Synthesis, characterization and biological evaluation of 7-substituted- 4-((1-aryl-1H-1,2,3-triazol-4-yl) methyl)-2H-benzo[b][1,4]oxazin- 3(4H)-ones as anticancer agents

Article abstract of DOI:10.1007/s00044-016-1616-9

A series of novel 1,2,3-triazole-2H-benzo[b][1,4]oxazin-3(4H)-ones (4a–4k and 5a–5g) were designed, synthesized and screened for their anticancer activity against MCF-7 (breast) and HeLa (cervical) cell lines using [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphen

Full text of DOI:10.1007/s00044-016-1616-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1616-9
ORIGINAL RESEARCH
Synthesis, characterization and biological evaluation of 7-substituted-
4-((1-aryl-1H-1,2,3-triazol-4-yl) methyl)-2H-benzo[b][1,4]oxazin-
3(4H)-ones as anticancer agents
Vasudeva Reddy Nagavelli1 Satheesh Kumar Nukala1 Sirassu Narsimha1
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Kumara Swamy Battula1 Savitha Jyostna Tangeda1 Yellu Narasimha Reddy2
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Received: 23 February 2016 / Accepted: 15 June 2016
© Springer Science+Business Media New York 2016
Abstract A series of novel 1,2,3-triazole-2H-benzo[b][1,4]
oxazin-3(4H)-ones (4a–4k and 5a–5g) were designed, syn-
thesized and screened for their anticancer activity against
MCF-7 (breast) and HeLa (cervical) cell lines using
[3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]
assay. Among all the tested compounds, 5b and 5c exhib-
ited good cytotoxic activity against two cancer cell lines
with IC50 values nearer to the standard drug, cisplatin. The
most active compounds 5b and 5c were further evaluated
for their in vivo anticancer activity against Ehrlich Ascites
Carcinoma-bearing mice and compound 5c found to possess
potential cytotoxic activity as compared with the reference
drug cisplatin. Molecular docking studies were also carried
out to complement the experimental results.
important advances have been achieved in the development
of various cancer static drugs for antitumor chemotherapy.
The search for new lead compounds of simple structure,
exhibiting optimal in vivo antitumor potency and new
mechanisms of action, is today’s one of the most intensely
pursued goals of modern medicinal chemistry and is a
continuous process till the goal is reached.
The chemistry of 1,4-oxazinones and their derivatives have
received considerable attention due to their synthetic and
effective biological importance (Fig. 1). For example, a large
number of 1,4-oxazinones have been incorporated into a wide
variety of therapeutically interesting drug candidates posses-
sing antibacterial (Sugimoto et al.,1990), anticancer (Rajitha
et al., 2011; Xue-Wen et al., 2014), anticonvulsant (Zhong-Tai
et al., 2008) and antithromobotic activities (Buckman et al.,
1998). The literature survey reveals that [1,4]-oxazines are also
known as 5-HT6 receptor antagonists (Zhao et al., 2007),
bladder-selective potassium channel openers (Chiu et al.,
2001), dual selective serotonin reuptake inhibitors and 5-
HT1A receptor (Zhou et al., 2006), dopamine agonists (Jones
et al., 1984) and inhibitors of PI3Kinase (Lanni et al., 2007).
Some substituted [1,4]-oxazines are also related to blocking
the TXA2 receptor and activate the PGI2 receptor (Ohno et al.,
2006). On the other hand, over the past two decades, 1,2,3-
triazole derivatives have received attention not only in organic
chemistry but also in medicinal chemistry due to their easy
synthesis by click chemistry bearing attractive features, as well
as numerous biological activities such as antibacterial (Holla
et al., 2005), H1-antihistamine (Buckle et al., 1986), anti-HIV
(Velazquez et al., 1998), anticonvulsant (Kelley et al., 1995),
antimalarial (Sunny et al., 2011), anticancer (Chen et al., 2008;
Cindy et al., 2013) and anti-inflammatory activities (Rao et al.,
2014). Such compounds have also been reported as β3-
selective adrenergic receptor agonists (Brockunier et al.,
2000), kinase inhibitors (Olesen et al., 2003) and other enzyme
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Keywords 1,4-Benzoxazine 1,2,3-Triazoles Click
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chemistry Anticancer activity Molecular docking
Introduction
The identification of novel structures for effective treatment
of cancer is still a major challenge to medicinal chemistry
researchers (Gibbs JB, 2000; Bach et al., 2007; Siegel et al.,
2013). Cancer is a chronic disease and the major cause of
human mortality in the world today. Over recent decades,
* Vasudeva Reddy Nagavelli
vasujac3@gmail.com
1
Department of Chemistry, Kakatiya University, Warangal 506009,
India
2
Department of Pharmacology and Toxicology, Kakatiya
University, Warangal 506009, India

Med Chem Res
Fig. 1 Some of biologically
active 1,4-benzoxazine
derivatives
Fig. 2 Some of 1,2,3-triazole
ring containing drugs in the
market
inhibitors (Krasinski et al., 2005). In particular, by combining
1,2,3-triazoles with other pharmacophore via click chemistry,
a number of compounds with potent biological active com-
pounds were synthesized (Stefely et al., 2010; Reddy et al.,
2011; Singh et al., 2012). 1,2,3-triazole moiety containing
drug molecules such as tazobactam (Bennet et al., 1991),
cefatrizine (Stilwell et al., 1975) and carboxyamidotriazole
(Soltis et al., 1996) are available (Fig. 2).
Based on all the above considerations and as an extension
of our studies on the development of novel 1,2,3-triazoles
(Narsimha et al., 2014, 2016; Kumaraswamy et al., 2016;
Ranjith et al., 2015; Suresh et al., 2015), we report the
synthesis and anticancer activity of 1,2,3-triazole-substituted-
2H-benzo[b][1,4]oxazin-3(4H)-one hybrids (Fig. 3).
Fig. 3 Design strategy for new 1,4-benzoxazine derived 1,2,3-triazole
hybrids
mesh. Melting points were determined using a Cintex appa-
ratus. Elemental analysis was measured by means of Perki-
nElmer 2400 CHN elemental analyzer. Infrared (IR) spectra
were obtained on a PerkinElmer BX serried FTIR 5000 spec-
trometer using KBr pellet. A 500-MHz nuclear magnetic
resonance (NMR) spectrometer was used to acquire 1H-NMR
spectra. The instrument was set at 125 MHz for acquiring
13C NMR spectra. Coupling constant (J) values are presented
in Hertz and spin multiples are given as s (singlet), d (doublet),
Experimental
Chemistry and biology
All the solvents and the starting materials were purchased from
commercial sources and used without further purification.
Column chromatography was performed on silica gel 60–120

Med Chem Res
t (triplet) and m (multiplet). Mass spectra were recorded by
using Electrospray Ionisation–Mass Spectrometry (ESI–MS).
MHz, CDCl3): δ 8.063 (s, 1H, triazole), 7.660 (s, 2H, Ar),
7.354–7.523 (m, 2H, Ar), 7.104–7.223 (m, 2H, Ar), 5.214
(s, 2H, N–CH2), 4.626 (s, 2H, O–CH2): 13C NMR (125
MHz, dimethyl sulfoxide (DMSO)): δ 163.6, 145.7, 143.6,
136.8, 131.2, 127.9, 125.2, 123.9, 122.1, 119.3, 117.3,
General procedure for the preparation of 7-bromo-2H-
benzo[b][1,4]oxazin-3(4H)-one (2)
116.6, 114.8, 67.0, 36.2; ESI–MS (m/z)
:
455
[M+H]+. Anal. cal. for C17H11BrCl2N4O2: C, 44.96; H,
2.44; N, 12.34; found: C, 44.88; H, 2.37; N, 12.25.
To a mixture of 2-amino-5-bromophenol (26.59 mmol),
Triethyl benzyl ammonium chloride (TEBA) (30 mmol) and
NaHCO3 (120 mmol) in Tetrahydrofuran (THF) (30 mL)
was added a solution of 2-chloroacetyl chloride (53.2
mmol) in THF (10 mL) at 0–5 °C. The reaction mixture was
stirred for 2 h at room temperature and then heated to 60 °C
for 12 h under inert atmosphere. After completion of reaction,
the resulting mixture was concentrated under vacuum and
then 50 mL of water was added. The precipitate was col-
lected and washed with hexane to afford 7-bromo-2H-benzo
[b][1,4]oxazin-3(4H)-one as a yellow solid (52 % yield).
ESI–MS (m/z): 229[M+H]+.
7-Bromo-4-((1-(3,5-dimethylphenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4b)
m.p.: 131–133 °C; IR (KBr): 3127 (CH, triazole), 3096
1
(CH, Ar), 1663 (C=O), 1592 (C=C) cm−1; H NMR (500
MHz, CDCl3): δ 8.529 (brs, 1H, triazole),7.653 (brs, 1H),
7.469 (brs, 2H, Ar), 7.384 (s, 1H, Ar), 7.307 (brs, 1H, Ar),
7.182 (brs, 1H, Ar), 5.357 (brs, 2H, N–CH2), 4.767 (s, 2H,
O–CH2), 2.507 (s, 6H, Ar–CH3), 13C NMR (125 MHz,
DMSO): δ 163.7, 145.7, 143.1, 139.3, 136.3, 129.9, 127.9,
125.2, 121.5, 119.2, 117.5, 117.3, 114.8, 67.0, 36.3, 20.7;
ESI–MS (m/z): 414 [M+H]+. Anal. cal. for C19H17BrN4O2:
C, 55.22; H, 4.15; N, 13.56; found: C, 55.16; H, 4.21;
N, 13.49.
General procedure for the preparation of 7-bromo-4-(prop-
2-yn-1-yl)-2H-benzo[b][1,4] oxazin-3(4H)-one (3)
A mixture of 7-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one
(2) (0.035 mol) and Cs2CO3 (0.07 mol) in N,N-dimethylfor-
mamide (DMF) (50 mL) was treated with 3-bromoprop-1-yne
(0.045 mol) at room temperature for 1 h. After completion of
the reaction by Thin layer chromatography (TLC), the reaction
mixture was poured carefully into ice-cold water (50 mL). The
resulted solid was filtered off, washed with excess water and
7-Bromo-4-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4c)
m.p.: 168–170 °C; IR (KBr): 3136 (CH, triazole), 3094
1
(CH, Ar), 1680 (C=O), 1587 (C=C) cm−1; H NMR (500
1
dried under vacuum for 2 h. Yield (79 %): H NMR (500
MHz, CDCl3): δ 7.965 (s, 1H, triazole), 7.599 (d, J = 8.8
Hz, 2H, Ar), 7.519 (d, J = 8.6 Hz, 1H, Ar), 7.112–7.221 (m,
2H, Ar), 7.002 (d, J = 8.8 Hz, 2H, Ar), 5.220 (s, 2H,
N–CH2), 4.631 (s, 2H, O–CH2), 3.859 (s, 3H, O–CH3),
13C NMR (125 MHz, DMSO): δ 163.7, 159.2, 145.7, 143.0,
129.8, 127.9, 125.2, 121.6, 119.2, 117.4, 114.7, 67.0, 55.5,
36.2; ESI–MS (m/z): 416 [M+H]+. Anal. cal. for
C18H15BrN4O3: C, 52.06; H, 3.64; N, 13.49; found:
C, 52.17; H, 3.57; N, 13.42.
MHz, CDCl3) δ 7.148–7.238 (m, 2H, Ar), 7.061 (d, J = 8.5
Hz, 1H, Ar), 4.677 (d, J = 2.2 Hz, 2H, N–CH2), 4.642
(s, 2H, O–CH2), 2.280 (t, J = 2.2 Hz, 1H, CH); ESI–MS
(m/z): 267 [M+H]+. Anal. cal. for C11H8BrNO2: C, 49.65; H,
3.03; N, 5.26; found: C, 49.73; H, 3.11; N, 5.22.
General procedure for the preparation of 4a–4k
To a stirred solution of alkyne (3) (1.6 mmol) and aryl azide
(2.0 mmol) in THF (15 mL) was added CuI (10 mol %) and the
reaction mixture was stirred at room temperature for 8–10 h.
After completion of the reaction, the reaction mixture was
diluted with water (15 mL) and the product was extracted with
ethylacetate (2 × 15 mL). The combined organic layer was
washed with brine and dried over anhydrous Na2SO4. After
filtration, the solvent was evaporated under vacuum and the
crude product obtained was purified by column chromatography
(hexane/ethyl acetate gradient) to afford the title compound.
7-Bromo-4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4d)
m.p.: 157–159 °C; IR (KBr): 3130 (CH, triazole), 3088
1
(CH, Ar), 1672 (C=O), 1590 (C=C) cm−1; H NMR (500
MHz, CDCl3):
δ
8.061 (s, 1H, triazole), 7.764
(brs, 1H, Ar), 7.621 (d, J = 7.3 Hz, 1H, Ar), 7.389–7.526
(m, 3H, Ar), 7.174 (m, 2H, Ar), 5.227 (s, 2H, N–CH2),
4.638 (s, 2H, O–CH2): 13C NMR (125 MHz, DMSO):
δ 163.6, 145.7, 143.5, 137.4, 134.1, 131.5, 128.4, 127.8,
125.2, 121.9, 119.6, 119.2, 118.5, 117.3, 114.8, 67.0,
36.2; ESI–MS (m/z): 420 [M+H]+. Anal. cal. for
C17H12BrClN4O2: C, 48.65; H, 2.88; N, 13.35; found:
C, 48.56; H, 2.93; N, 13.27.
7-Bromo-4-((1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4a)
m.p.: 154–156 °C; IR (KBr): 3131 (CH, triazole), 3092
1
(CH, Ar), 1672 (C=O), 1593 (C=C) cm−1; H NMR (500

Med Chem Res
7-Bromo-4-((1-(4-butylphenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4e)
7.484 (d, J = 8.6 Hz, 2H, Ar), 7.192 (dd, J = 2.1, 6.5 Hz, 1H,
Ar), 7.143 (d, J = 2.1 Hz, 1H, Ar), 5.240 (s, 2H, N–CH2),
4.642 (s, 2H, O–CH2): 13C NMR (125 MHz, DMSO):
δ 163.6, 145.7, 143.6, 136.8, 131.2, 130.6, 130.3, 129.9,
127.9, 125.2, 124.8, 123.9, 122.1, 119.3, 117.3, 116.6, 114.8,
67.0, 36.2; ESI–MS (m/z): 454 [M+H]+. Anal. cal. for
C18H12BrF3N4O2: C, 47.70; H, 2.67; N, 12.36; found:
C, 47.64; H, 2.73; N, 12.27.
m.p.: 124–126 °C; IR (KBr): 3132 (CH, triazole), 3096 (CH,
Ar), 1669 (C=O), 1589 (C=C); 1H NMR (500 MHz,
CDCl3): δ 8.322 (s, 1H, triazole), 7.567–7.801 (m, 3H, Ar)
7.366–7.493 (m, 2H, Ar), 7.268–7.362 (m, 2H, Ar), 5.348
(s, 2H, N–CH2), 4.757 (s, 2H, O–CH2), 2.785 (t, J = 7.7 Hz,
2H, Ar–CH2–CH2–CH2–CH3), 1.653–1.808 (m, 2H,
Ar–CH2CH2–CH2–CH3), 1.404–1.562 (m, 2H, Ar–CH2–
CH2–CH2–CH3), 1.056 (t, J = 7.3 Hz, 3H, Ar–CH2–
CH2–CH2–CH3): 13C NMR (125 MHz, DMSO): δ 163.7,
145.7, 143.1, 134.3, 129.5, 127.9, 125.2, 121.6, 119.8, 119.2,
117.4, 114.8, 67.0, 36.2, 34.1, 32.8, 21.6, 13.6: ESI–MS (m/
z): 442 [M+H]+. Anal. cal. for C21H21BrN4O2: C, 57.15; H,
4.80; N, 12.70; found: C, 57.07; H, 4.84; N, 12.61.
7-Bromo-4-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4i)
m.p.: 187–189 °C; IR (KBr): 3130 (CH, triazole), 3095
1
(CH, Ar), 1672 (C=O), 1594 (C=C) cm−1; H NMR (500
MHz, CDCl3): δ 8.052 (s, 1H, triazole), 7.549–7.820
(m, 4H, Ar) 7.484 (d, J = 8.6 Hz, 1H, Ar), 7.091–7.251
(m, 2H, Ar), 5.213 (s, 2H, N–CH2), 4.652 (s, 2H,
O–CH2): 13C NMR (125 MHz, DMSO) δ163.7, 148.5,
145.8, 145.3, 141.9, 127.9, 125.7, 125.2, 124.4, 122.3,
119.3, 117.5, 115.1, 114.8, 110.1, 67.0, 56.9, 56.7,
36.3: ESI–MS (m/z): 465 [M+H]+. Anal. cal. for
C17H12Br2N4O2: C, 43.99; H, 2.61; N, 12.07; found:
C, 43.92; H, 2.57; N, 12.12.
7-Bromo-4-((1-(4-chloro-3,5-dimethoxyphenyl)-1H-1,2,3-
triazol-4-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4f)
m.p.: 221–223 °C; IR (KBr): 3137 (CH, triazole), 3097
1
(CH, Ar), 1681 (C=O), 1596 (C=C); H NMR (500 MHz,
CDCl3): δ 8.225 (s, 1H, triazole), 7.542 (d, J = 8.4 Hz, 1H,
Ar) 7.453 (brs, 1H, Ar), 7.062–7.248 (m, 3H, Ar), 5.241
(s, 2H, N–CH2), 4.630 (s, 2H, O–CH2), 3.904 (s, 3H,
–OCH3), 3.848(s, 3H, –OCH3): 13C NMR (125 MHz,
DMSO): δ 163.7, 148.5, 145.8, 145.3, 141.9, 127.9, 125.7,
125.2, 124.4, 122.3, 119.3, 117.5, 115.1, 114.8, 110.1,
67.0, 56.9, 56.7, 35.9: ESI–MS (m/z): 480 [M+H]+. Anal.
cal. for C19H16BrClN4O4: C, 47.57; H, 3.36; N, 11.68;
found: C, 47.66; H, 3.28; N, 11.61.
7-Bromo-4-((1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4j)
m.p.: 201–203 °C; IR (KBr): 3139 (CH, triazole), 3098
1
(CH, Ar), 1680 (C=O), 1590 (C=C) cm−1; H NMR (500
MHz, CDCl3): δ 7.928–8.324 (m, 1H-triazole and 2H-Ar,
3H), 7.526–7.806 (m, 6H, Ar), 7.200–7.419 (m, 2H, Ar),
5.420 (s, 2H, N–CH2), 4.743 (s, 2H, O–CH2):
13C NMR (125 MHz, DMSO): δ 163.7, 145.8, 142.3, 133.5,
133.0, 128.3, 128.0,127.9, 127.8, 127.0, 126.3, 123.8,
121.8, 119.3, 117.5, 114.8, 67.1, 36.2; ESI–MS (m/z): 436
[M+H]+. Anal. cal. for C21H15BrN4O2: C, 57.95; H, 3.47;
N, 12.87; found: C, 57.82; H, 3.38; N, 12.81.
7-Bromo-4-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4g)
m.p.: 161–163 °C; IR (KBr): 3128 (CH, triazole), 3083
1
(CH, Ar), 1678 (C=O), 1592 (C=C) cm−1; H NMR (500
MHz, CDCl3): δ 8.049 (s, 1H, triazole), 7.557–7.784
(m, 4H, Ar), 7.485 (d, J = 8.6 Hz, 1H, Ar), 7.110–7.242
(m, 2H, Ar), 5.220 (s, 2H, N–CH2), 4.638 (s, 2H, O–CH2):
13C NMR (125 MHz, DMSO): δ 163.7, 148.4, 145.7, 143.7,
137.0, 131.4, 127.8, 125.9, 125.2, 123.1, 122.2, 119.3,
117.3, 114.8, 114.6, 67.0, 36.2; ESI–MS (m/z): 404
[M+H]+. Anal. cal. for C17H12BrFN4O2: C, 50.64; H, 3.00;
N, 13.90; found: C, 50.57; H, 3.13; N, 13.82.
7-Bromo-4-((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4k)
m.p.: 177–181 °C; IR (KBr): 3122 (CH, triazole), 3086
(CH, Ar), 1671 (C=O), 1595 (C=C) cm−1; 1H NMR (500
MHz, CDCl3):
δ 8.591 (s, 1H, triazole), 8.314
(d, J = 7.7 Hz, 1H, Ar), 8.086–8.258 (m, 2H, Ar), 7.751
(t, J = 8.1 Hz, 1H, Ar), 7.470 (d, J = 8.4 Hz, 1H, Ar),
7.089–7.235 (m, 2H, Ar), 5.253 (s, 2H, N–CH2), 4.651
(s, 2H, O–CH2): 13C NMR (125 MHz, DMSO): δ 163.7,
148.4, 145.7, 143.7, 137.0, 131.4, 127.8, 125.9,
125.2, 123.1, 122.2, 119.3, 117.3, 114.8, 114.6, 67.0,
36.2: ESI–MS (m/z): 431 [M+H]+. Anal. cal. for
C17H12BrN5O4: C, 47.46; H, 2.81; N, 16.28; found:
C, 47.53; H, 2.73; N, 16.35.
7-Bromo-4-((1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-
4-yl)methyl)-2H-benzo[b][1,4] oxazin-3(4H)-one (4h)
m.p.: 173–175 °C; IR (KBr): 3130 (CH, triazole), 3093
1
(CH, Ar), 1675 (C=O), 1589 (C=C) cm−1; H NMR (500
MHz, CDCl3): δ 8.125 (s, 1H, triazole), 7.999 (s, 1H, Ar),
7.943 (d, J = 7.9 Hz, 1H, Ar), 7.643–7.740 (m, 2H, Ar),

Med Chem Res
General procedure for the preparation of 5a–5g
MHz, CDCl3) δ 8.194(s, 1H, triazole), 7.589–7.935
(m, 2H), 7.395–7.684 (m, 4H), 7.208–7.364 (m, 2H), 7.006
(d, J = 8.0 Hz, 2H), 5.267 (s, 2H, N–CH2), 4.655 (s, 2H,
O–CH2), 3.858 (s, 3H, O–CH3); 13C NMR (125 MHz,
DMSO): δ 163.7, 160.1, 145.8, 143.6, 136.7, 133.5, 131.4,
130.2, 127.8, 125.3, 123.7, 122.3, 119.5, 117.6, 116.8,
114.9, 114.8, 67.0, 55.6, 36.2; ESI–MS (m/z): 482 [M+H]+.
Anal. cal. for C24H18Cl2N4O3: C, 59.89; H, 3.77; N, 11.64;
found: C, 59.96; H, 3.82; N, 11.57.
A stirred solution of compound 4a (0.5 g, 0.92 mmol),
arylboronic acid (1.4 mmol) and sodium carbonate
(2.7 mmol) in DMF-H2O (10 mL) was purged with argon
gas for 30 min. Then, tetrakis(triphenylphosphine)palladium
(5 mol %) was added to the reaction mixture under argon
gas and the mixture was stirred for 24 h at 120 °C. After
completion of the reaction, the reaction mixture was filtered
through a celite pad and washed with ethylacetate (20 mL).
The organic layer was separated, washed with brine, dried
over anhydrous Na2SO4 and the solvent was removed under
reduced pressure to afford crude product. The latter was
purified by column chromatography using silica gel
(100–200 mesh) and ethyl acetate–hexane gradient
(30–40 %) to afford the pure desired product.
7-(4-Chlorophenyl)-4-((1-(3,5-dichlorophenyl)-1H-1,2,3-
triazol-4-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (5d)
m.p.: 207–209 °C; IR (KBr): 3141 (CH, triazole), 3075 (CH,
1
Ar), 1678 (C=O), 1591 (C=C) cm−1; H NMR (500 MHz,
CDCl3)
δ
8.284 (s, 1H, triazole), 7.882–8.015
(m, 3H, Ar), 7.104–7.283 (m, 4H, Ar), 7.201–7.395 (m, 3H,
Ar), 5.402 (s, 2H, N–CH2), 4.703 (s, 2H, O–CH2);
13C NMR (125 MHz, DMSO): 163.6, 145.6, 143.6, 139.1,
136.7, 133.4, 131.4, 129.8, 129.0, 127.8, 125.3, 123.8, 122.1,
119.2, 117.4, 116.7, 114.8, 67.1, 36.3; ESI–MS
(m/z): 486 [M+H]+. Anal. cal. for C23H15Cl3N4O2: C, 56.87;
H, 3.11; N, 11.53; found: C, 56.79; H, 3.06; N, 11.58.
4-(4-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)
benzamide (5a)
m.p.: 241–243 °C; IR (KBr): 3360(–NH), 3210(–NH),
3130 (CH, triazole), 3088 (CH, Ar), 1680 (C=O), 1594
1
(C=C) cm−1; H NMR (500 MHz, CDCl3) δ 8.049 (s, 1H,
triazole), 7.872 (d, J = 7.9 Hz, 2H, Ar), 7.575–7.716
(m, 4H, Ar), 7.237–7.343 (m, 4H, Ar), 6.168 (brs, 1H, –NH),
5.794 (brs, 1H, –NH), 5.297 (s, 2H, N–CH2), 4.690 (s, 2H,
O–CH2); 13C NMR (125 MHz, DMSO): δ 169.1, 163.6,
145.7, 145.5, 143.6, 136.8, 133.5, 131.2, 130.4, 128.3, 127.9,
125.2, 123.9, 122.1, 119.3, 117.3, 116.6, 114.8, 67.0, 36.2;
ESI–MS (m/z): 495 [M+H]+. Anal. cal. for C24H17Cl2N5O3:
C, 58.31; H, 3.47 N, 14.17; found: C, 58.36; H, 3.42 N, 14.24.
4-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
(4-fluorophenyl)-2H-benzo[b]1,4]oxazin-3(4H)-one (5e)
m.p.: 189–191 °C; IR (KBr): 3153 (CH, triazole), 3080
(CH, Ar), 1681 (C=O), 1593 (C=C) cm−1; 1H NMR
(500 MHz, CDCl3) δ 8.197 (s, 1H, triazole), 7.912–8.043
(m, 3H, Ar), 7.412–7.743 (m, 4H, Ar), 7.203–7.384 (m, 3H,
Ar), 5.385 (s, 2H, N–CH2), 4.663 (s, 2H, O–CH2);
13C NMR (125 MHz, DMSO): 163.6, 161.9, 145.6, 143.6,
136.5, 136.7, 131.4, 129.3, 129.1, 127.8, 125.3, 123.8,
122.6, 122.5, 122.1, 119.2, 117.4, 116.7, 116.6, 114.8,
67.1, 36.3; ESI–MS (m/z): 470 [M+H]+. Anal. cal. for
C23H15Cl2FN4O2: C, 58.86; H, 3.22; N, 11.94; found:
C, 58.79; H, 3.29; N, 11.88.
4-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
(3-methoxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (5b)
m.p.: 197–199 °C; IR (KBr): 3122 (CH, triazole), 3075
1
(CH, Ar), 1684 (C=O), 1594 (C=C) cm−1; H NMR (500
MHz, CDCl3) δ 8.146 (s, 1H, triazole), 7.523–7.823
(m, 5H, Ar), 7.352–7.480 (m, 3H, Ar), 7.258 (s, 1H, Ar),
7.154 (d, J = 2.2 Hz, 1H, Ar), 5.302 (s, 2H, N–CH2), 4.653
(s, 2H, O–CH2), 3.884 (s, 3H, O–CH3); 13C NMR (125
MHz, DMSO): 163.7, 158.3, 146.8, 145.6, 143.6, 136.9,
131.3, 130.4, 127.8, 125.3, 123.8, 122.2, 120.4, 119.2, 117.4,
116.5, 114.8, 113.9, 113.4 67.0, 55.6, 36.2 ; ESI–MS (m/z):
482 [M+H]+. Anal. cal. for C24H18Cl2N4O3: C, 59.89;
H, 3.77; N, 11.64; found: C, 59.93; H, 3.72; N, 11.69.
4-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
(naphthalen-1-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (5f)
m.p.: 247–249 °C; IR (KBr): 3140 (CH, triazole), 3078(CH,
1
Ar), 1679 (C=O), 1590 (C=C) cm−1; H NMR (500 MHz,
CDCl3) δ 8.324 (s, 1H, triazole), 7.862–8.096 (m, 4H, Ar),
7.602–7.751 (m, 3H, Ar), 7.334–7.502 (m, 4H, Ar),
7.201–7.286(m, 2H, Ar), 5.357 (s, 2H, N–CH2), 4.723
(s, 2H, O–CH2); 13C NMR (125 MHz, DMSO): 163.6,
145.7, 143.6, 136.9, 136.8, 134.5, 133.2, 131.2, 128.4,
127.9, 126.4,127.5, 125.6, 125.3, 125.2, 123.9, 122.1,
119.3, 117.3, 116.6, 114.8, 67.0, 36.2; ESI–MS (m/z): 502
[M+H]+. Anal. cal. for C27H18Cl2N4O2: C, 64.68; H, 3.62;
N, 11.17; found: C, 64.61; H, 3.68; N, 11.24.
4-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
(4-methoxyphenyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (5c)
m.p.: 221–223 °C; IR (KBr): 3144 (CH, triazole), 3082
1
(CH, Ar), 1671 (C=O), 1592 (C=C) cm−1; H NMR (500

Med Chem Res
4-((1-(3,5-Dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-
In vivo anticancer bioassay
phenyl-2H-benzo[b][1,4]oxazin-3(4H)-one (5g)
Adult female Swiss albino mice (Mahaveer Enterprises,
Hyderabad, India) 8 weeks old (mean weight in the range of
20–25 g) were selected and housed in polypropylene cages
in a room, where the congenial temperature of 27 1 °C
and 12 h light and dark cycles were maintained. The ani-
mals were allowed to acclimatize to the environment for
7 days, and supplied with a standard pellet diet and water ad
libitum. All procedures for using animals were reviewed
and approved by the Institutional Animal Ethical Commit-
tee of Kakatiya University (IAEC/28/UCPSc/KU/2015).
The animals were divided into seven groups (n = 10). The
normal group was not inoculated with tumor cells, while
six groups were injected with EAC cells (0.2 mL of
2 × 106 cells per mice) intraperitoneally. This was taken as
day ‘0’, and the experimental treatment started 24 h later.
From the first day, 100 μL per mouse per day of sterile
saline was administered intraperitoneally to the negative
control group (EAC-bearing mice). The compounds 5b and
5c at doses of 5 and 10 mg kg−1 were administered each day
to the treated groups, and the standard drug cisplatin at a
dose of 5 mg kg−1 was administered to each animal from the
positive control group. The pharmacological treatment las-
ted for 9 days. Fourteen days after the treatment, five mice
from each group were killed for the study of antitumor
activity. The rest of the animal groups were kept to check
the mean survival time (MST) of EAC tumor-bearing hosts.
The antitumor effects of the compounds were determined by
the change in body weight, MST and percentage increased
life span (% ILS). The MST of each group containing five
mice was identified by recording the mortality on a daily
basis for 30 days, and the % ILS was calculated using the
following equations MST = (day of the first death + day of
the last death)/2; and ILS (%) = ((MST of treated group/
MST of control group)−1) x 100. The effect of compounds
5b and 5c was also assessed by the determination of
the body weight, tumor volume, packed cell volume and
viable tumor cell count of EAC-bearing mice by the trypan
blue incorporation method.
m.p.: 202–204 °C; IR (KBr): 3122 (CH, triazole), 3086
1
(CH, Ar), 1724 (C=O), 1595 (C=C) cm−1; H NMR (500
MHz, CDCl3) δ 8.056 (s, 1H, triazole), 7.858–8.016
(m, 3H, Ar), 7.423–7.671 (m, 4H, Ar), 7.081–7.382 (m, 3H,
Ar), 6.782 (d, J = 8.0 Hz, 1H, Ar), 5.357 (s, 2H, N–CH2),
4.723 (s, 2H, O–CH2); 13C NMR (125 MHz, DMSO):
163.6, 145.6, 143.6, 140.9, 136.8, 129.7, 129.2, 129.0,
131.3, 127.9, 125.2, 123.8, 122.1, 119.2, 117.3, 116.6,
114.9, 67.1, 36.3; ESI–MS (m/z): 451 [M+H]+. Anal. cal.
for C23H16Cl2N4O2: C, 61.21; H, 3.57; N, 12.41; found:
C, 61.28; H, 3.50; N, 12.47.
In vitro anticancer bioassay
All the synthesized compounds were evaluated for their
in vitro cytotoxic activity against two different cancer cell
lines such as breast cancer cell line (MCF-7) and cervical
carcinoma cell line (HeLa). The human cancer cell lines
MCF-7 (breast cancer) and HeLa (cervical) were obtained
from National Centre for Cell Sciences, Pune, India. Cell
viability in the presence of the test samples was measured
by the MTT-microcultured tetrazolium assay. This assay
is a quantitative colorimetric method for the determina-
tion of cell cytotoxicity. The assessed parameter is the
metabolic activity of viable cells. Metabolically active
cells reduce pale yellow tetrazolium salt (MTT) to a dark-
blue water-insoluble formazan, which can be directly
quantified after solubilization with DMSO. The absor-
bance of the formazan directly correlates with the number
of viable cells. MCF-7 and HeLa cells were plated into a
96-well plate at a density of 1 × 104 cells per well. Cells
were grown overnight in the full medium and then
switched to the low-serum media. DMSO was used as a
control. After 48 h of treatment with different con-
centrations of test compounds, the cells were incubated
with MTT (2.5 mg mL−1) in the CO2 chamber for 2 h. The
medium was then removed and 100 μL of DMSO was
added into each well to dissolve formazan crystals. After
thoroughly mixing, the plates were read at 570 nm for
optical density, which is directly correlated with cell
quantity. The results were represented as percentage of
cytotoxicity/viability. All the experiments were carried
out in triplicate. The IC50 values were calculated from the
percentage of cytotoxicity and compared with the refer-
ence drug cisplatin. The relationship between surviving
fraction and drug concentration was plotted to obtain the
survival curve of MCF-7 and HeLa. The response para-
meter calculated was the IC50 value, which corresponds
to the concentration required for 50 % inhibition of cell
viability.
In silico anticancer bioassay
To explore and support the anticancer mechanism, docking
studies for the synthesized compounds 4a–4k and 5a–5g
were performed. This drug designing tool is helpful to
investigate and to gain a deep insight into the mode of
binding interactions of each of these ligands with the
receptor sites of insulin-like growth factor-binding proteins
(IGFBPs), and also to determine the best in silico con-
formation. Docking of the synthesized ligands was
employed by using Lamarckian Genetic Algorithm (LGA;
Morris et al., 1998) inculcated in the docking program

Med Chem Res
Scheme 1 Reagents and
reaction conditions: (i) 2-
Chloroacetyl chloride, NaHCO3,
TEBA, THF, reflux, 12 h;
(ii) propargyl bromide, Cs2CO3,
acetone, RT, 1 h; (iii) aryl azide,
CuI, THF, RT, 8–10 h
Scheme 2 Reagents and reac-
tion conditions: (i) ArB(OH)2
/Pd(PPh3)4, Na2CO3, DMF-
H2O, 120oC, 24 h
AutoDock 4.2. The co-crystallized structure of target
enzyme IGFBPs (PDB id: 2DSQ) was obtained from Pro-
tein Data Bank (RCSB) (http://www.rcsb.org/pdb). To carry
out in silico studies, the 2D structures of the synthesized
ligands (4a–4k and 5a–5g) were drawn in ChemBioOffice
2012 and converted to energy minimized 3D structures in
pdb file format using MarvinSketch (ChemAxon). The
target protein file was prepared by leaving only the residues
associated with protein by using Discovery Studio 4.0
Visualizer. AutoDock 1.5.6 (MGL tools-1.5.6) tool was
used to prepare target protein file that involves, assigning
AD4 type atoms, calculating Gasteiger charges for every
atom of the macromolecule, and addition of polar hydro-
gen’s to the macromolecule, an essential step to correct the
calculation of partial charge by keeping all other values as
default. The binding site of protein identification was car-
ried out using CastP (serversts-fw.bioengr.uic.edu/castp/
calculation.php). Docking simulations for the compounds
4a–4k and 5a–5g were performed against the active site of
IGFBPs enzyme. Then finally docking results were visua-
lized using Maestro elements tutorial 1.8.
the presence of NaHCO3 in dry THF afforded the corre-
sponding 7-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one (2) in
good yield. The target intermediate 7-bromo-4-(prop-2-yn-1-
yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (3) was prepared by
treating 7-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one with 3-
bromoprop-1-yne in the presence of Cs2CO3 in acetone
(Haider et al., 2013). The key step of the synthesis, i.e., 1,3-
dipolar cyclo addition of terminal alkyne 3 with different
aryl azides using a catalytic amount of copper iodide at room
temperature, afforded the corresponding 1,4-di-substituted
1,2,3-triazoles (4a–4k) in good to excellent yields ranging
from 65 to 88% (Himo et al., 2005; Scheme 1; Fig. 4).
The structures of all the synthesized compounds (4a–4k)
were established with the aid of their spectral (IR, NMR and
mass) and elemental (C, H and N) analysis. Analytical and
spectral data of all the synthesized compounds were in full
agreement with the proposed structures and also discussed
for a representative compound 4c. From the IR spectrum,
the appearance of a broad absorption band at 3136 cm−1
(C–H, triazole) and sharp bands at 1680 cm−1 (C=O) and
1587 cm−1 (C=C) was indicative of the formation of the
1
title compound. From the H NMR spectrum, the appear-
ance of proton signals at
δ 7.965 (CH, triazole),
Result and Discussion
Chemistry
7.002–7.599 (m, Ar–H), 5.220 (N–CH2), 4.631 (O–CH2)
and 3.859 ppm (O–CH3 protons), and from the 13C NMR
spectrum, the presence of signals at 163.7 ppm (–C=O
carbon), 159.2 ppm (C–OCH3 carbon), 55.5 ppm
(Ar–OCH3 carbon) and 36.2 ppm (N–CH2 carbon) con-
firmed the formation of title compound. The M+2 ion peak
(416) from the mass spectrum as well as elemental analyses
data [C, 52.06; H, 3.64; N, 13.49] further confirmed the
The synthesis of compounds (4a–4k and 5a–5g) was
accomplished by a synthetic sequence shown in Scheme 1
and Scheme 2. Scheme 1 and Scheme 2 The cyclization of 5-
bromo-2-aminophenol (1) with 2-chloroacetyl chloride in

Med Chem Res
Fig. 4 Docking pose of 5b with Insulin-like growth factor-binding proteins (IGFBPs) with 2D, 3D interactions and 3D surface interaction
(represented in molecular cloud) with the enzyme
formation of the title compound. The compounds 5a–5g
were synthesized by using C–C bond coupling of 4a with
different arylboronic acids in the presence of palladium
catalyst as shown in Scheme 2 (Cornelia et al., 2015).
From the IR spectrum of compound 5a, the appearance
of broad absorption bands in the region 3360–3210 cm−1
(CO–NH2) and sharp absorption bands at 3130 cm−1
(CH, triazole), 1680 cm−1 (C=O) and 1594 cm−1 (C=C)
was indicative of the formation of the title compound. From
title compound. Similarly, the M+H ion peak (495) from the
mass spectrum and elemental analysis data [C, 58.36; H,
3.42 N, 14.24] confirmed the formation of the pure com-
pound 5a.
In vitro cytotoxic activity
All the newly synthesized compounds (4a–4k and 5a–5g)
were evaluated for in vitro cytotoxic activity against the two
human cancer cell lines, such as cervical carcinoma cell line
(HeLa) and breast cancer cell line (MCF-7), according to
the MTT assay method (Shekan et al., 1990; Monks et al.,
1991). Cisplatin was used as a reference drug and the results
1
the H NMR spectrum, the appearance of proton signals at
δ 8.049 (CH, triazole), 7.237–7.872 (Ar–H), 6.168 (brs, 1H,
–NH), 5.794 (brs, 1H, –NH), 5.297 (s, 2H, N–CH2) and
4.690 (s, 2H, O–CH2) was the evidence for the formation of

Med Chem Res
are presented in Tables 1 and 2. From Table 1, it is clear that
some of the compounds showed good activity against tumor
cell lines. Among all the synthesized compounds 4a, 4b
and 4f showed the highest cytotoxicity towards both
the cancer cell lines. The potential activity of these com-
pounds (4a, 4b and 4f) may be attributed due to the
presence of 3,5-disubstituted and 3,4,5-trisubstitued phenyl
groups on the triazole ring (both electron withdrawing and
electron releasing groups). The rest of the compounds
exhibited moderate to poor cytotoxic activity against the
two cancer cell lines.
The presence of mono substituted groups on the triazole
ring did not influence the cytotoxic activity much. The
better activity of compound 4a has encouraged us to syn-
thesize the compounds (5a–5g) by replacing bromine with
different aryl groups using palladium catalyzed Suzuki
coupling reaction. The compounds (5a–5g) were also
screened for their in vitro cytotoxic activity against MCF-7
and HeLa and the results are presented in Table 2.
From Table 2, it is clear that compound 5b, which con-
tains 3-methoxyphenyl group on the seventh position of
1,4-benzoxazine, exhibited potent activity against both
MCF-7 and HeLa cell lines with IC50 values 17.08 0.8
and 15.38 0.5 μg, respectively. These results are comparable
to the standard drug, cisplatin. Similarly, the introduction of
4-methoxyphenyl group on the 1,4-benzoxazine (5c) sig-
nificantly enhanced the cytotoxicity against the two cancer
cell lines with IC50 values 21.78 1.1 and 19.46 0.8 μg,
respectively. The compounds derived from benzamide,
4-chloro phenyl and 4-fluoro phenyl groups on the 1,
4-benzoxazine moiety (5a, 5d and 5e) showed good cytotoxic
activity against the two cancer cell lines with IC50 values
ranging from 22.28 1.2 to 29.16 1.1 μg, respectively.
Remaining compounds 5f and 5g showed moderate activity.
From these results, it can be concluded that the presence of
methoxyphenyl group on the 1,4-benzoxazine ring enhanced
anticancer activity. It is important to mention that on overall
comparison of data from Tables 1 and 2, the 7-aryl groups
enhanced the cytotoxic activity.
Table 1 Cytotoxic activity of 7-bromo-4-((1-aryl-1H-1,2,3-triazol-4-
yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (4a–4k) on human
cancer cell lines (in vitroa (IC50 μg mL−1))
Product
MCF-7
HeLa
4a
47.18 1.5
52.28 1.0
67.08 1.1
94.10 1.8
117.98 2.0
54.13 1.2
132.5 1.7
55.72 1.5
154.5 2.4
117.16 1.4
146.17 1.7
4.68 0.2
43.93 1.1
57.11 1.2
69.89 1.5
83.94 1.5
109.19 1.9
58.14 1.8
124.10 1.2
61.10 1.0
128.7 1.6
104.16 1.2
138.14 1.9
3.87 0.1
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
Cisplatin
a
Values are expressed as mean SEM
Table 2 Cytotoxic activity of 7-aryl-4-((1-(3,5-dichlorophenyl)-1H-
1,2,3-triazol-4-yl)methyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (5a–5g)
on human cancer cell lines (in vitroa (IC50 μg mL−1))
Product
MCF-7
HeLa
5a
22.28 1.2
17.08 0.8
21.78 1.1
27.18 1.0
29.16 1.1
34.98 0.7
42.02 1.2
4.68 0.2
24.10 1.0
15.38 0.5
19.46 0.8
25.93 0.7
26.11 1.0
36.19 1.1
38.87 1.0
3.87 0.1
5b
5c
In vivo cytotoxic activity
5d
5e
The potent cytotoxic compounds 5b and 5c were evaluated
for in vivo studies in EAC-bearing mice by using the liquid
tumor model (Seruga et al., 2000; Shimura et al., 2000;
Hazxa et al., 2002; Rajanarendar et al., 2012). The effect
of the compounds 5b and 5c in two different doses
5f
5g
Cisplatin
a
Values are expressed as mean SEM
Table 3 In vivo anticancer activity of 1,2,3-triazole derivatives 5b and 5c on EAC-bearing micea
Parameter
EAC control
(5 × 106 cells)
Cisplatin
5b
5b
5c
5c
(5 mg kg−1
)
(5 mg kg−1
)
(10 mg kg−1
)
(5 mg kg−1
)
(10 mg kg−1
)
Increased body weight (g)
Mean survival time (days)
Increased in life span (% ILS)
Tumor volume (mL)
13.4 0.6
13.34 0.7
—
3.83 0.28
28.7 0.5
116.30
11.22 0.42
14.2 0.46
10.28
9.2 0.82
18.8 0.52
35.42
8.54 0.32
16.3 0.82
28.53
7.02 0.36
22 0.56
68.94
11.23 0.85
2.98 0.6
7.7 0.67
3.82 0. 20
0.39 0.1
0.93 0.3
10.8 0.5
2.4 0.8
8.84 0.4
1.76 0.6
5.16 0.35
8.03 0.37
1.93 0.4
4.92 0.12
5.18 0.45
1.5 0.8
3.47 0.81
Packed cell volume (mL)
Viable tumor cell count (× 107 cells mL−1
a
)
6.98 0.72
All values are expressed as mean SEM

Med Chem Res
Table 4 Effect of 1,2,3-triazole derivatives 5b and 5c on biochemical and hematological parameters in EAC-bearing micea
Treatment
Parameters
Hemoglobin
(mg %)
RBC
WBC
Lymphocytes
(%)
Neutrophils
(%)
Monocytes
(%)
(million mm−3
)
(103 cellsmm−3
)
Normal
12.9 0.06
5.77 0.52
10.87 0.92
5.96 0.63
7.02 0.79
8.08 0.86
9.30 1.10
5.13 0.18
2.72 0.1
5.10 0.8
3.08 0.06
3.30 0.15
3.12 0.06
3.88 0.08
7.67 0.56
22.13 0.45
9.6 0.53
67 0.97
26 0.6
30 1.56
78.3 1.31
32 0.37
65 0.48
51 0.84
54 0.85
39 0.97
1.8 0.67
3.25 0.4
1.67 0.26
2.33 0.46
1.96 0.53
2.09 0.36
1.82 0.18
Tumor control
Cisplatin (5 mg kg−1
5b (5 mg kg−1
5b (10 mg kg−1
5c (5 mg kg−1
5c (10 mg kg−1
)
67.5 0.83
28 0.58
40 0.85
31 0.93
49 0.75
)
19.9 0.34
18.80 0.29
18.48 0.35
13.92 0.47
)
)
)
aAll values are expressed as mean SEM
Table 5 Autodock binding
energies, no. of hydrogen bonds
and residues involved in
hydrogen bonding interaction of
ligands for IGFBPs (PDB id:
2DSQ)
S. no. Compound Binding
energy
Inhibition
constant
) (microM-nanoM) bonds
No. of
Residues involved
hydrogen in hydrogen bonding
(kcal mol−1
1
4a
4b
4c
4d
4e
4f
−8.03
1.30 microM
1.75 microM
5.98 microM
1.32 microM
2.30 microM
4.73 microM
3.06 microM
7.56 microM
2.15 microM
113.28 nM
716.97 nM
84.96 nM
1
2
3
1
2
2
3
1
1
2
2
4
3
2
4
2
3
1
TYR207
2
−7.86
−7.13
−8.02
−7.69
−7.27
−7.52
−6.99
−7.73
−9.48
−8.38
−9.65
−10.22
−9.55
−8.49
−9.61
−10.01
−9.86
PHE230, SER189
3
TYR229, PHE230, ASN225
GLY22
4
5
ASN225, CYS226
HIS5, PHE25
6
7
4g
4h
4i
ARG190,SER189,PHE230
PHE25
8
9
ASN225
10
11
11
12
13
14
15
16
17
4j
ARG28, TYR207
4k
5a
5b
5c
5d
5e
5f
PHE25, GLU2
ASP20, ARG190, HIS188, SER189
TYR207, ARG52, ARG58
LYS183, ASN182
CYC226, ARG190, HIS188, SER189
ASN182, LYS183
ARG190, CYS226, PHE230
TYR207
32.27 nM
100.73 nM
602.24 nM
90.20 nM
45.91 nM
5g
58.75 nM
(5 and 10 mg kg−1) on body weight, MST, % increase
lifespan, tumor volume, packed cell volume and viable tumor
cell count were studied. From Table 3, it is evident that
compound 5c has shown significant activity in both the
doses, and decreased the body weight of EAC-bearing mice,
whereas the compound 5b has exhibited significant activity
only at 10 mg kg−1. Compound 5c significantly increased the
MST, and decreased the tumor volume, packed cell volume
and viable cell count in both the doses, (5 and 10 mg kg−1),
whereas 5b only in higher dose (10 mg kg−1) has shown
similar results. On day 14, the biochemical and hematolo-
gical parameters, with regard to hemoglobin level, ery-
throcytes and leukocytes counts were compared with EAC
control groups, standard drug cisplatin treated groups, and
the groups injected with the compounds 5b and 5c. As shown
in Table 4, the biochemical and hematological parameters, in
the group treated with the compound 5c, have been recovered
completely to the normal values. These results could indicate
macrophage activation and inhibition of vascular perme-
ability with the comparison of tumor control. The compounds
5b and 5c increased the hemoglobin and red blood cell
levels. Similarly, 5b and 5c decreased the white blood cell
levels, when compared with tumor control. The treatment
with 5b and 5c brought back the differential leukocyte count
more or less to the normal levels. This indicates that these
tested compounds possess protective action on hemopoietic
system. These results suggest that compound 5c is more
active in in vivo anticancer studies.

Med Chem Res
Fig. 5 Docking pose of 5f with Insulin-like growth factor-binding proteins (IGFBPs) with 2D, 3D interactions and 3D surface interaction
(represented in molecular cloud) with the enzyme
In silico anticancer bioassay
atom and remaining hydrophobic interactions through
π–π stacking with aromatic rings to amino acids. Simi-
larly, compound 5f has three hydrogen bonding interac-
tion with ARG190, CYS226, PHE230 amino acids and
remaining hydrophobic interaction through π–π stacking
with aromatic ring to amino acid. The best docked
orientations of synthesized ligands were shown in Figs. 4
and 5. The binding energies, inhibition constants and
hydrogen bond interactions of all the compounds were
tabulated in Table 5. These results revealed a variety of
binding modes that may provide a sufficient explanation
between docking scores and in vitro results of anticancer
activity.
All inhibitors were compared out of ten docking runs. The
docking studies revealed that all the synthesized mole-
cules exhibited good to excellent binding energies
towards the receptor active pocket ranging from −6.99 to
−10.22 kcal mol−1 (Table 5). Among all the compounds,
5b and 5f were energetically favored for IGFBPs
active site, and are exhibiting bonds with amino acids of
active pocket of the receptor and considered as the best
docking poses. Interactions of 5b has three hydrogen
bonding interaction with TYR207, ARG52, ARG58
amino acids by two oxygen-atoms, one triazole nitrogen

Med Chem Res
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Conclusion
In conclusion, we report the synthesis of novel 1,2,3-triazoles-
2H-benzo[b][1,4]oxazin-3(4H)-one hybrids using Cu(I) cata-
lyzed [3 + 2] cycloaddition reaction followed by Suzuki
reaction. All the newly synthesized compounds were evaluated
for in vitro anticancer activity against the two human cancer
cell lines MCF-7 and HeLa. The compounds 5b and 5c have
shown potent activity against the two cancer cell lines. The
compounds 5b and 5c were further evaluated for their in vivo
anticancer activity against EAC-bearing mice. Compound 5c
proved as a potential anticancer agent as compared with the
reference drug. The docking studies revealed that all the
synthesized compounds have shown moderate to good binding
energies. By effecting a simple modification in structure, it can
be considered as a future drug candidate for cancer therapy.
Acknowledgments Financial assistance from the UGC-MRP (F.No.
42-372/2013), New Delhi, India, is greatly acknowledged. Narsimha
and Satheesh are thankful to the Council of Scientific and Industrial
Research-University Grant Commission (CSIR-UGC, New Delhi) for
the award of senior research fellowship.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interests.
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