Benzaldehyde Schiff bases regulation to the metabolism, hemolysis, and virulence genes expression in vitro and their structure-microbicidal activity relationship

Article abstract of DOI:10.1016/j.ejmech.2015.04.042

There is an urgent need to develop new antibacterial agents because of multidrug resistance by bacteria and fungi. Schiff bases (aldehyde or ketone-like compounds) exhibit intense antibacterial characteristics, and are therefore, promising candidates as antibacterial agents. To investigate the mechanism of action of newly designed benzaldehyde Schiff bases, a series of high-yielding benzaldehyde Schiff bases were synthesized, and their structures were determined by NMR and MS spectra data. The structure-microbicidal activity relationship of derivatives was investigated, and the antibacterial mechanisms were investigated by gene assays for the expression of functional genes in vitro using Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The active compounds were selective for certain active groups. The polar substitution of the R₂ group of the amino acids in the Schiff bases, affected the antibacterial activity against E. coli and S. aureus; specific active group at the R₃ or R₄ groups of the acylhydrazone Schiff bases could improve their inhibitory activity against these three tested organisms. The antibacterial mechanism of the active benzaldehyde Schiff bases appeared to regulate the expression of metabolism-associated genes in E. coli, hemolysis-associated genes in B. subtilis, and key virulence genes in S. aureus. Some benzaldehyde Schiff bases were bactericidal to all the three strains and appeared to regulate gene expression associated with metabolism, hemolysis, and virulence, in vitro. The newly designed benzaldehyde Schiff bases possessed unique antibacterial activity and might be potentially useful for prophylactic or therapeutic intervention of bacterial infections.

Full text of DOI:10.1016/j.ejmech.2015.04.042

Accepted Manuscript
Benzaldehyde Schiff bases regulation to the metabolism, hemolysis, and virulence
genes expression in vitro and their structure–microbicidal activity relationship
Lei Xia, Yu-Fen Xia, Li-Rong Huang, Xiao Xiao, Hua-yong Lou, Tang-Jingjun Liu,
Wei-Dong Pan, Heng Luo
PII:
S0223-5234(15)30005-2
10.1016/j.ejmech.2015.04.042
EJMECH 7857
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 June 2014
Revised Date: 14 April 2015
Accepted Date: 18 April 2015
Please cite this article as: L. Xia, Y.-F. Xia, L.-R. Huang, X. Xiao, H.-y. Lou, T.-J. Liu, W.-D. Pan, H. Luo,
Benzaldehyde Schiff bases regulation to the metabolism, hemolysis, and virulence genes expression
in vitro and their structure–microbicidal activity relationship, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.04.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Benzaldehyde Schiff bases regulation to the metabolism, hemolysis,
and virulence genes expression in vitro and their
structure–microbicidal activity relationship
Lei Xiaa,b, Yu-Fen Xiaa,c, Li-Rong Huanga, b, Xiao Xiaoa, Hua-yong Loua, Tang-Jingjun
Liua, Wei-Dong Pana,*, Heng Luoa,*
a The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy
of Sciences, Guiyang 550002, Guizhou, China
b Guizhou University, Guiyang 550025, Guizhou, China
c
The second affiliated Hospital of Guiyang College of Traditional Chinese Medicine, Guiyang
550003, Guizhou, China
Abstract: There is an urgent need to develop new antibacterial agents because of multidrug
resistance by bacteria and fungi. Schiff bases (aldehyde or ketone-like compounds) exhibit intense
antibacterial characteristics, and are therefore, promising candidates as antibacterial agents. To
investigate the mechanism of action of newly designed benzaldehyde Schiff bases, a series of
high-yielding benzaldehyde Schiff bases were synthesized, and their structures determined by NMR
and MS spectra data. The structure–microbicidal activity relationship of the derivatives was
investigated, and the antibacterial mechanisms were investigated by gene assays for the expression of
functional genes in vitro using Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The
*
Corresponding author (Heng Luo) Tel: +86 0851-3876210; Fax +86 0851-3876210; E-mail:
luoheng71050@aliyun.com. (Wei-Dong Pan) Tel: +86 0851-3805348; Fax +86 0851-3805348; E-mail:
wdpan@163.com.
Abbreviations: E. coli, Escherichia coli; S. aureus, Staphylococcus aureus; B. subtilis, Bacillus subtilis; KOH,
potassium hydroxide; MBC, minimum bactericidal concentration; CFU, colony forming unit; ELISA, enzyme
linked immunosorbent assay; RT-PCR, reverse transcription PCR; LB, Luria-Bertani; .
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active compounds were selective for certain active groups. The polar substitution of the R2 group of
the amino acids in the Schiff bases, affected the antibacterial activity against E. coli and S. aureus;
specific active group at the R3 or R4 groups of the acylhydrazone Schiff bases could improve their
inhibitory activity against these three tested organisms. The antibacterial mechanism of the active
benzaldehyde Schiff bases appeared to regulate the expression of metabolism-associated genes in E.
coli, hemolysis-associated genes in B. subtilis, and key virulence genes in S. aureus. Some
benzaldehyde Schiff bases were bactericidal to all the three strains and appeared to regulate gene
expression associated with metabolism, hemolysis, and virulence, in vitro. The newly designed
benzaldehyde Schiff bases possessed unique antibacterial activity and might be potentially useful for
prophylactic or therapeutic intervention of bacterial infections.
Keywords: Antibacterial activity; Benzaldehyde Schiff base; Synthesis; Regulating gene expression
1. Introduction
Currently, multidrug resistance in several bacteria is one of the major causes of increasing rate
of mortality associated with infectious diseases in humans [1]. The main reason behind this problem
is lack of effective methods and drugs for treatment [2-3], and hence, there is an urgent need of
developing new antibacterial agents with novel and more efficient mechanisms of action [4]. Schiff
bases, named after Hugo Schiff, exhibit a broad range of physicochemical and biological activities
[5]. Structurally, they are aldehyde or ketone-like compounds in which the carbonyl group (C=O) is
replaced by an imine or azomethine group; they are formed by the reaction of primary amines with
aldehydes or ketones under specific conditions. Previous studies have established that the presence of
imine or azomethine subunits in various natural, natural-derived, and non-natural compounds was
critical to their biological activities [6-8]. Many Schiff bases also reversibly bind with oxygen,
catalyze the hydrogenation of olefins, coordinate with and show fluorescent variability with some
toxic metals [9-11]. In addition, some Schiff bases also exhibit a broad range of biological activities,
such as antifungal, antibacterial, antimalarial, antiproliferative, anti-inflammatory, antiviral, and
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antipyretic properties [12-13].
In recent years, hydrazone derivatives have received attention due to their various biological
properties and their wide application in medicinal chemistry. The biological activity of these
compounds was attributed to the presence of the (–CONHN=CH–) moiety. Some studies have
successfully identified that several hydrazide-hydrazone derivatives exhibited broad spectrum of
biological activities such as anticonvulsant [14], antidepressant [15], antimicrobial [16-17], antitumor
[18-19], analgesic, and anti-inflammatory [20] activities. Our aim was to confirm whether Schiff
bases, derived from benzaldehyde, possessed antibacterial property involved regulating genes related
to metabolism, hemolysis and virulence expression. Therefore, we designed and synthesized a series
of benzaldehyde Schiff bases based on the acylhydrazone structure, and assessed their antibacterial
activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Bacillus subtilis
(B.subtilis). In addition, the expression of some genes associated with metabolism, hemolysis and
virulence was determined at the transcriptional level in bacteria treated with selected derivatives,
using quantitative real-time reverse transcriptase PCR (RT-PCR). The results presented here validate
the antibacterial efficacy and structure–activity relationships of benzaldehyde Schiff bases in vitro
and also supported the development of these Schiff bases and related systemic antibacterial
benzaldehyde Schiff bases as potential antibacterial agents, targeting genes that regulate metabolism,
hemolysis, and virulence.
2. Results and discussion
2.1. Chemistry
Benzaldehyde Schiff bases were produced when the amidogen of amino acids was ground with
2-hydroxybenzaldehyde, in the presence of an alkali [21]. The synthetic pathway and chemical
structures of benzaldehyde Schiff bases (compounds 1a–1c) were outlined in Scheme 1. The starting
compounds of benzaldehyde Schiff bases: L-phenylalanine, L-tyrosine, and L-phenylalanine methyl
ester are commercially available. The target compounds could be generated by grinding the mixture
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of amino acid and KOH, which was followed by grinding the mixture with 2-hydroxybenzaldehyde
over a period of 30 minutes. The synthesis of acylhydrazone Schiff bases (compounds 2a–2t) were
described in Scheme 2. The starting materials, 4-methoxyphenylacetic acid or 4-chlorophenylacetic
acid were suspended in methanol, into which thionyl chloride (SOCl2) was added to generate methyl
ester I, which was then hydrazinolysized to produce acylhydrazide II [22, 23]. Isoniazide and
salicylhydrazide are commercially available. Twenty acylhydrazone Schiff bases were synthesized
via condensation of the acylhydrazide II with corresponding 2-hydroxybenzaldehyde by the previous
strategy [21, 24]. The structures of compounds 1a–1c and 2a–2t were determined on the basis of
NMR and ESI-MS data.
2.2. Antibacterial activity of benzaldehyde Schiff bases
Preliminary evaluation of the antibacterial activity was performed by assessing the anti-growth
activity of 100 ꢀmol/L purified benzaldehyde Schiff bases (results are summarized in Fig. 1 and
Table 1). Compounds which could inhibit ≥50% bacterial growth at a concentration of 100 µmol/L
were chose for further analysis. It could be observed that compounds 1b, 2c, and 2q exhibited better
activity against E. coli; especially 1b and 2c possessed similar antibacterial activity to that of control.
Similar inhibitory activity was observed for compounds 2p and 2r against B. subtilis. In addition, it
was also found that five compounds, 1c, 2d, 2f, 2h, and 2t, could selectively suppress the growth of
S. aureus, and similar anti-growth activity could be observed for 1c, 2d and 2t on the tested strain.
However, further studies are required to confirm the antibacterial activity of these compounds by
determining their MBC values.
The bacteria were incubated in LB medium containing compounds of varied concentrations for
8 hours at 37 °C, with shaking, to determine whether the screened compounds demonstrated
antibacterial activity of the compounds at a concentration of <100 ꢀmol/L. The optical density was
measured at 450 nm after 8 hours of incubation to calculate the inhibitory activity and MBC value of
every compound. The MBC value of each screened compound for each bacterium was defined as the
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lowest concentration of the compound that reduced growth by 1% as compared to the inoculum
control seeded with normal strains [25, 26]. By the concentration inhibited curves (Fig. 2) and MBC
(Table 2) values, it can be concluded that there were some benzaldehyde Schiff bases, which
inhibited the growth of three bacteria in a concentration-dependent manner. Nevertheless, the
inhibitory activity of compounds 1b and 2c against E. coli had no evident difference at different
concentrations. In addition, a moderate difference was seen in the MBC values of 1b, 2c, and control,
which indicated that 1b and 2c exhibited an inhibitory effect on the growth of E. coli, similar to
ampicillin, which is routinely used in clinical practice. Likewise, compound 2p showed a closed
inhibitory activity for B. subtilis, and two compounds 2d and 2t displayed a perfect antibacterial
activity for S. aureus, when compared with that of streptomycin.
2.3. Analysis on structure–activity relationships
Firstly, the potent inhibitory action of compound 1b on the growth of E. coli suggested that
higher polar substitution at R2 might improve the antibacterial activity for the amino acid-containing
Schiff bases. Meanwhile, the substitution at R2 by less polar groups (1c) might improve their
inhibitory activity on S. aureus.
Secondly, in case of acylhydrazone Schiff base series, their inhibitory activity against the
growth of E. coli was improved by the meta-substitution of a bromine-group at R4. The substitution
of chloride(s) at both the R3 and R4 positions might improve their inhibitory activity against the
growth of B. subtilis. As far as the antibacterial activity against S. aureus is concerned, compounds
2d and 2t were found to be the most active ones, which indicated that the presence of
electron-deficient aromatic rings, such as pyridinyl or para-chloro-phenyl groups at R3 and
electron-donating groups (lipophilic as well), such as 3,5-di-tert-butyl or 3,4-benzyl on the phenyl
ring of R4 might be potential pharmacophores for the inhibitory activity against S. aureus of the
acylhydrazone Schiff base.
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Finally, the most important point is that all these active compounds showed good selectivity in
their action against the three different bacteria and the selectivity was related to the presence of
specific active groups, which implied that they might possess different mechanisms of action on
different bacteria, respectively.
2.4. Effect of the compounds on the expression of the genes related to metabolism in E. coli
The effect of mechanism of the screened compounds on the growth of E. coli was determined
by measuring the expression of metabolism associated genes cysB, acrA, tsh, iroD, and the
housekeeping gene gapA. Real-time RT-PCR, (Fig. 3A) and semi-quantitative RT-PCR (using
agarose gel, Fig. 3B) were used for determining the expression level of the above-mentioned genes.
Results showed that compound 1b had significantly high (P < 0.01) inhibitory effect, 2c had a
significant (P < 0.05) promotion, and 2q displayed a slight (P < 0.05) inhibitory effect for the
expression of cysB (a structural gene of the transcriptional regulator CysB in E. coli that activates the
expression of the genes involved in the cysteine biosynthetic pathway [27, 28]. The above findings
demonstrated that three benzaldehyde Schiff bases inhibited the growth of bacteria (probably through
up or down regulation of cysB), and further interfered with the genes involved in the cysteine
biosynthetic pathway.
In order to evaluate the effects of the three screened compounds on the membranes of E. coli,
the expression level of acrA at the transcriptional level was assayed by RT-PCR. The acrA gene maps
between proC and purE at 10.5 min on the E. coli chromosome, contributes to the integrity of E. coli
membranes [29], and is responsible for the susceptibility to the basic dyes, detergents, and certain
antibiotics [30]. The results revealed that not every compound could affect the expression of acrA;
however, only the compound 2c and 2q could significantly (P < 0.01) promote and slightly inhibit
the expression of the gene, respectively compared to the control gene gapA. The up or down
regulation for the expression of E. coli membrane gene, suggests that Schiff bases might
differentially regulate a large number of membrane genes and then change the integrity and
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permeability, but not every compound can regulate the gene expression, that might be linked with the
structural and chemical properties of the compounds [29, 30].
We analyzed the tsh gene expression of E. coli following treatment with three Schiff bases
derivatives, because the limitation of the compounds to the virulence reflected directly the
pharmaceutical effect. The tsh gene encodes a temperature-sensitive hemagglutinin [31] and resides
on transmissible R plasmids encoding aerobactin and colicin V that is associated with avian E. coli
virulence [32–34]. The expression of the tsh gene was significantly (P < 0.01) decreased upon
treatment with three compounds, and this reduction was not due to a significant decrease in bacterial
burden; this indicated that the three Schiff bases were involved in the down regulation of expression
of tsh gene, and in inhibiting the biosynthesis of temperature-sensitive hemagglutinin in E. coli.
Similar results were found in the expression of IroD gene as the tsh gene in E. coli. The IroD gene,
belongs to the salmochelin gene cluster that encodes a cytoplasmic esterase with hydrolytic activity
that preferentially cleaves iron-free enterobactin [35]. IroD protein can hydrolyze the ester bonds of
both enterobactin and drug molecules, such as salmochelin, which is required for subsequent iron
release from the contained iron drugs [36, 37]. The data suggested that the three Schiff bases had
antimicrobial activity against E. coli in vitro and may be related with the reduced expression of IroD
to limit the hydrolytic activity of cytoplasmic esterase.
2.5. Effect of the compounds on the expression of hemolysis-associated genes in B. subtilis
The pathogenicity of the bacteria was closely related with hemolysis vitality because of the
hemolysin as an important virulence factor of pathopoiesis [38, 39]. There exists an increasing threat
to the human health with the wide application of B. subtilis and the direct relationship of its
production with human life. The whole genome sequencing of B. subtilis has been completed, and
the hemolysis-associated genes from the genome annotation were yqhC, yhdP, yhdT, yqhB, yugS, and
ytjA [40]. Except for the ytjA gene that was identified as hemolysis gene of B. subtilis [41], the
functions of the other several genes have not been identified yet. The change in expression level of
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yqhC, yhdP, yhdT, yqhB, and yugS genes was assayed, compared to the control housekeeping gene
16sRNA, of the test strain after treatment with two compounds by RT-PCR (Fig. 4A) and
semi-quantitative RT-PCR methods (Fig. 4B). Results showed that the expressions of five hemolysis
genes were significantly (P < 0.01) decreased when regulated by the treatment of 2p and 2r,
suggesting that the hemolysis genes were strongly involved in the inhibition of B. subtilis.
2.6. Effect of the compounds on the expression of virulence genes in S. aureus
In the results, it was found that the five Schiff base compounds were obtained had a better
inhibitory action towards the growth of S. aureus. To determine whether the screened compounds
had any influence on the expression of virulence factors, S. aureus were incubated with a sublethal
dose of the compounds (50 ꢀmol/L) and 8 hours post-exposure transcript abundance of key S. aureus
virulence factors saeR, hla, sbi, and mecA was measured (gyrB housekeeping gene as control) by
RT-PCR (Fig. 5A) and semi-quantitative RT-PCR methods (Fig. 5B). The results showed that there
were significant differences (P < 0.01) in the inhibitory action for the expression of saeR gene by
compounds 1c, 2d, 2f and 2h. Transcript abundance of saeR gene, the regulatory gene component of
the global virulence regulatory system SaeR/S, which is essential for the development of
staphylococcal skin lesions in mice [42], was upregulated about two-fold higher with other
compounds as compared to the control in S. aureus. The transcript expression of hla gene, which
encodes for α-toxin, a virulence factor responsible for dermonecrosis in mouse skin infections [43],
was up regulated by compounds 1c, 2d, 2f and 2t, and was markedly down regulated by 2h. The
transcript expression of sbi, a gene encoding an immunomodulatory protein that is important in
antibody and complement evasion [44] was markedly up regulated by 1c, 2h and 2t. In addition, the
expression of mecA gene that encodes an altered penicillin-binding protein 2a to confer resistance to
β-lactam antibiotics was analyzed using the same methods. The transcript expression level of mecA
[45] was upregulated by 1c with varying degrees; it was down regulated two-fold higher degree by
2d, 2h, and 2t than that in controls. The reduction in transcript expression of virulence genes of S.
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aureus, including the SaeR/S virulence regulatory system, suggests that the Schiff bases may
differentially regulate a considerably large number of S. aureus virulence genes, which may support
the hypothesis that the inhibitory effect of Schiff bases to S. aureus may correlate with the up- or
down regulation of the virulence gene expression.
3. Experimental sections
3.1. General
Varian Inova-400 spectrometer (USA) was employed to record the NMR spectra with
tetramethylsilane as an internal standard, and MS studies were carried out on a HP1100-MSD
spectrometer (ESI-MS mode) (USA). Column chromatography was performed using silica gel
(200–300 & 300–400 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, P R China).
Chemicals used in all the experiments performed during the study were of the analytical reagent
grade (AR). Organic solvents (spectroscopically pure from Acros, USA) used in biological screening
included ethyl alcohol, diethylether, and dimethylformamide (DMF). Deionization of water was done
entirely in glass equipments and this water was normally used in all preparations. Luria-Bertani (LB)
broth growth media was purchased from Oxoid (Basingstoke, UK); ampicillin and streptomycin were
obtained from Sigma; Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus)
ATCC 25923, and Bacillus subtilis (B. subtilis) ATCC 6051 were obtained from the National
Institute for the Control of Pharmaceutical and Biological Products (Beijing, PR China).
3.2. General procedure for synthesis of amino acid-containing Schiff bases
Compound 1a was synthesized according to the method described by Zhong et al. [46]. In brief,
L-phenylalanine (1.0 g, 6.1 mmol) and potassium hydroxide (KOH) (0.406 g, 7.3 mmol) were
ground together in an agate mortar to obtain a viscous mixture; 2-hydroxybenzaldehyde (0.8 mL, 7.3
mmol) was added drop-by-drop to the mixture, which was ground for 30 minutes prior to addition of
50 mL methanol to dissolve it homogenously; the mixture was then transferred to another reaction
vessel. Next, diethylether (30 mL) was added slowly into this mixture until a yellow precipitate was
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formed, which was filtered and washed with diethylether to obtain the new compound 1a.
3.2.1. Potassium-(S)-2-(2-hydroxybenzylideneamino)-3-phenylpropanoic (1a)
Yellow solid; yield: 95.4%; IR (KBr) νmax:3418, 3025, 1632, 1487, 1375, 1144, 1082 cm-1;
1H-NMR (CD3OD, 400 MHz) δ (ppm): 3.03 (m, 1H, 3-Ha), 3.36 (m, 1H, 3-Hb), 4.05 (m, 1H, 2-CH),
6.66 (m, 1H, 5′-H), 6.77 (m, 1H, 3′-H), 6.96 (m, 1H, 5-H), 7.05 (m, 1H, 7-H), 7.13 (m, 1H, 9-H),
7.17-7.25 (m, 4H, 6, 8, 4′, 6′-H), 7.86 (m, 1H, -N=CH); 13C-NMR (CD3OD, 125 MHz) δ (ppm): 36.1,
77.8, 116.9, 120.6, 123.6, 124.9, 127.7, 128.6, 132.3, 136.9, 160.8, 161.1, 175.4; ESI-MS: m/z 292.0
[M+Na]+.
3.2.2. Potassium-(S)-2-(2-hydroxybenzylideneamino)-3-(4-hydroxy)-phenylpropanoic (1b)
The title compound was obtained by condensing
L-tyrosine with salicylaldehyde, as a brown
1
solid with yield of 44.6%. IR (KBr) νmax:3421, 2925, 1636, 1484, 1376, 1150, 1105 cm-1; H-NMR
(CD3OD, 400 MHz) δ (ppm): 2.86 (m, 1H, 3-Ha), 3.48 (m, 1H, 3-Hb), 3.99 (m, 1H, 2-CH), 6.57 (m,
2H, 8-H), 6.64 (d, 1H, J = 8.0 Hz, 3′-H), 6.73 (d, 1H, J = 8.0 Hz, 6′-H), 6.89 (d, 2H, J = 8.4 Hz, 5,
9-H), 7.01 (m, 1H, 4′-H), 7.23 (m, 1H, 5′-H), 7.69 (s, 1H, -N=CH); 13C-NMR (CD3OD, 125 MHz) δ
(ppm): 37.1, 75.8, 114.3, 116.9, 120.6, 123.6, 128.4, 128.7, 132.1, 155.1, 159.2, 159.7, 176.4;
ESI-MS: m/z 308.0 [M+Na]+.
3.2.3. (S)-2-(2-hydroxybenzylideneamino)-3-phenylpropanoic acid methyl ester (1c)
The title compound was obtained by condensing
L-phenylalanine methyl ester with
salicylaldehyde, as yellow oil with yield of 84.4%. IR (KBr) νmax:2926, 1743, 1630, 1493, 1458,
1
1230, 1202 cm-1; H-NMR (CD3OD, 400 MHz) δ (ppm): 3.12 (m, 1H, 3-Ha), 3.36 (m, 1H, 3-Hb),
3.75 (s, 3H, 1-OCH3), 4.16 (m, 1H, 2-CH), 6.83 (m, 1H, 5′-H), 6.96 (d, 1H, J =8.2, 3′-H), 7.09 (m,
1H, 7-H), 7.14 (d, 2H, J =8.6, 5, 9-H), 7.19 (m, 1H, 4′-H), 7.24 (d, 2H, J =8.6, 6, 8-H), 7.32 (d, 1H, J
13
=8.2, 6′-H), 7.96 (s, 1H, -N=CH); C-NMR (CD3OD, 125 MHz) δ (ppm): 37.5, 50.3, 72.9, 116.4,
120.7, 123.6, 124.9, 126.4, 127.5, 130.1, 136.9, 157.8, 158.4, 172.7; ESI-MS: m/z 306.1 [M+Na]+.
3.3. General synthetic procedure for acylhydrazone Schiff bases
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An esterification procedure devised by Park et al. was used to synthesize methyl eater I [22], as
follows: 4-methoxy benzene acetic acid (10.0 g, 60.0 mmol) was suspended in methanol (100 mL)
and thionyl chloride (26 mL, 361.1 mmol) was added into the solution placed over an ice bath
(-10 °C). The reaction mixture was refluxed at 90 °C for 3 hours under argon, and then excess
methanol and thionyl chloride were evaporated. The crude product was purified by crystallization
(methanol/ethylacetate) and 4-methoxy benzene methyl acetate (9.84 g), with a yield of 90%, was
obtained.
Further, 4-methoxy benzene methyl acetate (9.84 g, 54 mmol) and hydrazine hydrate (27 mL,
300 mmol) were dissolved in methanol (100 mL). The mixture was refluxed at 80°C for 5 hours.
After cooling down to room temperature, the white crystal was filtered and washed with cold
methanol. 4-methoxy phenylacetyl hydrazine (4.66 g), with yield of 43.0% was obtained as a white
crystal by crystallization using methanol. Another compound named 4-chlorobenzene oxygen acetyl
hydrazine (white needle like crystals) was synthesized by the same procedure with a final yield of
73.1%.
Twenty acylhydrazone Schiff bases were synthesized as described previously [21, 24]. Under an
argon atmosphere, a solution of 2-hydroxybenzaldehyde (5 mmol) in 20 mL anhydrous ethanol was
added in acylhydrazine II (5 mmol; suspended in 30 mL anhydrous ethanol). The mixture was then
refluxed for 4–8 h at 80°C and monitored by TLC. The resulting solid was filtered and washed with
cold ethanol after cooling down to room temperature. Recrystallization in ethanol provided the final
derivatives 2a–2t.
3.3.1. N'-(2-hydroxybenzylidene)-isoniazide (2a)
The title compound 2a was synthesized by condensing isoniazide with 2-hydroxybenzaldehyde.
Light green crystals; yield: 53.2%; IR (KBr) νmax:3454, 3183, 3005, 1683, 1617, 1490, 1408, 1291,
1
1160 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.81 (d, 1H, J = 9.2 Hz, 3′-H), 6.93 (m, 1H,
5′-H), 7.30 (m, 1H, 4′-H), 7.59 (d, 1H, J = 6.0 Hz, 6′-H), 7.82 (d, 1H, J = 6.0 Hz, 3-H), 7.83 (d, 1H, J
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= 6.0 Hz, 5-H), 8.66 (s, 1H, CH=N), 8.77 (d, 1H, J = 6.0 Hz, 2-H), 8.78 (d, 1H, J = 6.0 Hz, 6-H);
13C-NMR (DMSO-d6, 125 MHz) δ (ppm):116.6, 118.8, 119.6, 121.7, 129.4, 131.9, 140.1, 149.1,
150.5, 157.6, 161.5; ESI-MS: m/z 264.0 [M+Na]+.
3.3.2. N'-(2-hydroxy-5-nitrobenzylidene)-isoniazide (2b)
The title compound 2b was synthesized by condensing isoniazide with
2-hydroxy-5-nitrobenzaldehyde. Orange powder; yield: 84.1%; IR (KBr) νmax:3454, 3230, 3071,
1658, 1549, 1483, 1439, 1298, 1081 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 7.09 (d, 1H, J =
9.2 Hz, 3′-H), 7.81 (d, 1H, J = 6.0 Hz, 3-H), 7.83 (d, 1H, J = 6.0 Hz, 5-H), 8.15 (m, 1H, 4′-H), 8.57
(s, 1H, 6′-H), 8.72 (s, 1H, CH=N), 8.77 (d, 1H, J = 5.2 Hz, 2-H), 8.78 (d, 1H, J = 5.2 Hz, 6-H);
13C-NMR (DMSO-d6, 125 MHz) δ (ppm):117.3, 120.2, 121.7, 123.6, 127.1, 140.1, 140.1, 145.2,
150.5, 161.9, 162.7; ESI-MS: m/z 309.0 [M+Na]+.
3.3.3. N'-(5-bromo-2-hydroxybenzylidene)-isoniazide (2c)
The title compound 2c was synthesized by condensing isoniazide with
5-bromo-2-hydroxybenzaldehyde. Light green crystals, yield: 76.5%; IR (KBr) νmax:3454, 3175,
1
3001, 1676, 1618, 1475, 1410, 1285, 1066 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.92 (d,
1H, J = 8.4 Hz, 3′-H), 7.46 (m, 1H, 4′-H), 7.84 (d, 1H, J = 6.0 Hz, 3-H), 7.85 (d, 1H, J = 6.0 Hz,
5-H), 7.86 (s, 1H, 6′-H), 8.66 (s, 1H, CH=N), 8.80 (d, 1H, J = 6.0 Hz, 6-H), 8.81 (d, 1H, J = 6.0 Hz,
2-H); 13C-NMR (DMSO-d6, 125 MHz) δ (ppm):110.7, 118.8, 121.4, 121.6, 130.2, 134.0, 140.0,
140.1, 146.4, 150.5, 156.5, 161.6; ESI-MS: m/z 342.0 [M+Na]+.
3.3.4. N'-(3,5-di-tert-butyl-2-hydroxybenzylidene)-isoniazide (2d)
The title compound was obtained as solid white crystals by condensing isoniazide with
3,5-di-tert-butyl-2-hydroxybenzaldehyde. Yield: 57.2%; IR (KBr) νmax:3441, 3191, 2960, 2866, 1656,
1
1611, 1436, 1361, 1250, 1067 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 1.27 (s, 9H, 3′-t-Bu),
1.39 (s, 9H, 5′-t-Bu), 7.21 (s, 1H, 4′-H), 7.31 (s, 1H, 6′-H), 7.82 (d, 1H, J = 6.0 Hz, 3-H), 7.83 (d, 1H,
J = 6.0 Hz, 5-H), 8.56 (s, 1H, CH=N), 8.78 (d, 1H, J = 6.0 Hz, 2-H), 8.79 (d, 1H, J = 6.0 Hz, 6-H);
12

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13C-NMR (DMSO-d6, 125 MHz) δ (ppm): 31.8, 34.7, 115.6, 120.6, 121.9, 126.3, 135.4, 138.2, 147.0,
150.2, 153.5, 161.6; ESI-MS: m/z 376.1 [M+Na]+.
3.3.5. N'-(2-hydroxybenzylidene)-salicylhydrazide (2e)
The title compound 2e was obtained by condensing salicylhydrazide with
2-hydroxybenzaldehyde. White powder; yield: 82.8%; IR (KBr) νmax: 3454, 3100, 1620, 1559, 1454,
1
1376, 1231, 1158 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.89 (m, 1H, 3-H), 6.93 (m, 1H,
3′-H), 6.96 (m, 1H, 5′-H), 6.99 (m, 1H, 5-H), 7.30 (m, 1H, 4′-H), 7.44 (m, 1H, 4-H), 7.55 (d, 1H, J =
9.6 Hz, 6′-H), 7.88 (d, 1H, J = 9.2 Hz, 6-H), 8.67 (s, 1H, CH=N); 13C-NMR (DMSO-d6, 125 MHz) δ
(ppm):115.7, 116.5, 117.3, 118.7, 119.1, 119.5, 128.7, 129.5, 131.7, 134.0, 149.0, 157.6, 159.0, 164.5;
ESI-MS: m/z 279.0 [M+Na]+.
3.3.6. N'-(5-bromine-2-hydroxybenzylidene)-salicylhydrazide (2f)
The title compound 2f was synthesized by condensing salicylhydrazide with
5-bromo-2-hydroxybenzaldehyde in yield of 82.6%. White powder; IR (KBr) νmax:3442, 3087, 1629,
1
1527, 1459, 1338, 1237, 1103 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.92 (d, 1H, J = 8.4
Hz, 3-H), 6.96 (d, 1H, J = 8.8 Hz, 3′-H), 6.99 (m, 1H, 5-H), 7.44 (m, 1H, 4′-H), 7.48 (m, 1H, 4-H),
13
7.80 (s, 1H, 6′-H), 7.88 (d, 1H, J = 9.2 Hz, 6-H), 8.64 (s, 1H, CH=N); C-NMR (DMSO-d6, 125
MHz) δ (ppm):115.8, 117.2, 117.4, 119.9, 123.9, 126.8, 128.8, 134.2, 140.0, 145.2, 159.0, 162.7,
164.9; ESI-MS: m/z 357.0 [M+Na]+.
3.3.7. N'-(2-hydroxy-5-nitrobenzylidene)-salicylhydrazide (2g)
The title compound 2g was synthesized by condensing salicylhydrazide with
2-hydroxy-5-nitrobenzaldehyde in yield of 82.1%. Light yellow powder; IR (KBr) νmax:3418, 3195,
1716, 1614, 1483, 1456, 1232, 1091 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.94 (d, 1H, J =
8.4 Hz, 3-H), 6.97 (m, 1H, 5-H), 7.10 (d, 1H, J = 8.4 Hz, 3′-H), 7.44 (m, 1H, 4-H), 7.86 (s, 1H, 6′-H),
13
8.15 (d, 1H, J = 8.4 Hz, 4′-H), 8.55 (s, 1H, 6-H), 8.72 (s, 1H, CH=N); C-NMR (DMSO-d6, 125
MHz) δ (ppm):110.6, 115.8, 117.4, 118.8, 119.2, 121.3, 128.8, 130.5, 133.9, 134.1, 146.4, 156.6,
13

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160.0, 164.8; ESI-MS: m/z 324.0 [M+Na]+.
3.3.8. N'-(3,5-Di-tert-butyl-2-hydroxybenzylidene)-salicylhydrazide (2h)
The title compound 2h was synthesized by condensing salicylhydrazide with
3,5-di-tert-butyl-2-hydroxybenzaldehyde in yield of 57.6%. White lumpy crystals; IR (KBr)
νmax:3236, 3074, 2956, 2907, 1796, 1641, 1233, 1098 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm):
1.27 (s, 9H, 3′-t-Bu), 1.40 (s, 9H, 5′-t-Bu), 6.96 (d, 1H, J = 7.6 Hz, 3-H), 6.99 (m, 1H, 5-H), 7.23 (s,
1H, 6′-H), 7.31(s, 1H, 4′-H), 7.45 (m, 1H, 4-H), 7.86 (d, 1H, J = 9.2 Hz, 6-H), 8.61 (s, 1H, N=CH);
13C-NMR (DMSO-d6, 125 MHz) δ (ppm):31.6, 34.9, 116.2, 117.0, 119.7, 121.7, 126.3, 126.9, 131.5,
135.4, 138.2, 144.0, 152.5, 157.4, 161.2; ESI-MS: m/z 391.1 [M+Na]+.
3.3.9. N'-(2-hydroxybenzylidene)-2-(4-chlorophenyl-oxyl)-acetyl hydrazide (2i)
The title compound 2i was synthesized by condensing 2-(4-chlorophenyl-oxyl)-acetyl hydrazide
with 2-hydroxybenzaldehyde with yield of 92%. White soild; IR (KBr) νmax:3443, 2924, 1680, 1641
1
cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 4.68 (s, 2H, 2-CH2), 6.83 (d, 1H, J = 8.6 Hz, 5-H),
6.85 (d, 1H, J = 8.6 Hz, 9-H), 6.93 (m, 1H, 5′-H), 7.02 (d, 1H, J = 6.4 Hz, 3′-H), 7.29 (d, 1H, J = 8.8
Hz, 6-H), 7.35 (d, 1H, J = 8.8 Hz, 8-H), 7.51 (m, 1H, 4′-H), 7.69 (d, 1H, J = 7.2 Hz, 6′H), 8.53 (s, 1H,
13
N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm): 69.0, 116.2, 116.8, 117.5, 120.4, 125.5, 126.5,
128.9, 130.4, 144.0, 154.2, 155.2, 169.0; ESI-MS: m/z 328.1 [M+Na]+.
3.3.10. N'-(2-hydroxy-5-nitrobenzylidene)-2-(4-methoxyphenyl)-acetyl hydrazide (2j)
The title compound 2j was synthesized by condensing 2-(4-methoxyphenyl)-acetyl hydrazide
with 2-hydroxybenzaldehyde with yield of 90%. Yellow solid; IR (KBr) νmax:3442, 3302, 2931, 1653,
1607 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.69 (s, 3H, 6-OCH3), 3.88 (s, 2H, 2-CH2), 6.86
(d, 1H, J = 9.2 Hz, 5-H), 6.88 (d, 1H, J = 8.4 Hz, 7-H), 7.05 (d, 1H, J = 9.2 Hz, 4-H), 7.06 (d, 1H, J
= 8.4 Hz, 8-H), 7.22 (d, 1H, J = 9.2 Hz, 3′-H), 8.14 (d, 1H, J = 8.4 Hz, 4′-H), 8.24 (s, 1H, 6′-H), 8.47
13
(s, 1H, N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm): 42.2, 55.4, 114.2, 116.7, 119.3, 125.3,
127.7, 128.4, 130.3, 140.2, 146.0, 159.2, 167.2, 171.0; ESI-MS: m/z 352.0 [M+Na]+.
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3.3.11. N'-(5-bromo-2-hydroxybenzylidene)-2-(4-methoxyphenyl)-acetyl hydrazide (2k)
The title compound 2k was obtained by condensing 2-(4-methoxyphenyl)-acetyl hydrazide with
5-bromo-2-hydroxybenzaldehyde with yield of 43%. White solid; IR (KBr) νmax:3668, 3200, 3059,
1
1667, 1610 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.71 (s, 3H, 6-OCH3), 3.86 (s, 2H,
2-CH2), 6.85 (d, 1H, J = 8.8 Hz, 5-H), 6.88 (d, 1H, J = 8.8 Hz, 7-H), 6.94 (d, 1H, J = 8.8 Hz, 3′-H),
7.17 (d, 1H, J = 8.8 Hz, 4-H), 7.19 (d, 1H, J = 8.8 Hz, 8-H), 7.51 (d, 1H, J = 8.8 Hz, 4′-H), 7.88 (s,
13
1H, 6′-H), 8.34 (s, 1H, N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm): 42.4, 55.7, 110.4, 114.6,
119.2, 120.6, 127.7, 130.6, 132.0, 135.3, 146.0, 159.5, 160.1, 169.7; ESI-MS: m/z 385.0 [M+Na]+.
3.3.12. N'-(5-chloro-2-hydroxybenzylidene)-2-(4-methoxyphenyl)-acetyl hydrazide (2l)
The title compound 2l was obtained by condensing 2-(4-methoxyphenyl)-acetyl hydrazide with
5-chloro-2-hydroxybenzaldehyde with yield of 84%. White crystals; IR (KBr) νmax:3454, 3199, 3059,
1
1668, 1612 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.69 (s, 2H, 2-CH2), 3.70 (s, 3H,
6-OCH3), 3.86 (s, 1H, 2′-CH2), 6.85 (d, 1H, J = 8.8 Hz, 5-H), 6.87 (d, 1H, J = 8.8 Hz, 7-H), 6.91 (d,
1H, J = 8.8 Hz, 3′-H), 7.17 (d, 1H, J = 8.8 Hz, 4-H), 7.19 (d, 1H, J = 8.8 Hz, 8-H), 7.20 (d, 1H, J =
13
8.8 Hz, 4′-H), 7.27 (s, 1H, 6′-H), 8.35 (s, 1H, N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm):
40.2, 55.2, 113.9, 118.2, 120.7, 123.1, 127.3, 130.2, 130.4, 130.8, 144.8, 157.9, 158.2, 167.1;
ESI-MS: m/z 341.0 [M+Na]+.
3.3.13. N'-(3,5-di-tert-butyl-2-hydroxybenzylidene)-2-(4-methoxyphenyl)-acetyl hydrazide (2m)
The title compound 2m was obtained by condensing 2-(4-methoxyphenyl)-acetyl hydrazide
with 3,5-di-tert-2-hydroxybenzaldehyde with yield of 50%. White crystals; IR (KBr) νmax:3454, 3199,
1
2957, 1650, 1613 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 1.25 (s, 9H, 3′-t-Bu), 1.36 (s, 9H,
5′-t-Bu), 3.48 (s, 2H, 2-CH2), 3.71 (s, 3H, 6-OCH3), 6.85 (d, 1H, J = 8.8 Hz, 5-H), 6.88 (d, 1H, J =
8.8 Hz, 7-H), 7.16 (s, 1H, 4′-H), 7.21 (d, 1H, J = 8.0 Hz, 4-H), 7.23 (d, 1H, J = 8.0 Hz, 8-H), 7.26 (s,
13
1H, 6′-H), 8.32 (s, 1H, N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm): 31.3, 34.4, 40.5, 55.8,
114.6, 117.5, 123.6, 127.5, 128.2, 130.2, 137.7, 146.0, 153.7, 159.5, 169.1; ESI-MS: m/z 419.2
15

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[M+Na]+.
3.3.14. N'-(2-hydroxybenzylidene)-2-(4-methoxyphenyl)-acetyl hydrazide (2n)
The title compound 2n was obtained by condensing 2-(4-methoxyphenyl)-acetyl hydrazide with
2-hydroxybenzaldehyde with yield of 74%. White crystals; IR (KBr) νmax:3500, 3166, 3036, 1662,
1614 cm-1;1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.70 (s, 3H, 6-OCH3), 3.85 (s, 2H, 2-CH2), 6.84
(d, 1H, J = 8.8 Hz, 5-H), 6.89 (d, 1H, J = 8.8 Hz, 7-H), 7.18 (d, 1H, J = 8.8 Hz, 3′-H), 7.20 (m, 1H,
5′-H), 7.22 (d, 1H, J = 8.4 Hz, 4-H), 7.26 (d, 1H, J = 8.4 Hz, 8-H), 7.49 (m, 1H, 4′-H), 7.66 (d, 1H, J
13
= 9.2 Hz, 6′-H), 8.37 (s, 1H, N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm): 42.3, 55.6, 113.5,
116.8, 118.5, 120.4, 126.6, 127.2, 128.6, 131.4, 146.0, 156.2, 158.5, 170.1; ESI-MS: m/z 307.1
[M+Na]+.
3.3.15. N'-(3,4-di-benzyloxy-benzylidene)-2-(4-methoxyphenyl)-acetyl hydrazide (2o)
The title compound 2o was obtained by condensing 2-(4-methoxyphenyl)-acetyl hydrazide with
3,4-di-benzyloxy-benzaldehyde with yield of 82%. White crystals; IR (KBr) νmax:3669, 3216, 3007,
1
1725, 1663 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.70 (s, 3H, 6-OCH3), 3.84 (s, 2H,
2-CH2), 5.16 (s, 2H, 3′-OCH2Ph), 5.18 (s, 2H, 4′-OCH2Ph), 6.82 (d, 1H, J = 8.4 Hz, 5-H), 6.86 (d,
1H, J = 8.4 Hz, 7-H), 7.11 (d, 1H, J = 9.6 Hz, 5′-H), 7.15 (d, 1H, J = 8.4 Hz, 4-H), 7.18 (d, 1H, J =
8.4 Hz, 8-H), 7.21(d, 1H, J = 9.6 Hz, 6′-H), 7.28-7.40 (m, 10H, 3′, 4′-OCH2Ph), 7.43 (s, 1H, 2′-H),
8.08 (s, 1H, N=CH); 13C-NMR (DMSO-d6, 125 MHz) δ (ppm): 41.6, 54.8, 71.1, 111.8, 114.5, 122.5,
127.1, 127.9, 128.9, 130.4, 130.6, 136.6, 146.2, 149.7, 151.9, 159.2, 170.8; ESI-MS: m/z 503.1
[M+Na]+.
3.3.16. N'-(2-hydroxy-5-nitrobenzylidene)-2-(4-chlorophenyl-oxyl)-acetyl hydrazide (2p)
The title compound 2p was obtained by condensing 2-(4-chlorophenyl-oxyl)-acetyl hydrazide
with 2-hydroxy-5-nitrobenzaldehyde with yield of 81%. Yellow powder; IR (KBr) νmax:3284, 3071,
1721, 1660 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 4.68 (s, 2H, 2-CH2), 7.09 (d, 1H, J = 7.6
Hz, 5-H), 7.11 (d, 1H, J = 7.6 Hz, 9-H), 7.35 (d, 1H, J = 8.4 Hz, 3′-H), 7.60 (d, 1H, J = 7.6 Hz, 6-H),
16

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7.61 (d, 1H, J = 7.6 Hz, 8-H), 7.94 (d, 1H, J = 8.4 Hz, 4′-H), 8.40 (s, 1H, 6′-H), 8.79 (s, 1H, N=CH);
13C-NMR (DMSO-d6, 125 MHz) δ (ppm): 68.6, 116.7, 117.3, 119.2, 125.3, 126.3, 128.6, 130.9,
140.6, 146.2, 156.2, 167.2, 171.3; ESI-MS: m/z 372.0 [M+Na]+.
3.3.17. N'-(5-bromo-2-hydroxybenzylidene)-2-(4-chlorophenyl-oxyl)-acetyl hydrazide (2q)
The title compound was obtained by condensing 2-(4-chlorophenyl-oxyl)-acetyl hydrazide with
5-bromo-2-hydroxybenzaldehyde with yield of 78%. White solid; IR (KBr) νmax:3645, 2978, 1683,
1
1630 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 4.68 (s, 2H, 2-CH2), 6.85 (d, 1H, J = 8.4 Hz,
3′-H), 6.93 (d, 1H, J = 9.6 Hz, 5-H), 7.02 (d, 1H, J = 9.6 Hz, 9-H), 7.30 (d, 1H, J = 8.4 Hz, 4′-H),
7.37 (d, 1H, J = 9.6 Hz, 6-H), 7.41 (d, 1H, J = 9.6 Hz, 8-H), 7.73 (s, 1H, 6′-H), 8.49 (s, 1H, N=CH);
13C-NMR (DMSO-d6, 125 MHz) δ (ppm): 69.0, 110.5, 117.1, 118.0, 119.8, 125.8, 128.9), 130.6,
134.4, 146.2, 154.7, 165.1, 171.0; ESI-MS: m/z 405.0 [M+Na]+.
3.3.18. N'-(5-chloro-2-hydroxy-benzylidene)-2-(4-chlorophenyl-oxyl)-acetyl hydrazide (2r)
The title compound was obtained by condensing 2-(4-chlorophenyl-oxyl)-acetyl hydrazide with
5-chloro-2-hydroxybenzaldehyde in yield of 78%. White solid; IR (KBr) νmax:3732, 2982, 1683,
1
1610 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 4.67 (s, 2H, 2-CH2), 6.89 (d, 1H, J = 6.8 Hz,
3′-H), 6.93 (d, 1H, J = 8.0 Hz, 5-H), 7.00 (d, 1H, J = 8.0 Hz, 9-H), 7.25 (d, 1H, J = 6.8 Hz, 4′-H),
7.30 (d, 1H, J = 10.0 Hz, 6-H), 7.34 (d, 1H, J = 10.0 Hz, 8-H), 7.60 (s, 1H, 6′-H), 8.48 (s, 1H,
13
N=CH); C-NMR (DMSO-d6, 125 MHz) δ (ppm): 68.7, 117.4, 118.2, 119.6, 123.4, 126.8, 130.6,
131.7, 133.5, 146.0, 156.3, 158.9, 171.0; ESI-MS: m/z 361.0 [M+Na]+.
3.3.19. N'-(3,5-di-tert-butyl-2-hydroxy-benzylidene)-2-(4-chlorophenyl-oxyl)-acetyl hydrazide (2s)
The title compound was obtained by condensing 2-(4-chlorophenyl-oxyl)-acetyl hydrazide with
3,5-di-tert-butyl-2-hydroxybenzaldehyde in yield of 30%. White solid; IR (KBr) νmax:3680, 3004,
1
1726, 1661 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 1.25 (s, 9H, 3′-t-Bu), 1.37 (s, 3H,
5′-t-Bu), 4.72 (s, 2H, 2-CH2), 7.02 (d, 1H, J = 9.2 Hz, 5-H), 7.03 (d, 1H, J = 9.2 Hz, 9-H), 7.19 (s,
1H, 6′-H), 7.29 (s, 1H, 4′-H), 7.35 (d, 1H, J = 8.8 Hz, 6-H), 7.37 (d, 1H, J = 8.8 Hz, 8-H), 8.47 (s, 1H,
17

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N=CH); 13C-NMR (DMSO-d6, 125 MHz) δ (ppm): 32.7, 34.4, 69.0, 117.4, 117.7, 122.8, 126.2, 128.3,
129.7, 137.9, 146.0, 153.5, 156.2, 171.2; ESI-MS: m/z 439.3 [M+Na]+.
3.3.20. N'-(3,4-di-benzyloxy-benzylidene)-2-(4-chlorophenyl-oxyl)-acetyl hydrazide (2t)
The title compound was obtained by condensing 2-(4-chlorophenyl-oxyl)-acetyl hydrazide with
3,4-di-benzyloxy-benzaldehyde in yield of 79%. White solid; IR (KBr) νmax:3668, 3216, 3007, 1708,
1
1512 cm-1; H-NMR (DMSO-d6, 400 MHz) δ (ppm): 4.63 (s, 2H, 2-CH2), 5.15 (s, 2H, 3′-OCH2Ph),
5.16 (s, 2H, 4′-OCH2Ph), 6.92 (d, 1H, J = 9.2 Hz, 5′-H), 6.99 (d, 1H, J = 8.4 Hz, 9-H), 7.09 (d, 1H, J
= 8.4 Hz, 5-H), 7.13 (d, 1H, J = 8.0 Hz, 6-H), 7.19 (d, 1H, J = 8.0 Hz, 8-H), 7.27-7.43 (m, 10H, 3′,
13
4′-OCH2Ph), 7.44 (d, 1H, J = 9.2 Hz, 6′-H), 7.86 (s, 1H, 2′-H), 8.19 (s, 1H, N=CH); C-NMR
(DMSO-d6, 125 MHz) δ (ppm): 68.1, 70.6, 111.8, 116.2, 121.4, 125.4, 127.1, 128.9, 130.6, 130.9,
136.7, 146.8, 149.5, 152.1, 156.2, 171.0; ESI-MS: m/z 523.0 [M+Na]+.
3.4. Turbidimetry test
The antibacterial activity of the test compounds were assessed in vitro by turbidimetric assays
[47-48]. Test compounds were prepared in 0.1 mol/L DMSO and diluted with LB, the final
concentration of DMSO was maintained at ≤1% (v/v). The bacteria were seeded at a concentration of
1 × 105 CFU/mL in 96-well microculture plates (90 ꢀL/well) containing LB; thereafter, the solutions
of prepared compounds were added to each well to obtain a final concentration of 100 ꢀM.
Thereafter, the microplates were vigorously shaken on a vibrating platform for 20 hours at 37°C.
Further, the plates were then examined in an ELISA plate reader at 450 nm for absorbance, to
determine the growth inhibitory activity. Results were expressed as the mean values of the three
independent variables.
Inhibitory activity = [(ODcontrol − ODsample )/ODcontrol ]×100%
ODcontrol was the optical density of bacteria suspension added the vehicle. ODsample was the optical
density of bacteria suspension treated with tested compounds.
The MBC values were measured by broth microdilution method in the 96-well microculture
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plates [49]. The bacteria were seeded in 96-well microculture plates at the concentration of 1×105
CFU/mL in LB, in a series of different concentrations of tested compounds and incubated for 20
hours at 37°C. The minimum bactericidal concentration was the lowest concentration of compound
whose absorbance by ELISA was parallel to the control in three independent experiments.
3.5. In vitro gene expression
Bacteria (2 × 107 CFU/mL) were resuspended in LB containing 50
ꢀmol/L compounds or in LB
alone. Samples were incubated for 8 hours at 37 °C and, the total RNA was harvested and confirmed
by TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR), as described by the
report [42]. The relative quantification of genes was determined by changes in expression of
transcripts relative to their expression in untreated bacteria. Samples were normalized to the
housekeeping gene gapA of E. coli, 16sRNA gene of B. subtilis, and gyrB gene of S. aureus. Data
were analyzed as change in transcript level after treatment with the screened compounds. The
sequences of the primers used for analysis are listed in Table 3.
3.6. Statistical analysis
The data were expressed as mean ± SD of the number of experiments indicated, and analyzed
using the SPSS 19.0 Windows Students version software (SPSS Inc., Chicago, USA). The
comparisons between multiple treatments were made with analysis of variance (ANOVA). One-way
ANOVA followed by a student’s t-test was performed to assess the statistical significance of
difference between control and chemotherapeutic agents treated for all the measurements. A
difference of P < 0.05 was considered statistically significant.
Acknowledgment
The authors are deeply thankful to the staff members of the analytical center for spectral
analyses and activity test centre for antibacterial activity of the Laboratory of Chemistry for Natural
Products, Guizhou Province, China and the Chinese Academy of Sciences. This research was
supported by the National Science Foundation of China (NSFC, Project No. 81160390) and the
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Science and Technology Department of Guizhou Province (QKHRZ [2011]33 and QSZHZ
[2010]71).
Appendix A. Supplementary data.
These data include NMR spectra of all new derivatives.
References
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Figure captions
Fig. 1. The inhibition of series of benzaldehyde Schiff bases on the growth of three bacteria in vitro. The
compounds were added into 96-well microculture plates containing the bacteria at a concentration of 1×105
CFU/mL in LB. The plates were analyzed in an ELISA plate reader at 450 nm after cultures were shaken on a
vibrating platform for 8 h at 37°C. The concentration of the compounds was 100 ꢀmol/L. Data were pooled from
three independent experiments, same as the below results.
Fig. 2. The concentration inhibited curves of the screened compounds against three bacterial strains in vitro.
Different concentration of tested compounds were added to 96-well microculture plates containing (A) the E. coli,
(B) B. subtilis and (C) S. aureus strains at concentration of 1×105 CFU/mL in LB, respectively. The plates were
measured in an ELISA plate reader at 450 nm after cultures were shaken on a vibrating platform for 8 h at 37°C.
The inhibition rate (%) was calculated with the results of the turbidimetry test (as described in Methods section).
Fig.3. The screened compounds altered gene expression of E. coli in vitro. (A) Change in the folds of four
metabolism genes were analyzed by real-time RT-PCR. (B) The expression of four genes was assayed used
semi-quantitative RT-PCR. The 2 × 107 CFU/mL E. coli was incubated with the 50 ꢀmol/L compounds for 8 h. Data
23

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were normalized to the transcript abundance of gapA gene, and fold change was relative to E. coli incubated in
medium alone (time matched).
Fig. 4. The screened compounds altered the expression of hemolysis-associated gene in B. subtilis in vitro. (A)
Changes in the folds of five hemolysis genes were analyzed by real-time RT-PCR. (B) The transcript expression
level of five genes was assayed used semi-quantitative RT-PCR. The 2 × 107 CFU/mL B. subtilis was incubated
with 50 ꢀmol/L compounds for 8 h. Data were normalized to the transcript abundance of 16sRNA gene, and fold
change was relative to B. subtilis incubated in medium alone (time matched). Data were pooled from three
experiments.
Fig. 5. The screened compounds altered the expression of S. aureus virulence genes in vitro. (A) Changes in the
folds of four virulence genes were analyzed by real-time RT-PCR. (B) The transcript expression level of four genes
was assayed used semi-quantitative RT-PCR. The 2 × 107 CFU/mL S. aureus was incubated with 50 ꢀmol/L
compounds for 8 h. Data were normalized to the transcript abundance of gyrB gene, and fold change was relative to
S. aureus incubated in medium alone (time matched). Data were pooled from three experiments.
Table 1. Anti-bacterial activity of compounds against the three strains at concentration of 100
ꢀ
mol/L in vitro.
Inhibition rate (%)
Compound
E.coli
B. subtilis
S. aureus
22.72 ± 4.38
96.04 ± 8.35
14.55 ± 1.32
6.81 ± 1.16
41.82 ± 1.28
95.84 ± 7.24
13.43 ± 1.07
11.80 ± 3.04
17.95 ± 4.01
18.30 ± 3.98
13.86 ± 3.95
ND
4.38 ± 1.23
4.21 ± 1.39
ND
ND
ND
1a
1b
1c
2a
2b
2c
2d
2e
2f
86.93 ± 3.10
ND
ND
ND
ND
ND
ND
95.55 ± 4.36
ND
81.92 ± 3.79*
ND
ND
ND
ND
70.93±5.73**
2g
2h
ND
24

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0.23 ± 3.92
5.57 ± 3.89
ND
ND
ND
ND
2i
ND
2j
1.01 ± 1.98
ND
ND
2k
ND
ND
2l
ND
ND
ND
2m
ND
ND
ND
ND
2n
ND
ND
2o
ND
96.39 ± 3.96
15.95 ± 2.39
83.18 ± 2.38*
19.71 ± 3.98
6.21 ± 2.69
-
1.39 ± 1.36
ND
2p
73.17 ± 4.32**
3.08 ± 3.66
10.48 ± 3.63
17.56 ± 3.6
99.34 ± 3.26
-
2q
ND
2r
2s
ND
99.46 ± 4.39
-
2t
Ampicillin
Streptomycin
94.12 ± 6.56
97.05 ± 4.24
Values were mean ± standard deviation of three independent experiments. The bacteria were seeded in 96-well
microculture plates at a concentration of 1×105 CFU/mL in LB. Test compounds and positive compound solution
were then added to the well to obtain the final concentration of 100 ꢀmol/L, respectively. The growth inhibitory
activity was measured in an ELISA plate reader at 450 nm after cultures were shaken on a vibrating platform at
*
37 °C for 8 h. P < 0.05 and **P < 0.01 compared with the control, respectively. ND: Antibacterial activity not
detected. Data were pooled from three independent experiments, same as the below results.
Table 2. MBC values of the tested compounds against three bacteria strains in vitro.
Bacteria
Compound
MBC (ꢀmol/L)
1.6 ± 0.3
3.2 ± 0.6
1b
2c
2q
E. coli
18.4 ± 2.3**
1.8 ± 0.7
Ampicillin
2.8 ± 0.6
2p
2r
B. subtilis
S. aureus
10.8 ± 1.3**
2.1 ± 0.5
Streptomycin
13.8 ± 3.9**
3.1 ± 0.6
1c
2d
2f
2h
2t
16.6±1.9**
19.0 ± 1.3**
1.4 ± 0.5
Streptomycin
1.3 ± 0.6
Values were mean ± standard deviation of three independent experiments. Series of different concentration of tested
compounds were added to a 96-well microculture plate containing the bacteria culture at concentration of 1 × 105
25

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CFU/mL. The plates were analyzed by an ELISA plate reader at 450 nm after cultures were shaken on a vibrating
platform for 8 h at 37°C.
** P < 0.01 compared with the control.
Table 3. List of the primers used in this study.
Gene
Primer
F: 5’-GTCTAGAACTGCGTTATCTGGCCGATG-3’
R: 5’-ACCCGGGTCCTTCCGCTGTTGATGA-3’
F: 5’-CCACCATTACCACCACCATCAC-3’
R: 5’-ACCGCCGAACTTCAACACTC-3’
F: 5’-TTATTCTCTTCGCTACAG-3’
cysB
acrA
tsh
E. coli
R: 5’-GATGACAGGCTACCGACAG -3’
F: 5’-TCCTGGTTGGGTTGAATA-3’
iroD
gapA
16sRNA
yqxC
yhdP
yugS
yhdT
yqhB
gyrB
saeR
hla
R: 5’-CAGCCAGAGGCCCACTA-3’
F: 5’-CAGAACATCATCCCGTCCTCTAC-3’
R: 5’-TACCAGTCAGTTTGCCATTCAGTT-3’
F:5’-GCGTGAGTGATGAAGGTTT-3’
R:5’-GCCGTGGCTTTCTGGTTA-3’
F: 5’-TATTAGTGGAAAGAGGGCT-3’
R: 5’-GCCTTCCTGTCCCTCTCC-3’
F: 5’-GGCATTTTTCGTGGCATC-3’
B. subtilis
R: 5’-GGTCAATTAACACTTTTCC-3’
F: 5’-GAATATTTATCAGCCTGTCA-3’
R: 5’-GATTTCCCCGACAATTTC-3’
F: 5’-GCGTGCTCATTGCTTTAAC-3’
R: 5’-CCTCAAGAATATCCTCTGTC-3’
F: 5’-GCTTTAGGACTGGGATGG-3’
R: 5’-CAGGCTCAGCGTCTATGT-3’
F: 5’-CAAATGATCACAGCTTTGGTACAG-3’
R: 5’-CGGCATCAGTCATAATGACGAT-3’
F: 5’CTGCCAAAACACAAGAACATGATAC-3’
R: 5’-ATTTACGCCTTAACTTTAGGTGCAGAT-3’
F: 5’-CAACAACACTATTGCTAGGTTCCATATT-3’
R: 5’-CCTGTTTTTACTGTAGTATTGCTTCCA-3’
F: 5’-ACTGATTAACCCAGTACAGATCCTTTC-3’
R: 5’-TCCAAACTTTGTTTTTCGTGTCTTT-3’
S. aureus
mecA
26

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F: 5’-ATACATCAAAACATTACGCGAACAC-3’
R: 5’-CTGGGTTCTTGCTGTCTTTAAGTG-3’
sbi
Fig. 1. The inhibition of series of benzaldehyde Schiff bases on the growth of three bacteria in vitro. The
compounds were added into 96-well microculture plates containing the bacteria at a concentration of 1×105
CFU/mL in LB. The plates were analyzed in an ELISA plate reader at 450 nm after cultures were shaken on a
vibrating platform for 8 h at 37°C. The concentration of the compounds was 100 ꢀmol/L. Data were pooled from
three independent experiments, same as the below results.
27

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Fig. 2. The concentration inhibited curves of the screened compounds against three strains in vitro. Different
concentration of tested compounds were added to 96-well microculture plates containing (A) the E. coli, (B) B.
subtilis and (C) S. aureus strains at concentration of 1×105 CFU/mL in LB, respectively. The plates were measured
in an ELISA plate reader at 450 nm after cultures were shaken on a vibrating platform for 8 h at 37°C. The
inhibition rate (%) was calculated with the results of the turbidimetry test (as described in Methods section).
28