Myeloperoxidase inhibitory and antioxidant activities of (E)-2-hydroxy-α-aminocinnamic acids obtained through microwave-assisted synthesis

Article abstract of DOI:10.3390/ph14060513

Myeloperoxidase (MPO) is an enzyme present in human neutrophils, whose main role is to provide defenses against invading pathogens. However, highly reactive oxygen species (ROS), such as HOCl, are generated from MPO activity, leading to chronic diseases. Herein, we report the microwave-assisted synthesis of a new series of stable (E)-(2-hydroxy)-α-aminocinnamic acids, in good yields, which are structurally analogous to the natural products (Z)-2-hydroxycinnamic acids. The radical scavenging activity (RSA), MPO inhibitory activity and cytotoxicity of the reported compounds were evaluated. The hydroxy derivatives showed the most potent RSA, reducing the presence of DPPH and ABTS radicals by 77% at 0.32 mM and 100% at 0.04 mM, respectively. Their mechanism of action was modeled with BDE_OH, IP and ?E_H-L theoretical calculations at the B3LYP/6 ? 31 + G(d,p) level. Compounds showed in vitro inhibitory activity of MPO with IC₅₀ values comparable to indomethacin and 5-ASA, but cytotoxicities below 15% at 100–200 μM. Docking calculations revealed that they reach the amino acid residues present in the distal cavity of the MPO active site, where both the amino and carboxylic acid groups of the α-aminopropenoic acid arm are structural requirements for anchoring. (E)-2-hydroxy-α-aminocinnamic acids have been synthesized for the first time with a reliable method and their antioxidant properties demonstrated.

Full text of DOI:10.3390/ph14060513

pharmaceuticals
Article
Myeloperoxidase Inhibitory and Antioxidant Activities of
(E)-2-Hydroxy-α-aminocinnamic Acids Obtained through
Microwave-Assisted Synthesis
Astrid Rivera-Antonio 1,2, Martha Cecilia Rosales-Hernández 2 , Irving Balbuena-Rebolledo 1,3
José Martín Santiago-Quintana 1,2 , Jessica Elena Mendieta-Wejebe 2 , José Correa-Basurto 3
Juan Benjamín García-Vázquez 3 , Efrén Venancio García-Báez 1 and Itzia I. Padilla-Martínez 1,
,
,
*
1
Laboratorio de Química Supramolecular y Nanociencias, Unidad Profesional Interdisciplinaria
de Biotecnología, Instituto Politécnico Nacional, Avenida Acueducto s/n, Barrio la Laguna Ticomán,
Ciudad de México 07340, Mexico; astridmriveraa@gmail.com (A.R.-A.); irving.balbu@gmail.com (I.B.-R.);
martinsnqn3@gmail.com (J.M.S.-Q.); efren1003@yahoo.com.mx (E.V.G.-B.)
Laboratorio de Biofísica y Biocatálisis, Sección de Estudios de Posgrado e Investigación, Escuela Superior
de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Salvador Díaz Mirón s/n, Casco de
Santo Tomas, Ciudad de México 11340, Mexico; marcrh2002@yahoo.com.mx (M.C.R.-H.);
jesmenwej@yahoo.com (J.E.M.-W.)
Laboratorio de Diseño y Desarrollo de Nuevos Fármacos e Innovación Biotecnológica, Escuela Superior
de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n, Col. Casco de Santo Tomas,
Ciudad de México 11340, Mexico; corrjose@gmail.com (J.C.-B.); benjagv_5202@hotmail.com (J.B.G.-V.)
Correspondence: ipadillamar@ipn.mx
2
3
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Rivera-Antonio, A.;
Rosales-Hernández, M.C.; Balbuena-
Rebolledo, I.; Santiago-Quintana, J.M.;
Mendieta-Wejebe, J.E.; Correa-
Basurto, J.; García-Vázquez, J.B.;
García-Báez, E.V.; Padilla-Martínez,
I.I. Myeloperoxidase Inhibitory and
Antioxidant Activities of (E)-2-
Hydroxy-α-aminocinnamic Acids
Obtained through Microwave-
Assisted Synthesis. Pharmaceuticals
2021, 14, 513. https://doi.org/
10.3390/ph14060513
*
Abstract: Myeloperoxidase (MPO) is an enzyme present in human neutrophils, whose main role is
to provide defenses against invading pathogens. However, highly reactive oxygen species (ROS),
such as HOCl, are generated from MPO activity, leading to chronic diseases. Herein, we report
the microwave-assisted synthesis of a new series of stable (E)-(2-hydroxy)-α-aminocinnamic acids,
in good yields, which are structurally analogous to the natural products (Z)-2-hydroxycinnamic
acids. The radical scavenging activity (RSA), MPO inhibitory activity and cytotoxicity of the reported
compounds were evaluated. The hydroxy derivatives showed the most potent RSA, reducing the
presence of DPPH and ABTS radicals by 77% at 0.32 mM and 100% at 0.04 mM, respectively. Their
mechanism of action was modeled with BDEOH, IP and
31 + G(d,p) level. Compounds showed in vitro inhibitory activity of MPO with IC50 values
comparable to indomethacin and 5-ASA, but cytotoxicities below 15% at 100–200 M. Docking
calculations revealed that they reach the amino acid residues present in the distal cavity of the MPO
active site, where both the amino and carboxylic acid groups of the -aminopropenoic acid arm are
structural requirements for anchoring. (E)-2-hydroxy- -aminocinnamic acids have been synthesized
∆EH-L theoretical calculations at the B3LYP/6
−
µ
Academic Editor: Thierry Besson
α
Received: 13 April 2021
Accepted: 11 May 2021
Published: 27 May 2021
α
for the first time with a reliable method and their antioxidant properties demonstrated.
Keywords: (Z)-2-hydroxycinnamic acid; (E)-dehydro-Phe; hypochlorous acid; antioxidants; 3-
acetamidocoumarins; 3-aminocoumarins
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Myeloperoxidase (MPO) is a hemeprotein member of the peroxidase family that can
be found in the azurophil granules of polymorphonuclear neutrophils. The crucial role of
MPO in the antimicrobial activity of neutrophils relies on the production of hypochlorous
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
acid (HOCl) [
not only chlorination of lipids, nucleic acids and carbohydrates but also the deamination of
amino acids [ ], which are strongly associated with chronic degenerative diseases such as
1]. This strong oxidant species is able to cross the cell membrane, promoting
2,3
atherosclerosis [2,4], cancer [5], Alzheimer’s, Parkinson’s [6] and rheumatoid arthritis [1].
The design of new MPO inhibitors has been based on their diverse modes of action
such as complex II accumulation, suicide substrates and reversible union to the native
Pharmaceuticals 2021, 14, 513. https://doi.org/10.3390/ph14060513
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 513
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enzyme. There are some MPO inhibitors, such as 5-aminosalicylic acid (5-ASA) [
7], dap-
sone, tryptamines and indomethacin, which are considered as inhibitors that cause the
accumulation of complex II (peroxidase deficient) [ ]. Therefore, in order to promote
8,9
complex II accumulation, these types of inhibitors first react with complex I; the latter
being more reactive than the former with two and one oxidation equivalents, respectively,
compared to the native MPO [10]. In contrast, suicide substrates such as 4-aminobenzoic
acid hydrazide (4-ABAH), benzoic acid hydrazide (BAH) and 2-thioxanthins can be cat-
egorized as irreversible inhibitors, which either destroy or modify the covalent bond of
the heme group present in the MPO [11]. On the other hand, reversible inhibitors such as
salicylhydroxamic acid (SHA) and aromatic hydroxamates compete with the substrate for
the active site of the enzyme but without modifying it.
Chronic degenerative diseases have long been associated with oxidative stress, mainly
derived from the enzymatic production of reactive oxygen species (ROS) [12,13]. Generally,
synthetic molecules designed to counteract the free radical effects are derived from com-
pounds of natural origin and some are structurally similar. Polyphenols constitute a class of
antioxidants which are found in the daily diet, commonly known as phytochemicals, where
hydroxycinnamic acids are found. Caffeic, ferulic and isoferulic acids are synthesized
within some plants such as coffee, cinnamon, citrus fruits and grapes, among others [14];
they are also found in wines [15]. Some of the reported pharmacological properties of
cinnamic acid and its derivatives have anti-inflammatory, antimicrobial, antimalarial [16],
antidepressant [17], anticancer and antioxidant activities, as well as neuroprotective ef-
fects [18].
The antioxidant properties of both cinnamic acid and its hydroxy derivatives can be
explained in terms of two important structural features: the presence of a phenol moiety
and the propenoic acid on the side chain. Both mentioned moieties have been shown
to confer an improved electron delocalization of the backbone upon reaction with free
radicals, thus avoiding the stage of radical propagation during the oxidation process [19].
The carboxylic acid functional group and the phenolic ring, in hydroxycinnamic acids,
can be found in opposite positions or in the same plane around the double bond. In the
first case, cinnamic acids are referred to as E and in the second case as Z isomers, Figure 1.
2-Hydroxycinnamic acids are a group of compounds of natural origin, very abundant in
some foods and appreciated for their antioxidant properties which can be mainly found
in the E configuration, whereas the Z configuration is scarce. As a natural product, (Z)-2-
hydroxycinnamic acid has been isolated in very small quantities [20,21]. Recent studies
in lung cancer showed that (Z)-cinnamic acid prevents metastasis to a greater extent than
the (E)-cinnamic acid [22]. It is worth mentioning that the (E) isomers are more abundant
in nature, presumably because they are more stable than the (Z) isomers [23]. There are
reported methods for the synthesis of (Z)-2-hydroxycinnamic acids, but the yields are low,
at 5–10%, and purification by chromatography is necessary, with the main product being
coumarin [24].
Nowadays, the pharmaceutical industry has integrated green chemistry into its pro-
cesses to obtain products free of solvents and heavy metals [25]. One of the modern
green methodologies is microwave-assisted chemical synthesis, which uses the property
of polar molecules to transform electromagnetic energy into heat to save time and energy,
generating fewer by-products and therefore improving yields, compared to conventional
methods [26].
Herein, we present the synthesis of a family of (E)-2-hydroxy-α-aminocinnamic
acid derivatives obtained through microwave-assisted chemical synthesis in good yields,
Figure 1. These compounds are analogous to dehydro-amino acids which are highly
valuable synthetic precursors [27] and particularly to (E)-dehydro-phenylalanine whose
(Z) isomer has recently been used as hydrogelator [28]. In this work, the title com-
pounds have been envisaged as antioxidants due to their structural analogy with (Z)-
2-hydroxycinnamic acid. The structures of the abovementioned compounds and that of
contrasting/inhibitory compounds used in the assays are depicted in Figure 1. Their an-

Pharmaceuticals 2021, 14, 513
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tioxidant capabilities were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
2,2-azino-bis(3-ethylbenzothiazolin)-6-sulfonic acid (ABTS) methods, as long as molecular
orbital calculations at the B3LYP/6–31 + G(d,p) level of theory allowed us to rational-
ize their mechanism of action. Selected compounds exhibited in vitro inhibitory activity
against MPO whose interaction in the binding site was modeled by docking calculations.
Finally, the cytotoxicity of this set of compounds was evaluated in a 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
HOOC
HOOC
NH2
COOH
OH
(E)-2-hydroxycinnamic acid
OH
OH
(E)-2-hydroxy- -aminocinnamic acid
(Z)-2-hydroxycinnamic acid
HOOC
NH2
H2N
COOH
(E)-dehydro-phenylalanine
(Z)-dehydro-phenylalanine
COOH
O
H2N
COOH
OH
HN NH2
O
N
H2N
O
Cl
4-ABAH
5-ASA
Indomethacin
Figure 1. Structures of (E)- and (Z)-2-hydroxycinnamic acids, (E)-2-hydroxy-α-aminocinnamic acid, (E)- and (Z)-dehydro-
phenylalanine, 5-aminosalicylic acid (5-ASA), indomethacin and 4-aminobenzoic acid hydrazide (4-ABAH) studied herein.
2. Results and Discussion
2.1. Chemistry
Cinnamic acid derivatives 2a
from the corresponding 3-acetamidocoumarins 1a
–
j
were obtained in moderate to good yields (51–95%)
29]. Compounds 1a were synthe-
–
j
[
–j
sized following a reported method [30] with modifications in the quantities of the reagents
used and reaction time and starting from glycine instead of N-acetylglycine, resulting in
higher yields (62–86%) than those reported elsewhere (40–69%) [31].
The general procedure to obtain 2a–j, as switterions, is shown in Scheme 1, which
consists of the microwave-assisted acid hydrolysis of the 3-acetamidocoumarins 1a–j,
followed by neutralization with NaHCO3. The reaction conditions were optimized for
each compound since they depend on the nature of the substituent. The concentration
of the H2SO4 aqueous solution, temperature and reaction time are the main factors that
determine the progress of the reaction.

Pharmaceuticals 2021, 14, 513
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5-15 %
+
-OOC
NH3
O
8
H2SO4 v/v
120-160 °C
15-20 min
1.
6
1
7
NH
O
6
5
4
G
Mw
G
2
7
O
OH
8
2. NaHCO (aq)
3
3
2a-j
1a-j
G
G (yield %)
1a
1c
1b
1d
1f 7-AcO
1h 6-AcO
1j 6-NH2
H
6-Cl
6-Br
6-F
2a H (68)
2b 5-Br (51)
2c
2e
2d
2f
5-Cl (63)
3-OEt (82)
5-F (75)
4-OH (67)
1e 8-OEt
1g 6-OMe
1i 6-NO2
2g 5-OMe (74) 2h 5-OH (95)
2i 5-NO2 (86) 2j 5-NH2 (56)
Scheme 1. Microwave-assisted hydrolysis of acetamidocoumarins 1a
acids 2a–j.
–j to produce (E)-2-hydroxy-α-aminocinnamic
1
The H-NMR spectra of the isolated reaction mixtures, for the conversion of com-
◦
pound 1f to 2f at 120 C and 5% v/v concentration of H2SO4 in water, with increasing
reaction times, are shown in Figure 2. The complete conversion required 15 min of reaction
at the specified conditions, however, this set of spectra allows us to observe the reaction in-
termediates. After 5 min of reaction time, the hydrolyzed ester
I–f and the aminocoumarin
II are observed. The mixture progresses to the complete lactone ring opening, leading to
–
f
compound 2f. The above result confirms that compound 2f can also be obtained starting
from the corresponding aminocoumarin, as sketched in Scheme 2. A summary of the
optimized reaction conditions is listed in Table 1. In general, milder conditions are required
for acetamidocoumarins substituted with electron-donating groups in comparison to those
substituted with electron-withdrawing groups.
Figure 2. 1H-NMR spectra (DMSO-d6) showing the effect of reaction time on the conversion of the starting compound 1f (~)
◦
◦
to 2f, at a reaction temperature of 120 C and 5% v/v of H2SO4 in water. The intermediates
I
–
f
(ˆ) and II
–f
( ) are observed
in 5 min of reaction time.

Pharmaceuticals 2021, 14, 513
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Scheme 2. Observed intermediates I–f and II–f in the acid hydrolysis of 1f to 2f.
Table 1. Optimized reaction conditions to obtain compounds 2a–2j using microwaves.
Reaction Conditions
Compounds
Aq. H2SO4 conc.
Temperature
Time
(min)
◦
(% v/v)
( C)
2f
5
120
120
160
15
15
20
2a, 2e, 2g, 2h, 2j
2b, 2c, 2d, 2i
15
15
One of the characteristic structural features of compounds 2a
of the vinyl proton H7, as shown in Scheme 1. It appears in the 6.95–7.26 ppm range
as a singlet, which in the corresponding spectra of the starting 1a , is shifted to higher
–j
is the 1H-NMR signal
–
j
frequencies, usually beyond 8.60 ppm [32] and in the aminocoumarins it appears at lower
frequencies in the 6.59–6.71 ppm range [29]. The synthesis of 3-aminocoumarins, by
the acid hydrolysis of 3-acetamidocoumarins, has been reported elsewhere [29], using
H2SO4 solution (70%) by both microwaves and reflux. In this context, compounds 2a
are proposed as the natural final product of hydrolysis. However, we observed by 1H-
NMR that compounds 2a are cyclized with dehydration in highly concentrated H2SO4
–j
–j
solutions to regenerate the corresponding 3-aminocoumarin (Figures S1 and S2). This
transformation requires the phenolic –OH and the –COOH groups on the same side around
the double bond to perform the lactone ring cyclization. This experimental behavior
supports the (E) configuration of compounds 2a
carried out successfully even though the (Z) derivatives of the cinnamic acid are reported
to naturally occur in low yields or with little stability [21 22 24] points to the amino group
as the structural factor that provides the observed stability of 2a , particularly allowing
–j. The fact that the synthesis of 2a–j was
,
,
–j
the formation of the switterionic form which prevents lactonization to the corresponding
aminocoumarin.
2.2. DPPH and ABTS Free Radical-Scavenging Activity
The antioxidant activity of the cinnamic derivatives 2a–j was evaluated using DPPH
and ABTS tests, using 5-ASA as the reference compound. These tests are widely used to
evaluate the removal of free radicals from natural and synthetic compounds; the results of
both tests are depicted in Figure 3. In general, the antioxidant capacity evaluated through
the DPPH test, using the highest tested concentration of 0.32 mM (see the complete results
in Figures S3 and S4), decreased in the following order: 5-ASA = 2i
> 2f = 2h > 2g > 2j > 2e
>
2b 2c 2a 2d. The radical scavenging activity (RSA) of compound 2i is the same of 5-
=
>
>
ASA (p < 0.0001) and compounds 2f and 2h reach up to 77% RSA, becoming almost as good
as 5-ASA. The value of DPPH RSA of 5-ASA is similar to the reported values of 85–90%
at 0.1–1.0 mM concentrations [33] and 80–85% at 0.20–0.41 mM concentrations [
9]. The
performances of compounds 2f 2h and 2i in the DPPH test are comparable to resveratrol–
,
salicylate analogs (90% RSA, 0.20 mM) [34] and caffeic acid (94% RSA, 0.11 mM) [35], the
latter being considered by some as the best cinnamic acid antioxidant [36].

Pharmaceuticals 2021, 14, 513
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Figure 3. Comparison of the radical-scavenging activity of compounds 2a–j vs. 5-ASA at a concentration of 0.32 mM and
0.04 mM in the DPPH and ABTS assays, respectively (* p < 0.05, *** p < 0.0001). The assays were performed in triplicate.
DPPH RSA (%): 31.6 2.6 (2a), 42.7 2.4 (2b), 38.3 5.4 (2c), 14.1 7.5 (2d), 54.8 3.7 (2e), 79.6 1.4 (2f), 72.5 1.8
2g), 77.1 1.5 (2h), 86.7 3.4 (2j) and 90.4 3.4 (5-ASA). ABTS RSA (%): 98.9 1.3 (2a 98.7 1.2 (2b),
93.1 ± 3.4 (2c), 17.7 0.4 (2e), 99.4 0.0, (2f), 60.0 5.3 (2g), 68.3 ± 1.8 (2h), 93.5
±
±
±
±
±
±
±
(
±
±
4.3 (2i), 59.5
±
±
±
),
±
±
2.0 (2d), 95.4
and 98.9 ± 1.3 (5-ASA).
±
±
±
±
0.4 (2i), 96.8 ± 0.3 (2j)
On the other hand, compounds 2a
in the ABTS test, being as active as 5-ASA (p < 0.0001). The herein obtained value of ABTS
RSA of 5-ASA is similar to the reported value of 98% at 0.05 Mm [ ]. Compounds 2c 2e
,
2b
,
2f and 2j exhibited
≈
100% of RSA at 0.04 mM
9
,
and 2i are within the 93.1–95.4% range, 2g and 2h are in the 60.0–68.3% range and the
less effective 2d has 17.7% RSA (see the complete results in Figures S5 and S6). Therefore,
their performance in the ABTS test is better than the reported values for caffeic (93% RSA,
0.14 mM) [35] (45% RSA, 0.01 mM), sinapic (50%, 0.01 mM) and ferulic (40%, 0.01 mM)
acids [37].
Compounds 2f
antioxidant tests; all of them are substituted with electron-donating groups with the
exception of compound 2i (5-NO2). In general, the RSAs of compounds 2a are related
to the possibility of -type delocalization of the unpaired electron in the whole structure.
–j show performances higher than 60% in both the DPPH and ABTS
–
j
π
In particular, the role played by the exocyclic conjugated double bond, carboxylic acid
and the –NH2 group in the stabilization of the 2-OH radical is depicted in Figure 4a.
Further, the highest antioxidant ability of 2f is explained by the radical delocalization
of the 4-OH group into the α-aminopropenoic fragment, Figure 4b. Compound 2h is
structurally comparable with synthetic salicylate derivatives whose RSA performance
was demonstrated to depend on the 1,4-disposition between the two –OH groups in the
molecule [33,34]. The stabilizing resonance structures, including the oxidation products
of compound 2h, are depicted in Figure 4c, ending with the formation of quinoid type
products which can act as cytotoxic agents in diseases such as cancer, as reported for caffeic
acid [38]. The results herein obtained are in agreement with those which point out that at
last two phenolic –OH groups are required to confer antioxidant properties, but this quality
is more dependent on their relative disposition [36]. Finally, the RSA of compound 2i
substituted with the strong electro-withdrawing nitro group, is due to the increased acidity
of the 2-OH group, facilitating the radical formation and also stabilizing it, Figure 4d. It
is worth noting that the fluorine atom (2d) has the worst RSA in both DPPH and ABTS
tests, in contrast to theoretical predictions (vide infra). This behavior could be associated
with the high electronegativity of the fluorine atom, accompanied by its limited electronic
delocalization possibilities.
,

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Figure 4. Resonant contributors to the stabilization of free radicals in compounds (a) 2a, (b) 2f, (c) 2h and (d) 2i.
2.3. Molecular Orbital Calculations and Antioxidant Mechanism
The DPPH and ABTS tests are useful colorimetric probes accepting hydrogen atoms
or electrons supplied by the antioxidant compounds. However, none of them allow
discriminating between the hydrogen atom transfer (HAT) and single electron transfer
(SET) as the most accepted mechanisms for phenols [39]. In this context, theoretical
calculations at the B3LYP/6–31 + G(d,p) level of theory were performed in order to gain
a better understanding of the operating antioxidant mechanism of compounds 2a
bond dissociation enthalpy of the 2-OH group (BDEOH), the ionization potential (IP), EH
(energy of the HOMO), EL (energy of LUMO) and EH-L (HOMO-LUMO gap) in vacuum
–j. The
∆
were calculated. The results are shown in Table 2. 5-ASA was used as the reference
compound to determine the reliability of our calculations. All the calculated values for
5-ASA are in agreement with the reported ones [33].
BDEOH is related to the HAT mechanism. It describes the thermodynamic stability
of the O–H bond and small BDEOH values are associated with increased susceptibility to
homolytic O-H bond dissociation. The calculated BDEOH values for compounds 2a–2j are
in the 52.99–81.13 kcal mol−1 range. The smallest value of 52.99 kcal mol−1 associated
with the bromine compound 2b is underestimated and therefore omitted from discussion.
Then, compounds 2d
in the 61.95–65.50 range), followed by the less active 2g
>
2a
>
2e
>
2h
>
2f can be ranked as the most active (BDEOH values
2c 2i 2j (BDEOH values
>
>
>
in the 73.01–81.13 range) through the HAT mechanism. The values for the most active
group are smaller than those reported for caffeic acid (72.25–73.95 kcal mol−1), ferulic
acid (81.36–75.08 kcal mol−1) and p-coumaric acid (81.18 -79.86 kcal mol−1) [40
,41] and
even smaller than ascorbic acid (75.4 kcal mol−1) [42]. As far as we are concerned, these
values are among the smallest reported for antioxidant compounds [43], indicating a great
propensity to react by the HAT mechanism. The OR (R = H, Me, Et) groups favor small
BDEOH values of the 2-OH: the 5-OH (2h) group is better than 4-OH (2f) and the OR group
is also more favorable in C-3 (2e) than in C-5 (2g). These structure trends are in agreement
with those observed by others in hydroxycinnamic acids [44].
IP describes the ability of compounds in electron donation, and smaller values of
IP are related to a more active SET mechanism [45]. In this context, compounds 2a–2j

Pharmaceuticals 2021, 14, 513
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can be sorted into three groups according to their calculated IP values. The most active
group through the SET mechanism is represented by 2f
group by the halogen substituted compounds 2b 2d and the less active group by 2j
2g, showing IP values in the 135.67–138.84, 141.24–145.54 and 150.22–156.95 kcal mol−1
>
2e
>
2h
>
2a, a moderately active
-
>
2i
>
ranges, respectively. The IP values of the first group of compounds are similar to p-
coumaric acid (130.43 kcal mol−1) and other cinnamic acids [44], but smaller than ascorbic
acid (152.0 kcal mol−1) or cinnamaldehyde (154.9 kcal mol−1) [46], pointing to the good
propensity of 2a, 2e, 2f and 2h to act through the SET mechanism.
In addition, EH, EL and
reactivity of the molecule. The lower the
from ground state to excited state and the higher the chemical reactivity. The calculated
EH-L value of compound 2i is the smallest (3.92 eV), but comparable to the value of
5-ASA (3.94 eV) and sinapic acid (3.8 Ev) [45], whereas the EH-L values of the rest of the
∆
EH-L (HOMO–LUMO gap) values are related to the chemical
∆
EH-L value, the easier the electronic transition
∆
∆
compounds are in the 4.53–4.81 eV range, slightly higher than those calculated for natural
hydroxycinnamic acids (4.1–4.5 eV).
Table 2. BDEOH, IP, EH, EL and
and compounds 2a–j.
∆EH-L calculated values at B3LYP/6–31 + G(d,p) in vacuum of 5-ASA
−1
−1
)
Comp.
BDEOH (kcal mol
)
IP (kcal mol
165.87
EH (eV) EL (eV)
∆EH-L (eV)
3.85
5-ASA [33]
92.60
95.44
62.02
52.99
75.68
61.95
62.79
−5.66
−5.79
−5.80
−5.93
−5.90
−5.90
−5.76
−1.81
−1.85
−1.05
−1.33
−1.27
−1.24
−0.95
5-ASA
2a
165.71
3.94
138.84
4.75
2b
141.24
4.60
2c
143.20
4.63
2d
145.54
4.66
2e
134.50
4.81
65.50
2f
2g
2h
135.67
156.95
136.42
−5.61
−5.55
−5.58
−0.96
−1.02
−1.05
4.65
4.53
4.53
55.14 a
73.01
62.85
53.95 b
2i
2j
79.29
81.13
152.65
150.22
4-OH; b 5-OH.
−6.42
−5.39
−2.51
−0.78
3.91
4.61
a
In general, the calculated BDEOH and IP values of compounds 2a
–
j
are in agreement
with a greater propensity to react by the HAT mechanism. However, compounds 2a
, 2e,
2f
and 2h are predicted to be the most active by either of the two mechanisms with larger
stabilities than natural hydroxycinnamic acids.
Finally, it is worth mentioning that theoretical calculations predict a strong change in
the conformation adopted by the
The C2-C1-C7-C8 torsion angle changes from the 143–147◦ range in 2a
range in the radicals. Then, the planar -aminopropenoic acid arm and the bent con-
α
-aminopropenoic acid arm when radicals are formed.
–
j
to the 20–34◦
α
formation adopted by radicals and cation radicals strongly resemble the structure of the
3-aminocoumarins, favoring the delocalization of the nitrogen atom lone pair to the phenyl
ring which might contribute to their stabilization, Figure 5 [45]. A complete list of C2-C1-
C7-C8 torsion angles is given in Table S1.

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Figure 5. Predicted conformations of (
a
)
2a, (b
)
2a radical and (
c)
2a cation radical. The torsion angle
C2-C1-C7-C8 is shown.
2.4. Docking of 2a–j with MPO
Molecular docking was performed with the aim to support the choice of MPO. Three
different conformers of the MPO from molecular dynamics (MD) simulation were used [47].
The validation of the method was carried out with N-acetylglucosamine, reaching an RMSD
value of 1.194 Å in agreement with accepted values [48] (Figure S7). The calculated binding
free energy (
G values are more favorable at the MPO conformation adopted at 10 ns than at 5 or 0 ns
(native structure). The calculated 6.5 to 6.0 range.
G values (kcal mol−1) are in the
∆G) and interactions in the binding site are listed in Table 3. In general, the
∆
∆
−
−
The largest values correspond to those compounds bearing halogens 2b (5-Br) and 2c
(5-Cl). This result contrasts with reported results which mention that halide substituents
in tested molecules to inhibit MPO have no effect on it [49]. Docking calculations predict
that compounds 2a–j bind to the MPO, forming complexes as stable as those of 4-ABAH, a
well-known irreversible inhibitor, and 5-ASA, the reference compound.
Table 3. Values of
∆
G (kcal mol−1) of the compounds 2a
–
j
in the 10 ns conformation of the MPO enzyme and their
interactions with the amino acid residues.
−1
Comp.
∆G (kcal mol
)
Interaction Residue
HEM605, Q91, D94, R239, M243
4-ABAH
−6.0
−6.1
−6.3
−6.5
−6.4
−6.3
−6.3
−6.1
−6.2
−6.1
−6.3
−6.0
5-ASA
2a
HEM605, Q91, D94, R239, E242, M243, L246, H336
HEM605, Q91, D94, R239, M243, L246
2b
2c
HEM605, Q91, D94, R239, M243, L246
HEM605, Q91, D94, R239, M243, L246
2d
2e
HEM605, Q91, D94, R239, S240, M243, L246
HEM605, Q91, D94, R239, E242, M243, L246, H336, F365
HEM605, Q91, D94, R239, M243, L246, H336
HEM605, Q91, D94, R239, M243, L246
2f
2g
2h
2i
HEM605, Q91, D94, R239, E242, M243, L246, H336, F365
HEM605, Q91, D94, R239, M243
2j
HEM605, Q91, D94, R239, M243, L246
In previous studies [47], the enzyme-binding sites were identified as being formed by
the amino acid (aa) residues Q91, H95, R239, D94, F99, E242, R333, F366, F407 and HEM605,
where Q91, H95, R239 and four water molecules make up the distal cavity of the MPO,
associated with the halogenation cycle. Docking calculations predict that compounds 2a–j,
4-ABAH and 5-ASA interact with the prosthetic heme group (HEM605) and also with
the aa residues present in both the main binding site and the distal cavity: Q91, R239,
D94, E242 and M243. These results are in agreement with those obtained in silico with
triazolopyrimidines which bind into the active site in a reversible manner, avoiding the

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oxidation of the halides by the MPO [50]. The 3D structure of one homodimer of the
MPO forming a complex with compound 2c is depicted in Figure 6, where the type of
non-covalent interactions is appreciated.
Figure 6. (a) 3D structure of one homodimer of the MPO forming a complex with compound 2c. (b) The main hydrogen
bonding interactions, between the carboxylic acid and amino groups with the aa residues glutamine (Q91) and aspartic acid
(D94), respectively, in addition to the π-stacking interaction between 2-hydroxyphenyl ring with the HEM605, are shown.
2.5. MPO Enzymatic Activity of Peroxidation and Chlorination
Peroxidation and chlorination enzymatic activities of the MPO, in the presence of
compounds 2a
with the exception of 2d, inhibit the peroxidation activity of the MPO. An inhibition larger
than 50% is exhibited by compounds 2a 2e 2f and 2h at 100 M. Particularly, the inhibition
–j, were tested in vitro. The results are shown in Figure 7. All compounds,
,
,
µ
of MPO by 2e and 2h reaches values as high as 79%, comparable to 5-ASA. The IC50 of
this set of compounds was estimated using a semi-logarithmic correlation of % inhibition
against the log of the concentration (3, 6, 12, 25, 50, 100, 200, 400
graphs are depicted in Figure S8. There are no significant differences between the IC50
values ( M) of 2f (30 5), 2h (26 2) and indomethacin (29 2), whose IC50 values are
smaller than those corresponding to 2a (45 4), 2e (43 4) and 5-ASA (39 2). These
results are in agreement with those obtained in DPPH and ABTS tests.
The chlorination test shows that compounds 2c 2f and 2i exhibit a significant decrease
in the enzymatic activity of chlorination, in the range of 20–40%, with 2c being the best
MPO-inhibiting compound, showing the same activity as 5-ASA at 100 M, Figure 8. These
µM), the dose–response
µ
±
±
±
±
±
±
,
µ
results indicate that the analyzed compounds, including 5-ASA, are not specific for MPO
inhibition, therefore, they could be good candidates for other peroxidases which do not
carry out the halogenation cycle, such as COX inhibitors.

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Figure 7. Comparison of the MPO peroxidation inhibitory activity of compounds 2a
4-ABAH at 100
Peroxidation inhibition values (%): 54.4
1.1 (2g), 77.1
–
j
vs. 5-ASA and
µ
M concentration (* p < 0.05, *** p < 0.001). The assays were performed in triplicate.
±
3.1 (2a), 48.4 4.1 (2b), 40.5
±
±
4.4 (2c), 4.3 ± 0.1 (2d)
,
79.7 ± 6.6 (2e)
,
63.0 ± 1.5 (2f), 33.0
±
±
3.9 (2h), 19.6
±
0.1 (2i), 38.3
±
0.1 (2j) and
74.4 ± 2.9 (5 − ASA).
100
***
75
50
25
0
***
***
***
***
***
***
***
***
***
i
j
f
g
2
a
2
c
e
2
b
2
d
2
h
A
H
2
2
2
2
2
S
A
A
B
-
A
5
-
4
[100 M]
Figure 8. Comparison of the MPO chlorination inhibitory activity of compounds 2a
4-ABAH at 100 M concentration (*** p < 0.001). The assays were performed in triplicate. Chlorination
inhibition values (%): 11.6 0.1 (2a), 15.3 0.4 (2b), 40.2 1.2 (2c), 12.4 0.1 (2d), 13.2 0.1 (2e),
29.6 0.3 (2f), 13.1 0.5 (2g), 18.3 0.6 (2h), 25.8 0.5 (2i), 12.5 0.1 (2j) and 36.6 0.5 (5 ASA).
–j vs. 5-ASA and
µ
±
±
±
±
±
±
±
±
±
±
±
−

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2.6. Cell Viability
Compounds 2a
–j
exhibit antioxidant capabilities and MPO inhibitory activity. Thus,
in order to gain insight into the cytotoxic potential of these derivatives, they were tested
at 12.5–200 M on the fibroblast cell line NIH/3T3. The cell viability was measured by
performing an MTT assay with the maximum pattern cell activity occurring at 100% of
MTT reduction. Results at 100 and 200 M are shown in Figure 9. The complete data can
be found in Table S2. In general, the cytotoxicity of compounds 2a 2i is low. The bromine
derivative 2b was found to be the most cytotoxic, diminishing the cell viability up to 19%
at 200 M. The rest of the compounds maintain the cell viability above 85%, and the effect
is unrelated to the concentration.
µ
µ
–
µ
Figure 9. Percentage of cell viability of NIH/3T3 cells after treatment with compounds 2a–i at 100–200 µM. * p < 0.05.
3. Materials and Methods
Substituted salicylaldehydes, sodium acetate, glycine, acetic anhydride, 2,2-diphenyl-
1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazolin)-6-sulfonic acid (ABTS),
phenyl-N-tert-butylnitrone (PBN), 5-aminosalicylic acid (5-ASA), hydrogen peroxide so-
lution 30 wt % in H2O (ACS reagent), taurine ( 99% purity), DTNB ( 99% purity), or-
α-
≥
≥
tho-dianisidine and solvents were analytical grade and used as received (Sigma-Aldrich).
Myeloperoxidase of human polymorphonuclear leukocytes (EC number 1.11.1.7, >95%
purity) from Millipore® Merk was used. The synthetic procedures and characterization of
compounds 1a–j are described in the Supplementary Materials.
The microwave-assisted synthesis of compounds 2a–j was carried out in a Monowave
300 synthesis reactor from Anton Paar operating at 850 W. IR spectra were recorded neat at
◦
25 C with a Perkin Elmer Spectrum GX series with an FT system spectrophotometer using
1
the ATR device. Melting points were measured in an Electrothermal IA 91000 device. H
and 13C NMR spectra were acquired on a Varian Mercury NMR spectrometer operating
at 300 MHz (1H, 300.08; 13C, 75.46 MHz) or Bruker Avance DPX-400, using DMSO-d6
as a solvent. All chemical shift values (δ) are reported in parts per million (ppm), using
as a reference the residual solvent peak (1H,
δ δ 39.52) and coupling constants
2.50; 13C,
nJ(H–H) in Hz. Multiplicity of the signals are expressed as: s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet) or br (broad) (Figures S9–S38).
3.1. Synthetic Procedures and Characterization of Compounds 2a–j
(E)-(2-hydroxy)-α-aminocinnamic acid (2a). First, 0.300 g of N-(2-oxo-2H-chromen-3-yl) ac-
etamide (1.48 mmol), 1a, were dissolved in 18 mL of a recently prepared aqueous H2SO4
solution of 15% (v/v) inside a 30 mL microwave vial. The reaction was allowed to pro-
◦
ceed for 15 min at 120 C and 1200 rpm. Thereafter, the reaction was neutralized with
NaHCO3 (0.70 g), and the resulting solid was then filtered and washed with distilled water
(10 mL) to afford 2a as a white powder in 68% yield (0.180 g, 1.00 mmol), m.p. 202 ◦C, Rf:

Pharmaceuticals 2021, 14, 513
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1
0.68 hexane/ethyl acetate (AcOEt) 1:1. H-NMR
δ
: 7.46 (dd, 1H, 3J = 7.5, H6), 7.26 (m, 2H,
H5,3), 7.23 (m, 1H, H4), 6.95 (s, 1H, H7). 13 C-NMR
δ: 161.0 (C=O), 148.7 (C2), 146.5 (C8),
126.0 (C4), 125.7 (C6), 124.9 (C5), 123.12 (C1), 115.9 (C3), 112.3 (C7). IR (cm−1): 3043 (N-H),
1681 (C=O), 1456, 735.
(E)-(5-Bromo-2-hydroxy)-α-aminocinnamic acid (2b). The synthesis was carried out as pre-
viously indicated for derivative 2a, starting from 0.300 g (1.06 mmol) of 1b under the
following conditions: 160 ◦C for 20 min, to yield 0.140 g (0.540 mmol, 51%) of a brown
1
solid, m.p. 243 ◦C, Rf: 0.76 hexane/AcOEt 1:1. H-NMR
δ
: 7.82 (d, 1H, 4J = 2.3, H6), 7.52
(dd, 1H, 3J = 8.6, 4J = 2.5, H4), 7.31 (d, 1H, 3J = 8.7, H3), 7.09 (s, 1H, H7) 10.7 (s, 1H, OH).
NMR 13
: 158.3 (C=O), 148.6 (C2), 143.0 (C8), 130.4 (C4), 128.8 (C6), 123.3 (C1), 118.3 (C5),
116.8 (C3), 114.0 (C7). IR (cm−1): 3355 (N-H), 1690 (C=O), 1384, 771.
C
δ
(E)-(5-Chloro-2-hydroxy)- -aminocinnamic acid (2c). The synthesis was carried out as previ-
α
ously indicated for derivative 2b, starting from 0.300 g (1.26 mmol) of 1c to yield 0.170 g
1
(0.790 mmol, 63%) of a brown solid, m.p. 252 ◦C, Rf: 0.81 hexane/AcOEt 1:1. H-NMR:
δ
10.7 (br, 1H, OH), 7.64 (d, 1H, 4J = 2.0, H6), 7.09 (s, 1H, H7), 7.39 (m, 2H, H4, 3). 13 C-NMR:
δ
158.3 (C=O), 148.1 (C2), 143.0 (C8), 128.9 (C5), 127.6 (C4), 125.8 (C6), 122.8 (C1), 117.9 (C3),
114.1 (C7). IR cm−1: 3381 (N-H), 1720 (C=O), 1518, 919, 835.
(E)-(5-Fluoro-2-hydroxy)-α-aminocinnamic acid (2d). It was synthesized as indicated for
compound 2b, starting from 0.300 g (1.36 mmol) 1d to yield 0.200 g (1.01 mmol, 75%) of
1
a brown solid m.p. 242 ◦C, Rf: 0.84 hexane/AcOEt 1:1. H-NMR:
δ
7.43 (dd, 3JH-F = 9.1,
4J = 3, H6), 7.39 (dd, 4JH-F = 5, 3J = 9.1, H3), 7.22 (ddd, 3JH-F = 9.0, 3J = 8.6, 4J = 3, H4), 7.08 (s,
1H, H7). 13 C-NMR:
δ
160.1 (C=O), 158.5 (C2), 145.8 (C8), 143.2 (C5), 122.6 (3JC-F = 10.3, C1),
117.8 (3JC-F = 9.1, C3), 114.8 (2JC-F = 24.8, C4), 114.3 (C7), 112.0 (2JC-F = 25, C6). IR cm−1
:
3115 (N-H), 1676 (C=O), 1518, 748.
(E)-(3-Ethoxy-2-hydroxy)-α-aminocinnamic acid (2e). It was synthesized as indicated for
compound 2a, starting from 0.300 g (1.20 mmol) of 1e to yield 0.220 g (0.990 mmol, 82%) of
1
a brown solid, m.p. 139 ◦C, Rf: 0.70 hexane/AcOEt 1:1. H-NMR:
δ 10.4 (br, 1H, OH), 7.16
(t, 1H, 3J = 8.1, H5), 7.07 (s, 1H, H7), 7.04 (m, 2H, H4, 6), 4.11(c, 2H, CH2, 3J = 7.0), 1.37 (t,
3H, CH3, 3J = 7.0).13C- NMR:
δ 158.6 (C=O), 145.9 (C2), 142.3 (C8), 138.7 (C3), 125.0 (C6),
121.7 (C1), 118.1 (C5), 115.5 (C7), 111.4 (C4), 64.5 (CH2), 15.0 (CH3). IR cm−1: 3352 (N-H,
1692 (C=O), 1472, 764.
(E)-(2,4-dihydroxy)-α-aminocinnamic acid (2f). It was synthesized as indicated for compound
2a, starting from 0.300 g (1.15 mmol) of compound 1f and 18 mL of a 5% v/v H2S◦O4
◦
solution, at 120 C, 10 min to yield 0.180 g (0.920 mmol, 67%) of a brown solid, m.p. 272 C,
1
Rf: 0.77 hexane/AcOEt 1:1. H-NMR:
δ
10.0 (br, 2H, OH), 7.35 (d, 1H, 3J = 8.5, H6), 7.06 (s,
1H, H7), 6.74 (dd, 1H, 3J = 8.5, 4J = 2.3, H5), 6.69 (d, 1H, 4J = 2.3, H3). 13C NMR:
δ 159.2
(C=O), 158.2 (C2), 151.0 (C4), 139.1 (C8), 127.7 (C6), 116.6 (C7), 113.6 (C5), 112.7 (C1),
102.4 (C3). IR cm−1: 3278 (OH, N-H), 1696 (C=O), 1454, 826.
(E)-(2-hydroxy-5-methoxy)-α-aminocinnamic acid (2g). It was synthesized as indicated for
compound 2a, starting with 0.300 g (1.29 mmol) of 1g to yield 0.200 g (0.95 mmol, 74%)
1
of a brown solid. m.p. 154 ◦C, Rf: 0.67 hexane/AcOEt 1:1. H- NMR:
δ
10.4 (br, 1H, OH),
7.24 (d, 1H, 3J = 9.0, H4), 7.05 (d, 1H, 4J = 3.2, H6), 6.98 (s, 1H, H7) 6.93 (dd, 1H, 3J = 8.8,
4J = 3.0, H3), 3.75 (s, 3H, CH3).13C- NMR:
δ 159.4 (CO2), 156.3 (C2, 5), 143.6 (C8), 122.4 (C6),
116.9 (C7), 114.4 (C4), 114.2 (C1), 109.1 (C3), 56.0 (OCH3). IR cm−1: 3360 (N-H), 1680 (C=O),
1400, 765.
(E)-(2,5-dihydroxy)-α-aminocinnamic acid (2h). It was synthesized as indicated for compound
2a, starting from 0.300 g (1.15 mmol) of 1h to yield 0.200 g (1.02 mmol, 95%) of a brown
1
solid, m.p. 211 ◦C. Rf: 0.79 hexane/AcOEt 1:1. H- NMR:
δ 10.2, 9.5 (br, 1H each, OH),
7.14 (d, 1H, 3J = 8.7, H3), 7.00 (s, 1H, H7), 6.81 (d, 1H, 4J = 2.6, H6), 6.76 (dd, 1H, 3J = 8.8,
4J = 2.6, H4). 13C-NMR:
159.4 (C=O), 154.8 (C2), 143.3 (C8), 143.5 (C5), 122.0 (C1), 117.3
(C3), 116.3 (C4), 115.8 (C7), 111.5 (C6). IR cm−1: 3249 (OH, NH), 1690 (C=O), 1456, 766.
δ

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(E)-(2-hydroxy-5-nitro)-
α
-aminocinnamic acid (2i). It was synthesized as indicated for com-
pound 2b, starting from 0.300 g (1.21 mmol) of 1i to yield 0.230 g (1.04 mmol, 86%) of a
1
brown solid, m.p. 240 ◦C, Rf: 0.7 hexane/AcOEt 1:1. H-NMR
δ
8.54 (d, 1H, 4J = 2.7, H6),
8.16 (dd, 1H, 3J = 8.9, 4J = 2.7, H4), 7.54 (d, 1H, 3J = 9.1, H3), 7.26 (s, 1H, H7). 13C NMR:
δ
158.1 (CO2), 153.4 (C2), 144.5 (C8), 143.9 (C5), 122.9 (C4), 122.6 (C6), 122.3 (C1), 117.7 (C3),
114.3 (C7). IR cm−1: 3456 (N-H), 1717 (C = O), 1518, 1343, 772.
(E)-(5-amino-2-hydroxy)-
α-aminocinnamic acid (2j). It was synthesized as indicated for com-
pound 2a, starting from 0.300 g (1.37 mmol) of 1j to yield 0.150 mg (0.770 mmol, 56%) of a
◦
1
brown solid, m.p. 246 C, Rf: 0.43 hexane/AcOEt 1:1. H-NMR:
6.96 (s, 1H, H7), 6.67 (dd, 1H, 3J = 8.2, 4J = 2.3, H4), 6.61 (d, 1H, 4J = 2.3, H6). 13C NMR:
δ
7.07 (d, 1H, 3J = 8.2, H3),
δ
158.9 (CO2), 145.7 (C2), 141.5 (C8), 141.2 (C5), 121.0 (C7), 116.2 (C3), 115.4 (C1), 115.0 (C4),
108.9 (C6). IR cm−1: 3369 (N-H), 3225 (O-H), 1719 (C=O), 1654, 1409, 1200, 117, 1047, 873.
3.2. DPPH Assay (2,2-Diphenyl-1-picrylhydrazyl)
One hundred microliters of DPPH 0.20 mM in absolute methanol and 100 µL of the
appropriate compound (0.32, 0.16, 0.08, 0.04, 0.02, 0.01 mM final concentrations) dissolved
in DMSO were poured into a 96-well plate. The mixtures were incubated for 30 min at
room temperature and protected from light. The absorbance was recorded at 517 nm in
a transparent 96-well test microplate (Multiskan-EX Thermo Scientific, Thermo Fisher
Scientific, Waltham, MA, USA). The results are expressed as percentage of DPPH radical
present for each concentration of derivatives. To determine the antioxidant activity of each
compound, the percentage of the DPPH radical-scavenging activity was calculated by the
following equation: [1
−
(A1
−
A2)/(ADPPH
−
AS)]
×
100, where: A1 = Absorbance of the
compound with DPPH, A2 = Absorbance of the compound, ADPPH = Absorbance of DPPH
(diluted 1:1 with solvent) and AS = Absorbance of DMSO.
3.3. ABTS Test (2,2-Azino-bis(3-ethylbenzothiazolin)-6-sulfonic Acid)
ABTS dissolved in water to a 7.00 mM final concentration was allowed to react for
16 h at room temperature in the dark with an aqueous solution of potassium persulfate
2.45 mM to produce the ABTS radical cation (ABTS+ •) before use. In a 96-well plate,
100
µ
L of the appropriate compound 2a
–
2j (0.040, 0.020, 0.010, 0.005, 0.0025 and 0.0012 mM
final concentrations) dissolved in water was mixed with either 100
µ
L of diluted ABTS+ •
solution or solvent. The reaction was allowed to proceed for 30 min at room temperature
and was protected from light. The absorbance was recorded at 734 nm in a transparent
96-well test microplate (Multiskan-EX Thermo Scientific) [51].
The antioxidant activity was calculated as the percentage of the ABTS cationic radical-
scavenging activity with the test compound, by the following equation: [1
−
(A1
−
A2)/(AABTS AS)] 100, where: A1 = Absorbance of the compound with ABTS;
−
×
A2 = Absorbance of the compound with solvent, AABTS = Absorbance of ABTS (diluted 1:1
with solvent), AS = Absorbance of DMSO. The reference used for DPPH and ABTS tests
was 5-ASA.
3.4. MPO Enzymatic Activity of Peroxidation
The enzymatic activity of peroxidation was conducted as has been described previ-
ously [52] with modifications. Briefly, hydrogen peroxide (30%) was mixed with ortho-
dianisidine in phosphate buffer (PBS) 50 mM, pH 6.0 to a final concentration of 0.0050%
(1632
of MPO (0.0126 units/
centrated DMSO solution of compounds 2a
and 3 mM final concentrations per well) and 180
oxide solution (616 M/1469 M final concentrations per well). Control wells were treated
with 10 L of DMSO. The reaction was allowed to react for 30 min and the absorbance was
measured at 460 nm. This assay was performed in triplicate.
µM) and 167
µ
g/mL (684
µ
M), respectively. In a 96-well plate, 5
L of PBS and 10 L of the appropriate con-
, 5-ASA or 4-ABAH (200, 100, 50, 25, 12.5, 6
L of the ortho-dianisidine–hydrogen per-
µL of a stock solution
µL) were mixed with 5
µ
µ
–
j
µ
µ
µ
µ

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3.5. MPO Enzymatic Activity of Chlorination
The enzymatic activity of chlorination was determined by the production of HOCl,
using the taurine–chloramine assay, where HOCl production is detected indirectly by
oxidizing 5-thio-2-nitrobenzoic acid (TNB), following a reported procedure [53]. TNB was
prepared starting from DTNB, and 20 mg of DTNB (1.0 mM) and 40 mg of NaBH4 (20 mM)
were mixed in 50 mL of PBS at pH = 7.4 and incubated for 30 minutes at room temperature.
Then, a dilution was made by taking 430 µL of the TNB solution and 4570 µL of PBS, whose
measured absorbance at 412 nm of 0.88 units is equivalent to 1.36 mM of TNB. The assay
was performed in a 96-well plate, and into each well were placed: 0.126 units of MPO
(10
(10 mM final concentration). Ten microliters of 2.0 mM DMSO solutions of compounds
2a , 5-ASA or 4-ABAH (100 M final concentration) and 7 L of 0.1% hydrogen peroxide
(10.3 mM final concentration) were incubated for 10 min. Control wells were treated with
10 L of DMSO instead. The reaction was stopped with 7 L of catalase 4 U/ L, 10.5 L of
µL), 53 µL of PBS 10 mM at pH = 7.4 added to 300 mM NaCl, 7 µL of 0.15 M taurine
–
j
µ
µ
µ
µ
µ
µ
TNB and 95.5 µL of water. The absorbance was measured at 412 nm after 10 min.
3.6. Cell Viability
The cell line NIH/3T3 was used, following reported procedures [54,55]. The cells were
cultured in Dulbecco’s modified Eagle medium (DMEM) with phenol red, supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were cultured
◦
at 37 C in a humidified incubator containing 5% CO2. The cells were detached using 1 mL
of trypsin 1% and 4 mL of PBS-EDTA. Afterwards, the cells were counted, and seeded in
96-well plates at 10
and 200 M of each compound in a final volume of 100
were dedicated for control cells treated only with medium or medium with DMSO. After
×
103 cells/well. After 24 h, the cells were treated with 12, 25, 50, 100
µ
µL (0.2% of DMSO), specific wells
48 h, the medium was removed and replaced by 20
and the cells were incubated for 3 h, at 37 C under CO2. After that, the BS/MTT was
µ
L of PBS containing MTT (0.5 mg/mL),
◦
discarded, and the formazan crystals produced were solubilized with DMSO (100 µL/well).
The optical density was measured at 550 nm using a microplate reader (Multiskan-EX
Thermo Scientific). Results are expressed as percentage of cell viability relative to control.
The experiments were performed in triplicate.
3.7. Docking MPO
Validation of the method was performed with N-acetylglucosamine, reaching an
RMSD value of 1.194 Å [48]. Snapshots (0, 5 and 10 ns) of different conformations of MPO
(PDB: 1DNU) were obtained from MD simulations [47]. All ligands were 3D prepared
in ChemSketch and optimized using Gaussian 98 software employing the AM1 method.
All possible torsions and partial charges were identified and calculated using AutoDock
Tools 1.5.6 software. All polar hydrogens were incorporated to the protein along with the
Kollman charges for all atoms. All other parameters were maintained at their default values.
The protein binding and scanning sites were prepared using a GRID-based method. A box
of 126 Å3 was employed with a spacing of 0.375 Å centered on the iron atom of the heme
group using the conformation at 10 nm. All simulations employed the hybrid Lamarckian
genetic algorithm with an initial population of 100 randomly placed individuals and
1 × 100 evaluations. Results were analyzed with Discovery Studio Visualizer 19.1 software,
to obtain both the binding energy (
amino acid residues of MPO.
∆G) and the interactions of compounds (2a–2j) with
3.8. Molecular Orbital Calculations
Calculations were performed as reported [56–58], and a brief summary is described.
All molecules and radicals were initially modeled under the semi-empirical method PM3.
Geometry optimizations and frequency calculations were performed using DFT calcula-
tions at the B3LYP/6–31 + G(d,p) level of theory for parent molecules 2a
–j, radicals and
cation radicals. Vibrational frequencies were scaled by a factor of 0.973 to obtain the scaled

Pharmaceuticals 2021, 14, 513
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zero-point energy. The thermal correction of the enthalpy was computed by a single-point
calculation with B3LYP/6–31 + G(d,p) level of theory. The total enthalpy at 298.15 K was
calculated as the sum of the thermal correction of the enthalpy and the total electronic
energy. The binding dissociation enthalpy values of the O-H bond (BDEOH) were calculated
according to the formula BDEOH = HR + HH
generated by H-abstraction, HH is the enthalpy of the H-atom and HP is the enthalpy of
the corresponding parent compound 2a . The enthalpy value of 0.49764 Hartree was
used for the hydrogen atom in all calculations of the BDE. The IP values were obtained
according to the equation IP = ER+ EP, where ER+ is the energy of the cation radical, EP
is the energy of the corresponding parent compound 2a . All calculations were performed
−
HP, where HR is the enthalpy of the radical
–
j
−
−
–
j
with the Gaussian 09 molecular package [59] in a computer with an unlocked AMD FX
processor. All the calculations refer to the gas phase. The Z-matrices, total energy and
imaginary frequencies are in the Supplementary Materials.
3.9. Statistical Analysis
One-way ANOVA was used for all the assays and a maximum value of p < 0.05 was
considered statistically significant. DPPH, ABTS, MPO enzyme activity of peroxidation
and cell viability assays were performed in triplicate whereas the MPO enzyme activity
of chlorination assay was performed in duplicate and the results were standardized and
expressed as the mean ± standard error (S.E.). A Tukey post hoc test was used.
4. Conclusions
The microwave-assisted synthesis of a family of (E)-2-hydroxy-α-aminocinnamic
acids, analogous to (Z)-2-hydroxycinnamic acids, was successfully achieved in moderate to
good yields in a diluted H2SO4 aqueous solution. The results obtained in the present study
have shown that five (2f
and ABTS free radicals under in vitro conditions, with activities similar to those reported
for caffeic acid. The conjugated -aminopropenoic acid skeleton and the substituents on
the phenyl ring play an important role in their observed antioxidant properties, with those
with a second ED group being the most active. The propensity of compounds 2a to
react through the HAT mechanism rather than the SET mechanism was predicted from
BDEOH, IP and EH-L theoretical calculations, with compounds 2a 2e, and 2h being
the most active by either of the two mechanisms. Furthermore, we have demonstrated
that compounds 2a 2e 2f and 2h showed in vitro peroxidation inhibitory activity of
–j) of the ten studied compounds can effectively scavenge DPPH
α
–
j
∆
,
2f
,
,
MPO with IC50 values comparable to indomethacin and 5-ASA but only the chlorinated
derivative 2c was capable of decreasing the in vitro chlorination activity of the MPO by
40% while the cytotoxicity of the whole set of compounds was below 15% at 100–200
µ
M. Docking calculations allowed for establishing that both amino and carboxylic acid
groups of the -aminopropenoic acid arm are required for anchoring to the MPO active site,
forming stable complexes comparable to 4-ABAH-MPO and 5-ASA-MPO. To the best of
our knowledge, (E)-2-hydroxy- -aminocinnamic acids have been synthesized for the first
α
α
time with a reliable method and their antioxidant properties demonstrated, introducing
the possibility to contrast the pharmacological properties between configurational isomers.
Finally, the pharmacological properties of these compounds, related to the generation of
oxidant species, will be the subject of further research.
Supplementary Materials: The following are available on line at https://www.mdpi.com/article/
10.3390/ph14060513/s1, Figures S1 and S2: 1H-NMR spectra showing the effect of the H2SO4
concentration on the conversion of 1i to 2i and 1a to 2a; Figures S3 and S4: Percentage of DPPH RSA
with compounds 2a–j; Figures S5 and S6: Percentage of ABTS RSA with compounds 2a–j; Figure S7:
Validation of the molecular docking protocols for MPO with N-Acetyl-D-glucosamine; Figure S8:
Percentage inhibition of MPO peroxidation activity with compounds 2a
,
–
2e
. Table S1: C2-C1-C7-C8
in their neutral, radical and cation radical forms: Table S2: Cell
viability assays; Procedures of acetamidocoumarins synthesis 1a . MO calculations at B3LYP/6-
, 2f, 2h, Indomethacin
1
and 5-ASA; Figures S9–S38: H, 13C NMR and IR spectra of compounds 2a
j
torsion angles of compounds 2a–j
–
j

Pharmaceuticals 2021, 14, 513
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31 + G(d,p), energies, imaginary frequencies and Z-matrix of 2a
–
j
in neutral, radical and cation
radical forms.
Author Contributions: Methodology, calculations, formal analysis, original draft preparation,
A.R.-A.; methodology I.B.-R.; calculations, J.M.S.-Q., E.V.G.-B.; conceptualization, supervision, fund-
ing acquisition review and editing, M.C.R.-H.; supervision, review and editing, J.E.M.-W., J.B.G.-V.;
methodology, software, J.C.-B.; conceptualization, supervision, funding acquisition, data curation,
original draft preparation, review and editing, I.I.P.-M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by CONACYT, grant number 255354 and Secretaría de Investi-
gación y Posgrado del Instituto Politécnico Nacional, grant number 20201274.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare that they have no known competing financial interest or
personal relationships that could have appeared to influence the work reported in this paper. The
funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in
the writing of the manuscript, or in the decision to publish the results.
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