Synthesis and Evaluation of Anti-inflammatory N-Substituted 3,5-Bis(2-(trifluoromethyl)benzylidene)piperidin-4-ones

Article abstract of DOI:10.1002/cmdc.201600606

A total of 24 N-substituted 3,5-bis(2-(trifluoromethyl)benzylidene)piperidin-4-one derivatives were synthesized via aldol condensation, and their anti-inflammatory activities were evaluated. These compounds were found to have no significant cytotoxicity a

Full text of DOI:10.1002/cmdc.201600606

DOI: 10.1002/cmdc.201600606
Full Papers
Synthesis and Evaluation of Anti-inflammatory N-
Substituted 3,5-Bis(2-
(trifluoromethyl)benzylidene)piperidin-4-ones
Zixin Xie, Zaikui Zhang, Shufang Yu, Donghua Cheng, Huan Zhang, Chao Han, Handeng Lv,
and Faqing Ye*[a]
A total of 24 N-substituted 3,5-bis(2-(trifluoromethyl)benzylide-
ne)piperidin-4-one derivatives were synthesized via aldol con-
densation, and their anti-inflammatory activities were evaluat-
ed. These compounds were found to have no significant cyto-
toxicity against mouse bone marrow cells in vitro. However,
some compounds, such as c6 (N-(3-methylbenzoyl)-3,5-bis-(2-
(trifluoromethyl)benzylidene)piperidin-4-one) and c10 (N-(2-
chlorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piperi-
din-4-one), displayed potent anti-inflammatory activity by in-
hibiting lipopolysaccharide (LPS)-stimulated tumor necrosis
factor (TNF)-a, interleukin-6 (IL-6), IL-1b, prostaglandin E2
(PGE2), and nitric oxide (NO) production in RAW 264.7 cells.
Treatment with c6 or c10 at 2.5 or 10 mgkgꢀ1 significantly de-
creased the paw edema induced by carrageenan in rats, and
the anti-inflammatory effects of these compounds were found
to be better than those of celecoxib or indomethacin as well
as their parent compound C66 (2,6-bis-(2-(trifluoromethyl)ben-
zylidene)cyclohexanone). Pharmacokinetic analysis indicated
that c6 has better bioavailability than curcumin. Therefore,
these compounds may be valuable leads for the development
of new anti-inflammatory drugs.
Introduction
Curcumin is one active ingredient of Rhizoma Curcuma and
has a variety of biological activities, such as anti-inflammatory,
antitumor, antioxidant, antimicrobial, and free-radical scaveng-
ing.[1–8] However, curcumin’s limited bioavailability, lack of
water solubility, and rapid metabolism hinder its clinical
application.[3,9,10] Notably, curcumin contains an unstable b-di-
ketone motif, which is a key transformation.[11] An approach to
overcome these problems is to generate improved synthetic
curcumin analogues with potent activity.[12] Our previous stud-
ies have generated different types of curcumin derivatives, in-
cluding C66, F35, and others (Figure 1).[13,14–16] These com-
pounds display anti-inflammatory activity by downregulating
inflammatory cytokine production.[17–24] C66 is an o-trifluoro-
methyl benzyl cyclohexanone curcumin analogue and can in-
hibit interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a)
production.[17,25,26] Further analysis suggests that the trifluoro-
inflammatory mediators, such as nitric oxide (NO), prostaglan-
din E2 (PGE2), IL-1b, TNF-a, among others. Therefore, inhibition
of their production during the inflammatory process is impor-
tant for the control of harmful tissue injury.[4,28–32]
Previous studies have shown that piperidone derivatives
have excellent anti-inflammatory activity.[33,34] In this study, we
designed and synthesized 24 new derivatives of C66 by trans-
forming the cyclohexanone ring into substituted piperidone in
the search for more suitable candidate compounds for clinical
study (Scheme 1). Subsequently, we evaluated their anti-inflam-
matory activity and pharmacokinetics. Our data indicate that
two compounds, c6 and c10, have potent anti-inflammatory
activity, with c6 having better bioavailability than curcumin
in vivo.
methyl and bis-olefinic sites are potential areas for C66 to in- Results and Discussion
teract with biological macromolecules. Hence, modulation of
Chemistry
these groups in C66 may enhance its anti-inflammatory activi-
ty.
The target compounds of a1–a5 were synthesized by mixing
The inflammatory response is an essential process in com-
5 mmol 2-(trifluoromethyl)benzaldehyde with 2.5 mmol N-R1
piperidinone at 88C in ethanol using 3–4 drops of 40% NaOH
as catalyst to yield a1–a5 at 95, 85, 89.5, 87.8, and 90.4%, re-
spectively (Scheme 2).[35] Compounds b1–b9 were synthesized
by two-step reactions. First, we obtained some related inter-
mediates by mixing 1 mmol piperidone hydrochloride and
1 mmol benzyl bromides with a variety of substituted groups
(R2) in the presence of 2 mmol anhydrous K2CO3 in acetonitrile
at 208C. Subsequently, compounds b1–b9 were synthesized
via the same approach as used for a1–a5 with yields of 65.8,
bating infection, but can also induce tissue injury that may
lead to cardiovascular diseases, rheumatoid arthritis, bronchitis,
diabetes, neurodegenerative disorders, and cancers.[27] In re-
sponse to extracellular stimuli, macrophages can produce pro-
[a] Dr. Z. Xie, Z. Zhang, S. Yu, D. Cheng, H. Zhang, C. Han, H. Lv, Prof. Dr. F. Ye
School of Pharmaceutical Sciences, Wenzhou Medical University, Chashan
Campus Chashan University Town, Wenzhou 325035 (China)
E-mail: yfq664340@163.com
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Figure 1. Reported curcumin derivatives C66 and F35, and general structure of the target compounds in this study.
Scheme 1. Overview of the 24 curcumin derivatives synthesized in this study.
groups (R3) to achieve 61.2, 70.5, 60.5, 65.4, 61.2, 60.5, 49.8,
43.0, 51.0, and 49.6% yields, respectively (Scheme 4).
When the synthetic methods of series b were applied to ser-
ies c, we found that the majority of byproducts and miscellane-
ous spots were located near the bottom of the silica plate by
TLC monitoring, and were difficult to purify. Thus, the subse-
quent synthesis was modified (Scheme 2). In this case, prod-
ucts with high purity were obtained, and then separated and
purified by recrystallization. All series of compounds were fully
characterized and identified by LC–MS, NMR, IR, and UV spec-
troscopic analyses.
Scheme 2. Synthesis of compounds a1–a5.
70.2, 57.2, 68.7, 68.7, 66.4, 69.2, 68.7, and 61.3%, respectively
(Scheme 3). Compounds c1–c10 were synthesized by reacting
a1–a5 and benzoyl chloride with a variety of substituted
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Scheme 3. Synthesis of compounds b1–b9.
Scheme 4. Synthesis of compounds c1–c10.
Biological evaluation
Enzyme-linked immunosorbent assay (ELISA)
We next examined the effect of different compounds on LPS-
induced TNF-a and IL-6 production in RAW 264.7 cells in vitro
by ELISA. Stimulation with LPS induced high levels of TNF-
a and IL-6 production in RAW 264.7 cells (Figure 3). In contrast,
treatment with compounds a3, c1, c2, c5, c6, and c10 signifi-
cantly decreased the levels of TNF-a and IL-6 in the cells by
>50%, relative to those in the cells with LPS stimulation
alone; the inhibitory effects of these six compounds were even
greater than that of their parent compound C66. Furthermore,
other compounds, b7 and b9, also significantly decreased the
levels of IL-6 production in RAW 264.7 cells, compared with
the cells with LPS stimulation alone.
Toxic effect
To determine the toxicity of individual compounds, mouse
bone marrow cells were treated in triplicate with individual
compounds at 10 or 20 mm for 24 h and the cell viability was
determined by CCK-8 assay (Figure 2). There was no significant
difference in the percentages of surviving cells among the dif-
ferent groups, as compared with that in the control cells that
had been treated with curcumin regardless of the doses of in-
dividual compounds. These data indicate that those com-
pounds had no significant toxicity against mouse bone
marrow cells in vitro.
Figure 2. The target compounds have no significant cytotoxicity against mouse bone marrow cells in vitro. Mouse bone marrow cells (1.0ꢁ104 cells per well)
were treated in triplicate with individual compounds at A) 20 or B) 10 mm for 24 h, and the cell viability in individual wells was determined by CCK-8 assay.
Data are the meanꢁSD of each group of cells from three separate experiments.
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Figure 3. ELISA analysis of the levels of cytokines induced by LPS stimulation in RAW 264.7 cells. RAW 264.7 cells were stimulated in triplicate with
100 ngmLꢀ1 LPS in the presence or absence of individual compounds at 10 mm for 24 h, and the levels of A) TNF-a and B) IL-6 in the supernatants of each
group of cells were determined by ELISA. Data are the meanꢁSD of each group of cells from three independent experiments; *p<0.05, **p<0.01,
***p<0.001 vs. C66 with LPS stimulation alone.
Furthermore, RAW 264.7 cells were stimulated with LPS in
the absence or presence of 1–15 mm a3, c1, c5, c6 or c10 for
24 h, and the levels of IL-1b and PGE2 in the supernatants of
cultured cells were determined by ELISA (Figure 4A,B). Treat-
ment with any one of compounds significantly decreased the
levels of IL-1b and PGE2 production in RAW 264.7 cells in
a dose-dependent manner, and compounds c6 and c10 dis-
played the most potent inhibitory activity with IC50 <2.5 mm.
In addition, treatment with a3 did not inhibit LPS-stimulated
NO production in RAW 264.7 cells, determined by Griess reac-
tion assay (data not shown). However, treatment with either
c1, c5, c6 or c10 significantly mitigated LPS-stimulated NO pro-
duction in RAW 264.7 cells, and the inhibitory effects of c6 and
c10 were superior to those of positive control compounds dex-
amethasone or acetylsalicylic acid (ASA; Aspirin) as well as
their parent compound C66 under our experimental conditions
(Figure 4C). Collectively, these data demonstrate that those
compounds have potent anti-inflammatory activity.
ized, treated orally with vehicle alone, c6 or c10 at 2.5 or
10 mgkgꢀ1 or positive controls (2.5 mgkgꢀ1 of celecoxib or in-
domethacin) by gavage and then injected subcutaneously with
0.1 mL of 1% carrageenan into the plantar side of the side
paw of each rat. The paw edema of individual rats was exam-
ined by volume displacement method before and 0.5, 1, 2, 3,
4, 5, 6, 7 and 8 h after induction. Significant inhibition of the
paw swelling by these compounds was observed at 2 h post-
treatment. Treatment with 2.5 and 10 mgkgꢀ1 c6 inhibited the
paw swelling by 16.33 and 59.55%, respectively (Figure 5).
Treatment with c10 at 2.5 or 10 mgkgꢀ1 inhibited the paw
swelling by 20.07 and 67.41%, respectively. The inhibitory
effect of c6 and c10 was stronger than that of celecoxib and
similar to that of indomethacin. In addition, intravenous ad-
ministration of 2.5 mgkgꢀ1 of c6 or c10 also significantly inhib-
ited the paw swelling within a short period post-injection.
Hence, c6 and c10 had potent anti-inflammatory activity
in vivo.
Rat model of carrageenan-induced paw edema
Pharmacokinetics studies
To determine the potential anti-inflammatory effect of these
compounds in vivo, male Sprague–Dawley rats were random-
The bioavailability of the target compound c6 in rats was de-
termined by high-performance liquid chromatography (HPLC,
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Figure 4. Effect of compounds on the levels of LPS-stimulated IL-1b, PGE2, and NO production in RAW 264.7 cells. RAW 264.7 cells were stimulated with LPS
in the presence or absence of various doses of the indicated compounds for 24 h, and the levels of A) IL-1b and B) PGE2 in the supernatants of cultured cells
were determined by ELISA; C) NO levels were determined by Griess assay. The cells in medium alone without LPS stimulation served as negative control,
whereas LPS-stimulated cells in the presence of 10 mm dexamethasone (Dex) or Aspirin (asp) served as positive controls. L: Lipopolysaccharides (LPS). Data
are the meanꢁSD of each group of cells from three independent experiments.
Figure 5. Anti-inflammatory activity of c6 and c10 in a rat model of paw edema induced by carrageenan. A) Oral administration (2.5 mgkgꢀ1, 10 mgkgꢀ1) of
c6. B) Oral administration (2.5 mgkgꢀ1, 10 mgkgꢀ1) of c10. C) Intravenous injection with c6 and c10. Celecoxib and indomethacin at 2.5 mgkgꢀ1 served as
positive controls. Data are the meanꢁSD of each group of cells from three independent experiments.
Table 1). Intravenous injection with c6 resulted in plasma con-
centration Cmax of 4.6ꢁ1.9 mgLꢀ1, tmax of 2.1ꢁ1.1 min and half-
life of 28.3 min. Furthermore, oral administration with c6 result-
ed in a plasma concentration Cmax of 0.9ꢁ0.6 mgLꢀ1, a tmax of
15.5ꢁ2.4 min, and a half-life of 86.1 min. Compound c6
showed a slight advantage, because the absolute bioavailabil-
ity of orally administered c6 was 63.6%, which was higher
than 4.13% of curcumin.
Structure–activity relationship analysis
Analysis of the structure–activity relationship (SAR) indicated
that compounds with a common aliphatic chain and N-ethyl,
such as a3, exhibited potent inhibitory activity toward TNF-
a and IL-6, but no inhibition on NO production, nor the inhibi-
tion of IL-1b and PGE2. Furthermore, the activities of class
b compounds are affected by the type of substituents on the
N position distinctly (see b1–b9); different substituted benzyl
groups were introduced at the N position, which did not make
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Experimental Section
Table 1. Essential pharmacokinetic parameters.[a]
General methods. Unless otherwise noted, all commercially avail-
able solvents and reagents were used without further purification.
The reagents and anhydrous solvents were purchased from Alad-
din and Sinopharm Chemical Reagents. Melting points were deter-
mined using an XRC-1 micro-melting point apparatus. Thin-layer
chromatography (TLC, Rf values) was performed on F254 or silica gel
plates F254 (0.2 mm thick), and the results were visualized after ex-
posure to UV light. The IR spectra were recorded by a Bruker Nico-
let 670FT-IR spectrophotometer using KBr pellets on a Nicolet
Impact 410 instrument. NMR spectra were recorded on a Bruker
Avance 600 (1H, 300 MHz; 13C, 600 MHz) spectrometer at 300 K,
and samples were dissolved in [D6]DMSO or CDCl3 using tetrame-
thylsilane (TMS) as an internal reference. Chemical shifts are report-
ed in ppm relative to TMS, and J values are expressed in Hz. Split-
ting patterns are designated as follows: s, singlet; d, doublet; t,
triplet. Mass spectra were performed on an Agilent-1200 LC–MS in-
strument.
Parameter
intravenous
c6
curcumin[36]
per os
curcumin[36]
c6
83.6
AUC0–t [mgLꢀ1 minꢀ1
AUC0–1 [mgLꢀ1 minꢀ1
R-AUC [%]
]
72.0
74.6ꢁ9.6
73.3ꢁ12.5
86.4ꢁ12.9
4.1
]
73.2
79.6
40.8
41.9
28.3
1.6
104.6ꢁ11.9
101.5
63.6
110.0
139.8
86.1
17.6
0.1
75
MRT0–t [min]
MRT0–1 [min]
t1/2 [min]
–
–
–
–
1.6ꢁ0.5
0.6ꢁ0. 3
0.1ꢁ0.0
5.0ꢁ0.0
3.6ꢁ0.5
–
13.2ꢁ2.2
129.3ꢁ26.6
2.4ꢁ0.4
VZ/F [Lkgꢀ1
]
Clz [Lminꢀ1 kgꢀ1
tmax [min]
]
0.04
2.1ꢁ1.1
4.6ꢁ1.9
8.4
15.5ꢁ2.4 32.1ꢁ4.9
Cmax [mgLꢀ1
]
0.9ꢁ0.6
0.7ꢁ0.1
C0 [mgLꢀ1
Clast [mgLꢀ1
Dose [mgkgꢀ1
]
–
0.0
10.0
–
–
]
0.0
–
]
3.0
10.0
200.0
[a] Data are the meanꢁSD of each group of cells from three independent
experiments.
General procedure for the preparation of compounds a1–5: A
solution of 2-(trifluoromethyl)benzaldehyde (5 mmol) in anhydrous
ethanol (15–50 mL) was mixed with anhydrous N-R1 piperidinone
(2.5 mmol) and 3–4 drops of 40% NaOH as catalyst. The mixture
was stirred at 88C for 12 h. After completion of reaction (TLC), the
solid precipitate was filtered. The precipitates were washed with
50% ethanol, and the crude products were dried under vacuum. If
there was no precipitated solid, the reactions were adjusted to
neutral pH with 10% HCl, and then the methods described above
were reiterated. The residue was purified by column chromatogra-
phy on silica gel [petroleum ether/EtOAc] to obtain a1–5 as yellow
powders.
any contribution to the anti-inflammatory activities. Surprising-
ly, the anti-inflammatory activities of class c compounds, in-
cluding those toward TNF-a, IL-6, IL-1b, PGE2, and NO, were
dependent on the phenylamide at the N position, and were af-
fected by different substituted groups on the benzene ring, in-
cluding electron-withdrawing Cl, F, NO2, and electron-donating
ꢀCH3, and others. Comparing c3, c4, c5, c7 and c8, in which
different types of groups were introduced at C4 of the ben-
zene ring, c3 and c5 were superior to others, which was due
to the electron-donating group ꢀCH3 or ꢀOCH3; activity was
therefore increased by introducing electron-donating groups
rather than electron-withdrawing groups at this position. How-
ever, there was little effect on whether electron-donating or
-withdrawing groups were introduced at C3, such as c6 or c1.
Comparing c9 and c10, in which electron-withdrawing groups
were introduced at C2, the most potent c10 has 2-chloroben-
zamide at the N position. It is possible that introducing benza-
mide at the N position and a carbonyl group may improve the
polarity of the compounds, which may facilitate cell entry.
N-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one
(a1):
Yellow solid, yield 95%; mp: 118.9–120.58C; 1H NMR (600 MHz,
[D6]DMSO): d=7.99 (s, 2H, C=CHꢁ2), 7.74 (d, J=7.80 Hz, 2H, Arꢀ
Hꢁ2), 7.56–7.58 (m, 2H, ArꢀHꢁ2), 7.45–7.47 (m, 2H, ArꢀHꢁ2),
7.23–7.25 (m, 2H, ArꢀHꢁ2), 3.85 (s, 2H, ꢀCH2), 3.84 (s, 2H, ꢀCH2),
1.62 ppm (br, 1H, ꢀNH); 13C NMR (600 MHz, [D6]DMSO): d=187.2,
140.1, 137.0, 134.0, 132.2, 130.7, 126.1, 126.4, 123.1, 46.6 ppm; ESI-
MS [M+H]+ 412.5; Anal. calcd for C21H15F6NO: C 62.41, H 4.98, F
29.01, N 3.61; found: C 62.02, H 3.98, F 28.41, N 5.59.
N-methyl-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one
(a2): Yellow solid, yield 85%; mp: 120.1–122.38C; 1H NMR
(600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.74 (d, J=7.8 Hz,
2H, ArꢀHꢁ2), 7.57–7.58 (m, 2H, ArꢀHꢁ2), 7.47–7.48 (m, 2H, Arꢀ
Hꢁ2), 7.25–7.28 (m, 2H, ArꢀHꢁ2), 3.51 (s, 2H, ꢀCH2), 3.50 (s, 2H, ꢀ
CH2), 2.29 ppm (s, 3H, ꢀCH3); 13C NMR (600 MHz, [D6]DMSO): d=
186.0, 143.0, 140.1, 131.9, 128.7, 126.1, 125.0, 122.0, 58.8, 45.0 ppm;
ESI-MS [M+H]+ 426.4; Anal. calcd for C22H17F6NO: C 63.13, H 4.83,
F 27.05, N 4.90; found: C 64.02, H 4.13, F 26.43, N 5.42.
Conclusions
A total of 24 N-substituted 3,5-bis(2-(trifluoromethyl)benzylide-
ne)piperidin-4-one derivatives were synthesized. These com-
pounds had no clear toxicity against mouse bone marrow
cells, and at 10–20 mm, some of them, particularly c6 and c10,
were found to have potent anti-inflammatory activity by inhib-
iting LPS-stimulated TNFa, IL-6, IL-1b, PGE2, and NO produc-
tion in rat macrophages. Furthermore, oral or intravenous ad-
ministration of c6 or c10 significantly decreased inflammatory
paw edema in rats induced by carrageenan. Pharmacokinetic
studies indicated that compound c6 has better bioavailability
than curcumin in vivo. Therefore, c6 and c10 may be valuable
leads for further development of new anti-inflammatory re-
agents. Detailed follow-up studies on the mechanisms of
action of these derivatives are underway, and will be reported
in due course.
N-Ethyl-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one
(a3): Yellow solid, yield 89.5%; mp: 109.4–112.18C; 1H NMR
(600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.74 (d, J=7.8 Hz,
2H, ArꢀHꢁ2), 7.57–7.59 (m, 2H, ArꢀHꢁ2), 7.45–7.47 (m, 2H, Arꢀ
Hꢁ2), 7.27–7.28 (m, 2H, ArꢀHꢁ2), 3.59 (s, 4H,CH2 ꢁ2), 2.44–2.45
(m, 2H, ꢀCH2), 0.90 ppm (t, J=8.40 Hz, 3H, ꢀCH3); 13C NMR
(600 MHz, [D6]DMSO): d=186.6, 140.1, 135.2, 134.3, 132.9, 130.7,
128.4, 126.4, 124.9, 123.1, 53.5, 49.9, 12.4 ppm; ESI-MS [M+H]+
440.4; Anal. calcd for C23H19F6NO: C 63.91, H 4.99, F 27.15, N 3.95;
found: C 64.07, H 4.83, F 26.23, N 4.87.
N-Propyl-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one
1
(a4): Yellow solid, yield 87.8%; mp: 87.8–90.28C; H NMR (600 MHz,
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[D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.74 (d, J=7.8 Hz, 2H, Arꢀ
Hꢁ2), 7.55–7.57 (m, 2H, ArꢀHꢁ2), 7.46–7.48 (m, 2H, ArꢀHꢁ2),
7.26–7.28 (m, 2H, ArꢀHꢁ2), 3.59 (s, 4H,CH2 ꢁ2), 2.33–2.35 (m, 2H,
propyl-H), 1.25–1.28 (m, 2H, propyl-H), 0.77 ppm (t, J=7.20 Hz, 3H,
propyl-H); 13C NMR (600 MHz, [D6]DMSO): d=186.7, 135.6, 135.3,
134.3, 130.8, 128.7, 126.4, 124.9, 123.1, 54.1, 53.9, 20.4, 12.4 ppm;
ESI-MS [M+H]+ 454.5; Anal. calcd for C24H21F6NO: C 64.70, H 5.60,
F 26.06, N 3.64; found: C 64.57, H 4.89, F 25.99, N 4.55.
N-(4-trifluoromethylbenzyl)-3,5-bis-(2-(trifluoromethyl)benzylide-
ne)piperidin-4-one (b4): Yellow solid, yield 68.7%; mp: 121.2–
123.78C; H NMR (600 MHz, [D6]DMSO): d=8.06 (s, 2H, C=CHꢁ2),
7.57 (d, J=8.40 Hz, 2H, ArꢀHꢁ2), 7.42–7.45 (m, 6H, ArꢀH), 7.11–
7.26 (m, 4H, ArꢀH), 3.62 (s, 2H, PhꢀCH2), 3.58 ppm (s, 4H, CH2 ꢁ2);
13C NMR (600 MHz, [D6]DMSO): d=186.4, 141.8, 136.6, 135.0, 125.2,
124.9, 124.7, 123.1, 53.5, 59.3 ppm; ESI-MS [M+H]+ 570.0; Anal.
calcd for C29H20F9NO: C 62.12, H 3.67, F 30.11, N 4.10; found: C
63.16, H 3.98, F 30.23, N 2.63.
1
N-Benzyl-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one
(a5): White solid, yield 90.4%; mp: 105.6–108.98C; 1H NMR
(600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.70 (d, J=
7.20 Hz, 2H, ArꢀHꢁ2), 7.40–7.44 (m, 6H, ArꢀH), 7.12–7.15 (m, 5H,
ArꢀH), 3.48 (s, 4H, CH2 ꢁ2), 3.60 ppm (s, 2H, PhꢀCH2); 13C NMR
(600 MHz, [D6]DMSO): d=186.9, 137.5, 135.7, 134.3, 126.4, 124.9,
123.1, 54.4, 53.8, 60.0 ppm; ESI-MS[M+H]+ 502.5; Anal. calcd for
C28H21F6NO: C 68.24, H 4.93, F 23.26, N 3.21; found: C 67.95, H 5.01,
F 23.22, N 3.82.
N-(4-bromobenzyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piper-
idin-4-one (b5): White solid, yield 58.4%; mp: 146.1–149.28C;
1H NMR (600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.47 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.41–7.45 (m, 4H, ArꢀH), 7.27–7.29 (m,
2H, ArꢀH), 7.10 (d, J=7.2 Hz, 2H, ArꢀH), 7.02 (d, J=7.2 Hz, 2H,
ArꢀH), 3.58 (s, 4H, CH2 ꢁ2), 3.48 ppm (s, 2H, PhꢀCH2); 13C NMR
(600 MHz, [D6]DMSO): d=186.1, 135.1, 134.7, 132.3, 129.6, 126.4,
124.9, 123.1, 53.4, 59.4 ppm; ESI-MS [M+H]+ 579.2; Anal. calcd for
C28H20F6NO: C 68.21, H 4.92, F 23.02, N 3.85; found: C 68.22, H 4.98,
F 23.22, N 3.58.
General procedure for the preparation of compounds b1–9: To
obtain some related N-R2-piperidone intermediates, a solution of
piperidone hydrochloride (1 mmol) in acetonitrile (10 mL) was
mixed with anhydrous K2CO3 (2 mmol). The mixture was stirred at
208C for 4 h. After completion of reaction (TLC), the products were
dried with acetonitrile, added water into the mixture and stirred
overnight, followed by extracting with EtOAc (50 mLꢁ3) to obtain
N-R2-piperidone intermediates. Subsequently, b1–b9 were obtained
with the same reaction of a1–a5, but the N-R1 piperidinone was
changed into above N-R2-piperidone intermediates to obtain b1–
b9 as pale-yellow powders.
N-(4-chlorobenzyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piper-
idin-4-one (b6): White solid, yield 66.4%; mp: 142.5–145.88C;
1H NMR (600 MHz, [D6]DMSO): d=8.10 (s, 2H, C=CHꢁ2), 7.47 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.41–7.45 (m, 6H, ArꢀH), 7.13 (d, J=
7.8 Hz, 2H, ArꢀH), 7.07 (d, J=7.8 Hz, 2H, ArꢀH), 3.58 (s, 4H, CH2 ꢁ
2), 3.48 ppm (s, 2H, PhꢀCH2); 13C NMR (600 MHz, [D6]DMSO): d=
186.8, 136.2, 135.9, 134.3, 123.1, 132.8, 126.4, 124.9, 53.4, 59.1 ppm;
ESI-MS [M+H]+ 536.1; Anal. calcd for C28H20ClF6NO: C 63.03, H
3.92, Cl 6.88, F 21.72, N 4.45; found: C 62.98, H 4.05, Cl 6.92, F
22.34, N 3.71.
N-(3,5-dimethoxybenzyl)-3,5-bis-(2-(trifluoromethyl)benzylide-
ne)piperidin-4-one (b1): Yellow solid, yield 65.8%; mp: 108.7–
110.18C; 1H NMR (600 MHz, [D6]DMSO): d=8.06 (s, 2H, C=CHꢁ2),
7.72 (d, J=7.80 Hz, 2H, ArꢀHꢁ2), 7.45–7.47 (m, 2H, ArꢀHꢁ2),
7.40–7.42 (m, 2H, ArꢀHꢁ2), 7.16–7.18 (m, 2H, ArꢀHꢁ2), 6.33 (d,
J=2.4 Hz, 2H, ArꢀH), 6.25 (s, 1H, ArꢀH), 3.75 (s, 6H, ꢀOCH3 ꢁ2),
3.60 (s, 2H, PhꢀCH2), 3.489 ppm (s, 4H, CH2 ꢁ2); 13C NMR (600 MHz,
[D6]DMSO): d=186.8, 161.9, 160.5, 141.4, 140.1, 136.0, 133.2, 124.9,
123.1, 53.6, 59.9, 55.4 ppm; ESI-MS [M+H]+ 562.7; Anal. calcd for
C30H25F6NO3: C 67.92, H 6.02, F 23.02, N 3.04; found: C 67.59, H
5.90, F 24.00, N 2.51.
N-(4-fluorobenzyl)-3,5-bis(2-(trifluoromethyl)benzylidene)piperi-
din-4-one (b7): Yellow solid, yield 69.2%; mp: 154.2–156.78C;
1H NMR (600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.47 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.41–7.44 (m, 6H, ArꢀH), 7.10 (d, J=
7.80 Hz, 2H, ArꢀH), 7.02 (d, J=7.80 Hz, 2H, ArꢀH), 3.58 (s, 4H,
CH2 ꢁ2), 3.48 ppm (s, 2H, PhꢀCH2); 13C NMR (600 MHz, [D6]DMSO):
d=186.2, 150.4, 139.2, 135.2, 135.1, 126.1, 123.8, 122.9, 53.3,
58.6 ppm; ESI-MS [M+H]+ 520.4; Anal. calcd for C28H20F7NO: C
64.74, H 4.14, F 26.03, N 5.09; found: C 65.54, H 4.05, F 27.92, N
2.49.
N-(3-nitrobenzyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piperi-
din-4-one (b8): Yellow solid, yield 68.7%; mp: 116.2–118.38C;
1H NMR (600 MHz, [D6]DMSO): d=8.08 (s, 2H, C=CHꢁ2), 7.99 (d,
J=7.20 Hz, 2H, ArꢀHꢁ2), 7.70 (d, J=7.20 Hz, 2H, ArꢀHꢁ2), 7.41–
7.45 (m, 6H, ArꢀH), 7.12–7.14 (m, 2H, ArꢀH), 3.62 (s, 4H, CH2 ꢁ2),
3.62 ppm (s, 2H, PhꢀCH2); 13C NMR (600 MHz, [D6]DMSO): d=
186.5, 148.3, 140.0, 135.1, 132.3, 126.4, 123.5, 122.5, 53.4, 58.6 ppm;
ESI-MS [M+H]+ 547.4; Anal. calcd for C28H20F6N2O3: C 65.54, H
3.99, F 22.86, N 7.61; found: C 65.90, H 4.05, F 23.73, N 6.32.
N-(3-fluorobenzyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piperi-
din-4-one (b2): Yellow solid, yield 70.2%; mp: 140.3–142.58C;
1H NMR (600 MHz, [D6]DMSO): d=8.10 (s, 2H, C=CHꢁ2), 7.71 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.41–7.43 (m, 3H, ArꢀH), 7.14–7.15 (m,
3H, ArꢀH), 7.11–7.13 (m, 2H, ArꢀH), 6.97–6.98 (m, 2H, ArꢀH),
3.53 ppm (s, 6H, NCH2 ꢁ3); 13C NMR (600 MHz, [D6]DMSO): d=
186.0, 161.2, 150.1, 145.1, 140.1, 135.1, 126.4, 122.5, 122.2, 58.1,
53.2 ppm; ESI-MS [M+H]+ 536.1; Anal. calcd for C28H20F7NO: C
65.04, H 3.74, F 26.09, N 5.13; found: C 65.71, H 4.01, F 26.11, N
4.17.
N-(2-nitrobenzyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piperi-
din-4-one (b9): Yellow solid, yield 61.3%; mp: 104.2–107.38C;
1H NMR (600 MHz, [D6]DMSO): d=8.08 (s, 2H, C=CHꢁ2), 7.70–7.80
(m, 2H, ArꢀH), 7.37–7.48 (m, 8H, ArꢀH), 7.15–7.17 (m, 2H, ArꢀH),
3.90 (s, 2H, PhꢀCH2), 3.58 ppm (s, 4H, CH2 ꢁ2); 13C NMR (600 MHz,
[D6]DMSO): d=186.5, 149.3, 134.9, 134.8, 126.3, 124.7, 123.3, 58.9,
53.6 ppm; ESI-MS [M+H]+ 547.4; Anal. calcd for C28H20F6N2O3: C
65.23, H 3.89, F 24.74, N 6.14; found: C 66.02, H 4.01, F 24.99, N
4.98.
N-(3-chlorobenzyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piper-
idin-4-one (b3): Yellow solid, yield 57.2%; mp: 142.1–144.78C;
1H NMR (600 MHz, [D6]DMSO): d=8.07 (s, 2H, C=CHꢁ2), 7.67 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.40–7.42 (m, 3H, ArꢀH), 7.17–7.19 (m,
3H, ArꢀH), 6.90–6.92 (m, 2H, ArꢀH), 6.84–6.86 (m, 2H, ArꢀH),
3.48 ppm (s, 6H, NCH2 ꢁ3); 13C NMR (600 MHz, [D6]DMSO): d=
186.1, 161.3, 160.2, 140.2, 138.2, 135.1, 134.7, 132.6, 128.6, 126.4,
53.4, 59.8 ppm; ESI-MS [M+H]+ 536.1; Anal. calcd for
C28H20ClF6NO: C 63.07, H 3.98, Cl 7.03, F 23.05, N 2.87; found: C 63.
57, H 3.89, Cl 7.44, F 22.39, N 2.71.
General procedure for the preparation of compounds c1–10: A
solution of a1 (0.3 mmol) in anhydrous dichloromethane (10 mL)
was mixed with DMAP (3 mg) and trimethylamine (5 mL). The mix-
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ture was stirred at 08C for 0.5 h, and added dropwise to a variety
of substituted benzoyl chlorides (0.6 mmol) dissolved in dichloro-
methane. After completion of reaction (TLC), the solution was
dried in vacuo and the crude product was successively washed
with 10% HCl, saturated Na2CO3, saturated NaCl, and extracted
with EtOAc (50 mLꢁ3), followed by concentration in vacuo to give
crude products. The residue was purified by column chromatogra-
phy on silica gel [petroleum ether/EtOAc] to obtain c1–c10 as
yellow powders.
[D6]DMSO): d=186.1, 170.8, 139.4, 138.4, 134.2, 134.2, 126.4, 124.8,
123.0, 39.6, 21.2 ppm; ESI-MS [M+H]+ 530.3; Anal. calcd for
C29H21F6NO2: C 65.08, H 5.50, F 23.53, N 5.89; found: C 66.73, H
4.90, F 22.50, N 5.87.
N-(4-fluorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c7): Yellow solid, yield 49.8%; mp: 121.3–123.78C;
1H NMR (600 MHz, [D6]DMSO): d=8.10 (s, 2H, C=CHꢁ2), 7.76 (d,
J=8.40 Hz, 2H, ArꢀH), 7.16–7.20 (m, 6H, ArꢀH), 6.74–6.76 (m, 4H,
ArꢀH), 3.60 ppm (s, 4H, NCH2 ꢁ2); 13C NMR (600 MHz, [D6]DMSO):
d=185.8, 172.1, 136.0, 134.6, 134.5, 134.3, 124.2, 122.9, 122.0,
49.1 ppm; ESI-MS [M+H]+ 534.3; Anal. calcd for C28H18F7NO2: C
64.34, H 4.10, F 24.90, N 6.66; found: C 65.00, H 3.98, F 25.03, N
5.99.
N-(3-chlorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c1): Yellow solid, yield 61.2%; mp: 156.2–162.38C;
1H NMR (600 MHz, [D6]DMSO): d=8.06 (s, 2H, C=CHꢁ2), 7.68 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.46–7.55 (m, 6H, ArꢀH), 7.00–7.18 (m,
2H, ArꢀH), 6.94–6.96 (m, 2H, ArꢀH), 4.40 ppm (s, 4H, NCH2 ꢁ2);
13C NMR (600 MHz, [D6]DMSO): d=185.7, 169.0, 136.0, 134.6, 130.1,
126.3, 124.9, 123.0, 121.2, 39.6 ppm; ESI-MS [M+H]+ 550.0; Anal.
calcd for C28H18ClF6NO2: C 63.16, H 3.60, Cl 6.85, F 21.73, N 4.66;
found: C 62.96, H 3.87, Cl 6.90, F 21.93, N 4.34.
N-(4-bromobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c8): Yellow solid, yield 43.0%; mp: 126.8–128.48C;
1H NMR (600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.47 (d,
J=7.80 Hz, 2H,ArꢀH), 7.41–7.45 (m, 4H, ArꢀH), 7.10–7.27 (m, 4H,
ArꢀH), 7.30–7.32 (m, 2H, ArꢀH), 3.57 ppm (s, 4H, NCH2 ꢁ2);
13C NMR (600 MHz, [D6]DMSO): d=186.1, 170.8, 138.4, 134.2, 131.9,
126.7, 124.8, 123.9, 123.0, 47.3, 21.2 ppm; ESI-MS [M+H]+ 594.0;
Anal. calcd for C28H18BrF6NO2: C 58.18, H 3.95, Br 13.94, F 20.18, N
3.75; found: C 57.38, H 4.05, Br 14.44, F 19.98, N 4.15.
N-(2,3-chlorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c2): Yellow solid, yield 70.5%; mp: 151.1–155.98C;
1H NMR (600 MHz, [D6]DMSO): d=8.04 (s, 2H, C=CHꢁ2), 7.63 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.35–7.50 (m, 3H, ArꢀH), 6.87–7.15 (m,
6H, ArꢀH), 4.19 ppm (s, 4H, NCH2 ꢁ2); 13C NMR (600 MHz,
[D6]DMSO): d=185.5, 164.0, 136.5, 135.1, 134.7, 134.1, 124.6, 124.6,
122.8, 50.9 ppm; ESI-MS [M+H]+ 584.5; Anal. calcd for
C28H17Cl2F6NO2: C 58.55, H 3.03, Cl 12.93, F 20.51, N 4.98; found: C
58.81, H 2.97, Cl 13.13, F 19.99, N 5.10.
N-(2-methylbenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c9): Yellow solid, yield 51.0%; mp: 121.4–124.08C;
1H NMR (600 MHz, [D6]DMSO): d=8.08 (s, 2H, C=CHꢁ2), 7.73–7.75
(m, 4H,ArꢀH), 7.44–7.46 (m, 3H, ArꢀH), 6.93–7.03 (m, 5H, ArꢀH),
3.87 (s, 4H, NCH2 ꢁ2), 2.17 ppm (s, 3H, ꢀCH3); 13C NMR (600 MHz,
[D6]DMSO): d=186.9, 169.0, 137.2, 134.8, 134.1, 133.7, 124.1, 123.8,
122.1, 50.1 ppm; ESI-MS [M+H]+ 530.5; Anal. calcd for
C29H21F6NO2: C 67.74, H 5.40, F 22.43, N 4.43; found: C 65.09, H
4.88, F 24.53, N 5.50.
N-(4-methylbenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c3): Yellow solid, yield 60.5%; mp: 125.6–128.38C;
1H NMR (600 MHz, [D6]DMSO): d=8.08 (s, 2H, C=CHꢁ2), 7.74–8.00
(m, 4H, ArꢀH), 7.42–7.45 (m, 2H, ArꢀH), 7.07–7.23 (m, 4H, ArꢀH),
6.85–6.87 (m, 2H, ArꢀH), 3.10 (s, 4H, NCH2 ꢁ2), 2.31 ppm (s, 3H, ꢀ
CH3); 13C NMR (600 MHz, [D6]DMSO): d=186.2, 170.8, 143.0, 140.4,
139.4, 134.9, 126.4, 124.9, 123.0, 39.6, 21.8 ppm; ESI-MS [M+H]+
530.5; Anal. calcd for C29H21F6NO2: C 65.99, H 4.90, F 22.53, N 6.58;
found: C 66.78, H 4.89, F 23.03, N 5.30.
N-(2-chlorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c10): Yellow solid, Yield 49.6%; mp: 151.2–153.98C;
1H NMR (600 MHz, [D6]DMSO): d=8.06 (s, 2H, C=CHꢁ2), 7.68 (d,
J=7.8 Hz, 2H, ArꢀH), 7.44–7.55 (m, 5H, ArꢀH), 7.24–7.26 (m, 1H,
ArꢀH), 7.17–7.19 (m, 1H, ArꢀH), 6.96–7.09 (m, 3H, ArꢀH), 4.41 ppm
(s, 4H, NCH2 ꢁ2); 13C NMR (600 MHz, [D6]DMSO): d=186.9, 169.0,
137.2, 134.8, 134.1, 133.7, 124.1, 123.8, 122.1, 50.1 ppm; ESI-MS
[M+H]+: 550.0; Anal. calcd for C28H18ClF6NO2: C 63.16, H 3.50, Cl
7.45, F 22.73, N 3.16, found: C 62.98, H 3.77, Cl 7.80, F 22.90, N
2.55.
N-(4-chlorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c4): Yellow solid, yield 65.4%; mp: 155.1–157.28C;
1H NMR (600 MHz, [D6]DMSO): d=8.09 (s, 2H, C=CHꢁ2), 7.77 (d,
J=7.80 Hz, 2H, ArꢀHꢁ2), 7.10–7.20 (m, 6H, ArꢀH), 7.02–7.05 (m,
4H, ArꢀH), 3.58 ppm (s, 4H, NCH2 ꢁ2); 13C NMR (600 MHz,
[D6]DMSO): d=185.8, 172.1, 134.3, 132.6, 132.0, 131.5, 124.2, 122.9,
122.0, 49.1 ppm; ESI-MS [M+H]+ 550.0; Anal. calcd for
C28H18ClF6NO2: C 62.19, H 3.89, Cl 6.45, F 23.09, N 4.38; found: C
61.88, H 4.30, Cl 6.05, F 22.73, N 5.04.
Cell viability assay: Cell viability was assessed using the Cell
Counting Kit-8 (CCK-8) assay kit. Mouse bone marrow cells (Chem-
Bio Research Center, Wenzhou, China) at 1.0ꢁ104 cells per well
were cultured in 96-well plates in RPMI 1640 medium at 378C for
12 h. The cells were treated in triplicate with vehicle alone or 20 or
10 mm each compound for 24 h. During the last 4-h incubation,
cells were exposed to 10 mL CCK-8 reagent. The resulting yellow
formazan color was assayed at l 570 nm using a microplate spec-
trophotometer. The cells in medium alone served as negative con-
trol, while cells that had been treated with the same concentra-
tions of curcumin served as positive control. The percentages of
viable cells relative to the negative control cells were determined.
N-(4-methoxybenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c5): Yellow solid, yield 61.15%; mp: 123.0–125.38C;
1H NMR (600 MHz, [D6]DMSO): d=8.09 (s, 2H, C=CHꢁ2), 7.75 (d,
J=7.20 Hz, 2H, ArꢀHꢁ2), 7.45–7.47 (m, 2H, ArꢀH), 7.14–7.16 (m,
4H, ArꢀH), 6.98 (d, J=8.4 Hz, 2H, ArꢀH), 6.55 (d, J=8.4 Hz, 2H,
ArꢀH), 3.89 (s, 4H, NCH2 ꢁ2), 3.73 ppm (s, 3H,ꢀOCH3); 13C NMR
(600 MHz, [D6]DMSO): d=186.2, 170.5, 161.2, 135.1, 134.0, 130.9,
124.9, 123.1, 121.4, 55.4, 55.7 ppm; ESI-MS [M+H]+ 546.3; Anal.
calcd for C29H21F6NO3: C 65.45, H 4.83, F 21.93, N 7.79; found: C
64.89, H 4.88, F 22.90, N 7.33.
Measurement of TNF-a, IL-1b, PGE2, NO, and IL-6. RAW 264.7
cells (ChemBio Research Center) were stimulated in triplicate with
100 ngmLꢀ1 of LPS in the presence or absence of individual com-
pounds at 10 or 20 mm for varying periods. The levels of TNF-a, IL-
1b, PGE2, and IL-6 in the supernatants of cultured cells were deter-
mined by ELISA using specific kits (eBio-Science, San Diego, CA,
USA) according to the manufacturer’s instructions. The levels of
N-(3-methylbenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)pi-
peridin-4-one (c6): Yellow solid, yield 60.5%; mp: 125.6–128.38C;
1H NMR (600 MHz, [D6]DMSO): d=8.06 (s, 2H, C=CHꢁ2), 7.69–7.72
(m, 2H, ArꢀH), 7.44–7.55 (m, 5H, ArꢀH), 6.93–7.02 (m, 5H, ArꢀH),
3.87 (s, 4H, NCH2 ꢁ2), 2.17 ppm (s, 3H,ꢀCH3); 13C NMR (600 MHz,
&
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ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Papers
NO were determined by Griess reaction assay using the Griess Re-
agent System (Promega, USA). The total amount of an inflammato-
ry factor in the supernatants was normalized to the total amounts
of proteins in the viable cell pellets.
work was supported by a grant from the Science and Technology
Project of Wenzhou (Y20150110).
Keywords: anti-inflammatory activity · curcumin analogues ·
piperidones · synthesis
Carrageenan-induced rat model of paw edema. A rat model of
paw edema was induced by carrageenan.[37–39] Briefly, male Spra-
gue–Dawley rats at 150–200 g body weights were obtained from
ChemBio Research Center, and randomized into groups. The rats
were fed with vehicle by gavage and subcutaneously with 0.1 mL
of 1% carrageenan freshly prepared into their plantar side of the
side paw as the model group. Other rats were fed with 2.5 mgkgꢀ1
c6 or c10 as the experimental groups or with 2.5 mgkgꢀ1 celecoxib
or indomethacin as the positive controls. After drug treatment,
these rats were injected subcutaneously with 1% carrageenan. The
paw edema in individual rats was measured by volume displace-
ment method (toe volume meter YLS-7A) before and 0.5, 1, 2, 3, 4,
5, 6, 7 and 8 h post-induction. The percentage of inhibition was
calculated by Equation (1):
[1] B. B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Adv. Exp. Med. Biol.
2007, 595, 1–75.
[2] R. C. Srimal, B. N. Dhawan, J. Pharm. Pharmacol. 1973, 25, 447–452.
[3] O. Naksuriya, S. Okonogia, R. M. Schiffelersc, W. E. Hennink, Biomaterials
2014, 35, 3365–83.
[4] Y. Gu, H.-M. Lee, N. Napolitano, M. Clemens, Y. Zhang, T. Sorsa, Y. Zhang,
F. Johnson, L. M. Golub, Mediators Inflammation 2013, 2013, 329740.
[5] C. Zeng, P. Zhong, Y. Zhao, K. Kanchana, Y. Zhang, Z. A. Khan, S. Chakra-
barti, L. Wu, J. Wang, G. Liang, J. Mol. Cell. Cardiol. 2015, 79, 1–12.
[6] S. Shishodia, M. M. Chaturvedi, B. B. Aggarwal, Curr. Probl. Cancer 2007,
31, 243–305.
[7] B. B. Aggarwal, B. Sung, Trends Pharmacol. Sci. 2009, 30, 85–94.
[8] J.-K. Lin, S.-Y. Lin-Shiau, Proc. Natl. Sci. Counc. Repub. China Part B Life
Sci. 2001, 25, 59–66.
[9] P. Anand, A. B. Kunnumakkara, R. A. Newman, B. B. Aggarwal, Mol.
Pharm. 2007, 4, 807–818.
[10] Y.-J. Wang, M.-H. Pan, A.-L. Cheng, L.-I. Lin, Y.-S. Ho, C.-Y. Hsieh, J.-K. Lin,
J. Pharm. Biomed. Anal. 1997, 15, 1867–1876.
[11] O. Firuzi, R. Miri, M. Tavakkoli, L. Saso, Curr. Med. Chem. 2011, 18, 3871–
3888.
[12] V. Sundararajan, R. J. Rosengren, K. Greish in Curcumin: Synthesis, Emerg-
ing Role in Pain Management and Health Implications (Ed.: D. L. Pouli-
quen), Nova Science Publishers, New York, 2014, Chapter 14, 347–378.
[13] J. Z. Wu, Y. Zhang, Y. Cai, J. Wang, B. Weng, Q. Tang, X. Chen, Z. Pan, G.
Liang, S. Yang, Bioorg. Med. Chem. 2013, 21, 3058–3065.
[14] J. Wu, J. Li, Y. Cai, Y. Pan, F. Ye, Y. Zhang, Y. Zhao, S. Yang, X. Li, G. Liang,
J. Med. Chem. 2011, 54, 8110–8123.
[15] P. Yu, L. Dong, Y. Zhang, W. Chen, S. Xu, Z. Wang, X. Shan, J. Zhou, Z.
Liu, G. Liang, Eur. J. Med. Chem. 2015, 94, 436–446.
[16] J. Feng, B. Xiao, W. Chen, T. Ding, L. Chen, P. Yu, F. Xu, H. Zhang, Z. Liu,
G. Liang, Chem. Biol. Drug Des. 2015, 86, 753–763.
[17] Y. Wang, Y. Wang, M. Luo, H. Wu, L. Kong, Y. Xin, W. Cui, Y. Zhao, J.
Wang, G. Liang, L. Miao, L. Cai, Biochim. Biophys. Acta Mol. Basis Dis.
2015, 1852, 34–46.
[18] Y. Pan, Y. Wang, L. Cai, Y. Cai, J. Hu, C. Yu, J. Li, Z. Feng, S. Yang, X. Li, G.
Liang, Br. J. Pharmacol. 2012, 166, 1169–1182.
ꢀ
ꢁ
ðValueexperimentalÞꢀðValuebasal
Þ
Inhibition ½%ꢂ ¼ 1ꢀ
ꢃ 100
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ðValuemodelÞꢀðValuebasal
Þ
In addition, some rats were intravenously injected with vehicle
alone or with 2.5 or 10 mgkgꢀ1 c6 or c10 and then injected subcu-
taneously with 1% carrageenan. The paw edema in individual rats
was measured before drug injection and every 10 min up to
60 min post-induction.
In vivo pharmacokinetics studies. The bioavailability of com-
pound c6 in rats was analyzed by HPLC.[40] Briefly, c6 was suspend-
ed in 0.5% CMC-Na to achieve a concentration of 0.75% for oral
gavage and dissolved in 10% DMSO-NS as a final concentration of
10 mgmLꢀ1 for intravenous injection. Male Sprague–Dawley rats at
150–200 g body weight were food-fasted overnight and adminis-
trated with 0.5 mL of 0.75% c6 in CMC-Na or injected intravenous-
ly with 0.5 mL of c6 solution (n=3 per group). Their tail vein blood
samples were collected before and 5, 15, 30, 60, 90, 120, 240, 360,
480 and 1440 min (0.3 mL per time point) after drug administra-
tion. The contents of c6 in blood were determined by HPLC.
Statistical methods. Student’s t test was used to determine the
differences between sets of data. Statistics were analyzed using
GraphPad Prism 5 (GraphPad, San Diego, CA, USA); p<0.05 was
considered significant.
[19] G. Liang, S. Yang, H. Zhou, L. Shao, K. Huang, J. Xiao, Z. Huang, X. Li,
Eur. J. Med. Chem. 2009, 44, 915–919.
[20] W. Li, M. Wu, L. Tang, Y. Pan, Z. Liu, C. Zeng, J. Wang, T. Wei, G. Liang,
Toxicol. Appl. Pharmacol. 2015, 282, 175–183.
[21] Q. Fang, L. Zhao, Y. Wang, Y. Zhang, Z. Li, Y. Pan, K. Kanchana, J. Wang,
C. Tong, D. Li, G. Liang, Toxicol. Appl. Pharmacol. 2015, 282, 129–138.
[22] L. Dong, S. Zheng, Y. Zhang, X. Jiang, J. Wu, X. Zhang, X. Shan, D. Liang,
S. Ying, J. Feng, G. Liang, MedChemComm 2015, 6, 1544–1553.
[23] P. Zou, J. Zhang, Y. Xia, K. Kanchana, G. Guo, W. Chen, Y. Huang, Z.
Wang, S. Yang, G. Liang, Oncotarget 2015, 6, 5860.
[24] Y. Pan, Y. Wang, Y. Zhao, K. Peng, W. Li, Y. Wang, J. Zhang, S. Zhou, Q.
Liu, X. Li, L. Cai, G. Liang, Diabetes 2014, 63, 3497–3511.
[25] Y. Pan, J. Cell. Mol. Med. 2014, 18, 231–241.
Author Contributions
The manuscript was written through the contributions of all listed
authors. All authors have approved the final version of this manu-
script.
[26] Y. Liu, Y. Wang, X. Miao, S. Zhou, Y. Tan, G. Liang, Y. Zheng, Q. Liu, J. Sun,
L. Cai, J. Cell. Mol. Med. 2014, 18, 1203–1212.
Notes
[27] P. Libby, Nutr. Rev. 2007, 65, S140–S146.
[28] R. Jin, L. Liu, S. Zhang, A. Nanda, G. Li, J. Cardiovasc. Transl. Res. 2013, 6,
834–851.
The authors declare no competing financial interest.
[29] D. N. Mori, D. Kreisel, J. N. Fullerton, D. W. Gilroy, D. R. Goldstein, Immu-
nol. Rev. 2014, 258, 132–144.
Acknowledgements
[30] S. Yung, K. F. Cheung, Q. Zhang, T. M. Chan, Clin. Dev. Immunol. 2013,
2013, 317682.
The authors thank the ChemBio Research Center of Wenzhou
Medical University and Keygen Biotech for their help and support.
We also thank the Analysis and Test Center of Wenzhou Medical
University for IR, NMR, and ESI-MS analyses. This manuscript has
been edited and proofread by Medjaden Bioscience Ltd. This
[31] C. Kizil, N. Kyritsis, M. Brand, EMBO Rep. 2015, 16, 416–426.
[32] S. I. Grivennikov, F. R. Greten, M. Karin, Cell 2010, 140, 883–899.
[33] L. Li, M. Chen, F.-C. Jiang, Bioorg. Med. Chem. 2016, 24, 1853–1865.
[34] A. Anthwal, K. Singh, M. S. M. Rawat, A. K. Tyagi, A. Haque, I. Ali, D. S.
Rawat, Anti-Cancer Agents Med. Chem. 2016, 16, 841–851.
ChemMedChem 2017, 12, 1 – 11
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Full Papers
[35] A. Anthwal, K. Singh, M. S. M. Rawat, A. K. Tyagi, B. B. Aggarwal, D. S.
Rawat, RSC Adv. 2014, 4, 28756–28764.
[40] X. Xu, H. Jiang, Y. Yu, G. Hu, R. Ge, Y. Cai, Lat. Am. J. Pharm. 2015, 34,
585–589.
[36] L. K. Zhang, X. Wang, Chin. Pharmacol. Bull. 2011, 27, 1458–1462.
[37] C. A. Winter, E. A. Risley, G. W. Nuss, Exp. Biol. Med. 1962, 111, 544–547.
[38] C. J. Morris, Methods Mol. Biol. 2003, 225, 115–121.
Manuscript received: December 3, 2016
Revised: December 25, 2016
[39] V. Hajhashemi, M. Minaiyan, H. R. Banafshe, A. Mesdaghinia, A. Abed,
Iran. J. Basic Med. Sci. 2015, 18, 654–658.
Accepted Article published: && &&, 0000
Final Article published: && &&, 0000
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FULL PAPERS
Taking the heat off: The 24 curcumin
analogues in this study were found to
have no toxicity toward mouse bone
marrow cells, and some of them
Z. Xie, Z. Zhang, S. Yu, D. Cheng,
H. Zhang, C. Han, H. Lv, F. Ye*
&& – &&
showed potent anti-inflammatory activi-
ty by inhibiting LPS-stimulated TNFa, IL-
6, IL-1b, PGE2, and NO production in rat
macrophages. Pharmacokinetic studies
indicated that compound c6 has better
bioavailability than curcumin in vivo.
Both c6 and c10 may be valuable leads
for the development of new anti-inflam-
matory agents.
Synthesis and Evaluation of Anti-
inflammatory N-Substituted 3,5-Bis(2-
(trifluoromethyl)benzylidene)piperi-
din-4-ones
ChemMedChem 2017, 12, 1 – 11
www.chemmedchem.org
11
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ