Synthesis, crystal structure and anti-inflammatory properties of curcumin analogues

Article abstract of DOI:10.1016/j.ejmech.2008.01.031

Curcuminoids have been reported to possess multifunctional bioactivities, especially the ability to inhibit proinflammatory induction. Since it has been suggested that the seven-carbon β-diketone linker in curcumin is responsible for its instability, nine mono-carbonyl five-carbon linker containing analogues were designed and synthesized. Their bioactivity against lipopolysaccharide-induced TNF-α amd IL-6 secretion was evaluated by using mouse J774.1 macrophages. The results showed that the 3′-methoxyl plays an important role in bioactivity and cyclohexanone containing analogues exhibited stronger inflammatory inhibition than acetone and cyclopentanone analogues. Subsequently the most active analogue 3c was determined using single-crystal X-ray diffraction. X-ray analysis and comparison with curcumin reveals that the presence of cyclohexanone in 3c, which remotely resembles the 6-membered ring in the enol tautomer in curcumin, may play an important role in the bioactivity. It is suggested that five-carbon linker analogues containing a cyclohexane ring which are synthetically assessable may be pharmacologically important.

Full text of DOI:10.1016/j.ejmech.2008.01.031

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 915e919
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, crystal structure and anti-inflammatory
properties of curcumin analogues
Guang Liang a,b, Shulin Yang b, Huiping Zhou c, Lili Shao a,
Kexin Huang a, Jian Xiao a, Zhifeng Huang a, Xiaokun Li a,b,
*
a School of Pharmacy, Wenzhou Medical College, Wenzhou 325035, China
b College of Chemistry, Nanjing University of Science and Technology, Nanjing 210094, China
c Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA
Received 11 December 2007; received in revised form 15 January 2008; accepted 16 January 2008
Available online 3 February 2008
Abstract
Curcuminoids have been reported to possess multifunctional bioactivities, especially the ability to inhibit proinflammatory induction. Since it
has been suggested that the seven-carbon b-diketone linker in curcumin is responsible for its instability, nine mono-carbonyl five-carbon linker
containing analogues were designed and synthesized. Their bioactivity against lipopolysaccharide-induced TNF-a amd IL-6 secretion was eval-
uated by using mouse J774.1 macrophages. The results showed that the 30-methoxyl plays an important role in bioactivity and cyclohexanone
containing analogues exhibited stronger inflammatory inhibition than acetone and cyclopentanone analogues. Subsequently the most active
analogue 3c was determined using single-crystal X-ray diffraction. X-ray analysis and comparison with curcumin reveals that the presence
of cyclohexanone in 3c, which remotely resembles the 6-membered ring in the enol tautomer in curcumin, may play an important role in
the bioactivity. It is suggested that five-carbon linker analogues containing a cyclohexane ring which are synthetically assessable may be phar-
macologically important.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Curcumin analogue; Anti-inflammation; X-ray diffraction; Cyclohexanone
1. Introduction
Curcumin (Fig. 1), a yellow compound isolated from tur-
meric, possesses multifunctional pharmacological applications
in a variety of diseases such as liver fibrosis, inflammation,
cardiovascular disease and cancer [4e6]. Curcumin has been
reported to alter the cytokine profiles [7,8]. Curcumin potently
inhibited the production of TNF-a and IL-12 in a dose-depen-
dent manner from mouse macrophages stimulated by lipopoly-
saccharide (LPS) and reduced the LPS-induced activation of
NF-kB [9,10]. However, pre-clinical and clinical studies
showed that curcumin possessed several disadvantages such
as instability, poor bioavailability and fast metabolism [11].
It is suggested that the presence of the methylene group and
b-diketone moiety contributes to the instability of curcumin
under physiological conditions [12,13]. Therefore, the deletion
of the b-diketone moiety may contribute to the enhancement
of stability of curcumin compounds. We present here the syn-
thesis of nine mono-carbonyl analogues of curcumin, as well
Proinflammatory cytokines are involved in the pathogenesis
of a variety of diseases such as immunological disease and
inflammation. For example, interleukin-6 (IL-6) and tumor
necrosis factor-alpha (TNF-a) are two multifunctional proin-
flammatory cytokines involved in the pathogenesis of cardio-
vascular diseases, cancer and neurodegenerational disease
through a series of cytokine signaling pathways [1,2]. The
inhibition of release of cytokines becomes a major focus of
current drug development and an important method for evalu-
ating the bioactivity of drugs [3].
* Corresponding author. School of Pharmacy, Wenzhou Medical College,
Wenzhou 325035, Zhejiang, China. Tel.: þ86 139 68801352; fax: þ86 577
86699350.
E-mail address: cuiliang1234@163.com (X. Li).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.031

916
G. Liang et al. / European Journal of Medicinal Chemistry 44 (2009) 915e919
H
O
O
O
O
1
1
7
7
H3CO 3'
OCH3
H3CO 3'
OCH3
3
3
5
5
3''
3''
4'
4'
4''
4''
HO
OH
HO
OH
Curcumin
Fig. 1. Chemical structure of curcumin, where the a,b-unsaturated b-diketone (heptadieneedione) moiety undergoes ketoeenol tautomerism and forms a hydrogen
bond containing 6-membered ring [14].
as their pharmacological screening in inhibition of LPS-induced
TNF-a and IL-6 secretion in macrophages. The crystal struc-
ture of (2E,6E )-2,6-bis(4-fluorobenzylidene) cyclohexanone
(3c), which has the highest bioactivity among analogues, is
reported by a single-crystal X-ray diffraction. The difference
in crystal structures of curcumin and 3c is analyzed.
linker chains in structures of 1b and 1c, only 1c reached a com-
parable activity to curcumin. This result indicates that (1) the 30-
methoxyl plays an important role in bioactivity, and (2) the
six-member ring containing linker of mono-canbonyl analogues
may be favorable to the inflammatory inhibition. With the
same electron-withdrawing fluoride displacing hydroxyl group,
compounds 3ae3c were synthesized and all of them showed
elevated activity against LPS-induced inflammatory response.
Especially, from 3a to 3c, an increasing trend similar to hydroxyl
analogues 1 appeared and 3c exhibited the highest ability in
analogues 3, reaching 65.3% (TNF-a) and 54.8% (IL-6).
Our data given above showed that the cyclohexanone con-
taining linker may play an important role in the bioactivity of
analogues. To demonstrate the structure of cyclohexanone
linker, an X-ray crystal structure of 3c, (2E,5E )-2,5-bis(4-flu-
orobenzylidene) cyclohexanone was determined and the atom
labeling scheme is presented in Fig. 3, as well as that of cur-
cumin for comparison according to Parimita et al. [15]. The
selected bond lengths and bond angles are given in Table 1.
Single-crystal X-ray structure analysis reveals that there are
two 3c molecules in one unit cell with different bond lengths
and angles. No intermolecular hydrogen bond and interaction
appear. All H atoms were located in difference Fourier maps
and were refined freely in idealized positions, with Ce
2. Results and discussion
Compounds 3ae3c were synthesized (Scheme 1) by direct
coupling of p-fluorobenzaldhyde with the three ketones, ace-
tone, cyclopentanone and cyclohexanone in alkaline media.
The synthesis of compounds 1 and 2, shown in Scheme 1, be-
gins by the protection of 4-hydroxybenzaldehyde with tetrahy-
dropyran-2-yl to afford compound 6. Aldol condensation of 6
with acetone, cyclopentanone and cyclohexanone gave com-
pounds 4ae4c and 5ae5c, respectively. Compounds 1 (or 2)
were subsequently obtained by hydrolysis of 4 (or 5) using
a catalytic amount of p-toluenesulfonic acid. A diaryl structure
is confirmed by the absence of methyl protons in the 1H NMR
spectra of a class compounds and the absence of two methy-
lene protons in the spectra of b and c class compounds.
These nine analogues containing five-carbon linker with
mono-carbonyl were evaluated their anti-inflammatory pro-
perties through inhibiting the LPS-induced TNF-a and IL-6
release in macrophages [14]. As shown in Fig. 2, the mono-
carbonyl compounds 2ae2c exhibited enhanced inhibition
on both TNF-a and IL-6 than curcumin. In the structure of cur-
cumin, the hydrogen bonds are formed between 40-hydroxyl
and 30-methoxyl, reducing the molecular polarity of curcumin.
The 30-methoxyl-deleting compound 1a, however, exhibited
an opposing activity to enhance the release of inflammatory
cytokines induced by LPS. Although it was reduced when the
cyclopentanone and cyclohexanone were introduced as rigid
˚
˚
H ¼ 0.93 A for aromatic H and vinyl H or 0.96 A for methy-
lene H. In 3c, the dihedral angle between the two least-square
planes passing through benzene rings is 34.2(6)ꢀ, while that of
curcumin is 19.1(3)ꢀ. Compound 3c possesses more rigid
structure than curcumin due to the introduction of cyclohexa-
none. The lengths of CeF bonds including C1eF1, C18eF2,
C21eF3 and C38eF4 are similar to that of CeO bonds of cur-
cumin in the corresponding position (data showed in Table 1).
In one of 3c molecule in unit cell, the C12 is symmetrically
placed between atoms C11 and C13 and the 6-membered
O
R
CHO
R
O
CHO
R
O
R
O
i
ii
n
HO
O
O
O
6
4a -4c; 5a -5c
O
1a : n = 0, R = H ; 2a : n = 0, R = OCH3
R
R
iii
1b : n = 2, R = H ; 2b : n = 2, R = OCH3
1c: n = 3, R = H ; 2c: n = 3, R = OCH3
n
HO
CHO
OH
O
O
3a : n = 0
/ CH3OH
NaOCH3
3b : n = 2
3c: n = 3
F
n
n
F
F
Scheme 1. Synthesis of presented compounds. Reagents and conditions: (i) 3,4-dihydro-a-pyran, pyridine-PTSA, CH2Cl2, rt; (ii) acetone, cyclopentanone or
cyclohexanone, NaOH/EtOH, rt; (iii) PTSA, MeOH, rt.

G. Liang et al. / European Journal of Medicinal Chemistry 44 (2009) 915e919
917
250
200
150
100
50
TNF-alfa
IL-6
*
*
*
*
0
Fig. 2. Inhibition of LPS-induced TNF-a and IL-6 by curcumin and its analogues in J774A.1 macrophages. Cells were pre-treated with curcumin or its analogues
(10 mM) for 3 h, then treated with LPS (0.5 mg/ml) for 24 h. TNF-a (A) and IL-6 (B) levels in the culture media were measured by ELISA. The results were ex-
pressed as percent of LPS control. Each bar represents mean ꢁ S.E. of three independent experiments (*n > 5 and p < 0.01).
ring of cyclohenxanone was positioned with C11eC13 dis-
˚
tance being 2.483 A, similar to the 6-membered ring in curcu-
min formed by hydrogen bonding among H23, O2 and O3, in
˚
which the O2eO3 distance is 2.455 A. In the other molecule
˚
of the cell, the C31eC32 length is 1.506(7) A while the C32e
Recent reports indicated that b-diketone moiety may lead to
instability of curcumin and affect the metabolic profile. There-
fore, it is suggested that five-carbon linker analogues contain-
ing a cyclohexane ring may be important for the development
of anti-inflammatory curcumin drugs.
˚
C33 length is 1.522(7) A. This result reveals that the structure
of cyclohexanone in 3c is similar to 6-membered ring formed
by hydrogen bonding in curcumin. Although the conformation
of drugs in micro-environment is decisive to activity, close
structure may easily result in close conformation. So, the struc-
tural similarity may play an important role in the bioactivity.
4. Experimental
1H NMR spectra were recorded on a Varian INOVA-400
spectrometer. Electron-spray ionization mass spectra in posi-
tive mode (ESI-MS) data were recorded on a Bruker Esquire
3000þ spectrometer. Melting points were determined on
a FishereJohns melting apparatus and are corrected. Column
chromatography purifications were carried out on Silica Gel
60 (E. Merck, 70e230 mesh).
3. Conclusion
In conclusion, our data show that the 30-methoxyl group is
important for activity and cyclohexanone containing ana-
logues exhibited higher anti-inflammatory activity than ace-
tone and cyclopentanone analogues. The structure of
cyclohexanone moiety is similar to 6-membered ring formed
by b-diketone tautomer in curcumin, which may render 1c
and 3c stronger ability on inhibition of TNF-a and IL-6.
4.1. Synthesis
A solution of 3,4-dihydro-a-pyran (0.2 mol) in dichlorome-
thane (10 mL) was added dropwise to a well-stirred suspen-
sion of p-hydroxylbenzaldehyde (0.1 mol) and pyridinium
Fig. 3. The crystal structure of 2c (left) in a unit cell and curcumin (right) [15] with labeled non-hydrogen atom, showing displacement ellipsoids at the 50%
probability level. Dashed lines indicate intramolecular hydrogen bonding. (To see curcumin in Lit. [15].)

918
G. Liang et al. / European Journal of Medicinal Chemistry 44 (2009) 915e919
Table 1
Selected bond lengths (A) and bond angles ( )
ꢀ
˚
Bond
Distance
Bond
Distance
Bond
Distance
F1eC1
1.364 (6)
1.353 (6)
1.356 (6)
1.362 (6)
1.224 (6)
1.218 (5)
C8eC9
1.487 (7)
1.500 (7)
1.495 (7)
1.497 (7)
1.511 (7)
1.511 (7)
C28eC33
C28eC29
C29eC30
C30eC31
C31eC32
C32eC33
1.497 (7)
1.498 (7)
1.504 (7)
1.499 (7)
1.506 (7)
1.522 (7)
F2eC18
F3eC21
F4eC38
O1eC9
O2eC29
C8eC13
C9eC10
C10eC11
C11eC12
C12eC13
Bond
Angle
Bond
Angle
Bond
Angle
C7eC8eC9
116.0 (4)
C11eC12eC13
C8eC13eC12
C27eC28eC33
C27eC28eC29
C34eC30eC31
110.5 (4)
113.3 (4)
124.6 (5)
115.7 (4)
124.6 (5)
C34eC30eC29
C30eC31eC32
C31eC32eC33
C28eC33eC32
117.1 (4)
110.7 (4)
109.5 (4)
112.0 (4)
C7eC8eC13
C14eC10eC9
C14eC10eC11
C10eC11eC12
125.7 (5)
117.8 (4)
124.2 (5)
111.0 (4)
p-toluenesulfonate (catalytic) in dichloromethane (40 mL). The
mixture was stirred at room temperature for 3 h and monitored
by TLC, and then washed with saturated NaHCO3 solution
(50 mL ꢂ 3) and brine (50 mL ꢂ 3) and dried over Na2SO4.
Evaporation of the solvent affords crude product which was
purified by column chromatography on silica gel (hexane/ethyl
acetate, 8:2, as eluent) to give the protective compound 6
(0.09 mol). A mixture of 6 (10 mmol) and acetone, cyclopen-
tanone or cyclohexanone (5 mmol) in ethanol (10 mL) was
stirred at rt for 10 min and 18% (w/v) NaOCH3 in methanol
(1.5 mL, 5 mmol) was added dropwise. After stirring for 2 h,
the resulting precipitate was filtered. The protected derivative
4 (10 mmol) was suspended in ethanol (20 mL) and treated
with a catalytic amount of p-toluenesulfonic acid. After stir-
ring at room temperature for 10 h, 100 ml cold water was
added and the mixture was filtered. The residue was washed
with cold water and then recrystallized from C2H5OH/H2O
(9:1) to obtain pure 1a, 1b or 1c. Compounds 2 were obtained
by the same way.
4.1.4. 1,5-Bis(4-hydroxy-3-methoxyphenyl)penta-1,
4-dien-3-one (2a)
Yellow powder, 85% yield, mp 87e89 ꢀC [Lit. [17] 84e
86 ꢀC].
4.1.5. 2,5-Bis(4-hydroxy-3-methoxybenzylidene)
cyclopentanone (2b)
Yellow powder, 76% yield, mp 214 ꢀC [Lit. [16] 212e
214 ꢀC].
4.1.6. 2,6-Bis(4-hydroxy-3-methoxybenzylidene)
cyclohexanone (2c)
Yellow powder, 67% yield, mp 174e176 ꢀC [Lit. [16]
178e179 ꢀC].
To a solution of 10 mmol 4-fluorobenzaldehyde in dried
EtOH (10 ml) was added 5 mmol acetone, cyclopentanone or
cyclohexanone. The solution was stirred at room temperature
for 10 min, followed by dropwise addition of 18% (w/v)
NaOCH3 in methanol (1.5 ml, 5 mmol). The mixture was
stirred at rt for 2 h. The resulting precipitate was filtered and
washed with water, cold ethanol, and then cold acetone and
dried in vacuum. The solid was purified by chromatography
over silica gel using CH2Cl2/CH3OH as the eluent affording
compounds 3ae3c.
4.1.1. 1,5-Bis(4-hydroxyphenyl)penta-1,4-dien-3-one (1a)
Orange crystal, 92% yield, mp 246e248 ꢀC [Lit. [16] 243e
245 ꢀC].
4.1.2. 2,5-Bis(4-hydroxybenzylidene)cyclopentanone (1b)
Yellow crystal, 89.3% yield, mp >300 ꢀC. 1H NMR
(DMSO-d6) d: 2.98 (4H, s, CH2eCH2), 6.88 (4H, d,
J ¼ 8.4 Hz, AreH2,6 ꢂ 2), 7.33 (2H, s, AreCH]C ꢂ 2),
7.53 (4H, d, J ¼ 8.4 Hz, AreH3,5 ꢂ 2), 10.01 (2H, br, Are
OH ꢂ 2). ESI-MS m/z: 291.13 (M ꢃ 1)þ, calcd for
C19H16O3: 292.33.
4.1.7. 1,5-Bis(4-fluorophenyl)penta-1,4-dien-3-one (3a)
Yellow crystal, 67% yield, mp 152 ꢀC [Lit. [18] 150e
152 ꢀC].
4.1.8. 2,5-Bis(4-fluorobenzylidene)cyclopentanone (3b)
Yellow crystal, 88% yield, mp 212e214 ꢀC [Lit. [19]
210 ꢀC].
4.1.3. 2,6-Bis(4-hydroxybenzylidene)cyclohexanone (1c)
Yellow crystal, 59% yield, mp >300 ꢀC. 1H NMR (DMSO-
d6) d: 1.71 (2H, m), 2.84 (4H, s, CeCH2 ꢂ 2), 6.83 (4H, d,
J ¼ 6.0 Hz, AreH2,6 ꢂ 2), 7.40 (4H, d, J ¼ 6.0 Hz, Ar-
H3,5 ꢂ 2), 7.53 (2H, s, AreCH]C ꢂ 2), 9.50 (2H, br, Are
OH ꢂ 2). ESI-MS m/z: 305.18 (M ꢃ 1)þ, calcd for
C20H18O3: 306.36.
4.1.9. 2,6-Bis(4-fluorobenzylidene)cyclohexanone (3c)
The product was recrystallized from ethanol/water (9:1),
1
yellow crystal, 69% yield, mp 156 ꢀC. H NMR (CDCl3) d:
1.81 (2H, q, J ¼ 12 Hz, CH2), 2.90 (4H, t, J ¼ 5.4 Hz,
CH2 ꢂ 2), 7.12 (4H, m, AreH2,6 ꢂ 2), 7.45 (4H, m, Are
H3,5 ꢂ 2), 7.76 (2H, s, AreCH]C ꢂ 2). ESI-MS m/z:
311.11 (M þ 1)þ, calcd for C20H16F2O: 310.34.

G. Liang et al. / European Journal of Medicinal Chemistry 44 (2009) 915e919
919
Table 2
Crystal data of analogue 6
were determined by ELISA using mouse TNF-a and mouse
IL-6 ELISA MaxÔ Set Deluxe Kits (Biolegend, USA). The
tests were performed according to the manufacturer’s instruc-
tion. The cells collected were added into the total lysis buffer
(TriseHCl, 20 mM; NP40, 1%; NaCl, 150 mM; EDTA, 2 mM;
SDS, 0.1%; NaF, 20 mM; Na3VO4, 20 mM; H2O) and vo-
taxed, and then centrifuged at 8000 rpm for 5 min to extract
the total proteins. The total protein concentrations of the viable
cell pellets were determined using Bio-Rad protein assay re-
agents. Total amounts of the TNF-a and IL-6 in the media
were normalized to the total protein amount of the viable
cell pellets. Each compound was repeatedly tested in three in-
dependent experiments.
Crystal data
a ¼ 78.479 (2)ꢀ
l ¼ 0.71073 A
˚
C20H16F2O
FW ¼ 310.33
b ¼ 74.498 (2)ꢀ
g ¼ 86.226 (2)ꢀ
Cell parameters
from 1584 reflections
3
˚
Triclinic, P-1
V ¼ 1554.9 (3) A
Hall symbol: -P1
Z ¼ 4
q ¼ 2.51e24.26ꢀ
m ¼ 0.097 mmꢃ1
T ¼ 298 (2) K
˚
a ¼ 9.6947 (11) A
F000 ¼ 648
b ¼ 11.8447 (12) A Dx ¼ 1.326 Mg mꢃ3
˚
˚
c ¼ 14.3422 (15) A
Mo Ka radiation
0.25 ꢂ 0.16 ꢂ 0.14 mm
Data collection [20,21]
Tmin ¼ 0.976
8202 Independent
reflections, 3778
reflections
h ¼ ꢃ9 / 11;
k ¼ ꢃ13 / 14;
l ¼ ꢃ12 / 17
Tmix ¼ 0.991
with I > 2s(I )
qmin ¼ 1.75ꢀ
qmax ¼ 25.02ꢀ
Rint ¼ 0.0295
Acknowledgement
Refinement [21]
R[F2 > 2s(F2)]
¼ 0.1024
This work was supported by the Zhejiang Provincial Pro-
gram for the Cultivation of High-level Innovative Health tal-
ents (LXK), General Grants of Zhejiang administration of
Chinese Traditional Medicine (2007CA079, 2007CA080),
and NIH grants AI-68432 and AT-04148 (H. Zhou), USA.
5414 Reflections
(D/s)max ¼ 0.000
ꢃ3
wR(F2) ¼ 0.3438
S ¼ 1.021
415 Parameters
Drmax ¼ 0.176 e A
˚
w ¼ 1/[s2(F2o) þ (0.2253P)2
þ 0.4116P], where
P ¼ (F2o þ 2F2c)/3
Drmin ¼ ꢃ0.214 e A
ꢃ3
˚
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4.2. Crystal data and structure determination
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˚
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or vehicle control for 3 h, followed by treatment with LPS
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