Antioxidant properties of phenolic Schiff bases: Structure-activity relationship and mechanism of action

Article abstract of DOI:10.1007/s10822-013-9692-0

Phenolic Schiff bases are known for their diverse biological activities and ability to scavenge free radicals. To elucidate (1) the structure-antioxidant activity relationship of a series of thirty synthetic derivatives of 2-methoxybezohydrazide phenolic Schiff bases and (2) to determine the major mechanism involved in free radical scavenging, we used density functional theory calculations (B3P86/6-31+(d,p)) within polarizable continuum model. The results showed the importance of the bond dissociation enthalpies (BDEs) related to the first and second (BDE_d) hydrogen atom transfer (intrinsic parameters) for rationalizing the antioxidant activity. In addition to the number of OH groups, the presence of a bromine substituent plays an interesting role in modulating the antioxidant activity. Theoretical thermodynamic and kinetic studies demonstrated that the free radical scavenging by these Schiff bases mainly proceeds through proton-coupled electron transfer rather than sequential proton loss electron transfer, the latter mechanism being only feasible at relatively high pH.

Full text of DOI:10.1007/s10822-013-9692-0

J Comput Aided Mol Des (2013) 27:951–964
DOI 10.1007/s10822-013-9692-0
Antioxidant properties of phenolic Schiff bases: structure–activity
relationship and mechanism of action
•
El Hassane Anouar Salwa Raweh Imene Bayach Muhammad Taha
•
•
•
•
Mohd Syukri Baharudin Florent Di Meo Mizaton Hazizul Hasan
•
•
•
•
•
Aishah Adam Nor Hadiani Ismail Jean-Frederic F. Weber Patrick Trouillas
´ ´
Received: 12 October 2013 / Accepted: 9 November 2013 / Published online: 16 November 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Phenolic Schiff bases are known for their
diverse biological activities and ability to scavenge free
radicals. To elucidate (1) the structure–antioxidant activity
relationship of a series of thirty synthetic derivatives of
2-methoxybezohydrazide phenolic Schiff bases and (2) to
determine the major mechanism involved in free radical
scavenging, we used density functional theory calculations
(B3P86/6-31?(d,p)) within polarizable continuum model.
The results showed the importance of the bond dissociation
enthalpies (BDEs) related to the first and second (BDEd)
hydrogen atom transfer (intrinsic parameters) for rational-
izing the antioxidant activity. In addition to the number
of OH groups, the presence of a bromine substituent
plays an interesting role in modulating the antioxidant
activity. Theoretical thermodynamic and kinetic studies
demonstrated that the free radical scavenging by these
Schiff bases mainly proceeds through proton-coupled
electron transfer rather than sequential proton loss electron
transfer, the latter mechanism being only feasible at rela-
tively high pH.
Keywords Schiff bases ꢀ Antioxidant ꢀ DFT ꢀ
Kinetics ꢀ Free radical scavenging ꢀ BDE ꢀ
Structure–activity relationship
Introduction
Natural and synthetic phenols including flavonoids, phenolic
acids, stilbenoids and curcuminoids have been described as
powerful antioxidants, being able to efficiently scavenge
free radicals [1–3]. Schiff bases form an important class of
synthetic phenolic compounds, substituted by a hydrazone
Electronic supplementary material The online version of this
article (doi:10.1007/s10822-013-9692-0) contains supplementary
material, which is available to authorized users.
N. H. Ismail
Faculty of Applied Sciences, Universiti Teknologi MARA,
40450 Shah Alam, Malaysia
E. H. Anouar (&) ꢀ S. Raweh ꢀ I. Bayach ꢀ M. Taha ꢀ
M. S. Baharudin ꢀ N. H. Ismail ꢀ J.-F. F. Weber
Atta-ur-Rahman Institute for Natural Product Discovery (RiND),
Universiti Teknologi MARA, Kampus Puncak Alam,
42300 Bandar Puncak Alam, Shah Alam, Selangor DE, Malaysia
e-mail: anouarelhassane@yahoo.fr
P. Trouillas
Laboratoire de Chimie des Materiaux Nouveaux, Universite de
´
Mons, Place du Parc, 20, 7000 Mons, Belgium
´
S. Raweh ꢀ M. H. Hasan ꢀ A. Adam ꢀ J.-F. F. Weber
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak
Alam Campus, Shah Alam, Malaysia
P. Trouillas
Regional Center of Advanced Technologies and Materials,
Department of Physical Chemistry, Faculty of Science, Palacky
University, 17. Listopadu 1192/12, 77146 Olomouc, Czech
Republic
´
I. Bayach
LCSN-Faculte de Pharmacie, Universite de Limoges,
´
2 rue du Dr Marcland, 87000 Limoges, France
´
F. Di Meo ꢀ P. Trouillas (&)
´
´
INSERM, UMR-S850/Faculte de Pharmacie, Universite de
Limoges, 2 rue du Dr Marcland, 87000 Limoges, France
e-mail: patrick.trouillas@unilim.fr
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J Comput Aided Mol Des (2013) 27:951–964
(iv) AF (Adduct Formation)
ArXꢁH þ Rꢀ ! ½ArXHꢁRꢂꢀ! metabolites or ArXꢀ þ RH
moiety. They are known for their various fields of applica-
tion including inorganic chemistry, biological and analytical
chemistry [4, 5]. Several studies focused on their biological
activities as antibacterial [6–9], anticancer [5], and anti-
fungal activities [10, 11]. Recently, we reported the antile-
ishmanial activity of the series of synthetic Schiff bases
studied in the present work (Fig. 1) [12]. Schiff bases also
showed potent antioxidant activity to scavenge free radicals.
In two recent studies, we reported the antioxidant activity of
acylhydrazide and 2,4,6-trichlorophenylhydrazine Schiff
bases as DPPH radical and super oxide anion scavengers
[13, 14]. Free radical scavenging capacity of (poly)phenols
is generally attributed to the hydrogen atom lability of the
OH groups attached to aromatic rings (Ar) [3, 15–17];
however in some other antioxidants, NH and SH groups may
provide labile hydrogen [18–24]. Antioxidants (ArX–H)
may scavenge free radicals (Rꢀ) by H atom transfer through
one of the four mechanisms:
ð6Þ
This mechanism is relatively specific to ꢀOH free radicals.
It has been observed in radiolytic solutions. The radical may
add on double bonds and aromatic rings. This mechanism is
not considered in this work. As often shown in the literature,
intrinsic parameters including BDE, IP and electron transfer
enthalpy rationalize free radical scavenging capacities [3,
15, 16, 31] as the thermodynamic balance of the first three
mechanisms is the same (DGCPET = DGET–PT = DGSPLET).
However to tackle the mechanisms of action, kinetic
parameters should be considered [3, 24, 29, 32–34].
In the present study, we elucidate the structure–antioxi-
dant activity relationship for a series of 30 synthetic deriv-
atives of 2-methoxybezohydrazide phenolic Schiff bases
(Fig. 1). The Schiff bases differ from the nature of the
R-(aromatic) moiety attached to the nitrogen atom engaged
in the –N=C double bond (Fig. 1). The compounds can be
subdivided into three classes, namely (1) compounds 1–25,
where the aromatic ring is a benzene substituted with dif-
ferent OH, OCH3, halogen, COOCH3 and NO2 groups, (2)
compounds 26–28, in which R is an unsubstituted pyridine
attached at the three different possible positions and (3)
compounds 29–30, in which R is an unsaturated five-
membered ring containing one heteroatom. The positive or
negative contributions of different descriptors (e.g., number
and position of OH groups, BDE, double (BDEd), IP, spin
density distribution, intramolecular hydrogen bonds, pre-
sence of electron donating or withdrawing groups, and
structural parameters) have been examined. The elucidation
of the mechanism of action is based on a combined exper-
imental and theoretical approach. For the theoretical
approach, transition state and Marcus–Levich–Jortner theo-
ries were used to evaluate atom (PCET mechanism) and
electron transfer (SPLET mechanism), respectively.
(i) Hydrogen atom transfer (HAT) and proton-coupled
electron transfer (PCET)
ArXꢁH þ Rꢀ ! ArXꢀ þ RꢁH
ð1Þ
This is the direct HAT, which is purely governed by the
homolytic bond dissociation enthalpy (BDE) of X–H; note
that the lower the BDE, the more important the role of the
corresponding XH group in the antioxidant activity. PCET
is distinguished from the pure HAT as it involves several
molecular orbitals in an H-bonding pre-reaction complex
[25–28]. The proton transfer is coupled to the electron
transfer that occurs from a lone pair of the antioxidant to
the singly occupied molecular orbital (SOMO) of the free
radical.
(ii) ElectronTransfer–ProtonTransfer(ET–PT)orSequential
ET–PT (SET–PT)
ArXꢁH þ Rꢀ ! ArXHþꢀ þ Rꢁ ! ArXꢀ þ RꢁH
ð2Þ
The first step of this reaction leads to the formation of
the radical cation ArXH?ꢀ, which easily undergoes
heterolytic dissociation of X-H bond leading to the same
final products than those yielded by PCET. The first step is
mainly governed by the ionization potential (IP) of the
antioxidant.
Methodology
Experimental synthesis
(iii) Sequential Proton-Loss-Electron-Transfer (SPLET)
The N-benzylidene-2-methoxybenzohydrazide phenolic
derivatives were synthesized by refluxing mixtures of
2 mmol 2-methoxy-benzohydrazide and different alde-
hydes in methanol and in the presence of catalytical
amount of acetic acid for 3 h, according to the previously
described method [12]. The reaction progress was moni-
tored by thin layer chromatography (TLC); after comple-
tion, the solvent was evaporated under vacuum to afford
the crude products, which were further re-crystallized in
ArXꢁH ! ArOꢁ þ Hþ
ArXꢁ þ Rꢀ ! ArXꢀ þ Rꢁ
Rꢁ þ Hþ ! RH
ð3Þ
ð4Þ
ð5Þ
In this mechanism, a proton is lost prior to electron transfer
from the subsequent anion to the free radical. SPLET is
strongly favoured under alkaline conditions, which may help
in the proton loss of the first step [29, 30].
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J Comput Aided Mol Des (2013) 27:951–964
953
R3
Fig. 1 Structures of the
synthesized Schiff bases
R2
R4
R5
3
2
OCH3
O
4
1
N
5
N
6
R6
H
Monohydroxylated
Monomethoxylated
11: R3 = OCH3
Halogenated
1: R2 = OH
2: R3 = OH
3: R4 = OH
18: R3 = Br, R4 = OH
19: R3 = Br, R4 = Cl
20: R4 = F
12: R4 = OCH3
Dimethoxylated
21: R4 = Cl
Dihydroxylated
4: R2 = R3 = OH
5: R2 = R4 = OH
6: R2 = R5 = OH
7: R3 = R4 = OH
8: R3 = R5 = OH
13: R3 = R4 = OCH3
14: R3 = R5 = OCH3
22: R2 = I, R3 = OH, R4 = OCH3
Other substitution
23: R4 = COOCH3
24: R4 = NO2
1-OH and 1-OCH3
15: R2 = OH, R4 = OCH3
16: R2 = OH, R5 = OCH3
17: R3 = OH, R4 = OCH3
No substitution
Trihydroxylated
25: Ri = H (i = 2 to 6)
9: R2 = R4 = R6 = OH
10: R3 = R4 = R5 = OH
N
OCH3
O
N
OCH3
O
N
OCH3
O
N
N
N
N
N
H
N
H
H
26
27
28
OCH3
O
O
OCH3
O
S
N
N
N
H
N
H
29
30
General computational details
methanol. Needle-like pure products in good to excellent
[0.40 g (78 %)–0.58 (92 %)] yields were obtained [12].
Geometry optimization and frequency calculations have been
carried out using density functional theory (DFT) as imple-
mented in Gaussian09 [36]. To elucidate the structure–anti-
oxidant activity relationship of polyphenols, the hybrid
functional B3P86 appears reliable [15, 37, 38]. Increasing the
number of polarization and diffuse functions have no signifi-
cant effects on theoretical results, especially on BDEs and IPs
[3, 15], therefore the calculations were performed with the
6-31?G(d,p) basis set. The ground states (GSs) geometries
were confirmed by the absence of any imaginary frequency,
while for the transition states (TSs) of PCET one imaginary
frequency was obtained, which corresponds to H atom transfer.
It has been shown that the B3P86 hybrid functional underes-
timates activation energy (DG#) especially for hydrogen atom
transfer reactions [29, 39]; in this course, MPWB1K appears
more recommended even if overestimation are sometimes
observed [40, 41]. The kinetic calculations of the PCET
mechanism were performed with both (U)B3P86/6-31?G
(d,p) and MPWB1K/6-31?G(d,p) methods.
DPPH free radical scavenging capacity
The antioxidant capacity was evaluated as the capacity of
the 30 different compounds to scavenge the 1,1-diphenyl-
2-picrylhydrazil (DPPH) free radical, following the Blois’
method [35]. The reaction mixture contains 5 lL of test
sample (1 mM in DMSO) and 95 lL of DPPH (Sigma,
300 lM) in ethanol; it was placed into a 96-well microtiter
plate and incubated at 37 °C for 30 min. The UV/Vis light
absorbance was measured at 515 nm on microtiter plate
reader (Molecular Devices, CA, and USA). The percentage
of free radical scavenging was determined with respect to
DMSO control. Following free radical scavenging versus
compound concentration, IC50 was measured, which rep-
resent the concentration of compounds that scavenge 50 %
of DPPH radicals (i.e., 50 % of absorbance at 515 nm).
Propyl gallate was used as a positive control. All chemicals
used were of analytical grade (Sigma, USA).
123

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J Comput Aided Mol Des (2013) 27:951–964
Solvent effects were taken into account implicitly using
successful approach to circumvent the use of expensive
post-HF methods [45–47]. The B3P86-D2 functional was
recently re-parameterized, reaching accuracy to evaluate
dispersion effects in the non-covalent complexes involving
natural polyphenols [48]. The geometries of the pre-reac-
tion complexes were obtained at the B3P86-D2/Def2-
TZVPP level of theory for all possible H-bond and [m–p]
complexes, i.e., either towards the OH groups or towards
aromatic and N–N p-bonds. All calculations were carried
out using Gaussian09 [36], Orca 2.8.1 [49] and NWCHEM
6.1.1 [50] packages for PCET, SPLET and electronic
coupling calculations, respectively. When available, the
RIJCOSX (resolution of identity and chain-of-sphere)
approximations were used, allowing large speed-up of
calculations for a minimal error [51].
the polarizable continuum model (PCM). In PCM models,
the substrate is embedded into a shape-adapted cavity
surrounded by a dielectric continuum characterized by its
dielectric constant (eMeOH = 32.613) [42]. Using an
explicit solvent were investigated by other authors [43],
confirming that PCM succeeded in providing a reasonably
accurate description of BDE, the main descriptor of anti-
oxidant capacity. Hybrid models (i.e., one or two mole-
cules in the surrounding of the OH groups ? PCM) were
also tested for quercetin, an antioxidant, showing only
slight differences in terms of BDE when compared to pure
PCM calculations, while computational time was dramat-
ically increased [16].
Pre-reaction complexes, descriptions of non-covalent
interactions and electron transfer kinetics
Results and discussion
The rate constants of electron transfer (i.e., for ET–PT and
SPLET mechanism) were evaluated within the Marcus–
Levich–Jortner (kLJ–Marcus) formalism according the fol-
Experimentally-based (DPPH scavenging)
structure–activity relationship
lowing expression:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
As usually observed for polyphenols, at least one phenolic
OH group is required to provide an active compound
(namely, 1–10, 15–18, 22 as seen in Table 1); the other
compounds without OH group are inactive (namely, 11–14,
19–21, 23–30). The position of OH groups is of crucial
importance to modulate antioxidant capacity. Based on the
measured IC50 values (Table 1), the active phenolic Schiff
bases can be divided into three classes, depending on the
number and position of the OH groups.
4p2
1
kLJꢁMarcus
¼
ꢀ V2RP
h
4pkSkbT
(
"
#)
À
Á
2
DGꢄ þ kS þ t0 ꢀhx
4kSkbT
0
St
X
ꢃ
ꢀ expðꢁSÞ ꢀ exp ꢁ
t0 !
t0
where DG° is the Gibbs energy difference of ET reactions
(i.e., Eqs. (2) or (4) for SET–PT and SPLET, respectively),
kS is the outer-shell reorganisation energy attributed to the
solvent, VRP is the electronic coupling, S is the Huang-Rhys
factor and m’ is the vibrational quantum number. The sum
runs over all effective vibrational modes, namely in our case
aromatic C–C, phenolic C–O and C–N bond stretching.
These bonds are the most probable reaction coordinates
involved in electron transfer. DG°, kS, S were calculated
following the previous study on free radical scavenging of
quercetin [29]. VRP was calculated from the pre-reaction
complex geometries. They were calculated following the
Farazdel et al. [44] approach with a Def2-TZVP basis set:
(i) One OH-group compounds (compounds 1–3, IC50
ranging from 0.6 to 1.1 lg/mL)—In this case, com-
pound 3 with a para-OH group is a stronger antiox-
idant than 1 having an ortho-OH group, being as well
more active than 2 with a meta-OH group.
(ii) Two OH-group compounds (compounds 4–8, IC50
ranging from 0.2 to 0.9 lg/mL)—The following order
in terms of antioxidant activity is observed
7 & 4 \5 \ 8 & 6. In the active phenolic Schiff
bases 7 and 4, the two OH groups are ortho to each
other, which makes the compounds particularly active
(IC50 about 0.2 lg/mL). This positive contribution of
the catechol moiety is in agreement with the general
literature on the structure–antioxidant activity relation-
ship of polyphenols [1, 3, 15, 16]. Compound 5, having
two OH groups meta to each other, one being in para
with respect to the N=C double bond is less active.
Compounds 6 and 8, having two OH groups meta to
each other (no OH group at C4), are even much less
active (Table 1). This highlights the importance of the
para substitution in the benzene moiety.
ðERþEPÞ
HRP ꢁ SRP
2
VRP
¼
1 ꢁ S2RP
where HRP is the total reactant–product interaction energy,
SRP is the reactant–product overlap, ER and EP are the
electronic energies of reactants and products, respectively.
Pre-reaction complexes are of major importance in ET
mechanisms since they drive the bimolecular mechanisms.
These complexes involved either H-bond or [m–p] disper-
sive interactions which are poorly described by classical
hybrid functionals. Dispersion-corrected DFT-D is a
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J Comput Aided Mol Des (2013) 27:951–964
955
Table 1 BDEs, IPs, spin density on the oxygen atom from which the hydrogen atom is removed, as obtained with PCM-B3P86/6-31?G (d,p),
and experimental IC50 of the 30 Schiff bases
Schiff base
BDEd (Kcal/mol)
IP (eV)
Spin densitya
IC50 (lg/mL)
NH
2-OH
3-OH
4-OH
5-OH
6-OH
1
96.5
97.7
95.7
96.7
94.7
96.4
95.8
97.9
94.8
95.7
97.7
95.6
95.6
97.7
94.7
97.2
95.5
96.6
99.0
97.4
97.7
–
83.8
–
–
–
–
–
–
–
–
–
–
–
–
84.9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6.4
6.6
6.3
6.5
6.2
6.3
6.2
6.6
6.1
6.2
6.6
6.3
6.2
6.6
6.2
6.2
6.2
6.4
6.7
6.6
6.6
–
0.27
0.37
0.32
0.31
0.25
0.31
0.25
0.34
0.23
0.27
0.50
0.46
–
0.90 0.045
1.1 0.05
0.65 0.045
0.22 0.045
0.34 0.045
0.92 0.045
0.20 0.045
0.91 0.0045
0.35 0.045
0.30 0.045
No activity
[[2
2
87.1
–
–
3
–
83.2
–
4
77.5
82.4
78.8
–
79.7
–
5
–
82.2
76.1
–
6
–
82.2
7
78.8
–
8
–
86.6
–
87.4
9
83.9
83.0
72.3
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
a
80.2
–
81.68
–
–
–
–
–
–
–
–
–
–
[[2
–
–
–
–
0.50
0.28
0.31
0.33
0.26
0.53
0.49
0.49
–
[[2
84.1
79.2
–
–
–
0.50 0.071
1.01 0.045
0.8 0.002
0.22 0.0045
[[2
–
–
–
86.0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
82.2
–
–
–
–
–
–
–
[[2
–
–
–
[[2
–
–
–
1.63 0.21
[[2
98.6
99.6
97.5
98.4
98.5
99.4
94.9
95.1
–
–
–
6.8
7.0
6.6
6.9
6.8
7.0
6.4
6.4
0.45
0.50
0.51
0.57
0.51
0.54
0.45
0.44
–
–
–
[[2
–
–
–
[[2
–
–
–
[[2
–
–
–
[[2
–
–
–
[[2
–
–
–
[[2
–
–
–
[[2
Spin density of the active OH site
(iii) Three OH-group compounds (compounds 9–10)—
Compound 10 is a stronger antioxidant than 9, is
particularly due to the adjacent three OH groups.
to both 17 (3-OH, 4-OCH3), and 18 (3-Br, 4-OH). The
methoxylation significantly reduces the antioxidant activity
(Table 1), while interestingly the bromine at C3 makes
compound 18 as active as 7 (having the active catechol
moiety).
The main trend observed here is that compounds of the
first class are less active than those of the other two classes.
Moreover the presence of two OH groups ortho to each
other and one OH group para is of crucial importance to
increase the activity. An additional third OH group is not
mandatory to increase the antioxidant capacity, as already
observed for myricetin with respect to quercetin [1].
Another interesting feature derived from this series of
compounds concern the effect of bromination and meth-
oxylation of phenolic ring. To emphasize this effect on the
antioxidant activity, compound 7 (3-OH, 4-OH) is compared
Theoretical rationalization
The free radical scavenging capacity has been extensively
correlated to O–H BDEs, rationalized by spin density dis-
tribution and stability of the phenoxyl radical (ArXꢀ with
X=O) formed after HAT. In principle, N–H BDE may also
correlate with the free radical scavenging capacity, since
the hydrogen of these groups may be labile according to the
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J Comput Aided Mol Des (2013) 27:951–964
Fig. 2 Spin density distribution
of the radicals obtained after
first HAT transfer (a) 2-OH,
3-OH, 4-OH groups for
compounds 1, 2 and 3 (b) 2-OH,
4-OH, 2-OH, 4-OH and 3-OH
groups for compounds 4, 5, 6, 7
and 8 (c) 3-OH and 4-OH
groups for compounds 17 and
18
chemical neighboring. The inactive compounds (11–14,
19–21, 23–30) having no OH group, and thus no O–H
BDE, could have only been active due to the NH group.
However all N–H BDEs of this series of compounds are
higher than 90 kcal/mol (Table 1), making these com-
pounds inefficient to scavenge DPPH. Indeed, the BDE of
DPPH-H is ca. 80 kcal/mol [38, 52], thus for these com-
pounds the thermodynamic balance of Eq. 1 is positive
with DPPH.
In the first class of active compounds (1–3), the low
antioxidant activity of 2 is in agreement with the relatively
high BDE of the ortho-2-OH group (87.1 kcal/mol)
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957
Table 2 BDEd and IPd of Schiff bases, as calculated with PCM-B3P86/6-31?G(d,p)
Schiff base
BDEd (Kcal/mol)
NH 2-OH
88.7
IC50 (lg/mL)
3-OH
4-OH
5-OH
6-OH
IPd (eV)
1-(2-OH)
2-(3-OH)
3-(4-OH)
4-(2-OH)
5-(4-OH)
6-(2-OH)
7-(4-OH)
8-(3-OH)
9-(4-OH)
10-(4-OH)
15-(2-OH)
16-(2-OH)
17-(3-OH)
18-(4-OH)
22-(3-OH)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6.5
7.1
6.4
6.5
6.4
6.2
6.2
7.1
6.4
6.2
6.4
6.1
6.3
6.4
–
0.90 0.045
1.1 0.05
112.1
86.1
91.8
84.9
90.7
87.3
113.3
84.6
88.1
86.3
91.8
100.6
86.8
–
–
–
–
–
–
–
–
–
0.65 0.045
0.22 0.045
0.34 0.045
0.92 0.045
0.20 0.045
0.91 0.0045
0.35 0.045
0.30 0.045
0.50 0.071
1.01 0.045
0.8 0.002
0.22 0.0045
1.63 0.21
–
76.7
–
–
94.1
–
–
–
–
–
68.8
–
–
75.9
–
–
–
–
107.9
–
95.0
–
–
–
83.6
–
76.6
–
79.1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
compared to those of the meta-3-OH and para-4-OH
groups (83.8 and 83.1 kcal/mol, respectively). The BDE is
related to the capacity of the phenoxy radical (ArOꢀ)
formed after HAT (Eq. 1) to stabilize by p-conjugation.
The higher BDE obtained for the 2-OH group is attributed
to a lower spin density delocalization in the corresponding
ArOꢀ (Fig. 2a-middle) compare to the better p-conjugation
observed when HAT occurs at 3-OH (Fig. 2a-left and
right). Regarding both compounds 1 and 3, the BDEs are
similar (difference in BDE ca. 0.5 kcal/mol as seen in
Table 1), while the latter compound is more active as free
radical scavenger (Table 1). This shows that BDE cannot
be the only descriptor to fully rationalize slight antioxidant
activities. In this case, the spin density distribution better
explain this difference i.e., ArOꢀ obtained after HAT from 3
exhibits a better electron delocalization (Fig. 2a-right). In
next section this is even better rationalized with another
secondary descriptor.
importance of the catechol moiety in the antioxidant
activity.
In the third class, the Schiff base 10 is more active than
9. This is attributed to the low BDE of 4-OH in 10
(72.3 kcal/mol) (Table 1).
The decrease in free radical scavenging capacity
observed from 7 to 17 (after methylation of 4-OH) is
simply attributed to the loss of the most active group
[having the lowest BDE and no delocalization of spin
density (Fig. 2c-left)]. When methylation occurs on the
other OH groups, the effects on BDE is lower and the free
radical scavenging capacity is not significantly reduced
(Table 1).
The substitution of position 3 by a bromine (compound
18) slightly decreases the 4-OH BDE compare to com-
pound 3, having only one OH group at C4 (Table 1) and
increases spin delocalization in the related phenoxy radical
compare to compound 7 (Fig. 2c-right). This agrees per-
fectly with a relatively good free radical scavenging
capacity (Table 1), showing the role of bromine substitu-
tion in enhancing the antioxidant activity.
In the second class of active compounds (4–8), the role
of the catechol moiety (compounds 4 and 7) is rationalized
by the low BDE values, namely 76.1 and 77.5 kcal/mol for
2-OH (4) and 4-OH (7), respectively. The low BDEs
obtained in the catechol moiety is attributed to the stabil-
ization of the corresponding ArOꢀ by spin delocalization
(Fig. 2b) and intramolecular H-bonding. In compound 7,
the 4-OH group has a lower BDE (76.1 kcal/mol) than the
3-OH group (78.8 kcal/mol), in agreement with the better
spin density delocalization when HAT occurs from the
former group (Fig. 2b). Compare to 4 and 7, the Schiff
bases 5, 6 and 8 exhibit higher BDEs, as correlated by the
higher IC50 values (Table 1). This again exemplified the
Double BDE analysis
The free radical scavenging capacity can be rationalized by
HAT from one of the active OH group, but it has also been
explained that a second HAT may occurs from active
compounds (to scavenge a second free radical). This has
been rationalized by the double BDE (BDEd) descriptor. In
case, the calculated BDEs have similar values, BDEd may
appear as an efficient descriptor to rationalize slight
123

958
J Comput Aided Mol Des (2013) 27:951–964
Table 3 Gibbs energies of the HAT and ET processes (DGHAT and DGET) of Schiff bases with the DPPH and CH3OOꢀ free radical, as obtained
with B3P86/6-31?G (d,p)
Schiff base
HAT mechanism
ET mechanism
DGHAT with DPPH
DGHAT with CH3OOꢀ
Gas Solvent
DGET with DPPH
DGET with CH3OOꢀ
Gas
Solvent
Gas
Solvent
Gas
Solvent
(a) First HAT
1
2.9
7.5
10.8
6.8
-1.6
-0.5
2.8
88.0
26.3
29.4
23.8
26.2
21.9
24.1
21.7
29.2
21.4
23.5
28.8
–
143.1
146.5
141.0
142.6
137.1
140.5
139.5
146.4
134.2
138.7
144.5
137.9
143.4
142.7
134.8
138.6
136.4
143.5
150.8
148.8
148.6
–
45.7
48.7
-1.2
45.6
41.3
43.5
41.1
48.6
40.8
42.9
48.1
–
2
7.1
4.1
2.6
-0.4
-8.6
-3.5
-7.8
-9.1
1.7
91.3
85.8
87.4
81.9
85.4
84.4
91.2
79.1
83.6
89.4
82.8
79.2
87.6
79.7
83.5
81.3
88.4
95.7
93.7
93.5
–
3
-1.2
-6.5
-2.3
-5.6
-7.9
2.3
4
-4.1
0.9
1.5
5
5.7
6
-1.3
-4.6
6.2
2.3
7
0.2
8
10.3
6.6
9
2.7
-1.8
-12.6
11.3
10.0
10.3
11.4
-0.7
-5.7
0.8
-1.4
-10.7
12.5
10.4
10.2
12.4
-0.4
-8.0
1.3
10
-8.1
15.8
14.5
14.8
15.9
7.4
-2.7
20.5
18.5
18.2
20.4
7.7
11
12
13
20.7
28.1
20.0
21.0
20.1
25.9
32.3
30.0
30.5
–
40.4
47.5
39.3
40.4
39.4
39.8
51.6
49.4
49.8
–
14
15
16
-1.2
5.4
0.0
17
9.3
18
14.9
16.0
15.8
16.0
–
6.0
10.5
11.5
11.3
11.5
–
-2.1
13.0
12.4
12.7
–
19
21.0
20.4
20.7
–
20
21
22
23
16.3
17.1
15.8
14.6
16.3
16.2
13.7
13.6
21.3
21.2
20.3
20.8
21.2
21.7
18.0
18.2
11.8
12.6
11.4
10.1
11.8
11.8
9.2
13.3
14.2
12.4
12.8
13.2
13.7
10.0
10.2
95.6
104.2
92.9
97.3
98.1
103.3
88.5
88.5
33.2
37.4
30.2
35.2
33.7
38.2
24.5
25.0
150.7
159.3
148.0
152.4
153.2
158.4
143.6
143.7
52.5
56.8
49.5
54.6
53.0
57.6
43.8
44.3
24
25
26
27
28
29
30
9.1
(b) Second HAT
1
7.0
33.5
10.2
-0.4
18.8
-12.5
-0.8
32.3
5.3
11.9
35.2
9.4
1.0
27.5
-1.6
21.7
91.8
106.9
90.3
92.0
88.7
84.5
85.8
107.1
87.6
85.3
88.7
28.0
42.5
25.7
29.5
25.0
21.4
23.4
43.9
26.7
21.2
27.5
146.9
161.9
145.4
147.0
143.7
139.5
140.8
162.1
142.6
140.3
143.7
42.0
56.5
39.7
43.4
38.9
35.4
37.3
57.8
40.6
35.2
41.5
2
3
4.2
-3.9
-14.0
4.6
4
-0.5
18.1
-7.6
-0.6
30.3
9.3
-6.4
12.8
5
6
-18.5
-6.8
26.3
-21.0
-14.1
16.9
7
8
9
-0.8
-6.5
-0.5
-4.2
-13.4
-3.7
10
15
-0.5
5.5
0.1
9.7
123

J Comput Aided Mol Des (2013) 27:951–964
959
Table 3 continued
Schiff base
HAT mechanism
ET mechanism
DGHAT with DPPH
DGHAT with CH3OOꢀ
Gas Solvent
DGET with DPPH
DGET with CH3OOꢀ
Gas
Solvent
Gas
Solvent
Gas
Solvent
16
17
18
10.1
15.7
24.2
9.8
4.1
2.3
10.8
81.7
22.1
25.1
26.5
136.7
142.1
145.4
36.1
39.1
40.5
21.6
3.9
15.6
87.1
90.4
-2.1
-3.6
Table 4 Free energies of activation (DG#) and rate constants (kTST, kTST/W and kTST/ST) of HAT mechanism of the active Schiff bases with the
CH3OO (Rꢀ) free radical, as obtained with hybrid functionals B3P86 and MPWB1K
No. of OH in
aromatic ring
ArO-H ? Rꢀ ? ArOꢀ ?
RH (ArO-H = 1–10)
DG#
KTST
KTST/W
KTST/ST
B3P86
1-OH
1
2
12.26
15.09
11.99
10.73
11.47
9.39
6.5 9 103
5.5 9 10
1.0 9 104
8.6 9 104
2.5 9 104
8.3 9 105
2.2 9 106
1.9 9 102
2.2 9 104
2.2 9 104
2.9 9 104
2.2 9 102
5.2 9 104
2.3 9 105
1.2 9 105
3.3 9 106
9.3 9 106
1.1 9 103
1.0 9 105
3.9 9 104
6.5 9 103
5.5 9 10
1.0 9 104
8.6 9 104
2.5 9 104
8.3 9 105
2.2 9 106
1.9 9 102
2.2 9 104
2.2 9 104
3
2-OH
4
5
6
7
8.82
8
14.36
11.55
11.53
3-OH
9
10
MPWB1K
1-OH
1
2
22.44
23.76
23.20
22.00
22.26
17.97
18.10
24.16
21.09
20.02
2.3 9 10-4
2.5 9 10-5
6.4 9 10-5
4.8 9 10-4
3.1 9 10-4
4.3 9 10-1
3.5 9 10-1
1.3 9 10-5
2.2 9 10-3
1.4 9 10-2
1.0 9 10-3
1.0 9 10-4
3.2 9 10-4
1.3 9 10-3
1.5 9 10-3
1.7 9 10
2.3 9 10-4
2.5 9 10-5
6.4 9 10-5
4.8 9 10-4
3.1 9 10-4
4.3 9 10-1
3.5 9 10-1
1.3 9 10-5
2.2 9 10-3
1.4 9 10-2
3
2-OH
3-OH
4
5
6
7
1.5 9 10
8
7.1 9 10-5
1.1 9 10-2
2.3 9 10-2
9
10
Table 5 Structural parameters of the transition state of active Schiff bases and the peroxyl CH3OOꢀ obtained with B3P86 functional
˚
Distance (A)
Schiff base
Angle (°)
Torsional angle (°)
Ca–Oa
Oa–Ha
Ha–Or
Or–Or
Ca–Oa–Ha
Oa–Ha–Or
Ha–Or–Or
Or–Or–Cr
Ca–Oa–
Ha–Or
Oa–Ha–
Or–Or
Ca–Oa–
Or–Or
1
2
3
4
5
6
7
1.302
1.311
1.304
–
1.181
1.161
1.139
–
1.204
1.214
1.257
–
1.378
1.378
1.380
–
120
115
119
–
169
167
170
–
105
104
105
–
110
109
109
–
180
94
-180
-102
179
0
-10
-179
–
0
–
0
0
0
–
1.305
1.313
1.314
1.127
1.103
1.110
1.275
1.307
1.295
1.381
1.376
1.370
119
118
118
170
169
171
106
106
106
109
109
109
-180
180
-180
180
-180
180
123

960
J Comput Aided Mol Des (2013) 27:951–964
Table 5 continued
˚
Distance (A)
Schiff base
Angle (°)
Torsional angle (°)
Ca–Oa
Oa–Ha
Ha–Or
Or–Or
Ca–Oa–Ha
Oa–Ha–Or
Ha–Or–Or
Or–Or–Cr
Ca–Oa–
Ha–Or
Oa–Ha–
Or–Or
Ca–Oa–
Or–Or
8
1.304
1.305
–
1.173
1.126
–
1.212
1.278
–
1.380
1.382
–
120
118
–
170
170
–
105
106
–
109
109
–
167
-178
–
-170
178
–
-3
0
9
10
–
a: refers to the atom of antioxidant, r: refers to atom of peroxyl radical
Table 6 Relative energy stabilization (DE) of both studied anion 7-[3H?] and 7-[4H?], internal ki and external ks reorganization energies
(kcal mol-1), electronic coupling VRP (kcal mol-1), Gibbs energy of the reaction DG° (kcal mol-1), SPLET rate constants k (M-1 s-1) for all
H-bond and [m–p] complexes
Anion
DE
0.00
Conformation
ki
ks
VRP
DG°
kSPLET
7-[3H?]
[m–p]Abot
[m–p]Atop
[m–p]Bbot
[m–p]Btop
[HB-4]
14.78
14.78
14.78
14.78
14.78
14.08
14.08
14.08
14.08
14.08
20.22
14.30
4.31
0.12
1.53
1.45
0.27
1.54
3.71
4.61
3.90
1.50
0.43
21.96
21.89
21.53
21.70
22.01
21.90
21.83
22.18
21.68
21.98
1.4 9 10-6
5.6 9 10-5
3.0 9 10-17
1.5 9 10-30
5.7 9 10-5
1.7 9 10-3
2.6 9 10-3
1.9 9 10-8
9.3 9 10-5
5.2 9 10-6
2.70
14.32
18.33
25.00
7.29
7-[4H?]
-2.60
[m–p]Abot
[m–p]Atop
[m–p]Bbot
[m–p]Btop
[HB-3]
14.61
13.85
Fig. 3 Geometries of the PCET
transition states between the
Schiff bases 4 (left) and 7
(right), and the peroxyl radical
CH3OOꢀ
123

J Comput Aided Mol Des (2013) 27:951–964
961
nature of the free radical itself. In terms of thermodynamics,
the radical scavenging depends on the Gibbs energy of
Eq. 1. Here we focus on DPPH and a prototype of peroxy
radical (CH3OOꢀ). The free radical scavenging of DPPH
usually correlate relatively well with that of peroxy radicals
even if both radicals are chemically different [31]. The
thermodynamic balance is calculated for both reactions:
ArOꢁH þ DPPHꢀ ! ArOꢀ þ DPPHꢁH
ð10Þ
ArOꢁH þ CH3OOꢀ ! ArOꢀ þ CH3OOꢁH
ð100Þ
In order to tackle the mechanism of action of free radical
scavenging, the kinetics of the limiting step should be
evaluated. Here only the kinetics of CH3OOꢀ scavenging
was evaluated. Regarding the large Gibbs energy of the ET
step in the SET–PT process (higher than 40 kcal/mol as
seen in Table 4), this mechanism appears unlikely, as
usually described in the literature [3]. The competition may
occur between PCET and SPLET.
Fig. 4 Correlation between BDE and free activation energy (DG#)
for peroxyl radical scavenging by active Schiff bases (1–10)
variations of the antioxidant activity. BDEd appeared par-
ticularly adequate to differentiate the antioxidant activity
of a series of synthetic oligomers of guaiacol [3]. BDEd
values of the second and third classes (two and three OH
groups, respectively) of Schiff bases are reported in
Table 2. To highlight the potent role of this descriptor,
compounds 8 and 17 were considered. After HAT from
20-OH (having very similar BDEs in both compounds), the
BDEds from the NH group are 113.3 and 100.6 kcal/mol
(Table 2), respectively, which agrees with the better
activity for the latter compound. Another example concerns
compounds 1 and 3, for which the O–H BDEs are very
similar (difference lower than 0.6 kcal/mol, as seen in
Table 1) while the N–H BDEd difference is 2.6 kcal/mol in
favor of compound 3 (Table 2), in agreement with the
better antioxidant activity of 3 (Table 3).
The former mechanism is described by the transition
state theory. Tables 5 and 6 report the rate constants, Gibbs
energies of activation and structural parameters of transi-
tion states (distance, angles and torsion angles). In the
transition state, the hydrogen atom is approximately loca-
ted between the phenoxyl and peroxyl radicals (Fig. 3).
As explained above the B3P86 hybrid functional under-
estimates Gibbs energy of activation, while MPWB1K give
a better description of PCET [29, 39]. Therefore the values
reported in Table 5 only provide a range. In this process,
tunneling appears crucial thus rate constants are more rele-
vant than DG# in order to discuss on PCET. The rate con-
stant of the most active compounds (e.g., compound 7) is
ranging from 10-1 to 106 M-1 s-1 (Table 5), probably
within an intermediate value. Compounds having only one
OH group (1–3) are 103 less active than 7. Interestingly,
BDE correlates with DG# (R2 = 0.99) when the two most
Mechanism of free radical scavenging
The capacity of antioxidant to scavenge free radicals
depends on intrinsic parameters as BDE but also on the
Fig. 5 HOMOs distribution
with electron abstraction a from
7-[3H?] to 7-[3Hꢀ] and b from
7-[4H?] to 7-[4Hꢀ]
123

962
J Comput Aided Mol Des (2013) 27:951–964
active compounds are excluded (Fig. 4). Regarding these
two compounds, the intramolecular H-bonding may strongly
influence kinetics.
Compare to quercetin, an antioxidant of reference, the
SPLET rate constants are significantly lower. Therefore,
the present series of compounds are not expected to be
powerful free radical scavenging by ET process, even from
the deprotonated (activated) forms. The p-delocalization in
the Schiff bases studied here is less extended than in
quercetin, thus significantly decreasing the positive ener-
getic contributions (e.g., internal reorganization ki, DG°)
along the electron transfer process.
The mechanism of antioxidant activity of polyphenols is
known to be dependent on pH [29, 30], due to the capacity
of these compounds to deprotonate, leading to an anion
having a better capacity to release an electron to the free
radical (SPLET mechanism as described in Eqs. 3–5).
SPLET is investigated on the most active compound
studied here (7). In this case, only the two OH groups of the
catechol moiety (3-OH and 4-OH) can be deprotonated.
From our calculations, the 4-OH group appears slightly
more acidic than 3-OH by 2.6 kcal/mol (Table 6); here we
quote 7-[4H?] and 7-[3H?] the deprotonated form of 7 at
C4 and C3, respectively.
Conclusion
Based on a series of synthetized Schiff bases, the present
study has confirmed the role of the catechol moiety and
the importance of the O–H BDE descriptor. Other minor
descriptors succeeded in explaining slight differences in
antioxidant activity, namely spin density distribution and
BDEd. The latter descriptor elegantly rationalizes the role
of the NH group that, according to the chemical struc-
ture, may provide a hydrogen atom for a second HAT.
Bromine substitution may slightly enhance antioxidant
activity. The free radical scavenging capacity of these
compounds mainly proceeds by PCET rather than
SPLET, the latter mechanism being only feasible at rel-
atively high pH.
As expected, IP of the deprotonated form is lower than that
of parent molecule by 1.32 and 1.27 eV for 7-[3H?] and 7-
[4H?], respectively. No significant difference is observed
between the electron donor capacities of 7-[3H?] and 7-[4H?]
(IP = 4.14 and 4.19 eV, respectively). This highlights that the
electron donor capacity of 7 is related to the catechol moiety
and not only one specific OH group.
The electron transfer towards the free radical is expected
only if a non-covalent pre-reaction complex is formed
between the antioxidant and the free radical. Two types of
pre-reaction complexes may exist, both involving the
O-atom lone pair of the free radical (Fig. 1 in SI). The first
type involves a H-bond with the OH group of the antiox-
idant ([HB]-type complex), as observed in the PCET
mechanism; the second involves a [m–p] dispersive inter-
action with aromatic p-electrons (i.e., [m–p]-type). It must
be stressed that quantum calculations did not allow
obtaining [m–p]-type interactions with N–N p-electrons.
From these pre-reaction complexes, the calculated SPLET
rate constants are in the range from 10-30 to 10-3 M-1 s-1
(Table 6). These rate constants strongly depend on (1) the
electron donor anion (i.e., 7-[3H?] and 7-[4H?]) and (2) the
geometry of pre-reaction complexes (Fig. 1 in SI). The 7-
[4H?] anion exhibits the highest rate constants (10-3 and
10-5 M-1 s-1 for 7-[3H?] and 7-[4H?], respectively).
Moreover the 7-[4H?] anion and the subsequent (post-ET)
phenoxyl radical (i.e., 7-[4Hꢀ]) show a better p-conjugation
than 7-[3H?] (Fig. 5). Furthermore, the electronic coupling
is significantly different between the reactions of both anions
(Table 6). AccordingtoVRP formula, the lowerthemolecular
orbital (MO) modification along the reaction, the higher the
reactant–product overlap SRP, the higher the electron cou-
´
Acknowledgments The authors thank the ‘‘Conseil Regional du
Limousin’’ for CALI (CAlcul en LImousin) and Atta-ur-Rahman
Institute for Natural Product Discovery, Universiti Teknologi for
computing facilities. The authors thank the ‘‘Pharmacology and
Toxicology Research Laboratory-Faculty of Pharmacy, Universiti
Teknologi MARA, Puncak Alam Campus, Malaysia’’ for antioxidant
facilities. P.T. gratefully acknowledges the support by the Operational
Program Research and Development for Innovations–European
Regional Development Fund (project CZ.1.05/2.1.00/03.0058 of the
Ministry of Education, Youth and Sports of the Czech Republic). I.B.
and P.T. gratefully thank the ‘Association Djerbienne de France’
(ADF) for the financial support.
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pling VRP and therefore the higher the rate constant kSPLET
.
The highest occupied MO (HOMO) modification from 7-
[4H?] to 7-[4Hꢀ] is low while HOMOs of 7-[3H?] and 7-
[3Hꢀ] have the same location but with opposite sign (Fig. 5),
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the corresponding rate constants.
123

J Comput Aided Mol Des (2013) 27:951–964
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