Synthesis, molecular docking and biological evaluation of metronidazole derivatives as potent Helicobacter pylori urease inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.09.018

Fourteen metronidazole derivatives (compounds 3a-f and 4b-h) have been synthesized by coupling of metronidazole and salicylic acid derivatives. All of them are reported for the first time. Their chemical structures are characterized by ¹H NMR,

Full text of DOI:10.1016/j.bmc.2009.09.018

Bioorganic & Medicinal Chemistry 17 (2009) 7531–7536
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular docking and biological evaluation of metronidazole
derivatives as potent Helicobacter pylori urease inhibitors
*
Wen-Jun Mao, Peng-Cheng Lv, Lei Shi, Huan-Qiu Li, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, 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:
Received 13 July 2009
Revised 8 September 2009
Accepted 10 September 2009
Available online 15 September 2009
Fourteen metronidazole derivatives (compounds 3a–f and 4b–h) have been synthesized by coupling of
metronidazole and salicylic acid derivatives. All of them are reported for the first time. Their chemical
structures are characterized by 1H NMR, MS, and elemental analysis. The inhibitory activities against Hel-
icobacter pylori urease have been investigated in vitro and many compounds have showed promising
potential inhibitory activities of H. pylori urease. The effect of compounds 4b (IC50 = 26
lM) and 4g
(IC50 = 12 M) was comparable with that of acetohydroxamic acid, a well known H. pylori urease inhibitor
l
Keywords:
Metronidazole
Docking
Inhibitor
H. pylori urease
used as a positive control. The experimental values of IC50 showed that inhibitor was potent urease inhib-
itor. A docking analysis using the AUTODOCK 4.0 program could explain the inhibitory activities of com-
pound 4g against H. pylori urease.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
imidazoles have been designed as inhibitors against urease. Metro-
nidazole in combination of other drugs has been used for the treat-
Urease (urea amidohydrolase; E.C. 3.5.1.5) is an enzyme that
catalyzes hydrolysis of urea to ammonia and carbamate, which is
the final step of nitrogen metabolism in living organisms.1 It is
widely distributed in a variety of bacteria such as Helicobacter py-
lori and Proteus mirabilis. H. pylori is a Gram-negative microaero-
philic bacterium that infects up to 50% of the world’s human
population.2 Urease that comes from H. pylori is now accepted as
a major cause of peptic ulcers. Many urease inhibitors have been
investigated in the past decades, like phosphorodiamidates,
hydroxamic acid derivatives, and imidazoles,3 but part of them
are prevented from using in vivo because of their toxicity or
instability and part of them had side effects. Thus, seeking novel
urease inhibitors with good bioavailability and low toxicity is
significative.
ment of infections caused by H. pylori,10 but it has not been
reported as efficient inhibitor against H. pylori urease. Our interest
in this area is to design H. pylori urease inhibitory active metroni-
dazole derivatives at the pendant hydroxy group. Salicylic acid
derivatives have wild biological activities. Some salicylic acid
derivatives, such as anacardic acids and salicylhydroxamic acids
(Scheme 2), were reported as urease inhibitors.11,12 Herein several
substituted salicylic acids were selected to join together with met-
ronidazole and 14 new nitroimidazoles of two series were synthe-
sized to test their H. pylori urease inhibitory activities. Fourteen
nitroimidazoles were evaluated for their inhibitory activities
against H. pylori urease. One nitroimidazole with the lowest IC50
value was chosen to dock with H. pylori urease using AUTODOCK 4.0.13
Nitroimidazole derivative compounds exhibited broad variety
of biological activities including antimicrobial,4 antitubercular,5
and urease inhibitors.6 More attention has been focused on
exploring novel biological properties of nitroimidazole derivative
2. Results and discussion
2.1. Synthesis
compounds.
Metronidazole
(1-[2-hydroxyethyl]-2-methyl-5-
Two series of compounds were designed and synthesized from
metronidazole and salicylic acid derivatives for H. pylori urease
inhibitors by the routes outlined in Scheme 3. The MET-OTs (2-
(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methylbenzenesulfo-
nate, compound 2) was synthesized by the method in our previous
paper.14 In different reaction conditions, two series of products,
including 3a–f and 4a–h, were first synthesized from MET-OTs
and substituted salicylic acids. The research results showed that
the ratio of MET-OTs and substituted salicylic acids and reaction
temperature were the key factors of the synthesis of compounds
nitroimidazole) (Scheme 1), one of the important nitroimidazole
derivatives, has been widely used as an antimicrobial medicine.
During recent years, the structure modification at the pendant hy-
droxy group of metronidazole has received much attention.7
Our previous research about the synthesis of new H. pylori ure-
ase inhibitors from nature products has been reported.8,9 Many
* Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.018

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W.-J. Mao et al. / Bioorg. Med. Chem. 17 (2009) 7531–7536
CH3
N
COOH
HO
OH
N
(a)
NO2
COOH
(b)
Scheme 1. Chemical structure of metronidazole.
HO
HO
3a–f or 4a–h. When the ratio of the amount of substance of MET-
OTs and substituted salicylic acid was 1:1.1, reaction temperature
was 70–80 °C and the reaction time was 14–16 h, compounds 3a–f
were obtained. When the ratio of the amount of substance of MET-
OTs and substituted salicylic acids is 2:1.1, reaction temperature
was 100–110 °C and the reaction time was 20–22 h, compounds
4a–h were obtained. All compounds were fully characterized by
spectroscopic method and elemental analysis. All the nitroimidaz-
oles 3a–f and 4a–h were synthesized for the first time.
COOH
(c)
(d)
OH
O
OH
2.2. Biological activity
N
H
Fourteen nitroimidazoles 3a–f and 4a–h were evaluated for
their inhibitory activities against H. pylori urease. Percent inhibi-
tion at a 1 mM concentration of compounds 3a–f and 4a–h was ini-
tially determined, and the results were reported in Table 1. Most of
Scheme 2. Chemical structures of some anti-urease salicylic acid derivatives:
anacardic acids (a–c), salicylhydroxamic acid (d).
the nitroimidazoles showed good inhibitory activities, especially
nitroimidazole 4b (IC50 = 26
lM) and 4g (IC50 = 12
lM) exhibited
COOH
OH
O
R3
R1
Cl
S
O
S
R3
R2
R2
COOH
O
O
N
O
CH3
OH
N
N
N
O
N
N
i
ii
NO2
R1
NO2
NO2
1
2
3a:R1=H,R2=CH3,R3=H
3b:R1=H,R2=NH2,R3=H
COOH
OH
1
2
3
3c
:R =NO2,R =H,R =NO2
3d:R1=I,R2=H,R3=I
iii
R3
R1
3e:R1=Br,R2=H,R3=Br
R2
1
2
3
3f
:R =Cl,R =H,R =Cl
O2N
N
N
O
4a:R1=H,R2=CH3,R3=H
4b:R1=H,R2=H,R3=Br
O2N
O
1
2
3
4c
:R =H,R =H,R =I
N
R1
N
4d:R1=H,R2=H,R3=SO3
4e:R1=H,R2=H,R3=CH3
O
R2
1
2
3
4f
:R =CH3,R =H,R =H
R3
4g:R1=H,R2=H,R3=Cl
4h:R1=H,R2=Cl,R3=H
4a-h
Scheme 3. General method of the preparation of compounds 2, 3a–f, and 4a–h. Reagents and conditions: (i) CH2Cl2, TEA, 0 °C, 5 h; (ii) DMF, K2CO3, 70–80 °C, 14–16 h; (iii)
DMF, K2CO3, 100–110 °C, 22–24 h.

W.-J. Mao et al. / Bioorg. Med. Chem. 17 (2009) 7531–7536
7533
Table 1
position of the structures of uninhibited and inhibited H. pylori ure-
ase reveals a flap motion of the segment composed of residues
Percent inhibition of compounds 1 (MET), 2 (MET-OTs), 3a–f and 4a–h against H.
pylori urease at the concentration of 1 mM
a
313–a346 forming a helix-turn-helix motif which opens the ac-
tive site when the inhibitor is bound and closes it when not boun.18
In the X-ray structures available for the H. pylori urease, the two
nickels were coordinated by His136, His138, Kcx219, His248,
His274, Asp362, and water molecules.19–21 The residues of active
site are showed together with nickel ions (NI3001 and NI3002)
by AUTODOCK 4.0 in Figure 1.
Compounds
Percent inhibition (%)
b
3a
3b
3c
3d
3e
3f
—
38.3
41.5
53.9
56.1
56.8
—
78.1
91.2
38.3
93.9
78.7
61.2
48.4
42.1
95.3
82.2
Among the several known urease inhibitors, phosphorodiami-
dates were the most efficient. Phenylphosphorodiamidate (Scheme
4) inhibited the enzyme even when the molar concentration of the
compound was comparable to that of the urease.3 On the basis of
the crystal structure of H. pylori urease (entry 1E9Z in the Protein
Data Bank), the binding mode of phenylphosphorodiamidate in the
enzyme active site was modeled (Fig. 2). In the binding model, amino
hydrogen of His221 and amino formed hydrogen bond with the oxy-
gen atoms of phenylphosphorodiamidate (length of the hydrogen
bond: His221N–Hꢀ ꢀ ꢀOphenylphosphorodiamidate = 2.167 Å; angle of the
hydrogen bond: His221N–Hꢀ ꢀ ꢀOphenylphosphorodiamidate = 171.6°). The
benzene ring of phenylphosphorodiamidate may form hydrophobic
interactions with Ala168, Ala169, and Ala365.
Metronidazole
MET-OTs
AHAa
4a
4b
4c
4d
4e
4f
4g
4h
a
Used as a positive control.
These compounds were found as inactive.
b
The binding model of compound 4g and H. pylori urease was
depicted in Figure 3 and the enzyme surface model was showed in
Figure 4. All the amino acid residues which had interactions with
H. pylori urease were showed. In the binding model, the nitro
oxygen of compound 4g formed hydrogen bond with the amino
hydrogen of His221 similar to phenylphosphorodiamidate as shown
potent in vitro inhibitory activities, which were comparable to ace-
tohydroxamic acid (AHA), a reversible urease inhibitor (Table 2).3
In general, compounds 4a–h with two imidazole groups exhibited
better inhibitory activities than compounds 3a–f. The comparison
of the inhibitory activities of the synthetic nitroimidazoles and
metronidazole indicated that the introduction of the substituted
salicylic acids onto the 2-hydroxyethyl was responsible for the
activities of the compounds.
In nitroimidazoles 3d–f, compound 3e exhibited better inhibi-
tory activity than 3d and the activity of compound 3f was the high-
est. At the same time, in nitroimidazoles 4b, 4c, and 4g, the order of
inhibitory activities showed the potency of 4g > 4b > 4c. It was
possibly due to the difference between the electro affinities of
chlorine, bromine, and iodine. This result indicated that the inhib-
itory activities increased with the increase of electron withdrawing
ability of R3. In addition, compound 4f (R1 = CH3) and compound 4a
(R2 = CH3) showed weaker inhibitory activity than compound 4e
(R3 = CH3), which indicated that the position of the substitute
group influenced the inhibitory activity. Compounds with halogen
atoms in the benzene ring showed better inhibitory activity than
those with methyl substitutes.
in Figure
2
(length of the hydrogen bond: His221N–
Hꢀ ꢀ ꢀO4g = 1.752 Å; angle of the hydrogen bond: His221N–
Hꢀ ꢀ ꢀO4g = 160.5°). Moreover, hydrophobic interactions probably
existed between compound 4g and Ala168, Ala169, and Ala365.
The novel interaction, in comparison to phenylphosphorodiami-
date—urease complex, occurred in the model, the hydrogen bond
formed by the oxygen atom and the NH of the inhibitor amide group
with Met366 (length of the hydrogen bond: Met366N–
Hꢀ ꢀ ꢀO4g = 1.975 Å; angle of the hydrogen bond: Met366N–H
Generally, nitroimidazoles 4b and 4g based on metronidazole
and salicylic acids exhibited good inhibitory activities against H.
pylori urease (Table 2), which attracted our interest in further
structure modification of them as lead compounds for new urease
inhibitors or anti-H. pylori agents.
2.3. Molecular docking study
Bacterial ureases, including H. pylori urease, are large hetero-
polymeric metalloproteins with nickel (II) ions present in their ac-
tive sites.15,16 A significant amino acid sequence similarity has
been observed between all ureases of a bacterial origin.17 A super-
Figure 1. The active site of urease showed by AUTODOCK 4.0.
Table 2
Inhibition of Helicobacter pylori urease by compounds 4b and 4g
NH2
O
Compound
IC50 (lM)
4b
4g
26
12
17
3
1
2
P
NH2
Acetohydroxamic acid
O
IC50 = 50% inhibitory concentration represents the mean S.D. from dose–response
curves of at least three experiments.
Scheme 4. Chemical structure of phenylphosphorodiamidate.

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W.-J. Mao et al. / Bioorg. Med. Chem. 17 (2009) 7531–7536
Figure 4. The enzyme surface model of structure of compound 4g—H. pylori urease
complex.
their inhibitory activities against H. pylori urease. Compound 4g,
2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl 5-chloro-2-(2-(5-ni-
tro-1H-imidazol-1-yl) ethoxy) benzoate, showed the most potent
inhibitory activity with IC50 of 12 lM against the urease. The intro-
duction of hydrophobic and electron-withdrawing halogeno
groups was conducive to the inhibitory activity. Docking simula-
tion was performed to position compound 4g into the H. pylori ure-
ase active site to determine the probable binding conformation and
the result indicated that compound 4g is a potent inhibitor of H.
pylori urease.
Figure 2. Computed structure of phenylphosphorodiamidate—H. pylori urease
complex (colored by atom: carbons—gray; nitrogens—blue; oxygens—red; chlo-
rine—green; phosphorus—purple). Hydrogen bonds are indicated as green dotted
lines.
4. Experiment part
4.1. Materials
All the NMR spectra were recorded on a Bruker DPX 300 model
spectrometer in DMSO-d6. Chemical shifts (d) for 1H NMR spectra
were reported in parts per million to residual solvent protons.
Melting points were measured on a Boetius micro melting point
apparatus. The ESI-MS spectra were recorded on a Mariner System
5304 Mass spectrometer. All chemicals and reagents used in cur-
rent study were of analytical grade. All the substituted salicylic
acids were purchased from Aladdin Company, Shanghai China,
and metronidazole was purchased from Changzhou Dongsheng
Company, Changzhou China. Protease inhibitors (Complete mini
EDTA-free) were purchased from Roche Diagnostics GmbH
(Mannheim, Germany) and brucella broth was from Becton–Dick-
inson (Cockeysville, MD). Horse serum was from Hyclone (Utah,
America).
4.2. Bacteria
H. pylori (ATCC 43504; American Type Culture Collection,
Manassas, VA) was grown in brucella broth supplemented with
10% heat-inactivated horse serum for 24 h at 37 °C under microaer-
obic conditions (5% O2, 10% CO2, and 85% N2), as previously
described.22
Figure 3. Compound 4g (colored by atom: carbons—gray; nitrogens—blue; oxy-
gens—red; chlorine—green) is bound into H. pylori urease (entry 1E9Z in the Protein
Data Bank). The dotted lines show the hydrogen bond.
4.3. Preparation of H. pylori urease
ꢀ ꢀ ꢀO4g = 140.8°). The result of molecular docking study could explain
the inhibitory activities of compound 4g against H. pylori urease.
For urease inhibition assays, 50 mL broth cultures
(2.0 ꢁ 108 CFU/mL) were centrifuged (5000g, 4 °C) to collect the
bacteria, and after washing twice with phosphate-buffered saline
(pH 7.4), the H. pylori precipitation was stored at ꢂ80 °C. H. pylori
was returned to room temperature, and after addition of 3 mL of
distilled water and protease inhibitors, sonication was performed
for 60 s. Following centrifugation (15,000g, 4 °C), the supernatant
3. Conclusions
Two series of new nitroimidazoles reacting by metronidazole
and salicylic acid derivatives were synthesized and assayed for

W.-J. Mao et al. / Bioorg. Med. Chem. 17 (2009) 7531–7536
7535
was desalted through SephadexG-25 column (PD-10 columns,
Amersham–Pharmacia Biotech, Uppsala, Sweden). The resultant
crude urease solution was added to an equal volume of glycerol
and stored at 4 °C until use in the experiment.
added. The reaction mixture was stirred at 70–80 °C for 14–16 h.
After the reaction mixture was vacuum distillated, the target prod-
uct was separated by column chromatography. When MET-OTs
(4 mmol) and K2CO3 (5 mmol) were dissolved in DMF (20 mL),
and one kind of the substituted salicylic acid (2.2 mmol) in DMF
(10 mL) was added, then the reaction mixture was stirred at
110 °C for 22–24 h, compound 4a–h were obtained.
4.4. Measurement of urease activity
The assay mixture, containing 25
lL (4 U) of H. pylori urease and
25
l
L of the test compound, was preincubated for 3 h at room tem-
4.6.1. 4-Methyl-2-(2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethoxy)benzoic acid (3a)
perature in a 96-well assay plate. Urease activity was determined
by measuring ammonia production using the indophenol method
as described by Weatherburn.23
White solid, mp 143–144 °C. 1H NMR (300 MHz, DMSO-d6):
2.29 (s, 3H,); 2.46 (s, 3H); 4.65 (t, J = 4.77 Hz, 2H); 4.72 (t,
J = 4.77 Hz, 2H); 6.77–6.79 (m, 2H); 7.49 (d, J = 8.04 Hz, 1H); 8.00
(s, 1H); 10.25 (s, 1H). ESI-MS C14H15N3O5 [M+H]+ 306.1. Anal. Calcd
for C14H15N3O5: C, 55.08; H, 4.95; N 13.76. Found: C, 55.30; H, 4.94;
N, 13.83.
4.5. Protocol of docking study
Molecular docking of compound 4g into the three-dimensional
X-ray structure of H. pylori urease (PDB code: 1E9Z) was carried out
using the AUTODOCK software package (version 4.0) as implemented
through the graphical user interface AUTODOCKTOOLS (ADT 1.4.6).24
The graphical user interface AUTODOCKTOOLS was employed to set-
up the enzymes: all hydrogens were added, Gasteiger charges were
calculated and nonpolar hydrogens were merged to carbon atoms.
The Ni initial parameters are set as r = 1.170 Å, q = +2.0, and van der
Waals well depth of 0.100 kcal/mol.25 For macromolecules, gener-
ated pdbqt files were saved.
4.6.2. 4-Amino-2-(2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethoxy)benzoic acid (3b)
Brown solid, mp 188–189 °C. 1H NMR (300 MHz, DMSO-d6):
2.49 (s, 3H); 4.58 (t, J = 4.95 Hz, 2H); 4.70 (t, J = 4.95 Hz, 2H);
5.98 (s, 1H); 6.10 (s, J = 8.76 Hz, 1H); 6.20 (s, 2H); 728 (d,
J = 8.76 Hz, 1H); 7.98 (s, 1H); 10.51 (s, 1H). ESI-MS C13H14N4O5
[M+H]+ 307.2. Anal. Calcd for C13H14N4O5: C, 50.98; H, 4.61; N,
18.29. Found: C, 50.84; H, 4.69; N, 18.40.
The 3D structures of ligand molecules were built, optimized
(PM3) level, and saved in Mol2 format with the aid of the molecu-
lar modeling program SPARTAN (Wavefunction Inc.). These partial
charges of Mol2 files were further modified by using the ADT pack-
age (version 1.4.6) so that the charges of the nonpolar hydrogens
atoms assigned to the atom to which the hydrogen is attached.
The resulting files were saved as pdbqt files.
AUTODOCK 4.0 was employed for all docking calculations. The
AUTODOCKTOOLS program was used to generate the docking input files.
In all docking a grid box size of 60 ꢁ 60 ꢁ 60 points in x, y, and z
directions was built, the maps were centered on N1 atom of the
4.6.3. 2-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-3,5-
dinitrobenzoic acid (3c)
Yellow solid, mp 155–156 °C. 1H NMR (300 MHz, DMSO-d6):
2.50 (s, 3H); 4.51 (t, J = 4.80 Hz, 2H); 4.61 (t, J = 4.80 Hz, 2H);
8.02 (s, 1H); 8.30 (d, J = 3.45 Hz, 1H); 8.48 (d, J = 3.45 Hz, 1H).
ESI-MS C13H11N5O9 [M+H]+ 382.1. Anal. Calcd for C13H11N5O9: C,
40.95; H, 2.91; N, 18.37. Found: C. 40.98; H, 2.96; N, 18.30.
4.6.4. 3, 5-Diiodo-2-(2-(2-methyl-5-nitro-1H-imidazol-1-yl
ethoxy) benzoic acid (3d)
Kcx 219 in the catalytic site of the protein. A grid spacing of
Brown solid, mp 175–176 °C. 1H NMR (300 MHz, DMSO-d6):
2.55 (s, 3H); 4.18 (t, J = 4.89 Hz, 2H); 4.76 (t, J = 4.89 Hz, 2H);
7.80 (s, 1H); 8.05 (s, 1H); 8.11 (s, 1H) ESI-MS C13H11I2N3O5
[M+H]+ 544.1. Anal. Calcd for C13H11I2N3O5: C, 28.75; H, 2.04; I,
46.74; N 7.74. Found: C, 28.73; H, 2.05; I, 46.79; N, 7.72.
0
0.375 ÅA (approximately one forth of the length of carbon–carbon
covalent bond) and a distances-dependent function of the dielec-
tric constant were used for the calculation of the energetic map.
Ten runs were generated by using Lamarckian genetic algorithm
searches. Default settings were used with an initial population of
50 randomly placed individuals, a maximum number of 2.5 ꢁ 106
energy evaluations, and a maximum number of 2.7 ꢁ 104 genera-
4.6.5. 3,5-Dibromo-2-(2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethoxy) benzoic acid (3e)
tions. A mutation rate of 0.02 and a crossover rate of 0.8 were cho-
White solid, mp 167–169 °C. 1H NMR (300 MHz, DMSO-d6):
2.53 (s, 3H); 4.19 (t, J = 4.86 Hz, 2H); 4.76 (t, J = 4.86 Hz, 2H);
7.64 (s, 1H); 8.01 (s, 1H); 8.07 (s, 1H). ESI-MS C13H11Br2N3O5
[M+H]+ 449.9. Anal. Calcd for C13H11Br2N3O5: C, 34.77; H, 2.47;
Br, 35.59; N, 9.36. Found: C, 34.60; H, 2.47; Br, 35.71; N, 9.38.
0
sen. Results differing by less than 0.5 ÅA in positional root–mean–
square deviation (RMSD) were clustered together and the results
of the most favorable free energy of binding were selected as the
resultant complex structures.
4.6. Compounds
4.6.6. 3,5-Dichloro-2-(2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethoxy)benzoic acid (3f)
General procedure of preparation of compounds 2 (MET-OTs)
and nitroimidazoles 3a–f and 4a–h: metronidazole (3.14 g,
20 mmol) and Et3N (3.0 mL, 22 mmol) were dissolved in CH2Cl2
White solid, mp 153–155 °C. 1H NMR (300 MHz, DMSO-d6):
2.51 (s, 3H); 4.16 (t, J = 4.89 Hz, 2H); 4.73 (t, J = 4.89 Hz, 2H);
7.56 (s, 1H); 7.94 (s, 1H); 8.01 (s, 1H). ESI-MS C13H11Cl2N3O5
[M+H]+ 360.1. Anal. Calcd for C13H11Cl2N3O5: C, 43.35; H, 3.08;
Cl, 19.69; N, 11.67. Found: C, 43.41; H, 3.10; Cl, 19.61; N, 11.64.
(20 mL),
and
4-methyl-benzenesufonyl
chloride
(3.83 g,
20.1 mmol) in CH2Cl2 (10 mL) was added. The reaction mixture
was stirred at 0 °C for 5 h followed by the addition of 30 mL ice
water, and then the layer was separated and the aqueous layer
was extracted with ethyl acetate (2 ꢁ 30 mL). The organic layer
was combined and washed with saturated NaHCO3, and dried with
anhydrous Na2SO4 for 0.5 h. Removal of the solvent gave a slight-
yellow crystal of compound 2 (MET-OTs). The compounds 3a–f
were prepared by following known procedure, MET-OTs (2 mmol)
and K2CO3 (5 mmol) were dissolved in DMF (20 mL), and one kind
of the substituted salicylic acid (2.2 mmol) in DMF (10 mL) was
4.6.7. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl4-methyl-2-
(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzoate (4a)
White solid, mp 159–160 °C. 1H NMR (300 MHz, DMSO-d6):
2.28 (s, 3H); 2.38 (s, 3H); 2.47 (s, 3H); 4.36 (t, J = 4.71 Hz, 2H);
4.50 (t, J = 4.77 Hz, 2H); 4.65 (m, 4H); 6.82 (d, J = 7.86 Hz, 1H);
6.98 (s, 1H); 7.39 (d, J = 7.86 Hz, 1H); 8.01 (s, 1H); 8.03 (s, 1H).
ESI-MS C20H22N6O7 [M+H]+ 459.1. Anal. Calcd for C20H22N6O7: C,
52.40; H, 4.84; N, 18.33. Found: C, 52.42; H, 4.87; N, 18.30.

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W.-J. Mao et al. / Bioorg. Med. Chem. 17 (2009) 7531–7536
4.6.8. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl5-bromo-2-
(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzoate (4b)
Brown solid, mp 160–161 °C. 1H NMR (300 MHz, DMSO-d6):
2.26 (s, 3H); 2.39 (s, 3H); 4.39 (t, J = 4.74 Hz, 2H); 4.52
(t, J = 4.77 Hz, 2H); 4.65 (m, 4H); 7.13 (d, J = 8.97 Hz, 1H); 7.59
(s, 1H); 7.66 (d, J = 8.97 Hz, 1H); 8.00 (s, 1H); 8.03 (s, 1H). ESI-
MS C19H19BrN6O7 [M+H]+ 553.1. Anal. Calcd for C19H19BrN6O7: C,
43.61; H, 3.66; Br, 15.27; N, 16.06. Found: C, 43.55; H, 3.62; Br,
15.32; N, 16.12.
(dd, J1 = 9.00 Hz, J2 = 2.55 Hz,1H); 8.00 (s, 1H); 8.03 (s, 1H). ESI-MS
C19H19ClN6O7 [M+H]+ 479.3. Anal. Calcd for C19H19ClN6O7: C,
47.66; H, 4.00; Cl, 7.40; N, 17.55. Found: C, 47.59; H. 4.02; Cl, 7.43;
N, 17.51.
4.6.14. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl4-chloro-2-
(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzoate (4h)
White solid, mp 101–102 °C. 1H NMR (300 MHz, DMSO-d6):
2.36 (s, 3H); 2.45 (s, 3H); 4.44 (t, J = 2.94 Hz, 2H); 4.52 (t,
J = 2.942 Hz, 2H); 4.66 (m, 4H); 7.10 (d, J = 5.10 Hz, 1H); 7.29 (s,
1H); 7.49 (d, J = 5.10 Hz,1H); 8.00 (s, 1H); 8.03 (s, 1H). ESI-MS:
C19H19ClN6O7 [M+H]+ 479.1. Anal. Calcd for C19H19ClN6O7: C,
47.66; H, 4.00; Cl, 7.40; N, 17.55. Found: C, 47.70; H, 3.98; Cl,
7.38; N, 17.56.
4.6.9. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl5-iodo-2-(2-
(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzoate (4c)
Brown solid, mp 166–168 °C. 1H NMR (300 MHz, DMSO-d6):
2.42 (s, 3H); 4.37 (t, J = 4.74 Hz, 2H); 4.49 (t, J = 4.92 Hz, 2H);
4.65 (m, 4H); 6.98 (d, J = 8.76 Hz, 1H); 7.71 (d, J = 2.01 Hz, 1H);
7.78 (dd, J1 = 8.76 Hz, J2 = 2.01 Hz,1H); 7.98 (s, 1H); 8.01 (s, 1H).
ESI-MS C19H19IN6O7 [M+H]+ 571.1. Anal. Calcd for C19H19IN6O7: C,
40.01; H, 3.36; I, 22.25; N, 14.74. Found: C, 40.05; H, 3.38; I,
22.15; N, 14.76.
Acknowledgment
The work was financed by from National Natural Science Foun-
dation of China (Project 30772627).
4.6.10. 4-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-3-((2-
(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)carbonyl)
benzenesulfonic acid (4d)
References and notes
1. Vassiliou, S.; Grabowiecka, A.; Kosikowska, P.; Yiotakis, A.; Kafarski, P.; Berlicki,
L. J. Med. Chem. 2008, 51, 5736.
2. Covacci, A.; Telford, L. J.; Giudice, G. D.; Parsonnet, J.; Rappuoli, R. Science 1999,
284, 1328.
3. Amtul, Z.; Atta-ur-Rahman; Siddiqui, A. R.; Choudhary, I. M. Curr. Med. Chem.
2002, 9, 1323.
4. Beena; Kumar, N.; Rohilla, R. K.; Roy, N.; Rawat, D. S. Bioorg. Med. Chem. Lett.
2009, 19, 1396.
5. Kim, P.; Zhang, L.; Ujjini, H.; Manjunatha; Singh, P.; Patel, S.; Jiricek, J.; Keller, H.
T.; Boshoff, I. H.; Barry, E. C.; Dowd, S. C. J. Med. Chem. 2009, 52, 1317.
6. Silva, A.; Munhoz, A.; Santos, J.; Camargo, R.; Castro, L.; Menegon, R.; Vilegas,
W.; Ferreira, A.; Varanda, E.; Chung, C. M. Patent, 142, 463721, 405359, 2005.
7. Mallia, B. M.; Mathur, A.; Subramanian, S.; Banerjee, S.; Sarma, H. D.;
Venkatesh, M. Bioorg. Med. Chem. Lett. 2005, 15, 3398.
8. Xiao, Z. P.; Shi, D. H.; Li, H. Q.; Zhang, L. N.; Xu, C.; Zhu, H. L. Bioorg. Med. Chem.
2007, 15, 3703.
9. Li, H. Q.; Xiao, Z. P.; Luo, Y.; Yan, T.; Lv, P. C.; Zhu, H. L. Eur. J. Med. Chem. 2009,
44, 2246.
10. Falagas, M. E.; Walker, A. M.; Jick, H.; Ruthazer, R.; Griffith, J.; Snydman, D. R.
Clin. Infect. Dis. 1998, 26, 384.
Yellow solid, mp 140–141 °C. 1H NMR (300 MHz, DMSO-d6):
2.73 (s, 3H); 2.89 (s, 3H); 4.29 (t, J = 4.774 Hz, 2H); 4.52 (t,
J = 4.89 Hz, 2H); 4.70 (m, 4H); 7.39 (d, J = 8.82 Hz, 1H); 7.57 (s,
1H); 7.68 (d, J = 8.82 Hz, 1H); 7.99 (s, 1H); 8.03 (s, 1H). ESI-MS
C19H20N6O10S [M+H]+ 525.2. Anal. Calcd for C19H20N6O10S: C,
43.51; H, 3.84; N, 16.02; S, 6.11. Found: C, 43.57; H, 3.85; N,
16.01; S, 6.08.
4.6.11. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl5-methyl-2-
(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzoate (4e)
Yellow solid, mp 155–156 °C. 1H NMR (300 MHz, DMSO-d6):
2.73 (s, 3H); 2.89 (s, 3H); 4.29 (t, J = 4.774 Hz, 2H); 4.52 (t,
J = 4.89 Hz, 2H); 4.70 (m, 4H); 7.39 (d, J = 8.82 Hz, 1H); 7.57 (s,
1H); 7.68 (d, J = 8.82 Hz, 1H); 7.99 (s, 1H); 8.03 (s, 1H). ESI-MS
C20H22N6O7 [M+H]+ 459.2. Anal. Calcd for C20H22N6O7: C, 52.40;
H, 4.84; N, 18.33. Found: C, 52.41; H, 4.84; N, 18.35.
11. Kubo, J.; Lee, J. R.; Kubo, I. J. Agric. Food Chem. 1999, 47, 533.
12. Hassan, S. S. M.; El-Bahnasawy, R. M.; Rizk, N. M. Anal. Chim. Acta. 1997, 351, 91.
13. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
4.6.12. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl3-methyl-2-
(2-(2-methyl-5-nitro-1H-imidazol-1-yl ethoxy benzoate (4f)
White solid, mp 144–147 °C. 1H NMR (300 MHz, DMSO-d6):
1.99 (s, 3H); 2.40 (s, 3H); 2.50 (s, 3H); 4.08 (t, J = 4.95 Hz, 2H);
4.51 (t, J = 4.95 Hz, 2H); 4.68 (m, 4H); 7.10 (d, J = 7.68 Hz, 1H);
7.40 (m, 2H); 8.03 (s, 1H); 8.07 (s, 1H). ESI-MS C20H22N6O7
[M+H]+ 459.3. Anal. Calcd for C20H22N6O7: C, 52.40; H, 4.84; N,
18.33. Found: C, 52.46; H, 4.86; N 18.30.
14. Cao, P.; Fang, R. Q.; Li, H. Q.; Zhu, H. L. Chin. J. Struct. Chem. 2007, 26, 1320.
15. Dixon, N. E.; Gazzola, C.; Blakeley, R. L.; Zerner, B. J. Am. Chem. Soc. 1975, 97,
4131.
16. Ermler, U.; Grabarse, W.; Shima, S.; Goubeaud, M.; Thauer, R. K. Curr. Opin.
Struct. Biol. 1998, 8, 749.
17. Jabri, E.; Karplus, P. A. Biochemistry 1996, 35, 10616.
18. Ha, N.; Oh, S.; Sung, J. Y.; Cha, K. A.; Lee, M. H.; Oh, B. H. Nat. Struc. Biol. 2001, 8,
505.
19. Estiu, G.; Suárez, D.; Merz, K. M. J. R. J. Comput. Chem. 2006, 27, 1231.
20. Pearson, M. A.; Park, I.; Schaller, R. A.; Michel, L. O.; Karplus, P. A.; Hausinger, R.
P. Biochemistry 2000, 39, 8575.
21. Krajewska, B.; Zaborska, W. Bioorg. Chem. 2007, 35, 355.
22. Li, H. Q.; Xu, C.; Li, H. S.; Shi, L.; Xiao, Z. P.; Zhu, H. L. ChemMedChem 2007, 2,
1361.
23. Weatherburn, M. W. Anal. Chem. 1967, 39, 971.
24. Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. J. Comput. Chem. 2007, 28,
1145.
4.6.13. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl5-chloro-2-
(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzoate (4g)
White solid, mp 150–151 °C. 1H NMR (300 MHz, DMSO-d6): 2.39
(s, 3H); 2.44 (s, 3H); 4.39 (t, J = 4.74 Hz, 2H); 4.52 (t, J = 4.92 Hz, 2H);
4.66 (m, 4H); 7.20 (d, J = 9.00 Hz, 1H); 7.47 (d, J = 2.55 Hz, 1H); 7.58
25. Musiani, F.; Arnofi, E.; Casadio, R.; Ciurli, S. J. Biol. Inorg. Chem. 2001, 6, 300.