Design, synthesis and antimicrobial activities of nitroimidazole derivatives containing 1,3,4-oxadiazole scaffold as FabH inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.05.050

Nitroimidazoles and their derivatives have drawn continuing interest over the years because of their varied biological activities, recently found application in drug development for antimicrobial chemotherapeutics and antiangiogenic hypoxic cell radiosensitizers. In order to search for novel antibacterial agents, we designed and synthesized a series of secnidazole analogs based on oxadiazole scaffold (4-21). Among these compounds, 4 and 7-21 were reported for the first time. These compounds were tested for antibacterial activities against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. This new nitroimidazole derivatives class demonstrated strong antibacterial activities. Escherichia coli β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitory assay and docking simulation indicated that the compounds 2-(2-methoxyphenyl)-5-((2- methyl-5-nitro-1H-imidazol-1-yl)methyl)-1,3,4-oxadiazole (11) with MIC of 1.56-3.13 μg/mL against the tested bacterial strains and 2-((2-methyl-5- nitro-1H-imidazol-1-yl)methyl)-5-(2-methylbenzyl)-1,3,4-oxadiazole (12) with MIC of 1.56-6.25 μg/mL were most potent inhibitors of Escherichia coli FabH.

Full text of DOI:10.1016/j.bmc.2012.05.050

Bioorganic & Medicinal Chemistry 20 (2012) 4316–4322
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and antimicrobial activities of nitroimidazole
derivatives containing 1,3,4-oxadiazole scaffold as FabH inhibitors
Yao Li a, , Yin Luo a, , Yang Hu a, Di-Di Zhu a, Shuai Zhang a, Zhi-Jun Liu a, Hai-Bin Gong b,
,
⇑
Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b Xuzhou Central Hospital, Xuzhou 221009, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitroimidazoles and their derivatives have drawn continuing interest over the years because of their var-
ied biological activities, recently found application in drug development for antimicrobial chemothera-
peutics and antiangiogenic hypoxic cell radiosensitizers. In order to search for novel antibacterial
agents, we designed and synthesized a series of secnidazole analogs based on oxadiazole scaffold
(4–21). Among these compounds, 4 and 7–21 were reported for the first time. These compounds were
tested for antibacterial activities against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and
Staphylococcus aureus. This new nitroimidazole derivatives class demonstrated strong antibacterial
activities. Escherichia coli b-ketoacyl-acyl carrier protein synthase III (FabH) inhibitory assay and docking
simulation indicated that the compounds 2-(2-methoxyphenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
Received 27 March 2012
Revised 21 May 2012
Accepted 22 May 2012
Available online 30 May 2012
Keywords:
Nitroimidazole
Oxadiazole
FabH inhibitors
Antibacterial activities
yl)methyl)-1,3,4-oxadiazole (11) with MIC of 1.56–3.13
2-((2-methyl-5-nitro-1H-imidazol-1-yl)methyl)-5-(2-methylbenzyl)-1,3,4-oxadiazole (12) with MIC of
1.56–6.25 g/mL were most potent inhibitors of Escherichia coli FabH.
Ó 2012 Elsevier Ltd. All rights reserved.
lg/mL against the tested bacterial strains and
l
1. Introduction
reaction between a CoA-attached acetyl group and an ACP-at-
tached malonyl group, yielding acetoacetyl-ACP as its final
product.11
Infections triggered by some pathogenic microorganisms can
bring illnesses even a fatal one.1 Although many kinds of antibac-
terial agents were discovered and used for clinical treatment, the
incidences of drug resistance of microorganisms to antibacterial
agents were constantly reported.2,3 Therefore, the development
of new types of antibacterial agents is a very important task and
much of the research effort is oriented to the design of new anti-
bacterial agents with high efficiency.4
FabH proteins from both 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 essen-
tially invariant in various bacterial FabH molecules.12–14 These
facts suggest the idea that FabH can be used as an effective molec-
ular target for the development of new antibacterial agents, since it
regulates the fatty acid biosynthesis rate via an initiation pathway
and its substrate specificity is a key factor in membrane fatty acid
composition.15–17
Nitroimidazoles and their derivatives have drawn continuing
interest over the years because of their varied biological activities,
recently found application in drug development for antimicrobial
chemotherapeutics and antiangiogenic hypoxic cell radiosensitiz-
ers.18–20 Importantly, the toxicology and metabolism of nitroimi-
dazoles, particularly secnidazole, have been characterized.18
In recent years, different kinds of targets in key areas of the bac-
terial cell cycle have been studied that would be a new approach
against the problem of drug resistance. One of the most attractive
biochemical pathways to be used as the target for new antibacte-
rial agents is the fatty acid biosynthesis (FAB). This pathway has
been demonstrated to be essential for bacteria cell survival.5,6
A
key enzyme in this pathway is b-ketoacyl-acyl carrier protein syn-
thase III (FabH), which is the enzyme responsible for the first reac-
tion in the pathway and plays an important regulatory role.7,8 The
enzyme initiates the fatty acid elongation cycles,9 and participates
in the feedback regulation of the biosynthetic pathway via product
inhibition.10 As shown in Figure 1 FabH catalyzes the condensation
Secnidazole (a,2-dimethyl-5-nitro-1H-imidazole-1-ethanol) is par-
ticularly effective in the treatment of amebiasis, giardiasis, tricho-
moniasis, and bacterial vaginosis21 as it is rapidly and completely
absorbed after oral administration and has a longer terminal elim-
ination half-life (17–29 h) than commonly used drugs in this
class.21 In these cases, the treatment with secnidazole is shorter
⇑
Corresponding authors. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.050

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4316–4322
4317
Figure 1. FabH-catalyzed initiation reaction of fatty acid biosynthesis.11
and significantly more effective than the treatment using other
imidazole drugs and the adverse effects are not very drastic.23 As
a result, excellent works have been published on the activities
and pharmacokinetics of secnidazole derivatives and their determi-
nation in pharmaceutical is of great importance.18–23
by the reaction of 2-methyl-5-nitro-1H-imidazole in excess ethyl
-chloroacetate and then hydrazinolysis of ethyl ester group by
hydrazine hydrate.
a
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
was
then reacted with substituted benzoic acids and phenylacetic acids
to prepare the corresponding nitroimidazole derivatives 4–21. The
chemical structures of these nitroimidazole derivatives were sum-
marized in Table 1. These compounds gave satisfactory elementary
analyses ( 0.4%). 1H NMR and ESI MS spectra data was consistent
with the assigned structures. Among these compounds, 4 and
7–21 were reported for the first time. Compounds 4–12 were
nitroimidazole derivatives prepared by substituted benzoic acids
with 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide. Com-
pounds 13–21 were nitroimidazole derivatives prepared by
substituted phenylacetic acids with 2-(2-methyl-5-nitro-1H-imi-
dazol-1-yl)acetohydrazide.
In order to search for novel antibacterial agents, we designed
and synthesized a series of secnidazole analogs based on oxadiaz-
ole scaffold. We chose oxadiazole moiety because it was found in a
variety of biologically substance and it showed widely biological
activities such as anticancer activities, antidepressant properties
and so on.23–28 However, to our knowledge, few reports have been
dedicated to the FabH inhibitory activities of nitroimidazole deriv-
atives owing oxadiazole groups. Herein, to keep on research on
antibacterial compounds with FabH inhibitory activity, we de-
scribed the synthesis and structure–activity relationship (SAR) of
a new series of nitroimidazole derivatives owing oxadiazole group,
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.
2.2. Biological activity
2.2.1. Antimicrobial activity
All the synthesized compounds (4–21) were screened for their
antibacterial activities against two Gram-negative bacterial
strains: E. coli and P. aeruginosa and two Gram-positive bacterial
strains: B. subtilis and S. aureus by MTT method. The MICs (mini-
mum 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 Anti-
microb. Agents Chemother. 2004, 48, 3093)30 under identical condi-
tions for comparison. The results revealed that most of
the synthesized compounds exhibited significant antibacterial
2. Results and discussion
2.1. Chemistry
Compound 3 was synthesized by the routes outlined in Scheme
1 according to Mirzaei et al.’s method.29 Compound 3 was prepared
N
N
N
ClCH2COOC2H5
H2NNH2H2O
MeOH
NO2
NO2
O
N
N
H
N
NO2 K2CO3, acetone
N
H
NH2
O
O
2
3
1
N
R1
NO2
COOH
N
POCl3
R1
O
N
N
N
NO2
4-12
N
N
H
N
NO2
NH2
N
POCl3
COOH
O
O
R2
R2
N
N
13-21
Scheme 1. Synthesis of nitroimidazole derivatives 4–21.

4318
Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4316–4322
Table 1
Table 3
Structures of compounds 4–21
E. coli FabH inhibitory activities of selected compounds 4–6
a
Compounds
R1
Compounds
R2
Compounds
E. coli FabH IC50
(
lM)
Hemolysis LC30 (mg/mL)
4
5
6
7
8
9
10
11
12
4-F
4-Cl
4-Br
3-F
13
14
15
16
17
18
19
20
21
4-F
4-Cl
4-Br
3-F
4
5
6
8
18.6
28.3
43.8
16.9
40.5
9.4
4.3
5.1
2.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
3-Br
3-Br
9
4-NO2
3-OCH3
2-OCH3
2-CH3
4-NO2
3-OCH3
2-OCH3
2-CH3
10
11
12
DDCP
a
Lytic concentration 30%.
activities. Among the 18 synthetic new nitroimidazole derivatives,
the benzoic acid derivatives 4–6 and 8–12, whose MIC values rang-
electron-withdrawing substituents (such as F, Cl, Br), and their
MICs value range from 6.25 to 25 g/mL. We proposed that elec-
tron-donating groups on benzoic acid component were conducive
to the antibacterial activity and compounds with electron-with-
drawing halogen groups on benzoic acid component were not
favorable for activity.
Moreover, according to the data in Table 2, among derivatives
with electron-donating substituents, compounds 10–12 with
substituted methoxy and methyl group on benzoic acid component
exhibited significant antibacterial activities with MICs of 1.56–
ing from 1.56 to 25
than phenylacetic acid derivatives 13–21, with MICs values rang-
ing from 6.25 to 50 g/mL, which showed the introduction of ben-
lg/mL, displayed higher antibacterial potencies
l
l
zoic acid lead to the increase of the antibacterial activity.
Out of the eight nitroimidazole derivatives (compound 4–6 and
8–12), compound 11 and 12 exhibited most potent activities with
MIC values of both 1.56
are superior to the positive control kanamycin with corresponding
MIC of 3.13 g/mL and equivalent to the positive control DDCP
with corresponding MIC of 1.56 g/mL. Besides, the data showed
compound 11 and 12 displayed significant activities with MIC val-
ues of 1.56, 1.56, 3.13 g/mL and 3.13, 1.56, 6.25 g/mL against P.
lg/mL against E. coil ATCC35128, which
l
l
12.5
substituted methoxy and methyl group on phenylacetic acid com-
ponent showed less potent activity with MIC of 12.5–50 g/mL.
lg/mL against E. coil ATCC35128. Compound 19–21 with
l
l
l
fluorescence, B. subtilis, S. aureus, respectively, indicating that they
possessed broad-spectrum antibacterial activities.
Based on the data obtained, we found that compounds with a
methoxy group on the 2-position of benzoic acid displayed higher
These results demonstrated that the synthetic nitroimidazole
derivatives with substituted electron-donating group on benzoic
acid component showed more potent antibacterial activity than
those of on phenylacetic acid component.
antibacterial activity with MIC values of 1.56
ATCC35128 than compounds with a methoxy group on the 3-posi-
tion of benzoic acid with MIC values of 12.5 g/mL and compounds
with a methoxy group on the 2-position of phenylacetic acid with
MIC values of 50 g/mL. And the varieties of substitutes of benzoic
acid and phenylacetic acid such as halogen, methyl and methoxyl
also lead to the different antibacterial activities of these nitroimi-
dazole derivatives. Among them, the derivatives which have elec-
tron-donating substituents (such as CH3, OCH3) exhibited more
potent activities against E. coil ATCC35128 than those have
lg/mL against E. coil
2.2.2. E. coli FabH inhibitory activity
l
The E. coli FabH inhibitory potency of the selected compounds
4–6 and 8–12 was examined and the results were summarized in
Table 3. As shown in Table 3, among the tested compounds, com-
pounds 11 and 12 showed potent inhibitory activities with IC50 of
l
4.3 and 5.1
tive control DDCP27 with IC50 of 2.1
displayed moderate inhibitory activities with IC50 ranging from 9.4
to 43.8 M. It suggested that the introduction of benzoic acid and
lM, respectively, which were comparable to the posi-
l
M. Other tested compounds
l
Table 2
Antibacterial activities of synthetic compounds
Compounds
Minimum inhibitory concentrations (lg/mL)
Gram-negative
Pseudomonas aeruginosa
Gram-positive
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
4
5
6
7
8
9
10
11
12
13
14
15
12.5
12.5
25
>100
6.25
12.5
12.5
1.56
1.56
25
25
12.5
25
>100
25
12.5
12.5
1.56
3.13
12.5
12.5
25
12.5
12.5
12.5
>100
6.25
25
6.25
1.56
1.56
50
25
25
50
>100
25
25
12.5
3.13
6.25
25
25
50
50
50
50
50
16
17
18
>100
25
25
>100
6.25
25
>100
25
25
>100
50
50
19
20
21
25
50
12.5
3.13
1.56
12.5
12.5
25
3.13
3.13
25
25
50
1.56
12.5
50
25
25
1.56
25
Kanamycin
DDCP

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4316–4322
4319
Table 4
molecular docking result, along with the biological assay data, sug-
gesting that compounds 11 and 12 was a potential inhibitor of
FabH.
The docking calculation of the synthesized compounds (4–21)
Compounds CDOCKER
INTERATION ENERGY
Gb (kcal/mol)
Compounds CDOCKER
INTERATION ENERGY
Gb (kcal/mol)
D
D
3. Conclusion
4
5
6
7
8
9
10
11
12
À31.5284
À31.3187
À30.6726
À26.9831
À32.2031
À30.9375
À32.7019
À33.2629
À32.9295
13
14
15
16
17
18
19
20
21
À29.7027
À27.9217
À27.0913
À27.0399
À30.2138
À29.0306
À29.171
In summary, a series of novel nitroimidazole derivatives 4–21
were synthesized and tested for their inhibitory activities against
E. coli, P. fluorescence, B. subtilis and S. aureus. Many of them exhib-
ited potent antibacterial and E. coli FabH inhibitory activities. Par-
ticularly, Compounds 11 and 12 were proved to be the most potent
compounds. Preliminary SARs and molecular modeling study pro-
vided 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.
À28.2003
À30.2406
the formation of nitroimidazole and oxadiazole structure were
essential for the FabH inhibitory activity. It also can be seen from
Table 3 that the selected compounds displayed low hemolytic
activity.
4. Experiments
4.1. Materials and measurements
All chemicals (reagent grade) used were commercially avail-
able. Melting points (uncorrected) were determined on a X-4 MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra were ob-
tained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were recorded on a Bruker PX500 or DPX300 spec-
trometern at 25 °C with TMS and solvent signals allotted as inter-
nal standards. Chemical shifts were reported in ppm (d). Elemental
analyses were performed on a CHN-O-Rapid instrument and were
within 0.4% of the theoretical values.
2.2.3. Binding model of compounds 11, 12 and E. coli FabH
Molecular docking of the synthesized compounds and E. coli
FabH was performed on the binding model based on the E. coli
FabH–CoA complex structure (1HNJ.pdb).31 The coordinate of the
input site sphere is 28.4494, 9.90349, 33.4428 and the radius of
the sphere is 11 Å.
All docking runs were applied LigandFit Dock protocol of Dis-
covery Studio 3.1. The docking calculation of the synthesized com-
pounds was showed in Table 4. The interaction energy of the
compounds and their antibacterial activity showed the corre-
sponding results. Among the docking calculation of the synthe-
sized compounds, compounds 11 and 12 showed the lowest
interaction energy. The binding model of compounds 11, 12 and
E. coli FabH was depicted in Figures 2 and 3. In the binding model,
compound 11 was nicely bound to the FabH kinase with four inter-
action bonds. The end amino of Asn274, His244 and Asn247 were
respectively formed four hydrogen bonds interaction with oxygen
atom of oxadiazole ring of compound 11. Besides, the hydrogen of
Asn274 and His244 were respectively formed four hydrogen bonds
interaction with oxygen atom of nitro group of compound 12. This
4.2. General method of synthesis of nitroimidazole derivatives
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide
(1–3)
was prepared according the previous procedure by Mirzaei et
al.29 To a suspension of 2-methyl-5-nitroimidazole (0.01 mol) in
ethyl
a-chloroacetate (0.1 mol), propionic acid (6.66 mL) was
added and refluxed for 16 h. The mixture was filtered and concen-
trated under reduced pressure to get compound 2. To a stirring
solution of hydrazine hydrate (6.5 mL) in an ice bath, a solution
of 2 (0.03 mol) in methanol (40 mL) was added slowly (10 min).
Figure 2. Binding model of compound 11 and E. coli FabH. H-bonds are displayed as dashed lines.

4320
Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4316–4322
Figure 3. Binding model of compound 12 and E. coli FabH. H-bonds are displayed as dashed lines.
The stirring was continued for 4.5 h at 0 °C under argon atmo-
sphere. The white precipitate which formed in À10 °C was filtered
and recrystallized from chloroform to give compound 3.
(C13H11FN5O3, [M+H]+). Anal. Calcd for C13H10FN5O3: C, 51.49; H,
3.32; N, 23.09. Found: C, 51.51; H, 3.30; N, 23.09.
An equimolar mixture of 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)acetohydrazide (0.001 mol) and substituted carboxylic acid-
s(0.001 mol) in phosphoryl chloride(15 mL) was refluxed for
10–16 h. Then reaction mixture was cooled, poured into ice-cold
water and neutralized with 20% NaHCO3 solution. The resultant so-
lid was filtered, washed with water and recrystallized from ethanol
to give nitroimidazoles derivatives 4–21.
4.2.5. 2-(3-Bromophenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (8)
Mp: 214–216 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.43 (s, 3H),
5.72 (s, 2H), 7.14 (d, J = 3.42 Hz, 2H), 7.26 (d, J = 8.49 Hz, 2H),
7.81 (d, J = 3.42 Hz, 1H), 8.45 (s, 1H). MS (ESI): 365.15
(C13H11BrN5O3, [M+H]+). Anal. Calcd for C13H10BrN5O3: C, 42.88;
H, 2.77; N, 21.94. Found: C, 42.86; H, 2.79; N, 21.93.
4.2.1. 2-(4-Fluorophenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (4)
4.2.6. 2-((2-Methyl-5-nitro-1H-imidazol-1-yl)methyl)-5-(4-
nitrophenyl)-1,3,4-oxadiazol (9)
Mp: 229–230 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.44 (s, 3H),
5.75 (s, 2H), 7.45 (t, J = 8.61 Hz, 2H), 8.04 (t, J = 8.40 Hz, 2H), 8.46
(s, 1H). MS (ESI): 304.25 (C13H11FN5O3, [M+H]+). Anal. Calcd for
Mp: 214–216 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.34 (s, 3H),
4.97 (s, 2H), 8.09 (d, J = 8.76 Hz, 2H), 8.34 (d, J = 10.62 Hz, 3H).
MS (ESI): 331.26 (C13H11N6O5, [M+H]+). Anal. Calcd for
C13H10FN5O3: C, 51.49; H, 3.32; N, 23.09. Found: C, 51.51; H,
C14H10N6O5: C, 47.28; H, 3.05; N, 25.45. Found: C, 47.30; H, 3.04;
3.33; N, 23.07.
N, 25.42.
4.2.2. 2-(4-Chlorophenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (5)
4.2.7. 2-(3-Methoxyphenyl)-5-((2-methyl-5-nitro-1H-imidazol-
1-yl)methyl)-1,3,4-oxadiazole (10)
Mp: 225–226 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.49 (s, 3H),
5.75 (s, 2H), 7.56 (d, J = 8.43 Hz, 2H), 7.96 (d, J = 9.39 Hz, 2H),
8.46 (s, 1H). MS (ESI): 320.70 (C13H11ClN5O3, [M+H]+). Anal. Calcd
for C13H10ClN5O3: C, 48.84; H, 3.15; N, 21.91. Found: C, 48.85; H,
3.13; N, 21.90.
Mp: 216–217 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.49 (s, 3H),
3.84 (s, 3H), 5.76 (s, 2H), 7.49–7.67 (m, 3H), 7.79 (d, J = 9.72 Hz,
1H), 8.47 (s, 1H). MS (ESI): 316.28 (C14H14N5O4, [M+H]+). Anal.
Calcd for C14H13N5O4: C, 53.33; H, 4.16; N, 22.21. Found: C,
53.35; H, 4.14; N, 22.22.
4.2.3. 2-(4-Bromophenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (6)
4.2.8. 2-(2-Methoxyphenyl)-5-((2-methyl-5-nitro-1H-imidazol-
1-yl)methyl)-1,3,4-oxadiazole (11)
Mp: 220–223 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.45 (s, 3H),
5.68 (s, 2H), 7.69–7.80 (m, 2H), 7.84–7.93 (m, 2H), 8.46 (s, 1H).
MS (ESI): 365.15 (C13H11BrN5O3, [M+H]+). Anal. Calcd for
Mp: 185–186 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.48 (s, 3H),
3.86 (s, 3H), 5.76 (s, 2H), 7.10–7.15 (m, 2H), 7.26 (t, J = 8.34 Hz,
1H), 7.68 (d, J = 8.40 Hz, 1H), 8.45 (s, 1H). MS (ESI): 317.28
(C14H14N5O4, [M+H]+). Anal. Calcd for C14H13N5O4: C, 53.33; H,
4.16; N, 22.21. Found: C, 53.36; H, 4.15; N, 22.23.
C13H10BrN5O3: C, 42.88; H, 2.77; N, 21.94. Found: C, 42.87; H,
2.78; N, 21.92.
4.2.4. 2-(3-Fluorophenyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (7)
4.2.9. 2-((2-Methyl-5-nitro-1H-imidazol-1-yl)methyl)-5-(2-
methylbenzyl)-1,3,4-oxadiazole (12)
Mp: 185–186 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.50 (s, 3H),
5.76 (s, 2H), 7.12 (d, J = 8.76 Hz, 1H), 7.92 (d, J = 8.79 Hz, 2H),
7.81 (d, J = 8.40 Hz, 1H), 8.46 (s, 1H). MS (ESI): 304.25
Mp: 233–236 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.49 (s, 3H),
2.69 (s, 3H), 5.75 (s, 2H), 7.21 (d, J = 7.53 Hz, 2H), 7.81–7.89 (m,
2H), 8.44 (s, 1H). MS (ESI): 300.28 (C14H14N5O3, [M+H]+). Anal.

Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4316–4322
4321
Calcd for C14H13N5O3: C, 56.18; H, 4.38; N, 23.40. Found: C, 56.16;
4.2.18. 2-((2-Methyl-5-nitro-1H-imidazol-1-yl)methyl)-5-(2-
H, 4.35; N, 23.43.
methylbenzyl)-1,3,4-oxadiazole (21)
Mp: 218–220 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.36 (s, 3H),
2.54 (s, 3H), 3.52 (s, 2H), 4.52 (s, 2H), 7.29 (d, J = 8.04 Hz, 2H),
7.83 (d, J = 8.22 Hz, 2H), 8.29 (s, 1H). MS (ESI): 314.31
(C15H16N5O3, [M+H]+). Anal. Calcd for C15H15N5O3: C, 57.50; H,
4.83; N, 22.35. Found: C, 76.53; H, 4.80; N, 22.32.
4.2.10. 2-(4-Fluorobenzyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (13)
Mp: 264–265 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.33 (s, 3H),
3.57 (s, 2H), 4.91 (s, 2H), 7.20–7.34 (m, 4H), 8.29 (s, 1H). MS
(ESI): 318.28 (C14H13FN5O3, [M+H]+). Anal. Calcd for C14H12FN5O3:
C, 53.00; H, 3.81; N, 22.07. Found: C, 52.98; H, 3.82; N, 22.08.
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 meth-
od recommended by National Committee for Clinical Laboratory
Standards (NCCLS).32
4.2.11. 2-(4-Chlorobenzyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (14)
Mp: 255–256 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.23 (s, 3H),
3.56 (s, 2H), 4.84 (s, 2H), 7.23–7.36 (m, 4H), 8.28 (s, 1H). MS
(ESI): 334.73 (C14H13ClN5O3, [M+H]+). Anal. Calcd for
C14H12ClN5O3: C, 50.39; H, 3.62; N, 20.99. Found: C, 50.36; H,
In vitro activities of the compounds were tested in Nutrient
broth (NB) for bacteria by the twofold 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 °C. The bacterial suspension was adjusted with sterile sal-
ine to a concentration of 1 Â 104–105 CFU. The tested compounds
and reference drugs were prepared by twofold serial dilution to ob-
tain the required concentrations of 100, 50, 25, 12.5, 6.25 and
3.64; N, 20.96.
4.2.12. 2-(4-Bromobenzyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (15)
Mp: 225–227 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.27 (s, 3H),
3.46 (s, 2H), 4.91 (s, 2H), 7.17–7.30 (m, 2H), 7.51 (t, J = 9.24 Hz,
2H) 8.33 (t, J = 9.60 Hz, 1H). MS (ESI): 379.18 (C14H13BrN5O3,
[M+H]+). Anal. Calcd Anal. Calcd for C14H12BrN5O3: C, 44.46; H,
3.20; N, 18.52. Found: C, 44.48; H, 3.21; N, 18.50.
3.13 lg/mL. The tubes were incubated in BOD incubators at
37 1 °C for bacteria. The MICs were recorded by visual observa-
tions after 24 h (for bacteria) of incubation. Kanamycin and DDCP
were used as standards for bacterial. The observed MICs are pre-
sented in Table 2.
4.2.13. 2-(3-Fluorobenzyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (16)
Mp: 221–223 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.33 (s, 3H),
3.57 (s, 2H), 4.93 (s, 2H), 7.20 (d, J = 8.40 Hz, 2H), 7.35 (d, J
=8.40 Hz, 2H) 8.33 (s, 1H). MS (ESI): 316.28 (C14H13FN5O3,
[M+H]+). Anal. Calcd for C14H12FN5O3: C, 50.39; H, 3.62; N, 20.99.
Found: C, 50.37; H, 3.64; N, 20.97.
4.4. 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 indi-
vidually 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
4.2.14. 2-(3-Bromobenzyl)-5-((2-methyl-5-nitro-1H-imidazol-1-
yl)methyl)-1,3,4-oxadiazole (17)
Mp: 215–218 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.23 (s, 3H),
3.46 (s, 2H), 4.84 (s, 2H), 7.23–7.35 (m, 4H), 8.29 (s, 1H). MS
(ESI): 379.18 (C14H13BrN5O3, [M+H]+). Anal. Calcd for
supplemented with kanamycin (30 lg/mL). Sodium dodecyl sul-
fate–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
C14H12BrN5O3: C, 44.46; H, 3.20; N, 18.52. Found: C, 44.43; H,
3.22; N, 18.53.
30 lg/mL of kanamycin and grown overnight at 37 °C, 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 °C, and then induced with 0.5 mM isopropyl b-D-thiogalactopy-
ranoside (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 superna-
tant 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 FabHs were concentrated up to
2 mg/mL and stored at À80 °C in 20 mM Tris, pH 7.6, 100 mM NaCl,
1 mM DTT, and 20% glycerol for enzymatic assays.
4.2.15. 2-((2-Methyl-5-nitro-1H-imidazol-1-yl)methyl)-5-(4-
nitrobenzyl)-1,3,4-oxadiazole (18)
Mp: 225–227 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.37 (s, 3H),
4.44 (s, 2H), 5.65 (s, 2H), 7.63 (d, J = 8.79 Hz, 2H), 8.22 (d,
J = 8.76 Hz, 2H) 8.39 (s, 1H). MS (ESI): 345.28 (C14H13N6O5,
[M+H]+). Anal. Calcd for C14H12N6O5: C, 48.84; H, 3.51; N, 24.41.
Found: C, 48.86; H, 3.50; N, 24.39.
4.2.16. 2-(3-Methoxybenzyl)-5-((2-methyl-5-nitro-1H-imidazol-
1-yl)methyl)-1,3,4-oxadiazole (19)
Mp: 226–228 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.28 (s, 3H),
3.45 (s, 2H), 3.71 (s, 3H), 4.86 (s, 2H), 6.83–6.87 (m, 3H), 7.13 (d,
J = 9.24 Hz, 1H), 8.28 (s, 1H). MS (ESI): 330.31 (C15H16N5O4,
[M+H]+). Anal. Calcd for C15H15N5O4: C, 54.71; H, 4.59; N, 21.27.
Found: C, 54.74; H, 4.57; N, 21.28.
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 mM
4.2.17. 2-(2-Methoxybenzyl)-5-((2-methyl-5-nitro-1H-imidazol-
1-yl)methyl)-1,3,4-oxadiazole (20)
Tris, 2 mM DTT, 10 mM MgCl2, 600 lM CoA, and 0.2 lM ACPS
Mp: 201–203 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.31 (s, 3H),
3.49 (s, 2H), 3.75 (s, 3H), 4.52 (s, 2H), 6.84–6.90 (m, 3H), 7.22 (d,
J = 8.40 Hz, 1H), 8.28 (s, 1H). MS (ESI): 330.31 (C15H16N5O4,
[M+H]+). Anal. Calcd for C15H15N5O4: C, 54.71; H, 4.59; N, 21.27.
Found: C, 54.72; H, 4.58; N, 21.25.
was incubated for 1 h at 37 °C. The pH of the reaction was then ad-
justed 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 gradi-
ent over 25 column volumes.

4322
Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 4316–4322
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0.5 mM DTT, 0.25 mM MgCl2, and 2.5
with 1 nM FabH, and H2O was added to 15 mL. After 1 min incuba-
tion, a 2 L mixture of 25 M acetyl-CoA and 0.75
Ci [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
l
L reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
20992.
l
M holo-ACP were mixed
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l
l
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%.
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 compounds 11 and 12 into the three-dimen-
sional X-ray structure of FabH was carried out using CDOCKER
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The work was financed by a grant (No. J1103512) from National
Natural Science Foundation of China.
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