Synthesis of 2-Aryl-4H-3,1-Benzoxazin-4-ones: A Class of α-Chymotrypsin Inhibitors

Article abstract of DOI:10.14233/ajchem.2014.16108

Twenty one derivatives of 2-aryl-4H-3,1-benzoxazin-4-one were synthesized and their potential therapeutically significance and structureactivity relationship were tested against α-chymotrypsin. Majority of synthesized compounds showed significant in vitro α-chymotrypsin inhibitory properties having IC₅₀values in the range of 5.42 ± 1.66 - 41.27 ± 1.33 μM, whereas standard inhibitor chymostatin have IC50 value 7.13 ± 1.06 μM. In the present series compounds 2-(2-fluorophenyl)-4H-3,1-benzoxazin-4-one (3h), 2-(2-bromophenyl)-4H-3,1- benzoxazin-4-one (3n) and 2-(1-naphthyl)-4H-3, 1-benzoxazin-4-one (3t) with IC₅₀values 7.22 ± 0.75, 6.99 ± 0.29 and 5.42 ± 1.66 μM, respectively were found to be most active members of series, even better than standard inhibitor a-chymostatin.

Full text of DOI:10.14233/ajchem.2014.16108

Asian Journal of Chemistry; Vol. 26, No. 15 (2014), 4561-4565
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.16108
Synthesis of 2-Aryl-4H-3,1-Benzoxazin-4-ones: A Class of α-Chymotrypsin Inhibitors
1,*
1
2
1
1
ZULFIQAR ALI KHAN , NOSHABA AFZAL , ZAIB HUSSAIN , SYED ALI RAZA NAQVI , AYESHA BARI ,
3
4
5
1
SOHAIL ANJUM SHAHZAD , MUHAMMAD YAR , NASIR MAHMOOD , SHAZIA ANWER BUKHARI ,
1
1
3
1
ASIM MANSHA , AMEER FAWAD ZAHOOR , ABDUR RAHMAN KHAN and MATLOOB AHMAD
1Department of Chemistry, Government College University, Faisalabad-38000, Pakistan
2Institute of Chemistry, University of the Punjab, Lahore-54000, Pakistan
3Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan
4Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore-54000, Pakistan
5Department of Allied Health Sciences and Chemical Pathology, University of Health Sciences, Lahore-54000, Pakistan
*Corresponding author: Tel: +92 3326243829; E-mail: zulfiqarchem@gmail.com
Received: 27 July 2013;
Accepted: 3 February 2014;
Published online: 16 July 2014;
AJC-15525
Twenty one derivatives of 2-aryl-4H-3,1-benzoxazin-4-one were synthesized and their potential therapeutically significance and structure-
activity relationship were tested against α-chymotrypsin. Majority of synthesized compounds showed significant in vitro α-chymotrypsin
inhibitory properties having IC50 values in the range of 5.42 1.66 – 41.27 1.33 µM, whereas standard inhibitor chymostatin have IC50
value 7.13 1.06 µM. In the present series compounds 2-(2-fluorophenyl)-4H-3,1-benzoxazin-4-one (3h), 2-(2-bromophenyl)-4H-3,1-
benzoxazin-4-one (3n) and 2-(1-naphthyl)-4H-3, 1-benzoxazin-4-one (3t) with IC50 values 7.22 0.75, 6.99 0.29 and 5.42 1.66 µM,
respectively were found to be most active members of series, even better than standard inhibitor α-chymostatin.
Keywords: 2-Aryl-4H-3,1-benzoxazin-4-ones, in vitro α-chymotrypsin inhibitory activity, Structure-activity relationship.
known to be essential for viral replication has become target
for anti-HCV drugs9. So search for new effective inhibitors of
serine proteases is an urgent need for the drug development.
In the recent past we explored many classes of compounds for
their potential use in medicinal chemistry as well as their new
INTRODUCTION
Enzyme inhibition is an essential area of chemical biology
since studies in this field have already led to the discovery of
ample variety of drugs helpful in curing a number of diseases.
Specific inhibitors act together with enzymes and block their
activity towards their subsequent natural substrates. The
treatment of a number of physiological conditions by enzyme
inhibitor molecules reflects their importance as drugs1.Although
the physiological role of serine proteases inhibitors have clearly
been established. It has been considered that they are ingredient
of plant's natural defence system against insect predation and
function by inhibiting insect proteinases2-5. Hence these inhi-
bitors have gained concern as apparent sources of engineered
resistance against pests and pathogens for transgenic plants
expressing heterologous inhibitors3-6. Tobacco plants trans-
formed with gene coding serine protease inhibitor has been
revealed to posses' insect pest resistance6. Bowman-Birk inhi-
bitors have also been shown to be competent tumor suppressor
agents in vivo and in vitro7. Serine proteases such as chymo-
trypsin and trypsin are involved in the devastation of certain
fibrous proteins8. Chronic infection by hepatitis C virus can
lead to the progressive liver injury, cirrhosis and liver cancer.
A chymotrypsin like serine protease known NS3 protease is
synthetic methodology developments10-16
During our preliminary studies on benzoxazinones, it has
been observed that they have property to inhibit chymotrypsin17,18
.
.
A variety of biological activities has been associated with 2-
substituted benzoxazin-4-ones such as antifungal, antibac-
terial19-24 and antielastase properties25. They can be used for
the treatment of obesity26 and also found to be novel specific
puromycin-sensitive aminopeptidase inhibitors27. Recently this
class of compounds reported to show a strong dual inhibitory
effect on human neutrophil elastase release28.
The promising therapeutic potential of this class of comp-
ounds prompted us to devise, synthesize and biologically screen
a series of aryl structural variants of 2-substituted-4H-3,1-
benzoxazin-4-one against α-chymotrypsin.
EXPERIMENTAL
IR spectra (KBr discs or MeOH) were recorded on a Bruker
FT-IR IFS48 spectrophotometer. EI mass spectra data were
recorded with various MAT 711 (70 eV) spectrophotometers

4562 Khan et al.
Asian J. Chem.
and data are tabulated as m/z. 1H NMR spectra were recorded
in CDCl3 using Bruker AC400 (300 MHz and 400 MHz)
spectrophotometer, respectively. Chemical shifts are reported
in δ (ppm) and coupling constants are given in Hz. Melting
points were determined on a Büchi 434 melting point apparatus
and were uncorrected. The progress of all reactions was moni-
tored by TLC, which was performed on 2 × 5 cm aluminum
sheets precoated with silica gel 60F254 to a thickness of 0.25 mm
(Merck). The chromatograms were visualized under ultraviolet
light (254-366 nm) or iodine vapours. All the carboxylic acids
are commercially available (Flulka Aldrich).
1H NMR (400 MHz, CDCl3): δ 8.21 (d, 1H, J = 7.9 Hz, H-5),
8.2 (d, 2H, J = 8.2, H-2´/6´), 7.79 (t, 1H, J = 7.9 Hz, H-6),
7.64 (d, 1H, J = 7.9 Hz, H-8), 7.47 (t, 1H, J = 7.9 Hz, H-7),
7.29 (d, 2H, J = 8.2 Hz, H-3´/5´), 2.44 (s, 3H, CH3); EIMS:
m/z (%) 237 (95, M+), 193 (31), 146 (10), 119 (100), 91 (42),
76 (3 %). Anal. calcd. for C15H11NO2 (237.253): C, 75.94; H,
4.67; N, 5.90. Found: C, 75.95; H, 4.65; N, 5.88.
2-(2-Nitrophenyl)-4H-3,1-benzoxazin-4-one (3e):Yield:
70 % (1.87 g, 7 mmol); m.p.: 191 °C (lit.29 m.p. = 193 °C); IR
1
(KBr, νmax, cm-1): 1768, 1636, 1531, 1346, 1225; H NMR
(400 MHz, DMSO-d6): δ 8.20 (d, 1H, J = 7.8, H-5), 8.14 (d,
1H, J = 7.5 Hz, H-6´), 8.08 (d, 1H, J = 7.5, H-3´), 7.99 (t, 1H,
J = 7.8 Hz, H-6), 7.93 (t, 1H, J = 7.5 Hz, H-5´), 7.88 (t, 1H, J
= 7.8 Hz, H-7), 7.72 (d, 1H, J = 7.8, H-8), 7.69 (t, 1H, J = 7.5
Hz, H-4´); EIMS: m/z (%) 268 (M+, 62), 146 (29), 133 (100),
118 (2), 76 (15). Anal. calcd. for C14H8N2O4 (268.224): C,
62.69 %; H, 3.01 %; N, 10.44 %. Found: 62.71; 3.02; N, 10.46.
2-(3-Nitrophenyl)-4H-3,1-benzoxazin-4-one (3f):Yield:
82 % (2.2 g, 8.2 mmol), m.p.: 165 °C (lit.29 m.p. = 166 °C); IR
(KBr, νmax, cm-1): 1756, 1626, 1530, 1474, 1346, 1220, 1036;
1H NMR (400 MHz, DMSO-d6): δ 8.85 (s, 1H, H-2´), 8.56 (d,
1H, J = 7.6 Hz, H-4´), 8.47 (d, 1H, J = 7.6 Hz, H-6´), 8.17 (d,
1H, J = 8.0 Hz, H-5), 7.98 (t, 1H, J = 8.0 Hz, H-6), 7.89 (t, 1H,
J = 8.0 Hz , H-7), 7.80 (d, 1H, J = 8.0 Hz , H-8), 7.66 (t, 1H,
J = 7.6 Hz , H-5´); EIMS: m/z (%) 268 (M+, 100), 222 (13),
150 (34), 146 (64), 104 (28), 76 (26); Anal. calcd. for
C14H8N2O4 (268.224): C, 62.69 %; H, 3.01 %; N, 10.44. Found:
C, 62.67, H, 3.03, N, 10.46.
General procedure for synthesis of 2-substituted-4H-
3,1-benzoxzin-4-one (3a-3u)29: To a 100 mL round bottom
flask containing the solution of anthranilic acid (1.37 g, 0.01
mol) in pyridine (30 mL) was cooled to 0 °C in an ice bath
followed by the careful addition of acid chloride (0.02 mol).
An exothermic reaction occurred. The reaction mixture was
stirred for 5 min at 0 °C. The ice bath was removed and the
reaction mixture was allowed to warm slowly to room tempe-
rature. The reaction mixture was further stirred for 0.5 h at
room temperature. The reaction mixture was poured into ice
cold water (200 mL) and precipitates were filtered off. The
residue was washed free of pyridine with cold water (3 × 60
mL) and dried. Benzoxazin-4-one crystallized from ethanol
as white prismatic needles. 4-Methyl-N-[2-(4-oxo-4H-3,1-
benzoxazin-2-yl)phenyl]-benzenesulfonamide (3u) was also
synthesized with similar procedure in which p-toluene sulpho-
nylchloride was used instead of acid chloride.
2-Phenyl-4H-3,1-benzoxazin-4-one (3a): Yield: 90 %
(2 g, 8.96 mmol); m.p.: 111 °C (lit.29 m.p. = 123 °C); IR (KBr,
2-(4-Nitrophenyl)-4H-3,1-benzoxazin-4-one (3g):
Yield: 78 % (2.01 g, 7.8 mmol); m.p.: 198 °C (lit.29 m.p. =
203 °C29); IR (KBr, νmax, cm-1): 1768, 1596, 1519, 1437, 1354,
1254; 1H NMR (400 MHz, DMSO-d6): δ 8.41 (d, 2H, J = 8.9
Hz, H-3´/5´), 8.30 (1H, d, J = 8.2Hz, H-5), 8.14 (2H, d, J =
8.9 Hz, H-2´/6´ ), 7.98 (1H, t, J = 8.2 Hz, H-6), 7.77 (d, 1H,
J = 8.2 Hz, H-8), 7.67 (t, 1H, J = 8.2 Hz, H-7); EIMS: m/z (%)
268 (M+, 100), 222 (4), 146 (74), 104 (58), 76 (86 %). Anal.
calcd. for C14H8N2O4 (268.224): C, 62.69 %; H, 3.01 %; N,
10.44 %. Found: C, 62.68; H, 3.02; 10.45.
1
ν
max, cm-1): 1764.5, 1616, 1572, 1472; H NMR (400 MHz,
DMSO-d6): δ 8.20 (d, 1H, J = 7.1 Hz, H-5 ), 7.94 (t, 1H, J =
7.1 Hz, H-7 ), 7.72 (1H, d, J = 7.1 Hz, H-8), 7.64 (t, 1H, J =
7.1 Hz, H-6), 8.15 (d, 2H, J = 7.8 Hz, H-2´,6´), 7.57-7.61 (m,
3H, H-3´/4´/5´); EIMS: m/z (%) 223 (M+, 100), 146 (16), 105
(52), 76 (5 %);Anal. calcd. for C14H9NO2 (223.227): C, 75.33;
H, 4.06; N, 6.27. Found: C, 75.30; H, 4.07; N, 6.30.
2-(2-Methylphenyl)-4H-3,1-benzoxazin-4-one (3b):Yield:
68 % (1.61 g, 6.79 mmol), m.p.: 112 °C (lit.29 m.p. = 114 °C); IR
(KBr, νmax, cm-1 ): 2960, 1758, 1611, 1470, 1218; 1H NMR (400
MHz, DMSO-d6): δ 8.16 (1H, d, J = 7.8 Hz, H-5), 7.97-7.90 (m,
2H, H-4´/6´), 7.70 (d, 1H, J = 7.8, H-8), 7.64 (t, 1H, J = 7.8 Hz,
H-6), 7.50 (t, 1H, J = 7.8 Hz, H-7), 7.32-7.38 (m, 2H, H-3´/5´),
2.44 (3H, s, CH3); EIMS: m/z 237 (M+, 100), 146 (7), 119 (58),
118 (30), 91 (33 %). Anal. calcd. for C15H11NO2 (237.253): C,
75.94; H, 4.67; N, 5.90. Found: C, 75.95; H, 4.66; N, 5.88.
2-(3-Methylphenyl)-4H-3,1-benzoxazin-4-one (3c):Yield:
65 % (1.54 g, 6.5 mmol); m.p.: 104-106 °C (lit.30 m.p. = 112-
115 °C); IR (KBr, νmax, cm-1): 2957, 1757, 1602, 1474, 1321,
1260; 1H NMR (400 MHz, CDCl3): δ 8.23 (d, 1H, J = 7.8 Hz, H-
5), 8.10 (d, 1H, J = 7.5 Hz, H-6´), 7.81 (t, 1H, J = 7.8 Hz, H-6),
7.68 (d, 1H, J = 7.8 Hz, H-8), 7.49 (t, 2H, J = 8.1 Hz, H-7/5´),
7.39 (d, 1H, J = 7.3 Hz, H-4´), 7.37 (s, 1H, H-2´), 2.43 (s, 3H,
CH3); EIMS: m/z (%) 237 (M+, 90), 193 (25), 146 (10), 119
(100), 91 (35), 76 (5%). Anal. calcd. for C15H11NO2 (237.253):
C, 75.94; H, 4.67; N, 5.90. Found: C, 75.92; H, 4.66; N, 5.88.
2-(4-Methylphenyl)-4H-3,1-benzoxazin-4-one (3d):
Yield: 86 % (2.04 g, 8.6 mmol); m.p.: 149 °C (lit.29 m.p. =
155 °C); IR (KBr, νmax, cm-1): 2963, 1757, 1608, 1473, 1315;
2-(2-Fluorophenyl)-4H-3,1-benzoxazin-4-one (3h):
Yield: 53 % (1.27 g, 5.3 mmol); m.p.: 110 °C (lit.29 m.p. =
115 °C); IR (KBr, νmax, cm-1): 1772, 1625, 1606, 1477, 1320,
1278; 1H NMR (400 MHz, CDCl3): δ 8.24 (d, 1H, J = 7.8 Hz,
H-5), 8.11 (t, 1H, J = 7.6 Hz, H-4´), 7.83 (t, 1H, J = 7.8 Hz, H-
6), 7.7 (d, 1H, J = 7.8 Hz, H-8), 7.51-7.56 (m, 2H, H-6´/7),
7.27 (t, 1H, J = 7.6 Hz, H-3´), 7.20 (1H, t, J = 7.6 Hz, H-5´);
EIMS: 241(M+, 100), 213 (9), 197 (98), 146 (85), 95 (70), 76
(15 %).Anal. calcd. for C14H8NO2F (241.217): C, 69.71 %; H,
3.34 %; N, 5.81 %. Found: C, 69.69; H, 3.36; N, 5.81.
2-(3-Fluorophenyl)-4H-3,1-benzoxazin-4-one (3i):
Yield: 69 % (1.66 g, 6.9 mmol), m.p.: 133 °C; IR (KBr, νmax
,
1
cm-1): 1766, 1615, 1477, 1445, 1323, 1260; H NMR (400
MHz, CDCl3): 8.23 (d, 1H, J = 7.8 Hz, H-5), 8.08 (d, 1H, J =
7.8 Hz, H-8), 7.97-8.012 (m, 1H, H-2´), 7.82 (t, 1H, J = 7.8
Hz, H-7), 7.68 (d, 1H, J = 7.6 Hz, H-6´), 7.51(t, 1H, J = 7.95
Hz, H-5´), 7.46 (t, 1H, J = 8 Hz, H-6), 7.25-7.27 (m, 1H, H-
4´); EIMS: m/z (%) 241 (100 %, M+), 213 (5 %), 197 (76 %),
146 (36 %), 95 (38 %), 76 (5 %). Anal. calcd. for C14H8NO2F
(241.217): C, 69.71 %; H, 3.34 %; N, 5.81 %. Found: C, 69.72;
H, 3.36; N, 5.83.

Vol. 26, No. 15 (2014)
Synthesis of 2-Aryl-4H-3,1-Benzoxazin-4-ones: A Class of α-Chymotrypsin Inhibitors 4563
2-(4-Fluorophenyl)-4H-3,1-benzoxazin-4-one (3j):
Yield: 97 % (2.34 g, 9.7 mmol); m.p.: 168 °C (lit.29 m.p. = 170
°C); IR (KBr, νmax, cm-1): 1764, 1601, 1508, 1472, 1321, 1231;
1H NMR (300 MHz, CDCl3): δ 8.30 (d, 1H, J = 6.5 Hz, H-5),
8.21 (1H, d, J = 6.5 Hz, H-8), 7.81 (t, 1H, J = 6.5 Hz, H-6),
7.66 (d, 2H, J = 5.9 Hz, H-2´/6´), 7.54 (t, 1H, J = 6.5 Hz, H-
7), 7.14-7.17 (m, 2H, H-3´/ 5´); EIMS: m/z (%) 241 (M+, 31),
146 (7), 123 (100), 95 (40), 76 (4 %). Analysis calcd. for
C14H8NO2F (241.217): C, 69.71 %; H, 3.34 %; N, 5.81 %.
Found: C, 69.69; H, 3.36; N, 5.82.
7.69 (d, 2H, J = 7.9 Hz, H-8/6´), 7.52 (t, 1H, J = 8 Hz, H-6),
7.34 (t, 1H, J = 7.6 Hz, H-5´), 7.32 (d, 1H, J = 7.6 Hz, H-4´).
EIMS: m/z (%) 303 (M+, 97), 301 (100), 257 (29), 259 (38),
222 (6), 183 (47), 185 (48), 146 (21), 76 (11 %). Anal. calcd.
for C14H8NO2Br (302.123): C, 55.66 %; H, 2.67 %; N, 4.64 %.
Found: C, 55.68; H, 2.66; N, 4.63.
2-(4-Bromophenyl)-4H-3,1-benzoxazin-4-one (3p):
Yield: 80 % (2.41 g, 8.0 mmol); m.p.: 182 °C (lit.29 m.p. = 184
°C); IR (KBr, νmax, cm-1): 1764, 1619, 1480, 1320, 1256; 1H
NMR (400 MHz, CDCl3): δ 8.23 (d, 1H, J = 7.8 Hz, H-5),
8.16 (d, 2H, J = 8.6 Hz, H-2´/ 6´), 7.81 (t, 1H, J = 7.8, H-7),
7.67 (d, 1H, J = 7.8 Hz, H-8), 7.63 (d, 2H, J = 8.6 Hz, H-3´5´),
7.51 (t, 1H, J = 7.8 Hz, H-6); EIMS: m/z (%) 303 (M+, 98),
301 (100), 155 (46), 157 (45), 146 (38) 118 (2), 104 (5), 102
(5), 76 (42), 64 (5); Anal. calcd. for C14H8NO2Br (302.123):
C, 55.66; H, 2.67; N, 4.64. Found: C, 55.65; H, 2.68; N, 4.65.
2-(2-Methoxyphenyl)-4H-3,1-benzoxazin-4-one (3q):
Yield: 75 % (1.9 g, 7.5 mmol); m.p.: 129 °C (lit.30 m.p. = 132-
133 °C); IR (KBr, νmax, cm-1): 1769, 1607, 1490, 1467, 1255;
1H NMR (400 MHz, CDCl3): δ 8.24 (d, 1H, J = 7.9 Hz, H-5),
7.84 (d, 1H, J = 7.6 Hz, H-6´), 7.79 (d, 1H, J = 7.6 Hz, H-4´),
7.70 (d, 1H, J = 7.9 Hz, H-8), 7.51 (t, 1H, J = 7.9 Hz, H-7),
7.48 (t, 1H, J = 7.9 Hz, H-6), 7.0-7.07 (m, 2H, H-3´/5´), 3.93
(s, 3H, -OCH3); EIMS: 253 (M+, 64,), 224 (26), 209 (3), 146
(8), 135 (63), 133 (23), 119 (100), 77 (25), 76 (4 %); Anal.
calcd. for C15H11NO3 (253.253): C, 71.14; H, 4.38; N, 5.53.
Found: C, 71.12; H, 4.40; N, 5.54.
2-(2-Chlorophenyl)-4H-3,1-benzoxazin-4-one (3k):
Yield: 64 % (1.64 g, 6.4 mmol); m.p.: 136 °C (lit.29 m.p. = 138
°C); IR (KBr, νmax, cm-1): 1769, 1622, 1474, 1316, 1272, 1225;
1H NMR (300 MHz, CDCl3): δ 8.26 (d, 1H, J = 6.1 Hz, H-5),
7.87 (d, 1H, J = 6.8 Hz, H-6´), 7.84 (t, 1H, J = 6.1 Hz, H-7),
7.7 (d, 1H, J = 6.1 Hz, H-8), 7.57 (t, 1H, J = 6.1 Hz , H-6),
7.51 (d, 1H , J = 6.8 Hz, H-3´), 7.45 (t, 1H , J = 6.8 Hz, H-5´),
7.38 (t, 1H , J = 6.8 Hz, H-4´); EIMS: m/z (%) 259 (M+, 15),
257 (44), 213 (31), 215 (11), 146 (21), 139 (47), 141 (19),
113 (6), 146 ( 21), 76 (5); Anal. calcd. for C14H8NO2Cl
(257.672): C, 65.26 %); H, 3.13 %; N, 5.44 %. Found: C,
65.24; H, 3.15; N, 5.46.
2-(3-Chlorophenyl)-4H-3,1-benzoxazin-4-one (3l):
Yield: 87 % (2.19 g, 8.5 mmol); m.p.: 153 °C (lit.30 m.p. =
156-157 °C); IR (KBr, νmax, cm-1): 1761, 1601, 1566, 1473,
1416; 1H NMR (400 MHz, CDCl3): δ 8.30 (s, 1H, H-2´), 8.23
(1H, d, J = 7.8 Hz, H-5), 8.17 (d, 1H, J = 8.1 Hz, H-6´), 7.81
(t, 1H, J = 7.8 Hz, H-7), 7.68 (d, 1H, J = 7.8 Hz, H-8), 7.50-
7.55 (m, 2H, H-4´/5´), 7.42 (t, 1H, J = 7.8 Hz, H-6); EIMS:
m/z (%) 259 (M+, 36), 257 (100), 213 (61), 215 (21), 146 (41),
139 (67), 141 (21), 113 (14), 111 (43), 76 (10 %); Analysis
calcd. for C14H8NO2Cl (257.672): C, 65.26 %; H, 3.13 %; N,
5.44 %. Found: C, 65.28; H, 3.12; N, 5.44.
2-(4-Methoxyphenyl)-4H-3,1-benzoxazin-4-one (3r):
Yield: 94 % (2.38 g, 9.4 mmol), m.p.: 150 °C (lit.29 m.p. = 148
°C); IR (KBr, νmax, cm-1): 1757, 1606, 1568, 1472, 1259, 1168;
1H NMR (400 MHz, CDCl3): δ 8.24 (d, 1H, J = 8.5 Hz, H-5),
8.2 (d, 1H, J = 8.5 Hz, H-8), 7.82 (t, 1H, J = 8.5 Hz, H-7),
7.63 (d, 2H, J = 8 Hz, H-2´/6´), 7.45 (t, 1H, J = 8.5 Hz, H-6),
6.99 (d, 1H, J = 8 Hz, H-3´/5´), 3.88 (s, 3H, OCH3); EIMS:
m/z (%) 253 (M+, 23), 209 (5), 146 (3), 135 (100), 107 (8),
76(3), 64 (9 %). Anal. calcd. for C15H11NO3 (253.253): C,
71.14, H, 4.38, N, 5.53. Found: C, 71.14; H, 4.39; N, 5.51.
2-(3, 4, 5-Trimethoxyphenyl)-4H-3,1-benzoxazin-4-one
(3s). Yield: 40 % (1.25 g, 4 mmol); m.p.: 181 °C (lit.30 m.p. =
185 °C); IR (KBr, νmax, cm-1): 1754, 1607, 1504, 1349, 1292,
1131; 1H NMR (300 MHz, CDCl3): δ 8.20 (1H, d, J = 8.1 Hz,
H-5), 7.80 (t,1H, J = 8.1 Hz, H-7), 7.66 (d, 1H, J = 8.1, H-8),
7.53 (s, 2H, H-2´/6´), 7.48 (t, 1H, J = 8.1 Hz, H-6), 3.92 (s,
9H, -OCH3); EIMS: m/z (%) 313 (M+, 100), 268 (5), 223 (2),
195 (28), 193 (3), 167 (6), 146 (30), 76 (2). Anal. calcd. for
C17H15NO5 (313.305): C, 65.17; H, 4.83; N, 4.47; found: C,
65.18; H, 4.82; N, 4.47.
2-(4-Chlorophenyl)-4H-3,1-benzoxazin-4-one (3m):
Yield: 85 % (2.18 g, 8.49 mmol); m.p.: 192 °C (lit.29 m.p. = 190
1
°C); IR (KBr, νmax, cm-1): 1770, 1633, 1484, 1316, 1255; H
NMR (400 MHz, CDCl3): δ 8.01 (d, 1H, J = 7.9 Hz, H-5), 7.83
(2H, d, J = 7.4 Hz, H-2´/6´), 7.46 (t, 1H, J = 7.9, H-6), 7.34 (d,
1H, J = 7.9 Hz, H-8), 7.27(d, 2H, J = 7.4 Hz, H-3´/5´), 7.01 (t,
1H, J = 7.9 Hz, H-7); EIMS: m/z (%) 259 (M+, 27), 257 (81),
213 (37), 215 (13), 146 (14), 139 (100), 141 (32), 113 (12), 111
(37), 76 (8 %).Anal. calcd. for C14H8NO2Cl (257.672): C, 65.26
%; H, 3.13 %; N, 5.44 %. Found: C, 65.26; H, 3.14; N, 5.43.
2-(2-Bromophenyl)-4H-3,1-benzoxazin-4-one (3n):
Yield: 87 % (2.63 g, 8.7 mmol); m.p.: 115 °C (lit.29 m.p. = 118
°C); IR (KBr, νmax, cm-1): 1767, 1626, 1474, 1315, 1270, 1224;
1H NMR (400 MHz, CDCl3): δ 8.26 (d, 1H, J = 8 Hz, H-5),
7.82-7.86 (m, 2H, H-3´/5´), 7.71 (d, 2H, J = 7.9 Hz, H-8/6´),
7.56 (t, 1H, J = 8 Hz, H-7), 7.42 (t, 1H, J = 7.4, H-4´), 7.35 (t,
1H, J = 8 Hz, H-6); EIMS: m/z (%) 303 (M+, 97), 301 (100),
257 (33), 259 (32), 222 (4), 183 (47), 185 (45), 146 (25), 76
(8 %). Anal. calcd. for C14H8NO2Br (302.123): C, 55.66 %; H,
2.67 %; N, 4.64 %. Found: C, 55.68; H, 2.66; N, 4.63.
2-(1-Naphthyl)-4H-3,1-benzoxazin-4-one (3t):Yield: 95
% (2.59 g, 9.5 mmol); m.p.: 136-137 °C (lit.30 m.p. = 138-140
°C); IR (KBr, νmax, cm-1): 1747, 1596, 1510, 1471, 1320, 1223;
1H NMR (300 MHz, CDCl3): δ 8.98 (d, 1H, J = 8.6 Hz, H-8´),
8.93 (d, 1H, J = 8.6 Hz, H-2´), 8.37 (d, 1H, J = 7.3 Hz, H-4´),
8.20 (d, 1H, J = 7.6 Hz, H-5 ), 8.13 (d, 1H, J = 7.6 Hz, H-8),
8.07(d, 1H, J = 8.6 Hz, H-5´), 7.99 (t, 1H, J = 8.6 Hz, H-7´),
7.79 (t, 1H, J = 7.6 Hz, H-7), 7.73-7.65 (m, 3H, H-6/6´/3´);
EIMS: m/z (%) 273 (M+, 20), 153 (4), 146 (7), 155 (100), 127
(67), 76 (4); Anal. calcd. for C18H11NO2 (273.285): C, 79.11;
H, 4.06; N, 5.13. Found: C, 79.11; H, 4.08; N, 5.11.
2-(3-Bromophenyl)-4H-3,1-benzoxazin-4-one (3o):
Yield: 68 % (2.05 g, 6.8 mmol); m.p.: 165 °C (lit.30 m.p. =
160-161 °C); IR (KBr, νmax, cm-1): 1765, 1625, 1475, 1317,
1
1270, 1224; H NMR (300 MHz, CDCl3): δ 8.46 (s, 1H, H-
2´), 8.22 (d, 1H, J = 7.9 Hz, H-5), 7.85 (t, 1H, J = 7.9, H-7),

4564 Khan et al.
Asian J. Chem.
O
4-Methyl-N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]-
benzenesulfonamide (3u): Yield: 55 % (1.8 g, 4.59 mmol);
m.p.: 212 °C (lit.30 m.p. = 216 °C); IR (KBr, νmax, cm-1): 1778,
1704, 1604, 3446, 1570, 1474; 1H NMR (400 MHz, CDCl3):
δ 11.92 (s, 1H, NH), 8.17 (d, 1H, J = 8.5 Hz, H-6´), 7.97
(d,1H, J = 7.7 Hz, H-5) 7.69 (t, 1H, J = 7.7 Hz, H-7), 7.56 (d,
1H, J = 7.7 Hz, H-8), 7.52 (d, 2H, J = 8 Hz, H-2´´/6´´), 7.44 (t,
1H, J = 7.7 Hz, H-6´), 7.25 (d, 2H, J = 8 Hz, H-3´´/H-5´´),
7.21 (t, 1H, J = 8.5 Hz, H-4´) 7.11 (d, 2H, J = 8.5 Hz, H-5´/
3´), 2.27 (s, 3H, CH3); EIMS: 392.2 (M+ , 100), 377 (3), 237
(66), 146 (4), 91 (20 %). CHN analysis calcd. for C21H16N2O4
(392.429): C, 64.27; H, 4.11; N, 7.14. Found: C, 64.28; H,
4.13; N, 7.12.
COOH
O
Pyridine
O
+
0°C to r.t.
R
Cl
2a-t
N
3a-t
R
NH2
1
Compound R
Compound R
C6H5-
-CH3C6H4- 3l
-CH3C6H4- 3m
-CH3C6H4- 3n
o
m-ClC6H4-
-ClC6H4-
3a
3b
3c
3d
3e
3f
3k
o
m
p
o
-ClC6H4-
-BrC6H4-
p
o
-NO2C6H4-
-NO2C6H4-
m
-BrC6H4-
-BrC6H4-
-OCH3C6H4-
-OCH3C6H4-
3o
3p
3q
3r
3s
3t
m
p
o
p
p
o
-NO2C6H4-
-FC6H4-
3g
3h
3i
Chymotrypsin inhibition assay: The α-chymotrypsin
inhibitory activity of compounds was performed by method
of Cannel et al.2. Chymotrypsin (9 units/mL of 50 mM Tris-
HCl buffer pH 7.6; Sigma Chemical Co. USA) was pre-incu-
bated with the compounds for 20 min at 25 °C. The substrate
solution of 100 µL volume (N-succinyl-phenylalanine-p-
nitroanilide, 1 mg/mL of 50 mM Tris-HCl buffer pH 7.6) was
added to start the enzyme reaction. The absorbance of released
p-nitroaniline was continuously monitored at 410 nm until a
significant color change had achieved. The final DMSO con-
centration in the reaction mixture was 7 %.All the experiments
were carried out at least in triplicates and the results represent
the mean S.E.M. (standard error of the mean). Chymostatin
was used as a standard inhibitor for α-chymotrypsin inhibition.
m
-FC6H4-
-FC6H4-
3,4,5-(OCH3)3C6H2-
1-Naphthyl
p
3j
Scheme-I: Synthesis of 2-aryl-4H-3,1-benzoxazin-4-ones
O
O
O
O
S
O
COOH
S
O
HN
Pyridine
Cl
+
0°C to r.t.
NH2
N
3u
CH3
H3C
2u
1
Scheme-II: Synthesis of 4-Methyl-N-[2-(4-oxo-4H-3,1-benzoxazin-2-
yl)phenyl]-benzenesulfonamide
TABLE-1
α-CHYMOTRYPSIN INHIBITORY ACTIVITY (IC50)
OF 2-ARYL-4H-3,1-BENZOXAZIN-4-ONES (3a-3u)
IC50 SEMa
Compound
RESULTS AND DISCUSSION
Entry
R
No.
(µM)
11.54 1.73
41.27 1.33
35.0 1.86
13.26 0.79
N.A.b
1
2
C6H5-
3a
Anthranilic acid (1) was treated with aryl chlorides (2a-2t)
in the presence of excess of pyridine to give 2-substituted
benzoxazin-4-one (3a-3t) in moderate to excellent yields
according to the literature protocol29 (Scheme-I). The acid
chlorides, where not commercially available, were prepared
by the action of thionyl chloride on the appropriate carboxylic
acid. The failure of aliphatic acyl halides to produce benzo-
xazinones is most likely due to ready hydrolysis of initially
formed 2-alkyl-4H-3,1-benzoxazin-4-one31.
o-CH3C6H4-
m-CH3C6H4-
p-CH3C6H4-
o-NO2C6H4-
m-NO2C6H4-
p-NO2C6H4-
o-FC6H4-
3b
3
3c
4
3d
5
3e
6
19.66 1.31
N.A.b
3f
7
3g
8
7.22 0.75
14.92 0.33
39.21 0.34
21.01 0.13
39.69 0.52
14.53 0.35
6.99 0.29
38.06 1.93
31.06 0.96
12.62 1.09
35.01 2.33
N.A.b
3h
9
m-FC6H4-
3i
10
11
12
13
14
15
16
17
18
19
20
21
p-FC6H4-
3j
o-ClC6H4-
3k
Similarly, anthranilic acid (1) reacts with p-tolylsulphonyl
chloride (2u) in the presence of excess of pyridine to give 2-
substituted benzoxazin-4-one (3u) having sulfonamide moiety
in good yield (Scheme-II). This reaction probably proceeds
via a mixed carboxylic acid-sulphonic acid anhydride inter-
mediate29.
m-ClC6H4-
p-ClC6H4-
3l
3m
o-BrC6H4-
3n
m-BrC6H4-
p-BrC6H4-
3o
3p
o-OCH3C6H4-
p-OCH3C6H4-
3,4,5-(OCH3)3C6H2-
1-Naphthyl
Sulfonamide
Standard
3q
in vitro α-Chymotrypsin inhibitory activity: A small
library of 2-substituted-4H-3,1-benzoxazin-4-one (3a-3u) were
synthesized and their potential biological significance were
investigated as α-chymotrypsin inhibitors. Most of them showed
significant activity against α-chymotrypsin. in vitro α-Chymo-
trypsin inhibiton assay of benzoxazinone derivatives were
performed according to liteature protocol2 and a list of inhibi-
tory activities results in term of IC50 values are summarized in
Table-1. α-Chymostatin was used as standard inhibitor with
IC50 value 7.13 1.06 µM (Table-1). Further standard inhibitor
(α-Chymostatin) is a tetra-peptide analogue and a potent
inhibitor of a number of proteinases including the serine
proteinases, chymotrypsin and streptomyces griseus proteinase
A and several cysteine proteinases32.
3r
3s
5.42 1.66
16.23 1.99
7.13 1.06
3t
3u
Chymostatinc
Notes: aSEM standard error of the mean; bN.A. Not active against
enzyme α-chymotrypsin; cStandard inhibitors of enzyme
α-chymotrypsin
Among these 21 compounds investigated for α-chymo-
trypsin inhibition, 6 compounds 3t, 3n, 3h, 3a, 3q and 3d
showed promising results in comparison to standard inhibitor
(Table-1). Any how certain variations were noted in results
for example compounds 3e and 3g were found to be inactive
having polar nitro group at ortho- and para-positions but

Vol. 26, No. 15 (2014)
Synthesis of 2-Aryl-4H-3,1-Benzoxazin-4-ones: A Class of α-Chymotrypsin Inhibitors 4565
compound 3f having nitro group at meta-position showed
moderate activity with IC50 = 19.66 1.31 µM (Table-1).
Whereas compound 3s was also found to be inactive with three
adjacent methoxy groups (Table-1).
molecule inhibitors which are effective and specific for α-
chymotrypsin inhibition. Of this series, the most potent com-
pounds 2-(2-fluorophenyl)-4H-3,1-benzoxazin-4-one (3h),
2-(2-bromophenyl)-4H-3,1-benzoxazin-4-one (3n) and 2-(1-
naphthyl)-4H-3,1-benzoxazin-4-one (3t) may act as a potential
lead molecules for the future research in the field of α-chymo-
trypsin inhibition.
Compounds 2-naphthyl-4H-3,1-benzoxazin-4-one (3t)
and 2-(2-bromophenyl)-4H-3,1-benzoxazin-4-one (3n)
showed exciting α-chymotrypsin enzyme inhibitory activity
with IC50 values 5.42 1.66 µM and 6.99 0.29 µM respec-
tively, whereas the standard inhibitor has IC50 = 7.13 1.06
µM. The 2-phenyl-4H-3,1-benzoxazin-4-one (3a) showed
good inhibitory activity with IC50 = 11.54 1.73 µM (Table-1).
When phenyl group 3a was replaced by naphthyl group in
compound 3t (IC50 = 5.42 1.66 µM), activity was almost
doubled. The enhancement in activity may be attributed to
steric bulk and prolongation of conjugation by naphthyl moiety.
The α-chymotrypsin enzyme inhibitory activity in compounds
3b, 3c and 3d having methyl moiety was gradually enhanced
from ortho- to para-positions (Table-1). This is might be due
to positive inductive effect of methyl substituent at ortho and
para position. The complete reverse order of α-chymotrypsin
enzyme inhibitory activity was observed in case of highly
electronegative fluoro-substituent in compounds 3h, 3i and 3j
with negative inductive effect (Table-1). The 2-(2-fluorophenyl)-
4H-1,3-benzoxazin-4-one (3h) was found to be most active
among the fluoro-substituents with IC50 = 7.22 0.75 µM and
its para-analogue 3j was found to be least active with IC50 =
39.21 0.34 µM while meta analogue 3i showed medium
inhibitory activity (Table-1). Among the compounds 3k, 3l
and 3m with chloro-substituent at ortho-, meta- and para-
positions, respectively, its para-analogue 3m was found to be
most active member with IC50 = 14.53 0.35 µM whereas its
meta-analogue 3l was least active (IC50 = 39.69 0.52 µM).
This trend of activity may be explained on the basis of linear
geometry of para-substituted phenyl ring. The 2-(2-bromo-
phenyl)-4H-3,1-benzoxazin-4-one (3n) was found to be most
active (IC50 = 6.99 0.29 µM) among bromo-substituents
(Table-1). Compound 3q having postive mesomeric effect of
methoxy at ortho-position showed very similar inhibitory
activity to compound 3d having methyl group at para-position.
A marked decrease in activity was observed in case of 3r
having methoxy group at para-position. The compound 4u
having p-tolylsulfonyl group with sulfonamide moiety (IC50
= 16.23 1.99 µM) showed similar activity as 3i (IC50 = 14.92
0.33 µM) (Table-1).
ACKNOWLEDGEMENTS
The authors are thankful to Higher Education Commission
of Pakistan (HEC) for munificent financial support under IPFP
/TTS program.
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