Pharmacological evaluation and docking studies of α,β- unsaturated carbonyl based synthetic compounds as inhibitors of secretory phospholipase A2, cyclooxygenases, lipoxygenase and proinflammatory cytokines

Article abstract of DOI:10.1016/j.bmc.2014.05.052

Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α,β- unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A₂ (sPLA₂), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six α,β-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA₂ activity, with IC₅₀ values in the range of 2.19-8.76 μM. Nine compounds 1-4 and 10-14 displayed inhibition of COX-1 with IC₅₀ values ranging from 0.37 to 1.77 μM (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these α,β-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents.

Full text of DOI:10.1016/j.bmc.2014.05.052

Accepted Manuscript
Pharmacological Evaluation and Docking Studies of α, β-Unsaturated Carbonyl
Based Synthetic Compounds as Inhibitors of Secretory Phospholipase A2, Cy-
clooxygenases, Lipoxygenase and Proinflammatory Cytokines
Syed Nasir Abbas Bukhari, Gianluigi Lauro, Ibrahim Jantan, Giuseppe Bifulco,
M.W. Bin Amjad
PII:
S0968-0896(14)00411-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.052
BMC 11613
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 April 2014
21 May 2014
22 May 2014
Please cite this article as: Bukhari, S.N.A., Lauro, G., Jantan, I., Bifulco, G., Bin Amjad, M.W., Pharmacological
Evaluation and Docking Studies of α, β-Unsaturated Carbonyl Based Synthetic Compounds as Inhibitors of
Secretory Phospholipase A2, Cyclooxygenases, Lipoxygenase and Proinflammatory Cytokines, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.052
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Graphical Abstract
Pharmacological Evaluation and Docking Studies of α, β-Unsaturated Carbonyl Based
Synthetic Compounds as Inhibitors of Secretory Phospholipase A2, Cyclooxygenases,
Lipoxygenase and Proinflammatory Cytokines
Syed Nasir Abbas Bukhari, Gianluigi Lauro, Ibrahim Jantan, Giuseppe Bifulco, M.W. Bin
Amjad
1

Pharmacological Evaluation and Docking Studies of α, β-Unsaturated Carbonyl Based
Synthetic Compounds as Inhibitors of Secretory Phospholipase A2, Cyclooxygenases,
Lipoxygenase and Proinflammatory Cytokines
Syed Nasir Abbas Bukhari1*, Gianluigi Lauro2, Ibrahim Jantan1*, Giuseppe Bifulco2, M.W. Bin
Amjad1
1. Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan
Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
2. Dipartimento di Farmacia,Università di Salerno, Via Giovanni Paolo II 132, 84084
Fisciano (SA), Italy.
* Authors to whom correspondence should be addressed;
E mail: (SNAB) snab@pharmacy.ukm.my, snab_hussaini@yahoo.com ;
(IJ) ibj@pharmacy.ukm.my
Tel: +6-01123695295
Fax: +6-0326983271
2

ABSTRACT
Arachidonic acid and its metabolites have generated high level of interest among researchers due
to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid
metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α, β-
unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory
activity on secretory phospholipase A2 (sPLA2), cyclooxygenases (COX), soyabean
lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six
α, β-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of
sPLA2 activity, with IC50 values in the range of 2.19 to 8.76 µM. Nine compounds 1-4 and 10-14
displayed inhibition of COX-1 with IC50 values ranging from 0.37 to 1.77 µM (lower than that of
reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The
compounds 10-14 exhibited strong inhibitory activity against LOX enzyme. All compounds were
evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the
macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were
found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular
docking experiments were performed to clarify the molecular aspects of the observed COX and
LOX inhibitory activities of the investigated compounds. Present findings increases the
possibility that these α, β-unsaturated carbonyl based compounds might serve as beneficial
starting point for the design and development of improved anti-inflammatory agents.
KEYWORDS: Tumor necrosis factor alpha; lipopolysaccharides; curcumin; Claisen-
Schmidt condensation.
3

1. INTRODUCTION
Arachidonic acid and its metabolites have generated great interest due to their important role in
inflammation. The important targets for the design of novel and safe anti-inflammatory agents
include the inhibition of enzymes involved in arachidonic acid metabolism as secretory
phospholipase A2 (sPLA2); cyclooxygenase (COX) and lipoxygenase (LOX) and also pro-
inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The
metabolism of arachidonic acid leads to the formation of lipid mediators. Phospholipases A2
(PLA2s) are abundant enzymes that catalyze the hydrolysis of ester bond at the sn-2 position of
glycerophospholipids. The released fatty-acid, such asarachidonic acid (AA), may be
enzymatically metabolized into strong pro-inflammatory mediators known as eicosanoids
(prostaglandins, leucotrienes and thromboxanes), though the lyso-phospholipid, other product of
the PLA2 catalyzed reaction in the case of lyso-platelet-activating-factor (lyso-PAF), might be
converted into PAF (another renowned pro-inflammatory mediator) by the enzyme PAF-
acetyltransferase. The suppression of production of lipid mediators has long been considered as
therapeutical strategy due to their involvement in various pathological processes1.
The first two steps in the biosynthesis of prostaglandins (PGs) are catalyzed by
cyclooxygenase (COX) from the substrate arachidonic acid. At least two forms of this enzyme
are known to exist2-3. Among them, COX-1 is constitutively expressed and maintains normal
physiologic function and the PGs produced by this enzyme play a protective role. The other form
cyclooxygenase-2 (COX-2), is an inducible form and its expression is affected by several stimuli
including mitogens, oncogenes, tumorpromoters, and growth factors3. LOX enzymes catalyze
arachidonic acid into leukotrienes and lipoxins. These mediators play a vital role during
inflammation. Leukotrienes and prostaglandins are known to enhance the inflammatory reaction
4

whereas; the lipoxins have primarily anti-inflammatory effect. The important role of arachidonic
acid and its metabolites in cancer biology and various types of cardiovascular diseases has
developed interest amongst researchers. Latest research in the field of inflammation has given
valuable information for the better understanding of modulation of arachidonic acid metabolism.
The inhibition of enzymes involved in arachidonic acid metabolism has been deliberated as the
synergistic anti-inflammatory effect with enhanced spectrum of activity4.
Inflammation is a common phenomenon that is related to several diseases including
cardiovascular diseases and cancer5-7. The pro-inflammatory cytokines, IL-6 and TNF-α are
involved in the pathogenesis of numerous inflammatory disorders including rheumatoid arthritis
(RA), inflammatory bowel disease, osteoarthritis, psoriasis, endotoxemia and/or toxic shock
syndrome8-16. Regardless of pro-inflammatory attributes, these cytokines have widespread
functions for retaining the normal cellular physiology. TNF-α can induce apoptosis and secretion
of cytokines such as IL-1, IL-6 and IL-10 and can also activate T cells and other inflammatory
cells. Conversely, an excess of TNF-α and IL-6 is attributed to the development of several
human diseases as well as inflammatory disorders. The inhibition of cytokines, particularly TNF-
α, has been successful in several clinical trials for the treatment of RA. Hence, the inhibition of
TNF-α, pro-inflammatory cytokines, and over-expressions of cytokines has been recognized as
an attractive target for the design and development of novel anti-inflammatory agents17-20
.
According to literature, curcumin and related α, β-unsaturated carbonyl based compounds
exhibit a variety of pharmacological activities including potent antioxidant21, anti-
inflammatory22-24
,
antiviral24,
antibacterial25,
antifungal26,
antitubercular27
and
immunomodulatory properties28. Recently, we have reported the synthesis and effects of
curcumin-like α, β-unsaturated carbonyl based compounds on secretory phospholipase A2,
5

cyclooxygenases, lipoxygenase, microsomal prostaglandin E Synthase-1 and immune system
respectively28-29. Some compounds were found to be potent anti-inflammatory agents. These
interesting findings encouraged us to synthesize a novel series of α, β-unsaturated carbonyl based
compounds and evaluate their effects on enzymes involved in arachidonic acid metabolism and
pro-inflammatory cytokines. Molecular docking experiments were also performed to clarify the
molecular basis of the observed COX and LOX inhibitory activities of the investigated α, β-
unsaturated carbonyl based compounds.
2. RESULTS AND DISCUSSION
2.1.
Chemistry
Thirty novel curcumin related compounds (ten different types) were synthesized as reported
before28-29. The α, β-unsaturated carbonyl based compounds (1-7; 11-17; 21-27) were
synthesized by a Claisen-Schmidt condensation28 between various ketones and the appropriate
aryl aldehyde ata molar ratio of 1:2 in ethanol in the presence of NaOH. Moreover, some
compounds were synthesized in acetic acid by passing through the mixture of dry HCl gas. Nine
compounds (8-10; 18-20; 28-30) related to chalcone analogues were synthesized by similar
Claisen-Schmidt condensation at a molar ratio 1:1 of ketone and aldehyde (Scheme 1). Eight
compounds (7,8, 10, 17, 18, 20, 27,28) were synthesized by using both NaOH and dry HCl gas
mixture. The catalytic system using HCl gas mixture gave all compounds more efficiently, with
high percentage yield as compared to the catalytic system involving NaOH. A mixture ofseveral
unidentified products was also formed when NaOH was used and extensive purification was
required (column chromatography). Synthesized compounds were purified by recrystallization
and column chromatography using appropriate solvent systems. All thirty synthesized
6

compounds were characterized by using different spectroscopic techniques. Elemental analysis
of C, H, N, and melting points of the compounds were also determined.
2.2.
Inhibition of secretory phospholipase A2-V
The inhibition of PLA2 represents a key strategy for the prevention of inflammation. The
inhibitory potency of all compounds (at a concentration of 1.25 to 20 µg/mL) against sPLA2 was
evaluated by an in vitro enzymatic photometric assay based on Ellman’s method (Table 1).
Curcumin and dexamethasone were positive controls. Curcumin which was used as reference
compound inhibited the sPLA2 activity by 81.57% (IC50 11.10 µM). The previous studies on
other isoforms of PLA2 reported that curcumin inhibited the activity of PLA2, and the isoforms
of PLA2 enzymes showed more than 70% homology30-31. Compounds 2, 3, 4, 12, 13 and 14
strongly inhibited sPLA2-V in a dose dependent manner (Figure 1), with IC50 2.19 to 8.76µM,
which were lower or comparable to curcumin (10.12 µM).
2.3.
Inhibition of COX-1 and COX-2
Modulation of arachidonic acid metabolism by inhibiting COX-1 and COX-2 is considered as
one of the anti-inflammatory mechanisms. The cyclooxygenase (COX-1 and COX-2) inhibitory
activity of α, β-unsaturated carbonyl based compounds was determined by in vitro COX-inhibitor
screening assay at a concentration of 40 µg/mL31. The IC50 values were calculated and presented
in Table 1. The COX-1 and COX-2 inhibitory effect of these α, β-unsaturated carbonyl based
compounds was compared with pure curcumin. The α, β-unsaturated carbonyl based compounds
showed affinity towards COX-1, with compounds (1, 2, 3, 4, 10, 11, 12, 13 and 14) exhibiting
strong inhibitory effect (81.79 - 92.22%) on the activity of COX-1. Curcumin was used as a
reference compound while Indomethacin served as positive control. Most of the compounds
7

exhibited better COX-1 inhibitory activity than that of curcumin with IC50 0.37 to 21.54 µM,
which was lower than that of curcumin (29.55 µM); however the inhibitory activity of compound
20 was comparable with curcumin with IC5029.33 µM. The compound 4 showed highest COX-1
inhibition (IC50 0.37 µM) followed by compound 11 (IC50 0.42 µM). The compounds were less
efficient in suppressing the activity of COX-2, as compared to COX-1. Six compounds (2, 7, 10,
13, 14 and 18) exhibited COX-2 inhibitory activity with IC50<15 µM. Compound 13 showed
highest COX-2 inhibition with IC50 7.88 µM. Majority of compounds displayed better COX-2
inhibitory activity with IC50<100 µM. It was observed that the compounds inhibited COX
activity in a dose-dependent manner. These results suggest that α, β-unsaturated carbonyl based
compounds act as inhibitors of COX enzymes and exhibited anti-inflammatory effects.
2.4.
In vitro inhibition of soybean lipoxygenase
We evaluated the ability of our synthesized compounds to inhibit soybean lipoxygenase.
Curcumin was used as reference. Curcumin has been reported to inhibit 5-LOX activity both in
in vitro and in vivo models and was used as reference in this assay32-33. Katsori et al. reported the
inhibitory effect of curcumin on soybean LOX 34. The inhibitory effect of our newly synthesized
compounds (at concentration of 2.5 to 40 µg/mL) on LOX was determined (Table 1). The
preliminary screening revealed that compounds 4, 10, 11, 12, 13 and 14 strong inhibitors of LOX
with percentage inhibitions ranging from 65 - 85 %. DMSO was used as negative control and did
not affect the activity of enzyme. At 40 µg/mL, compounds 4 and 10 (67.57 and 69.52 %) were
equipotent to curcumin (68.28%). The compounds 10, 11, 12, 13 and 14 had IC50 values ranging
from 29.28 to 52.22 µM, which were lower or comparable to that of curcumin (59.52 µM). The
compound 11 exhibited strongest LOX inhibitory activity (82.72%) among all the compounds,
8

with IC5029.28 µM. Hence, it can be concluded that the compounds inhibited LOX activity in a
dose-dependent manner (Figure 2).
2.5.
Inhibition of TNF-α and IL-6 release in LPS-stimulated macrophages
Lipopolysaccharide (LPS) is a well-studied immune stimulator that induces a systemic
inflammation response 35, particularly, the expression of proinflammatory cytokines, such as
TNF-α and IL-6. The inhibitory effect of new compounds against LPS-induced TNF-α and IL-6
release in mouse macrophages (RAW 264.7) was evaluated. The macrophages were pre-treated
with 10 µM compounds for 2 h followed by the incubation with 0.5 µg/ml LPS for 22 h.
Enzyme-linked immunosorbant assays (ELISA) was performed to detect the amount of TNF-α
and IL-6 in media.
The results of the enzyme-linked immunosorbant assay of α, β-unsaturated carbonyl based
compounds are presented in Figure 3. Compounds 2, 3, 4, 7, 12, 13 and 14 exhibited strongest
percentage inhibitory activity (73 to 91 %) against LPS-induced TNF-α expression, while
compounds 3, 4, 6, 12, 13, 14 and 16 showed strong inhibitory effects (76 - 91 %) against IL-6
release as compared to the LPS-control. Compounds 3, 4, 13 and 14 were more potent than
curcumin at the same concentration in inhibiting LPS-induced TNF-α and IL-6 expression.
Compounds 3 and 14, displayed the strongest inhibitory effect (91%) on LPS-induced TNF-α
and IL-6 release among all compounds, compared to the LPS-control. On the other hand, three
compounds (9, 27 and 28) showed immunostimulatory effects and increased the release of TNF-
α from stimulated macrophages and similarly three compounds (19, 28 and 30) showed increase
in release of IL-6.
2.6.
Structure-activity relationship analysis of α, β-unsaturated carbonyl based
compounds
9

All α, β-unsaturated carbonyl based compounds were evaluated for their inhibitory effects on
sPLA2, COX and LOX enzymes (Table 1) and on release of TNF-α and IL-6 (Figure 3). The
analysis of results and structure-activity relationship (SAR) revealed that compounds 3, 4, 13 and
14 exhibited strong inhibitory effects on four enzymes of arichidonic acid metabolism and on
release of TNF-α and IL-6 from macrophages.
After analyzing SAR, it can be observed that that the linkers derived from ketones during
synthesis play important roles in terms of inhibitory activity. Variable activity trends were seen
amongst different series of α, β-unsaturated carbonyl based compounds when either linear or
cyclic linkers were used between the two aromatic rings of compounds. Generally, N-methyl-4-
piperidone linker containing compounds (4, 14, 24) showed potent anti-inflammatory activities,
moderate activity was exhibited by compounds (3, 13, 23) with 4-piperidone linker, whereas
three compounds (2, 12, 22) with tetrahydropyran-4-one displayed good inhibition power, yet it
was lesser as compared to compounds having 4-piperidone linker. Bis-derivatives, possessing
either1-Benzyl-4-piperidone (5, 15, 25), acetone (6, 16, 26) or cyclopentanone (7, 17, 27) linker,
were less active as compared to the others, however compound 7 exhibited good inhibition of IL-
6 and COX-1. Compounds 4 and 14 containing N-methyl-4-piperidone linker were observed to
be strong inhibitors of sPLA2, COX-1, LOX and cytokines. Synthetic α, β-unsaturated carbonyl
based compounds possessing the 4-piperidone moiety (3and 13) also showed good inhibitory
activity but comparatively weaker as compared to N-methyl-4-piperidone linker, except
compound 3 that exhibited more inhibition of TNF-α release in contrast to compound 4.
Compounds 23 and 24 containing 4-piperidone and N-methyl-4-piperidone linker did not show
strong anti-inflammatory activities in any assay that can be result of pyrrolidine introduction at
10

position 4 of the aromatic rings. It can be further concluded that the presence of pyrolidine at
position 4 of compounds (21-30) rendered the compounds only slightly active.
Effects of substitution of various groups on aromatic rings of compounds were also
compared. It was seen that introduction of 2-nitro-4-dimethylamine combination on the aromatic
rings enhanced the anti-inflammatory activity by increased inhibition of sPLA2, COX-2, LOX,
IL-6, and TNF-α. In all the three groups (based on substitution pattern 1-10, 11-20,21-30),
compounds bearing diethoxymethyl group at position 4 of rings showed significantly reduced
anti-inflammatory activity except 3 and 4 which showed more COX-1 inhibition in comparison
to compound 13 and 14. Moreover, the presence of pyrolidine at position 4 of compounds (21-
30) resulted in compounds which were not highly active.
2.7.
Docking studies
2.7.1. COX-1/COX-2
Molecular docking experiments were employed in order to clarify the molecular basis of the
observed COX inhibitory activities of the investigated curcumin-like α, β-unsaturated carbonyl
based compounds. All the compounds were docked into the 3-D COX-1 and COX-2 structures
complexed with indometacin (PDB codes: 1PGG36 for COX-1, and 4COX37 for COX-2, focusing
the conformational search in the related ligand binding sites (LBDs).
Regarding COX-1 predicted complexes, we generally observed a suitable
accommodation of the compounds in the related LBD. All the active compounds are able to
deeply occupy the binding cavity, establishing at the same time both polar and hydrophobic
interactions. In these cases, we found the presence of H-bonds between the investigated
compounds and Ser530, and/or Tyr355, and/or Arg120, thus respecting the binding mode
11

reported by Selvam et al. for other curcumin analogues38. The remaining hydrophobic parts of
the investigated compounds are involved in vdW interactions with the other residues belonging
to the COX-1 LBD, namely Leu93, Tyr355, Leu357, Tyr385, Leu384, Phe518, Ile523. In the
binding pose of the most active compound 4 is represented (Figure 4).
Compound 4 establishes several hydrophobic interactions along the whole COX-1 LBD.
In the most external part of the binding site, we found vdW contacts between the terminal
(diethoxymethyl)-benzyl part of 4 and Ile89, Leu93, Trp100, Leu112, Leu115, Val116, and
Leu357. The central core of the molecule is involved in a hydrogen bond between the lactone
oxygen and terminal -OH in the side chain of Tyr355 and in polar contacts with Arg120, while
other hydrophobic interactions are detectable with Tyr348 and Leu359. The other terminal
(diethoxymethyl)-benzyl substituent, located in the internal part of the COX-1 LBD, establishes
a H-bond with Ser530 and vdW contacts with Val349, Leu352, Tyr385, Trp387, Ile523, Gly526
and Ala527 (Figure 4).
Concerning inactive compounds, docking models highlight the lack of fundamental
interactions with the protein counterpart (Supporting Information)
As known, COX-2 differs from COX-1 for several amino acidic substitutions in the
related LBD (V523I, V434I, R513H, and A516S). The main structural differences between the
two isoforms are related to the first two modifications, and in particular the presence of two
valins instead of isoleucins determines a larger space in the COX-2 LBD that can be exploit for
the design of selective inhibitors. We generally observed a decrement of the experimental
inhibitory activity of the investigated compounds that are generally less fitted in the larger COX-
2 binding site. For example, compound 4 is in this case placed in the LBD with a reduced shape
12

complementarity, establishing at the same time less contacts with the receptor counterpart.
Moreover, an inversion of the central core is also observable, determining the loss of the H-bond
with Tyr355 (Figure 5).
Compounds 5, 9, 11, 22, 24 and 29 showed a selective inhibition for COX-1, being
inactive to COX-2. Once again, this can be ascribed to the larger COX-2 LBD, that determines
the occupation of the additional space behind the channel generated by the V523I substitution
instead of the deeper part of the binding groove. For example, compound 11 oriented one of its
4-(dimethylamino)-2-nitro-benzyl group in this additional cavityin the binding site, losing the
interactions with Ser530 and the related close residues (Figure-supporting information).
2.7.2. Docking on Soybean Lipoxygenase (LOX)
As reported by Jankun et al.,39-40 curcumin is able to interact with soybean LOX binding its
central cavity, close to the residues directly contacting with Iron cofactor. The different crystal
structures of this protein are characterized by a restricted internal space, that makes necessary the
use of flexible docking procedures to rightly dock the investigated compounds41. In particular,
starting from crystal structure of soybean LOX (PDB code1HU9), five key residues were chosen
to be flexible during docking experiments: Ser510, Gln514, Trp519, Arg562, and Phe576.
Compound 11, showing the better inhibitory activity on the investigated target, occupies
this binding cavity establishing several polar (Ser510, His513, His523, Glu527, Arg562, Thr575,
Gln716, Arg726, Asp766) and hydrophobic interactions (Tyr207, Leu565, Ile770, Leu773),
while one of the nitro groups of the molecule points to the iron cofactor (Figure 6).
Active compounds not showing nitro groups are able to bind iron in different ways; for
example, compound 10 can interact with the cofactor or with the terminal -(dietoxymehyl)phenil
13

group, or thanks to the diketo-moiety on the opposite part of the molecule (Figure 7). Also in this
case, compounds showing the pyrrolidine terminal groups are inactive, and this is mainly due to
their minor flexibility preventing the productive interaction with the metal in the LBD (e.g.,
compound 27, Figure in supporting information).
3. CONCLUSIONS
Herein, we report the pharmacological evaluation and molecular modeling of thirty novel α, β-
unsaturated carbonyl based synthetic compounds. We evaluated their inhibitory activities against
the enzymes involved in biosynthesis of arachidonic acid and its metabolites. Among these
compounds, numerous derivatives were able to inhibit the activity of PLA2, COX, LOX, IL-6
and TNF-α. The most potent enzyme and cytokine inhibitors in this series correspond to N-
methyl-4-piperidone and 4-piperidone moieties. Inhibition of these enzymes and cytokines by α,
β-unsaturated carbonyl based compounds highlights the anti-inflammatory property of these
compounds. It increases the possibility that curcumin related α, β-unsaturated carbonyl based
might serve as a starting point for the design and development of new and improved anti-
inflammatory agents.
4. EXPERIMENTAL SECTION
4.1.
Materials
All reagents and chemicals were purchased from Sigma-Aldrich, Merck and Acros Organics
(above 98% purity) and were used without further purification. Chemicals used in sPLA2 assay
include 1,2-bis(heptanoylthio)-phosphatidylcholine, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB),
and recombinant human PLA2-V from Cayman Chemicals U.S.A. CaCl2, KCl, and HCl were
purchased from Merck, Germany. Curcumin, dimethyl sulphoxide (DMSO), ethylene diamine
14

tetra acetic acid (EDTA), reduced glutathione (GSH) and SnCl2 were obtained from Sigma-
Aldrich Steinheim, Germany. Cyclooxygensae activity was determined by using a COX Inhibitor
Screening Kit from Cayman Chemicals U.S.A. Soyabean lipooxygenase, arachidonic acid and
potassium hydroxide were also obtained from Cayman Chemicals U.S.A.TNF-α and IL-6 in the
media were determined by ELISA using mouse TNF-α (Catalogue No. 500850) and mouse IL-6
(Catalogue No. 583371) ELISA Kits (Cayman, USA). Mouse RAW 264.7 macrophages were
obtained from Abcam, UK (Cambridge, ENG). Cell culture reagents and fetal bovine serum
(FBS) were obtained from Sigma Aldrich and FBS was heat-inactivated for 30 min at 65°C. LPS
purchased from Sigma was dissolved in PBS. Synthetic compounds were dissolved in DMSO.
4.2.
General Procedures
1
13
JEOL ECP spectrometer operating at 500 MHz was used to record H and C NMR spectra,
with Me4Si as internal standard and CDCl3 or DMSO-d6 as the solvents. Electrospray ionization
mass spectrometry (ESI-MS) on MicroTOF-Q mass spectrometer (Bruker) recorded high
resolution mass spectra (HRMS) of the compounds. Microanalyses of the compounds were
carried out on Fison EA 1108 elemental analyzer. KBr disc method was used to record infrared
spectra on a Perkin Elmer 400 (FTIR) spectrometer. Silica gel 60 (230-400mesh) (Merck) was
used for flash column chromatography, whereas thin layer chromatography (TLC) was carried
out on pre-coated silica plates (kiesel gel 60 F254, BDH). Melting points of the compounds were
determined on an electrothermal instrument.
4.3.
Synthesis of α, β-Unsaturated Carbonyl based Compounds
Twenty one α, β-unsaturated carbonyl based compounds (1-7; 11-17; 21-27) were synthesized by
coupling of suitable aromatic aldehyde with the ten types of ketones at a molar ratio of 1:2,
15

whereas nine (8-10; 18-20; 28-30) were synthesized at a ratio of 1:1, under base catalyzed
Claisen–Schmidt condensation reaction. Scheme 1 demonstrates the synthesis of α, β-unsaturated
carbonyl based compounds. The appropriate aromatic aldehyde (20 mmol, 2equivalant) and
suitable ketone (10 mmol, 1 equivalent) were mixed and dissolved in ethanol in a round
bottomed flask and stirred at 5°C for a few minutes. A 40% NaOH solution in ethanol was added
drop wise into this solution and the resulting mixture was allowed to stir at room temperature (27
°C) for 1-24 h. The appearance of precipitate and color changes of the reaction mixture acted as
indicative markers of product formation. The reaction was monitored by TLC and on
completion; the reaction was stopped by adding acidified ice. The α, β-unsaturated carbonyl
based compounds were isolated by either column chromatography or recrystallization.
4.3.1. 2,6-Bis[4-(diethoxymethyl)benzylidene]cyclohexanone (1)
1
Yellow crystals (2.59 g, 54%). mp: 112-114 ˚C; H NMR (500 MHz, CDCl3) δ: 7.89 (s, 2H),
7.29 (d, J=8Hz, 4H), 6.82 (d, J=8Hz, 4H), 5.89 (s, 2H), 3.42 (q, J=7.5, 8H), 2.32 (t, J=12.0 Hz,
13
4H), 1.82 (m, 2H), 1.24 (t, J=7.5, 12H); C NMR (500 MHz, CDCl3) δ: 186.4, 152.9, 144.4,
136.5, 132.5, 127.3, 126.5, 101.1, 55.0, 28.3, 27.8, 16.2;HRMS (ESI) m/z: 479.68 [M+H]+,
Microanalysis calculated for C30H38O5 (478.62), C: 75.28%, H: 8.00%. Found C: 75.42%, H:
8.12%.
4.3.2. 3,5-Bis-(4-diethoxymethyl-benzylidene)-tetrahydro-pyran-4-one (2)
Pale Yellow crystals (3.06 g, 64%). mp: 106-108 ˚C;1H NMR (500 MHz, CDCl3) δ: 7.85 (s, 2H),
7.32 (d, J=8Hz, 4H), 6.95 (d, J=8Hz, 4H), 5.52 (s, 2H), 3.62 (q, J=7.5, 8H), 2.69 (s, 4H), 1.18 (t,
13
J=8, 12H); C NMR (500 MHz, CDCl3) δ: 187.2, 152.7, 143.8, 136.7, 131.9, 126.2, 125.2,
16

101.5, 62.4, 55.2, 16.1; HRMS (ESI) m/z: 481.65 [M+H]+, Microanalysis calculated for
C29H36O6 (480.59), C: 72.48%, H: 7.55%. Found C: 72.62%, H: 7.72%.
4.3.3. 3,5-Bis[4-(diethoxymethyl)benzylidene]piperidin-4-one (3)
Yellow solid (2.89 g, 60%). mp: 101-102 ˚C;1H NMR (500 MHz, CDCl3) δ: 7.68 (s, 2H), 7.12
(d, J=7.5Hz, 4H), 6.93 (d, J=7.5Hz, 4H), 5.65 (s, 2H), 3.36 (q, J=7.5, 8H), 2.65 (s, 4H), 1.27 (t,
13
J=7.0, 12H); C NMR (500 MHz, CDCl3) δ: 185.2, 145.1, 144.5, 138.5, 136.2, 128.1, 127.8,
102.1, 55.1, 48.7, 16.4 ;HRMS (ESI) m/z: 480.71 [M+H]+, Microanalysis calculated for
C29H37NO5 (479.61), C: 72.62%, H: 7.78%, N: 2.92%. Found C: 72.71%, H: 7.86%, N: 3.10%.
4.3.4. 3,5-Bis[4-(diethoxymethyl)benzylidene]-1-methyl-piperidin-4-one (4)
Light Yellow solid (2.12 g, 43%).mp: 142-144 ˚C;1H NMR (500 MHz, CDCl3) δ: 7.63 (s, 2H),
7.32 (d, J=8Hz, 4H), 7.15 (d, J=8Hz, 4H), 5.59 (s, 2H), 3.29 (q, J=7.5, 8H), 2.71 (s, 4H), 2.12 (s,
3H), 1.20 (t, J=7.0, 12H);13C NMR (500 MHz, CDCl3) δ: 189.7, 148.2, 147.5, 139.8, 135.1,
128.4, 127.1, 101.4, 56.6, 51.2, 40.1, 16.7 ;HRMS (ESI) m/z: 494.65 [M+H]+, Microanalysis
calculated for C30H39NO5 (493.63), C: 72.99%, H: 7.96%, N: 2.84%. Found C: 72.95%, H:
7.99%, N: 2.87%.
4.3.5. 1,5-Bis-(4-diethoxymethyl-phenyl)-penta-1,4-dien-3-one (6)
Pale brownish solid (1.96 g, 45%). mp: 98-100 ˚C;1H NMR (500 MHz, CDCl3) δ: 7.82 (d,
J=6.5Hz, 4H),7.71 (d, J=8Hz, 2H) , 7.49 (d, J=6.5Hz, 2H), 7.16 (d, J=7Hz, 4H), 5.25 (s, 2H),
13
3.52 (q, J=7Hz, 8H), 1.17 (t, J=7.0, 12H); C NMR (500 MHz, CDCl3) δ: 190.5, 152.2, 148.6,
139.7, 136.4, 135.2, 132.8, 102.2, 55.5, 16.9 ;HRMS (ESI) m/z: 439.62 [M+H]+, Microanalysis
calculated for C27H34O5 (438.56), C: 73.94%, H: 7.81%. Found C: 74.12%, H: 7.89%.
17

4.3.6. 2,6-Bis-(4-dimethylamino-2-nitro-benzylidene)-cyclohexanone (11)
White powder (2.78 g, 62%). mp: 139-140 ˚C;δ: 7.94 (s, 2H), 7.55 (d, J=8Hz, 2H), 7.41 (d,
J=8Hz, 2H), 7.13 (s, 2H), 3.15 (s, 12H), 2.35 (t, J=12.0 Hz, 4H), 1.87 (m, 2H) ; 13C NMR (500
MHz, CDCl3) δ: 190.7, 149.5, 145.7, 145.6, 140.1, 128.2, 118.6, 117.6, 106.5, 46.8, 29.1,
27.5;HRMS (ESI) m/z: 451.64 [M+H]+, Microanalysis calculated for C24H26N4O5 (450.49), C:
63.99%, H: 5.82%, N: 12.44%. Found C: 64.12%, H: 5.72%, N: 12.42%.
4.3.7. 3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]tetrahydro-pyran-4-one (12)
White powder (2.92 g, 65%). mp: 188- 190 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.89 (s, 2H), 7.46
13
(d, J=8Hz, 2H), 7.14 (d, J=8Hz, 2H), 7.05 (s, 2H), 3.13 (s, 12H), 2.93 (s, 4H); C NMR (500
MHz, CDCl3) δ: 190.5, 149.2, 146.9, 145.1, 139.9, 128.4, 118.9, 118.1, 107.8, 65.5, 46.2;HRMS
(ESI) m/z: 475.52 [M+Na]+, Microanalysis calculated for C23H24N4O6 (452.46), C: 61.05%, H:
5.35%, N: 12.38%. Found C: 61.24%, H: 5.59%, N: 12.42%.
4.3.8. 3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]piperidin-4-one (13)
Light yellow solid (2.28 g, 51%). mp: 192- 194 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.82 (s, 2H),
13
7.72 (d, J=8Hz, 2H), 7.21 (d, J=8Hz, 2H), 6.92 (s, 2H), 3.17 (s, 12H), 3.13 (s, 4H); C NMR
(500 MHz, CDCl3) δ: 189.4, 149.7, 148.6, 142.5, 140.7, 127.3, 118.8, 118.1, 106.5, 49.4,
46.9;HRMS (ESI) m/z: 452.52 [M+H]+, Microanalysis calculated for C23H25N5O5 (451.48), C:
61.19%, H: 5.58%, N: 15.51%. Found C: 61.42%, H: 5.62%, N: 15.89%.
4.3.9. 3,5-Bis[4-(dimethylamino)2-nitro-benzylidene]-1-methyl-piperidin-4-one (14)
White powder (2.56 g, 55%). mp: 181- 182 ˚C; 1H NMR (500 MHz, CDCl3) δ: 7.76 (s, 2H), 7.45
(d, J=8Hz, 2H), 7.23 (d, J=8Hz, 2H), 7.13 (s, 2H), 3.21 (s, 12H), 3.03 (s, 4H), 2.19 (s, 3H); 13C
18

NMR (500 MHz, CDCl3) δ: 187.5, 148.9, 148.2, 143.2, 141.8, 126.7, 119.6, 118.9, 106.2, 44.5,
46.1, 38.8;HRMS (ESI) m/z: 466.72 [M+H]+, Microanalysis calculated for C24H27N5O5 (465.50),
C: 61.92%, H: 5.85%, N: 15.04%. Found C: 61.99%, H: 5.91%, N: 15.19%.
4.3.10. 1,5-Bis-(4-dimethylamino-2-nitro-phenyl)-penta-1,4-dien-3-one (16)
Pale yellow solid (2.97 g, 68%). mp: 177-179 ˚C;1H NMR (500 MHz, CDCl3) δ: 7.70 (d, J=6Hz,
2H), 7.68 (d, J=8Hz, 2H), 7.44 (d, J=6Hz, 2H),7.25 (d, J=8Hz, 2H), 7.12 (s, 2H), 3.15 (s, 12H);
13C NMR (500 MHz, CDCl3) δ: 187.5, 150.9, 148.6, 144.4, 132.8, 128.1, 117.5, 113.8, 107.9,
46.7; HRMS (ESI) m/z: 409.45 [M-H]+, Microanalysis calculated for C21H22N4O5 (410.42), C:
61.45%, H: 5.40%, N: 13.65%. Found C: 61.48%, H: 5.62%, N: 13.77%.
4.4.
Secretory Phospholipase A2 -V (sPLA2-V) Activity Assay
The effect of α, β-unsaturated carbonyl based compounds on the activity of sPLA2 enzyme was
investigated by a photometric assay based on Ellman’s method42. Briefly, the hydrolysis of sn-
2ester bond of the substrate 1,2-bis(heptanoylthio)-glycerophosphocholine by PLA2-V resulted
in exposure of free thiols. These thiols triggered the alteration of DTNB (5,5–dithio-bis-(2-
nitrobenzoic acid) to 2-nitro-5-thiobenzoic acid, which was detected photometrically at 405 nm.
Afterwards, the assay was proceeded in an aqueous buffer solution (pH 7.5) containing KCl(94
mM), CaCl2 (9mM), Tris (24 mM) and Triton-X 100 (280 BM ). Prior to the start of assay,
substrate and PLA2-V were re- suspended in assay buffer, and DTNB was dissolved in a solution
of Tris-HCl (pH 8).The enzyme and DTNB yielded final concentrations of 100 ng/mL and 87
BM, respectively. The assay was performed in 96-well microliter plates, containing DTNB,
substrate solution and the respective test substance. The maximum (100%) activity of the
19

enzyme was calculated by adding substrate and enzyme only. DMSO was used as negative
control and was inactive at the concentration used in the assay (1.7% v/v).
4.5.
Cyclooxygenase Assay
The effect of α, β-unsaturated carbonyl based compounds on COX-1and COX-2 was determined
by using a COX Inhibitor Screening Kit (Catalog No 560131) from Cayman Chemicals, Ann
Arbor, Michigan, U.S.A. The reaction mixture was prepared with 1µM heme and COX-1 (ovine)
or COX-2 (human recombinant) enzymes in 100mM Tris-HCl buffer, and then pre-incubated for
10 min in a water bath (37 °C) at pH 8.0. The reaction was started by adding 10µl arachidonic
acid (final concentration in reaction mixture 100µM). After 2 min, the reaction was terminated
by adding 1M HCl and lastly PGE2 was quantified by ELISA. The compounds were dissolved in
DMSO and diluted to desired concentration with potassium phosphate buffer. After transferring
the compounds to 96 well plate coated with a mouse anti-rabbit IgG, tracer prostaglandin
acetylcholine esterase and primary antibody (mouse anti PGE2) were added and the plates were
kept for overnight. Thereafter, the reaction mixtures were removed and the wells were washed
with 10mM potassium phosphate buffer containing 0.05% Tween 20. Ellman’s reagent (200µl)
was then added to all the wells and were incubated at room temperature for 60 min, until the
control wells gave an OD=0.3-0.8 at 412 nm. A standard curve with PGE2 was generated from
the same plate, which was used to calculate the PGE2 levels produced in the presence of test
compounds. The results were expressed as percentage relative to control (solvent treated
samples). All these experiments were performed in triplicate and values normally agreed within
10%.
4.6.
Lipoxygenase Activity Assay
20

The effect of α, β-unsaturated carbonyl based compounds on purified LOX enzyme was
investigated by a standard colorimetric assay43. Soybean lipoxygenase was used as enzyme
source. The assay was performed in 0.1 M Tris-HCl buffer solution (pH 7). Before the start of
the assay, LOX enzyme was re-suspended in buffer. The substrate was dissolved in equal volume
of potassium hydroxide, vortexed and diluted to get the concentration of 1 mM. The assay was
performed in 96-well microliter plates at room temperature containing substrate, enzyme and test
substance. The maximum (100%) activity of enzyme was calculated by adding the substrate and
enzyme. To determine the activity, 90 BL of LOX and 10 BL of respective test sample was
added. The reaction was initiated by adding substrate solution to all wells for 5 min. After that,
100 BL of chromogen solution was added to all wells for 5 min to stop enzyme catalysis. In the
next step,the absorbance was measured at 490 nm by using Tecan® Infinite Pro 200microplate
reader.
4.7.
Cell treatment and ELISA assay for TNF-α and IL-6
Mouse RAW 264.7 macrophages were incubated in DMEM supplemented with 10% FBS, 1%
Pen Strep at 37°C with 5% CO2. The cells were pre-treated with 10 µM α, β-
unsaturated carbonyl based compounds or vehicle control for 2 h, and then treated with LPS (0.5
µg/ml) for 22 h. The culture medium and cells were collected separately after the treatment. The
medium collected was centrifuged at 1000 rpm for 10 min. The levels of TNF-α and IL-6 in the
medium were measured by mouse TNF-α and mouse IL-6 ELISA Kits (Cayman, USA). The
supernatant obtained after centrifugation was separated and stored at -80°C until use. The cells
were washed with PBS and harvested with cell lysis buffer (Tris-HCl 20 mM, NP40 1%, NaCl
150 mM, EDTA 2 mM,Na3VO4 200 mM, SDS 0.1%, NaF 20 mM). The mixed liquor was
shaken vigorously for 10 min in lysis buffer at 0°C. After centrifugation at 12,000 rpm for 30
21

min at 4°C, the total protein was collected and the concentrations were calculated using Bio-Rad
protein assay reagents. The total amount of inflammatory factor in the medium was normalized
to the total protein amount of the viable cell pellets.
4.8.
Computational details
The chemical structures of investigated compounds were built with Maestro (version 9.6)44 Build
Panel and then prepared with LigPrep software. We applied an optimization (Conjugate
Gradient, 0.05 Å convergence threshold) of the structures to identify possible three-dimensional
starting models of each compounds for the subsequent docking calculations. Then, all the
structures were converted in the .pdbqt format using OpenBabel software (version 2.3.2)45,
adding Gasteiger charges.
Protein 3D models of COX-1, COX-2, and Soybean LOX were prepared starting from the X-ray
structures in the Protein Data Bank database (PDB codes: 1PGG for COX-1, 4COX for COX-2,
and 1HU9 for Soybean LOX).Water molecules were removed, and .pdb files obtained were then
processed with Autodock Tools 1.5.6 and converted in .pdbqt format, merging non polar
hydrogens and adding Gasteiger charges. Charge deficit was spread over all atoms of related
residues.
Docking calculations were performed using the Autodock-Vina software46. In the configuration
files linked to 3D structures of the proteins, we specified coordinates and dimensions along x,y,z
axes of the grid related to the site of presumable pharmacological interest, with spacing of 1.0 Å
between the grid points. In particular, for COX-1 target, we set a grid of 24×20×24 and centered
at 27.631 (x), 33.575 (y), and 210.355 (z). Regarding COX-2, the grid was centered at 25.453
(x), 17.467 (y), 17.793 (z), and with dimensions of 20×22×22.The exhaustiveness value was set
22

to 64, saving 30 conformations as maximum number of binding modes. Regarding Soybean
LOX target, we employed Autodock-Vina flexible docking procedure, choosing residues Ser510,
Gln514, Trp519, Arg562, and Phe576 as flexible and the rest of the protein as rigid. We set a
grid of 224×28×20 and centered at 21.156 (x), 3.403 (y), and 20.652 (z), setting the
exhaustiveness to 64, and saving 30 conformations as maximum number of binding modes.
For all the investigated compounds, all open-chain bonds were treated as active torsional bonds.
Docking results were analyzed with Autodock Tools 1.5.6. Illustrations of the 3D models were
generated using Autodock Tools 1.5.647,VMD48 and Maestro software.
4.9.
Statistical analysis
All the experiments were performed in triplicates and data presented as the mean standard
error of mean (S.E.M.). The IC50 values were calculated by using Graph Pad Prism 5 software.
Data was analyzed using a one-way analysis of variance (ANOVA) for multiple comparisons. P
< 0.05 was considered to be statistically significant.
ACKNOWLEDGMENTS
Syed Nasir Abbas Bukhari thanks the Universiti Kebangsaan Malaysia for postdoctoral contract.
This work was supported by University Kebangsaan Malaysia (Grant No: UKM-DLP-2013-013)
Notes
The authors declare no competing financial interest.
23

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Schrödinger, LLC:. New York, NY, 2013.
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