Facile Synthesis and In Vitro Nitric Oxide Production Inhibitory Activity of Benzoxazoles

Article abstract of DOI:10.1002/bkcs.11465

A Facile synthesis of natural benzoxazoles, nocarbenzoxazoles F (1), G (5) and their derivatives (2–4 and 6–8) was achieved from the commercially available inexpensive precursors with overall yields ranging from 15 to 49%. Our strategy to access this fami

Full text of DOI:10.1002/bkcs.11465

Article
DOI: 10.1002/bkcs.11465
BULLETIN OF THE
H. J. Park et al.
KOREAN CHEMICAL SOCIETY
Facile Synthesis and In Vitro Nitric Oxide Production Inhibitory
Activity of Benzoxazoles
Hyeong Jin Park,† Jin-Kyung Kim,‡ and Jong-Gab Jun†,
*
†Department of Chemistry and Institute of Applied Chemistry, Hallym University, Chuncheon 24252,
South Korea. *E-mail: jgjun@hallym.ac.kr
‡Department of Biomedical Science, College of Natural Science, Catholic University of Daegu,
Gyeungsan-Si 38430, South Korea
Received March 5, 2018, Accepted April 5, 2018
A Facile synthesis of natural benzoxazoles, nocarbenzoxazoles F (1), G (5) and their derivatives (2–4 and
6–8) was achieved from the commercially available inexpensive precursors with overall yields ranging
from 15 to 49%. Our strategy to access this family of benzoxazoles was enabled by POCl3-mediated
cyclodehydration, selective and/or complete demethylation and reduction as the key steps. Their in vitro
nitric oxide (NO) inhibitory effect was further evaluated as an indicator of anti-inflammatory activity in
LPS-induced RAW 264.7 cells and found to display weak to moderate activity in a concentration-
dependent manner without marked cytotoxicity. Overall, compound 8 (53.5% at 10 μM; IC50 = 8.17 μM)
followed by compound 6 (12.7% at 10 μM; IC50 = 28.26 μM) exhibited significant activity, being more
active than the positive control, L-NMMA (19.5% at 10 μM; IC50 = 18.77 μM).
Keywords: Benzoxazoles, Nocarbenzoxazoles F & G, Cyclodehydration, Nitric oxide, RAW-264,7
macrophages, Anti-inflammatory
Introduction
ibuprofen, corticosteroids, selective COX-2 inhibitors
(COXIBs), and antihistamines are commonly prescribed for
treatment of pain, fever and inflammation. These work
either by interrupting the synthesis or action of mediators
that drive the host’s response to injury. Though these
small-molecule inhibitors have contributed the essential
treatment for various inflammatory diseases, their use is
restricted by their common side effects, such as gastric
ulceration, joint destruction, renal toxicity, and cardiovascu-
lar disorders.6,7 Consequently, there is a need for novel
drugs that possess improved efficacy and minimal side
effect profiles to address these problems.
Benzoxazole, a benzene fused oxazole ring structure, is
an important heterocycle. Many of its derivatives are used
as building blocks for biochemicals and pharmaceuticals as
well as in other industrial applications such as pesticides,
dyes, textile auxiliaries, and plastics.8,9 The substituted ben-
zoxazoles have been shown to display diverse biological
activities, such as antibiotic, anticancer agent, antimicrobial,
antifungal, anti-parkinson, anti-inflammatory, anticonvul-
sant, immunosuppressive antiviral, and antiparasitic.8,9
Some benzoxazoles also exhibited enzyme inhibitory activ-
ity.10 In addition, some benzoxazoles are recognized as an
important scaffold in fluorescent probes such as anion and
metal cation sensors.11
Inflammation is the body’s local protective attempt to
endogenous and exogenous insults such as pathogens,
infection or disease. It can present as acute or chronic con-
dition.1 Although, this response enables the body to limit
invasions and damage after injury, long-term inflammation
has been linked to the development of several diseases and
conditions including cardiovascular diseases, rheumatoid
arthritis, cancer, bronchitis, diabetes, and neurodegenerative
disorders.2 The inflammatory process involves the release
of various chemical mediators such as cytokines (interleu-
kin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)),
histamines, prostaglandins (PGs), chemokines, complement
fragments and reactive molecules such as nitric oxide
(NO) released by leukocytes.3 Out of these, NO, a small
and transient-free radical generated by three similar nitric
oxide synthases (NOS) viz. endothelial-NOS, neuronal-
NOS and inducible-NOS, has a pivotal role in the patho-
genesis of inflammation.4 It acts as both a “friend and foe”
depending upon its concentration.5 NO has anti-
inflammatory effects under normal physiological
conditions. However, elevation of NO production by
inducible-NOS is implicated in many pathophysiological
processes. Therefore, pharmacological intervention of NO
production will generate massive impact on the future man-
agement of inflammation-based diseases.
Substituted benzoxazoles (1–8) under the current study
are depicted in Figure 1. Compounds 1 (nocarbenzoxazole
F) and 5 (nocarbenzoxazole G) were natural products iso-
lated12 from the halophilic strain Nocardiopsis lucentensis
Non-steroidal anti-inflammatory drugs (NSAIDs), includ-
ing over the-counter (OTC) drugs such as aspirin and
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and then refluxed for 12 h. After completion of the reac-
tion, the reaction mixture was cooled to room temperature,
poured into crushed ice and neutralized with aqueous satu-
rated NaHCO3 solution. The reaction mixture was extracted
with EtOAc (3 × 45 mL). The combined organic layer was
washed with H2O (2 × 20 mL) and brine (20 mL), dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude was purified by column chromatogra-
phy (EtOAc:hexane = 1:2) to afford the pure product 10
(1.01 g, 85%) as pale orange color solid; mp 96–98ꢀC;
Rf = 0.33 (EtOAc/hexane = 1/2); 1H NMR (300 MHz,
CDCl3) δ 7.44 (1H, d, J = 1.8 Hz), 7.41 (1H, dd, J = 7.8,
1.8 Hz), 6.75 (1H, d, J = 7.8 Hz), 4.32 (2H, q, J = 7.2 Hz)
1.37 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3) δ
167.0, 148.6, 134.1, 123.3, 122.2, 118.2, 114.6, 61.0, 14.7.
General Procedure for Substituted Benzoyl Chlorides
(14, 16, and 17) Preparation From Substituted Benzoic
Acids (11−13): SOCl2 (2 mL) was added to substituted
benzoic acid (0.55 mmol) under nitrogen atmosphere at
0ꢀC. The resulting mixture was warmed to 60ꢀC and stirred
for 2 h. After completion of the reaction, the reaction mix-
ture was cooled to room temperature and the excess SOCl2
was removed under reduced pressure. The resulting acid
chloride was kept under vacuum for 30 min and used in the
next step without further purification.
General Procedure for POCl3-Mediated Synthesis of
2-Aryl Benzo[d]oxazole-5-carboxylate: To a stirred solu-
tion of 10 (0.55 mmol) in anhydrous 1,4-dioxane (3 mL)
was added substituted benzoyl chloride (1.1 mmol, 2.0
equiv.) in anhydrous 1,4-dioxane (2 mL) at room tempera-
ture under nitrogen atmosphere. The reaction mixture was
then warmed to 90ꢀC and stirred for 3 h. At this stage,
POCl3 (0.16 mL, 1.65 mmol, 3.0 equivalents) was added
dropwise and stirring was continued further 12 h. After
completion of the reaction, the reaction mixture was cooled
to room temperature, solvent and the excess POCl3 was
removed under reduced pressure. EtOAc (25 mL) and H2O
(10 mL) were added to the crude compound and two layers
were separated. Aqueous layer was extracted with EtOAc
(2 × 20 mL). The combined organic layer was washed with
H2O (2 × 15 mL) and brine (15 mL), dried over anhydrous
Na2SO4 and concentrated in vacuo. The crude was purified
by column chromatography (EtOAc:hexane = 1:5–1:2) to
afford the pure product.
Figure 1. Structures of nocarbenzoxazoles F (1), G (5) and their
derivatives (2–4 and 6–8).
DSM 44048, whereas, compounds 2–4 and 6–8 are their
synthetic derivatives. Nocarbenzoxazole G (5) exhibited
selective activity against HepG2 and HeLa with IC50 values
of 3 and 1 μM, respectively.12 However, no report has been
issued on their anti-inflammatory activity and also the syn-
thesis of 3, 4, 6, and 8. Hence, as part of our on-going
efforts directed toward the synthesis of bioactive natural
products and their derivatives as inhibitors of nitric oxide
production,13 herein, we report a facile synthesis along with
in vitro biological assessment of the benzoxazoles 1–8.
Experimental
All commercially available reagents were purchased from
local sources and used as received unless stated otherwise.
All solvents used for reactions were freshly distilled from
proper dehydrating agents under nitrogen atmosphere. All
solvents used for chromatography were purchased and
directly used without further purification. Reactions pro-
gress was monitored by thin-layer chromatography (TLC)
on silica gel 60 F254 (Merck, Darmstadt, Germany) plates
and visualized by UV light (254 nm) and staining with
phosphomolybdic acid (PMA) and/or p-anisaldehyde. Silica
gel 60 (230–400 mesh, Merck, Darmstadt, Germany) was
used for purification of the obtained compounds by column
1
chromatography. H NMR spectra were recorded on Varian
Mercury-300 MHz FT-NMR (Palo alto, CA, USA) and
75 MHz for 13C, with the chemical shift (δ) reported in
parts per million (ppm) downfield relative to TMS and the
coupling constants (J) quoted in Hz. Peak splitting patterns
were abbreviated as s (singlet), d (doublet), t (triplet), q
(quartet), dd (doublet of doublet) and m (multiplet) and
CDCl3/DMSO‑d6/CD3COCD3/CD3OD was used as a sol-
vent and an internal standard. Low resolution and high res-
olution mass spectra were recorded on Agilent-5977E
(Agilent Technologies Inc., Santa Clara, California, USA)
and JMS-700 (JEOL Ltd., Tokyo, Japan) spectrometers,
respectively. Melting points were determined in glass capil-
laries under air with a MEL-TEMP II (Triad Scientific,
Manasquan, NJ, USA) apparatus and were uncorrected.
Ethyl 3-amino-4-hydroxybenzoate (10): To a stirred
solution of 3-amino-4-hydroxybenzoic acid (9) (1.0 g,
6.53 mmol) in EtOH (10 mL) was added concentrated
H2SO4 (3.0 mL) at 0ꢀC under nitrogen atmosphere. After
completion of addition, the reaction mixture was warmed
Ethyl 2-(4-methoxyphenyl)benzo[d]oxazole-5-carboxyl
ate (18): Yield: 55%; off-white solid; mp 156–158ꢀC;
Rf = 0.73 (EtOAc/hexane = 1/2); 1H NMR (300 MHz,
CDCl3) δ 8.38 (1H, s), 8.14 (2H, d, J = 8.7 Hz), 8.04 (1H,
d, J = 8.4 Hz), 7.53 (1H, d, J = 8.4 Hz), 6.99 (2H, d,
J = 8.7 Hz), 4.40 (2H, q, J = 7.2 Hz), 3.86 (3H, s), 1.42
(3H, t, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3) δ 166.3,
164.3, 162.6, 153.5, 142.4, 129.6, 127.2, 126.6, 121.5,
119.1, 114.5, 110.1, 61.3, 55.6, 14.6.
Ethyl 2-phenylbenzo[d]oxazole-5-carboxylate (19):
Yield: 60%; off-white solid; mp 102–104ꢀC; Rf = 0.54
1
(EtOAc/hexane = 1/3); H NMR (300 MHz, CDCl3) δ 8.42
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Synthesis and In Vitro Nitric Oxide Production Inhibitory
BULLETIN OF THE
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Activity of Benzoxazoles
KOREAN CHEMICAL SOCIETY
(1H, d, J = 0.9 Hz), 8.22–8.18 (2H, m), 8.06 (1H, dd,
J = 8.7, 1.8 Hz), 7.56–7.48 (4H, m), 4.40 (2H, q,
J = 7.2 Hz), 1.42 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz,
CDCl3) δ 166.1, 164.2, 153.6, 142.2, 131.9, 129.0, 127.8,
127.4, 127.0, 126.7, 122.0, 110.3, 61.3, 14.6.
(1H, d, J = 2.1 Hz), 7.54 (1H, dd, J = 8.4, 2.1 Hz), 6.92
(1H, d, J = 8.4 Hz), 4.34 (2H, q, J = 7.2 Hz), 1.35 (3H, t,
J = 7.2 Hz); 13C NMR (75 MHz, DMSO‑d6) δ 165.2,
164.1, 152.8, 149.8, 145.6, 141.8, 126.5, 125.9, 120.0,
119.9, 116.6, 116.0, 114.3, 110.6, 60.8, 14.2.
Ethyl 2-(2,4-dimethoxyphenyl)benzo[d]oxazole-5-carb
oxylate (20): Yield: 48%; off-white solid; mp 148–150ꢀC;
Rf = 0.61 (EtOAc/hexane = 1/2); 1H NMR (300 MHz,
CDCl3) δ 8.45 (1H, s), 8.08 (1H, J = 9.3 Hz), 8.03 (1H, d,
J = 9.3 Hz), 7.54 (1H, d, J = 8.7 Hz), 6.60 (1H, d,
J = 8.7 Hz), 6.58 (1H, s), 4.40 (2H, q, J = 6.9 Hz), 4.00
(3H, s), 3.87 (3H, s), 1.42 (3H, t, J = 6.9 Hz); 13C NMR
(75 MHz, CDCl3) δ 166.2, 163.7, 162.8, 159.9, 152.9,
142.2, 132.5, 126.8, 126.2, 121.6, 109.8, 108.4, 105.3,
99.0, 61.0, 56.1, 55.5, 14.4.
Ethyl 2-(3,4-dimethoxyphenyl)benzo[d]oxazole-5-carb
oxylate (21): Yield: 53%; off-white solid; mp 146–148ꢀC;
Rf = 0.47 (EtOAc/hexane = 1/2); 1H NMR (300 MHz,
CDCl3) δ 8.36 (1H, d, J = 1.8 Hz), 8.03 (1H, dd, J = 8.7,
1.8 Hz), 7.78 (1H, dd, J = 8.4, 2.1 Hz), 7.67 (1H, d,
J = 2.1 Hz),7.50 (1H, d, J = 8.7 Hz), 6.92 (1H, d,
J = 8.4 Hz), 4.39 (2H, q, J = 7.2 Hz), 3.98 (3H, s), 3.94
(3H, s), 1.42 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz,
CDCl3) δ 166.2, 164.3, 153.5, 152.3, 149.3, 142.3, 127.3,
126.6, 121.5, 121.4, 119.2, 111.1, 110.2, 110.1, 61.3, 56.3,
56.2, 14.6.
Ethyl
2-(2-hydroxy-4-methoxyphenyl)benzo[d]oxa-
zole-5-carboxylate (24): To a stirred solution of compound
21 (0.09 g, 0.27 mmol) in anhydrous CH2Cl2 (4 mL) was
added BCl3 (1.0 M in CH2Cl2, 0.28 mL) dropwise at 0ꢀC
under nitrogen atmosphere. The reaction mixture was
warmed to room temperature and stirred for 2 h. After com-
pletion of the reaction, the reaction mixture was cooled to
0ꢀC. 1.0 N HCl (1 mL) was added slowly to the reaction
mixture and stirred for 15 min. The mixture was then
extracted with CH2Cl2 (2 × 20 mL). The combined organic
layer was washed with H2O (2 × 10 mL) and brine
(10 mL), dried over anhydrous Na2SO4 and concentrated in
vacuo. The crude was purified by column chromatography
(EtOAc:hexane = 1:2) to afford the pure product 24
(0.08 g, 89%) as off-white solid. mp 154–156ꢀC; Rf = 0.70
(EtOAc/hexane = 1/2); 1H NMR (300 MHz, CDCl3) δ
11.33 (1H, s), 8.31 (1H, d, J = 1.8 Hz), 8.05 (1H, dd,
J = 8.4, 1.8 Hz), 7.84 (1H, d, J = 9.0 Hz), 7.54 (1H, d,
J = 8.4 Hz), 6.57 (1H, d, J = 2.1 Hz), 6.54 (1H, dd,
J = 9.0, 2.1 Hz), 4.41 (2H, q, J = 7.2 Hz), 3.85 (3H, s),
1.44 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3) δ
165.8, 164.4, 164.1, 160.8, 151.6, 140.2, 128.2, 127.5,
126.6, 120.4, 110.0, 107.8, 103.1, 101.2, 61.2, 55.5, 14.4.
General Procedure for Demethylation Using BBr3: To
a
stirred solution of ethyl 2-aryl benzo[d]oxazole-
5-carboxylate (0.2 mmol) in anhydrous CH2Cl2 (4 mL) was
added BBr3 (1.0 M in CH2Cl2, 0.3 mL, 1.5 equivalents)
dropwise at 0ꢀC under nitrogen atmosphere. The reaction
mixture was warmed to room temperature and stirred for
3 h. After completion of the reaction, the reaction mixture
was cooled to 0ꢀC. 1.0 N HCl (1 mL) was added slowly to
the reaction mixture, stirred for 15 min and extracted with
CH2Cl2 (2 × 20 mL). The combined organic layer was
washed with H2O (2 × 10 mL) and brine (10 mL), dried
over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude was purified by column chromatogra-
phy (EtOAc:hexane = 1:2–1:1) to afford the pure product.
[Note: 3.0 equivalents of BBr3 was employed in case of
compound 21].
Ethyl
2-(4-hydroxy-2-methoxyphenyl)benzo[d]oxa-
zole-5-carboxylate (25): To a stirred solution of 21
(0.09 g, 0.27 mmol) in anhydrous CH2Cl2 (4 mL) was
added AlCl3 (0.12 g, 0.92 mmol) under nitrogen atmo-
sphere. The reaction mixture was stirred for 48 h at room
temperature. After completion of the reaction, H2O (1 mL)
was added slowly to the reaction mixture and stirred for
15 min. The mixture was extracted with CH2Cl2
(2 × 25 mL). Combined organic layer was washed with
H2O (2 × 10 mL) and brine (10 mL), dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The
crude was purified by column chromatography (EtOAc:
hexane = 1:2) to afford the pure product 25 (0.04 g, 42%)
as off-white solid. mp 204–206ꢀC; Rf = 0.22 (EtOAc/hex-
1
Ethyl
2-(4-hydroxyphenyl)benzo[d]oxazole-
ane = 1/2); H NMR (300 MHz, CD3COCD3) δ 9.48 (1H,
5-carboxylate (22): Yield: 71%; off-white solid; mp
210–212ꢀC; Rf = 0.55 (EtOAc/hexane = 1/2); 1H NMR
(300 MHz, DMSO‑d6) δ 8.26 (1H, d, J = 1.5 Hz),
8.08–8.03 (3H, m), 7.68 (1H, d, J = 8.7 Hz), 6.95 (2H, d,
J = 8.4 Hz), 4.38 (2H, q, J = 7.2 Hz), 1.43 (3H, t,
J = 7.2 Hz); 13C NMR (75 MHz, DMSO‑d6) δ 167.5,
166.6, 163.1, 154.8, 143.3, 131.0, 128.6, 127.7, 121.6,
118.4, 117.2, 111.6, 62.6, 14.9.
s), 8.29 (1H, s), 8.04 (1H, d, J = 8.4 Hz), 8.00 (1H, d,
J = 8.4 Hz), 7.71 (1H, d, J = 8.7 Hz), 6.68 (1H, s), 6.63
(1H, d, J = 8.4 Hz), 4.38 (2H, q, J = 7.2 Hz), 3.94 (3H, s),
1.40 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz, CD3COCD3)
δ 166.3, 163.2, 161.3, 154.1, 143.1, 133.6, 127.8, 126.8,
121.4, 111.0, 108.8, 106.9, 100.5, 99.7, 61.6, 56.2, 14.7.
Ethyl
2-(2,4-dihydroxyphenyl)benzo[d]oxazole-
5-carboxylate (26): Following the procedure used for com-
pound 24 preparation, treatment of compound 25 (0.09 g,
0.29 mmol) with BCl3 (1.0 M in CH2Cl2, 0.58 mL) gave
the compound 26 (0.08 g, 97%) as off-white solid. mp
218–220ꢀC; Rf = 0.12 (EtOAc/hexane = 1/1); 1H NMR
(300 MHz, CD3COCD3) δ 11.19 (1H, s), 9.53 (1H, s), 8.27
Ethyl
2-(3,4-dihydroxyphenyl)benzo[d]oxazole-
5-carboxylate (23): Yield: 75%; off-white solid; mp
214–216ꢀC; Rf = 0.35 (EtOAc/hexane = 1/2); 1H NMR
(300 MHz, DMSO‑d6) δ 8.21 (1H, d, J = 1.8 Hz), 7.96
(1H, d, J = 8.4 Hz), 7.81 (1H, dd, J = 8.4, 1.8 Hz), 7.58
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Synthesis and In Vitro Nitric Oxide Production Inhibitory
BULLETIN OF THE
Article
Activity of Benzoxazoles
KOREAN CHEMICAL SOCIETY
(1H, d, J = 1.2 Hz), 8.06 (1H, dd, J = 8.4, 1.8 Hz), 7.85
(1H, d, J = 9.0 Hz), 7.76 (1H, d, J = 8.4 Hz), 6.59 (1H,
dd, J = 9.0, 1.8 Hz), 6.52 (1H, d, J = 1.8 Hz), 4.37 (2H, q,
J = 7.2 Hz), 1.40 (3H, t, J = 7.2 Hz); 13C NMR (75 MHz,
CD3COCD3) δ 166.0, 165.4, 163.9, 161.7, 152.4, 141.0,
129.6, 128.5, 127.3, 120.5, 111.2, 109.5, 103.8, 102.9,
61.8, 14.6.
General Procedure for Reduction of Ethyl 2-aryl
Benzo[d]oxazole-5-carboxylate: To a stirred solution of
ethyl 2-aryl benzo[d]oxazole-5-carboxylate (0.17 mmol) in
anhydrous THF (3 mL) was added LiAlH4 solution (1.0 M in
THF, 0.34 mL, 0.34 mmol, 2.0 equivalents) dropwise at 0ꢀC
under nitrogen atmosphere. The reaction was warmed to
room temperature and stirred for 30 min. After completion of
the reaction, the reaction mixture was cooled to 0ꢀC and the
excess LiAlH4 was quenched by the slow addition of aqueous
saturated Na2CO3 solution (1 mL) and stirred for 20 min.
The resulting suspension was filtered, washed with EtOAc
(40 mL). The filtrate was washed with H2O (2 × 8 mL) and
brine (8 mL), dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude was purified by column
chromatography (MeOH/CH2Cl2 = 1/10) to afford the pure
product [note: EtOAc:hexane = 1:2–1:1 was used for com-
pounds 2, 3 and 7 purification).
4-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)phenol
(nocarbenzoxazole F) (1): Yield: 95%; pale yellow color
solid; mp 230–232ꢀC; Rf = 0.25 (MeOH/CH2Cl2 = 1/10);
1H NMR (300 MHz, DMSO‑d6) δ 10.36 (1H, s), 8.01 (2H,
d, J = 7.5 Hz), 7.64 (1H, d, J = 8.1 Hz), 7.62 (1H, s), 7.29
(1H, d, J = 8.1 Hz), 6.94 (2H, d, J = 7.5 Hz), 5.35 (1H, br
s), 4.59 (2H, s); 13C NMR (75 MHz, DMSO‑d6) δ 162.8,
160.7, 148.9, 141.6, 139.2, 129.2, 123.3, 117.2, 116.9,
116.0, 109.9, 62.8; EI-MS m/z 241 (M+), 207 (base),
148, 119; HRMS: Calcd for C14H11NO3 (M+): 241.0739,
found: 241.0744.
2-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)-
5-methoxyphenol (4): Yield: 93%; pale yellow color solid;
mp 170–172ꢀC; Rf = 0.30 (MeOH/CH2Cl2 = 1/15); 1H
NMR (300 MHz, CDCl3) δ 7.90 (1H, d, J = 8.4 Hz), 7.68
(1H, d, J = 1.5 Hz), 7.55 (1H, d, J = 8.7 Hz), 7.36 (1H, d,
J = 8.4, 1.5 Hz), 6.63 (1H, d, J = 2.4 Hz), 6.59 (1H, dd,
J = 8.7, 2.4 Hz), 4.83 (2H,s), 3.88 (3H, s); 13C NMR
(75 MHz, CDCl3) δ 164.2, 163.7, 160.8, 148.6, 140.5,
138.1, 128.4, 124.1, 117.4, 110.5, 107.9, 103.8, 101.4,
65.5, 55.8; EI-MS m/z 271 (M+, base), 254, 240, 149;
HRMS: Calcd for C15H13NO4 (M+): 271.0845, found:
271.0851.
4-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)-
3-methoxyphenol (5): Yield: 94%; pale yellow color solid;
mp 214–216ꢀC; Rf = 0.18 (MeOH/CH2Cl2 = 1/10); 1H
NMR (300 MHz, CD3OD) δ 7.93 (1H, d, J = 8.7 Hz), 7.68
(1H, s), 7.56 (1H, d, J = 8.7 Hz), 7.35 (1H, d, J = 8.4 Hz),
6.60 (1H, d, J = 1.8 Hz), 6.54 (1H, dd, J = 8.4, 1.8 Hz),
4.72 (2H, s), 3.95 (3H, s); 13C NMR (75 MHz, CD3OD) δ
164.1, 161.9, 143.0, 139.8, 133.3, 125.2, 118.3, 111.1,
109.2, 107.8, 107.6, 100.6, 99.7, 65.3, 56.3; EI-MS m/z
271 (M+, base), 254, 240, 149; HRMS: Calcd for
C15H13NO4 (M+): 271.0845, found: 271.0834.
4-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)benzene-1,-
3-diol (6): Yield: 92%; pale yellow color solid; mp
1
224–226ꢀC; Rf = 0.21 (MeOH/CH2Cl2 = 1/10); H NMR
(300 MHz, CD3OD) δ 11.28 (1H, s), 10.45 (1H, s), 7.81
(1H, dd, J = 8.7, 2.1 Hz), 7.70 (1H, dd, J = 8.4, 1.8 Hz),
7.65 (1H, s), 7.33 (1H, d, J = 8.4 Hz), 6.50 (1H, d,
J = 8.7 Hz), 6.45 (1H, s), 5.38 (1H, t, J = 4.5 Hz), 4.60
(2H, d, J = 4.5 Hz); 13C NMR (75 MHz, CD3OD) δ 162.9,
162.6, 159.6, 147.3, 139.8, 139.4, 128.7, 123.5, 116.1,
110.1, 108.7, 102.8, 102.0, 62.7; EI-MS m/z 257 (M+,
base), 256 (M-H), 240, 228; HRMS: Calcd for C14H11NO4
(M+): 257.0688, found: 257.0692.
(2-Phenylbenzo[d]oxazol-5-yl)methanol (2): Yield:
(2-(2,4-Dimethoxyphenyl)benzo[d]oxazol-5-yl)metha-
97%; pale yellow color solid; mp 146–148ꢀC; Rf = 0.31
nol (7): Yield: 95%; white solid; mp 130–132ꢀC; Rf = 0.25
1
1
(MeOH/CH2Cl2 = 1/20); H NMR (300 MHz, CDCl3) δ
(EtOAc/hexane = 1/2); H NMR (300 MHz, CDCl3) δ 7.86
8.24–8.21 (2H, m), 7.73 (1H, s), 7.55–7.50 (4H, m), 7.35
(1H, d, J = 8.4 Hz), 4.80 (2H, s), 2.41 (1H, br s); 13C
NMR (75 MHz, CDCl3) δ 163.6, 150.3, 142.3, 137.9,
131.7, 129.0, 127.8, 127.2, 124.5, 118.6, 110.6, 65.5; EI-
MS m/z 225 (M+, base), 224 (M-H), 208, 194; HRMS:
Calcd for C14H11NO2 (M+): 225.0790, found: 225.0783.
(2-(2,4-Dimethoxyphenyl)benzo[d]oxazol-5-yl)metha-
nol (3): Yield: 92%; white solid; mp 134–136ꢀC; Rf = 0.3
(EtOAc/hexane = 1/2); 1H NMR (300 MHz, CDCl3) δ
8.05 (1H, d, J = 8.4 Hz), 7.70 (1H, s), 7.45 (1H, d,
J = 8.4 Hz), 7.25 (1H, d, J = 8.7 Hz), 6.60 (1H, dd,
J = 8.7, 2.1 Hz), 6.56 (1H, d, J = 2.1 Hz), 4.75 (2H, s),
3.99 (3H, s), 3.87 (3H, s), 2.62 (1H, br s); 13C NMR
(75 MHz, CDCl3) δ 163.6, 162.2, 160.0, 149.6, 142.3,
137.6, 132.5, 123.9, 118.4, 110.2, 109.0, 105.5, 99.2,
65.5, 56.3, 55.8; EI-MS m/z 285 (M+, base), 284 (M-H),
254, 148; HRMS: Calcd for C16H15NO4 (M+): 285.1001,
found: 285.1011.
(1H, dd, J = 8.1, 1.5 Hz), 7.75 (1H, d, J = 1.5 Hz), 7.73
(1H, s), 7.54 (1H, d, J = 8.4 Hz), 7.35 (1H, d, J = 8.1 Hz),
7.00 (1H, d, J = 8.4 Hz), 4.82 (2H, d, J = 6.0 Hz), 4.03
(3H, s), 3.99 (3H, s), 1.88 (1H, br t, J = 6.0 Hz); 13C NMR
(75 MHz, CDCl3) δ 162.9, 152.2, 150.4, 149.4, 142.6,
137.8, 124.1, 121.4, 119.9, 118.3, 111.2, 110.5, 110.3,
65.7, 56.4, 56.3; EI-MS m/z 285 (M+, base), 284 (M-H),
268, 163; HRMS: Calcd for C16H15NO4 (M+): 285.1001,
found: 285.0983.
4-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)benzene-1,-
2-diol (8): Yield: 91%; pale yellow color solid; mp
1
222–224ꢀC; Rf = 0.18 (MeOH/CH2Cl2 = 1/10); H NMR
(300 MHz, DMSO‑d6) δ 9.80 (1H, s), 9.53 (1H, s), 7.63
(H, d, J = 8.7 Hz), 7.62 (1H, s), 7.56 (1H, d, J = 2.1 Hz),
7.51 (1H, dd, J = 8.4, 2.1 Hz), 7.29 (1H, d, J = 8.7 Hz),
6.90 (1H, d, J = 8.4 Hz), 5.30 (1H, t, J = 4.2 Hz), 4.60
(2H, d, J = 4.2 Hz); 13C NMR (75 MHz, DMSO‑d6) δ
162.9, 149.2, 148.8, 145.6, 141.6, 139.1, 123.2, 119.4,
Bull. Korean Chem. Soc. 2018
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Synthesis and In Vitro Nitric Oxide Production Inhibitory
BULLETIN OF THE
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Activity of Benzoxazoles
KOREAN CHEMICAL SOCIETY
Table 1. Optimization study of cyclodehydration reaction between
10 and 14.a
117.3, 116.8, 116.0, 114.1, 109.8, 62.8; EI-MS m/z
257 (M+, base), 256 (M-H), 240, 228; HRMS: Calcd for
C14H11NO4 (M+): 257.0688, found: 257.0681.
Entry
Reagent
Solvent
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
POCl3
POCl3
POCl3
H2SO4
POCl3
POCl3
PCl5
DMF
1,4-dioxane
Pyridine
Ac2O
DMF
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
1,4-dioxane
0.5
0.5
0.5
1
3
3
3
3
3
3
15
15
NR
NR
16
29
NR
24
17
25
20
55
45
35
Results and Discussion
Our synthesis commenced with the esterification of the
commercially available 3-amino-4-hydroxybenzoic acid (9)
(Scheme 1). Treatment of compound 9 with EtOH in the
presence of H2SO4 under reflux condition afforded the
ethyl ester of substituted benzoic acid 10 in 85% yield.
Next, reaction of substituted benzoic acids 11–13 with
SOCl2 at 60ꢀC gave the corresponding acid chlorides 14,
16, and 17 respectively, which were used in the next step
immediately without any purification. Having the key frag-
ments in hand, next, we set out to explore the key cyclode-
hydration reaction using compounds 10 and 14 as model
substrates (Table 1). POCl3, PCl5, H2SO4, P2O5, SOCl2,
CH3SO3H, and CF3SO3H were screened as cyclodehydrat-
ing agents in different solvents such as DMF, acetic anhy-
dride, 1,4-dioxane and pyridine. All attempts to advance
P2O5
9
SOCl2
CH3SO3H
CF3SO3H
POCl3
POCl3
POCl3
10
11
12
13
14
a
3
15
12
24
Reaction conditions: 10 (0.2 mmol), 14 (0.4 mmol), reagent
(0.6 mmol), and solvent (1 mL) at 90ꢀC.
Isolated yields. NR, no reaction.
b
Scheme 1. Synthesis of nocarbenzoxazoles F (1), G (5) and their derivatives (2–4 and 6–8).
Bull. Korean Chem. Soc. 2018
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Synthesis and In Vitro Nitric Oxide Production Inhibitory
BULLETIN OF THE
Article
Activity of Benzoxazoles
KOREAN CHEMICAL SOCIETY
Table 2. NO production inhibitory effects of nocarbenzoxazole F (1) and its derivatives (2–4 and 6–8).
R1 R2
N
H
O
R3
O
NO production (% inhibition)a
Proliferationa
Compound
R1
R2
R3
1 μM
10 μM
IC50 (μM)
1 μM
10 μM
LPS
1
2
3
4
6
7
8
100.0 ꢁ 1.8
120.6 ꢁ 1.1**
107.7 ꢁ 2.5
117.7 ꢁ 0.7**
117.0 ꢁ 2.3**
115.0 ꢁ 1.3*
114.6 ꢁ 4.4
90.6 ꢁ 25.4
95.2 ꢁ 1.1
100.0 ꢁ 1.8
97.2 ꢁ 3.9*
92.2 ꢁ 2.7
92.6 ꢁ 1.7
96.6 ꢁ 1.5
87.3 ꢁ 0.7
110.4 ꢁ 2.5
46.5 ꢁ 3.3***
80.5 ꢁ 2.9**
H
H
OMe
OH
OH
H
H
H
H
H
H
OMe
OH
OH
H
26.24
29.11
27.31
21.01
28.26
39.91
8.17
111.9 ꢁ 2.1*
90.2 ꢁ 3.1
92.7 ꢁ 3.8
99.2 ꢁ 5.1
104.4 ꢁ 4.1
97.1 ꢁ 2.0
99.9 ꢁ 3.9
102.2 ꢁ 2.5
91.8 ꢁ 4.8
84.6 ꢁ 1.4**
103.1 ꢁ 1.1
98.9 ꢁ 4.1
98.1 ꢁ 5.1
92.2 ꢁ 4.0
90.9 ꢁ 6.2
100.7 ꢁ 1.8
OMe
OMe
OH
OMe
OH
H
L-NMMA
18.77
a
The results are reported as mean value ꢁ SEM for n = 3. Statistical significance is based on the difference when compared with LPS-treated
groups.
(*p < 0.05, **p < 0.01, ***p < 0.001).
the cyclodehydration met with either failure (Table 1, entry
3, 4, and 7) or low yielding of 18 (Table 1, entries 1,2,
5, 6, and 8–11). To our delight, POCl3 mediated cyclodehy-
dration in 1,4-dioxane accomplished the product 18 in 55%
yield (Table 1, entry 12). Either reducing or extending the
reaction time did not offer any improvement in the product
yield (Table 1, entries 13 and 14). Following this optimized
condition, benzoxazoles 19–21 were obtained respectively,
by the reaction of 10 with the substituted benzoyl chlorides
15–17.
Next, treatment of 18 and 21 with boron tribromide
(BBr3) solution (1.0 M in CH2Cl2) provided compounds 22
and 23 in good yields. Compound 20 underwent selective
demethylation using boron trichloride (BCl3) solution
(1.0 M in CH2Cl2) and aluminum trichloride (AlCl3) to
yield 24 and 25, respectively. Due to the chelation with the
benzoxazole ring nitrogen or oxygen atom, BCl3 extremely
effect the demethylation at 20-position rather than the 40-
position. As BCl3 does not selectively remove isolated aryl
methyl groups efficiently,14 we used AlCl3 to effect the
demethylation at 40-position and succeeded to certain
extent. Compound 25 further demethylated with BCl3 to
give the benzoxazole 26. Finally, ester group reduction of
22, 19, 20, 24–26, 21, and 23 using LiAlH4 offered the tar-
get benzoxazoles 1–8 in high yields (91–97%), respec-
tively. All the target compounds 1–8 were settled from
their NMR (1H- and 13C-) and MS data.
Due to the extremely short physiological half-life of NO
and existence of other scavenging molecules, NO levels
were measured indirectly by evaluation of its stable metabo-
lites nitrite and nitrate concentration by the spectrophotomet-
ric method using the Griess reaction.15 NG-Monomethyl-L-
arginine acetate (L-NMMA)16 was employed as a positive
control. Effect of compounds 1–8 on inhibition of NO pro-
duction by induced macrophages was monitored (Table 2).
Unfortunately, nocarbenzoxazole G (compound 5) was
highly unstable and easily decomposed within 24 h to get
the biological activity. The benzoxazoles had a concentra-
tion dependent inhibition on NO production induced by
LPS in macrophages. All the compounds reduced NO pro-
duction to less extent at the lowest concentration (1 μM).
However, compounds 6 and 8 had the best inhibitory activ-
ity at the highest concentration (10 μM) when compared
with the positive control, L-NMMA. In addition, cell viabil-
ity was assessed based on the reduction of the tetrazolium
salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT)17 into formazan crystals to discover whether
the inhibitory effects on NO production were due to the
cytotoxicity of the test compounds. The results showed that
compounds 1–4, 6–8 did not exhibit significant cytotoxicity
on RAW 264.7 cells at the concentrations evaluated. The
concentration required to inhibit the production of NO by
50% (IC50 value) were calculated by GraphPad Prism 4.0
software GraphPad Prism 4.0 software (GraphPad Software
Inc. La Jolla, California, USA) and displayed 26.24, 29.11,
27.31, 21.01, 28.26, 39.91, and 8.17, respectively. Taken
together with all these findings, it is worth noting that com-
pound 8 which is having a catechol moiety at 2-position of
benzoxazole scaffold exerted superior NO inhibitory activity
over the positive control, L-NMMA.
Inhibition of Nitric Oxide (NO) Production. The biolog-
ical activity of synthesized benzoxazoles 1–8 as inhibitors
of NO production has been evaluated by measuring the
amount of nitric oxide (NO) in lipopolysaccharide (LPS)-
stimulated RAW 264.7 cells, as an indicator of anti-
inflammatory activity, following our previous procedure.13b
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Synthesis and In Vitro Nitric Oxide Production Inhibitory
BULLETIN OF THE
Article
Activity of Benzoxazoles
KOREAN CHEMICAL SOCIETY
4. J. A. McCleverty, Chem. Rev. 2004, 104, 403.
Conclusion
5. J. Lei, Y. Vodovotz, E. Tzeng, T. R. Billiar, Nitric Oxide
2013, 35, 175.
6. R. J. Flower, Nat. Rev. Drug Discov. 2003, 2, 179.
7. I. L. Meek, M. A. F. J. van de Laar, H. E. Vonkeman, Phar-
maceuticals 2010, 3, 2146.
We have completed a facile synthesis of nocarbenzoxazoles
F (1), G (5) and their derivatives (2–4 and 6–8) using the
commercially inexpensive building blocks and in overall
yields of 15–49%. POCl3-mediated cyclodehydration,
selective and/or complete demethylation and reduction were
the key steps to the success of this synthetic pathway. Next,
these benzoxazoles were screened for their inhibitory effect
against NO production in LPS-induced RAW 264.7 cells
and were found to display week to moderate activity in a
concentration-dependent manner and without notable cyto-
toxicity. In particular, compounds 8 (53.5% at 10 μM;
IC50 = 8.17 μM) and 6 (12.7% at 10 μM; IC50 = 28.26 μM)
displayed pronounced inhibition of NO production, being
more active than the positive control, L-NMMA (19.5% at
10 μM; IC50 = 18.77 μM). The present study revealed that
the presence of a catechol moiety at 2-position was found
to contribute the most to benzoxazole 8 for NO inhibitory
activity.
8. B.
Maleki,
M.
Baghayeri,
S.
M.
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Acknowledgments. Publication cost of this article was
supported by the Korean Chemical Society.
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Bull. Korean Chem. Soc. 2018
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