Design, synthesis, and antibacterial evaluation of new Schiff's base derivatives bearing nitroimidazole and pyrazole nuclei as potent E. coli FabH inhibitors

Article abstract of DOI:10.1007/s11164-015-2018-1

New Schiff's base derivatives 5a-j have been synthesized by reaction between 5-aryloxypyrazole-4-carbaldehydes 3a-j and 2-(2-methyl-5-nitro-1Himidazol- 1-yl)acetohydrazide 4 in the presence of nickel (II) nitrate as a catalyst in ethanol at room temperatu

Full text of DOI:10.1007/s11164-015-2018-1

Res Chem Intermed
DOI 10.1007/s11164-015-2018-1
Design, synthesis, and antibacterial evaluation of new
Schiff’s base derivatives bearing nitroimidazole
and pyrazole nuclei as potent E. coli FabH inhibitors
Chetan B. Sangani1 Jigar A. Makwana1
•
•
Yong-Tao Duan1 Umesh P. Tarpada2
Yogesh S. Patel2 Ketan B. Patel Vivek N. Dave
•
•
2
2
•
•
•
Hai-Liang Zhu1
Received: 19 December 2014 / Accepted: 13 March 2015
ꢀ Springer Science+Business Media Dordrecht 2015
Abstract New Schiff’s base derivatives 5a–j have been synthesized by reaction
between 5-aryloxypyrazole-4-carbaldehydes 3a–j and 2-(2-methyl-5-nitro-1H-
imidazol-1-yl)acetohydrazide 4 in the presence of nickel (II) nitrate as a catalyst in
ethanol at room temperature with good yield (75–88 %). All compounds were tested
for antibacterial properties and inhibition of E. coli FabH. Of the compounds
studied, the majority of the compounds showed effective antibacterial properties
and inhibition of E. coli FabH activity. Compound 5i showed the most effective
inhibition (IC50 = 4.6 0.2 lM) by binding into the active site of the E. coli FabH
&
&
Chetan B. Sangani
chetansangani1986@yahoo.com
Hai-Liang Zhu
zhuhl@nju.edu.cn
Jigar A. Makwana
jigarsynthesis@yahoo.in
Yong-Tao Duan
duanyongtao860409@163.com
Umesh P. Tarpada
umeshtarpada@gmail.com
Yogesh S. Patel
dryspatel@gmail.com
Ketan B. Patel
kbp.gsc@gmail.com
Vivek N. Dave
davevivekdave@gmail.com
1
2
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
People’s Republic of China
Chemistry Department, Government Science College, Gandhinagar 382015, Gujarat, India
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C. B. Sangani et al.
receptor with minimum binding energy (DGb = -54.2961 kcal/mol). The binding
was stabilized by two hydrogen bonds, two p–p, and three p–cation interactions.
Keywords Pyrazole ꢀ Imidazol ꢀ Antibacterial activity ꢀ E. coli FabH
Introduction
An alarming increase in pathogenic resistance to existing first line standard drugs is
a serious problem in antimicrobial cure and necessitates continuing research into
new classes of antimicrobials [1]. Moreover, the progression of drug-resistant
strains has contributed to the inefficiency of the straight antimicrobial therapy. Thus,
there is enormous interest in antibacterial research, and we strongly believe that
there is an urgent call for the development of new drugs with divergent and unique
structure and probably with an unusual mechanism of action from that of existing
first line drugs. Consequently, the enormous significance of this area of research
continues to attract increasing numbers of medicinal chemists. In order to prevent
serious illness caused by microorganisms, the discovery of new types of
antibacterial agents is a crucial task at present. Fortunately, much of the research
effort is put into the design of new antibacterial agents with high efficiency [2].
In the recent 10 years, research has been focused toward new antibacterial agents
that may act through different targets in key areas of the bacterial cell cycle, in order
to surpass the problem of acquired resistance. Fatty acid synthesis (FAS) pathway in
bacteria is a promising target in the recent research, and fatty acid biosynthesis
(FAB) is a fundamental metabolic process for microorganisms and is essential for
cell viability and growth [3, 4]. b-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 [5]. 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 [6, 7]. On the other hand, some
novel compounds have 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 [8, 9]. FabH has proved to be a promising target
for the design of novel antimicrobial drugs because it adjusts and controls the fatty
acid biosynthesis rate in an initiation pathway, and its substrate specificity is a key
factor in membrane fatty acid composition [10–12]. 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, there is gaining interest in nitroimidazole
derivatives in drug research for antimicrobial chemotherapeutics and antiangiogenic
hypoxic cell radiosensitizers. The metabolism and toxicology of nitroimidazole
derivatives, particularly for secnidazole, have been characterized in recent reports [13,
14]. Secnidazole (a, 2-dimethyl-5-nitro-1H-imidazole-1-ethanol) is extraordinarily
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Design, synthesis and antibacterial evaluation of new Schiff’s…
effective in the treatment of giardiasis, amebiasis, and bacterial vaginosis. By oral
administration, secnidazole can be rapidly and completely absorbed and has a longer
terminal elimination half-life (17–29 h) than popular medication [15]. Also, the
treatment achieved with secnidazole is more effective and has fewer side effects [16].
Moreover, Schiff’s bases are compounds with the structure AC=NB, which are
usually synthesized from the condensation of active carbonyl groups and primary
amines. Schiff’s bases constitute an important class of biologically active drug
molecules that has attracted the attention of medicinal chemists due to their wide
range of pharmacological properties. These compounds are being synthesized as
drugs by many researchers in an effort to combat diseases with minimal toxicity and
maximal effects. The results of these predictions have provided a therapeutic
pathway to develop new effective biologically active Schiff’s base derivatives.
Many researchers have studied the synthesis, characterization, and structure–
activity relationship (SAR) of Schiff’s bases, and some were reported to have
antibacterial activities [17]. Further, Kim et al. reported the YKAs3003, a Schiff’s
base condensed by 4-hydroxy salicylaldehyde and cyclohexanamine, as a potent
inhibitor of Escherichia coli (E. coli) FabH with antimicrobial activity [18].
Encouraged by potential clinical applications of Schiff’s base derivatives and our
previous investigations on FabH inhibitory activity of nitroimidazole derivatives
[19], and another literature survey on pyrazole having the same activity [20], we
report here the synthesis and structure–activity relationship of a new series of
Schiff’s base derivatives having nitroimidazole and pyrazole nuclei in a single
scaffold and their antibacterial activities against Escherichia coli, Pseudomonas
aeruginosa (P. aeruginosa), Bacillus subtilis (B. subtilis), and Staphylococcus
aureus (S. aureus), as well as inhibitory activities against E. coli FabH.
Experiments
Materials and measurements
All chemicals and reagents used in the current study were of analytical grade.
Melting points (uncorrected) were determined on an XT4 MP apparatus (Taike
1
Corp., Beijing, China). All the H NMR and 13C NMR spectra were recorded on a
Bruker DPX300 model spectrometer in DMSO-d6 and chemical shifts were reported
in ppm (d). FTIR spectra (KBr) were run on a Nexus 870 FT-IR spectrophotometer.
ESI–MS spectra were recorded on a Mariner System 5304 mass spectrometer.
Elemental analyses were performed on a CHN-O-Rapid instrument. TLC was
performed on the glass backed silica gel sheets (Silica Gel 60 GF254) and visualized
in UV light (254 nm). Compound 3a–j was already reported by us [21].
General method for synthesis of Schiff’s base derivatives 5a–j
5-aryloxypyrazole-4-carbaldehydes 3a–j (1 mol) and 2-(2-methyl-5-nitro-1H-
imidazol-1-yl) acetohydrazide 4 (1.1 mol) and Ni(NO3)2ꢀ6H2O (10 mol %) were
mixed together in ethanol as a solvent. The reaction mixture was stirred at room
123

C. B. Sangani et al.
temperature for 2.5–3 h. After the completion of reaction (checked by TLC ethyl
acetate:hexane = 1:1), the solid compound was separated, filtered, washed well
with ethanol (10 mL) and water (10 mL), and finally dried and recrystallized from
ethanol to get the pure solid sample 5a–j. Physical, analytic, and spectroscopic
characterization data of the compounds 5a–j are presented hereafter.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N0-((3-methyl-5-phenoxy-1-phenyl-1H-
pyrazol-4-yl)methylene)acetohydrazide (5a) Yield: 81 %; mp 238–240 ꢁC; IR
(KBr, mmax, cm-1): 3325 (str. of –N–H), 3035 (ArC–H), 1685 (C=O stretching),
1215 cm-1 (C–O–C ether stretching);1H NMR (DMSO-d6) d ppm: 2.37, 2.48 (s,
6H, 2 9 CH3), 4.95 (s, 2H, CH2), 7.02–7.93 (m, 11H, Ar–H), 8.78 (s, 1H, CH=N),
11.20 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.32, 21.37 (Ar–CH3), 37.81 (CH2–
CO), 109.35, 118.78, 121.93, 124.24, 128.47, 128.95, 130.38, 132.18, 136.73,
139.54, 143.26 (–CH=N), 150.76, 152.37, 153.48, 157.59, (Ar–C), 175.92 (C=O);
Anal. Calcd. for C23H21N7O4 (459.46 g/mol): C, 60.12; H, 4.61; N, 21.34 (%);
Found: C, 60.40; H, 4.39; N, 21.54 (%); MS (m/z): 460 [M?H]?.
(E)-N0-((3-methyl-1-phenyl-5-(p-tolyloxy)-1H-pyrazol-4-yl)methylene)-2-(2-methyl-
5-nitro-1H-imidazol-1-yl)acetohydrazide (5b) Yield: 83 %; mp 249–251 ꢁC; IR
(KBr, mmax, cm-1): 3320 (str. of –N–H), 3025 (ArC–H), 1680 (C=O stretching),
1230 cm-1 (C–O–C ether stretching);1H NMR (DMSO-d6) d ppm: 2.33, 2.35, 2.52
(s, 9H, 3 9 CH3), 5.04 (s, 2H, CH2), 6.94–7.82 (m, 10H, Ar–H), 8.73 (s, 1H,
CH=N), 11.23 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.44, 20.90, 21.34 (Ar–
CH3), 37.84 (CH2–CO), 108.52, 117.09, 123.68, 124.85, 127.93, 129.07, 130.48,
132.52, 137.13, 139.30, 143.72 (–CH=N), 150.37, 152.72, 153.25, 157.38 (Ar–C),
175.75 (C=O); Anal. Calcd. for C24H23N7O4 (473.48 g/mol): C, 60.88; H, 4.90; N,
20.71 (%); Found: C, 60.59; H, 5.12; N, 20.83 (%); MS (m/z): 474 [M?H]?.
(E)-N0-((5-(4-methoxyphenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-2-
(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (5c) Yield: 84 %; mp
218-220 ꢁC; IR (KBr, mmax, cm-1): 3325 (str. of –N–H), 3040 (ArC–H), 1700
1
(C=O stretching), 1210 cm-1 (C–O–C ether stretching); H NMR (DMSO-d6) d
ppm: 2.45, 2.55 (s, 6H, 2 9 CH3), 3.42 (s, 3H, OCH3), 4.90 (s, 2H, CH2), 7.08–7.95
(m, 10H, Ar–H), 8.85 (s, 1H, CH=N), 11.18 (s, 1H, NH); 13C NMR (DMSO-d6) (d):
14.27, 21.29 (Ar–CH3), 37.76 (CH2–CO), 54.87 (Ar–OCH3), 107.93, 119.24,
122.90, 124.18, 127.98, 129.26, 130.32, 132.42, 137.45, 139.78, 143.16 (–CH=N),
151.78, 152.24, 153.90, 157.52 (Ar–C), 176.05 (C=O); Anal. Calcd. for
C24H23N7O5 (489.48 g/mol): C, 58.89; H, 4.74; N, 20.03 (%); Found: C, 59.65;
H, 4.84; N, 20.40 (%); MS (m/z): 490 [M?H]?.
(E)-N0-((5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-2-
(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (5d) Yield: 77 %; mp
267–269 ꢁC; IR (KBr, mmax, cm-1): 3335 (str. of –N–H), 3025 (ArC–H), 1690
1
(C=O stretching), 1220 cm-1 (C–O–C ether stretching); H NMR (DMSO-d6) d
ppm: 2.38, 2.50 (s, 6H, 2 9 CH3), 4.99 (s, 2H, CH2), 6.90–7.87 (m, 10H, Ar–H),
8.80 (s, 1H, CH=N), 11.21 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.40, 21.38
(Ar–CH3), 37.92 (CH2–CO), 108.20, 118.62, 122.47, 127.68, 128.45, 129.15,
130.48, 132.93, 138.02, 139.96, 143.81 (–CH=N), 149.11, 153.15, 154.38, 158.38
123

Design, synthesis and antibacterial evaluation of new Schiff’s…
(Ar–C), 175.68 (C=O); Anal. Calcd. for C23H20FN7O4 (477.45 g/mol): C, 57.86; H,
4.22; N, 20.54 (%); Found: C, 58.98; H, 4.53; N, 20.75 (%); MS (m/z): 478
[M?H]?.
(E)-N0-((5-(4-chlorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-2-
(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (5e) Yield: 80 %; mp
210–212 ꢁC; IR (KBr, mmax, cm-1): 3345 (str. of –N–H), 3035 (ArC–H), 1685
1
(C = O stretching), 1230 cm-1 (C–O–C ether stretching); H NMR (DMSO-d6) d
ppm: 2.40, 2.49 (s, 6H, 2 9 CH3), 5.05 (s, 2H, CH2), 6.95–7.86 (m, 10H, Ar–H),
8.74 (s, 1H, CH=N), 11.15 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.35, 21.42
(Ar–CH3), 37.89 (CH2–CO), 106.94, 117.33, 122.47, 124.85, 128.09, 129.27,
130.28, 132.69, 137.24, 139.25, 143.19 (–CH=N), 150.64, 152.62, 153.37, 157.95
(Ar-C), 175.86 (C=O); Anal. Calcd. for C23H20ClN7O4 (493.90 g/mol): C, 55.93; H,
4.08; N, 19.85 (%); Found: C, 55.68; H, 4.43; N, 19.64 (%); MS (m/z): 494
[M?H]?.
(E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N0-((3-methyl-5-phenoxy-1-p-tolyl-1H-
pyrazol-4-yl)methylene)acetohydrazide (5f) Yield: 79 %; mp 254–256 ꢁC; IR
(KBr, mmax, cm-1): 3330 (str. of -N–H), 3025 (ArC–H), 1690 (C=O stretching),
1225 cm-1 (C–O–C ether stretching);1H NMR (DMSO-d6) d ppm: 227, 2.35, 2.45
(s, 9H, 3 9 CH3), 5.96 (s, 2H, CH2), 6.96–7.81 (m, 10H, Ar–H), 8.83 (s, 1H,
CH=N), 11.22 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.28, 21.17, 21.49 (Ar–
CH3), 37.94 (CH2–CO), 107.16, 117.19, 122.23, 124.50, 128.44, 129.83, 130.47,
132.38, 136.92, 139.47, 143.47 (–CH=N), 149.22, 152.69, 153.58, 157.73 (Ar–C),
176.21 (C=O); Anal. Calcd. for C24H23N7O4 (473.48 g/mol): C, 60.88; H, 4.90; N,
20.71 (%); Found: C, 61.12; H, 4.62; N, 20.93 (%); MS (m/z): 474 [M?H]?.
(E)-N0-((3-methyl-1-p-tolyl-5-(p-tolyloxy)-1H-pyrazol-4-yl)methylene)-2-(2-methyl-
5-nitro-1H-imidazol-1-yl)acetohydrazide (5g) Yield: 76 %; mp 279–281 ꢁC; IR
(KBr, mmax, cm-1): 3335 (str. of –N–H), 3015 (ArC–H), 1695 (C=O stretching),
1230 cm-1 (C–O–C ether stretching);1H NMR (DMSO-d6) d ppm: 2.20, 2.35, 2.43,
2.51 (s, 12H, 4 9 CH3), 5.10 (s, 2H, CH2), 6.99–7.80 (m, 9H, Ar–H), 8.82 (s, 1H,
CH=N), 11.19 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.25, 20.93, 21.11, 21.46
(Ar–CH3), 37.80 (CH2–CO), 109.13, 116.26, 122.65, 124.35, 128.26, 129.96,
131.25, 132.50, 137.32, 139.62, 143.75 (–CH=N), 150.16, 152.28, 153.82, 157.30
(Ar–C), 175.93 (C=O); Anal. Calcd. for C25H25N7O4 (487.51 g/mol): C, 61.59; H,
5.17; N, 20.11 (%); Found: C, 61.85; H, 5.40; N, 20.37 (%); MS (m/z): 488
[M?H]?.
(E)-N0-((5-(4-methoxyphenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)methylene)-2-
(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (5h) Yield: 88 %; mp
262–264 ꢁC; IR (KBr, mmax, cm-1): 3330 (str. of –N–H), 3025 (ArC–H), 1690
1
(C=O stretching), 1225 cm-1 (C–O–C ether stretching); H NMR (DMSO-d6) d
ppm: 2.29, 2.37, 2.48 (s, 9H, 3 9 CH3), 3.41 (s, 3H, OCH3), 5.02 (s, 2H, CH2),
6.92–7.78 (m, 9H, Ar–H), 8.70 (s, 1H, CH=N), 11.25 (s, 1H, NH); 13C NMR
(DMSO-d6) (d): 14.50, 21.22, 21.30 (Ar–CH3), 37.85 (CH2–CO), 54.85 (Ar–OCH3),
108.82, 116.87, 123.11, 124.74, 128.20, 129.31, 130.53, 132.74, 137.78, 139.19,
143.87 (–CH=N), 150.45, 152.70, 153.74, 157.25 (Ar–C), 175.82 (C=O); Anal.
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C. B. Sangani et al.
Calcd. for C25H25N7O4 (503.51 g/mol): C, 59.63; H, 5.00; N, 19.47 (%); Found: C,
59.29; H, 4.77; N, 19.64 (%); MS (m/z): 504 [M?H]?.
(E)-N0-((5-(4-fluorophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)methylene)-2-
(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (5i) Yield: 77 %; mp
224–226 ꢁC; IR (KBr, mmax, cm-1): 3340 (str. of –N–H), 3015 (ArC–H), 1680
(C=O stretching), 1215 cm-1 (C–O–C ether stretching);1H NMR (DMSO-d6) d
ppm: 2.32, 2.39, 2.47 (s, 9H, 3 9 CH3), 5.08 (s, 2H, CH2), 7.05–7.97 (m, 9H, Ar–
H), 8.84 (s, 1H, CH=N), 11.23 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.37, 21.15,
21.43 (Ar–CH3), 37.72 (CH2–CO), 108.21, 115.37, 121.98, 125.67, 128.01, 129.23,
131.14, 132.46, 137.56, 139.73, 143.22 (–CH=N), 152.24, 153.46, 154.37, 157.35
(Ar–C), 175.77 (C=O); Anal. Calcd. for C24H22FN7O4 (491.47 g/mol): C, 58.65; H,
4.51; N, 19.95 (%); Found: C, 58.81; H, 4.73; N, 20.62 (%); MS (m/z): 492
[M?H]?.
(E)-N0-((5-(4-chlorophenoxy)-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)methylene)-2-
(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (5j) Yield: 75 %; mp
242–244 ꢁC;IR (KBr, mmax, cm-1): 3325 (str. of –N–H), 3020 (ArC–H), 1685
(C=O stretching), 1220 cm-1 (C–O–C ether stretching);1H NMR (DMSO-d6) d
ppm: 2.23, 2.42, 2.50 (s, 9H, 3 9 CH3), 5.05 (s, 2H, CH2), 7.03–7.90 (m, 9H, Ar–
H), 8.82 (s, 1H, CH=N), 11.18 (s, 1H, NH); 13C NMR (DMSO-d6) (d): 14.45, 21.18,
21.44 (Ar–CH3), 37.90 (CH2–CO), 107.90, 116.78, 122.44, 124.79, 128.84, 129.30,
130.47, 132.59, 137.22, 139.80, 143.51 (–CH=N), 151.47, 153.63, 154.25, 157.85
(Ar-C), 176.12 (C = O); Anal. Calcd. for C24H22ClN7O4 (507.93 g/mol): C, 56.75;
Table 1 In vitro antibacterial activity of compounds 5a–j
Compounds
Minimum inhibitory concentrations (lg/mL)
Gram-positive
Gram-negative
Bacillus
subtilis
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
aeruginosa
5a
50
25
[50
50
[50
[50
25
5b
25
3.13
50
5c
12.5
6.25
25
6.25
25
5d
50
3.13
25
5e
[50
25
6.25
12.5
25
5f
[50
12.5
25
3.13
12.5
[50
25
5g
6.25
25
5h
3.13
3.13
50
5i
25
12.5
[50
1.56
25
5j
25
50
Kanamycin B
DDCP
1.56
12.5
3.13
1.56
3.13
3.13
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Design, synthesis and antibacterial evaluation of new Schiff’s…
H, 4.37; N, 19.30 (%); Found: C, 57.05; H, 4.58; N, 19.17 (%); MS (m/z): 508
[M?H]?.
Antibacterial activity assay
The antibacterial activities of the synthesized compounds 5a–j were tested against
Gram-negative bacterial strains (E. coli ATCC 25922 and P. aeruginosa ATCC
27853) and Gram-positive bacterial strains (B. subtilis ATCC 530 and S. aureus
ATCC 25923) using the method recommended by National Committee for Clinical
Laboratory Standards (NCCLS) [220]. In vitro activities of the compounds were
tested in nutrient broth (NB) for bacteria using twofold serial dilution.
Seeded broth (broth containing microbial spores) was prepared in NB from 24 h
old bacterial cultures on nutrient agar (Hi-media) at 37 1 ꢁC. The bacterial
suspension was adjusted with sterile saline to a concentration of 1 9 104–105 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 lg/mL.
The tubes were incubated in BOD incubators at 37 1 ꢁC for bacteria. The
minimum inhibitory concentrations (lg/mL) MICs were recorded by visual
observations after 24 h (for bacteria) of incubation. Kanamycin B and DDCP were
used as standards for bacterial. The observed MICs are presented in Table 1.
E. coli FabH purification and activity assay
Full-length E. coli acyl carrier protein (ACP), acyl carrier protein synthase (ACPS),
and b-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
B (30 lg/mL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–
PAGE) analysis was used to screen colonies for over-expression of proteins. One
such positive colony was used to inoculate 10 mL of LB medium with 30 lg/mL of
kanamycin B and was grown overnight at 37 ꢁC, 1 mL of which was used to
inoculate 100 mL LB medium supplemented with 30 mg/mL of kanamycin B. The
culture was shaken for 4 h at 37 ꢁC, and then induced with 0.5 mM isopropyl b-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 FabH were lysed by
sonication in 20 mM Tris, pH 7.6, 5 mM imidazole, and 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 mM Tris, pH 7.6, 1 mM
DTT, and 100 mM NaCl. Purified FabH was concentrated up to 2 mg/mL and
stored at -80 ꢁC 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
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volume of 50 mL, 50 mg ACP, 50 mM Tris, 2 mM DTT, 10 mM MgCl2, 600 lM
CoA, and 0.2 lM ACPS was incubated for 1 h at 37 ꢁC. 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 with Source Q-15 ion exchange
chromatography using a 0–500 mM NaCl gradient over 25 column volumes.
In a final 20 lL reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0, 0.5 mM DTT,
0.25 mM MgCl2, and 2.5 lM holo-ACP were mixed with 1 nM FabH, and H2O was
added to 15 mL. After 1 min incubation, a 2 lL mixture of 25 lM acetyl-CoA and
0.75 lCi [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 pellet was washed with 10 % ice-cold
TCA and resuspended with 5 lL of 0.5 M NaOH. 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 %.
Scheme 1 Synthesis of compounds 5a–j
123

Design, synthesis and antibacterial evaluation of new Schiff’s…
Docking simulations
The crystal structures of E. coli FabH (PDB code: 1HNJ) was obtained from the
Protein Data Bank (http://www.rcsb.org). Molecular docking of compounds into the
three-dimensional X-ray structure of FabH was carried out using Ligand Fit Dock
protocol of Discovery Studio 3.1.
Results and discussion
Chemistry
Schiff’s base derivatives 5a–j have been synthesized by reaction between
5-arloxypyrazole-4-carbaldehydes [21] 3a–j and 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)acetohydrazide 4 [23] in presence of nickel (II) nitrate as a catalyst in ethanol at
room temperature in good yield (75–88 %) (Scheme 1). The reaction was also
attempted using AcOH [24, 25], and conc. H2SO4 [26]; however, some shortcom-
ings were observed such as incomplete reaction, long reaction time, loss of yield,
and purification problems. Therefore, we applied Ni(NO3)2ꢀ6H2O as a catalyst [27]
for this reaction to avoid such drawbacks as well as to develop an easy and efficient
method for the synthesis of new Schiff’s base derivatives 5a–j.
1
The structures of all the new synthesized compounds were established by H
NMR, 13C NMR, FT-IR, elemental analysis, and molecular weight of compounds
confirmed by mass spectrometry. Mass spectroscopy of compounds showed
molecular ion peak [M?H]? corresponding to the exact mass.
Biological evaluation
In vitro antibacterial and E. Coli FabH inhibitory activity
All the synthesized compounds 5a–j were screened for antibacterial activities
against two Gram-negative bacterial strains, E. coli ATCC 25922 and Pseudomonas
aeruginosa ATCC 27853 (P. aeruginosa); and two Gram-positive bacterial strains,
Bacillus subtilis ATCC 530 (B. subtilis) and Staphylococcus aureus ATCC 6538 (S.
aureus), by serial dilution. The MICs (minimum inhibitory concentrations) of the
compounds against these bacteria are presented in Table 1. Kanamycin B and
DDCP were taken as reference compound under identical conditions for
comparison.
Upon investigation of antibacterial activity (Table 1), it has been observed that
the majority of the compounds have shown effective activity against used strains.
Against Gram-negative bacteria E. coli, compounds 5h (MIC = 3.13 lg/mL) and 5i
(MIC = 3.13 lg/mL) showed more effective activity than other compounds of the
series and comparable activity to kanamycin B (MIC = 3.13 lg/mL), while
compounds 5d (MIC = 3.13 lg/mL) and 5f (MIC = 3.13 lg/mL) showed compa-
rable activity to both reference compounds (MIC = 3.13 lg/mL) against Gram-
negative bacteria P. aeruginosa. Against Gram-positive bacterial S. aureus,
123

C. B. Sangani et al.
compounds 5b (MIC = 3.13 lg/mL), 5g (MIC = 6.25 lg/mL), and 5i
(MIC = 12.5 lg/mL) showed more effective activity, and compounds 5a
(MIC = 25 lg/mL), 5f (MIC = 25 lg/mL), and 5h (MIC = 25 lg/mL) showed
comparable activity as compared to DDCP (MIC = 25 lg/mL). Compounds 5d
(MIC = 6.25 lg/mL) showed more effective activity and compound 5g
(MIC = 12.5 lg/mL) showed comparable activity as compared to DDCP
(MIC = 12.5 lg/mL) against B. subtilis. Of the compounds studied for E. coli
FabH inhibitory activity (Table 2), compounds 5h (IC50 = 6.1 0.9 lM) and 5i
(IC50 = 4.6 0.2 lM) showed the most potent activity as compared to other
compounds of the series and less active than DDCP (IC50 = 2.1 0.1 lM).
Structure activity relationship (SAR) was carried out from E. coli FabH
inhibitory and antibacterial activities. According to the activity data, it has been
observed that the change in R1 and R2 substitution may lead to change in the activity
against employed strains as well as E. coli FabH. Compounds 5h (R1 = CH3,
R2 = OCH3), and 5i (R1 = CH3, R2 = F) showed potent activity against E. coli by
introducing –CH3 at R1 position in compounds 5c (R1 = H, R2 = OCH3) and 5d
(R1 = H, R2 = F) respectively. Against P. aeruginosa, R2 = F and H may
responsible for the good activity. The activity was completely lost as compared
to the reference compounds by introducing other functional groups at the R2
position. Compounds with R1 = H (5d) became more effective than R1 = CH3 (5i)
against B. subtilis, while the reverse trend has been observed in the case of activity
against S. aureus. Moreover, reviewing and comparing the activity data, it is worth
mentioning that the antibacterial and activity against E. coli FabH of the target
compounds depends not only on the bicyclic heteroaromatic pharmacophore, but
also on the nature of the substituent.
Molecular docking
Molecular docking of all compounds and E. coli FabH was performed on the
binding model based on the E. coli FabH-CoA complex structure (1HNJ.pdb) [28].
All docking runs were applied Ligand Fit Dock protocol of Discovery Studio 3.1.
Table 2 Escherichia coli FabH
inhibitory activities of
compounds 5a–j
Compounds
IC50 (lM)
5a
51.4 1.4
37.1 0.3
17.5 0.7
42.2 2.1
12.0 0.1
19.4 0.6
21.3 1.2
6.1 0.9
4.6 0.2
16.7 0.3
2.1 0.1
5b
5c
5d
5e
5f
5g
5h
5i
5j
DDCP
123

Design, synthesis and antibacterial evaluation of new Schiff’s…
Table 3 Banding energy of
Compounds
CDOCKER interaction
energy –DGn (kcal/mol)
synthesized compounds 5a–j
5a
5b
5c
5d
5e
5f
42.2099
45.2938
47.745
45.8859
48.6371
47.7319
45.9751
49.1384
54.2961
46.7888
5g
5h
5i
5j
The binding energy calculation of the synthesized compounds is mentioned in
Table 3. Among them, compound 5i showed the lowest interaction energy
(DGb = -54.2961 kcal/mol). The binding model of compound 5i and E. coli FabH
is depicted in Figs. 1 and 2. In the binding model, compound 5i was nicely bound to
the FabH kinase with two hydrogen bonds, two p–p and three p–cation interactions.
Among two hydrogen bonds, one formed between O-atom of –NO2 and THR28
˚
with distance: 2.12849 A; DHA angle: 150.5ꢁ and HAY angle: 144.8ꢁ, while
another one formed between F-atom of aryloxypyrazole nucleus and ARG36 with
˚
distance: 2.46349 A; DHA angle: 126.5ꢁ and HAY angle: 95.9ꢁ. Two p–p
interactions formed between imidazole ring and TRP32 with distance 3.66368 and
˚
4.0845 A. Among three p–cation interactions, one formed between imidazole ring
˚
and TRP32 with distance 3.65463 A, the second between the imidazole ring and
˚
ARG151 with distance 4.07692 A, the third with phenoxy ring of pyrazole nucleus
Fig. 1 2D binding model of compound 5i into the active site of FabH
123

C. B. Sangani et al.
Fig. 2 Surface model of compound 5i into the active site of FabH (3D-model)
˚
and ARG249 with distance 5.83549 A. This molecular docking result, along with
the biological assay data, suggests that compound 5i proved to be a potential
inhibitor of E. coli FabH.
Conclusion
In conclusion, a new series of Schiff’s base derivatives with nitroimidazole and
pyrazole nuclei was synthesized by reaction between 5-aryloxypyrazole-4-car-
baldehydes and 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide in the pres-
ence of nickel (II) nitrate as a catalyst in ethanol at room temperature with a good
yield. This synthetic strategy allows the assimilation of two promising bioactive
nuclei in a single scaffold with an easy method. Reviewing the biological activity
data, it has been concluded that the majority of the compounds are found to be the
most effective against applied bacterial strains. Compound 5i showed the most
effective inhibition by binding into the active site of the E. coli FabH receptor with
minimum binding energy. According to this, it is worth mentioning that the Schiff’s
base derivatives with nitroimidazole and pyrazole nuclei have become a vital area of
antibacterial and E. coli FabH inhibition medicine research.
Acknowledgments The work was financed by National Natural Science Foundation of China (No.
J1103512).
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