Design, synthesis and biological evaluation of 2-(2-aryl-morpholino-4-yl) ethyl esters of indomethacin as potential cyclooxygenase-2 (COX-2) inhibitors

Article abstract of DOI:10.1002/cjoc.201200196

A number of novel 2-(2-arylmorpholino-4-yl)ethyl 1-(4-chlorobenzoyl)-5- methoxy-2-methyl-1H-indol-3-acetate hydrochlorides were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds exhibited moderate to good selective COX-2 inhibition, and subtle structural changes in the substituents on the side chain of the ester moiety altered the inhibitory properties significantly. 2-[2-(4-Butoxyphenyl) morpholino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate hydrochloride (1f), showed good selective COX-2 inhibitory activity (Selective index (SI) 182), which is comparative with celecoxib (SI 214), a COX-2 inhibitor of diarylpyrazoles. While 2-[2-(2,4-dichloro-5-fluorophenyl)morpholino-4-yl] ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate hydrochloride (1g), showed greater selective COX-2 inhibitory activity (SI 358) than celecoxib. Both compounds were identified as compromising derivatives in this class to reduce the side effects generated by nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin.

Full text of DOI:10.1002/cjoc.201200196

FULL PAPER
DOI: 10.1002/cjoc.201200196
Design, Synthesis and Biological Evaluation of 2-(2-Aryl-
morpholino-4-yl)ethyl Esters of Indomethacin as
Potential Cyclooxygenase-2 (COX-2) Inhibitors†
Shi, Leia(石磊)
Hu, Aixi*,a(胡艾希)
Xu, Jiangping*,b(徐江平)
Jiang, Yipingb(蒋毅萍)
a College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, China
b Department of Pharmacology, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou,
Guangdong 510515, China
A
number of novel 2-(2-arylmorpholino-4-yl)ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
acetate hydrochlorides were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition prop-
erties in vitro. Many of these compounds exhibited moderate to good selective COX-2 inhibition, and subtle struc-
tural changes in the substituents on the side chain of the ester moiety altered the inhibitory properties significantly.
2-[2-(4-Butoxyphenyl)morpholino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate hydro-
chloride (1f), showed good selective COX-2 inhibitory activity (Selective index (SI) 182), which is comparative
with celecoxib (SI 214), a COX-2 inhibitor of diarylpyrazoles. While 2-[2-(2,4-dichloro-5-fluorophenyl)mor-
pholino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate hydrochloride (1g), showed greater
selective COX-2 inhibitory activity (SI 358) than celecoxib. Both compounds were identified as compromising de-
rivatives in this class to reduce the side effects generated by nonsteroidal anti-inflammatory drugs (NSAIDs) indo-
methacin.
Keywords indomethacin, 2-(2-arylmorpholino-4-yl)ethanol, synthesis, COX-2 selective inhibition, antioxidant
Introduction
matory and antioxidant activity. Kourounakis and
co-workers[8] synthesized a variety of 2,6-di-tert-bu-
tylphenol, which are amides or amines of morpholine
residues, some of which exhibited antiinflammatory,
LOX inhibitory and antioxidant activities. Thus, we
suppose that COX-2 inhibitors bearing morpholine de-
rivatives could reduce the cardiovascular side effects
after using COX-2 inhibitors for a long time.
Based on our research of NSAID naproxen deriva-
tives[9] and phenethyl 2-(1-(4-chlorobenzoyl)-5-meth-
oxy-2-methyl-1H-indol-3-yl)acetate (A)[10], in this work,
the morpholine moiety[11] was inserted into compound A,
which has shown good antiinflammatory and antioxidant
activity. That can be useful for the design of antiathero-
sclerotic agents (Scheme 1). We felt that the substituted
2-(2-arylmorpholino-4-yl)ethyl ester moiety might be of
great value to explore deeply, as it is capable of inhibiting
COX-2 selectively through forming additional hydrogen
bonds by occupying the additional side pocket of COX-2
enzyme and reducing cardiovascular side effects by the
antioxidant activity of morpholine moiety. These com-
pounds were tested in vitro for the evaluation of their
COX-2 selective inhibitory activities.
Traditional nonsteroidal anti-inflammatory drugs
(NSAIDs) remain the most commonly used medications
for the treatment of many inflammatory diseases, how-
ever, the toxicity of NSAIDs substantially limit their
long term use.[1] The essential fatty acid arachidonic
acid (AA) which is released from membrane phospho-
lipids by various stimuli is metabolized into inflamma-
tory factors protaglandins (PGs), thromboxane A2
(TXA2) and leukotries (LTs).[2,3] Cyclooxygenases
(COXs) and lipoxygenase (LOX) play important roles
during the two metabolic routes, thus exploitation of
dual-targeted compounds may be a new method in
NSAIDs pharmacology.[4-6] The results on lipid peroxi-
dation largely correlate with the effect on LOX. Re-
cently, many synthetic chemists have focused their at-
tention on the derivatives of NSAIDs with antioxidant
activities. Zhang group[7] reported a series of conjugates
of indomethacin with phenolic antioxidants which
showed potent antioxidants in vitro enhanced antiin-
flammatory activity as well as reduced ulcerogenic po-
tency.
Morpholine analogues have shown good anti-inflam-
*
E-mail: axhu@hnu.edu.cn. and jpx@fimmu.com; Tel./Fax: 0086-0731-88822275/88713642 or 0086-020-61648236
Received February 27, 2012; accepted May 17, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200196 or from the author.
Dedicated to Professor Jun Zhou on the occasion of his 80th birthday.
†
Chin. J. Chem. 2012, 30, 1339—1344
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1339

Shi et al.
FULL PAPER
Scheme 1 Synthesis of 2-(2-arylmorpholino-4-yl)ethyl esters of indomethacin hydrochlorides
MeO
MeO
R
CH2CO2CH2CH2
CH2CO2CH2CH2C6H5
Me
N
R
N
N
Me
O
N
Ar
O
O
O
Cl
Cl
Ar
1
A
Ar
R
O
O
a
b
Br
Ar
R
MeO
O
NCH2CH2OH
2a－2m
R
CH2CO2CH2CH2
HCl
R
N
O
d
MeO
MeO
N
Me
CH2CO2H
Me
CH2COCl
Me
Ar
c
O
N
N
Cl
O
1a－1m
O
Cl
Cl
3
2a, 1a: R＝H, Ar＝C6H5; 2b, 1b: R＝H, Ar＝4-CH3C6H4; 2c, 1c: R＝H, Ar＝4-C2H5C6H4; 2d, 1d: R＝H, Ar＝4-CH3OC6H4; 2e, 1e: R＝H, Ar＝
4-(CH3)2CHOC6H4; 2f, 1f: R＝H, Ar＝4-CH3(CH2)3OC6H4; 2g, 1g: R＝H, Ar＝2,4-Cl2-5-FC6H2; 2h, 1h: R＝H, Ar＝2-Cl-4-(4-ClC6H4O)C6H3; 2i,
1i: R＝H, Ar＝6-CH3O-2-C10H6; 2j, 1j: R＝H, Ar＝6-CH3O-5-Cl-2-C10H5; 2k, 1k: R＝H, Ar＝6-CH3O-5-Br-2-C10H5; 2l, 1l: R = CH3, Ar＝
6-CH3O-2-C10H6; 2m, 1m: R＝CH3, Ar＝4-C6H5CH2OC6H4
Reagents and conditions: (a) CuBr2, EtOH, reflux, 6 h; (b) (i) HN(CH2CH2OH)2, N-methylpyrrolidin-2-one (NMP), 60 °C, 2 h; (ii) HCOOH, 180
oC, 18 h; (c) (COCl)2, CH2Cl2, 0 °C, 3 h; (d) (i) THF, Et3N, r.t., 6 h; (ii) HCl(g), Et2O then (CH3)2CO.
Indomethacin acid chloride (3)
Experimental
Indomethacin (3 mmol) and oxalyl chloride (1 mL)
in dichloromethane (8 mL) were stirred at 0 ℃ for 3 h.
The excess oxalyl chloride and dichloromethane were
distilled off under reduced pressure, then the residue
was washed with hexane (25 mL×3) and dried under
vacuum to give 3 as pale yellow solid. Yield 92%, m.p.
126—128 ℃, which is in accordance with reference
[12] values (Yield 95%, m.p. 125—127 ℃).
All reagents and solvents were of commercial qual-
ity and used without further purification. Reactions were
performed under the protection of nitrogen and were
monitored by thin-layer chromatography (TLC) on sil-
ica gel plates, visualizing with ultraviolet light or iodine
spray. Flash chromatography was performed on silica
gel (200—400 mesh) using commercially available pe-
troleum ether and ethyl acetate. Melting points (m.p.)
were taken in open capillaries and uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were
obtained with a Bruker 400 MHz spectrometer. Chemi-
cal shifts (δ) are relative to tetramethylsilane multiplic-
ities and given as s (singlet), d (doublet), t (triplet), and
m (multiplet) as well as br (broad). Coupling constants
(J) are given in Hz. Infrared spectra were recorded on
an FT-IR spectrometer. Mass spectra were recorded on
a Thermo Finnigan LCQ Advantage ion-trap mass spec-
trometer. Microanalysis were performed using a C H N
S/O analyzer. Elemental data are within ±0.2%. The
following drugs and chemicals were purchased and used:
sodium thioglycollate (DIFCO), calcium cation iono-
phore A23187, lipopolysaccharides (LPS), dimethyl
sulfoxide (DMSO), Celecoxib (SIGMA), new calf se-
rum (NCS, 5%, GIBCO), square-free kit (PGE2 and
6-keto-PGF1α, Institute of Immune Technology of East
Asia).
General procedure for the preparation of 2-(2-aryl-
morpholino-4-yl)ethyl 1-(4-chlorobenzoyl)-5-meth-
oxy-2-methyl-1H-indol-3-acetate
1a—1m
hydrochlorides
Indomethacin acid chloride prepared before was
dissolved in 8 mL of tetrahydrofuran, then the solution
of 2-(2-arylmorpholino-4-yl)ethanols 2a — 2m (4.5
mmol) as well as triethylamine (1 mL) was slowly
added. The reaction mixture was stirred at room tem-
perature for 6 h and filtered to remove triethylamine
hydrochloride. The filtrate was distilled under reduced
pressure to remove tretrahydrofuran, then the residue
was dissolved with ethyl acetate and washed with
－
1
aqueous sodium hydroxide (1.0 mol•L ), dried over
anhydrous sodium sulfate, filtered and evaporated under
reduced pressure. The free base was obtained as oily
product by flash chromatography, eluting with petro-
leum ether/ethyl acetate. Then the oily product was dis-
solved in proper ether and treated with ethereal hydro-
1340
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1339—1344

Design, Synthesis and Biological Evaluation of 2-(2-Arylmorpholino-4-yl)ethyl Esters
gen chloride, followed by filtration and residue was
washed with acetone to give the hydrochlorides.
1H, C4H7NO-6-He), 4.68 (s, 2H, CH2CO), 5.17 (d, J＝
10.4 Hz, 1H, C4H7NO-2-H), 6.86—7.68 (m, 11H, C6H4,
C6H4, C6H3), 13.67 (s, 1H, NH); IR (KBr) ν: 3436, 2966,
2336, 1745, 1674, 1614, 1591, 1476, 1458, 1243, 1218,
2-(2-Phenylmorpholino-4-yl)ethyl 1-(4-chloroben-
zoyl)-5-methoxy-2-methyl-1H-indol-3-acetate hydro-
chloride (1a): White solid, yield 72%; m.p. 184—186
－
＋
1
1160, 1100 cm ; ESI-MS m/z: 576 (M －HCl). Anal.
calcd for C32H34Cl2N2O6: C 62.64, H 5.59, N 4.57;
found C 62.59, H 5.65, N 4.59.
1
℃; H NMR (CDCl3, 400 MHz) δ: 2.35 (s, 3H, CH3),
2.61 (br, 2H, C4H7NO-3,5-Ha), 3.21 (m, 2H, NCH2),
3.35 (d, J＝12 Hz, 1H, C4H7NO-5-He), 3.49 (d, J＝11.2
Hz, 1H, C4H7NO-3-He), 3.80 (s, 2H, OCH2), 3.85 (s, 3H,
OCH3), 3.93 (d, J＝11.2 Hz, 1H, C4H7NO-6-Ha), 4.38 (t,
J＝12.8 Hz, 1H, C4H7NO-6-He), 4.68 (s, 2H, CH2CO),
5.24 (d, J＝10 Hz, 1H, C4H7NO-2-H), 6.68—7.68 (m,
12H, C6H5, C6H4, C6H3), 13.76 (s, 1H, NH); IR (KBr) ν:
3435, 3092, 1730, 1690, 1594, 1479, 1417, 1261, 1227,
2-[2-(4-Isopropoxyphenylmorpholino-4-yl)ethyl [1-
(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
acetate hydrochloride (1e): White solid, yield 57%; m.p.
1
218—219 ℃; H NMR (CDCl3, 400 MHz) δ: 1.32 (d,
J＝6 Hz, 6H, 2CH3), 2.37 (s, 3H, CH3), 2.61 (br, 2H,
C4H7NO-3,5-Ha), 3.21 (br, 2H, NCH2), 3.33 (d, J＝11.2
Hz, 2H, C4H7NO-5-He), 3.44 (d, J＝11.6 Hz, 2H,
C4H7NO-3-He), 3.81 (s, 2H, OCH2), 3.85 (s, 3H, OCH3),
3.90 (dd, J＝2.8, 2.8 Hz, 1H, C4H7NO-6-Ha), 4.37 (t,
J＝12.4 Hz, 1H, C4H7NO-6-He), 4.53 (heptet, 1H,
(CH3)2CHO), 4.68 (s, 2H, CH2CO), 5.15 (d, J＝10.8 Hz,
1H, C4H7NO-2-H), 6.68—7.68 (m, 11H, C6H4, C6H4,
C6H3), 13.69 (s, 1H, NH); IR (KBr) ν: 3081, 2987, 2289,
1742, 1671, 1634, 1599, 1480, 1449, 1322, 1251, 1147,
－
＋
1
1140 cm ; ESI-MS m/z: 546 (M －HCl). Anal. calcd
for C32H34Cl2N2O5: C 63.81, H 5.53, N 4.80; found C
63.79, H 5.52, N 4.75.
2-[2-(4-Methylphenylmorpholino-4-yl)ethyl [1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate
hydrochloride (1b): White solid, yield 49%; m.p. 170—
1
172 ℃; H NMR (CDCl3, 400 MHz) δ: 2.33 (s, 3H,
－
＋
1
CH3), 2.35 (s, 3H, indole-2-CH3), 2.59 (br, 2H,
C4H7NO-3,5-Ha), 3.20 (m, 2H, NCH2), 3.34 (d, J＝12.4
Hz, 1H, C4H7NO-5-He), 3.46 (d, J ＝ 12 Hz, 1H,
C4H7NO-3-He), 3.80 (s, 2H, OCH2), 3.85 (s, 3H, OCH3),
3.91 (dd, J＝3.2, 3.2 Hz, 1H, C4H7NO-6-Ha), 4.38 (t,
J＝12.0 Hz, 1H, C4H7NO-6-He), 4.68 (s, 2H, CH2CO),
5.19 (d, J＝9.2 Hz, 1H, C4H7NO-2-H), 6.68—7.68 (m,
11H, C6H5, C6H4, C6H3), 13.72 (s, 1H, NH); IR (KBr) ν:
3440, 2958, 1738, 1674, 1607, 1478, 1455, 1261, 1239,
1085 cm ; ESI-MS m/z: 604 (M －HCl). Anal. calcd
for C34H38Cl2N2O6: C 63.81, H 5.53, N 4.80; found C
63.88, H 5.40, N 4.79.
2-[2-(4-Butoxyphenylmorpholino-4-yl)ethyl [1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate
hydrochloride (1f): White solid, yield 46%; m.p. 179—
1
180 ℃; H NMR (CDCl3, 400 MHz) δ: 0.97 [t, J＝6.8
Hz, 3H, CH3(CH2)3], 1.48 (m, 2H, CH3CH2), 1.75 (m,
2H, CH3CH2CH2), 2.36 (s, 3H, CH3), 2.60 (br, 2H,
C4H7NO-3,5-Ha), 3.21 (br, 2H, NCH2), 3.33 (d, J＝12.0
Hz, 1H, C4H7NO-5-He), 3.44 (d, J＝11.6 Hz, 1H,
C4H7NO-3-He), 3.80 (s, 2H, C3H7CH2O), 3.85 (s, 3H,
indole-5-OCH3), 3.92 (s, 2H, OCH2), 3.94 (d, J＝10.8
Hz, 1H, C4H7NO-6-Ha), 4.37 (t, J＝12.4 Hz, 1H,
C4H7NO-6-He), 4.68 (s, 2H, CH2CO), 5.15 (d, J＝10.4
Hz, 1H, C4H7NO-2-H), 6.68—7.22 (m, 11H, C6H4,
C6H4, C6H3), 13.65 (s, 1H, NH); IR (KBr) ν: 3098, 2948,
2234, 1752, 1672, 1593, 1525, 1474, 1411, 1332, 1252,
－
＋
1
1163 cm ; ESI-MS m/z: 560 (M －HCl). Anal. calcd
for C31H32Cl2N2O5: C 64.21, H 5.47, N 4.69; found C
64.33, H 5.44, N 4.68.
2-[2-(4-Ethylphenylmorpholino-4-yl)ethyl 1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate
hydrochloride (1c): White solid, yield 22%; m.p. 186—
1
189 ℃; H NMR (CDCl3, 400 MHz) δ: 1.21 (t, J＝7.6
Hz, 3H, CH3), 2.35 (s, 3H, indole-2-CH3), 2.58—2.68
(m, 4H, PhCH2, C4H7NO-3,5-Ha), 3.22 (m, 2H, NCH2),
3.35 (d, J＝10.8 Hz, 1H, C4H7NO-5-He), 3.48 (d, J＝
10.8 Hz, 1H, C4H7NO-3-He), 3.80 (s, 2H, OCH2), 3.85
(s, 3H, OCH3), 3.91 (d, J＝12.4 Hz, 1H, C4H7NO-6-Ha),
4.38 (t, J＝12.4 Hz, 1H, C4H7NO-6-He), 4.68 (s, 2H,
CH2CO), 5.20 (d, J＝10.8 Hz, 1H, C4H7NO-2-H),
6.68—7.68 (m, 11H, C6H5, C6H4, C6H3), 13.69 (s, 1H,
NH); IR (KBr) ν: 3435, 2962, 1744, 1669, 1603, 1478,
－
＋
1
1149, 1102 cm ; ESI-MS m/z: 618 (M －HCl). Anal.
calcd for C35H40Cl2N2O6: C 64.12, H 6.15, N 4.27;
found C 64.15, H 6.12, N 4.20;
2-[2-(2,4-Dichloro-5-fluorophenyl)morpholino-4-yl]-
ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-acetate hydrochloride (1g): White solid, yield
43%; m.p. 171—174 ℃; 1H NMR (CDCl3, 400 MHz) δ:
2.36 (s, 3H, CH3), 2.59 (br, 2H, C4H7NO-3,5-Ha),
3.21—3.37 (m, 4H, C4H7NO-3,5-He, NCH2), 3.81 (s,
2H, OCH2), 3.85 (s, 3H, OCH3), 3.95 (d, J＝12 Hz, 1H,
C4H7NO-6-Ha), 4.45 (s, 1H, C4H7NO-6-He), 4.69 (s, 2H,
CH2CO), 5.52 (d, J＝8.0 Hz, 1H, C4H7NO-2-H), 6.68—
7.68 (m, 9H, C6H4, C6H3, C6H2), 14.06 (s, 1H, NH); IR
(KBr) ν: 3436, 2928, 2336, 1736, 1679, 1612, 1455,
－
＋
1
1455, 1262, 1241, 1161 cm ; ESI-MS m/z: 574 (M －
HCl). Anal. calcd for C33H36Cl2N2O5: C 64.71, H 5.93,
N 4.58; found C 64.78, H 5.99, N 4.56.
2-[2-(4-Methoxyphenylmorpholino-4-yl)ethyl 1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate
hydrochloride (1d): White solid, yield 47%; m.p. 185—
1
187 ℃; H NMR (CDCl3, 400 MHz) δ: 2.36 (s, 3H,
－
1
CH3), 2.59 (br, 2H, C4H7NO-3,5-Ha), 3.21 (br, 2H,
NCH2), 3.34 (d, J＝11.2 Hz, 2H, C4H7NO-5-He), 3.44
(d, J＝11.2 Hz, 2H, C4H7NO-3-He), 3.80 (s, 2H, OCH2),
3.81 (s, 3H, OCH3), 3.85 (s, 3H, indole-5-OCH3), 3.91
(d, J＝11.6 Hz, 1H, C4H7NO-6-Ha), 4.38 (t, J＝8.8 Hz,
1389, 1257, 1237, 1167, 1144 cm ; ESI-MS m/z: 632
(M＋ －HCl－1). Anal. calcd for C31H29Cl4N2O5: C
55.44, H 4.36, N 4.18; found C 55.58, H 4.31, N 4.19.
2-[(2-Chloro-4-(4-chlorophenoxy)phenyl)-morpho-
lino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-
Chin. J. Chem. 2012, 30, 1339—1344
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Shi et al.
FULL PAPER
methyl-1H-indol-3-acetate hydrochloride (1h): White
solid, yield 33%; m.p. 166—168 ℃; H NMR (CDCl3,
4.75; found C 58.21, H 4.83, N 4.79.
1
2-[3-Methyl-2-(6-methoxynaphthalen-2-yl)-morpho-
lino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-acetate hydrochloride (1l): White
400 MHz) δ: 2.36 (s, 3H, CH3), 2.51 (br, 2H,
C4H7NO-3,5-Ha), 3.21 (br, 2H, NCH2), 3.35 (d, J＝10.4
Hz, 1H, C4H7NO-5-He), 3.65 (d, J＝12.8 Hz, 1H,
C4H7NO-3-He), 3.81 (s, 2H, OCH2), 3.85 (s, 3H, OCH3),
3.94 (d, J＝12.4 Hz, 1H, C4H7NO-6-Ha), 4.45 (t, J＝
11.6 Hz, 1H, C4H7NO-6-He), 4.69 (s, 2H, CH2CO), 5.53
(d, J＝10.4 Hz, 1H, C4H7NO-2-H), 6.68—7.68 (m, 14H,
C6H4, C6H4, C6H3, C6H3), 14.03 (s, 1H, NH); IR (KBr) ν:
3435, 2952, 2435, 1721, 1685, 1608, 1591, 1480, 1457,
1
solid, yield 22%; m.p. 145—147 ℃; H NMR (CDCl3,
400 MHz) δ: 1.27 (d, J＝6.4 Hz, 3H, CH3), 2.44 (s, 3H,
CH3), 2.72 (br, 1H, C4H7NO-3-H), 3.08 (br, 2H, NCH2),
3.31 (d, J＝11.2 Hz, 1H, C4H7NO-5-Ha), 3.67 (br, 1H,
C4H7NO-5-He), 3.77 (dd, J ＝ 10.4, 2.0 Hz, 1H,
C4H7NO-6-Ha), 3.81 (s, 2H, OCH2), 3.86 (s, 3H,
indole-5-CH3O), 3.93 (s, 3H, CH3O), 4.46 (br, 1H,
C4H7NO-6-He), 4.66 (s, 2H, CH2CO), 5.01 (br, 2H,
C4H7NO-2-H), 6.69—7.75 (m, 13H, C10H6, C6H4, C6H3),
13.47 (s, 1H, NH); IR (KBr) ν: 3309, 2953, 2630, 1686,
1635, 1609, 1486, 1452, 1373, 1267, 1223, 1174, 1126,
－
1
1400, 1272, 1247, 1122, 1092 cm ; ESI-MS m/z: 708
(M＋ －HCl＋1). Anal. calcd for C37H34Cl4N2O6: C
59.69, H 4.60, N 3.76; found C 59.78, H 4.56, N 3.77.
2-[2-(6-Methoxynaphthalen-2-yl)morpholino-4-yl]-
ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-acetate hydrochloride (1i): White solid, yield
30%; m.p. 101—102 ℃; 1H NMR (CDCl3, 400 MHz) δ:
2.36 (s, 3H, CH3), 2.47 (br, 2H, C4H7NO-3,5-Ha),
2.92—3.28 (m, 4H, NCH2, C4H7NO-3,5-He), 3.73 (s,
2H, OCH2), 3.83 (s, 3H, indole-5-CH3O), 3.91 (s, 3H,
CH3O), 3.97 (d, J＝8 Hz, 1H, C4H7NO-6-Ha), 4.01 (br,
1H, C4H7NO-6-He), 4.48 (s, 2H, CH2CO), 4.91 (m, 3H,
OCH2, C4H7NO 2-H), 6.64—7.72 (m, 13H, C10H6, C6H4,
C6H3), 13.49 (s, 1H, NH); IR (KBr) ν: 3439, 2992, 2929,
1744, 1676, 1603, 1477, 1467, 1360, 1324, 1291, 1236,
－
＋
1
1080, 1026, 979 cm ; ESI-MS m/z: 660 (M －HCl).
Anal. calcd for C31H32Cl2N2O5: C 65.58, H 5.65, N 4.13;
found C 65.54, H 5.69, N 4.10.
2-[3-Methyl-2-(4-benzyloxyphenyl)morpholino-4-
yl]-ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-acetate hydrochloride (1m): White solid, yield
17%; m.p. 122—125 ℃; 1H NMR (CDCl3, 400 MHz) δ:
1.21 (br, 3H, CH3), 2.43 (s, 3H, CH3), 2.68 (br, 1H,
C4H7NO-3-H), 2.95 (br, 2H, NCH2), 3.15 (br, 1H,
C4H7NO-5-Ha), 3.70 (br, 1H, C4H7NO-5-He), 3.83—
3.86 (br, 5H, OCH2, CH3O), 3.93 (s, 1H, C4H7NO-6-Ha),
4.41 (br, 1H, C4H7NO-6-He), 4.65 (s, 2H, CH2CO), 4.82
(br, 1H, C4H7NO-2-H), 5.06 (s, 2H, PhCH2O), 6.68—
7.68 (m, 16H, C6H5, C6H4, C6H4, C6H3), 13.37 (s, 1H,
－
1
1215, 1184, 1089, 1072, 1036 cm ; ESI-MS m/z: 626
(M＋－HCl). Anal. calcd for C36H36Cl2N2O6: C 65.16, H
5.47, N 4.22; found C 65.13, H 5.42, N 4.28.
2-[2-(6-Methoxy-5-chloronaphthalen-2-yl) morpho-
lino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-acetate hydrochloride (1j): White
NH); IR (KBr) ν: 3339, 2932, 2609, 1737, 1681, 1611,
1514, 1477, 1458, 1239, 1175, 1146, 1089 cm－
;
1
＋
ESI-MS m/z: 640 (M － HCl). Anal. calcd for
C31H32Cl2N2O5: C 66.57, H 5.73, N 3.98; found C 66.46,
H 5.76, N 3.95
1
solid, yield 33%; m.p. 225—227 ℃; H NMR (CDCl3,
400 MHz) δ: 2.37 (s, 3H, CH3), 2.66 (br, 2H, C4H7NO-
3,5-Ha), 3.31—3.55 (m, 4H, NCH2, C4H7NO-3,5-He),
3.81 (s, 2H, OCH2), 3.85 (s, 3H, indole-5-CH3O), 3.99
(br, 1H, C4H7NO-6-Ha), 4.05 (s, 3H, CH3O), 4.46 (br,
1H, C4H7NO-6-He), 4.71 (br, 2H, CH2CO), 5.44 (br, 1H,
C4H7NO-2-H), 6.68—8.22 (m, 12H, C10H5, C6H4, C6H3),
13.78 (s, 1H, NH); IR (KBr) ν: 3436, 2958, 2326, 1746,
1669, 1604, 1479, 1455, 1356, 1340, 1279, 1243, 1228,
COX-1/2 inhibitory assay
Peritoneal macrophages were obtained from mice
that had been euthanized by cervical dislocation. The
peritonea of the animals were surgically exposed using a
midline incision. An experimental group of mice had
been administered an intraperitoneal injection of 2 mL
of 30 mg/mL sodium thioglycollate 4 d prior to the sac-
rifice. Following removal of the carotid artery, the ex-
cised tissue was immersed in 75% ethanol for 1—2 min;
the peritoneal fluid was then harvested by injecting
D-Hanks into the peritoneal cavity and subsequent sy-
ringe aspiration. Cell suspensions were pelleted by cen-
trifugation and washed with D-Hanks for twice.
－
1
1162, 1140, 1093, 1072, 1036 cm ; ESI-MS m/z: 667
(M＋－HCl). Anal. calcd for C36H35Cl3N2O6: C 61.94, H
5.05, N 4.01; found C 61.98, H 5.11, N 3.94.
2-[2-(6-Methoxy-5-bromonaphthalen-2-yl)-morpho-
lino-4-yl]ethyl 1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-acetate hydrochloride (1k): White
1
solid, yield 45%; m.p. 229—230 ℃; H NMR (CDCl3,
Cells were incubated in PRMI 1640 complete me-
dium supplemented with 5% NCS for 4 h at 37 ℃ with
5% CO2 in a humidified chamber.
The mouse peritoneal macrophages were seeded in
48-well plates (1×109 L/well), cultured for 2 h. Cul-
tures were removed and washed twice with D-Hanks,
PRMI 1640 complete medium supplemented with 5%
NCS was added into each well, then processed accord-
ing to the following groups: (1) negative control group
of DMSO (control); (2) A23187 (final concentration 1
400 MHz) δ: 2.36 (s, 3H, CH3), 2.66 (br, 2H, C4H7NO-
3,5-Ha), 3.28—3.56 (m, 4H, NCH2, C4H7NO-3,5-He),
3.81 (s, 2H, OCH2), 3.85 (s, 3H, indole-5-CH3O), 3.98
(br, 1H, C4H7NO-6-Ha), 4.04 (s, 3H, CH3O), 4.46 (br,
1H, C4H7NO-6-He), 4.71 (br, 2H, CH2CO), 5.43 (br, 1H,
C4H7NO-2-H), 6.68—8.22 (m, 12H, C10H5, C6H4, C6H3),
13.80 (s, 1H, NH); IR (KBr) ν: 3460, 2957, 2326, 1742,
1668, 1606, 1479, 1456, 1357, 1327, 1274, 1163, 1142,
－
＋
1
1095, 1068, 1037 cm ; ESI-MS m/z: 706 (M －HCl).
Anal. calcd for C36H35BrCl2N2O5: C 58.24, H 4.75, N
1342
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1339—1344

Design, Synthesis and Biological Evaluation of 2-(2-Arylmorpholino-4-yl)ethyl Esters
－
－
1
1
μmol•L ); (3) A23187＋10a—10g (5 μmol•L L); (4)
A23187＋celecoxib (1 μmol•L－1). Experiments were
performed at least three times in triplicate. The perito-
neal macrophages, drugs or solvents were incubated for
1 h at 37 ℃ with 5% CO2, then A23187 was added
(final concentration 1 μmol•L ) and incubated for 1 h
under the same conditions. The supernatant was col-
lected and the concentration of 6-keto-PGF1α was tested
with square-free kit marked with 125I. The standard
curve was made following the method supplied, and the
concentration of the tested samples was calculated ac-
cording to the measured values respectively.
Table 1 Selected examples of COX inhibitory activity and se-
lectivity
IC50/(μmol•L－
)
1
Compd. Selectivity index (COX-1/COX-2)
COX-2 COX-1
89.4 ＞100
0.076 3.72
0.031 1.56
0.071 2.13
－
1
1a
＞1.1
49
1b
1c
50
1d
30
1e
1f
58
0.13
7.52
182
358
214
0.084 15.3
0.038 13.6
1g
The COX-2 inhibitory activity processed according
to the following four groups: (1) Negative control group
of DMSO (control); (2) LPS (final concentration 1
Celecoxib
0.05
10.7
－
1
mg/L); (3) LPS＋10a—10g (5 μmol•L ); (4) LPS＋
compounds showed that the conversion of the free acid
group of non-selective inhibitor indomethacin to the
2-(2-arylmorpholino-4-yl)ethyl ester gives rise to com-
pounds possessing various degrees of COX-2 selectivity.
Nonsubstituted phenyl derivative 1a which showed
weak COX-1 and COX-2 inhibitory activity differs from
other compounds with substituents on the phenyl ring.
The results are similar with the reported unsubstituted
benzyl esters of 4- or 6-chloroindomethacin which also
showed weak inhibitory activity.[18]
－
1
celecoxib (1 μmol•L ). The peritoneal macrophages,
drugs or solvents were also incubated for 1 h at 37 ℃
with 5% CO2, then LPS was added (final concentration
1 mg/L) and incubated for 9 h under the same condi-
tions. The supernatant was collected and the concentra-
tion of PGE2 was tested with square-free kit marked
with 3H.
Results and Discussion
Replacement of phenyl by 4-methylphenyl group
(1b, IC50＝0.076 μmol•L－1) increased the selectivity
and potency further against COX-2. 4-Ethylphenyl de-
rivative (1c, IC50＝0.031 μmol•L－1) showed greater
COX-2 inhibitory activity than 4-methylphenyl deriva-
tive (1b) probably because the larger group ethylphenyl
in size possessed the side pocket of COX-2 isoenzyme
more easily. A variety of alkoxylphenyl moieties (1d—
1f) were examined and all induced moderate to good
COX-2 selectivity. Among the alkoxyl derivatives,
Preparation of the intermediate 2-(2-arylmorpholino-
4-yl)ethanols 2a—2m followed the method reported
before by us,[9,13] arylalkylones were brominated via
copper(II) bromide in the presence of ethanol as solvent
affording α-phenacylbromides. Reaction of phenacyl-
bromides with 2,2'-azanediyl-diethanol gave hemiketals,
formic acid (88%) was added, then intermediates 2a—
2m formed via a Leuckart-Wallach reaction.[14]
Then indomethacin acid chloride (3) was reacted
with 2-(2-phenylmorpholino-4-yl)ethanol (2a) without
purification in the presence of tetrahydrofuran (THF)
and triethylamine to obtain the ester as an oily product,
which was converted into its hydrochloride derivative
1a as white solid. The other products 1b—1m were ob-
tained in a similar way by reacting their respective es-
ters with hydrochloride. All the compounds were char-
acterized by their spectra data.
－
1
4-(n-BuO)phenyl derivative (1f, IC50＝15.3 μmol•L )
showed least COX-1 inhibitory activity and greatest
COX-2 selective inhibitory activity (SI 182), which is
comparative with celecoxib (SI 214). Compared with
the alkyl replacement derivatives, the alkoxyl deriva-
tives showed higher selectivity with little variation of
COX-2 inhibitory. The reason may be that: (1) the
alkoxyl replacement with bigger size than alkyl group
leading to greater steric hindrance hindered the binding
with the side pocket of COX-2; (2) the hydrogen bonds
between alkoxyl and amino acid residues strengthened
the binding, the proportion of the two sides led to the
different performances of 1d—1f. The least COX-2 in-
All of the new compounds synthesized were tested
in vitro initially at 10.0 μmol•L－ for selectivity and
1
potency against human COX-1 and COX-2 enzymes.
On the basis of their in vitro efficacy, selected com-
pounds 1a—1g were tested against COX-1 at 0.1, 1.0,
10.0 and 100 μmol•L－ 1, respectively, while against
－
1
hibitory activity of 1e (IC50＝0.13 μmol•L ) may be
because that 4-isopropoxyphenyl replacement caused a
too big steric hindrance which hindered the binding with
the side pocket. It means that only the replacement with
proper size could enhance the binding.
－
1
COX-2 at 0.01, 0.1, 1.0 and 10.0 μmol•L , respectively.
Celecoxib was used as reference compound for the in
vitro assay. The concentration of the selected/promising
compounds for 50% inhibition of COX-1 and COX-2
(IC50) was calculated using the software (SPSS). The
results are shown in Table 1.
Remarkably, selectivity was observed for strong
electron-withdrawing group such as fluoro and chloro
atoms, compound 1g possessing 2,4-dichloro-5-fluoro-
phenyl group showed better COX-2 inhibitory activity
and selectivity over celecoxib. We suppose that strong
Indomethacin is a non-selective inhibitor of COX
isozymes [COX-2 SI＜1, the ratio of IC50(COX-1) to
IC50(COX-2)].[15-17] Biological evaluation of the title
Chin. J. Chem. 2012, 30, 1339—1344
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1343

Shi et al.
FULL PAPER
hydrogen bonds were formed between fluoro, chloro
atoms and amino acid residues in the side pocket of
COX-2 enzyme.
ject of Changsha, Hunan Province (No. 0901077-31) for
the financial support for this project.
This study revealed that the replacement of the
phenyl could lead to increased selectivity and potency
further against COX-2. The steric hindrance produced
by phenyl replacement hindered the binding with the
COX-1 and promoted the binding with COX-2, however,
too big replacement (4-isopropoxyphenyl) hindered the
binding with the side pocket too. The fluoro, chloro at-
oms in the replacement could strongly strengthen the
selectivity and potency further against COX-2. Totally,
the selectivity and potency against COX-2 were deter-
mined by the steric hindrance caused by the replacement,
and the hydrogen bonds formed between the replace-
ment and the amino acids residues as well.
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Acknowledgement
We thank the Key Scientific and Technological Pro-
(Zhao, C.)
1344
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1339—1344