Anti-Helicobacter pylori agents endowed with H2-antagonist properties

Article abstract of DOI:10.1016/S0960-894X(00)00671-5

New anti-Helicobacter pylori (H. pylori) agents endowed with H₂-antagonists properties were obtained by combining the lamtidine derived pharmacophoric group with the antibiotic calvatic acid. All the compounds were tested for their irreversible H₂-antagonist properties and for their ability to inhibit 20 H. pylori strains, two of them metronidazole resistant. The most active derivative (compound 4) displayed antimicrobial activity similar to metronidazole.

Full text of DOI:10.1016/S0960-894X(00)00671-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 403±406
Anti-Helicobacter pylori Agents Endowed with H -Antagonist
2
Properties
a
b
b
b,
Giovanni Sorba, Massimo Bertinaria, Antonella Di Stilo, Alberto Gasco, *
c
c
d
Maria M. Scaltrito, Maria I. Brenciaglia and Francesco Dubini
aDipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universit aÁ degli Studi del Piemonte Orientale,
Viale Ferrucci 33, I-28100 Novara, Italy
b
Dipartimento di Scienza e Tecnologia del Farmaco, Universit aÁ degli Studi di Torino, Via Pietro Giuria 9, I-10125 Torino, Italy
cIstituto di Microbiologia, UniversitaÁ degli Studi di Milano, Via Pascal 36, I-20133 Milano, Italy
dI Cattedra di Microbiologia Clinica, UniversitaÁ La Sapienza, Piazzale Aldo Moro 5, I-00186 Roma, Italy
Received 29 June 2000; accepted 24 November 2000
AbstractÐNew anti-Helicobacter pylori (H. pylori) agents endowed with H
lamtidine derived pharmacophoric group with the antibiotic calvatic acid. All the compounds were tested for their irreversible H
2
-antagonists properties were obtained by combining the
2
-
antagonist properties and for their ability to inhibit 20 H. pylori strains, two of them metronidazole resistant. The most active
derivative (compound 4) displayed antimicrobial activity similar to metronidazole. # 2001 Elsevier Science Ltd. All rights reserved.
Today it is widely accepted that Helicobacter pylori
H. pylori), a spiral microaerophilic S-shaped Gram
drugs, such as H -receptor antagonists or proton
2
(
pump inhibitors. A number of problems are associated
with multiple therapy, among these drug resistance,
side e€ects and non-compliance. Therefore great
interest is devoted to novel agents suitable for a single
therapy treatment. A possible strategy for obtaining
these products is to combine an antisecretory phar-
macophore with an anti-H. pylori agent in a single
molecule. Recently we found that calvatic acid 1,
negative bacterium which colonises the gastric mucosa,
is a major causative factor of a number of gastric
pathologies such as gastritis, peptic ulcers and certain
1
gastric cancers. Multiple therapeutic regimens are gen-
erally used for the treatment of infections by this
2
microrganism; all of which imply the frequent combi-
nation of antimicrobial agents with antisecretory
*
0
Corresponding author. Tel.: +39-11-6707670; fax: +39-11-6707687; e-mail: gasco@pharm.unito.it
960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00671-5

4
04
G. Sorba et al. / Bioorg. Med. Chem. Lett. 11 (2001) 403±406
Scheme 1.
Block a is similar to the moiety present in lamtidine, a
well known H -antagonist, and identical to the moiety
2
present in a new class of ligands with high anity and
4
selectivity for the histamine H -receptor. Block b is a
2
suitable polymethylene spacer and block c is represented
by the acyl residue of calvatic acid. Here we report the
synthesis, irreversible H -antagonist properties and the
2
anti-H. pylori activity of these derivatives.
The synthetic pathway used to obtain the ®nal com-
pounds is reported in Scheme 1. Amines 6±8 were syn-
4
an antibiotic isolated from culture broth of the gastro-
mycete Calvatia lilacina (Berk.) Henn. P., and some of
its analogues display potent action against many strains
of H. pylori, including two that are metronidazole
resistant. On these bases we designed structures 2, 3, 4
of general formula I.
thesized by action of appropriate alkanediamine on 5,
according to a method reported in the literature and
modi®ed in some aspects. These intermediates were
allowed to react with the activated ester 1a, obtained
from calvatic acid, N-hydroxysuccinimide and N,N -
dicyclohexyl-carbodiimide (DCC), to a€ord the expec-
3
0
ted ®nal compounds 2±4. The pharmacological pro®le
of the products at histamine H -receptor was assessed
2
on isolated guinea pig right atria. Irreversible inactiva-
tion of H -binding sites was studied by incubating the
2
compounds for various times at ®xed concentrations.
The tissues were then washed free of drug, and hista-
mine cumulative concentration±response curve was
recorded. At 30 mM concentration, after 20 min of incu-
bation with guinea pig right atrium, 2 was able to shift
the curve of histamine irreversibly to the right without
reducing the maximal response (Fig. 1). A progressive
reduction of this response was observed after 80 min
(
20%) and 3 h (50%) of incubation, respectively.
Working at a lower concentration (10 mM) an irrever-
sible rightwards shift of histamine curve without reduc-
tion of the maximal response occurred (data not
shown). The length increase of the spacer in 2 was
accompanied by a drop of the irreversible action. In
fact, after 3 h of incubation a less marked shift to the
right of the histamine curve and only 10% and 30%
reduction of the maximum was observed for derivatives
3 and 4, respectively (Fig. 2). The irreversible block of
Figure 1. Concentration±response curves for histamine (HA) after
incubation with
2 (30 mM) at di€erent times: & (control); ~
(
t=20 min); ! (t=80 min); ^ (t=180 min).

G. Sorba et al. / Bioorg. Med. Chem. Lett. 11 (2001) 403±406
405
receptor by these compounds could be explained by the
presence of cyano-NNO-azoxy function which is able to
react in physiological conditions with -SH groups. In
Activity against H. pylori was assessed on 20 clinical
strains, two of these (NCTC 11637 and 102R) were
metronidazole resistant. Metronidazole was taken as
reference. The results expressed as minimal inhibitory
5
fact, studies with a series of tetramine disulphides rela-
ted to benextramine indicate that a thiol group able to
bind irreversibly the disulphide moiety of the drugs is
concentrations MIC50 and MIC , namely the minimal
90
concentration needed to inhibit 50% and 90% of the
used strains, respectively, are reported in Table 1. The
antibacterial activity of our compounds is much lower
than calvatic acid activity, but comparable to the one
shown by metronidazole. The activity of the synthesized
hybrids ranks the order 4>3>2. The most active com-
pound (4) shows a MIC50 value four times higher than
metronidazole, by contrast its MIC90 value is four times
lower than the reference. Furthermore compound 4
displays good activity against the two metronidazole
resistant strains. In conclusion the compounds described
in the present work represent a new class of products
6
present on the surface of the histamine H -receptor.
2
This process may be expected to occur in two con-
secutive steps, as clearly shown for other irreversible
7
antagonists at other receptors. The ®rst is the reversible
interaction of the lamtidine part of the drugs, able to
recognise the receptor, with the target active sites; the
second the covalent bond formation between cyano-
NNO-azoxy function and an adjacent thiol group.
Thus, the drop of the irreversible activity observed on
increasing the length of the spacer could be due to the
fact that the shorter the chain the better the placement
of the reactive N(O)NCN function at the -SH group.
Obviously, during the walk to the receptor the com-
pounds could also react with other -SH groups. How-
ever, this interaction should only in¯uence their
accessibility to the target but not the selectivity for it,
this latter being due to the lamtidine moiety. In fact
calvatic acid, when tested alone with the same protocol
endowed with both H -antagonist and anti-H. pylori
2
activity. In vivo studies to evaluate the ability of 2±4 to
exert both antisecretory and anti-H. pylori activity are
being conducted.
Experimental8
Synthesis
(
30 mM, 3 h), did not display any e€ect on H -receptor.
2
0
00
N-(Aminoalkyl)-N -cyano-N -(3-(3-(1-piperidylmethyl)-
phenoxy)propyl)guanidines 6±8. A solution of 5 (1.96 g,
5
.00 mmols) in a mixture of ethyl acetate (20 mL) and
methanol (5 mL) was added dropwise to a stirred solu-
tion of the appropriate diaminoalkane (50 mmol). The
reaction mixture was stirred at room temperature for
3
h. The solvent was removed under reduced pressure,
and the residue ¯ash chromatographed on silica gel,
eluting ®rst with a mixture of dichloromethane/metha-
nol 9:1 and then with a mixture of dichloromethane/
methanol:32% ammonia 8:2:0.3. The products were
obtained as oils and were pure enough for further
reactions (6,7 yield 75%; 8 yield 60%). Analytical
samples were obtained as oxalates and recrystallized
.
.
from methanol/isopropanol. 6 2H C O H O, mp
2
2
4
2
ꢀ ꢀ
. .
58±159 C dec.; 7 2H C O 0.5H O, mp 184±185 C
2 2 4 2
dec.; 8 2H C O 0.5H O, mp 174±175 C dec.
2
1
ꢀ
.
.
2
2
4
Figure 2. Concentration±response curves for histamine (HA) after
80 min of incubation with: & (control); * (3, 30 mM); ! (4, 30 mM).
1
0
00
N-Cyano-N -(4-(cyano-NNO-azoxy)benzamido)alkyl-N -
3-(3-(1-piperidylmethyl)phenoxy)propyl)guanidines 2±4.
N-Hydroxysuccinimide (1.05 g, 9.10 mmol) and DCC
(
(
(
Table 1. Minimal inhibitory concentration (MIC) of compounds 1,
, 3, 4 and metronidazole against 20 H. pylori strains, including two
metronidazole resistant strains
1.87 g, 9.10 mmol) were added to a stirred solution of 1
1.34 g, 7.00 mmol) in dry THF (75 mL) at room tem-
perature. After 1 h of stirring the residue of dicyclohex-
ylurea was ®ltered o€ and the appropriate amine 6±8
2
a
MIC (mg/mL)
(
7.00 mmol) dissolved in dry THF (20 mL) was added to
Compound
All strains
Metronidazole
resistant
the solution of the active ester 1a. The reaction mixture
was stirred for 2 h at room temperature and ®ltered. The
solvent was then removed under reduced pressure and
the residue dissolved in 100 mL of dichloromethane
containing 1 mL of methanol. The organic solution was
washed with a saturated solution of sodium bicarbo-
nate, with brine, with water and then dried on anhy-
drous magnesium sulphate. The residue obtained after
solvent removal was puri®ed by ¯ash chromatography
®rst eluting with a mixture of ethyl acetate/methanol
Rangeb
MIC50 MIC90 NCTC11637 102R
1
2
3
<0.0039±0.062 0.016
0.031
16
4
2
8
0.0078
16
0.031
0.25±16
0.25±8
0.25±2
8
2
1
0.25
8
8
2
8
4
1
4
Metronidazole
0.031±16
16
a
MICs are expressed as mg/mL of calvatic acid (see Experimental).
Range: range of minimal inhibitory concentrations.
b

4
06
G. Sorba et al. / Bioorg. Med. Chem. Lett. 11 (2001) 403±406
9
8
5
5:5, and then with a mixture of ethyl acetate/methanol
0:20. The obtained products (yields: 2, 40%; 3, 60%; 4,
0%) were transformed into the corresponding oxalates,
in a gas incubator (Haereus). Before use the media were
always preincubated under the same microaerobic con-
ditions for a minimum of 2 h in order to allow them to
equilibrate, and none of the cultures were kept in the air
for more than 15 min.
recrystallized from methanol/ether and dried for 4 days
in vacuo at 60 C. Decomposition maxima were recor-
ꢀ
ded by DSC analysis (Perkin±Elmer DSC 7).
ꢀ
C NMR (DMSO-d ): 21.8, 22.7, 28.6, 38.5, 39.3, 40.6,
.
.
H C O 0.5H O, decomposition maximum 161 C,
2
Minimal inhibitory concentration (MIC) determination.
MICs of all the compounds and metronidazole, taken
as reference, were determined using the agar dilution
method. In brief, all the substances were dissolved in
dimethylsulphoxide (DMSO) and serial double dilutions
were performed for calvatic acid 1 ranging from 128 to
0.0039 mg/mL. All remaining compounds including
metronidazole, employed in equimolar concentration to
1, were diluted in agar medium in serial double dilution.
The plates of Columbia agar with horse serum and yeast
extract containing antimicrobial agents were prepared
on the day of use. The inoculum was prepared as fol-
lows: a suspension of 72 h growth of each strain on agar
plates was made in Wilkins Chalgren broth (Difco) at a
2
2
4
2
1
3
6
5
1
1
2.0, 59.4, 65.4, 111.0, 115.4, 116.8, 118.2, 123.2, 123.3,
28.9, 129.9, 132.3, 140.6, 146.3, 158.7, 159.6, 165.0,
ꢀ
C NMR (DMSO-d ): 22.1, 23.1, 26.3, 26.6, 28.8, 38.4,
.
65.2; 3 H C O H O, decomposition maximum 135 C,
.
2
2
4
2
1
3
6
3
1
1
9.2, 40.9, 52.3, 59.9, 65.3, 111.0, 114.9, 116.5, 118.3,
23.3, 122.8, 128.8, 129.7, 133.5, 140.9, 146.2, 158.7,
.
59.4, 164.5, 165.0; 4 H C O 0.5H O decomposition
.
2
2
4
2
ꢀ
13
maximum 137 C, C NMR (DMSO-d ): 22.2, 23.2,
6
2
6
1
6.0, 26.3, 28.8, 28.8, 28.9, 38.5, 39.1, 41.0, 52.4, 60.1,
5.3, 111.0, 114.8, 116.5, 118.3, 122.7, 123.3, 128.8,
29.7, 133.8, 140.9, 146.2, 158.7, 159.4, 164.4, 164.8.
ꢀ
turbidity equivalent to N 0.5 Mac Farland standard.
The plates were inoculated using a multipoint inocu-
Biological Assays
lator (Denley A 400 PBI) dispensing 5 mL and incubated
at 37 C for 72 h under microareobic conditions (10%
ꢀ
CO in gas incubator). The MIC was de®ned as the
Guinea pig right atria
2
Guinea pigs (350±400 g) were sacri®ced by cervical dis-
location followed by exanguination. The right atria
were rapidly dissected from the ventricles, cleaned of
excess tissue and hung vertically in the organ bath con-
taining oxygenated Ringer±Locke solution of the fol-
lowing composition (mM): NaCl 154; KCl 5.4;
lowest concentration capable of inhibiting any visible
bacterial growth.
Acknowledgements
.
.
CaCl 2H O 1.5; NaH PO H O 0.25; NaHCO 4.3;
2
This work was supported by a grant from MURST,
Studi e Ricerche Finalizzate 40%, Roma.
2
2
4
2
3
ꢀ
glucose 8.3 (31 C). A stabilization period of 60 min was
allowed before a cumulative concentration±response
curve to histamine was performed. During this equili-
bration period the bathing solution was changed every
30 min. The preparations were incubated with antagonists
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1
1637, 102R) were metronidazole resistant. The strains
ꢀ
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Laboratories) supplemented with 10% horse serum (Ser-
7
8
. Satisfactory analyses (Æ0.4%) were obtained for all new
1
omed) and 0.25% Bacto yeast extract (Difco) incubated for
ꢀ
compounds; H NMR spectra are in keeping with the
proposed structures.
72 h at 37 C under microaerobic conditions (10% CO )
2