Synthesis of phenylpiperazine derivatives of 1,4-benzodioxan as selective COX-2 inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2016.09.023

1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-substituted-phenylpiperazine moiety was prepared and has been found to be a new and selective ligand for the enzyme cyclooxygenase-2 (COX-2). The biological activity of compound 3k as anti-inflammatory agent was further investigated both in vitro and in vivo. Notably, compound 3k exhibited the best anti-inflammatory activity among the eleven designed compounds with no toxicity, as determined by the ulcerogenic activity. Computational docking studies also showed that compound 3k has interaction with COX-2 key residues in the active site. Compound 3k maybe a new anti-inflammatory lead-candidate as powerful and novel non-ulcerogenic.

Full text of DOI:10.1016/j.bmc.2016.09.023

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of phenylpiperazine derivatives of 1,4-benzodioxan as
selective COX-2 inhibitors and anti-inflammatory agents
⇑
⇑
Juan Sun , Su Wang, Gui-Hua Sheng, Zhi-Min Lian, Han-Yu Liu, Hai-Liang Zhu
School of Life Sciences, Shandong University of Technology, Zibo 255049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-substituted-phenylpiperazine moiety was prepared
and has been found to be a new and selective ligand for the enzyme cyclooxygenase-2 (COX-2). The bio-
logical activity of compound 3k as anti-inflammatory agent was further investigated both in vitro and
in vivo. Notably, compound 3k exhibited the best anti-inflammatory activity among the eleven designed
compounds with no toxicity, as determined by the ulcerogenic activity. Computational docking studies
also showed that compound 3k has interaction with COX-2 key residues in the active site. Compound
3k maybe a new anti-inflammatory lead-candidate as powerful and novel non-ulcerogenic.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 12 July 2016
Revised 9 September 2016
Accepted 10 September 2016
Available online xxxx
Keywords:
Anti-inflammatory scaffold
Cyclooxygenase-2
1,4-Benzodioxan
Phenylpiperazine
1. Introduction
On the other hand the phenylpiperazine group, as a known
pharmacophore, it has
a wide range of activities such as
Inflammation is a normal response to any noxious stimulus that
threatens the host and may vary from a localized response to a
generalized one.1 However, uncontrolled inflammatory cascades
are responsible for various diseases such as osteoarthritis, multiple
sclerosis, inflammatory bowl diseases and diabetic nephropathy,2
tumor initiation, and malignant progression.3 The nonsteroidal
anti-inflammatory drugs (NSAIDs) are used worldwide for thera-
peutic intervention of pain and inflammation. These achieve their
anti-inflammatory action through an inhibitory effect on cyclooxy-
genase enzyme (COX), a protein essential for prostaglandin biosyn-
thesis from arachidonic acid. COX exists under two isoforms,
namely COX-1 and COX-2.4 In general terms, COX-1 is found in
healthy populations and has mainly a physiological role in the kid-
neys and the stomach. In contrast, the mainly inducible COX-2 is
involved in the production of prostaglandins mediating pain and
supporting the inflammatory process.5,6 The side effects such as
gastric ulcer and gastrointestinal bleeding associated with the tra-
ditional NSAIDs (nonselective inhibitors of COX-1 and COX-2) has
led intense efforts in search for potent and selective COX-2 inhibi-
tors which could provide anti-inflammatory drugs with fewer
risks.7–11 Several classes of compounds having selective COX-2
inhibitory activity have been reported in the literature such as
SC-55812 and celecoxib.13
antitumor,14–17 anti-inflammatory18–21 and antibacterial activi-
ties.22,23 Besides, it is reported that 1,4-benzodioxan afforded a
new scaffold for small molecular anti-inflammatory activity.24–26
Serious of derivatives containing 1,4-benzodioxan template were
designed and synthesized in our lab,27,28 and among them, com-
pounds A and B (Fig. 1) showed good anti-inflammatory activity.
According to the binding models of these compounds, we can dis-
covery that the 1, 4-benzodioxan scaffold is nicely bonded with
amino acids in the active domain, with H-bonds and other
hydrophobic interactions. This is further evidence of the impor-
tance of the 1, 4-benzodioxan ring as anti-inflammatory skeleton.
Recently it is reported that 1,4-benzodioxan directly inhibits
COX-2 with efficacy comparable to that of the representative drug,
parent caffeic acid.29 Such earlier work on the COX-2 inhibiting
activity of 1,4-benzodioxan afforded a new scaffold for small-
molecular COX-2 inhibitors, which should offer opportunities for
various kinds of structural development.
Thus our main objective is to design novel phenylpiperazine
derivatives of 1,4-benzodioxan as specific inhibitors of COX-2 in
the hope that these molecules may be further explored as powerful
and novel non-ulcerogenic anti-inflammatory lead-candidates. Our
strategy is intended to obtain potent anti-inflammatory activity
with selective inhibition of COX-2 using traditional medicinal
chemistry techniques motivated by the comparative modeling of
COX-2 complexed with C (IC50COX-2 = 7.5 lM) in the available
pharmacophore (Fig. 2).
⇑
Corresponding authors.
http://dx.doi.org/10.1016/j.bmc.2016.09.023
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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J. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
the substituent at different positions led to different activity, and
the potency order was para ? meta. However, introducing of CF3
moieties, afforded 3k with a remarkable increase in anti-inflamma-
tory activities. Interestingly, compound 3j with pyridine ring
showed moderate activity. Meanwhile, dose–effect relationships
experiment have been conducted of compound 3k, and the results
is given in Table 2. There was a dose-dependent inhibition of paw
edema in rat by compound 3k.
Figure 1. The structures of compounds A and B.
2.3. Analgesic activity
2. Results and discussion
2.1. Chemistry
In this method animals are individually placed on a hot plate
maintained at constant temperature (55 1 °C) and the reaction
of animals, such as licking of the paw or jump response is taken
as the end point. A cut-off time of 15 s was taken as maximum
analgesic response to avoid injury to the paws. The reaction time
of animals at 30, 60 and 90 min after compound administration
is presented in Table 3. In this assay, it was observed that com-
pound 3k was the strongest inhibitor.
The structures of 1-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
methyl)-4-substituted-phenylpiperazine were established on the
basis of 1H NMR. The synthesis of compounds 3a–3k followed
the general pathway outlined in Scheme 1. They were all prepared
in four steps. Firstly, a solution of 1,2-dibromoethane in acetone
was added dropwise to a stirring protocatechualdehyde in acetone
to obtain compound 1, using concentrated sulfuric acid as catalyst.
Secondly, compound 2 was synthesized in ethanol by treating
compound 1 with sodium borohydride. Thirdly, compounds 2
was treated in stirring CH2Cl2 with boron tribromide for 8 h to
get compound 3. Lastly, compound 3 were conducted to react with
substituted phenylpiperazine, together with K2CO3 and tetrabuty-
lammonium bromide at 82 °C in acetonitrile. Subsequent purifi-
caiton with recrystallization was conducted and the refined
compounds were finally abtained. All of the synthetic compounds
gave satisfactory analytical and spectroscopic data, which in full
accordance with their depicted structures.
2.4. Ulcerogenic activity
The mucosal damage for each stomach was examined with a
magnifying lens for the presence of macroscopically visible lesions.
The results of ulcerogenic activity are given in Figure 3. The result
was showed that compound 3k was almost no ulcer.
2.5. Cyclooxygenase inhibition assay
It was found that compound 3k was obtained as a potent and
slightly COX-2-selective inhibitor. According to the above ratio-
nale, the compound 3k synthesized in this work was evaluated
for the ability to inhibit COX-1 and COX-2. IC50 are determined
and are means of two determinations acquired and the deviation
from the mean is <10% of the mean value. The selectivity index
(S1 values) was defined as IC50 (COX-1)/IC50 (COX-2). The result
showed that compound 3k exhibited potent inhibition against
2.2. Anti-inflammatory activity
The anti-inflammatory activity of eleven newly synthesized
compounds was determined by carrageenan induced paw edema
method. The results were expressed as% inhibition of oedema over
the untreated control group. The results of anti-inflammatory stud-
ies are given in Table 1. Based on the data, we can concluded that
COX-2 (IC50 = 0.2
lM) compared to the inhibition for COX-1
(IC50 = 8.35 M) as listed in Table 4.
l
Figure 2. Design strategy of the target compounds.
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J. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
O
OH
OH
CHO
CHO
O
O
OH
Sodium borohydride
Dibromoethane
O
1
2
HN
N R
O
Boron tribromide
N
Br
N
O
R
O
3a-3k
3
3a: R=3-ClPh
3b: R=4-MeOPh
3c: R=Ph
3d: R=-CH-2Ph
3e: R=4-NO2Ph
3f: R=3-MeOPh
3g: R=2,3-ClPh
3h: R=2,4-CH3Ph
3i: R=4-FPh
3j: R=2-Py
3k: R=3-triFPh
Scheme 1. General synthesis of compounds 3a–3k.
Table 1
The anti-inflammatory activity at different time intervals and ulcer indices of compounds 3a–3k and compared to diclofenac and Na CMC
Compounds
R
% Of edema inhibition (% mean SEM)
3 h
1 h
5 h
3a
3b
3c
3d
3e
3f
3g
3h
3-ClPh
4-MeOPh
Ph
CH2-2Ph
4-NO2Ph
3-MeOPh
2,3-ClPh
2,4-CH3Ph
4-FPh
31 0.51
71 1.23
28 0.59
15 1.02
68 2.12
45 2.67
18 0.98
16 1.14
62 0.10
40 5.31
78 0.13
42 0.62
0
37 1.50
88 3.21
35 0.92
27 1.93
79 4.05
62 2.76
28 0.51
28 2.34
82 3.65
55 3.12
92 2.45
79 1.77
0
24 1.56
63 2.31
25 0.76
19 2.44
65 4.25
40 0.78
13 0.13
11 0.51
57 3.18
35 2.86
68 4.02
50 4.65
0
3i
3j
2-Py
3-TriFPh
—
3k
Diclofenac
Na CMC
—
Table 2
Inhibitory effect of compounds on carrageenan induced paw edema in rat at different time
Time (h)
% Of edema inhibition (%)
Diclofenac (mg/kg)
3k (mg/kg)
10
20
10
5
1
3
5
41 0.78
80 2.05
52 3.05
78 1.72
95 3.52
75 1.03
76 1.12
91 1.98
68 3.67
56 2.34
73 4.12
50 0.78
Table 3
The tested compounds at the dose of 10 mg/kg body weight in 0.5% Na CMC were given as suspension orally to animals and observed the reaction time of animals at 30, 60 and
90 min after compound administration
Compounds
Pain threshold (s)
0
0.5 h
1 h
1.5 h
3b
3e
3i
3k
21.2 5.5
19.7 3.8
19.1 4.2
18.8 3.1
19.1 2.5
24.6 3.7
23.4 5.0
21.5 4.5
26.4 4.7
24.1 3.3
23.3 4.2
22.6 5.2
20.7 2.7
24.7 4.1
23.5 4.3
22.3 3.9
21.2 4.5
19.9 4.6
23.1 3.8
22.3 3.5
Diclofenac
2.6. Docking studies
simulation was also performed to explore the probable binding
modes of these compounds.
2.6.1. Molecular docking
The model of compound 3k docked with COX-2 is depicted in
Figure 4. In the binding model, compound 3k is nicely bound to
the active site of the COX-2 by one hydrogen bond with His90,
A docking study was performed iteratively in this study, and the
general workflow was described in the method section. In modify-
ing the phenylpiperazine derivatives of 1,4-benzodioxan scaffold in
silico, molecular docking was carried out to fit the scaffold mole-
cules into the activity pocket of COX-2. Afterwards, docking
screening was employed to filter the candidates for better binding
potential. When the lead compound was finally validated and con-
comitantly modified to produce the target compounds, docking
one
p-cation bond with Arg513 and one p-sigma bond with
Ser353. The 1,4-benzodioxan scaffold at the pocket terminal
played a conserved role in stabilizing the docking, and balanced
its placing with the key amino acid His90, by forming proper H-
bonds. The hydrophobic residues Leu352, Phe518 and Val349 sur-
round the piperazine ring. Meanwhile, the trifluoromethyl group is
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J. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
thus obtained concludes that all the analogs have good ADMET
properties and can be considered for further evaluation. These
compounds are predicted to be devoid of any mutagenic or car-
cinogenic properties, all of them show negligible discriminant
score (Fig. 5).
2.7. Single crystal X-ray diffraction
Crystals of compound 3k (CCDC 1487466) were obtained from
methanol solution. Figure 6 shows a perspective view of the mono-
meric unit with the atomic numbering schemes. Crystallographic
data, details of data collection and structure refinement parame-
ters are listed in Table 5. Single crystal of 3k
(0.35 mm ꢀ 0.33 mm ꢀ 0.27 mm) was mounted on a D-8 venture
diffractometer equipped with graphite-monochromated MoKa
(k = 0.71073 Å) radiation. For 3k, a total of 17702 reflections were
collected, of which 3456 were unique with Rint = 0.067 and 1754
observed reflections with I > 2r (I) were used in the succeeding
structure calculations. The final cycle of refinement of full matrix
least-squares was converged to R = 0.1633 and wR = 0.2563. The
highest and lowest residual peaks in the final difference Fourier
map are 0.673 and ꢁ0.354 e/Å3, respectively.
3. Conclusion
Figure 3. The mucosal damage for each stomach was examined with various
concentrations of diclofenac and 3k. (a) diclofenac, 10 mg/kg; (b)3k, 5 mg/kg; (c)
3k, 10 mg/kg; (d) 3k, 20 mg/kg.
In conclusion, a series of 1-((2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)methyl)-4-substituted-phenylpiperazine derivatives have been
synthesized and evaluated for their anti-inflammatory activities.
According to the data presented in Table 1 and molecular docking
results, it could be concluded that the activity of the tested com-
pounds may be correlated to the variation and modifications of
structure. Compound 3k containing 3-triFPh substituent displayed
the most potent anti-inflammatory activities among the synthe-
sized compounds, no-ulcerogenic side effect as well.
Table 4
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of the designed compounds
Compound
IC50
(lM)
SI
COX-1
COX-2
3k
Celecoxib
8.35
15.13
0.12
0.04
69.58
>300
Moreover, compound 3k demonstrated the most potent inhibi-
tory activity against COX-2 with IC50 of 0.12 lM. Docking simula-
nicely binded through hydrophobic interactions in a hydrophobic
cavity formed by Tyr348, Val349, Tyr385, Trp387, Ala527,
Gly526. Furthermore, other weak interactions, such as van der
Waals and carbon–hydrogen bonds, also contributed to the binding
affinity of 3k with COX-2. The molecular docking suggests that
compound 3k may be a potential COX-2 ligand.
tion was performed to position compound 3k into the COX-2
active site to determine the probable binding conformation and
the result indicated that compound 3k was a potent inhibitor of
COX-2. Besides, all of the compounds showed druglike ADMET
properties. Given the unforeseen structural differences within the
active site of some pathogenic enzymes, the key to discover inhibi-
tors with anti-inflammatory activity lies in a detailed understand-
ing of the COX-2 active sites.
2.6.2. All of the phenylpiperazine derivatives of 1,4-benzodi-
oxan are non-toxic
The phenylpiperazine derivatives of 1,4-benzodioxan were then
subjected to ADMET prediction using TOPKAT and the information
Further studies on the COX-2 inhibition ability of this com-
pound, new structural data were guiding further modifications of
Figure 4. Binding mode of compound 3k with COX-2 (PDB code: 3NTG).
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J. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 5. ADMET properties predicted for eleven novel compounds. Compounds located inside the innermost oval have the best results. The eleven compounds were as
follows: 3d, 3g, 3h(3k), 3a, 3i, 3c, 3f(3b), 3j, 3e (from top to bottom, from left to right).
4. Materials and methods
4.1. General
All of the synthesized compounds were chemically character-
ized by thin layer chromatography (TLC), proton nuclear magnetic
resonance (1H NMR) and elemental microanalyses (CHN). 1H NMR
spectra were measured on a Bruker AV-300 spectrometer at 25 °C
and referenced to Me4Si. Chemical shifts were reported in ppm (d)
using the residual solvent line as internal standard. Splitting pat-
Figure 6. Crystal structure of compound 3k.
terns are designed as s, singlet; d, doublet; t, triplet; m, multiplet.
Melting points were determined on a WRS-1B apparatus (Jingke
Corp., Shanghai, China) and are as read. Analytic thin-layer chro-
matography (TLC) was performed on the glass-backed silica gel
sheets (silica gel 60 Å GF254). All compounds were detected using
UV light (254 nm or 365 nm).
Table 5
Crystallographic data, details of data collection and structure refinement parameters
Compound
3k
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C
20H21F3N2O2
378.39
Monoclinic
P21/c
10.696(3)
11.176(3)
15.488(4)
90
100.548(7)
90
100.548(7)
4
1.381
1.9–25.8
792
4.2. General method for the preparation of target compounds
4.2.1. (2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methanol (1)
b (Å)
c (Å)
Sodium borohydride (0.38 g, 10 mmol) was added to a solution
of
2,3-dihydrobenzo[b][1,4]dioxine-6-carbaldehyde
(1.64 g,
a
(°)
b (°)
(°)
10 mmol) in anhydrous ethanol (29 mL) and the reaction mixture
was stirred at room temperature for 2 h while monitoring by
TLC. The reaction mixture was concentrated under reduced pres-
sure and then extracted with ethyl acetate and distilled water.
The organic layer was then dried over anhydrous Na2SO4, concen-
trated and purified by recrystallization.
c
V (Å)
Z
Dcalc/g cmꢁ3
h range (°)
F(000)
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmꢁ1
17702/3456
1754/0.067/244
0.110
0.0817/0.2090
0.1633/0.2563
1.019
4.2.2. 6-(Bromomethyl)-2,3-dihydrobenzo[b][1,4]dioxine (2)
Boron tribromide (2.6 g, 10 mmol) was slowly added to a solu-
tion of 1 (1.66 g, 10 mmol) in anhydrous dichloromethane (10 mL)
and the mixture was stirred at room temperature for 8 h while
monitoring by TLC. The mixture was then concentrated under
reduced pressure and saturated sodium bicarbonate solution was
added to pH7. The product was obtained by precipitation.
)
R1/WR2 [I > 2
r
(I)]
R1/WR2 (all date)
GOOF
the current series with the aim to improve both enzymatic inhibi-
tion and physical properties.
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J. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.2.3. General procedure for the synthesis of compounds 3a–3k
(3)
Cl2N2O2, [M+H]+). Anal. Calcd. for C19H19Cl2N2O2: C, 60.17; H,
5.32; N, 7.39. Found: C, 60.27; H, 5.27, N, 7.36.
Sodium carbonate (1 mmol) was added to a solution of substi-
tuted-piperazine (1 mmol) in anhydrous acetonitrile (20 mL) and
the reaction mixture was stirred at room temperature for 30 min.
Compound 2 (1 mmol) was added and the reaction mixture was
stirred at 82 °C for 5 h while monitoring by TLC. The mixture was
then filtered, the filtrate was evaporated under reduced pressure
and the crude products were recrystallized from methanol to
abtain pure compounds 3a–3k.
4.2.3.8. 1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(2,4-
dimethylphenyl)piperazine (3h).
Yield: 68%; mp: 118.8–
119.7 °C; 1H NMR (300 MHz, CDCl3): 1.631 (s, 3H), 2.191 (s, 3H),
2.525 (s, 4H), 2.779–2.934(m, 4H), 3.280–3.393 (m, 2H), 4.166–
4.170(m, 4H), 6.740–6.914 (m, 6H). MS (ESI): 338 (C21H26N2O2,
[M+H]+). Anal. Calcd. for C21H25N2O2: C, 74.52; H, 7.74; N, 8.28.
Found: C, 74.71; H, 7.64, N, 8.24.
4.2.3.1. 1-(3-Chlorophenyl)-4-((2,3-dihydrobenzo[b][1,4]dioxin-
4.2.3.9. 1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(4-
6-yl)methyl)piperazine (3a).
Yield: 60%; mp: 96.0–96.3 °C;
fluorophenyl)piperazine (3i).
Yield: 78%; mp: 99.3–99.9 °C;
1H NMR (300 MHz, CDCl3): 2.563–2618 (m, 4H), 3.234–3.376 (m,
4H), 3.415 (s, 2H), 4.257 (s, 4H), 6.765–6.823 (m, 3H), 7.123–
7.238 (m, 2H), 7. 327–7.338 (m, 2H). MS (ESI): 345 (C19H21ClN2O2,
[M+H]+). Anal. Calcd. for C19H20ClN2O2: C, 66.18; H, 6.14; N, 8.12.
Found: C, 66.25; H, 6.18, N, 8.01.
1H NMR (300 MHz, CDCl3): 2.553–2.575 (m, 4H), 3.402–3.259 (m,
6H), 4.255–4.259 (m, 4H), 6.812–6.833 (m, 3H), 7.126–7.129 (m,
1H), 7.238–7.265 (m, 2H), 7.338–7.340 (m, 1H). MS (ESI): 328 (C19
21FN2O2, [M+H]+). Anal. Calcd. for C19H20FN2O2: C, 69.49; H, 6.45;
N, 8.53. Found: C, 69.32; H, 6.49, N, 8.58.
-
H
4.2.3.10.
(pyridin-2-yl)piperazine (3j).
1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-
Yield: 68%; mp: 225.6–
4.2.3.2. 1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(4-
methoxyphenyl)piperazine (3b).
Yield: 66%; mp: 88.1–
226.2 °C; 1H NMR (300 MHz, CDCl3): 2.512–2.542 (m, 4H), 3.442–
3.458 (d, J = 8.4 Hz, 2H), 3.510–3.535 (m, 4H), 4.192–4.240 (m,
4H), 6.567–6.624 (m, 2H), 6.804 (s, 2H), 6.863 (s, 1H), 7.410–
7.423 (m, 1H), 8.164–8.181 (m, 1H). MS (ESI): 394 (C23H30N4O2,
[M+H]+). Anal. Calcd. for C23H39N4O2: C, 70.02; H, 7.66; N, 14.20.
Found: C, 70.18; H, 7.61, N, 14.16.
88.3 °C; 1H NMR (300 MHz, CDCl3): 2.592–2.623 (m, 4H), 3.077–
3.110 (m, 4H), 3.472 (s, 2H), 3.703–3.760 (m, 3H), 4.249 (s, 4H),
6.813–6.902 (m, 7H). MS (ESI): 340 (C20H24N2O3, [M+H]+). Anal.
Calcd. for C20H23N2O3: C, 70.56; H, 7.11; N, 8.23. Found: C, 70.66;
H, 7.15, N, 8.09.
4.2.3.3.
phenylpiperazine (3c).
1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-
Yield: 71%; mp: 114.3–114.8 °C; 1H
4.2.3.11. 1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(3-
(trifluoromethyl)phenyl)piperazine (3k).
Yield: 79%; mp:
NMR (300 MHz, CDCl3): 2.504–2.632 (m, 4H), 3.223–3.298 (m,
4H), 3.519–3.543 (m, 2H), 4.219 (s, 4H), 6.808–6.834 (m, 2H),
6.921–6.953 (m, 2H), 7.012–7.124 (m, 3H), 7.287–7.367 (m, 1H).
MS (ESI): 310 (C19H22N2O2, [M+H]+). Anal. Calcd. for C19H21N2O2:
C, 73.52; H, 7.14; N, 9.03. Found: C, 73.63; H, 7.15, N, 8.91.
88.0–88.6 °C; 1H NMR (300 MHz, CDCl3): 2.574–2.607 (m, 4H),
2.574–2.607 (m,4H), 3.212–3.246 (m,4H), 3.415–3.474 (m, 2H),
4.190–4.250 (m,4H), 6.806–6.837 (m, 2H), 6.870 (s, 1H), 7.023–
7.092 (m, 3H), 7.296–7.349 (m,1H). MS (ESI): 378 (C20H21F3N2O2,
[M+H]+). Anal. Calcd. for C20H20F3N2O2: C, 63.48; H, 5.59; N, 7.40.
Found: C, 63.33; H, 5.61, N, 7.46.
4.2.3.4. 1-Benzhydryl-4-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
methyl)piperazine (3d).
Yield: 73%; mp: 120.0–120.7 °C; 1H
NMR (300 MHz, CDCl3): 1.681 (s, 4H), 3.403–3.464 (m, 6H), 4.216
(s, 5H), 6.763–6.805 (m, 3H), 7.148–7.172 (d, J = 9.6 Hz, 2H),
7.218–7.267 (m, 4H), 7.337–7.402(d, J = 10 Hz, 4H). MS (ESI): 400
(C26H28N2O2, [M+H]+). Anal. Calcd. for C26H27N2O2: C, 77.97; H,
7.05; N, 6.99. Found: C, 77.79; H, 7.18, N, 7.04.
4.3. Screening of anti-inflammatory activity in vivo
This experiment was conducted in accordance with the guide-
line issued by the State Food and Drug Administration (SFDA of
China). The animals were housed and cared for in accordance with
the guidelines established by the National Science Council of
Republic China. All experimental protocols were approved by Ani-
mal Care and Use Committee of Nanjing University. Male mice, 35–
40 days old and weighing 18–22 g, were supplied by Shanghai Lab-
oratory Animal Limited Company. The mice were raised in air-con-
ditioned rooms under controlled lighting (12 h lighting/day) and
were fed with standard laboratory food and water ad libitum.
The synthesized compounds were evaluated for their anti-
inflammatory activity using carrageenan-induced paw edema
method in rats described by Winter et al.30 The test compounds
and diclofenac were administered orally at a dose level of 10 mg/
kg (suspended in 0.5% Na CMC given p.o.) 30 min before car-
rageenan (0.1 mL of 1% (w/v)) injection at the right hind paw of
male rats. The thickness of both paws was measured at different
time intervals of 1, 3 and 5 h after carrageenan injection. The
anti-inflammatory activity of the tested compounds and diclofenac
was calculated as the percentage decrease in edema thickness
induced by carrageenan and was determined using the following
formula:
4.2.3.5. 1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(4-
nitrophenyl)piperazine (3e).
Yield: 70%; mp: 168.4–
168.8 °C; 1H NMR (300 MHz, CDCl3): 2.559–2.593 (m, 4H), 3.401–
3.461(m, 6H), 4.254 (s, 4H), 6.779–6.859 (s, 5H), 8.090–8.121 (m,
2H). MS (ESI): 355 (C19H21N3O2, [M+H]+). Anal. Calcd. for
C19H20N3O4: C, 64.21; H, 5.96; N, 11.82. Found: C, 64.15; H, 5.98,
N, 11.86.
4.2.3.6. 1-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(3-
methoxyphenyl)piperazine (3f).
Yield: 63%; mp: 127.3–
127.9 °C; 1H NMR (300 MHz, CDCl3): 2.556–2.591 (m, 4H), 3.412–
3.510 (m, 6H), 3.708 (s, 3H), 4.255–4.259 (m, 4H), 6.814–6.852
(m, 3H), 7.123–7.165 (m, 2H), 7.337–7.339 (m, 1H), 7.423–7.431
(m, 1H). MS (ESI): 340 (C20H24N2O3, [M+H]+). Anal. Calcd. for
C20H23N2O3: C, 70.56; H, 7.11; N, 8.23. Found: C, 70.66; H, 7.15,
N, 8.09.
4.2.3.7.
1-(2,3-Dichlorophenyl)-4-((2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)methyl)piperazine (3g).
Yield: 63%; mp: 104.9–
Percentage of oedema inhibition ¼ 100 ꢁ ðVtest=VcontrolÞ ꢀ 100;
105.3 °C; 1H NMR (300 MHz, CDCl3): 2.611 (s, 4H), 3.043 (s, 2H),
3.352–3.463 (m, 4H), 4.240(s, 4H), 6.801(s, 1H), 6.862(s, 1H),
where, Vcontrol = volume of paw oedema in control group; Vtest = vol-
ume of paw oedema in the test compounds in treated group.
6.919–6.951(m, 2H), 7.113–7.131(m, 2H). MS (ESI): 378 (C19H20
-
Please cite this article in press as: Sun, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.023

J. Sun et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
The results were expressed as% inhibition of oedema over the
4.8. Statistical analysis
untreated control group.
Statistical analysis was performed with SPSS Version 11.0
statistic software package. Data were expressed as means stan-
dard deviation (SD). Comparisons between groups were performed
with analysis of non-parametric test. A value of P < 0.05 was con-
sidered statistically significant.
4.4. Analgesic activity
Compounds which showed good anti-inflammatory activity
were further tested for their analgesic activity using Eddy’s hot
plate method.31 In this method heat is used as a source of pain.
The tested compounds at the dose of 10 mg/kg body weight in
0.5% Na CMC were given as suspension orally to animals and
observed the reaction time of animals at 30, 60 and 90 min after
compound administration.
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Please cite this article in press as: Sun, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.023