Convenient one-pot synthesis, anti-mycobacterial and anticancer activities of novel benzoxepinoisoxazolones and pyrazolones

Article abstract of DOI:10.1016/j.ejmech.2014.02.042

Series of new benzoxepinoisoxazolones 4a-d and pyrazolones 6a-t were prepared by the cyclocondensation of substituted (E)-ethyl 3-oxo-2,3- dihydrobenzo[b]oxepine-4-carboxylates 3a-d with hydroxylamine hydrochloride and phenylhydrazine hydrochlorides 5a-k.

Full text of DOI:10.1016/j.ejmech.2014.02.042

European Journal of Medicinal Chemistry 76 (2014) 460e469
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Convenient one-pot synthesis, anti-mycobacterial and anticancer
activities of novel benzoxepinoisoxazolones and pyrazolones
G. Saidachary a, K. Veera Prasad a, D. Divya b, Ashita Singh b, U. Ramesh b, B. Sridhar c,
,
B. China Raju a
*
a Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Center for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
c Laboratory of X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of new benzoxepinoisoxazolones 4aed and pyrazolones 6aet were prepared by the cyclo-
condensation of substituted (E)-ethyl 3-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylates 3aed with hy-
droxylamine hydrochloride and phenylhydrazine hydrochlorides 5aek. Synthesized compounds were
screened for their in vitro anti-mycobacterial activity and anticancer activity. Ten compounds displayed
good anti-mycobacterial activity, among these; compound 4d and 6b found to be potent when compared
to standard drug isoniazid. Eleven compounds displayed good anticancer activity and compounds 4bed
displayed equipotent activity on HeLa cell lines when compared to standard drug doxorubicin. Activation
of caspase-3 and caspase-9 has been measured for compounds 4bed on HeLa cell lines (apoptosis). This
is the first report assigning in vitro anti-mycobacterial, anticancer and structureeactivity relationship for
this new class of benzoxepinoisoxazolones and pyrazolones.
Received 4 September 2013
Received in revised form
13 February 2014
Accepted 14 February 2014
Available online 15 February 2014
Keywords:
Benzoxepinoisoxazolones
Benzoxepinopyrazolones
Cyclocondensation
Ó 2014 Elsevier Masson SAS. All rights reserved.
Anti-mycobacterial activity
Anticancer activity
1. Introduction
compromises host defense and allows latent infection to reactivate
or render individuals more susceptible to tuberculosis. New drugs
Isoxazolones and pyrazolones are an important heterocyclic
compounds and drug substances in pharmaceutical industry due to
their potential biological activities [1]. Phenazone, propyphena-
zone, ampyrone and metamizole sodium are useful pyrazolone
drugs as antipyretic and analgesic [2], whilst edaravone (MCI-186)
has been used for brain disorders [3] and myocardial ischemia [4]
(Fig. 1). Few isoxazolones were also reported as effective p38
MAP kinase inhibitors [5].
Tuberculosis [6] is chronic infective disease caused by Myco-
bacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium,
Mycobacterium microti, Mycobacterium africanum, Mycobacterium
leprae and Gram-positive pathogens of higher vertebrates. Rifam-
picin, isoniazid, pyrazinamide, ethambutol and streptomycin are
the first line drugs used for tuberculosis and the treatment involves
two months of isoniazid, pyrazinamide, ethambutol, and rifam-
picin; followed by rifampicin and isoniazid for four months.
Increasing of drug resistant associated with HIV infection which
are very much needed to reduce time therapy for cure and to treat
multidrug resistant (MDR) M. tuberculosis strains. Cancer [7], the
uncontrolled and rapid proliferation of abnormal cells is the leading
cause of human deaths, despite of its progress in chemical biology
and pharmacology. Therefore, there is an urgent need to develop
more potent new chemical entities for M. tuberculosis and cancer.
Our studies on biologically active heterocyclic compounds [8]
and recent focus on feasible reactions of carbonyl compounds/sal-
icylaldehydes with 3-oxobutanoates bearing chloro or trifluoro
substituents. In this context, we studied the reactivity of carbonyl
compounds with ethyl 4,4,4-trifluoro-3-oxobutanoate using
piperidine in CH2Cl2 at room temperature to provide a series of (E)-
a,b-unsaturated esters and ketones [9a]. We also studied the
reactivity of various salicylaldehydes with ethyl 4-chloro-3-
oxobutanoate using piperidine to provide 2H-chromenes [9b],
interim these derivatives were successfully converted to corre-
sponding 2,3-dihydrobenzoxepine-4-carboxylates [9c]. Further,
2H-chromenes were utilized for the preparation of chromeno-
pyrrolones and 1H-1,2,3-triazolylchromenopyrrolones in very good
yields [10]. In this study we report series of substituted new ben-
zoxepinoisoxazolone, pyrazolone derivatives, their anti-
* Corresponding author.
E-mail address: chinaraju@iict.res.in (B. China Raju).
http://dx.doi.org/10.1016/j.ejmech.2014.02.042
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
461
mycobacterial and anticancer activities as well as structureeactiv-
ity relationship in relation to various substitutions present on
benzoxepine/phenylhydrazines for the first time.
Electron withdrawing groups have an advantage over electron
donating groups in terms of yields (Scheme 2).
After having achieved the synthesis of benzoxepinoisoxazolones
4aed, next we extended this protocol to prepare benzox-
epinopyrazolones 6aet. Condensation of 3a with phenylhydrazine
hydrochloride 5a in presence of acetic acid in ethanol under reflux
conditions provided 2-phenyl-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one 6a as orange color solid in 80% yield. The re-
action was preceded by the formation of phenylhydrazone inter-
mediate followed by in situ cyclization afforded 6a. Next, the
reactions were carried out with substituted benzoxepinecarbox-
ylates 3aed and phenylhydrazine hydrochlorides 5aek under
optimized conditions to provide series of benzoxepinopyrazolones
6bet (Scheme 3). Thus synthesized compounds are new and well
characterized by spectral data (Supporting Information).
2. Chemistry
Scheme 1 illustrates the synthesis of (E)-ethyl 3-oxo-2,3-
dihydrobenzo[b]oxepine-4-carboxylates 3aed [9c]. Salicylalde-
hydes 1aed with ethyl 4-chloro-3-oxobutanoate under optimized
conditions provided 2H-chromenes 2aed. Wittig reaction of 2ae
d with (1-ethoxycarbonylethylidene)triphenylphosphorane pro-
vided benzoxepinecarboxylates 3aed. These derivatives are an
important synthons having an olefin ester and carbonyl functional
group similar to 1,3-dicarbonyl compounds, hence, these functional
groups can be utilized for the preparation of various new hetero-
cyclic compounds. Accordingly, the initial reaction was performed
with benzoxepinecarboxylate 3a and hydroxylamine hydrochloride
in presence of acetic acid in ethanol under reflux conditions for 3 h
(Scheme 2) and to provide 3H,10H-benzo[6,7]oxepino[3,4-c]iso-
xazol-3-one 4a as pale yellow color solid in 72% yield. The reaction
proceeds initial formation of an oxime intermediate followed by in
situ cyclization with the elimination of ethanol afforded 4a. The IR
spectrum of compound 4a has shown absorption bands at
nmax ¼ 1710 and 1560 cmꢀ1 corresponds to carbonyl group and an
imine double bond. The 1H NMR spectrum shown a characteristic
3. Biology
The benzoxepinoisoxazolones 4aed and pyrazolones 6aet were
screened for their in vitro anti-mycobacterial activity against
Mycobacterium smegmatis ATCC 14468 (MC2) by broth dilution
method [12] and compared with the standard drugs rifampicin and
isoniazid. GI50 values were calculated (concentration of compounds
inhibiting growth by 50%) in triplicate. Further, anticancer activity
was also screened for all the synthesized compounds against a
panel of three human cancer cell lines such as lung (A549), prostate
(DU-145), cervical (HeLa) and non cancerous human embryonic
kidney cells HEK 293 by the standard MTT assay method and
compared with the standard drug doxorubicin [13].
singlet resonated at
zoxepine; another singlet at
lene protons. The multiplet at
¼ 7.54 ppm correspond to aromatic protons. Compound 4a was
further analyzed by 13C NMR spectroscopy, signal at
¼ 168.84 ppm
corresponds to carbonyl ester group and another characteristic
signal at
d
¼ 7.74 ppm corresponds to C-5 of the ben-
¼ 5.02 ppm corresponds to methy-
¼ 7.20e7.32 ppm and doublet at
d
d
d
d
4. Results and discussion
d
¼ 66.78 ppm corresponds to OCH2 of the benzox-
epinoisoxazolone. Compound 4a was shown molecular ion MS (ESI)
m/z 202 [M þ H]þ. Finally, the compound 4a was crystallized [11] in
ethanol and its structure was confirmed by X-ray crystallography
(Fig. 2). Under optimized conditions, benzoxepinoisoxazolone de-
rivatives 4bed was prepared from the corresponding benzox-
epinones 3bed with hydroxylamine hydrochloride in good yields.
4.1. Anti-mycobacterial activity of benzoxepinoisoxazolones and
pyrazolones
The GI50 values for compounds 4aed and 6aet were tabulated
in Table 1; based on GI50 values the structureeactivity relationships
were discussed below. The compound having phenyl substitution
Fig. 1. Structures of isoxazolones and pyrazolones drugs.

462
G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
Scheme 1. Synthesis of compounds 3aed.
Scheme 2. Synthesis of compounds 4aed.
substituted benzoxepinoisoxazolone 4d displayed potent anti-
mycobacterial activity than benzoxepinopyrazolone 6d. The
presence of methyl/methoxy substitutions present on phenyl-
hydrazines 6feh could not display activity, however, dimethyl
substituted derivative 6i (GI50: 19.35
tivity when compared to mono substituted derivative 6e (GI50
49.27 g/mL). Further, it is interesting to note that, the para (6e)
mg/mL) displayed good ac-
:
m
substitution has an impact in displaying the activity when
compared to ortho (6h) and meta (6g). Except fluoro derivative 6j
(GI50: 38.74 mg/mL), the other halo substituted derivatives 6ken
could not display the anti-mycobacterial activity. The presence of
methoxy substitution on benzoxepine and methyl/bromo sub-
stitution on phenylhydrazine 6oep could not display the activity.
The bromo/phenyl substitution on benzoxepine at 6th position
and methyl on phenylhydrazine at 4th position 6q, 6r (GI50
:
42.40 g/mL/GI50: 34.01 g/mL) displayed moderate activity,
m
m
however, the presence of bromo substitution on phenylhydrazine
at 4th position 6set, the anti-mycobacterial activity was drasti-
cally reduced. It is interesting to note that the EWG present on
benzoxepine and EDG present on phenylhydrazine displayed
good activity when compared to EDG on benzoxepine and EWG
on phenylhydrazine (Fig. 3).
Fig. 2. The molecular structure of 4a, with the atom-numbering scheme. Displacement
ellipsoids are drawn at the 30% probability level.
4d (GI50: 9.1
when compared to standard drug isoniazid (GI50: 7.94
Compound 4a (GI50: 21.36 g/mL) and methoxy substituted deriv-
ative 4c (GI50: 28.52 g/mL) displayed moderate anti-mycobacterial
m
g/mL) displayed potent anti-mycobacterial activity
mg/mL).
m
m
activity, however, bromo substituted derivative 4b could not
display the activity. The results indicate that the presence of phenyl
substitution on benzoxepine 4d has an advantage in improving the
activity when compared to bromo 4b and methoxy 4c substitutions
(Fig. 3).
4.2. Anticancer activity of benzoxepinoisoxazolones and
pyrazolones
The in vitro results of anti-mycobacterial activity prompted us to
evaluate their anticancer activity of benzoxepinoisoxazolones 4ae
d, pyrazolones 6aet and IC50 values of these derivatives were
tabulated in Table 1. Compound 4a selectively displayed moderate
anticancer activity on DU-145 and HeLa cell lines. Compounds 4be
Anti-mycobacterial activity of benzoxepinopyrazolones 6aet
was discussed below. Compound having bromo substitution on
benzoxepine 6b (GI50
mycobacterial activity in comparison with standard drug isoni-
azid (GI50: 7.94 g/mL). Compound 6a (GI50: 23.19 g/mL) and
methoxy substituted derivative 6c (GI50: 15.65 g/mL) displayed
: 11.89 mg/mL) displayed potent anti-
d (IC50: 9.03e10.12
selectively on HeLa cell line when compared to standard drug
doxorubicin (IC50: 6.94 M) and displayed good activity (IC50
28.84e49.11 M) on A549, DU-145 and HEK 293. Interestingly,
compounds 4bed shown potent cytotoxicity selectively on HeLa
mM) displayed potent anticancer activity
m
m
m
m
:
good anti-mycobacterial activity, however, phenyl substituted
derivative 6d drastically reduced the activity. The phenyl
m

G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
463
Scheme 3. Synthesis of compounds 6aet.
Table 1
Anti-mycobacterial and anticancer activities of benzoxepinoisoxazolones 4aed and benzoxepinopyrazolones 6aet.
Compounds
MSa (GI50 in
m
g/mL)b
Cytotoxicity (IC50 in
A549
m
M)c
DU-145
HEK 293
HeLa
4a
4b
4c
4d
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
21.36
NAd
28.52
9.1
23.19
11.89
15.65
NAd
NAd
84.83 ꢁ 11.18
33.59 ꢁ 2.47
28.84 ꢁ 0.48
28.91 ꢁ 2.22
NAd
NAd
85 ꢁ 4.96
10.12 ꢁ 0.59
10.05 ꢁ 1.09
9.03 ꢁ 0.33
NAd
46.49 ꢁ 5.99
49.11 ꢁ 1.33
29.70 ꢁ 0.41
NAd
47.73 ꢁ 2.09
48.16 ꢁ 0.53
39.19 ꢁ 2.38
NAd
NAd
122.94 ꢁ 3.98
NAd
NAd
NAd
NAd
NAd
167.18 ꢁ 3.5
116.60 ꢁ 8.99
NAd
NAd
NAd
49.27
NAd
152.33 ꢁ 63.85
67.55 ꢁ 1.79
NAd
NAd
NAd
NAd
133.23 þ 18.74
NAd
NAd
166 ꢁ 23.75
NAd
NAd
NAd
NAd
NAd
63.90 ꢁ 19.08
153.73 ꢁ 44.63
109 ꢁ 4.57
NAd
19.35
38.74
NAd
84.29 ꢁ 10.67
NAd
NAd
NAd
NAd
NAd
NAd
178.94 ꢁ 38.89
215.01 ꢁ 18.45
244.39 ꢁ 52.06
NAd
97.00 ꢁ 12.72
89.39 ꢁ 12.10
NAd
NAd
190 ꢁ 18.52
NAd
NAd
NAd
NAd
NAd
NAd
NAd
117.05 ꢁ 0.13
117.03 ꢁ 3.22
NAd
NAd
NAd
NAd
199.18 ꢁ 21.10
33.30 ꢁ 3.32
162.73 ꢁ 14.57
NAd
138.36 ꢁ 2.97
129.19 ꢁ 6.31
NAd
161.41 ꢁ 18.60
180.24 ꢁ 24.98
42.4
34.01
NAd
NAd
NAd
NAd
78.17 ꢁ 7.63
149.22 ꢁ 24.69
185.48 ꢁ 8.94
NAd
NAd
NAd
NAd
NAd
118.44 ꢁ 18.81
139.59 ꢁ 8.85
e
NAd
NAd
Rifampicin
Isoniazid
Doxorubicin
1.04
7.94
e
e
e
e
e
e
e
e
7.47 ꢁ 0.45
8.19 ꢁ 0.56
7.41 ꢁ 0.36
6.94 ꢁ 0.47
a
Mycobacterium smegmatis ATCC 14468 (MC2).
Concentration of compounds inhibiting growth by 50%.
b
c
IC50 values are indicated as mean ꢁ SD (standard deviation) of three independent experiments.
d
No activity.
cancer cell lines and 4 fold less toxicity towards non cancerous cells
HEK 293. The reason may be due to induced programmed cell death
(apoptosis), therefore to confirm, we have measured activation of
caspase-3 and caspase-9 (leads apoptotic cancer cell death) for
compounds 4bed on exposure of HeLa cells. Results indicating that
the exposure of cells induces activation of both caspase-3 and
caspase-9 up to 40 and 20 fold respectively (Fig. 4). Taken together,
activation of caspase-3 and caspase-9 suggest that these

464
G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
Fig. 3. Illustration of model derivatives as potent anti-mycobacterial and anticancer activities.
compounds 4bed inhibit cancer cell growth by inducing activation
of apoptosis pathway.
Pyrazolones having bromo/methoxy/phenyl substitutions at 6th
position on benzoxepine 6aed did not show promising anticancer
shown moderate activity on DU-145. Further, the presence of
bromo substitution on benzoxepine as well as on phenylhydrazine
6s did not display anticancer activity; however, the presence of
phenyl substitution on benzoxepine 6t displayed mild activity on
A549 and DU-145 cell lines.
Overall in present series (4aed, 6aet), the compounds 4d, 6b
displayed potent anti-mycobacterial activity and 4a, 4c, 6a, 6c, 6e,
6iej, 6qer displayed good to moderate activity. Compounds 4be
d shown potent anticancer activity selectively on HeLa cell lines
and good activity on A549, DU-145 and HEK 293 cell lines. Com-
pounds 6i and 6p selectively displayed good to moderate anti-
cancer activity on A549, however, compounds 6f, 6h, 6kel and 6r
selectively displayed moderate activity on DU-145. Taken together,
compounds 6aec, 6e, 6iej, and 6q selectively displayed towards
anti-mycobacterial activity than anticancer.
activity. The methoxy 6f (IC50: 67.55
63.90 M) substituted derivatives selectively displayed moderate
activity on DU-145, however, dimethyl substituted derivative 6i
(IC50: 84.29 M) selectively shown moderate activity on A549. The
fluoro 6k (IC50: 97.00 M) and chloro substituted pyrazolones 6l
(IC50: 89.39 M) shown selectively moderate activity on DU-145.
mM) and methyl 6h (IC50:
m
m
m
m
The presence of electron donating substitutions such as methoxy
on benzoxepine, methyl on phenylhydrazine (6o) displayed mod-
erate activity on all the tested cell lines, however, bromo substi-
tution on phenylhydrazine 6p (IC50: 33.30
mM) enhances and
selectively displayed good anticancer activity on A549 cell line. The
compound having methyl substitution on phenylhydrazine and
bromo substitution on benzoxepine 6q displayed mild activity on
A549, however, phenyl substitution on benzoxepine 6r selectively
5. Conclusion
In conclusion, benzoxepinoisoxazolones 4aed pyrazolones 6ae
t were synthesized by cyclocondensation of (E)-ethyl 3-oxo-2,3-
dihydrobenzo[b]oxepine-4-carboxylates 3aed with hydroxyl-
amine/phenylhydrazine hydrochlorides for the first time. All the
compounds were screened for their in vitro anti-mycobacterial
activity against M. smegmatis strain and anticancer activity
against A549, DU-145, HeLa and HEK 293 by standard methods and
compared with standard drugs. Compounds 4d and 6b displayed
potent anti-mycobacterial activity and compounds 4bed shown
potent cytotoxicity against cervical cancer (HeLa). Activation of
caspases has been measured for compounds 4bed on HeLa cell
lines since these derivatives have shown very good anticancer ac-
tivity (apoptosis). These compounds are not known so far in the
literature along with the synthetic methodology applied, and the
assignment of the in vitro anti-mycobacterial, anticancer activity in
this report is new aspect. The present study reveals that the ben-
zoxepinoisoxazolones, pyrazolones are potent compounds and it
may serve as a model compounds for design and development of
therapeutics based agents. The synthesis and biological activities of
other benzoxepine derived heterocyclic compounds are under
investigation.
6. Experimental section
6.1. General
Salicylaldehydes, ethyl 4-chloro-3-oxobutanoate, phenylboronic
acid and palladium catalyst were procured from SigmaeAldrich.
Phenylhydrazine hydrochlorides, piperidine, and solvents were
obtained from local suppliers. Wittig reagent was prepared in the
Fig. 4. Determination of caspase-3 and caspase-9 activities induced by compounds
4bed.

G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
465
laboratory as per standard procedures. Reactions were monitored
by thin layer chromatography (TLC) on pre-coated silica gel 60 F254
(mesh); spots were visualized under UV light. Column chromato-
graphic separations were carried out on silica gel (60e120 mesh).
Melting points were determined on a Mettler-Temp apparatus and
are uncorrected. An IR spectrum was recorded with a Thermo
Nicolet Nexus 670 spectrometer. 1H NMR and 13C NMR spectra were
recorded on a Gemini 200 MHz, Bruker Avance 300 MHz spec-
(0.06 mL, 0.06 mmol) were added at room temperature and the
resulting mixture was refluxed for 3 h. After completion of the
reaction (TLC), the solvent was removed under reduced pressure.
The section mixture was diluted with water and extracted with
ethyl acetate, the organic layer was dried over Na2SO4, solvent was
removed under reduced pressure, and the residue was purified by
column chromatography using silica gel (hexane/ethyl acetate)
provided 3H,10H-benzo[6,7]oxepino[3,4-c]isoxazol-3-one 4a in
72% yield as pale yellow color solid. Similarly, compounds 4b
(methoxy), 4c (bromo) and 4d (phenyl) were synthesized from
corresponding 3-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate
derivatives 3bed.
trometers. Chemical shifts (d) are quoted in parts per million and
are referenced to tetramethylsilane (TMS) as internal standard.
EIMS obtained on 7070H spectrometer operating at 70 eV using a
direct inlet system. HRMS were carried out on Agilent 6510 Q-TOF
LC/MS instrument.
6.4.1. 3H,10H-Benzo[6,7]oxepino[3,4-c]isoxazol-3-one (4a)
6.2. General procedure for synthesis of 2,2,3-trisubstituted 2H-
Yield: 72%; pale yellow solid; m.p.: 154e156 ꢂC; 1H NMR
chromenes (2aed)
(300 MHz, CDCl3):
d
5.04 (s, 2H, OCH2), 7.23 (d, 1H, J ¼ 7.9 Hz, ar-
omatic), 7.28 (t, 1H, J ¼ 7.9 Hz, aromatic), 7.55 (d, 2H, J ¼ 6.9 Hz,
Ethyl 4-chloro-3-oxobutanoate (1 mmol) was added slowly to a
stirred solution of salicylaldehyde (1a, 1 mmol) and piperidine
(30 mol%) in DCM (2 mL) at room temperature over a period of
15 min. The mixture was stirred for another 8 h at the same tem-
perature. After completion of the reaction (TLC), the crude product
was subjected to column chromatography purification to provide
2H-chromene 2a. Similarly 2bed was prepared from corresponding
substituted salicylaldehydes 1bed.
aromatic), 7.74 (s, 1H, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d
66.78, 120.12, 122.88, 125.34, 125.72, 135.68, 136.19, 145.28,
159.79, 160.47, 168.84 ppm; IR (KBr): 1759, 1710, 1644, 1560, 1210,
1008, 759 cmꢀ1; MS (ESI): m/z 202 [M þ H]þ.
6.4.2. 6-Methoxy-3H,10H-benzo[6,7]oxepino[3,4-c]isoxazol-3-one
(4b)
Yield: 75%; orange color solid; m.p: 150e152 ꢂC; 1H NMR
(300 MHz, CDCl3):
d 3.84 (s, 3H, OCH3), 4.98 (s, 2H, OCH2), 6.98 (d,
6.2.1 .Ethyl
carboxylate (2a). Ref. [9b]
6.2.2 .Ethyl
mene-3-carboxylate (2b): Ref. [9b]
6.2.3 .Ethyl 6-bromo-2-(chloromethyl)-2-hydroxy-2H-chromene-
3-carboxylate (2c). Ref. [9b]
2-(chloromethyl)-2-hydroxy-2H-chromene-3-
2-(chloromethyl)-2-hydroxy-6-methoxy-2H-chro-
1H, J ¼ 3.0 Hz, aromatic), 7.08 (dd, 1H, J ¼ 3.0, 9.0 Hz, aromatic), 7.16
(d,1H, J ¼ 9.1 Hz, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 55.83,
66.93, 118.78, 120.34, 121.77, 123.75, 126.05, 145.17, 154.34, 156.29,
159.91, 168.79 ppm; IR (KBr): 1766, 1645, 1564, 1483, 1280, 1009,
772 cmꢀ1; MS (ESI): m/z 232 [M þ H]þ.
6.2.4. Ethyl 2-(chloromethyl)-2-hydroxy-6-phenyl-2H-chromene-
3-carboxylate (2i). Ref. [9b]
6.4.3. 6-Bromo-3H,10H-benzo[6,7]oxepino[3,4-c]isoxazol-3-one
(4c)
Yield: 80%; pale yellow color solid; m.p: 167e169 ꢂC; 1H NMR
(300 MHz, CDCl3):
d
5.03 (s, 2H, OCH2), 7.12 (d, 1H, J ¼ 8.5 Hz, ar-
6.3. General procedure for the synthesis of (E)-ethyl 3-oxo-2,3-
dihydrobenzo[b]oxepine-4-carboxylates (3aed)
omatic), 7.62 (dd, 1H, J ¼ 2.5, 6.6 Hz, aromatic), 7.66 (d, 1H,
J ¼ 2.3 Hz, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 66.85,
117.75, 121.25, 124.62, 127.17, 130.36, 134.11, 137.93, 143.36, 159.27,
168.22 ppm; IR (KBr): 1766, 1647, 1472, 1244, 1210, 1134, 1008,
760 cmꢀ1; MS (ESI): m/z 280 [M þ H]þ.
A mixture of ethyl 2-(chloromethyl)-2-hydroxy-2H-chromene-
3-carboxylate
ethoxycarbonylethylidene)triphenylphosphorane
(2a,
268
mg,
1.0
mmol)
and
(506
(1-
mg,
1.4 mmol) in DCM or benzene (4 mL) were refluxed for 6 h under
nitrogen atmosphere. After completion of the reaction (TLC), the
solvent was removed under reduced pressure, crude residue was
purified by column chromatography using silica gel (hexane/ethyl
acetate 9:1) afforded (E)-ethyl 3-oxo-2,3-dihydrobenzo[b]oxepine-
4-carboxylate 3a (190 mg, 82% yield) as yellow liquid. Similarly
compounds 3bed was synthesized from corresponding 2bed un-
der optimized conditions.
6.4.4. 6-Phenyl-3H,10H-benzo[6,7]oxepino[3,4-c]isoxazol-3-one
(4d)
Yield: 78%; yellow color solid; m.p: 190e192 ꢂC; 1H NMR
(300 MHz, CDCl3):
d
5.07 (s, 2H, OCH2), 7.30 (d, 1H, J ¼ 8.3 Hz, ar-
omatic), 7.42 (d, 1H, J ¼ 6.8 Hz, aromatic), 7.49 (t, 1H, J ¼ 6.8 Hz,
aromatic), 7.56 (d, 2H, J ¼ 7.6 Hz, aromatic), 7.71e7.78 (m, 2H, ar-
omatic) 7.81 (s, 1H, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d
66.83, 120.28, 123.29, 125.82, 126.79, 128.09, 129.08, 130.05,
130.22, 134.14, 134.32, 134.44, 134.75, 138.56, 145.34, 159.68,
6.3.1. (E)-Ethyl 3-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate
(3a). Ref. [9c]
6.3.2. (E)-Ethyl 7-methoxy-3-oxo-2,3-dihydrobenzo[b]oxepine-4-
carboxylate (3b). Ref. [9c]
168.76 ppm; IR (KBr): 1752, 1645, 1479, 1216, 1015, 849, 760 cmꢀ1
;
MS (ESI): m/z 278 [M þ H]þ.
6.5. General procedure for the preparation of
6.3.3. (E)-Ethyl
7-bromo-3-oxo-2,3-dihydrobenzo[b]oxepine-4-
benzoxepinopyrazolones (6aet)
carboxylate (3c). Ref. [9c]
Compound (E)-ethyl 3-oxo-2,3-dihydrobenzo[b]oxepine-4-
carboxylate (3a, 100 mg, 0.4 mmol) was dissolved in absolute
ethanol (3 mL), phenylhydrazine hydrochloride (5a, 62 mg,
0.4 mmol) and acetic acid (0.06 mL, 0.06 mmol) were added at
room temperature, and the resulting mixture was refluxed for 4 h.
The reaction was monitored by TLC and after completion of the
reaction (TLC), the reaction mixture was brought to room temper-
ature and solvent was removed under reduced pressure; diluted
6.4. General procedure for the preparation of
benzoxepinoisoxazolones (4aed)
(E)-Ethyl 3-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylate (3a,
100 mg, 0.4 mmol) was dissolved in absolute ethanol (3 mL), hy-
droxylamine hydrochloride (30 mg, 0.4 mmol) and acetic acid

466
G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
with water and extracted with ethyl acetate. The organic layer was
dried over Na2SO4, solvent was removed under reduced pressure,
and the residue was purified by column chromatography using
silica gel (hexane/ethyl acetate) provided 6a in 80% yield. Similarly
other benzoxepinopyrazolones 6bet were prepared from corre-
sponding 3-oxo-2,3-dihydrobenzo[b]oxepine-4-carboxylates 3be
d with substituted phenylhydrazine hydrochlorides 5bek.
aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 21.56, 68.72, 115.87,
119.22, 122.57, 124.79, 125.87, 126.33, 127.81, 128.66, 134.58, 135.94,
138.11, 138.75, 141.08, 148.91, 160.27, 163.07 ppm; IR (KBr): 2921,
1694, 1642, 1597, 1491, 1342, 1293, 1205, 1138, 1019, 770 cmꢀ1; MS
(ESI): m/z 291 [M þ H]þ.
6.5.6. 2-(4-methoxyphenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6f)
6.5.1. 2-Phenyl-2,10-dihydro-3H-benzo[6,7]oxepino[3,4-c]pyrazol-
3-one (6a)
Yield: 67%; brick red color solid; m.p: 176e178 ꢂC; 1H NMR
(300 MHz, CDCl3):
d 3.82 (s, 3H, OCH3), 5.04 (s, 2H, OCH2), 6.96 (d,
Yield: 80%; orange color solid; m.p: 206e208 ꢂC; 1H NMR
2H, J ¼ 9.1 Hz, aromatic), 7.22 (t, 2H, J ¼ 5.7 Hz, aromatic), 7.49 (dt,
1H, J ¼ 1.8, 7.9 Hz, aromatic), 7.59 (dd, 1H, J ¼ 1.1, 7.5 Hz, aromatic),
7.73 (s, 1H, aromatic), 7.80 (d, 2H, J ¼ 9.1 Hz, aromatic) ppm; 13C
(300 MHz, CDCl3):
d
5.06 (s, 2H, OCH2), 7.22 (d, 2H, J ¼ 7.6 Hz, ar-
omatic), 7.26 (d, 1H, J ¼ 7.6 Hz, aromatic), 7.43 (t, 2H, J ¼ 8.3 Hz,
aromatic), 7.50 (dt,1H, J ¼ 1.5, 7.5 Hz, aromatic), 7.57 (dd,1H, J ¼ 2.3,
7.6 Hz, aromatic), 7.75 (s, 1H, aromatic), 7.94 (dd, 2H, J ¼ 1.5, 8.3 Hz,
NMR (75 MHz, CDCl3):
d
55.44, 68.74, 114.01 ꢃ 2C, 120.59 ꢃ 2C,
122.58, 124.79, 126.38, 127.88, 131.59, 134.55, 135.94, 141.03, 148.69,
156.99, 160.29, 162.84 ppm; IR (KBr): 2926, 1685, 1639, 1593, 1504,
1453, 1239, 1146, 1115, 1032, 828, 761 cmꢀ1; MS (ESI): m/z 307
[M þ H]þ; HRMS (ESI): m/z calcd for C18H15O3N2 [M þ H]þ
307.10745; found 307.10772.
aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d
68.78, 118.67 ꢃ 2C,
122.64, 124.86, 125.06, 126.39, 12 7.85, 128.87 ꢃ 2C, 134.65, 135.99,
138.27, 141.18, 149.04, 160.36, 163.16 ppm; IR (KBr): 2923, 2853,
1696, 1646, 1592, 1497, 1341, 1289, 1209, 1147, 1010, 761 cmꢀ1; MS
(ESI): m/z 277 [M þ H]þ; HRMS (ESI): m/z calcd for C17H13O2N2
[M þ H]þ 277.09692; found 277.09715.
6.5.7. 2-(3-methylphenyl)-2,10-dihydro-3H-benzo [6,7]oxepino
[3,4-c]pyrazol-3-one (6g)
6.5.2. 6-Bromo-(2-phenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6b)
Yield: 77%; orange color solid; m.p: 194e196 ꢂC; 1H NMR
(300 MHz, CDCl3): d 2.40 (s, 3H, CH3), 5.05 (s, 2H, OCH2), 7.03 (d, 1H,
Yield: 88%; red color solid; m.p: 154e156 ꢂC; 1H NMR (300 MHz,
J ¼ 7.6 Hz, aromatic), 7.19e7.29 (m, 2H, aromatic), 7.33 (d, 1H,
CDCl3):
d
5.04 (s, 2H, OCH2), 7.11 (d,1H, J ¼ 8.3 Hz, aromatic), 7.23 (d,
J ¼ 7.6 Hz, aromatic), 7.46e7.58 (m, 3H, aromatic), 7.74 (brs, 2H,
1H, J ¼ 7.6 Hz, aromatic), 7.43 (t, 2H, J ¼ 8.3 Hz, aromatic), 7.58 (dd,
1H, J ¼ 3.0, 9.1 Hz, aromatic), 7.63 (s, 1H, aromatic), 7.68 (d, 1H,
J ¼ 2.3 Hz, aromatic), 7.92 (dd, 2H, J ¼ 1.5, 9.1 Hz, aromatic) ppm; IR
(KBr): 2922, 2852, 1701, 1473, 1337, 1218, 1110, 772 cmꢀ1; MS (ESI):
m/z 355, 357 [M þ H]þ.
aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 21.58, 68.75, 115.19,
119.26, 122.60, 124.81, 125.89, 126.37, 127.85, 128.68, 134.59, 135.96,
138.15, 138.77, 141.08, 148.92, 160.31, 163.01 ppm; IR (KBr): 2922,
2853, 1700, 1642, 1599, 1457, 1344, 1215, 1152, 1006, 771, 721 cmꢀ1
;
MS (ESI): m/z 291 [M þ H]þ.
6.5.3. 6-Methoxy-(2-phenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6c)
6.5.8. 2-(2-methylphenyl)-2,10-dihydro-3H-benzo[6,7]oxepino[3,4-
c]pyrazol-3-one (6h)
Yield: 70%; red color solid; m.p: 176e178 ꢂC; 1H NMR (300 MHz,
Yield: 68%; pale yellow color solid; m.p: 128e130 ꢂC; 1H NMR
CDCl3):
d
3.74 (s, 3H, OCH3), 4.90 (s, 2H, OCH2), 6.91 (d, 1H,
(300 MHz, CDCl3):
(m, 5H, aromatic), 7.46e7.58 (m, 2H, aromatic), 7.77 (s, 1H, aro-
matic) ppm; 13C NMR (75 MHz, CDCl3):
18.44, 68.81, 122.58,
124.81, 126.41, 126.53, 126.60, 127.00, 128.48, 131.12, 133.98, 134.58,
135.22, 135.95, 141.38, 148.82, 160.32, 163.62 ppm; IR (KBr): 2922,
2853, 1700, 1642, 1599, 1457, 1344, 1215, 1006, 771 cmꢀ1; MS (ESI):
m/z 291 [M þ H]þ.
d 2.32 (s, 3H, CH3), 5.02 (s, 2H, OCH2), 7.18e7.36
J ¼ 1.5 Hz, aromatic), 6.95 (d, 1H, J ¼ 3.0 Hz, aromatic), 7.05 (d, 1H,
J ¼ 8.3 Hz, aromatic), 7.12 (t, 1H, J ¼ 7.6 Hz, aromatic), 7.34 (t, 2H,
J ¼ 7.6 Hz, aromatic), 7.59 (s, 1H, aromatic), 7.85 (dd, 2H, J ¼ 1.5,
d
9.0 Hz, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 55.73, 68.87,
118.23 ꢃ 2C, 120.77, 123.48, 125.02, 126.75, 128.05, 128.85, 129.25,
129.40, 138.19, 141.07, 149.22, 154.27, 155.96, 163.01 ppm; IR (KBr):
2921, 2852, 1686 1642, 1562, 1289, 1054, 753 cmꢀ1; MS (ESI): m/z
307 [M þ H]þ.
6.5.9. 2-(3, 4-dimethylphenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6i)
6.5.4. 2,6-Diphenyl-2,10-dihydro-3H-benzo[6,7]oxepino[3,4-c]
pyrazol-3-one (6d)
Yield: 66%; orange color solid; m.p: 200e202 ꢂC; 1H NMR
(300 MHz, CDCl3):
d 2.26 (s, 3H, CH3), 2.37 (s, 3H, CH3), 5.04 (s, 2H,
Yield: 82%; red color solid; m.p: 238e240 ꢂC; 1H NMR (300 MHz,
OCH2), 7.17 (d, 1H, J ¼ 8.9 Hz, aromatic), 7.23 (d, 1H, J ¼ 6.8 Hz,
aromatic), 7.49 (dt, 1H, J ¼ 1.5, 7.9 Hz, aromatic), 7.56 (dd, 1H, J ¼ 1.1,
7.7 Hz, aromatic), 7.64 (dd, 1H, J ¼ 1.5, 8.1 Hz, aromatic), 7.67 (s, 1H,
aromatic), 7.73 (s, 1H, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
CDCl3):
d
5.09 (s, 2H, OCH2), 7.21 (t, 1H, J ¼ 7.1 Hz, aromatic), 7.29 (d,
1H, J ¼ 8.1 Hz, aromatic), 7.39e7.49 (m, 5H, aromatic), 7.57 (d, 1H,
J ¼ 8.1 Hz, aromatic), 7.64e7.72 (m, 2H, aromatic), 7.76 (s, 1H, ar-
omatic), 7.76 (d, 1H, J ¼ 2.3 Hz, aromatic), 7.82 (s, 1H, aromatic), 7.95
d
19.26, 19.96, 68.77, 116.40, 120.00, 122.58, 124.77, 126.40, 127.95,
(d, 2H, J ¼ 8.1 Hz, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d
68.88,
129.84, 133.52, 134.49, 135.92, 136.02, 137.13, 140.87, 148.72, 160.29,
162.96 ppm; IR (KBr): 2967,1697,1646,1605,1496,1339,1210,1006,
811, 766 cmꢀ1; MS (ESI): m/z 305 [M þ H]þ; HRMS (ESI): m/z calcd
for C19H17O2N2 [M þ H]þ 305.12805; found 305.12845.
118.67, 123.07, 125.06, 126.80, 127.86, 128.38, 128.54, 128.86, 129.01,
131.92, 131.98, 132.11, 133.13, 134.18, 141.15, 148.90, 159.61,
163.10 ppm; IR (KBr): 2917, 2846, 1697, 1483, 1333, 1219, 772 cmꢀ1
;
MS (ESI): m/z 353 [M þ H]þ.
6.5.10. 2-(4-fluorophenyl)-2,10-dihydro-3H-benzo[6,7]oxepino[3,4-
c]pyrazol-3-one (6j)
6.5.5. 2-(4-methylphenyl)-2,10-dihydro-3H-benzo[6,7]oxepino[3,4-
c]pyrazol-3-one (6e)
Yield: 81%; yellow color solid; m.p: 208e210 ꢂC; 1H NMR
Yield: 68%; orange color solid; m.p: 194e196 ꢂC; 1H NMR
(300 MHz, CDCl3):
d
5.05 (s, 2H, OCH2), 7.11 (t, 2H, J ¼ 8.5 Hz, ar-
(300 MHz, CDCl3):
d
2.41 (s, 3H, CH3), 5.05 (s, 2H, OCH2), 7.02 (d, 1H,
omatic), 7.18e7.28 (m, 2H, aromatic), 7.49 (d, 1H, J ¼ 7.6 Hz, aro-
J ¼ 7.4 Hz, aromatic), 7.21 (d, 1H, J ¼ 7.7 Hz, aromatic), 7.26 (t, 1H,
J ¼ 6.9 Hz, aromatic), 7.32 (d, 1H, J ¼ 7.7 Hz, aromatic), 7.50 (t, 1H,
J ¼ 7.7 Hz, aromatic), 7.56 (d, 1H, J ¼ 7.4 Hz, aromatic), 7.74 (brs, 3H,
matic), 7.57 (d,1H, J ¼ 7.4 Hz, aromatic), 7.75 (s,1H, aromatic), 7.87e
7.94 (m, 2H, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 68.758,
115.46, 115.76, 117.77, 120.42, 120.52, 122.71, 124.71, 124.95, 126.37,

G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
467
127.69, 134.84, 136.10, 141.56, 149.13, 158.33, 160.42, 161.57 ppm; IR
6.5.16. 6-Methoxy-2-(4-bromophenyl)-2,10-dihydro-3H-benzo[6,7]
oxepino[3,4-c]pyrazol-3-one (6p)
Yield: 67%; red color solid; m.p: 206e208 ꢂC; 1H NMR (300 MHz,
CDCl3): 3.84 (s, 3H, OCH3), 4.99 (s, 2H, OCH2), 6.92 (d, 2H,
(KBr): 2924, 2853, 1706, 1644, 1506, 1228, 1210, 1148, 1106, 824,
764 cmꢀ1; MS (ESI): m/z 295 [M þ H]þ.
d
J ¼ 8.3 Hz, aromatic), 7.04 (dd, 2H, J ¼ 3.0, 9.0 Hz, aromatic), 7.34 (d,
1H, J ¼ 8.3 Hz, aromatic), 7.52 (d, 1H, J ¼ 8.3 Hz, aromatic), 7.70 (s,
1H, aromatic), 7.86 (d, 1H, J ¼ 9.1 Hz, aromatic) ppm; 13C NMR
6.5.11. 2-(3-fluorophenyl)-2,10-dihydro-3H-benzo[6,7]oxepino[3,4-
c]pyrazol-3-one (6k)
Yield: 84%; yellow color solid; m.p: 207e209 ꢂC; 1H NMR
(75 MHz, CDCl3):
d
55.78, 68.87, 117.80, 118.74, 119.93 ꢃ 2C, 120.99,
(300 MHz, CDCl3):
d
5.05 (s, 2H, OCH2), 6.89 (dt, 1H, J ¼ 2.5, 8.3 Hz,
123.57, 126.71, 131.84 ꢃ 2C, 134.86, 137.36, 141.42, 149.54, 154.35,
156.06, 163.05 ppm; IR (KBr): 2924, 2846, 1708, 1647, 1564, 1487,
1337, 1289, 1271, 1192, 1171, 1010, 815, 770 cmꢀ1; MS (ESI): m/z 385
[M þ H]þ; HRMS (ESI): m/z calcd for C18H14O3N2Br [M þ H]þ
385.01855; found 385.01823.
aromatic), 7.24 (d, 1H, J ¼ 7.7 Hz, aromatic), 7.26 (dd, 1H, J ¼ 1.1,
7.7 Hz, aromatic), 7.32e7.42 (m, 1H, aromatic), 7.47e7.60 (m, 2H,
aromatic), 7.72e7.82 (m, 3H, aromatic) ppm; 13C NMR (75 MHz,
CDCl3):
d 60.692, 105.62, 105.98, 111.44, 111.72, 113.65, 122.68,
124.93, 126.29, 127.55, 130.17, 134.88, 136.01, 141.65, 149.41, 160.39,
164.50 ppm; IR (KBr): 2917, 2851, 1711, 1646, 1593, 1561, 1482, 1453,
1347, 1214, 767 cmꢀ1; MS (ESI): m/z 295 [M þ H]þ; HRMS (ESI): m/z
calcd for C17H12O2N2F [M þ H]þ 295.08752; found 295.08773.
6.5.17. 6-Bromo-2-(4-methylphenyl)-2,10-dihydro-3H-benzo[6,7]
oxepino[3,4-c]pyrazol-3-one (6q)
Yield: 79%; red color solid; m.p: 222e224 ꢂC; 1H NMR (300 MHz,
CDCl3):
d
2.36 (s, 3H, CH3), 5.02 (s, 2H, OCH2), 7.10 (d, 1H, J ¼ 8.7 Hz,
6.5.12. 2-(4-Chlorophenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6l)
aromatic), 7.22 (d, 2H, J ¼ 8.3 Hz, aromatic), 7.56 (dd, 1H, J ¼ 2.3,
8.7 Hz, aromatic), 7.61 (s, 1H, aromatic), 7.67 (d, 1H, J ¼ 1.5 Hz, ar-
omatic), 7.77 (d, 2H, J ¼ 8.1 Hz, aromatic) ppm; 13C NMR (75 MHz,
Yield: 78%; orange color solid; m.p: 224e226 ꢂC; 1H NMR
(300 MHz, CDCl3): d 5.05 (s, 2H, OCH2), 7.32 (m, 3H, aromatic), 7.53
CDCl3):
d 20.97, 68.89, 116.66, 117.19, 118.73, 124.43, 128.02, 128.93,
(m, 2H, aromatic), 7.75 (s, 1H, aromatic), 7.92 (d, 2H, J ¼ 7.7 Hz,
aromatic) ppm; IR (KBr): 2922, 2851, 1703, 1643, 1591, 1491, 1340,
1204, 1146, 1006, 820, 770 cmꢀ1; MS (ESI): m/z 311, 313 [M þ H]þ;
HRMS (ESI): m/z calcd for C17H12O2N2Cl [M þ H]þ 311.05798; found
311.05818.
129.42, 134.94, 135.64, 136.87, 137.58, 139.05, 148.29, 157.77, 159.22,
162.59 ppm; IR (KBr): 2922, 2853, 1687, 1646, 1510, 1475, 1336,
1216, 818, 772 cmꢀ1; MS (ESI): m/z 391 [M þ Na]þ.
6.5.18. 2-(4-Methylphenyl)-6-phenyl-2,10-dihydro-3H-benzo[6,7]
oxepino[3,4-c]pyrazol-3-one (6r)
Yield: 78%; red color solid; m.p: 176e178 ꢂC; 1H NMR (300 MHz,
6.5.13. 2-(3-chlorophenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6m)
CDCl3):
d
2.36 (s, 3H, CH3), 5.08 (s, 2H, OCH2), 7.23 (d, 1H, J ¼ 8.3 Hz,
Yield: 83%; yellow color solid; m.p: 182e184 ꢂC; 1H NMR
aromatic), 7.28 (d, 1H, J ¼ 10.5 Hz, aromatic), 7.41 (d, 1H, J ¼ 6.8 Hz,
aromatic), 7.48 (t, 2H, J ¼ 7.5 Hz, aromatic), 7.57 (d, 2H, J ¼ 7.6 Hz,
aromatic), 7.71 (dd, 1H, J ¼ 2.3, 8.3 Hz, aromatic), 7.75 (d, 1H,
J ¼ 2.3 Hz, aromatic), 7.80 (t, 3H, J ¼ 8.3 Hz, aromatic) ppm; IR (KBr):
2961, 2917, 1680, 1644, 1511, 1476, 1339, 1148, 1009, 815, 755,
690 cmꢀ1; MS (ESI): m/z 367 [M þ H]þ.
(300 MHz, CDCl3):
d
5.05 (s, 2H, OCH2), 7.16 (td, 1H, J ¼ 1.9, 7.9 Hz,
aromatic), 7.22 (d, 1H, J ¼ 7.9 Hz, aromatic), 7.26 (dd, 1H, J ¼ 1.9,
7.6 Hz, aromatic), 7.34 (t,1H, J ¼ 8.1 Hz, aromatic), 7.48e7.58 (m, 1H,
2H, aromatic), 7.75 (s, 1H, aromatic), 7.91 (td, 1H, J ¼ 1.9, 8.3 Hz,
aromatic), 8.00 (t, 1H, J ¼ 2.1 Hz, aromatic) ppm; 13C NMR (75 MHz,
CDCl3): d 68.70, 116.31, 118.44, 122.68, 124.93, 126.30, 127.51, 129.93,
6.5.19. 6-Bromo-2-(4-bromophenyl)-2,10-dihydro-3H-benzo[6,7]
oxepino[3,4-c]pyrazol-3-one (6s)
134.63, 134.89, 136.11, 139.11, 139.33, 141.69, 149.47, 160.42,
163.19 ppm; IR (KBr): 2920, 2851,1708,1643,1598,1481,1432,1342,
1212, 1152, 1081, 769 cmꢀ1; MS (ESI) : m/z 311, 313 [M þ H]þ.
Yield: 90%; red color solid; m.p: 228e230 ꢂC; 1H NMR (300 MHz,
CDCl3):
d
5.02 (s, 2H, OCH2), 7.11 (d,1H, J ¼ 8.7 Hz, aromatic), 7.53 (d,
2H, J ¼ 9.1 Hz, aromatic), 7.58 (dd, 1H, J ¼ 2.5, 9.1 Hz, aromatic), 7.63
(s, 1H, aromatic), 7.68 (d, 1H, J ¼ 2.3 Hz, aromatic), 7.85 (d, 2H,
6.5.14. 2-(4-Bromophenyl)-2,10-dihydro-3H-benzo[6,7]oxepino
[3,4-c]pyrazol-3-one (6n)
J ¼ 8.9 Hz, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
d 68.71,117.84,
Yield: 76%; orange color solid; m.p: 228e230 ꢂC; 1H NMR
119.96, 122.67, 124.94, 126.31, 127.57, 131.85, 134.87, 136.11, 137.39,
141.64, 149.38, 160.41, 163.12 ppm; IR (KBr): 2922, 2851, 1704, 163,
1492, 1471, 1337, 1213, 1142, 1009, 820, 772 cmꢀ1; MS (ESI): m/z 455
[M þ Na]þ.
(300 MHz, CDCl3):
d
5.04 (s, 2H, OCH2), 7.22 (dd, 1H, J ¼ 1.5, 8.3 Hz,
aromatic), 7.26 (t, 1H, J ¼ 4.5 Hz, aromatic), 7.48e7.58 (m, 4H, ar-
omatic), 7.75 (s, 1H, aromatic), 7.85e7.90 (m, 2H, aromatic) ppm; IR
(KBr): 2923, 1695, 1639, 1593, 1487, 1342, 1291, 1206, 1144, 1008,
823, 770 cmꢀ1; MS (ESI): m/z 355, 357 [M þ H]þ; HRMS (ESI): m/z
calcd for C17H12O2N2Br [M þ H]þ 355.00800; found 355.00767.
6.5.20. 2-(4-Bromophenyl)-6-phenyl-2,10-dihydro-3H-benzo[6,7]
oxepino[3,4-c]pyrazol-3-one (6t)
Yield: 79%; red color solid; m.p: 238e240 ꢂC; 1H NMR (300 MHz,
6.5.15. 6-Methoxy-2-(4-methylphenyl)-2,10-dihydro-3H-benzo
[6,7]oxepino[3,4-c]pyrazol-3-one (6o)
CDCl3):
d
5.07 (s, 2H, OCH2), 7.30 (d, 1H, J ¼ 8.3 Hz, aromatic), 7.42
(d, 1H, J ¼ 7.6 Hz, aromatic), 7.49 (t, 2H, J ¼ 7.6 Hz, aromatic), 7.55 (t,
2H, J ¼ 9.1 Hz, aromatic), 7.72 (d, 2H, J ¼ 9.1 Hz, aromatic), 7.76 (s,
1H, aromatic), 7.82 (s, 1H, aromatic), 7.89 (d, 2H, J ¼ 8.3 Hz, aro-
Yield: 63%; red color solid; m.p: 190e192 ꢂC; 1H NMR (300 MHz,
CDCl3):
d 2.36 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.99 (s, 2H, OCH2),
7.02 (brs, 1H, aromatic), 7.05 (d, 1H, J ¼ 3.0 Hz, aromatic), 7.14 (dd,
1H, J ¼ 0.8, 9.1 Hz, aromatic), 7.23 (d, 2H, J ¼ 8.3 Hz, aromatic), 7.68
(s, 1H, aromatic), 7.79 (d, 2H, J ¼ 8.3 Hz, aromatic) ppm; 13C NMR
matic) ppm; IR (KBr): 2917, 2846, 1697, 1483, 1333, 1219, 772 cmꢀ1
;
MS (ESI): m/z 431 [M þ H]þ.
(75 MHz, CDCl3):
d
55.73, 68.89, 118.62, 118.68 ꢃ 2C, 120.62, 123.46,
6.6. X-ray crystallography
126.77, 128.15, 129.33 ꢃ 2C, 134.69, 135.74, 140.79, 148.96, 154.22,
155.92, 162.89 ppm; IR (KBr): 2920, 2851, 1695, 1508, 1487, 1338,
1289, 1270, 1215, 1053, 815, 772 cmꢀ1; MS (ESI): m/z 321 [M þ H]þ;
HRMS (ESI): m/z calcd for C19H17O3N2 [M þ H]þ 321.12299; found
321.12337.
X-ray data for the compound 4a was collected at room tem-
perature using a Bruker Smart Apex CCD diffractometer with
graphite monochromated MoK
scan method. Preliminary lattice parameters and orientation
ꢀ
a
radiation (
l
¼ 0.71073 A) with
u-

468
G. Saidachary et al. / European Journal of Medicinal Chemistry 76 (2014) 460e469
matrices were obtained from four sets of frames. Integration and
scaling of intensity data was accomplished using SAINT program.
The structure was solved by direct methods using SHELXS97 [11]
and refinement was carried out by full-matrix least-squares tech-
nique using SHELXL97. Anisotropic displacement parameters were
included for all non-hydrogen atoms. All H atoms were positioned
geometrically and treated as riding on their parent C atoms [Ce
individual cell lines further continued to grow in complete medium
under standard conditions. After 18 h to treat cells with test mol-
ecules, aliquots of 2
mL of the compound dilutions were added to
the appropriate microtiter wells already containing 198
mL of fresh
medium with cells, resulting in the required final drug concentra-
tions. As a standard reference drug we have used doxorubicin in all
assay plates served as internal control. For each, compound four
ꢀ
H ¼ 0.93e0.97 A and Uiso(H) ¼ 1.2Ueq(C)].
concentrations (0.1, 1, 10 and 100
done in triplicate wells. Plates were incubated further for 48 h and
assay was terminated by the addition of 10 L of 3-(4, 5-
mM) were evaluated and each was
6.6.1. Crystal data for compound 4a
m
C
11H7NO3, M ¼ 201.18, colorless block, 0.21 ꢃ 0.18 ꢃ 0.11 mm3,
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
5 mg/mL) and incubated for 60 min at 37 ꢂC. The plates were air-
dried and the dye bound to the cells was subsequently solubilized
orthorhombic, space group Fdd2 (No. 43), a ¼ 19.4953 (19),
3
ꢀ
ꢀ
b ¼ 24.910 (2), c ¼ 7.3577 (7) A, V ¼ 3573.1 (6) A , Z ¼ 16,
Dc ¼ 1.496 g/cm3, F000 ¼ 1664, CCD Area Detector, MoK
a
radiation,
in 100 mL of DMSO, and the absorbance was red on a multimode
l
¼ 0.71073 A, T ¼ 294 (2) K, 2qmax ¼ 50.0ꢂ, 8379 reflections
reader (Tecan) at a wavelength of 560 nm is directly proportional to
cell growth. Percent growth was calculated for test wells relative to
control wells. The above determinations were repeated three times.
The growth inhibitory effects of the compounds were analyzed by
generating dose response curves as a plot of the percentage sur-
viving cells versus drug concentration. Sensitivity of the cancer
cells to the drug treatment was expressed in terms of the concen-
tration of drug required for 50% inhibition of cell growth (IC50).
ꢀ
collected, 857 unique (Rint
¼
0.0220). Final GooF
¼
1.183,
R1 ¼ 0.0259, wR2 ¼ 0.0677, R indices based on 844 reflections with
I
m
> 2s 1 restraint,
(I) (refinement on F2), 137 parameters,
¼ 0.111 mmꢀ1. CCDC 914544 contains supplementary crystallo-
graphic data for the structure. Email: deposit@ccdc.cam.ac.uk.
6.7. Biology activity
6.7.1. In vitro anti-mycobacterial activity using growth inhibition
assay by turbidometry
6.7.3. Determination of caspase-3 and caspase-9 activities for
compounds 4be4d
Anti-mycobacterial activity of the synthesized compounds was
performed with Mycobacterium smegmatis strain using growth in-
hibition assay by turbidometry. Briefly, isolated single colonies of
M. smegmatis MC2 155 (ATCC 14468) from 7H10 agar plate were
grown overnight in Middlebrook 7H9 medium (0.47% Middlebrook
7H9 broth base, 10% ADS, 0.2% glycerol, and 0.1% Tween-80) to mid
exponential phase at 37 ꢂC. Subsequently, 5 mL of Middlebrook 7H9
broth were inoculated with the overnight grown culture and
allowed to grow at 37 ꢂC to early log phase (OD600 z 0.3). For anti-
Caspase-3 and caspase-9 activities were measured 48 h after
treating the HeLa cells with selected compounds or DMSO using
specific fluorogenic substrates Ac-DEVD-AMC and Ac-LEHD-AFC,
for caspase-3 and -9 respectively. After treatment, harvested cells
were lysed with caspase lysis buffer (50 mM HEPES, 5 mM CHAPS,
5 mM DTT, pH 7.5) and then incubated with the respective substrate
(Ac-DEVD-AMC/Ac-LEHD-AFC) in 20 mM HEPES (pH 7.5), 0.1%
CHAPS, 2 mM EDTA and 5 mM DTT at 37 ꢂC for 2 h. The release of
AMC and AFC was analyzed by fluorimeter using an excitation/
emission wavelength of 380/460 nm (for AMC), 400 ⁄ 505 nm (for
AFC) is directly proportional to caspase-3 and -9 activity respec-
tively. The observed fluorescence values were normalized with
total protein concentration estimated by Bradford method and the
relative caspase activities were calculated as the ratio of values
microbial assay, 98
was dispensed into 96-well microtiter plate. To each well 2
test compound was added to attain a final concentration of 6.25,
12.5, 25 and 50 L of
M, and allowed to grow at 37 ꢂC for 32 h 240
mL of 1:1000-folds dilution of secondary culture
mL of
m
m
sterile water were added to each well of the peripheral rows of 96-
well plate to minimize media evaporation during assay incubation.
between mock treated and treated cells. Doxorubicin (10
used as a positive control (Fig. 4).
mM) was
Bacterial growth was assessed after 32
h of incubation by
measuring turbidity at 600 nm OD600 values using TECAN Infinite
200 PROÔ (Tecan Instruments, Switzerland). Positive controls were
included in every assay plates using stock solutions of INH (10 mg/
mL, HiMedia) and rifampicin (10 mg/mL, HiMedia) to achieve the
Acknowledgments
The authors thank Dr. (Ms.) M. Lakshmi Kantam, Director and Dr.
S. Chandrasekhar, Head, Natural Products Chemistry Division, CSIR-
IICT for their constant encouragement and support. G.S. thank CSIR,
K.V.P. thank UGC, New Delhi, for their fellowships. B. China Raju
acknowledges CSIR, New Delhi for financial support through the
programme ORIGIN of XII five year plan (CSC0108). U. Ramesh ac-
knowledges Open source drug discovery (OSDD) and smile projects
of CSIR (CSC0111) for biological screening of compounds.
final concentration of 0.5, 1, 2, 4, 8 and 16
mg/mL for INH and 0.25,
0.5, 1, 2, 4 and 8 g/mL for rifampicin. Additional controls DMSO
m
(solvent without compound) and medium without inoculums were
included in all the assay plates avoiding intra assay variability. The
results were analyzed as the percentage of growth inhibition. All
the experiments were carried out in triplicates and results were
reported as ꢁSD. From the percentage of the growth inhibition, the
GI50 values are calculated and presented in Table 1.
Appendix A. Supplementary data
6.7.2. Anticancer assay using MTT
The synthesized compounds have been evaluated for their
in vitro cytotoxicity in four different cell lines. All cell lines used in
this study were purchased from the American Type Culture
Collection (ATCC, USA). A549 (human lung carcinoma epithelial),
HeLa (human epithelial cervical cancer), DU-145 (Prostate cancer)
and HEK 293 were grown in standard Dulbecco’s modified Eagles
medium containing 10% FBS in a humidified atmosphere of 5% CO2
at 37 ꢂC. Cells were trypsinized when sub-confluent from T75
flasks/90 mm dishes and seeded in 96 well plates at a density of
5000e10000 cells/well depending on the doubling time of
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.042.
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