Design, synthesis and study of nitrogen monoxide donors as potent hypolipidaemic and anti-inflammatory agents

Article abstract of DOI:10.3390/molecules25010019

Inflammation and oxidative stress are involved in cardiovascular diseases. Nitrogen monoxide participates in the regulation of endothelial processes. Thus, derivatives of classic nonsteroidal anti-inflammatory drugs (NSAIDs), trolox or cinnamic acids esterified with 2-(nitrooxy)ethanol were designed and studied. It was found that the nitrogen monoxide (NO) releasing activity was comparable to that of S-nitroso-N-acetylpenicillamine. The nitrooxy derivatives decreased potently lipid indices in the plasma of hyperlipidaemic rats (30-85%). All compounds presented increased anti-inflammatory activity in vivo, inhibiting carrageenan-induced rat paw oedema as high as 76%, up to six times higher than that of the parent acids. Lipoxygenase inhibitory activity was significant for most of them, although the parent molecules exerted a minor effect (IC₅₀ > 0.2 mM). Those compounds incorporating an antioxidant structure inhibited rat microsomal membrane lipid peroxidation strongly and possessed radical scavenging activity. These results indicated that the described compounds could act at different targets in multifactorial diseases, further limiting the possible adverse effects of drug combinations.

Full text of DOI:10.3390/molecules25010019

molecules
Article
Design, Synthesis and Study of Nitrogen Monoxide
Donors as Potent Hypolipidaemic and
Anti-Inflammatory Agents
Panagiotis Theodosis-Nobelos 1,2,*, Georgios Papagiouvanis 1,2, Maria Pantelidou 1,
Panos N. Kourounakis 2, Chrysoula Athanasekou 3 and Eleni A. Rekka 2,
*
1
Department of Pharmacy, School of Health Sciences, Frederick University, Nicosia 1036, Cyprus;
gpapagiou@gmail.com (G.P.); hsc.pm@frederick.ac.cy (M.P.)
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki,
54124 Thessaloniki, Greece; panoskur@pharm.auth.gr
2
3
Institute of Nanoscience & Nanotechnology, NCSR “Demokritos”, 15310 Agia Paraskevi Attikis, Athens, Greece
;
c.athanasekou@inn.demokritos.gr
*
Correspondence: nobelos1988@yahoo.gr (P.T.-N.); rekka@pharm.auth.gr (E.A.R.); Tel.: +30-2310997614
(P.T.-N. & E.A.R.); Fax: +30-2310997852 (P.T.-N. & E.A.R.)
Academic Editors: Alessandra Montalbano, Virginia Spanò and Paola Barraja
Received: 8 November 2019; Accepted: 17 December 2019; Published: 19 December 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract:
Inflammation and oxidative stress are involved in cardiovascular diseases.
Nitrogen monoxide participates in the regulation of endothelial processes. Thus, derivatives
of classic nonsteroidal anti-inflammatory drugs (NSAIDs), trolox or cinnamic acids esterified with
2-(nitrooxy)ethanol were designed and studied. It was found that the nitrogen monoxide (NO)
releasing activity was comparable to that of S-nitroso-N-acetylpenicillamine. The nitrooxy derivatives
decreased potently lipid indices in the plasma of hyperlipidaemic rats (30–85%). All compounds
presented increased anti-inflammatory activity in vivo, inhibiting carrageenan-induced rat paw
oedema as high as 76%, up to six times higher than that of the parent acids. Lipoxygenase inhibitory
activity was significant for most of them, although the parent molecules exerted a minor effect
(IC50 > 0.2 mM). Those compounds incorporating an antioxidant structure inhibited rat microsomal
membrane lipid peroxidation strongly and possessed radical scavenging activity. These results
indicated that the described compounds could act at different targets in multifactorial diseases, further
limiting the possible adverse effects of drug combinations.
Keywords: NO donors; synthesis; antioxidants; hyperlipidaemia; anti-inflammatory agents;
lipoxygenase inhibition
1. Introduction
Oxygen-derived free radicals and inflammation are associated with a number of pathological
conditions, including Alzheimer’s, Parkinson’s, cardiovascular, metabolic, gastrointestinal and
autoimmune diseases [1].
Increased production of reactive oxygen species (ROS) can promote vascular dysfunction and
atherogenesis. In patients with hyperlipidaemia and coronary artery disease, activation of NADPH
oxidase and xanthine oxidase generates superoxide anion radical, contributing to oxidative stress.
Oxidised low density lipoprotein cholesterol (LDL) is atherogenic. Hypercholesterolemia upregulates
angiotensin II type 1 (AT1) receptors, further enhancing oxidative stress and vasoconstriction [2].
Classical non-steroidal anti-inflammatory drugs (NSAIDs) are widely used, although the
produced non selective cyclooxygenase (COX) -1 and -2 inhibition is associated with gastric and renal
Molecules 2020, 25, 19; doi:10.3390/molecules25010019
www.mdpi.com/journal/molecules

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complications [3]. Lipoxygenase (LOX) inhibitors can exert anti-inflammatory activity and limit the
undesirable effects of COX inhibition [
3
]. The role of lipoxygenases on the generation of atherosclerotic
] is under investigation.
plaques [ ] and their implication in the inflammation of lipid tissue [6
4,5
Therefore, the interrelation of inflammation, hyperlipidaemia and ROS production has become evident.
Nitrogen monoxide (NO) participates in neuronal plasticity, memory and learning processes,
vascular tone, inhibition of platelet aggregation and neutrophil chemotaxis. In cases of inflammatory and
oxidative conditions, like diabetes, hypertension, hypercholesterolemia and atherosclerosis, uncoupling
of nitrogen oxide synthase (NOS), propagation of oxidative stress, neutrophil and macrophage
infiltration are observed [
7]. The latter phenomena could be reversed by the administration of
hypolipidaemic agents that could limit endothelial NOS uncoupling [8]. Furthermore, the reduction
of the gastrointestinal toxicity of NSAIDs with the incorporation of NO releasing moieties has been
reported [3,9].
In view of the aforementioned evidence, adequate antioxidant and/or anti-inflammatory activity,
combined with NO releasing potential could be considered useful in the treatment of complex diseases,
such as those affecting the cardiovascular system. Therefore, we designed and synthesised a series
of 2-(nitrooxy)ethanol esters with classic NSAIDs (compounds
trolox (( )-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), compound
((E)-3-phenylacrylic acid) compound , or BHCA ((E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic
acid), compounds , a cinnamic acid derivative possessing the molecular characteristics of both
1,
3
–
5), the well-known antioxidant
±
9
and cinnamic acid
6
7
butylated hydroxytoluene (BHT) and cinnamic acid (Figure 1). 2-(Nitooxy)ethanol was selected,
since we found that a chain of two carbon atoms between the carboxylate and the nitric ester groups
favoured activity [
9]. Two more compounds, one representing the NSAIDs group (2) and one from the
antioxidant acids set (
8), with a p-nitrophenyl moiety, were synthesised for comparison, due to their
comparable lipophilic and electron withdrawing character.
NO2
O
O
ONO2
O
O
2
1
ONO2
O
O
O
N
O
3
Cl
O
O
O
ONO2
ONO2
O
O
O
5
4
O
ONO2
O
6
NO2
O
O
ONO2
O
O
HO
HO
7
8
HO
O
ONO2
O
O
9
Figure 1. Structures of the synthesised compounds.

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Compound
1
is reported in the literature [10] as an agent promoting wound healing, without
synthetic details or structure characterisation. Compound
5 has been synthesised previously [11]
via a different method (nitration of 2-bromoethanol with a 70% HNO3-95% H2SO4 mixture gave
nitrooxyethyl bromide, which yielded the final product after condensation with ketoprofen in the
presence of Cs2CO3) and was tested for anti-proliferative activity using prostate cancer cells. All other
compounds are novel. Compounds 1, 2, 5 and 9 are racemic.
The effect of the synthesised compounds on lipidaemic indices in the plasma of hyperlipidaemic
rats was examined and the NO releasing ability was determined. The antioxidant activity, expressed
as the inhibition of rat hepatic microsomal membrane lipid peroxidation, as well as the interaction
with the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was evaluated for selected compounds.
The effects on carrageenan-induced rat paw oedema and the in vitro activity of soybean lipoxygenase
were examined.
2. Results and Discussion
2.1. Synthesis
As shown in Scheme 1, the starting material
2-chloroethanol [ ]. Compound or 2-(4-nitrophenyl)ethanol was esterified with (
(compounds ), indomethacin (compound ), (+) naproxen (compound ), (
(compound ), cinnamic acid (compound ), BHCA (compounds ) trolox (compound
methods, in good yields (up to 80%). An excess of the corresponding alcohol was used due to the
electron withdrawing effect of the nitrooxy group. The application of method for the synthesis of the
trolox derivative failed to give the expected product, possibly due to steric reasons. Compound
was obtained (yield 74%) using CDI (method B), since this reaction is less sterically hindered.
i
was prepared using silver nitrate and
) ibuprofen
) ketoprofen
) using three
9
i
±
1,
2
3
4
±
5
6
7
,
8
9
A
9
9
Cl
ONO2
+
AgNO3
HO
HO
i
O
ONO2
ONO2
1, 3-7, 9
HO
O
A) DCC/DMAP/CHCl3
B) CDI/THF
R
O
R
OH
or
+
C) oxalylchloride/Et3N/CHCl3
or
NO2
NO2
O
HO
R
O
2, 8
Scheme 1. Synthesis of compounds
(2 mmol) or 2-(4-nitrophenyl)ethanol (2 mmol, compound
in CHCl3, 4 h, r.t. B) Compound : Trolox (1 mmol), CDI, (1.1 mmol), tetrahydrofuran, stirring, 4 h.
1–
9. A) Compounds
1,
3,
4,
5,
7,
8: Acid (1 mmol), 2-nitroxy-ethanol
8), DCC (1.5 mmol), DMAP (0.1 mmol),
9
2-Nitrooxy-ethanol (2 mmol), 24 h, r.t. C) Compounds 2, 6. Acid (1 mmol), oxalylchloride (3 mmol),
0–4 ◦C, CHCl3, stirring, 3 h. 2-Nitrooxy-ethanol (2 mmol), triethylamine (1.1 mmol), 3 h, r.t.
2.2. Hypolipidaemic Effect
The pleiotropic effects of statins, used as hypolipidaemic drugs, include antioxidant ability and
direct increase of eNOS activity, in addition to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase [12]. Considering the aforementioned, compounds
1, 3–7 and 9 were tested for
anti-dyslipidaemic activity in Triton-induced hyperlipidaemia in rats. The systemic administration

Molecules 2020, 25, 19
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of Triton-WR1339 (tyloxapol) to rats induces a biphasic elevation of plasma cholesterol, atherogenic
VLDL cholesterol and LDL cholesterol, as well as triglyceride levels. In phase I, plasma lipid levels
increased sharply, after 24 h. Agents shown to be active in phase I are considered to interfere with
cholesterol biosynthesis [13,14]. Results are shown in Table 1.
Table 1. Effect of the nitrooxy compounds, simvastatin, ibuprofen and ketoprofen on Triton WR1339
(tyloxapol) induced hyperlipidaemia.
% Reduction
Compound
Dose (i.p.) (µmol/kg)
TC a
LDL-C c
TG b
1
3
4
5
6
7
9
150
150
150
150
150
150
150
150
300
500
c
62.2 **
71.7 **
78.2 ***
46.6 **
70.9 ***
65.5 **
55.4 ***
73.0 ***
41.0 ***
53.0 ***
30.1 **
31.8 **
38.9 ***
40.2 ***
54.4 ***
45.7 ***
30.0 ***
-
80.0 *
62.3 *
84.6 ***
57.8 ***
74.8 ***
60.6 *
65.0 ***
70.0 ***
41.6 ***
25.5 ***
Simvastatin
Ibuprofen
Naproxen
38.0 ***
43.5 ***
a
b
TC: Total cholesterol; TG: Triglycerides; LDL-C: LDL cholesterol. Tyloxapol: 200 mg/kg, i.p. Significant
difference from hyperlipidaemic control group: * p < 0.01, ** p < 0.005, *** p < 0.001 (Student’s t test).
All tested compounds strongly decreased lipidaemic indices, especially cholesterol levels (LDL
and total, 61–85% and 55.4–78.2%, respectively). The animals appeared normal, macroscopically and
by autopsy, 24 h post administration. The activity of compounds was higher than that of the relevant
parent acids at a much lower dose. In this experiment, it was confirmed that, although simvastatin
profoundly lowered cholesterol, being comparable to the synthesised compounds, it had no effect on
plasma triglyceride levels. Indeed, it has been reported that statins mainly lower serum LDL levels,
whereas a serum triglyceride decrease is a minor effect [15]. The synthesised compounds significantly
decreased triglyceride plasma concentrations as well (30–54.4%).
2.3. Nitrogen Monoxide Release
The ability of compounds
in Table 2. Compounds and
increase in the amount of released NO was observed with increasing compound concentration.
With the small deviation of compounds and , compounds showed similar NO donating ability
reaching up to 24 M NO/100 M compound. NO release from S-nitroso-N-acetylpenicillamine
1,
3–
7
and
9 to release NO in vitro, at different concentrations is shown
2
8 were not included, because they were not possible NO donors. A linear
4
7
µ
µ
(SNAP), a known potent NO donor, was found to be 56.3µM / 100 µM.
Table 2. In vitro nitrogen monoxide release.
NO Release (µM)/Compound
Compound
1
3
4
5
6
7
9
500
250
125
62.5
31.25
µ
µ
µ
M
M
M
116
82
40
17
7
102
58
28
13
5
73
37
17
7
100
57
26
11
4
98
51
22
11
5
77
38
17
6.5
2
93
49
23
10
3
µ
M
µM
2.5
All determinations (using the Griess reagent) were performed at least in triplicate and the standard deviation was
always within ±10% of the mean value.

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Compounds with NO-releasing activity could be useful especially in cardiovascular complications.
It is known that the NO precursor L-arginine reduced atherosclerosis in LDL receptor knockout
hypercholesterolaemic mice, and this activity was inhibited by NOS inhibitors [16].
2.4. Lipoxygenase Inhibitory Activity
Lipoxygenases form the second major path of arachidonic acid metabolism. In atherogenic plaques
considerable lipoxygenase activity has been observed [6], implicated in adipose tissue inflammation
and atherosclerosis [17].
Although there are several lipoxygenase enzymes, it is believed that all enzymes share a common
mechanism, i.e., the stereo- and region- specific peroxidation of arachidonic or linoleic acid by molecular
oxygen [18]. Soybean lipoxygenase 1-B can use arachidonic acid as a substrate, with about 15% of the
activity using linoleic acid.
The ability of compounds to inhibit lipoxygenase, presented as IC50 values towards soybean
lipoxygenase 1-B, using linoleic acid as a substrate, after 7 min of incubation, is demonstrated in Table 3.
The IC50 of nordihydroguaiaretic acid (NDGA), an antioxidant compound acting as a nonspecific
inhibitor of lipoxygenase, was also included as a reference together with ibuprofen, ketoprofen
and trolox. When linoleic acid was used at 1 mM, a concentration 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 is
diminishing by increasing substrate concentration.
Table 3. Effect of compounds
lipophilicity (clogP) b.
1–
9, ibuprofen, ketoprofen, trolox and NDGA on lipoxygenase a, and their
Compound
IC50 (µM)
clogP
1
2
3
4
5
6
7
8
9
46
24
107
>>300
86
4.65
5.92
5.15
3.78
3.73
3.18
6.16
7.44
4.16
220
44
10.5
120
200
220
>>300
1.3
Ibuprofen
Ketoprofen
Trolox
NDGA
3.10
a
b
After 7 min of incubation; NDGA: nordihydroguaiaretic acid. clogP: ClogP for windows v. 4.0, BioByte Corp
(BioByte Corporation, Claremont, CA, U.S.A.).
The majority of the compounds showed considerable activity, except for compound
4.
Trolox derivative
compounds and
to the higher lipophilicity of the former compound (Table 3). However, the clogP value of
9
8
appeared active while trolox itself had an IC50 higher than 300
were more active than and , respectively. At first sight, this might be attributed
(5.92) was
µM. Interestingly,
2
1
7
2
very close to that of compound 7 (6.16), but 2 had about double the activity of 7. It has been reported
that a 4-nitro group on a phenyl ring is among selective groups for 5-lipoxygenase inhibition [19].
Furthermore, di-tert-butylphenol derivatives, like tebufelone and darbufelone, are considered to
comprise a class of dual COX-2/5-LOX inhibitors [19]. It was previously shown by us [20] that this
structural moiety contributed to increased lipoxygenase inhibitory activity.
Among the nitric ester derivatives, it seems convincing that the more rigid molecules are less active.
Thus, compound
4
was practically inactive, while compound
5, although equally lipophilic with
4
, was quite active. Similarly, activity was lower for compounds
3
and . Since we showed that
9

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these derivatives acted as competitive inhibitors of lipoxygenase, they were expected to occupy the
hydrophobic cavity at the active site, which was intended for the flexible molecules of arachidonic
or linoleic acid. Therefore, more flexible, lipophilc molecules are more active than the less flexible
analogues, and the least lipophilc compound 6 had low activity.
2.5. Antioxidant and Radical Scavenging Activity
The antioxidant activity of compounds was estimated by the inhibition of rat hepatic microsomal
membrane lipid peroxidation, induced by Fe+2/ascorbate, estimated as 2-thiobarbituric acid (TBA)
reactive material, and by their interaction with the N-centred 1,1-diphenyl-2-picrylhydrazyl (DPPH)
stable free radical [14]. Compounds
was less than 25% at 1 mM). The percent interaction with DPPH and the IC50 values of compounds
on rat hepatic microsomal membrane lipid peroxidation, after 45 min of incubation is shown in Table 4.
1–6 had minor antioxidant activity (inhibition of lipid peroxidation
7–9
Table 4. Interaction of compounds 7–9 and trolox, at various concentrations, with DPPH (200 µ
M) a
and their effect on lipid peroxidation b.
Percent Interaction with DPPH
Compound
Inhibition of Lipid Peroxidation IC50 (µM)
200 µM
100 µM
50 µM
7
8
9
87
88
92
92
49
57
90
90
21
23
55
38
41
150
2.3
25
Trolox
a After 30 min of incubation. b After 45 min of incubation. Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid. All determinations were performed at least in triplicate and the standard deviation was always within
of the mean value.
±
10%
Compound 9 was almost an eleven-fold stronger inhibitor of lipid peroxidation than trolox and
of similar activity with trolox as a DPPH radical scavenger. It is widely known (e.g., [20]) that the
antioxidant activity of phenolic compounds is due to the phenolic hydrogen abstraction and can be
enhanced by the extended conjugation of the chroman ring or the cinnamic structure, resulting in
radical stabilisation. This effect may be further improved by the proper lipophilicity, which would
permit an effective approach to the lipid phase.
The time course of lipid peroxidation, as affected by various concentrations of 9 is shown in Figure 2.
A long lag period of about 20 min, characteristic of chain-breaking antioxidants, was observed.
1.8
1.6
1.4
1.2
control
1.0
2,0 μΜ
0.8
2,5 μΜ
0.6
3,0 μΜ
0.4
5,0 μΜ
0.2
0.0
0
10
20
30
40
Time (min)
Figure 2. Effect of compound 9 (various concentrations) on the time course of lipid peroxidation.

Molecules 2020, 25, 19
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Compound 7 showed considerable activity in both experiments. The lower protective effect of 8
against lipid peroxidation, compared to , can be attributed to the lower solubility of in the aqueous
environment of the experiment. Compounds and had about the same reducing activity in the
7
8
7
8
ethanolic medium of the DPPH test. Taken together, it could be suggested that these compounds may
be able to offer protection against a free radical attack by direct inhibition of lipid peroxidation as well
as by inhibiting lipoxygenase, considered to play a significant contributory role to atherosclerosis [21].
2.6. In Vivo Anti-Inflammatory Activity
The carrageenan-induced paw oedema is a well-known and widely used model of acute
inflammation. The response to carrageenan inflammation may be divided into three phases. In the
first phase (after 1.5 h), histamine and serotonin are released, the second phase (1.5–2.5 h after
carrageenan administration) is mediated by kinins, whereas, in the third phase, more than two hours
post administration, neutrophil infiltration, prostaglandin production and release of pro-inflammatory
cytokines are involved [22]. In this work, oedema was measured 3.5 h post injection.
The effect of the synthesised compounds on acute inflammation, applying the carrageenan paw
oedema model, as well as the anti-inflammatory activity of some classic NSAIDs used as a reference, is
shown in Table 5.
Table 5.
Effect of compounds 1–9, ibuprofen, indomethacin, ketoprofen and naproxen on
carrageenan-induced rat paw oedema a.
Compound
% Oedema Reduction
1
2
76 **
53 *
3
4
5
6
7
8
9
29 **
70 **
75 **
61 **
55 **
51 **
57 **
36 *
Ibuprofen
Indomethacin
Naproxen
Ketoprofen
42 *
11 *
47 *
a
The effect on oedema is expressed as percent oedema reduction in comparison to controls, which received
carrageenan only. All compounds were administered i.p. at 0.15 mmol/kg of body weight. Significant difference
from control: * p < 0.005, ** p < 0.001 (Student’s t test).
The synthesised compounds demonstrated more than 50% oedema inhibition, except for
compound
for the naproxen derivative
3
. This increase, compared with the parent NSAIDs was more than six fold higher
, while and were about two times more active than ibuprofen and
4
1
5
ketoprofen, respectively. It seems that esterification with 2-(nitrooxy)ethanol generally increased the
anti-inflammatory effect of the NSAIDs. This molecular modification also added anti-inflammatory
activity to the antioxidant acids and cinnamic acid. It has been previously reported by us that esters or
amides of several NSAIDs, e.g., with 2-methoxy-4-methyl-phenol or thiomorpholine, enhanced the
anti-inflammatory activity of the parent molecules [23
potent anti-inflammatory agents if they are esterified, e.g., with cinnamyl alcohol [20], while butylated
hydroxytoluene (BHT) has been found devoid of any anti-inflammatory activity [ ]. It has also been
shown than the effect of a number of NSAID esters is not due to hydrolysis of the ester group [23].
,24] and that antioxidant acids such as trolox yield
9

Molecules 2020, 25, 19
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3. Materials and Methods
3.1. General
All commercially available chemicals of the appropriate purity were purchased from Merck
(Kenilworth, NJ, U.S.A.) or Sigma ((St. Louis, MO, U.S.A.). The IR spectra were recorded on a
Perkin Elmer Spectrum BX FT-IR spectrometer (Waltham, MA, U.S.A.). The 1H NMR and 13C NMR
spectra were recorded using a BRUKER Avance III-300 MHz (Billerica, MA, U.S.A.) or an AGILENT
DD2-500 MHz ((Santa Clara, CA, U.S.A.) spectrometer. Chemical shifts were reported in
δ (ppm) and
signals were given as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Melting points (mp) were
determined with a MEL-TEMPII apparatus, Laboratory Devices, Sigma-Aldrich (Milwaukee WI, U.S.A)
and were uncorrected. The microanalyses were performed on a Perkin-Elmer 2400 CHN elemental
analyser (Waltham, MA, U.S.A.). Thin-layer chromatography (TLC silica gel 60 F254 aluminium sheets,
Merck (Kenilworth, NJ, U.S.A.) was used to follow the reactions and the spots were visualised under
UV light.
3.2. Synthesis
3.2.1. 2-Nitrooxy-Ethanol [9]
Silver nitrate (35 mmol) was added to a solution of 2-chloroethanol (23 mmol) in acetonitrile
(40 mL). The reaction mixture was stirred at room temperature overnight and was light protected.
Then, the reaction mixture was filtered and the volatiles were removed under reduced pressure.
The residue was dissolved in diethyl ether and washed with saturated NaCl solution. The organic
layer was dried over calcium chloride, filtered, and concentrated.
Pale yellow oil, yield 16%. IR (film) v: 3400, 2900, 1630 cm−1 1H NMR (CDCl3)
δ: 2.00 (s, 1H,
.
OH), 3.90–4.00 (t, J = 5 Hz, 2H, CH2OH), 4.50–4.60 (t, J = 5 Hz, 2H, CH2ONO2).
3.2.2. General Procedures for the Synthesis of Compounds 1–9
(A) Compounds
1
,
3
,
4
,
5
,
7
,
8: The corresponding acid (1 mmol) and 2-nitrooxy-ethanol
) were mixed in CHCl3.
(2 mmol) or 2-(4-nitrophenyl)ethanol (2 mmol, for compound
8
Then, N,N’-dicyclohexylcarbodiimide (DCC, 1.5 mmol) and N,N-dimethylaminopyridine (DMAP,
0.1 mmol) were added and dissolved in the same solvent. The mixture was stirred for 4 h at
ambient temperature. The final compounds were isolated with flash column chromatography using
mixtures of petroleum ether and ethyl acetate.
(B) Compound 9: Trolox (1 mmol) and carbonyldiimidazole (CDI, 1,1 mmol) in tetrahydrofuran
were mixed, stirred for 45 min and 2-nitrooxy-ethanol (2 mmol) was added and stirred for 24 h at
ambient temperature. The solvent was distilled off, the residue was dissolved in ethyl acetate, washed
with water, 5% aqueous HCl and NaHCO3 solutions and dried (Na2SO4). The final compound was
isolated with flash column chromatography using mixtures of petroleum ether and ethyl acetate
as eluents.
(C) Compounds
2
and
6
: The corres◦ponding acid (1 mmol) was dissolved in CHCl3 and
oxalylchloride (3 mmol) was added at 0–4 C. After stirring for 3 h, the volatile compounds were
distilled off. 2-nitrooxy-ethanol (2 mmol) and triethylamine (1.1 mmol), in the same solvent, were added
◦
at 0–4 C and left for 3 h. The mixture was filtered, washed with water, 5% aqueous NaHCO3,
saturated NaCl solutions and dried (Na2SO4). The final compounds were isolated with flash column
chromatography with mixtures of petroleum ether and ethyl acetate as eluents.
(
±
)-2-(Nitrooxy)ethyl 2-(4-isobutylphenyl)propanoate (
ether/ethyl acetate, 40/1). Viscous transparent liquid, yield 85 %. IR (film)
2957, 2910, 2870 (C-H alkyl), 1739 (C=O ester), 1634 (N=O), 1513 (C-C aromatic) cm−1
(CDCl3), : 0.93 (d, 6H, J = 6.6 Hz, -CH-(CH3)2), 1.53 (d, 3H, J = 7.2 Hz, O=C-CH-C 3), 2.00–1.81
(m, 1H, -CH2C -(CH3)2), 2.48 (d, 2H, J = 7.1 Hz, -C 2CH-(CH3)2), 3.75 (q, 1H, J = 7.1 Hz,
1
): Flash column chromatography (petroleum
: 3052, 3024 (C-H aromatic),
1H NMR
ν
.
δ
H
H
H

Molecules 2020, 25, 19
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O=C-C
H
-CH3), 4.45–4.29 (m, 2H, -O-CH2-C
H
2ONO2), 4.73–4.55 (m, 2H, -O-C
H
2-CH2ONO2), 7.12 (d,
2H, J = 8.0 Hz, phenyl C3, C5), 7.21 (d, 2H, J = 8.0 Hz, phenyl C2, C6). 13C NMR (CDCl3)
δ
: 18.32 (1C,
H-(CH3)2), 44.85 (1C, O=C- H-CH3),
H2-CH2ONO2), 70.30 (1C, -O-CH2- H2ONO2), 127.06 (2C,
phenyl C2, C6), 129.39 (2C, phenyl C3, C5), 137.05 (1C, phenyl C4), 140.78 (1C, phenyl C1), 174.34 (1C,
O- =O). Anal. Calculated for C15H21NO5x0.3H2O: C, 59.91; H, 7.24; N, 4.66. Found: C, 59.73; H, 7.15;
N, 4.86.
)-4-Nitrophenethyl 2-(4-isobutylphenyl)propanoate (
ether/ethyl acetate, 25/1 and gradually 10/1). Viscus liquid, yield 75%. IR (nujol)
1606, 1599 (C-C aromatic), 1623 (N=O) cm−1.1H NMR (CDCl3)
: 0.94 (d, 6H, J = 6.5 Hz, -CH-(CH3)2),
1.48 (d, 3H, J = 7.1 Hz, O=C-CH-C 3), 1.89 (dt, 1H, J = 13.2, 6.5 Hz, -CH2C -(CH3)2), 2.50 (d,
2H, J = 7.1 Hz, -C 2CH-(CH3)2), 2.99 (t, 2H, J = 6.2 Hz, -O-CH2-C 2-Ar), 3.67 (q, 1H, J = 7.1 Hz,
O=C-CH-C 3), 4.50–4.23 (m, 2H, -O-C 2-CH2-Ar), 7.09 (d, 2H, J = 7.7 Hz, phenyl C3, C5), 7.14 (d,
2H, J = 7.9 Hz, phenyl C2, C6), 7.21 (d, 2H, J = 8.0 Hz, nitrophenyl C3, C5), 8.07 (d, 2H, J = 8.0 Hz,
nitrophenyl C2, C6). 13C NMR (CDCl3)
: 18.22 (1C, O=C-CH- H3), 22.34 (2C, -CH2CH-( H3)2), 30.18
(1C, -CH2 H-(CH3)2), 34.83 (1C, O-CH2-CH2-), 44.95 (1C, O=C- H-CH3), 45.06 (1C, - H2CH-(CH3)2),
O=C-CH-
45.00 (1C, -
C
H3), 22.35 (2C, -CH2CH-(
C
H3)2), 30.15 (1C, -CH2
C
C
C
H2CH-(CH3)2), 60.35 (1C, -O-
C
C
C
(±
2): Flash column chromatography (petroleum
ν
: 1723 (C=O ester),
δ
H
H
H
H
H
H
δ
C
C
C
C
C
63.93 (1C, O-CH2-CH2-), 123.53 (2C, nitrophenyl C3, C5), 127.12 (2C, phenyl C2, C6), 129.31 (2C,
nitrophenyl C2, C6), 129.67 (2C, phenyl C3, C5), 137.39 (1C, phenyl C4), 140.73 (1C, phenyl C1),
145.68 (1C, nitrophenyl C4), 146.69 (1C, nitrophenyl C1), 174.41 (1C, O-C=O). Anal. Calculated for
C21H25NO4x0.9H2O: C, 67.84; H, 7.27; N, 3.77. Found: C, 67.86; H, 7.46; N, 3.74.
2-(Nitrooxy)ethyl 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate (3): Flash column
chromatography (petroleum ether/ethyl acetate, 10/1).Yellow powder, yield 50%, mp 83–84 ◦C. IR (nujol)
1
ν
: 1743 (C=O ester), 1684 (C=O amide), 1604, 1590 (C-C aromatic) cm−1. H NMR (CDCl3)
δ
: 2.41 (s,
2ONO2),
2-CH2ONO2), 6.71 (dd, 1H, J = 9.0, 2.4 Hz, indole C6), 6.91 (d, 1H, J = 9.0 Hz,
indole C7), 6.98 (d, 1H, J = 2.4 Hz, indole C4), 7.50 (d, 2H, J = 8.6 Hz, chloro-benzoyl C3, C5), 7.69 (d, 2H,
J = 8.6 Hz, chloro-benzoyl C2, C6). 13C NMR (CDCl3)
: 13.27 (1C, H3-C-N-), 30.02 (1C, - H2-C=O),
55.69 (1C, H3-O-), 60.77 (1C, -O- H2-CH2ONO2), 70.19 (1C, -O-CH2- H2ONO2), 101.09 (1C, indole
3H, C
4.75–4.60 (m, 2H, -O-C
H3-C-N-), 3.73 (s, 2H, -CH2-C=O), 3.87 (s, 3H, -O-CH3), 4.46–4.38 (m, 2H, -O-CH2-CH
H
δ
C
C
C
C
C
C4), 111.74 (1C, indole C3), 111.80 (1C, indole C6), 114.98 (1C, indole C7), 129.12 (2C, chloro-benzoyl
C2, C6), 130.40 (1C, indole C3a), 130.77 (1C, indole C7b), 131.17 (2C, chloro-benzoyl C3, C5), 133.79 (1C,
chloro-benzoyl C1), 136.09 (1C, chloro-benzoyl C4), 139.31 (1C, indole C2), 156.08 (1C, indole C5), 168.28
(1C, chloro-benzoyl C=O), 170.40 (1C, acetate C=O). Anal. Calculated for C21H19ClN2O7x0.45H2O: C,
55.44; H, 4.41; N, 6.16. Found: C, 55.46; H, 4.54; N, 6.06.
(+)-2-(Nitrooxy)ethyl 2-(6-methoxynaphthalen-2-yl)propanoate (4): Flash column chromatography
◦
(petroleum ether/ethyl acetate, 8/1 and gradually 4/1). White powder, yield, 55%, mp 64–65 C.
1
IR (nujol)
J = 7.1 Hz, C
O-CH2-C 2ONO2), 4.70–4.54 (m, 2H, O-C
1H, J = 6.8 Hz, naphthyl C5), 7.80–7.64 (m, 3H, naphthyl C3, C8, C10). 13C NMR (CDCl3)
O=C-CH- H3), 45.20 (1C, O=C- H-CH3), 55.30 (1C, -O-CH3), 60.44 (1C, -O- H2-CH2ONO2), 70.28
(1C, -O-CH2- H2ONO2), 105.56 (1C, naphthyl C5), 119.07 (1C, naphthyl C7), 125.97 (1C, naphthyl
ν
: 1727 (C=O ester), 1625 (N=O), 1604 (C-C aromatic) cm−1. H NMR (CDCl3)
δ
: 1.63 (d, 3H,
-C=O), 4.48–4.28 (m, 2H,
2-CH2ONO2), 7.20–7.05 (m, 2H, naphthyl C2, C7), 7.41 (d,
: 18.37 (1C,
H
3-CH-C=O), 3.95 (s, 3H, -O-CH3), 4.06–3.87 (m, 1H, CH3-CH
H
H
δ
C
C
C
C
C10), 126.01 (1C, naphthyl C9), 127.25 (1C, naphthyl C2), 128.88 (1C, naphthyl C3), 129.26 (1C, naphthyl
C8), 133.76 (1C, naphthyl C4), 139.96 (1C, naphthyl C1), 157.71 (1C, naphthyl C6), 174.27 (1C, C=O).
Anal. Calculated for C16H17NO6x0.2H2O: C, 59.51; H, 5.43; N, 4.34. Found: C, 59.51; H, 5.17; N, 4.65.
(±
)-2-(Nitrooxy)ethyl 2-(3-benzoylphenyl)propanoate (5): Flash column chromatography (petroleum
ether/ethyl acetate, 5/1). Viscous transparent liquid, yield 46%. IR (nujol) : 1740 (C=O ester),
1658, 1636 (N=O), 1598, 1581 (C-C aromatic) cm−1 1H NMR (CDCl3),
: 1.59 (d, 3H, J = 7.1 Hz,
O=C-CH-C 3), 3.86 (q, 1H, J = 7.0 Hz, O=C-C -CH3), 4.48–4.28 (m, 2H, -O-CH2-C 2ONO2), 4.65 (s,
2H, -O-C : 19.65 (1C, O=C-CH- H3),
2-CH2ONO2), 7.86–7.42 (m, 9H, aromatic). 13C NMR (CDCl3)
45.33 (1C, O=C- H-CH3), 60.35 (1C, -O- H2-CH2ONO2), 70.64 (1C, -O-CH2- H2ONO2), 128.26 (2C,
ν
.
δ
H
H
H
H
δ
C
C
C
C

Molecules 2020, 25, 19
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benzoyl C3, C5), 128, 31 (1C, phenyl C2), 128,73 (2C, benzoyl C2, C6), 129, 35 (1C, phenyl C4), 130, 84
(1C, phenyl C5), 131, 22 (1C, phenyl C6), 131,74 (1C, benzoyl C4), 137,12 (1C, benzoyl C1), 140.78 (1C,
phenyl C1), 142.61 (1C, phenyl C3), 175.12 (1C, O-C=O), 190,83 (1C, C=O ketone). Anal. Calculated for
C18H17NO6x0.2H2O: C, 62.32; H, 5.05; N, 4.03. Found: C, 62.32; H, 5.13; N, 4.19.
(E)-2-(Nitrooxy)ethyl cinnamate (6): Flash column chromatography (petroleum ether/ethyl
acetate, 40/1). Viscous transparent liquid, yield 42%. IR (film)
ν
: 1717 (C=O ester), 1636 (N=O),
2ONO2), 4.98–4.70
-CH-C=O), 7.53–7.40 (m, 3H, aromatic C3, C4,
C5), 7.68–7.54 (m, 2H, aromatic C2, C6), 7.75 (d, 1H, 16.0 Hz, -CH-C
-C=O). 13C NMR (CDCl3)
59.78 (1C, O- H2-CH2ONO2), 70.44 (1C, O-CH2- H2ONO2), 116.24 (1C, -CH- H-C=O), 126.05 (1C,
phenyl C1), 125.87 (2C, phenyl C2, C6), 126.33 (2C, phenyl C3, C5), 128.46 (1C, phenyl C4), 146.50 (1C,
H-CH-C=O), 167.75 (1C, -CH-CH- =O). Anal Calculated for C11H11NO5: C, 55.70; H, 4.67; N, 5.90.
Found: C, 55.28; H, 4.37; N, 5.95.
1
1578, 1496 (C-C aromatic) cm−1. H NMR (CDCl3),
δ: 4.68–4.46 (m, 2H, -O-CH2-C
H
(m, 2H, -O-CH2-CH2ONO2), 6.47 (d, 1H, 16.0 Hz, -CH
H
δ:
C
C
C
-
C
C
(E)-2-(Nitrooxy)ethyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylate (7): Flash column chromatography
(petroleum ether/ethyl acetate, 20/1). White powder, yield 71%, mp 80 ◦C. IR (nujol)
ν
: 3569 (O-H), 1711
1
(C=O ester), 1630 (N=O), 1596 (C-C aromatic) cm−1. H NMR (CDCl3),
δ
(ppm): 1.48 (s, 18H, -CH3),
2-CH2ONO2), 5.59 (s, 1H, Ar-OH), 6.32
-C=O), 7.40 (s, 2H, phenyl), 7.66 (d, 1H, J = 15.9 Hz, -C
-CH-C=O). 13
H3)3), 34.32 (2C, -(CH3)3), 59.96 (1C, O- H2-CH2ONO2), 70.76 (1C,
H-C=O), 125.47 (1C, phenyl C1), 125.65 (2C, phenyl C2, C6),
H-CH-C=O), 156.44 (1C, phenyl C4), 166.92 (1C, -CH-CH- =O).
4.55–4.47 (m, 2H, O-CH2-C
H
2ONO2), 4.80–4.72 (m, 2H, O-C
H
(d, 1H, J = 15.9 Hz, -CH=C
H
H
C
NMR (CDCl3)
O-CH2- H2ONO2), 113.29 (1C, -CH-
136.40 (2C, phenyl C3, C5), 147.19 (1C, -
δ: 30.12 (6C, C-(
C
C
C
C
C
C
C
Anal. Calculated for C19H27NO7: C, 62.45; H, 7.45; N, 3.83. Found: C, 62.03; H, 7.17; N, 4.18.
(E)-4-Nitrophenethyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylate (8): Flash column chromatography
(petroleum ether/ethyl acetate, 10/1). Yellow powder, yield 60%, mp 156–157 ◦C. IR (nujol)
ν
: 3626
1
(O-H), 1706 (C=O ester), 1629 (N=O), 1606, 1591 (C-C aromatic) cm−1. H NMR (CDCl3)
δ: 1.49 (s,
18H, -CH3), 3.16 (t, 2H, O-CH2-C
1H, J = 15.9 Hz, -CH=C -C=O), 7.38 (s, 2H, phenyl C2, C6), 7.46 (d, 2H, J = 7.5 Hz, nitrophenyl
C2, C6), 7.66 (d, 1H, J = 15.9 Hz, -C
=CH-C=O), 8.21 (d, 2H, J = 7.5 Hz, nitrophenyl C3, C5). 13C
NMR (CDCl3) : 30.12 (6C, C-( H3)3), 34.31 (2C, -(CH3)3), 35.13 (1C, O-CH2- H2-Ar), 63.64 (1C,
O- H2-CH2-Ar), 113.89 (1C, -CH- H-C=O), 123.60 (2C, phenyl C2, C6), 125.51 (2C, nitrophenyl C3,
C5), 125.53 (1C, phenyl C1), 129.76 (2C, nitrophenyl C2, C6), 136.40 (2C, phenyl C3, C5), 145.89 (1C,
H-CH-C=O), 146.52 (1C, nitrophenyl C4), 146.87 (1C, nitrophenyl C1), 156.32 (1C, phenyl C4), 167.20
H2-Ar), 4.50 (t, 2H, O-CH2-CH2-Ar), 5.54 (s, 1H, Ar-OH), 6.27 (d,
H
H
δ
C
C
C
C
C
-C
(1C, -CH-CH-C=O). Anal. Calculated for C25H31NO5: C, 70.57; H, 7.34; N, 3.29. Found: C, 70.34; H,
7.67; N, 3.51.
(
±
)-2-(Nitrooxy)ethyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (9):
Flash column
◦
chromatography (petroleum ether/ethyl acetate, 40/1). White powder, yield 54%, mp 95–97 C.
IR (nujol)
1
ν
: 3532 (O-H), 1741 (C=O ester), 1649, 1634 (N=O) cm−1. H NMR (CDCl3),
δ
(ppm):1.65
(s, 3H, chromane C1), 2.00–1.86 (m, 1H, chromane C2 axial), 2.20, 2.19, 2.09 (3s, 9H, -CH3 aromatic),
2.52–2.41 (m, 1H, chromane C2 equatorial), 2.74–2.53 (m, 2H, chromane C3), 4.44–4.24 (m, 2H,
-O-CH2-C
(3C, - H3 aromatic), 20.87 (1C, chromane C3), 25.32 (1C, chromane C1-
60.46 (1C, O- H2-CH2ONO2), 70.28 (1C, O-CH2- H2ONO2), 77.19 (1C, chromane C1), 116.80 (1C,
H
2ONO2), 4.60–4.47 (m, 2H, -O-C
H
2-CH2ONO2). 13C NMR (CDCl3)
H3), 30.61 (1C, chromane C2),
δ: 11.17, 11.71, 12.13
C
C
C
C
chromane C4), 118.42 (1C, chromane C7), 121.24 (1C, chromane C8), 122.64 (1C, chromane C5), 145.40
(1C, chromane C6), 145.47 (1C, chromane C9), 173.63 (1C, C=O). Anal. Calculated for C16H21NO7
x2H2O: C, 51.20; H, 6.71; N, 3.73. Found: C, 51.31; H, 6.47; N, 3.87.
3.3. Biological Evaluation
κ
-Carrageenan and lipoxygenase type I-B from soybean were purchased from Sigma
(St. Louis, MO, U.S.A.). For the in vivo experiments, Wistar rats (180–220 g, 3–4 months old) were
kept in the Centre of the School of Veterinary Medicine (EL54 BIO42), Aristotelian University of

Molecules 2020, 25, 19
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Thessaloniki, which is registered by the official state veterinary authorities (presidential degree 56/2013,
in harmonization with the European Directive 2010/63/EEC). The experimental protocols were approved
by the Animal Ethics Committee of the Prefecture of Central Macedonia (no. 270079/2500).
3.3.1. Effect on Plasma Cholesterol, Triglyceride and LDL-Cholesterol Levels
Hyperlipidaemia was induced by the i.p. administration of Triton WR 1339 (200 mg/kg) to rats.
The examined compounds (0.15 mmol/kg) were administered i.p. one hour later. Blood was taken from
the aorta after 24 h, for the determination of plasma total cholesterol, LDL-cholesterol and triglyceride
concentrations, using commercial kits [13].
3.3.2. In Vitro NO Release
Due to their poor aqueous solubility, compounds 1, 3-7 and 9 were dissolved in 7/3 (v/v) mixture of
DMSO/water at various concentrations and incubated overnight at room temperature in the presence
of cadmium. S-nitroso-N-acetylpenicillamine (SNAP) was dissolved either in DMSO/water 7/3 or in
water and treated as above.
Aliquots were taken from each sample and added to an equal volume of
N-naphthylaminoethylamine (0.2%) and sulfanilamide (2%) solution in 3 N hydrochloric acid
(Griess reagent). Appropriate sodium nitrite solutions in the same DMSO/water mixture were
used for the construction of the standard curve and values were identical to those obtained with
aqueous solutions. Nitric oxide release was estimated spectrophotometrically (540 nm) [
9]. NO release
from SNAP (100 M in DMSO/water 7/3 and in water) was found to be 56.3 and 56.7 M, respectively,
µ
µ
further verifying that the addition of DMSO did not influence the experiment.
3.3.3. In Vitro Evaluation of Lipoxygenase Activity
The reaction mixture contained the examined compounds (in absolute ethanol, 1–300
µM),
soybean lipoxygenase (in saline, 250 u/mL) and sodium linoleate (100
pH 9.0. The reaction was monitored for 7 min at 28 C, recording the absorbance at 234 nm.
Nordihydroguaiaretic acid (NDGA) was used as a reference.
µM), in Tris–HCl buffer,
◦
For the estimation of the type of inhibition, the described experiments were repeated, using
sodium linoleate at a concentration (1 mM) higher than the saturating substrate concentration [23].
3.3.4. In Vitro Lipid Peroxidation
The peroxidation of heat-inactivated (90◦, 90 s) rat liver microsomal fraction was induced by
ascorbic acid (0.2 mM) and ferrous sulphate (10
µM). The studied compounds, in dimethylsulfoxide,
were added at concentrations 1
µ
M–1 mM. Aliquots were taken from the incubation mixture (37 ◦C)
for 45 min. Lipid peroxidation was assessed spectrophotometrically (535/600 nm) as 2-thiobarbituric
acid reactive material. All compounds and solvents were found not to interfere with the assay [25].
3.3.5. In Vitro Interaction with the Stable Radical 1,1-Diphenyl-2-Picrylhydrazyl (DPPH)
Compounds (in absolute ethanol, final concentrations 25–200
µ
M) were added to an ethanolic
±
solution of DPPH (final concentration 200
was recorded after 30 min.
µM) at room temperature (22
2 ◦C). Absorbance (517 nm)
3.3.6. Carrageenan-Induced Paw Oedema
The tested compounds (in water with a few drops of Tween 80) were given i.p. (0.15 mmol/kg) to
rats, just after the i.d. injection of 0.1 mL of an aqueous carrageenan solution (1% w/v) in the hind paw
of rats. The produced oedema, after 3.5 h, was estimated as paw weight increase [24].

Molecules 2020, 25, 19
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4. Conclusions
There are a number of reports in the literature concerning derivatives of NSAIDs with NO releasing
ability. Those compounds were designed mainly as gastroprotective agents, counteracting the gastric
irritation caused by the classical NSAIDs [26]. In addition, various derivatives of antioxidants, such
as butylated hydroxytoluene, vitamin E and trolox, acting as NO donors, have been synthesised and
tested as vasodilating agents in in vitro experiments [27–29].
In the present investigation, the designed new compounds were synthesised and tested for
hypolipidaemic activity in vivo, a process that is close to the medical practice. This activity is also
correlated with their in vivo effect on acute inflammation, their ability to act as NO donors, lipoxygenase
inhibitors and antioxidants in vitro. Among the tested compounds, the naproxen derivative
demonstrated strong hypolipidaemic (80% reduction of total cholesterol) and anti-inflammatory
(70% reduction of rat paw oedema) activity. The butylated hydroxycinnamic acid analogue is a
potent hypolipidaemic (66% reduction of total cholesterol), anti-inflammatory (55% reduction of rat
paw oedema) and antioxidant (lipid peroxidation inhibition, IC50 41 M) compound. The ester of
butylated hydroxycinnamic acid with 4-nitrophenylethyl alcohol inhibits lipoxygenase (IC50 10.5 M)
and is an anti-inflammatory agent (51% reduction of rat paw oedema).
4
7
µ
8
µ
The successful combination of antioxidant, anti-inflammatory and NO releasing activities is
important for compounds that would acquire a series of biological properties able to prevent or
restore a number of pathological changes in conditions like cardiovascular and inflammatory disorders.
We hope that the described multifunctional compounds may assist towards the development of agents
against multicausal disorders, which otherwise would require multiple drug therapies to address the
various pathological disturbances.
Author Contributions: Investigation, P.T.-N., G.P., C.A.; Supervision, E.A.R.; Writing—review & editing, M.P.
and P.N.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: G. Papagiouvannis 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.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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