Synthesis and antioxidant activity of polyhydroxylated trans-restricted 2-arylcinnamic acids

Article abstract of DOI:10.3390/molecules20022555

A series of sixteen polyhydroxylated trans-restricted 2-arylcinnamic acid analogues 3a-p were synthesized through a one-pot reaction between homophthalic anhydrides and various aromatic aldehydes, followed by treatment with BBr3. The structure of the newly synthesized compounds was confirmed by spectroscopic methods and the configuration around the double bond was unequivocally estimated by means of gated decoupling ¹³C-NMR spectra. It was shown that the trans-cinnamic acid fragment incorporated into the target compounds' structure ensures the cis-configuration of the stilbene backbone and prevents further isomerization along the carbon-carbon double bond. The antioxidant activity of compounds 3a-p was measured against 1,1-diphenyl-2-picrylhydrazyl (DPPH^?), hydroxyl (OH^?) and superoxide (O₂^?-) radicals. The results obtained showed that the tested compounds possess higher activities than natural antioxidants such as protocatechuic acid, caffeic acid and gallic acid. Moreover, it was shown that a combination of two different and independently acting fragments of well-known pharmacological profiles into one covalently bonded hybrid molecule evoke a synergistic effect resulting in higher than expected activity. To rationalize the apparent antioxidant activity and to establish the mechanism of action, a SAR analysis and DFT quantum chemical computations were also performed.

Full text of DOI:10.3390/molecules20022555

Molecules 2015, 20, 2555-2575; doi:10.3390/molecules20022555
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antioxidant Activity of Polyhydroxylated
trans-Restricted 2-Arylcinnamic Acids
Mitko Miliovsky 1, Ivan Svinyarov 1, Elena Prokopova 2, Daniela Batovska 2, Simeon Stoyanov 2
and Milen G. Bogdanov 1,*
1
Faculty of Chemistry and Pharmacy, University of Sofia “St. Kliment. Ohridski”, 1,
James Bourchier Blvd., Sofia 1164, Bulgaria; E-Mails: lishpera_87@abv.bg (M.M.);
ivansvinyarov@abv.bg (I.S.)
2
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Science,
Acad. G. Bonchev Str. Bl. 9, Sofia 1113, Bulgaria; E-Mails: elenatodorova90@gmail.com (E.P.);
danibat@orgchm.bas.bg (D.B.); s_stoyanov@orgchm.bas.bg (S.S.)
* Author to whom correspondence should be addressed; E-Mail: mbogdanov@chem.uni-sofia.bg;
Tel.: +395-2-8161-228; Fax: +359-2-962-54-38.
Academic Editor: D. Hadjipavlou-Litina
Received: 30 October 2014 / Accepted: 26 January 2015 / Published: 2 February 2015
Abstract: A series of sixteen polyhydroxylated trans-restricted 2-arylcinnamic acid
analogues 3a–p were synthesized through a one-pot reaction between homophthalic
anhydrides and various aromatic aldehydes, followed by treatment with BBr3. The structure
of the newly synthesized compounds was confirmed by spectroscopic methods and the
configuration around the double bond was unequivocally estimated by means of gated
decoupling 13C-NMR spectra. It was shown that the trans-cinnamic acid fragment incorporated
into the target compounds’ structure ensures the cis-configuration of the stilbene backbone
and prevents further isomerization along the carbon–carbon double bond. The antioxidant
activity of compounds 3a–p was measured against 1,1-diphenyl-2-picrylhydrazyl (DPPH●),
hydroxyl (OH●) and superoxide (O2●▬) radicals. The results obtained showed that the tested
compounds possess higher activities than natural antioxidants such as protocatechuic acid,
caffeic acid and gallic acid. Moreover, it was shown that a combination of two different and
independently acting fragments of well-known pharmacological profiles into one covalently
bonded hybrid molecule evoke a synergistic effect resulting in higher than expected activity.
To rationalize the apparent antioxidant activity and to establish the mechanism of action, a
SAR analysis and DFT quantum chemical computations were also performed.

Molecules 2015, 20
Keywords: homophthalic anhydride; ROS; antioxidants; phenolic acids; hybrid molecules;
2556
synergy
1. Introduction
Various Reactive Oxygen Species (ROS) such as superoxide radical anion (O2●▬), hydroxyl radical
(HO●) and hydrogen peroxide (H2O2) are essential cellular components, enzymatically generated in
aerobic living organisms, which play an important role in different physiological and pathological
processes. Particularly, at low levels, ROS take part in signal transduction, gene transcription and
regulation of soluble guanylate cyclase activity [1–3]. In contrast, the accumulation of excessive ROS,
mainly due to external influences such as radiation, ultraviolet light, cigarette smoke, pathogens, drugs,
etc., can inflict damage upon cellular macromolecules—DNA, proteins and lipids—thus contributing to
the development of various diseases, including diabetes mellitus, atherosclerosis, myocardial infarction,
ischemia, epilepsy, anemia and carcinogenesis, to name just a few [4–7]. Healthy organisms are able to
maintain the balance between the available reactive species and the antioxidant systems, but in some
pathological circumstances, the endogenous antioxidants are not enough to deal with the increased levels
of ROS [8,9]. Thus, in order for the level of excessive ROS to be reduced, and so the oxidative damage
can be suppressed, the need for additional intake of exogenous antioxidants can be suggested [10,11].
Indeed, various natural and synthetic phenolic antioxidants such as stilbenoids, coumarins, flavonoids
and carboxylic acids are part of our everyday diet, and it is nowadays truly believed that they are
responsible for the prevention of many diseases associated with the oxidative stress [12–19]. However,
the beneficial effect of these antioxidants seems to be conditioned by their limited distribution
throughout the body, mainly due to their low lipophilicity [20,21]. To overcome this, experiments aimed
at the synthesis of new antioxidants with improved properties from a pharmacological standpoint have
been performed. Particularly, Borges and co-workers showed that the antioxidant properties of natural
phenolic acids can be improved by introduction of functionalities which increase their lipophilicity [22–29].
Moreover, they have shown [30] that hybrid molecules consisting of gallic and cinnamic acid fragments
(see Figure 1) possess significantly improved absorption, distribution, metabolism, and excretion
(ADME) properties, which suggests their potential as drugs for the treatment of oxidative stress
associated diseases.
Recently we reported a convenient approach for the synthesis of polyhydroxy cis-restricted stilbenes,
structurally based on two independent fragments such as protocatechuic acid and cinnamic acid (Figure 1),
and demonstrated that the synthesized compounds possess a triple biological action as potent radical
scavengers, antifungal agents and tyrosinase inhibitors at micromolar concentrations [31]. Furthermore,
we showed that a combination of two different and independently acting fragments of well-known
pharmacological profiles into one covalently bonded hybrid molecule evokes a synergistic effect
resulting in higher than expected activity.

Molecules 2015, 20
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HOOC
HOOC
HOOC
HOOC
R3
R
R5
R4
R1
R2
R
R
R
R
gallic acid: R1 = R2 = R3 = OH
protocatechuic acid:
cinnamic acid: R4 = R5 = H
p-coumaric acid: R4 = OH; R5 = H
caffeic acid: R4 = R5 = OH
R = H or OH
R1 = R2 = OH; R3 = H
Target compounds
in this study
Figure 1. Structure of natural phenolic acids targeted as active fragments for the synthesis
of hybrid antioxidants.
In continuation of this preliminary study, herein we present the synthesis of a more complete series
of hybrid derivatives composed of natural phenolic acids. Their antioxidant capacity was evaluated by
means of in vitro screening tests against 1,1-diphenyl-2-picrylhydrazyl (DPPH●), hydroxyl (OH●) and
superoxide (O2●▬) radicals. The results obtained showed that most of the tested compounds are highly
effective in micromolar concentrations, possessing higher activities than the conventional reference
antioxidants. To rationalize the apparent antioxidant activity and to establish the mechanism of action,
DFT quantum chemical computations and SAR analysis, which take into account the number and position
of the hydroxyl groups in the carbon skeleton were also performed.
2. Results and Discussion
2.1. Chemistry
The compounds studied herein were obtained by means of a one-pot procedure for the synthesis of
polyhydroxylated cis-restricted stilbenes recently published by us [31]. In brief (see Scheme 1), the
method used consists of sequential reactions between homophthalic anhydrides 1 and aromatic
aldehydes 2 to produce diastereomeric mixtures of the corresponding methoxylated 3-aryl-3,4-dihydro-
isocoumarin-4-carboxylic acids [32,33], which, after treatment with BBr3, give the target
polyhydroxylated compounds 3a–p [31]. This way, a series of hybrid homologues consisting of benzoic,
protocatechuic, cinnamic, caffeic, p-coumarinc, or gallic acids fragments were obtained in short reaction
times (10–60 min).
Scheme 1. One-pot synthesis of polyhydroxylated trans-restricted 2-arylcinnamic acid.
Numbering in the general formula 3 is used for NMR spectra description.

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Table 1. Substitution patterns, number of hydroxyl groups, type of phenolic fragments, and antioxidant activity (EC50 ± SD) of compounds
3a–p and standard antioxidants.
Antioxidant Activity a
Type of Phenolic
Fragments in
Rings A/B
Total
OH
Comp.
R1
R2
R3
R4
R5
R6
DPPH●
TEC50 b (min)
nd c
O2 ▬ (µM)
HO● (µM)
●
EC50 (µM)
na c
3a
3b
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0
1
1
2
2
2
2
3
2
3
3
4
4
4
4
5
1
2
2
3
2
–/–
na
201.0 ± 8.0
133.7 ± 16.1
134.2 ± 5.6
120.9 ± 9.8
122.9 ± 2.0
108.9 ± 14.0
59.4 ± 0.4
100.6 ± 2.1
118.7 ± 2.6
79.1 ± 0.6
64.5 ± 0.8
68.9 ± 0.8
58.8 ± 1.1
84.6 ± 2.3
42.6 ± 1.8
58.6 ± 0.9
109.6 ± 7.8
73.0 ± 1.8
117.7 ± 1.8
146.9 ± 4.0
135.7 ± 13.1
OH
H
–/phenol
na
nd
na
3c
H
H
H
OH
OH
H
–/phenol
na
nd
na
3d
H
H
H
OH
OH
–/resorcinol
–/hydroQ c
41.30 ± 0.35
6.74 ± 0.30
6.26 ± 0.58
5.81 ± 0.29
4.47 ± 0.07
7.62 ± 0.28
8.04 ± 0.32
7.34 ± 0.25
5.43 ± 0.36
3.33 ± 0.04
3.52 ± 0.05
2.76 ± 0.12
2.09 ± 0.11
9.34 ± 0.07
9.48 ± 0.17
8.85 ± 0.24
5.32 ± 0.34
13.28 ± 0.54
32.25 ± 2
3.5 ± 0.1
4 ± 0.1
na
3e
H
H
OH
H
na
3f
H
H
H
OH OH
–/catechol
372.9 ± 8.5
109.7 ± 5.3
11.1 ± 0.1
233.6 ± 0.9
238.0 ± 5.4
230.7 ± 4.9
269.2 ± 12.8
nd
3g
H
H
H
OH
OH
H
OH
OH
H
H
H
–/catechol
2.75 ± 0.1
3 ± 0.1
3h
H
H
OH
H
–/pyrogallol
catechol/–
3i
OH
OH
OH
OH
OH
OH
OH
OH
–
OH
OH
OH
OH
OH
OH
OH
OH
–
H
31.75 ± 1.5
26 ± 0.75
33 ± 2
3j
H
H
H
OH
H
catechol/phenol
catechol/phenol
catechol/resorcinol
catechol/hydroQ c
catechol/catechol
catechol/catechol
catechol/pyrogallol
phenol
3k
H
OH
OH
H
H
3l
H
H
OH
OH
26.5 ± 0.75
10 ± 0.5
13 ± 0.5
6.5 ± 0.25
9 ± 0.75
5.75 ± 0.25
9 ± 0.75
28.25 ± 1.25
10 ± 0.5
10 ± 0.5
3m
3n
OH
H
H
H
OH OH
137.2 ± 3.9
64.9 ± 1.9
11.3 ± 0.5
na
3o
H
OH
OH
–
OH
OH
–
H
H
–
3p
OH
–
Trolox
CA
PCA
GA
CA:PCA
–
–
H
OH
–
OH
–
H
–
catechol
126 ± 10.6
233.5 ± 3.0
29.1 ± 1.0
143.9 ± 5.7
OH
–
OH
–
–
catechol
–
–
–
–
pyrogallol
–
–
–
–
–
–
catechol + catechol
a
Results are presented as a mean value of three measurements and the corresponding p-values of all possible pairs of compounds are given as supporting information
(Tables S1–S3); b TEC50—the time required for each compound to reach equilibrium at concentration equal to EC50; c “na” refers to “not active”, “nd” refers to “not determined”,
“–” refers to “not applicable”, and “hydroQ” refers to hydroquinone.

Molecules 2015, 20
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All compounds 3a–p (whose substitution patterns are given in Table 1) were isolated in pure form
after flash-chromatography purification and their structures were elucidated by means of spectral
13
methods (see Section 3.2). In the case of 1H-NMR, C-NMR and IR methods, the spectra were taken
for the synthesized compounds, while their mass spectra were obtained after derivatization with ethereal
diazomethane solution prior to analysis. It is noteworthy that when ortho-methoxy-benzaldehydes were
used as starting materials, the compounds of type 3 were obtained in low yields (ca. 14%–32%) due to
their tendency to undergo a subsequent rearrangement leading to coumarins as main products [34].
Nevertheless, the fast precipitation of the residue after the purification step resulted in the isolation of
these compounds in pure form, which allowed us to assess the influence of the ortho-phenolic group in
the cinnamic acid fragment on the antioxidant activity. In order to elucidate the configuration of the
herein synthesized hybrid molecules an approach based on the heteronuclear coupling constants was
applied, since it is known that the vicinal heteronuclear coupling constants exhibit a Karplus type
relationship between their value and the dihedral angle between the interacting nuclei [35]. This
approach has already proved successful and accurate for configurational studies of similar compounds,
even when the diastereomers with the opposite configuration are not available [31,33]. The results
obtained proved that the incorporated trans-cinnamic acid fragment into the target compounds structure
ensures cis-configuration of the stilbene backbone and prevents further isomerization along
the carbon–carbon double bond. For more detailed discussion on the configuration and conformation of
compounds 3a–p, see Section 2.3.
2.2. In Vitro Antioxidant Capacity Assays
The antioxidant capacity of the hybrid molecules 3a–p was measured against 1,1-diphenyl-2-
picrylhydrazyl (DPPH●), hydroxyl (HO●) and superoxide (O2●▬) radicals. The analyses were performed
as described in the Experimental Section and the activities were expressed as EC50 ± SD (µM) values.
The averages were statistically compared and this was taken into account throughout the following
discussion. A level of confidence higher than 95% (p < 0.05) was considered as statistically significant.
The individual p-values of each pair of compounds are given as supporting information (Tables S1–S3).
For a comparison purpose, well-known antioxidants such as Trolox, protocatechuic acid (PCA), caffeic
acid (CA) and gallic acid (GA) were used as reference compounds. The results obtained are presented
in Table 1, and for the sake of simplicity, the aromatic rings of the benzoic acid and cinnamic acid
fragments are respectively named as ring A and ring B.
2.2.1. DPPH● Radical Scavenging Assay
The DPPH●-scavenging assay has been successfully applied to evaluate the activity of diverse natural
and synthetic antioxidants [36–39], it is easy to perform, highly reproducible and therefore, this method
was initially employed to assess the radical scavenging activity of the hybrid molecules synthesized by
us. The following observed order: 3d < CA ≈ Trolox < PCA < 3j < 3i ≈ 3k < 3e ≈ 3f < 3g < 3l ≈ GA <
3h < 3n < 3m < 3o < 3p suggests, that except for 3a–c which showed no activity, the group of compounds
tested demonstrated comparable or even higher radical scavenging activity than the standards used. As
it can be seen from Table 1, 3a–c, which possess no or a single OH group on ring B, are not able to
scavenge DPPH radicals, the latter confirming the importance of the number of the OH groups for the

Molecules 2015, 20
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antioxidant activity. Thus, despite it is the least active compound in the series, compound 3d showed an
EC50 = 41.3, and this can be attributed to the presence of only two meta-orientated OH groups (a
resorcinol-like fragment) in ring B. Compounds 3e–g, each of them possessing two OH groups capable of
forming ortho- or para-quinone structures in the same fragment, showed slightly higher activity
(EC50 = 5.81–6.74) than Trolox (EC50 = 9.34), PCA (EC50 = 8.85) and CA (EC50 = 9.48). Similarly, 3i–k,
possessing two or three OH groups, showed EC50 = 7.34–8.04, and this can be attributed to the presence
of the same catechol moiety in ring A, which seems to be responsible for the apparent activity.
Compound 3h contains a pyrogallol fragment (three neighboring OH groups) and 3l combines cathechol
and resorcinol fragments (2 × 2 OH groups), which suggests an increased radical scavenging ability.
Indeed, their EC50 values (4.47 and 5.43 for 3h and 3l, respectively) were found close to that of GA
(EC50 = 5.32), and thus, the distribution of the OH groups in the basic carbon backbone can be considered
of an immense importance in modulating the radical scavenging activity. Considering compounds 3a–h,
which differ only by the number of OH groups present in ring B, it can be seen that the presence of a
single OH group either at the 2 or 4 position in ring B (3b and 3c, respectively) did not affect the activity
of 3a, but the addition of a second or a third OH group did do so, significantly in some cases, and thus
compounds 3e–h possess one order of magnitude lower EC50 values compared to 3d. Unlike compounds
3е–3l, which have only one structural fragment capable of scavenging free radicals effectively, 3m–p
possess another one in addition to the catechol fragment in ring A. This way, they are the most active
compounds in the series with EC50 = 3.33, 3.52, 2.76 and 2.09, respectively. The latter suggests a
synergistic effect resulting from the combination of two active fragments in one hybrid molecule, which
can be rationalized by a comparison of the activity of 3o to that of CA, PCA and their equimolar mixture.
As it can be seen, the whole is better than the parts, since the hybrid molecule possesses a three- to
fourfold lower EC50 value due to its facilitated hydrogen transfer and better radical stabilization.
2.2.2. O2●▬ Radical Anion Scavenging Activity
Superoxide anion radical is one of the most dangerous ROS. It is the first free radical generated after
oxygen uptake in living cells, and it can undergo further enzyme-catalyzed transformations into other
harmful ROS such as hydrogen peroxide and hydroxyl radical [40]. In the present study, the radical
scavenging activity of compounds 3a–p toward O2●▬ was estimated by means of a method proposed by
Nishikimi et al. [41], but with slight modifications. According to this method, the superoxide anion
radical, formed from an oxygen molecule by the phenazine methosulfate (PMS)/nicotinamide adenine
dinucleotide (NADH) system, reduces nitroblue tetrazolium (NBT) to a blue colored formazan, which
can be quantified spectrophotometrically at 560 nm. A short retrospection of the results presented in
Table 1, indicates that the ability of a particular molecule to scavenge O2●▬ is simultaneously dependent
on the number and distribution of the OH groups into the basic carbon backbone. As it can be seen,
Trolox and compounds 3a–e, possessing no, one or two OH groups in ring B, show no activity. The
latter suggests that the presence of “isolated” OH groups, regardless their number, is not enough to evoke
activity and this can be rationalized by consideration of the results for compounds 3d–g. Although each
of them possesses two OH groups in ring B, 3d and 3e are not active, whereas 3f and 3g exhibit EC50 = 373.0
and 109.7, respectively. Consequently, the presence of a catechol fragment can be considered necessary
for superoxide anion radical scavenging activity. Moreover, it seems that the position of the catechol

Molecules 2015, 20
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fragment is also important, since 3g and CA (possessing similar substitution patterns in ring B), are
nearly fourfold more active than 3f. Further, PCA and compounds 3i–l, each of them possessing the
same catechol moiety into ring A, showed similar EC50 values and so the increasing number of “isolated”
OH groups in the cinnamic part again does not alter the apparent activity, until an additional catechol
fragment does not appear in ring B. This way, the combination of two active fragments in 3n and 3o
leads to a significant activity increase in comparison with that demonstrated by the individual parts or
their equimolar mixture. Another interesting result deserves additional attention, namely, that the highest
activity was demonstrated by compounds 3h and 3p, both of them containing pyrogallol fragments in
ring B. As it can be seen, the two compounds show similar to each other and threefold lower EC50 values
than that demonstrated by the strongest standard antioxidant—gallic acid—and this can be attributed to
the presence of an extended π-conjugated system providing better delocalization. The latter also suggests
that in case of several fragments capable of scavenging free radicals, the apparent activity can be
dominated by those fragments which better stabilize the radicals formed. In sum, the catechol and
pyrogallol fragments, as well as an extended π-conjugated system, can be considered as fragments of an
immense importance when one intends to synthesize effective antioxidants capable of scavenging
superoxide anion radicals.
2.2.3. HO● Radical Scavenging Activity
Hydroxyl radical (HO●) can be considered as a highly aggressive ROS, since it is able to cause
oxidative damage to almost any biological molecule, including proteins, DNA, nucleic acid, etc. [42].
One of the most applied in vitro methods for determination of the scavenging activity of a substance
towards this species is based on the spectrophotometric measurement at 440 nm of the level of radical-
induced oxidation of N,N-dimethyl-4-nitrosoaniline to p-nitrodimethylaniline, while a constant flux of
HO● is generated by a Fe3+/EDTA/H2O2/ascorbic acid system [43]. In contrast to the results obtained by
the other two methods employed in the present study, the scavenging activities of compounds 3a–p
towards HO● seem to be determined by the basic carbon backbone. As it can be seen, even compound
3a (possessing no OH groups) showed satisfactory potential and this can be attributed to the ability of
the carbon-carbon double bond to participate in reactions with HO●. Moreover, the introduction of a
single or two, but “isolated”, phenolic groups, increases the activity of 3a, and thus 3b–e demonstrated
●▬
nearly twofold lower EC50 values comparable with those of PCA and GA. However, as in case of O2
,
the catechol fragment can be considered important, since the introduction of such a fragment additionally
increased the activity of 3f and 3g. Similarly, the series of compounds follows the tendency for enhanced
activity due to the presence of an extended π-conjugated system, and this can be rationalized by a
comparison of the EC50 values of 3g and 3i, which resemble that of CA and PCA, respectively.
Interestingly, the inclusion of a third OH group in ring B (pyrogallol fragment) in 3h lowers its activity
in comparison to 3g, but the same behavior is also observed for the parent standards PCA and GA.
Consequently, the introduction of a pyrogallol fragment is not necessary, since the activity of 3h is
slightly lower than that of 3b–e, which possess reduced numbers of OH groups in ring B. The latter
observation is noteworthy, since phenolic compounds with a single hydroxyl groups are less susceptible
to oxidation reactions, and this advantage can be considered of immense importance from an industrial
standpoint. Regarding compounds 3i–p, each of them possessing the same catechol moiety in ring A and

Molecules 2015, 20
2562
continuously increasing number of OH groups in ring B, it can be said that they obey the same tendency
as 3a–g, the latter underlining the importance of the catechol fragment to evoke antioxidant activity
towards the HO●.
2.3. Quantum Chemistry Computations
Three radical scavenging mechanisms stand out amongst the others proposed in the literature (see
Figure 2). Two of them include hydrogen atom transfer (1)—the HAT-mechanism, and single-electron
transfer (2)—the SET-PT-mechanism [44–48]. The HAT mechanism is based on homolytic dissociation
of the O–H bond in phenolic compounds, and SET mechanism takes place in two steps—ionization,
followed by a proton transfer. The third mechanism (SPL-ET) also proceeds in two steps [49,50], which
include deprotonation at the first step followed by sequential electron transfer in the second.
H
PT-mechanism
H is PDE
SET-mechanism
ArOH
R
Δ
ΔH is IP
2)
1)
3)
-e-
R
R
ArO
R-H
ArOH
HAT-mechanism; H is BDE
Δ
H
H
ET-mechanism
SPL-mechanism
R
ArO
ΔH is ETE
ΔH is PA
e-
Figure 2. Probable reaction mechanisms of radical scavenging by phenolic compounds.
In many cases two or more mechanisms can operate simultaneously during the antioxidant action,
and, since the reaction path (1) is related to the bond dissociation energy—BDE; (2) to the ionization
potential—IP, and proton dissociation enthalpy—PDE; and (3) to the proton affinity—PA, and electron
transfer enthalpy—ETE, the calculation and comparison of these parameters can help distinguish the
most probable reaction mechanism [51,52]. Therefore, in order to explain the apparent antioxidant
activity and to assess the influence of the substitution pattern, the geometry and physical descriptors of
compounds 3i, 3k, 3o and 3p were determined by means of DFT quantum chemical computations at the
IEF-PCM (U)B3LYP/6-31+G(d) and (U)B3LYP/6-311++G(d,p) levels of theory. The computed data
for all possible conformers of the corresponding molecule, radical, cation-radical and anion-radical
species of the compounds of interest was found consistent with that experimentally obtained for a similar
compound in a solid state [33], and showed that the carbon backbone is not planar and is composed of
two independent fragments, e.g., protocatechuic and cinnamic acid fragments (ring A and ring B,
respectively). The dihedral angle between the planes of these two parts (φ1, Figure 3) is in the range of
73–75°, the latter suggesting an absence of conjugation between them, and the aromatic ring in the
cinnamic acid fragment is out of the plane with ca. 15–18° (φ2, Figure 3). Furthermore, it was found that
the most stable species possess hydroxyl groups coplanar to the aromatic rings, orientated to each other
in a way to form as much as possible intramolecular hydrogen bonds, and that the conversion of the

Molecules 2015, 20
2563
molecule species into radicals, radical-cations and radical-anions does not lead to significant changes in
the preferred conformations.
H
H
O
H
O
H
O
H
H
O
H
O
H
O
383.8
~375
384.1
398.6
~375
~375
~375
385.8
O
O
O
O
O
O
O
O
O
O
C
C
C
C
C
C
C
C
ϕ1
ϕ1
ϕ1
ϕ1
ϕ2
ϕ2
ϕ2
ϕ2
312.6
311.9
312.9
312.2
H
O
H
O
H
O
H
O
318.4
318.3
H
318.2
318.3
O
H
O
O
H
O
H
311.4
H
O
O
O
278.8
H
295.1
H
322.3
3k H
309.6
O
304.7
O
O
H
H
3i
3o
3p
Figure 3. BDEs of compounds 3i, 3k, 3o and 3p for a homolytic dissociation of different
bonds. Calculations are performed at IEF-PCM (U)B3LYP/6-31+G(d) level of theory.
The computed enthalpies for mechanisms (1)–(3), i.e., BDE; IP; PDE; PA; ETE, are listed in
Table 2. As it can be seen the BDE values are two- to threefold lower than the IP and PA, which suggests
that the HAT mechanism is favored for the compounds under study and will always predominate over
SET-PT and SPL-ET. As a rule of thumb, the HAT mechanism supposes a homolytic dissociation of the
O–H bond, and more rarely, of a N–H and C–H bond. Therefore, when multiple groups are capable of
participating in a reaction, the probability of a particular group to be “chosen” by the attacking radical
can be assessed by a comparison of the corresponding BDEs. According to the values depicted in Figure 3,
the BDEs of phenolic O–H groups, carboxylic O–H groups and C–H bonds are in the 278–323, 370–399,
and 460–470 kJ·mol−1 range, respectively. Consequently, it can be concluded that the phenolic O–H
groups are preferably attacked by selective radicals such as DPPH (BDE = 348 kJ·mol−1).
Table 2. IEF-PCM B3LYP/6-311++G** computed enthalpies (kJ·mol−1) of reaction
mechanisms (1)–(3).
Compound BDE a IP b PDE c PA d ETE e
3i
3k
3o
3p
324.4 826.2 169.0 759.2 684.8
323.7 803.8 192.5 760.3 683.1
306.4 796.1 189.2 749.3 676.6
290.2 795.2 118.3 677.4 732.3
a Bond Dissociation Energy; b Ionization Potential; c Proton Dissociation Enthalpy; d Proton Affinity; e Electron
Transfer Enthalpy.
It is noteworthy that the influence of the substitution pattern of the target molecule is also of immense
importance for the stability of the radicals produced, since the presence of groups capable of enhancing
the resonance stabilization will facilitate the homolytic dissociation. Thus, due to the absence of phenolic
groups in the cinnamic fragment in compound 3i, participation in the reaction of the protocatechuic
fragment, and more precisely the OH group meta-orientated toward the carboxylic function, will be
preferred. This way, 3i showed similar antioxidant activity compared to that of PCA. Similarly, the
addition of a single OH group at 4 position in the cinnamic fragment in compound 3k did not
significantly affect the activity and this can be attributed to the absence of a neighboring group assuming
additional stabilization. The importance of the latter can be rationalized by taking into account the BDEs

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of OH group at 4 position in the cinnamic fragment in compounds 3o and 3p, where the presence of one
or two groups capable of stabilizing the resulting radical lowers the corresponding enthalpies, and hence
facilitates the reaction to take place at this position. Based on the above arguments, it can be concluded
that the major factor responsible for the apparent antioxidant activity of the compounds under study is
the presence of at least one catechol fragment, regardless of which aromatic ring it is, and that in case
where multiple centers are able to react, the presence of an extended π-conjugated system providing
better delocalization, and thus stabilization of the radicals formed, will determine the preferred group to
be attacked. This conclusion can also be supported by calculation of the spin densities, which show the
probability a single electron to be found around given atom or atom groups. Moreover, since spin
densities show the single electron distribution through the basic structure, they can serve as descriptors
of the received radicals’ stability.
To obtain these parameters for the radicals of compounds 3i, 3k, 3o and 3p we employed two
approaches, namely Mulliken population analysis and Natural population analysis, and the results
obtained are listed in Table 3. A short analysis of the data shows that the electron density in all cases is
not concentrated only in the radical centre, but it is rather distributed either to the protocatechuic or to
the cinnamic acid fragments, which in turn confirms the important role of these two fragments to evoke
high antioxidant activities.
Table 3. Distribution of spin densities into radicals of compounds 3i, 3k, 3o and 3p.
Mulliken Spin Density
Natural Spin Density
Compound
Radical Protocatechuic Cinnamic
Radical Protocatechuic Cinnamic
Centre
0.359
0.337
0.268
0.287
Fragment
0.602
Fragment
0.039
Centre
0.284
0.269
0.198
0.218
Fragment
0.609
Fragment
0.107
3i
3k
3o
3p
0.608
0.055
0.666
0.065
0.011
0.721
0.020
0.782
0.038
0.675
0.021
0.761
3. Experimental Section
3.1. General Remarks
All chemicals used in this study were purchased from Sigma-Aldrich (FOT, Sofia, Bulgaria). The
organic solvents were of analytical grade. Melting points were determined on a Kofler microscope
Boetius PHMK 0.5 (VEB Kombinat Nagema, Radebeul, Germany). All reactions were monitored by
means of thin layer chromatography (TLC) on pre-coated polyesters sheets POLIGRAM® SIL G/UV254
(Merck, Darmstadt, Germany) and the spots were visualized with UV light. The column chromatography
was performed on Horizon High Performance FLASH chromatography system (HPFC) with cartridges
filled with Silica gel 60 (230–400 mesh, MACHEREY-NAGEL, Düren, Germany). The IR analyses
were carried out on Cary 630 FTIR spectrophotometer (Agilent Technologies Inc., Santa Clara, CA,
USA) equipped with diamond ATR-1 bounce and are reported in reciprocal centimeters. NMR spectra
were recorded on а Bruker Avance II+ (600 MHz and 151 MHz for 1H and 13C, respectively) and Bruker
Avance III HD (500 MHz and 126 MHz for 1H and 13C, respectively) using DMSO-d6 as а solvent and
TMS as an internal standard. The chemicals shifts (δ) are given in ppm and J values are reported in Hz.

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Mass spectra were taken on Agilent 6890 series GC fitted with a HP-5MS capillary column (5%
phenylmethylsiloxane, 30m × 250 μm × 0.25 μm, TEAM, Sofia, Bulgaria) with helium carrier gas
(constant flow rate: 0.8 mL/min) coupled with an Agilent 5973 mass selective detector. Temperature
programme: T(inlet): 250 °C, Oven: 150 °C (1 min), 150–300 °C (10 °C/min), 300 °C (5 min). Mass
spectra: electron impact (EI+) mode (70 eV). In case of 1H-NMR, 13C-NMR and IR methods, the spectra
were taken for the synthesized compounds, while their mass spectra were obtained after derivatization
with diazomethane etherealsolution prior to analysis (see Section 3.2.2). The radical scavenging and
antioxidant activity assays were performed on Thermo Evolution 60S UV/Vis spectrophotometer
(ACM2, Sofia, Bulgaria).
3.2. Chemistry
3.2.1. General Procedure for One-Pot Synthesis of Polyhydroxy (E)-2-(1-Carboxy-2-phenylvinyl)-benzoic
Acids 3a–p
An equimolar mixture of homophtalic anhydride (1a) or 6,7-dimethoxyhomophthalic anhydride (1b),
aldehyde 2a–h and 4-dimethylaminopyridine (DMAP) in dry dichloromethane (10 mL) was stirred at
room temperature while the consumption of the reagents was monitored by TLC. At the end of the
reaction (ca. 10 min), boron tribromide (1.6 M solution in dichloromethane) was slowly added (one
equivalent per heteroatom) and the reaction mixture was stirred additionally for 10–60 min. At the end
of the reaction, the mixture was poured over ice, stirred for 10 min and acidified with conc. HCl (pH = 1).
The aqueous layer was then saturated with NaCl and the products were extracted with ethyl acetate. The
organic phase was washed with brine to a constant pH, dried over anhydrous Na2SO4 and the solvent
was evaporated under reduced pressure. The residue was further purified by column chromatography
(mobile phase: acetone/cyclohexane/formic acid—1/1/0.01) and the products 3a–p were isolated after
crystallization from appropriate mixture of solvents. Compounds 3a, 3i, 3k, 3o and 3p were available
from our previous works [31,33].
(E)-2-(1-Carboxy-2-(2-hydroxyphenyl)vinyl)benzoic Acid (3b)
Oil (0.32 g, 32%) after HPFC from the reaction of homophthalic anhydride (1a, 0.57 g, 3.52 mmol),
2-methoxybenzaldehyde (2b, 0.48 g, 3.52 mmol), 4-dimethylaminopyridine (0.43 g, 3.52 mmol) and
boron tribromide (17.6 mL, 28.14 mmol) in dichloromethane (10 mL). Pale yellow crystals (0.18 g, 56%)
after crystallization from 1:2 dichloromethane-acetone. M.p. = 180–182 °C; IR (ATR diamond, cm−1):
1
ν = 3480 (OH), 3300–2250 (ОH, COOH), 1675 (C=O, COOH), 1630 (C=C); H-NMR (500 MHz,
DMSO-d6): δ = 12.54 (bs, 2H, 2 × COOH), 9.96 (s, 1H, OH), 7.98 (d, 1H, J = 8.8 Hz, H8), 7.91 (s, 1H),
7.46–7.39 (m, 2H), 6.99 (dd, 2H, J = 8.1, 6.4 Hz), 6.83 (d, 1H, J = 8.1 Hz), 6.44–6.33 (m, 2H); 13C-NMR
(126 MHz, DMSO-d6): δ = 168.1 (C1a), 167.7 (C7), 156.6 (C), 138.6 (C), 133.2 (C), 132.2 (=CH–),
131.7 (=CH–), 131.5 (C), 131.2 (=CH–), 130.3 (=CH–), 130.0 (=CH–), 129.6 (=CH–), 127.7 (=CH–),
121.7 (C), 118.5 (=CH–), 115.6 (=CH–); EIMS: m/z (%) = 326 (54) [M]+, 295 (100), 267 (43), 234 (44),
220 (41), 206 (26), 178 (66), 165 (61), 152 (42), 133 (35), 119 (21), 91 (21).

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(E)-2-(1-Carboxy-2-(4-hydroxyphenyl)vinyl)benzoic Acid (3c)
Oil (0.89 g, 89%) after HPFC from the reaction of homophthalic anhydride (1a, 0.57 g, 3.52 mmol),
4-methoxybenzaldehyde (2c, 0.43 mL, 3.52 mmol), 4-dimethylaminopyridine (0.43 g, 3.52 mmol) and
boron tribromide (17.6 mL, 28.14 mmol) in dichloromethane (10 mL). Pale yellow crystals (0.46 g,
52%) after crystallization from 1:5 dichloromethane-ethyl. M.p. = 166–167 °C; IR (ATR diamond, cm−1):
1
ν = 3625 (OH), 3400–2200 (ОH, COOH), 1650 (C=O, COOH); H-NMR (600 MHz, DMSO-d6):
δ = 12.48 (bs, 2H, 2 × COOH), 9.79 (s, 1H, OH), 8.01 (dd, 1H, J = 7.6, 1.1 Hz, H6), 7.54 (s, 1H, H8),
7.51 (td, 1H, J = 7.5, 1.5 Hz, H4), 7.47 (td, 1H, J = 7.5, 1.5 Hz, H5), 7.08 (dd, 1H, J = 7.6, 1.1 Hz, H3), 6.79
(d, 2H, J = 8.7 Hz, H2' and H6'), 6.55 (d, 2H, J = 8.7 Hz, H3' and H5'); 13C-NMR (151 MHz, DMSO-d6): δ
= 168.2 (C1a), 167.6 (C7), 158.2 (C), 138.7 (C), 136.6 (C), 132.4 (=CH–), 132.0 (2 × =CH–), 131.3 (C),
131.2 (=CH–), 131.0 (C), 130.5 (=CH–), 127.8 (=CH–), 125.6 (=CH–), 115.3 (2 × =CH–); EIMS: m/z
(%) = 326 (96) [M]+, 267 (63), 251 (33), 235 (100), 181 (20), 165 (45), 151 (48).
(E)-2-(1-Carboxy-2-(2,4-dihydroxyphenyl)vinyl)benzoic Acid (3d)
Oil (0.23 g, 23%) after HPFC from the reaction of homophthalic anhydride (1a, 0.54 g, 3.33 mmol),
2,4-dimethoxybenzaldehyde (2d, 0.55 g, 3.33 mmol), 4-dimethylaminopyridine (0.41 g, 3.33 mmol) and
boron tribromide (18.7 mL, 29.97 mmol) in dichloromethane (10 mL). Pale yellow crystals (0.18 g, 80%)
after crystallization from 1:2 dichloromethane-acetone. M.p. = 175–177 °C; IR (ATR diamond, cm−1):
1
ν = 3500 (OH), 3300–2250 (ОH, COOH), 1675 (C=O, COOH), 1625 (C=C); H-NMR (500 MHz,
DMSO-d6): δ = 12.35 (bs, 2H, 2 × COOH), 9.87 (s, 1H, OH), 9.58 (s, 1H, OH), 7.96 (dd, 1H, J = 7.7,
1.4 Hz, H6), 7.88 (s, 1H, H8), 7.46 (td, 1H, J = 7.5, 1.5 Hz, H4), 7.41 (td, 1H, J = 7.5, 1.5 Hz, H5), 7.03
(dd, 1H, J = 7.5, 1.2 Hz, H3), 6.29 (d, 1H, J = 2.4 Hz, H3'), 6.14 (d, 1H, J = 8.7 Hz, H6'), 5.81 (dd, 1H,
J = 8.7, 2.4 Hz, H5'); 13C-NMR (126 MHz, DMSO-d6): δ = 168.4 (C1a), 167.8 (C7), 159.4 (C), 158.3
(C), 139.2 (C), 132.2 (=CH–), 131.7 (=CH–), 131.5 (C), 131.3 (=CH–), 130.5 (=CH–), 130.3 (=CH–),
129.2 (C), 127.4 (=CH–), 113.0 (C), 106.7 (=CH–), 102.1 (=CH–); EIMS: m/z (%) = 356 (100) [M]+,
325 (45), 297 (29), 265 (21), 250 (30), 235 (26), 181 (36), 165 (40), 152 (34), 133 (20).
(E)-2-(1-Carboxy-2-(2,5-dihydroxyphenyl)vinyl)benzoic Acid (3e)
Orange solid foam (0.14 g, 14%) after HPFC from the reaction of homophthalic anhydride (1a, 0.54 g,
3.33 mmol), 2,5-dimethoxybenzaldehyde (2e, 0.55 g, 3.33 mmol), 4-dimethylaminopyridine (0.41 g,
3.33 mmol) and boron tribromide (18.7 mL, 29.97 mmol) in dichloromethane (10 mL). M.p. = 108–110 °C;
IR (ATR diamond, cm−1): ν = 3480 (OH), 3300–2250 (ОH, COOH), 1675 (C=O, COOH), 1630 (C=C);
1H-NMR (500 MHz, DMSO-d6): δ = 12.50 (s, 2H, 2 × COOH), 9.23 (s, 1H, OH), 8.44 (s, 1H, OH), 7.99
(dd, 1H, J = 7.4, 1.6 Hz, H6), 7.85 (s, 1H, H8), 7.44 (td, 2H, J = 7.4, 1.6 Hz, H4 and H5), 6.98 (dd, 1H,
J = 7.4, 1.6 Hz, H3), 6.63 (d, 1H, J = 8.7 Hz, H3'), 6.46 (dd, 1H, J = 8.7, 2.9 Hz, H4'), 5.83 (d, 1H,
J = 2.9 Hz, H6'); 13C-NMR (126 MHz, DMSO-d6): δ = 168.2 (C1a), 167.7 (C7), 149.4 (C), 149.0 (C),
138.6 (C), 132.9 (C), 132.2 (=CH–), 131.9 (=CH–), 131.4 (C), 131.2 (=CH–), 130.3 (=CH–), 127.6
(=CH–), 122.1 (C), 117.3 (=CH–), 116.0 (=CH–), 115.7 (=CH–); EIMS: m/z (%) = 356 (79) [M]+, 325
(100), 297 (24), 265 (22), 250 (47), 207 (52), 179 (22), 165 (40), 152 (45), 139 (24), 59 (25).

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(E)-2-(1-Carboxy-2-(2,3-dihydroxyphenyl)vinyl)benzoic Acid (3f)
Brown solid foam (0.20 g, 20%) after HPFC from the reaction of homophthalic anhydride (1a, 0.54 g,
3.33 mmol), 2,3-dimethoxybenzaldehyde (2f, 0.55 g, 3.33 mmol), 4-dimethylaminopyridine (0.41 g,
3.33 mmol) and boron tribromide (18.7 mL, 29.97 mmol) in dichloromethane (10 mL). M.p. = 137–138 °C;
1
IR (ATR diamond, cm−1): ν = 3630 (OH), 3500–2200 (ОH, COOH), 1670 (C=O, COOH); H-NMR
(500 MHz, DMSO-d6): δ = 12.52 (bs, 2H, 2 × COOH), 9.47 (s, 1H, OH), 8.85 (s, 1H, OH), 7.97 (dd,
1H, J = 6.8, 2.3 Hz, H6), 7.93 (s, 1H, H8), 7.46–7.38 (m, 2H, H4 and H5), 6.99 (dd, 1H, J = 6.7 Hz, H3),
6.61 (dd, 1H, J = 7.8, 1.4 Hz, H6'), 6.22 (t, 1H, J = 7.9 Hz, H5'), 5.84 (dd, 1H, J = 8.0, 1.1 Hz, H4');
13C-NMR (126 MHz, DMSO-d6): δ = 168.1 (C1a), 167.7 (C7), 145.2 (C), 145.1 (C), 133.0 (C), 132.1
(=CH–), 132.0 (=CH–), 131.4 (C), 131.3 (=CH–), 130.2 (=CH–), 127.6 (=CH–), 122.5 (C), 120.0 (=CH–),
118.2 (=CH–), 115.3 (=CH–); EIMS: m/z (%) = 356 (23) [M]+, 325 (100), 265 (25), 165 (21).
(E)-2-(1-Carboxy-2-(3,4-dihydroxyphenyl)vinyl)benzoic Acid (3g)
Oil (0.51 g, 51%) after HPFC from the reaction of homophthalic anhydride (1a, 0.54 g, 3.33 mmol),
3,4-dimethoxybenzaldehyde (2g, 0.55 g, 3.33 mmol), 4-dimethylaminopyridine (0.41 g, 3.33 mmol) and
boron tribromide (18.7 mL, 29.97 mmol) in dichloromethane (10 mL). Beige crystals (0.35 g, 68%) after
crystallization from 1:2 petroleum ether-ethyl acetate. M.p. = 192–194 °C; IR (ATR diamond, cm−1):
1
ν = 3575 (OH), 3500–2250 (ОH, COOH), 1670 (C=O, COOH), 1610 (C=C); H-NMR (600 MHz,
DMSO-d6): δ = 12.45 (bs, 2H, 2 × COOH), 9.34 (s, 1H, OH), 8.81 (s, 1H, OH), 8.01 (dd, 1H, J = 7.5,
1.3 Hz, H6), 7.51 (td, 1H, J = 7.5, 1.3 Hz, H4), 7.46 (td, 1H, J = 7.5, 1.3 Hz, H5), 7.45 (s, 1H, H8), 7.07
(dd, 1H, J = 7.5, 1.3 Hz, H3), 6.53 (d, 1H, J = 8.8 Hz, H5'), 6.33 (td, 2H, J = 4.3, 1.9 Hz, H2' and H6');
13C-NMR (151 MHz, DMSO-d6): δ = 168.2 (C1a), 167.6 (C7), 146.7 (C), 144.8 (C), 138.8 (C), 137.0
(C), 132.4 (=CH–), 131.3 (=CH–) 131.2 (C), 130.9 (C), 130.5 (=CH–), 127.8 (=CH–), 126.1 (=CH–),
122.9 (=CH–), 117.5 (=CH–), 115.3 (=CH–); EIMS: m/z (%) = 356 (99) [M]+, 297 (30), 265 (100), 238
(28), 221 (20), 181 (37), 165 (35), 152 (35), 139 (20).
(E)-2-(1-Carboxy-2-(3,4,5-trihydroxyphenyl)vinyl)benzoic Acid (3h)
Oil (0.74 g, 74%) after HPFC from the reaction of homophthalic anhydride (1a, 0.51 g, 3.16 mmol),
3,4,5-trimethoxybenzaldehyde (2h, 0.62 g, 3.16 mmol), 4-dimethylaminopyridine (0.39 g, 3.16 mmol)
and boron tribromide (19.8 mL, 31.62 mmol) in dichloromethane (10 mL). Yellow crystals (0.27 g, 36%)
after crystallization from 1:5 dichloromethane-ethyl acetate. M.p. = 158–160 °C; IR (ATR diamond,
cm−1): ν = 3640 (ОH), 3580–2180 (ОH, COOH), 1665 (C=O, COOH), 1630 (C=C); 1H-NMR (600 MHz,
DMSO-d6): δ = 12.45 (s, 2H, 2 × COOH), 8.79 (s, 2H, 2 × OH), 8.54 (s, 1H, OH), 8.00 (dd, 1H, J = 7.6,
1.3 Hz, H6), 7.49 (td, 1H, J = 7.6, 1.3 Hz, H4), 7.45 (td, 1H, J = 7.6, 1.3 Hz, H5), 7.35 (s, 1H, H8), 7.05
13
(dd, 1H, J = 7.6, 1.3 Hz, H3), 5.94 (s, 2H, H2' and H6'); C-NMR (151 MHz, DMSO-d6): δ = 168.3
(C1а), 167.7 (C7), 145.5 (3 × C), 138.7 (C), 137.3 (C), 134.8 (=CH–), 132.3 (C), 131.3 (=CH–), 130.5
(=CH–), 127.7 (C), 125.0 (=CH–), 109.9 (3 × =CH–); EIMS: m/z (%) = 386 (100) [M]+, 311 (43), 295
(35), 183 (20), 139 (24).

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(E)-2-(1-Carboxy-2-(2-hydroxyphenyl)vinyl)-4,5-dihydroxybenzoic Acid (3j)
Оil (0.23 g, 23%) after HPFC from the reaction of 6,7-dimethoxyhomophthalic anhydride (1b, 0.70 g,
3.16 mmol), 2-methoxybenzaldehyde (2b, 0.43 g, 3.16 mmol), 4-dimethylaminopyridine (0.39 g,
3.16 mmol) and boron tribromide (19.8 mL, 31.62 mmol) in dichloromethane (10 mL). Yellow crystals
(0.18 g, 77%) after crystallization from 1:2 dichloromethane-acetone. M.p. = 179–180 °C; IR (ATR
diamond, cm−1): ν = 3670–2160 (ОH, COOH), 1656 (C=O, COOH), 1620 (C=C); 1H-NMR (600 MHz,
DMSO-d6): δ = 11.94 (bs, 2H, 2 × COOH), 9.69 (s, 1H, OH), 9.37 (s, 1H, OH), 9.08 (s, 1H, OH), 7.80
(s, 1H, H8), 7.43 (s, 1H, H6), 7.00 (t, 1H, J = 7.5 Hz, H4'), 6.82 (d, 1H, J = 7.7 Hz, H6'), 6.53 (d, 1H,
13
J = 7.7 Hz, H3'), 6.46 (t, 1H, J = 7.5 Hz, H5'), 6.33 (s, 1H, H3); C-NMR (151 MHz, DMSO-d6):
δ = 168.3 (C1a), 167.1 (C7), 156.3 (C), 149.0 (C), 144.1 (C), 133.5 (C), 131.2 (=CH–), 130.4 (C), 129.5
(2 × =CH–), 122.2 (C), 121.7 (C), 118.6 (=CH–), 118.0 (=CH–), 117.8 (=CH–), 115.5 (=CH–); EIMS:
m/z (%) = 386 (100) [M]+, 355 (43), 327 (80), 295 (63), 281 (31), 265 (38), 251 (24), 237 (20), 223 (28),
205 (29), 193 (55), 181 (44), 165 (51), 152 (46), 139 (38), 119 (21), 91 (33), 77 (30), 59 (58), 51 (25).
(E)-2-(1-Carboxy-2-(2,4-dihydroxyphenyl)vinyl)-4,5-dihydroxybenzoic Acid (3l)
Оil (0.16 g, 16%) after HPFC from the reaction of 6,7-dimethoxyhomophthalic anhydride (1b, 0.67 g,
3.01 mmol), 2,4-dimethoxybenzaldehyde (2d, 0.50 g, 3.01 mmol), 4-dimethylaminopyridine (0.37 g,
3.01 mmol) and boron tribromide (20.7 mL, 33.11 mmol) in dichloromethane (10 mL). Pale yellow
crystals (0.13 g, 81%) after crystallization from 1:2 dichloromethane-acetone. M.p. = 182–183 °C; IR
(ATR diamond, cm−1): ν = 3640 (OH), 3560 (OH), 3500–2160 (ОH, COOH), 1670 (C=O, COOH), 1615
(C=C); 1H-NMR (600 MHz, DMSO-d6): δ = 11.98 (bs, 2H, 2 × COOH), 9.81 (s, 1H, OH), 9.56 (s, 2H,
2 × OH), 9.26 (bs, 1H, OH), 7.74 (s, 1H, H8), 7.41 (s, 1H, H6), 6.34 (s, 1H, H3), 6.30–6.28 (m, 2H, H3'
and H6'), 5.89 (dd, 1H, J = 8.7, 2.3 Hz, H5'); 13C-NMR (151 MHz, DMSO-d6): δ = 168.6 (C1a), 167.1
(C7), 159.0 (C), 157.8 (C), 148.9 (C), 143.8 (C), 131.7 (C), 130.4 (C), 130.2 (=CH–), 129.7 (C), 121.7
(C), 117.9 (=CH–), 117.8 (=CH–), 113.4 (=CH–), 106.6 (=CH–), 102.1 (=CH–); EIMS: m/z (%) = 416
(100) [M]+, 357 (38), 325 (24), 181 (81), 149 (25), 59 (39).
(E)-2-(1-Carboxy-2-(2,5-dihydroxyphenyl)vinyl)-4,5-dihydroxybenzoic Acid (3m)
Oil (0.21 g, 21%) after HPFC from the reaction of 6,7-dimethoxyhomophthalic anhydride (1b, 0.67 g,
3.01 mmol), 2,5-dimethoxybenzaldehyde (2e, 0.50 g, 3.01 mmol), 4-dimethylaminopyridine (0.37 g,
3.01 mmol) and boron tribromide (20.7 mL, 33.11 mmol) in dichloromethane (10 mL). Pale yellow
crystals (0.15 g, 71%) after crystallization from 1:2 dichloromethane-acetone. M.p. = 173–174 °C; IR
(ATR diamond, cm−1): ν = 3600 (OH), 3520–2160 (ОH, COOH), 1675 (C=O, COOH); 1H-NMR (600 MHz,
DMSO-d6): δ = 12.09 (s, 2H, 2 × COOH), 9.58 (s, 1H, OH), 9.28 (s, 1H, OH), 9.18 (s, 1H, OH), 8.52 (s,
1H, OH), 7.73 (s, 1H, H8), 7.43 (s, 1H, H6), 6.62 (d, 1H, J = 8.7 Hz, H3'), 6.47 (dd, 1H, J = 8.7, 3.0 Hz,
H4'), 6.31 (s, 1H, H3), 5.97 (d, 1H, J = 3.0 Hz, H6'); 13C-NMR (151 MHz, DMSO-d6): δ = 168.7 (C1a),
167.3 (C7), 149.4 (C), 149.1 (C), 149.0 (C), 144.2 (C), 133.2 (C), 131.2 (C), 130.6 (C), 122.5 (=CH–),
121.5 (C), 118.1 (=CH–), 117.8 (=CH–), 117.1 (=CH–), 115.9 (=CH–), 115.8 (=CH–); EIMS: m/z (%)
= 416 (100) [M]+, 385 (71), 357 (41), 325 (55), 310 (28), 295 (21), 283 (24), 267 (40), 253 (21), 225
(25), 209 (23), 193 (26), 181 (38), 165 (21), 149 (28), 139 (24), 59 (70).

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(E)-2-(1-Carboxy-2-(2,3-dihydroxyphenyl)vinyl)-4,5-dihydroxybenzoic Acid (3n)
Oil (0.37 g, 37%) after HPFC from the reaction of 6,7-dimethoxyhomophthalic anhydride (1b, 0.67 g,
3.01 mmol), 2,3-dimethoxybenzaldehyde (2f, 0.50 g, 3.01 mmol), 4-dimethylaminopyridine (0.37 g,
3.01 mmol) and boron tribromide (20.7 mL, 33.11 mmol) in dichloromethane (10 mL). Beige crystals
(0.20 g, 54%) after crystallization from 1:2 dichloromethane-acetone. M.p. = 180–182 °C; IR (ATR
diamond, cm−1): ν = 3630 (OH) 3550–2160 (ОH, COOH), 1670 (C=O, COOH); 1H-NMR (600 MHz,
DMSO-d6): δ = 12.11 (bs, 2H, 2 × COOH), 9.57 (s, 1H, OH), 9.45 (s, 1H, OH), 9.27 (s, 1H, OH), 8.79
(s, 1H, OH), 7.80 (s, 1H, H8), 7.41 (s, 1H, H6), 6.61 (dd, 1H, J = 7.9, 1.2 Hz, H6'), 6.31 (s, 1H, H3),
13
6.29 (t, 1H, J = 7.9 Hz, H5'), 5.98 (dd, 1H, J = 7.9, 1.2 Hz, H4'); C-NMR (151 MHz, DMSO-d6):
δ = 168.6 (C1а), 167.3 (C7), 149.1 (C), 145.2 (C), 145.0 (C), 144.2 (C), 133.4 (C), 131.4 (C), 130.8 (C),
122.8 (=CH–), 121.5 (C), 120.1 (=CH–), 118.3 (=CH–), 118.0 (=CH–), 117.9 (=CH–), 115.1 (=CH–);
EIMS: m/z (%) = 416 (56) [M]+, 385 (65), 357 (23), 325 (100), 298 (20), 267 (27), 193 (31), 181 (24),
139 (20), 59 (50).
3.2.2. General Procedure for Derivatization of Polyhydroxy (E)-2-(1-Carboxy-2-phenylvinyl)benzoic
Acids 3a–p
To each compound (1 mg), ethereal diazomethane solution was slowly added until complete
dissolution. The end of the reaction was determined by means of TLC. After that, the solvent was
evaporated under reduced pressure, the residue was dissolved in dichloromethane and analyzed as
described in Section 3.1.
3.3. In Vitro Antioxidant Capacity Assays
3.3.1. 1,1-Diphenyl-2-picrylhydrazyl Radical Scavenging Assay (DPPH●)
A methanolic solution (100 μM) of the DPPH● radical was prepared daily and protected from light.
Absorbance was recorded to check the stability of the radical throughout the time of analysis. Five
different concentrations of DPPH● radical (in the range 100–10 μM) were also prepared every day and
a linear relationship between radical concentration and absorbance was established. The effect of tested
compounds on the DPPH● absorbance was estimated by using a modified procedure described by
Brand-Williams [53]. Different analyte concentrations dissolved in methanol (0.5 mL) were added to
DPPH● methanolic solution (0.5 mL). Absorbance at 518 nm was recorded at different time intervals
until the reaction reached equilibrium (plateau). The initial absorbance was close to 0.600 in all cases.
The blank reference cuvette contained methanol. All measurements were performed in triplicate. Five
different concentrations of each phenolic compound have been assayed in order to check the linearity of
response and to establish the antioxidant activity values in the adequate linear range. The end of the
reaction for each compound is simultaneously dependent on its reactivity and concentration, therefore
this issue was addressed by introduction of an additional parameter—TEC50—the time required to reach
equilibrium at a concentration of compound at EC50.

Molecules 2015, 20
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●▬
3.3.2. Superoxide Anion Radical Scavenging Assay (O2
)
A NADH/PMS/NBT system was used to determine the superoxide anion scavenging activities of the
compounds as described by Suzumura et al. [54]. Briefly, generation of superoxide anion was measured
in a reaction mixture containing 600 μM NADH, 150 μM NBT and 30 μM PMS in phosphate buffered
saline (PBS) at pH = 7.4. The reduction of NBT was followed by measuring the change in absorbance
at 560 nm for 10 min, a period in which the absorbance increases linearly. Test compounds with different
concentrations were prepared in methanol and added to the reaction mixture, whereafter their activity
was determined in comparison with the control sample which did not contain the test compound.
3.3.3. Hydroxyl Radical Scavenging Assay (HO●)
Hydroxyl radical scavenging activity was measured as described by Kunchandy and Rao [55], with
slight modifications. Hydroxyl radical generated through Fenton reaction, can bleach p-nitroso-dimethyl
aniline (p-NDA) specifically. Scavenging activity was measured by the extent of inhibition of bleaching
of p-NDA in presence of the test compound. To a reaction mixture containing FeCl3/EDTA (0.1/0.1 mM),
H2О2 (2 mM), p-NDA (0.01 mM), different concentrations of test compound in phosphate buffer pH = 7.4
(20 mM), was added ascorbic acid (0.1 mM) to give final volume of 1.5 mL. Absorbance was measured
at 440 nm. Percentage scavenging was calculated from the control where not test compound was present.
3.4. Statistical Analysis
All analyses were performed in triplicate. Results were expressed as mean EC50 ± SD (μM). Statistical
analyses of the data were performed with one-way analysis of variance (ANOVA) from summary data.
The averages were statistically compared and this was taken into account in the discussion of the antioxidant
capacity. A level of confidence higher than 95% (p < 0.05) was considered as statistically significant.
3.5. Theoretical Approach
All computations were performed by means of the Gaussian-09 program package [56]. The
geometries of all possible conformers of the studied species were fully optimized by application of
B3LYP hybrid functional [57] on 6-31+G(d) and 6-31++G(d,p) basis sets. The geometries of open shell
species (radicals and cation-radicals) were optimized on the same theory levels by unrestricted wave
functions (UB3LYP). The stationary points found on the potential energy hypersurfaces for each
structure, were characterized using the standard harmonic vibrational analysis. The absence of imaginary
frequencies confirmed that the stationary points corresponded to a minimum on the potential
hypersurfaces. Since the free radical-scavenging activity strongly depends on medium polarity, solvent
effect was taken into account in this study. For this aim, Integral Equation Formalism version of
Tomasi’s Polarizable Continuum Model (IEF-PCM) [58] was used for all geometry optimizations and
calculation of descriptors. We choose water as solvent to represent real biological systems. All enthalpies
were calculated for 298 K. The values of BDE, IP, PDE, PA, ETE were determined from total enthalpies
of the individual species, as follows:
BDE = H(ArO●) + H(H●) − H(ArOH)
(1)

Molecules 2015, 20
2571
IP = H(ArOH+●) + H(e−) − H(ArOH)
PDE = H(ArO●) + H(H+) − H(ArOH+●)
PA = H(ArO−) + H(H+) − H(ArOH)
(2)
(3)
(4)
(5)
ETE = H(ArO●) + H(e−) − H(ArO−)
Besides enthalpies of ArO●, ArOH+●, ArO− the enthalpies of hydrogen radical H● and proton
were also calculated at the same level of theory. The value of enthalpy of hydrated electron was
taken from the literature [59]. The spin densities were obtained from Mulliken and from Natural
Population Analysis.
4. Conclusions
In this work, we have evaluated the antioxidant capacity of a series of hybrid molecules, composed
of natural phenolic acids such as benzoic, protocatechuic, cinnamic, p-coumaric, caffeic, or gallic acids,
against DPPH●, HO● and O2●▬ radicals. The results obtained showed that most of the tested compounds
are highly effective in micromolar concentrations, possessing higher activities than well-known natural
antioxidants. Based on the performed SAR analysis and DFT quantum chemical computations, it was
deduced that, in comparison with their number, the distribution of the OH groups in the basic carbon
backbone contributes in a greater extent to the radical scavenging activity, which is consistent with
previously described relationships [60]. It was also shown that the major factor responsible for the
apparent activity of the compounds under study is the presence of at least one catechol fragment,
regardless in which aromatic ring it is on, and that in case of multiple centers are able to react, the
presence of an extended π-conjugated system providing better delocalization, and thus stabilization of
the radicals formed, will determine the preferred group to be attacked. In sum, the catechol fragment
was shown to be of higher impact toward the scavenging activity against OH● radical, whereas pyrogallol
fragment – toward O2●▬. On the other hand, the absence of correlations between the results obtained by
different methods suggests that different mechanism of action can operate, and that the lack of activity
against DPPH● does not mean that the compounds do not possess antioxidant activities and vice versa.
Based on the above reasoning, it can be concluded that the synthesis of hybrid antioxidants is credible,
and that additional work toward the evaluation of their potential as drug-candidates for preventing
oxidative stress associated diseases is worth to be performed.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/02/2555/s1.
Acknowledgments
The financial support of the National Science Fund of Bulgaria at the Ministry of Education, Youth
and Science (projects DMU-03-10/2011) and Sofia University Fund (project 029/2013), as well as the
computational facilities kindly provided by Petko Ivanov, are greatly acknowledged by the authors.

Molecules 2015, 20
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Author Contributions
M. Miliovsky was responsible for the synthesis, purification, isolation and characterization of
compounds 3a–p; M. Miliovsky and I. Svinyarov contribute equally to the antioxidant activity
measurements toward DPPH● and HO●; E. Prokopova and D. Batovska conducted measurements toward
O2●▬; S. Stoyanov performed the quantum chemical computations; M.G. Bogdanov designed the
research and wrote the paper. All authors have approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2015 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 license
(http://creativecommons.org/licenses/by/4.0/).