent cell lines, and not selective towards any particular cell line.
The substitution of the electron releasing groups such as methyl
and methoxy on the phenyl ring present either at 5-position
(10c) or 7-position (10d and 10e) increased the anticancer activ-
ity. But the introduction of electron withdrawing chloro group
(10b) decreased the activity moderately. The replacement of ben-
zene by furan or thiophene ring at position-5 (10i and 10j) en-
hanced the cytotoxic activity remarkably. These results suggest
that bis-isoxazolo[4,5-b]azepines bearing furan and thiophene
rings at position-5 played a vital role in the modulation of cyto-
toxicity among all the tested compounds 10a–j. Compound 10j
(bearing thiophene ring) is most cytotoxic towards all the three
cancer cell lines.
ium was then removed, and 100 lL of DMSO was added each well
to dissolve formazan crystals, which is the metabolite of MTT. After
thoroughly mixing, the plate was read at 490 nm for optical
density that is directly correlated with cell quantity. The cytotoxic
effects of the compounds were calculated as percentage inhibition
in cell growth as per the formula. % Cytotoxicity = 1 ꢁ [(O.D. in
sample well)/(O.D. in control well)] ꢀ 100.
In vivo cytotoxic bioassay: Adult female Swiss albino mice
(Mahaveer Enterprises, Hyderabad, India) of 8 weeks old (mean
weight in the range of 20–25 g) were selected and housed in poly-
propylene cages in a room, where the congenial temperature of
27 1 °C and 12 h light and dark cycles were maintained. The ani-
mals were allowed to acclimatize to the environment for 7 days,
and supplied with a standard pellet diet and water ad libitum.
All procedures using animals were reviewed and approved by the
Institutional Animal Ethical Committee of Kakatiya University.
The animals were divided into seven groups (n = 10). The normal
group was not inoculated with tumor cells, while six groups were
injected with EAC cells (0.2 mL of 2 ꢀ 106 cells/mice) intraperito-
neally. This was taken as day ‘0’, and the experimental treatment
started 24 h later. From the 1st day, 100 lL/mouse per day of ster-
ile saline was administered intraperitoneally to the negative con-
trol group (EAC-bearing mice). Compounds 10i and 10j at doses
Rajanarendar
