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

Molecules 2015, 20, 2555-2575; doi:10.3390/molecules20022555

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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.

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HOOC

R³

R

R⁵

R⁴

R¹

R²

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

0

1

2

3

2

3

4

5

1

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

OH

H

–/phenol

na

nd

na

3d

H

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

OH

H

na

3f

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

OH

H

OH

H

–/catechol

2.75 ± 0.1

3 ± 0.1

3h

H

OH

H

–/pyrogallol

catechol/–

3i

OH

–

OH

–

H

31.75 ± 1.5

26 ± 0.75

33 ± 2

3j

H

OH

H

catechol/phenol

catechol/resorcinol

catechol/hydroQ ^c

catechol/catechol

catechol/pyrogallol

phenol

3k

H

OH

H

3l

H

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

3m

3n

OH

H

OH OH

137.2 ± 3.9

64.9 ± 1.9

11.3 ± 0.5

na

3o

H

OH

–

OH

–

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); ^bTEC₅₀—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 EC₅₀values (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 EC₅₀values 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 EC₅₀value 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 EC₅₀values 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 EC₅₀values

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 EC₅₀values 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 EC₅₀values 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

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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

ArO

R-H

ArOH

HAT-mechanism; H is BDE

Δ

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

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molecule species into radicals, radical-cations and radical-anions does not lead to significant changes in

the preferred conformations.

H

O

H

O

H

O

H

O

H

O

H

O

383.8

~375

384.1

398.6

~375

385.8

O

C

ϕ₁

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

H

O

H

311.4

H

O

278.8

H

295.1

H

322.3

3k ^H

309.6

O

304.7

O

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⁻¹range, 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 ^aIP ^bPDE ^cPA ^dETE ^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

^aBond Dissociation Energy; ^bIonization Potential; ^cProton Dissociation Enthalpy; ^dProton Affinity; ^eElectron

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

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-d₆as 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 Na₂SO₄and 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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Article Doi

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

DOI: 10.3390/molecules20022555

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