Antiradical and antioxidative activity of azocalix[4]arene derivatives: Combined experimental and theoretical study

Article abstract of DOI:10.3390/molecules24030485

Developing antioxidants with high efficiency is fundamentally important for the protection of living cells and engineering materials against oxidative damage. In this present study, two azocalix[4]arene derivatives were synthesized via a diazo coupling re

Full text of DOI:10.3390/molecules24030485

molecules
Article
Antiradical and Antioxidative Activity of
Azocalix[4]arene Derivatives: Combined
Experimental and Theoretical Study
Jiaqi Ni 1,2, Lilin Lu 1,2,
*
and Yi Liu 2,3
1
State Key Laboratory of Refractories and Metallurgy, Wuhan University of Science and Technology,
Wuhan 430081, China; 15671569690n@sina.com
Hubei Province Key Laboratory of Coal Conversion and New Carbon Materials, School of
Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, China;
yiliuchem@whu.edu.cn
State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine,
College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, China
Correspondence: lulilin@wust.edu.cn
2
3
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 15 January 2019; Accepted: 28 January 2019; Published: 29 January 2019
Abstract: Developing antioxidants with high efficiency is fundamentally important for the protection
of living cells and engineering materials against oxidative damage. In this present study,
two azocalix[4]arene derivatives were synthesized via a diazo coupling reaction between calix[4]arene
and diazonium salts. Their antiradical and antioxidative performances were evaluated by hydroxyl
radical scavenging and pyrogallol autoxidation inhibition experiments. Combined with theoretical
studies, the antiradical and antioxidative mechanisms have been explored. The results demonstrated
that these two azocalix[4]arene derivatives both exhibited remarkable antiradical and antioxidative
activity. The macrocyclic framework of the calix[4]arene and para-azo substituent group at the
upper rim of calix[4]arene contributed synergistically and importantly to its excellent antiradical and
antioxidant activity.
Keywords: azocalix[4]arene; antiradical; antioxidant; mechanism; density functional theory
1
. Introduction
Reactive oxygen species (ROS) are widely found in a number of life-sustaining biochemical
processes in living cells, such as aerobic metabolism [1]. Furthermore, these types of reactive species
are involved in many material fields (e.g., polymers and lubricants) and industries (e.g., food industry).
Oxidative degradation resulting from reactive species is a general phenomenon that occurs in living
cells and engineering materials, leading to many severe problems, such as cardiovascular disease,
cancer, chronic inflammatory diseases [2], and a sharp deterioration of the mechanical properties of
engineering materials. Therefore, developing efficient antioxidants to scavenge reactive oxygen species
and to alleviate oxidative damage has always been an imperative research issue for many decades.
Calixarene, which are the cyclic oligomers obtained by the condensation of formaldehyde
with p-alkylphenols, has been considered to be the third generation supramolecular host molecule
after crown ethers and cyclodextrins. It has been extensively used in molecular recognition, ion
sensing and drug delivery [3–5]. Due to the hindered phenol molecular structure, calixarene and
its derivatives were also reported to be part of a class of excellent antioxidants with inherent
antioxidative activities. p-tertbutyl-calix[4]arene was reported to be effective light and heat stabilizers
for polyolefins [
6
,7]. The radiation stability of polypropylene can be significantly improved by
p-tert-butylcalix[n]arene [8]. Low density polyethylenes and high density polyethylenes could
Molecules 2019, 24, 485; doi:10.3390/molecules24030485
www.mdpi.com/journal/molecules

Molecules 2019, 24, 485
2 of 11
be effectively stabilized by p-tertbutyl-calix[4]arene and p-tertbutyl-calix[6]arene, respectively [9].
Calix[n]arenes that feature sulphonate groups above and/or below the plane of the macrocycle have
intrinsic antioxidant capacity and antibacterial activity [10]. Four dihydropyrimidine moieties were
grafted at the upper rim of calix[4]arene, which produced calix[4]arene-based dihydropyrimidines
that possess amplified antiradical activity in comparison to the corresponding monomers [11].
Furthermore, a calix[4]arene-like tetramer of resveratrol was reported to exhibit significantly
improved antioxidative activity with respect to resveratrol [12]. Calixarene-based antioxidant
C-methylcalix[4]resorcinarene also exhibited superior antioxidative efficiency in natural rubber
vulcanizates compared to that of classical antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT) [13].
In addition, calix[4]arene has also been used for rigid molecular scaffolds to cluster hydroxycinnamic
acid in order to improve radical scavenging and antioxidative activity [14].
In this work, two azocalix[4]arene derivatives (Figure 1), which were namely 5,11,17,23-tetrakis[(p-carb
oxyphenyl)azo]-25,26,27,28-tetrahydroxycalix[4]arene (1) and 5,11,17,23-tetrakis[(3-pyridine)azo]-25,26,27,
2
8-tetra hydroxycalix[4]arene (
2
), were synthesized via a diazo coupling reaction between calix[4]arene and
1
diazonium salts. It was then characterized by H-NMR and IR (Figure 2). Their antiradical capacities were
evaluated by hydroxyl radical ( OH) scavenging experiments and their antioxidative activities were
·
tested by a pyrogallol autoxidation inhibiting experiment. To elucidate the antiradical and antioxidative
mechanisms, combined experimental and theoretical studies were also performed to explore the
necessary pharmacophores that are responsible for their antiradical and antioxidative activities.
The effect of the calix[4]arene framework and azo substituent group at the upper rim of calix[4]arene
on the antiradical and antioxidative activities were experimentally investigated by comparison with
calix[4]arene (
and 3-aminopyridine-azo-phenol (
studied on the basis of hydrogen abstraction transfer (HAT) and sequential proton loss electron transfer
SPLET) pathways.
3
) and the corresponding monomer model compounds, p-carboxyphenyl-azo-phenol (
4)
5). The antiradical and antioxidative mechanisms were theoretically
(
Figure 1.
Chemical structures of azocalix[4]arene derivatives 1 and 2, calix[4]arene (3),
p-carboxyphenyl-azo-phenol (4) and 3-aminopyridine-azo-phenol (5).
Figure 2. Synthesis route of azocalix[4]arene derivatives 1 and 2.

Molecules 2019, 24, 485
3 of 11
2
. Results and Discussion
2
.1. Hydroxyl Radical Scavenging Activity
In this work, hydroxyl radicals (
·
OH) were produced via irradiation decomposition of
H O [15,16] before being reacted with terephthalic acid to form a fluorescent compound
2
2
hydroxyterephthalic acid (HTA, λem 425 nm) [17,18]. The reactions are described as follows:
2
54 nm UV irradiation
H O −−−−−−−−−−−−→ 2•OH
(1)
(2)
2
2
•
OH + terephthalic acid (TA) → HTA(fluorescent)
In the antiradical experiment, the antioxidant competes with terephthalic acid to trap hydroxyl
radicals, thus inhibiting the production of HTA and leading to a decrease in fluorescence intensity.
The results of hydroxyl radical scavenging of azocalixarene derivatives
As shown in Figure 3, a significant decrease in fluorescence intensity at 425 nm was induced by
azocalixarene derivatives and . The hydroxyl radical scavenging efficiency was calculated according
to the following equation:
1 and 2 are shown in Figure 3.
1
2
d = (I − I )/I × 100%
(3)
0
f
0
where the I0 and I represent the fluorescence intensity before and after the addition of
f
azocalixarene, respectively.
Figure 3. Hydroxyl radical scavenging efficiency as a function of the concentration ratio of
[
2
azocalixarene]/[
, respectively.
·
OH] where cycle (
#
) and triangle ( ) represent azocalix[4]arene derivatives 1 and
4
Figure 3 shows the plot of hydroxyl radical scavenging efficiency (d) against the concentration ratio
of [azocalixarene]/[ OH]. At the [azocalixarene]/[ OH] ratio of 0.02, the hydroxyl radical scavenging
efficiency of and are approximately 54.3% and 32.5%, respectively. When the concentration
ratio increased to approximately 0.2, the hydroxyl radical scavenging efficiencies increased to 71.7%
and 63.2%, respectively. Compared with compound , the calix[4]arene derivative showed higher
hydroxyl radical scavenging activity, especially at a low concentration range. For instance, at the
azocalixarene]/[ OH] ratio of 0.01, the hydroxyl radical scavenging efficiency of is about 45%,
which is almost three-fold of that (approximately 15%.) of , demonstrating that compound possesses
greater antiradical capacity than compound . These results demonstrated that the two investigated
azocalixarene derivatives both exhibited significant scavenging activity for hydroxyl radicals.
·
·
1
2
2
1
[
·
1
2
1
2
2
.2. Pyrogallol Autoxidation Inhibition Activity
The autoxidation of pyrogallol in an alkaline aqueous solution has been extensively observed
in previous works [19,20]. In this present study, pyrogallol autoxidation experiments have also been

Molecules 2019, 24, 485
4 of 11
performed and the UV–vis absorption spectrum was constantly measured (displayed in Figure 4).
An increasing absorption peak at 320 nm was observed and the absorbance reached its maximum after
pyrogallol autoxidation for 30 min. In this work, the pyrogallol autoxidation inhibition experiments of
azocalixarene derivatives were allowed to proceed for 30 min before the absorbance at 320 nm was
measured to calculate the inhibition efficiency E(%) according to the following equation:
A − A
0
E% =
× 100%
(4)
A
0
where A was the absorbance at 320 nm after autoxidation for 30 min when no antioxidant was added
0
and A was the absorbance after azocalixarene was added as an antioxidant.
Figure 4. Absorption spectrum of pyrogallol in Tris-HCl buffer solution (pH = 8.2) after autoxidation
for 0.5, 4.0, 8.0, 12.0, 16.0, 20.0, 24.0, 28.0 and 32.0 min (1–9).
The performances of azocalixarene derivatives in the pyrogallol autoxidation inhibition
experiment are displayed in Figure 5. As shown in Figure 5, the investigated azocalix[4]arene
derivatives
When [azocalixarene]/[pyrogallol] ratio was equal to 0.01, the inhibition efficiencies reach
about 25% and 18% for and , respectively. With an increase in the concentration ratio
1 and 2 both exhibited remarkable activity in pyrogallol autoxidation inhibition.
1
2
of [azocalixarene]/[pyrogallol], the inhibition efficiency increases correspondingly. When the
concentration ratio increased to 0.07, the inhibition efficiency increased to 36.9% and 31.8%, respectively,
for
compared to compound
indicating that the azocalixarene derivatives 1 possess more excellent antioxidative capacity.
1
and
2. Similarly, compound
1 exhibited higher activity in the inhibition of pyrogallol autoxidation
2
over the whole testing range of [azocalixarene]/[pyrogallol] ratios,
Figure 5. Pyrogallol autoxidation inhibition efficiency as a function of the concentration ratio of
[
1
azocalixarene]/[pyrogallol] where cycle (
and 2, respectively.
#
) and triangle ( ) represent azocalix[4]arene derivatives
4

Molecules 2019, 24, 485
5 of 11
The performance of calix[4]arene (
phenol (
were also investigated and compared to each other. An [antioxidant]/[
antioxidant]/[pyrogallol] of 0.05 were selected, respectively, for hydroxyl radical scavenging and
3
), p-carboxyphenyl-azo-phenol (
4
) and 3-aminopyridine-azo-
5) in hydroxyl radical scavenging and pyrogallol autoxidation inhibition experiments
·
OH] ratio of 0.1 and
[
pyrogallol autoxidation inhibition experiments under identical experimental conditions. The results
manifested that calix[4]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol exhibited
negligible antiradical capacity and antioxidative activity. The hydroxyl radical scavenging efficiencies
of calix[4]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol were 2.8%, 5.2% and
3
.6%, respectively. In pyrogallol autoxidation inhibition experiments, the inhibition efficiencies of these
compounds were 1.9%, 1.6% and 1.4%, respectively. These results demonstrated that the macrocyclic
framework of calix[4]arene or azo group was not the crucial factor that is individually responsible for
determining the capacity of azocalix[4]arene derivatives. It was the synergistic effect of the macrocyclic
framework of calix[4]arene and azo group at the upper rim of calix[4]arene that might result in the
significant antiradical and antioxidative activity of azocalixarene derivatives 1 and 2.
2
.3. Phenolic O–H BDEs, Proton Affinities (PAs) and Electron Transfer Enthalpies of Phenolate Anions
For phenolic antioxidants, their radical scavenging and antioxidative activity rely significantly on
their ability to transfer their phenolic H-atom to radical species in order to break the chain reaction.
Generally, there are two pathways for H-atom transfer, which are namely the hydrogen abstraction
transfer (HAT) and sequential proton loss electron transfer (SPLET) [21,22]. In the former case,
the antioxidant donates a H-atom via the homolytic dissociation of the phenolic O–H bond, which is
under the control of the O–H bond dissociation enthalpy (BDE). The lower O–H BDE has a weaker
phenolic O–H bond and favors the O–H homolytic dissociation donating the H-atom. In the latter
case, the H-atom transfer process is governed by the acidity of phenolic O–H, which is generally
characterized by the proton affinity (PA) of the corresponding phenolate anion. A lower PA means
the stronger O–H acidity, which is more favorable for the heterolytic dissociation. Compared with
a neutral molecule, the produced phenolate anion has the lower ionization potential, which facilitates
the sequential electron transfer process to produce the phenoxyl radical.
Considerable efforts have been devoted to the measurement of O–H BDE using versatile
techniques, such as photoacoustic calorimetry [23] and EPR spectroscopy [24
methods have also been extensively used for evaluating the O–H BDE [26
DFT calculations have been performed at the BP86/6-311+G(d,p) level of theory to calculate the O–H
BDEs and the PAs of phenolate anions of azocalixarene derivatives and in an aqueous solution
in order to explore the antiradical and antioxidative mechanisms [12 28]. For comparison, the O–H
BDEs of calix[ ]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol and the PAs of
,
25]. Quantum mechanical
,
27]. In this present work,
1
2
,
4
phenolate anions of them are also calculated. All of the results are listed in Table 1.
Table 1. The calculated phenolic O–H bond dissociation enthalpies (BDEs) and proton affinities (PAs)
of phenolate anions of investigated chemical systems at BP86/6-311+G(d,p) level of theory.
Phenolate Anion
Compounds
O–H BDEs (kcal/mol)
PAs (kcal/mol)
ETEs (kcal/mol)
Calix[4]arene
p-carboxyphenyl-azo-phenol
-aminopyridine-azo-phenol
82.17 *
81.26
81.42
76.39 *
77.97 *
276.59
278.57
279.54
265.85
266.70
117.74
115.21
114.41
122.89
123.64
3
1
2
*
For calix[4]arene compounds, only one phenolic hydroxyl was arbitrarily selected to be studied by DFT calculations.
As shown in Table 1, the calculated O–H BDE of calix[4]arene, p-carboxyphenyl-azo-phenol
and 3-aminopyridine-azo-phenol is 82.17, 81.26 and 81.42 kcal/mol, respectively. In azocalix[4]arene
derivatives 1 and 2, the O–H BDEs were 76.39 and 77.97 kcal/mol, respectively. They are distinctly

Molecules 2019, 24, 485
6 of 11
lower than those of calix[4]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol.
This indicated that the phenolic O–H bonds in azocalix[4]arene derivatives and were significantly
1
2
weakened, thus facilitating H-atom being donated from the phenolic O–H group to break the chain
reaction of radicals. This result is consistent with the above-mentioned experimental findings that
azocalix[4]arene derivatives showed superior hydroxyl radical scavenging capacity compared to
calix[4]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol.
The proton affinities and electron transfer enthalpy of phenolate anions were also investigated
to explore the acidity of phenolic O–H and electron loss capacity of phenolate anions. For phenolate
anions of all investigated compounds, the calculated electron transfer enthalpy is significantly lower
than the proton affinity, indicating that the first proton dissociation is the rate-determined step of the
sequential proton loss electron transfer (SPLET) process. For calix[4]arene, p-carboxyphenyl-azo-phenol
and 3-aminopyridine-azo-phenol, their proton affinities were calculated to be 276.59, 278.57 and
2
79.54 kcal/mol, respectively. The calculated proton affinities of azocalix[4]arene derivatives 1 and 2
are 265.85 kcal/mol and 266.70 kcal/mol, respectively. The distinct decrease in the proton affinity of
azocalix[4]arene derivatives with respect to those of calix[4]arene, p-carboxyphenyl-azo-phenol and
3-aminopyridine-azo-phenol indicated the stronger acidity of the O–H group, which would facilitate
the production of phenolate anions.
As concluded from the above-mentioned results, the synergistic effect of macrocyclic framework
of calix[4]arene and azo group at the upper rim of calix[4]arene is responsible for the lower O–H BDEs
and proton affinity of the phenolate anion, thus resulting in the excellent radical scavenging activity
and pyrogallol autoxidation inhibition ability of azocalix[4]arene derivatives 1 and 2.
−
In previous works, the superoxide anion radical (
·
O2 ) has been confirmed to be involved
in the autoxidation of pyrogallol [29] and inhibition of pyrogallol autoxidation was widely used
to measure the capacity of antioxidants to scavenge superoxide anion radicals [20]. In this work,
the investigated azocalixarene derivatives exhibited excellent activity for pyrogallol autoxidation
inhibition, which might be explained by the strong acidity of their O–H group. Firstly, the negatively
charged superoxide anion radical, which was produced during pyrogallol autoxidation, accepted the
proton donated by azocalix[4]arene derivatives to form a neutral radical
was transferred from the phenolate anion of the azocalixarene derivative to
·
O H. Secondly, the electron
2
·
O H and subsequently
2
quenched the anion. As shown by the relatively higher PA of phenolate anion with respect to O–H BDE
in Table 1, the investigated azocalix[4]arene derivatives possess more excellent activity in hydroxyl
radical scavenging than in superoxide anion radical scavenging, thus exerting higher efficiency in the
hydroxyl radical scavenging experiment but relatively lower efficiency in the pyrogallol autoxidation
inhibition experiment. This is consistent with the experimental performance as the highest efficiency
of hydroxyl radical scavenging for the investigated azocalix[4]arene derivatives was about 71.7%,
whereas the highest efficiency of pyrogallol autoxidation inhibition was only about 36.9%.
2
.4. Spin Density Distribution Analysis of Phenoxyl Radicals
The stability of the phenoxyl radical produced via the HAT or SPLET process is another
important index for antioxidants. It is desirable that a phenoxyl radical is produced with enough
stability because otherwise it may not be able to easily induce a new chain reaction between itself
and substrates. The unpaired electron distribution in phenoxyl radical plays an important role in
determining its stability. More delocalized unpaired electrons in the radical creates more stability in
the radical. In this present study, a spin density distribution analysis was performed and displayed in
Figure 6 to explore the unpaired electron delocalization in the phenoxyl radical of azocalix[4]arene
derivatives. Furthermore, the phenoxyl radicals of calix[4]arene, p-carboxyphenyl-azo-phenol and
3
-aminopyridine-azo-phenol were also investigated and compared.

Molecules 2019, 24, 485
7 of 11
Figure 6. Spin density distribution in the phenoxyl radicals of azocalix[4]arene derivatives
calix[4]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol.
1 and 2,
As shown in Figure 6, in the phenoxyl radicals of p-carboxyphenyl-azo-phenol and
3-aminopyridine-azo-phenol, the unpaired electron is mainly confined to the remaining phenolic
oxygen atom, carbon atom in benzene ring and the nitrogen atom in the azo group. The spin
density on the phenolic O-atom are 0.170 and 0.193, respectively. In the phenoxyl radical of
calix[4]arene, the unpaired electron was confined to the remaining phenolic O-atom and the
connected phenol ring, which results in the spin density of 0.290 on the remaining phenolic O-atom.
In phenoxyl radicals of azocalix[4]arene derivatives 1 and 2, the spin density on the remaining phenolic
O-atom decreased to 0.115 and 0.178, respectively. These values are significantly lower than the
corresponding spin density in the phenoxyl radicals of calix[4]arene, p-carboxyphenyl-azo-phenol
and 3-aminopyridine-azo-phenol. These results demonstrated that the unpaired electron in phenoxyl
radicals is more delocalized while the macrocyclic framework of calix[4]arene and azo substituent
group at the upper rim of calixarene contributed synergistically and importantly to the delocalization
of unpaired electrons.
3
. Materials and Methods
3
5
.1. Synthesis of 5,11,17,23-tetrakis[(p-carboxyphenyl)azo]-25,26,27,28-tetrahydroxycalix[4]arene (1) and
,11,17,23-tetrakis[(3-pyridine) azo]-25,26,27,28-tetrahydroxycalix[4]arene (2)
p-tert-butylcalix[4]arene was synthesized according to the method described in literature [30].
Calix[4]arene was prepared by the debutylation of p-tertbutylcalix[4]arene [31]. The azocalix[4]arene
derivatives ( and ) were synthesized via a diazo coupling reaction between calix[4]arene and
1
2
diazonium salts [32–34].
In a typical synthesis, 0.685 g of p-aminobenzoic acid crystal was added to 10 mL of water
while constantly stirring and heating. After it was dissolved, the solution was cooled down to
◦
5
C in an ice bath before a solution of 0.345 g NaNO dissolved in 2.5 mL of water was added.
2
The solution was acidified by a diluted aqueous HCl solution. After half an hour of stirring,
approximately 0.2 g of urea was added and the solution was stirred for a further 10 min to obtain
the solution of diazonium salt. The diazonium salt solution was added into a cold solution of 1.0 g
(2.36 mmol) calix[4]arene and 2.46 g (30 mmol) of anhydrous sodium acetate in 26 mL of MeOH-DMF
(5:8 v/v) in order to produce a red suspension. This red reactant was allowed to couple for two
hours in an ice bath, which was subsequently acidified by 150 mL of a HCl aqueous solution and
◦
warmed at 60 C for 30 min to produce a reddish viscous solid. After being filtered out, the solid
was washed with water and methanol several times before being dried under vacuum to obtain
5
,11,17,23-tetrakis[(p-carboxyphenyl)azo]-25,26,27,28-tetrahydroxy-calix[4]arene (
1
). We obtained
1
the following results. H-NMR(DMSO-D),
ArOH), 7.1~7.8(m, 24H, Ar), 8~14(s, 4H, COOH). IR,
δ
(ppm): 3.8 and 4.0 (d, 8H,ArCH Ar), 8.0~8.1(m, 4H,
2
−1
−
1
−1
ν
: 3421 cm and 3190 cm (OH), 1602 cm
−
1
(N=N), 1408 cm (C=O/ArCOOH). In a similar procedure, 0.77 g (10 mmol) 3-aminopyridine

Molecules 2019, 24, 485
8 of 11
was used to synthesize 5,11,17,23-tetrakis[(3-pyridine) azo]-25,26,27,28-tetrahydroxycalix[4]arene (2).
1
H-NMR(MeOD), δ(ppm): 4.6 and 4.9 (d, 8H,ArCH Ar), 8.6 (s, 4H, ArOH), 6.8~8.1 (m, 24H, Ar).
2
− −1 −1 −1
1
IR, ν: 3176 cm (OH), 1594 cm (N=N), 1676cm (Ar), 1649cm (pyridine). For comparison,
p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol were also synthesized in a similar way
via the diazo coupling reaction between phenol and diazonium salts. All chemicals and solvents
used in this work were of analytical grade and used without further purification unless otherwise
mentioned. Furthermore, doubly distilled water was used for the solution preparation.
3
.2. Hydroxyl Radical Scavenging and Pyrogallol Autoxidation Inhibition
Hydroxyl radicals were produced via the irradiation decomposition of H O under 254-nm UV
2
2
light. The amount of hydroxyl radicals was determined on the basis of the fluorescence intensity of
hydroxyterephthalic acid (HTA) [35]. The details of the procedure are provided as follows. A total of
100
µL of H O solution (20 mM), a certain volume of azocalixarene derivatives solution (0.02 mM)
2 2
and 100
µL of terephthalic acid (TA) solution (2 mM) were mixed before the final volume of mixed
solution was adjusted to 5 mL. After stirring for thirty seconds, the solution was transferred into a 1-cm
quartz cell and irradiated directly under 254-nm UV light for 20 min at room temperature and the
fluorescence intensity (λex 312 nm) at 425 nm was measured immediately. The pyrogallol autoxidation
inhibition performances of the investigated samples were tested according to the following procedure.
We mixed 0.3 mL of a pyrogallol aqueous solution (3 mM), 4.5 mL of a Tris-HCl buffer solution (50 mM,
pH = 8.2) and a certain volume of azocalixarene derivatives solution (0.02mM) before the final volume
◦
was adjusted to 9 mL. After being kept in a water bath at 25 C for 30 min, the absorbance at 320 nm
was measured. In control experiments, the azocalixarene derivatives have been replaced by other
compounds, such as calix[4]arene, p-carboxyphenyl-azophenol and 3-pyridine-azophenol.
3
.3. Density Functional Theory Study of the Antiradical and Antioxidative Mechanism
Geometries of azocalixarene derivatives ( and ), related phenoxyl radicals and phenolate anions
were optimized by employing a generalized gradient approximation using the BP86 functional [36 37
1
2
,
]
and 6-31G(d) basis set. Unrestricted formulation was applied for open-shell radical species.
The energies of all investigated chemical systems were refined by performing single-point calculations
at BP86/6-311+G(d,p) level of theory in an aqueous solution. A polarizable continuum model
(PCM) was used to simulate the aqueous solution environment with the dielectric constant of
78.3553. The antioxidative and antiradical activities of azocalixarene derivatives were theoretically
studied based on the hydrogen abstraction transfer (HAT) mechanism and sequential proton loss
electron transfer (SPLET) mechanism. Zero-point corrected energies and enthalpies were selected to
calculate the O–H bond dissociation enthalpy (BDE), the proton affinity (PA) and electron transfer
enthalpy (ETE) of phenolate anions according to the method described in previous works [21,22].
All computations in this work are performed with the Gaussian 09W suite of programs (Gaussian, Inc.,
Pittsburgh, PA, USA) [38].
4
. Conclusions
In conclusion, azocalix[4]arene derivatives
1
and 2 have been synthesized via the diazo
coupling reaction between calix[4]arene and diazonium salts. Hydroxyl radical scavenging and
pyrogallol autoxidation inhibition experiments demonstrated that the two investigated azocalix[4]arene
derivatives both exhibited remarkable antiradical and antioxidative activity. The compound
1 showed
higher activity in hydroxyl radical scavenging and pyrogallol autoxidation inhibition, with the
highest hydroxyl radical scavenging efficiency and pyrogallol autoxidation inhibition efficiency
found to be about 71.7% and 36.9%, respectively. Combined experimental and theoretical studies
revealed that the macrocyclic framework of calix[4]arene and para-azo substituent group at the
upper rim of calix[4]arene mainly contributed to their antiradical and antioxidant activity due to
the poor antiradical and antioxidative performance of calix[4]arene, p-carboxyphenyl-azo-phenol and

Molecules 2019, 24, 485
9 of 11
3
-aminopyridine-azo-phenol. DFT calculations at BP86/6-311+G(d,p) level of theory demonstrated
that phenolic O–H BDEs and proton affinities of phenolate anions of azocalix[4]arene derivatives
1
3
and
-aminopyridine-azo-phenol. Spin density analysis revealed that the spin density on the remaining
and was distinctly lower
2 were significantly lower than that in calix[4]arene, p-carboxyphenyl-azo-phenol and
phenolic O-atom in the phenoxyl radicals of azocalix[4]arene derivatives
1
2
than those in calix[4]arene, p-carboxyphenyl-azo-phenol and 3-aminopyridine-azo-phenol. This was
indicative of a higher degree of unpaired electron delocalization and more stable phenoxyl radicals
in the phenoxyl radicals of azocalix[4]arene derivatives
1 and 2. These results confirmed that the
synergetic effect of macrocyclic framework of calix[4]arene and para-azo substituent group at the
upper rim of calix[4]arene was responsible for the excellent antiradical and antioxidative activity of the
investigated azocalix[4]arene derivatives. Thus, these derivatives would be a promising antioxidant
candidate to protect living cells and engineering materials against radical and oxidative damage.
Author Contributions: Conceptualization, L.L.; Data curation, L.L.; Formal analysis, J.N., L.L. and Y.L.;
Funding acquisition, L.L.; Investigation, J.N. and L.L.; Methodology, J.N. and Y.L.; Project administration, L.L.;
Supervision, L.L.; Validation, Y.L.; Writing–original draft, J.N.; Writing–review & editing, L.L. and Y.L.
Funding: This research was funded by National Natural Science Foundation of China [21671154, U1732147] and
Natural Science Foundation of Hubei Province [2015CFC840].
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to
publish the results.
References
1
.
Mathews, C.K.; van Holde, K.E.; Ahern, K.G. Biochemistry, 3rd ed.; Part IV; Benjamin Cummings:
San Francisco, CA, USA, 2001.
2
.
Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.D.; Mazur, M.; Telser, J. Free radicals and antioxidants in
normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44–84. [CrossRef]
[PubMed]
3
4
5
6
7
.
.
.
.
.
Ikeda, A.; Shinkai, S. Novel Cavity Design Using Calix[n]arene Skeletons: Toward Molecular Recognition
and Metal Binding. Chem. Rev. 1997, 97, 1713–1734. [CrossRef] [PubMed]
Mokhtari, B.; Pourabdollah, K.; Dalali, N. Analytical applications of calixarenes from 2005 up-to-date. J. Incl.
Phenom. Macrocycl. Chem. 2011, 69, 1–55. [CrossRef]
Pur, F.N. Calixdrugs: Calixarene-based cluster of established therapeutic drug agents. Mol. Divers. 2016, 20, 781–787.
[CrossRef]
Seiffarth, K.; Schulz, M.; Gormar, G.; Bachmann, J. Calix[n]arenes-New light stabilizers for polyolefins.
Polym. Degrad. Stab. 1989, 24, 73–80. [CrossRef]
Chennakesavulu, K.; Sreedevi, P.; Basaria, M.R.; Reddy, G.R.; Sasipraba, T.; Raju, G.B.; Prabhakar, S.
Thermal decomposition mechanism of p-tert-butyl-calix[n]arenes. Thermochim. Acta 2014, 575, 55–63.
[CrossRef]
8
9
1
1
1
1
.
Feng, W.; Yuan, L.; Zheng, S.; Huang, G.; Qiao, J.; Zhou, Y. The effect of p-tert-butylcalix[n]arene on
γ-radiation degradation of polypropylene. Radiat. Phys. Chem. 2000, 57, 425–429. [CrossRef]
Jipa, S.; Zaharescu, T.; Setnescu, R.; Setnescu, T.; Dumitru, M.; Gorghiu, L.M.; Mihalcea, I.; Bumbac, M.
Effect of calixarenes on thermal stability of polyethylenes. Polym. Degrad. Stab. 2003, 80, 203–208. [CrossRef]
.
0. Stephens, E.K.; Tauran, Y.; Coleman, A.W.; Fitzgerald, M. Structural requirements for anti-oxidant activity of
calix[n]arenes and their associated anti-bacterial activity. Chem. Commun. 2015, 51, 851–854. [CrossRef]
1. Pur, F.N.; Dilmaghani, K.A. New Antiradical Clusters Synthesized Using the First Green Biginelli Reactions
of Calix[4]Arene. Pharm. Chem. J. 2016, 50, 80–82. [CrossRef]
2. Lu, L.; Ruan, Z.; Ni, J.; Chen, J.; Shu, H.; Wang, Y.; Liu, Y. Improvement of antioxidative activity of resveratrol
by calix[4]arene-like tetramer: A theoretical study. Comput. Theor. Chem. 2019, 1148, 1–7. [CrossRef]
3. Li, H.; Zhong, Y.; Wu, W.; Zhang, L.; Lai, X.; Zeng, X. Phenolic antioxidants based on calixarene: Synthesis, structural
characterization and antioxidative properties in natural rubber. J. Appl. Polym. Sci. 2017, 134, 45144. [CrossRef]

Molecules 2019, 24, 485
10 of 11
1
1
1
4. Consoli, G.M.L.; Galante, E.; Daquino, C.; Granata, G.; Cunsolo, F.; Geraci, C. Hydroxycinnamic acid clustered by
a calixarene platform: Radical scavenging and antioxidant activity. Tetrahedron Lett. 2006, 47, 6611–6614. [CrossRef]
5. Baxendale, J.H.; Wilson, J.A. The photolysis of hydrogen peroxide at high light intensities. Trans. Faraday Soc.
1
957, 53, 344–356. [CrossRef]
6. Chenna, M.; Messaoudi, K.; Drouiche, N.; Lounici, H. Study and modeling of the organophosphorus
compound degradation by photolysis of hydrogen peroxide in aqueous media by using experimental
response surface design. J. Ind. Eng. Chem. 2016, 33, 307–315. [CrossRef]
1
1
1
2
2
2
2
7. Šnyrychová, I.; Hideg, E. The first application of terephthalate fluorescence for highly selective detection of
hydroxyl radicals in thylakoid membranes. Funct. Plant Biol. 2007, 34, 1105–1111. [CrossRef]
8. Zhao, J.; Dong, W.; Zhang, X.; Chai, H.; Huang, Y. FeNPs@Co O hollow nanocages hybrids as effective
3
4
peroxidase mimics for glucose biosensing. Sens. Actuators B Chem. 2018, 263, 575–584. [CrossRef]
9. Siegel, S.M.; Siegel, B.Z. Autoxidation of Pyrogallol: General Characteristics and Inhibition by Catalase.
Nature 1958, 181, 1153–1154. [CrossRef]
0. Li, X. Improved Pyrogallol Autoxidation Method: A Reliable and Cheap Superoxide-Scavenging Assay
Suitable for All Antioxidants. J. Agric. Food Chem. 2012, 60, 6418–6424. [CrossRef]
1. Lu, L.; Zhu, S.; Zhang, H.; Li, F.; Zhang, S. Theoretical study of complexation of resveratrol with cyclodextrins
and cucurbiturils: Structure and antioxidative activity. RSC Adv. 2015, 5, 14114–14122. [CrossRef]
2. Lu, L.; Zhu, S.; Zhang, H.; Zhang, S. Improvement of antioxidative activity of resveratrol by elongating
conjugated chain: A DFT theoretical study. Comput. Theor. Chem. 2013, 1019, 39–47. [CrossRef]
3. Wayner, D.D.M.; Lusztyk, E.; Page, D.; Ingold, K.U.; Mulder, P.; Laarhoven, L.J.J.; Aldrich, H.S. Effects of
Solvation on the Enthalpies of Reaction of tert-Butoxyl Radicals with Phenol and on the Calculated O–H
Bond Strength in Phenol. J. Am. Chem. Soc. 1995, 117, 8737–8744. [CrossRef]
2
2
2
4. Lucarini, M.; Pedrielli, P.; Pedulli, G.F.; Cabiddu, S.; Fattuoni, C. Bond Dissociation Energies of O H Bonds
−
in Substituted Phenols from Equilibration Studies. J. Org. Chem. 1996, 61, 9259–9263. [CrossRef]
5. Lucarini, M.; Pedulli, G.F.; Cipollone, M. Bond Dissociation Enthalpy of.alpha.-Tocopherol and Other
Phenolic Antioxidants. J. Org. Chem. 1994, 59, 5063–5070. [CrossRef]
6. Wright, J.S.; Carpenter, D.J.; McKay, D.J.; Ingold, K.U. Theoretical Calculation of Substituent Effects on the
O
−
H Bond Strength of Phenolic Antioxidants Related to Vitamin E. J. Am. Chem. Soc. 1997, 119, 4245–4252.
[CrossRef]
2
2
2
3
3
3
3
3
7. Kosinova, P.; Meo, F.D.; Anouar, E.H.; Duroux, J.L.; Trouillas, P. H-atom acceptor capacity of free radicals
used in antioxidant measurements. Int. J. Quantum Chem. 2011, 111, 1131–1142. [CrossRef]
8. Borgohain, R.; Guha, A.K.; Pratihar, S.; Handique, J.G. Antioxidant activity of some phenolic aldehydes and
their diimine derivatives: A DFT study. Comput. Theor. Chem. 2015, 1060, 17–23. [CrossRef]
9. Marklund, S.; Marklund, G. Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol
and a Convenient Assay for Superoxide Dismutase. Eur. J. Biochem. 1974, 47, 469–474. [CrossRef]
0. Gutsche, C.D.; Iqbal, M.; Stewart, D. Calixarenes. 19. Syntheses procedures for p-tert-butylcalix[4]arene.
J. Org. Chem. 1986, 51, 742–745. [CrossRef]
1. Gutsche, C.D.; Lin, L.G. Calixarenes 12: The synthesis of functionalized calixarenes. Tetrahedron 1986, 42, 1633–1640.
[CrossRef]
2. Morita, Y.; Agawa, T.; Nomura, E.; Taniguchi, H. Syntheses and NMR behavior of calix[4]quinone and
calix[4]hydroquinone. J. Org. Chem. 1992, 57, 3658–3662. [CrossRef]
3. Xing, B.; Choi, M.; Zhou, Z.; Xu, B. Spontaneous Enrichment of Organic Molecules from Aqueous and Gas
Phases into a Stable Metallogel. Langmuir 2002, 18, 9654–9658. [CrossRef]
4. Lu, L.; Zhu, S.; Liu, X.; Xie, Z.; Yan, X. Highly selective chromogenic ionophores for the recognition of
chromium(III) based on a water-soluble azocalixarene derivative. Anal. Chim. Acta 2005, 535, 183–187.
[CrossRef]
3
3
5. Armstrong, W.A.; Facey, R.A.; Grant, D.W.; Humphreys, W.D. A tissue-equivalent chemical dosimeter
sensitive to 1 rad. Can. J. Chem. 1963, 41, 1575–1577. [CrossRef]
6. Becke, A.D. Density-functional exchange-energy approximation with correct asymptotic behavior.
Phys. Rev. A 1988, 38, 3098–3100. [CrossRef]

Molecules 2019, 24, 485
11 of 11
3
3
7. Perdew, J.P. Density-functional approximation for the correlation energy of the inhomogeneous electron gas.
Phys. Rev. B 1986, 33, 8822–8824. [CrossRef]
8. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G.A.; et al. Gaussian 09; Revision B.02; Gaussian, Inc.: Pittsburgh, PA, USA, 2009.
Sample Availability: Samples of the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).