Synthesis and pharmacological evaluations of novel 2H-benzo[b][1,4]oxazin- 3(4H)-one derivatives as a new class of anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.07.045

The synthesis of novel 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives has been carried out using trifluoroacetic anhydride/phosphoric acid mediated C-C bond forming reaction as a key step. This method does not require the use of environmentally harmful AlCl₃ or moisture sensitive acid chloride. A number of compounds containing the benzooxazinone moiety attached to a five-membered central heterocyclic ring was synthesized and tested for their anti-cancer properties in vitro against three cell lines e.g. A549 (lung), DLD-1 (colorectal adenocarcinoma) and MV4-11 (acute myeloid leukemia). Some of them showed anti-cancer activities along with a number of reference compounds tested. Few of them showed promising anti-leukemic properties. A brief Structure-Activity-Relationship study within the series is presented. An imidazole derivative 9c containing benzene ring with a para-CF₃ group at C-2 position was identified as a potent anti-leukemic agent.

Full text of DOI:10.1016/j.ejmech.2011.07.045

European Journal of Medicinal Chemistry 46 (2011) 4887e4896
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluations of novel 2H-benzo[b][1,4]
oxazin-3(4H)-one derivatives as a new class of anti-cancer agents
,
Chittipaka Rajitha a b, P.K. Dubey b, Venkataiah Sunku a, F. Javier Piedrafita c,
**
,
d,
*
Venugopal Rao Veeramaneni a , Manojit Pal
a Indus BioSciences Private Limited, Plot No 72/A, Part 2 Phase -1, IDA Jeedimetla, Hyderabad 500055, India
b Department of Chemistry, JNT University, Kukatpally, Hyderabad 500072, India
c Torrey Pines Institute for Molecular Studies, 3550 General Atomics Ct San Diego, CA 92121, USA
d Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of novel 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives has been carried out using tri-
fluoroacetic anhydride/phosphoric acid mediated CeC bond forming reaction as a key step. This method
does not require the use of environmentally harmful AlCl3 or moisture sensitive acid chloride. A number
of compounds containing the benzooxazinone moiety attached to a five-membered central heterocyclic
ring was synthesized and tested for their anti-cancer properties in vitro against three cell lines e.g. A549
(lung), DLD-1 (colorectal adenocarcinoma) and MV4-11 (acute myeloid leukemia). Some of them showed
anti-cancer activities along with a number of reference compounds tested. Few of them showed
promising anti-leukemic properties. A brief StructureeActivity-Relationship study within the series is
presented. An imidazole derivative 9c containing benzene ring with a para-CF3 group at C-2 position was
identified as a potent anti-leukemic agent.
Received 1 June 2011
Received in revised form
24 July 2011
Accepted 26 July 2011
Available online 3 August 2011
Keywords:
Benzooxazinone
CeC bond
Acylation
Ó 2011 Elsevier Masson SAS. All rights reserved.
1,2-Diaryl-1-ethanone
Cancer
1. Introduction
initial findings on the synthesis and pharmacological evaluation of
a series of compound C structurally related to A as potential anti-
Triaryl substituted heterocyclic class of compounds represented
by structure A (Fig. 1) have attracted considerable interest in the
development of potential anti-cancer agents. For example, imid-
azole derivative SB-590885 possessing a 2,3-dihydro-1H-inden-1-
one oxime substituent has been identified as a potent and
extremely selective inhibitor of the B-Raf kinase [1]. B-Raf muta-
tions have been identified in many types of cancer, especially in
malignant melanomas and thyroid cancers [2], and several agents
targeting B-Raf have been reported. 2H-Benzo[b][1,4]oxazin-3(4H)-
one derivatives on the other hand have been reported to be useful
chemical entities for treating and/or preventing various human
diseases [3]. In our continuing effort to identify novel compounds of
potential pharmacological interest [4] we became interested to
build a library of small molecules incorporating the 2H-benzo[b]
[1,4]oxazin-3(4H)-one moiety (e.g. compound C, Fig. 1) for assess-
ing their anti-cancer properties in vitro. Herein, we report our
cancer agents.
A number of methods have been reported for the synthesis of
diverse series of compounds related to A. One of the commonly
utilized strategy for the synthesis of triaryl substituted heterocyclic
compounds involved the use of appropriately functionalized 1,2-
diaryl-1-ethanone D (Fig. 2) that could also be the precursor for
compound C. These ketones are prepared via an array of methods
including (a) synthesis from
a-aminonitrile [5e7], (b) acylation in
the presence of Lewis acids [8e10], or CF3SO3H [11], or palladium
catalysts [12], (d) synthesis using Grignard reagent [13], (e) pinacol
rearrangement catalyzed by AlCl3 [14], (f) benzotriazole-assisted
method [15], (g) self coupling of phenylacetic acid in the presence
of PPA [16,17], (h) a multistep sequences consisting of Perkin
condensation and Crutius rearrangement [18], and (i) synthesis via
clay catalyzed rearrangement of phenyloxiranes [19]. However, the
simplest one being the FriedeleCrafts acylation [18] of arenes using
arylacetyl chloride or acid in the presence of stoichiometric amount
of AlCl3 or ZnCl2ePOCl3 mixture. Since this process leads to the
formation of environmentally harmful gaseous HCl thus a more
convenient method was developed by reacting arylacetic acids with
appropriate arenes in the presence of trifluoroacetic anhydride/
* Corresponding author. Tel.: þ91 40 6657 1500; fax: þ91 40 6657 1581.
** Corresponding author. Tel.: þ91 40 2319 7766; fax: þ91 40 2309 1692.
E-mail addresses: venu@indusbio.com (V.R. Veeramaneni), manojitpal@
rediffmail.com (M. Pal).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.045

4888
C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
N
Ar
Ar
H
N
O
Hetero
O
cycle
N
N
H
O
Hetero
cycle
R
NMe2
Ar
N
Ar
OH
C
B
A
Fig. 1. Triaryl substituted heterocycles (A), anti-cancer agent SB-590885 (B) and the general structure of proposed small molecule library (C).
phosphoric acid [20]. In further continuation of our earlier work we
became interested in the application of this CeC bond forming
reaction as a key step to synthesize our target compounds. To the
best of our knowledge this methodology has not been explored
earlier for the preparation of compounds represented by C.
year, and therefore discovery and development of suitable agents to
treat various types of cancer is highly desirable. We have used A549
(NSCLC or Non small cell lung cancer), DLD-1 (colorectal adenocar-
cinoma) and MV4-11 (acute myeloid leukemia) cells obtained from
ATCC (American Type Culture Collection) for our in vitro assay. The
effect of test compounds on cell viability was measured using
a
colorimetric
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-
2. Results and discussion
diphenyltetrazolium bromide] assay after 3 days of treatment in
culture medium containing 5% FBS. The percentages of cell viability
(calculated with respect to control cells grown in the presence of
0.1% v/v DMSO) for new and reference compounds at two concen-
2.1. Chemistry
The key starting material 3 required for our synthesis was
readily prepared from 2-nitrophenol (1) via a two-step process
(Scheme 1). Thus reaction of 2-nitrophenol (1) with ethyl bro-
moacetate followed by reduction and in situ cyclization of the
resulting ether derivative 2 provided the desired benzooxazinone 3.
Subsequent reaction of compound 3 with phenylacetic acid in the
presence of H3PO4/(CF3CO)2O provided the ketone 4 in good yield.
Notably, the present H3PO4/(CF3CO)2O mediated CeC bond
formation represents a straightforward, efficient and environ-
mental friendly process and does not require the use of moisture
sensitive acyl halides.
The compound 4 was then converted to the 3-(dimethylamino)
acrylaldehyde derivative (5) which on treatment with appropri-
ately substituted hydrazines provided triaryl substituted pyrazoles
(6) (Scheme 2). In order to prepare a range of isoxazole derivatives
the benzooxazinone 3 was reacted with an array of arylacetic acids
to give the ketone 4aee (Scheme 3). These ketones were then
converted to the compounds 5aee which along with 5 provided the
desired compound 7aef when treated with hydroxylamine
hydrochloride.
trations (1.0 and 20 mM for test compounds whereas 1.0 and 10 mM
for reference compounds) are presented in Table 1. The reference
compounds used in our assay include Gefitinib [an epidermal
growth factor receptor (EGFR) inhibitor], Imatinib (or Gleevec, an
inhibitor of tyrosine kinase activities), Vandetanib [or Zactima, an
antagonist of the vascular endothelial growth factor receptor
(VEGFR) and the EGFR], Erlotinib (or Tarceva, a tyrosine kinase
inhibitor which acts on the EGFR), Sunitinib [or Sutent, a multi-
targeted receptor tyrosine kinase (RTK) inhibitor] and Sorafenib
[or Nexavar, an inhibitor of several Tyrosine protein kinases (VEGFR
and PDGFR) and Raf (Rapidly Accelerated Fibrosarcoma)]. All the
experiments were performed twice with triplicate data points. It is
evident from Table 1 that at the concentration of 20
9e, 9d and 9c showed comparable activities to the reference
compounds Vandetanib, Sunitinib and Sorafenib (all at 10 M)
against A549 cells. The other compounds that showed activities
against A549 cells include 6g, 6e, 6a, 6b and 6c. Similarly, all the
compounds except 9b and 6d were found to be active against DLD-1
mM compounds
m
cells when tested at 20
obtained when all these compounds were tested against MV4-11
cells at 20 M. Indeed a number of compounds e.g. 9a, 9e, 9d, 9f
mM. However, impressive results were
The imidazole derivatives were prepared (Scheme 4) from
compound 5 that was initially oxidized to the 1,2-diketone 8.
Condensation of compound 8 with appropriate aldehydes in the
presence of NH4OAc afforded the desired products 9.
m
and 9c showed significant anti-proliferative properties in this assay
in compared to the reference compounds. A graphical presentation
of percentage of cell viability after 3 days treatment of MV4-11 cells
with most active compounds is shown in Chart 1. Overall, imidazole
derivatives (9) were found to be superior to pyrazoles (6) in terms of
their in vitro activities. Among the imidazole derivatives the
compounds containing benzene ring with a para-substituent or no
substituent at C-2 position was found to be superior to others. The
CF3 group was found to be best among all the para-substituents
examined and compound 9c was identified as a potent anti-
leukemic agent. Notably, leukemia a cancer of blood-forming cells in
the bone marrow affects approximately 3,00,000 people worldwide
[22] and hence there is a need for the identification of new anti-
leukemic agents. The compounds presented here therefore have
medicinal value.
2.2. Pharmacology
Having prepared a divers series of compounds based on 2H-
benzo[b][1,4]oxazin-3(4H)-one we then tested all these compounds
for their potential anti-cancer properties in vitro. Cancer is the
second leading cause of death [21] worldwide after cardiovascular
diseases, according to WHO. Indeed, lung, breast, stomach, liver and
colorectal cancers cause the most cancer deaths worldwide each
Ar1
Ar2
O
Ar1
Ar2
O
Z
C
3. Conclusions
D
In conclusion, we have described the design and synthesis of
Fig. 2. Strategy for the synthesis of C from ketone D.
a series of novel 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as

C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
4889
O
O
O
O
O
OH
a
c
b
O
N
H
O
N
H
O
NO2
NO2
4
3
2
1
Scheme 1. Reagents and condition: (a) K2CO3, BrCH2CO2Et, acetone, 50 ꢀC, 1 h (92%); (b) Fe powder, CH3CO2H, 0 ꢀC for 3 h, room temp for 2 h, reflux for 2 h (91%); (c) C6H5CH2CO2H,
H3PO4/(CF3CO)2O, room temp, 1 h (85%).
potential anti-cancer agents. Multi-step synthesis of these
compounds was carried out using trifluoroacetic anhydride/phos-
phoric acid mediated CeC bond forming reaction as a key step. This
method does not require the use of environmentally harmful AlCl3
or moisture sensitive acid chloride. Thus acylation of benzoox-
azinone ring under these conditions followed by appropriate
reaction sequences afforded a range of tri and diaryl substituted
heterocyclic compounds containing the benzooxazinone moiety
attached to a five-membered central ring. Some of the compounds
prepared were tested for their potential anti-cancer properties
in vitro against A549 (lung), DLD-1 (colorectal adenocarcinoma)
and MV4-11 (acute myeloid leukemia) cell lines. Few of them
showed promising anti-proliferative properties especially against
leukemia cells. An imidazole derivative containing benzene ring
with a para-CF3 group at C-2 position was identified as a potent
anti-leukemic agent. Overall, the present series of heterocyclic
compounds represent a potential scaffold for the development of
potent anti-leukemic agents.
added drop wise. After completion of addition, temperature was
raised to 50 ꢀC and stirred for 1 h. The reaction mixture was cooled
and the solid separated out was filtered and washed with acetone.
Filtrate was concentrated in reduced pressure. The crude was par-
titioned between methyl tertiary butyl ether and water. The organic
layer was extracted with ethyl acetate (2 ꢁ 20 mL), washed with
water (20 mL), dried over anhydrous sodium sulfate and concen-
trated to give the title compound (15.4 g, yield 92%); mp 39e40 ꢀC;
1H NMR (CDCl3, 400 MHz):
d
7.85 (1H, d, J ¼ 8.2 Hz, ArH), 7.50 (1H, t,
J ¼ 7.8 Hz, ArH), 7.10 (2H, d, J ¼ 7.7 Hz, ArH), 4.75 (2H, s, OCH2),
4.30e4.20 (2H, m, OCH2), 1.30 (3H, t, J ¼ 7.3 Hz, CH3); m/z (CI) 224
(M þ 1, 100%).
4.1.3. Synthesis of 4H-benzo [1,4]oxazin-3-one (3)
To a solution of 2 (15.4 g 0.068 mol) in acetic acid (75 mL), Fe
powder (38.4 g, 0.68 mol) was added portion wise for 3 h at 0 ꢀC.
The reaction was exothermic. The reaction mixture was allowed to
stir at room temperature for 2 h and heated to reflux for 2 h. After
completion of the reaction, the reaction mixture was cooled to
room temperature, filtered on celite and the cake was washed with
acetic acid. Acetic acid was concentrated to ¼ of its volume and
diluted with water (75 mL). The solid separated was filtered,
washed with water (20 mL) and dried to give the title compound
4. Experimental section
4.1. Chemistry
(7.5 g, yield 91%); mp 173e174 ꢀC; 1H NMR (CDCl3, 400 MHz):
d 9.95
4.1.1. General methods
(1H, s, NH), 7.00e6.80 (4H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI) 150
(M þ 1, 100%).
Unless stated otherwise, reactions were performed under
nitrogen atmosphere. Reactions were monitored by thin layer
chromatography (TLC) on silica gel plates (60 F254), visualizing
with ultraviolet light or iodine spray. Flash chromatography was
performed on silica gel (100e200 mesh) using distilled hexane,
ethyl acetate, dichloromethane. 1H NMR spectra were determined
in CDCl3 or DMSO-d6 solution by using Varian 400 MHz spec-
4.1.4. Synthesis 6-phenylacetyl-4H-benzo [1,4]-oxazin-3-one (4)
Phosphoric acid (3.94 g, 0.04 mol) and TFAA (18.6 mL, 0.132 mol)
were added to the mixture of 3 (5 g, 0.033 mol) and phenylacetic
acid (6.73 g, 0.04 mol), at 0 ꢀC. After completion of addition, the
mixture was allowed to warm to room temperature and stirred for
1 h. The reaction mixture was cooled to 0 ꢀC and adjusted the pH to
7 by adding the saturated NaHCO3 solution. The precipitated solid
was filtered, washed with water and dried to furnish 7.5 g (85%) of
the desired product, mp 188e189 ꢀC; 1H NMR (CDCl3, 400 MHz):
trometer. Proton chemical shifts (
d) are relative to tetramethylsi-
lane (TMS,
d
¼ 0.00) as internal standard and expressed in ppm.
Spin multiplicities are given as s (singlet), d (doublet), t (triplet) and
m (multiplet) as well as b (broad). Coupling constants (J) are given
in hertz. Infrared spectra were recorded on an FT-IR spectrometer
(Perkin Elmer). Melting points were determined using melting
point apparatus and are uncorrected. MS spectra were obtained on
a mass spectrometer.
d
10.85 (1H, bs, NH), 7.70 (1H, d, J ¼ 7.8 Hz, ArH), 7.50 (1H, s, ArH),
7.40e7.20 (5H, m, ArH), 6.95 (1H, d, J ¼ 7.8 Hz, ArH), 4.65 (2H, s,
CH2), 4.20 (2H, s, CH2); m/z (CI) 266 (M-1, 100%).
4.1.5. Synthesis 6-(3-dimethylamino-2-phenyl-acrolyl)-4H-benzo
[1,4]-oxazin-3-one (5)
DMFeDMA (1.07 g, 0.0089 mol) was added to a solution of 6-
phenyl-4H-benzo [1,4]-oxazin-3-one (2 g, 0.0074 mol) in toluene
(10 mL) and heated to reflux for 0.5 h. After sometime the solution
4.1.2. Synthesis of triaryl substituted pyrazoles (6)
4.1.2.1. Synthesis of (2-nitrophenoxy) ethyl acetate (2). To a stirred
solution of 2-nitrophenol (10 g, 0.071 mol) in acetone (25 mL),
K2CO3 (21.5 g, 0.156 mol) was added at room temperature and
stirred for 15 min. Then ethyl bromoacetate (13.2 g, 0.079 mol) was
Scheme 2. Reagents and condition: (a) DMFeDMA, toluene, reflux, 0.5 h (76%); (b) RNHNH2$HCl, EtOH, 40e50 ꢀC, 15 min.

4890
C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
O
O
O
O
N
c
N
H
O
N
H
O
O
b
a
O
N
H
O
N
3
R
R
R
7
4a-e
5a-e
4a; 5a; 7a; R=4-fluorophenyl
4b; 5b; 7b
; R=2-chlorophenyl
4c; 5c; 7c
; R=3-chlorophenyl
4d; 5d; 7d
; R=3-methylphenyl
4e; 5e; 7e
; R=4-bromophenyl
4; 5; 7f
; R=phenyl
Scheme 3. Reagents and condition: (a) RCH2CO2H, H3PO4/(CF3CO)2O, room temp, 1 h; (b) DMFeDMA, toluene, reflux, 0.5 h; (c) NH2OH$HCl, EtOH, 40e50 ꢀC, 15 min.
became clear and solid was precipitated out. The solid separated
was filtered, washed with toluene (15 mL) and dried to give the title
compound (1.75 g, yield 76%) as light yellow solid; mp 194e195 ꢀC;
d 10.85 (1H, s, NH), 7.80 (1H, s, ]CH),
7.65 (1H, s, ArH), 7.30e7.00 (5H, m, ArH), 6.60e6.45 (2H, m, ArH),
4.55 (2H, s, OCH2), 2.80 (6H, s, N(CH3)2); m/z (CI) 321 (M-1, 100%).
695; 1H NMR (DMSO-d6):
d 10.65 (1H, s, eNH), 8.00 (1H, s, ArH),
7.35e7.10 (8H, m, ArH), 6.95 (2H, d, J ¼ 8.4 Hz, ArH), 6.80e6.60 (2H,
m, ArH), 4.60 (2H, s, OCH2), 3.75 (3H, s, OCH3); m/z (CI) 396 (M-1,
100%); HPLC 98.9%, column: Intersil C-18 (150 ꢁ 4.6 mm), mobile
phase A: water, mobile phase B: acetonitrile, gradient (A/%B): 90/
10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention
time 11.6 min.
1H NMR (CDCl3, 400 MHz):
4.1.6. General procedure for the synthesis of triaryl pyrazoles (6)
To
a
solution of 6-(3-dimethylamino-2-phenyl-acrolyl)-4H-
4.1.6.4. 6-(4-Phenyl-1-o-tolyl-1H-pyrazol-3-yl)-2H-benzo[b][1,4]
oxazin-3(4H)-one (6d). Yield 83.3%; white solid, mp 144e146 ꢀC;
IR (KBr, cmꢂ1): 3208, 1701, 1491, 1372, 1039, 764, 697; 1H NMR
benzo [1,4]-oxazin-3-one (0.00065 mol) in ethanol, substituted
phenylhydrazine hydrochloride (0.00071 mol) in ethanol was
added slowly. After completion of addition, the mixture was heated
to 40e50 ꢀC in a water bath. After 15 min, the solution became clear
and solid was precipitated out. The solid separated was filtered,
washed with ethanol and dried to give the expected product.
(DMSO-d6):
d 10.70 (1H, s, NH), 8.10 (1H, s, ArH), 7.40e7.15 (9H, m,
ArH), 6.85 (1H, d, J ¼ 8.8 Hz, ArH), 6.75e6.68 (2H, m, ArH), 4.60
(2H, s, OCH2), 2.00 (3H, s, CH3); m/z (CI) 382 (M þ 1, 97%); HPLC
98.9%, column: Intersil C-18 (150 ꢁ 4.6 mm), mobile phase A:
water, mobile phase B: acetonitrile, gradient (A/%B): 90/10, 10/90,
10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention time
11.7 min.
4.1.6.1. 6-(1-(4-Chlorophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-benzo
[b][1,4]oxazin-3(4H)-one (6a). Yield 88%; white solid. mp > 270 ꢀC;
IR (KBr, cmꢂ1): 3480, 1702, 1494, 1044, 826, 769, 696; 1H NMR
(DMSO-d6):
d
10.65 (1H, s, ArH), 8.05 (1H, s, ArH), 7.45 (2H, d,
4.1.6.5. 6-(1-(4-Bromophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-benzo
J ¼ 8.2 Hz, ArH), 7.35e7.18 (7H, m, ArH), 6.95 (1H, d, J ¼ 8.2 Hz, ArH),
6.80e6.60 (2H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI) 400 (M-1,
100%); HPLC 98.1%, column: Intersil C-18 (150 ꢁ 4.6 mm), mobile
phase A: water, mobile phase B: acetonitrile, gradient (A/%B): 90/
10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention
time 12.7 min.
[b][1,4]oxazin-3(4H)-one
mp > 270 ꢀC: IR (KBr, cmꢂ1): 3470, 1703, 1491, 1044, 823, 769, 697;
1H NMR (DMSO-d6):
10.65 (1H, s, NH), 8.10 (1H, s, ArH), 7.60 (2H,
d, J ¼ 8.7 Hz, ArH), 7.35e7.15 (7H, m, ArH), 6.95 (1H, d, J ¼ 8.2 Hz,
ArH), 6.88e6.70 (2H, m, ArH), 4.65 (2H, s, OCH2); m/z (CI) 448
(M þ 2, 100%); HPLC 97.0%, column: Intersil C-18 (150 ꢁ 4.6 mm),
mobile phase A: water, mobile phase B: acetonitrile, gradient (A/%
B): 90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm,
retention time 11.8 min.
(6e). Yield
86.2%;
white
solid,
d
4.1.6.2. 6-(1-(4-Fluorophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-benzo
[b][1,4]oxazin-3(4H)-one (6b). Yield 88%; white solid. mp > 270 ꢀC;
IR (KBr, cmꢂ1): 3195, 1703, 1588, 1219; 1H NMR (DMSO-d6):
d 10.65
(1H, s, ArH), 8.05 (1H, s, ArH), 7.38e7.10 (9H, m, ArH), 6.95 (1H, d,
J ¼ 8.2 Hz, ArH), 6.80e6.60 (2H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI)
4.1.6.6. 6-(1-(3,5-Dichlorophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (6f). Yield 85%; white solid, mp
189e190 ꢀC; IR (KBr, cmꢂ1): 3447, 1705, 1583, 1046, 807, 765, 693;
286 (M
þ
1, 100%); HPLC 98.0%, column: Intersil C-18
(150 ꢁ 4.6 mm), mobile phase A: water, mobile phase B: acetoni-
trile, gradient (A/%B): 90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/
min, UV 254 nm, retention time 11.9 min.
1H NMR (DMSO-d6):
d 10.75 (1H, s, ArH), 8.10 (1H, s, ArH), 7.80 (1H,
s, ArH), 7.30e7.10 (7H, m, ArH), 6.95 (1H, d, J ¼ 8.1 Hz, ArH),
6.80e6.65 (2H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI) 436 (M þ 1,
100%); HPLC 98.2%, column: Intersil C-18 (150 ꢁ 4.6 mm), mobile
phase A: water, mobile phase B: acetonitrile, gradient (A/%B): 90/
10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention
time 14.0 min.
4.1.6.3. 6-(1-(4-Methoxyphenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (6c). Yield 86%; white solid, mp
242e243 ꢀC; IR (KBr, cmꢂ1): 3480, 1702, 1510, 1249, 1042, 830, 766,
O
O
9a; R=phenyl
O
O
a
b
N
H
O
N
9b
; R=2,6-dichlorophenyl
; R=4-trifluoromethylphenyl
; R=4-methoxyphenyl
; R=4-fluorophenyl
N
H
O
5
R
9c
9d
9e
9f
N
H
; R=7-azaindole-3-yl
8
9
Scheme 4. Reagents and condition: (a) NaIO4, THFeH2O, room temp, 15 min; (b) RCHO, NH4OAc, CH3CO2H, reflux, 2 h.

C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
4891
Table 1
The percentages of cell viability noted for 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives and reference compounds.
Entry
Compounds (9 and 6)
A549 (Lung)
DLD-1 (Colon)
MV4-11
(Leukemia)
1
m
M
20
mM
1
m
M
20
m
M
1
m
M
20 mM
O
H
N
N
H
O
O
1
9a
9b
9e
95.6
97.4
97.1
58.5
68.3
37.5
98.2
97.8
94.8
47.3
61.9
29.5
104.6
108.7
108.2
7.6
N
Cl
H
N
Cl
F
N
H
O
O
2
41.1
N
O
H
N
N
H
3
1.1
N
O
H
N
MeO
N
H
O
4
9d
100.8
38.1
96.0
19.1
108.1
1.2
N
O
N
H
N
N
H
O
5
9f
100.3
86.8
97.7
53.5
110.8
8.9
HN
N
O
H
N
CF3
N
H
O
6
9c
102.8
21.7
95.0
12.1
110.8
0.6
N
O
N
N
H
O
N
7
6g
92.2
59.7
77.0
47.7
117.2
97.9
O
N
N
H
O
N
54.2
8
6d
94.8
85.3
92.2
62.9
99.4
(continued on next page)

4892
C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
Table 1 (continued )
Entry
Compounds (9 and 6)
A549 (Lung)
DLD-1 (Colon)
MV4-11
(Leukemia)
1
m
M
20
m
M
1
m
M
20
m
M
1
m
M
20 mM
O
N
Br
Cl
N
H
O
N
9
6e
6a
81.5
84.4
58.8
52.4
66.4
61.9
54.2
47.1
107.8
107.8
91.5
91.9
O
N
N
H
O
N
10
O
N
F
N
H
O
N
11
12
6b
78.2
93.1
56.4
70.2
64.4
90.3
45.2
52.2
107.3
105.1
92.6
50.5
O
Cl
N
N
H
O
N
6f
Cl
O
O
N
N
H
O
N
13
14
6c
95.6
84.4
42.6
92.2
87.0
35.6
106.1
59.9
F
O
HN
N
Cl
Gefitinib
61.5a
65.7a
92.4
7.0a
N
O
O
N
N
H
N
N
O
N
98.6a
89.9
91.5a
105.0
82.0a
HN
N
15
Imatinib
108.3
N
N
N
O
O
N
16
Vandetanib
88.7
41.7a
88.6
37.9a
84.1
1.6a
HN
F
Br

C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
4893
Table 1 (continued )
Entry Compounds (9 and 6)
A549 (Lung)
DLD-1 (Colon)
MV4-11
(Leukemia)
1
m
M
20
mM
1
m
M
20
m
M
1
m
M
20 mM
O
O
O
O
N
N
17
Erlotinib
83.0
68.5a
85.7
55.4a
98.3
16.8a
HN
O
NH
18
Sunitinib
102.5
37.4a
92.9
52.7a
1.4
0.5a
N
H
N
F
O
N
H
CF3
O
Cl
O
O
NHCH3
19
Sorafenib
103.2
28.6a
93.4
28.0a
1.6
0.7a
N
N
N
H
H
Sorafenib
a
The assay was carried out using the compound at 10
m
M.
4.1.7. Synthesis of isoxazole derivatives (7)
ArH), 7.18e7.05 (3H, m, ArH), 4.70 (2H, s, OCH2), 4.30 (2H, m, ]
CeCH2Ph); m/z (CI) 284 (M-1, 100%).
4.1.7.1. Typical procedure for the synthesis 6-phenylacetyl-4H-benzo
[1,4]-oxazin-3-one. Phosphoric acid (3.15 g, 0.032 mol) and TFAA
(21.8 mL, 0.104 mol) were added to the mixture of 4H-Benzo [1,4]
oxazin-3-one (4 g, 0.026 mol) and substituted phenylacetic acid
(1.1eq), at 0 ꢀC. After completion of addition, the mixture was
allowed to warm to room temperature and stirred for 1 h. The
reaction mixture was cooled to 0 ꢀC and adjusted the pH to 7 by
adding the saturated NaHCO3 solution. The precipitated solid was
filtered, washed with water and dried to give the desired product.
4.1.7.3. 6-(2-(2-Chlorophenyl)acetyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (4b). Yield 82%; white solid; 1H NMR (DMSO-d6):
d 10.85 (1H, s,
NH), 7.90e7.70 (1H, m, ArH), 7.53 (1H, s, ArH), 7.55e7.25 (4H, m,
ArH), 7.05 (1H, d, J ¼ 8.4 Hz, ArH), 4.69 (2H, s, OCH2), 4.42 (2H, s, ]
CeCH2ePh); m/z (CI) 300 (M-1, 100%).
4.1.7.4. 6-(2-(3-Chlorophenyl)acetyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (4c). Yield 82.3%; white solid; 1H NMR (DMSO-d6):
d 10.75 (1H,
4.1.7.2. 6-(2-(4-Fluorophenyl)acetyl)-2H-benzo[b][1,4]oxazin-3(4H)-
s, NH), 7.75e7.65 (1H, m, ArH), 7.55 (1H, s, ArH), 7.40e7.25 (3H, m,
ArH), 7.20 (1H, d, J ¼ 7.2 Hz, ArH), 7.17 (1H, d, J ¼ 7.2 Hz, ArH), 4.67
(2H, s, OCH2), 4.32 (2H, s, ]CeCH2ePh); m/z (CI) 300 (M-1, 100%).
one (4a). Yield 80%; white solid; 1H NMR (DMSO-d6):
d 10.85 (1H, s,
NH), 7.74 (1H, d, J ¼ 8.4 Hz, ArH), 7.52 (1H, s, ArH), 7.35e7.20 (2H, m,
4.1.7.5. 6-(2-(3-Methylphenyl)acetyl)-2H-benzo[b][1,4]oxazin-
3(4H)-one (4d). Yield 80.6%; white solid; 1H NMR (DMSO-d6):
d
10.85 (1H, s, NH), 7.72 (2H, d, J ¼ 2.2 Hz, ArH), 7.55 (1H, s, ArH),
7.20e7.15 (1H, m, ArH), 7.15e7.00 (4H, m, ArH), 4.69 (2H, s, OCH2),
4.16 (2H, s, ]CeCH2ePh), 2.33 (3H, s, CH3); m/z (CI) 280 (M-1,100%).
4.1.7.6. 6-(2-(4-Bromophenyl)acetyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one (4e). Yield 81.5%; white solid; 1H NMR (DMSO-d6):
d 10.85 (1H,
s, NH), 7.74 (1H, d, J ¼ 8.4 Hz, ArH), 7.41e7.57 (3H, m, ArH),
7.30e7.10 (2H, m, ArH), 7.05 (1H, d, J ¼ 8.3 Hz, ArH), 4.69 (2H, s,
OCH2), 4.27 (2H, s, ]CeCH2ePh); m/z (CI) 344 (M-2, 100%).
4.1.8. Typical procedure for the synthesis 6-(3-dimethylamino-2-
phenyl-acrolyl)-4H-benzo [1,4]-oxazin-3-one
DMFeDMA (2 g, 0.0074 mol) was added to a solution of
substituted 6-phenyl-4H-benzo [1,4]-oxazin-3-one (1.07 g,
0.0089 mol) in toluene and heated to reflux on an oil bath for 0.5 h.
Chart 1. Three days treatment of MV4-11 cells with most active compounds.

4894
C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
After sometime the solution became clear and immediately solid
was precipitated out. The solid was filtered, washed with toluene
and dried to give the desired product.
4.1.9.3. 6-(4-(3-Chlorophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa-
zin-3(4H)-one (7c). Yield 92%; White solid; mp. 178e180 ꢀC; IR
(KBr, cmꢂ1): 3447, 1692, 1599, 1491, 1396, 780; 1H NMR (DMSO-d6):
d
10.80 (1H, bs, NH), 8.85 (1H, s, ArH), 7.58 (1H, s, ArH), 7.46 (2H, d,
4.1.8.1. (Z)-6-(3-(Dimethylamino)-2-(4-fluorophenyl)acryloyl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (5a). Yield 70%; white solid; 1H
J ¼ 5.0 Hz, ArH), 7.38 (1H, s, ArH), 7.20e7.00 (3H, m, ArH), 4.68 (2H,
s, OCH2); m/z (CI) 325 (M-1, 100%); HPLC 97.9%, column: Intersil C-
18 (150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water, mobile
phase B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10,
flow rate: 1.0 mL/min, UV 215 nm, retention time 11.8 min.
NMR (DMSO-d6):
d 10.85 (1H, s, NH), 7.25 (1H, s, ArH), 7.15e7.05
(4H, m, ArH), 7.05 (1H, s, Me2NeCH]), 6.95e6.80 (2H, m, ArH),
4.70 (2H, s, OCH2), 2.70 (6H, s, N(CH3)2); m/z (CI) 291 (M þ 1,
100%).
4.1.9.4. 6-(4-(3-Methylphenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa-
zin-3(4H)-one (7d). Yield 89%; White solid; mp 191e193 ꢀC; IR
(KBr, cmꢂ1): 3448, 1698, 1498, 1387, 1039, 699; 1H NMR (DMSO-d6):
4.1.8.2. (Z)-6-(3-(Dimethylamino)-2-(2-chlorophenyl)acryloyl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (5b). Yield 77.9%; white solid; 1H
NMR (DMSO-d6):
d
10.85 (1H, s, NH), 7.40 (1H, d, J ¼ 3.7 Hz, ArH),
d 10.80 (1H, bs, NH), 8.85 (1H, s, ArH), 7.40e7.30 (2H, m, ArH), 7.22
7.35e7.15 (4H, m, ArH), 7.10 (1H, s, Me2NeCH]), 7.00e6.85 (2H, m,
ArH), 4.69 (2H, s, OCH2), 2.70 (6H, s, N(CH3)2); m/z (CI) 355 (M-1,
100%).
(3H, s, ArH), 7.10e6.98 (2H, m, ArH), 4.67 (2H, s, OCH2), 2.33 (3H, s,
CH3); m/z (CI) 307 (M þ 1, 100%); HPLC 97.4%, column: Intersil C-18
(150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase
B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow
rate: 1.0 mL/min, UV 230 nm, retention time 11.7 min.
4.1.8.3. (Z)-6-(3-(Dimethylamino)-2-(3-chlorophenyl)acryloyl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (5c). Yield 74%; white solid; 1H NMR
(DMSO-d6):
d
10.85 (1H, s, NH), 7.38e7.20 (3H, m, ArH), 7.17 (1H, s,
4.1.9.5. 6-(4-(4-Bromophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa-
zin-3(4H)-one (7e). Yield 91.3%; White solid; mp 205e206 ꢀC; IR
(KBr, cmꢂ1): 3448, 1690, 1490, 1388, 812; 1H NMR (DMSO-d6):
Me2NeCH]), 7.10e6.85 (4H, m, ArH), 4.60 (2H, s, OCH2), 2.70 (6H,
s, N(CH3)2); m/z (CI) 355 (M-1, 100%).
d
10.85 (1H, bs, eNH), 8.85 (1H, s, ArH), 7.64 (2H, d, J ¼ 8.4 Hz, ArH),
4.1.8.4. (Z)-6-(3-(Dimethylamino)-2-(3-methylphenyl)acryloyl)-2H-
7.39 (2H, d, J ¼ 8.4 Hz, ArH), 7.10e7.00 (3H, m, ArH), 4.67 (2H, s,
OCH2); m/z (CI) 369 (M-2, 100%); HPLC 97.5%, column: Intersil C-18
(150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase
B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow
rate: 1.0 mL/min, UV 230 nm, retention time 12.1 min.
benzo[b][1,4]oxazin-3(4H)-one (5d). Yield 75%; white solid; 1H
NMR (DMSO-d6):
d 10.85 (1H, s, NH), 7.20e7.15 (2H, m, ArH), 7.00
(1H, s, Me2NeCH]), 6.95e6.80 (5H, m, ArH), 4.69 (2H, s, OCH2),
2.25 (3H, s, CH3), 2.70 (6H, s, N(CH3)2); m/z (CI) 335 (M-1, 100%).
4.1.8.5. (Z)-6-(3-(Dimethylamino)-2-(4-bromophenyl)acryloyl)-2H-
4.1.9.6. 6-(4-Phenylisoxazol-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one
benzo[b][1,4]oxazin-3(4H)-one (5e). Yield 70%; white solid; 1H NMR
(7f). Yield 87%; White solid; mp 166e167 ꢀC; IR (KBr, cmꢂ1): 3134,
(DMSO-d6):
d
10.85 (1H, s, NH), 7.25 (1H, s, ArH), 7.15e7.05 (4H, m,
1700, 1382, 763, 689; 1H NMR (DMSO-d6):
d 10.85 (1H, bs, eNH),
ArH), 7.05 (1H, s, (CH3)2NCH]), 6.95e6.80 (2H, m, ArH), 4.70 (2H, s,
OCH2), 2.70 (6H, s, N(CH3)2); m/z (CI) 403 (M þ 2, 100%).
8.90 (1H, s, ArH), 7.55e7.35 (5H, m, ArH), 7.20 (1H, s, ArH),
7.15e7.00 (2H, m, ArH), 4.70 (2H, s, OCH2); m/z (CI) 291 (M-1,100%);
HPLC 99.2%, column: Intersil C-18 (150 ꢁ 4.6 mm), mobile phase A:
0.05% TFA in water, mobile phase B: acetonitrile, gradient (T/%B):
90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 230 nm,
retention time 10.9 min.
4.1.9. Typical procedure for the synthesis of isoxazole
To
a solution of 6-(3-dimethylamino-2-phenyl-acrolyl)-4H-
benzo [1,4]-oxazin-3-one (0.00065 mol) in ethanol, hydroxylamine
hydrochloride (0.00071 mol) in ethanol was added slowly. After
completion of addition, the mixture was heated to 40e50 ꢀC on
water bath. After 15 min, the solution became clear and immedi-
ately solid precipitated out. The solid was filtered, washed with
ethanol and dried.
4.1.10. Preparation of imidazole derivatives (9)
4.1.10.1. Synthesis of 1-(3-oxo-3,4-dihydro-2H-benzo [1,4]oxazin-6-
yl)-2-phenyl-ethane-1,2-dione (8). To
a
suspension of 6-(3-
dimethyl amino-2-phenyl acronyl)-4H-benzo{1,4}-oxazine-3-one
(1.5 g, 0.0046 mol) in a mixture of THF and H2O (9:1) was added
NaIO4 (3.98 g, 0.018 mol) portion wise for a duration of 15 min at
room temp. After completion of addition, the mixture was allowed
to stir for 2 h. After completion of the reaction (indicated by TLC/1:1
EtOAcehexane) the solid separated was filtered and washed with
ethyl acetate (2 ꢁ 10 mL). The filtrate was collected and treated
with cold water (15 mL). The organic layer was collected, dried over
anhydrous Na2SO4 and concentrated. The residue was purified by
column chromatography using EtOAcehexane (1:1) to give the
4.1.9.1. 6-(4-(4-Fluorophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxazin-
3(4H)-one (7a). Yield 90%; white solid; mp 176e177 ꢀC; IR (KBr,
cmꢂ1): 3464,1694,1498, 1398,1221, 843, 810, 542; 1H NMR (DMSO-
d6): d 10.85 (1H, bs, eNH), 8.85 (1H, s, ArH), 7.55e7.40 (2H, m, ArH),
7.38e7.25 (2H, m, ArH), 7.10e7.00 (3H, m, ArH), 4.67 (2H, s, OCH2);
m/z (CI) 311 (M þ 1, 98%); HPLC 97.0%, column: Intersil C-18
(150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase
B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow
rate: 1.0 mL/min, UV 230 nm, retention time 11.1 min.
desired product (yield 77%); 1H NMR (DMSO-d6):
d 10.80 (1H, bs,
eNH), 7.93 (2H, d, J ¼ 7.0 Hz, ArH), 7.85e7.75 (1H, m, ArH),
7.70e7.60 (3H, m, ArH), 7.50 (1H, d, J ¼ 2.0 Hz, ArH), 7.15 (1H, d,
J ¼ 8.4 Hz, ArH), 4.86 (2H, s, OCH2); m/z (CI) 280 (M-1, 100%).
4.1.9.2. 6-(4-(2-Chlorophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa-
zin-3(4H)-one (7b). Yield 92%; White solid; mp 247e248 ꢀC; IR
(KBr cmꢂ1): 3447, 1654, 1508; 1H NMR (DMSO-d6):
d 10.80 (1H,
bs, NH), 8.85 (1H, s, ArH), 7.63 (1H, d, J ¼ 7.6 Hz, ArH), 7.55e7.38
(3H, m, ArH), 7.10 (1H, s, ArH), 7.10e6.85 (2H, m, ArH), 4.61 (2H,
s, OCH2); m/z (CI) 325 (M-1, 100%); HPLC 99.2%, column: Intersil
C-18 (150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water,
mobile phase B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/
90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention time
11.3 min.
4.1.11. Typical procedure for the synthesis of imidazole derivatives
To a solution of 1-(3-oxo-3,4-dihydro-2H-benzo [1,4]oxazin-6-
yl)-2-phenyl-ethane-1,2-dione (0.1 g, 0.0003 mol) in acetic acid
(10 mL) was added ammonium acetate (0.23 g, 0.003 mol) followed
by an appropriate aldehyde (0.0003 mol) slowly. The mixture was
heated to reflux for 2 h. After completion of the reaction the
mixture was poured into crushed ice (20 g) and extracted with

C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
4895
CH2Cl2 (3 ꢁ 20 mL). The organic layers were collected, combined,
dried over anhydrous Na2SO4 and concentrated. The residue was
purified by column chromatography using EtOAcehexane (3:2) to
give desired product.
ArH), 7.57 (2H, d, J ¼ 6.7 Hz, ArH), 7.45e6.90 (8H, m, ArH), 4.60 (2H,
s, OCH2); m/z (CI) 408 (M þ 1,100%); HPLC 98.5%, column: Intersil C-
18 (150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water, mobile
phase B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10,
flow rate: 1.0 mL/min, UV 230 nm, retention time 7.4 min.
4.1.11.1. 6-(2,4-Diphenyl-1H-imidazol-5-yl)-2H-benzo[b][1,4]oxazin-
3(4H)-one (9a). Yield 86%; white solid; mp 158e159 ꢀC; IR (KBr,
cmꢂ1): 3448, 1684, 1490, 1380, 775, 697; 1H NMR (DMSO-d6):
4.2. Pharmacology: In vitro cell proliferation assay
d
12.60 (1H, bs, NH), 10.70 (1H, bs, NH), 8.05 (2H, d, J ¼ 7.2 Hz, ArH),
A549 (NSCLC), DLD-1 (colorectal adenocarcinoma) and MV4-11
(acute myeloid leukemia) cells were obtained from ATCC and
grown as recommended. The effect of test compounds on cell
viability was measured using a colorimetric MTT assay after 3 days
of treatment in culture medium containing 5% FBS. A549 and DLD-1
cells were seeded in a 96 well plate at a density of 5000 cells per
7.65e6.80 (11H, m, ArH), 4.60 (1H, s, OCH2), 4.56 (1H, s, OCH2); m/z
(CI) 368 (M þ 1, 100%); HPLC 98.4%, column: Intersil ODS-3 C-18
(150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase
B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow
rate: 1.0 mL/min, UV 230 nm, retention time 7.4 min.
well (in 100 ml medium) the day before treatment. MV4-11 cells
4.1.11.2. 6-(2-(2,4-Dichlorophenyl)-4-phenyl-1H-imidazol-5-yl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (9b). Yield 80%; white solid; mp
154e156 ꢀC; IR (KBr, cmꢂ1): 3421, 2925, 1696, 1490, 1388, 787, 698;
were seeded at 40,000 cells per well immediately before treatment.
All the test compounds were prepared in DMSO as 20 mM stock
solutions and diluted in RPMI as 20X solutions to achieve the
1H NMR (DMSO-d6):
d 12.50 (bs, 1H, eNH), 10.80 (1H, bs, eNH),
desired final concentrations up to a maximum of 20
mM, with
7.25e7.75 (8H, m, ArH), 7.15 (1H, s, ArH), 6.90e7.10 (2H, m, ArH),
4.62 (2H, s, OCH2); m/z (CI) 436 (M þ 1, 100%); HPLC 97.6%, column:
Intersil ODS C-18 (150 ꢁ 4.6 mm), mobile phase A: 0.05% TFA in
water, mobile phase B: acetonitrile, gradient (T/%B): 90/10, 10/90,
10/90, 90/10, flow rate: 1.0 mL/min, UV 215 nm, retention time
7.7 min.
0.1% v/v DMSO. At the end of treatment, cells were incubated with
0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide for 4 h, followed by solubilization of the formazan crystals
with 100 ml 10% SDS in 10 mM HCl. Absorbance at 595 nm was
measured and the percentage of cell viability was calculated with
respect to control cells grown in the presence of 0.1% v/v DMSO.
Experiments were performed twice with triplicate data points.
4.1.11.3. 6-(4-Phenyl-2-(4-(trifluoromethyl)phenyl)-1H-imidazol-5-
yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (9c). Yield 85%; white solid;
mp 164e166 ꢀC; IR (KBr, cmꢂ1): 3255, 1685, 1327, 1124, 1066,
Acknowledgments
697; 1H NMR (DMSO-d6):
d 12.95 (1H, bs, NH), 10.75 (1H, d, NH),
The authors thank management of Indus BioSciences Private
Limited for encouragement and support.
8.30 (2H, d, J ¼ 8.1 Hz, ArH), 7.85 (2H, d, J ¼ 8.2 Hz, ArH),
7.65e7.20 (6H, m, ArH), 7.15e6.80 (2H, m, ArH), 4.65 (1H, s,
OCH2), 4.55 (1H, s, OCH2); m/z (CI) 434 (M-1, 100%); HPLC 99.3%,
column: Intersil ODS-3 C-18 (150 ꢁ 4.6 mm), mobile phase A:
0.05% TFA in water, mobile phase B: acetonitrile, gradient (T/%B):
90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 230 nm,
retention time 8.7 min.
References
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4.1.11.4. 6-(2-(4-Methoxyphenyl)-4-phenyl-1H-imidazol-5-yl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (9d). Yield 85%; white solid; mp
134e135 ꢀC; IR (KBr, cmꢂ1): 3447, 2923, 1654, 1458, 419; 1H NMR
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4919e4938;
(DMSO-d6): d 12.50 (bs, 1H, eNH), 10.75 (1H, bs, eNH), 8.00 (1H, d,
J ¼ 8.7 Hz, ArH), 7.65e7.20 (6H, m, ArH), 7.10e6.80 (4H, m, ArH),
4.60 (2H, s, OCH2), 3.85 (3H, s, OCH3); m/z (CI) 398 (M þ 1, 100%);
HPLC 97.4%, column: Intersil C-18 (150 ꢁ 4.6 mm), mobile phase A:
0.05% TFA in water, mobile phase B: acetonitrile, gradient (T/%B):
90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 215 nm,
retention time 7.5 min.
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4.1.11.5. 6-(2-(4-Fluorophenyl)-4-phenyl-1H-imidazol-5-yl)-2H-
benzo[b][1,4]oxazin-3(4H)-one (9e). Yield 87%; white solid. mp
130e131 ꢀC; IR (KBr, cmꢂ1): 3186, 1693, 1498, 1409, 692, 509. 1H
NMR (DMSO-d6): d 12.65 (1H, bs, NH), 10.70 (1H, bs, NH), 8.20e8.05
(2H, m, ArH), 7.65e7.20 (8H, m, ArH), 7.10e6.80 (2H, m, ArH), 4.65
(1H, s, OCH2), 4.55 (1H, s, OCH2); m/z (CI) 384 (M-1, 100%); HPLC
98.7%, column: Intersil ODS-3 C-18 (150 ꢁ 4.6 mm), mobile phase
A: 0.05% TFA in water, mobile phase B: acetonitrile, gradient (T/%B):
90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 230 nm,
retention time 7.6 min.
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zol-5-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (9f). Yield 83%; white
solid; mp 201e202 ꢀC; IR (KBr, cmꢂ1): 3198, 1695, 1492, 1405, 771,
698, 521; 1H NMR (DMSO-d6):
d
12.10 (1H, bs, eNH), 10.70 (1H, s,
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eNH), 8.78 (1H, d, J ¼ 7.6 Hz, ArH), 8.40 (1H, bs, NH), 8.13 (1H, s,

4896
C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896
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