Synthesis of some 2(3H)-benzoxazolone derivatives and their in-vitro effects on human leukocyte myeloperoxidase activity

Article abstract of DOI:10.1002/ardp.200500106

Myeloperoxidase (MPO), a heme protein expressed by polymorphonuclear leukocytes, generates potent oxidants which are proposed to contribute to tissue damage during inflammation and certain pathogenesis such as neurodegenerative disorders. In this study, twenty ω-[2-oxo-3H-benzoxazol-3-yl]-N- phenylacetamide and propionamide derivatives having substituents of different lipophilic and electronic nature on the N-phenyl ring were synthesized to evaluate the inhibitory effects on in vitro leukocyte MPO chlorinating activity. The most active compounds in the series were the derivatives bearing 2-methyl and 4-nitro substituent on the N-phenyl ring.

Full text of DOI:10.1002/ardp.200500106

Arch. Pharm. Chem. Life Sci. 2005, 338, 405−410
DOI 10.1002/ardp.200500106
405
Synthesis of Some 2(3H)-Benzoxazolone Derivatives
and their in-vitro Effects on Human Leukocyte
Myeloperoxidase Activity
Zeynep Soyera, Meral Basb, Aysun Pabuccuoglub, Varol Pabuccuoglua
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University Bornova, Izmir, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Ege University Bornova, Izmir, Turkey
Myeloperoxidase (MPO), a heme protein expressed by polymorphonuclear leukocytes, generates potent
oxidants which are proposed to contribute to tissue damage during inflammation and certain patho-
genesis such as neurodegenerative disorders. In this study, twenty ω-[2-oxo-3H-benzoxazol-3-yl]-N-
phenylacetamide and propionamide derivatives having substituents of different lipophilic and elec-
tronic nature on the N-phenyl ring were synthesized to evaluate the inhibitory effects on in vitro
leukocyte MPO chlorinating activity. The most active compounds in the series were the derivatives
bearing 2-methyl and 4-nitro substituent on the N-phenyl ring.
Keywords: 2(3H)-Benzoxazolone; Anilide; Myeloperoxidase
Received: April 12, 2005; Accepted: June 17, 2005
Introduction
and multiple sclerosis, MPO has become an important
therapeutic target in recent years [4, 7, 8]. It has been re-
ported that development of MPO inhibitor with high speci-
ficity and low toxicity is a challenging goal for the future [9].
Myeloperoxidase (MPO, EC 1.11.1.7) is an enzyme found in
polymorphonuclear neutrophils and monocytes and certain
populations of tissue macrophages. It belongs to the family
of peroxidase enzymes such as lactoperoxidase, eosinophil
peroxidase, and thyroid peroxidase. Beside its regular per-
oxidative activity, MPO has the ability to catalyze the con-
version of hydrogen peroxide and chloride to hypochlorous
acid (HOCl), which has powerful antimicrobial and cyto-
toxic activity as a potent oxidant [1Ϫ3]. MPO seems to play
an important role in the defense system against invading
microorganisms and certain tumor killing actions by the
products derived from the MPO-H2O2-ClϪ reaction known
as chlorinating activity of the enzyme [1, 4]. HOCl produced
by MPO also contributes to the tissue damage caused by
neutrophils at the sites of inflammation through inactivating
enzymes, cross-linking proteins, oxidizing susceptible amino
acids, and chlorinating lipids and tyrosyl residues [3, 5]. The
activity of many antiinflammatory drugs has been, at least
partly, attributed to the inhibition of leukocyte MPO [5].
MPO also oxidizes various functional groups such as phe-
nols, anilines, and β-diketons to free radicals by its
peroxidative activity, which may partly account for the toxic
metabolites of certain drugs [1, 5]. Since the involvement of
reactive bioproducts produced by chlorinating activity of
MPO in a wide variety of disorders such as atherosclerosis,
neurodegenerative diseases including Alzheimer’s disease
The above mentioned state encouraged us to begin a syn-
thetic program toward exploring the new molecules as a
starting point to develop highly specific MPO inhibitors. In
this preliminary study, we report the synthesis and the ef-
fects on in vitro human leukocyte MPO chlorinating activity
of some 2(3H)-benzoxazolone derivatives (Table 1).
Results and Discussion
Chemistry
As illustrated in Scheme 1, the title compounds were pre-
pared in a two-step synthesis. In the first step, according to
the reported procedure [10], 2-chloroacetyl and 3-chloropro-
pionyl chlorides reacted with appropriately substituted ani-
lines and yielded ω-chloroanilides. In the second step, those
intermediates were refluxed with 2(3H)-benzoxazolone in
acetone to furnish the title compounds. 2(3H)-Benzoxazo-
lone was prepared by reaction of o-aminophenol with urea
as described in the literature [11]. The structures of the title
1
compounds were confirmed by elemental analysis, H- and
13C-NMR, IR, and ESI-MS data (Tables 1 and 2). The re-
flux times, yields, and melting points are summarized in
Table 1. The six derivatives of the title compounds, namely
1 [12, 13], 2 [14], 3, 5, 9, 11 [12], were reported previously.
Compounds 12 and 14 are listed compounds with registry
number CASRN 263386-37-4 and CASRN 263160-21-0
Correspondence: Varol Pabuccuoglu, Pharmaceutical Chemistry
Department, Faculty of Pharmacy, Ege University Bornova 35100,
Izmir, Turkey. Phone: ϩ90 232 339-9931, Fax: ϩ90 232 388-5258,
e-mail: papuccuogluv@pharm.ege.edu.tr
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406
Pabuccuoglu et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 405−410
Table 1. Reflux time, yield, melting point, formula, IR, and ESI-Ms data of the title compounds.
Comp. n
R
Reflux Yield
Mp.
[°C]
Formula
IR [cm؊1
]
ESI-MS m/z (% intensity)
[h]
[%]
1
2
1
2
H
H
5
18
7
5
5
65
61
40
36
43
54
92
90
56
195†
165
C15H12N2O3 3332, 1762, 1693
C16H14N2O3 3347, 1768, 1686
C16H14N2O3 3266, 1764, 1666
C16H14N2O3 3275, 1788, 1669
C16H14N2O3 3322, 1772, 1679
C17H16N2O3 3234, 1757, 1644
C17H16N2O3 3293, 1757, 1653
C17H16N2O3 3252, 1766, 1661
C15H11ClN2O3 3257, 1762, 1672
268 (3, Mϩ), 148 (100)
282 (15, Mϩ), 134 (100)
3
1 o-CH3
1 m-CH3
1 p-CH3
2 o-CH3
2 m-CH3 55
2 p-CH3
1
1
1
2
2
2
205‡
184
282 (9, Mϩ), 148 (100)
4
282 (4, Mϩ), 148 (100)
5
214§
206
282 (3, Mϩ), 148 (100)
6
30
296 (23, Mϩ), 295 (100)
7
154-156
174
296 (14, Mϩ), 134(100)
8
34
7
5
296 (14, Mϩ), 134(100)
9
o-Cl
m-Cl
p-Cl
o-Cl
m-Cl
p-Cl
203$
304 (2, [Mϩ2]ϩ), 302 (8, Mϩ), 148 (100)
304 (4, [Mϩ2]ϩ), 302 (11, Mϩ), 148 (100)
304 (1, [Mϩ2]ϩ), 302 (3, Mϩ), 148 (100)
318 (7, [Mϩ2]ϩ), 316 (24, Mϩ), 315 (100)
318 (5, [Mϩ2]ϩ), 316 (17, Mϩ), 134 (100)
318 (6, [Mϩ2]ϩ), 316 (16, Mϩ), 134 (100)
10
11
12
13
14
44 208Ϫ210 C15H11ClN2O3 3334, 1763, 1744,1688
5
57 218Ϫ220& C15H11ClN2O3 3329, 1783, 1682
30
50
55
8
91
76
87
158
161
184
C16H13ClN2O3 3231, 1783, 1756,1654
C16H13ClN2O3 3306, 1735, 1679
C16H13ClN2O3 3294, 1767, 1659
15 1 o-NO2
16 1 m-NO2
17 1 p-NO2 6.5
18 2 o-NO2 18
19 2 m-NO2 18
20 2 p-NO2 17
62 199Ϫ200 C15H11N3O5 3345, 1760, 1698, 1509,1348 313 (14, Mϩ), 148 (100)
6
95
84
81
96
70
270
263
149
214
241
C15H11N3O5 3327, 1764, 1695, 1536,1341 313 (17, Mϩ), 312 (100)
C15H11N3O5 3301, 1769, 1693, 1506,1344 313 (22, Mϩ), 148 (100)
C16H13N3O5 3230, 1759, 1671, 1522,1353 327 (18, Mϩ), 326 (100)
C16H13N3O5 3339, 1747, 1687, 1517,1343 327 (19, Mϩ), 326 (100)
C16H13N3O5 3340, 1751, 1695, 1485,1335 327 (19, Mϩ), 326 (100)
†
202°C from ethanol/water, Ref. [13], 211°C from ethanol, Ref. [12 ]; ‡ 232Ϫ234°C Ref. [12], § 223Ϫ224°C from ethanol Ref. [12]; $ 208°C
&
from ethanol Ref. [12]; 227Ϫ229°C from ethanol Ref. [12].
due to nitro group stretching were observed in the spectra
of compounds 15Ϫ20 (Table 1).
Concerning 1H-NMR spectral data, an intriguing feature
was some unexpected minor signals in acetamide deriva-
tives, except compound 9, in which minor signals were unde-
tectable. Those unexpected minor signals resembled the
major ones. Tarikogullari et al. [12], reported similar data
for acetamide derivatives 1, 3, 5, 9, and 11. In our study,
the highest major/minor signal ratios were observed with
methyl-substituted acetamide derivatives 3, 4, 5 (Table 2).
Scheme 1.Synthesis of the title compounds.
Those minor signals would be due to two different rotamer
1
formations. In order to clarify the situation, the H-NMR
spectra of compounds 3, 4, and 5 were re-recorded in
CDCl3/DMSO-d6 solvent mixture, since solvent polarity-de-
pendent behavior of rotamer mixtures is well documented
[15Ϫ17]. As seen in Table 2, the spectra run in CDCl3/
DMSO-d6 did not produce any minor signals. This solvent
dependent behavior obviously proved that the title com-
pounds with acetamide derivative, existed as two different
rotamers in DMSO-d6. Similar situation were observed in
13C-NMR spectra of acetamide compounds, indicating the
respectively, but corresponding scientific reference data are
not available.
The IR spectral data presented the expected characteristic
absorption bands indicating the amide carbonyl (between
1644Ϫ1698 cmϪ1), amide N-H (between 3231Ϫ3247 cmϪ1),
and 2(3H)-benzoxazolone carbonyl stretching (between
1735Ϫ1788 cmϪ1). Additional diagnostic absorption bands
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Arch. Pharm. Chem. Life Sci. 2005, 338, 405−410
Table 2. NMR data of title compounds.
Synthesis of 2(3H)-Benzoxazolone Derivatives and MPO Activity
407
Comp. Major/Minor NMR
ratio†
1H NMR (DMSO-d6): 2.18 (s, 3H, CH3, min.), 2.20 (s, 3H, CH3), 4.45 (d, 1H, J ϭ 17.6, CH2, min.), 4.54 (d, 1H,
J ϭ 17.6, CH2, min.), 4.75 (s, 2H, CH2), 6.82 (td, 1H, min.), 6.95 (dd, 1H, J ϭ 1.5, 8.2, min.), 7.06Ϫ7.37 (m, 8H,
Ar-H),
7.06Ϫ7.37 (m, 6H, min.), 9.81 (brs, NH).
3
1.2/1
1H NMR (CDCl3؉DMSO-d6): 2.09 (s, 3H, CH3), 4.54 (s, 2H, CH2), 6.94Ϫ7.08 (m, 7H, Ar-H), 7.32Ϫ7.42 (m,
1H, Ar-H), 8.99 (brs, NH).
13C NMR: 18.0, 18.3 (min.), 45.3 (min.), 52.7, 110.0, 110.2, 117.5, 119.6, 123.0 (min.), 124.4, 124.6 (min.), 127.5,
129.50, 129.55, 129.58, 129.7, 131.4, 131.9 (min.), 136.9, 155.1, 170.1.
1H NMR (DMSO-d6): 2.25 (s, 3H, CH3), 2.34 (s, 3H, CH3, min.), 4.43 (s, 2H, CH2, min.), 4.70 (s, 2H, CH2),
6.82 (td, 1H, min.), 6.87 (dd, 1H, min.), 6.94 (dd, 1H, min.), 7.15Ϫ7.16 (m, 1H, min.), 7.17Ϫ7.18 (m, 2H, Ar-H),
7.20Ϫ7.22 (m, 2H, Ar-H), 7.20Ϫ7.22 (m, 1H, Ar-H, min.), 7.26 (dd, 1H, J ϭ 1.4, 7.6, Ar-H), 7.33Ϫ7.42 (m, 3H,
Ar-H), 7.33Ϫ7.42 (m, 3H, Ar-H, min.), 10.41 (brs, NH).
4
1.4/1
1H NMR (CDCl3؉DMSO-d6): 2.14 (s, 3H, CH3), 4.48 (s, 2H, CH2), 6.73 (d, 1H, J ϭ 7.0, Ar-H), 6.87 (d, 1H,
J ϭ 7.8, Ar-H), 6.93Ϫ7.05 (m, 4H, Ar-H),7.16 (d, 1H, J ϭ 7.8, Ar-H), 7.28Ϫ7.30 (m, 1H, Ar-H), 9.62 (brs, NH).
13C NMR: 21.5 (min.), 21.8, 45.3, 52.3 (min.), 110.2, 110.3, 117.1, 117.6 (min.), 120.5, 123.0, 124.2 (min.), 124.6,
125.1 (min.), 127.9, 129.30 (min.), 129.32 (min.), 129.35, 129.5 (min.), 129.6 (min.), 132.2, 132.9 (min.), 138.7,
139.0 (min.), 139.1, 142.7, 154.8, 155.0 (min.), 165.2, 170.0 (min.).
1H NMR (DMSO-d6): 2.23 (s, 3H, CH3), 2.33 (s, 3H, CH3, min.), 4.42 (s, 2H, CH2, min.), 4.68 (s, 2H, CH2),
6.85 (td, 1H, min.), 6.93 (dd, 1H, J ϭ 1.2, 8.2, min.), 7.10 (d, 2H, J ϭ 8.2, Ar-H), 7.13 (d, 1H, min.), 7.15 (dd,
1H, min.), 7.19 (td, 1H, J ϭ 1.2, 7.8, Ar-H), 7.25 (dd, 1H, J ϭ 1.2, 7.8, Ar- H), 7.34Ϫ7.35 (m, 1H, Ar-H),
7.36Ϫ7.37 (m, 1H, Ar-H), 7.43 (d, 2H, J ϭ 7.4, Ar-H), 10.29 (brs, NH).
1H NMR (CDCl3؉DMSO-d6): 2.15 (s, 3H, CH3), 4.48 (s, 2H, CH2), 6.89 (dd, 1H, J ϭ 1.0, 7.6, Ar-H), 6.93 (d,
2H, J ϭ 8.2, Ar-H), 6.97 (dd, 1H, J ϭ 1.4, 7.8, Ar-H), 7.01 (dd, 1H, J ϭ 1.4, 7.6, Ar- H), 7.04Ϫ7.07 (m, 1H, Ar-
H), 7.30 (d, 2H, J ϭ 8.2, Ar-H), 9.58 (brs, NH).
5
6
1.7/1
13C NMR: 21.1, 21.4 (min.), 45.3, 52.4 (min.), 110.2, 110.3, 117.5 (min.), 120.0, 123.0, 124.2 (min.), 124.6, 127.2,
129.5 (min.), 129.6 (min.), 129.91, 129.96, 130.3 (min.), 132.3, 133.4, 136.6, 138.1 (min.), 142.7, 154.1 (min.),
154.8, 155.1 (min.), 165.0, 170.1 (min.).
1H NMR (DMSO-d6): 2.01 (s, 3H, CH3), 2.81 (t, 2H, J ϭ 6.6, α-H), 4.11 (t, 2H, J ϭ 6.6, β-H), 7.03 (td, 1H,
J ϭ 1.2, 7.4, Ar-H), 7.07Ϫ7.14 (m, 3H, Ar-H), 7.19 (td, 1H, J ϭ 1.2, 7.8, Ar-H), 7.23 (d, 1H, J ϭ 7.8 Ar-H),
7.28 (dd, 1H, J ϭ 1.4, 7.2, Ar-H), 7.30 (dd, 1H, J ϭ 0.8, 8.6, Ar-H), 9.39 (brs, NH).
13C NMR: 18.3, 34.6, 39.4, 110.2, 110.3, 122.8, 124.5, 125.8, 125.9, 126.5, 130.9, 131.7, 132.4, 136.7,
142.7,154.3, 169.3.
Ϫ
1H NMR (DMSO-d6): 4.44 (s, 2H, CH2, min.), 4.72 (s, 2H, CH2), 6.85 (td, 1H, min.), 6.94 (dd, 1H, min.), 7.13(td,
2H, J ϭ 1.2, 7.8, Ar-H), 7.20 (td, 1H, J ϭ 1.2, 7.8, Ar-H), 7.26 (dd, 1H J ϭ 1.2, 7.8, Ar-H), 7.32Ϫ7.37 (m, 2H,
Ar-H), 7.42 (dt, 1H, J ϭ 1.2, 8.2, Ar-H), 7.48Ϫ7.56 (m, 1H, min.), 7.75 (t, 1H J ϭ 2.0, Ar-H), 10.58 (brs, NH).
13C NMR: 45.4, 52.4 (min.), 110.2, 110.4, 117.5 (min.), 118.4, 119.5, 119.9 (min.), 123.1, 123.9 (min.), 124.2,
124.7, 126.0 (min.), 127.1 (min.), 128.5 (min.), 129.4 (min.), 129.7 (min.), 131.2 (min.), 131.3, 132.2, 133.5 (min.),
133.9, 134.3 (min.), 140.5, 142.7, 154.0 (min.), 154.5 (min.), 154.8, 165.7, 169.8 (min.).
10
12
9/1
1H NMR (DMSO-d6): 2.86 (t, 2H, Jϭ6.8, α-H), 4.11 (t, 2H, J ϭ 6.8, β-H), 7.08Ϫ7.21 (m, 3H, Ar-H), 7.25Ϫ7.32
(m, 3H, Ar-H), 7.42 (dd, 1H, J ϭ 1.2, 8.0, Ar-H), 7.55 (d, 1H, J ϭ 7.6, Ar-H), 9.66 (brs, NH).
Ϫ
13C NMR: 34.6, 39.2, 110.2, 110.3, 122.8, 124.5, 127.1, 127.3, 128.0, 130.1, 131.6, 135.3, 142.7, 154.3, 169.7.
1H NMR (DMSO-d6): 4.48 (s, 2H, CH2, min.), 4.77 (s, 2H, CH2), 6.86 (td, 1H, min.), 6.94 (dd, 1H, min.), 7.14
(td, 1H, J ϭ 1.6, 7.8, Ar-H), 7.20 (dd, 1H, J ϭ 1.0, 7.7, Ar-H), 7.28 (dd, 1H, J ϭ 1.0, 7.6, Ar-H), 7.37 (d, 1H,
J ϭ 7.8, Ar-H), 7.62 (t, 1H, J ϭ 8.2, Ar-H), 7.81 (t, 1H, min.), 7.89 (dd, 1H, J ϭ 2.0, 7.8, Ar-H), 7.93 (dd, 1H,
J ϭ 2.3, 8.2, Ar-H),8.26 (dd, 1H, min.), 8.36 (t, 1H, min.), 8.57 (t, 1H, J ϭ 2.1, Ar-H), 10.89 (brs, NH).
13C NMR: 50.2, 57.1 (min.), 113.1 (min.), 115.0, 115.2, 118.9, 122.2 (min.), 123.7, 124.6 (min.), 126.6, (min.),
127.9, 128.5 (min.), 129.4, 130.7, 134.1 (min.), 134.6 (min.), 135.8, 136.9, 138.2 (min.), 138.7, (min.), 144.9, 147.4,
153.2(min.), 153.4, 158.7 (min.), 159.1 (min.), 159.5, 170.9, 174.5 (min.).
16
18
11/1
1H NMR (DMSO-d6): 2.84 (t, 2H, Jϭ6.6, α-H), 4.08 (t, 2H, J ϭ 6.6, β-H), 7.09 (td, 1H, J ϭ 1.2, 7.8, Ar-H),
7.17 (td, 1H, J ϭ 1.2, 7.6, Ar-H), 7.26 (dd, 1H, J ϭ 1.0, 8.0, Ar-H), 7.30 (dd, 1H, J ϭ 1.0, 8.4, Ar-H),7.34 (dd,
1H, J ϭ 1.4, 7.2, Ar-H), 7.49 (dd, 1H, J ϭ 1.6, 8.2, Ar-H), 7.65 (td, 1H, J ϭ 1.6, 7.8, Ar-H), 7.89 (dd, 1H, J ϭ
1.6, 8.2, Ar-H), 10.36 (brs, NH).
Ϫ
13C NMR: 34.6, 38.8, 110.1, 110.2, 112.8, 124.5, 125.5, 126.1, 126.2, 131.5, 131.6, 134.6, 142.7, 143.2, 154.3, 169.6
†
According to the ratio of methylene signal in rotamers.
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408
Pabuccuoglu et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 405−410
Table 3. The inhibitor effects of title compounds on human
leukocyte MPO chlorinating activity.
rotamer existence. However, the propionamides did not
cause any rotamer formation in NMR spectra recorded in
DMSO-d6. The rotamer formation in acetamide derivatives
could be related to the restricted rotation of amide N-CO
bond, which is known to produce E/Z rotamers [17]. In gen-
Compound
[1 mM]
% Inhibition of MPO activity
(Mean SD) (n ؍ 5)
1
eral, H-NMR and 13C-NMR spectra in either series dem-
1
18.50 2.07
58.06 3.50
90.80 1.40
73.40 2.20
30.50 1.80
11.90 1.10
63.80 1.60
2.10 0.10
26.14 0.60
22.80 1.90
0
29.80 2.20
85.20 1.50
26.60 1.50
22.30 3.20
30.50 3.60
63.00 1.70
79.20 1.40
52.20 3.70
94.10 2.50
96.7 0.00
onstrated the expected resonance frequencies suggesting the
structural confirmation. The representative examples of
NMR data of title compounds are summarized at Table 2.
2
3
4
5
ESI-MS spectra with negative polarity provided the ions
accounting for molecular weights of the title compounds.
Main fragmentation seems to occur at α-position of amide
carbonyl (α-cleavage) to furnish m/z 148 and 134 ions corre-
6
7
8
9
10
sponding to [C8H6NO2]ϩ and [C7H4NO2]ϩ respectively.
,
11
Those two ions, derived from 2(3H)-benzoxazolone portion
of amide bond, were the base peaks of title compounds ex-
cept 6, 12, 16, 18-20 (Table 1).
12
13
14
15
16
17
Biology
18
19
20
In vitro MPO-chlorinating activity inhibition of 2(3H)-
benzoxazolone derivatives was determined on human poly-
morphonuclear leukocyte isolated from venous blood of
healthy volunteers according to the reported procedure [1].
The assay is based on the chlorination of taurine with the
MPO/H2O2/ClϪ system. The reaction of taurine with hypo-
chlorous acid produces taurine chloramine, which is meas-
ured by reaction with 5-thio-2-nitrobenzoic acid (TNB).
Yellow TNB is oxidized to colorless 5,5’-dithiobis-2-nitro-
benzoic acid (DTNB), which was detected spectrophoto-
metrically at 412 nm.
aminopyrine
ring has produced diverse biological activities and many de-
rivatives of 2(3H)-benzoxazolone have been known to pos-
sess analgesic and anti-inflammatory properties [18Ϫ24].
Substituents on N-phenyl ring with diverse electronic and
liphophilic properties were chosen and their contributions
to the activity were assessed in terms of their position on
N-phenyl ring.
Experimentally, five measurements were performed for each
compound. Aminopyrine, a potent inhibitor of MPO ac-
tivity, was used as standard [5]. Results were expressed as%
inhibition of MPO-chlorinating activity at 1 mM concen-
tration of tested compounds (Table 3).
According to our assay system, the title compounds, except
compound 11, exhibit varying inhibition percentage on the
chlorination activity of MPO (Table 3). In general, propion-
amide derivatives with chlorine and nitro substituents and
non-substituted ones are more active than the acetamide
counterparts whereas methyl substitution renders the aceta-
mide derivatives more active than the propionamide anal-
ogues. Methyl and chlorine exhibit similar contributions in
either series in terms of substitution pattern. Their inhi-
bition percentage decreases in the order of o > m > p in
acetamides and m > o > p in case of propionamides. A nitro
substituent produces the most active compounds when it is
in para position. The inhibition activity of nitro-substituted
compounds decreases in the order p > m > o in acetamides
and p > o > m in propionamides.
The taurine chlorination assay was also used to investigate
HOCl scavenging capacity for compounds 1 and 20 at con-
centration studied in MPO chlorination assay. Experiments
were performed according to the reported procedure [6].
Five measurements were realized for each compound. The
compounds studied did not interfere with the chlorination
of taurine.
Discussion
Because of the many known effects of HOCl, modulation
of the MPO function has a considerable potential for thera-
peutic intervention in numerous pathologies [4, 7, 9]. There-
fore, our attention focuses on the chlorination activity of
MPO as a possible therapeutic target. Our title compounds
are ω-[2-oxo-3H-benzoxazol-3-yl]-N-phenylacetamide and
propionamide derivatives since the 2(3H)-benzoxazolone
Among the substituents studied, a methyl group in ortho
and meta positions in acetamides, a nitro group in ortho and
para, or a chlorine atom in meta positions in propionamides
produce the most active compounds in their series. In either
series, the nitro group, an electron-withdrawing substituent,
optimized the activity for the para position. The inhibition
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Arch. Pharm. Chem. Life Sci. 2005, 338, 405−410
Synthesis of 2(3H)-Benzoxazolone Derivatives and MPO Activity
409
Isolation of polymorphonuclear leukocyte
capacity of 3 and 20 as the most active compounds with
inhibition percentage over 90% are comparable with that
of aminopyrine used as standard. According to the HOCl-
scavenging activity experiment, both of the compounds
were unable to react directly with hypochloride, thus con-
firming their MPO-specific inhibition of chlorination.
Human polymorphonuclear leukocytes were isolated from freshly
sampled venous blood of healthy volunteers by using dextran (6%
dextran in 0.9% NaCl solution) and heparin (25000 IU/mL) [25,
26]. MPO was solubilized by homogenizing leukocytes with deter-
gent (HETAB)-containing buffer [2].
MPO chlorinating activity
Those results suggest that ω-[2-oxo-3H-benzoxazol-3-yl]-N-
phenylacetamide and propionamide derivatives can be start-
ing compounds to obtain potent inhibitors of MPO-chlorin-
ating activity. Our effort will continue to do so.
MPO-chlorinating activity was determined by measuring the chlori-
nation of taurine with the MPO/H2O2/Cl- system [1]. This assay is
based on the reaction of hypochlorous acid with taurine to produce
taurine chloramine, which is measured by reacting taurine chlor-
amine with 5-thio-2-nitrobenzoic acid (TNB) [27]. Yellow TNB is
oxidized to colorless 5,5’-dithiobis-2-nitro benzoic acid (DTNB),
which was detected spectrophotometrically at 412 nm. TNB was
prepared by raising the pH of a solution of 2 mM DTNB in buffer
to 12 and readjusting to pH 7.4 as previously described by Kettle
and Winterbourn [1]. The stock solution of compounds 1Ϫ20 was
prepared at 10 mM concentration in DMF fresh daily. The reaction
mixture contained the following reagents in a final volume of 1 mL:
15 µL of leukocyte homogenate (including 30 µg protein), 585 µL
of 10 mM KH2PO4/KOH buffer containing 300 mM NaCl, at pH
7.4, 100 µL of 150 mM taurine, 100 µL of tested compound’s stock
solution (final concentration 1 mM), and 100 µL of distilled water.
The assay was initiated by the addition of 100 µL of 1 mM H2O2.
The mixture was incubated 5 min at 37°C and the reaction was
stopped by addition of 100 µL of catalase (4U/mL). After the reac-
tion stopped, 750 µL of TNB and 2.150 mL of distilled water were
added and absorbance was measured at 412 nm [1, 6, 27]. Five
measurements were performed under each set of experimental con-
ditions.
Acknowledgments
This study was supported by research grants from Ege Uni-
versity (project number: 2001/Ecz/19) and The Scientific
and Technical Research Council of Turkey (TUBITAK;
project number: SBAG/AYD 456).
Experimental
Chemistry
Melting points were determined on a Büchi 510 Melting point ap-
paratus (Büchi Labortechnik, Flawil, Switzerland) and are uncor-
rected. The IR spectra of compounds were recorded as potassium
bromide pellets on a Jasco FT/IR-400 spectrometer (Jasco, Tokyo,
Japan). The NMR spectra were recorded on a Varian AS 400 FT-
NMR (Varian Inc., Palo Alto, CA, USA) using DMSO-d6 and
CDCl3/DMSO-d6 mixture (0.6 mL /5 drops) as solvent. Chemical
shifts were reported in parts per million (δ). J values were given in
Hz. LC-MS spectra (-ESI-MS) were measured on an Agilent 1100
MSD spectrometer (Palo Alto, CA, USA). Elemental analyses
(C, H, N) were performed by TUBITAK Analytical Laboratory
Ankara, Turkey.
HOCl Scavenging Activity
The experiment was performed according to the reported procedure
[6]. The reaction mixture contained the following reagents in a final
volume of 1 mL: 100 µL of 150 mM taurine, 100 µL of tested com-
pound’s stock solution (final concentration 1 mM), 700 µL of 10
mM KH2PO4/KOH buffer containing 140 mM NaCl, at pH 7.4.
The assay was initiated by the addition of 100 µL of NaOCl,
600 µM (final concentration 60 µM). The mixture was incubated
10 min at 37°C. After the addition 750 µL of TNB 0.45 mM (stock
TNB solution was diluted to 0.45 mM) and 2.250 mL of water, the
absorbance was recorded at 412 nm. The results compared with the
compound-free reference.
2(3H)-Benzoxazolone [11] and ω-chloroanilides [10] were prepared
according to the reported procedures.
Synthesis of ω-[2-oxo-3H-benzoxazol-3-yl]-N-phenylacetamide and
propionamide derivatives 1Ϫ20
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points are reported in Table 1.
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