Metronidazole-deoxybenzoin derivatives as anti-Helicobacter pylori agents with potent inhibitory activity against HPE-induced interleukin-8

Article abstract of DOI:10.1002/cmdc.201000126

A series of new metronidazole-deoxybenzoin derivatives were synthesized and evaluated for their antimicrobial activity against Helicobacter pylori. Highly selective anti-H. pylori activity was also observed in synthesized compounds. Compound 34 exhibited

Full text of DOI:10.1002/cmdc.201000126

MED
DOI: 10.1002/cmdc.201000126
Metronidazole–Deoxybenzoin Derivatives as Anti-
Helicobacter pylori Agents with Potent Inhibitory Activity
against HPE-Induced Interleukin-8
Yin Luo, Huan-Qiu Li, Yang Zhou, Zi-Lin Li, Tao Yan, and Hai-Liang Zhu*[a]
A series of new metronidazole–deoxybenzoin derivatives were
synthesized and evaluated for their antimicrobial activity
against Helicobacter pylori. Highly selective anti-H. pylori activity
was also observed in synthesized compounds. Compound 34
exhibited the most potent activity, similar to the positive con-
trol amoxicillin. Furthermore, compounds 17 and 34 were able
to significantly decrease H. pylori water extract (HPE)-induced
production of interleukin-8 (IL-8) in gastric mucosal cells, which
did not show any effect on the cell viability.
Introduction
Helicobacter pylori is a gram-negative microaerophilic bacteri-
um that colonizes at the surface of the gastric epithelium be-
neath the mucus. It can cause many gastroduodenal diseases
such as gastritis, gastric and duodenal ulcers, and even gastric
cancer.[1] In the clinical setting, the increasingly extensive use
of antibiotics to treat H. pylori leads to many serious side ef-
fects. The most serious one among these being the drug resist-
ance of H. pylori strains.[2] Metronidazole used to be one of the
most effective agents against H. pylori, but now it is unlikely to
have great therapeutic potential for this bacterium in the clinic
because of the increasing resistance of most H. pylori strains.[3]
Hence, there are unmet medical needs for novel, efficacious,
and selective eradication therapies that minimize resistance
problems.
exhibit estrogenic and antimicrobial activities. In our recent re-
search about the functional natural products and structural
modifications, we synthesized some new flavonoid derivatives.
They exhibited potent inhibition of HPE-induced release of IL-
8.[3] As important derivatives of flavonoid, deoxybenzoins are
also considered to have good bioactivity in this process. Our
interest in this area is to join metronidazole with deoxyben-
zoins to develop a series of new deoxybenzoin derivatives, in-
tending to obtain compounds that minimize resistance prob-
lems and exhibit bioactivity through the inhibition of IL-8 pro-
duction.
Results and Discussion
Recently more attention has been paid to the pathological
reaction to the infection of H. pylori in host cells, which leads
to inflammation and then many gastroduodenal diseases.[4]
Some anti-H. pylori agents such as ecabet sodium and rebami-
pide have been investigated and subsequently proved to have
the ability to suppress inflammation relating to H. pylori infec-
tion and this would be a potential therapeutic target for the
treatment of H. pylori infection.[5]
Chemistry
A series of deoxybenzoins were synthesized using phenols and
derivatives from phenylacetic acids by the routes outlined in
Scheme 1. Equimolar polyhydric phenol and substituents of
phenylacetic acid were dissolved in boron trifluoride diethyl
etherate, which also played the role of catalyst. The solution
was boiled under reflux at 808C for 2 h and then precipitated
with a saturated solution of sodium acetate. The product was
filtered from the solution and recrystallized with methanol/
water. Deoxybenzoins 3–17 were obtained in high yields.
Compound 19 should be prepared before synthesis of the
target compounds 20–34. Metronidazole and 4-toluenesulfonyl
chloride were dissolved in CH2Cl2 in the presence of triethyl-
amine. The solution was agitated at room temperature for 12 h
and then neutralized with sodium hydrogen carbonate. Com-
pound 19 was obtained by means of extraction.
Investigations revealed that the attachment of H. pylori to
gastric epithelial cells induces the production of interleukin
(IL)-8, which in turn causes the activation and recruitment of
neutrophils to the site of infection.[6] IL-8 is not only a very
potent endogenous monocyte/neutrophil chemoattractant but
also a potent secretagogue and activator of the phagocyte
NADPH-oxidase, inducing the release of a large number of pro-
teases and reactive oxygen metabolites, which finally leads to
inflammation.[7,8] IL-8 is an important approach to combat in-
flammation and infectious disease and would be a target to
treat H. pylori-induced disease.[9,10]
[a] Dr. Y. Luo,+ Dr. H.-Q. Li,+ Dr. Y. Zhou, Z.-L. Li, Dr. T. Yan, Prof. H.-L. Zhu
State Key Laboratory of Pharmaceutical Biotechnology
Nanjing University, Nanjing 210093 (PR China)
Fax: (+86)25-8359 2572
E-mail: zhuhl@nju.edu.cn
[+] These authors contributed equally to this work.
The structure modification at the pendant hydroxy group of
metronidazole has received much attention during recent
years and many metronidazole derivatives exhibit high anti-
H. pylori activity.[11] Flavonoids are important compounds which
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Metronidazole–Deoxybenzoin Derivatives
Biological activity
Anti-H. pylori activity
The metronidazole–deoxybenzoin derivatives 20–34 were eval-
uated for antimicrobial activity against H. pylori. Six H. pylori
strains, including two standards (ATCC 43504 and SS1) and
four clinical strains, were used, and the results are reported in
Table 2.
Table 2. Minimum inhibitory concentrations of compounds 20–34
against H. pylori strains.
Scheme 1. Synthesis of deoxybenzoins 3–26: a) BF3·OEt2, reflux, 808C,
NaOAc (satd), recrystallization, 80%.
Compd
MIC [mm]
ATCC
SS1
H. pylori
43504
Clin.1
125
Clin.2
Clin.3
Clin.4
Compound 19 was then dissolved in N,N-dimethylforma-
mide and reacted with potash at 808C for 15 min. Then deoxy-
benzoins were added into the reaction system, and target
compounds 20–32 were obtained after 6 h boiling under
reflux (Scheme 2 and Table 1). In most isoflavones and deoxy-
benzoins, the alkylation of the hydroxy groups was very selec-
tive; the hydroxy group at the R1 position was more active.[12]
Thus, for compounds 20, 23, 26, 29, and 32, only one of two
free phenolic hydroxy groups in the starting deoxybenzoin
were alkylated in good yields.
20
21
22
23
24
25
26
27
28
29
30
31
32
125
125
30
120
60
7.24
109
109
125
125
30
120
60
7.24
109
109
>250
125
60
120
60
7.24
109
109
125
125
30
120
120
7.24
109
109
125
125
30
120
120
14.47
109
109
6.55
>243
121
>234
117
62.5
30
120
60
7.24
109
109
6.55
13.11
6.55
6.55
6.55
>243
>243
117
>243
121
117
58
121
121
117
58
121
121
117
58
>243
>243
117
58
117
33
117
117
117
117
117
117
34
1.76
146
0.14
1.76
146
0.14
1.76
>585
1.07
0.88
>585
1.07
1.76
>585
1.07
1.76
>585
1.07
MTZ[a]
AMX[b]
[a] Metronidazole. [b] Amoxicillin.
Compounds 22, 24, 25, 28, 32 and 34 showed lower MIC
values against H. pylori strains than metronidazole. In particu-
lar, they exhibited good biological activity against the four an-
timicrobial strains which had resistance to metronidazole.
Compound 34 had the greatest activity among the metronida-
zole–deoxybenzoin derivatives and was similar to the positive
control amoxicillin.
Scheme 2. Synthesis of compounds 20–34: a) 4-toluenesulfonyl chloride,
Et3N, CH2Cl2, NaHCO3, room temperature; b) K2CO3, DMF, reflux, 808C.
The comparison of the inhibitory activities of compounds in
Table 2 indicate that metronidazole–deoxybenzoin derivatives
(22, 25, 28 and 34) with two hydroxy groups on the center
phenyl ring demonstrated better inhibitory activities than
those with one hydroxy group (the other compounds in
Table 2). The difference between anti-H. pylori activity among
compounds with the same substituent group R4 showed hy-
droxy groups present on the aromatic ring may be responsible
for inhibitory activity.
Table 1. Structures of compounds 20–34.[a]
Compd
R1
R2
R3
R4
Compd
R1
R2
R3
R4
20
21
22
23
24
25
26
27
28
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
H
OH
OH
H
H
F
F
F
Cl
Cl
Cl
Br
Br
Br
29
30
31
32
33
34
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
H
H
OCH3
OCH3
OCH3
NO2
NO2
NO2
OH
OH
H
OH
OH
H
OH
OH
H
OH
OH
Any compounds with a methoxy group (29–31) showed
weak activity. It is likely that the electrophilic activity of the
substituent group R4 also plays a role in the inhibitory activity
of the compounds. Additionally, the intermediate deoxyben-
zoins were also evaluated for anti-H. pylori activity. The deoxy-
benzoins generally exhibited weaker activity than homologous
metronidazole–deoxybenzoin derivatives, indicating that the
conjunction of metronidazole increased the inhibitory activity.
OH
OH
OH
OH
[a] See Scheme 2 for scaffold structures.
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H.-L. Zhu et al.
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Moreover, selectivity of compounds 20–34 for E. coli, P. fluo-
rescence, B. subtilis, and S. aureus was also evaluated. All the
compounds 20–34 shown low antibacterial activity and dis-
played potent selectivity against H. pylori (Table 3).
addition of metronidazole, compound 34, and compound 17.
Compound 34 significantly decreased IL-8 levels in a dose-de-
pendent manner at concentrations of 15, 30, and 60 mm (P<
0.001), and the lowest IL-8 production was observed with com-
pound 34 at 60 mm.
The reason to test metronidazole and compound 17 was
that compound 34 possesses the structure of the two chemi-
cals. Comparatively, compound 34 showed a more potent in-
hibitory activity against HPE-induced IL-8 production than
both metronidazole at all concentrations (P<0.001) and com-
pound 17 at concentrations of 30 mm (P<0.05) and 60 mm (P<
0.01).
Table 3. Antimicrobial activity of synthetic compounds 20–34.
Compd
MIC [mm]
Gram-negative
P. fluorescence
Gram-positive
B. subtilis
E. coli
S. aureus
20
21
22
23
24
125
125
120
>250
120
125
125
>250
120
>250
120
125
>250
>240
>250
>240
125
125
>250
>240
>250
>240
125
Cell viability evaluation
25
125
The results shown in Figure 2 demonstrate that metronidazole,
compound 17, and compound 34 did not affect cell viability at
the concentrations tested (15, 30, and 60 mm).
26
27
28
29
30
31
32
62.5
62.5
125
121.5
62.5
31.2
50
62.5
125
125
125
121.5
125
125
125
>250
>250
125
121.5
125
125
125
121.5
62.5
62.6
50
125
125
33
34
KAN[a]
125
120
3.13
125
120
3.13
>250
120
>250
>240
1.56
1.56
[a] Kanamycin.
IL-8 assessment
Compound 34 with the strongest anti-H. pylori activity was se-
lected to act against the IL-8 production induced by HPE in
gastric mucosal cells. The results are shown in Figure 1.
HPE alone stimulated gastric mucosal cells to produce IL-8
as much as 1010.5 pgmLÀ1. Various dose-dependent attenua-
tions of HPE-induced IL-8 production were observed with the
Figure 2. Effects of various agents on cell viability. Human gastric mucosal
cells were incubated with metronidazole, compound 17, or compound 34 at
serial concentrations of 15, 30, and 60 mm. The MTT assay was performed
48 h later. Results are the mean ÆSEM of 4–6 experiments. V: cell viability;
comparison of all the agents at 15, 30, and 60 mm versus control.
Acute oral toxicity test
The tested animals appeared normal immediately after admin-
istration and did not exhibit any indication of acute toxicity for
14 days afterward. The body weights in treated mice were sim-
ilar to that of the control mice dosed with an equal volume of
vehicle. The result indicated that compound 34 was nontoxic.
Figure 1. Inhibitory activities of compounds 17 and 34 on IL-8 production
induced by H. pylori water extract (HPE). Human gastric mucosal cancer cells
were pre-incubated with or without metronidazole, compound 17, or com-
pound 34 in serial concentrations of 15, 30, and 60 mm for 1 h and then
stimulated by 10% HPE (v/v) for 12 h. The levels of IL-8 in the culture super-
natant were determined by ELISA. Basal: cells without incubation of HPE;
control: cells incubated with HPE but without any agents. *P<0.05;
#P<0.01.
Discussion
In recent years, H. pylori strains susceptible to amoxicillin, clari-
thromycin, or metronidazole are generally easily eradicated,
whereas those resistant to clarithromycin or metronidazole are
much more difficult to clear. Consequently, these findings are
indeed major drivers for developing novel anti-H. pylori agents.
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Metronidazole–Deoxybenzoin Derivatives
In this work, compounds 22, 24, 25, 28, 32 and 34 showed
low MIC values against H. pylori strains, especially the strains
resistant to metronidazole.
34 followed by stimulation of HPE. At the same time, com-
pound 34 at the same concentration exhibited little effect on
gastric mucosal cell viability, as shown in Figure 2, which can
exclude the disturbance of its cytotoxic activity to gastric mu-
cosal cells. It implied that compound 34 has the ability to de-
crease the HPE-induced IL-8 production by a direct influence
on the gastric cells.
H. pylori infection leads to different clinical and pathological
outcomes in humans, including chronic gastritis, peptic ulcer
disease, and gastric neoplasia. Until now, complete eradiation
of H. pylori is the most effective therapy, which stimulates re-
search to find more new anti-H. pylori natural or synthetic
agents. However, recently more attention has been paid to the
pathological reaction to the infection of H. pylori in host cells,
which leads to inflammation and then many gastroduodenal
diseases. Some anti-H. pylori agents such as ecabet sodium[13]
and rebamipide[14] have been investigated and subsequently
proved to have the ability to suppress the inflammation relat-
ing to H. pylori infection and this would be a potential thera-
peutic target for the treatment of H. pylori infection.
Considering that compound 34 possesses the structure of
metronidazole and compound 17, the two chemicals were
tested in this model to assess their effects on the intracellular
cascade of the IL-8 production in gastric mucosal cells. The re-
sults demonstrated that the group with the addition of metro-
nidazole showed little inhibitory activity of HPE-induced IL-8
production compared with the same concentration of com-
pound 34, indicating that metronidazole has no effect on the
gastric mucosal cells infected with H. pylori, except its direct
antibacterial activity. On the other hand, compound 17 could
significantly decrease the IL-8 production in gastric mucosal
cells stimulated by HPE which suggests that deoxybenzoin can
directly influence the intracellular cascade of IL-8 production.
Notably, genistein can specifically inhibit tyrosine kinase ac-
tivity, which in turn enables it to be a useful tool to elucidate
the role of tyrosine phosphorylation in cells.[17] To the best our
knowledge, tyrosine phosphorylation plays a crucial role in the
process of activation of NF-kB, which is the key signal pathway
relating to the production of IL-8 in H. pylori-infected gastric
mucosal cells. The adherence and colonization of H. pylori in
the host cells activates NF-kB as a consequence of phosphory-
lation by tyrosine kinase, and induces nuclear translocation of
NF-kB, which is followed by increased IL-8 mRNA and protein
levels.[19,20] Deoxybenzoin, as the derivative of genistein, can in-
hibit IL-8 production in gastric mucosal cells stimulated by
HPE. It can be concluded that compound 34 inhibits the IL-8
level through inhibiting tyrosine kinase activity.
It is known that the key pathophysiological event in H. pylori
infection is initiation and continuance of an inflammatory re-
sponse. Following adherence to the host cell, H. pylori triggers
this inflammatory process, in which many cytokines, such as
TNF-a, IL-1, and IL-8, are implicated. In particular, IL-8 shows a
potent chemotactic activity for neutrophils and accordingly
plays an important role in this process. IL-8 induction in
H. pylori-infected gastric epithelial cells is universal and specific,
and may be an important factor which is responsible for the
neutrophil activation and infiltration, and consequently, for the
inflammation-associated mucosal injury and carcinogenesis.
In the current study, in addition to the common anti-
H. pylori screening of a series of synthesized compounds, we
also emphasized pharmacological evaluation of a novel com-
pound concerning its effect on the pathological reaction to
the infection of H. pylori in gastric mucosal cells, which showed
a promising and exciting result. Compound 34 with the stron-
gest anti-H. pylori activity was selected to investigate its effect
on IL-8 production induced by H. pylori in gastric mucosal cells.
Compound 17 as the precursor was also added to the test in
order to contrast with compound 34. As reported earlier, sever-
al methods can be used to induce the expression of IL-8 in
gastric epithelial cells, including inflammatory cytokines (for ex-
ample, TNF-a and IL-1), live H. pylori,[15] the extract or surface
proteins of this bacterium,[16] and other inflammatory pro-
teins.[17] In this experiment, to rule out the influence of com-
pound 34 on the live bacteria and investigate its effect on the
intracellular cascade of the IL-8 production, HPE rather than
the live bacteria was employed as the stimulating factor
throughout the experiment.
Therefore, the inhibition of tyrosine kinase can down-regu-
late IL-8 expression in transcriptional and translational levels in
the gastric cells stimulated by the bacterium and subsequently
attenuates the inflammatory response. In our experiment, de-
oxybenzoin significantly decreased HPE-induced IL-8 produc-
tion in gastric mucosal cells. It can be suggested that deoxy-
benzoin attenuate HPE-induced IL-8 expression probably
through inhibiting tyrosine phosphorylation and subsequently
down-regulating the nuclear translocation of NF-kB.
Compound 34, structurally characterized by metronidazole
and deoxybenzoin, has the biological activities of the two
chemicals, which was never observed in a simple mixture of
metronidazole and deoxybenzoin as shown in our experiment.
Its potent anti-H. pylori activity is much stronger than metroni-
dazole and may be due to the structural modification of met-
ronidazole.
As shown in Figure 1, the amount of IL-8 significantly in-
creased in the supernatant of gastric mucosal cells culture
medium after addition of HPE, an extract of H. pylori contain-
ing the main surface protein of this bacterium. This result was
consistent with some other reports,[18] suggesting the proteins
existing in the surface of H. pylori could activate the intracellu-
lar signal pathway and stimulate the inflammatory activity in
host cells.
On the other hand, the structure of deoxybenzoin in com-
pound 34 makes it possible to deduce that the significant de-
crease in IL-8 production might be attributed to the inhibition
of tyrosine kinase, similar to the mode of action of deoxyben-
zoin. Consequently, compound 34 is likely to be a potent ther-
apy for H. pylori-infection because of its two independent ac-
tions, the efficient anti-H. pylori activity and the potent inhibi-
A significant and dose-dependent decrease of HPE-induced
IL-8 production in gastric mucosal cell culture supernatant can
be observed when the cells are pre-incubated with compound
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H.-L. Zhu et al.
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J=7.3 Hz, 1H), 4.57 (s, 2H), 4.31 (s, 2H), 3.82 (s, 2H), 2.51 (s, 3H);
13C NMR (500 MHz, [D6]DMSO): d=13.7, 41.5, 43.6, 65.8, 102.5,
108.4, 112.7, 115.4, 131.9, 132.4, 133.8, 138.2, 153.4, 160.6, 162.5,
165.5, 198.2 ppm; MS (ESI) C20H18FN3O5 [M+H]+ 400.1; Anal. calcd
for C20H18FN3O5: C 60.15, H 4.54, F 4.76, N 10.52%, found: C 60.03,
H 4.38, F 5.64, N 10.58%.
tion of IL-8 production. The latter might be achieved via tyro-
sine kinase inhibition, which should be investigated for further
study.
Conclusions
2-(4-fluorophenyl)-1-(2-hydroxy-4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)ethanone (21): Yield: 73%; Rf =0.21
(MeOH/CH3Cl 1:4); mp: 148–1498C; H NMR (300 MHz, [D6]DMSO):
In conclusion, a series of metronidazole–deoxybenzoin deriva-
tives were synthesized for the first time and evaluated for anti-
microbial activity against H. pylori. Among these, compounds
22, 24, 25, 28, 32 and 34 exhibited potent in vitro activities,
especially against the four clinically resistant strains of H. pylori.
Compound 34 showed much stronger activity than metronida-
zole and was similar to the positive control amoxicillin. Further-
more, compound 34 significantly decreased HPE-induced IL-8
production in gastric mucosal cells. Based on this study, we
concluded that compound 34 would be a promising agent for
the treatment of H pylori-related diseases.
1
d=10.78 (s, 1H), 8.12 (s, 1H), 8.01 (d, J=6.3 Hz 2H), 7.85 (d, J=
6.2 Hz, 1H), 7.56 (d, J=7.1 Hz, 1H), 7.32 (d, J=6.1 Hz, 2H), 6.65 (d,
J=7.7 Hz, 1H), 4.58 (s, 2H), 4.32 (s, 2H), 3.78 (s, 2H), 2.53 (s, 3H);
13C NMR (500 MHz, [D6]DMSO): d=13.7, 41.5, 43.9, 65.4, 102.2,
107.3, 112.5, 115.4, 131.5, 132.1, 132.8, 138.2, 152.4, 165.2, 167.1,
167.7, 202.2 ppm; MS (ESI) C20H18FN3O5 [M+H]+ 400.1; Anal. calcd
for C20H18FN3O5: C 60.15, H 4.54, F 4.76, N 10.52%, found: C 60.07,
H 4.33, F 5.60, N 10.52%.
1-(2,3-dihydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-fluorophenyl)ethanone (22): Yield: 81%; Rf =0.35
1
(MeOH/CH3Cl 1:4); mp: 154–1558C; H NMR (300 MHz, [D6]DMSO):
Experimental Section
d=11.44 (s, 1H), 11.03 (s, 1H), 8.15 (s, 1H), 8.01 (d, J=6.4 Hz 2H),
7.79 (d, J=6.8 Hz, 1H), 7.40 (d, J=6.1 Hz, 2H), 6.65 (d, J=8.3 Hz,
1H), 4.44 (s, 2H), 4.30 (s, 2H), 3.81 (s, 2H), 2.55 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.6, 41.5, 43.8, 66.2, 101.6, 111.5, 115.4,
128.9, 131.9, 132.4, 138.2, 146.7, 153.1, 160.4, 161.8, 165.2,
202.5 ppm; MS (ESI) C20H18FN3O6 [M+H]+ 416.1; Anal. calcd for
C20H18FN3O6: C 57.83, H 4.37, F 4.57, N 10.12%, found: C 57.91, H
4.32, F 4.55, N 10.09%.
Chemistry
All NMR spectra were recorded on a Bruker DRX 500 or DPX 300
model spectrometer in [D6]DMSO. Chemical shifts (d) for 1H NMR
spectra are reported in ppm to residual solvent protons. Melting
points were measured on a Boetius micro melting point apparatus.
The ESIMS spectra were recorded on a Mariner System 5304 mass
spectrometer. All chemicals and reagents used were of analytical
grade. Phenols and para-substituted phenylacetic acids were pur-
chased from Nanjing Huakang Co., Nanjing (China), and metroni-
dazole was purchased from Changzhou Dongsheng Co., Changz-
hou (China). TLC was run on silica gel coated aluminum sheets
(silica gel 60 GF254, E. Merck, Germany) and visualized under UV
light (l 254 nm).
2-(4-chlorophenyl)-1-(3-hydroxy-4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)ethanone (23): Yield: 75%; Rf =0.28
1
(MeOH/CH3Cl 1:4); mp: 133–1348C; H NMR ([D6]DMSO): d=11.08
(s, 1H), 8.02 (s, 1H), 7.91 (d, J=5.7 Hz 2H), 7.81 (d, J=6.5 Hz, 1H),
7.66 (d, J=7.0 Hz, 1H), 7.35 (d, J=6.5 Hz, 2H), 6.85 (d, J=8.7 Hz,
1H), 4.48 (s, 2H), 4.31 (s, 2H), 3.82 (s, 2H), 2.43 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.7, 41.5, 43.6, 65.8, 102.5, 108.4, 112.7,
128.6, 128.8, 131.9, 132.4, 133.8, 134.5, 138.2, 153.4, 160.6, 162.5,
198.8 ppm; MS (ESI) C20H18ClN3O5 [M+H]+ 416.1; Anal. calcd for
C20H18ClN3O5: C 57.77, H 4.36, Cl 8.53, N 10.11%, found: C 57.68, H
4.33, Cl 8.50, N 10.01%.
General method for compound synthesis
General procedure for the preparation of deoxybenzoins 3–17:[21]
A
phenol (0.050 mol) and an arylacetic acid (0.05 mol) were dissolved
into freshly distilled BF3·OEt2 (10 mL) under Ar. The mixture was
stirred at 808C and then poured into an ice bath. The resulting
mixture was extracted with EtOAc(aq) (100 mL), and the organic
layer was washed with NaHCO3(aq), dried (Na2SO4), and evaporated.
The residue was purified by column chromatography on silica gel,
using a mixture of CH2Cl2 and CH3OH.
2-(4-chlorophenyl)-1-(2-hydroxy-4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)ethanone (24): Yield: 68%; Rf =0.19
(MeOH/CH3Cl 1:4); mp: 138–1398C; H NMR ([D6]DMSO): d=10.88
1
(s, 1H), 8.04 (s, 1H), 7.98 (d, J=6.4 Hz 2H), 7.71 (d, J=6.4 Hz, 1H),
7.63 (d, J=7.4 Hz, 1H), 7.25 (d, J=7.2 Hz, 2H), 6.79 (d, J=9.1 Hz,
1H), 4.58 (s, 2H), 4.27 (s, 2H), 4.02 (s, 2H), 2.54 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.7, 41.5, 43.9, 65.4, 102.2, 107.3, 112.5,
128.6, 128.8, 131.5, 132.1, 133.5, 134.5, 138.2, 152.4, 165.2, 167.1,
202.2 ppm; MS (ESI) C20H18ClN3O5 [M+H]+ 416.1; Anal. calcd for
C20H18ClN3O5: C 57.77, H 4.36, Cl 8.53, N 10.11%, found: C 57.85, H
4.32, Cl 8.48, N 10.06%.
General procedure for the preparation of compound 19: Metroni-
dazole (0.05 mol) and 4-toluenesulfonyl chloride (0.05 mol) were
dissolved in CH2Cl2 (10 mL) in the presence of Et3N. The solution
was agitated at room temperature for 12 h and then neutralized
with NaHCO3. Compound 19 was obtained by means of extraction.
2-(4-chlorophenyl)-1-(2,3-dihydroxy-4-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethoxy)phenyl)ethanone (25): Yield: 77%; Rf =0.37
(MeOH/CH3Cl 1:4); mp: 151–1528C; H NMR ([D6]DMSO): d=11.37
General procedure for the preparation of compounds 20–34: Com-
pound 19 (0.05 mol) was dissolved in DMF (10 mL) and reacted
with potash at 808C for 15 min. Then the deoxybenzoin (0.05 mol)
was added to the reaction system, and the target compounds
were formed after 6 h boiling at reflux as shown in Scheme 2.
1
(s, 1H), 11.13 (s, 1H), 8.06 (s, 1H), 8.01 (d, J=6.6 Hz 2H), 7.69 (d,
J=6.9 Hz, 1H), 7.36 (d, J=6.1 Hz, 2H), 6.77 (d, J=8.8 Hz, 1H), 4.53
(s, 2H), 4.30 (s, 2H), 4.01 (s, 2H), 2.57 (s, 3H); 13C NMR (500 MHz,
[D6]DMSO): d=13.6, 41.5, 43.8, 66.2, 101.6, 111.5, 128.6, 128.9,
131.1, 131.9, 132.4, 134.5, 138.2, 146.7, 153.1, 160.4, 161.8,
202.3 ppm; MS (ESI) C20H18ClN3O6 [M+H]+ 431.1; Anal. calcd for
C20H18ClN3O6: C 55.63, H 4.20, Cl 8.21, N 9.73%, found: C 55.53, H
4.15, Cl 8.15, N 9.66%.
2-(4-fluorophenyl)-1-(3-hydroxy-4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)ethanone (20): Yield: 76%; Rf =0.26
1
(MeOH/CH3Cl 1:4); mp: 144–1458C; H NMR (300 MHz, [D6]DMSO):
d=11.41 (s, 1H), 8.05 (s, 1H), 7.99 (d, J=6.1 Hz 2H), 7.81 (d, J=
5.8 Hz, 1H), 7.64 (d, J=7.3 Hz, 1H), 7.37 (d, J=6.6 Hz, 2H), 6.71 (d,
1114
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ChemMedChem 2010, 5, 1110 – 1116

Metronidazole–Deoxybenzoin Derivatives
2-(4-bromophenyl)-1-(3-hydroxy-4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)ethanone (26): Yield: 65%; Rf =0.25
(MeOH/CH3Cl 1:4); mp: 174–1758C; H NMR ([D6]DMSO): d=10.96
(500 MHz, [D6]DMSO): d=13.6, 41.5, 43.8, 55.5, 66.2, 102.8, 113.1,
117.6, 130.1, 131.3, 132.6, 133.7, 138.2, 146.7, 153.1, 159.5, 160.4,
161.6, 201.9 ppm; MS (ESI) C21H21N3O7 [M+H]+ 428.1; Anal. calcd
for C21H21N3O7: C 59.01, H 4.95, N 9.83%, found: C 58.92, H 4.91, N
9.85%.
1
(s, 1H), 8.03 (s, 1H), 7.86 (d, J=6.2 Hz 2H), 7.78 (d, J=6.1 Hz, 1H),
7.66 (d, J=6.8 Hz, 1H), 7.32 (d, J=6.2 Hz, 2H), 6.83 (d, J=9.2 Hz,
1H), 4.51 (s, 2H), 4.30 (s, 2H), 3.92 (s, 2H), 2.53 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.7, 41.5, 43.6, 65.8, 103.1, 108.8, 113.4,
120.2, 130.3, 131.5, 132.1, 133.8, 134.8, 138.6, 153.4, 160.6, 162.5,
199.3 ppm; MS (ESI) C20H18BrN3O5 [M+H]+ 460.0; Anal. calcd for
C20H18BrN3O5: C 52.19, H 3.94, Br 17.36, N 9.13%, found: C 52.12, H
3.91, Br 17.32, N 9.14%.
1-(3-hydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-nitrophenyl)ethanone (32): Yield: 67%; Rf =0.20
1
(MeOH/CH3Cl 1:4); mp: 164–1658C; H NMR ([D6]DMSO): d=10.83
(s, 1H), 8.07 (s, 1H), 7.93 (d, J=5.7 Hz, 2H), 7.78 (d, J=6.8 Hz, 1H),
7.61 (d, J=6.8 Hz, 1H), 7.42 (d, J=6.0 Hz, 2H), 6.85 (d, J=8.7 Hz,
1H), 4.56 (s, 2H), 4.32 (s, 2H), 4.08 (s, 2H), 2.47 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.6, 41.5, 43.6, 65.8, 103.1, 108.6, 113.3,
125.2, 130.3, 132.1, 133.8, 138.6, 139.5, 146.8, 153.4, 160.4, 162.4,
199.6 ppm; MS (ESI) C20H18N4O7 [M+H]+ 427.1; Anal. calcd for
C20H18N4O7: C 56.34, H 4.26, N 13.14%, found: C 56.26, H 4.22, N
13.11%.
2-(4-bromophenyl)-1-(2-hydroxy-4-(2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethoxy)phenyl)ethanone (27): Yield: 75%; Rf =0.22
1
(MeOH/CH3Cl 1:4); mp: 167–1688C; H NMR ([D6]DMSO): d=11.26
(s, 1H), 8.01 (s, 1H), 7.96 (d, J=5.7 Hz 2H), 7.75 (d, J=7.1 Hz, 1H),
7.53 (d, J=6.8 Hz, 1H), 7.30 (d, J=6.6 Hz, 2H), 6.79 (d, J=8.2 Hz,
1H), 4.61 (s, 2H), 4.29 (s, 2H), 3.94 (s, 2H), 2.51 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.6, 41.5, 43.9, 65.4, 103.1, 108.6, 112.8,
120.3, 130.1, 131.2, 132.3, 133.5, 134.8, 138.2, 152.4, 165.2, 167.1,
202.2 ppm; MS (ESI) C20H18BrN3O5 [M+H]+ 460.0; Anal. calcd for
C20H18BrN3O5: C 52.19, H 3.94, Br 17.36, N 9.13%, found: C 52.15, H
3.90, Br 17.31, N 9.08%.
1-(2-hydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-nitrophenyl)ethanone (33): Yield: 72%; Rf =0.16
1
(MeOH/CH3Cl 1:4); mp: 171–1728C; H NMR ([D6]DMSO): d=10.96
(s, 1H), 8.11 (s, 1H), 8.02 (d, J=6.7 Hz, 2H), 7.73 (d, J=6.2 Hz, 1H),
7.64 (d, J=6.8 Hz, 1H), 7.39 (d, J=6.3 Hz, 2H), 6.77 (d, J=8.3 Hz,
1H), 4.48 (s, 2H), 4.30 (s, 2H), 3.88 (s, 2H), 2.53 (s, 3H); 13C NMR
(500 MHz, [D6]DMSO): d=13.7, 41.5, 43.9, 65.4, 103.1, 108.4, 112.5,
125.3, 130.1, 132.3, 133.5, 138.2, 140.4, 146.8, 152.4, 165.2, 167.1,
202.1 ppm; MS (ESI) C20H18N4O7 [M+H]+ 427.1; Anal. calcd for
C20H18N4O7: C 56.34, H 4.26, N 13.14%, found: C 56.31, H 4.23, N
13.09%.
2-(4-bromophenyl)-1-(2,3-dihydroxy-4-(2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethoxy)phenyl)ethanone (28): Yield: 79%; Rf =0.41
1
(MeOH/CH3Cl 1:4); mp: 171–1728C; H NMR ([D6]DMSO): d=11.18
(s, 1H), 11.02 (s, 1H), 8.11 (s, 1H), 8.03 (d, J=7.1 Hz, 2H), 7.76 (d,
J=7.1 Hz, 1H), 7.33 (d, J=6.4 Hz, 2H), 6.78 (d, J=8.1 Hz, 1H), 4.48
(s, 2H), 4.29 (s, 2H), 3.92 (s, 2H), 2.46 (s, 3H); 13C NMR (500 MHz,
[D6]DMSO): d=13.6, 41.5, 43.8, 66.2, 102.8, 113.1, 120.2, 130.3,
131.1, 132.2, 132.6, 134.8, 138.2, 146.7, 153.1, 160.4, 161.7,
202.1 ppm; MS (ESI) C20H18BrN3O6 [M+H]+ 476.0; Anal. calcd for
C20H18BrN3O6: C 50.44, H 3.81, Br 16.78, N 8.82%, found: C 50.38, H
3.79, Br 16.77, N 8.79%.
1-(2,3-dihydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-nitrophenyl)ethanone (34): Yield: 68%; Rf =0.13
1
(MeOH/CH3Cl 1:4); mp: 156–1578C; H NMR ([D6]DMSO): d=11.06
(s, 1H), 10.95 (s, 1H), 8.08 (s, 1H), 7.79 (d, J=5.9 Hz, 2H), 7.65 (d,
J=6.8 Hz, 1H), 7.42 (d, J=6.2 Hz, 2H), 6.81 (d, J=8.9 Hz, 1H), 4.50
(s, 2H), 4.31 (s, 2H), 3.93 (s, 2H), 2.48 (s, 3H); 13C NMR (500 MHz,
[D6]DMSO): d=13.6, 41.5, 43.8, 66.2, 102.8, 113.1, 125.2, 130.5,
132.2, 132.6, 134.1, 138.2, 145.5, 146.7, 153.1, 160.6, 161.3,
202.1 ppm; MS (ESI) C20H18N4O8 [M+H]+ 443.1; Anal. calcd for
C20H18N4O8: C 54.30, H 4.10, N 12.66%, found: C 54.33, H 4.15, N
12.68%.
1-(3-hydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-methoxyphenyl)ethanone (29): Yield: 81%; Rf =0.16
1
(MeOH/CH3Cl 1:4); mp: 132–1338C; H NMR ([D6]DMSO): d=10.26
(s, 1H), 8.07 (s, 1H), 7.96 (d, J=6.3 Hz, 2H), 7.82 (d, J=7.1 Hz, 1H),
7.63 (d, J=6.9 Hz, 1H), 6.91 (d, J=6.6 Hz, 2H), 6.78 (d, J=8.2 Hz,
1H), 4.53 (s, 2H), 4.31 (s, 2H), 3.87 (s, 2H), 3.31 (s,3H), 2.53 (s, 3H);
13C NMR (500 MHz, [D6]DMSO): d=13.7, 41.5, 43.6, 55.6, 65.8, 103.1,
108.8, 113.4, 117.4, 129.5, 131.7, 132.3, 133.6, 138.6, 153.4, 159.5,
160.8, 162.7, 199.8 ppm; MS (ESI) C21H21N3O6 [M+H]+ 412.1; Anal.
calcd for C21H21N3O6: C 61.31, H 5.14, N 10.21%, found: C 61.23, H
5.08, N 10.17%.
Cell culture
Human gastric mucosal cancer cells were grown in Ham’s F12
medium containing 10% fetal bovine serum, l-glutamine (2 mm),
penicillin G (100 UmLÀ1), and streptomycin (100 mgmLÀ1). Cell cul-
tures were maintained at 378C in a humidified atmosphere of 95%
air and 5% CO2.
1-(2-hydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-methoxyphenyl)ethanone (30): Yield: 77%; Rf =0.29
1
(MeOH/CH3Cl 1:4); mp: 149–1508C; H NMR ([D6]DMSO): d=10.64
(s, 1H), 8.01 (s, 1H), 7.86 (d, J=6.1 Hz 2H), 7.72 (d, J=6.7 Hz, 1H),
7.56 (d, J=7.3 Hz, 1H), 6.82 (d, J=6.5 Hz, 2H), 6.74 (d, J=8.4 Hz,
1H), 4.45 (s, 2H), 4.27 (s, 2H), 3.75 (s, 2H), 3.26 (s,3H), 2.51 (s, 3H);
13C NMR (500 MHz, [D6]DMSO): d=13.6, 41.5, 43.9, 55.6, 65.4, 103.1,
108.6, 112.8, 117.3, 129.8, 131.1, 132.3, 133.5, 138.2, 152.4, 159.7,
165.4, 167.2, 202.2 ppm; MS (ESI) C21H21N3O6 [M+H]+ 412.1; Anal.
calcd for C21H21N3O6: C 61.31, H 5.14, N 10.21%, found: C 61.29, H
5.12, N 10.22%.
H. pylori culture
The H. pylori strains used in this study were two standard strains
[ATCC 43504 and H. pylori mouse-adapted strain Sydney strain 1
(SS1)], and four clinical isolates of H. pylori (1–4), which were ob-
tained from antral biopsies of child and adult patients hospitalized
at Jiangsu People’s Hospital in Nanjing. All strains were cultured on
Columbia Agar (BioMerieux, France) supplemented with 7% sheep
blood and cultured for 3 days at 378C under microaerophilic condi-
tions with high humidity as detailed elsewhere.[22]
1-(2,3-dihydroxy-4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-
phenyl)-2-(4-methoxyphenyl)ethanone (31): Yield: 72%; Rf =0.21
1
(MeOH/CH3Cl 1:4); mp: 141–1428C; H NMR ([D6]DMSO): d=11.24
(s, 1H), 10.93 (s, 1H), 8.07 (s, 1H), 7.95 (d, J=6.5 Hz, 2H), 7.79 (d,
J=6.6 Hz, 1H), 7.63 (d, J=7.1 Hz, 1H), 6.74 (d, J=8.2 Hz, 1H), 4.52
(s, 2H), 4.33 (s, 2H), 4.06 (s, 2H), 3.34 (s,3H), 2.43 (s, 3H); 13C NMR
ChemMedChem 2010, 5, 1110 – 1116
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1115

H.-L. Zhu et al.
MED
derived, and a P value of <0.05 was considered statistically signifi-
Antimicrobial activity
cant.
Antimicrobial activity against H. pylori was determined by the agar
dilution method recommended by the National Committee for
Clinical Laboratory (NCCL)[22] standards. In vitro test strains
(ATCC 43504, SS1, and the clinical strains 1–4) were cultured for
3 days, then the anti-H. pylori agents were tested by twofold serial
dilutions of the compounds ranging from 100–0.05 mgmLÀ1 with
an initial cell count of ~106 CFUmLÀ1. The bacterial suspensions
(200 mL) were inoculated on each Columbia infusion blood agar
plate containing serial twofold dilutions of all compounds with the
concentration of DMSO <1% (v/v). After incubation for 3 days,
minimum inhibitory concentration (MIC) values were determined
as the lowest concentrations of the agents that visibly inhibited
bacterial growth inhibition. The antibacterial activity of the synthe-
sized compounds was tested against E. coli, P. fluorescence, B. sub-
tilis, and S. aureus using Mꢁller–Hinton (MH) medium: casein hy-
drolysate (17.5 g), soluble starch (1.5 g), beef extract (1000 mL),
The MIC values of the test compounds were determined by a col-
orimetric method using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT).[21]
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (30772627), the China Postdoctoral Science
Foundation (200902513), the Jiangsu National Science Founda-
tion (BK2009239), and the Anhui National Science Foundation
(No. 070416274X).
Keywords: deoxybenzoins · Helicobacter pylori · HPE-induced
IL-8 · metronidazole
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IL-8 assessment
Briefly, human gastric mucosal cancer cell line, grown for 2 days on
24-well plates to ~80% confluence in Ham’s F12 medium, were
washed three times with serum-free Ham’s F12 medium, and then
pre-incubated with metronidazole, compound 17, and compound
34 in serial concentrations at 15, 30, and 60 mm with the concen-
tration of DMSO <1% for 1 h. After that cells were incubated with
10% HPE (v/v) for 12 h. The supernatants were aspirated, centri-
fuged at 500 g for 10 min, and then stored at À808C. The levels of
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Acute oral toxicity of compound 34
Acute oral toxicity studies were performed according to the
method of Vasudevan et al.[23] Briefly, mice of either sex, selected
by a random sampling technique, were employed in this study.
Mice (n=3) in each group were fasted for 4 h with free access to
water only. Compound 6, suspended with carboxymethyl cellulose,
(CMC, 0.5% w/v) was administered orally at doses of 50, 250, or
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Statistical analysis
One-way analysis of variance (ANOVA) was performed to compare
Received: March 26, 2010
differences between groups. Two-tailed probability (P) values were
Published online on May 5, 2010
1116
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1110 – 1116