Synthesis, molecular modeling and structural characterization of vanillin derivatives as antimicrobial agents

Article abstract of DOI:10.1016/j.molstruc.2013.01.071

Two vanillin derivatives have been designed and synthesized and their biological activities were also evaluated for antimicrobial activity. Their chemical structures are characterized by single crystal X-ray diffraction studies, ¹H NMR, MS, and

Full text of DOI:10.1016/j.molstruc.2013.01.071

Journal of Molecular Structure 1039 (2013) 214–218
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, molecular modeling and structural characterization
of vanillin derivatives as antimicrobial agents
Juan Sun a, Yong Yin a, Gui-Hua Sheng a, Zhi-Bo Yang b, Hai-Liang Zhu a,b,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b School of Life Sciences, Shandong University of Technology, Shandong 255049, People’s Republic of China
h i g h l i g h t s
"
"
"
The synthesis, crystal structure and antibacterial activities of these compounds have not been reported so far.
Our current findings are completely new.
Their biological activities are also evaluated for FtsZ inhibitory activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Two vanillin derivatives have been designed and synthesized and their biological activities were also
evaluated for antimicrobial activity. Their chemical structures are characterized by single crystal X-ray
diffraction studies, 1H NMR, MS, and elemental analysis. Structural stabilization of them followed by
intramolecular as well as intermolecular H-bonds makes these molecules as perfect examples in molec-
ular recognition with self-complementary donor and acceptor units within a single molecule. Docking
simulations have been performed to position compounds into the FtsZ active site to determine their
probable binding model. Compound 3a shows the most potent biological activity, which may be a prom-
ising antimicrobial leading compound for the further research.
Received 28 September 2012
Received in revised form 11 January 2013
Accepted 29 January 2013
Available online 14 February 2013
Keywords:
Vanillin derivatives
Schiff base
Antibacterial activity
FtsZ inhibitors
H-bond
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
after Hugo Schiff [11], have also been shown to exhibit a broad
range of biological activities [12–18]. FtsZ, an essential protein
Although several classes of antibacterial agents are presently
available, resistance in most of the pathogenic bacteria to these
drugs constantly emerges. In order to prevent this serious medical
problem, the elaboration of new types of antibacterial agents is a
very important task [1]. Vanillin (4-hydroxy-3-methoxybenzalde-
hyde), a dietary flavoring agent, has been reported to show antiox-
idant and anti-mutagenic activities, and has also been proved to be
an anticarcinogen against a variety of chemical and physical agents
[2–6]. Vanillin is considered to be one of the most widely appreci-
ated flavor compounds, with an odor threshold for humans equal
to 11.8 Â 10À14 M, and has the unique characteristic that, even at
high doses, the flavor is still pleasant [7]. Besides its flavor quali-
ties, vanillin exhibits the antimicrobial potential and has been used
as a natural food preservative [8]. In old medicinal literature, vanil-
la was described as a remedy for fevers [9,10]. Schiff bases, named
for bacterial viability [19–21], is considered to be the most critical
component of the division machinery. It is a highly conserved and
potentially broad-spectrum antibacterial target, it does have struc-
tural and functional homology, suggesting that FtsZ may also be
amenable to inhibitor development. The growing efforts to dis-
cover hybrid drugs resulting from the combination of pharmaco-
phoric moieties of different known lead. Hence, the impressive
results of Vanillin derivatives and various phenylamine fueled
our interest in combining two scaffolds and exploring their possi-
bilities as potential anti-FtsZ agents.
2. Experimental
2.1. Chemistry
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin layer chro-
matography (TLC) on Merck pre-coated silica GF254 plates. Melt-
⇑
Corresponding author at: State Key Laboratory of Pharmaceutical Biotechnol-
ogy, Nanjing University, Nanjing 210093, People’s Republic of China.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.01.071

J. Sun et al. / Journal of Molecular Structure 1039 (2013) 214–218
215
Scheme 1. Synthesis route of the title compounds.
ing points (uncorrected) were determined on a XT4MP apparatus
(Taike Corp., Beijing, China). ESI mass spectra were obtained on a
Mariner System 5304 mass spectrometer, and 1H NMR spectra
were collected on a Bruker DPX300 spectrometer at room temper-
ature with TMS and solvent signals allotted as internal standards.
Chemical shifts are reported in ppm (d). Elemental analyses were
performed on a CHN-O-Rapid instrument, and were within 0.4%
of the theoretical values.
crystal structure was solved by direct methods using SIR92 and re-
fined by full-matrix least-squares method using SHELXL97 [22,23].
All non-hydrogen atoms were refined anisotropically and hydro-
gen atoms have been refined in the riding mode on their carrier
atoms wherever applicable.
2.3. Molecular docking study
The synthetic route to target compounds (3a and 3b) is shown
in Scheme 1.
Automated docking studies were carried out using Discovery
Studio (version 3.1) as implemented through the graphical user
interface DS-CDocker protocal.
2.1.1. Synthesis of 4-formyl-2-methoxyphenyl acetate (2)
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 11.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2009)], then they were energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The Gasteiger-Hückel charges of ligands were assigned. The crystal
structures of EGFR protein (PDB code: 2VAM) [24] complex were
retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/
To a stirred and cooled fuming nitric acid (12 mL) was added
slowly compound 4-formyl-2-methoxyphenyl acetate (2.4 g) over
2 h. The dark brown reaction mixture was stirred for 5 h and then
poured into crushed ice. The yellow precipitate was filtered off and
washed with cold water (300 mL), to form compound 2.
2.1.2. General method of synthesis of vanillin derivatives (3a and 3b)
An equivocal compound 2 (1 mmol) and substituted aromatic
amine (1 mmol) in methanol or acetonitrile was stirred for 4–6 h
at room temperature, then reaction mixture was concentrated un-
der reduced pressure, recrystallized from ethanol to give vanillin
derivatives 3a and 3b.
Compound 3a
2.1.2.1. (E)-2-Methoxy-4-(((4-methoxyphenyl)imino)methyl)-3-nitro-
phenyl acetate (3a). Mp: 114–115 °C. Yield: 75%. 1H NMR
(300 MHz, CDCl3): 2.39–2.40 (m, 3H), 3.83 (s, 3H), 3.94 (s, 3H),
6.91–6.94 (m, 2H), 7.22–7.26 (m, 2H), 7.32–7.34 (m, 1H), 7.95 (s,
1H), 8.34 (s, 1H). MS (ESI): 345(C17H17N2O6, [M + H]+). Anal. Calcd
for C17H16N2O6: C, 59.30; H, 4.68; N, 8.14; Found: C, 59.52; H, 4.79;
N, 8.02.
Compound 3b
2.1.2.2. (E)-4-(((2,6-Diisopropylphenyl)imino)methyl)-2-methoxy-3-
nitrophenyl acetate (3b). Mp: 147–149 °C. Yield: 90%. 1H NMR
(300 MHz, CDCl3): 1.16–1.17 (m, 12H), 2.41(s, 3H), 3.2.85–2.90
(m, 2H), 3.96 (s, 3H), 7.71–7.76 (m, 3H), 7.26 (s, 1H), 7.37–7.39
(d, J = 6.0 Hz, 1H), 8.08 (s, 1H). MS (ESI): 399 (C22H27N2O5,
[M + H]+). Anal. Calcd for C22H264N2O5: C, 66.32; H, 6.58; N, 7.03;
Found: C, 66.19; H, 6.61; N, 7.17.
2.2. X-ray crystallography
Single crystal X-ray diffraction data was collected on a Bruker
D-8 venture diffractometer at room temperature (293 K). The X-
ray generator was operated at 50 kV and 35 mA using Mo Ka radi-
ation (k = 0.71073 Å). The data was collected using SMART soft-
ware package. The data were reduced by SAINT-PLUS, an
empirical absorption correction was applied using the package
SADABS and XPREP were used to determine the space group. The
Fig. 1. Molecular structures of the title compounds with atomic numbering
scheme.

216
J. Sun et al. / Journal of Molecular Structure 1039 (2013) 214–218
ton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef
Compound 3a
extract 1000 mL). The MICs (minimum inhibitory concentrations)
of the test compounds were determined by a colorimetric method
using the dye MTT (3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyl tet-
razoliumbromide). A stock solution of the synthesized compound
(100 lg/mL) in DMSO was prepared and graded quantities of the
test compounds were incorporated in specified quantity of steril-
ized liquid MH medium. A specified quantity of the medium con-
taining the compound was poured into microtitration plates.
Suspension of the microorganism was prepared to contain approx-
imately 105 cfu/mL and applied to microtitration plates with seri-
ally diluted compounds in DMSO to be tested and incubated at
37 °C for 24 h. After the MICs were visually determined on each
of the microtitration plates, 50 lL of PBS (phosphate buffered sal-
ine 0.01 mol/L, pH 7.4, Na2HPO4Á12H2O 2.9 g, KH2PO4 0.2 g, NaCl
8.0 g, KCl 0.2 g, distilled water 1000 mL) containing 2 mg of MTT/
mL was added to each well. Incubation was continued at room
temperature for 4–5 h. The content of each well was removed,
Compound 3b
and 100 lL of isopropanol containing 5% 1 mol/L HCl was added
to extract the dye. After 12 h of incubation at room temperature,
the optical density (OD) was measured with a microplate reader
at 550 nm.
2.5. Inhibition of FtsZ polymerization
The FtsZ was expressed and purified with the following modifi-
cation [25]. A polymerization/depolymerization step was added
before preparative gel filtration. This improved purification gave
a protein preparation that was >99% pure with a 1:1 M ratio of
GDP to FtsZ.
The polymerization and depolymerization of purified FtsZ at
0.5 mg/mL. Compounds were initially evaluated at 100 lM. If inhi-
bition was observed, the compounds were retested at several con-
centrations. The percentage control activity was calculated by
comparison with an assay without compound. Three independent
curves were run. Quantitative analysis of the FtsZ GTPase enzy-
matic reaction was done by monitoring the oxidation of NADH as
a decrease in absorbance at 340 nm.
Fig. 2. Crystal packing of the title compounds.
pdb/home/home.do). All bound waters and ligands were elimi-
nated from the protein and the polar hydrogens and the Koll-
man-united charges were added to the proteins.
2.4. Antibacterial assay
3. Results and discussion
The antibacterial activity of the synthesized compounds was
tested against Bacillus subtilis, Escherichia coli, Pseudomonas aeru-
ginosa and Staphylococcus aureus using MH medium (Mueller–Hin-
3.1. Crystal structures of compounds 3a and 3b
Crystals of three compounds were obtained from methanol
solution. Fig. 1 shows a perspective view of the monomeric unit
with the atomic numbering scheme, and Fig. 2 depicts the intramo-
lecular and intermolecular hydrogen bonds. Crystallographic data,
details of data collection and structure refinement parameters are
listed in Table 1. The hydrogen bond lengths and bond angles are
given in Table 2.
Table 1
Crystallographic data, details of data collection and structure refinement parameters.
Compound
3a
3b
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C
17H16N2O6
C22H26N2O5
398.45
Orthorhombic
Pbca
19.0075(15)
8.7528(7)
26.320(2)
90
344.32
Monoclinic
P21/c
7.1969(7)
13.6667(13)
16.9664(15)
90
98.621(3)
90
Single crystals of 3a (0.32 mm  0.27 mm  0.25 mm) and 3b
(0.38 mm  0.26 mm  0.25 mm) were mounted on a D-8 venture
b (Å)
Table 2
c (Å)
(o)
Hydrogen bond lengths (Å) and bond angles (°).
a
b (o)
90
90
Compounds D–H. . .A
d(D–
H)
d(H. . .A) d(D. . .A)
\DHA
c
(o)
V (Å)
1649.9(3)
4
1.386
4378.8(6)
8
1.209
Z
3a
3b
C(7)–H(7). . .N(1)
C(7)–H(7). . .N(1)
C(14)–
0.93
0.93
0.98
2.56
2.57
2.38
3.4092(18) 153
D calc/g cmÀ3
2.887(2)
2.838(3)
100
108
h Range (°)
F (000)
2.8, 34.3
720.0
2.6–26.5
1696
H(14). . .N(2)
C(17)–
H(17). . .N(2)
C(20)–
H(20A). . .O(5)
C(22)–
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmÀ1
19195/4562
3236/0/229
0.107
45684/4490
3438/
0.086
0.98
0.96
0.96
2.43
2.59
2.53
2.931(3)
3.146(4)
3.484(4)
111
117
171
)
R1/WR2 [I > 2
R1/WR2 (all date)
GOOF
r
(I)]
0.0461/0.1271
0.0732/0.1271
1.043
0.0610/0.1708
0.0806/0.1708
1.053
H(22A). . .O(5)

J. Sun et al. / Journal of Molecular Structure 1039 (2013) 214–218
217
Compound 3a
Compound 3b
Fig. 3. 2D molecular docking modeling with 2VAM.
diffractometer equipped with graphite-monochromated MoKa
(k = 0.71073 Å) radiation. For 3a, a total of 19195 reflections were
collected, of which 4562 were unique with Rint = 0.020 and 3236
wR = 0.1708. The highest and lowest residual peaks in the final dif-
ference Fourier map are 0.32 and À0.30 e/Å3, respectively.
In the crystal structure of compound 3a, there are two benzene
rings in the molecule. C(1), C(2), C(3), C(4), C(5) and C(6) form the
first plane with the mean deviation of 0.0059 Å, defined as plane I;
Similarly, C(8), C(9), C(10), C(11), C(12) and C(13) forms the second
plane with the mean deviation of 0.0110 Å, defined as plane II. The
dihedral angle between plane I and plane II is 150.9°. In the crystal
structure of compound 3b, there are two benzene rings in the mol-
ecule. C(1), C(2), C(3), C(4), C(5) and C(6) form the first plane with
the mean deviation of 0.0026 Å, defined as plane I; Similarly, C(8),
C(9), C(10), C(11), C(12) and C(13) forms the second plane with the
observed reflections with I > 2r (I) were used in the succeeding
structure calculations. The final cycle of refinement of fullmatrix
least-squares was converged to R = 0.0461 and wR = 0.1271. The
highest and lowest residual peaks in the final difference Fourier
map are 0.22 and À0.16 e/Å3, respectively. For 3b, a total of
45,684 reflections were collected, of which 4490 were unique with
Rint = 0.033 and 3438 observed reflections with I > 2r (I) were used
in the succeeding structure calculations. The final cycle of refine-
ment of fullmatrix least-squares was converged to R = 0.0610 and

218
J. Sun et al. / Journal of Molecular Structure 1039 (2013) 214–218
3.4. Experimental protocol of docking study
Compound 3a
Molecular docking of the synthesized compounds and FtsZ was
performed on the binding model based on the FtsZ protein com-
plex structure (2VAM.pdb). The binding poses of active compounds
are selected through CDOCKER INTERACTION ENERGY [26]. Dock-
ing algorithm utilized: CDOCKER algorithm; definition of binding
site: 39.172, À4.4254, 8.9296; radius: 9; scoring function: CDOC-
KER interaction energy; rigid receptor: PDB code 2VAM; flexible li-
gand docking: YES; cluster analysis of docking poses: ten optimal
poses were retained.
The binding model of compounds and FtsZ was depicted in
Figs. 3 and 4. In the binding model, compound 3a was nicely bound
to the FtsZ with five hydrogen interaction bonds. Moreover, the p-
cation interactions were existed between benzene-ring and amino
acids Arg 143. Besides, compound 3b was nicely bound to the FtsZ
with two hydrogen interaction bonds.
4. Conclusion
In summary, two vanillin derivatives 3a and 3b were synthe-
sized and tested for their inhibitory activities against E. coli, Pseu-
domonas aeruginosa fluorescence, B. subtilis and S. aureus. Both of
them exhibited potent antibacterial and FtsZ inhibitory activities.
Particularly, Compound 3a was proved to be the more potent com-
pound. Molecular modeling study provided further insight into
interactions between the enzyme and its ligand.
Compound 3b
Acknowledgment
This work was financed by National Natural Science Foundation
of China (No. J1103512).
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3.3. Inhibition of FtsZ polymerization
The GTPase FtsZ inhibitory potency of the selected compounds
was examined and among the tested compounds, compounds 3a
showed potent inhibitory activities with polymerization ID50 of
2.1 lM than 3b (ID50 = 7.6 lM).