Synthesis and evaluation of 2-(2-arylmorpholino)ethyl esters of ibuprofen hydrochlorides as COX-2 and serotonin reuptake inhibitors

Article abstract of DOI:10.1002/ardp.201300279

Based on the positive effects of COX-2 inhibitors on depressive symptoms and the desirable physicochemical and biological properties of the morpholine group, a series of novel 2-(2-arylmorpholino)ethyl esters of ibuprofen hydrochlorides were designed, synthesized, and tested for their COX-2 inhibitory and serotonin reuptake inhibitory activities in vitro. The structure-activity relationships of the 2-(2-arylmorpholino)ethyl esters of ibuprofen hydrochlorides as dual COX-2 and serotonin reuptake inhibitors were determined and discussed in detail. The biological assays indicated that five of the compounds possess good COX-2 selectivity (selectivity index COX-1/COX-2 42.8-158.1). The compound 2-[2-(4-benzyloxyphenyl)morpholino]ethyl 2-(4-iso-butylphenyl)-propanoate hydrochloride (1k) shows better COX-2 inhibitory activity (IC₅₀ = 0.78 μM) than ibuprofen (IC ₅₀ = 7.6 μM), and it simultaneously possesses favorable serotonin reuptake inhibitory activity. A series of novel 2-(2-arylmorpholino)ethyl esters of ibuprofen were tested for their COX-2 inhibitory and serotonin reuptake inhibitory activities in vitro. Most of the compounds possess good COX-2 selectivity. Compound 1k shows better COX-2 inhibitory activity than ibuprofen and possesses favorable serotonin reuptake inhibitory activity. According to the results of the biological assays, 2-arylmorpholine-substituted ibuprofen could be used as a core structure to design novel dual COX-2 and serotonin reuptake inhibitors.

Full text of DOI:10.1002/ardp.201300279

Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
89
Full Paper
Synthesis and Evaluation of 2-(2-Arylmorpholino)ethyl Esters
of Ibuprofen Hydrochlorides as COX-2 and Serotonin
Reuptake Inhibitors
Jie Dou1, Lei Shi1, Aixi Hu1, Minyu Dong1, Jiangping Xu2, Ailin Liu3, and Yiping Jiang2
1
College of Chemistry and Chemical Engineering, Hunan University, Changsha, China
Department of Pharmacology, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou,
2
China
3
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Med College, Beijing,
China
Based on the positive effects of COX-2 inhibitors on depressive symptoms and the desirable
physicochemical and biological properties of the morpholine group, a series of novel 2-(2-
arylmorpholino)ethyl esters of ibuprofen hydrochlorides were designed, synthesized, and tested for
their COX-2 inhibitory and serotonin reuptake inhibitory activities in vitro. The structure–activity
relationships of the 2-(2-arylmorpholino)ethyl esters of ibuprofen hydrochlorides as dual COX-2 and
serotonin reuptake inhibitors were determined and discussed in detail. The biological assays indicated
that five of the compounds possess good COX-2 selectivity (selectivity index COX-1/COX-2 42.8–158.1).
The compound 2-[2-(4-benzyloxyphenyl)morpholino]ethyl 2-(4-iso-butylphenyl)-propanoate hydro-
chloride (1k) shows better COX-2 inhibitory activity (IC50 ¼ 0.78 mM) than ibuprofen (IC50 ¼ 7.6 mM),
and it simultaneously possesses favorable serotonin reuptake inhibitory activity.
Keywords: 2-(2-Arylmorpholino)ethyl ester hydrochloride / COX-2 inhibitors / Ibuprofen / Serotonin reuptake inhibitors /
Synthesis
Received: July 25, 2013; Revised: September 6, 2013; Accepted: September 11, 2013
DOI 10.1002/ardp.201300279
Introduction
which have been found to negatively regulate GR function
through their signaling pathways.
Accumulating evidence demonstrates that depression is
associated with increased inflammatory drive, and provoking
an acute inflammatory response in healthy humans can result
in depression-like behaviors [1–4]. Depression and inflamma-
tion are also common among cancer patients. Systemic
inflammation may result directly from the tumor, which can
release proinflammatory cytokines, or it may arise as cancer
cells, which are identified as a kind of foreign body by the
immune system [2, 5]. A plausible biological pathway linking
inflammation and depression is the activation of indoleamine
2,3-dioxygenase [6]. In addition, impaired glucocorticoid
receptor (GR) function has been characterized by increased
circulation and expression of proinflammatory cytokines,
Celecoxib, a classic COX-2 inhibitor, has also been
demonstrated to significantly increase nuclear localization
of the GR, which expands its potential clinical application to
multiple disorders associated with impaired GR function
including major depression [7]. Moreover, the inhibition of
COX-2 was reported to limit the impact of stressors. Treatment
with celecoxib prevented the deregulation of the hypotha-
lamic pituitary adrenal (HPA) axis, in particular with regard
to the increase of cortisol, one of the key biological features
associated with depression, while treatment with a COX-1
inhibitor did not prevent HPA-axis dysregulation [7, 8]. In
addition, in a randomized, double-blind trial in patients with
major depression, celecoxib plus fluoxetine resulted in a
statistically significant better outcome related to improve-
ments in major depression symptoms compared with
fluoxetine-alone monotherapy [9]. Accordingly, a clinical
antidepressant effect of rofecoxib was observed in patients
with osteoarthritis among which 15% had a comorbid
Correspondence: Dr. Aixi Hu, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, China.
E-mail: axhu@hnu.edu.cn
Fax: þ86 731 88713642
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90
J. Dou et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
depressive syndrome, as evaluated by a specific depression
self-report, a significant decrease (from 15% to 3% of patients)
in the rate of substantive depression during therapy with
rofecoxib [10]. Taken together, it is not difficult to predict that
the potential COX-2 inhibitors that possess both considerable
anti-inflammatory activity and possible antidepressant
activity will be widely used in the treatment of multiple
disorders associated with inflammation and depression and
even in the auxiliary treatment of cancer.
Morpholine analogs have shown good anti-inflammatory
and antioxidant activity. Kourounakis and co-workers [11]
synthesized a variety of 2,6-di-tert-butylphenol, which are
amides or amines of morpholine residues, some of which
exhibited anti-inflammatory and antioxidant activities.
Naproxen [12] and indomethacin [13] as traditional non-
steroidal anti-inflammatory drugs (NSAIDs) were modified
with 2-(2-arylmorpholino)ethanols, which have shown better
COX-2 inhibitory activities in vitro and lower gastrointestinal
irritation side effects by occupying the additional side pocket
of the COX-2 enzyme and the antioxidant activity of the
morpholine moiety.
and provided a pharmaceutical composition. This research
makes it possible to use morpholine derivatives as antide-
pressant drugs.
Based on the positive effects of COX-2 inhibitors on
depressive symptoms and on the desirable physicochemical
and biological properties of the morpholinyl group, the free
carboxylic moiety of a NSAID ibuprofen, which is restricted
due to its gastrointestinal irritation side effects, after long-
term use [26] was modified with 2-(2-arylmorpholino)ethyl
esters to increase its COX-2 inhibitory activity based on the
structural differences between COX-1 and COX-2 [27–30]. We
suppose that these novel COX-2 inhibitors could exhibit good
stability and show both favorable COX-2 inhibitory activity
and antidepressant property (Fig. 2). Then, a series of
compounds were obtained and tested in vitro for the
evaluation of their COX-2 inhibitory and antidepressant
activities. And according to the biological assay, these
derivatives of ibuprofen show both good COX-2 inhibitory
activity and antidepressant property. The liposolubility and
bioavailability of these compounds in in vivo tests will be the
subject of a separate report soon.
Morpholine ring possessing specially chemical and physico-
chemical properties was widely utilized to modify biological
properties of various drugs. Reboxetine [14], moclobe-
mide [15], and viloxazine [16], known as good antidepressants,
The target molecules 2-(2-arylmorpholino)ethyl ester of
ibuprofen hydrochlorides (1a–1l) were synthesized using the
sequence of reactions illustrated in Scheme 1.
all share a morpholine scaffold in their structure (Fig. 1). It has Results and discussion
been demonstrated that reboxetine, as an a-aryloxybenzyl
derivative of morpholine, is metabolized via oxidation of the
morpholine ring, O-dealkylation of the ethoxyphenoxy ring,
and hydroxylation. Some evidences also suggest that,
compared with fluoxetine, reboxetine may be more effective
in severely depressed patients in improving social behavior.
Limited long-term data suggest that reboxetine may prevent
relapse in patients who respond to short-term therapy [17, 18].
Other novel morpholine derivatives with potent antidepres-
sant effects were also reported recently [19–22]. One study [23]
reported the pharmacological and biochemical properties of a
new compound, 2-(7-indeyloxymethyl)morpholine hydrochlo-
ride, which has been confirmed possessing less effect on the
uptake of either serotonin (5-HT) or norepinephrine (NE). This
compound has a novel profile as an antidepressant agent,
which is quite different from that of either viloxazine or
tricyclic compounds. Two US patents [24, 25] also reported the
structure and synthesis of a series of morpholine derivatives
Synthesis
Ibuprofen was converted into 2-(4-iso-butylphenyl)propanoyl
chloride (3) through the reaction with thionyl chloride. And
2-(2-phenylmorpholino)ethanol (2a) formed via the Leuckart–
Wallach reaction [31] without purification was reacted with
the compound (3) in the presence of THF and Et3N. Then the
obtained ester as an oily product was converted into its
hydrochloride derivative (1a) as a white solid. Other products
(1b–1l) were obtained in the similar way by reacting their
respective esters with hydrogen chloride. We found that the
substituent Ar has great influence on the cyclization reaction.
The compounds (1g–1l) substituted with naphthyl or
benzyloxyphenyl have a higher yield than those substituted
with phenyl; the order of yield is 6-MeO-2-C10H6 >
4-PhCH2OC6H4 > C6H5. The synthesis method was high-yield,
safe, operationally simple, and cost-effective, meeting with
the requirements of contemporary organic synthesis, and was
Figure 2. The structure design on morpholine-substituted ibuprofen
derivatives expected to be dual-targeted compounds.
Figure 1. Morpholine scaffold in common antidepressants.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
Ibuprofen Derivatives as COX-2 and 5-HT Reuptake Inhibitors
91
Scheme 1. Synthesis of the molecules. Reagents and conditions: (a) CuBr2, EtOH, reflux, 6 h; (b) (i) HN(CH2CH2OH)2, NMP, 60°C, 2 h;
(ii) HCO2H, 180°C, 18 h; (c) SOCl2, C6H6, reflux, 5 h; and (d) (i) THF, Et3N, rt, 24 h; (ii) HCl (g), Et2O then (CH3)2CO.
reported in [13]. All of the compounds were characterized by
1H NMR and elemental analysis.
factor in maintaining potent COX-2 inhibitory activity.
Increasing the size of Ar groups appropriately is benefit to
improving the selectivity index (COX-1/COX-2), while an
oversize group often results in the decrease in COX-1 and
COX-2 inhibitory activity, as the IC50 shown in Table 1.
6-Methoxy naphthyl substituted morpholine derivative (1j)
shows the best COX-2 inhibitory activity (IC50 ¼ 0.038 mM).
This may be due to the morpholino-3-methyl, which made
compound 1j suitable for taking the side pocket of COX-2.
Plausibly, it may increase the biological activity when
CH3– replaced in R instead of H– according to the comparison
of COX-2 inhibitory activity of 1j and 1g. In addition,
compound 1h (IC50 ¼ 0.075 mM) shows better COX-2 inhibitory
activity than compound 1i (IC50 ¼ 0.16 mM). Replacement of
bromine atom with chlorine could increase the selectivity,
and it is probably because chlorine atom could form hydrogen
bonds with amino acid residues more easily than bromine
atom. 4-Benzyloxyphenyl substituted morpholine derivative
(1k) shows good COX-2 inhibitory and selectivity.
Biological in vitro evaluation
All of the new compounds synthesized were tested in vitro
initially at 10 mM for selectivity and potency against human
COX-1 and COX-2 enzymes. On the basis of their in vitro
efficacy, selected compounds (1g–1l) were tested against
COX-1 at 0.1, 1.0, 10.0, and 100 mM, respectively, while against
COX-2 at 0.01, 0.1, 1.0, and 10.0 mM, respectively. The IC50
values for COX-1 and COX-2 were determined for selected/
promising compounds. Celecoxib was used as control group
for the in vitro COXs inhibitory assay. The concentration of the
selected/promising compounds for 50% inhibition of COX-1
and COX-2 (IC50) was calculated by the software SPSS. The
results are shown in Table 1.
COX-2 inhibitory activity and selectivity
Ibuprofen is a non-selective inhibitor of COX isozymes; the
IC50 values of COX-2 and COX-1 are 7.6 and 20 mmol/L,
respectively [32]. Biological evaluation of the target molecules
showed that the conversion of the free acid group of the non-
selective inhibitor ibuprofen to 2-(2-arylmorpholino)ethyl
esters gives rise to compounds possessing good COX-2
selectivity (selectivity index COX-1/COX-2 42.8–158.1).
According to the analysis of the result of biological activity
assay, the possible structure–activity relationship of com-
pounds 1a–1l is shown in Fig. 3. On the whole, choosing a
suitable size of Ar groups, which has been confirmed by the
comparison between phenyl-, naphthyl-, and benzyloxy-
phenyl-substituted derivatives according to 1a–1l, is a major
Serotonin reuptake inhibitory activity
The compounds (1g–1l) possessing favorable COX-2 inhibitory
activity were tested in vitro for the inhibition of SERT at the
concentration of 10 mg/L. Fluoxetine was used as control
group for the in vitro SERT inhibitory assay (Table 1). Results
revealed that several compounds also displayed substantial
serotonin reuptake inhibition. The compound 1h (inhibition
of SERT 30.0%) also shows better SERT inhibitory activity than
compound 1i (inhibition of SERT 5.5%), and it can be supposed
that the mechanism of chlorine atom in SERT inhibition is
similar to its function in COX-2 inhibition. Moreover, the
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J. Dou et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
Table 1. The COX inhibitory activity and the inhibition rate of SERT of compounds 1a–1l.
IC50 (mmol/L)
Selectivity index
(COX-1/COX-2)
Inhibition on SERT (%)
(at 10 mg/L)
Ar
R
Compd
COX-2
COX-1
C6H5
4-MeC6H4
4-EtC6H4
4-MeOC6H4
H
H
H
H
H
H
H
H
H
1a
1b
1c
1d
1e
1f
1g
1h
–
–
>100
>100
>100
>100
>100
>100
0.09
0.075
0.16
0.038
0.78
>100
>100
>100
>100
>100
>100
3.85
1.76
25.3
1.76
47.2
–
–
–
–
–
–
4-i-PrOC6H4
–
–
2, 4-Cl2-5-FC6H2
6-MeO-2-C10H6
5-Cl-6-MeO-2-C10H5
5-Br-6-MeO-2-C10H5
6-MeO-2-C10H6
4-PhCH2OC6H4
4-PhCH2OC6H4
–
–
42.8
43.4
158.1
46.3
60.5
0.88
214.0
2.6
19.4
30.0
5.5
12.1
49.9
14.6
1i
1j
1k
CH3
H
CH3
1l
78.4
0.05
7.6
69.5
10.7
20.0
Celecoxib
Ibuprofen [32]
Fluoxetine
55.0
compound 1k shows good SERT inhibitory activity (inhibition
of SERT 49.9%), which is related to its COX-2 inhibitory activity.
promising compound among these scaffolds, the further
exploration in vivo model of which will be reported in future
publications. What is more, we have formed the structure–
activity relationship of 2-(2-arylmorpholino)ethyl ester of
ibuprofen hydrochlorides as dual COX-2 and SERT inhibitors,
which will be vital for further development of more effective
drugs and a significant reference for similar research.
Conclusion
With the aim of developing novel compounds possessing both
COX-2 and SERT inhibitory activity, we have designed,
synthesized, and tested a series of 2-(2-arylmorpholino)ethyl
ester of ibuprofen hydrochlorides, and some of them,
especially 1h and 1k, displayed dual COX-2 and SERT
inhibitory activities. The compound 1k shows good COX-2
selective inhibitory property and its anti-inflammatory
activity is higher than that of ibuprofen. At the same time,
compound 1k possesses favorable antidepressant activity
compared with fluoxetine. It is identified as the most
Experimental
Instruments
All reagents and solvents were of commercial quality and used
without further purification. Reactions were performed under
the protection of nitrogen and monitored by thin-layer
chromatography (TLC) on silica gel plates, visualizing with
ultraviolet light or iodine spray. Flash chromatography was
Figure 3. The structure–activity relationship of
compounds 1a–11.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
Ibuprofen Derivatives as COX-2 and 5-HT Reuptake Inhibitors
93
performed on silica gel (200–400 mesh) using commercially
available petroleum ether, ethyl acetate. Melting points (m.p.)
were taken in open capillaries and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were obtained with
a Bruker 400 MHz spectrometer. Chemical shifts (d) are relative to
tetramethylsilane (TMS, d ¼ 0.00) as internal standard and
expressed in ppm, and signals are given as follows. Spin
multiplicities are given as s (singlet), d (doublet), t (triplet), and
m (multiplet) as well as br (broad). Coupling constants (J) are given
in Hertz. Microanalysis was performed using a C H N S/O analyzer.
Elemental data are within ꢀ0.2%. Sodium thioglycollate (Difco).
Calcium cation ionophore A23187, lipopolysaccharides (LPS),
dimethyl sulfoxide (DMSO), cecoxib (Sigma), new calf serum (NCS,
5%, Gibco), square-free kit (PGE2 and 6-keto-PGF1a, Institute of
Immune Technology of East Asia). 2-(2-Arylmorpholino)ethanols
were synthesized following [12].
1.83 (m, 1H, CH), 2.05–2.24 (m, 2H, C4H7NO 5-H), 2.34 (s, 3H, CH3),
2.43 (dd, J ¼ 2.0 Hz, J ¼ 6.8 Hz, 2H, CH2), 2.59–2.69 (m, 3H, NCH2,
C4H7NO 3-Ha), 2.84–2.87 (m, 1H, C4H7NO 3-He), 3.67–3.75 (m, 2H,
CH, C4H7NO 6-Ha), 3.90–3.94 (m, 1H, C4H7NO 6-He), 4.21–4.24
(bm, 2H, OCH2), 4.44–4.246 (bm, 1H, C4H7NO 2-H), 7.04–7.26
(m, 8H, C6H4, C6H4). Elemental analysis found C, 70.03; H, 8.12;
N, 3.11; C26H36O3NCl requires C, 70.01; H, 8.14; N, 3.14.
2-[2-(4-Ethylphenyl)morpholino]ethyl 2-(4-iso-butylphenyl)-
propanoate hydrochloride (1c)
White solid, m.p. 134–136°C, yield 35.1%. 1H NMR (CDCl3,
400 MHz), d: 0.89 (dd, J ¼ 0.8 Hz, J ¼ 6.8 Hz, 6H, 2 ꢁ CH3), 1.22
(t, J ¼ 8.0 Hz, 3H, CH3), 1.48 (m, 3H, CH3), 1.83 (m, 1H, CH), 2.22–
2.28 (m, 2H, C4H7NO 5-H), 2.43 (dd, J ¼ 1.2 Hz, J ¼ 7.2 Hz, 2H, CH2),
2.60–2.69 (m, 5H, CH2, NCH2, C4H7NO 3-Ha), 2.87–2.90 (m, 1H
C4H7NO 3-He), 3.68–3.75 (m, 2H, CH, C4H7NO 6-Ha), 3.91 (bm, 1H,
C4H7NO 6-He), 4.23–4.26 (bm, 2H, OCH2), 4.48 (bm, 1H, C4H7NO
2-H), 7.03–7.26 (m, 8H, C6H4, C6H4). Elemental analysis found C,
70.54; H, 8.28; N, 3.05; C27H38O3NCl requires C, 70.49; H, 8.33;
N, 3.04.
Synthesis of the title compounds
Synthesis of 2-(4-iso-butylphenyl)propanoyl chloride (3)
Ibuprofen (1.95 mmol) and thionyl chloride (5.48 mmol) in
benzene (5 mL) were refluxed for 5 h, until the evolution of
hydrogen chloride and sulfur oxide ceased. The excess thionyl
chloride and benzene were distilled off under reduced pressure,
to give yellow liquid 2-(4-iso-butylphenyl)propanoyl chloride (3).
2-[2-(4-Methoxyphenyl)morpholino]ethyl 2-(4-iso-
butylphenyl)propanoate hydrochloride (1d)
Yellow oil, yield 40.6%, 1H NMR (CDCl3, 400 MHz), d: 0.89 (dd,
J ¼ 0.8 Hz, J ¼ 6.8 Hz, 6H, 2 ꢁ CH3), 1.48 (d, J ¼ 6.8 Hz, 3H, CH3),
1.83 (m, 1H, CH), 2.04–2.26 (m, 2H, C4H7NO 5-H), 2.43
(d, J ¼ 6.8 Hz, 2H, CH2), 2.57–2.68 (m, 3H, NCH2, C4H7NO 3-Ha),
2.81–2.84 (m, 1H, C4H7NO 3-He), 3.67–3.74 (m, 2H, CH, C4H7NO
6-Ha), 3.82 (s, 3H, OCH3), 3.89–3.92 (m, 1H, C4H7NO 6-He), 4.17–
4.26 (m, 2H, OCH2), 4.41 (bm, 1H, C4H7NO 2-H), 6.85–7.26 (m, 8H,
C6H4, C6H4). Elemental analysis found C, 67.58; H, 7.81; N, 3.05;
C26H36O4NCl requires C, 67.59; H, 7.85; N, 3.03.
General procedure for the synthesis of 2-(2-arylmorpholino)-
ethyl ester of ibuprofen hydrochlorides 1a–1l
2-(4-iso-Butylphenyl)propanoyl chloride (3) prepared before was
dissolved in 10 mL THF; then the solution of 2-(2-arylmorpholino)
ethanols (2a–2l, 3.9 mmol) as well as triethylamine (1 mL) was
slowly added. The reaction mixture was stirred at room
temperature for 24 h and filtered to remove triethylamine
hydrochloride. The filtrate was distilled under reduced pressure
to remove tetrahydrofuran; then the residue was dissolved in
ethyl acetate and washed in sodium carbonate (6%), dried over
anhydrous sodium sulfate, filtered, and evaporated under
reduced pressure. The free base was obtained as oily product
by flash chromatography, eluting with petroleum ether/ethyl
acetate. Then the oily product was dissolved in proper ether
and treated with ethereal hydrogen chloride, followed by
filtration, and the residue was washed with acetone to give
hydrochlorides.
2-[2-(4-iso-Propoxyphenyl)morpholino]ethyl 2-(4-iso-
butylphenyl)propanoate hydrochloride (1e)
Yellow oil, yield 33.9%. 1H NMR (CDCl3, 400 MHz), d: 0.89
(dd, J ¼ 0.8 Hz, J ¼ 6.8 Hz, 6H, 2 ꢁ CH3), 1.32 (d, J ¼ 6.4 Hz, 6H,
2 ꢁ CH3), 1.48 (d, J ¼ 6.8 Hz, 3H, CH3), 1.82 (m, 1H, CH), 2.10–2.28
(m, 1H, C4H7NO 5-Ha), 2.43 (d, J ¼ 6.8 Hz, 2H, CH2), 2.68–2.72
(m, 3H, NCH2, C4H7NO 3-Ha), 3.12 (m, 1H, C4H7NO 5-He), 3.42–3.54
(bm, 1H, C4H7NO 3-He), 3.68–3.87 (m, 3H, C4H7NO 6-H, CH),
4.19–4.24 (m, 2H, OCH2), 4.38–4.42 (m, 1H, C4H7NO 2-H), 4.54
(m, J ¼ 6.4 Hz, 1H, CH), 6.80–7.26 (m, 8H, C6H4, C6H4). Elemental
analysis found C, 68.58; H, 8.21; N, 2.85; C28H40O4NCl requires C,
68.62; H, 8.23; N, 2.86.
2-(2-Phenylmorpholino)ethyl 2-(4-iso-butylphenyl)-
propanoate hydrochloride (1a)
Yellow solid, m.p. 119–122°C, yield 54.1%. 1H NMR (CDCl3,
400 MHz), d: 0.81–0.91 (m, 6H, 2 ꢁ CH3), 1.48 (dd, J ¼ 2.8 Hz,
J ¼ 7.2 Hz, 3H, CH3), 1.83 (m, 1H, CH), 2.22–2.28 (m, 2H, C4H7NO
5-H), 2.42 (m, 2H, CH2), 3.00–3.24 (bm, 4H, NCH2, C4H7NO 3-H),
3.70 (m, 1H, CH), 3.90 (tt, J ¼ 8.8 Hz, J ¼ 2.8 Hz, 1H, C4H7NO 6-Ha),
4.12 (bs, 1H, C4H7NO 6-He), 4.53 (bs, 2H, OCH2), 4.94 (br, 1H,
C4H7NO 2-H), 7.10–7.70 (m, 9H, C6H4, C6H5). Elemental analysis
found C, 69.48; H, 7.91; N, 3.25; C25H34O3NCl requires C, 69.51; H,
7.93; N, 3.24.
2-[2-(2,4-Dichloro-5-fluorophenyl)morpholino]ethyl 2-(4-
iso-butylphenyl)propanoate hydrochloride (1f)
White solid, m.p. 185–188°C, yield 63.5%. 1H NMR (CDCl3,
400 MHz), d: 0.88–0.93 (m, 6H, 2 ꢁ CH3), 1.48 (dd J ¼ 7.6 Hz,
J ¼ 5.6 Hz, 3H, CH3), 1.83 (m, 1H, CH), 2.18–2.29 (bs, 1H, C4H7NO 5-
Ha), 2.37–2.45 (m, 2H, CH2), 2.52–2.61 (bs, 1H, C4H7NO 5-He),
2.98–3.10 (bm, 1H, C4H7NO 3-Ha), 3.17–3.24 (m, 2H, NCH2), 3.57
(d, 1H, J ¼ 9.2 Hz, C4H7NO 3-He), 3.67–3.73 (m, 1H, CH), 3.85–3.97
(m, 1H, C4H7NO 6-Ha), 4.37–4.46 (m, 1H, C4H7NO 6-He), 4.70
(t, J ¼ 7.2 Hz, 2H, OCH2), 5.47 (t, 1H, J ¼ 7.2 Hz, C4H7NO 2-H),
6.92–7.49 (m, 6H, C6H4, C6H2), 14.05 (bs, 1H, HCl). Elemental
analysis found C, 57.88; H, 6.01; N, 2.72; C25H31O3NFCl3 requires
C, 57.87; H, 6.02; N, 2.70.
2-[2-(p-Tolyl)morpholino]ethyl 2-(4-iso-butylphenyl)-
propanoate hydrochloride (1b)
Yellow oil, yield 56.1%. 1H NMR (CDCl3, 400 MHz), d: 0.89 (dd,
J ¼ 0.8 Hz, J ¼ 6.8 Hz, 6H, 2 ꢁ CH3), 1.48 (d, J ¼ 6.8 Hz, 3H, CH3),
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94
J. Dou et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
2-[2-(6-Methoxynaphthalen-2-yl)morpholino]ethyl 2-(4-iso-
butylphenyl)propanoate hydrochloride (1g)
71.40; H, 7.51; N, 2.63; C32H40O4NCl requires C, 71.42; H, 7.49; N,
2.60.
White solid, m.p. 168–170°C, yield 45.0%. 1H NMR (CDCl3,
400 MHz), d: 0.84–0.90 (m, 6H, 2 ꢁ CH3), 1.47 (m, 3H, CH3), 1.71–
1.80 (m, 1H, CH), 2.31–2.38 (m, 2H, CH2), 2.44 (bs, 1H, C4H7NO 5-
Ha), 2.62 (bs, 1H, C4H7NO 5-He), 3.16–3.19 (bm, 1H, C4H7NO 3-Ha),
3.19–3.25 (m, 2H, NCH2), 3.43–3.55 (dd, 1H, J ¼ 0.8 Hz, J ¼ 4.0 Hz,
C4H7NO 3-He), 3.66–3.71 (br, 1H, CH), 3.97–3.99 (m, 1H, C4H7NO
6-Ha), 3.93 (s, 3H, OCH3), 4.38–4.41 (m, 1H, C4H7NO 6-He), 4.30–
4.74 (m, 2H, OCH2), 5.32 (t, 1H, J ¼ 7.2 Hz, C4H7NO 2-H), 6.84–7.78
(m, 10H, C6H4, C10H6). Elemental analysis found C, 70.37; H, 7.51;
N, 2.75; C30H38O4NCl requires C, 70.36; H, 7.48; N, 2.74.
2-[2-(4-Benzyloxyphenyl)-3-methylmorpholino]ethyl 2-(4-
iso-butylphenyl)propanoate hydrochloride (1l)
1
Yellow oil, yield 54.5%. H NMR (CDCl3, 400MHz), d: 0.76 (m, 3H,
CH3), 0.88–0.90 (m, 6H, 2ꢁ CH3), 1.49 (m, 3H, CH3), 1.82 (m, 1H, CH),
2.42 (m, 2H, CH2), 2.52–2.61 (m, 2H, NCH2), 2.72 (m, 1H, C4H6NO
5-Ha), 3.01 (m, 1H, C4H6NO 5-He), 3.65–3.72 (m, 3H, OCH2, CH),
3.81 (m, 1H, C4H6NO 3-H), 3.95 (m, 1H, C4H6NO 2-H),
4.12–4.31 (m, 2H, C4H6NO 6-H), 5.06 (d, 2H, PhCH2O), 6.92–7.41
(m, 13H, 2ꢁ C6H4, C6H5). Elemental analysis found C, 71.76; H, 7.64;
N, 2.55; C33H42O4NCl requires C, 71.78; H, 7.67; N, 2.54.
2-[2-(5-Chloro-6-methoxynaphthalen-2-yl)morpholino]-
ethyl 2-(4-iso-butylphenyl)propanoate hydrochloride (1h)
Pale white solid, m.p. 119–122°C, yield 59.5%, 1H NMR (CDCl3,
400 MHz), d: 0.77–0.86 (m, 6H, 2 ꢁ CH3), 1.33–1.38 (d, J ¼ 7.6 Hz,
3H, CH3), 1.80 (m, 1H, CH), 2.09–2.22 (m, 2H, CH2), 3.20–3.25 (bm,
3H, NCH2, C4H7NO 5-Ha), 3.57–3.64 (m, 2H, C4H7NO 5-He, CH),
3.74–3.83 (m, 2H, C4H7NO 3-H), 4.01 (s, 3H, OCH3), 4.05–4.12
(m, 2H, OCH2), 4.20–4.24 (m, 1H, C4H7NO 6-Ha), 4.46 (bs, 1H,
C4H7NO 6-He), 5.07 (m, 1H, C4H7NO 2-H), 6.84–8.13 (m, 9H, C6H4,
C10H5), 11.56 (bs, 1H, HCl). Elemental analysis found C, 65.89; H,
6.81; N, 2.54; C30H37O4NCl2 requires C, 65.93; H, 6.82; N, 2.56.
Biological assay
COX-1/2 inhibitory assay
The methods that followed [13] were shown as follows.
Peritoneal macrophages were obtained from mice that were
euthanized by cervical dislocation. The peritonea of the animals
were surgically exposed using a midline incision. An experimen-
tal group of mice were administered an intraperitoneal injection
of 2 mL of 3% m/v sodium thioglycollate four days prior to the
sacrifice. Following the removal of the carotid artery, the excised
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 syringe aspiration. Cell suspensions
were pelleted by centrifugation and washed with D-Hanks
two times.
2-[2-(5-Bromo-6-methoxynaphthalen-2-yl)morpholino]-
ethyl 2-(4-iso-butylphenyl)propanoate hydrochloride (1i)
Brown solid, m.p. 168–171°C, yield 71.2%, 1H NMR (CDCl3,
400 MHz), d: 0.84–0.90 (m, 6H, 2 ꢁ CH3), 1.47 (d, J ¼ 7.6 Hz, 3H,
CH3), 1.75 (m, 1H, CH), 2.28–2.38 (m, 2H, CH2), 2.62 (bs, 1H,
C4H7NO 5-Ha), 3.08–3.25 (bm, 3H, NCH2, C4H7NO 5-He), 3.45–3.55
(br, 1H, C4H7NO 3-Ha), 3.62–3.80 (m, 2H, C4H7NO 3-He, CH), 3.93–
4.01 (m, 1H, C4H7NO 6-Ha), 4.05 (s, 3H, OCH3), 4.40 (m, 1H,
C4H7NO 6-He), 4.70 (m, 2H, OCH2), 5.37 (br, 1H, C4H7NO 2-H), 6.82–
8.26 (m, 9H, C6H4, C10H5), 13.63 (bs, 1H, HCl). Elemental analysis
found C, 60.95; H, 6.41; N, 2.33; C30H37O4NClBr requires C, 60.97;
H, 6.37; N, 2.31.
Cells were incubated in PRMI 1640 complete medium
supplemented with 5% NCS for 4 h at 37°C 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. Cultures were removed
and washed twice with D-Hanks. PRMI 1640 complete medium
supplemented with 5% NCS was added into each well, then
processed according to the following groups: (i) negative
control group of DMSO (control); (ii) A23187 (final concentration
1 mmol/L); (iii) A23187 þ 10a–10g (5 mmol/L); (iv) A23187 þ
celecoxib (1 mmol/L). Experiments were performed at least three
times in triplicate. The peritoneal macrophages, drugs, or
solvents were incubated for 1 h at 37°C with 5% CO2; then
A23187 was added (final concentration 1 mmol/L) and incubated
for 1 h under the same conditions. The supernatant was collected
and the concentration of 6-keto-PGF1a 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 according to the measured values, respectively.
The COX-2 inhibitory activity was processed according to the
following four groups: (i) negative control group of DMSO
(control); (ii) LPS (final concentration 1 mg/L); (iii) LPS þ 10a–10g
(5 mmol/L); (iv) LPS þ celecoxib (1 mmol/L). The peritoneal macro-
phages, drugs, or solvents were also incubated for 1 h at 37°C
with 5% CO2; then LPS was added (final concentration 1 mg/L) and
incubated for 9 h under the same conditions. The supernatant
was collected and the concentration of PGE2 was tested with
2-[2-(6-Methoxynaphthalen-2-yl)-3-methylmorpholino]-
ethyl 2-(4-iso-butylphenyl)propanoate hydrochloride (1j)
1
Pale yellow solid, m.p. 184–187°C, yield 87.6%. H NMR (CDCl3,
400 MHz), d: 0.72 (dd, J ¼ 6.0 Hz, J ¼ 8.8 Hz, 3H, CH3), 0.84–0.87 (m,
6H, 2 ꢁ CH3), 1.47 (d, J ¼ 7.6 Hz, 3H, CH3), 1.79 (m, 1H, CH), 2.36–
2.42 (m, 2H, CH2), 2.54–2.62 (m, 2H, NCH2), 2.68–2.72 (m, 1H,
C4H6NO 5-Ha), 2.91–3.02 (m, 1H, C4H6NO 5-He), 3.65–3.75 (m, 2H,
CH, C4H6NO 3-H), 3.81 (s, 5H, OCH2, OCH3), 4.14 (m, 1H, C4H6NO 6-
Ha), 4.21 (m, 1H, C4H6NO 6-He), 7.03–7.68 (m, 10H, C6H4, C10H6).
Elemental analysis found C, 70.79; H, 7.61; N, 2.65; C31H40O4NCl
requires C, 70.77; H, 7.66; N, 2.66.
2-[2-(4-Benzyloxyphenyl)morpholino]ethyl 2-(4-iso-
butylphenyl)propanoate hydrochloride (1k)
1
Pale yellow solid, m.p. 147–149°C, yield 61.2%, H NMR (CDCl3,
400 MHz), d: 0.88–0.91 (m, 6H, 2 ꢁ CH3), 1.48 (m, 3H, CH3), 1.83 (m,
1H, CH), 2.42 (d, 2H, J ¼ 7.6 Hz, CH2), 2.23 (m, 1H, C4H7NO 5-Ha),
3.00–3.23 (m, 3H, NCH2, C4H7NO 3-Ha), 2.54 (m, 1H, C4H7NO 5-He),
3.37 (m, 1H, C4H7NO 3-He), 3.70 (m, 1H, CH), 3.86 (m, 1H, C4H7NO
6-Ha), 4.20 (m, 1H, C4H7NO 6-He), 4.68 (bs, 2H, OCH2), 5.08 (d, 2H,
PhCH2O), 5.10–5.13 (m, 1H, C4H7NO 2-H), 6.96–7.42 (m, 13H,
2 ꢁ C6H4, C6H5), 13.59 (bs, 1H, HCl). Elemental analysis found C,
3
square-free kit marked with H.
Serotonin reuptake inhibitory assay
RBL-2H3 cell line, purchased from China Center for Type Culture
Collection (CCTCC), was maintained in 85% MEM with Earle’s
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Arch. Pharm. Chem. Life Sci. 2014, 347, 89–95
Ibuprofen Derivatives as COX-2 and 5-HT Reuptake Inhibitors
95
salts supplemented with 1.5 g/L NaHCO3, 0.1 mM nonessential
amino acids, 1.0 mM sodium pyruvate, and 2.0 mM L-glutamine
with 15% bovine calf serum at 37°C. The cells were washed with
D-Hanks twice until they achieved logarithmic phase in good
condition; then 0.125% trypsin–EDTA solution was added and
placed for 5 min at 37°C. Cell suspensions were pelleted by
centrifugation and purified liquid was removed; then cells were
incubated in PRMI 1640 complete medium and resuspended
at 6 ꢁ 105 per milliliter.
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CACO-2 cell line, purchased from National Center for
Pharmaceutical Screening (NCPC), was maintained under the
same conditions just as RBL-2H3 cells did.
(Suppl. 14), 3.
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wavelength) and 605 nm (emission wavelength) after the cells
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The inhibition ratio was calculated as follows: (cpm of
sample ꢂ cpm of background)/(cpm of maximum ꢂ cpm of
background).
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This work was supported by the key project of Natural Science
Foundation of Hunan province (Grant No.07JJ3022).
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The authors have declared no conflict of interest.
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