European Journal of Medicinal Chemistry 46 (2011) 4887e4896

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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,
Venugopal Rao Veeramaneni a, **, Manojit Pal d, *
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-
Received 1 June 2011 fluoroacetic anhydride/phosphoric acid mediated CeC bond forming reaction as a key step. This method
Received in revised form does not require the use of environmentally harmful AlCl3 or moisture sensitive acid chloride. A number
24 July 2011
of compounds containing the benzooxazinone moiety attached to a five-membered central heterocyclic
Accepted 26 July 2011
Available online 3 August 2011
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
Keywords:
Benzooxazinone
promising anti-leukemic properties. A brief StructureeActivity-Relationship study within the series is
CeC bond presented. An imidazole derivative 9c containing benzene ring with a para-CF3 group at C-2 position was
Acylation identified as a potent anti-leukemic agent.
1,2-Diaryl-1-ethanone Ó 2011 Elsevier Masson SAS. All rights reserved.
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 cancer agents.
by structure A (Fig. 1) have attracted considerable interest in the A number of methods have been reported for the synthesis of
development of potential anti-cancer agents. For example, imid- diverse series of compounds related to A. One of the commonly
azole derivative SB-590885 possessing a 2,3-dihydro-1H-inden-1- utilized strategy for the synthesis of triaryl substituted heterocyclic
one oxime substituent has been identified as a potent and compounds involved the use of appropriately functionalized 1,2-
extremely selective inhibitor of the B-Raf kinase [1]. B-Raf muta- diaryl-1-ethanone D (Fig. 2) that could also be the precursor for
tions have been identified in many types of cancer, especially in compound C. These ketones are prepared via an array of methods
malignant melanomas and thyroid cancers [2], and several agents including (a) synthesis from a-aminonitrile [5e7], (b) acylation in
targeting B-Raf have been reported. 2H-Benzo[b][1,4]oxazin-3(4H)- the presence of Lewis acids [8e10], or CF3SO3H [11], or palladium
one derivatives on the other hand have been reported to be useful catalysts [12], (d) synthesis using Grignard reagent [13], (e) pinacol
chemical entities for treating and/or preventing various human rearrangement catalyzed by AlCl3 [14], (f) benzotriazole-assisted
diseases [3]. In our continuing effort to identify novel compounds of method [15], (g) self coupling of phenylacetic acid in the presence
potential pharmacological interest [4] we became interested to of PPA [16,17], (h) a multistep sequences consisting of Perkin
build a library of small molecules incorporating the 2H-benzo[b] condensation and Crutius rearrangement [18], and (i) synthesis via
[1,4]oxazin-3(4H)-one moiety (e.g. compound C, Fig. 1) for assess- clay catalyzed rearrangement of phenyloxiranes [19]. However, the
ing their anti-cancer properties in vitro. Herein, we report our 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
* Corresponding author. Tel.: þ91 40 6657 1500; fax: þ91 40 6657 1581.
** Corresponding author. Tel.: þ91 40 2319 7766; fax: þ91 40 2309 1692.
formation of environmentally harmful gaseous HCl thus a more
E-mail addresses: [email protected] (V.R. Veeramaneni), manojitpal@ convenient method was developed by reacting arylacetic acids with
rediffmail.com (M. Pal). appropriate arenes in the presence of trifluoroacetic anhydride/

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 O
N
Hetero O
cycle N N O
R Hetero H
NMe 2 cycle
N Ar
Ar
OH

A B C
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 year, and therefore discovery and development of suitable agents to
became interested in the application of this CeC bond forming treat various types of cancer is highly desirable. We have used A549
reaction as a key step to synthesize our target compounds. To the (NSCLC or Non small cell lung cancer), DLD-1 (colorectal adenocar-
best of our knowledge this methodology has not been explored cinoma) and MV4-11 (acute myeloid leukemia) cells obtained from
earlier for the preparation of compounds represented by C. ATCC (American Type Culture Collection) for our in vitro assay. The
effect of test compounds on cell viability was measured using
2. Results and discussion a colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay after 3 days of treatment in
2.1. Chemistry culture medium containing 5% FBS. The percentages of cell viability
(calculated with respect to control cells grown in the presence of
The key starting material 3 required for our synthesis was 0.1% v/v DMSO) for new and reference compounds at two concen-
readily prepared from 2-nitrophenol (1) via a two-step process trations (1.0 and 20 mM for test compounds whereas 1.0 and 10 mM
(Scheme 1). Thus reaction of 2-nitrophenol (1) with ethyl bro- for reference compounds) are presented in Table 1. The reference
moacetate followed by reduction and in situ cyclization of the compounds used in our assay include Gefitinib [an epidermal
resulting ether derivative 2 provided the desired benzooxazinone 3. growth factor receptor (EGFR) inhibitor], Imatinib (or Gleevec, an
Subsequent reaction of compound 3 with phenylacetic acid in the inhibitor of tyrosine kinase activities), Vandetanib [or Zactima, an
presence of H3PO4/(CF3CO)2O provided the ketone 4 in good yield. antagonist of the vascular endothelial growth factor receptor
Notably, the present H3PO4/(CF3CO)2O mediated CeC bond (VEGFR) and the EGFR], Erlotinib (or Tarceva, a tyrosine kinase
formation represents a straightforward, efficient and environ- inhibitor which acts on the EGFR), Sunitinib [or Sutent, a multi-
mental friendly process and does not require the use of moisture targeted receptor tyrosine kinase (RTK) inhibitor] and Sorafenib
sensitive acyl halides. [or Nexavar, an inhibitor of several Tyrosine protein kinases (VEGFR
The compound 4 was then converted to the 3-(dimethylamino) and PDGFR) and Raf (Rapidly Accelerated Fibrosarcoma)]. All the
acrylaldehyde derivative (5) which on treatment with appropri- experiments were performed twice with triplicate data points. It is
ately substituted hydrazines provided triaryl substituted pyrazoles evident from Table 1 that at the concentration of 20 mM compounds
(6) (Scheme 2). In order to prepare a range of isoxazole derivatives 9e, 9d and 9c showed comparable activities to the reference
the benzooxazinone 3 was reacted with an array of arylacetic acids compounds Vandetanib, Sunitinib and Sorafenib (all at 10 mM)
to give the ketone 4aee (Scheme 3). These ketones were then against A549 cells. The other compounds that showed activities
converted to the compounds 5aee which along with 5 provided the against A549 cells include 6g, 6e, 6a, 6b and 6c. Similarly, all the
desired compound 7aef when treated with hydroxylamine compounds except 9b and 6d were found to be active against DLD-1
hydrochloride. cells when tested at 20 mM. However, impressive results were
The imidazole derivatives were prepared (Scheme 4) from obtained when all these compounds were tested against MV4-11
compound 5 that was initially oxidized to the 1,2-diketone 8. cells at 20 mM. Indeed a number of compounds e.g. 9a, 9e, 9d, 9f
Condensation of compound 8 with appropriate aldehydes in the and 9c showed significant anti-proliferative properties in this assay
presence of NH4OAc afforded the desired products 9. 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
2.2. Pharmacology derivatives (9) were found to be superior to pyrazoles (6) in terms of
their in vitro activities. Among the imidazole derivatives the
Having prepared a divers series of compounds based on 2H- compounds containing benzene ring with a para-substituent or no
benzo[b][1,4]oxazin-3(4H)-one we then tested all these compounds substituent at C-2 position was found to be superior to others. The
for their potential anti-cancer properties in vitro. Cancer is the CF3 group was found to be best among all the para-substituents
second leading cause of death [21] worldwide after cardiovascular examined and compound 9c was identified as a potent anti-
diseases, according to WHO. Indeed, lung, breast, stomach, liver and leukemic agent. Notably, leukemia a cancer of blood-forming cells in
colorectal cancers cause the most cancer deaths worldwide each 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
Ar1 O Ar1 O medicinal value.
C
Ar 2 Ar 2 Z 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
OH a O O c O
O b N O
H
NO2 NO2 N O
H
2 3 4
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 added drop wise. After completion of addition, temperature was
compounds was carried out using trifluoroacetic anhydride/phos- raised to 50  C and stirred for 1 h. The reaction mixture was cooled
phoric acid mediated CeC bond forming reaction as a key step. This and the solid separated out was filtered and washed with acetone.
method does not require the use of environmentally harmful AlCl3 Filtrate was concentrated in reduced pressure. The crude was par-
or moisture sensitive acid chloride. Thus acylation of benzoox- titioned between methyl tertiary butyl ether and water. The organic
azinone ring under these conditions followed by appropriate layer was extracted with ethyl acetate (2  20 mL), washed with
reaction sequences afforded a range of tri and diaryl substituted water (20 mL), dried over anhydrous sodium sulfate and concen-
heterocyclic compounds containing the benzooxazinone moiety trated to give the title compound (15.4 g, yield 92%); mp 39e40  C;
1
attached to a five-membered central ring. Some of the compounds H NMR (CDCl3, 400 MHz): d 7.85 (1H, d, J ¼ 8.2 Hz, ArH), 7.50 (1H, t,
prepared were tested for their potential anti-cancer properties J ¼ 7.8 Hz, ArH), 7.10 (2H, d, J ¼ 7.7 Hz, ArH), 4.75 (2H, s, OCH2),
in vitro against A549 (lung), DLD-1 (colorectal adenocarcinoma) 4.30e4.20 (2H, m, OCH2), 1.30 (3H, t, J ¼ 7.3 Hz, CH3); m/z (CI) 224
and MV4-11 (acute myeloid leukemia) cell lines. Few of them (M þ 1, 100%).
showed promising anti-proliferative properties especially against
leukemia cells. An imidazole derivative containing benzene ring 4.1.3. Synthesis of 4H-benzo [1,4]oxazin-3-one (3)
with a para-CF3 group at C-2 position was identified as a potent To a solution of 2 (15.4 g 0.068 mol) in acetic acid (75 mL), Fe
anti-leukemic agent. Overall, the present series of heterocyclic powder (38.4 g, 0.68 mol) was added portion wise for 3 h at 0  C.
compounds represent a potential scaffold for the development of The reaction was exothermic. The reaction mixture was allowed to
potent anti-leukemic agents. 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
4. Experimental section room temperature, filtered on celite and the cake was washed with
acetic acid. Acetic acid was concentrated to ¼ of its volume and
4.1. Chemistry diluted with water (75 mL). The solid separated was filtered,
washed with water (20 mL) and dried to give the title compound
4.1.1. General methods (7.5 g, yield 91%); mp 173e174  C; 1H NMR (CDCl3, 400 MHz): d 9.95
Unless stated otherwise, reactions were performed under (1H, s, NH), 7.00e6.80 (4H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI) 150
nitrogen atmosphere. Reactions were monitored by thin layer (M þ 1, 100%).
chromatography (TLC) on silica gel plates (60 F254), visualizing
with ultraviolet light or iodine spray. Flash chromatography was 4.1.4. Synthesis 6-phenylacetyl-4H-benzo [1,4]-oxazin-3-one (4)
performed on silica gel (100e200 mesh) using distilled hexane, Phosphoric acid (3.94 g, 0.04 mol) and TFAA (18.6 mL, 0.132 mol)
ethyl acetate, dichloromethane. 1H NMR spectra were determined were added to the mixture of 3 (5 g, 0.033 mol) and phenylacetic
in CDCl3 or DMSO-d6 solution by using Varian 400 MHz spec- acid (6.73 g, 0.04 mol), at 0  C. After completion of addition, the
trometer. Proton chemical shifts (d) are relative to tetramethylsi- mixture was allowed to warm to room temperature and stirred for
lane (TMS, d ¼ 0.00) as internal standard and expressed in ppm. 1 h. The reaction mixture was cooled to 0  C and adjusted the pH to
Spin multiplicities are given as s (singlet), d (doublet), t (triplet) and 7 by adding the saturated NaHCO3 solution. The precipitated solid
m (multiplet) as well as b (broad). Coupling constants (J) are given was filtered, washed with water and dried to furnish 7.5 g (85%) of
in hertz. Infrared spectra were recorded on an FT-IR spectrometer the desired product, mp 188e189  C; 1H NMR (CDCl3, 400 MHz):
(Perkin Elmer). Melting points were determined using melting d 10.85 (1H, bs, NH), 7.70 (1H, d, J ¼ 7.8 Hz, ArH), 7.50 (1H, s, ArH),
point apparatus and are uncorrected. MS spectra were obtained on 7.40e7.20 (5H, m, ArH), 6.95 (1H, d, J ¼ 7.8 Hz, ArH), 4.65 (2H, s,
a mass spectrometer. CH2), 4.20 (2H, s, CH2); m/z (CI) 266 (M-1, 100%).

4.1.2. Synthesis of triaryl substituted pyrazoles (6) 4.1.5. Synthesis 6-(3-dimethylamino-2-phenyl-acrolyl)-4H-benzo

4.1.2.1. Synthesis of (2-nitrophenoxy) ethyl acetate (2). To a stirred [1,4]-oxazin-3-one (5)
solution of 2-nitrophenol (10 g, 0.071 mol) in acetone (25 mL), DMFeDMA (1.07 g, 0.0089 mol) was added to a solution of 6-
K2CO3 (21.5 g, 0.156 mol) was added at room temperature and phenyl-4H-benzo [1,4]-oxazin-3-one (2 g, 0.0074 mol) in toluene
stirred for 15 min. Then ethyl bromoacetate (13.2 g, 0.079 mol) was (10 mL) and heated to reflux for 0.5 h. After sometime the solution

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 O c N N O
a O b
N O H O H
3 H N
R R
R
7
4a-e 5a-e
4a; 5a; 7a; R=4-f luorophenyl
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 695; 1H NMR (DMSO-d6): d 10.65 (1H, s, eNH), 8.00 (1H, s, ArH),
was filtered, washed with toluene (15 mL) and dried to give the title 7.35e7.10 (8H, m, ArH), 6.95 (2H, d, J ¼ 8.4 Hz, ArH), 6.80e6.60 (2H,
compound (1.75 g, yield 76%) as light yellow solid; mp 194e195  C; m, ArH), 4.60 (2H, s, OCH2), 3.75 (3H, s, OCH3); m/z (CI) 396 (M-1,
1
H NMR (CDCl3, 400 MHz): d 10.85 (1H, s, NH), 7.80 (1H, s, ]CH), 100%); HPLC 98.9%, column: Intersil C-18 (150  4.6 mm), mobile
7.65 (1H, s, ArH), 7.30e7.00 (5H, m, ArH), 6.60e6.45 (2H, m, ArH), phase A: water, mobile phase B: acetonitrile, gradient (A/%B): 90/
4.55 (2H, s, OCH2), 2.80 (6H, s, N(CH3)2); m/z (CI) 321 (M-1, 100%). 10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention
time 11.6 min.
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]
benzo [1,4]-oxazin-3-one (0.00065 mol) in ethanol, substituted oxazin-3(4H)-one (6d). Yield 83.3%; white solid, mp 144e146  C;
phenylhydrazine hydrochloride (0.00071 mol) in ethanol was IR (KBr, cm1): 3208, 1701, 1491, 1372, 1039, 764, 697; 1H NMR
added slowly. After completion of addition, the mixture was heated (DMSO-d6): d 10.70 (1H, s, NH), 8.10 (1H, s, ArH), 7.40e7.15 (9H, m,
to 40e50  C in a water bath. After 15 min, the solution became clear ArH), 6.85 (1H, d, J ¼ 8.8 Hz, ArH), 6.75e6.68 (2H, m, ArH), 4.60
and solid was precipitated out. The solid separated was filtered, (2H, s, OCH2), 2.00 (3H, s, CH3); m/z (CI) 382 (M þ 1, 97%); HPLC
washed with ethanol and dried to give the expected product. 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,
4.1.6.1. 6-(1-(4-Chlorophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-benzo 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention time
[b][1,4]oxazin-3(4H)-one (6a). Yield 88%; white solid. mp > 270  C; 11.7 min.
IR (KBr, cm1): 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), [b][1,4]oxazin-3(4H)-one (6e). Yield 86.2%; white solid,
6.80e6.60 (2H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI) 400 (M-1, mp > 270  C: IR (KBr, cm1): 3470, 1703, 1491, 1044, 823, 769, 697;
1
100%); HPLC 98.1%, column: Intersil C-18 (150  4.6 mm), mobile H NMR (DMSO-d6): d 10.65 (1H, s, NH), 8.10 (1H, s, ArH), 7.60 (2H,
phase A: water, mobile phase B: acetonitrile, gradient (A/%B): 90/ d, J ¼ 8.7 Hz, ArH), 7.35e7.15 (7H, m, ArH), 6.95 (1H, d, J ¼ 8.2 Hz,
10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention ArH), 6.88e6.70 (2H, m, ArH), 4.65 (2H, s, OCH2); m/z (CI) 448
time 12.7 min. (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/%
4.1.6.2. 6-(1-(4-Fluorophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-benzo B): 90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm,
[b][1,4]oxazin-3(4H)-one (6b). Yield 88%; white solid. mp > 270  C; retention time 11.8 min.
IR (KBr, cm1): 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, 4.1.6.6. 6-(1-(3,5-Dichlorophenyl)-4-phenyl-1H-pyrazol-3-yl)-2H-
J ¼ 8.2 Hz, ArH), 6.80e6.60 (2H, m, ArH), 4.60 (2H, s, OCH2); m/z (CI) benzo[b][1,4]oxazin-3(4H)-one (6f). Yield 85%; white solid, mp
286 (M þ 1, 100%); HPLC 98.0%, column: Intersil C-18 189e190  C; IR (KBr, cm1): 3447, 1705, 1583, 1046, 807, 765, 693;
1
(150  4.6 mm), mobile phase A: water, mobile phase B: acetoni- H NMR (DMSO-d6): d 10.75 (1H, s, ArH), 8.10 (1H, s, ArH), 7.80 (1H,
trile, gradient (A/%B): 90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/ s, ArH), 7.30e7.10 (7H, m, ArH), 6.95 (1H, d, J ¼ 8.1 Hz, ArH),
min, UV 254 nm, retention time 11.9 min. 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
4.1.6.3. 6-(1-(4-Methoxyphenyl)-4-phenyl-1H-pyrazol-3-yl)-2H- phase A: water, mobile phase B: acetonitrile, gradient (A/%B): 90/
benzo[b][1,4]oxazin-3(4H)-one (6c). Yield 86%; white solid, mp 10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention
242e243  C; IR (KBr, cm1): 3480, 1702, 1510, 1249, 1042, 830, 766, time 14.0 min.

O O

a O b 9a; R=phenyl
N O N N O 9b; R=2,6-dichlorophenyl
5 H R H 9c ; R=4-trifluoromethylphenyl
O N 9d; R=4-methoxyphenyl
H 9e ; R=4-fluorophenyl
9f ; 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 mM 20 mM 1 mM 20 mM 1 mM 20 mM

O
H
N
N O
1 H 9a 95.6 58.5 98.2 47.3 104.6 7.6
N

O
Cl
H
Cl N
N O
2 H 9b 97.4 68.3 97.8 61.9 108.7 41.1
N

O
H
F N
N O
3 H 9e 97.1 37.5 94.8 29.5 108.2 1.1
N

O
H
MeO N
N O
4 H 9d 100.8 38.1 96.0 19.1 108.1 1.2
N

O
N H
N
5
N O 9f 100.3 86.8 97.7 53.5 110.8 8.9
HN H
N

O
H
CF3 N
N O
6 H 9c 102.8 21.7 95.0 12.1 110.8 0.6
N

N
N N O
7 H 6g 92.2 59.7 77.0 47.7 117.2 97.9

N
N N O
8 H 6d 94.8 85.3 92.2 62.9 99.4 54.2

(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 mM 20 mM 1 mM 20 mM 1 mM 20 mM

Br N
N N O
9 H 6e 81.5 58.8 66.4 54.2 107.8 91.5

Cl N
N N O
10 H 6a 84.4 52.4 61.9 47.1 107.8 91.9

F N
N N O
11 H 6b 78.2 56.4 64.4 45.2 107.3 92.6

Cl O

N
N N O
12 H 6f 93.1 70.2 90.3 52.2 105.1 50.5
Cl

O N
N N O
13 H 6c 95.6 42.6 92.2 35.6 106.1 59.9

O HN Cl
14 Gefitinib 84.4 61.5a 87.0 65.7a 92.4 7.0a
N O
N

O N

N
H
N N

15 HN N Imatinib 108.3 98.6a 89.9 91.5a 105.0 82.0a

N
O N

16
N Vandetanib 88.7 41.7a 88.6 37.9a 84.1 1.6a
O
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 mM 20 mM 1 mM 20 mM 1 mM 20 mM

O O N

N
17 O O Erlotinib 83.0 68.5a 85.7 55.4a 98.3 16.8a
HN

NH

18 Sunitinib 102.5 37.4a 92.9 52.7a 1.4 0.5a

N N
F H
O
N
H

CF3 O
Cl O
O NHCH3
19 N Sorafenib 103.2 28.6a 93.4 28.0a 1.6 0.7a
N N
H H

Sorafenib
a
The assay was carried out using the compound at 10 mM.

4.1.7. Synthesis of isoxazole derivatives (7) ArH), 7.18e7.05 (3H, m, ArH), 4.70 (2H, s, OCH2), 4.30 (2H, m, ]
4.1.7.1. Typical procedure for the synthesis 6-phenylacetyl-4H-benzo CeCH2Ph); m/z (CI) 284 (M-1, 100%).
[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] 4.1.7.3. 6-(2-(2-Chlorophenyl)acetyl)-2H-benzo[b][1,4]oxazin-3(4H)-
oxazin-3-one (4 g, 0.026 mol) and substituted phenylacetic acid one (4b). Yield 82%; white solid; 1H NMR (DMSO-d6): d 10.85 (1H, s,
(1.1eq), at 0  C. After completion of addition, the mixture was NH), 7.90e7.70 (1H, m, ArH), 7.53 (1H, s, ArH), 7.55e7.25 (4H, m,
allowed to warm to room temperature and stirred for 1 h. The ArH), 7.05 (1H, d, J ¼ 8.4 Hz, ArH), 4.69 (2H, s, OCH2), 4.42 (2H, s, ]
reaction mixture was cooled to 0  C and adjusted the pH to 7 by CeCH2ePh); m/z (CI) 300 (M-1, 100%).
adding the saturated NaHCO3 solution. The precipitated solid was
filtered, washed with water and dried to give the desired product. 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,
one (4a). Yield 80%; white solid; 1H NMR (DMSO-d6): d 10.85 (1H, s, ArH), 7.20 (1H, d, J ¼ 7.2 Hz, ArH), 7.17 (1H, d, J ¼ 7.2 Hz, ArH), 4.67
NH), 7.74 (1H, d, J ¼ 8.4 Hz, ArH), 7.52 (1H, s, ArH), 7.35e7.20 (2H, m, (2H, s, OCH2), 4.32 (2H, s, ]CeCH2ePh); m/z (CI) 300 (M-1, 100%).

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,
Chart 1. Three days treatment of MV4-11 cells with most active compounds. 0.0089 mol) in toluene and heated to reflux on an oil bath for 0.5 h.
4894 C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896

After sometime the solution became clear and immediately solid 4.1.9.3. 6-(4-(3-Chlorophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa-
was precipitated out. The solid was filtered, washed with toluene zin-3(4H)-one (7c). Yield 92%; White solid; mp. 178e180  C; IR
and dried to give the desired product. (KBr, cm1): 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- J ¼ 5.0 Hz, ArH), 7.38 (1H, s, ArH), 7.20e7.00 (3H, m, ArH), 4.68 (2H,
benzo[b][1,4]oxazin-3(4H)-one (5a). Yield 70%; white solid; 1H s, OCH2); m/z (CI) 325 (M-1, 100%); HPLC 97.9%, column: Intersil C-
NMR (DMSO-d6): d 10.85 (1H, s, NH), 7.25 (1H, s, ArH), 7.15e7.05 18 (150  4.6 mm), mobile phase A: 0.05% TFA in water, mobile
(4H, m, ArH), 7.05 (1H, s, Me2NeCH]), 6.95e6.80 (2H, m, ArH), phase B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10,
4.70 (2H, s, OCH2), 2.70 (6H, s, N(CH3)2); m/z (CI) 291 (M þ 1, flow rate: 1.0 mL/min, UV 215 nm, retention time 11.8 min.
100%).
4.1.9.4. 6-(4-(3-Methylphenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa-
4.1.8.2. (Z)-6-(3-(Dimethylamino)-2-(2-chlorophenyl)acryloyl)-2H- zin-3(4H)-one (7d). Yield 89%; White solid; mp 191e193  C; IR
benzo[b][1,4]oxazin-3(4H)-one (5b). Yield 77.9%; white solid; 1H (KBr, cm1): 3448, 1698, 1498, 1387, 1039, 699; 1H NMR (DMSO-d6):
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, (3H, s, ArH), 7.10e6.98 (2H, m, ArH), 4.67 (2H, s, OCH2), 2.33 (3H, s,
ArH), 4.69 (2H, s, OCH2), 2.70 (6H, s, N(CH3)2); m/z (CI) 355 (M-1, CH3); m/z (CI) 307 (M þ 1, 100%); HPLC 97.4%, column: Intersil C-18
100%). (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
4.1.8.3. (Z)-6-(3-(Dimethylamino)-2-(3-chlorophenyl)acryloyl)-2H- rate: 1.0 mL/min, UV 230 nm, retention time 11.7 min.
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-
Me2NeCH]), 7.10e6.85 (4H, m, ArH), 4.60 (2H, s, OCH2), 2.70 (6H, zin-3(4H)-one (7e). Yield 91.3%; White solid; mp 205e206  C; IR
s, N(CH3)2); m/z (CI) 355 (M-1, 100%). (KBr, cm1): 3448, 1690, 1490, 1388, 812; 1H NMR (DMSO-d6):
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,
benzo[b][1,4]oxazin-3(4H)-one (5d). Yield 75%; white solid; 1H OCH2); m/z (CI) 369 (M-2, 100%); HPLC 97.5%, column: Intersil C-18
NMR (DMSO-d6): d 10.85 (1H, s, NH), 7.20e7.15 (2H, m, ArH), 7.00 (150  4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase
(1H, s, Me2NeCH]), 6.95e6.80 (5H, m, ArH), 4.69 (2H, s, OCH2), B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow
2.25 (3H, s, CH3), 2.70 (6H, s, N(CH3)2); m/z (CI) 335 (M-1, 100%). rate: 1.0 mL/min, UV 230 nm, retention time 12.1 min.

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, cm1): 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, 8.90 (1H, s, ArH), 7.55e7.35 (5H, m, ArH), 7.20 (1H, s, ArH),
OCH2), 2.70 (6H, s, N(CH3)2); m/z (CI) 403 (M þ 2, 100%). 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:
4.1.9. Typical procedure for the synthesis of isoxazole 0.05% TFA in water, mobile phase B: acetonitrile, gradient (T/%B):
To a solution of 6-(3-dimethylamino-2-phenyl-acrolyl)-4H- 90/10, 10/90, 10/90, 90/10, flow rate: 1.0 mL/min, UV 230 nm,
benzo [1,4]-oxazin-3-one (0.00065 mol) in ethanol, hydroxylamine retention time 10.9 min.
hydrochloride (0.00071 mol) in ethanol was added slowly. After
completion of addition, the mixture was heated to 40e50  C on 4.1.10. Preparation of imidazole derivatives (9)
water bath. After 15 min, the solution became clear and immedi- 4.1.10.1. Synthesis of 1-(3-oxo-3,4-dihydro-2H-benzo [1,4]oxazin-6-
ately solid precipitated out. The solid was filtered, washed with yl)-2-phenyl-ethane-1,2-dione (8). To a suspension of 6-(3-
ethanol and dried. 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
4.1.9.1. 6-(4-(4-Fluorophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxazin- NaIO4 (3.98 g, 0.018 mol) portion wise for a duration of 15 min at
3(4H)-one (7a). Yield 90%; white solid; mp 176e177  C; IR (KBr, room temp. After completion of addition, the mixture was allowed
cm1): 3464, 1694, 1498, 1398, 1221, 843, 810, 542; 1H NMR (DMSO- to stir for 2 h. After completion of the reaction (indicated by TLC/1:1
d6): d 10.85 (1H, bs, eNH), 8.85 (1H, s, ArH), 7.55e7.40 (2H, m, ArH), EtOAcehexane) the solid separated was filtered and washed with
7.38e7.25 (2H, m, ArH), 7.10e7.00 (3H, m, ArH), 4.67 (2H, s, OCH2); ethyl acetate (2  10 mL). The filtrate was collected and treated
m/z (CI) 311 (M þ 1, 98%); HPLC 97.0%, column: Intersil C-18 with cold water (15 mL). The organic layer was collected, dried over
(150  4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase anhydrous Na2SO4 and concentrated. The residue was purified by
B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow column chromatography using EtOAcehexane (1:1) to give the
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),
4.1.9.2. 6-(4-(2-Chlorophenyl)isoxazol-3-yl)-2H-benzo[b][1,4]oxa- 7.70e7.60 (3H, m, ArH), 7.50 (1H, d, J ¼ 2.0 Hz, ArH), 7.15 (1H, d,
zin-3(4H)-one (7b). Yield 92%; White solid; mp 247e248  C; IR J ¼ 8.4 Hz, ArH), 4.86 (2H, s, OCH2); m/z (CI) 280 (M-1, 100%).
(KBr cm1): 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 4.1.11. Typical procedure for the synthesis of imidazole derivatives
(3H, m, ArH), 7.10 (1H, s, ArH), 7.10e6.85 (2H, m, ArH), 4.61 (2H, To a solution of 1-(3-oxo-3,4-dihydro-2H-benzo [1,4]oxazin-6-
s, OCH2); m/z (CI) 325 (M-1, 100%); HPLC 99.2%, column: Intersil yl)-2-phenyl-ethane-1,2-dione (0.1 g, 0.0003 mol) in acetic acid
C-18 (150  4.6 mm), mobile phase A: 0.05% TFA in water, (10 mL) was added ammonium acetate (0.23 g, 0.003 mol) followed
mobile phase B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/ by an appropriate aldehyde (0.0003 mol) slowly. The mixture was
90, 90/10, flow rate: 1.0 mL/min, UV 254 nm, retention time heated to reflux for 2 h. After completion of the reaction the
11.3 min. 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, ArH), 7.57 (2H, d, J ¼ 6.7 Hz, ArH), 7.45e6.90 (8H, m, ArH), 4.60 (2H,
dried over anhydrous Na2SO4 and concentrated. The residue was s, OCH2); m/z (CI) 408 (M þ 1, 100%); HPLC 98.5%, column: Intersil C-
purified by column chromatography using EtOAcehexane (3:2) to 18 (150  4.6 mm), mobile phase A: 0.05% TFA in water, mobile
give desired product. 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, 4.2. Pharmacology: In vitro cell proliferation assay
cm1): 3448, 1684, 1490, 1380, 775, 697; 1H NMR (DMSO-d6):
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
7.65e6.80 (11H, m, ArH), 4.60 (1H, s, OCH2), 4.56 (1H, s, OCH2); m/z (acute myeloid leukemia) cells were obtained from ATCC and
(CI) 368 (M þ 1, 100%); HPLC 98.4%, column: Intersil ODS-3 C-18 grown as recommended. The effect of test compounds on cell
(150  4.6 mm), mobile phase A: 0.05% TFA in water, mobile phase viability was measured using a colorimetric MTT assay after 3 days
B: acetonitrile, gradient (T/%B): 90/10, 10/90, 10/90, 90/10, flow of treatment in culture medium containing 5% FBS. A549 and DLD-1
rate: 1.0 mL/min, UV 230 nm, retention time 7.4 min. cells were seeded in a 96 well plate at a density of 5000 cells per
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- were seeded at 40,000 cells per well immediately before treatment.
benzo[b][1,4]oxazin-3(4H)-one (9b). Yield 80%; white solid; mp All the test compounds were prepared in DMSO as 20 mM stock
154e156  C; IR (KBr, cm1): 3421, 2925, 1696, 1490, 1388, 787, 698; solutions and diluted in RPMI as 20X solutions to achieve the
1
H 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), 0.1% v/v DMSO. At the end of treatment, cells were incubated with
4.62 (2H, s, OCH2); m/z (CI) 436 (M þ 1, 100%); HPLC 97.6%, column: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
Intersil ODS C-18 (150  4.6 mm), mobile phase A: 0.05% TFA in bromide for 4 h, followed by solubilization of the formazan crystals
water, mobile phase B: acetonitrile, gradient (T/%B): 90/10, 10/90, with 100 ml 10% SDS in 10 mM HCl. Absorbance at 595 nm was
10/90, 90/10, flow rate: 1.0 mL/min, UV 215 nm, retention time measured and the percentage of cell viability was calculated with
7.7 min. 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; Acknowledgments
mp 164e166  C; IR (KBr, cm1): 3255, 1685, 1327, 1124, 1066,
697; 1H NMR (DMSO-d6): d 12.95 (1H, bs, NH), 10.75 (1H, d, NH), The authors thank management of Indus BioSciences Private
8.30 (2H, d, J ¼ 8.1 Hz, ArH), 7.85 (2H, d, J ¼ 8.2 Hz, ArH), Limited for encouragement and support.
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%,
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(i) M. Pal, Tetrahedron 65 (2009) 433e447;
4.1.11.5. 6-(2-(4-Fluorophenyl)-4-phenyl-1H-imidazol-5-yl)-2H- (j) M. Pal, N.K. Swamy, P.S. Hameed, S. Padakanti, K.R. Yeleswarapu, Tetra-
hedron 60 (2004) 3987e3997.
benzo[b][1,4]oxazin-3(4H)-one (9e). Yield 87%; white solid. mp [5] S.F. Dyke, E.P. Tiley, A.W.C. White, D.P. Gale, Tetrahedron 31 (1975)
130e131  C; IR (KBr, cm1): 3186, 1693, 1498, 1409, 692, 509. 1H 1219e1222 and references cited therein.
NMR (DMSO-d6): d 12.65 (1H, bs, NH), 10.70 (1H, bs, NH), 8.20e8.05 [6] R. Olivera, R. SanMartin, E. Dominguez, X. Solans, M.K. Urtiaga, M.I. Arriortua,
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(1H, s, OCH2), 4.55 (1H, s, OCH2); m/z (CI) 384 (M-1, 100%); HPLC [8] N.I. Al-Maharik, A.A.K. Seppo, I. Mutikainen, K. Waehaelae, J. Org. Chem. 65
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A: 0.05% TFA in water, mobile phase B: acetonitrile, gradient (T/%B):
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4896 C. Rajitha et al. / European Journal of Medicinal Chemistry 46 (2011) 4887e4896

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