A specific anti-Helicobacter pylori agent NE2001: Synthesis and its effect on the growth of H. pylori

Article abstract of DOI:10.1016/S0960-894X(03)00547-X

The synthesis and anti-Helicobacter pylori activity of a novel agent NE2001, 4-(4-methylbenzyl)-4′-[guanidinomethylbenzoyloxy] biphenyl-4-carboxylate hydrochloride, are described. NE2001 had a specific inhibitory effect on the growth of H. pylori preceded by the suppression DNA synthesis in the cell. The effects of NE2001 on RNA and protein syntheses in H. pylori were also examined.

Full text of DOI:10.1016/S0960-894X(03)00547-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2703–2707
A Specific Anti-Helicobacter pylori Agent NE2001: Synthesis and
Its Effect on the Growth of H. pylori
Ni Cheng,a Jian-Shu Xie,b Min-Yue Zhang,a Chang Shua and De-Xu Zhua,*
aState Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093,
PR China
bShanghai East Best Biopharmaceutical Enterprises Co., Ltd., Shanghai, PR China
Received 2 April 2003;revised 21 May 2003;accepted 23 May 2003
Abstract—The synthesis and anti-Helicobacter pylori activity of a novel agent NE2001, 4-(4-methylbenzyl)-40-[guanidino-
methylbenzoyloxy] biphenyl-4-carboxylate hydrochloride, are described. NE2001 had a specific inhibitory effect on the growth of
H. pylori preceded by the suppression DNA synthesis in the cell. The effects of NE2001 on RNA and protein syntheses in H. pylori
were also examined.
# 2003 Elsevier Ltd. All rights reserved.
It has been widely accepted that Helicobacter pylori, a
Gram negative spiral bacterium is an important bacterial
pathogen which induces chronic gastritis and is asso-
ciated with gastroduodenal ulcer, adenocarcinoma of the
distal stomach, and gastric lymphoma in humans.1
Recently, the World Health Organization classified H.
pylori as a group 1 carcinogen responsible for its leading
role in the development of gastric cancer.2 The most
recent guidelines proposed in the Maastricht 2-2000
Consensus Report recommend the use of proton pump
inhibitors (PPI) along with antibacterial agent to eradi-
cate H. pylori.3 Since oral administration of metronida-
zole, PPI, and clarithromycin and amoxicillin was put
to use, the infection in up to 80–90% of the cases have
been cured. However, the application of antibacterial
agents causes a serious problem which induces the
resistant strain of H. pylori to the reagent. Actually, the
resistant strains to metronidazole, clarithromycin and
amoxicillin have already been reported.4 Another severe
problem is caused by the administration of PPI and
antibacterial agent, that is, PPI can induce indigestion
and large amounts of antibacterial agent can result in
severe destruction of the bacterial flora in digestive tract.
Therefore, it is important to find out new types of active
compounds for the eradication of H. pylori. When we
were screening various compounds, we discovered that a
novel compound 4-(4-methylbenzyl)-40-[guanidinomethyl-
benzoyloxy] biphenyl-4-carboxylate hydrochloride, NE-
2001, specifically inhibited the growth of H. pylori in
vitro.
The synthetic route for preparing NE2001 is shown in
Scheme 1. The minimum inhibitory concentrations (MIC,
mg/mL) of NE2001 and several related compounds
against H. pylori, Staphylococcus aureus and Escherichia
coli are shown in Table 1. The results indicate that no
intermediates showed any anti-bacterial activity
(MIC>64 mg/mL), except intermediate 6 that had weak
activity against H. pylori, while NE2001 showed potent
anti-H. pylori activity (MIC 0.8 mg/mL). The structure–
activity relationships (SAR) of NE2001 were also stud-
ied and the results are shown in Table 1. NE2001
showed about 10-fold improved activity compared to B,
which indicated when compound A and B linked by an
ester bond, the anti-H. pylori activity significantly
increased. Compound G linked by an ester bond showed
some anti-H. pylori activity while compound H linked by
an amide bond had none anti-H. pylori efficiency. These
results indicated that the ester bond may play an impor-
tant role in the activity of NE2001. The MICs for
NE2001, amoxicillin and metronidazole against 12 clin-
ical isolates of H. pylori and other common bacteria are
shown in Table 2. NE-2001 inhibited the growth of all
the tested H. pylori strains with the MIC ranging from
0.4 to 1.6 mg/mL. No strain resistant against NE2001
was found among the 12 clinical isolates of H. pylori,
*Corresponding author. Tel.:+86-25-359-2405;fax:+86-25-6637621;
e-mail: zjq@nju.edu.cn
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00547-X

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N. Cheng et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2703–2707
Scheme 1.
Table 1. Antibacterial spectrum of NE2001 and related compounds
Compd
Chemical structure
MIC (mg/mL)a
H. pylori ATCC43504
S. aureus 209P JC
E. coli K-12
>64
NE2001
0.8
>64
>64
>64
>64
>64
>64
>64
>64
>64
A
B
>64
8
>64
>64
>64
>64
>64
>64
>64
>64
C
D
E
>64
>64
>64
>64
16
F
G
H
>64
aMIC (mg/mL): Columbia agar+7% defibrinated sheep blood for H. pylori, 37 ꢀC, 72 h, agar dilution method;Mueller–Hinton agar for other bacteria.

N. Cheng et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2703–2707
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and NE2001 showed no antibacterial activity against
the selected bacteria as compared to amoxicillin. The
selective anti-H. pylori activity of NE2001 may be con-
ferred by its preferential penetrability into the outer
membrane of H. pylori cells, as shown for hydrophobic
compounds of small molecular size.5 As the anti-
bacterial spectrum of NE2001 is mainly restricted to H.
pylori, its oral use will not cause the disturbance of
normal gastrointestinal microflora.
The effects of NE2001 on the growth of H. pylori,
DNA, RNA, and protein syntheses were examined. The
Table 2. Antibacterial activity of NE2001, amoxicillin and metronidazole against Helicobacter pylori and other common bacteria
Organism
Strain
MIC (mg/mL)a
Amoxicillin
0.05
NE2001
0.8
Metronidazole
64
Helicobacter pylori
ATCC43504
Clinical isolates
HP001
0.4
0.8
0.8
0.8
0.8
0.8
1.6
0.4
1.6
0.8
0.8
0.8
0.025
0.05
0.05
0.05
0.05
0.05
0.025
0.05
0.05
0.05
0.025
0.025
4
2
8
HP003
HP004
HP007
HP008
HP016
HP017
HP018
HP019
16
32
32
64
8
32
128
32
16
HP020
HP021
HP022
Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Escherichia coli
Klebsiella pneumoniae
Morganella morganii
209P JC
ATCC1228
ATCC29212
K12
NCTC9632
KONO
>64
>64
>64
>64
>64
>64
0.2
0.78
0.78
1.6
>100
>100
>100
>100
>100
>100
50
>100
aMIC (mg/mL): Columbia agar+7% defibrinated sheep blood for H. pylori, 37 ꢀC, 72 h, agar dilution method;Mueller–Hinton agar for other bacteria.
Figure 1. Inhibitory effects of NE2001 on the growth of H. pylori (a) and on the uptake of [3H] thymidine;(b) [5- 3H] uridine;(c) or l-[4,5-3H]
leucine;(d) into DNA, RNA or protein without ( *) or with 1.6 mg/mL (&), 3.2 mg/mL (~) or 6.4 mg/mL (^) NE2001.

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N. Cheng et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2703–2707
growth of H. pylori was determined by an increase in
turbidity at 600 nm. NE2001 dose-dependently inhibited
the growth of H. pylori after 2 h of incubation, and the
cell growth was completely suppressed at a concen-
tration of 6.4 mg/mL (Fig. 1a). Fig. 1b shows the effect
of NE2001 on the uptake of [3H] thymidine into DNA.
Without NE2001 the incorporation increased time-
dependently and NE2001 dose-dependently suppressed
the uptake of [3H] thymidine into DNA after an hour of
incubation. The addition of the above 3.2 mg/mL of
NE2001 immediately reduced the uptake. Figure 1c and
d shows the effects of NE2001 on RNA and protein
synthesis respectively. NE2001 also showed an inhibi-
tory effect on RNA and protein synthese in the cells
performed in a dose-dependent manner similar to the
patterns observed in the growth of H. pylori (Fig. 1a).
The results shown in Figure 1 suggest that the suppres-
sion of the cell growth by NE2001 is due to the inhibi-
tion of DNA replication, because the suppression of
DNA replication by the inhibitor precedes that of cell
growth. But the exact target of NE2001 action remains
to be investigated.
(200.0 mmol), sodium methoxide 1.0 g (4.0 mmol), tol-
uene 20.0 mL, under N2 prevention, were heated to
reflux 2.5 h. During the reaction, additional 20.0 mL
toluene was added to bring out the resultant methanol
under reflux. Then it was cooled to room temperature,
and 10 mL acetic acid and ice 40 g were added to adjust
the pH=5. The obtained organic layer was con-
centrated under reduced pressure to remove the solvent
and the excess 4-methylbenzylalcohol. Then it was
cooled to obtain brownish oil and left to stand to pro-
duce the crude crystals slowly. The crude crystals were
recrystallized from toluene/n-hexane. A white crystal, 4-
(4-methylbenzyl)-40-hydroxybiphenyl-4-carboxylate 7.3 g
1
was obtained (yield 71%) H NMR (500 MHz, CDCl3):
d 2.35 (s, 3H), 5.35 (s, 2H), 6.90 (d, 2H), 7.15 (d, 2H),
7.35 (d, 2H), 7.50 (d, 2H), 7.60 (d, 2H), 8.10 (d, 2H).
4-(4-methylbenzyl)-40-[guanidinomethylbenzoyloxy]
bi-
phenyl-4-carboxylate hydrochloride 7. A suspension of
4-(4-methylbenzyl)-40 -hydroxybiphenyl-4-carboxylate,
2.42 g (0.010 mol), 4-guanidinomethyl benzoic acid
hydrochloride 2.3 g (0.010 mol) and dicyclohexyl-
cabondiimide 4.1 g (0.020 mol) in pyridine/DMF 150
mL was stirred at room temperature for 48 h, after the
removal of insoluble materials by filtration. The filtrate
was evaporated to dryness and residue solid was treated
with 0.1N hydrochloric acid (50 mL) and ether (50 mL).
The aqueous layer was washed with ether again and
concentrated to 20 mL, the resulting crystals were
recrystallized in ethanol/hexane, giving 4-(methylbenzyl)-
40[guanidinomethylbenzoyloxy] biphnyl-4-carboxylate
hydrochloride, 2.9 g (yield 55%) LC/MS=494 (M+H).
Experimental
Synthesis
4-Guanidinomethyl benzoic acid hydrochloride 2. 2N
NaOH solution 72 mL was added to a solution of
methyl isothiourea disulfate 20.0 g (0.14 mol) in 36 mL
water cooled in ice batch, and stirred. Then 21.0 g
(0.138 mol) 4-aminomethylbenzoic acid in 140 mL 2N
NaOH solution was added drop wise. The mixture was
left to stand overnight at room temperature and then
chilled in ice water for 1 h. The precipitated white crys-
tals were filtered off and washed with cold water. The
filtrate was dissolved in warm 1N HCL and insoluble
material was removed by filtration. The solution was
concentrated in vacuum to crystallize. The colorless
prisms crystallized when the solution was cooled, and
then was filtered and dried, giving 4-guanidinomethyl
benzoic acid hydrochloride 22.1 g (yield 70%) LC/
MS=194(M+H)
Antimicrobial Activity
Bacterial strains
Twelve clinical isolates of H. pylori and strain ATCC
43504 were used. The identification of clinical isolates
was based on microaerophilic growth requirement,
morphology, Gram stain and oxidase, catalase, and
rapid urease reactions.6 Six reference strains of different
species were also used. All strains were stored at ꢁ80 ꢀC
until susceptibility testing.
4-Methyl-40-hydroxybiphenyl-4-carboxylate 4. A solu-
tion of 4-(4-hydroxyphenyl) benzoic acid 21.4 g (0.1
mol) in 500 mL absolute methanol in a flask equipped
Soxhlet apparatus filled with A4 molecular sieve. Then
2.0 mL concentrated sulfuric acid was dropped. The
mixture refluxed for 72 h. After the removal the solvent
by vacuum, the residual oil was dissolved in 100 mL
toluene and washed to pH=7 with water. The organic
layer was dried by MgSO4 and filtered. The obtained
filter liquor was added to a certain quantity of activated
charcoal heated to reflux for 10–15 min and filtered.
The solvent was removed to obtain a white crystal,
4-methyl-40-hydroxybiphenyl-4-carboxylate 18.2 g (yield
80%).
Susceptibility testing
The MICs of NE2001 and other compounds for H.
pylori were determined by an agar dilution method
using Columbia agar (Difco Co.), which was added with
7% defibrinated sheep blood. In brief, all the com-
pounds were dissolved in dimethylsulphoxide (DMSO)
and serial double dilutions were performed. H. pylori
cultured in Brucella broth medium containing 5% fetal
bovine serum under microaerophilic condition (5% O2,
10% CO2 and 85% N2) at 37 ꢀC for 24 h, and the
number of bacteria thereof was adjusted to about
108CFU/mL. This bacteria solution was painted in each
square about 2 cm with inoculating line loop. The plates
were cultured under microaerophilic condition for
three days at 37 ꢀC. Minimum concentration which
completely inhibit the growth of the bacteria was judged
4-(4-Methylbenzyl)-40-hydroxybiphenyl-4-carboxylate 6.
A suspension of 4-methyl-4-hydroxybiphenyl-4-carboxy-
late 9.0 g (40.0 mmol), 4-methylbenzylalcohol 24.4 g

N. Cheng et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2703–2707
2707
as the value of MIC. The MICs for other common bac-
teria were also determined by the agar dilution method
using Mueller–hinton agar (Difco Co.).
Acknowledgements
This work was supported by a grant from National
Natural Science Foundation of China, No. 30271555.
Determination of DNA, RNA and protein syntheses in
H. pylori
References and Notes
H. pylori cells were grown in brucella broth medium
containing 5% fetal bovine serum with or without dif-
ferent concentration of NE2001 under microaerophilic
conditions at 37 ꢀC. The uptake of [3H] thymidine,
[5-3H] uridine or L-[4,5-3H] leucine (Amersham Phar-
macia Biotech, UK) into DNA, RNA or protein was
determined in a way described by Joselean-Petit.7 In
brief, a volume of 0.4 mL of the growing H. pylori
suspension was withdrawn and incubated with 20 mL
radiolabelled thymidine, uridine or leucine (100 mCi/
mL) for 30 min under the same condition. Adding 0.4
mL of 10% trichloroacetic acid stopped the reaction.
After 30 min, precipitates were collected on a 0.45 mm
Millipore filter and rinsed with 5% trichloroacetic
acid. Filter were dried and counted by liquid scintilla-
tion on a 1400 WinSpectral scintillation counter
(Wallac).
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