Effects of novel diarylpentanoid analogues of curcumin on secretory phospholipase A2, cyclooxygenases, lipo-oxygenase, and microsomal prostaglandin e synthase-1

Article abstract of DOI:10.1111/cbdd.12280

Arachidonic acid and its metabolites have generated a heightened interest due to their significant role in inflammation. Inhibiting the enzymes involved in arachidonic acid metabolism has been considered as the synergistic anti-inflammatory effect. A series of novel curcumin diarylpentanoid analogues were synthesized and evaluated for their inhibitory effects on activity of secretory phospholipase A₂, cyclooxygenases, soybean lipo-oxygenase as well as microsomal prostaglandin E synthase-1. Among the curcumin analogues, compounds 3, 6, 9, 12, and 17 exhibited strong inhibition of secretory phospholipase A₂ activity, with IC₅₀ values ranging from 5.89 to 11.02 μm. Seven curcumin analogues 1, 3, 6, 7, 9, 11, and 12 showed inhibition of cyclooxygenases-2 with IC₅₀ values in the range of 46.11 to 94.86 μm, which were lower than that of curcumin. Compounds 3, 6, 7, 12, and 17 showed strong inhibition of lipo-oxygenase enzyme activity. Preliminary screening of diarylpentanoid curcumin analogues for microsomal prostaglandin E synthase-1 activity revealed that four diarylpentanoid curcumin analogues 5, 6, 7, and 13 demonstrated higher inhibition of microsomal prostaglandin E synthase-1 activity with IC₅₀ ranging from 2.41 to 4.48 μm, which was less than that of curcumin. The present results suggest that some of these diarylpentanoid analogues were able to inhibit the activity of these enzymes. This raises the possibility that diarylpentanoid analogues of curcumin might serve as useful starting point for the design of improved anti-inflammatory agents. A series of novel diarylpentanoid analogues of curcumin was synthesized by direct coupling of the appropriate aromatic aldehyde with three ketones, namely cyclohexanone, acetone, and cyclopentanone, under base-catalyzed Claisen-Schmidt condensation reaction. These analogues were screened for their inhibitory effects on the activity of secretory phospholipase A2 (sPLA2), cyclooxygenases, soybean lipo-oxygenase as well as microsomal prostaglandin E synthase-1 using in vitro assays.

Full text of DOI:10.1111/cbdd.12280

Chem Biol Drug Des 2014; 83: 670–681
Research Article
Effects of Novel Diarylpentanoid Analogues of
Curcumin on Secretory Phospholipase A2,
Cyclooxygenases, Lipo-oxygenase, and Microsomal
Prostaglandin E Synthase-1
A number of studies have shown the involvement of
aberrant arachidonic acid metabolism in inflammation.
Membrane phospholipids, the main source of arachidonic
acid, are hydrolyzed by secretory phospholipase A2
(sPLA2); the liberated arachidonic acid is additionally
metabolized by cyclooxygenase (COX) and lipo-oxygen-
ase (LOX) (1). Metabolism of arachidonic acid gives rise
to key lipid mediators. COX enzymes catalyze the first
step in the biosynthesis of prostaglandin and thrombox-
ane. Lipo-oxygenase enzymes catalyze arachidonic acid
leading to the production of leukotrienes and lipoxins.
These mediators play a fundamental role during inflam-
mation. Leukotrienes and prostaglandins are recognized
to uphold the inflammatory reaction, while the lipoxins
are mainly anti-inflammatory in nature. Arachidonic acid
and its metabolites have newly generated a heightened
interest owing to increase in evidence regarding their
important role in cancer biology and various types of car-
diovascular diseases. New advances in the field of
inflammation have given insight into the modulation of
arachidonic acid metabolism. Inhibiting the enzymes
involved in arachidonic acid metabolism has been con-
sidered as the synergistic anti-inflammatory effect with
enhanced spectrum of activity (2). Therefore, inhibitors of
these enzymes have initially been of interest for the treat-
ment of inflammation.
Waqas Ahmad, Endang Kumolosasi, Ibrahim
Jantan*, Syed N. A. Bukhari and Malina Jasamai
Drug and Herbal Research Centre, Faculty of Pharmacy,
Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul
Aziz 50300 Kuala Lumpur, Malaysia
*Corresponding author: Ibrahim Jantan, profibj@gmail.com
Arachidonic acid and its metabolites have generated a
heightened interest due to their significant role in
inflammation. Inhibiting the enzymes involved in ara-
chidonic acid metabolism has been considered as the
synergistic anti-inflammatory effect. A series of novel
curcumin diarylpentanoid analogues were synthesized
and evaluated for their inhibitory effects on activity
of secretory phospholipase A2, cyclooxygenases,
soybean lipo-oxygenase as well as microsomal prosta-
glandin E synthase-1. Among the curcumin analogues,
compounds 3, 6, 9, 12, and 17 exhibited strong inhibi-
tion of secretory phospholipase A2 activity, with IC50
values ranging from 5.89 to 11.02 lM. Seven curcumin
analogues 1, 3, 6, 7, 9, 11, and 12 showed inhibition of
cyclooxygenases-2 with IC50 values in the range of
46.11 to 94.86 lM, which were lower than that of curc-
umin. Compounds 3, 6, 7, 12, and 17 showed strong
inhibition of lipo-oxygenase enzyme activity. Prelimin-
ary screening of diarylpentanoid curcumin analogues
for microsomal prostaglandin
E synthase-1 activity
revealed that four diarylpentanoid curcumin analogues
5, 6, 7, and 13 demonstrated higher inhibition of micro-
somal prostaglandin E synthase-1 activity with IC50
ranging from 2.41 to 4.48 lM, which was less than that
of curcumin. The present results suggest that some of
these diarylpentanoid analogues were able to inhibit
the activity of these enzymes. This raises the possibil-
ity that diarylpentanoid analogues of curcumin might
serve as useful starting point for the design of
improved anti-inflammatory agents.
Curcumin
1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hept
adiene-3,5-dione,diferuloylmethane) is a natural phenolic
compound, isolated as a yellow pigment from turmeric
(dry rhizomes of Curcuma longa), which is frequently used
as a food colorant, spice, and conventional medicine in
India, China, and some Asian countries. Curcumin is
renowned for its anti-inflammatory effects, and over the
past few decades, its several biological and pharmacologi-
cal activities have been reported. As per recent reviews,
curcumin is a multitarget pleiotropic agent, showing a wide
range of biological activities (3). Curcumin and its deriva-
tives exhibit a wide variety of pharmacological activities, viz.
antibacterial, antifungal, antiviral, anti-HIV anti-inflammatory,
anti-Parkinson’s, anti-Alzheimer’s, anti-angiogenesis, free
radical scavenging activity, antirheumatic, antimalarial,
anticancer, antiprotozoan, antimutagenic, wound treat-
ment, hepatoprotective activity, anti-leishmanial activity,
Key words: chemical biology, drug discovery, enzymatic
mechanism
Received 3 September 2013, revised 23 November 2013 and
accepted for publication 6 January 2014
670
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12280

Anti-inflammatory Effects of Diarylpentanoids
antibacterial, antiviral, antioxidant, and amyloid heart
disease (4–6).
Screening Kit from Cayman Chemicals. Soybean lipo-oxy-
genase, arachidonic acid, and potassium hydroxide were
also obtained from Cayman Chemicals. Human recombinant
microsomal PGE synthase-1 enzyme and prostaglandin H2
(PGH2) were purchased from Cayman Chemicals. Prosta-
glandin E2 (PGE2) EIA kit used to quantify the PGE2 concen-
tration was also purchased from Cayman Chemicals.
Curcumin has been revealed to inhibit the metabolism of
arachidonic acid and the activities of cyclooxygenase-2
(COX-2), lipo-oxygenase, proinflammatory cytokines,
inducible nitric oxide (iNOS), protein kinases, transcription
factors such as nuclear factor-kB, and release of steroids
(7,8). Curcumin has been shown to inhibit COX-2 expres-
sion in gastrointestinal cancer cells and mouse skin (9). A
number of previous studies have also indicated that curcu-
min affects the formation of COX- and LOX-dependent
metabolites and decreases activities of PLA2 (10). Curcu-
min is recognized to have a poor bioavailability, as orally
administered curcumin goes through hepatic conjugation,
resulting in the formation of glucuronides and sulfates,
whereas systemic administration causes it to undergo
reduction (11). Due to these limitations, its potential use as
a therapeutic agent is seriously affected. As a result, plen-
tiful approaches have been undertaken to synthesize new
analogues and derivatives to enhance bioavailability of
curcumin by altering its molecular structure, that is, elimi-
nating the unstable b-diketone moiety and modifying the
heptadiene linker while retaining the phenolic OH groups
(12,13). Latest reports have demonstrated that the b-dike-
tone moiety of curcumin is a specific substrate for liver
aldoketo reductases. The presence of b-diketone moiety
may add to the rapid metabolism of curcumin, thus limiting
its beneficial effects in many types of disease. Recent
studies have illustrated that the curcumin analogues are of
great chemical and pharmacological interest owing to their
anti-inflammatory properties (14).
General methods
1H and 13C NMR spectra were recorded with a JEOL ECP
spectrometer operating at 500 MHz, with Me4Si as internal
standard and CDCl3 or DMSO-d6 as the solvent. High-
resolution mass spectra (HRMS) were determined by the
electrospray ionization mass spectrometry (ESI-MS) on
MicroTOF-Q mass spectrometer (Bruker, Bremen, Ger-
many). Microanalyses data were obtained from the Fison
EA 1108 elemental analyzer (Thermo Fisher Scientific, Wal-
tham, MA, USA). Infrared spectra were recorded using
KBr disc on a Perkin Elmer 400 (FTIR) spectrometer
(Waltham, MA, USA). Flash column chromatography was
performed with silica gel 60 (230–400 mesh) (Merck), and
thin-layer chromatography (TLC) was carried out on pre-
coated silica plates (kiesel gel 60 F254, BDH). Melting
points were determined on an electrothermal instrument
and are uncorrected. Compounds were visualized by illu-
mination under ultraviolet (UV) light (254 nm) or by the use
of vanillin stain followed by charring on a hotplate.
General procedure for synthesis of diarylpentanoid
analogues
The diarylpentanoid analogues of curcumin were synthe-
sized by direct coupling of the appropriate aromatic alde-
hyde with the three ketones, namely cyclohexanone,
acetone, and cyclopentanone, at a molar ratio of 1:2,
under base-catalyzed Claisen–Schmidt condensation
reaction. Figure 1 illustrates the general synthesis of diaryl-
pentanoids. Briefly, the appropriate aromatic aldehyde
(20 mmol, two equivalent) and the appropriate ketone
(10 mmol, one equivalent) were mixed and dissolved in
15 mL of ethanol in a single-necked round-bottomed flask
and stirred at 5 °C for a few minutes. Into this solution, a
40% NaOH solution in ethanol was then added drop wise
over several minutes. The mixture was then allowed to stir
at room temperature (27 °C) for 1–24 h. The appearance
of precipitate and color changes of the reaction mixture
served as an indicative marker of product formation. Reac-
tion was monitored by TLC, and on completion, the reac-
tion was quenched by adding acidified ice to the mixture.
The diarylpentanoid curcumin analogues were isolated by
column chromatography or recrystallization.
Thus, a series of 17 curcumin-like diarylpentanoids has
been synthesized, derived from the chemical structure of
curcumin by eliminating the b-diketone and modifying it
into conjugated double bonds, that is, two identical
aromatic ring regions separated by five carbon linkers (Fig-
ure 1). To the best of our knowledge, curcumin-related di-
arylpentanoid analogues have not been investigated for
their effects on enzymes of arachidonic acid metabolism.
Therefore, we evaluated these compounds for their effects
on the sPLA2, COX, LOX, and microsomal prostaglandin E
synthase-1 (mPGES-1).
Methods and Materials
Materials
Chemicals used in sPLA2 assay include 1,2-bis(heptanoyl-
thio)-phosphatidylcholine, 5,5-dithiobis (2-nitrobenzoic acid)
(DTNB), and recombinant human PLA2-V from Cayman
Chemicals, Ann Arbor, MI, USA. CaCl2, KCl, and HCl were
purchased from Merck, Darmstadt, Germany. Curcumin,
dimethyl sulphoxide (DMSO), ethylenediaminetetraacetic
acid (EDTA), reduced glutathione (GSH), and SnCl2 were
obtained from Sigma-Aldrich (Steinheim, Germany). Cyclo-
oxygenase activity was determined using a COX Inhibitor
2,6-Bis[4-(methylaminoethanol)benzylidine]
cyclohexanone (1)
Cyclohexanone (1.00 mL, 10 mmol) with N-methyl-N-
(2-hydroxyethyl)-4-aminobenzaldehyde (3.58 g, 20 mmol)
Chem Biol Drug Des 2014; 83: 670–681
671

Ahmad et al.
Figure 1: Synthesis scheme of
diarylpentanoid analogues of
curcumin.
were reacted to obtain compound 1 as dark red solids
(2.89 g, 63%). mp: 80–81 °C; 1H NMR (500 MHz,
CDCl3) d: 7.99 (s, 2H), 7.50 (d, J = 8 Hz, 4H), 6.76 (d,
J = 8 Hz, 4H), 3.89 (t, J = 13.0, 4H), 3.62 (t, J = 7.5,
4H), 3.13 (s, 6H), 2.61 (t, J = 24.0 Hz, 4H), 1.70 (m, 2H);
13C NMR (500 MHz, CDCl3) d: 190.42, 153.94, 147.42,
137.77, 132.60), 125.30, 111.77, 60.01, 54.36, 39.18,
28.77, 23.60; IRVmax=cmÀ1 (ATR) 3347.5, 1583.92,
1520.85, 1241.43; HRMS (ESI) m/z: 421.24 [M+H]+;
microanalysis calculated for C26H32N2O3 (420.54),
C:74.26%, H:7.67%, N:6.66%. Found C: 74.42%, H:
7.62%, N: 6.99%.
crystals of 2,6-bis(2,3-dimethoxybenzylidene)cyclohexa-
none (3.23 g, 82%). 1H NMR (500 MHz, CDCl3) d: 7.95
(s, 2H), 7.07 (d, J = 8 Hz, 2H), 6.94 (m, J = 4 Hz, 4H),
3.90 (s, 6H), 3.84 (s, 6H), 2.95 (t, J = 24 Hz, 4H), 1.37
(m, J = 21 Hz, 2H); 13C NMR (500 MHz, CDCl3) d:
190.32, 152.88, 148.37, 137.55, 132.48, 130.49,
123.44, 122.17, 112.80, 61.13, 55.88, 28.73, 23.31;
IRVmax=cmÀ1 (ATR) 1576.5, 1471.78, 1257.27; HRMS (ESI)
m/z 417.16 [M+Na]+; microanalysis calculated for
C24H26O5 (394.46), C:73.08%, H:6.64%. Found C:
73.58%, H: 6.77%.
2,6-Bis(4-diethyl-aminobenzylidene)cyclohexanone
(5)
Cyclohexanone (1 mL, 10 mmol) and 4-diethylaminobenz-
aldehyde (3.54 g, 20 mmol) were reacted to yield light
red solid powder of 2,6-bis(4-diethyl-aminobenzylidene)
2,6-Bis(2,3-dimethoxybenzylidene)cyclohexanone
(3)
Cyclohexanone (1 mL, 10 mmol) and 2,3-dimethoxybenz-
aldehyde (3.32 g, 20 mmol) were reacted to give yellow
672
Chem Biol Drug Des 2014; 83: 670–681

Anti-inflammatory Effects of Diarylpentanoids
(1E,4E)-1,5-Bis[2-methyl-4(N-ethyl-N-
ethylacetonitrile)aniline]-1,4-pentadiene-3-one
(12)
cyclohexanone (2.12 g, 51%). 1H NMR (500 MHz, CDCl3)
d: 8.03 (s, 2H), 7.69 (d, J = 8 Hz, 4H), 6.59 (d,
J = 8.5 Hz, 4H), 3.94 (q, J = 6.5 Hz, 8H), 2.33 (t,
J = 7 Hz, 4H), 1.61 (m, J = 11 Hz, 2H), 1.32 (t,
J = 6.5 Hz, 4H).; 13C NMR (500 MHz, CDCl3) d: 189.73,
152.21, 148.09, 124.48, 130.77, 110.95, 44.56, 29.06,
23.79, 12.40; IRVmax=cmÀ1 (ATR) 2974.2, 1588.15, 1522.45,
1272.49; HRMS (ESI) m/z: 417.28 [M+H]+.
The reaction of acetone (0.73 mL, 10 mmol) with 2-methyl-
N-ethyl-N-(2-cyanoethyl)-4-aminobenzaldehyde
(4.32 g,
20 mmol), followed by purification (by column chromatogra-
phy) yielded a dark red solid compound 12 (2.95 g, 64%).
mp: 178–179 °C; 1H NMR (500 MHz, CDCl3) d: 7.82 (d,
J = 15.5 Hz, 2H), 7.70 (d, J = 4.8 Hz 2H), 6.65 (d,
J = 15.5 Hz, 2H), 6.63 (d, J = 2.4 Hz, 2H), 6.52 (s, 2H), 3.66
(q, J = 9 Hz, 8H), 2.75 (t, J = 7.2 Hz, 4H), 2.38 (s, 6H), 1.12
(t, J = 2.6 Hz, 6H); 13C NMR (500 MHz, CDCl3) d: 187.85,
148.94, 140.88, 139.86, 128.74, 128.65, 123.07, 119.94,
113.17, 110.50, 56.26, 44.61, 23.07, 16.24, 12.46;
IRVmax=cmÀ1 (ATR) 1746.0, 1516.42, 1048.69-1351.89;
HRMS (ESI) m/z: 455.27 [M+H]+; microanalysis calculated
for C29H34N4O (454.61), C: 76.62%, H: 7.54%, N: 12.32%.
Found C: 76.69%, H: 7.79%, N: 12.41%.
2,6-Bis[2-methyl-4(N-ethyl-N-ethylacetonitrile)
aniline]cyclohexanone (6)
Cyclohexanone (1 mL, 10 mmol) and 2-methyl-N-ethyl-N-
(2-cyanoethyl)-4-aminobenzaldehyde (4.32 g, 20 mmol)
were reacted to give a dark red solid compound 6 (2.55 g,
52%). mp: 180–181 °C; 1H NMR (500 MHz, CDCl3) d: 7.61
(s, 2H), 6.71 (d, J = 2.4 Hz, 2H), 6.70 (d, J = 3.0 Hz, 2H),
6.61 (s, 2H), 3.71 (t, J = 6.6 Hz, 4H), 3.49 (q, J = 7.2 Hz,
4H), 2.77 (t, J = 6.6 Hz, 4H), 2.50 (m, 6H), 2.36 (s, 6H), 1.11
(t, J = 7.2 Hz, 6H); 13C NMR (500 MHz, CDCl3) d: 190.41,
151.21, 142., 139.54, 139.21, 134.77, 123.58, 119.51,
113.76, 109.45, 55.55, 44.71, 29.24, 28.81, 20.24, 16.15,
12.47; IRVmax=cmÀ1 (ATR) 1583.92, 1520.85, 1241.43; HRMS
(ESI) m/z: 495.27 [M+H]+; microanalysis calculated for
2,5-Bis[4-(methylaminoethanol)benzylidene]
cyclopentanone (13)
This compound was obtained by reacting cyclopentanone
(0.88 mL, 10 mmol) with N-methyl-N-(2-hydroxyethyl)-4-
aminobenzaldehyde (3.58 g, 20 mmol). Compound 13
was isolated as red solid (2.92 g, 72%). mp: 221–223 °C;
1H NMR (500 MHz, CDCl3) d: 7.97 (s, 2H), 7.56 (d,
J = 8.0 Hz, 4H), 7.35 (d, J = 9 Hz, 4H), 3.93 (t,
J = 8.0 Hz, 4H), 3.62 (t, J = 11.5 Hz, 4H), 3.52 (s, 4H),
2.99 (s, 6H); 13C NMR (500 MHz, CDCl3) d: 195.92,
154.84, 137.83, 136.97, 136.51, 134.00, 123.72, 61.37,
55.92, 38.38, 26.74; IRVmax=cmÀ1 (ATR) 3369.5, 1741.10,
1169.92; HRMS (ESI) m/z: 407.17 [M+H]+; microanalysis
Calculated for C25H30N2O3 (406.52), C: 73.86%, H:
7.44%, N: 6.89%. Found C: 73.69%, H: 7.65%, N:
6.53%.
C
32H38N4O (494.67), C: 77.70%, H: 7.74%, N: 11.33%.
Found C: 77.91%, H: 7.78%, N: 11.42%.
(1Z,4Z)-1,5-Bis [4-(methylaminoethanol)phenyl]-
1,4-pentadiene-3-one (7)
Acetone (0.73 mL, 10 mmol) and N-methyl-N-(2-hydroxy-
ethyl)-4-aminobenzaldehyde (3.58 g, 20 mmol) were
reacted, and a dark brown powder of (1Z,4Z)-1,5-Bis [4-
(methylaminoethanol)phenyl]-1,4-pentadiene-3-one (2.12 g,
55%) was obtained. RF 0.56 (EtOAc-PE 1:3 v/v); mp: 80–
81 °C; 1H NMR (500 MHz, CDCl3) d: 7.74 (d, J = 8.5 Hz,
4H), 7.51 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 6.79
(d, J = 9.0 Hz, 2H), 6.76 (d, J = 6.0 Hz, 2H), 3.88 (t,
J = 4.0 Hz, 4H), 3.62 (m, 4H), 3.14 (s, 3H), 3.09 (s, 3H), 2.36
(s, 2H); 13C NMR (500 MHz, CDCl3) d: 190.32, 153.90,
151.40, 130.17, 122.44, 121.43, 112.10, 60.20, 60.13,
38.93; IRVmax=cmÀ1 (ATR) 3391.9, 1746.0, 1516.42, 1048.69-
1351.89; HRMS (ESI) m/z: 381.21 [M+H]+.
2,5-Bis(4-diethylaminobenzylidene)cyclopentanone
(17)
Cyclopentanone (0.88 mL, 10 mmol) and 4-diethylamino-
benzaldehyde (3.54 g, 20 mmol) were reacted to give
compound 17 as dark brown powder (2.76 g. 69%). 1H
NMR (500 MHz, CDCl3) d: 8.03 (s, 2H), 7.53 (d, J = 4 Hz,
4H), 6.71 (d, J = 8.5 Hz, 4H), 3.43 (q, J = 7 Hz, 4H), 3.08
(s, 4H), 1.22 (t, J = 7 Hz, 12H); 13C NMR (500 MHz,
CDCl3) d: 196.02, 148.30, 133.49, 123.49, 133.08,
132.88; 111.33, 44.46, 26.69, 12.66; IRVmax=cmÀ1 (ATR)
2977.8, 1575.07, 1513.89, 1264.85; MS (ESI) m/z: 403.22
[M+H]+; microanalysis calculated for C27H34N2O (402.57),
C: 80.55%, H: 8.51%, N: 6.96%. Found C: 80.48%, H:
8.27%, N: 6.77%.
(1Z,4Z)–1-5-Bis[2,3(dimethoxy)phenyl]-1,4-
pentadien-3-one (9)
Acetone (0.73 mL, 10 mmol) and 2, 3-dimethoxybenzalde-
hyde (3.32 g, 20 mmol) were reacted to yield pale yellow
solid (1Z,4Z)–1-5-bis[2,3(dimethoxy)phenyl]-1,4-pentadien-
3-one (1.32 g, 37%). 1H NMR (500 MHz, CDCl3) d: 8.06
(d, J = 16 Hz, 2H), 7.27 (d, J = 7.5 Hz, 2H), 7.18 (d,
J = 16 Hz, 2H), 7.11 (t, J = 5 Hz, 2H), 6.99 (d,
J = 7.5 Hz, 2H), 3.91 (s, 6H); 13C NMR (500 MHz, CDCl3)
d: 189.72, 153.20, 148.83, 137.92, 129.10, 126.94,
124.23, 119.14, 114.14, 61.36, 55.92; IRVmax=cmÀ1 (ATR)
2974.2, 1715.59, 1469.64, 1369.16; HRMS (ESI) m/z:
291.17 [M+H]+.
Secretory phospholipase A2 –V (sPLA2-V) activity
assay
Human recombinant sPLA2-V was employed as an
enzyme source. The activity of sPLA2 enzyme was
Chem Biol Drug Des 2014; 83: 670–681
673

Ahmad et al.
determined by a photometric assay based on the Ellman’s
method (15). In brief, the hydrolysis of sn-2 ester bond of
the substrate 1,2-bis(heptanoylthio)-glycerophosphocholine
by PLA2-V was followed by the exposure of free thiols.
These thiols activated the alteration of DTNB (5,5–dithio-
bis-(2-nitrobenzoic acid) to 2-nitro-5-thiobenzoic acid,
which was detected photometrically at 405 nm. Later, the
assay was progressed in an aqueous buffer solution (pH
7.5) containing KCl (94 mM), CaCl2 (9 mM), Tris (24 mM),
and Triton-X 100 (280 lM). Earlier to the assay, substrate
and PLA2-V were resuspended in assay buffer, and DTNB
was dissolved in an aqueous solution of Tris–HCl (pH 8).
Enzyme and DTNB yielded final concentrations of 100 ng/
mL and 87 lM, correspondingly. Assays were carried out
in 96-well microliter plates, at room temperature containing
DTNB, substrate solution, and the respective test sub-
stance. 100% activity of the enzyme was calculated by
adding substrate and enzyme only. Dimethyl sulphoxide
served as negative control and was inactive at the concen-
tration used in the assay (1.7% v/v).
nated by adding 1 M HCl, and finally, PGE2 was quantified
by an ELISA method. The test compounds were dissolved
in DMSO and diluted to the desired concentration with
potassium phosphate buffer of pH 7.4. Following transfer
to a 96-well plate coated with a mouse anti-rabbit IgG, the
tracer prostaglandin acetylcholine esterase and primary
antibody (mouse anti-PGE2) were added. Plates were then
incubated at room temperature overnight, reaction mix-
tures were removed, and the wells were washed with
10 mM potassium phosphate buffer containing 0.05%
Tween-20. Ellman’s reagent (200 lL) was added to each
well, and the plate was incubated at room temperature for
60 min and measured the absorbance at 412 nm using
Tecanꢀ Infinite Pro 200 microplate reader. A standard
curve with PGE2 was generated from the same plate,
which was used to quantify the PGE2 levels produced in
the presence of test samples. Percent inhibition was cal-
culated by the comparison of compound treated to control
incubations. All determinations were performed in dupli-
cate.
Lipo-oxygenase activity assay
Microsomal prostaglandin E synthase-1 activity
assay
The inhibitory activity of the compounds against purified
LOX enzyme was determined by a standard colorimetric
assay (16). Soybean lipo-oxygenase was employed as an
enzyme source. The assay was progressed in a 0.1 M
Tris–HCl buffer solution at pH 7. Immediately preceding
the assay, LOX enzyme was resuspended in assay buffer.
Substrate was dissolved in equal volume of potassium
hydroxide, vortexed, and diluted with the HPLC grade
water to obtain the final concentration of 1 mM. Assays
were performed in 96-well microliter plates at room tem-
perature containing substrate, enzyme, and respective test
substance. 100% activity of enzyme was calculated by
adding the substrate and enzyme only. 90 lL of LOX and
10 lL of respective test sample were added to determine
the activity. The reaction was started by adding substrate
solution to all wells and incubated for 5 min. Then, 100 lL
of chromogen solution was added to all wells to stop
enzyme catalysis and incubated for 5 min. After that, the
plate cover was removed and the absorbance was mea-
sured at 490 nm using Tecanꢀ Infinite Pro 200 microplate
reader (Mannedorf, Switzerland). Dimethyl sulphoxide
served as a negative control. This solvent was inactive at
the concentration used in the assay (1.7% v/v).
Microsomal prostaglandin
E synthase-1 activity was
assessed, as described previously (18). Human recombi-
nant mPGES-1 was used as enzyme source. Prostaglan-
din H2 (PGH2) served as the substrate for the enzyme
mPGES-1. Reduced glutathione was also employed,
which served as catalyst for the enzyme. Hundred millimo-
lar KHPO4 buffer at pH 7.0 supplemented with 2 mM
EDTA and 2.5 mM reduced GSH served as assay buffer.
Microsomal prostaglandin E synthase-1 activity was deter-
mined using 96-well non-binding plate. 100 lL of reaction
buffer containing mPGES-1 was added to each well of a
96-well non-binding plate. Afterward, the test samples
were added to their respective wells. The 96-well plate
was covered with the plate sealer and incubated for
30 min at 20 °C. After incubation, the enzyme reaction
was initiated by adding 20 lL of cold 2.8 lM PGH2 to all
wells and incubated again for 30 seconds at room tem-
perature. At set time, the reaction was quenched by add-
ing 20 lL of SnCl2 solution in 1 N HCl. The activity of
mPGES-1 enzyme was calculated by measuring the con-
centration of product (PGE2). Prostaglandin E2 concentra-
tion was measured by enzyme-linked immunosorbent
assay. The procedure was performed according to proto-
col supplied by the manufacturer. The sample was trans-
ferred to a 96-well plate coated with a goat antimouse
IgG. Afterward, 50 lL of tracer prostaglandin acetylcholine
esterase and 50 lL of monoclonal antibody were added
and incubated for 60 min at room temperature. Reaction
mixtures were removed and washed with the washing buf-
fer. Ellman’s reagent (200 lL) was added to each well and
incubated at room temperature in the dark on an orbital
shaker for 60–90 min, and the absorbance of the solution
at 420 nm was measured using a plate reader. All deter-
minations were performed in duplicate.
Cyclooxygenase activity assay
The inhibitory effects of the curcumin analogues on COX-1
and COX-2 activities were determined using a COX inhibi-
tor screening kit (17). Reaction mixtures were prepared in
100 mM Tris–HCl buffer, pH 8.0 containing 1 mM heme
and COX-1 (ovine) or COX-2 (human recombinant) and
preincubated for 10 min in a water bath at 37 °C. The
reaction was initiated by the addition of 10 lL arachidonic
acid. Final concentration of arachidonic acid in reaction
mixture was 100 lM. After 2 min, the reaction was termi-
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Chem Biol Drug Des 2014; 83: 670–681

Anti-inflammatory Effects of Diarylpentanoids
Table 1: Inhibition of secretory phospholipase A2-V (sPLA2-V)
Statistical analysis
activity by curcumin analogues
All the experiments are conducted three times, and all
data are presented as the mean Æ standard error of mean
(SEM). The IC50 values were calculated using GraphPad
Prism 5 software (Graphpad Software, Inc., La Jolla, CA,
USA). The values were obtained from at least three deter-
minations. Data were analyzed using a one-way analysis
of variance (ANOVA) for multiple comparisons. p < 0.05 was
considered to be statistically significant.
Compounds
sPLA2-V% inhibition
IC50 (lM)
1
79.01 Æ 1.59
49.44 Æ 0.90
83.64 Æ 2.10
48.96 Æ 1.20
79.89 Æ 2.30
82.92 Æ 1.80
80.13 Æ 2.03
51.68 Æ 1.10
86.05 Æ 2.40
44.73 Æ 0.89
75.74 Æ 3.15
86.93 Æ 4.10
70.78 Æ 0.97
42.80 Æ 2.20
74.09 Æ 1.78
22.11 Æ 5.20
78.69 Æ 1.19
81.57 Æ 1.56
77.66 Æ 1.03
15.90 Æ 0.71
53.28 Æ 2.50
11.02 Æ 0.50
94.71 Æ 4.23
12.09 Æ 1.68
5.89 Æ 0.20
16.36 Æ 0.52
51.88 Æ 2.30
10.38 Æ 1.69
125.38 Æ 7.23
16.06 Æ 1.59
5.98 Æ 0.43
19.13 Æ 0.98
83.21 Æ 5.10
16.45 Æ 0.52
ND
2
3
4
5
6
7
8
Results and Discussion
9
10
11
12
Inhibition of sPLA2-V
Eicosanoids are the oxygenated metabolites of arachidonic
acid (AA) with broad implications in a number of diseases.
Upon stimulation of neutrophils, PLA2 cleaves arachidonic
acid from membrane phospholipids at sn-2 position and
generates lysophospholipid and free fatty acid. Subse-
quently, the free fatty acid is converted into biologically
active prostaglandins, prostacyclins, thromboxanes, and
leukotrienes, collectively known as eicosanoids (19). The
release of arachidonic acid by PLA2 from membrane phos-
pholipids is typically the rate-limiting step for further arachi-
donic acid metabolism and eicosanoid generation. In this
context, there is an increased support for strategies that
prevent inflammatory reaction. Mixed results have been
achieved by inhibiting selective pathways of eicosanoid pro-
duction, that is, the LOX and COX pathways (20). Numerous
types of PLA2s are found in human system. Among these
PLA2 enzymes, sPLA2s play a key role in the pathogenesis
of inflammatory diseases, and elevated levels of sPLA2s are
detected in many inflammatory conditions (21). So, the inhi-
bition of PLA2 is an important strategy for the prevention of
inflammation. Thus, the effects of diarylpentanoid curcumin
analogues at concentrations of 1.25–20 lg/mL on the activ-
ity of sPLA2 were determined by photometric assay based
on Ellman’s method. Their percentage inhibitions (%) are
shown in Table 1. Ten curcumin analogues 1, 3, 5, 6, 7, 9,
11, 12, 15, and 17 strongly inhibited the activity of sPLA2
with percentage inhibition ranging from 74.09% to 86.93%.
Dimethyl sulphoxide was used as a control and was inactive
at the concentration used in the assay (1.70% v/v). Curcu-
min and dexamethasone were used as positive controls.
The IC50 values of the compounds on the inhibition of PLA2
are shown in Table 1. Curcumin was used as a reference
compound, which inhibited the activity of sPLA2 by 81.57%
(IC50 11.10 lM). Several previous studies had been con-
ducted for the other isoforms of PLA2, which indicated that
curcumin decreased activities of PLA2, and various isoforms
of PLA2 enzymes exhibited more than 70% homology
(17,22). Compounds 3, 5, 6, 9, 12, and 17 strongly inhibited
sPLA2-V in a dose-dependent manner (Figure 2), with IC50
values in the range of 5.89–12.09 lM, which are lower or
comparable to that of curcumin (11.10 lM).
13
14
15
16
17
Curcumin
Dexamethasone
10.18 Æ 0.74
11.10 Æ 0.30
0.61 Æ 0.01
Values are the mean Æ SD; n = 3. ND, not determined.
(2-cyanoethyl)-4-amino, diethylamino, and N-methyl-N-
(2-hydroxyethyl)-4-amino substitutions at position
benzene ring showed good inhibition of sPLA2 activity in
all three types of curcumin analogues bearing acetone,
cyclopentanone, and cyclohexanone. However, 2-methyl-
4 of
N-ethyl-N-(2-cyanoethyl)-4-amino
bearing
analogues
demonstrated stronger inhibition of sPLA2, in fact almost a
twofold improvement in inhibition of sPLA2 relative to
curcumin. Compounds of cyclohexanone ring system were
more active than cyclopentanone or acetone bearing
analogues. Among these three, the most active was com-
pound 6, which belongs to cyclohexanone ring system.
Methoxylation at positions 2 and 3 of both phenyl rings in
acetone and cyclohexanone demonstrated stronger inhibi-
tory activity; however, methoxylation at same position in
cyclopentanone system did not show such strong inhibi-
tion. The introduction of 4-tert-butyl substitution reduced
the inhibitory effect in all three types of curcumin analogues
bearing acetone, cyclopentanone, and cyclohexanone.
Inhibition of cyclooxygenases
The enzyme cyclooxygenase (COX) catalyzes the first two
steps in the biosynthesis of prostaglandins (PGs) from the
substrate arachidonic acid. Prostaglandins play important
roles in health as well as in disease state, in the gastro-
intestinal tract, renal, skeletal, and ocular systems. Two
isoforms of this enzyme exist, which are COX-1 and COX-
2 (23). The constitutive COX-1 is well recognized to be
housekeeping enzyme and is responsible for maintaining
normal physiological function; in contrast, COX-2 is an
inducible enzyme and its expression is activated by a
diversity of stimuli such as mitogens, oncogenes, tumor pro-
moters, growth factors, tissue damage, and inflammatory
Structure–activity analysis of the diarylpentanoid analogues
indicated that compounds bearing 2-methyl-N-ethyl-N-
Chem Biol Drug Des 2014; 83: 670–681
675

Ahmad et al.
Figure 2: Concentration-
dependent inhibitory effects of
diarylpentanoid analogues on
activity of secretory phospholipase
A2-V. Graph represents the %
inhibition by most active
compounds along with curcumin.
*p < 0.05, **p < 0.01, ***p < 0.001
are significant difference with
respect to curcumin. The data
shown are an average of three
independent experiment, and
values are mean Æ SD.
conditions (23). The inhibition of COX-1 results in undesir-
able side-effects, whereas COX-2 inhibition provides thera-
peutic effects in pain, inflammation, cancer, and
neuropathologic conditions like Alzheimer’s and Parkinson
diseases (24,25). The inhibition of the enzymes COX-1 and
COX-2 is considered as one of the mechanisms of anti-
inflammatory actions. The inhibitory effect of curcumin
analogues on the cyclooxygenase activity was determined
at concentration of 40 lg/mL for both isoforms of cycloox-
ygenase, that is, COX-1 and COX-2. COX-1 and COX-2
inhibitory activity was evaluated using the COX inhibitor
screening assay kit in vitro (17). Percentage inhibition and
IC50 were calculated, and the results are exhibited in the
Table 2. The COX-1 and COX-2 inhibitory effect of these
diarylpentanoid analogues was compared with that of pure
curcumin.
Table 2: Inhibition of COX-1 and COX-2 activity by curcumin analogues
COX% inhibition
IC50 (lM)
Compounds
COX-1
COX-2
COX-1
COX-2
1
89.41 Æ 0.67
71.51 Æ 1.07
84.34 Æ 0.87
52.68 Æ 1.33
74.97 Æ 0.95
82.55 Æ 0.38
88.20 Æ 0.59
68.50 Æ 1.23
79.82 Æ 1.02
49.29 Æ 2.01
78.88 Æ 1.21
86.62 Æ 0.57
72.46 Æ 1.04
66.51 Æ 1.45
77.90 Æ 0.96
46.65 Æ 2.48
75.35 Æ 1.73
83.06 Æ 1.43
94.06 Æ 0.45
61.73 Æ 1.02
39.02 Æ 1.23
63.51 Æ 0.33
32.55 Æ 2.31
41.98 Æ 0.67
55.78 Æ 0.87
67.91 Æ 1.33
34.24 Æ 1.43
58.20 Æ 0.34
22.52 Æ 2.01
51.93 Æ 0.77
65.13 Æ 0.65
44.27 Æ 1.03
31.55 Æ 1.45
36.04 Æ 0.46
33.83 Æ 0.25
44.88 Æ 1.12
51.53 Æ 2.6
75.11 Æ 0.13
22.08 Æ 0.42
43.22 Æ 0.28
33.33 Æ 0.17
194.0 Æ 3.14
38.52 Æ 0.48
16.65 Æ 0.35
23.62 Æ 0.38
53.24 Æ 0.76
41.51 Æ 0.49
194.6 Æ 2.40
37.70 Æ 0.31
17.40 Æ 0.60
35.86 Æ 0.24
60.01 Æ 0.97
40.24 Æ 0.36
319.64 Æ 4.12
31.96 Æ 0.54
31.31 Æ 0.45
0.21 Æ 0.03
46.55 Æ 1.35
ND
2
3
53.23 Æ 1.27
4
ND
5
ND
6
57.75 Æ 0.58
56.57 Æ 1.37
ND
7
8
9
10
70.71 Æ 1.36
ND
11
12
94.86 Æ 2.16
46.11 Æ 1.4 8
ND
ND
ND
13
14
15
16
ND
ND
17
Curcumin
Indomethacin*
96.36 Æ 1.43
3.24 Æ 0.01
*30 lM concentration.
Values are the mean Æ SD; n = 3. ND, not determined.
676
Chem Biol Drug Des 2014; 83: 670–681

Anti-inflammatory Effects of Diarylpentanoids
The result showed that the curcumin analogues had a
affinity toward COX-1. Five compounds (1, 3, 6, 7, and
12) exhibited strong inhibitory effect on activity of COX-1
with extent of inhibition ranging from 82.55% to 89.41%.
Effect of pure curcumin was also observed on the activity
of COX-1 and was used as a reference compound. Indo-
methacin served as positive control. Four compounds (1,
6, 7, and 12) illustrated better COX-1 inhibitory activity
than that shown by curcumin with IC50 values in the range
of 16–22 lM, which was on the lower side than that of
curcumin (31.31 lM), whereas the inhibitory activity of
compound 17 was comparable with the curcumin with an
IC50 value of 31.96 lM. Compound 6 exhibited the highest
COX-1 inhibition (IC50 16.65 lM) followed by 12 (IC50
17.40 lM). The COX-1 inhibitory effect of 6 was twofold
when compared to curcumin (IC50 31.31 lM).
Arachidonic acid is a substrate for the lipo-oxygenases.
Lipo-oxygenase is a family of non-heme iron-containing di-
oxygenases. The upregulation of LOX has been associated
with various adverse effects (26). Lipo-oxygenase family
exists in three isoforms: LOX-5, LOX-12, and LOX-15.
Together, they catalyze the deoxygenation of arachidonic
acid into hydroperoxyeicosatetraenoic acids (HpETEs). In
the end, this is followed by their conversion to their corre-
sponding hydroxyeicosatetraenoic acids (HETEs), resulting
in the formation of the leukotrienes (LKs), lipoxins (LOs),
and hepoxilins (HOs) (27). Inhibitors of LOX have attracted
attention initially as potential agents for the treatment for
inflammatory and allergic diseases and certain types of
cancer. For this reason, we evaluated the ability of curcu-
min analogues to inhibit soybean lipo-oxygenase, which
converts linoleic to 13-hydroperoxylinoleic acid. Soybean
lipo-oxygenase is inhibited by non-steroidal anti-inflamma-
tory drugs (NSAIDs) in a qualitatively similar manner to that
of the rat mast cell lipo-oxygenase and can be used as a
reliable screen for such activity (28).
In case of COX-2, in contrast to COX-1, curcumin ana-
logues were less efficient in suppressing the activity of
COX-2. Seven compounds (1, 3, 6, 7, 9, 11, and 12)
exhibited COX-2 inhibitory activity with IC50 values in the
range of 46.11–94.86 lM. COX-2 inhibitory activity of these
compounds was better than that of curcumin (IC50
96.36 lM). The highest COX-2 inhibition was exhibited by
compound 12 with an IC50 value of 46.11, which was two-
fold greater when compared to curcumin (IC50 96.36 lM).
Thus, analogue 12 showed significant enhancement in the
selectivity toward COX-2 enzyme (COX-2/COX-1 = 2.6)
when compared to curcumin (COX-2/COX-1 = 3.07).
Compounds 1, 3, 6, and 7 also showed better selectivity
toward COX-2. It was observed that the compounds inhib-
ited COX activity in a dose-dependent manner; there was
an increase in the inhibitory activity of compounds with
increase in the concentration. The results of the present
study suggest that curcumin analogues act as non-selec-
tive inhibitors of COX enzymes and showed anti-inflamma-
tory effects. The structure–activity relationship studies
(SAR) revealed that diarylpentanoid analogues (5, 11, and
17) with diethylamine group at position 4 of both phenyl
rings showed better COX-1 inhibition without having effect
on COX-2. The conversion of diethylamine groups at posi-
tion 4 of phenyl ring into methoxy groups (3 and 9)
improved both COX-1 and COX-2 inhibitory activity; how-
ever, compound 15 with similar substitution pattern but
contains cyclopentanone linker did not show improvement
toward COX-2. The presence of N-methyl-N-(2-hydroxy-
ethyl)-4-amino at position 4 of the both phenyl rings of
cyclohexanone and acetone diarylpentanoid analogues
resulted in compounds 1 and 7, which exhibited strong
COX-2 inhibitory activity than curcumin. Compound 12
acetone diarylpentanoid analogue with 2-methyl-N-ethyl-
N-(2-cyanoethyl)-4-amino substitution at position 4 of both
phenyl rings showed strongest COX-2 inhibition.
Curcumin was used as a reference compound. Curcumin
has also been reported to inhibit 5-LOX activity both in
in vitro and in vivo models (6,29). Inhibitory effect of curcu-
min on soybean LOX has also been reported in a study
conducted by Katsori et al. (28). The effects of the diaryl-
pentanoid analogues on the activity of LOX were determined
at concentration ranging 2.5–40 lg/mL, and their percent-
age inhibitions are shown in Table 3. Preliminary screening
of the compounds on the activity of LOX showed that
compounds 3, 6, 7, 9, 12, 15, and 17 strongly inhibited the
activity of LOX with percentage inhibitions ranging from
Table 3: Inhibition of lipo-oxygenase activity by curcumin ana-
logues
Lipo-oxygenase%
Compounds
inhibition
IC50 (lM)
1
54.45 Æ 1.95
36.03 Æ 2.97
77.42 Æ 1.41
36.27 Æ 3.72
42.82 Æ 2.72
80.53 Æ 1.43
62.57 Æ 1.51
38.21 Æ 2.03
70.29 Æ 1.49
30.12 Æ 2.44
52.04 Æ 1.88
75.10 Æ 1.14
52.27 Æ 2.58
23.16 Æ 3.68
62.17 Æ 1.77
27.86 Æ 4.86
62.29 Æ 1.86
66.29 Æ 1.59
94.65 Æ 1.48
82.44 Æ 0.71
ND
47.83 Æ 0.51
ND
2
3
4
5
ND
6
30.02 Æ 0.21
56.42 Æ 0.52
ND
7
8
9
10
62.86 Æ 1.69
ND
11
12
95.07 Æ 4.24
41.13 Æ 0.43
89.51 Æ 0.98
ND
13
14
15
16
63.76 Æ 1.84
ND
17
Curcumin
NDGA*
56.76 Æ 0.74
57.77 Æ 0.21
9.23 Æ 0.33
Inhibition of lipo-oxygenase
Eicosanoids are oxygenated metabolites of arachidonic
acid (AA) with broad implications in a variety of diseases.
*16 lM concentration.
Values are the mean Æ SD; n = 3. ND, not determined.
Chem Biol Drug Des 2014; 83: 670–681
677

Ahmad et al.
64.2% to 80.53%. Dimethyl sulphoxide was used as
negative control and did not show any effect on activity of
enzyme. Compounds 7 and 15 (62.57% and 62.17%) were
equipotent to curcumin (66.29%) at 40 lg/mL. Among the
analogues tested, 3, 6, 9, and 12 were the strong inhibitors
with IC50 values ranging from 30.02 to 62.86 lM, which are
lower or comparable to the value for curcumin (57.7 lM).
transformed to PGH2 by COX, and it is this PGH2 which
then acts as substrate for microsomal PGE synthase-1
(mPGES-1) and gets converted to PGE2. mPGES-1 is the
major PGE2 synthase under pathological conditions related
to inflammation and cancer (30). mPGES-1 expressed in
response to pro-inflammatory stimuli results in increased
formation of PGE2 during inflammation (31). Latest
advances in genetic and pharmacologic inhibition of
mPGES-1 indicate a crucial role of mPGES-1 in the
development and maintenance of inflammatory disorders,
pain, fever, and cardiovascular diseases, and suggest
mPGES-1 inhibitors as alternatives to non-steroidal anti-
inflammatory drugs showing comparable anti-inflammatory
effectiveness while being essentially free of severe side-
effects (30,32).
Among all curcumin analogues,
6 exhibited highest
inhibitory activity. The extent of % inhibition was 80.53 with
an IC50 value of 30.02 lM. From the results, it can be
concluded that the compounds inhibited LOX activity in a
dose-dependent manner, that is, as the concentration of
the compound increased, there was a decrease in the
activity of enzyme and vice versa, as shown in Figure 3.
Structure–activity analysis of the diarylpentanoid analogue
(compounds 3, 5, and 15) indicates that the presence of
methoxy groups at positions 2 and 3 on the phenyl ring
demonstrated stronger inhibitory activity against LOX.
Among the diarylpentanoid analogue, compound bearing
2-methyl-N-ethyl-N-(2-cyanoethyl)-4-amino groups at posi-
tion 4 of both phenyl rings (compounds 6 and 12) exhib-
ited the strongest inhibition of LOX activity than the
curcumin. The presence of diethylamino group at position
4 (compound 17) of both phenyl rings also shows inhibi-
tion of LOX in cyclopentanone ring system. However, sub-
stitution of phenyl rings with 4-tert-butyl produced
compounds with lower inhibitory activity.
The effect of curcumin analogue on mPGES-1 activity was
assessed, as described previously (18). Purified human
recombinant mPGES-1 was used as enzyme source to
investigate the direct effects of curcumin analogue on the
activity of mPGES-1. PGE2 concentration was assessed
by ELISA. Pure curcumin effect was also determined and
used as a reference. Curcumin has been shown to lower
production of PGE2 both in vitro and in vivo in earlier
demonstrations (33,34). Well-known mPGES-1 inhibitor
MK-886 was used as control. All curcumin analogues
were screened to assess the effect on the activity of
mPGES-1 at concentrations ranging from 0.625 to 10 lg/mL.
Dimethyl sulphoxide was used as a solvent to dissolve the
curcumin analogue and also as negative control. Percent-
age inhibitions and IC50 values were calculated. Results
are demonstrated in the Table 4 in the form of % inhibition
and IC50 values. The curcumin analogues inhibited the
activity of the mPGES-1. The% inhibition of mPGES-1
Inhibition of microsomal prostaglandin E synthase-
1
Prostaglandin E2 is a potent lipid mediator, that is, closely
related with inflammation and cancer. Arachidonic acid is
Figure 3: Concentration-
dependent inhibitory effects of
diarylpentanoid analogues on
activity of lipo-oxygenase. Graph
represents the % inhibition by most
active compounds along with
curcumin. *p < 0.05, **p < 0.01 are
significant difference with respect to
curcumin. The data shown are an
average of three independent
experiment, and values are
mean Æ SD.
678
Chem Biol Drug Des 2014; 83: 670–681

Anti-inflammatory Effects of Diarylpentanoids
Table 4: Inhibition of microsomal prostaglandin
(mPGES-1) activity by curcumin analogues
E synthase-1
effectively inhibited PGE2 formation and mPGES-1 directly
with IC50 values ranging from 1 to 5 lM (35,36). So, our
findings are somehow consistent with previous studies.
The four diarylpentanoid curcumin analogues (5, 6, 7, and
13) demonstrated strong inhibition of mPGES-1 activity
with IC50 values ranging from 2.41 to 4.48 lM, which was
less than that of curcumin (4.88 lM). While the inhibitory
activity of compound 1 was comparable to curcumin,
compound 1 showed almost similar IC50 value (4.96 lM).
Among all tested curcumin analogues, compound 5 exhib-
ited the strongest inhibitory effect with 85.20% reduction
in mPGES-1 activity and with an IC50 of 2.41 lM, followed
by 13, 7, and 6, respectively. The results indicate that the
potency of compound 5 to inhibit the mPGES-1 was
twofold greater when compared to curcumin (4.88 lM).
The results showed that the compounds inhibit activity of
mPGES-1 in a dose-dependent manner, that is, as the
concentration of the samples increased the activity of
mPGES-1 decreased and vice versa (Figure 4).
mPGES-1%
Compounds
inhibition
IC50 (lM)
1
81.38 Æ 2.83
69.38 Æ 0.83
74.07 Æ 1.54
56.07 Æ 1.49
85.20 Æ 2.89
79.92 Æ 2.83
80.02 Æ 2.50
48.72 Æ 1.67
59.44 Æ 3.63
42.77 Æ 2.44
71.75 Æ 2.67
67.65 Æ 0.83
82.12 Æ 2.83
51.96 Æ 1.44
67.24 Æ 2.20
70.21 Æ 1.83
79.93 Æ 2.20
83.17 Æ 1.63
91.83 Æ 2.20
4.96 Æ 0.47
8.95 Æ 1.03
7.34 Æ 0.76
2
3
4
20.27 Æ 2.11
2.41 Æ 0.24
4.48 Æ 0.80
4.23 Æ 0.26
36.99 Æ 4.32
14.75 Æ 2.25
60.71 Æ 5.85
6.98 Æ 0.79
7.89 Æ 0.21
3.78 Æ 0.24
24.48 Æ 3.22
10.43 Æ 1.84
7.48 Æ 1.89
5.48 Æ 0.74
4.88 Æ 0.54
1.91 Æ 0.8
5
6
7
8
9
10
11
12
13
14
15
16
17
Curcumin
MK 886*
Structure–activity analysis of the diarylpentanoid analogues
indicated that compounds (5 and 17) bearing diethylamino
substitutions at position 4 of both phenyl rings were
showing strong inhibition of mPGES-1 activity. In fact, the
4-diethylamino analogue (compound 5) was the most
potent compound among the 17 diarylpentanoid analogues
tested. Compound 11, which has a similar substitution
pattern on its phenyl ring structures but contains an acetone
linker, showed weaker activity than the latter. The presence
*30 lM concentration.
Values are the mean Æ SD; n = 3.
activity varied for different analogues at different concen-
trations. Compounds (1, 5, 6, 7, 13, and 17) showed
strong inhibitory effect on mPGES-1 activity with IC50
values ranging from 2.4 to 5.4 lM, respectively. The effects
of pure curcumin were also investigated on mPGES-1
activity. It was observed that curcumin inhibited mPGES-1
activity in a concentration-dependent manner, with an IC50
value of 4.88 lM. Previous studies showed that curcumin
of
N-methyl-N-(2-hydroxyethyl)-4-amino
substitutions
at position 4 of both phenyl rings resulted in a compounds
(1, 7, 13) with strong inhibitory activity. Substitution of
2-methyl-N-ethyl-N-(2-cyanoethyl)-4-amino at position 4 of
both phenyl rings (compound 6) produced a compound
Figure 4: Concentration-
dependent inhibitory effects of
diarylpentanoid analogues on
activity of microsomal prostaglandin
E synthase-1. Graph represents the
% inhibition by most active
compounds along with curcumin.
*p < 0.05, **p < 0.01, ***p < 0.001
are significant difference with
respect to curcumin. The data
shown are an average of three
independent experiment, and
values are mean Æ SD.
Chem Biol Drug Des 2014; 83: 670–681
679

Ahmad et al.
with strong inhibition of mPGES-1 activity. Compound 12,
which has a similar substitution pattern on its phenyl ring
structures but contains an acetone linker, showed weaker
activity than the latter.
7. Shakibaei M. (2007) Suppression of NF-kB activation
by curcumin leads to inhibition of expression of
cyclooxygenase-2 and matrix metalloproteinase-9 in
human articular chondrocytes; Implications for the
treatment of osteoarthritis. Biochem Pharmacol;73:
1434–1445.
Conclusions
8. Kohli K., Ali J., Ansari M., Raheman Z. (2005) Curcu-
min; a natural anti-inflammatory agent. Indian J Phar-
macol;37:141–147.
9. Chun K.S., Keum Y.S., Han S.S., Song Y.S., Kim S.H.,
Surh Y.J. (2003) Curcumin inhibits phorbol ester-
induced expression of cyclooxygenase-2 in mouse skin
through suppression of extracellular signal regulated
kinase activity and NF-kB activation. Carcinogene-
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promises. Mol Pharm;4:807–818.
In summary, we presented a series of novel diarylpenta-
noid analogues of curcumin and evaluated their potential
inhibitory activities against the enzymes involved in bio-
synthesis of arachidonic acid and its metabolites. Out of
the series employed, several derivatives were able to
inhibit the activity of PLA2, COX, LOX, and mPGES-1.
Inhibition of these enzymes by diarylpentanoid analogues
enlightens the anti-inflammatory property of these com-
pounds. It raises the possibility that diarylpentanoid ana-
logues of curcumin might serve as a useful starting
point for the design of new and improved anti-inflamma-
tory agents.
Acknowledgments
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analysis for b-diketone cleavage by the non-heme
The work was supported by a grant (No. 05-01-02-
SF1022) from the Ministry of Agriculture Malaysia.
Fe2+ dependent dioxygenase dke1.
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