Quantum-Chemical Studies to Approach the Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs: Synthesis, Molecular Structure, Hirshfeld and Density Functional Theory Analyses

Article abstract of DOI:10.1002/bkcs.10132

In the present study, five nonphenolic (E)-N′-benzylidenebenzohydrazides including three new compounds were synthesized and evaluated for their free radical scavenging activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH). X-Ray analysis of a single cryst

Full text of DOI:10.1002/bkcs.10132

Article
DOI: 10.1002/bkcs.10132
BULLETIN OF THE
M. S. Alam and D.-U. Lee
KOREAN CHEMICAL SOCIETY
Quantum-Chemical Studies to Approach the Antioxidant Mechanism of
Nonphenolic Hydrazone Schiff Base Analogs: Synthesis, Molecular
Structure, Hirshfeld and Density Functional Theory Analyses
*
Mohammad Sayed Alam and Dong-Ung Lee
Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea.
*E-mail: dulee@dongguk.ac.kr
Received October 10, 2014, Accepted December 3, 2014, Published online January 30, 2015
In the present study, five nonphenolic (E)-N0-benzylidenebenzohydrazides including three new compounds
were synthesized and evaluated for their free radical scavenging activities using 2,2-diphenyl-1-picrylhydrazyl
(DPPH). X-Ray analysis of a single crystal of (E)-N0-(4-chlorobenzylidene)benzohydrazide (3c) revealed a
triclinic, space group P-1 structure with a trans configuration around the azomethine ( C2 N2 ) double
bond. The three-dimensional Hirshfeld surfaces and the related two-dimensional fingerprint plots were also
drawn to study the plausible intermolecular interactions. Density functional calculations of structures, elec-
tronic densities, frontier molecular orbitals modeling, and Mulliken charge analysis of all compounds were
performed at the B3LYP/6-311G level of theory. Theoretical vibrational frequencies were predicted and com-
pared with experimental values, and results supported the validity of optimized geometry of noncrystalline
compounds. All synthesized compounds showed significant DPPH radical scavenging activity, although com-
pound 3d exhibited greatest antioxidant activity with an IC50 value of 11 μM. The results of DFT analysis were
used to explain the proposed antioxidant mechanism of (E)-N0-benzylidenebenzohydrazide analogs. This anal-
ysis revealed that protons attached to N, O, and C atoms possessing high negative charge are involved in the
production of free radicals that scavenge DPPH. Moreover, the antioxidant activities of (E)-N0-
benzylidenebenzohydrazide analogs correlated well with HOMO–LUMO energy difference of molecules.
Keywords: Benzylidenebenzohydrazides, Crystal structure, Antioxidant activity, DFT-B3LYP calculation
Introduction
are capable of affording protection against potentially fatal
diseases caused by oxidative DNA damage. The antioxidant
mechanismofphenolicsarewelldocumentedandinvolvepro-
cesses, such as, hydrogen atom transfer (HAT), proton-
coupled electron transfer (PCET), electron transfer–proton
transfer (ET–PT), sequential proton loss single electron trans-
fer (SPLET), sequential electron–proton transfer (SEPT), and
adduct formation.16–20 For this reason, it is also important to
determine mechanisms responsible for the activities of non-
phenolic antioxidants with the aims of improving potency
and discovering new antioxidant agents to fight against
the fatal diseases caused by free radicals. In the present
study, we report the antioxidant mechanism of nonphenolic
(E)-N0-benzylidenebenzohydrazide analogs together with
the synthesis, crystal structure, and Hirshfeld analyses.
The structural geometries, electronic densities, and frontier
molecular orbitals (FMOs) modeling of the compounds
were studied at the B3LYP/6-311G level of theory to explain
the antioxidant mechanism of nonphenolic (E)-N0-
benzylidenebenzohydrazide analogs.
Hydrazide–hydrazoneSchiff bases aretheclassofcompounds
with an azomethine ( N CH ) active pharmacophore and
have significant biological activities. This class of compounds
has been reported to exhibit diverse biological activities, such
as, antimicrobial,1 antioxidant,2 anticancer,3 antitubercular,4
anticonvulsant,5 anti-inflammatory,6 antifungal,7 antiviral,8
and antibacterial9 activities. Recently, Ayman et al.10
described the pro-angiogenic and anticancer activities of
Schiff bases of N-valproyl-4-aminobenzoyl hydrazide.
HydrazoneSchiffbases arealsousedasligandstoformvariety
of complexes with transition and inner transition metals that
have wide-ranging biological activities.11–13 Not surprisingly
these biological activities and applications of hydrazone
Schiff bases have attracted the attention of many researchers.
Oxidative stress generates various reactive oxygen species
(ROS) that can cause DNA damage leading to deleterious bio-
logical consequences, which include genetic mutation, carcin-
ogenesis, and cell death.14,15 Free radicals have also been
reported to play important roles during aging, cardiovascular
diseases, rheumatoid arthritis, and other pathologies. Antiox-
idants can neutralize free radicals and provide protection
against diseases caused by ROS. Therefore, much research
effort is being expended to identify potential antioxidants that
Experimental
General. All commercially available reagents and solvents
were purchased from Sigma-Aldrich (St. Louis, MO, USA)
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Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs
KOREAN CHEMICAL SOCIETY
and used without further purification. The melting points
(uncorrected) of synthesized compounds were determined
using a Stuart SMP3 apparatus (Bibby Sci. Ltd., Staffordshire,
UK). FT-IR spectra were obtained using a Bruker Tensor
37 spectrometer and KBr discs. NMR spectra were recorded
using a Bruker 400 MHz spectrometer (Billerica, MA,
USA) indimethyl sulfoxide. Massspectrawereacquired using
a Jeol JMS700 high-resolution mass spectrometer (Tokyo,
Japan) at the Korea Basic Science Center (Daegu, Korea).
Elemental analyses (C, H, and N) were performed on a
PerkinElmer 2400 II CHN elemental analyzer (PerkinElmer,
Waltham, MA, USA).
General Procedure for the Preparation of (E)-N0-
Benzylidenebenzohydrazide Analog (3a–e). Compounds
3a–e were prepared as described in a previous report.2 Briefly,
to a stirred solution of benzohydrazide (2, 10 mM) in 30 mL
ethanol, a suitably substituted benzaldehyde (10 mM) in 20
mL ethanol was added slowly and refluxed for 1.5–2.5 h.
On reaction completion (progress was monitored by TLC),
reaction mixtures were cooled to room temperature and fil-
tered to give solid crude products, which were then crystal-
lized from ethanol to provide the pure compounds. The IR,
1H NMR, mass spectrometry, and elemental analysis data of
compounds 3a–e are provided as supporting information.
Crystal Structure Determination. The solvent loss tech-
nique was used to grow white plate-shaped crystals of 3c.
A single crystal of suitable size (0.23 mm × 0.11 mm× 0.05
mm) was chosen for the X-ray diffraction study. Data were
collected at a temperature of 193(2) K from a Bruker SMART
CCD area detector diffractometer21 operating with graphite
monochromated radiation MoKα (λ = 0.71073 Å). A total of
22,530 reflections were collected, of which 3007 (−7 ≤ h ≤
7, −10 ≤ k ≤ 10, −19 ≤ l ≤ 19) were treated as observed. The
structure was solved by direct methods using SHELXTL.22
Full-matrix least-squares refinement using SHELXTL with
isotropic displacement parameters for all nonhydrogen atoms
converged the residual to R1 = 0.1342. Subsequent refine-
ments23 were carried out using anisotropic thermal parameters
for nonhydrogen atoms. Hydrogen atoms were fixed at chem-
ically acceptable positions and were allowed to ride on parent
atoms at a C H distance of 0.9500 Å. The final refinement
converged to R = 0.0442, wR = 0.1202, w = 1/[σ2(Fo)2 +
(0.0680P)2 + 0.42P], where R = Σ(|Fo| − |Fc||)/Σ|Fo|, Fo =
observed structure factor, Fc = calculated structure factor, P
2
= (Fo + 2Fc2)/3, σ = 1.053, (Δ/σ)max < 0.001, (Δρ)max
=
0.347 and (Δρ)min = −0.318 e/Å3.
2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Activ-
ity. Free radical-scavenging activities of the synthesized com-
pounds were assayed as described by Blois24 with some
modifications using 2,2-diphenyl-1-picrylhydrazyl (DPPH).
Briefly, to 0.1 mL samples of compounds 3a–e at different
concentrations in ethanol, 4 mL of 1.5 × 10−5 M DPPH solu-
tion was added, thoroughly mixed, and left to stand at room
temperature in a dark place for 30 min. Solution absorbances
were then measured at 520 nm, and used to calculate DPPH
radical scavengingactivities(%)usingthefollowing equation:
DPPH radical−scavenging activity ð%Þ =
½ðAbsorbance of the control−Absorbance of the sampleÞ=
Absorbance of the controlÞ × 100ꢀ
Computational Studies. Themoleculargeometriesofthe(E)-
N0-benzylidenebenzohydrazide analogs (3a–e) were optimized
by MM+ molecular modeling and by using semi-empirical
molecular orbital AM1 methods.25 Density functional theory
(DFT) calculations with a hybrid functional B3LYP (Becke's
three parameter nonlocal exchange functional along with the
Lee-Yang-Parr correlation function)26,27 at basis set 6-311G
were then performed using the GAMESS interface in Chem-
Bio3D ultra ver. 14.0 (PerkinElmer). No imaginary frequencies
were obtained during vibrational frequency calculations, indi-
cating that all structures were stable. After optimization, Mulli-
ken charge and properties of FMOs of all compounds were
analyzed using results calculated at the B3LYP/ 6-311G level.
Hirshfeldsurfaces and the related two-dimensional (2D)finger-
print plots were calculated using Crystal Explorer 3.1.28
Results and Discussion
Synthesis, Crystal Structure, and Hirshfeld Analyses. The
synthetic pathway of hydrazone Schiff base analogs (3a–e) is
shown in Scheme 1. Products were obtained in excellent yields
3a. R1, R2 = H, R3 = H; 3b. R1 = H, R2 , R3 = OCH ; 3c. R1, R2 = H, R3 = Cl;
3d. R1, R2 = H, R3 = N(CH3)2; 3e. R1 = CHO, R2, R3 = H.
Scheme 1. The synthetic pathway of hydrazone Schiff base analogs (3a–e).
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Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs
KOREAN CHEMICAL SOCIETY
(81–96%). Compounds 3b, 3d, and 3e are new. The syntheses
ofcompounds3aand3chavealreadybeenreported;29 however,
the crystal structure, Hirshfeldanalyses of compound 3c, aswell
as DFT studies and antioxidant activity of compounds 3a–e,
have not yet been reported. The structures of the synthesized
compounds were elucidated using IR, 1H NMR, mass spectra,
and elemental analyses, which are provided as supporting infor-
mation. The IR spectra of compounds 3a–e showed an absorp-
tion band around the 3174–3437 cm−1 region resulting from
NH stretch. The stretching absorption band of >C O
appeared at 1641–1667 cm−1. 1H NMR spectra of compounds
3a–e showed singlet protons at 8.13–8.70 ppm corresponding
to the CH N proton and at 11.00–11.94 ppm for the
N NH proton. Aromatic and other functional group pro-
tons appeared in their usual positions. The EI-MS spectra of
3a–e showed molecular ion peaks with intensities of 22–35%.
Among five compounds (3a–e), 3c was analyzed with X-
ray diffractometer system, because only this compound could
be crystallized as single white crystals by slow evaporation
from ethanol and other compounds were obtained in powder
form. The displacement ellipsoid plot of 3c, at a 50%
probability and with the numbering scheme is shown in
Figure 1(a). X-Ray diffraction crystal data and refinements
are presented in Table 1. Selected bond lengths and angles
are listed in Table 2. Detailed atomic coordinates, thermal
parameters, and torsion angles are detailed in supplementary
materials. Compound 3c crystallized in the triclinic,
space group P-1 with a = 5.3742(2) Å, b = 7.7205(3) Å,
c = 14.9609(6) Å, α = 100.2540(16)ꢁ, β = 92.6700(15)ꢁ, γ =
90.6900(16)ꢁ, V = 610.04(4) Å3, Z = 2, Dc = 1.408 Mg/m3, F
(000) = 268, μ = 0.301 mm−1, R = 0.0422, and wR = 0.1202.
In 3c, two phenyl rings were found to be linked by a open-
chain carbonyl-hydrazone ( C N NH CO ) system, in
which C2, N2, N1, C1, and O1 atoms were almost in the same
plane with dihedral angle of −3.3(3)ꢁ between O(1) C(1) N
(1) N(2) and of −10.5(4)ꢁ between C(2) N(2) N(1) C(1).
As was expected, 3c adopts a trans configuration about the
N2 C2 double bond. Furthermore, the p-chlorophenyl
ring of 3c is nearly coplanar with the plane formed by
C2 N2 with a dihedral angle of −15.2(3)ꢁ. The second
Table 1. Crystal data and structure refinement for (E)-N0-(4-
chlorobenzylidene)benzohydrazide (3c).
Empirical formula
Formula weight
Temperature
C14H11ClN2O
258.70
193(2) K
Wavelength
Crystal system
Space group
0.71073 Å
Triclinic
P-1
Unit cell dimensions
a = 5.3742(2) Å
b = 7.7205(3) Å
α = 100.2540(16)ꢁ
β = 92.6700(15)ꢁ
c = 14.9609(6) Å γ = 90.6900(16)ꢁ
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data
collection
610.04(4) Å3
2
1.408 Mg/m3
0.301 mm−1
268
0.23 × 0.11 × 0.05 mm3
2.68–28.34ꢁ
Index ranges
−7 ≤ h ≤ 7, −10 ≤ k ≤ 10, −19 ≤ l ≤ 19
Reflections collected
Independent reflections
Completeness to theta =
28.34ꢁ
22530
3007 [R(int) = 0.0421]
98.6%
Absorption correction
Max. and min.
transmission
Semi-empirical from equivalents
0.9851 and 0.9340
Refinement method
Full-matrix least-squares on F2
Data/restraints/parameters 3758/0/201
Goodness-of-fit on F2
1.053
Final R indices [I > 2σ(I)] R1 = 0.0422, wR2 = 0.1202
R indices (all data) R1 = 0.0534, wR2 = 0.1342
Largest diff. peak and hole 0.347 and −0.318 e/Å
CCDC deposit number CCDC 1017656
Figure 1. (a) The molecular structure of (E)-N0-(4-chlorobenzyli-
dene)benzohydrazide (3c), showing 50% probability displacement
ellipsoids and the atom-numbering scheme. (b, c) Packing arrange-
ments in the crystal structure of 3c in different views. Blue dashed
lines indicate hydrogen bonds.
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Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs
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phenyl ring (C3 C8) of 3c is significantly rotated out of the
plane formed by the remainder of the molecule at an angle
of −32.1(2)ꢁ. The torsion angle values of N(2) C(2) C
(9) C(10) and N(1) C(1) C(3) C(4) were 166.30(16)ꢁ
and 147.57(16)ꢁ, respectively. Figure 1(b) and (c) shows the
crystal packing arrangement of 3c in different views and inter-
molecular hydrogen bonding interactions. The bond lengths
and angles related to H-bonding are summarized in Table 3.
Briefly, the H-atom attached to N1 is involved in hydrogen
bond formation with the O(1)-atom of another molecule to
form a layer. As shown in Figure 2(b), molecules in different
layer are juxtaposed. No H-bonds were observed between
layers, and thus, crystal formation appears to depend of weak
van der Waals forces.
Hirshfeld surface analyses30 are a unique approach to visu-
alize and explore for the individual types of intermolecular
interactions within the crystal structure. Hirshfeld surface
mapped with dnorm using red–white–blue color scheme where
it is indicating shorter contacts, vdW contacts and longer con-
tacts, respectively. Fingerprint plots generated based on the de
anddi distanceswherede isthedistancefromtheHirshfeld sur-
face to the nearest atom outside the surface and di is the dis-
tance from the Hirshfeld surface to the nearest atom inside
the surface. Hirshfeld surface analysis and the associated 2D
fingerprint plots of 3c were performed using CrystalExplorer
software. The Hirshfeld surfaces of compound 3c have been
mapped over dnorm (−0.5 to 1.5 Å) and showing the deep
red circular depressions visible in the front and back views
of surfaces are indicating hydrogen bonding contacts
(Figure 2). The strong interactions between C H and N H
and carbonyl O atoms can be seen in the Hirshfeld surface
as red areas. Some significant π–π interactions are also
observed in Figure 2(c), showing red and blue triangles repre-
senting the presence of π–π stacking. In the 2D fingerprint plot
of 3c appeared four distinct spikes indicating different interac-
tions can occur between two different molecules in the
crystal structure (Figure 3). The visible complementary
regions in the fingerprint plots showing one molecule acts
as a donor (de > di) and the other as an acceptor (de < di).
The C Hꢂ ꢂ ꢂO, C Hꢂ ꢂ ꢂN, C Hꢂ ꢂ ꢂCl and C Hꢂ ꢂ ꢂC inter-
molecular interactions appear as one spike each in the 2D fin-
gerprint plots for compound 3c.
Table2. Selectedbondlengths(Å)andangles(ꢁ)asdeterminedbyX-
ray crystallography for (E)-N0-(4-chlorobenzylidene)
benzohydrazide (3c).
Bond lengths (Å)
Bond angles (ꢁ)
C(1) O(1)
C(1) N(1)
C(1) C(3)
N(1) N(2)
N(1) H(1)
N(2) C(2)
C(2) C(9)
C(3) C(4)
C(3) C(8)
C(4) C(5)
C(5) C(6)
C(6) C(7)
C(7) C(8)
C(9) C(14)
C(9) C(10)
C(10) C(11)
C(11) C(12)
C(12) C(13)
C(12) Cl(1)
C(13) C(14)
1.223(2)
1.359(2)
1.502(2)
1.3847(18) C(1) N(1) N(2)
0.8800
O(1) C(1) N(1)
O(1) C(1) C(3)
N(1) C(1) C(3)
123.92(15)
121.80(15)
114.22(14)
120.82(14)
119.6
C(1) N(1) H(1)
N(2) N(1) H(1)
C(2) N(2) N(1)
N(2) C(2) C(9)
C(4) C(3) C(8)
C(4) C(3) C(1)
C(8) C(3) C(1)
C(5) C(4) C(3)
C(6) C(5) C(4)
C(5) C(6) C(7)
C(6) C(7) C(8)
C(7) C(8) C(3)
1.281(2)
1.470(2)
1.389(2)
1.396(2)
1.389(2)
1.388(3)
1.390(3)
1.394(2)
1.395(2)
1.401(2)
1.388(2)
1.383(2)
1.390(2)
119.6
113.27(14)
121.39(15)
119.50(15)
117.31(14)
123.18(14)
120.53(15)
120.02(16)
119.80(15)
120.28(15)
119.85(15)
DFT Calculations, Vibrational Frequencies, FMO Ener-
gies, and Mulliken Charge Distributions. DFT has received
much interest as reliable tool for the quantum-chemical calcu-
lations of molecular structures and biological properties of
molecules. In the present study, quantum-chemical calcula-
tions were carried out using DFT at the B3LYP/6-311G level
of theory to determine the properties of FMOs and Mulliken
charge distributions to explain the mechanism responsible
for the DPPH free-radical scavenging activities of the nonphe-
nolic hydrazone Schiff base analogs (3a–e). The optimized
geometric parameters of compounds 3a–e at the B3LYP/6-
311G level are provided in Tables S1 and S2 (Supporting
information). The calculated bond lengths and angles of com-
pounds 3a–e were within normal ranges,31 but showed small
deviations from experimental values (Table 2), presumably
because theoretical calculations are based on the isolated
C(14) C(9) C(10) 119.05(15)
C(14) C(9) C(2)
122.82(15)
118.11(15)
1.7407(16) C(10) C(9) C(2)
1.390(2)
C(11) C(10) C(9) 120.62(15)
C(12) C(11) C(10) 119.09(15)
C(11) C(12) C(13) 121.59(15)
C(11) C(12) Cl(1) 119.01(13)
C(13) C(12) Cl(1) 119.40(13)
C(12) C(13) C(14) 118.87(15)
C(13) C(14) C(9) 120.76(15)
Table 3. Hydrogen-bond geometries (Å, ꢁ) in (E)-N0-(4-
chlorobenzylidene)benzohydrazide (3c)
D H (Å) Hꢂ ꢂ ꢂA (Å) Dꢂ ꢂ ꢂA (Å) D Hꢂ ꢂ ꢂA (ꢁ)
Figure 2. Hirshfeld map with dnorm (front view, a and back view, b),
where deep red circle indicating hydrogen bonding contacts, and
shape index (c), where red (δ−) and blue (δ+) surfaces showing π–π
stacking, noncovalent attractive force for compound 3c.
D Hꢂ ꢂ ꢂA
N(1) H(1)ꢂ ꢂ ꢂO(1)i 0.88
2.33
3.1434(19)
154.2
Symmetry code: (i) x − 1, y, and z.
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Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs
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molecules in the gaseous phase whereas experimental results
are obtained in the solid state. The theoretical structures of the
hydrazone Schiff base analogs (3a–e) are shown in Figure 4.
The vibrational frequencies of compounds 3a–e were pre-
dicted using DFT at the B3LYP/6-311G level at a spin multi-
plicity of one; no imaginary frequencies were obtained,
indicating that the structures are in stable form. Some selected
experimental and calculated harmonic frequencies for com-
pounds 3a–e are listed in Table S3. Figure S1 provides a
comparison of the experimental and simulated infrared spectra
of 3d, a N,N0-dimethyl analog of (E)-N0-benzylidenebenzohy-
drazide, which demonstrated high antioxidant activity.
According to the results presented in Table S3 and
Figure S1, predicted vibrational frequencies are in well agree-
ment with the experimental values, indicating the validity of
the optimized structure of the synthesized compounds.
FMOs play an important part in predicting molecular reac-
tivity and properties. Single point energies and FMOs were
Figure 3. 2D fingerprint plots of compound 3c, full (a); showing reciprocal contacts and resolved into: Oꢂ ꢂ ꢂH (b), Nꢂ ꢂ ꢂH (c), Clꢂ ꢂ ꢂH (d), Cꢂ ꢂ ꢂH
(e), and Hꢂ ꢂ ꢂH (f ) showing the percentage of contact contributed to the total Hirshfeld surface area of molecules. Black arrows show spikes for
different interactions. di is the closest internal distance from a given point on the Hirshfeld surface and de is the closest external contacts.
Figure 4. Theoretical geometric structures of 3a (a), 3b (b), 3c (c), 3d (d), and 3e (e), and the atom numbering scheme.
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Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs
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determined using quantum chemical calculations. In these cal-
culations, 29atomsinvolving257basisfunctionsand59occu-
pied orbitals for 3a, 37 atoms involving 321 basis functions
and 75 occupied orbitals for 3b, 29 atoms involving 275 basis
functions and 67 occupied orbitals for 3c, 37 atoms involving
311 basis functions and 71 occupied orbitals for 3d, and
31 atoms involving 283 basis functions and 66 occupied orbi-
tals for 3e, were identified. The energy levels of HOMO and
LUMO for 3a, 3b, 3c, 3d, and 3e are provided in Figure 5. The
total energies of 3a (−724 a.u.), 3b (−953 a.u.), 3c (−1184 a.
u.), 3d (−858 a.u.), and 3e (−838 a.u.), and the energies of
HOMO and LUMO including neighboring orbitals were all
negative, indicating that all compounds are stable. HOMO
and its vicinal orbitals act as nucleophiles, whereas LUMO
and its vicinal orbitals act as electrophiles.
The Mulliken charge distributions of all atoms were calcu-
lated based on the optimized geometries of compounds 3a–e;
results are presented in Table 4. According to this analysis, the
electro-negativities of bonding atoms play an important role in
the distribution of Mulliken charge. As was expected, H and
C bonded with O and N atom were all positive, whereas azo-
methine and imine N atoms, O bonded with C atom, and
C bonded with H atom were all negative. So, the hetero atoms
N9 and O17 for 3a, 3c, and 3d, N9, O17, O18, and O20 for 3b,
and N9, O17, and O19 for 3e could act as electron donors and
coordinate with metals which is consistent with our previous
study.2
Antioxidant Activities. Compounds 3a–e were evaluated for
free radical scavenging activities using DPPH. The details of
the DPPH antioxidant assay data are provided in Table S4. As
presented in Table 5, all compounds exhibited significant
DPPH radical scavenging activity. In particular, 3d showed
greatest antioxidant activity with an IC50 value of 11.0 μM.
The unsubstituted hydrazone Schiff base, that is, (E)-N0-
benzylidenebenzohydrazide (3a) showed lowest activity with
an IC50 value of 360 μM. The 3,4-dimethoxy analog (3b, IC50
= 164 μM) was ~2.2 times more active than 3a, and the
4-chloro analog (3c, IC50 = 264 μM) was ~1.4 times more
active. The 4-N,N-dimethylamino analog (3d), which contain-
ing a stronger electron-donating group than the other analogs
exhibited ~33 times more activity than 3a, while the 2-formyl
analog (3e, IC50 = 114 μM) containing a stronger electron-
withdrawing group exhibited ~11 times less activity than
3d, but ~threefold more activity than 3a. All the compounds
showed lower activity than ascorbic acid (IC50 = 0.87 μM),
a standard antioxidant agent.
Computational Studies. Phenolic Schiff bases have been
reported to be strong antioxidants and powerful free-radical
scavengers.32,33 Because the antioxidant mechanism of phe-
nolic antioxidants has been well established, it would be inter-
esting to examine relationships between the electronic
properties and molecular structures of nonphenolic hydrazone
Schiff base antioxidants. To explain the DPPH free-
radical scavenging mechanism of hydrazone Sciff base ana-
logs 3a–e, DFT calculations were carried out at the B3LYP/
6-311G level to determine the electron densities, energies,
FMO maps, and Mulliken charge distributions of the atoms
composing the synthesized compounds.
The Mulliken charge distributions of all atoms in 3a–e
(Table 4) showed that the following atoms and bonds are bear-
ing the higher values of negative charge: N9 (−0.570) and O17
(−0.403) for 3a, N9 (−0.562), O17 (−0.402), O18 C21
(−0.535, −0.292), and O20 C19 (−0.538, −0.294) for
3b, N9 (−0.570), and O17 (−0.398) for 3c, N9 (−0.571),
O17 (−0.411) and C19 N18 C20 (−0.355, −0.664,
−0.354) for 3d, and N9 (−0.563), O17 (−0.396), and O19
(−0.369) for 3e. This result indicates that these atom groups
may be subjected to oxidation or loss of hydrogen attached
to the N, O, or C atoms. Additionally, C2 (−0.261) attached
to the chlorine atom in 3c, C1 (−0.198) and C3 (−0.206) in
3d, and C3 (−0.208) in 3e also bear moderate negative charge,
Figure 5. FMOs contour plots (HOMO and LUMO) and the corre-
sponding energies of 3a (a), 3b (b), 3c (c), 3d (d), and 3e (e). Different
surface colors represent opposite signs of the wave function.
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Table 4. Mulliken charges (e) for compounds 3a–e.
3a 3b
3c
3d
3e
Atom
Charge/e
Atom
Charge/e
Atom
Charge/e
Atom
Charge/e
Atom
Charge/e
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
N(8)
−0.172
−0.119
−0.171
−0.141
−0.0772
−0.0731
−0.008
−0.132
−0.570
0.490
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
N(8)
−0.164
0.264
0.214
−0.157
−0.044
−0.056
−0.013
−0.136
−0.562
0.486
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
N(8)
−0.072
−0.261
−0.073
−0.145
−0.064
−0.075
−0.001
−0.141
−0.570
0.500
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
N(8)
−0.198
0.376
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
N(8)
−0.131
−0.118
−0.208
−0.102
−0.022
−0.111
0.024
−0.148
−0.563
0.494
−0.206
−0.152
−0.057
−0.090
0.001
−0.158
−0.571
0.498
N(9)
N(9)
N(9)
N(9)
N(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
O(17)
H(18)
H(19)
H(20)
H(21)
H(22)
H(23)
H(24)
H(25)
H(26)
H(27)
H(28)
H(29)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
O(17)
O(18)
C(19)
O(20)
C(21)
H(22)
H(23)
H(24)
H(25)
H(26)
H(27)
H(28)
H(29)
H(30)
H(31)
H(32)
H(33)
H(34)
H(35)
H(36)
H(37)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
O(17)
Cl(18)
H(19)
H(20)
H(21)
H(22)
H(23)
H(24)
H(25)
H(26)
H(27)
H(28)
H(29)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
O(17)
N(18)
C(19)
C(20)
H(21)
H(22)
H(23)
H(24)
H(25)
H(26)
H(27)
H(28)
H(29)
H(30)
H(31)
H(32)
H(33)
H(34)
H(35)
H(36)
H(37)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
O(17)
C(18)
O(19)
H(20)
H(21)
H(22)
H(23)
H(24)
H(25)
H(26)
H(27)
H(28)
H(29)
H(30)
H(31)
−0.121
−0.107
−0.162
−0.118
−0.190
−0.021
−0.403
0.155
0.156
0.154
0.159
0.184
0.148
0.314
0.183
0.155
0.154
0.152
0.180
−0.123
−0.106
−0.162
−0.118
−0.191
−0.018
−0.402
−0.535
−0.294
−0.538
−0.292
0.165
0.166
0.185
0.149
0.312
0.184
0.155
0.154
0.152
−0.123
−0.106
−0.162
−0.116
−0.191
−0.023
−0.398
−0.007
0.186
0.184
0.167
0.191
0.152
0.317
0.185
0.157
0.157
0.154
0.180
−0.123
−0.109
−0.164
−0.119
−0.191
−0.024
−0.411
−0.664
−0.355
−0.354
0.170
0.168
0.148
0.175
0.141
0.312
0.181
0.152
0.152
0.149
0.182
0.195
0.178
0.196
0.197
0.178
0.197
−0.121
−0.107
−0.162
−0.118
−0.190
−0.022
−0.396
0.177
−0.369
0.164
0.162
0.170
0.190
0.212
0.323
0.185
0.156
0.155
0.153
0.180
0.144
0.179
0.191
0.206
0.175
0.192
0.202
0.175
Table 5. DPPH radical scavenging activity of compounds 3a–e.
and thus, could undergo oxidation. The N9 (−0.570) atom of
3a, which bears a high electron density, and is attached to a
hydrogen atom could generate a proton free radical to neutral-
ize DPPH radicals, as shown in Figure 6 (Path I). Because
hydrazone Schiff bases usually show keto-enol tautomerism34
in solution and the O17 (−0.403) atom of 3a bears high elec-
tron density, 3a could also exert antioxidant activity via the
mechanism shown in Figure 6 (Path II). In compound 3b,
the carbons of the two methoxyl groups, C19 (−0.294) and
C21 (−0. 292), bear high electron density, and thus, could pro-
duce proton free radicals to neutralize DPPH. So, 3b could
exert its antioxidant activity according to the mechanism
Compound
IC50 (μM)
360
3a
3b
164
3c
264
3d
11
3e
114
Ascorbic acid
0.87
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Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs
KOREAN CHEMICAL SOCIETY
shown inFigures 6and7. Therefore, theantioxidant activity of
3b is predicted to be higher than that of 3a, which is consistent
with our experimental data. In compound 3c, C2 (−0.261),
which is connected with the chlorine atom (−0.007) bears high
negative charge, andthus, in addition to the mechanism shown
in Figure 6, compound 3c might also neutralize DPPH radicals
via homolysis of the C2 Cl18 bond as the chlorine is more
electronegative than the carbon. However, compound 3d
might exert its antioxidant activity via the formation of proton
radicals attached to C19 (−0.355) and C20 (−0.354) (Figure 8)
and via the mechanism shown in Figure 6. Whereas, O19
(−0.369) and C3 (−0.208) in compound 3e bear high negative
charge, which might enable protons attached to C18 and C2 to
form proton radicals and scavenge DPPH.
Maps of the FMOs and of the energies of compounds 3a–e
are shown in Figure 5. The energy differences between HOMO
and LUMO for 3a–e were 6.19, 5.32, 5.218, 3.808, and 4.323
eV, respectively. Due to the lower energy difference between
HOMO and LUMO for compound 3d, the benzylidenebenzo-
hydrazide radical would be more stable through resonance after
the generation of proton free radicals than the corresponding
radicals of 3a–c and 3e. However, the correlation coefficient
(r2) between HOMO–LUMO energy differences and the anti-
oxidant activities of compounds 3a–e was found to be 0.90
Figure 6. The proposed DPPH radical scavenging mechanism of (E)-
N0-benzylidenebenzohydrazide (3a).
Figure 7. The proposed DPPH radical scavenging mechanism of (E)-N0-(3,4-dimethoxybenzylidene)benzohydrazide (3b).
Figure 8. The proposed DPPH radical scavenging mechanism of (E)-N0-(4-(dimethylamino)benzylidene)benzohydrazide (3d).
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Crystallographic Data Center (CCDC), 12 Union Road,
Cambridge, CB2 1EZ, UK; fax: +44(0) 1222–336033; e-mail:
deposit@ccdc.cam.ac.uk; www.ccdc.cam.ac.uk/Community/
Requestastructure/Pages/DataRequest.aspx
400
3a
R2 = 0.90
n = 5
300
200
100
0
3c
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Conclusion
Nonphenolic (E)-N0-benzylidenebenzohydrazide analogs
were synthesized and their antioxidant activity was evaluated
using DPPH. A single crystal of (E)-N0-(4-chlorobenzylidene)
benzohydrazide (3c) was obtained using the solvent loss tech-
nique and X-ray diffraction analysis revealed that it had tri-
clinic symmetry (P-1) and a trans configuration around the
azomethine ( C2 N2 ) double bond. Weak nonclassical
intermolecular N Hꢂ ꢂ ꢂO hydrogen bonds were observed on
crystal layers and the crystal unit cell showed adjacent juxapo-
sitioned crystal layers. Hirshfeld analyses reveal the close
Oꢂ ꢂ ꢂH, Nꢂ ꢂ ꢂH, Clꢂ ꢂ ꢂH, and Cꢂ ꢂ ꢂH contacts and π–π stacking
in the crystal structure. All compounds showed significant
DPPH radical scavenging activity, and of these, compound
3d showed greatest activity with an IC50 value of 11 μM,
although this was lower than that of the standard antioxidant,
ascorbic acid (IC50, 0.87 μM). DFT studies revealed that pro-
tons attached to N, O, and C atoms with high negative charge
might produce proton free radicals to neutralize DPPH. The
study also indicates that low energy differences between
HOMO and LUMO help to stabilize benzylidenebenzohydra-
zide radicals and increase activity. A good correlation was
observed between HOMO–LUMO energy differences and
the antioxidant activities of compounds 3a–e.
Acknowledgments. Dr. Ha-Jin Lee at Korea Basic Science
Institute (Western Seoul Center) is acknowledged for the
X-ray analyses. Publication cost of this paper was supported
by the Korean Chemical Society.
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KOREAN CHEMICAL SOCIETY
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