Dual antioxidant structures with potent anti-inflammatory, hypolipidemic and cytoprotective properties

Article abstract of DOI:10.1016/j.bmcl.2017.09.054

Novel amide derivatives of trolox, 3,5-di-tert-butyl-4-hydroxybenzoic acid, (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid and cinnamic acid with cysteamine and L-cysteine ethyl ester were synthesised. In four cases, the disulfide derivatives were

Full text of DOI:10.1016/j.bmcl.2017.09.054

Accepted Manuscript
Dual antioxidant structures with potent anti-inflammatory, hypolipidemic and
cytoprotective properties
Panagiotis Theodosis-Nobelos, Chrysoula Athanasekou, Eleni A. Rekka
PII:
S0960-894X(17)30963-0
https://doi.org/10.1016/j.bmcl.2017.09.054
BMCL 25318
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2017
20 September 2017
27 September 2017
Please cite this article as: Theodosis-Nobelos, P., Athanasekou, C., Rekka, E.A., Dual antioxidant structures with
potent anti-inflammatory, hypolipidemic and cytoprotective properties, Bioorganic & Medicinal Chemistry
Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.09.054
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1
Dual antioxidant structures with potent anti-inflammatory, hypolipidemic and cytoprotective
properties.
Panagiotis Theodosis-Nobelos1, Chrysoula Athanasekou2, Eleni A. Rekka1*
1
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of
Thessaloniki, Thessaloniki 54124, Greece.
2
Institute of Nanoscience & Nanotechnology, NCSR “Demokritos”, 15310 Agia Paraskevi
Attikis, Athens, Greece.
* Author for correspondence:
Eleni A. Rekka
Tel.: (+30) 2310997614
Fax: (+30)2310997852
E-mail: rekka@pharm.auth.gr
Keywords
Antioxidants; anti-inflammatory agents; dyslipidemia; paracetamol toxicity.

2
Dual antioxidant structures with potent anti-inflammatory, hypolipidemic and cytoprotective
properties.
Panagiotis Theodosis-Nobelos1, Chrysoula Athanasekou2, Eleni A. Rekka1*
1
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of
Thessaloniki, Thessaloniki 54124, Greece.
2 Institute of Nanoscience & Nanotechnology, NCSR “Demokritos”, 153 10 Agia Paraskevi
Attikis, Athens, Greece.
Abstract
Novel amide derivatives of trolox, 3,5-di-tert-butyl-4-hydroxybenzoic acid, (E)-3-(3,5-di-tert-
butyl-4-hydroxyphenyl)acrylic acid and cinnamic acid with cysteamine and L-cysteine ethyl
ester were synthesised. In four cases, the disulfide derivatives were also isolated and tested.
All compounds were examined for antioxidant activity, expressed as their ability to inhibit
lipid peroxidation and to scavenge free radicals. They were found to demonstrate up to 17
fold better activity than that of the parent antioxidant acids. They could reduce acute
inflammation up to 87%. The most active antioxidant compounds were further tested for their
in vivo hypolipidemic effect, which ranged from 47% to 73%, and for their ability to protect
the liver against oxidative toxicity caused by high paracetamol dose. The disulfide derivatives
of 3,5-di-tert-butyl-4-hydroxybenzoic acid and cinnamic acid had no antioxidant activity and
presented equal or lower anti-inflammatory effect than their thiol analogues, indicating that
their molecular characteristics may not permit biological barrier penetration.

3
Reactive oxygen species (ROS) are implicated in cell signalling,1 playing an essential role in
signal transduction. However, oxidative stress, i.e. imbalance of antioxidant to oxidant ratio in
favour of the latter, is involved in various pathological conditions such as obesity,
cardiovascular disease, cancer and aging.2 During the inflammatory process, mast and white
blood cells are attracted to the lesion leading to respiratory burst.3; 4 In inflammatory diseases
high levels of ROS are produced and accumulated.5; 6
ROS are involved in the oxidation of low density lipoprotein (LDL) to oxLDL, which
respectively mediates the generation of more ROS.7 oxLDL is not recognized by the LDL
receptors, but interacts with scavenger receptors leading to high lipid levels in blood and high
affinity with macrophages in the atheromatic lesions.8; 9; 10 LDL is susceptible to oxidation and
glycosylation, by oxidised sugars, forming Schiff bases. High levels of LDL and especially
oxidized LDL are linked to atherogenesis resulting in the activation of blood inflammatory
cells and the formation of foam cells11. Furthermore, abnormal levels of mitochondrial
reactive oxygen species are implicated in the initiation and development of diabetes12 and
normalisation of these levels can improve diabetic complications.13 Oxidative deregulation
due to high fat intake may lead to insulin resistance14 and propagation of the metabolic
syndrome.
Butylated hydroxytoluene (BHT) and vitamin E, well known antioxidants, can inhibit
inflammation and reduce biological stress.15; 16 Cinnamic acid derivatives possess antioxidant,
anti-inflammatory and antitumour activities.17; 18 Cysteamine and L-cysteine belong to the low
molecular weight thiols (LMWTs), able to chelate heavy metals19 and to act as antioxidants
through direct binding to radicals20 or through interaction with transition metals. Polyphenolic
compounds may also cause oxidation of transition metal-carrying groups21 and LMWTs
demonstrate a protective antioxidant effect in equimolar concentration with the phenolic
compound.
In view of all the above, a series of amides of antioxidant acids with cysteamine or L-cysteine
ethyl ester were synthesised (Fig. 1). The acids were trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), BHBA (3,5-di-tert-butyl-4-hydroxybenzoic acid),
CA (cinnamic acid, (E)-3-phenylprop-2-enoic acid) and BHCA ((E)-3-(3,5-di-tert-butyl-4-
hydroxyphenyl)acrylic acid). The disulfide reaction products of BHBA and cinnamic acid
with the mentioned thiols were also isolated.
The ability of these compounds to inhibit lipid peroxidation and to interact with DPPH was
tested. Their inhibitory effects on carrageenan induced rat paw oedema and on the in vitro
activity of soybean lipoxygenase were examined. Selected structures were tested for
hypolipidemic activity and protection against paracetamol hepatotoxicity in vivo.

4
Fig. 1. Structures of the synthesised compounds.
The compounds were synthesised using 1,1΄- carbonyldiimidazole (compounds 1-6, 9, 10) or
via the intermediate acid chlorides (compounds 7, 8, 11, 12), followed by the reaction with

5
the appropriate amine. They were obtained at various yields (6–78%). The low yields are
mainly due to the concomitant oxidation of thiols to disulfides. In some cases, an effort to
purify the product by recrystallisation resulted in a further oxidation of the thiols to disulfides,
thus affecting the final yields. The disulfide derivatives 9-12 were isolated and tested for
comparison.
The interaction of compounds with the lipophilic, electron accepting, N-centred 1,1-diphenyl-
2-picrylhydrazyl (DPPH) stable free radical is a common assay for antioxidant activity and an
expression of the reducing ability of compounds. The interaction of compounds 1-8, at
various concentrations, with a standard concentration of DPPH is given in Table 1.
Table 1
Interaction of compounds 1-8, at various concentrations, with DPPH (200 μΜ)a
Percent interactionb
Compound
200 μΜ 100 μΜ 50 μΜ
93
93
92
96
85
85
86
86
92
91
60
70
45
46
59
42
61
61
34
37
20
23
31
27
1
2
3
4
5
6
7
8
a After 30 min of incubation.
b
Percent interaction = 100 – , where Ao: Absorbance of DPPH alone; Ai:
Absorbance after the addition of the compound.
All determinations were performed at least in triplicate and standard deviation is always
within ±10% of the mean value.
This interaction seems to be concentration dependent and the chromane compounds 1 and 2
possess higher activity than the other phenolic derivatives (3-6). The higher activity of
compounds 3 and 4 than that of 5 and 6 is probably due to the more extended conjugation of
the phenylacrylic system, resulting in radical stabilisation, after the phenolic hydrogen
abstraction. Compounds 7 and 8, having only one reducing group (-SH), possess about the
same, or slightly increased activity compared with compounds 5 and 6. However, compounds
7 and 8 have a smaller polar area (Table 2) than 5 and 6. It could be suggested that the

6
absence of one antioxidant group could be counterbalanced by the decrease of the polar area,
facilitating the necessary contact with DPPH. Compounds 9-12 were inactive in this
experiment. The lack of activity of 9 and 10, despite their phenolic groups, may similarly be
related to their increased polar area and their bulky structure.
The effect of compounds 1-6 and of the parent acids, trolox and BHBA, on rat hepatic
microsomal membrane lipid peroxidation, expressed as IC50 values after 45 min of incubation,
is shown in Table 3. Compounds 7-12 showed no activity in this experiment.
Table 2
Calculated physicochemical properties of the synthesised compounds: Molecular Weight
MW), total polar surface area (TPSA), number of H donors and acceptors, lipophilicity
(clogP) and violations of Lipinski’s “rule of 5” a
Compounds MW
TPSA (Å2) H acceptors H donors clogP
violations
1
309.42
58.56
84.86
49.33
75.63
49.33
75.63
29.10
55.40
98.66
151.26
58.20
110.81
66.76
57.53
37.30
57.53
26.02
4
6
3
5
3
5
2
4
6
4
4
8
4
3
2
3
1
2
2
2
2
2
2
1
1
4
2
2
2
2
2
1
2
2
3.46
4.12
5.22
5.67
4.67
5.15
2.24
2.69
9.61
10.59
4.44
14.99
3.08
5.01
2.24
5.02
-0.25
0
0
1
1
0
0
0
0
2
2
0
2
0
0
0
0
0
2
381.49
335.50
407.57
309.47
381.54
207.29
279.35
616.92
761.04
412.57
524.64
250.29
250.33
148.16
276.37
3
4
5
6
7
8
9
10
11
12
Troloxb
BHBA
CA
BHCA
Cysteamine 77.15

7
CEE
149.21
52.32
3
2
-0.04
0
a
Calculated: TPSA: ChemBioDraw Ultra 12.0; clogP: clogP for windows v. 4.0, BioByte
Corp.
b
Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; BHBA: 3,5-di-tert-butyl-
4-hydroxybenzoic acid; CA: cinnamic acid; BHCA: (E)-3-(3,5-di-tert-butyl-4-
hydroxyphenyl)acrylic acid; CEE: cysteine ethyl ester.
Compounds 1 and 2 were even tenfold more active than trolox; 5 and 6 were more potent than
BHBA, possibly due to the ionisable carboxylic group of BHBA and further because 5 and 6
incorporate a second antioxidant moiety. However, cysteamine and cysteine ethyl ester have
been reported to inhibit lipid peroxidation only by 37% and 49%, respectively, at 1 mM,
under the same experimental conditions.22 Again, compounds 3 and 4 were more active than 5
and 6.
Table 3
Effect of the synthesised compounds 1–6, trolox and BHBA on lipid peroxidation
Lipid peroxidation
Compound
inhibition
IC50 (μΜ)a
1.6
3
1
2
30
3
40
4
45
5
200
25
6
Trolox
BHBA
250
a After 45 min of incubation; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid; BHBA: 3,5-di-tert-butyl-4-hydroxybenzoic acid.
An interesting observation was the inactivity of compounds 7-10. The lack of antioxidant
activity for 11 and 12 could be anticipated; however the diminished effect of 7 and 8 was not
expected, considering their potent interaction with DPPH (Table 1). This could be explained
by the relatively low lipophilicity of compounds 7 and 8 (Table 2), which may limit their

8
effective approach to the lipid membranes. It has been proposed that structural characteristics
offering antioxidant properties, in combination with favourable lipophilicity, usually
corresponding to a logP value close to 3, are important factors for protection against lipid
peroxidation.23 As far as compounds 9 and 10 are concerned, their too high lipophilicity and
large polar area may impede their easy access to the lipid phase.
The in vivo anti-inflammatory activity of the compounds was expressed as reduction of
oedema induced by carrageenan in rats.18 The carrageenan paw oedema, produced by the
subplantar injection of a 1% carrageenan suspension in saline, is a common model of acute
inflammation.24 In this model, inflammation is induced in two phases: histamine, serotonin
and pro-inflammatory cytokine release during the first phase is followed, at a later phase, by
increased prostaglandin production. Since the later phase of carrageenan oedema (2.5-6 h)
demonstrates the two main features of the inflammatory process, vascular response and
cellular emigration, the effect of the compounds was tested after 3.5 hours. The anti-
inflammatory activity of compounds, applying the carrageenan paw oedema model, as well as
the anti-inflammatory activity of ibuprofen, used as a reference, are shown in Table 4.
Table 4
Effect of compounds 1-8, 10, 11 and ibuprofen on carrageenan-induced rat paw oedemaa
% Paw weight
increase (± SD)
18.24 ± 7.94 *
% Oedema
reduction
67
Compound
1
2
23.47 ± 3.08 **
18.48 ± 9.45 **
17.84 ± 6.53 **
7.27 ± 3.06 **
39
67
61
87
3
4
5
15.78 ± 5.08 **
51.35 ± 8.14 *
54.33 ± 5.48 *
37.63 ± 7.15 **
22.54 ± 3.64 **
59
38
35
36
42
6
7
8
10
11

9
37.72 ± 4.15 **
36
Ibuprofen
a
The effect on oedema is expressed as percent of inhibition of oedema in comparison with
controls. All compounds were administered ip at 0.15 mmol/kg body weight.
Significant difference from control: * P<0.05, ** P ≤ 0.005 (Student’s t test).
All compounds presented remarkable activity. The cysteamine derivatives were better anti-
inflammatory agents than the L-cysteine analogues, a result that may be related to their better
antioxidant activity. The cysteamine derivative 5 was the most potent of all tested
compounds. It seems that the 3,5-di-tert-butyl-4-hydroxybenzoyl moiety contributes to the
anti-inflammatory activity, since we have reported that the amide of 3,5-di-tert-butyl-4-
hydroxybenzoic acid with thiomorpholine inhibited rat paw oedema considerably.25
Furthermore, tebufelone, bearing the same structural component, and other di-tert-
butylphenol derivatives demonstrate anti-inflammatory activity.26 Cysteamine and cysteine
ethyl ester have been reported to reduce carrageenan oedema by 15% and 25%, respectively,
at the same dose,27 whereas butylated hydroxytoluene (BHT) was found to be inactive.25
The disulfides 10 and 11 were less or equally active, compared with their respective thiols 6
and 7. The lower activity of 10, compared to 6, may be partially due to poor biological
membrane penetration, because of its highly lipophilic character, as indicated by the
calculated two violations of Lipinski’s empirical “rule of five” 28 (Table 2).
Soybean lipoxygenase, possessing structural and functional similarities with mammalian
lipoxygenases, is commonly used for the study of anti-inflammatory agents.29 The ability of
compounds to inhibit soybean lipoxygenase was determined; however, most compounds
presented diminished activity (tested at 100-300 μΜ). The most potent were compounds 3-6,
containing the 3,5-di-tert-butyl-4-hydroxyphenyl moiety, with IC50 160, 135, 121 and 67 μΜ,
respectively. Possibly this property contributes to their in vivo anti-inflammatory activity,
since they are weaker antioxidants than compounds 1 and 2.
Elevated plasma levels of cholesterol and triglycerides constitute the most important risk
factors for atherosclerosis, which has been characterised as a chronic inflammatory disease of
the arteries. Thus, the most potent antioxidant compounds 1 and 2, with significant anti-
inflammatory activity, were tested for anti-dyslipidemic activity in hyperlipidemic rats. The
applied Triton WR1339 induced hyperlipidemia is characterised by a dramatic increase of
serum cholesterol and especially triglyceride levels, reaching a peak twenty four hours post
administration.30
Results are shown in Table 5. Both compounds reduced plasma lipidemic indices profoundly,
about 50% to 70%. It could be suggested that the tested compounds act via their antioxidant

10
properties. It is known that suppression of ROS production may hinder the progression of
pathological conditions associated with hyperlipidemia, while increase of plasma membrane
free cholesterol augments ROS production.31
Table 5
Effect of compounds 1 and 2 on Triton WR1339 (tyloxapol) induced hyperlipidemia
Plasma concentration (mg/dL) ± SD
(% Reductiona)
Treatment
TC
TG
64.4 ± 5.0
127.6 ± 8.2
Saline
214.6 ± 13.6
733.4 ± 51.5
Triton
70.6 ± 10.2* (67.1)
58.0 ± 3.7* (73.0)
386.2 ± 16.3* (47.3)
203.2 ± 14.9* (72.3)
Triton+1
Triton+2
a Compared to the Triton-treated hyperlipidemic control group
TC: Total cholesterol; TG: Triglycerides
Triton: 200 mg/kg, ip; compounds: 150 μmol/kg, ip.
Significant difference from hyperlipidemic control: * P <0.001 (Student’s t test).
Paracetamol (acetaminophen), in therapeutic doses, is mainly metabolised in the liver by
UDP-glucuronosyltransferases and sulfotransferases, and to a small extent it is converted into
the highly reactive, cytotoxic N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450,
mainly CYP2E1. NAPQI is produced via one electron abstraction from paracetamol, followed
by deprotonation of the resulting cationic radical and a second electron transfer.32 At high
paracetamol doses, the conjugation route is saturated and the oxidative biotransformation
plays a critical role in the toxicity of paracetamol. NAPQI is detoxified via conjugation with
glutathione, thus, it may cause glutathione depletion as much as 90%, covalent binding to the
cysteine groups of proteins, eventually leading to oxidative hepatic injury.33
Table 6
Effect of compounds 1 and 2 on some enzymes in plasma after paracetamol-induced liver
injury.
Parameter a
Treatment

11
Saline
Paracetamol
78.2 ± 6.0
Paracetamol + 1
Paracetamol + 2
66.5 ± 5.3*
(15%)
28.3 ± 4.8**
(64%)
27.6 ± 3.2
GPT (U/L)
102.7 ±8.7**
(26%)
41.5 ± 4.7**
(70%)
36.9 ± 3.0
138.7 ± 9.1
GOT (U/L)
LDH (U/L)
404.7 ± 27.6**
(70%)
300.5 ± 39.9**
(78%)
181.7 ± 17.3
1343.0 ± 164.5
a
GPT: glutamate pyruvate transaminase, GOT: glutamate oxaloacetate transaminase, LDH:
lactate dehydrogenase.
* P < 0.01, ** P < 0.001, Student’s t test.
Numbers in parentheses denote per cent reduction compared to paracetamol-treated rats.
The results of the administration of paracetamol and compounds 1 and 2 on the hepatic
enzymes glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase
(GOT), as well as lactate dehydrogenase (LDH) in plasma, compared with the paracetamol
treated rats, are shown in Table 6. High paracetamol dose caused a profound increase of the
examined enzymes, indicative of a serious hepatic damage. Treatment with compound 1 had a
moderate protective effect on GPT and GOT, however, it caused a strong reduction of LDH
levels. Compound 2 reduced GPT and GOT to physiological values and produced a very
significant reduction of LDH towards normal condition.
In conclusion, as antioxidants are potentially able to protect the organism against radical
damage or to enhance the normal cellular defence against oxidative injury, a number of
antioxidant acids were amidated with the naturally occurring thiol derivatives cysteamine and
cysteine ethyl ester, and their effect on conditions which involve oxidative insult were
examined. Their in vitro strong antioxidant activity and their in vivo pleiotropic beneficial
effect against acute inflammation, hyperlipidemia and paracetamol-induced hepatic injury
may become useful in developing agents for the protection or treatment of similar disorders.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
P. Theodosis-Nobelos acknowledges the General Secretariat for Research and Technology
(GSRT) of Greece and the Hellenic Foundation for Research and Innovation (HFRI) for a
grant supporting his PhD research.
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Graphical abstract