5-(4H)-oxazolones and their benzamides as potential bioactive small molecules

Article abstract of DOI:10.3390/molecules25143173

The five membered heterocyclic oxazole group plays an important role in drug discovery. Oxazolones present a wide range of biological activities. In this article the synthesis of 4-substituted-2-phenyloxazol-5(4H)-ones from the appropriate substituted aldehydes via an Erlenmeyer-Plochl reaction is reported. Subsequently, the corresponding benzamides were produced via a nucleophilic attack of a secondary amine on the oxazolone ring applying microwave irradiation. The compounds are obtained in good yields up to 94percent and their structures were confirmed using IR, 1H-NMR, 13C-NMR and LC/MS data. The in vitro anti-lipid peroxidation activity and inhibitory activity against lipoxygenase and trypsin induced proteolysis of the novel derivatives were studied. Inhibition of carrageenin-induced paw edema (CPE) and nociception was also determined for compounds 4a and 4c. Oxazolones 2a and 2c strongly inhibit lipid peroxidation, followed by oxazolones 2b and 2d with an average inhibition of 86.5percent. The most potent lipoxygenase inhibitor was the bisbenzamide derivative 4c, with IC50 41 μM. The benzamides 3c, 4a-4e and 5c were strong inhibitors of proteolysis. The replacement of the thienyl moiety by a phenyl group does not favor the protection. Compound 4c inhibited nociception higher than 4a. The replacement of thienyl groups by phenyl ring led to reduced biological activity. Docking studies of the most potent LOX inhibitor highlight interactions through allosteric mechanism. All the potent derivatives present good oral bioavailability.

Full text of DOI:10.3390/molecules25143173

molecules
Article
5-(4H)-Oxazolones and Their Benzamides as Potential
Bioactive Small Molecules
Evangelos Mavridis, Eleftherios Bermperoglou, Eleni Pontiki and Dimitra Hadjipavlou-Litina *
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences,
Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; emavridis.pharm@gmail.com (E.M.);
lefterisberberoglou@gmail.com (E.B.); epontiki@pharm.auth.gr (E.P.)
* Correspondence: hadjipav@pharm.auth.gr; Tel.: +30-231-099-7627
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic editor: Rachid Skouta
Received: 26 June 2020; Accepted: 10 July 2020; Published: 11 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The five membered heterocyclic oxazole group plays an important role in drug
discovery. Oxazolones present a wide range of biological activities. In this article the
synthesis of 4-substituted-2-phenyloxazol-5(4H)-ones from the appropriate substituted aldehydes
via an Erlenmeyer–Plochl reaction is reported. Subsequently, the corresponding benzamides were
produced via a nucleophilic attack of a secondary amine on the oxazolone ring applying microwave
irradiation. The compounds are obtained in good yields up to 94% and their structures were confirmed
1
using IR, H-NMR, 13C-NMR and LC/MS data. The in vitro anti-lipid peroxidation activity and
inhibitory activity against lipoxygenase and trypsin induced proteolysis of the novel derivatives were
studied. Inhibition of carrageenin-induced paw edema (CPE) and nociception was also determined
for compounds 4a and 4c. Oxazolones 2a and 2c strongly inhibit lipid peroxidation, followed by
oxazolones 2b and 2d with an average inhibition of 86.5%. The most potent lipoxygenase inhibitor
was the bisbenzamide derivative 4c, with IC50 41 µM. The benzamides 3c, 4a–4e and 5c were strong
inhibitors of proteolysis. The replacement of the thienyl moiety by a phenyl group does not favor
the protection. Compound 4c inhibited nociception higher than 4a. The replacement of thienyl
groups by phenyl ring led to reduced biological activity. Docking studies of the most potent LOX
inhibitor highlight interactions through allosteric mechanism. All the potent derivatives present good
oral bioavailability.
Keywords: oxazolones; benzamides; antioxidant activities; anti-inflammatory activities; lipoxygenase
inhibition; lipid peroxidation; docking studies
1. Introduction
Current novel therapeutic approaches suggest that multifunctional compounds with diverse
biological properties offer significant advantages in the treatment of complicated diseases.
Since inflammation is a complicated phenomenon in which several different factors are implicated,
pleiotropic agents will offer additional beneficial effects. Inflammation is defined as a protective
mechanism and it is the natural response of the biological system to various stimuli. It is well-established
that excessive chronic inflammation is linked to reactive oxygen species, oxidative stress [1] leading
to several diseases. Consequently, inflammation and oxidative environment are in a vicious circle of
damaging healthy cells and increased antioxidant activity could only break into that circle.
The inflammatory process starts from arachidonic acid metabolism by three enzymatic
routes: cyclooxygenase (COX), lipoxygenase (LOX) and monooxygenase cytochrome P450 (CYP) to
eicosanoids [2]. The COX route leads to pro-inflammatory prostaglandins (PGs), thromboxane and other
prostanoids; while the 5-, 12- and 15-LOX route leads to 5-, 12- or 15-hydro(-pero)xy-eicosatetraenoic
acids [H(P)ETEs] and leukotrienes and lipoxins (LXs). Finally the cytochrome P450 monooxygenase
Molecules 2020, 25, 3173; doi:10.3390/molecules25143173
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3173
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route leads to different epoxyeicosatetraenoic acids (EETs) and diHETEs [3]. COX presents two isoforms
the constitutive form (COX-1) implicated in the physiological production of prostaglandins (PGs),
important for maintenance actions (e.g., stomach cytoprotection) and the inducible form (COX-2)
associated with inflammation [4].
The importance of five-membered heterocyclic rings, such as isoxazoline and isoxazole
is known in drug discovery [
flucloxacillin and oxacillin bear the 3-arylisoxazole moiety in the side chain [
mentions the importance of glucose-derived spiro-isoxazolines II against type 2 diabetes acting as
anti-hyperglycemic agents through glycogen phosphorylase (GP) inhibition [ ]. Moreover, roxifiban
5–
7]. Marketed antibiotics
I
such as cloxacillin, dicloxacillin,
8
]. Recent literature
9
acetate (III), is a well-known antiplatelet agent (platelet glycoprotein IIb/IIIa receptor antagonist) used
in percutaneous coronary intervention [10] while ISO-1 (IV) inhibits macrophage migration inhibitory
factor (MIF), a proinflammatory cytokine [11].
Furthermore, many oxazolone derivatives have been found to present potent cycloxygenase-2
(COX-2) inhibitory activity, such as the oxazolone derivative
4-phenyl ring, which also presented remarkable activity in vivo
V
[
bearing a sulfonamide moiety on the
12]. Additionally, the oxazole ring has
been linked with the COX-inhibition e.g., oxaprozin (VI) which is a non-selective COX-1 and COX-2
inhibitor [13], valdecoxib (VII) which is a non-selective COX-2 inhibitor [14] and parecoxib (VIII),
the sulfonamide-based prodrug of valdecoxib, the only parenterally administered coxib available to
date [14
,15]. In the literature a series of 4,5-diphenyl-2-oxo-3H-1,3-oxazole derivatives IX have been
reported presenting selective COX-2 inhibition [16] (Figure 1).
Figure 1. Representative biologically active oxazole-derivatives.
The oxazolone group is implicated as an intermediate in the synthesis of different compounds
such as amino alcohols, amides [17], amino acids [18 19] and dyes [18 20]. Oxazolones are heterocyclic
,
,
compounds which can be used as versatile building blocks in organic synthesis, as they contain
numerous reactive sites allowing a diversity of possible modifications. Their reactivity (nucleophilic
attack to the carbon atom at position 5 of the oxazolone ring) makes them excellent substrates for the

Molecules 2020, 25, 3173
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synthesis e.g., of enol acetate and benzoxazinone derivatives, phenylpyruvic acid, imidazolinones,
amino acids and triazinones [21,22].
Based on the above, a number of new benzamides was designed guided by quantitative
structure activity relationship (QSAR) results [23] and synthesized from appropriate substituted
5-(4H)-oxazolones. Benzamides
antiproteolytic-antiviral, antischistosomal and anti-inflammatory activities [24
5-(4H)-oxazolones show a variety of biological activities: analgesic, antimicrobial, antidiabetic,
A
(Figure 2) are biologically active agents demonstrating antibacterial,
–
27]. On the other hand
B
anti-inflammatory as well as inhibition of trypsin and tyrosinase [28–30] (Figure 2).
Figure 2. Structure of benzamides A and 4-substituted-2-phenyloxazol-5(4H)-ones B.
The design principle was aimed at combining the synergistic property of biological potent
ArC=C-C(=O) group to get new amides that might act as effective pleiotropic bioactive agents.
Yet another objective of the study was to evaluate the effect of steric and electronic parameters on
anti-inflammatory activities, to optimize the activity through systematic modification of the substituents
and to take under consideration isosterism and druglikeness properties.
2. Results and Discussion
2.1. Chemistry
The compounds were synthesized according to the general procedure presented in Scheme 1.
The condensation of glycine with benzoyl chloride leads to compound
1. Equimolar amounts of hippuric
acid ( ) and of the appropriate aldehyde were condensed with a stoichiometric amount of fused sodium
1
acetate, in the presence of acetic anhydride as the dehydrating agent [21]. An Erlenmeyer–Plochl
reaction was used for the preparation of 4-substituted-2-phenyloxazol-5(4H)-ones 2a–2f. Subsequently,
benzamides 3a, 3c, 3d and 4a–4e were produced via a nucleophilic attack of an appropriate secondary
amine (morpholine and piperazine, respectively) on the oxazolone ring. It should to be noticed that
under the experimental conditions, the nucleophilic attack of piperidine and ethanol on 2c resulted
in products 5c and 6c, respectively. For the synthesis of the compounds, known and/or modified
procedures and simple techniques were applied, which led to good yields. We focused on the use of
microwave irradiation in case of 2a, 2c and 2d. This is an environmentally friendly and rapid method,
with good results.
From the literature, itis generally accepted that in the Erlenmeyer-Plochl reaction [31] Z isomers
are obtained. Also, the configuration of the ring opened products is the same as that of the parent
oxazolone [32]. Compounds 2a
, 2c, 2d, 2e, 2f, 3a, 6c have been synthesized previously under different
experimental conditions [33
–
44]. The structures of the new compounds were confirmed by IR, 1H-NMR,
MS (ESI) and elemental analysis and they were found to be consistent with the proposed structures.
All the oxazolones presented a characteristic double absorption in the IR (KBr) [1792–1780 cm−1 (C=O)]
due to the Fermi resonance. Benzamides presented the expected 1H-NMR signals of olefinic protons
[δ
(ppm): 5.83–6.68] in the Z configuration of the double bond. The 1H-NMR value for the olefinic
proton of 6c was recorded at higher
δ
values (7.39 ppm). The 13C-NMR value for the carbonyl amide is
in accordance with the referred values in similar cases. The LC-MS results point to the presence of
[M + H]+, [M + Na]+, [M + K]+ and [2M + Na]+.

Molecules 2020, 25, 3173
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2a : Ar = C6H5, Yield = 67 %
Cl
O
O
O
Ar
ii
OH
2b : Ar = Br-C6H4-p-CH2O-C6H4, Yield = 83 %
i
HN
O
N
O
+
S
OH
NH2
2c : Ar =
, Yield = 66 %
O
1
2d : Ar = a-C10H7, Yield = 53 %
2
O
2e : Ar =
, Yield = 36 %
O
O
iii
2f : Ar = p-C6H5-O-C6H4-p, Yield = 12 %
NH
N
O
Ar
3a : Ar = C6H5, Yield = 68 %
O
Ar
N
O
S
3
3c : Ar =
, Yield = 26 %
O
O
3d : Ar = a-C10H7, Yield = 60 %
4a : Ar = C6H5, Yield = 94 %
NH
N
iv
2
N
O
Ar
4b : Ar = Br-C6H4-p-CH2O-C6H4, Yield = 31 %
HN
O
S
Ar
4c : Ar =
, Yield = 19 %
4
4d : Ar = a-C10H7, Yield = 92 %
O
4e : Ar =
, Yield = 54 %
S
v
NH
O
O
5c, Yield = 45 %
N
S
O
N
O
O
O
vi
NH
2c
6c, Yield = 19 %
O
S
Scheme 1. Synthetic route for ta◦rget compounds 2a–2f, 3a, 3c, 3d, 4a–e, 5b and 6b. Reagents and
conditions: (i) (1) 10% NaOH, 80 C, 30 min; (2) HCl, H2O (ii) Method A: ArCHO, Ac2O, AcONa,
reflux; Method B: ArCHO, Ac2O, AcONa, MW, 100 ◦C, 15 min; Method C: ArCHO, Ac2O, 5 mol.% I2,
MW, 90 ◦C, 20 min; (iii) Method D: morpholine, toluene, reflux; Method E: morpholine, AcOEt, MW,
80 ◦C, 15 min; (iv) Method F: piperazine, toluene, reflux; Method G: piperazine, toluene, TEA, MW,
110 ◦C, 10–20 min; Method H: piperazine, EtOH, rt; Method I: piperazine, MEG, MW, 120 ◦C, 5 min;
(v) piperidine, toluene, MW, 100 ◦C, 15 min; (vi) EtOH, 75 ◦C, 30 min.
2.2. In Silico Determination of Lipophilicity as Clog P
Lipophilicity is an important physicochemical property in relation to the biological activity and
biodistribution. We theoretically calculated the lipophilicity values of all compounds as clog P using
the CLOGP Program of Biobyte Corp. [45].
2.3. Biological Evaluation
Reactive oxygen species (ROS) are continuously produced as byproducts of cell metabolism.
Depending on their nature, some are highly toxic and rapidly detoxified by various cellular enzymatic
and non-enzymatic mechanisms. Their engagement in pathological processes is due to their extreme
reactivity and their tendency to initiate and participate in chain reactions. Detoxification of reactive
oxygen species is paramount to the survival of all aerobic life forms. Normally, a number of defense
mechanisms have been involved by organisms against these highly reactive species by using enzymes
and naturally occurring antioxidants. Antioxidants in general, even at low concentration, significantly
delay or prevent oxidation of easily oxidizable substrates.
Azo compounds, like AAPH, generating free radicals through spontaneous thermal decomposition
are useful for in vitro studies of free radical production. Production of conjugated diene hydroperoxide
by oxidation of sodium linoleate in an aqueous solution is monitored at 234 nm. This assay can
be used to follow oxidative changes and to understand the contribution of each tested compound.
Oxazolones 2a and 2c strongly inhibit lipid peroxidation, followed by oxazolones 2b and 2d (Table 1).

Molecules 2020, 25, 3173
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The average inhibition of these four compounds is 86.5%, demonstrating that the Ar-substituent (bulky
or not) doesn’t significantly affect the result. It seems that the presence of moiety Ar (Scheme 1) is
mainly correlated with the antioxidant result. The nature of the heteroatom O/S in Ar substituent in the
5-membered oxazolone ring (compounds 2c and 2e) influences the biological response. The replacement
of the thienyl group in 2c by a furyl group (2e) leads to a 31% decrease of the antioxidant ability. It must
be noticed that isosteric replacement of Ar = phenyl by a thienyl (2c, 91%) or a napthyl group (2d, 81%)
does not induce any dramatic change.
Among the group of morpholinylbenzamides 3c and 3d present higher inhibitory activity, whereas
compound 3a with Ar = phenyl exhibits 69% inhibition. Lipophilicity does not seem to play a role.
The stereochemistry of the molecules might be more important. Considering the piperazinyl group of
bisbenzamides, all the analogues present high anti-lipid peroxidation activity ranging from 82–99%,
with 4b
,
4c
, 4d in the 90–99% range. The replacement of the Ar substituent (phenyl by a furyl group
as in 4a
,
4e) does not influence the antioxidant ability. It seems that the Ar substituent does not
offer/contribute to the overall response. On the contrary the nature of the alicyclic ring does seem to
influence the anti-lipid peroxidation ability. Thus, the morpholinylbenzamide 3c is more potent (93%)
compared to the piperidinyl (5c, 72%). The piperazinyl bisbenzamides 4a–4e showed high protection
against lipid peroxidation.
It is well known that free radicals play an important role in inflammatory action [46]. In fact,
many inhibitors of LOX have been reported to act either as radical scavengers or as inhibitors of free
radical production, since lipoxygenation occurs via a carbon centered radical [47]. LOXs involvement
in membrane lipid peroxidation by forming hydroperoxides in the lipid bilayer, leads their inhibitors
to be considered as potential agents for the treatment of inflammatory diseases. Consequently,
LOX inhibitors with antioxidant properties could be expected to offer protection in inflammation and
lead to potentially effective drugs. Thus, we found interesting to test our compounds for their ability
to inhibit plant LOX activity, since it has been shown that its inhibition by NSAIDs is qualitatively
similar to their inhibition of the rat mast cell LOX and may be used as a simple qualitative screen for
such activity [48,49].
In our experiments the compounds showed either no (2a, 2b, 2c) or low (2d, 2e, 3a, 3c, 3d, 5c, 6c)
biological activity (<50%, Table 1). Among the tested compounds the most interesting representatives
were the piperazinyl bis-benzamides 4b–4e for which we were able to determine r IC50 values under our
experimental conditions. Therefore, it must be stressed that the presence of a piperazine, by forming
bisbenzamides, is crucial for the majority of our compounds in order to exert satisfactory biological
activity. Compounds 4b–4e highly inhibit LOX as well as lipid peroxidation. These results strengthen
the assumption that many inhibitors of LOX may act either as radical scavengers or as inhibitors of
free radical production. However, compounds 2a and 2c with high antioxidant activity did not present
any LOX inhibition.
The synthesized derivatives have been tested for their antityrosinase activity [50]. Tyrosinase is
an enzyme with a dinuclear copper centre widely distributed in Nature. Tyrosinase plays a critical role
in the biosynthesis of melanin in melanocytes, considering to be the key enzyme in coloring the skin,
hair, eyes [51]. Thus, inhibition of tyrosinase will offer to skin whitening effects and dipegmentation
after sunburn [52]. Oxazolones 2a
found to present any significant activity. Judging the activity of piperazinyl bisbenzamides 4a
seems that they are more potent with a range from 28–42 % (4c 4a 4d). Again, 4c was found to be
–
2e as well as the tested morpholinyl benzamides 3a
,
3c
,
3d were not
,
4c, 4d it
>
>
the most active compared to the corresponding oxazolone 2c and benzamide 3c. However, they are
less potent than kojic acid which has been used as a reference compound. This compound is the most
potent tyrosinase inhibitor till now. Thus, there is a need to search for new inhibitors.
Therefore, we tested our compounds for their ability to inhibit tyrosinase in combination to LOX
inhibition in order to have possible treatment in cases of hyperpigmentation in people suffering from
psoriasis, etc.

Molecules 2020, 25, 3173
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It is well known that serine proteases are associated with inflammatory responses, especially
trypsin which has been implicated in host defense reactions and tissue damage [28 30]. Trypsin is
–
a prime example of a serine protease that contains a catalytic triad consisting of histidine, aspartate
and serine units. These three amino acids form a charge relay that serves to make the active site serine
nucleophilic by modifying its electrostatic environment. The aspartate residue located in the catalytic
pocket of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine,
and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly
cleaves, through ‘’activated” serine, proteins at the C-terminal side of the amino acids lysine and
arginine except when either is bound to a C-terminal proline [28]. Inhibition of enzymatic proteolysis is
of great importance because proteolysis, apart from inflammation, plays a crucial role in the premature
activation of proteases themselves, resulting in the self-digestion of the pancreas, as well as in the life
cycle of herpes viruses [29]. Therefore, potential antiprotease activity of our compounds could lead to
multifunctional drugs.
Table 1. Theoretically calculated clog P values; Anti-lipid peroxidation (AAPH); In vitro lipoxygenase
(LOX) inhibitory activity (IC50 (µM) or % (100 µM)); In vitro inhibition of mushroom tyrosinase
(Tyr)—(TyrI% (100
µM)); In vitro inhibition of trypsin induced proteolysis (IC50 (µM) or % Trypsin
Inh—Iptr% (10 µM)).
LOX
IC50 (µM)
or % 100 µM
AAPH%
100 µM
TyrI%
100 µM
IC50 (µM) or Iptr%
10 µM
Compd.
Clog P *
2a
2b
2c
2d
2e
2f
3a
3c
3d
4a
4b
4c
4d
4e
5c
6c
NDGA
Trolox
3.70
6.25
3.34
4.86
2.87
5.80
2.82
2.47
3.99
6.03
11.13
5.33
8.38
4.39
3.60
3.21
95
79
91
81
60
no
69
93
91
82
99
93
90
83
72
59
-
no
no
no
49
15
no
100 µM
39
37
36
85 µM
41 µM
65 µM
88 µM
41
no
no
16
5
3
nt
nt
7
nt
33
12
42
28
17
13
5
33
8.25 µM
10 µM
7 µM
60 µM
nt
no
6.75 µM
nt
8 µM
9 µM
9.1 µM
8.5 µM
8.75 µM
6.7 µM
1 µM
-
36
0.45 µM, 93
-
-
-
93
-
IC50 = 2.81
µM
Kojic acid
-
-
-
Salicylic Acid
100 µM
* Theoretically calculated clog P values using the C-QSAR Program, Biobyte; no: no action under the experimental
conditions; nt: not tested
Bisbenzamides 4a–4e present high activity with minor differences among them. Amides of
morpholine 3c and piperidine 5c seem to be almost equipotent. However the difference in terms of the
acyclic amine did not lead to any change on the biological response. The small fluctuation in the rates
concerning the inhibitory ability of the above compounds (8–9.1
structural characteristics slightly influence their biological action. The benzamide 6c is the only ester
and presents the highest IC50 value (IC50 = 1 M), compared to the reference compound salicylic acid
(IC50 = 100 M). More studies have to be done in order to clarify the structural characteristics that
µM) denotes that their individual
µ
µ
determine the wide range of IC50 values (60–7µM for 2b–2e and 33% for 2a) (Table 1).
For the estimation of anti-inflammatory potential [30] of the tested compounds the rat carrageenin
induced paw edema assay was employed as a model for acute inflammation. Indomethacin,
as a reference drug, and our compounds were administered intraperitoneally in order to ensure systemic
response via fast bioavailability, while carrageenin’s intradermal administration, as inflammatory
agent, aimed to local response. The development of the edema induced by carrageenin has been

Molecules 2020, 25, 3173
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described as a biphasic event. The early phase of the inflammatory response, lasting 0.5–1.5 h, is chiefly
mediated by histamine and serotonin release inducing hyperalgesia, swelling and redness, while the
later phase, lasting 3–6 h, is sustained presumably by prostaglandin release. This model reliably
predicts the anti-inflammatory efficacy of the NSAIDs during the second phase, since non-steroidal
anti-inflammatory drugs (NSAIDs) present weak activity in the first phase. Therefore, using this
protocol we could evaluate our compounds as potential anti-inflammatory agents, as a result of
inhibition of prostaglandin amplification.
Compound 4c was chosen to be examined because it showed generally promising antioxidative
activity, anti-lipid peroxidation and LOX inhibition, while 4a was selected in order to delineate the
influence of the nature of the ring on the anti-inflammatory activity. Compound 4c and the reference
drug indomethacin seem to be equipotent, while compound 4a presents lower decrease at an equivalent
dose (Table 2). Thus, it is obvious that the replacement of thienyl by phenyl group does not favor
the protection against the CPE. Compounds 4a and 4c were tested for their peripheral nociception
using the writhing test (Figure 3). Despite the fact that acetic acid induced writhing test lacks of
specialization, it remains a remarkable method for assessing peripheral analgesic activity because of its
quickness, simplicity and reproducibility (Table 2).
Table 2. In vivo anti-inflammatory and analgesic activities of 4a and 4c
.
% inhibition of
carrageenin-induced rat paw edema (CPE%) and % inhibition of writhing responses (writhing
inhibition%).
Compd.
CPE (%) a
Writhing Inhibition (%) a
4a
4c
44 *
56 **
58 **
-
32
58
-
Indomethacin
Aspirin
77
* p < 0.01, ** p < 0.05; a Dose of the administered 0.0057 mmol/kg body weight.
Figure 3. % Anti-nociception measured every 5 min for 4a, 4c and aspirin.
Derivative 4c inhibited writhing responses more effectively than 4a. Thus the replacement of
the thienyl group by the phenyl ring led to reduced biological activity. Obviously these results are
in accordance with the compounds’ anti-inflammatory activity. According to Table 2, compound 4c
exhibited protective action from the very first moment, after the administration of acetic acid,
compared to the reference drug aspirin whereas compound 4a later, after the 15 min. These findings
point to a different pharmacokinetic behavior however more experiments are in progress to delineate
these results.

Molecules 2020, 25, 3173
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2.4. Molecular Properties Prediction—Lipinski “Rule of Five”
Pharmacokinetic properties should be considered in the design and synthesis of new bioactive
compounds. Nowadays the traditional experimental techniques have been replaced by the use of
computational tools. The predicted properties have been popular since they are taken faster and
without cost [53].
Herein, we obtained and entered in the online Molinspiration software version 2018.10 (www.
molinspiration.com) [54] the chemical structures and SMILES notations of the more potent representative
compounds 4a, 4b, 4c, 4d, 4e, 5c and 6c to calculate various molecular properties e.g., partition coefficient
(log P), topological polar surface area (TPSA), hydrogen bond donors and acceptors, rotatable bonds,
number of atoms, molecular weight and to define Lipinski’s rule of five violations, in an attempt to
evaluate their drug-likeness [55].
Following the Lipinski’s rule of five the presence of more than five H-bond donors, 10-H-bond
acceptors, molecular weight (MW) greater than 500 and the calculated log P values (milogP) higher
than 5, leads to poor absorption or permeation. It is a rule of thumb to define if a chemical structure
with a certain bioactivity presents drug-likeness. Thus, these properties would support its behavior
in humans as an active per os drug. Compounds 4a
They might present permeability problems. However, compounds 4c
,
b
,
d
with log P values > 5 violate the rule of 5.
and 5c 6c have lipophilicity
,
e
,
lower than five suggesting satisfactory permeability across cell membranes. On the contrary 4c has high
MW as well as compound 4a. Both present molecular weight higher than 500. Thus, these molecules
could not be transported, diffused and absorbed. The compounds of group 4 are bisbenzamides of
piperazine. Following the rule of Lipinski, replacement of the alicyclic ring by an aliphatic amine
e.g., ethylenediamine (-NHCH2CH2NH-) should result in a better metabolic profile for the compounds
of this group (Figure 4). Considering this modification compound 7 would be subjected to a possible
proteolysis/amidolysis in vivo leading to several products. Prediction of bioactivity of compound
7 by
Molinspiration [54] presents an interesting antiprotease score.
Figure 4. Suggested modifications—Compound 7.
The number of hydrogen bond acceptors and donors in compounds 4a–e, 5c and 6c obey Lipinski’s
rules. Topological polar surface area (TPSA) is referred as a significant indicator of bioavailability of
a molecule with certain biological activity. TPSA is correlated with the hydrogen bonding properties.
Compounds 4a
, b, c, d,
5c and 6c exhibit TPSA values well below the limit of 160 Å2, supporting the
presence of good bioavailability per os. The upper limit of TPSA is 90 Å2. The calculated results for
the 4a, b, c, d, e show that these structures are not able to cross the Blood Brain Barrier (TPSA value
should be 40 Å2). On the contrary compounds 5c and 6c are able due to their lower (less than 60 Å2)
values (Table 3).

Molecules 2020, 25, 3173
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Table 3. Molecular properties prediction-Lipinski “Rule of five. Drug likeness of the more potent
representative compounds.
TPSA b
No OH, NH d
Volume f
Compd.
milogP a
No Atoms
NoO,N c
No Violations
No Rotational Bonds e
MW g
4a
4b
4c
4d
4e
5c
6c
5.43
9.17
4.87
7.47
3.47
3.57
3.55
98.81
117.28
98.81
98.81
125.09
49.41
55.40
44
63
42
53
43
24
21
8
10
8
8
10
4
2
2
2
2
2
1
1
2
2
1
2
1
0
0
8
15
8
9
9
535.77
782.76
517.20
640.59
515.74
309.90
266.30
584.68
954.72
596.73
684.80
564.60
340.45
301.36
4
6
4
a
Logarithm of partition coefficient between-octanol and water (milogP); b Topological polar surface area (TPSA);
Number of hydrogen bond acceptors (n-ON); Number of hydrogen bond donors (n-OHNH); Number of
c
d
e
rotatable bonds (n-rotb); f Molecular Volume; g Molecular Weight.
2.5. Computational Studies—Docking Simulation Soybean Lipoxygenase
Molecular Modeling of the Synthesized Derivatives in Soybean LOX
The synthesized derivatives have been subjected to in silico docking in accordance with the
biological results. The favored docking position for the most promising bisbenzamide 4c is shown
in Figure 5. Compound 4c has an AutoDockVina score of -10.6 kcal/mol binding to soybean LOX (PDB
code: 3PZW). It is well known that the results from the in vitro inhibition of soybean lipoxygenase
represent experimental values while docking scores are based on algorithms and scoring function
calculations so a 1 to 1 correlation is difficult to achieve. Docking describes the ligand binds to the
protein. It seems that the novel compounds interact with the SLOX through allosteric interactions.
Compound 4c presents weak hydrogen bonds with ARG533 and THR529 with the two carbonyl groups
of the 1H-indene-1,3(2H)-dione template and hydrophobic ones with the rest of the molecule with
aminoacids LEU20, PHE108, VAL126, PHE143, LEU246, VAL520, TYR525 and ASP768. Since most
LOX inhibitors act as antioxidants or by scavenging free radicals [56] and the oxidation of the enzyme
occurs via a carbon-centered radical on a lipid chain, it is possible that compound 4c extends into the
hydrophobic domain by blocking the substrates to the binding site and thus preventing oxidation.
Figure 5. Preferred docking pose of 4c (depicted in grey) bound to soybean lipoxygenase (LOX-1).
3. Experimental Section
3.1. Materials and Instruments
All chemicals, solvents, chemical and biochemical reagents were of analytical grade and purchased
from commercial sources (Merck KGaA, Darmstadt, Germany, Fluka Sigma-Aldrich Laborchemikalien
GmbH, Hannover, Germany, Alfa Aesar, Karlsruhe, Germany and Sigma-Aldrich St. Louis, MO, USA)

Molecules 2020, 25, 3173
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and used without further purification. Soybean lipoxygenase, albumin, trypsin, sodium linoleate,
sodium linoleate, 2,2-azinobis-2-methyl-propanimidamine HCl (AAPH), mushroom tyrosinase were
obtained from Sigma Chemical, Co. (St. Louis, MO, USA), nordihydroguairetic acid (NDGA),
salycilic acid, kojic acid were purchased from the Aldrich Chemical Co. (Milwaukee, WI, USA).
Melting Points (uncorrected) were determined on a MEL-Temp II (Lab. Devices, Holliston,
MA, USA). For the in vitro tests, UV-Vis spectra were obtained on a Perkin-Elmer 554 double
beam spectrophotometer. Infrared spectra (film as Nujol mulls or KBr pellets) were recorded with
a Perkin-Elmer 597 spectrophotometer (Perkin-Elmer Corporation Ltd., Lane Beaconsfield, Bucks,
England). For the microwave irradiated reactions a 700 W CEM Microwave Discover System was used
(Matthews, North Carolina, MC USA).
The 1H Nucleic Magnetic Resonance (NMR) spectra were recorded at 300 MHz on an AM-300
spectrometer (Bruker Analytische Messtechnik GmbH, Rheinstetten, Germany) in CDCl3 or DMSO
using tetramethylsilane as an internal standard unless otherwise stated. 13C-NMR spectra were obtained
at 75.5 MHz on a Bruker AM-300 spectrometer in CDCl3 or DMSO solutions with tetramethylsilane
as internal reference unless otherwise stated. Chemical shifts are expressed in _ (ppm) and coupling
constants J in Hz. Mass spectra were determined on a LC-MS 2010 EV system (Shimadzu, Kyoto,
Japan) using MeOH as solvent. Elemental analyses for C and H gave values acceptably close to the
theoretical values ( 0.4%) in a Perkin-Elmer 240B CHN analyzer. Reactions were monitored by thin
±
layer chromatography on 5554 F254 Silica gel/TLC cards (Merck and Fluka Chemie GmbH Buchs,
Steinheim, Switzerland).
3.2. Chemistry General Procedure
3.2.1. Synthesis of N-Benzoylglycine Hippuric Acid (1)
Glycine (20 mmol) was dissolved in 20 mL of 10% NaOH and the mixture was stirred to ambient
temperature for 20 min. Afterward, 20 mmol of benzoyl chloride was added in drops into the flask
and the temperature of the reaction solution was raised to 80 ◦C for 30 min. Crushed ice was added
to the solution and concentrated HCl was added dropwise until the mixture was acidified (pH 2–3).
The white formed precipitate was collected by filtration and washed out with water (negative reaction
for chloride). The spectral data was in agreement with literature data [33–44].
3.2.2. Synthesis of (Z)-4-Arylidene-2-phenyloxazol-5(4H)-ones 2a–f
Method A: To a mixture of N-benzoylglycine (1) (1 mmol) and fused sodium acetate (1 mmol),
acetic anhydride (3 mmol) and the appropriate aldehyde (1 mmol) was added. The reaction mixture
was heated until melting, and then refluxed for 2 h. The reaction process was monitored by TLC.
The solution was poured into ice cold ethanol and then allowed to cool overnight. The formed
precipitate was collected by filtration, washed with ice cold ethanol and boiling water, successively,
and then recrystallized, if necessary, from aqueous ethanol or from water/aqueous ethanol mixture.
Method B: N-Benzoylglycine (1) (1 mmol), fused sodium acetate (1 mmol) and an appropriate
aldehyde (1 mmol) were mixed in acetic anhydride (3 mmol) contained in a 10 mL microwave
vial sealed by Teflon-lined rubber cap. The reaction mixture was irradiated by microwave at
300 W and 100 ◦C for 15 min, poured into ice cold ethanol and allowed to cool overnight,
giving a precipitate which was filtered, washed with ice cold ethanol and boiling water to afford
(Z)-4-arylidene-2-phenyloxazol-5(4H)-one derivatives 2a, 2c, 2d (Table 4).
Method C: A mixture of N-benzoylglycine (1 mmol), an appropriate aldehyde (1 mmol),
and molecular iodine (5 mol.%) instead of sodium acetate, in acetic anhydride (4 mmol) was irradiated
by microwave at 300 W and 90 ◦C for 20 min. The mixture was cooled to room temperature, ice cold
ethanol was added and the crude oxazolone was separated. The product was filtered and washed with
ice cold ethanol/boiling water to afford the pure product 2c (Table 4).

Molecules 2020, 25, 3173
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(Z)-4-Benzylidene-2-phenyloxazol-5(4H)-one (2a) [35
–
38
,
41]. Yield: 67%; Rf = 0.8 (CH3COOC2H5–
petroleum ether, 6:1); m.p. 165–167 ◦C; IR (KBr) (cm−1): 3066–3054 (arom. C-H), 1793, 1769 (C=O),
1652 (C=N), 1596 (C=C), 1553, 1449 (arom. C-C), 1295, 1230 (C-O); 1H-NMR (CDCl3),
(s, 1H, olefinic), 7.44–7.50 (m, 3H), 7.53 (t, 2H), 7.62 (t, 1H), 8.17–8.25 (m, 4H); 13C-NMR (CDCl3),
δ
(ppm): 7.21
:126.5
δ
(2C), 128.6 (2C), 128.7 (2C), 128.9, 129.0 (2C), 129.4, 131.2, 131.6, 131.7, 132.0, 164.7, 165.1;C16H11NO2
[M + H]+ =250. The spectral data were in agreement with the literature data.
(Z)-4-(4-((4-Bromobenzyl)oxy)benzylidene)-2-phenyl◦oxazol-5(4H)-one (2b). Yield: 83%; Rf = 0.8
(CH3COOC2H5–petroleum ether, 6:1); m.p. 199–200 C; IR (KBr) (cm−1): 3064–3054 (arom. C-H),
2919 (olefinic C-H), 1780, 1762 (C=O), 1655 (C=N), 1592 (C=C), 1509 (arom. C-C), 1298, 1252 (C-O);
1H-NMR (CDCl3),
δ
(ppm): 5.07–5.12 (m, 2H, aliphatic), 6.98–7.09 (m, 2H, arom.), 7.21 (s, 1H, olefinic),
7.31 (d, 2H, J = 9 Hz, arom.), 7.49–7.56 (m, 4H, arom.), 7.59 (t, 1H, arom.), 8.03–8.21 (m, 4H, arom.);
13C-NMR (CDCl3),
: 69.5, 115.5 (2C), 120.8, 127.6 (2C), 128.3(2C), 129.0 (2C), 129.1, 131.0 (2C), 131.2,
δ
131.4, 131.6, 132.0, 133.1(2C), 134.6, 161.1, 164.7, 165.2; Anal. C, H, N. (C23H16BrNO3) Calcd %: C: 63.61,
H: 3.71, N: 3.25; Found %: C: 63.59, H: 3.91, N: 3.41; [M]+ = 433, [M + H]+ = 434 (436), [M + Na]+ = 456.
(Z)-2-Phenyl-4-(thiophen-2-ylmethylene)oxazol-5(4H)-one (2c) [36,41]. Yield: 66%; Rf = 0.7
(CH3COOC2H5–petroleum ether, 6:1); m.p. 179–181 ◦C; IR (KBr) (cm−1): 3072–3060 (arom. C-H), 2.916
(olefinic C-H), 1791, 1769 (C=O), 1646 (C=N), 1598 (C=C), 1552, 1414 (arom. C-C), 1295.4, 1240.8 (C-O);
1H-NMR (CDCl3),
δ
(ppm): 7.15–7.19 (m, 1H, thienyl), 7.49 (s, 1H, olefinic), 7.51–7.56 (m, 2H, arom.),
7.57–7.61 (m, 1H, arom.), 7.63–7.66 (m, 1H, thienyl), 7.72 (d, 1H, J = 6 Hz, thienyl), 8.17 (d, 2H, J = 6
Hz, arom.); 13C-NMR (CDCl3),
δ: 123.6, 123.7, 126.2, 126.8, 127.8, 132.1, 133.0, 134.2, 134.6, 136.2, 144.4,
161.0, 168.5, 178.0; (C14H9NO2S) [M + H]+ = 256, [M + H + HCOOH]+ = 302. The spectral data were
in agreement with the literature data.
(Z)-4-(Naphthalen-1-ylmethylene)-2-phenyloxazol-5(4H)-one (2d) [34
(CH3COOC2H5–petroleum ether, 6:1); m.p. 169–171 ◦C; IR (KBr) (cm−1): 3058.8–3030.8 (arom. C-H),
1793, 1779 (C=O), 1637 (C=N), 1561 (C=C), 1297, 1245 (C-O); 1H-NMR (CDCl3),
(ppm): 7.56–7.61 (m,
3H), 7.62–7.67 (m, 3H), 7.93 (d, 1H, J = 6 Hz), 7.99 (d, 1H, J = 6 Hz), 8.16 (s, 1H, olefinic), 8.21–8.26 (m,
2H), 8.34 (d, 1H, J = 6 Hz), 9.04 (d, 1H, J = 6 Hz); 13C-NMR (CDCl3),
: 122.7, 125.5, 126.1 (3C), 126.5,
,39,40]. Yield: 53%; Rf = 0.7
δ
δ
126.8, 127.2, 128.3 (2C), 128.8, 129.4, 131.1, 131.6 (2C), 131.7, 133.2, 137.9, 159.8, 164.2; (C20H13NO2)
[M + H]+ =300, [M + Na]+ = 322, [M + K]+ = 338, [M + CH3OH + Na]+ = 354. The spectra data were
in agreement with the literature data.
(Z)-4-(Furan-2-ylmethylene)-2-phenyloxazol-5(4H)-one (2e) [36
,
41].
Yield: 36%; Rf = 0.6
◦
(CH3COOC2H5–petroleum ether, 6:1); m.p. 165–168 C; IR (KBr) (cm−1): 3109–3064 (arom. C-H),
1789 (C=O), 1648 (C=N), 1557 (C=C), 1491, 1454 (arom. C-C), 1296.3, 1231.1 (C-O); 1H-NMR (CDCl3),
δ
(ppm): 6.62–6.69 (m, 1H, furyl), 7.20 (s, 1H, olefinic), 7.22–7.28 (m, 1H, furyl), 7.51–7.57 (m, 2H,
arom.), 7.58–7.61 (m, 1H, arom.), 7.62–7.71 (m, 1H, furyl), 8.10–8.20 (m, 2H, arom.); 13C-NMR (CDCl3),
: 113.7, 118.2, 120.0 (2C), 128.3 (2C), 128.9, 131.3, 131.5, 133.1, 142.4, 146.5, 162.5, 165.0; C14H9NO3
[M + H]+ = 240, [M + 2CH3OH + H]+ = 304. The spectra data were in agreement with the literature data.
(Z)-4-(4-Phenoxybenzylidene)-2-phenyloxazol-5(4H)-one (2f) [43 44]. Yield: 12%; Rf = 0.9
(CH3COOC2H5–petroleum ether, 3:7); m.p. 169 ◦C; IR (Nujol) (cm−1) 3100 (arom. C-H), 1784 (C=O),
1654 (C=N); 1H-NMR (CDCl3),
(ppm): 7.11–7.24 (m, 4H arom. and 1H olefinic), 7.39–7.53 (m, 5H),
7.58–7.63 (m, 1H), 7.70 (d, 1H, J = 9 Hz), 8.02–8.07 (m, 3H); 13C-NMR (CDCl3)
: 113.0, 119.9, 120.7,
δ
,
δ
δ
121.4, 123.9, 124.2, 126.1, 127.3, 128.5, 128.9, 130.0, 130.9, 131.3 (2C), 133.5, 135.3, 136.4, 143.3, 160.0,
165.0, 169.9, 178.0; Anal. C, H, N. Calcd %: (C22H15NO3) C: 77.41, H: 4.38, N: 4.06; Found %: C: 77.63,
H: 4.38, N: 4.10. The spectral data were in agreement with the literature data.
3.2.3. Synthesis of (Z)-Morpholino-benzamides 3a, 3c, 3d
Method D: An equimolar quantity of arylideneoxazolone 2c and morpholine in dry toluene were
refluxed for about 2 h. The reaction process was monitored by TLC. Approximately 2/3 of the solvent
was removed under reduced temperature and the remaining mixture was allowed to cool overnight to

Molecules 2020, 25, 3173
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obtain solid precipitate. The precipitated solid was washed with 0.5N HCl to get the pure product 3c
(Table 4).
Method E: An equimolar quantity of arylideneoxazolones 2a, 2d and morpholine in ethyl acetate
(2 mL) were irradiated by microwave at 50 W and 80 ◦C for 15 min. The solution was evaporated
to dryness and washed with 0.5N HCl affording a residue that was recrystallized from aqueous
ethanol/water mixture.
(Z)-N-(3-morpholino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (3a) [35]. Yield: 68%; Rf = 0.5
(CH3COOC2H5–petroleum ether, 3:1); m.p. 135–138 ◦C; IR (Nujol) (cm−1): 3201 (N-H), 1657 (NH-C=O),
1646 (C=O), 1601 (C=C); 1H-NMR (CDCl3),
δ
(ppm): 3.76–3.85 (m, 8H, morpholino), 6.16 (s, 1H, olefinic),
7.29–7.34 (m, 1H), 7.38–7.46 (m, 6H), 7.49–7.54 (m, 1H), 7.77–7.82 (m, 2H), 8.05 (s, 1H, N-H); 13C-NMR
(CDCl3), : 47.70 (2C), 65.7, 66.5, 119.7, 127.4, 128.3 (2C), 128.5 (2C), 128.6 (2C), 128.7 (2C), 129.1, 132.1,
δ
134.0, 134.5, 166.1, 166.6; (C20H20N2O3) [M
[2M + Na]+ = 695. The spectral data were in agreement with the literature data.
−
H]+ = 335, [M + Na]+ = 359, [M + CH3OH + Na]+ = 391,
(Z)-N-(3-morpholino-3-oxo-1-(thiophen-2-yl)prop-1-en-2-yl)benzamide (3c). Yield: 26%; Rf = 0.6
(CH3COOC2H5–petroleum ether, 3:1); m.p. 184–186 ◦C; IR (Nujol) (cm−1): 3157 (N-H), 1662 (NH-C=O),
1
1639 (C=O), 1608 (C=C); H-NMR (CDCl3),
δ (ppm): 3.67–3.84 (m, 8H, morpholino), 6.43 (s, 1H,
olefinic), 7.02–7.11 (m, 1H, thienyl), 7.12–7.19 (m, 1H, thienyl), 7.34–7.40 (m, 1H, thienyl), 7.41–7.57
(m, 3H, arom.), 7.87–8.02 (m, 2H, arom.), 8.12 (s, 1H, N-H); 13C-NMR (CDCl3),
: 47.5 (2C), 66.6 (2C),
115.4, 124.3, 127.6 (2C), 128.8 (2C), 129.0 (2C), 129.1, 132.3, 133.0, 136.4, 165.7, 168.6; Anal. C, H, N.
δ
(C18H18N2O3S) Calcd % C: 63.14, H: 5.30, N: 8.18; Found %: C: 63.10, H: 5.57, N: 8.47; [M
[M + Na]+ = 365, [M + K]+ = 381, [2M + Na]+ = 707.
−
H]+ = 341,
(Z)-N-(3-morpholino-1-(naphthalen-1-yl)-3-oxoprop-1-en-2-yl)benzamide (3d). Yield: 60%; Rf = 0.5
(CH3COOC2H5–petroleum ether, 3:1); m.p.
135–137 ◦C; IR (Nujol) (cm−1): 3265 (N-H),
1649 (NH-C=O),1634 (C=O), 1607 (C=C); 1H-NMR (CDCl3),
(ppm): 3.75–3.93 (m, 8H, morpholino),
6.62–6.63 (m, 1H, olefinic), 7.33 (t, 2H), 7.45 (t, 1H), 7.50 (t, 1H), 7.53–7.57 (m, 2H), 7.59–7.64 (m, 3H),
7.86–7.92 (m, 2H), 7.96–7.97 (m, 1H), 8.09 (m, 1H, N-H); 13C-NMR (CDCl3),
: 47.8 (2C), 66.7 (2C), 118.7,
δ
δ
125.4, 125.7, 126.2, 126.7, 126.9, 127.5, 127.8, 128.4 (4C), 129.0, 131.7, 131.9, 132.6, 133.1,134.8, 166.2,
166.8;Anal. C, H, N. (C24H22N2O3) Calcd %: C: 74.59, H: 5.75, N: 7.25; Found %: C: 74.39, H: 5.87, N:
7.03; [M − H]+ = 385, [M + Na]+ = 409, [M + CH3OH + Na]+ = 441, [2M + Na]+ = 795.
3.2.4. Synthesis of (Z,Z0)-Piperazine-bisbenzamides 4a–4e
Method F: Arylidene oxazolones 2a–2e and half the stoichiometric amount of piperazine were
refluxed in dry toluene. The reaction progress was monitored by TLC. Approximately 2/3 of the solvent
was removed under reduced pressure and the remaining mixture was allowed to cool overnight to
obtain a solid precipitate. The precipitated solid was washed with 0.5N HCl to give the pure products
4a–4e (Table 4).
Method G: Arylidene oxazolones 2a, 2c, 2d (2 mmol) and piperazine (1 mmol) were mixed in dry
toluene (5 mL) with a catalytic amount of triethylamine, and irradiated by microwaves at 300 W, 110 ◦C
for 10–20 min (Table 4). The mixture was allowed to cool overnight, and then the crude bisbenzamide
thus separated was filtered, washed with 0.5N HCl and recrystallized, if necessary, from DMF/aqueous
ethanol mixture.
Method H: A solution of arylidene oxazolone 2a (2 mmol) and piperazine (1 mmol) in EtOH
(5 mL) was stirred at room temperature for 74 h. The ethanol was evaporated under reduced pressure
and the residue was triturated with diethyl ether to give the pure product 4a.
Method I: In a 10 mL microwave vial arylidene oxazolones 2c, 2d (2 mmol) with piperazine
(1 mmol), in ethylene glycol (MEG, 2 mL) were mixed and then capped. The mixture was irradiated by
microwaves at 300 W and 120 ◦C for 5 min. The contents were cooled to room temperature and then
poured into cold water, filtered to give crude products, which were further purified by recrystallization
from aqueous ethanol.

Molecules 2020, 25, 3173
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N,N0-((1Z,10Z)-piperazine-1,4-diylbis(3-oxo-1-phenylprop-1-ene-3,2-diyl))bisbenzamide (4a). Yield: 94%;
◦
Rf = 0.3(CH3COOC2H5–petroleum ether, 2:1); m.p. 265–268 C; IR (Nujol) (cm−1): 3204 (N-H),
1644 (NH-C=O), 1629 (C=O), 1606 (C=C); 1H-NMR (CDCl3),
(ppm): 3.81–4.18 (m, 8H, piperazine),
6.20 (s, 2H, olefinic), 7.30–7.35 (m, 2H), 7.36–7.48 (m, 12H), 7.50–7.54 (m, 2H), 7.74–7.85 (m, 4H),
8.19 (s, 2H, N-H); 13C-NMR (CDCl3),
: 43.8(4C), 126.1, 126.4, 126.5 (2C), 126.6 (2C), 127.0 (4C),
130.1 (8C), 132.3 (4C), 131.8 (2C), 132.9 (2C), 133.1 (2C), 163.4 (2C), 164.0 (2C); Anal. C, H, N.
δ
δ
(C36H32N4O4): Calcd % C: 73.95, H: 5.52, N: 9.58; Found %: C: 73.74, H: 5.24, N: 9.31; [M
−
H]+ = 583,
[M + H]+ = 585, [M + Na]+ = 607, [M + K]+ = 623.
N,N0-((1Z,10Z)-piperazine-1,4-diylbis(1-(4-((4-bromobenzyl)oxy)phenyl)-3-oxoprop-1-ene-3,2-diyl))-bis
-benzamide (4b). Yield: 31%; Rf = 0.7 (CH3COOC2H5–petroleum ether, 2:1); m.p. 285–286 ◦C; IR (Nujol)
(cm−1): 3208 (N-H), 1668 (NH-C=O), 1622 (C=O), 1609 (C=C); 1H-NMR (DMSO),
δ (ppm): 3.14–3.17
(m, 8H, piperazine), 5.02–5.10 (m, 4H), 6.44–6.58 (m, 2H, olefinic), 6.87–6.94 (m, 4H), 7.11–7.18 (m, 4H),
7.29–7.34 (m, 4H), 7.36–7.41 (m, 4H), 7.43–7.49 (m, 6H), 7.80–7.91 (m, 4H), 8.12 (s, 1H, N-H), 10.11–10.25
(m, 1H, N-H); 13C-NMR (DMSO),
δ: 47.8 (4C), 70.8 (2C), 115.7 (4C), 120.9(2C), 121.9 (2C), 124.7 (2C),
126.8 (2C), 128.5 (8C), 129.4 (4C), 130.7 (4C), 131.9 (2C), 132.0 (4C), 134.3 (2C), 134.5 (2C), 158.6 (2C),
166.2 (2C), 166.7 (2C);Anal. C, H, N. (C50H42Br2N4O6): Calcd % C: 62.90, H: 4.43, N: 5.87; Found %: C:
62.87, H: 4.56, N: 5.73; [M − H]+ = 953, [M + Na]+ = 977, [M + K]+ = 993.
N,N0-((1Z,10Z)-piperazine-1,4-diylbis(3-oxo-1-(thiophen-2-yl)prop-1-ene-3,2-diyl))bisbenzamide (4c).
◦
Yield: 19%; Rf = 0.3 (CH3COOC2H5–petroleum ether, 2:1); m.p. 292–294 C; IR (Nujol) (cm−1):
3188 (N-H), 1658 (NH-C=O),1638 (C=O), 1606 (C=C); 1H-NMR (CDCl3),
δ (ppm): 3.79–4.10 (m, 8H,
piperazine), 6.49 (s, 2H, olefinic), 7.08–7.11 (m, 2H, thienyl), 7.14–7.17 (m, 2H, thienyl), 7.38–7.42 (m,
2H, thienyl), 7.45–7.52 (m, 4H, arom.), 7.53–7.60 (m, 2H, arom.), 7.86–7.99 (m, 4H, arom.), 8.22 (s, 2H,
N-H); 13C-NMR (CDCl3),
δ: 47.6 (4C), 124.2 (2C), 126.3 (2C), 128.4 (2C), 128.5 (8C), 128.6 (2C),129.2 (2C),
132.0 (2C), 134.4 (2C), 139.0 (2C); 165.8 (2C), 166.2 (2C);Anal. C, H, N. (C32H28N4O4S2): Calcd % C:
64.41, H: 4.73, N: 9.39; Found %: C: 64.90, H: 4.71, N: 9.02; [M
−
H]+ = 595, [M + H]+ = 597, [M + Na]+
= 619, [M + K]+ = 635.
N,N0-((1Z,10Z)-piperazine-1,4-diylbis(1-(naphthalen-1-yl)-3-oxoprop-1-ene-3,2-diyl))bisbenzamide (4d).
Yield: 92%; Rf = 0.2 (CH3COOC2H5–petroleum ether, 2:1); m.p. 282–284 C; IR (Nujol) (cm−1):
◦
3210 (N-H), 1659 (broad, C=O), 1609(C=C), 1519(H-N-C=O); 1H-NMR (CDCl3),
δ
(ppm): 4.00–4.22
(m, 8H, piperazine), 6.68 (s, 2H, olefinic), 7.32–7.38 (m, 4H), 7.43–7.47 (m, 2H), 7.48–7.52 (m, 2H),
7.54–7.58 (m, 4H), 7.59–7.65 (m, 6H), 7.84–7.93 (m, 6H), 7.96–8.02 (m, 2H, N-H); Anal. C, H, N.
(C44H36N4O4) Calcd %: C: 77.17, H: 5.30, N: 8.18; 13C-NMR (CDCl3),
δ: 47.6 (4C), 118.8 (2C); 125.4 (2C),
125.6 (2C), 126.2 (2C), 126.8 (2C), 126.9 (2C), 127.4 (2C), 127.5 (2C), 128.5 (8C), 129.0 (2C), 131.8 (2C),
131.9 (2C), 132.6 (2C), 133.1 (2C), 134.4 (2C), 166.2 (2C), 166.7 (2C);Found %: C: 77.25, H: 5.15, N: 8.28;
[M − H]+ = 683, [M + Na]+ = 707, [M + K]+ = 723.
N,N0-((1Z,10Z)-piperazine-1,4-diylbis(1-(furan-2-yl)-3-oxoprop-1-ene-3,2-diyl))bisbenzamide (4e). Yield:
54%; Rf = 0.5 (CH3COOC2H5–petroleum ether, 2:1); m.p. 293–295 ◦C; IR (Nujol) (cm−1): 3435 (N-H),
1682 (NH-C=O), 1623 (C=O), 1561 (C=C); 1H-NMR (CDCl3),
δ
(ppm): 3.80–3.95 (m, 8H, piperazine),
5.83 (s, 2H, olefinic), 6.36–6.44 (m, 2H, furyl), 6.45–6.52 (m, 2H, furyl), 7.46–7.64 (m, 8H, arom. and
furyl), 7.86–7.97 (m, 4H, arom.), 9.31–9.47 (m, 2H, N-H); 13C-NMR (CDCl3),
: 41.4 (2C), 47.0 (2C),
δ
103.1 (2C), 111.4 (2C), 114.6 (2C), 127.4 (2C), 128.2 (8C), 132.3 (2C), 133.0 (2C), 142.7 (2C), 150.8 (2C),
164.3 (2C), 166.4 (2C); Anal. C, H, N. (C32H28N4O6): Calcd % C: 68.07, H: 5.00, N: 9.92; Found %: C:
68.26, H: 5.07, N: 10.01; [M − H]+ = 563, [M + Na]+ = 587, [M + K]+ = 603.
3.2.5. Synthesis of (Z)-N-(3-Oxo-3-(piperidin-1-yl)-1-(thiophen-2-yl)prop-1-en-2-yl)benzamide (5c)
Arylideneoxazolone 2c (1 mmol) and pip◦eridine (1 mmol) were mixed in dry toluene (5 mL)
and irradiated by microwaves at 300 W, 100 C for 15 min (Table 4). The mixture was allowed
to cool overnight, and then the solvent was removed under reduced pressure. The crude
product was recrystallized from aqueous ethanol to give pure product 5c. Yield: 45%; Rf = 0.7
(CH3COOC2H5–petroleum ether, 3:1); m.p. 181–182 ◦C; IR (Nujol) (cm−1): 3148 (N-H), 1657 (NH-C=O),

Molecules 2020, 25, 3173
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1636 (C=O), 1599 (C=C); 1H-NMR (CDCl3),
δ
(ppm): 1.65–1.73 (m, 6H, piperidine), 3.64–3.73 (m, 4H,
piperidine), 6.43 (s, 1H, olefinic), 7.06–7.10 (m, 1H, thienyl), 7.11–7.16 (m, 1H, thienyl), 7.37 (d, 1H, J = 6
Hz, thienyl), 7.43–7.50 (m, 2H, arom.), 7.54 (t, 1H, arom.), 7.94 (d, 2H, J = 6 Hz, arom.), 8.09 (s, 1H,
N-H); 13C-NMR (CDCl3),
δ: 24.7, 25.6 (2C), 47.2 (2C), 124.4, 126.9, 127.5, 128.7 (4C), 129.0,132.1, 133.1,
136.6, 140.1, 165.4, 174.7; Anal. C, H, N. (C19H20N2O2S): Calcd % C: 67.03, H: 5.92, N: 8.25; Found %: C:
67.13, H: 5.78, N: 8.35; [M + H]+ = 341, [M + Na]+ = 363, [M + K]+ = 379, [M + Na + MeOH]+ = 395,
[2M + Na]+ = 703.
3.2.6. Synthesis of (Z)-Ethyl 2-benzamido-3-(thiophen-2-yl)acrylate (6c)
Arylideneoxazolone 2c was heated in ethanol at 75 ◦C for 30 min, until pure compound 6c was
◦
produced. Yield: 19%; Rf = 0.2 (CH3COOC2H5–petroleum ether, 1:3); m.p. 156–159 C; IR (KBr)
(cm−1): 3279 (N-H), 1706 (C=O ester),1649 (NH-C=O), 1579 (C=C), 1513 (H-N-C=O); 1H-NMR (CDCl3),
δ
(ppm): 1.33 (t, 3H, ester), 4.30 (q, 2H, J = 6 Hz, ester), 7.07 (dd, 1H, J1 = 3 Hz, J2 = 3 Hz, thienyl),
7.34 (d, 1H, J1 = 3Hz, thienyl), 7.39 (s, 1H, olefinic), 7.44 (d, 1H, J2 = 3Hz, thienyl), 7.47–7.54 (m, 2H,
arom.), 7.55–7.63 (m, 1H, arom.), 7.86 (s, 1H, N-H), 7.96 (d, 2H, J = 9Hz, arom.); 13C-NMR (CDCl3),
14.5, 61.0, 124.1, 128.3, 128.4 (4C), 128.5, 129.2, 131.3, 132.0, 134.5, 139.2, 166.3, 167.8; Anal. C, H, N.
δ:
(C16H15NO3S): Calcd % C: 63.77, H: 5.02, N: 4.65; Found %: C: 63.45, H: 5.15, N: 4.51; [M
−
H]+ = 300,
[M + H]+ = 302, [M +N a]+ = 324, [M + K]+ = 340, [M + CH3OH + Na]+ = 356, [2M + Na]+ = 625.
The spectral data were in agreement with the literature data [33,42].
Table 4. Reaction data from the different synthetic methods of novel compounds.
Compd.
Method
Time (min)
Yield (%)
Compd.
Method
Time (min)
Yield (%)
2a
2a
2b
2c
2c
2c
2d
2d
2e
2f
A
B
A
A
B
C
A
B
A
A
E
120
15
120
120
15
20
120
15
67
56
83
37
66
19
53
38
36
12
68
26
3d
4a
4a
4a
4b
4c
4c
4c
4d
4d
4d
4e
E
F
G
H
F
F
G
I
15
75
10
4440
240
135
15
5
90
20
5
60
12
94
14
31
14
19
trace
10
92
120
120
15
F
G
I
3a
3c
trace
54
D
120
F
120
3.3. Biological Assays
3.3.1. Biological In Vitro Assays
Each in vitro experiment was performed at least in triplicate and the standard deviation of
absorbance was less than 10% of the mean. For the in vitro assays, a stock solution (1% DMSO in the
appropriate buffer with the tested compound diluted under sonification) was prepared from which
several dilutions were made with the appropriate buffer.
Inhibition of Linoleic Acid Lipid Peroxidation
The AAPH-induced oxidation of linoleic acid measures the ability of an antioxidant to prevent
oxidation of linoleic acid sodium salt induced by alkylperoxyl radicals, generated from the water soluble
azo compound 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH). The assay was performed
according to our previous publications [56–59]. Trolox was used as reference compound.
Soybean Lipoxygenase Inhibition Study
DMSO solution of the tested compound (stock solutions 10 mM) was incubated with sodium
linoleate (0.1 mM) and 0.2 mL of soybean lipoxygenase solution (1/9
×
10−4 w/v in saline) at room
temperature. The test follows our previous published methods [56 58 59]. The conversion of sodium
,
,

Molecules 2020, 25, 3173
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linoleate to 13-hydroperoxylinoleic acid was recorded at 234 nm and compared with the standard
inhibitor NDGA. Several concentrations were used to determine the IC50.
Tyrosinase Inhibition Assay
Ten
µL of compound was dissolved in DMSO (final concentration 100
µM) and incubated with
40
µ
L mushroom tyrosinase (5370 U/mg–1 mg/2.6 mL buffer) and 200
µ
L of L-Dopa (1.5 mM) in 0.1 M
◦
Na-phosphate buffer (pH = 6.5) for 20 min at 37 C. Then the enzyme reaction was monitored by
measuring the change in absorbance at 490 nm due to the formation of the DOPA-chrome and compared
with the standard inhibitor kojic acid [60].
Inhibition of Trypsin Induced Proteolysis
The tested compounds (10
with phosphate buffer (pH = 7.6) to give 0.005–0.1 mM solutions, and 100
were preincubated at 37 ◦C for 30 min. Then, 200
µ
L) were dissolved in DMSO (final concentration 100
L trypsin (1 mg/10 mL)
L of albumin (6 mg in 3 mL of distilled water) was
µM),
µ
µ
added and incubated again at 37 ◦C for 24 h. After the incubation 1 mL trichloroacetic acid (5% w/w)
was added to terminate the reaction. The mixture is centrifugated, the water layer is collected and
monitored at 280 nm [61].
3.3.2. Biological In Vivo Assays
The animals (Fisher 344 rats), which have been bred in our laboratory, were housed under
standard conditions and received a diet of commercial food pellets and water ad libitum during the
maintenance but they were entirely fasted during the experiment period. Both sexes were used while
females pregnant were excluded. Each group was composed of 6–15 animals. Our studies were
in accordance with recognized guidelines on animal experimentation (guidelines for the care and use
of laboratory animals published by the Greek Government 160/1991, based on EU regulations 86/609).
Rats were kept in the Centre of the School of Veterinary Medicine (EL54 BIO42), Aristotle University
of 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).
Inhibition of the Carrageenin-Induced Edema
Edema was induced in the right hind paw of the rats by the intradermal injection of 0.1 mL 2%
carrageenin in water we followed our previous published method [59]. Each group was composed of
five animals. The tested compounds were suspended in water (0.0057 mmol/kg body weight), with few
drops of Tween 80, ground in a mortar before use and were given intraperitoneally simultaneously.
The rats were euthanized 3.5 h after carrageenin injection. The change in paw weight was compared
with that in control animals (treated with water) and expressed as a percent inhibition of the edema CPE
% values. Indomethacin 0.0057 mmol/kg body weight was used as reference compound. Values CPE %
are the mean from two different experiments with a standard error of the mean less than 10 %.
Anti-Nociception—Writhing Test
In order to study the analgesic activity of the tested compound, pain is provoked with acetic acid.
Three groups for each compound were used with five animals per group. We followed our previous
presented method [62,63]. After treatment with the tested compounds (0.0057 mmol/kg body weight;
intraperitoneally) in group 1, 2nd group was used as a positive control (aspirin; 0.0057 mmol/kg body
weight i.p.) and another group (3rd) served as the control in which saline was administered (negative
control). After 30 min, 0.6% acetic acid (1 mL/kg body weight) was injected i.p. The number of writhes
was recorded for each animal every 5 min during a subsequent 30 min period.

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3.4. Molecular Properties Prediction-Lipinski “Rule of Five”
Compounds were subjected to molecular properties prediction, drug-likeness by Molinspiration
[54] (Table 3).
3.5. Computational Methods. Molecular Docking Studies on Soybean Lipoxygenase
UCSF Chimera was used for the visualization of the protein (PDB code: 3PZW) [64].
Water molecules were removed, missing residues were added with Modeller [65], hydrogen atoms
and AMBER99SB-ILDN charges were added, and the charge on iron was set to +2.0, with no restraint
applied to the iron atom and the ligands. OpenBabel was used to generate and minimize ligand 3D
coordinates using the MMFF94 force field [66]. Ligand topologies and parameters were generated by
AnteChamberPYthon Parser interfacE (ACPYPE) [67] using Antechamber [68]. Energy minimizations
were carried out using the AMBER 99SB-ILDN force field [69] with GROMACS 4.6. Docking was
performed with AutoDockVina (1.1.2) [70] applying a grid box of size 100 Å, 70 Å, 70 Å in X, Y,
Z dimensions. The generation of docking input files and the analysis of the docking results was
accomplished by UCSF-Chimera. Docking was carried out with an exhaustiveness value of 10 and
a maximum output of 20 docking modes.
4. Conclusions
The designed and synthesized benzamides derivatives of oxazolones present multi-target activity
against different targets. They exhibit anti lipid peroxidation activity, inhibition of lipoxygenase and
trypsin in vitro as well as anti-inflammatory and analgesic activities in vivo. In Table 5 the most
significant derivatives in terms of activities are given.
Table 5. Biological activities of the most potent derivatives.
LOX
IC50 (µM)
or % 100 µM
AAPH%
100 µM
TyrI%
100 µM
IC50 (µM) or Iptr%
10 µM
Compd.
CPE (%)
Writhing Inhibition (%)
3c
4a
4b
4c
4d
4e
5c
6c
93
82
99
93
90
83
72
59
39
36
7
6.75 µM
8 µM
9 µM
9.1 µM
8.5 µM
8.75 µM
6.7 µM
1 µM
-
44
-
56
-
-
-
-
-
32
-
58
-
-
-
-
33
12
42
28
17
13
5
85 µM
41 µM
65 µM
88 µM
41
36
The oxazolones 2a–2e as well as all the benzamides highly inhibit lipid peroxidation. In oxazolones
the Ar-substituent doesn’t significantly affect the results while in bisbenzamides the nature of the
alicyclic ring seems to influence the anti-lipid peroxidation ability. Among the morpholinylbenzamides,
compounds 3c and 3d present higher inhibitory activity.
As for the lipoxygenase inhibition the most interesting representatives were found to be the
piperazinyl bisbenzamides4b–4e, with compound 4c being the most active.
The piperazinyl bisbenzamides 4a
stronger inhibitor.
Compound 6c was found to be an exceptional potent trypsin inhibitor and it could be used
–4e exhibited inhibition of tyrosinase, with 4c being the
as a lead scaffold for antiprotease activity. The benzamides 3c, 4a–4e and 5c presented strong
antiprotease activity.
Compounds 4c and 4a, with the best activity in the above biological assays, were selected
to be tested as anti-inflammatory agents using the carrageenin-induced rat paw edema method.
Compound 4c seems to be equipotent in vivo to the reference drug indomethacin, while compound 4a at
the same dose presents lower decrease in the carrageenin paw edema and thus lower anti-inflammatory
activity. Compound 4c inhibited writhing better than 4a.

Molecules 2020, 25, 3173
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Compound 4c present the better multi-target abilities in vitro and in vivo and it can be considered
as a lead compound with a multifunctional profile. The pharmacokinetic prediction analysis led to
useful observations for the design and optimization of the ADME properties of the new derivatives.
All the potent derivatives (4a–e, 5c, 6c) present good oral bioavailability. The bis-benzamides are
not able to cross the blood brain barrier, while 5c and 6c display this ability. Theoretical structural
modifications are in progress to delineate the role of the piperazine ring in the bisbenzamides. All the
compounds have been subjected to docking studies showing that soybean LOX oxidation was prevented
by blocking into the hydrophobic domain the substrates to the active site with hydrogen bonds and
hydrophobic interactions.
Author Contributions: E.M. and E.B. working in this research synthesized and biologically evaluated the new
compounds as a part of their master thesis, D.H.-L. designed and supervised the research and contributed in the
writing, design, synthesis, biological evaluation and analysis of the data; E.P. contributed in the writing, biological
evaluation, docking studies and analysis of data. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments: E. Pontiki would like to thank A. Patsilinakos from the Department of Chemistry and Drug
Technologies, “Sapienza” University of Rome, Italy. Hadjipavlou would like to be thankful to Biobyte and A. Leo
for providing free access to Biobyte platform.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
AA: Arachidonic acid; AAPH: 2,20-azinobis(2-amidinopropane) hydrochloride; BBB: Blood Brain Barrier; Clog P:
Theoretical calculated lipophilicity; COX: Cycloxygenase; CPE: Carrageenin-induced rat paw edema; H(P)ETEs:
15-hydro(-pero)xy-eicosa-tetra-enoicacids; LOX: Lipoxygenase; LXs: Lipoxins; NDGA: Nordihydroguaretic acid;
NSAIDs: non-steroidal anti-inflammatory drugs; PGs: Prostaglandins; QSAR: Quantitative Structure Activity
Relationships; ROS: Reactive Oxygen Species; TEA: trimethylamine; W: Watt.
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