Diarylpentadienone derivatives (curcumin analogues): Synthesis and anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmcl.2017.02.056

A series of new (2E,4E)-1-(substitutedphenyl)-5-(substitutedphenyl)penta-2,4-dien-1-one derivatives were designed and synthesized. Compounds 3i, 3k were determined by X-ray. All of the compounds have been screened for their anti-inflammatory activity characterized by evaluating their inhibition against LPS-induced IL-6 and TNF-α release in cell RAW 264.7 stimulated with LPS. Compound 3i showed the highest anti-inflammatory activity on decreasing IL-6 and TNF-α. The further study showed that title compound 3i inhibited expression of proteins p-p65, iNOS, COX-2 LPS-induced. Immunofluorescence also revealed compound 3i could lightly reduce activation p65 in nuclei. These results indicate that compound 3i anti-inflammatory role may partly due to its inhibitory effect on the NF-κB signaling pathway.

Full text of DOI:10.1016/j.bmcl.2017.02.056

Accepted Manuscript
Diarylpentadienone derivatives (Curcumin analogues): synthesis and anti-in-
flammatory activity
Zhi Sen Wang, Liu Zen Cheng, Hai Pin Zhou, Xin Hua Liu, Fei Hu Chen
PII:
S0960-894X(17)30185-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.056
BMCL 24728
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
12 December 2016
13 February 2017
22 February 2017
Please cite this article as: Wang, Z.S., Cheng, L.Z., Zhou, H.P., Liu, X.H., Chen, F.H., Diarylpentadienone
derivatives (Curcumin analogues): synthesis and anti-inflammatory activity, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.056
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Diarylpentadienone derivatives (Curcumin analogues): synthesis and anti-
inflammatory activity
Zhi Sen Wang a, Liu Zen Cheng a, Hai Pin Zhou b, Xin Hua Liua,b,*, Fei Hu Chena, *
a School of Pharmacy, Anhui Medical University, Hefei, 230032, P. R. China
b School of Material Science Chemical Engineering, ChuZhou University, ChuZhou,
239000, PR China
Abstract: A series of new (2E,4E)-1-(substitutedphenyl)-5-(substitutedphenyl)penta-2,4
-dien-1-one derivatives were designed and synthesized. Compounds 3i, 3k were
determined by X-ray. All of the compounds have been screened for their anti-
inflammatory activity characterized by evaluating their inhibition against LPS-induced IL-6
and TNF-α release in cell RAW 264.7 stimulated with LPS. Compound 3i showed the
highest anti-inflammatory activity on decreasing IL-6 and TNF-α. The further study
showed that title compound 3i inhibited expression of proteins p-p65, iNOS, COX-2 LPS-
induced. Immunofluorescence also revealed compound 3i could lightly reduce activation
p65 in nuclei. These results indicate that compound 3i anti-inflammatory role may partly
due to its inhibitory effect on the NF-κB signaling pathway.
1

Keywords: Synthesis; diarylpentadienone; anti-inflammatory; IL-6
_________________
*Corresponding author.
Tel.: +86 551 65161115; fax: +86 551 65161115. E-mail: xhliuhx@163.com (Xin Hua Liu).
E-mail: fhchen@soho.com (Fei Hu Chen).
Curcumin, a naturally occurring phytochemical derived from the rootstalk of the turmeric
plant, has been extensively investigated for centuries in a variety of pharmaceutical
applications. 1-3 Previous studies reveal that curcumin, and its derivatives have various
pharmacological activities such as anti-inflammatory, 4-6 antioxidant 7-10 and anti-HIV. 11 It
has also been investigated for COX inhibitory activity using the bovine seminal vesicles,
microsomes and cytosol from homogenates of mouse. 12 However, due to its relatively
low bioavailability, the potential utility of curcumin is limited.
In order to develop the molecules with enhances properties and stability, number of
curcumin analogs/derivatives have been designed and synthesized. Kok Wai Lam group
has developed the synthesis of symmetrical curcumin analogues and evaluated their
effects on a variety of biological activities including anti-inflammatory and
immunomodulatory.13-17 Among them, a lot of results suggested that the unsymmetrical
form of demethoxycurcumin derivative might possess greater biological profile compared
to the symmetrical form of bisdemethoxcurcumin. 18-20 Motivated by the afore-
2

mentioned findings, we aim in this study was to find new, potent anti-inflammatory
agents with higher efficacy and better safety profiles, herein, we have synthesized a
series of unsymmetrical diarylpentadienone.
The synthesis of title compounds 3 (Scheme 1) started from substituted salicylaldehyde
and catalyzed by KOH at 5~10 oC was added distilled acetaldehyde, acidification with
acetic acid, recrystallized by the mixed solvent of ether and petroleum ether, the
compound 2 was obtained. Claisen-Schmidt condensation substituted phenyl ketone and
(E)-3-(3-substitued-phenyl) acrylaldehyde using mild catalyst piperidine, the ethanol used
as solvent, proved to be an efficient method for the synthesis of pentadienone title
compound 3. The general synthetic procedure process and spectral data of compound 3
can be found in the supporting information. 21
O
CHO
CHO
B
A
R2
R1
R1
3
2
1
Scheme 1. Synthesis of title compounds 3
Reagent and conditions: (A) KOH, C2H5OH, 5~10 oC, Acetaldehyde, 25 min. (B) substituted
phenyl ketone, piperidine, acetic acid, C2H5OH, reflux, 2 h.
R1= 2-OMe, R2=2-Cl (3a);
R1= H, R2=4-isopropylphenyl (3g);
3

R1= 2-OMe, R2=4-NO2 (3b);
R1= H, R2=4-NO2 (3h);
R1= 2-OMe, R2=3,4,5-3OMe (3c); R1= H , R2=2-OH (3i);
R1= 2-OMe, R2=2,4-2F (3d);
R1=H, R2=2-Cl (3e);
R1= H, R2=2,4-2F (3j);
R1=4-dimethylamino, R2=2-Cl (3k).
R1=H, R2=3,4,5-3OMe (3f);
The structures of compounds 3i and 3k were determined by X-ray crystallography. Crystal
data of 3i: Colorless crystals, yield, 84%; mp 176-177 oC; C17 H14 O2, Orthorhombic, space
group P2ac ; a = 10.8190(2), b = 7.9031(2), c = 30.1667(10) (Å); α =90, β = 90, γ =90 (°), V =
2578.36(12) nm3, T =290.92(10) K, Z =8, Dc = 1.289 g/cm3, F(000)= 1056, Reflections
collected / unique = 2351 / 1892, Data / restraints / parameters = 2351 / 0 / 173,
Goodness of fit on F2 =1.056, Fine, R1= 0.0276, wR(F2) = 0.0256.
Crystal data of 3k: Colorless crystals, yield, 79%; mp 146-148 oC; C19 H18 Cl N O, Monoclinic
, space group P21-1 ; a = 11.00289(17), b = 9.52147(16), c = 31.6909(5) (Å); α =90, β
=100.0337(15), γ =90 (°), V = 3269.28(9) nm3, T =290.92(10) K, Z =8, Dc = 1.267 g/cm3,
F(000)= 1312, Reflections collected / unique = 6061 / 5329, Data / restraints / parameters
= 6061 / 1 / 401, Goodness of fit on F2 =1.049, Fine, R1= 0.01792, wR(F2) = 0.0194. The
molecular structure of compounds 3i and 3k were shown in Figure 1. Crystallographic
data (excluding structure factors) for the structure had been deposited with the
Cambridge Crystallographic Data Center as supplementary publication No. CCDC-
1040096, 1040097. 23
4

3i
3k
Figure 1. ORTEP drawing of compounds 3i, 3k
The aqueous solubility of compounds 3a~3k has been determined by UV
spectrophotometer. 25 As presented in Table 1, compared to curcumin, compounds 3c, 3d
and 3i show better solubitily. The best soluble compound was (2E,4E)-5-(2-
methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)penta-2,4-dien-1-one(Compound 3c) with its
solubility 25.90 µg/mL, which was increased about 2-fold compared to curcumin.
5

However, compared with curcumin, the solubility of compounds 3g, 3j and 3k was poor.
26
Table 1 The solubility in of compounds 3a~3k
Compound
Solubility a
(µg/mL)
12.87
Compound
Solubility a
(µg/mL)
20.33
6.78
Curcumin
3f
3g
3h
3i
3a
3b
3c
3d
3e
9.45
13.94
13.22
23.50
7.86
25.90
24.80
3j
8.20
3k
7.55
a Values are the means of at least three independent determinations; errors are within
±20%.
To investigate the anti-inflammatory effects of compounds 3a~3k, pro-inflammatory
mediators IL-6 and TNF-α induced by LPS were analyzed in RAW264.7 cells by ELISA
(Figure 2). 27 Compared with curcumin, compounds 3c, 3f and 3i showed better anti-
inflammatory activity, among which, we found that compound 3i was the most potent
among the title compounds (inhibition rate up to ~50% compared to LPS) and was
selected for subsequent experiments. The preliminary SARs showed that substituent R2
6

possess large effect on the anti-inflammatory activity, Hydroxyl or methoxy group is
better than that of halogen substitution (Compound 3i, 3c, 3f).
Figure 2. Compounds 3a~3k (20 µM) inhibited LPS (1 µg/mL) induced pro-inflammatory
mediator IL-6 and TNF-α relative production in RAW264.7 cells. IL-6 and TNF-α levels in
7

culture supernatants were measured by ELISA. Curcumin was used as a positive control.
Data are representative of at least three independent experiments with similar results.
Data were presented as mean ±SD. ***p<0.001 compared to control group; # p<0.05, ##
p<0.01, ### p<0.001 compared to LPS group.
The NF-κB transcription factor family is a pleiotropic regulator of many cellular signaling
pathways, providing a mechanism for the cells in response to a wide variety of stimuli
linking to inflammation, which can activate the NF-κB signaling pathway. Subsequently,
NF-κB will be phosphorylated and the activated NF-κB will translocate from cytoplasm to
nucleus promoted transcription of various inflammatory marker genes, including those of
interleukins, cytokines, chemokines, iNOS, and COX-2. In order to understand whether
the effect of title compound 3i on LPS-induced NF-κB signaling, the relative protein levels
of p65, p-p65, iNOS and COX-2 were examined by Western blot. 28
The results showed that LPS significantly upregulated the expression of protein p-p65,
iNOS and COX-2 compared with normal group, however, the expression of above LPS-
induced proteins were inhibited when treated with compound 3i or bay 11-7082 (Figure
3).
8

Figure 3. Compound 3i inhibits activation of NF-κB induced by LPS in RAW 264.7 cells.
Cells were treated with compound 3i (10, 20, 30 µM) and Bay 11-7082 (0.3 µg/ml) for 12
h, and then stimulated by LPS (1ng/ml) for 3 h (p65, pp65) or for 12 h (iNOS, COX-2).
Subsequently, cell total protein was extracted from the cells. Westetn blot was used to
detect the expression of p65, p-p65, iNOS, COX-2. Bay 11-7082 was used for NF-κB
inhibitor. β-actin was chosen as an internal control. Data were presented as mean ± SD
obtained in three independent experiments. **p<0.01 vs normal group. ##p<0.01 vs LPS
group.
Furthermore, Immunofluorescence revealled LPS stimulated cells showed a clear and
positive labeling for activation p65 in nuclei, compared to bay 11-7082, compound 3i
could lightly reduce this effect (Figure 4). 29
NF-κB p65
DAPI
Merge
9

Figure 4. Immunofluorescence analysis
Representative immunofluorescence analysis performed on RAW264.7 using anti-
p65 (green) antibody after compound 3i treatment for 12 h and LPS treatment for 3 h.
Nuclei were counterstained with DAPI (blue). Merge contains the combined image of p65
immunostaining and DAPI staining (original magnification ×400).
In summary, based on finding new, efficient compounds with anti-inflammatory activity,
and to extend research on curcumin analogues, we designed some new (2E,4E)-1-
(substitutedphenyl)-5-(substitutedphenyl)penta-2,4-dien-1-one derivatives, followed by
10

chemical synthesis and biological evaluated for them. The results revealed that
compound 3i exhibited strong anti-inflammatory activity on decreasing IL-6. The
preliminary mechanism of the anti-inflammatory action indicated that title compound 3i
inhibited protein expression LPS-induced. Immunofluorescence revealed this compound
could lightly reduce activation p65 in nuclei. These results indicate that compound 3i anti-
inflammatory role may partly due to its inhibitory effect on the NF-κB signaling pathway
in LPS-induced RAW 264.7 cells.
Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (No.
21272008, 21572003), Science and Technological Fund of Anhui Province for Outstanding
Youth (1408085J04).
Supporting information
CCDC- 1040096, 1040097 contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge via the URL http://
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
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Z. A.; Sulaiman, M. R.; Lajis, N. H.; Israf, D. A. Eur. J. Pharmacol. 2010, 628, 247.
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12

15. Tham, C. L.; Lam, K. W.; Rajajendram, R.; Cheah, Y. K.; Sulaiman, M. R.; Lajis, N. H.;
Kim, M. K.; Israf, D. A. Eur. J. Pharmacol. 2011, 652, 136.
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21. General synthetic procedure process for compound 3: To a ethanol (20 ml) solution of
(E)-3-(3-substitued-phenyl) acrylaldehyde 2 (10 mmol) was added substituted phenyl
ketone (11 mmol), piperidine (10 mmol) and 5 dot acetic acid, the reaction mixture was
refluxed for 2 h. The mixture was cooled and collected by filtration and the crude residue
was purified by chromatography on SiO2 (petroleum ether / ethyl acetate, v:v= 1:3) to
give title compounds 3 (Scheme 1) as colorless solids. 22
3a: (2E,4E)-1-(2-chlorophenyl)-5-(2-methoxyphenyl)penta-2,4-dien-1-one, colorless
crystals, yield, 75%; mp 151-153 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.49 (dd, J = 7.7,
1.5 Hz, 1H), 7.39 (m, 3H), 7.31 (ddd, J=11.0, 8.6, 1.5 Hz, 1H), 7.27–7.24 (m, 1H), 7.24–7.17
(m, 1H), 7.01 (dd, J = 15.6, 11.1 Hz, 1H), 6.95–6.90 (m, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.77
(dd, J = 16.0, 7.8 Hz, 1H), 6.60 (d, J = 15.3 Hz, 1H), 3.82 (s, 3H). Anal. calcd for C18H15ClO2:
C, 72.36; H, 5.06%. Found: C, 72.61; H, 4.73%.
13

3b: (2E,4E)-5-(2-methoxyphenyl)-1-(4-nitrophenyl)penta-2,4-dien-1-one, colorless crystals,
yield, 80%; mp 161-162 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.36–8.31 (m, 2H), 8.12 –
8.07 (m, 2H), 7.70–7.61 (m, 1H), 7.55 (dd, J = 7.7, 1.6 Hz, 1H), 7.43 (d, J = 15.6 Hz, 1H),
7.35–7.32 (m, 1H), 7.11 (dd, J = 15.5, 11.2 Hz, 1H), 6.99 (dd, J = 18.3, 11.1 Hz, 2H), 6.92 (d,
J = 8.3 Hz, 1H), 3.91 (s, 3H). Anal. calcd for C18H15NO4: C, 69.89; H, 4.89; N, 4.53%. Found:
C, 70.22; H, 4.65; N, 4.17%.
3c: (2E,4E)-5-(2-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)penta-2,4-dien-1-one,
colorless crystals, yield, 76%; mp 150-152 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.59 (dd,
J = 7.6, 1.6 Hz, 1H), 7.24 (m, 1H), 7.04 (dd, J = 15.5, 11.1 Hz, 1H), 6.98 (dd, J = 18.2, 11.0
Hz, 2H), 6.89 (d, J = 8.1 Hz, 1H), 6.68 (m, 3H), 3.88 (s, 12H). Anal. calcd for C21H22O5: C,
71.17; H, 6.26 %. Found: C, 70.89; H, 6.55%.
3d: (2E,4E)-5-(2-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)penta-2,4-dien-1-one,
colorless crystals, yield, 70%; mp 147-148 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.82 (d,
J=8.1 Hz, 1H), 7.70 (dd, J = 7.6, 1.6 Hz, 1H), 7.20 (m, 1H), 7.02 (dd, J = 15.5, 11.1 Hz, 1H),
6.94 (dd, J = 18.2, 11.1 Hz, 1H), 6.83 (m, 3H), 6.70 (m, 3H), 3.92 (s, 3H). Anal. calcd for
C18H14F2O2: C, 71.99; H, 4.70 %. Found: C, 72.35; H, 4.99%.
3e: (2E,4E)-1-(2-chlorophenyl)-5-phenylpenta-2,4-dien-1-one, colorless crystals, yield,
77%; mp 156-158 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.77 (dd, J = 7.7, 1.5 Hz, 1H), 7.64
(d, J=8.1 Hz, 1H), 7.52 (d, J=8.1 Hz, 1H), 7.41 (m, 2H), 7.28 (m, 3H), 7.19 (m, 4H), 6.84 (d, J
= 8.2 Hz, 1H). Anal. calcd for C17H13ClO: C, 75.98; H, 4.88 %. Found: C, 76.39; H, 5.22%.
3f: (2E,4E)-5-phenyl-1-(3,4,5-trimethoxyphenyl)penta-2,4-dien-1-one, colorless crystals,
yield, 82%; mp 137-139 oC; 1H NMR (600 MHz, DMSO-d6) δ (ppm): 7.60 (d, J = 7.5 Hz, 2H),
7.54 (dd, J = 14.8, 9.2 Hz, 1H), 7.49 (d, J = 14.8 Hz, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.36 (t, J =
7.3 Hz, 1H), 7.34 (s, 2H), 7.24 (dd, J = 17.3, 12.4 Hz, 2H), 3.89 (s, 6H), 3.77 (s, 3H). Anal.
calcd for C20H20O4: C, 74.06; H, 6.21 %. Found: C, 74.00; H, 5.90%.
14

3g: (2E,4E)-1-(4-isopropylphenyl)-5-phenylpenta-2,4-dien-1-one, colorless crystals, yield,
74%; mp 170-172 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.89 (d, J = 8.5 Hz, 2H), 7.71 (dd,
J = 7.8, 1.6 Hz, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.20 (dd, J = 17.2, 12.3
Hz, 2H), 6.79-7.10 (m, 4H), 2.99 (m, 1H), 1.37 (s, 6H). Anal. calcd for C20H20O: C, 86.92; H,
7.29 %. Found: C, 87.17; H, 6.81%.
3h: (2E,4E)-1-(4-nitrophenyl)-5-phenylpenta-2,4-dien-1-one, colorless crystals, yield, 75%;
mp 180-182 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.34 (d, J = 8.6 Hz, 2H), 8.10 (d, J = 8.6
Hz, 2H), 7.64 (dd, J = 14.9, 10.1 Hz, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.42–7.33 (m, 3H), 7.11–
7.01 (m, 3H). Anal. calcd for C17H13NO3: C, 73.11; H, 4.69; N, 5.02%. Found: C, 73.45; H,
5.02; N, 4.79%.
3i: (2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-dien-1-one, colorless crystals, yield,
84%; mp 176-177 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 12.88 (s, 1H), 7.81 (dd, J = 7.7,
1.6 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.40 (m, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.17–7.25 (m,
4H), 6.89–7.03 (m, 4H). Anal. calcd for C17H14O2: C, 81.58; H, 5.64 %. Found: C, 81.44; H,
6.06%.
3j: (2E,4E)-1-(2,4-difluorophenyl)-5-phenylpenta-2,4-dien-1-one, colorless crystals, yield,
77%; mp 163-164 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.82-7.84 (m, 2H), 7.30 (d, J = 8.5
Hz, 2H), 7.22-7.24 (m, 3H), 7.16 (m, 1H), 6.88–6.97 (m, 3H), 6.69 (d, J = 15.4 Hz, 1H). Anal.
calcd for C17H12F2O: C, 75.55; H, 4.48 %. Found: C, 75.33; H, 4.55%.
3k: (2E,4E)-1-(2-chlorophenyl)-5-(4-(dimethylamino)phenyl)penta-2,4-dien-1-one,
colorless crystals, yield, 79%; mp 146-148 oC; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.77-
7.83 (m, 2H), 7.50 (d, J = 8.5 Hz, 1H), 7.38-7.46 (m, 2H), 7.18-7.24 (m, 3H), 6.95 (dd, J =
15.6, 11.2 Hz, 1H), 6.67 (d, J = 15.4 Hz, 1H), 6.48 (d, J = 8.5 Hz, 2H), 2.92 (s, 6H). Anal. calcd
for C19H18ClNO: C, 73.19; H, 5.82; N, 4.49%. Found: C, 73.56; H, 6.17; N, 4.11%.
22. 1H NMR spectrums were measured on a Mercury Plus-600 MHz spectrometer in CDCl3
solution with TMS as the internal standard. Elemental analyses were performed on a CHN
15

Rapid instrument, and were within ±0.4% of the theoretical values. Melting point was
measured on a XT4A Melting-Point apparatus with microscope and uncorrected. The
reagents employed were of analytical grade. The reactions were monitored by thin layer
chromatography (TLC) on Merck pre-coated silica GF254 plates.
23. Crystallographic studies: a colorless single crystal of title compounds 3i, 3k were
chosen for X-ray diffraction analysis performed on a BRUCKER SMART APEX-CCD
diffractometer equipped with a graphite monochromatic MoKa radiation (λ = 0.71073 A)
radiation at 290 K. The data set was corrected by SADABS program; the structure was
solved by direct methods with SHELXS-97 and refined by full-matrix least-squares method
on F2 with SHELXL-97. 24 The non-hydrogen atoms were refined anisotropically, and the
hydrogen atoms were added according to theoretical models. The structure was refined
by full-matrix least-squares method on F2 with SHELXT-97.
24. Sheldrick, G. M. SHELXTL-97, Program for Crystal Structure Solution and Refinement;
University of Göttingen: Göttingen, Germany, 1997.
25. Kim, M. K.; Park, K. S.; Yeo, W. S.; Bioorg. Med. Chem. 2009, 17, 1164-1171.
26. The solubility of compounds 3a~3k was determined by known method. Under UV
radiation, curcumin was monitored by UV at the wavelength of maximum absorbance
(418 nm) from the whole spectrum. Each tested compound (600 µg) was dissolved in 10
mL CH3OH. The solutions of the tested compounds had concentrations ranging from 5
µg/mL to 50 µg/mL. Different concentration solutions of each compound were
determined by UV scanning, and good linear relationship were obtained, then the
standard curve was completed. Each tested compound (1 mg) was ultrasound dissolved in
10 mL pure water for 1 h at room temperature. The solutions were stranded for 30 min
and centrifuged with speed 30000 r/min. The solution of each compound was determined
by UV scanning, absorbances were obtained. Through analysis standard curve, solubility
of all compounds was obtained.
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27. Cell culture: Mouse macrophage cell line RAW 264.7 was purchased from the Type
Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured
in DMEM medium supplemented with 10% FBS (Tianhang Biotechnology, Zhejiang,
China), 100 U/ml penicillin-G and 100 g/ml streptomycin (Beyotime, Shanghai, China) at
37 oC in an atmosphere of 5% CO2.
28. Western blot: The RAW 264.7 cells were plated at a density of 5×105 cells/well, which
were treated with compound 3i (10, 20, 30 µM) and Bay 11-7082 (0.3 µg/ml) for 12 h, and
then stimulated by LPS (1ng/ml) for 3 h (p65, pp65) or for 12 h (iNOS, COX-2).
Subsequently, cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China). Whole
extracts were prepared, and the protein concentrations were determined using a BCA
protein assay kit (Boster, Wuhan, China). Equal amounts of protein lysates (30 μg) were
separated by SDS-PAGE (10%, 80 V for 30 min and then 120 V for 60 min). The proteins
were transferred onto a PVDF membrane (Millipore Corp, Billerica, MA, USA). Then the
PVDF membranes will be incubated in TBS/Tween-20 containing 5% nonfat dry milk at 37
°C for 3 h. After blocking, the PVDF membranes were incubated with specific primary
antibodies overnight at 4 °C. Rabbit monoclonal antibodies against NF-κB p65, iNOS, COX-
2 (abcam, Cambridge, England), NF-κB phospho-p65 (Cell Signaling Technology, USA) and
mouse monoclonal anti-β-actin (ZSGB-BIO, Beijing, China) were used at 1:1000. Following
incubation with primary antibodies, blots were washed three times in TBS/Tween-20
before incubation at 37 °C for about 1 h in goat anti-mouse or goat anti-rabbit
horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, USA) conjugate antibody
at 1:10000 dilution in TBS/Tween-20 containing 5% nonfat dry milk. After extensive
washing in TBS/Tween-20 for another three times, the membranes were detected by the
enhanced chemiluminescence system. Proteins were visualized with ECL
chemiluminescent kit (ECL-plus, Thermo Fisher Scientific, Waltham, MA, USA).
Autoradiographs were scanned using a Image-Pro Plus Imaging analysis software (Media
Cybernetics, MD Rockville, USA).
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29. Immunofluorescence assay: Cells were pretreated with compound 3i and Bay 11-7082
(0.3 µg/mL) for 12 h before stimulation with 1 ng/ml LPS for 3 h. The cells were fixed with
ice-acetone for 15 min, permeabilized with 0.3% TritonX-100 in PBS for 15 min, and then
blocked with PBS (Boster, Wuhan, China) containing 5% bovine serum albumin (BSA,
Sigma, St. Louis, MO, USA) for 1 h. The cells were then incubated with the primary
antibody against NF-κB p65 (1:500) overnight at 4 °C, followed by detection with a FITC-
conjugated anti-rat IgG (Molecular Probes, Beijing, China) in the dark for 40 min at 37 °C.
Nuclear staining was incubated with 4', 6-diamidino-2-phenylindole, dilactate (DAPI;
Invitrogen, Carlsbad, CA, USA). Cells were washed and imaged using an inverted
fluorescence microscope (Olympus, Tokyo, Japan).
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Figure Captions
Table 1. The solubility in of compounds 3a~3k
Figure 1. ORTEP drawing of compounds 3i, 3k
Figure 2. Compounds 3a~3k (20 µM) inhibited LPS (1 µg/mL) induced pro-inflammatory
mediator IL-6 and TNF-α relative production
Figure 3. Compound 3i inhibits activation of NF-κB induced by LPS
Figure 4. Immunofluorescence analysis
Scheme 1. Synthesis of title compounds 3
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Graphical Abstract
Diarylpentadienone derivatives (Curcumin analogues): synthesis and anti-
inflammatory activity
New diarylpentadienone derivatives were synthesized. The results revealed that
compound 3i showed the highest anti-inflammatory activity on decreasing IL-6. The
further study showed that title compound 3i inhibited expression of proteins p-p65, iNOS,
COX-2 LPS-induced.
.
20

Highlights
A series of unsymmetrical diarylpentadienones were synthesized.
Title compound showed high anti-inflammatory activity on decreasing IL-6.
Title compound could lightly reduce activation p65 in nuclei.
21