Synthesis and antibacterial evaluation of novel Schiff's base derivatives of nitroimidazole nuclei as potent E. coli FabH inhibitors

Article abstract of DOI:10.1039/c4ra08567a

Series of novel Schiff's base derivatives have been synthesized by combining 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-formylbenzoate 5, 6 with aromatic/heterocyclic amine 7a-r, 8, 9a-r in ethanol. All compounds were evaluated for antibacterial assay and inhibition against E. coli FabH. Among the compounds studied, most of the compounds showed effective antibacterial and potential inhibitory activity against E. coli FabH. Compound 10q showed most potent inhibitory activity (IC₅₀ = 2.6883 μM) by binding tightly to the active site of the E. coli FabH receptor with minimum binding energy (ΔG_b = -55.3117 kcal mol^-1), in which molecular docking study indicated the binding mode was stabilized by one hydrogen bond and five π-π interactions. This journal is

Full text of DOI:10.1039/c4ra08567a

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Synthesis and antibacterial evaluation of novel
Schiff's base derivatives of nitroimidazole nuclei as
potent E. coli FabH inhibitors†
Cite this: RSC Adv., 2014, 4, 54217
Xin Zhang, Chetan B. Sangani, Li-Xin Jia, Pi-Xian Gong, Fang Wang, Jun-Fang Wang
and Hai-Liang Zhu*
Series of novel Schiff's base derivatives have been synthesized by combining 2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl 4-formylbenzoate 5, 6 with aromatic/heterocyclic amine 7a–r, 8, 9a–r in ethanol. All
compounds were evaluated for antibacterial assay and inhibition against E. coli FabH. Among the
compounds studied, most of the compounds showed effective antibacterial and potential inhibitory
activity against E. coli FabH. Compound 10q showed most potent inhibitory activity (IC50 ¼ 2.6883 mM)
Received 12th August 2014
Accepted 23rd September 2014
by binding tightly to the active site of the E. coli FabH receptor with minimum binding energy (DG
b
¼
DOI: 10.1039/c4ra08567a
ꢀ1
ꢀ
55.3117 kcal mol ), in which molecular docking study indicated the binding mode was stabilized by
www.rsc.org/advances
one hydrogen bond and five p–p interactions.
b-Ketoacyl-acyl carrier protein synthase III (FabH) is the
1
Introduction
key enzyme responsible for the rst reaction in the pathway
5
An alarming increment in pathogenic resistance to existing and plays an important regulatory role. FabH has also been

rst-line standard drugs is a serious problem in antimicrobial demonstrated to be essential for initiating the fatty acid
1
cure. Moreover, the progression of drug-resistant strains has elongation cycles and is involved in the feedback regulation of
6
,7
contributed to the inefficiency of the straight antimicrobial the biosynthetic pathway via product inhibition.
Some
therapy. This sets up an enormous interest in antibacterial novel compounds had been demonstrated to inhibit FabH
research and we strongly believe that there is an urgent call found in Gram-positive and Gram-negative bacteria,
for the development of novel antibacterial drugs with diver- including multi-drug resistant strains. FabH proteins from
gent and unique structures. Consequently, this area of Gram-positive and Gram-negative bacteria are highly
research is accorded an enormous signicance and continues conserved at the sequence and structural level whereas there
to attract considerable attention from an increasing number are no signicantly homologous proteins in humans. Impor-
of medicinal chemists. In order to prevent the serious medical tantly, the residues that comprise the active site are essen-
8
,9
problems caused by microorganisms, the discovery of new tially invariant in various bacterial FabH molecules. FabH
types of antibacterial agents is a crucial task at present. has been proved to be a promising target for the design of
Fortunately, considerable research effort is made for the novel antimicrobial drugs because it adjusts and controls the
2
design of new antibacterial agents with high efficiency.
fatty acid biosynthesis rate in an initiation pathway, and its
In the last 10 years, the research has been focused toward substrate specicity is a key factor in membrane fatty acid
1
0–12
new antibacterial agents, which may act through different kinds composition.
These facts indicate that small molecule
of targets in key areas of the bacterial cell cycle, to surpass the inhibitors of FabH enzymatic activity could be potential
problem of acquired resistance. The fatty acid synthesis (FAS) candidates for selective, nontoxic, and broad spectrum
pathway in bacteria is a promising target in the recent research antibacterials.
and fatty acid biosynthesis (FAB) is a fundamental metabolic
Because of varied biological activities, nitroimidazole deriv-
process for microorganisms and essential for cell viability and atives have gained constant interests in drug research for anti-
3
,4
growth.
microbial chemotherapeutics and antiangiogenic hypoxic cell
radiosensitizers. The metabolism and toxicology of nitro-
imidazole derivatives, particularly for secnidazole, have been
13,14
characterized in recent reports.
Secnidazole (a,2-dimethyl-5-
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
nitro-1H-imidazole-1-ethanol) is extraordinarily 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
2
8
†
1
10093, People's Republic of China. E-mail: zhuhl@nju.edu.cn; Fax: +86-25-
3592672; Tel: +86-25-83592672
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra08567a
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15
(
17–29 h) than popular medication. Also, the treatment ach- derivatives of nitroimidazole nuclei in a single scaffold and
ieved with secnidazole is more effective and displays fewer side their antibacterial activities against Escherichia coli, Pseudo-
16
effects.
monas aeruginosa (P. aeruginosa), Bacillus subtilis (B. subtilis) and
Moreover, Schiff bases are compounds with the structure of Staphylococcus aureus (S. aureus) and also E. coli FabH inhibitory
AC ¼ NB, which are usually synthesized from the condensation activities.
of active carbonyl groups and primary amines. Schiff's bases
constitute an important class of biologically active drug mole-
cules, which have attracted attention of medicinal chemists due
to their wide range of pharmacological properties. These
2
Results and discussion
2.1 Chemistry
compounds are being synthesized as drugs by many researchers Schiff's base derivatives 10a–r have been synthesized by
in order to combat diseases with minimal toxicity and maximal reaction between the intermediate 5 (Intermediate 5 can be
effects. These predictions have provided therapeutic pathways obtained by the simple two steps using compound 1 and
to develop new effective biologically active Schiff's base deriva- metronidazole 3) and various substituted aniline 7a-r in
tives. Many researchers have studied the synthesis, character- ethanol and when reuxed overnight provides good yield
ization and structure-activity relationship (SAR) of Schiff bases (45–87%) (Scheme 1). Moreover, the addition of 2-phenyl-
and some Schiff bases were reported to have antibacterial ethanamine
8
into the intermediate
5 could produce
17
activities.
Moreover, Kim and co-workers reported the target compound 11s in ethanol, when reuxed overnight.
YKAs3003, a Schiff base condensed by 4-hydroxy salicylaldehyde Other compounds 12t–w were derived from the reaction
and cyclohexanamine, as a potent inhibitor of Escherichia coli between compound 2 and secnidazole 4, subsequently inter-
18
(E. coli) FabH with antimicrobial activity.
acting with four substituted aniline 9t–w in ethanol, reuxed
Along the studies on FabH of our group based on nitro- overnight.
19
20
imidazole and Schiff's base, we report here the synthesis and
structure-activity relationship of a new series of Schiff base established by H NMR, elemental analysis, and molecular
The structures of all the new synthesized compounds were
1
Scheme 1 Synthesis of the titled compounds.
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ꢀ
1
weight of compounds conrmed by mass spectrometry. Mass (MIC ¼ 3.13 mg mL ) showed comparable activity to kanamycin
+
ꢀ1
spectroscopy of compounds showed molecular ion peak (M ) B (MIC ¼ 3.13 mg mL ). Against Gram-positive bacteria
ꢀ1
corresponding to the exact mass.
S. aureus, compound 10q (MIC ¼ 3.13 mg mL ) showed the
most effective activity. As well compounds 10b, 10f, 10k, 10o
ꢀ1
and 10p (MIC ¼ 6.25 mg mL ) showed comparable activity as
2.2 Biological activity
ꢀ1
compared to penicillin G (MIC ¼ 6.25 mg mL ) but less
2
.2.1 In vitro antibacterial and E. coli FabH inhibitory
ꢀ1
compared to kanamycin B (MIC ¼ 1.56 mg mL ). Compound
activity. All the synthesized compounds were screened for their
antibacterial activities against two Gram-negative bacterial
strains, i.e., E. coli ATCC 35218 and Pseudomonas aeruginosa
ATCC 13525 (P. aeruginosa), and two Gram-positive bacterial
strains, i.e., Bacillus subtilis ATCC 6633 (B. subtilis) and Staphy-
lococcus aureus ATCC 6538 (S. aureus) by serial dilution method.
The MICs (minimum inhibitory concentrations) of the
compounds against these bacteria were presented in Table 1.
Kanamycin B and penicillin G were taken as reference
compounds under identical conditions for comparison.
ꢀ1
1
0q (MIC ¼ 1.56 mg mL ) showed comparable activity and
compounds 10b, 10f, 10g, 10k, 10p, 10t and 12v (MIC ¼ 3.13 mg
ꢀ1
mL ) showed less activity as compared to penicillin G (MIC ¼
ꢀ1
1
.56 mg mL ) against B. subtilis. Of the compounds studied for
E. coli FabH inhibitory activity (Table 2), compounds 10q (IC50
¼
2
.6883 mM), 12v (IC50 ¼ 4.928 mM) and 10r (IC50 ¼ 5.5923 mM)
showed more potent activity as compared to secnidazole (IC50
¼
2
8.5 mM) and metronidazole (IC50 ¼ 17.6 mM) as well as other
compounds of the series.
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 R
substitution may lead to change in the activity against
employed strains as well as E. coli FabH. Compounds 10o,
0p, 10q, 10r, 12v and 12w having sulfonamide linkage
–SO NH) showed potent activity compared to other
compounds. Electron releasing group R ¼ 3-OCH in
compound 10f gave better activity than 10e (R ¼ 3-CH ), while
Upon investigation of antibacterial activity (Table 1), it has
been observed that a majority of the compounds have shown
effective activity against the strains used. Against Gram-negative
ꢀ1
bacteria E. coli, compound 10q (MIC ¼ 1.56 mg mL ) showed
more effective activity compared to penicillin G (MIC ¼ 3.13 mg
1
(
ꢀ
1
mL ) and comparable activity to kanamycin B (MIC ¼ 1.56 mg
ꢀ1
2
mL ), whereas compounds 10f, 10l, 10o, 10r, 12v and 12w (MIC
ꢀ
1
3
¼
3.13 mg mL ) showed comparable activity to penicillin G
1
3
(
MIC ¼ 3.13 mg mLꢀ ). Against P. aeruginosa, compound 10q
Table 1 The MICs (minimum inhibitory concentrations) of the compounds against these bacteria
ꢀ
1
Minimum inhibitory concentrations (mg mL ) of 10a–r, 11s, 12t–w
Gram-negative
Gram-positive
P. aeruginosa
B. subtilis
S. aureus
Compound
E. coli ATCC 35218
ATCC 13525
ATCC 6633
ATCC 6538
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
0g
0h
0i
0j
0k
0l
0m
0n
0o
0p
0q
0r
25
6.25
25
12.5
50
3.13
6.25
6.25
25
12.5
6.25
3.13
12.5
12.5
3.13
6.25
1.56
3.13
6.25
12.5
12.5
3.13
3.13
3.13
1.56
100
12.5
100
100
100
12.5
25
100
100
100
12.5
25
25
3.13
25
25
25
3.13
3.13
12.5
25
12.5
3.13
12.5
6.25
12.5
6.25
3.13
1.56
6.25
6.25
3.13
12.5
3.13
6.25
1.56
0.78
50
6.25
50
25
100
6.25
12.5
12.5
50
25
6.25
25
50
50
25
25
12.5
25
3.13
12.5
25
25
100
25
6.25
6.25
3.13
12.5
12.5
12.5
25
12.5
12.5
6.25
1.56
1s
2t
2u
2v
2w
12.5
6.25
3.13
Penicillin G
Kanamycin B
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Table 2 E. coli FabH inhibitory activities of compounds 10a–r, 11s, Table 3 Binding energy of synthesized compounds 10a–r, 11s, 12t–w
2t–w
1
R
CDOCKER interaction
energy – DG (kcal mol )
b
ꢀ1
ꢀ1
Compounds
IC50 (mM)
Hemolysis LC30 (mg ml
)
Compounds
(substitution)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
0g
0h
0i
0j
0k
0l
0m
0n
0o
0p
0q
0r
31.4738
9.9087
39.0767
30.8408
31.0767
6.4994
12.2729
17.3407
43.4738
30.7389
11.1688
11.9535
14.8985
28.0284
6.1688
6.9193
2.6883
5.5923
10.0336
10.0857
28.4504
4.928
6.8125
3.1542
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
4-CH
4-OCH
2-CH
2-OCH
3-CH
3-OCH
2-Cl
2-Br
2 F
4-Cl
4-Br
4 F
2,4-Cl
H
3
38.5262
45.0913
37.9892
39.1592
38.9233
48.5006
41.7271
40.6593
36.9981
39.2611
44.8312
42.0465
41.1015
39.9716
3
3
3
3
3
1
1
0o
0p
48.8312
1s
2t
2u
2v
2w
48.0807
DCCP
10q
55.3117
49.4077
there is no activity difference observed in 2-, 3- and 4-position
of –CH group. Secnidazole derivatives 12t–w gave better
3
activity than derivatives. Introduction of alkyl chain in 10r
compound 11s gave better activity than simple amine moiety
in compound 10n; moreover, different halogen groups at 2-, 3-
1
1
1s
2t
H
4-OCH
4-CH
3
44.9664
44.9143
39.5496
and 4-position gave different activity. The decreasing order of
3
activity in halogen compounds is (4-Br > 4-F > 2-Cl > 2-Br > 4-Cl 12u
2, 4-Cl > 2-F). Moreover, reviewing and comparing the
>
activity data, it is worthy to mention that the antibacterial
activity against E. coli FabH of the target compounds depends
not only on the heteroaromatic pharmacophore, but also on
the nature of the substituents.
1
1
2v
51.0720
48.1875
In addition, an acute oral toxicity test was conducted with
mice to determine the toxicity from a single dose via the oral
route. Based on the results (not listed), the single dose acute
oral LD50 (half maximal concentration of lethal dose) values of
the compounds (10b, 10f, 10q, 10r, 12v) are all greater than 5000
2w
ꢀ
1
mg kg of bodyweight.
.2.2 Molecular docking. Molecular docking of all interactions. Among hydrogen bonds formed between O-
compounds and E. coli FabH was performed on the binding atom of C]O (carbonyl group) and ARG151 with distance:
2
˚
ꢁ
ꢁ
model based on the E. coli FabH–CoA complex structure 1.948 A; DHA angle: 133.0 and HAY angle: 149.3 , one p–p
2
1
(
1HNJ.pdb). All docking runs were applied using Ligand Fit interaction formed between imidazole ring and ARG151 with
˚
Dock protocol of Discovery Studio 3.5. The binding energy distance 3.61222 A, two p–p interactions formed between
calculation of the synthesized compounds is listed in Table 3. imidazolering and TRP32 with distance 4.53889 A and
˚
˚
Among them, compound 10q showed the lowest interaction 5.83906 A and two p–p interactions formed between –NO
2
ꢀ
1
¼ ꢀ55.3117 kcal mol ). The binding model of group of imidazole ring and TRP32 with distance 3.57476 A˚
energy (DG
b
˚
compound 10q and E. coli FabH is depicted in Fig. 1 and 2. In and 4.05855 A. Moreover, the binding model of compound
the binding model, compound 10q was nicely bound to the 10f and E. coli FabH is depicted in Fig. 3 and 4. In the binding
FabH kinase with one hydrogen bond and ve p–p model, compound 10f was nicely bound to the FabH kinase
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Fig. 1 3D binding model of compound 10q into the active site of FabH.
Fig. 2 Surface model of compound 10q into the active site of FabH (3D-model).
Fig. 3 3D binding model of compound 10f into the active site of FabH.
with two hydrogen bonds and one p–p interaction. p–p interaction formed between imidazole ring and HIS244
˚
A hydrogen bond formed between O-atom of –OCH and with distance 6.62666 A. This molecular docking result,
3
˚
ꢁ
ARG151 with distance: 2.4297 A; DHA angle: 107.1 and HAY along with the biological assay data, suggests that
angle: 100.2 , while another one formed between O-atom compounds 10q and 10f prove to be potential inhibitors of
ꢁ
of C]O (carbonyl group) and ASN247 with distance: E. coli FabH.
˚
ꢁ
ꢁ
2
.3913 A; DHA angle: 125.3 and HAY angle: 117.0 . One
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Fig. 4 Surface model of compound 10f into the active site of FabH (3D-model).
spectroscopic characterization data of the compounds are pre-
sented hereaer.
3
Conclusions
New Schiff's base derivatives 10a–r, 11s and 12t–w have been
4.2.1 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-((p-
synthesized by reaction between 2-(2-methyl-5-nitro-1H-imidazol- tolylimino)methyl)benzoate (10a). White powder, yield: 83%.
ꢁ
1
1
-yl)ethyl 4-formylbenzoate 5, 6 and aromatic/heterocyclic amine Mp 191–193 C; H NMR (DMSO-d6) d ppm: 2.33, 2.48 (s, 6H, 2
in ethanol. This synthetic strategy allows the assimilation of two ꢂ CH ), 4.66–4.77 (t, 4H, 2 ꢂ CH ), 7.24–8.06 (m, 9H, ArHs),
promising bioactive nuclei in a single scaffold through an easy 8.73 (s, 1H, CH]N); anal. calcd for C21 (392.15 g
3
2
20 4 4
H N O
ꢀ
1
method. Reviewing the biological activity data, it has been mol ): C, 64.28; H, 5.14; N, 14.28 (%); found: C, 64.49; H, 5.22;
concluded that a majority of the compounds have been found N, 14.33 (%); MS (m/z): 392.1 (M ).
+
most effective against applied bacterial strains. Compound 10q
showed most effective inhibition by binding into the active site of methoxyphenyl)imino)methyl) benzoate (10b). White powder,
4.2.2 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4-
ꢁ
1
E. coli FabH receptor with a minimum binding energy. According yield: 87%. Mp 197–199 C; H NMR (DMSO-d6) d ppm: 2.48 (s,
to this, it is worthy to mention that the Schiff's base derivatives 3H, CH ), 3.79 (s, 3H, OCH ), 6.99–
), 4.65–4.77 (t, 4H, 2 ꢂ CH
having nitroimidazole nuclei have become a vital spot of anti- 8.05 (m, 9H, ArHs), 8.74 (s, 1H, CH]N); anal. calcd for
3
3
2
ꢀ
1
bacterial and E. coli FabH inhibition medicine research.
21 20 4 5
C H N O (408.41 g mol ): C, 61.76; H, 4.94; N, 13.72 (%);
+
found: C, 61.80; H, 5.12; N, 13.82 (%); MS (m/z): 408.1 (M ).
.2.3 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-((o-
tolylimino)methyl)benzoate (10c). White powder, yield: 75%.
4
4
Experiments
ꢁ
1
Mp 187–188 C; H NMR (DMSO-d6) d ppm: 2.35, 2.48 (s, 6H, 2
ꢂ CH ), 4.68–4.76 (t, 4H, 2 ꢂ CH ), 7.09–8.07 (m, 9H, ArHs),
All chemicals and reagents used in the current work were of 8.70 (s, 1H, CH]N); anal. calcd for C H N O (392.15 g
analytical grade. Melting points were determined using an XT4 mol ): C, 64.28; H, 5.14; N, 14.28 (%); found: C, 64.49; H, 5.22;
MP apparatus (Taike Corp., Beijing, China). All the H NMR and N, 14.33 (%); MS (m/z): 392.1 (M ).
4.1 Materials and measurements
3
2
2
1
20 4 4
ꢀ
1
1
+
1
3
C NMR spectra were recorded using a Bruker DPX300 model
spectrometer in DMSO-d6 and chemical shis were reported in methoxyphenyl)imino)methyl)benzoate (10d). White powder,
4.2.4 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((2-
ꢁ
1
ppm (d). ESI-MS spectra were recorded using a Mariner System yield: 80%. Mp 196–197 C; H NMR (DMSO-d6) d ppm: 2.48 (s,
304 mass spectrometer. Elemental analyses were performed 3H, CH ), 3.80 (s, 3H, OCH ), 4.68–4.76 (t, 4H, 2 ꢂ CH ), 6.95–
5
3
3
2
using a CHN–O-Rapid instrument. TLC was performed using 8.05 (m, 9H, ArHs), 8.63 (s, 1H, CH]N); anal. calcd for
ꢀ1
glass backed silica gel sheets (Silica Gel 60 GF254) and visual- C H N O (408.41 g mol ): C, 61.76; H, 4.94; N, 13.72 (%);
2
1
20 4 5
+
ized in UV light (254 nm).
found: C, 61.80; H, 5.12; N, 13.82 (%); MS (m/z): 408.1 (M ).
.2.5 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-((m-
4
tolylimino)methyl)benzoate (10e). White powder, yield: 75%.
Mp 185–187 C; H NMR (DMSO-d6) d ppm: 2.35, 2.48 (s, 6H, 2
4.2 General method for synthesis of Schiff's base derivatives
ꢁ
1
ꢂ CH ), 4.68–4.76 (t, 4H, 2 ꢂ CH ), 7.09–8.07 (m, 9H, ArHs),
2
-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-formylbenzoate 5, 6
3
2
8
.70 (s, 1H, CH]N); anal. calcd for C H N O (392.15 g
(1 mol) and aromatic/heterocyclic amine 7a–r, 8, 9t–w (1.1 mol)
21 20 4 4
ꢀ
1
mol ): C, 64.28; H, 5.14; N, 14.28 (%); found: C, 64.49; H, 5.22;
were mixed together in ethanol as a solvent. The reaction
mixture was stirred and reuxed overnight. Aer the comple-
tion of reaction (checked by TLC), the separated solid was
+
N, 14.33 (%); MS (m/z): 392.1 (M ).
4.2.6 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((3-
methoxyphenyl)imino)methyl)benzoate (10f). White powder,

ltered, washed well with ethanol (10 mL) and water (10 mL),
ꢁ
1
yield: 77%. Mp 195–197 C; H NMR (DMSO-d6) d ppm: 2.49
s, 3H, CH ), 3.81 (s, 3H, OCH ), 4.68–4.78 (t, 4H, 2 ꢂ CH ),
and nally dried and recrystallized from ethanol to get the pure
solid samples 10a–r, 11s, 12t–w. Physical, analytical, and
(
3
3
2
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.86–8.08 (m, 9H, ArHs), 8.74 (s, 1H, CH]N); anal. calcd for 4.79; N, 14.81 (%); found: C, 63.64; H, 4.63; N, 14.91 (%); MS (m/z):
ꢀ1
+
C H N O (408.41 g mol ): C, 61.76; H, 4.94; N, 13.72 (%); 378.1 (M ).
found: C, 61.80; H, 5.12; N, 13.82 (%); MS (m/z): 408.1 (M ).
2
1
20 4 5
+
4.2.15 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4-
4
.2.7 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((2- (N-(pyridin-2-yl)sulfamoyl)phenyl)imino)methyl)benzoate
ꢁ
1
chlorophenyl)imino)methyl)benzoate (10g). White powder, (10o). White powder, yield: 45%. Mp 234–235 C; H NMR (DMSO-
yield: 76%. Mp 199–200 C; H NMR (DMSO-d6) d ppm: 2.45 (s, d6) d ppm: 2.48 (s, 3H, CH
H, CH
ꢁ
1
3
), 4.69–4.76 (t, 4H, 2 ꢂ CH
2
), 6.87–8.08
NH); anal.
4 4 22 6 6
17ClN O (412.83 g calcd for C25H N O S (534.54 g mol ): C, 56.17; H, 4.15; N, 15.72
3
8
3
), 4.64–4.75 (s, 4H, 2 ꢂ CH
2
), 7.21–8.02 (m, 9H, ArHs), (m, 13H, ArHs), 8.72 (s, 1H, CH]N), 11.70 (s, 1H, SO
2
ꢀ1
.75 (t, 1H, CH]N); anal. calcd for C20
H
ꢀ
1
+
mol ): C, 58.19; H, 4.15; N, 13.57 (%); found: C, 58.25; H, 4.31; (%); found: C, 56.24; H, 3.93; N, 15.85 (%); MS (m/z): 534.1 (M ).
+
N, 13.62 (%); MS (m/z): 412.1 (M ).
4.2.16 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4-
4
.2.8 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((2- (N-(4-methylpyrimidin-2-yl)sulfamoyl)phenyl)imino)methyl)-
ꢁ
1
bromophenyl)imino)methyl)benzoate (10h). White powder, benzoate (10p). White powder, yield: 50%. Mp 245–246 C; H
ꢁ
1
yield: 78%. Mp 202–203 C; H NMR (DMSO-d6) d ppm: 2.45 (s, NMR (DMSO-d6) d ppm: 2.33, 2.48 (s, 6H, 2 ꢂ CH
3
), 4.68–4.76 (t,
), 6.91–8.34 (m, 11H, ArHs), 8.72 (s, 1H, CH]N),
(457.28 g 11.72 (s, 1H, SO NH); anal. calcd for C25 S (549.56 g
3
8
H, CH
3
), 4.64–4.75 (s, 4H, 2 ꢂ CH
2
), 7.21–8.02 (m, 9H, ArHs), 4H, 2 ꢂ CH
17BrN
2
.76 (t, 1H, CH]N); anal. calcd for C20
H
4
O
4
2
23 7 6
H N O
ꢀ
1
ꢀ1
mol ): C, 52.53; H, 3.75; N, 12.25 (%); found: C, 52.65; H, 3.81; mol ): C, 54.64; H, 4.22; N, 17.84 (%); found: C, 56.79; H, 4.12;
+
+
N, 12.42 (%); MS (m/z): 456.1 (M ).
.2.9 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((2-
N, 17.58 (%); MS (m/z): 549.1 (M ).
4
4.2.17 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4-

orophenyl)imino)methyl)benzoate (10i). White powder, yield: (N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)imino)
ꢁ
1
75%. Mp 198–199 C; H NMR (DMSO-d6) d ppm: 2.45 (s, 3H, methyl)benzoate (10q). White powder, yield: 54%. Mp 256–257
ꢁ
1
CH
3
), 4.64–4.75 (s, 4H, 2 ꢂ CH
2
), 7.21–8.02 (m, 9H, ArHs), 8.74
C; H NMR (DMSO-d6) d ppm: 2.24, 2.26, 2.47 (s, 9H, 3 ꢂ CH
3
),
ꢀ
1
(t, 1H, CH]N); anal. calcd for C20
H
17FN
4
O
4
(396.37 g mol ): C, 4.67–4.75 (t, 4H, 2 ꢂ CH
2
), 5.94–8.04 (m, 10H, ArHs), 8.71 (s, 1H,
2 25 7 6
60.60; H, 4.32; N, 14.13 (%); found: C, 60.65; H, 4.40; N, 14.20 CH]N), 11.75 (s, 1H, SO NH); anal. calcd for C26H N O S
+
ꢀ1
(%); MS (m/z): 396.4 (M ).
(563.59 g mol ): C, 55.41; H, 4.47; N, 17.40 (%); found: C, 55.31;
+
4
.2.10 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4- H, 4.25; N, 17.63 (%); MS (m/z): 563.2 (M ).
chlorophenyl)imino)methyl)benzoate (10j). White powder,
yield: 86%. Mp 201–202 C; H NMR (DMSO-d6) d ppm: 2.49 (s, (N-carbamimidoylsulfamoyl)phenyl)imino)methyl)benzoate
4.2.18 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4-
ꢁ
1
ꢁ
1
3
8
H, CH ), 4.68–4.78 (s, 4H, 2 ꢂ CH ), 7.35–8.08 (m, 9H, ArHs), (10r). White powder, yield: 51%. Mp 232–233 C; H NMR
3
2
.74 (t, 1H, CH]N); anal. calcd for C20
H
17ClN
4
O
4
(412.83 g (DMSO-d6) d ppm: 2.48 (s, 3H, CH
3
), 4.69–4.76 (t, 4H, 2 ꢂ CH
2
),
ꢀ
1
mol ): C, 58.19; H, 4.15; N, 13.57 (%); found: C, 58.25; H, 4.31; 6.72–8.09 (m, 12H, ArHs + NH
N, 13.62 (%); MS (m/z): 412.1 (M ).
2
), 8.73 (s, 1H, CH]N), 11.73 (s,
+
ꢀ1
1H, SO
2
NH); anal. calcd for C21
H
21
N
7
O
6
S (499.50 g mol ): C,
4
.2.11 (E)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4- 50.50; H, 4.24; N, 19.63 (%); found: C, 50.37; H, 4.06; N, 19.48
+
bromophenyl)imino)methyl)benzoate (10k). White powder, (%); MS (m/z): 499.1 (M ).
yield: 87%. Mp 203–204 C; H NMR (DMSO-d6) d ppm: 2.49 (s,
ꢁ
1
4.2.19 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-((phe-
3
8
H, CH ), 4.68–4.78 (s, 4H, 2 ꢂ CH ), 7.35–8.08 (m, 9H, ArHs), nethylimino)methyl)benzoate (11s). White powder, yield: 80%.
3
2
ꢁ
1
.74 (t, 1H, CH]N); anal. calcd for C H BrN O (457.28 g Mp 198–199 C; H NMR (DMSO-d6) d ppm: 2.47 (s, 3H, CH ), 2.95
2
0
17
4
4
3
ꢀ
1
mol ): C, 52.53; H, 3.75; N, 12.25 (%); found: C, 52.65; H, 3.81; (s, 2H, CH
2
), 3.86 (s, 2H, CH
2
), 4.65–4.76 (t, 4H, 2 ꢂ CH
2
), 7.17–
+
N, 12.42 (%); MS (m/z): 456.1 (M ).
.2.12 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((4-
orophenyl)imino)methyl)benzoate (10l). White powder, yield: found: C, 64.86; H, 5.64; N, 13.85 (%); MS (m/z): 406.2 (M ).
8.06 (m, 10H, ArHs), 8.35 (s, 1H, CH]N); anal. calcd for
ꢀ
1
4
22 22 4 4
C H N O (406.43 g mol ): C, 65.01; H, 5.46; N, 13.78 (%);
+

ꢁ
1
85%. Mp 199–200 C; H NMR (DMSO-d6) d ppm: 2.49 (s, 3H,
4.2.20 (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl
CH ), 4.68–4.78 (s, 4H, 2 ꢂ CH ), 7.35–8.08 (m, 9H, ArHs), 8.74 4-(((4-methoxyphenyl)imino)methyl)benzoate (12t). White
3
2
ꢀ
1
ꢁ
t, 1H, CH]N); anal. calcd for C H FN O (396.37 g mol ): C, powder, yield: 87%. Mp 204–205 C; H NMR (DMSO-d6) d
20 17 4 4
1
(
6
0.60; H, 4.32; N, 14.13 (%); found: C, 60.65; H, 4.40; N, 14.20 ppm: 1.57, 2.54 (s, 6H, 2 ꢂ CH ), 3.89 (s, 3H, OCH ), 4.48–
3
3
+
(%); MS (m/z): 396.4 (M ).
2
4.78 (d, 2H, CH ), 5.59–5.64 (t, 1H, CH), 6.99–8.04 (m, 9H,
4
.2.13 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(((2,5- ArHs), 8.58 (s, 1H, CH]N); anal. calcd for C22H N O
22 4 5
ꢀ
1
dichlorophenyl)imino)methyl)benzoate (10m). White powder, (422.43 g mol ): C, 62.55; H, 5.25; N, 13.26 (%); found: C,
yield: 65%. Mp 205–206 C; H NMR (DMSO-d6) d ppm: 2.45 (s, 62.41; H, 5.04; N, 13.14 (%); MS (m/z): 422.2 (M ).
H, CH ), 7.31–8.07 (m, 8H, ArHs), 8.66 4.2.21 (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl
s, 1H, CH]N); anal. calcd For C H C N O (447.27 g mol ): C, 4-((p-tolylimino)methyl)benzoate (12u). White powder, yield:
3.71; H, 3.61; N, 12.53 (%); found: C, 53.82; H, 3.49; N, 12.43 (%); 86%. Mp 203–205 C; H NMR (DMSO-d6) d ppm: 1.56, 2.43,
ꢁ
1
+
3
(
5
3
), 4.64–4.75 (t, 4H, 2 ꢂ CH
2
ꢀ
1
2
0
16 l2 4 4
ꢁ
1
+
MS (m/z): 446.1 (M ).
2.54 (s, 9H, 3 ꢂ CH ), 4.48–4.78 (d, 2H, CH ), 5.60–5.64 (t, 1H,
3
2
4
.2.14 (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-((phe- CH), 7.21–8.05 (m, 9H, ArHs), 8.56 (s, 1H, CH]N); anal. calcd
ꢀ1
22 4 4
nylimino)methyl)benzoate (10n). White powder, yield: 67%. Mp for C22H N O (406.43 g mol ): C, 65.01; H, 5.46; N, 13.78 (%);
89–190 C; H NMR (DMSO-d6) d ppm: 2.49 (s, 3H, CH
ꢁ
1
+
1
4
3
), found: C, 65.13; H, 5.30; N, 13.83 (%); MS (m/z): 406.2 (M ).
4.2.22 (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 4-
.68–4.78 (t, 4H, 2 ꢂ CH ), 7.28–8.09 (m, 10H, ArHs), 8.73 (s, 1H,
2
ꢀ1
18 4 4
CH]N); anal. calcd for C20H N O (378.38 g mol ): C, 63.48; H, (((4-(N-(4-methylpyrimidin-2-yl)sulfamoyl)phenyl)imino)methyl)
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ꢁ
1
benzoate (12v). White powder, yield: 45%. Mp 256–257 C; H supernatant was applied to a Ni-NTA agarose column, washed,
), 4.67– and eluted using a 5–500 mM imidazole gradient over 20 column
), 5.60–5.64 (t, 1H, CH), 5.94–8.04 (m, 11H, ArHs volumes. Eluted protein was dialyzed against 20 mM Tris, pH 7.6,
), 8.71 (s, 1H, CH]N), 11.75 (s, 1H, SO
NH); anal. calcd 1 mM DTT, and 100 mM NaCl. Puried FabHs were concentrated
NMR (DMSO-d6) d ppm: 2.24, 2.26, 2.47 (s, 9H, 3 ꢂ CH
3
4.75 (d, 2H, CH
NH
2
+
2
2
ꢀ
1
ꢀ1
ꢁ
for C26
H
25
N
7
O
6
S (563.59 g mol ): C, 55.41; H, 4.47; N, 17.40 up to 2 mg mL and stored at ꢀ80 C in 20 mM Tris, pH 7.6, 100
+
(
%); found: C, 55.31; H, 4.25; N, 17.63 (%); MS (m/z): 563.2 (M ). mM NaCl, 1 mM DTT, and 20% glycerol for enzymatic assays.
.2.23 (E)-1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-yl 4-
Puried ACP contains the apo-form that needs to be converted
((4-(N-carbamimidoylsulfamoyl)phenyl)imino)methyl)benzoate into the holo-form. The conversion reaction is catalyzed by acyl
4
(
(
(
ꢁ
1
12w). White powder, yield: 54%. Mp 240–241 C; H NMR carrier protein synthase (ACPS). In the nal volume of 50 mL, 50
DMSO-d6) d ppm: 1.46, 2.45 (s, 6H, 2 ꢂ CH
), 4.57–4.73 (d, 2H, mg ACP, 50 mM Tris, 2 mM DTT, 10 mM MgCl , 600 mM CoA, and
), 5.71 (t, 1H, CH), 7.37–8.08 (m, 11H, ArHs + NH
H, CH]N), 11.75 (s, 1H, SO NH); anal. calcd for C22
513.53 g mol ): C, 51.46; H, 4.51; N, 19.09 (%); found: C, 51.52; potassium phosphate. Holo-ACP was puried by fractionation of
3
2
ꢁ
CH
1
2
2
), 8.74 (s, 0.2 mM ACPS were incubated for 1 h at 37 C. The pH of the
23 7 6
H N O S
reaction was then adjusted to approximately 7.0 using 1 M
2
ꢀ1
(
+
H, 4.40; N, 19.20 (%); MS (m/z): 513.1 (M ).
the reaction mixture by Source Q-15 ion exchange chromatog-
raphy using a 0–500 mM NaCl gradient over 25 column volumes.
In a nal 20 mL reaction, 20 mM Na
.5 mM DTT, 0.25 mM MgCl , and 2.5 mM holo-ACP were mixed
.3.1 Antibacterial activity assay. The antibacterial activities with 1 nM FabH, and H O was added to 15 mL. Aer 1 min
2 4 2 4
HPO /NaH PO , pH 7.0,
4
.3 Biological assays
0
2
4
2
of the synthetic compounds were tested against two Gram-negative incubation, a 2 mL mixture of 25 mM acetyl-CoA and 0.75 mCi
bacterial strains, i.e., E. coli ATCC 35218 and P. aeruginosa ATCC [3H] acetyl-CoA was added for FabH reaction for 25 min. The
1
6
3525, and two Gram-positive bacterial strains, i.e., B. subtilis ATCC reaction was stopped by adding 20 mL of ice-cold 50% TCA,
633 and S. aureus ATCC 6538, using the method recommended by incubating for 5 min on ice, and centrifuging to pellet the
22
National Committee for Clinical Laboratory Standards (NCCLS).
In vitro activities of the compounds were tested in nutrient broth suspended with 5 mL of 0.5 M NaOH. The incorporation of the
NB) for bacteria by the twofold serial dilution method. 3H signal in the nal product was read by liquid scintillation.
protein. The pellet was washed with 10% ice-cold TCA and re-
(
Seeded broth (broth containing microbial spores) was When determining the inhibition constant (IC50), inhibitors
prepared in NB from 24 h old bacterial cultures on nutrient agar were added from a concentrated DMSO stock such that the nal
ꢁ
(
HiMedia) at 37 ꢃ 1 C. The bacterial suspension was adjusted concentration of DMSO did not exceed 2%.
4
5
with sterile saline to a concentration of 1 ꢂ 10 to 10 CFU. The
4.3.3 Acute oral toxicity assay. Five thousand milligrams of
tested compounds and reference drugs were prepared by the compounds (10b, 10f, 10q, 10r, 12v) per kilogram of body-
twofold serial dilution to obtain the required concentrations of weight were administered to 25 healthy rats by oral gavage,
ꢀ
1
1
00, 50, 25, 12.5, 6.25 and 3.13 mg mL . The tubes were incu- respectively. The animals were observed for mortality, signs of
ꢁ
bated in BOD incubators at 37 ꢃ 1 C for bacterial growth. The gross toxicity and behavioral changes at least once daily for 14
MICs were recorded by visual observations aer 24 h (for days. Bodyweights were recorded prior to administration and
bacteria) of incubation. Kanamycin B and penicillin G were again on days 7 and 14. All animals kept active and healthy during
used as standards for antibacterial activity. The observed MICs the entire study time. There were no signs of gross toxicity or
are presented in Table 1.
.3.2 E. coli FabH purication and activity assay. Full-
abnormal behavior.
4
length E. coli acyl carrier protein (ACP), acyl carrier protein syn-
thase (ACPS), and b-ketoacyl-ACP synthase III (FabH) were indi-
4.4 Docking simulations
vidually cloned into pET expression vectors with an N-terminal The crystal structures of E. coli FabH (PDB code: 1HNJ) were
His-tag (ACP, ACPS in pET19; FabH in pET28). All proteins were obtained from the Protein Data Bank (http://www.rcsb.org).
expressed in E. coli strain BL21 (DE3). Transformed cells were Molecular docking of compounds into the three-dimensional X-
grown on Luria–Bertani (LB) agar plates supplemented with ray structure of FabH was carried out using Ligand Fit Dock
ꢀ
1
kanamycin B (30 mg mL ). Sodium dodecyl sulfate poly- protocol of Discovery Studio 3.5.
acrylamide 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 mg
Acknowledgements
ꢀ
1
ꢁ
mL of kanamycin B and grown overnight at 37 C, 1 mL of
which was used to inoculate 100 mL LB medium supplemented
The work was nanced by a grant (no. J1103512) from the
National Natural Science Foundation of China.
ꢀ1
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-thio-
ꢁ
galactopyranoside (IPTG). The culture was grown for 4 h, and
harvested by centrifugation (30 min at 15 000 rpm).
Notes and references
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were lysed by sonication in 20 mM Tris, pH 7.6, 5 mM imidazole,
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54224 | RSC Adv., 2014, 4, 54217–54225
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