Synthesis and biological evaluation of curcumin-like diarylpentanoid analogues for anti-inflammatory, antioxidant and anti-tyrosinase activities

Article abstract of DOI:10.1016/j.ejmech.2009.03.020

A series of 46 curcumin related diarylpentanoid analogues were synthesized and evaluated for their anti-inflammatory, antioxidant and anti-tyrosinase activities. Among these compounds 2, 13 and 33 exhibited potent NO inhibitory effect on IFN-γ/LPS-activated RAW 264.7 cells as compared to l-NAME and curcumin. However, these series of diarylpentanoid analogues were not significantly inhibiting NO scavenging, total radical scavenging and tyrosinase enzyme activities. The results revealed that the biological activity of these diarylpentanoid analogues is most likely due to their action mainly upon inflammatory mediator, inducible nitric oxide synthase (iNOS). The present results showed that compounds 2, 13 and 33 might serve as a useful starting point for the design of improved anti-inflammatory agents.

Full text of DOI:10.1016/j.ejmech.2009.03.020

European Journal of Medicinal Chemistry 44 (2009) 3195–3200
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of curcumin-like diarylpentanoid analogues
for anti-inflammatory, antioxidant and anti-tyrosinase activities
,
b
,
b
,
,d
Ka-Heng Lee a, Farida Haryani Ab. Aziz a, Ahmad Syahida a , Faridah Abas c, Khozirah Shaari b
,
*
,
,d
Daud Ahmad Israf b e, Nordin Haji Lajis b
a Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b Institute of Biosciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
d Faculty of Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
e Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 46 curcumin related diarylpentanoid analogues were synthesized and evaluated for their anti-
Received 31 December 2008
Received in revised form
11 March 2009
Accepted 16 March 2009
Available online 26 March 2009
inflammatory, antioxidant and anti-tyrosinase activities. Among these compounds 2, 13 and 33 exhibited
potent NO inhibitory effect on IFN-g/LPS-activated RAW 264.7 cells as compared to L-NAME and cur-
cumin. However, these series of diarylpentanoid analogues were not significantly inhibiting NO scav-
enging, total radical scavenging and tyrosinase enzyme activities. The results revealed that the biological
activity of these diarylpentanoid analogues is most likely due to their action mainly upon inflammatory
mediator, inducible nitric oxide synthase (iNOS). The present results showed that compounds 2, 13 and
33 might serve as a useful starting point for the design of improved anti-inflammatory agents.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Anti-inflammatory
Antioxidant
Anti-tyrosinase
Diarylpentanoids
iNOS
1. Introduction
(iNOS) and excessive NO production may lead to chronic inflam-
mation and also associated with number of diseases such as
Nitric oxide (NO) is a free radical generated by nitric oxide
synthase (NOS) and served as an important mediator that involved
in the regulation of physiological and pathophysiological mecha-
nism in cardiovascular, nervous and immunological systems [1].
However, abnormal expression of inducible nitric oxide synthase
rheumatoid arthritis, diabetes, hypertension and septic shock [2].
Macrophages are known as major immune cells to express iNOS in
response to stimuli such as lipopolysaccharide (LPS) or pro-
inflammatory cytokine including tumour necrosis factor-alpha
(TNF-a), interleukin-1b (IL-1b) and interferon-gamma (IFN-g) [3].
Studies have shown that NO biosynthesis is regulated by a variety of
signaling mechanisms at the transcriptional and post-translation
levels in activated macrophages. The upstream signaling protein or
Abbreviations: AP-1, activator protein-1; DMEM, Dulbecco’s modified eagle’s
medium; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; DPPH, diphe-
transcriptional factors such as nuclear factor kappa-B (NF-kB),
activator protein-1 (AP-1) and mitogen activated protein kinase
(MAPK) are the main regulators of iNOS expression [1]. Therefore,
inhibition of iNOS expression or its activity as well as upstream
signaling regulatory protein has been a prime target for develop-
ment of new anti-inflammatory drugs.
nylpicrylhydrazine radical; HEMn, human epidermal melanocyte cell line; IC50
inhibitory concentration at 50%; IFN- , interferon-gamma; IL-1 , interleukin-1beta;
iNOS, Inducible nitric oxide synthase; -DOPA, -3,4-dihydroxyphenylalanine;
-NAME, NG-nitro- -arginine methyl ester; LPS, lipopolysaccharide; MAPK, mitogen
activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide; NF-
B, nuclear factor kappa-B; NO, nitric oxide; NOꢀ2 , nitrite; OD,
,
g
b
L
L
L
L
k
In addition, NO can interact with reactive oxygen species (ROS)
leading to formation of reactive nitrogen species (RNS) such as
peroxynitrite [OONOꢀ], S-nitrosothiols [RSNO] and nitrogen
dioxide [NOꢀ2 ] [4]. It is well known that overproduction of ROS and
RNS generated from activated macrophages are the potent
oxidizing agents causing DNA fragmentation and lipid oxidation,
thus resulting in oxidative stress and cell injury [5]. Moreover,
optical density; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide;
RAW 264.7, murine monocytic macrophages cell line; RNS, reactive nitrogen
species; ROS, reactive oxygen species; S.E.M., standard error of mean; SNP, sodium
nitroprusside; TNF-a, tumour necrosis factor-alpha.
* Corresponding author. Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: þ603
89466694; fax: þ603 89430913.
E-mail address: syahida@biotech.upm.edu.my (A. Syahida).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.020

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K.-H. Lee et al. / European Journal of Medicinal Chemistry 44 (2009) 3195–3200
overproduction of ROS is associated with inflammatory response by
regulating several activities of kinases, transcription factors and cell
death machinery [6]. Therefore, compounds with radical scav-
enging activities can be used in treating or preventing various
chronic inflammatory diseases.
O
O
O
R
R
R
EtOH
H
+
NaOH
1
11. R = 2-OMe, 3-OMe
12. R = 2-OMe, 4-OMe
13. R = 2-OMe, 5-OMe
14. R = 2-OMe, 6-OMe
15. R = 3-OMe, 4-OH
2. R = 2-OH
3. R = 3-OH
4. R = 4-OH
5. R = 2-Cl
6. R = 3-Cl
7. R = 4-Cl
8. R = 2F
On the other hand, tyrosinase is a copper containing enzyme
involved in melanin synthesis and widely distributed in nature.
Two reactions involved in melanin biosynthesis pathways cata-
lyzed by tyrosinase are hydroxylation of monophenols to o-phenols
and the oxidation of the o-phenols to o-quinones [7]. Tyrosinase
catalyzed the formation of quinones and mediated the develop-
ment of brown colour pigment through spontaneous polymeriza-
tion of highly reactive quinones [8]. Elevated levels of epidermal
pigmentation have been implicated in various dermatological
disorders, such as melasma, age spots and sites of actinic damage.
Hence, inhibitory action on tyrosinase activity is an important
target in treating pigmentation-related disorders and new whit-
ening agent development.
A number of dietary chemopreventive compounds such as
curcumin and flavonoids have been shown beneficial in treating of
inflammatory diseases or dermatological disorders respectively
[9,10]. Recent studies have shown that curcumin analogues are
compounds of great chemical and pharmacological interest due to
their potential antioxidant, anti-proliferation [11], anti-angiogen-
esis [12], anti-tumour [13] and anti-inflammatory properties [14].
Furthermore, studies have suggested that two aromatic regions of
curcumin might be critical for potential protein-ligand binding and
became a rational design of potential inhibitor (ligand) of protein
targets [12]. Therefore, a series of curcumin-like diarylpentanoid
analogues with two identical aromatic ring region separated by five
carbon spacers were synthesized and evaluated for their anti-
inflammatory, antioxidant and anti-tyrosinase activities.
16. R = 2-F, 4-OMe
17. R = 2-OMe, 3-OMe, 4-OMe
18. R = 2-OMe, 4-OMe, 6-OMe
19. R = 3-OMe, 4-OMe, 5-OMe
9. R = 3-F
10. R = 4-F
O
O
O
R
R
R
EtOH
NaOH
H
+
20
21. R = 2-OH
22. R = 4-OH
23. R = 2-Cl
24. R = 3-Cl
25. R = 4-Cl
26. R = 2F
29. R = 3-OMe, 4-OH
30. R = 2-F, 4-OMe
31. R = 2-OMe, 3-OMe
32. R = 2-OMe, 4-OMe
33. R = 2-OMe, 5-OMe
34. R = 2-OMe, 6-OMe
35. R = 2-OMe, 3-OMe, 4-OMe
36. R = 2-OMe, 4-OMe, 6-OMe
27. R = 3-F
28. R = 4-F
O
O
O
R
R
R
EtOH
H
+
NaOH
37. R = 2-Cl
38. R = 3-Cl
39. R = 4-Cl
40. R = 2F
41. R = 3-F
42. R = 4-F
43. R = 2-OMe, 3-OMe
44. R = 2-OMe, 5-OMe
45. R = 2-OMe, 3-OMe, 5-OMe
46. R = 3-OMe, 4-OMe, 5-OMe
Scheme 1. General synthesis of diarypentanoids analogues.
suppression of NO production by affecting the iNOS expression. In
contrast, compound 33 (Fig. 1c) demonstrated potent inhibitory
action of NO production without causing any cytotoxicity effect at
all concentrations tested (Fig. 1f). Interestingly, compounds 13 and
33 shared same substitution groups, where methoxyl group was
2. Chemistry
All compounds were synthesized by coupling the appropriate
aromatic aldehyde with acetone (1–19), cyclopentanone (20–36)
and cyclohexanone (37–46) under base catalyzed aldol condensa-
tion, using the ratio of ketone:aldehyde of 1:2. A straightforward
approach to desired analogues is summarized in Scheme 1. All
compounds were determined based on the analysis of spectro-
scopic data and comparison of these data with those of related
compounds. Detailed spectroscopic data of the synthesized
compounds can be obtained from us upon request.
Table 1
The IC50 values of anti-inflammatory, NO-scavenging, DPPH radical scavenging and
anti-tyrosinase activities of diarylpentanoid analogues.
Analogues
IC50
(mM)
Anti-inflammatory Direct NO
activity
DPPH radical
scavenging scavenging
Anti-tyrosinase
activity
activity
activity
3. Results and discussion
2
4
10
13
15
16
22
25
26
28
29
31
33
Curcumin
10.24 ꢁ 1.05
NA
NA
>500
>500
NA
NA
>500
NA
>500
NA
NA
NA
>500
NA
>500
>500
NA
NA
79.05 ꢁ 3.92
>250
NA
>250
>250
>250
>250
NA
The results of anti-inflammatory, NO scavenging, antioxidant
and anti-tyrosinase activities are shown in Table 1. Seven
compounds (2, 13, 16, 26, 28, 31, 33) showed NO production
13.64 ꢁ 0.41
NA
NA
257.35 ꢁ 23.3
33.13 ꢁ 9.28
NA
NA
NA
inhibitory effect on IFN-
values varying from 10.24 to 40.24
compounds, 2, 13 and 33 exhibited potent NO inhibitory effect with
IC50 value of 10.24 ꢁ1.05 M, 13.64 ꢁ 0.41 M and 13.66 ꢁ 0.61 M,
respectively as compared to positive control, -NAME (IC50 val-
ue ¼ 27.13 ꢁ 5.58 M) and curcumin (20.38 ꢁ 0.28 M).
Compounds 2 (Fig. 1a) and 13 (Fig. 1b) showed NO inhibitory effect
in dose-dependent manner, but showed toxicity effect at
concentrations of 25 M (Fig. 1d) and 50 M (Fig. 1e), respectively.
Recently, Weber et al. [14] have shown that compounds 2 and 13
have attenuated TNF- induced nuclear factor-kappa-B (NF- B)
activation from human embryonic kidney cells which transfected
with stable NF- B reporter. Transcriptional factor NF- B is one of
the upstream signaling proteins that involved in the regulation of
iNOS expression and the inactivation of NF- B indirectly caused the
g/LPS-activated macrophage cells with IC50
>500
NA
NA
m
M. Among the seven
42.04 ꢁ 4.72
42.01 ꢁ 1.19
NA
36.95 ꢁ 2.87
13.66 ꢁ 0.61
20.38 ꢁ 0.28
27.13 ꢁ 5.58
NA
NA
m
m
m
316.64 ꢁ 23.79 NA
L
NA
NA
NA
NA
NA
NT
NT
NT
m
m
NA
>500
NT
109.74 ꢁ 2.04
a
L
-NAMEa
NT
Quercetinb NT
NA
41.79 ꢁ 2.23
m
m
PTIOc
NT
0.57 ꢁ 0.27 NT
Kojic acidd NT
NT
NT
66.55 ꢁ 0.19
a
k
NA: not active; NT: not tested.
a
Positive control used for anti-inflammatory assay.
Positive control used for DPPH radical scavenging assay.
Positive control used for NO-scavenging assay.
Positive control used for anti-tyrosinase assay.
k
k
b
c
d
k

K.-H. Lee et al. / European Journal of Medicinal Chemistry 44 (2009) 3195–3200
3197
50
60
50
40
30
20
10
0
a
b
*
40
***
*
30
***
20
***
***
***
***
***
***
10
0
***
***
***
c
M
M
M
M
0
0.78 1.56 3.13 6.25 12.5 25
50 250
L-NAME
c
0
0.78 1.56 3.13 6.25 12.5 25
50 250
L-NAME
Compound 2
Compound 13
IFN-γ /LPS
IFN-γ /LPS
50
40
30
20
10
0
140
120
100
80
c
d
*
**
***
***
***
60
***
***
***
40
***
20
***
c
0
M
0
0.78 1.56 3.13 6.13 12.5 25
50 250
L-NAME
C
0
0.78 1.56 3.13 6.13 12.5 25
50 250
L-NAME
Compound 33
Compound 2
IFN-γ /LPS
IFN-γ /LPS
140
120
100
80
140
120
100
80
e
f
***
60
60
40
40
20
20
0
0
M
0
0.78 1.56 3.13 6.13 12.5 25
50 250
L-NAME
C
0
0.78 1.56 3.13 6.13 12.5 25 50 250
L-NAME
Compound 13
C
Compound 33
IFN-γ /LPS
IFN-γ /LPS
Fig. 1. Nitric oxide (NO) production level (a–c) and RAW 264.7 cell viability (d–f) in the presence of compounds 2, 13 and 33. C; basal level of nitrite concentration without IFN-g/
LPS-treatment. All values are mean ꢁ S.E.M. of three different experiments. *P < 0.05, **P < <0.01, ***P < 0.001 significantly different from the IFN-
g/LPS-treated control group
(second column).
present at positions 2 and 5 on both their phenyl ring structures. It
was further supported by Ko et al. [15] and Rojas et al. [16] who
showed that chalcone derivatives with methoxyl groups at posi-
tions 2 and 5 of their phenyl rings structure potently inhibiting NO
production in activated RAW 264.7 cells. Therefore, it was sug-
gested that 2,5-dimethoxyl substitution groups on phenyl rings are
significant factors responsible for NO inhibitory activity.
respectively. It was well known that the presence of hydroxyl group
on phenyl ring is a critical factor contributing radical scavenging
properties [19]. Concomitantly, these three analogues (2, 4, 22)
have hydroxyl group at position either 2 or 4 of both their phenyl
0.003
It was found that excessive RNS and ROS generation takes place
in many inflammation disorders [17]. In this study, the relationship
of scavenging activity and anti-inflammatory properties of the
analogues was studied. The stable free radical diphenylpicrylhy-
drazyl (DPPH) was used to estimate the antioxidant activity of the
diarylpentanoid analogues. Compounds that can donate
a hydrogen atom to the DPPH radical, then gives rise to the reduced
form of DPPH which will be considered as potential antioxidant
agents [18]. The present results showed that the majority of the
analogues displayed poor radical scavenging activity. However,
compounds 2, 4 and 22 showed NO scavenging and DPPH radical
scavenging activities less than 50% at highest concentration tested
0.002
***
***
***
0.001
***
0.000
M
C
3.91 7.81 15.6 32.5 62.5
125
230
Kojic Acid
Compound 4
Fig. 2. Tyrosinase enzyme activity in the presence of compound 4. C; tyrosinase
enzyme activity without inhibitor. All values are mean ꢁ S.E.M. of three different
experiments. ***P < 0.001 significantly different from control group (first column).
(IC50 values >500 mM) as compared to PTIO and quercetin,

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K.-H. Lee et al. / European Journal of Medicinal Chemistry 44 (2009) 3195–3200
O
5. Experimental
5.1. Chemical syntheses
OH
HO
4
Analytical TLC was carried out on silica gel F254 precoated
(0.2 mm thickness, Merck) plastic TLC sheets. The TLC plates were
spotted with samples using a fine glass capillary tube and devel-
oped in a chromatographic tank saturated with solvent vapour at
room temperature. Melting points were determined using a hot
stage melting point apparatus equipped with a microscope, XSP-12
model 500X, and are uncorrected. 1H NMR spectra were recorded
on a Varian Unity 500 spectrometer (Varian Inc., Palo Alto, CA) and
measured at 500 MHz. Deuterated chloroform (CDCl3), and
acetone-d6 were used as solvents. Chemical shifts were recorded in
O
HO
HO
OH
NH2
L-DOPA
Fig. 3. The structure of tyrosinase substrates, L-DOPA and compound 4.
rings, thus demonstrating their antioxidant effect. Moreover, the
methoxyphenolic moiety has also been reported to be an essential
structural feature contributing antioxidant activity [20]. Consis-
tently, compounds 15 and 29 exhibited improved DPPH radical
d
ppm. Mass spectra (MS) have been recorded on a Polaris Q
ThermoFinnigan (San Jose, CA) with ionization induced by electron
impacts (EIMS) at 70 eV.
scavenging activity with IC50 values of 257.35 ꢁ 23.3
mM and
5.1.1. General procedure for the preparation of analogues
A mixture of the aromatic aldehyde (20 mmol, 2 equiv) and the
appropriate ketone (10 mmol, 1 equiv) were dissolved in 15 ml of
ethanol in a single necked round bottomed flask and stirred for
several minutes at 5 ꢂC (ice bath). Into this solution 10 ml of a 40%
NaOH solution in water was then added drop wise over several
minutes. The mixture is then allowed to stir at room temperature
for approximately 10 h. The reaction was neutralized with a dilute
HCl solution to form a precipitate, which was then collected by
suction filtration. The product, obtained after removal of the
solvent under reduced pressure, was crystallized from an appro-
priate solvent.
316.64 ꢁ 23.79
m
M, respectively. The antioxidant activity of both 15
and 29 might be explained by the presence of 4-OH and 3-methoxy
substitutions which enhanced their radical scavenging activity by
the stabilized phenoxy radical.
On the other hand, tyrosinase plays a critical role in controlling
melanin biosynthesis [21]. Hence, tyrosinase became an important
target in treating pigmentation-related disorders and developing of
new skin whitening agents. In the present study, most of the
compounds did not show anti-tyrosinase property except for
compound 4, which has moderately inhibited tyrosinase enzyme
activity in a dose-dependent manner (Fig. 2) with IC50 value of
79.05 ꢁ 3.92
mM (Table 1). It was also suggested that the hydroxyl
group at position 4 of both phenyl rings of compound 4 might be
the important factor contributing to tyrosinase enzyme inhibition.
Nerya et al. [8] had demonstrated that chalcone derivatives with
hydroxyl group at position 4 (ring B) potentially inhibit tyrosinase
enzyme activity. The tyrosinase inhibitory activity can also be
explained by the resemblance of structure of compound 4 with
5.1.2. 1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3-one
(Compound 2)
Orange amorphous solid; Yield 75%; m.p. 155–157 ꢂC; (lit. [13]
m.p.155 ꢂC); 1H NMR (500 MHz, Acetone-d6)
d in ppm: 8.09 (d,
J ¼ 16.0 Hz, 2H), 7.72 (dd, J ¼ 8.0, 1.5 Hz, 2H), 7.34 (d, J ¼ 16.0 Hz,
2H), 7.27 (td, J ¼ 8.5, 1.5 Hz, 2H), 6.99 (d, J ¼ 8.5 Hz, 2H), 6.93 (t,
J ¼ 7.5 Hz, 2H). MS (EI): [M-H2O]þ at m/z 248.
tyrosinase substrate,
L-DOPA (Fig. 3). Thus, compound 4 might be
acting as a competitive inhibitor to
L-DOPA. Further test has been
performed to study the cytotoxicity effect of compound 4 on
human epidermal melanocyte cell line (HEMn). Unfortunately,
compound 4 exhibited high degree of cytotoxicity to HEMn (data
not shown), thus denying the suitability of its use as cosmetic or
therapeutic agent in humans. On the other hand, compounds 10, 15,
16, 22, and 25 inhibited less than 50% at highest concentration
5.1.3. 1,5-Bis(4-hydroxyphenyl)-1,4-pentadiene-3-one
(Compound 4)
Orange amorphous solid; Yield 12%; m.p. 246–247 ꢂC (lit. [13]
m.p. 244–246 ꢂC); 1H NMR (500 MHz, Acetone-d6)
d in ppm: 7.71
(d, J ¼ 16.0 Hz, 2H), 7.63 (d, J ¼ 8.0 Hz, 4H), 7.10 (d, J ¼ 16.0 Hz, 2H),
6.93 (d, J ¼ 8.0 Hz, 4H). MS (EI): [M]þ at m/z 266.
tested (IC50 values >250 mM).
5.1.4. 1,5-Bis(2,5-dimethoxyphenyl)-1,4-pentadiene-3-one
(Compound 13)
4. Conclusion
Yellow amorphous solid; Yield 90%; m.p. 104–105 ꢂC (lit. [14]
The present results showed that a series of curcumin-like dia-
rylpentanoid analogues were not significantly inhibiting NO scav-
enging, DPPH radical scavenging and tryrosinase enzyme activities.
Among the 46 compounds tested, three curcumin-like diary-
lpentanoid analogues which are 2, 13 and 33 showed more likely
active in inhibitory action upon NO production in macrophage cells
(RAW 264.7). The presence of 2,5-dimethoxylated phenyl rings on
both compounds 13 and 33, while 2-hydroxylated phenyl rings on
compound 2 might become the main factors contributing to their
potent anti-inflammatory property. It was suggested that the anti-
inflammatory property of these compounds (2,13, 33) might be due
to the inhibition of target protein (iNOS) or its upstream regulatory
protein [14], rather as radical scavengers or antioxidant agents.
Thus, compounds 2,13 and 33 provide a useful starting point for the
rational design of anti-inflammatory agents with improved phar-
macokinetic and pharmacodynamic properties.
m.p.105–106 ꢂC); 1H NMR (500 MHz, Acetone-d6)
d in ppm: 7.98 (d,
J ¼ 15.5 Hz, 2H), 6.88 (d, J ¼ 15.5 Hz, 2H), 6.84 (d, J ¼ 8.0 Hz, 4H),
6.82 (s, 2H), 3.96 (s, 6H, OCH3), 3.85 (s, 6H, OCH3). MS (EI): [M]þ at
m/z 354.
5.1.5. 2,6-Bis(2,5-dimethoxybenzylidene)cyclohexanone
(Compound 33)
Yellow amorphous solid; Yield 75%; m.p. 118–120 ꢂC; 1H NMR
(500 MHz, acetone-d6)
d
in ppm: 7.88 (s, 2H), 7.01 (d, J ¼ 8:0 Hz,
4H), 6.96 (s, 2H), 3.84 (s, 6H, OCH3), 3.80 (s, 6H, OCH3), 2.91 (t,
J ¼ 6.0 Hz, 4 H), 1.77 (q, J ¼ 6.0 Hz, 2H). MS (EI): [M]þ at m/z 394.
5.2. DPPH radical scavenging assay
The free radical scavenging activity assay was carried out
according to Tagashira and Ohtake [22] with some modification.

K.-H. Lee et al. / European Journal of Medicinal Chemistry 44 (2009) 3195–3200
3199
Briefly, stock solution of test compound was prepared at 50 mM
concentration in 100% DMSO. The solutions were serially diluted to
different concentrations in a 96-well microtiter plate, from 7.81 to
The cells were incubated under 5% CO2 at 37 ꢂC for 4 h. The formazan
crystals formed were dissolved inDMSOand the absorbancewasread
at 570 nm using a microplate reader. Cell viability was calculated by
following the formula:
500 mM. Then 5 mL of diphenylpicrylhydrazine radical (DPPH)
solution (1 mg/ml dissolved in methanol) was then added to each
well. The plate was shaken gently and incubated in the dark for
30 min. The absorbance was measured at 517 nm and percentage of
total radical scavenging activity was calculated based on the
formula:
ODsample
Percentage of cell viability ¼
ꢃ 100%
ODcontrol
5.7. Anti-tyrosinase assay
Percentage of total radical scavenging activity
The inhibitory effect of diarylpentanoid derivatives on tyrosi-
nase activity was carried out according to Khatib et al. [7] with
some modification. Equal volume of phosphate buffer (50 mM) and
tyrosinase (SIGMA) (333 units/ml) was added into 96-well micro-
titer plate. The test samples dissolved in absolute ethanol at
ODcontrol ꢀ ODsample
¼
ꢃ 100%
ODcontrol
5.3. NO scavenging activity
concentration of 100
mM were then added into each well. After
5 min incubation, -DOPA (6 mM) was added. The absorbance was
measured at 492 nm at the intervals of 30 s for 10 min. Anti-
tyrosinase activity was calculated by following the formula:
L
The direct NO scavenging assay was carried out according to
Mirkov et al. [23] with some modification. Briefly, 100 L of SNP
(10 mM) solution were incubated alone or in combination with
different concentrations of test compounds (7.81–500 M) for
60 min with light exposure. The nitrite levels of the mixture were
then determined by Griess assay. NO scavenging activity was
calculated following the formula:
m
m
Percentage of anti-tyrosinase activity
Slopecontrol ꢀ Slopesample
¼
ꢃ 100%
Slopecontrol
Percentage of NO scavenging activity
5.8. Statistical analysis
ODcontrol ꢀ ODsample
¼
ꢃ 100%
ODcontrol
All the experiments are conducted three times and all data are
presented as the mean ꢁ S.E.M. Differences between groups were
determined by one-way analysis of variance (ANOVA) followed by
Dunnett test. P values <0.05 were considered significant. The IC50
values were calculated using GraphPad Prism 5 software.
5.4. Cell cultures and treatment
The RAW 264.7 cells (ATCC) were cultured in a plastic culture
flask in Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 10% FBS and 1% penicillin/streptomycin under 5% CO2
at 37 ꢂC. Cells at confluency of 80–90% were scrapped out and
Acknowledgments
seeded into 96-well plate at 5 ꢃ104 cells/50
mL well. Attached cells
This study was financially supported by a grant from the
Ministry of Higher Education, Malaysia under the Fundamental
Research Grant Scheme (FRGS) (01-01-07-013FR). Ka-Heng Lee was
a recipient of Graduate Research Fellowship (GRF) scheme sup-
ported by UPM. Post-Doctoral Fellowship awarded to Faridah Abas
was highly appreciated.
were then induced with 100 U/ml of recombinant mouse inter-
feron-gamma (IFN-g) and 5 mg/ml of LPS (Escherichia coli, serotype
0111:B4) in the presence or absence of test compounds for 17–20 h.
The NO production was then determined by Griess assay. NO
inhibitory activity was calculated following the formula:
h
i
h
i
NOꢀ2
ꢀ NOꢀ2
NOꢀ2
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h
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