Synthesis, characterization, and anti-inflammatory activities of methyl salicylate derivatives bearing piperazine moiety

Article abstract of DOI:10.3390/molecules21111544

In this study, a new series of 16 methyl salicylate derivatives bearing a piperazine moiety were synthesized and characterized. The in vivo anti-inflammatory activities of target compounds were investigated against xylol-induced ear edema and carrageenan-induced paw edema in mice. The results showed that all synthesized compounds exhibited potent anti-inflammatory activities. Especially, the anti-inflammatory activities of compounds M15 and M16 were higher than that of aspirin and even equal to that of indomethacin at the same dose. In addition, the in vitro cytotoxicity activities and anti-inflammatory activities of four target compounds were performed in RAW264.7 macrophages, and compound M16 was found to significantly inhibit the release of lipopolysaccharide (LPS)-induced interleukin (IL)-6 and tumor necrosis factor (TNF)-α in a dose-dependent manner. In addition, compound M16 was found to attenuate LPS induced cyclooxygenase (COX)-2 up-regulation. The current preliminary study may provide information for the development of new and safe anti-inflammatory agents.

Full text of DOI:10.3390/molecules21111544

molecules
Article
Synthesis, Characterization, and Anti-Inflammatory
Activities of Methyl Salicylate Derivatives Bearing
Piperazine Moiety
Jingfen Li 1,†, Yong Yin 2,†, Lisheng Wang 2, Pengyun Liang 2, Menghua Li 2, Xu Liu 2,
Lichuan Wu 2,* and Hua Yang 2,
*
1
Department of Life Science, Huzhou Teachers’ College, Huzhou 313000, China; ljfljf@hutc.zj.cn
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China;
yinyong1220@163.com (Y.Y.); w_lsheng@163.com (L.W.); gxulpyun@163.com (P.L.);
lixiaomeng0108@163.com (M.L.); wendaoliuxu@163.com (X.L.)
2
*
†
Correspondence: wulichuan@126.com (L.W.); yanghua6316@sina.com (H.Y.); Tel.: +86-771-3270-732 (L.W.)
These authors contributed equally to this work.
Academic Editor: Diego Muñoz-Torrero
Received: 11 October 2016; Accepted: 11 November 2016; Published: 23 November 2016
Abstract: In this study, a new series of 16 methyl salicylate derivatives bearing a piperazine
moiety were synthesized and characterized. The in vivo anti-inflammatory activities of target
compounds were investigated against xylol-induced ear edema and carrageenan-induced paw edema
in mice. The results showed that all synthesized compounds exhibited potent anti-inflammatory
activities. Especially, the anti-inflammatory activities of compounds M15 and M16 were higher
than that of aspirin and even equal to that of indomethacin at the same dose. In addition,
the in vitro cytotoxicity activities and anti-inflammatory activities of four target compounds were
performed in RAW264.7 macrophages, and compound M16 was found to significantly inhibit the
release of lipopolysaccharide (LPS)-induced interleukin (IL)-6 and tumor necrosis factor (TNF)-
α
in a dose-dependent manner. In addition, compound M16 was found to attenuate LPS induced
cyclooxygenase (COX)-2 up-regulation. The current preliminary study may provide information for
the development of new and safe anti-inflammatory agents.
Keywords: inflammation; salicylate; piperazine; derivatives; anti-inflammatory activity
1. Introduction
Inflammation is a complex biological process that occurs when body tissues are exposed to
hazardous stimuli, such as irritants and pathogens [
health, and exaggerated and prolonged inflammation may cause various diseases, including arthritis,
sepsis, and even cancer [ ]. Presently, the most widely used drugs in inflammation treatment
are non-steroidal anti-inflammatory drugs (NSAIDs), accounting for 35% of the global market of
prescription of pain medications [ ]. The common NSAIDs, such as aspirin and indomethacin,
can inhibit cyclooxygenases (COX-1 and COX-2) [ ], which are involved in the catalyzation of
arachidonic acid to prostaglandins (PGs). Considering the significant toxicity of NSAIDs to the
1]. Inflammation seriously threatens human
2
3,4
5,6
gastrointestinal tract and kidney [7], it is of great importance and urgent need to develop new
anti-inflammation drugs [8].
Pharmaceuticals bearing a piperazine moiety are widely used in medicinal and pesticide
chemistry [ ]. Piperazine can effectively change the physicochemical properties of drugs and improve
9
their pharmacokinetic properties and biological activity. Piperazine derivatives can act as histamine
and serotonin receptor antagonists in the control of inflammation [10–12].
Molecules 2016, 21, 1544; doi:10.3390/molecules21111544
www.mdpi.com/journal/molecules

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Salicylates are a class of chemicals with favorable anti-inflammatory effects. A large number
of studies have indicated that methyl salicylate derivatives exhibited great anti-inflammatory
effects [13–15]. In the current study, methyl salicylate and piperazine were combined to synthesize
a new series of methyl salicylate derivatives, and their anti-inflammation activities were screened
in vitro and in vivo.
2. Results
2.1. Synthesis of Salicylate Derivatives Bearing Piperazine Moiety
The synthesis of target compounds is outlined in Scheme 1. Methyl salicylate (a) firstly reacted with
3-chloro-1,2-epoxypropane (b) to generate methyl-2-(oxiran-2-ylmethoxy) benzoate (c) (Scheme 1A,
upper). Meanwhile, various aryl carboxylic acids (d) reacted with thionyl chloride to obtain the
intermediate (e) which subsequently reacted with anhydrous piperazine (f) in the presence of acetic acid
at room temperature, generating intermediate (g) (Scheme 1A, bottom). Then, based on combination
principles, intermediate c reacted with g to generate products M1
yields ranging from 41% to 62% (Scheme 1A). For products M14
methyl-2-(oxiran-2-ylmethoxy) benzoate (c) in the presence of toluene to generate the target methyl
salicylate derivatives (M14 M16) (Scheme 1B), with yields ranging from 34% to 67%. All compounds
–
M13 in the presence of toluene, with
–
M16, raw material (h) reacted with
–
were characterized by microanalytical and spectral data.
Scheme 1. Synthetic routes of methyl salicylate derivatives. (A) Synthetic routes of compounds M1~M13.
(B) Synthetic routes of compounds M14~M16. Reagents and conditions: (i) K2CO3, acetone, reflux, 30 h,
yield = 85%; (ii) SOCl2, reflux, 2 h, yield = 71%–87%; (iii) Piperazine, AcOH, r.t., 3 h, yield = 64%–79%
;
(iv) toluene, reflux, 12 h, yield = 34%–67%.

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2.2. In Vivo Anti-Inflammatory Activities of Salicylate Derivatives Bearing Piperazine Moiety
The in vivo anti-inflammatory activities of target compounds were evaluated against
xylol-induced ear edema and carrageenan-induced paw edema in mice. All of the target compounds
were administered at a dose of 100 mg/kg, and aspirin (100 mg/kg) and indomethacin (5 mg/kg)
were used as standard controls. The results indicated that all compounds exhibited favorable
anti-inflammatory activity compared to aspirin, except for compound M10 (Table 1). Among the
tested compounds, M2
M16 exhibited the strongest anti-inflammatory activity. To further depict the dose-effect relationships
of the four compounds (M2 M14 M15, and M16), various doses (5, 10, and 20 mg/kg) of the
, M14, M15, and M16 were much more potent than others, and compound
,
,
four compounds, aspirin, and indomethacin were intragastrically administered. The results showed
that the anti-inflammatory activities of the four compounds were increased in a dose-dependent
manner (Figure 1). Especially, the anti-inflammatory activities of compounds M15 and M16 were
better than that of aspirin, and even equal to that of indomethacin at the same dose.
Table 1. Anti-inflammatory (in vivo) activity of the target compounds against xylol-induced ear edema
and carrageenan-induced paw edema in mice.
Ear Edema
Swelling Degree (mg)
Paw Edema
Swelling Degree (mg)
Dose
(mg/kg)
Dose
(mmol/kg)
Compound
Inhibition (%)
Inhibition (%)
Control
M1
M2
M3
M4
M5
M6
M7
M8
8.36 ± 2.50
2.82 ± 0.56 ***
2.29 ± 0.47 ***
2.42 ± 0.44 ***
2.70 ± 0.49 ***
4.61 ± 1.11 **
4.54 ± 1.16 **
5.03 ± 1.77 *
4.05 ± 1.55 **
4.63 ± 1.43 **
4.07 ± 1.08 **
3.74 ± 1.13 ***
4.31 ± 1.37 **
3.61 ± 1.17 ***
2.43 ± 0.75 ***
2.13 ± 1.09 ***
1.53 ± 0.55 ***
4.85 ± 1.70 *
4.96 ± 1.09 **
-
94.93 ± 8.46
-
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
5
66.25
72.65
71.09
67.67
44.82
45.70
39.80
51.55
44.65
51.30
55.31
48.40
56.80
70.97
74.48
81.73
41.96
40.66
59.31 ± 6.25 ***
43.44 ± 8.65 ***
50.58 ± 7.40 ***
54.82 ± 5.03 **
57.98 ± 4.94 **
61.04 ± 5.58 ***
64.58 ± 6.75 ***
59.60 ± 6.86 ***
59.22 ± 5.21 ***
79.22 ± 7.06 **
49.24 ± 7.79 ***
59.91 ± 7.72 ***
53.43 ± 6.01 ***
43.64 ± 6.37 ***
41.42 ± 7.07 ***
33.43 ± 6.92 ***
67.24 ± 8.68 ***
64.13 ± 8.91 ***
37.52
54.24
46.72
42.25
38.92
35.70
31.97
37.21
37.62
16.55
48.13
36.89
43.72
54.03
56.37
64.78
29.16
32.45
0.25
0.23
0.23
0.24
0.24
0.23
0.23
0.20
0.23
0.23
0.25
0.24
0.23
0.32
0.24
0.20
0.56
0.01
M9
M10
M11
M12
M13
M14
M15
M16
Aspirin
Indomethacin
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from the control value.
Figure 1. Target compounds showed dose-dependent inhibition on (
A) ear and (B) paw edema in vivo.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from the control value.
Piperazine-derived drugs are considered to be designer drugs which can be divided into
two classes: the benzylpiperazines and the phenylpiperazines [16]. Although piperazine-derived

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drugs have been considered to be safe [16], adverse effects, such as dizziness, headache,
hypersensitivity reactions, vomiting, and nausea have been reported from several experimental,
clinical, and epidemiological studies [17
bearing a piperazine moiety with the central nervous system (CNS) have been revealed, indicating
that drugs with the least CNS toxicity would be predicted to be those with low -aminobutyric
,18]. Presently, the structure-side-effect relationships of drugs
γ
acid (GABA)-binding ability and low overall lipophilicity [19]. Considering the potential toxicity of
piperazine drugs, it is of great importance to detect their metabolites in humans or animals. Metabolism
studies of piperazine designer drugs show that piperazine-derived drugs are mainly metabolized in the
liver. The main metabolites are N-(4-hydroxy-3-methoxybenzyl)piperazine for the benzylpiperazines
class, and hydroxyl-3-chloroaniline for phenylpiperazines, which finally turned into glucuronides
and/or sulfates [16]. Our in vivo study showed that the oral dose of 100 mg/kg did not cause any
gross behavioral alterations such as dizziness, respiratory distress, or mortality, indicating that the
lethal dose of the target compounds is above 100 mg/kg body weight. Certainly, for future follow
ups, we should carry out studies on acute toxicity, pharmacokinetics, and metabolism, especially for
compound M16.
2.3. Cytotoxicity Assays of Compounds M2, M14, M15, and M16
To reveal the cytotoxicity of M2
2,5-diphenyltetrazolium bromide) assay was conducted in mouse RAW264.7 macrophages. The results
indicated that compounds M2 M14, and M16 showed no significant cytotoxicity with concentrations
less than 75 M (Figure 2). Compound M15 displayed no significant cytotoxicity with concentrations
less than 25 M, but showed a great cytotoxicity at 50 M (Figure 2). Thus, we used 6.25, 12.5,
and 25 µM in the following ELISA (enzyme-linked immunoadsorbent assay) experiments.
, M14, M15, and M16, a MTT (3-(4,5-Dimethylthiazol-2-yl)-
,
µ
µ
µ
Figure 2. Cytotoxicity activities of M2, M14, M15, and M16 against RAW264.7 macrophages.
2.4. Inhibitory Screening against Lipopolysaccharide (LPS)-Induced Interleukin (IL)-6 and Tumor Necrosis
Factor (TNF)-α Release
TNF-
lipopolysaccharide (LPS) [20
TNF- or LPS subsequently induces the cells to release IL-6 [22]. In this study, the in vitro
anti-inflammatory effects of four target compounds were evaluated in the macrophage RAW264.7 cells
by measuring the expressions of TNF- and IL-6 through ELISA assays. RAW264.7 macrophages were
pretreated with different doses (6.25, 12.5, and 25 M) for 2 h and then exposed to LPS (1 g/mL)
for an additional 22 h. The levels of TNF- and IL-6 in the media were determined. The results
displayed that all four target compounds could inhibit the release of TNF- and IL-6. Especially,
compound M16 significantly inhibited TNF- and IL-6 release in a dose-dependent manner (Figure 3).
Compound M15 significantly inhibited TNF- and IL-6 release at 25 M. These findings suggested
α
and IL-6, which are important pro-inflammatory mediators, can be triggered by
,
21]. TNF- is released in macrophages upon LPS treatment. The secreted
α
α
α
µ
µ
α
α
α
α
µ

Molecules 2016, 21, 1544
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that compounds M15 and M16 exerted their anti-inflammatory effects by suppressing the production
of pro-inflammatory factors, which might partly explain their in vivo anti-inflammatory activities.
Figure 3. Target compounds inhibited lipopolysaccharide (LPS)-induced (
(TNF)- and ( ) interleukin (IL)-6 release in a dose-dependent manner in vitro. * p < 0.05, ** p < 0.01
significantly different from the LPS value.
A) tumor necrosis factor
α
B
2.5. Compound M16 Attenuates LPS Induced Cyclooxygenase-2 (COX-2) Up-Regulation
COX-2 is recognized as an inducible pro-inflammatory enzyme which is the rate-limiting
enzyme of prostanoids synthesis. Many NSAIDs exert their anti-inflammatory activities by inhibiting
COX-2 activity [23]. To further elucidate the underlying anti-inflammation mechanism of compound
M16, Western blot assays were performed to detect the expression of cyclooxygenase-1 (COX-1)
and cyclooxygenase-2 (COX-2). The results showed that COX-2 was up-regulated in RAW264.7
macrophages upon LPS stimulation. Pretreatment with M16 could attenuate LPS-induced COX-2
up-regulation (Figure 4). These results inferred that compound M16 might exert its anti-inflammatory
activities by down-regulating COX-2 expression and inhibiting the release of TNF-α and IL-6.
Figure 4. Compound M16 attenuated LPS induced cyclooxygenase (COX)-2 up-regulation.
3. Experimental Section
3.1. Synthetic Details of Intermediates and Target Compounds
For intermediate c, 20 mL methyl salicylate (0.15 mol) was dissolved in 250 mL acetone, and then
40 g anhydrous potassium carbonate (0.30 mol) was added into the solution. The solution was kept

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stirring at room temperature, and 100 mL 3-chloro-1,2-epoxypropane (1.26 mol) was added and stirred
at reflux for 30 h at 60 ◦C. The excess solvent was removed in vacuo, and then the crude product c
was dissolved in 200 mL toluene. Then, the solvent was sequentially washed with water (150 mL
5% sodium hydroxide (200 mL 2), and water (150 mL 2). Finally, the organic phase was dried
over anhydrous sodium sulfate overnight and then evaporated under vacuum.
×
2),
×
×
For intermediate g, 0.02 mol aryl carboxylic acid was dissolved in 20 mL thionyl chloride,
and the solution was stirred at reflux for 4 h. Then, 20 mL anhydrous chloroform was added into the
solution, and thionyl chloride was removed in vacuo under reduced pressure to give intermediate e.
Subsequently, intermediate e was dissolved in 10 mL chloroform and added dropwise into anthalazine
dissolved in acetic acid. The solution was kept stirring at room temperature for 3 h, and then was
alkalized in an ice bath with 20% sodium hydroxide till the pH value reached 9~10. Finally, the organic
phase was extracted with chloroform (50 mL
and then evaporated under vacuum.
×
5), dried over anhydrous sodium sulfate overnight,
For products M1–M13, intermediates c and g were dissolved in 80 mL toluene with a material
ratio (1:1.1, mol ratio). The solution was stirred at reflux for 10 h at 95 ◦C. Finally, the excess solvent
was evaporated off, and the residues were purified by silica gel-column chromatography with acetic
ether/petroleum ether (7:3, v:v) as eluent to afford the corresponding products M1–M13.
For M14 and M15, monosubstituted alkyl piperazine and intermediate c were dissolved in 80 mL
◦
toluene with a material ratio (1:1.1, mol ratio). The solution was stirred at reflux for 10 h at 95 C.
Finally, the excess solvent was evaporated off and the residues were filtered by silica gel-column
chromatography with acetic ether/petroleum ether (6:4, v:v) as eluent to afford the corresponding
products M14–M15.
For M16, anhydrous piperazine and intermediate c were dissolved in ◦80 mL toluene with
a material ratio (1:2.2, mol ratio). The solution was stirred at reflux for 10 h at 95 C. Finally, the excess
solvent was evaporated off and the residues were filtered by silica gel-column chromatography with
acetic ether/petroleum ether (6:4, v:v) as eluent to afford the corresponding product M16.
Compounds characterization data:
Methyl 2-(3-(4-benzoylpiperazin-1-yl)-2-hydroxypropoxy)benzoate M1: yellow liquid. IR (KBr,
3442 (νO-H); 3064 (νAr-H); 2943, 1498, 1444 (νC-H); 1721, 1628 (νC=O); H-NMR (CD3Cl, 600 MHz,
ν
cm−1):
1
δ
ppm): 2.42~2.67 (m, 6H, -CH2-), 3.44 (m, 2H, -CH2-), 3.80 (m, 2H, -NCH2-), 3.87 (s, 3H, -CH3), 4.02
(m, 2H, -OCH2-), 4.14 (m, 1H, -OH), 4.19 (m, 1H, -CH-), 7.00 (m, 2H, ArH), 7.39 (m, 5H, ArH), 7.45
(m, 1H, ArH), 7.81 (m, 1H, ArH). MS (ESI, m/z): 399.35 ([M + H]+). Elemental analysis: calcd. for
C22H26N2O5: C, 66.32; H, 6.58; N, 7.03; Found: C, 66.38; H, 6.90; N, 6.56.
Methyl 2-(3-(4-(4-chlorobenzoyl)piperazin-1-yl)-2-hydroxypropoxy)benzoate M2: yellow liquid. IR (KBr,
1
ν
cm−1): 3431 (νO-H); 3055 (νAr-H); 2943, 1498, 1445 (νC-H); 1718 (νC=O); H-NMR (CD3Cl,
δ
ppm):
2.57~2.68 (m, 6H, -CH2-), 3.44 (m, 2H, -CH2-), 3.79 (m, 2H, -NCH2-), 3.87 (s, 3H, -CH3), 4.02 (m,
2H, -OCH2-), 4.18~4.21 (m, 2H, -CH(OH)-), 7.01 (m, 2H, ArH), 7.34 (d, J = 8.4 Hz, 2H, ArH), 7.38 (d,
J = 8.4 Hz, 2H, ArH),7.46 (m, 1H, ArH), 7.81 (t, J = 3.9 Hz, 1H, ArH). MS (ESI, m/z): 433.35 ([M + H]+).
Elemental analysis: calcd. for C22H25ClN2O5: C, 61.04; H, 5.82; N, 6.47; Found: C, 61.08; H, 5.84;
N, 6.49.
Methyl 2-(3-(4-(2-chlorobenzoyl)piperazin-1-yl)-2-hydroxypropoxy)benzoate M3: yellow liquid. IR (KBr,
ν
cm−1): 3439 (νO-H); 3072 (νAr-H); 2934, 1486, 1453 (νC-H); 1727, 1633 (νC=O); 1H-NMR (CD3Cl,
δ ppm):
2.57~2.63 (m, 6H, -CH2-), 3.18~3.32 (m, 2H, -CH2-), 3.71 (m, 2H, -CH2-), 3.85 (s, 3H, -CH3), 4.02 (d,
J = 7.2 Hz, 2H, -CH2-), 4.12 (m, 1H, -OH), 4.16 (d, J = 2.4 Hz, 1H, -CH-), 6.98 (m, 2H, ArH), 7.26~7.32
(m, 3H, ArH), 7.37 (m, H, ArH), 7.44 (t, J = 7.8 Hz, 1H, ArH), 7.79 (m, 1H, ArH). MS (ESI, m/z): 433.29
([M + H]+). Elemental analysis: calcd. for C22H25ClN2O5: C, 61.04; H, 5.82; N, 6.47; Found: C, 61.02;
H, 5.88; N, 6.44.

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Methyl 2-(3-(4-(2-fluorobenzoyl)piperazin-1-yl)-2-hydroxypropoxy)benzoate M4: yellow liquid. IR (KBr,
cm−1): 3456 (νO-H); 3080 (νAr-H); 2942, 1498, 1427 (νC-H); 1735, 1642 (νC=O); 1H-NMR (CD3Cl,
ppm):
ν
δ
2.47~2.62 (m, 6H, -CH2-), 3.30 (m, 2H, -CH2-), 3.77 (m, 2H, -CH2-), 3.81 (s, 3H, -CH3), 3.98 (m, 1H,
-OH), 4.11~4.15 (m, 3H, -CHCH2-), 6.95 (t, J = 7.5 Hz, 2H, ArH), 7.03 (t, J = 9 Hz, 1H, ArH), 7.15 (t,
J = 7.5 Hz, 1H, ArH), 7.32 (m, 2H, ArH), 7.41 (t, J = 7.8 Hz, 1H, ArH), 7.52 (d, J = 7.8 Hz, 1H, ArH). MS
(ESI, m/z): 417.33 ([M + H]+). Elemental analysis: calcd. for C22H25FN2O5: C, 63.45; H, 6.05; N, 6.73;
Found: C, 63.48; H, 6.01; N, 6.6.
Methyl 2-(3-(4-(4-fluorobenzoyl)piperazin-1-yl)-2-hydroxypropoxy)benzoate M5: yellow liquid. IR (KBr,
ν
cm−1): 3439 (νO-H); 3072 (νAr-H); 2942, 1506, 1452 (νC-H); 1718, 1642 (νC=O); 1H-NMR (CD3Cl,
δ ppm):
2.50~2.70 (m, 6H, -CH2-), 3.41 (m, 2H, -CH2-), 3.75 (m, 2H, -CH2-), 3.84 (s, 3H, -CH3), 3.99 (m, 1H,
-CH-), 4.07~4.14 (m, 2H, -CH2-), 4.17 (m, 1H, -OH), 6.97 (m, 2H, ArH), 7.05 (t, J = 8.4 Hz, 2H, ArH),
7.38 (m, 2H, ArH), 7.43 (t, J = 7.8 Hz, 1H, ArH), 7.78 (d, J = 7.8 Hz, 1H, ArH). MS (ESI, m/z): 417.26
(
[M + H]+). Elemental analysis: calcd. for C22H25FN2O5: C, 63.45; H, 6.05; N, 6.73; Found: C, 63.51;
H, 6.08; N, 6.78.
Methyl 2-(2-hydroxy-3-(4-(3-nitrobenzoyl)piperazin-1-yl)propoxy)benzoate M6: yellow liquid. IR (KBr,
1
ν
cm−1): 3448 (νO-H); 3080 (νAr-H); 2925, 1532, 1452 (νC-H); 1726, 1642 (νC=O); H-NMR (CD3Cl,
δ
ppm): 2.54~2.70 (m, 6H, -CH2-), 3.42 (m, 2H, -CH2-), 3.81 (m, 2H, -CH2-), 3.85 (s, 3H, -CH3), 4.01 (t,
J = 7.5 Hz, 1H, -CH-), 4.08 (d, J = 3.6 Hz, 1H, -OH), 4.14~4.19 (m, 2H, -CH2-), 6.98 (t, J = 9.33 Hz, 2H,
ArH), 7.44 (t, J = 7.8 Hz, 1H, ArH), 7.59 (t, J = 7.8 Hz, 1H, ArH), 7.72 (d, J = 7.8 Hz, 1H, ArH), 7.79
(d, J = 7.8 Hz, 1H, ArH), 8.25 (d, J = 8.4 Hz, 2H, ArH). MS (ESI, m/z): 444.12 ([M + H]+). Elemental
analysis: calcd. for C22H25N3O7: C, 59.59; H, 5.68; N, 9.48; Found: C, 59.62; H, 5.65; N, 9.52.
Methyl 2-(2-hydroxy-3-(4-(4-nitrobenzoyl)piperazin-1-yl)propoxy)benzoate M7: yellow liquid. IR (KBr,
1
ν
cm−1): 3439 (νO-H); 3080 (νAr-H); 2942, 1524, 1444 (νC-H); 1727, 1634 (νC=O); H-NMR (CD3Cl,
δ
ppm): 2.58~2.74 (m, 6H, -CH2-), 3.38 (m, 2H, -CH2-), 3.81 (m, 2H, -CH2-), 3.86 (s, 3H,-CH3), 4.02 (m,
1H, -OH), 4.10~4.21 (m, 3H, -CHCH2-), 7.01 (m, 2H, ArH), 7.46 (m, 1H, ArH), 7.56 (m, 2H, ArH), 7.81
(m, 1H, ArH), 8.26 (m, 2H, ArH). MS (ESI, m/z): 444.14 ([M + H]+). Elemental analysis: calcd. for
C22H25N3O7: C, 59.59; H, 5.68; N, 9.48; Found: C, 59.63; H, 5.64; N, 9.53.
Methyl 2-(3-(4-(3,5-dinitrobenzoyl)piperazin-1-yl)-2-hydroxypropoxy)benzoate M8: yellow liquid. IR (KBr,
ν
cm−1): 3456 (νO-H); 3097 (νAr-H); 2926, 1548, 1445 (νC-H); 1727, 1650 (νC=O); 1H-NMR (CD3Cl,
δ
ppm):
2.61~2.78 (m, 6H, -CH2-), 3.45 (m, 2H, -CH2-), 3.84 (m, 2H, -CH2-), 3.87 (s, 3H, -CH3), 4.02~4.07 (m, 2H,
-CH2-), 4.17 (s, 1H, -CH-), 4.22 (m, 1H, -OH), 7.02 (m, 2H ArH,), 7.26 (s, 1H, ArH), 7.48 (t, J = 7.8 Hz
1H, ArH), 7.82 (d, J = 7.8 Hz, 1H, ArH), 8.60 (s, 2H, ArH), 9.09 (s, 1H, ArH). MS (ESI, m/z): 489.28
[M + H]+). Elemental analysis: calcd. for C22H24N4O9: C, 54.10; H, 4.95; N, 11.47; Found: C, 54.12;
,
(
H, 4.98; N, 11.44.
Methyl 2-(2-hydroxy-3-(4-(4-methoxybenzoyl)piperazin-1-yl)propoxy)benzoate M9: yellow liquid. IR (KBr,
1
ν
cm−1): 3431 (νO-H); 3072 (νAr-H); 2942, 1515, 1446 (νC-H); 1727, 1625 (νC=O); H-NMR (CD3Cl,
δ
ppm): 2.48~2.70 (m, 8H, -CH2-), 3.79 (m, 3H, -CH3), 3.84 (s, 3H, CH3), 4.00 (m, 1H, -OH), 4.15 (m,
3H, -CHCH2-), 6.87 (d, J = 7.8 Hz, 2H, ArH), 6.97 (t, J = 8.1 Hz, 2H, ArH), 7.34 (d, J = 7.8 Hz, 2H, ArH),
7.43 (t, J = 7.8 Hz, 1H, ArH), 7.78 (d, J = 7.8 Hz, 1H, ArH). MS (ESI, m/z): 429.30 ([M + H]+). Elemental
analysis: calcd. for C23H28N2O6: C, 64.47; H, 6.59; N, 6.54; Found: C, 64.43; H, 6.54; N, 6.58.
Methyl 2-(3-(4-(2-(3-fluorophenyl)acetyl)piperazin-1-yl)-2-hydroxypropoxy)benzoate M10: yellow liquid.
IR (KBr,
ppm): 2.36~2.61 (m, 6H, -CH2-), 3.42 (t, J = 5.1 Hz, 2H, -CH2-), 3.58~3.70 (m, 5H, -CHCH2-,-CH2-),
ν
cm−1): 3448 (νO-H); 3074 (νAr-H); 2951, 1486, 1445 (νC-H); 1718, 1642 (νC=O); 1H-NMR (CD3Cl,
δ
3.83 (s, 3H, -CH3), 3.98 (m, 1H, -OH), 4.08~4.15 (m, 2H, -CH2-), 6.89~6.98 (m, 5H, ArH), 7.24 (d,
J = 6.0 Hz, 1H, ArH), 7.42 (m, 1H, ArH), 7.78 (t, J = 3.9 Hz, 1H, ArH). MS (ESI, m/z): 431.40 ([M + H]+).
Elemental analysis: calcd. for C23H27FN2O5: C, 64.17; H, 6.32; N, 6.51; Found: C, 64.15; H, 6.38; N, 6.48.

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Methyl 2-(2-hydroxy-3-(4-(thiophene-2-carbonyl)piperazin-1-yl)propoxy)benzoate M11
:
yellow liquid.
1
IR (KBr,
(CD3Cl,
ν
cm−1): 3447 (νO-H); 3089 (νAr-H); 2934, 1523, 1452 (νC-H); 1727, 1625 (νC=O); H-NMR
δ
ppm): 2.53~2.64 (m, 6H, -CH2-), 3.68~3.80 (m, 4H, -CH2-), 3.83 (s, 3H, -CH3), 4.00 (m, 1H,
-OH), 4.05~4.18 (m, 3H, -CHCH2-), 6.98 (m, 3H, ArH), 7.24 (m, 1H, ArH), 7.40 (m, 2H, ArH), 7.77 (m,
1H, ArH). MS (ESI, m/z): 405.16 ([M + H]+). Elemental analysis: calcd. for C20H24N2O5S: C, 59.39;
H, 5.98; N, 6.93; Found: C, 59.44; H, 6.02; N, 6.89.
Methyl 2-(3-(4-cinnamoylpiperazin-1-yl)-2-hydroxypropoxy)benzoate M12: yellow liquid. IR (KBr,
3448 (νO-H); 3097 (νAr-H); 2934, 1527, 1453 (νC-H); 1718, 1642 (νC=O); H-NMR (CD3Cl,
ν
δ
cm−1):
ppm):
1
2.50~2.68 (m, 6H, -CH2-), 3.50~3.78 (m, 4H, -CH2-), 3.86 (s, 3H, -CH3), 4.01~4.12 (m, 2H, -CH(OH)-),
4.15~4.21 (m, 2H, -CH2-), 6.85 (d, J = 15.6 Hz, 1H, ArH), 6.97~7.00 (t, J = 7.2 Hz, 2H, -CH = CH-),
7.31~7.36 (m, 3H, ArH), 7.44 (t, J = 7.8 Hz, 1H, ArH), 7.49 (d, J = 7.2 Hz, 2H, ArH), 7.64 (d, J = 15 Hz,
1H, ArH), 7.80 (d, J = 7.8 Hz, H, ArH). MS (ESI, m/z): 425.31 ([M + H]+). Elemental analysis: calcd. for
C24H28N2O5: C, 67.91; H, 6.65; N, 6.60; Found: C, 67.88; H, 6.64; N, 6.64.
Methyl 2-(2-hydroxy-3-(4-(3-phenylpropanoyl)piperazin-1-yl)propoxy)benzoate M13: yellow liquid. IR (KBr,
ν
cm−1): 3456 (νO-H); 3038 (νAr-H); 2951, 1498, 1452 (νC-H); 1718, 1642 (νC=O); 1H-NMR (CD3Cl,
δ ppm):
2.41~2.61 (m, 8H, -CH2-), 2.95 (t, J = 7.8 Hz, 2H, -CH2-), 3.8 (t, J = 5.1 Hz, 2H, -CH2-), 3.63 (m, 2H,
-CH2-), 3.86 (s, 3H, -CH3), 4.01 (m, 1H, -OH), 4.10~4.19 (m, 3H, -CHCH2-), 6.98 (m, 2H, ArH), 7.19 (t,
J = 8.7 Hz, 3H, ArH), 7.27 (t, J = 7.5 Hz, 2H, ArH), 7.45 (m, 1H, ArH), 7.80 (d, J = 7.2 Hz, 1H, ArH).
MS (ESI, m/z): 427.34 ([M + H]+). Elemental analysis: calcd. for C24H30N2O5: C, 67.59; H, 7.09; N, 6.57;
Found: C, 67.56; H, 7.06; N, 6.61.
Methyl 2-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)benzoate M14: yellow liquid. IR (KBr,
3439 (νO-H); 3072 (νAr-H); 2951, 1486, 1453 (νC-H); 1718, 1608 (νC=O); 1H-NMR (CD3Cl,
ppm): 2.26
ν
cm−1):
δ
(s, 3H, -CH3), 2.30~2.70 (m, 10H, -CH2-), 3.85 (s, 3H, -CH3), 4.00 (m, 1H, -OH), 4.10~4.14 (m, 3H,
-CHCH2-), 6.97 (m, 2H, ArH), 7.43 (t, J = 7.8 Hz, 1H, ArH), 7.79 (m, 1H, ArH). MS (ESI, m/z): 309.20
([M + H]+). Elemental analysis: calcd. for C16H24N2O4: C, 62.32; H, 7.84; N, 9.08; Found: C, 62.29;
H, 7.88; N, 9.04.
Methyl 2-(3-(4-benzhydrylpiperazin-1-yl)-2-hydroxypropoxy)benzoate M15: yellow liquid. IR (KBr,
3456 (νO-H); 3114 (νAr-H); 2942, 1489, 1452 (νC-H); 1718, 1642 (νC=O); H-NMR (CD3Cl,
ν
δ
cm−1):
ppm):
1
2.36~2.70 (m, 10H, -CH2-), 3.87 (s, 3H, -CH3), 3.93~4.08 (m, 2H, -CH(OH)-), 4.09~4.16 (m, 2H, -CH2-),
4.23 (s, 1H, -CH-), 6.99 (t, J = 7.5 Hz, 2H, ArH), 7.17 (t, J = 7.8 Hz, 2H, ArH), 7.27 (m, 4H, ArH), 7.40~7.46
(m, 5H, ArH), 7.81 (d, J = 7.8 Hz, 1H, ArH). MS (ESI, m/z): 416.43 ([M + H]+). Elemental analysis: calcd.
for C28H32N2O4: C, 73.02; H, 7.00; N, 6.08; Found: C: 73.08; H, 7.03; N, 6.11.
Dimethyl 2,20-((piperazine-1,4-diylbis(2-hydroxypropane-3,1-diyl))bis(oxy))dibenzoate M16: yellow liquid.
1
IR (KBr,
(CD3Cl,
4.13 (m, 2
ν
cm−1): 3430 (νO-H); 3080 (νAr-H); 2934, 1506, 1453 (νC-H); 1727, 1633 (νC=O); H-NMR
δ
ppm): 2.54~2.72 (m, 2
×
6H, -CH2-), 3.85 (s, 2
×
3H, -CH3), 4.00 (t, J = 4.5 Hz, 2
×
1H, -OH),
1H, ArH). MS (ESI,
×
2H, -CH2-), 6.97 (m, 2
×
2H, ArH), 7.42 (m, 2
×
1H, ArH), 7.79 (m, 2
×
m/z): 503.35 ([M + H]+). Elemental analysis: calcd. for C26H34N2O8: C, 62.14; H, 6.82; N, 5.57; Found:
C, 62.18; H, 6.79; N, 5.54.
3.2. Cell Culture and Cytotoxicity Assays
Mouse RAW264.7 macrophages were purchased from American Type Culture Collection (ATCC)
and cultured in DMEM (Dulbecco modified Eag◦le medium) supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin–streptomycin at 37 C, in an atmosphere of 95% air and 5% CO2 under
humidified conditions. For cytotoxicity assay, cells were seeded in 96-well plates at the density of
2
×
104 cells per well and incubated for 16 h. The medium was removed, and the cells were then
incubated with different concentrations of synthesized compounds for indicated times, followed by
2–3 h treatment with MTT. After MTT removal, 150 L dimethyl sulfoxide (DMSO) was added into
each well and absorption values was measured at 490 nm using a SpectraMAX M5 plate reader
µ

Molecules 2016, 21, 1544
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(Molecular Devices Inc. San Francisco, CA, USA). Three independent experiments were conducted,
and samples were analyzed in triplicate for each experiment.
3.3. In Vivo Anti-Inflammatory Activity Assays in Ear and Paw Edema
The animal studies were approved by the Institutional Review Board of Guangxi Medical
University. All animal studies were conducted according to protocols approved by the Animal
Ethics Committee of Guangxi Medical University (Nanning, China). The anti-inflammatory activity
was evaluated on xylene-induced ear edema and carrageenan-induced mice paw edema [24]. Briefly,
the mice (20
±
2 g, 10/group) were fasted with free access to water for at least 16 h, and then randomly
divided into two groups: the dosage and control groups. Ear edema and paw edema was induced
by applying xylene on the left ear and subcutaneous injection of carrageenan in the left toe of each
mouse, respectively. One hour before edema induction, the mice were intragastrically administered
with target drugs in 1% carboxymethyl cellulose solution or control solvent. Three hours later, the mice
were euthanized by cervical dislocation, and right ears or paws were cut and weighed for swelling
degree and swelling inhibition calculation.
3.4. ELISA Assay
Mouse RAW264.7 macrophages were pretreated with compounds (M2
vehicle control for 2 h, then treated with LPS (1 g/mL) for 22 h. After treatment, the cells and media
were collected separately. The levels of IL-6 and TNF- in the media were determined using ELISA
kit (eBioscience, San Diego, CA, USA). The total protein was extracted from the collected cells and
, M14, M15, and M16) or
µ
α
determined using Bio-Rad protein assay. The amount of IL-6 and TNF-α was normalized to the total
protein amount.
3.5. Western Blot Assays
For Western blot assay, cells were lysed in RIPA (Radio-Immunoprecipitation Assay) buffer
supplemented with protease inhibitors. Proteins (20 g) were subjected to 6%–15% SDS-PAGE,
µ
electrophoresed, and transferred on to a nitrocellulose membrane. After blocking with 5% non-fat
milk in tris-buffered saline, the membrane was washed and incubated with the indicated primary and
secondary antibodies and detected using the Luminescent Image Analyser LSA 4000 (GE, Fairfield,
CO, USA).
3.6. Statistical Analysis
The data are presented as the mean
for significance using Student’s t-test of unpaired data. p values < 0.05 were considered
±
SD. Differences between data groups were evaluated
statistically significant.
4. Conclusions
In summary, a new series of methyl salicylate derivatives were synthesized and characterized.
The in vivo anti-inflammatory results revealed that all target compounds exhibited potent
anti-inflammatory activity compared to aspirin, except compound M10. In addition, compounds M2
,
M14 M15, and M16 exhibited strong anti-inflammatory activity in a dose-dependent manner in vivo
,
.
Especially, the anti-inflammatory activities of compounds M15 and M16 were higher than that of
aspirin, and even equal to that of indomethacin at the same dose. Furthermore, compound M16 could
significantly inhibit LPS-induced IL-6 and TNF-α release and down-regulate the expression of COX-2
in a dose-dependent manner. Our findings may provide information on potentially new and safe
anti-inflammatory agents for further studies.

Molecules 2016, 21, 1544
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Acknowledgments: The work was supported by the National Natural Science Foundation of Zhejiang Province of
China (No. Y12H300003), the high level innovation team and outstanding scholar project of Guangxi institutions
of higher education (gui jiao ren [2014] 49 hao), and the Postdoctoral Science Foundation of Guangxi Province of
China (Y201001928).
Author Contributions: The project was conceived by Lichuan Wu and Hua Yang. The experiments were designed
by Lichuan Wu. The experiments were conducted by Jingfen Li, Yong Yin, Lichuan Wu, Pengyun Liang, Hua Yang,
Menghua Li, and Xu Liu The manuscript was written by Lisheng Wang and Lichuan Wu.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).