Design, synthesis and antimicrobial activities evaluation of Schiff base derived from secnidazole derivatives as potential FabH inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.03.023

FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is critically important to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and Gram-negative bacteria. A series of novel secnidazole derivatives (1-20) were sy

Full text of DOI:10.1016/j.bmc.2013.03.023

Accepted Manuscript
Design, synthesis and antimicrobial activities evaluation of Schiff Base derived
from secnidazole derivatives as Potential FabH Inhibitors
Yao Li, Chang-Po Zhao, Meng-Yue Zhao, Yin Luo, Shuai Zhang, Zhi-Jun Liu,
Hai-Liang Zhu
PII:
S0968-0896(13)00236-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.03.023
BMC 10671
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 February 2013
12 March 2013
18 March 2013
Please cite this article as: Li, Y., Zhao, C-P., Zhao, M-Y., Luo, Y., Zhang, S., Liu, Z-J., Zhu, H-L., Design, synthesis
and antimicrobial activities evaluation of Schiff Base derived from secnidazole derivatives as Potential FabH
Inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.03.023
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Design, synthesis and antimicrobial activities evaluation of Schiff
Base derived from secnidazole derivatives as Potential FabH
Inhibitors
Yao Li a, ‡, Chang-Po Zhaoa, ‡, Meng-Yue Zhaoa, Yin Luoa, Shuai Zhanga, Zhi-Jun
Liua, Hai-Liang Zhua, *
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
*Corresponding author. Tel. & fax: +86-25-83592672; e-mail: zhuhl@nju.edu.cn.
‡ These two authors equally contributed to this paper.

Abstract
FabH, b-ketoacyl-acyl carrier protein (ACP) synthase III, is critically important to the
initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and
Gramnegative bacteria. A series of novel secnidazole derivatives (1-20) were
synthesized and fully characterized by spectroscopic methods and elemental analysis.
Among these compounds, 6, 8, 11, 13, 14, 16-20 were reported for the first time.
These compounds were tested for antibacterial activities against Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. The
compounds inhibitory assay and docking simulation indicated that compound 20
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(3,4,5-trimethylbenzylidene)acetohydra
zide with MIC of 3.13-6.25 μg/mL against the tested bacterial strains was a potent
inhibitor of Escherichia coli FabH.
Key words:
Secnidazole derivatives
Schiff base
FabH inhibitors
Antibacterial activities

Introduction
Microorganisms are almost everywhere in nature and infections caused by some
small pathogenic microorganisms could lead to illnesses even a fatal one. [1]Although
several class of antibacterial agents are still available and used for clinical treatment,
their advances in medical care are threatened by a natural phenomenon known as
“antimicrobial resistance”. The incidences of drug resistance of microorganisms to
antibacterial agents were constantly reported for the past few years.[2-5] In order to
prevent the serious medical problem caused by microorganisms, the discovery of new
types of antibacterial agents is a crucial task at present. Fortunately, much of the
research effort is made to the design of new antibacterial agents with high
efficiency.[6-7]
In recent 10 years, the research has been focused toward new antibacterial agents,
which may act through different kinds of targets in key areas of the bacterial cell
cycle, to surpass the problem of acquired resistance. The fatty acid synthesis (FAS)
pathway in bacteria is a promising target in the recent research and the fatty acid
biosynthesis (FAB) is a fundamental metabolic process for microorganisms and
essential for cell viability and growth.[8-9] β-ketoacyl-acyl carrier protein synthase III
(FabH) is the key enzyme responsible for the first reaction in the pathway and plays
an important regulatory role.[10] FabH has also been demonstrated to be essential for
initiating the fatty acid elongation cycles, and is involved in the feedback regulation
of the biosynthetic pathway via product inhibition.[11-12] On the other hand, some
novel compounds had been demonstrated to inhibit FabH from Gram-positive and
Gram-negative bacteria, including multi-drug resistant strains. FabH proteins from
Gram-positive and Gram-negative bacteria are highly conserved at the sequence and
structural level while there are no significantly homologous proteins in humans.
Importantly, the residues that comprise the active site are essentially invariant in
various bacterial FabH molecules. [13-14] FabH has been proved to be a promising
target for the design of novel antimicrobial drugs because it adjust and control the
fatty acid biosynthesis rate in an initiation pathway and its substrate specificity is a
key factor in membrane fatty acid composition.[15-17] These facts indicate that small

molecule inhibitors of FabH enzymatic activity could be potential candidates for
selective, nontoxic, and broad-spectrum antibacterials.
Because of varied biological activities, nitroimidazoles derivatives have gained
constant interests in drug research for antimicrobial chemotherapeutics and
antiangiogenic hypoxic cell radiosensitizers. The metabolism and toxicology of
nitroimidazoles derivatives, especially for secnidazole, have been characterized in
recent reports.[18-19] Secnidazole ( α, 2-dimethyl-5-nitro-1H- imidazole-1-ethanol ) is
extraordinary effective in the treatment of giardiasis， amebiasis and bacterial
vaginosis. By oral administration, secnidazole can be rapidly and completely absorbed,
and owns a longer terminal elimination half-life(17-29h) than popular medication.[20]
On this occasion, the treatment outcome with secnidazole is more effective than the
treatment using other imidazole drugs. Besides, the adverse effects of secnidazole are
less drastic than others.[21] To sum up, secnidazole derivatives have been proved to be
of great importance in pharmaceutical.
Schiff bases are the compounds with the structure of AC=NB, which are usually
synthesized from the condensation of active carbonyl groups and primary amines.
Lots of researchers studied the synthesis, characterization and structure-activity
relationship (SAR) of Schiff bases and some of Schiff bases were reported to own
antibacterial activities and.[22] Kim and coworkers reported the YKAs3003, a Schiff
base condensed by 4-hydroxy salicylaldehyde and cyclohexanamine as a potent
inhibitor of Escherichia coli (E.coli) FabH with antimicrobial activity.[23]
As a part of our research for new antibacterial drugs, a series of secnidazole
derivatives containing Schiff base had been designed and synthesized. We chose
Schiff base moiety as a part because it is a variety of biologically active substances,
especially with antimicrobial activity. Compounds with the structure of C=N are
known as Schiff bases, which are usually synthesized by the condensation of primary
amines and active carbonyl groups. Some Schiff bases were reported to possess
antibacterial, antifungal and antitumor activities.[22] Lots of researchers studied the
synthesis, characterization and structure-activity relationship (SAR) of Schiff
bases.[23-26] Y. Kim et. al reported the YKAs3003, a Schiff base condensed by

4-hydroxy salicylaldehyde and cyclohexanamine as a potential inhibitor of
Escherichia coli (E.coli) FabH with antimicrobial activity.[27]
In this study, we described the synthesis and structure-activity relationship of a new
series of secnidazole derivatives containing Schiff base, and studied their antibacterial
activities against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa),
Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) and E. coli FabH
inhibitory activities. Docking simulation was performed using the X-ray
crystallographic structure of the FabH of E.coli in complex with the most potent
inhibitor to explore the binding mode of the compound at the active site.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide was
prepared by the reaction of 2-methyl-5-nitro-1H-imidazole in excess ethyl
α-chloroacetate and then hydrazinolysis of ethyl ester group by hydrazine hydrate
according to Javad Mirzaei et al.’s method.[28]
Then 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide was reacted with
substituted benzaldehyde to prepare the corresponding secnidazole derivatives 1-20.
The chemical structures of these secnidazole derivatives were summarized in Table 1.
1
All these compounds were given satisfactory elementary analyses ( 0.4%). H NMR
and ESI MS spectra data was consistent with the assigned structures. Among these
compounds, 6, 8, 11, 13, 14, 16-20 were reported for the first time.
2.2. Biological activity
2.2.1. Antimicrobial activity
All the synthesized compounds (1-20) were screened for their antibacterial
activities against two Gram-negative bacterial strains: E. coli ATCC 25922 and
Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa), two Gram-positive bacterial
strains: Bacillus subtilis ATCC 530 (B. subtilis) and Staphylococcus aureus ATCC
6538 (S. aureus) by serial dilution method. The MICs (minimum inhibitory
concentrations) of the compounds against these bacteria were presented in Table 2.

Also included was the activity of reference compound Kanamycin and DDCP
(Compound 5 in Antimicrob. Agents Chemother 2004, 48, 3093-3102)[39] under
identical conditions for comparison. The results revealed that most of the synthesized
compounds exhibited significant antibacterial activities.
Compared with the other synthetic secnidazole derivatives, compound 20,
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(3,4,5-trimethylbenzylidene)acetohydra
zide, exhibited the most potent antibacterial activity with MIC of 3.13, 6.25, 3.13 and
3.13 μg/mL against E. coli, P. aeruginosa, B. subtilis and S. aureus, respectively,
which was similar to the broad-spectrum antibiotic Kanamycin corresponding MIC of
3.13, 3.13, 1.56 and 1.56 μg/mL. From the data we got, the varieties of substitutes of
benzaldehyde such as halogen, nitro, methyl and methoxyl lead to the different
antibacterial activities of these secnidazole derivatives. Structure-activity relationships
in these secnidazole derivatives proved that compounds with electron-donating
substituents (such as CH3, OCH3) (4, 7, 8, 19, 20), MICs value range from 3.13 to
12.5 μg/mL against E. coil ATCC 25922, exhibited more potential activities than
those with electron-withdrawing substituents (such as F, Cl, Br). Based on the
mention above, we presented ideas that electron-donating groups on benzaldehyde
component were beneficial to the antibacterial activity and compounds with
electron-withdrawing halogen groups on benzaldehyde component were not
conducive for the activity, especially for compound 20 with three substituted methyl
groups on benzaldehyde component.
According to Table 2, we found that compounds with an electron-donating group
on the 4-position of benzaldehyde component (4, 7, 19, 20), MICs value range from
3.13 to 12.5 μg/mL, displayed higher antibacterial activity against E. coil ATCC
25922 than compounds with an electron-donating group on the 3-position and
2-position of benzaldehyde component (13, 17), MICs value range from 25 to 50
μg/mL. From this trend, we suggested the fact that electron-donating groups on the
4-position of benzaldehyde component were beneficial to the antibacterial activity.
2.2.2. E. coli FabH inhibitory activity

The E. coli FabH inhibitory potencies of the synthetic oximes with best
antibacterial activities (4, 7, 8, 16, 19, 20) and weakest antibacterial activities (1, 15,
17) were examined and the results were summarized in Table 3. Most of the tested
compounds displayed prominent E. coli FabH inhibitory activities. Among them,
compound 20 showed the best inhibitory activity with IC50 of 2.2 μM; compound 1
showed the weakest inhibitory activity with IC50 of 58.3 μM. This result supported
the prominent antibacterial activity of 20.
Compounds 7 with para-substituted methyl group showed better E. coli FabH
inhibitory activity than compounds 1 with para-substituted F group on benzaldehyde
component. Compounds 4 with para-substituted methoxy group showed better E. coli
FabH inhibitory activity than compounds 17 with ortho-substituted methoxy group on
benzaldehyde component. The results of E. coli FabH inhibitory activities of the test
compounds were corresponding to the structure-activity relationships of their
antibacterial activities. This demonstrated that antibacterial activities of the synthetic
compounds were probably correlated to their FabH inhibitory activities.
2.2.3. Binding model of compound 11, 12 and E. coli FabH
Molecular docking of the compound 20 and E. coli FabH was performed on the
binding model based on the E. coli FabH-CoA complex structure (1HNJ.pdb). [30]
All docking runs were applied Ligand Fit Dock protocol of Discovery Studio 3.1.
The docking calculation of the synthesized compounds was showed in Table 4. The
interaction energy of the compounds and their antibacterial activity showed the
corresponding results. Among the docking calculation of the synthesized compounds,
compound 20 showed the lowest interaction energy. The binding model of compound
20 and E. coli FabH was depicted in Figure 1 and Figure 2. In the binding model,
compound 20 was nicely bound to the FabH kinase with 3 interaction bonds. The end
amino of Trp32, Arg151 and Gly152 were respectively formed one hydrogen bonds
interaction with oxygen atom and two PiCationMonitor of secnidazole ring of
compound 11. This molecular docking result, along with the biological assay data,
suggesting that compound 20 was a potential inhibitor of FabH.

3. Conclusion
To sum up, a series of novel nitroimidazole derivatives 1-20 were synthesized and
tested for their inhibitory activities against E. coli, P. aeruginosa, B. subtilis and S.
aureus. Among these compounds, compound 6, 8, 11, 13, 14, 16-20 were reported for
the first time. Many of them exhibited potent antibacterial and E.coli FabH inhibitory
activities. Particularly, Compound 20 (E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
N'-(3,4,5-trimethylbenzylidene)acetohydrazide with MIC of 3.13-6.25 μg/mL against
the tested bacterial strains were proved to be the most potent compounds. Preliminary
SARs and molecular modeling study provided further insight into interactions
between the enzyme and its ligand. The result provided valuable information for the
design of E. coli FabH inhibitors as antibacterial agents.
4. Experiments
4.1. Materials and Measurements
All chemicals (reagent grade) used were commercially available. Melting points
(uncorrected) were determined on a X-4 MP apparatus (Taike Corp., Beijing, China).
ESI mass spectra were obtained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were recorded on a Bruker PX500 or DPX300 spectrometern at 25 ºC
with TMS and solvent signals allotted as internal standards. Chemical shifts were
reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid
instrument and were within 0.4% of the theoretical values.
4.2. General method of synthesis of secnidazole derivatives
2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide was prepared according the
previous procedure by JavadMirzaei et al.[29]
An equimolar mixture of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(0.001 mol) and substituted benzaldehyde(0.001 mol) in methyl alcohol(15 mL) was
reacted for 3–6 h with ice-bath. The resultant solid was filtered, washed with water
and recrystallized from ethanol to give secnidazole derivatives 1-20.

(E)-N'-(4-fluorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (1)
mp: 259-260℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.31 (s, 3H), 5.39 (s, 2H),
7.27-7.32 (m, 2H), 7.78-7.83 (m, 2H), 8.06 (s, 1H), 8.27 (t, J=17.55Hz, 1H), 11.84 (s,
1H). MS (ESI):306.26 (C13H13FN5O3, [M+H]+). Anal. Calcd for C13H12FN5O3:
C, 51.15; H, 3.96; N, 22.94. Found: C, 51.17; H, 3.95; N, 22.93.
(E)-N'-(4-chlorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (2)
mp: 254-255℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.31 (s, 3H), 5.39 (s, 2H), 7.52
(t, J=10.45Hz, 2H), 7.76 (t, J=17.38Hz, 2H), 8.05 (s, 1H), 8.27 (t, J=18.65Hz, 1H),
11.89 (s, 1H). MS (ESI):322.72 (C13H13ClN5O3, [M+H]+). Anal. Calcd for
C13H12ClN5O3: C, 48.52; H, 3.76; N, 21.77. Found: C, 48.54; H, 3.75; N, 21.78.
(E)-N'-(4-bromobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (3)
mp: 229-230℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.31 (s, 3H), 5.39 (s, 2H),
7.63-7.72 (m, 4H), 8.04 (s, 1H), 8.29 (d, J=7.75Hz, 1H), 11.89 (s, 1H). MS
(ESI):367.17 (C13H13BrN5O3, [M+H]+). Anal. Calcd for C13H12BrN5O3: C, 42.64;
H, 3.30; N, 19.13. Found: C, 42.65; H, 3.31; N, 19.14.
(E)-N'-(4-methoxybenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydra
zide (4)
mp: 194-196℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.30 (d, J=5.17Hz, 3H), 3.81 (s,
3H), 5.37 (s, 2H), 7.02(d, J=4.42Hz, 2H), 7.65-7.70 (m, 2H), 8.00 (s, 1H), 8.30 (d,
J=5.80Hz, 1H), 11.70 (d, J=7.63Hz, 1H). MS (ESI):318.30 (C14H16N5O4, [M+H]+).
Anal. Calcd for C14H15N5O4: C, 52.99; H, 4.76; N, 22.07. Found: C, 52.98; H, 4.75;
N, 22.09.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(4-nitrobenzylidene)acetohydrazide
(5)
mp: 247-248℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.32 (s, 3H), 5.45 (s, 2H), 8.00
(t, J=9.87Hz, 2H), 8.17 (s, 1H), 8.29-8.35 (m, 3H), 12.13 (s, 1H). MS (ESI):333.27

(C13H13N6O5, [M+H]+). Anal. Calcd for C13H12N6O5: C, 46.99; H, 3.64; N,
25.29. Found: C, 46.98; H, 3.65; N, 25.26.
(E)-N'-(4-hydroxybenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydra
zide (6)
mp: 294-296℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 5.34 (s, 2H), 6.83
(d, J=4.31Hz, 2H), 7.55 (t, J=8.78Hz, 2H), 7.95 (s, 1H), 8.30 (d, J=3.12Hz, 1H), 9.94
(s, 1H), 11.64 (d, J=4.49Hz, 1H). MS (ESI):304.27 (C13H14N5O4, [M+H]+). Anal.
Calcd for C13H13N5O4: C, 51.48; H, 4.32; N, 23.09. Found: C, 51.49; H, 4.31; N,
23.07.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(4-methylbenzylidene)acetohydrazi
de (7)
mp: 208-210℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 2.34 (s, 3H), 5.33
(s, 2H), 6.75 (d, J=4.38Hz, 2H), 7.53 (t, J=7.86Hz, 2H), 7.91 (s, 1H), 8.31 (d,
J=2.30Hz, 1H), 11.54 (d, J=6.03Hz, 1H). MS (ESI):302.30 (C14H16N5O3, [M+H]+).
Anal. Calcd for C14H15N5O3: C, 55.81; H, 5.02; N, 23.24. Found: C, 56.81; H, 5.01;
N, 23.25.
(E)-N'-(4-hydroxybenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydra
zide (8)
mp: 277-278℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 2.97 (s, 6H), 5.33
(s, 2H), 6.74 (d, J=4.02Hz, 2H), 7.53 (t, J=7.68Hz, 2H), 7.92 (s, 1H), 8.30 (d,
J=2.30Hz, 1H), 11.54 (d, J=5.85Hz, 1H). MS (ESI):331.34 (C15H19N6O3, [M+H]+).
Anal. Calcd for C15H18N6O3: C, 54.54; H, 5.49; N, 25.44. Found: C, 54.55; H, 5.48;
N, 25.42.
(E)-N'-(3-fluorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (9)
mp: 222-223℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.29 (s, 3H), 5.41 (s, 2H), 7.28
(t, J=8.40Hz, 1H), 7.48-7.53 (m, 1H), 7.57 (d, J=3.83Hz, 1H), 7.62 (d, J=5.05Hz, 1H),
8.06 (s, 1H), 8.28 (t, J=16.63Hz, 1H), 11.93 (s, 1H). MS (ESI):306.26

(C13H13FN5O3, [M+H]+). Anal. Calcd for C13H12FN5O3: C, 51.15; H, 3.96; N,
22.94. Found: C, 51.18; H, 3.97; N, 22.93.
(E)-N'-(3-fluorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (10)
mp: 197-198℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.29 (s, 3H), 5.42 (s, 2H), 7.42
(t, J=7.80Hz, 1H), 7.63 (d, J=3.95Hz, 1H), 7.72 (d, J=3.83Hz, 1H), 7.99 (s, 1H), 8.06
(s, 1H), 8.03 (s, 1H), 8.27 (s, 1H), 11.94 (s, 1H). MS (ESI):367.17 (C13H13BrN5O3,
[M+H]+). Anal. Calcd for C13H12BrN5O3: C, 42.64; H, 3.30; N, 19.13. Found: C,
42.65; H, 3.31; N, 19.15.
(E)-N'-(3-fluorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (11)
mp: 259-260℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 5.43 (s, 2H),
7.46-7.51 (m, 2H), 7.67-7.71 (m, 1H), 7.86 (s, 1H), 8.05 (s, 1H), 8.27 (t, J=14.91Hz,
1H), 11.96 (s, 1H). MS (ESI):322.72 (C13H13ClN5O3, [M+H]+). Anal. Calcd for
C13H12ClN5O3: C, 48.53; H, 3.76; N, 21.77. Found: C, 48.55; H, 3.75; N, 21.78.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(3-nitrobenzylidene)acetohydrazide
(12)
mp: 256-257℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.30 (s, 3H), 5.46 (s, 2H),
7.46-7.51 (m, 1H), 7.67-7.71 (m, 2H), 8.28 (t, J=5.20Hz, 1H), 8.35 (d, J=11.28Hz,
1H), 8.56 (d, J=6.40Hz, 1H), 12.08 (s, 1H). MS (ESI):334.27 (C13H13N6O5,
[M+H]+). Anal. Calcd for C13H12N6O5: C, 46.99; H, 3.64; N, 25.29. Found: C,
46.98; H, 3.65; N, 25.27.
(E)-N'-(3-methoxybenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydra
zide (13)
mp: 165-165℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 3.81 (s, 3H),
5.40(s, 2H), 7.02 (d, J=4.58Hz, 1H), 7.26-7.44 (m, 3H), 8.02 (s, 1H), 8.30 (d,
J=3.93Hz, 1H), 11.85 (s, 1H). MS (ESI):318.30 (C14H16N5O4, [M+H]+). Anal.
Calcd for C14H16N5O4: C, 52.99; H, 4.76; N, 22.07. Found: C, 52.98; H, 4.75; N,

22.08.
(E)-N'-(2-chlorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (14)
mp: 257-258℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 5.41 (s, 2H),
7.42-7.49 (m, 2H), 7.55 (t, J=4.76Hz, 1H), 8.05 (t, J=4.76Hz, 1H), 8.30 (d, J=5.94Hz,
1H), 8.45 (s, 1H), 12.03 (s, 1H). MS (ESI):322.72 (C13H13ClN5O3, [M+H]+). Anal.
Calcd for C13H12ClN5O3: C, 48.53; H, 3.76; N, 21.77. Found: C, 48.52; H, 3.75; N,
21.78.
(E)-N'-(2-fluorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazi
de (15)
mp: 231-232℃. 1H NMR (500 MHz, DMSO-d6) δ: 2.30 (s, 3H), 5.40 (s, 2H), 7.31
(t, J=8.23Hz, 2H), 7.50 (t, J=6.25Hz, 1H), 7.98 (t, J=7.63Hz, 1H), 8.28 (d, J=3.38Hz,
1H), 8.45 (s, 1H), 11.95 (s, 1H). MS (ESI):306.26 (C13H13FN5O3, [M+H]+). Anal.
Calcd for C13H12FN5O3: C, 51.15; H, 3.96; N, 22.94. Found: C, 51.14; H, 3.94; N,
22.96.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(2-nitrobenzylidene)acetohydrazide
(16)
mp: 269-270℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.30 (s, 3H), 5.40 (s, 2H), 7.70
(t, J=7.77Hz, 1H), 7.84 (t, J=7.14Hz, 1H), 8.08-8.17 (m, 2H), 8.28 (s, 1H), 8.46 (s,
1H), 12.15 (s, 1H). MS (ESI):333.27 (C13H13N6O5, [M+H]+). Anal. Calcd for
C13H12N6O5: C, 46.99; H, 3.64; N, 25.29. Found: C, 46.97; H, 3.65; N, 25.31.
(E)-N'-(2-methoxybenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydra
zide (17)
mp: 279-280℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 3.86 (s, 3H), 5.38
(s, 2H), 7.01 (t, J=6.87Hz, 1H), 7.12 (d, J=4.22Hz, 1H), 7.41-7.46 (m, 1H), 7.89 (t,
J=3.86Hz, 1H), 8.29 (s, 1H), 8.40 (s, 1H), 11.80 (s, 1H). MS (ESI):318.30
(C14H16N5O4, [M+H]+). Anal. Calcd for C14H15N5O4: C, 52.99; H, 4.76; N,
22.07. Found: C, 52.99; H, 4.76; N, 22.07.

(E)-N'-(2,4-difluorobenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydr
azide (18)
mp: 273-275℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H), 5.41 (s, 2H),
7.54-7.57 (m, 1H), 7.75 (d, J=1.01Hz, 1H), 8.06 (d, J=4.31Hz, 1H), 8.27 (s, 1H), 8.39
(s, 1H), 12.07 (s, 1H). MS (ESI):324.25 (C13H12F2N5O3, [M+H]+). Anal. Calcd for
C13H13F2N5O3: C, 48.30; H, 3.43; N, 21.67. Found: C, 48.31; H, 3.41; N, 21.66.
(E)-N'-(3,4-dimethoxybenzylidene)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohy
drazide (19)
mp: 273-275℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.30 (s, 3H), 3.82 (s, 6H), 5.39
(s, 2H), 7.54-7.57 (m, 1H), 7.75 (d, J=1.01Hz, 1H), 8.06 (d, J=4.31Hz, 1H), 8.27 (s,
1H), 8.39 (s, 1H), 12.07 (s, 1H). MS (ESI):324.25 (C15H18N5O5, [M+H]+). Anal.
Calcd for C15H19N5O5: C, 51.87; H, 4.93; N, 20.16. Found: C, 51.88; H, 4.92; N,
20.17.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(3,4,5-trimethylbenzylidene)acetoh
ydrazide (20)
mp: 245-246℃. 1H NMR (300 MHz, DMSO-d6) δ: 2.31 (s, 3H), 3.70 (s, 3H), 3.83
(d, J=2.21Hz, 6H), 5.40 (s, 2H), 7.06 (s, 1H), 7.97 (s, 1H), 8.31 (s, 1H), 12.07 (s, 1H).
MS (ESI):330.35 (C16H20N5O3, [M+H]+). Anal. Calcd for C15H19N5O5: C, 58.35;
H, 5.81; N, 21.26. Found: C, 58.34; H, 5.83; N, 21.28.
4.3. Antibacterial activity
The antibacterial activities of the synthetic compounds were tested against two
Gram-negative bacterial strains: E. coli ATCC 25922 and P. aeruginosa ATCC 27853,
two Gram-positive bacterial strains: B. subtilis ATCC 530 and S. aureus ATCC 25923,
using method recommended by National Committee for Clinical Laboratory
Standards (NCCLS). [31]
In vitro activities of the compounds were tested in Nutrient broth (NB) for bacteria
by the two-fold serial dilution method. Seeded broth (broth containing microbial
spores) was prepared in NB from 24 h old bacterial cultures on nutrient agar

(Hi-media) at 37 1 ºC. The bacterial suspension was adjusted with sterile saline to a
concentration of 1 ×104-105 CFU. The tested compounds and reference drugs were
prepared by twofold serial dilution to obtain the required concentrations of 100, 50,
25, 12.5, 6.25 and 3.13 μg/mL. The tubes were incubated in BOD incubators at 37
1
ºC for bacteria. The MICs were recorded by visual observations after 24 h (for
bacteria) of incubation. Kanamycin and DDCP were used as standards for bacterial.
The observed MICs are presented in Table 2
4.4. E. coli FabH purification and activity assay
Full-length E. coli acyl carrier protein (ACP), acyl carrier protein synthase
(ACPS), and β-ketoacyl-ACP synthase III (FabH) were individually cloned into pET
expression vectors with an N-terminal His-tag (ACP, ACPS in pET19; FabH in
pET28).
All proteins were expressed in E. coli strain BL21 (DE3). Transformed cells were
grown on Luria-Bertani (LB) agar plates supplemented with kanamycin (30 μg/ml).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis
was used to screen colonies for overexpression of proteins. One such positive colony
was used to inoculate 10 ml of LB medium with 30 μg/ml of kanamycin and grown
overnight at 37 ºC, 1 ml of which was used to inoculate 100 ml LB medium
supplemented with 30 mg/ml of kanamycin. The culture was shaken for 4 h at 37 ºC,
and then induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). The
culture was grown for 4 h, and harvested by centrifugation (30 min at 15 000 rpm).
Harvested cells containing His-tagged ACP, ACPS, and FabHs were lysed by
sonication in 20 mM Tris, pH 7.6, 5 mM imidazole, 0.5 M NaCl and centrifuged at
20,000 rpm for 30 min. The supernatant was applied to a Ni-NTA agarose column,
washed, and eluted using a 5-500 mM imidazole gradient over 20 column volumes.
Eluted protein was dialyzed against 20 mMTris, pH 7.6, 1 mM DTT, and 100 mM
NaCl. Purified FabHs were concentrated up to 2 mg/mL and stored at -80 ºC in 20
mM Tris, pH 7.6, 100 mM NaCl, 1 mM DTT, and 20% glycerol for enzymatic assays.
Purified ACP contains the apo-form that needs to be converted into the holo-form.

The conversion reaction is catalyzed by ACP synthase (ACPS). In the final volume of
50 mL, 50 mg ACP, 50 mMTris, 2 mM DTT, 10 mM MgCl2, 600 μM CoA, and 0.2
μM ACPS was incubated for 1 h at 37 ºC. The pH of the reaction was then adjusted to
approximately 7.0 using 1 M potassium phosphate. Holo-ACP was purified by
fractionation of the reaction mixture by Source Q-15 ion exchange chromatography
using a 0-500 mM NaCl gradient over 25 column volumes.
In a final 20 μL reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0, 0.5 mM DTT, 0.25
mM MgCl2, and 2.5 μMholo-ACP were mixed with 1 nM FabH, and H2O was added
to 15 mL. After 1 min incubation, a 2 μL mixture of 25 μM acetyl-CoA and 0.75 μCi
[3H] acetyl-CoA was added for FabH reaction for 25 min. The reaction was stopped
by adding 20 mL of ice-cold 50% TCA, incubating for 5 min on ice, and centrifuging
to pellet the protein. The pelletwaswashed with 10% ice-cold TCA and resuspended
with 5 μL of 0.5 MNaOH. The incorporation of the 3H signal in the final product was
read by liquid scintillation. When determining the inhibition constant (IC50), inhibitors
were added from a concentrated DMSO stock such that the final concentration of
DMSO did not exceed 2%.
4.5. Docking simulations
The crystal structures of E. coli FabH (PDB code: 1HNJ) [31] was obtained from the
Protein Data Bank (http://www.rcsb.org). Molecular docking of compound 20 into the
three-dimensional X-ray structure of FabH was carried out using Ligand Fit Dock
protocol of Discovery Studio 3.1.
Acknowledgements
The work was financed by a grant (No. J1103512) from National Natural Science
Foundation of China.
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Legends for Figures and Schemes :
Scheme 1. Synthesis of nitroimidazole derivatives 1-20.
Figure 1. Binding model of compound 20 and E. coli FabH. H-bonds are displayed as
dashed lines.
Figure 2.The surface model structure of compound 20 binding model with FabH
complex
Scheme 1
Figure 1

Figure 2
Table 1
Structures of Compounds 1-20

Compounds
R
4-F
Compounds
R
1
2
11
12
13
14
15
16
17
18
19
20
3-Cl
4-Cl
3-NO2
3-OCH3
2-Cl
3
4-Br
4
4-OCH3
4-NO2
4-OH
4-CH3
4-N(CH3)2
3-F
5
2-F
6
2-NO2
7
2-OCH3
2,4-2F
8
9
3,4-2OCH3
3,4,5-3CH3
10
3-Br
Table 2
Antibacterial activities of synthetic compounds.
Compounds
Minimum inhibitory concentrations (μg/mL)
Gram-negative Gram-positive
Escherichia
Pseudomonas
Bacillus
Staphylococcus
coli
>100
50
aeruginosa
>100
50
subtilis
>100
>100
25
aureus
>100
>100
25
1
2
3
25
12.5
6.25
25
4
6.25
25
6.25
50
12.5
25
5
6
50
>100
6.25
12.5
50
>100
25
>100
12.5
25
7
6.25
12.5
50
8
12.5
25
9
25
10
11
25
50
>100
50
>100
50
25
50

12
25
25
50
50
>100
50
50
25
13
14
>100
>100
25
>100
>100
25
50
50
15
>100
12.5
>100
50
>100
12.5
>100
25
16
17
50
50
18
50
50
19
12.5
3.13
3.13
1.56
12.5
6.25
3.13
3.13
25
12.5
3.13
1.56
25
20
3.13
1.56
12.5
Kanamycin
DDCP
Table 3
E. coli FabH inhibitory activities of selected compound 4-6.
E. coli FabH inhibitory activities of top compounds
Compounds
IC50(μM)
58.3 3.9
4.5 0.3
6.2 0.4
15.9 0.9
47.5 4.2
19.8 1.4
42.4 3.5
8.8 0.7
2.3 0.2
2.1 0.1
1
4
7
8
15
16
17
19
20
DDCP
Table 4
The docking calculation of the synthesized compounds (1-20)

CDOCKER INTERATION
ENERGY
CDOCKER INTERATION
ENERGY
Compounds
Compounds
ΔGb (kcal/mol)
-26.1226
ΔGb (kcal/mol)
-29.8734
1
2
11
12
13
14
15
16
17
18
19
20
-28.7742
-29.7453
3
-32.7251
-30.8633
4
-33.9155
-28.5753
5
-32.0342
-28.3193
6
-28.5169
-33.0668
7
-33.8697
-28.5587
8
-33.4241
-30.2775
9
-31.2451
-33.7007
10
-29.0816
-35.0512

Design, synthesis and antimicrobial activities evaluation of Schiff Base
derived from secnidazole derivatives as Potential FabH Inhibitors
a, ‡
Yao Li , Chang-Po Zhao
a,
‡
a
, Xianhui Yang , Yin Luo , Zhuai Zhang , Zhi-Jun Liu ,
a
a
a
a,
Hai-Liang Zhu *
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China
FabH, b-ketoacyl-acyl carrier protein (ACP) synthase III, is critically important to the
initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and
Gramnegative bacteria. A series of novel secnidazole derivatives (1-20) were
synthesized and fully characterized by spectroscopic methods and elemental analysis.
Among these compounds, 6, 8, 11, 13, 14, 16-20 were reported for the first time.
These compounds were tested for antibacterial activities against Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. The
compounds inhibitory assay and docking simulation indicated that compound 20
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N'-(3,4,5-trimethylbenzylidene)acetohydraz
ide with MIC of 3.13-6.25 μg/mL against the tested bacterial strains was a potent
inhibitor of Escherichia coli FabH.