Esters of some non-steroidal anti-inflammatory drugs with cinnamyl alcohol are potent lipoxygenase inhibitors with enhanced anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmcl.2015.10.036

Novel esters of non steroidal anti-inflammatory drugs, α-lipoic acid and indol-3-acetic acid with cinnamyl alcohol were synthesised by a straightforward method and at high yields (60-98%). They reduced acute inflammation more than the parent acids and are potent inhibitors of soybean lipoxygenase. Selected structures decreased plasma lipidemic indices in Triton-induced hyperlipidemia to rats. Therefore, the synthesised compounds may add to the current knowledge about agents acting against various inflammatory disorders.

Full text of DOI:10.1016/j.bmcl.2015.10.036

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Esters of some non-steroidal anti-inflammatory drugs with cinnamyl
alcohol are potent lipoxygenase inhibitors with enhanced
anti-inflammatory activity
⇑
Panagiotis Theodosis-Nobelos, Malamati Kourti, Paraskevi Tziona, Panos N. Kourounakis, Eleni A. Rekka
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, Thessaloniki 54124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel esters of non steroidal anti-inflammatory drugs,
a
-lipoic acid and indol-3-acetic acid with cin-
Received 1 September 2015
Revised 12 October 2015
Accepted 13 October 2015
Available online xxxx
namyl alcohol were synthesised by a straightforward method and at high yields (60–98%). They reduced
acute inflammation more than the parent acids and are potent inhibitors of soybean lipoxygenase.
Selected structures decreased plasma lipidemic indices in Triton-induced hyperlipidemia to rats.
Therefore, the synthesised compounds may add to the current knowledge about agents acting against
various inflammatory disorders.
Keywords:
Non steroidal anti-inflammatory drugs
Cinnamyl esters
Ó 2015 Elsevier Ltd. All rights reserved.
Inflammation
Lipoxygenase inhibition
Dyslipidemia
Nonsteroidal anti-inflammatory drugs (NSAIDs) modulate the
arachidonic acid cascade and are widely used for the treatment
of pathophysiological inflammatory conditions. Eicosanoids are
synthesised from arachidonic acid via three pathways. Cycloxyge-
nase (COX) enzymes catalyse the formation of prostaglandins and
thromboxanes; the lipoxygenase (LOX) pathway leads to leuko-
trienes and lipoxins, while epoxyeicosatrienoic acids are products
of the cytochrome P450 enzymes. These lipid mediators generate
both inflammatory and anti-inflammatory responses, through a
complex communication between the arachidonic acid pathways.1
Classical NSAIDs are mainly non-selective COX inhibitors and con-
stitute the most commonly used approach for the treatment of
inflammatory conditions. This class of drugs, as well as the second
generation selective COX-2 inhibitors (coxibs) present gastroin-
testinal and cardiovascular undesired effects, evolved from an
imbalance in homeostatic eicosanoid production. In case of COX-
1/2 inhibition, a shift towards the LOX pathway is observed and
leukotrienes are also important causes of gastrointestinal irrita-
tion. Concerning LOX inhibition, up to now, zileuton is the only
5-LOX inhibitor used in the clinic for the treatment of asthma,
although there are some restrictions concerning its use.2,3
and a combination therapy with the co-administration of two or
more drugs seems to raise efficacy and safety issues, as well as
compliance problems, the rational design of multi-target agents
has gained interest lately.4,5 Tenidap, a dual COX/LOX inhibitor
and cytokine modulator, found to be more potent than diclofenac
in the treatment of rheumatoid arthritis,6 has been withdrawn
from the clinic because of liver and kidney toxicity. However, this
toxicity was attributed to reactive metabolites due to the thio-
phene moiety,7,8 and replacement of the thiophene ring gave ana-
logues with stronger activity and better gastric tolerance than
tenidap.9
In this communication, we used eight classical NSAIDs (Fig. 1)
known to be non-selective COX inhibitors, with negligible activity
on LOX.10 In addition, indole-3-acetic acid, a part of the indometha-
cin structure, as well as a-lipoic acid, known for its antioxidant and
anti-inflammatory activity,11,12 were included. All the above car-
boxylic acids were esterified with cinnamyl alcohol, giving the
final, novel compounds. Cinnamic acid and its analogues have been
studied for anti-inflammatory activity in a number of reports.
Especially, cinnamaldehyde has been found to decrease car-
rageenan-induced inflammation, suppress iNOS, COX-2 and NF-
Consequently, since the selective inhibition of one pathway in
the arachidonic acid cascade appears to cause undesired effects,
j
B expression,13 as well as TNF-a 14
The effects of these compounds on carrageenan induced rat paw
.
oedema and on the in vitro activity of soybean lipoxygenase were
examined. Selected structures were tested for hypolipidemic activ-
ity in vivo.
⇑
Corresponding author.
E-mail address: rekka@pharm.auth.gr (E.A. Rekka).
http://dx.doi.org/10.1016/j.bmcl.2015.10.036
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Theodosis-Nobelos, P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.036

2
P. Theodosis-Nobelos et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 1. Structures of the carboxylic acids used: (a) Ibuprofen; (b) Ketoprofen; (c) Naproxen; (d) Indomethacin; (e) Diclofenac; (f) Tolfenamic acid; (g) Flufenamic acid; (h)
Mefenamic acid; (i) 2-(1H-indol-3-yl)acetic acid; (j) 5-(1,2-Dithiolan-3-yl)pentanoic acid ( -lipoic acid).
a
Table 1
Effect of compounds 1–10 and parent NSAIDs on carrageenan-induced rat paw
oedemaa
Compound
% Oedema reduction
1
46⁄
68⁄⁄⁄
Comp. RCOOH
clogP* Comp. RCOOH
clogP
7.67
7.34
7.41
1 (0.30 mmol/kg)
Ibuprofen
2
Ketoprofen
3
Naproxen
4
Indomethacin
5
Diclofenac
6
Tolfenamic acid
7
Flufenamic acid
8
1
2
3
4
5
Ibuprofen
Ketoprofen
Naproxen
Indomethacin 6.96
Diclofenac 7.10
6.45
5.54
5.59
6
7
8
Tolfenamic acid
Flufenamic acid
Mefenamic acid
36
55⁄⁄⁄
47
60⁄⁄⁄
11⁄⁄
56⁄⁄
42⁄⁄
53
9
10
2-(1H-indol-3-yl)acetic acid 4.18
Lipoic acid 5.13
Figure 2. Synthesis of compounds and their lipophilicity (ClogP). DCC: N,N0-
dicyclohexylcarbodiimide; DMAP: 4-dimethylaminopyridine. Cinnamyl alcohol:
(2E)-3-phenylprop-2-en-1-ol. ⁄ClogP for windows v. 4.0, BioByte Corp.
37⁄⁄
42⁄⁄
24⁄⁄
48⁄⁄
19⁄
Compounds 1–10 were synthesised by
a straightforward
62⁄⁄⁄
44⁄⁄
33
method from the related acids and cinnamyl alcohol ((2E)-3-
phenylprop-2-en-1-ol), using DDC and a catalytic amount of
DMAP, at room temperature and with excellent yields (60–98%)
(Fig. 2).
Mefenamic acid
9
10
10 (0.30 mmol/kg)
Cinnamyl alcohol
48⁄⁄
57⁄⁄
13NS
Their effect on acute inflammation, applying the carrageenan
paw oedema model,15 as well as the anti-inflammatory activity
of the parent NSAIDs, under the same experimental conditions,
are shown in Table 1. The carrageenan-induced paw oedema is a
well-known and widely used model of acute inflammation. By this
model, a biphasic inflammation is induced. The early phase is
related to the release of histamine, serotonin, bradykinin and, to
a lesser extent, of COX activation, whereas, in the delayed phase,
more than one hours after administration, neutrophil infiltration,
further prostaglandin production and release of pro-inflammatory
cytokines are involved.16
Significant difference from control: NS(not significant) P > 0.3, ⁄P < 0.005, ⁄⁄P < 0.001
(Student’s t test).
The effect on oedema is expressed as percent of inhibition of oedema in com-
parison to controls. All compounds were administered ip, at a dose of 0.15 mmol/kg
of body weight. Each value represents the mean obtained from 6 to 8 animals.
a
dose-dependent, as indicated by the increase of activity of 1 and
10 at 300 lmol/kg. Compounds 1–8 were found to be more potent
than their individual parent NSAIDs, indicating a further enhance-
ment of the anti-inflammatory activity by the performed molecular
modification. Compound 10 reduced oedema by 48%, more than
All compounds could reduce paw oedema significantly, at
150 lmol/kg, ip, 3.5 h post injection. This effect seems to be
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P. Theodosis-Nobelos et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 2
Effect of compounds 1–10, cinnamyl alcohol and NDGA on lipoxygenasea
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Compound
IC50 (lM)
1
2
3
4
5
6
7
8
6.7
Control
1 µM
Comp. 1
22.5
40.5
9.0
78
9.6
9.0
38
116
11.3
250
1.3
IC50 6.7 μ
5 µM
10 µM
50 µM
9
10
Cinnamyl alcohol
NDGA
a
After 7 min of incubation; NDGA: nordihydroguaiaretic acid.
0
60 120 180 240 300 360 420
Time (sec)
most of the NSAIDs included in this experiment. Lipoic acid itself
has been reported17 to possess a weak activity, about 5% oedema
reduction, at similar doses (50 and 100 mg/kg, corresponding to
1.2
1.0
0.8
0.6
0.4
0.2
0.0
250 and 500 lmol/kg), under an analogous experimental setting.
Compound 9 has a moderate effect on acute inflammation, while
indol-3-acetic acid has no reported anti-inflammatory action. Cin-
namyl alcohol, tested under the same experimental conditions,
was found to have no significant effect on acute inflammation.
Soybean lipoxygenase (linoleate 13S-lipoxygenase) is often
used as a reliable screen for LOX inhibition.18 It has been reported
that arachidonic acid binding sites in soybean lipoxygenases share
almost the same similarity with animal 5-LOX.19
The ability of compounds to inhibit lipoxygenase, presented as
IC50 values towards soybean lipoxygenase after 7 min of incuba-
tion, is demonstrated in Table 2. The IC50 of nordihydroguaiaretic
acid (NDGA), an antioxidant compound acting as a nonspecific
inhibitor of lipoxygenase, is also included as a reference.
Control
2.5 µ
Comp. 7
IC50 9 μ
5 µM
10 µM
25 µM
50 µM
0
60
120 180 240 300 360 420
Time (sec)
Figure 3. Time course of lipoxygenase inhibition, as affected by various concen-
trations of compounds 1 and 7.
The time course of LOX inhibition, as affected by the most active
compounds 1 and 7 is shown in Figure 3.
All compounds inhibited lipoxygenase very significantly. Given
that the NSAIDs used here have insignificant or no effect on LOX, as
verified in this work and elsewhere,10,18,20 it is apparent that the
observed inhibition is a result of the esterification with cinnamyl
alcohol. This is further supported by the important inhibition
caused by the lipoic acid ester 10, and even of compound 9, consid-
ering that the parent carboxylic acids are inactive. However, cinn-
namyl alcohol itself has been found to offer a weak inhibition of
LOX, under the same experimental conditions.
Table 3
Effect of compounds 1, 5 and 7 on Triton WR1339 (tyloxapol) induced hyperlipidemia
Compound
% Reduction
TG
TCa
LDL-C
1
5
7
53.3⁄⁄
78.4⁄⁄
48.4⁄⁄
77.5⁄⁄
71.6⁄⁄
50.1⁄⁄
75.0⁄⁄⁄
76.8
65.1⁄⁄
When linoleic acid was used at a concentration of 1 mM, which
is higher than the saturating substrate concentration, no inhibition
was observed, under the same experimental conditions. These
results indicate that the examined compounds act as competitive
inhibitors of lipoxygenase, since inhibition can be overcome by
increasing substrate concentration.
Tyloxapol: 200 mg/kg, ip; compounds: 150 lmol/kg, ip. Significant difference from
hyperlipidemic control: ⁄P < 0.01, ⁄⁄P < 0.001 (Student’s t test).
Each group was composed of six to eight rats.
a
TC: total cholesterol; TG: triglycerides; LDL-C: LDL-cholesterol.
triglycerides constitute the most important risk factor for
atherosclerosis. Moreover, a direct involvement of hypercholes-
terolemia in 5-LOX activity has been reported.25 Thus, three potent
anti-inflammatory compounds, 1 and 7 found to be most active
LOX inhibitors, as well as 5, a derivative of diclofenac, a NSAID used
widely for myoskeletal and dental pain, migrains, moderate post-
traumatic and menstrual pain, were tested for anti-dyslipidemic
activity in hyperlipidemic rats. The Triton-WR1339 induced
hyperlipidemia is characterised by a dramatic increase of serum
cholesterol and especially triglyceride levels, the latter due to
surfactant-mediated inhibition of lipoprotein lipase activity. Tri-
ton-induced hyperlipidemia occurs twenty four hours after the
administration and causes significant increase in the concentra-
tions of atherogenic C-LDL, C-VLDL and IDL in mice and rats.26
Results are shown in Table 3. All tested compounds reduced
plasma lipidemic indices profoundly. The highest inhibition, more
Considering the results of the described in vivo and in vitro
experiments, it could be concluded that the enhanced activity of
1–8, compared with the parent NSAIDs may, at least in part, be
attributed to the offered LOX inhibition. Meclofenamic acid, a
NSAID, has been found to inhibit 5-LOX10,21 and it has been sug-
gested that this effect may add to the increased anti-inflammatory
activity of this drug.22 The contribution of LOX inhibition may be
further supported by the significant anti-inflammatory activity of
cinnamyl lipoate 10, in conjunction with its strong LOX inhibition.
Atherosclerosis has been characterised as a chronic inflamma-
tory disease of the arteries and leukotrienes derived from the 5-
LOX pathway mediate various inflammatory processes during
atherogenesis, leading to foam cell formation. Furthermore, it has
been suggested that atorvastatin, a hypolipidemic drug, signifi-
cantly alleviates atherosclerotic lesions by inhibiting the 5-LOX
pathway.23,24 Elevated plasma levels of cholesterol and
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Supplementary data associated with this article can be found, in
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Please cite this article in press as: Theodosis-Nobelos, P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.036