Lipid peroxidation inhibition study: A promising case of 1,3-di([1,1′-biphenyl]-3-yl)urea

Article abstract of DOI:10.1016/j.cbi.2020.109137

In the present study eighteen inhibitors of the hydrolytic enzymes of the endocannabinoid system were investigated for antioxidant activity using lipid peroxidation (LP) method. Among the assayed compounds ten belong to carbamates with phenyl [1,1′-biphenyl]-3-ylcarbamate (6), reported for the first time, and eight are retro-amide derivatives of palmitamine. Interestingly, results indicated that most of the tested compounds have good antioxidant properties. In particular, 1,3-di([1,1′-biphenyl]-3-yl)urea (3) shows IC₅₀ = 26 ± 6 μM comparable to ones obtained for standard antioxidants trolox and quercetin (IC₅₀ = 22 ± 6 μM and 23 ± 6 μM, respectively). Compound 3 was investigated further by means of DFT calculations, to clarify a possible mechanism of the antioxidant action. In order to estimate the capability of 3 to act as radical scavenger the structure was optimized at B3LYP/6–311++G** level and the respective bond dissociation enthalpies were calculated. The calculations in non-polar medium predicted as favorable mechanism a donation of a hydrogen atom to the free radical and formation of N-centered radical, while in polar solvents the mechanism of free radical scavenging by SPLET dominates over HAT H-abstraction. The possible radical scavenging mechanisms of another compound with potent antioxidant properties (IC₅₀ = 53 ± 12 μM), the retro-amide derivative of palmitamine (compound 18), was estimated computationally based on the reaction enthalpies of a model compound (structural analogue to 18). The computations indicated that the most favorable mechanisms are hydrogen atom transfer from the hydroxyl group in meta-position of the benzamide fragment in nonpolar medium, and proton transfer from the hydroxyl group in ortho-position of the benzamide fragment in polar medium.

Full text of DOI:10.1016/j.cbi.2020.109137

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Lipid peroxidation inhibition study: A promising case of 1,3-di([1,1′-biphenyl]-3-yl)urea
Jelena Lazarević, Andrija Šmelcerović, Jelena Zvezdanović, Denitsa Yancheva,
Silvana Casati, Roberta Ottria, Pierangela Ciuffreda
PII:
S0009-2797(20)30149-6
https://doi.org/10.1016/j.cbi.2020.109137
CBI 109137
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 30 January 2020
Revised Date: 27 April 2020
Accepted Date: 12 May 2020
Please cite this article as: J. Lazarević, A. Šmelcerović, J. Zvezdanović, D. Yancheva, S. Casati, R.
Ottria, P. Ciuffreda, Lipid peroxidation inhibition study: A promising case of 1,3-di([1,1′-biphenyl]-3-
yl)urea, Chemico-Biological Interactions (2020), doi: https://doi.org/10.1016/j.cbi.2020.109137.
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Authors contributions
J.L. and P.C. conceived and designed the study, S.C. and R.O. performed the chemical synthesis,
J.L. J.Z. and A.S. performed LP experiments, D.Y. performed theoretical calculations. All the
authors contributed to the paper writing.

Lipid peroxidation inhibition study: a promising case of 1,3-di([1,1′-biphenyl]-3-yl)urea
a
a
b
c
Jelena Lazarević *, Andrija Šmelcerović , Jelena Zvezdanović , Denitsa Yancheva , Silvana
d
d
d
Casati , Roberta Ottria , Pierangela Ciuffreda *
a
Department of Chemistry, Faculty of Medicine, University of Niš, Bulevar Dr Zorana Đinđića
8
1, 18000 Nis, Serbia
b
c
Faculty of Technology, University of Niš, Bulevar oslobođenja 124, 16000 Leskovac, Serbia
Institute of Organic Chemistry and Centre of Phytochemistry, Bulgarian Academy of
Sciences,Acad. G. Bonchev Str., Build. 9, 1113 Sofia, Bulgaria
d
Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi diMilano,
Via G.B. Grassi 74, 20157 Milano, Italy
*
Correspondence to:
Jelena Lazarević, e-mail: jelena217@yahoo.com; jelena.lazarevic@medfak.ni.ac.rs and
Pierangela Ciuffreda, e-mail: pierangela.ciuffreda@unimi.it
Abstract
In the present study eighteen inhibitors of the hydrolytic enzymes of the endocannabinoid system
were investigated for antioxidant activity using lipid peroxidation (LP) method. Among the
assayed compounds ten belong to carbamates with phenyl [1,1'-biphenyl]-3-ylcarbamate (6),
reported for the first time, and eight are retro-amide derivatives of palmitamine. Interestingly,
results indicated that most of the tested compounds have good antioxidant properties. In
particular, 1,3-di([1,1′-biphenyl]-3-yl)urea (3) shows IC50 = 26± 6 ꢀM comparable toones
obtained for standard antioxidants trolox and quercetin (IC50 = 22± 6 ꢀM and 23 ± 6 ꢀM,
respectively). Compound 3 was investigated further by means of ab initio calculations, to clarify
a possible mechanism of the antioxidant action. In order to estimate the capability of 3 to act as
radical scavenger the structure was optimized at B3LYP/6-311++Gꢁꢁ level and the respective
bond dissociation enthalpies were calculated. The calculations in non-polar medium predicted as
favorable mechanism a donation of a hydrogen atom to the free radical and formation of N-
centered radical, while in polar solvents dominate mechanism of free radical scavenging by
SPLET over HAT H-abstraction. The possible radical scavenging mechanisms of another
compound with potent antioxidant properties (IC50 =53 ± 12 ꢀM), the retro-amide derivative of
palmitamine, compound 18, was estimated computationally based on the reaction enthalpies of a

model compound (structural analogue to 18). The computations indicated that the most favorable
mechanisms are hydrogen atom transfer from the hydroxyl group in meta-position of the
benzamide fragment in nonpolar medium, and proton transfer from the hydroxyl group in ortho-
position of the benzamide fragment in nonpolar medium.
Keywords: antioxidant activity, lipid peroxidation inhibition, ab initio calculations,
endocannabinoid modulators, urea derivatives
Abbreviations
AAPH 2,2′-Azobis(2-methylpropionamidine) dihydrochloride
B3LYP Becke’s three-parameter non-local exchange in conjunction with Lee, Yang and Parr
correlation potentials
BDE Bond dissociation enthalpy
CDCl Deuterochloroform
3
CDI Carbonyldiimidazole
COMU
1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium
hexafluorophosphate
DFT Density-functional theory
DIPEA N,N-Diisopropylethylamine
DMAP 4-(Dimethylamino)pyridine
DNA Deoxyribonucleic acid
EtOAc Ethyl acetate
HAT Hydrogen atom transfer
IEFPCM Integral equation formalism polarizable continuum model

IP Ionization potential
LP Lipid peroxidation
MAGL Monoacylglycerol lipase
MDA Malondialdehyde
NAAA N-acylethanolamide acid amidase
NMR Nuclear magnetic resonance
PA Proton affinity
PL90 Phospholipon® 90
PUFA Polyunsaturated fatty acids
ROS Reactive oxygen species
SET Single electron transfer
SPLET Sequential proton loss electron transfer
TBA Thiobarbituric acid
TCA Trichloroacetic acid
TLC Thin-layer chromatography
1
. Introduction
Reactive oxygen species (ROS) are produced through enzymatic and non-enzymatic mechanisms
in cells and the homeostatic balance between the physiological and pathological concentration of
ROS can be altered not only after radiation and pollution exposure but also in stress conditions
and with incorrect diet [1]. ROS are highly reactive species that can attack various classes of
biomolecules including DNA, proteins and fatty acids, especially polyunsaturated fatty acids
(PUFA) [2]. Bringing to close connection with oxidative stress in vivo, lipid peroxidation (LP)

has received a great deal of attention. This free radical mediated chain reaction process, once
initiated results in an oxidative deterioration of polyunsaturated lipids [3]. Through a proven
disturbance process in membrane organization and a functional loss and modification of proteins
and DNA bases, LP has been implicated in the pathogenesis of various diseases and aging,
including atherosclerosis, cataract, rheumatoid arthritis and neurodegenerative disorders [4].
Moreover, correlation between the excessive production of reactive oxygen species (ROS) and
their role in cancer development is well known [5,6]. Consequently, the role of antioxidants has
received extensive attention and many natural and synthetic supplements and drugs with radical-
scavenging capacity have been explored [7]. Beyond the beneficial effects of antioxidants, we
should also recall the dark side of this class of molecules reported by recent literature. In the last
decades, indeed, several research findings present antioxidants as causative risk factor in various
human diseases as cardiovascular disease or diabetes [8]. Moreover, also the effects of traditional
antioxidant micronutrients as cancer chemoprevention agents remain unclear revealing both
benefits and detrimental effects or even resulted in harm [9,10]. On the contrary the mechanistic
link between oxidative stress and carcinogenesis is well documented, prompting further
researches. Additionally, special targets of LP processes are long chain fatty acids and the PUFA
arachidonic acid, the principal components of the endocannabinoids’ moiety, lipid signaling
mediators involved in a wide range of physiological and pathological conditions, comprising
cancer [11].
Starting from these considerations we decided to investigate if the monoacylglycerol lipase
(MAGL) and N-acylethanolamide acid amidase (NAAA) inhibitors, synthesized and analyzed in
our laboratories [12,13], could also have an additional antioxidant effect. The present study
reports antioxidant activity of 1-18 investigated by LP method. For the most active among the
studied compounds (compounds 3 and 18), theoretical calculations were preformed to clarify a
possible mechanism of an antioxidant action.
2
. Materials and methods
2
.1. Reagents and Materials

All of the reagents, standards and solvents used were of analytical reagent grade, obtained from
commercial sources and used without further purification (unless specified otherwise, all
chemicals were purchased from Merck (Darmstadt, Germany)).
Phospholipids (Phospholipon® 90 – PL90) were obtained by courtesy of Phospholipid GMBH,
Cologne, Germany. According to the specification, the PL90 mixture is composed of
phosphatidylcholine 98 % and lyso-phosphatidylcholine 2.1 %, where the phospholipids′ fatty
acid composition is palmitic acid 12 ± 2 %, stearic acid 3 ± 1 %, oleic acid 10 ± 3 %, linoleic
acid 66 ± 5 % and linolenic acid 5 ± 2 %; the peroxide value maximum is 1.3. Thiobarbituric
acid (TBA), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), trichloroacetic acid
(TCA), methanol, sodium hydroxyde and standards of caffeic acid, quercetin and trolox were
purchased from Sigma-Aldrich (St. Louis, MO).
2
.2. General synthetic procedures
Compounds 1 [14], 2-5 and 7-10 [13], and 11-18 [12] were synthesized following procedures as
described in Tarzia et al., 2003 [14], Lauria et al., 2018 [13] and Vago et al., 2017 [12],
respectively (Fig 1). Melting point determination and NMR spectra confirmed the structures.
Purity of all products (>98%) was verified by thin-layer chromatography and NMR
measurements [12–17]. The new compound 6 was synthesized according to procedure reported
by Lauria et al., 2018 [13] through Suzuki coupling reaction via the key biphenylamine
intermediate, involving phenylboronic acid, 3-bromoaniline and a suitable palladium catalyst
(intermediate yield 72%).
2
.2.1. Synthesis of phenyl [1,1'-biphenyl]-3-ylcarbamate (6)
Carbonyldiimidazole (CDI) (115 mg, 7.08 mmol, 4 eq.) and [1,10-biphenyl]-3-amine (300 mg,
.77 mmol, 1 eq.) were mixed in dry toluene (8 mL) and heated at reflux for 12 h under nitrogen
1
atmosphere. After cooling to room temperature white precipitates were collected by filtration.
The product, purified by chromatography on silica gel (petroleum ether:EtOAc, 90:10 v/v), was
obtained as a white solid in 82% yield. mp: 96-97 °C. TLC (petroleum ether: EtOAc, 80:20 v/v):
1
Rf=0.38; H NMR (CDCl
3
)δ 7.78 (1H, bs, 2-H), 7.63 (2H, d, J=7.3 Hz, 2', 6'-H), 7.49-7.35 (8H,
m, 3', 4', 5', 4, 5, 6, 2 x meta-H, 8 x Ar-H), 7.29 (1H, dd, J=8.7, 1.8 Hz, para-H), 7.23 (2H, d,
1
3
J=8.7 Hz, 2 x orto-H), 7.08 (1H, bs, NH); C NMR δ: 150.5 (CO), 142.3, 140.6, 137.8, 129.5,

1
2
29.4, 128.8, 127.6, 127.2, 125.8, 122.8, 121.7 and 117.6 (C-Ar). Anal. Calcd. for C19H15NO :
C, 78.87; H, 5.23; N, 4.84. Found: C, 78.51; H, 5.45; N, 4.71. For NMR spectra see
Supplementary data.
2
.3.Lipid peroxidation inhibition by thiobarbituric acid-malondialdehyde test
Lipid peroxidation (LP) and its inhibition in the presence of the tested compounds (1-18), was
measured by thiobarbituric acid-malondialdehyde (TBA–MDA) test according to the slightly
modified procedure described by Zvezdanovic et al. [18] and Mavrova et al. [19]. The
modification consisted in altered volume ratio of methanol solution of PL-90, aqueous solution
of hydrophilic thermal LP initiator AAPH and of methanol solution of the compounds tested (1-
1
8) allowing substances to be assayed at concentrations of 500 ꢀM in the final reaction mixture
(volume ratio 2:1:2 instead of 2:2:1, respectively). Experiment was designed so that methanol
concentration was set constant in all probes for which the absorbances were read.
The absorbance of TBA–MDA complex in the supernatant was read at 530 nm and used to
calculate the inhibition percentage of lipid peroxidation given by the equation:
Inhibition of lipid peroxidation (%) = 100 x (Ac-As)/(Ac-Ab)
Ac - the absorbance of control (methanol solution of PL90) which is treated with the AAPH and
TBA solution, As -the absorbance of sample (methanol solution of PL90/1-18) which is treated
with the AAPH and TBA solution and Ab - the absorbance of blank [(methanol solution of PL90
not treated by AAPH, but treated with TBA solution (monitoring MDA level in the lipid before
LP initiation by AAPH)].
Samples were assayed for LP-inhibitory activity and those showing inhibition greater than 50%
at 500 ꢀM were tested in a broader concentration range to allow calculation of IC values. The
5
0
same experiments were done by using known antioxidants as standards (trolox, quercetin and
caffeic acid).The standards were assayed at concentrations of 50 ꢀM (caffeic acid) and 80 ꢀM
(quercetin and trolox) in the final reaction mixture.
2
.4. DFT computations
The computational study of molecular geometry and reaction enthalpies was carried out by the
use of Gaussian 09 suite of programs [20]. Hybrid functional B3LYP in conjunction with 6-
3
11++G(d,p) basis set was used in all calculations [21,22]. The solvent effects were taken into

account by including the Integral Equation Formalism Polarizable Continuum Model (IEF-PCM)
in benzene and water [23]. The optimized structures were ascertained as minima on the potential
energy hypersurface based on analytic vibrational frequency computations. No imaginary
frequencies were found for all structures.
Dissociation enthalpy (BDE), ionization potential (IP) and proton affinity (PA) of the most stable
conformers were calculated at 298 K according to the procedure established by Klein et al. [24].
The enthalpy of hydrogen atom, H(H), in benzene and water were obtained using the same
+
-
functional and basis set. Solvation enthalpies of proton, H(H ), and electron, H(e ), were taken
from the literature [25].
3
. Results and discussion
Derivatives included in our present research (Fig. 1) were part of previous studies involving
either MAGL [9,11] or NAAA [10] inhibition testing, with exception of phenyl [1,1'-biphenyl]-
3
-ylcarbamate (6) which is a new compound, with spectroscopic data reported herein for the first
time (for details see Fig 1S). First group includes compounds 2-10 synthesized based on
structural modifications of the parent structure URB602 (1) [14], to evaluate new derivatives
potentially targeting MAGL [13]. According to these results [13], compounds 8 and 10 were
recognized as potent hMAGL inhibitors with IC 4.5 ± 0.7 and 7.9 ± 0.8 ꢀM respectively, while
5
0
a symmetric compound 3 appears to be an interesting MAGL activator. Second group, comprised
of retro-amide derivatives of palmitamine (11-18), was designed, synthesized and characterized
applying the coupling reaction conditions optimized for the synthesis of endogenous
cannabinoids [26,27] aiming towards identification of new NAAA inhibitors. Based on these
results, compounds 14, 16 and 18 were found to represent competitive inhibitors of hNAAA
characterized by interesting inhibitor activity (IC50 20 ± 0.9 µM, IC50 11 ± 1.0 µM and 38 ± 1.0
µM respectively) [12]. Both MAGL and NAAA inhibitors showed relevant anticancer activity on
melanoma and bladder cancer respectively. Given the involvement of oxidative stress in cancer
development and outcome [5,6], here we investigate the antioxidant activity of synthesized
inhibitors to evaluate a possible antioxidant effect in addition to the anticancer activity of these
compounds.
Lipid peroxidation inhibition effect of ten carbamates 1-10, among which compound 6 reported
for the first time, and eight retro-amide derivatives of palmitamine 11-18 was measured using the

method based on TBA-MDA assay, selecting caffeic acid, toxol and quercetin as positive
controls. TBA-MDA based method is the most commonly used spectrophotometric assay to
measure LP and takes advantage of a pink-colored MDA-(TBA) formation, an adduct
2
characterized by strong absorbance at 530 nm [2]. The assay is widely used and though having
limitations (e.g. does not monitor the total kinetic aspects of LP inhibition but rather the extent of
LP estimated after a fixed time, in-depth discussion is provided in excellent reviews [3,28-30]) is
recognized as a reliable estimator of LP [3,28-30] having commercial kits also available [28]. LP
experiments were performed, the obtained results were plotted and IC values were calculated
5
0
and reported in Table 1 and in Supplementary data (Fig. S1).
Fig. 1.Synthesis of the assayed compounds 1-18. Reagents and conditions: (a) Pd(PPh
3 4
) ,
Na CO , CH OH, reflux, 12 h; (b) CDI, DMAP, CH CN, reflux, overnight; (c) R -OH, CH CN,
2
3
3
3
1
3
reflux; d) CDI, DMAP,CH CN, reflux, overnight; e) COMU, CH Cl /CH CN (3/1), DIPEA, r.t.,
3
2
2
3
nitrogen atmosphere.
The obtained results indicated that most of the tested compounds have good antioxidant
properties, except compounds 11 and 14 having IC ˃ 500 ꢀM, and 7, 12 and 13 that have IC
50
50

comprised between 450 and 500 ꢀM. On value range of LP inhibition displayed by compounds
, 2, 4, 6, 8-10 and 15 (values between 133± 32 and 309± 72) a possible additive antioxidant
1
effect cannot be considered. Three interesting compounds are 5, 16 and 18 that showed LP
inhibition with IC50 below 100 ꢀM (85 ± 17, 82± 10and 53 ± 12 ꢀM, respectively, Table 1). The
most potent LP inhibition was expressed by compound 3 (IC = 26± 6 ꢀM), which gave a result
5
0
comparable to trolox and quercetin as antioxidant standards tested (Table 1) and, for this reason,
was further involved in theoretical calculations aiming the clarification of the mechanism at the
base of an antioxidant action.
Table 1. Lipid peroxidation inhibition effects of compounds 1-18 and of selected antioxidants
(
IC values given in ꢀM)
50
Compound
No.
1
2
3
4
5
6
7
LP
inhibition
271 ± 86
133± 32
26± 6
213± 44
85 ± 17
142± 33
458 ± 88
IC (ꢀM)
5
0
Compound
No.
8
250± 27
15
9
309± 72
16
10
158 ± 38
17
11
˃ 500
18
12
13
14
LP
inhibition
IC50 (ꢀM)
469 ± 158 488± 164
˃ 500
Compound
No.
Caffeic
Trolox
acid
Querceti
n
LP
inhibition
IC50 (ꢀM)
15± 3
22± 6
23 ± 6
176 ± 43
82± 10
179± 30
53 ± 12
Being influenced by electronic effects of the neighboring groups and the overall geometry of the
molecule, the conformation can be regarded as the first parameter of importance in analyzing the
antioxidant capacity of any molecule [31]. In order to determine the most stable molecular
geometry of compound 3 we have fully optimized the most probable conformations with
different orientation of the biphenyl fragments (Fig. 2) at IEFPCM-B3LYP/6-311++G** level of
theory in both, benzene and water. Based on the calculated relative energies, it was found that

the molecular structure is stabilized by the formation of short contacts between the amide O-
atom and the α-phenyl C-H bonds (conformer C1 in Fig. 2). However due to the possibility of
effective resonance stabilization in all the three conformers, the energy differences between them
are almost neglectful - less than 0.1 kJ/mol. Therefore, it could be expected that the different
conformers could interconvert freely into each other by rotation around the C-N bond in solution.
(C1) most stable
(C2) ∆E = 0.04 kJ/mol
(C3) ∆E = 0.09 kJ/mol
Fig. 2. DFT B3LYP/6-311++G** optimized structure of the most probable conformers of
compound 3 in benzene
The radical scavenging mechanism of compound 3 was modeled in both nonpolar and polar
mediaby calculating the reaction enthalpies for hydrogen atom transfer (HAT mechanism), single
electron transfer (SET) and sequential proton loss electron transfer (SPLET) at the same level of
theory. Nonpolar medium was represented by benzene while as a description of polar medium
water was used.
In nonpolar medium where the ionization is not supported by the solvent, the radical scavenging
usually proceeds by direct hydrogen atom transfer. In line with this, DFT calculations predicted
that for compound 3 the most favorable would be to donate a hydrogen atom to the free radical
and form a N-centered radical (Fig. 3). The corresponding N-H bond dissociation enthalpy
(BDE) in benzene is 367 kJ/mol.

Fig. 3. Hydrogen atom transfer mechanism of compound 3 in nonpolar medium
The BDE values of free radicals in benzene were estimated by the radicals of (Z)-4-
hydroperoxyhex-2-ene, (Z)-4-hydroxyhex-2-ene, methyl hydroperoxide, methanol, hydrogen
peroxide, and water and used to establish the reactivity of compound 3. The corresponding BDE
•
values are as follows: 339 kJ/mol ((Z)-4-hydroperoxyhex-2-enyl radical), 344 kJ/mol (CH
3
OO ),
•
•
3
53 kJ/mol (HOO ), 424 kJ/mol ((Z)-4-hydroxyhex-2-enyl radical), 418 kJ/mol (CH O ), 489
3
•
kJ/mol (HO ).
Comparing the BDE value of compound 3 with those of the model free radicals, it is obvious that
compound 3 could efficiently scavenges the lipid alkoxyl, methoxyl and hydroxyl radicals.
Therefore, despite it lacks chain-breaking capacity, compound 3 could exert protective effect by
deactivating the hydroxyl radicals and thus inhibiting the initiation of lipid peroxidation as well
as by deactivating the alkoxyl radicals LO formed from the reduction of lipid peroxides and thus
decreasing the harmful effects of the lipid peroxidation. Moreover, there are two sites for
hydrogen atom abstraction in the molecule of compound 3 with equal reactivity which enables
one molecule of the antioxidant to react with two free radicals. Well-known antioxidants that
also react via HAT mechanism are α-tocopherol, curcumin, epigallocatechin galate and caffeic
acid [18].
In water i.e. polar medium, the calculated proton affinity PA of compound 3 is 221 kJ/mol. This
value is markedly lower than the BDE value in water (364 kJ/mol) and ionization potential IP in
water (460 kJ/mol),and in this way the proton transfer from compound 3 becomes more
favorable than hydrogen atom donation or electron transfer. Based on that, it could be concluded
that free radical scavenging by SPLET mechanism is prevailing over HAT and SET, as
according to thermodynamics is the most feasible reaction route in polar medium (Fig. 4).

Similarly, to the H-atom abstraction, proton transfer is equally possible from the two N-H groups
of compound 3. It was shown that radical scavenging through homolytic or heterolytic N–H
bond cleavage depends on the medium polarity, substituent effects and other structural factors
[25,32,33]. On the other hand, diverse amide derivatives were shown to easily deprotonate in
polar medium [34–37].
Fig. 4. The most probable mechanism of antioxidant action of compound 3 in polar medium
(water)
In theoretical investigations a similar mechanism of antioxidant activity has been proposed for
flavonoids [31,38-41], effective bioactive compounds found in foodstuff, which overall intake
[
42] is considered to be negatively correlated with the incidence of some chronic diseases
including cardiovascular diseases, type II diabetes, neurodegenerative diseases, and cancers [43–
6].
4
The radical scavenging mechanisms of compound 18 characterized by the best antioxidant
properties among the second group of tested compounds,(retro-amide derivatives of

palmitamine) was characterized by a model analogue, where the C16 alkyl tail was replaced with
a C residue. The optimization of the most probable molecular structures with different possible
4
orientation of the amide, hydroxyl and alkyl groups and respective intramolecular hydrogen
bonding, at IEFPCM-B3LYP/6-311++G** level of theory in benzene and water, established the
most stable geometry of the neutral compound (Fig. 5) which was further used in the study of the
radical scavenging mechanisms.
(C1) most stable
(C2) ∆E = 0.98 kJ/mol
(C3) ∆E = 2.53 kJ/mol
(C4) ∆E = 17.83 kJ/mol
(C5) ∆E = 18.12 kJ/mol
(C6) ∆E = 22.44 kJ/mol
Fig. 5. DFT B3LYP/6-311++G** optimized structure of the most probable conformers of
compound 18 in benzene
The total energies (E ) and relative energies (∆E) of the radical and anionic forms of the model
tot
compound, collected in Table 2, showed that the formation of hydroxylic O6-radical is
thermodynamically favored in benzene, as well as water. On the ther hand, deprotonation of the
O3-H group is considerably more favorable than from O6-H and N1-H as it could be seen from
the relative stability of the formed anions and the respective proton affinities in benzene and
water (Table 2).
Table 2. Relative stability of the radical and anionic species, and reaction enthalpies for the
model analogue of 18 (total energies Etot - in Hartrees; the relative energies ∆E and reaction
enthalpies BDE, IP and PA - in kJ/mol) in benzene and water
Relative stability
Radical scavenging mechanisms

a
Species
Benzene
Water
Reaction enthalpies
Benzene
Water
b
E
tot
∆E
E
tot
∆E
Molecule
-708.624405
-708.002388
-708.632872
-708.010972
HAT mechanism
.
.
.
.
Radical O6
Radical O3
Radical N1
BDE (site 1 - O6 )
325
339
434
315
326
428
.
-707.997215 13.58
-707.961362 107.71
-708.007012 10.40
-707.968441 111.67
BDE (site 2 - O3 )
.
BDE (site 3 - N1 )
SET mechanism
IP
Radical cation -708.385589
-708.421397
622
452
SPLET mechanism
-
-
Anion O3
Anion O6
-708.131270
-708.172454
PA (site 1 - O3 )
407
427
448
192
207
237
-
-
-
-708.122939 21.87
-708.166598 15.37
-708.155070 45.64
PA (site 2 - O6 )
-
Anion N1
-708.115111 42.43
PA (site 3 - N1 )
a
b
Numbering acc. to Fig. 5A; in respect to the most stable form
Thus it could be concluded that in nonpolar medium the retro-amide derivative 18 would
deactivate the free radicals preferably by hydrogen atom transfer from the hydrohyl group in m-
position (Fig. 6). According to the calculated enthalpies the compound should be able to trap the
hydroxyl, alkoxyl and peroxyl radicals.
Fig. 6. Hydrogen atom transfer mechanism of compound 18 in nonpolar medium
In polar medium – water, the preferred mechanism would be SPLET by deprotonation at the o-
hydroxyl group (Fig. 7). In water, the calculated proton affinities PA from all possible sites of
compound 18 are much lower than the BDE values from any of the sites and the respective
ionization potential IP (Table 2).

Fig. 7. The most probable mechanism of antioxidant action of compound 18 in polar medium
water)
(
4
. Conclusion
In this study we evaluated the antioxidant activity on lipid peroxidation of eighteen inhibitors of
the hydrolytic enzymes of the endocannabinoid system. Most of the tested compounds showed
good antioxidant properties in our in vitro LP assay, with an interesting activity displayed by
compounds 16 and 18, the two retro-amide derivatives caring a phenyl and hydroxyl-phenyl
group linked to the palmitate. Surprisingly, compound 3 demonstrated an antioxidant activity
comparable to that of standard antioxidants, trolox and quercetin. The mechanisms based on
DFT calculations for compound 3 in non-polar medium predicted a donation of a hydrogen atom
to the free radical and formation of N-centered radical, while in polar solvents dominate

mechanism of free radical scavenging by SPLET over HAT H-abstraction. Compound 3, based
on comparison of the BDE value with those of the model free radicals, could efficiently scavenge
the lipid alkoxyl, methoxyl and hydroxyl radicals and could exert protective effect by
deactivating the hydroxyl radicals and thus inhibiting the initiation of LP, as well by deactivating
the alkoxyl radicals formed from the reduction of lipid peroxides, thus decreasing the harmful
effects of the LP. The computational estimation on the possible radical scavenging mechanisms
of the 18, carried out by a structural analogue where the C16-alkyl chain was replaced by C4-
chain, indicated that the most favorable mechanisms are hydrogen atom transfer from the
hydroxyl group in meta-position of the benzamide fragment in nonpolar medium, and proton
transfer from the hydroxyl group in ortho-position of the benzamide fragment in polar medium.
The computational estimations for BDE, IP and PA are not in congruence with the experimental
evidences provided. Given the fact that compound 18 is a phenolic compound, in contrary to 3
which should cleave N-H bonds in order to deactivate the free radicals, it is not surprising that
based on the computed values (BDE, IP and PA) 18 should be a better radical scavenger. The
antioxidant capacity of the phenolic compounds is strongly reduced when the reaction medium
consists of a solvent prone to the formation of hydrogen bonds with the phenolic compounds
[29,47]. Possible explanation for this fact may need to be sought in reaction medium we were
using (aqueous solution of methanol) or may be different lipophylicity of the samples tested. In
any event, these preliminary in vitro results suggest an interesting antioxidant role of the tested
compounds in addition to the anticancer effects.
Acknowledgments
This work was supported by Ministry of Education, Science and Technological Development of
the Republic of Serbia (Grant OI 172044 and TR 34012), Faculty of Medicine of the University
of Niš (Internal project No. 4) and by „Università degli Studi di Milano, Linea 2 – Azione A
2
017-2018” funding to R.O.
Conflict of interests
The authors declare no conflict of interests
Authors contributions

J.L. and P.C. conceived and designed the study, S.C. and R.O. performed the chemical synthesis,
J.L. J.Z. and A.S. performed LP experiments, D.Y. performed theoretical calculations. All the
authors contributed to the paper writing.
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Highlights
•
•
•
antioxidant activity was evaluated using lipid peroxidation (LP) method
activity of the most potent compound (3) was comparable with standard antioxidants
theoretical calculations were involved in order to clarify a possible mechanism of
action
•
appropriate models for HAT, SET and SPLET-mechanisms of the antioxidant activity
are proposed

Declaration of interests
x
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: