Anti-Helicobacter pylori activity of some newly synthesized derivatives of xanthone

Article abstract of DOI:10.1038/ja.2016.36

A series of 20 xanthone derivatives was synthesized and evaluated for anti-Helicobacter pylori (H. pylori) activity. Qualitative and quantitative in vitro tests using the Kirby-Bauer method (agar disc-diffusion method) were performed. The tested compounds were screened against clarithromycin- and/or metronidazole-resistant strains of H. pylori. As a reference, Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial strains were examined. On the basis of microbiological assays, xanthones can be considered as potential anti-H. pylori agents. They displayed significant activity against the examined strains, which was higher against the bacteria resistant to metronidazole than clarithromycin. The lowest MIC values ranging up to 20 mg l^-1 were observed for the following compounds: 3, 4, 8, 9, 12, 19 (against the metronidazole-resistant strains) and the compound 10 (against the clarithromycin-resistant strain). These preliminary results for screening of xanthone derivatives form a part of an ongoing study of the structure-activity relationships of a large group of compounds. Microbiological assays will be conducted afterwards to determine the mechanism of xanthones' action against H. pylori.

Full text of DOI:10.1038/ja.2016.36

The Journal of Antibiotics (2016), 1–10
2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16
www.nature.com/ja
&
ORIGINAL ARTICLE
Anti-Helicobacter pylori activity of some newly
synthesized derivatives of xanthone
1
1
1
2
2
Karolina Klesiewicz , Elżbieta Karczewska , Alicja Budak , Henryk Marona and Natalia Szkaradek
A series of 20 xanthone derivatives was synthesized and evaluated for anti-Helicobacter pylori (H. pylori) activity. Qualitative and
quantitative in vitro tests using the Kirby–Bauer method (agar disc-diffusion method) were performed. The tested compounds
were screened against clarithromycin- and/or metronidazole-resistant strains of H. pylori. As a reference, Gram-positive
(
Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial strains were examined. On the basis of microbiological
assays, xanthones can be considered as potential anti-H. pylori agents. They displayed significant activity against the examined
strains, which was higher against the bacteria resistant to metronidazole than clarithromycin. The lowest MIC values ranging up
to 20 mg l− were observed for the following compounds: 3, 4, 8, 9, 12, 19 (against the metronidazole-resistant strains) and the
compound 10 (against the clarithromycin-resistant strain). These preliminary results for screening of xanthone derivatives form a
part of an ongoing study of the structure–activity relationships of a large group of compounds. Microbiological assays will be
conducted afterwards to determine the mechanism of xanthones’ action against H. pylori.
1
The Journal of Antibiotics advance online publication, 30 March 2016; doi:10.1038/ja.2016.36
INTRODUCTION
structural similarity to the xanthone scaffold. These results encouraged
Xanthone derivatives comprise an interesting group of compounds us to synthesize xanthone derivatives with potential anti-H.
that, depending on the type of substituent and its localization in one of pylori activity.
the xanthone’s rings, exhibit diversified biological activity. Biological
H. pylori is a Gram-negative, spiral-shaped bacterium that inhabits
properties of synthetic xanthone derivatives have been investigated gastric mucosa of about 50% of human population worldwide.
1
since 1968, when Da Re et al. described, for the first time, central
stimulating and analeptic activities of synthetic aminoalkyl xanthone
derivatives. Since then, xanthone skeleton has been widely modified as
it is a potential pharmacophoric structure. Literature reports
confirmed broad spectrum of activity of both natural and synthetic
derivatives of xanthone (including antimalarial, antituberculotic,3
H. pylori is the main pathogenic factor of type B chronic gastritis.
Moreover, H. pylori infections are associated with peptic ulcer disease
or dyspepsia. Since the year 1994, the International Agency
for Research on Cancer of the World Health Organization
(IARC/WHO) concluded that H. pylori has been a class I carcinogen,
2
that has an important role in mucosa-associated lymphoid tissue
lymphoma and gastric cancer.12–18 Furthermore, many studies
revealed a relation between H. pylori infections and extra-gastric
diseases like iron-deficiency anemia, idiopathic thrombocytopenic
purpura, cardiovascular disorders, neurological diseases, as well as
obesity, diabetes mellitus, asthma or diseases of oral mucosa.12,19–21
Guidelines for the management of H. pylori infection and treatment
regimens are regularly issued by the European Helicobacter Study
Group. Standard treatment protocol includes a combination of three
kinds of drugs: a proton pump inhibitor, a cytoprotectant drug
and containing bismuth salts and an antibiotic or chemotherapeutic
4
5
antibacterial and antifungal activity).
Searching for biologically active structures in the group of xanthone
derivatives remains one of the main directions of the research carried
out at our Department of Bioorganic Chemistry. We synthesized and
number of xanthone derivatives that exhibited
activity, especially against Mycobacterium
tuberculosis or pathogenic bacteria and fungi. We have not
determined anti-Helicobacter pylori (H. pylori) activity of the
synthesized xanthone derivatives so far, and according to our literature
survey, only naturally occurring xanthones or flavones isolated from
evaluated
microbiological
a
6
7
8
9
10
Garcinia fusca, Artocarpus obtusus, Glycyrrhiza glabra
Cratoxylum arborescens showing anti-H. pylori activity were described. (clarithromycin, amoxicillin, metronidazole, tetracycline, levofloxacin,
1
1
Chimenti et al. of La Sapienza University of Rome also reported rifabutin). Despite 10–14 days of triple or quadruple therapy, a
anti-H. pylori activity of synthesized N-substituted derivatives of considerable increase of H. pylori resistance to the antimicrobial
2-oxo-2H-1-benzopyran-3-carboxamides which showed significant agents could be observed. Resistance to antimicrobials, especially to
1
2
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland and Department of Bioorganic Chemistry, Faculty of
Pharmacy, Jagiellonian University Medical College, Krakow, Poland
Correspondence: Dr N Szkaradek, Department of Bioorganic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, Krakow 30-688,
Poland.
E-mail: nszkarad@cm-uj.krakow.pl
Received 21 May 2015; revised 19 January 2016; accepted 23 February 2016

Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
2
clarithromycin, is a major therapeutic problem because the standard
treatment regimens become less effective.
The tested compounds exhibited high anti-H. pylori activity,
whereas their activity against methicillin-resistant S. aureus and E. coli
1
2,22
Discovery of novel antimicrobial compounds appears to be a good strains was lower. The latter strains were susceptible only to some of
solution to overcome therapeutic problems resulting from increasing the tested compounds and their inhibition zone diameters were
resistance of H. pylori strains to antibiotics and chemotherapeutics.
The use of new drugs is therefore an alternative to the common
antibiotics/chemotherapeutics and give chance to achieve a successful
eradication therapy. Xanthones are well-known for their antimicrobial
and antifungal activity, as well as anti-H. pylori activity.8
smaller.
Our results showed that among twenty tested xanthone derivatives,
fourteen exhibited activity against S. aureus ATCC 25922, compound
1
4 gave the largest zone of growth inhibition with a diameter of
,23
1
7 mm.
Herein we report the in vitro activity of some new structures against
H. pylori strains. Selected compounds were varied in order to find
potential structure–activity relationships. Taking into account the
structures of previously evaluated flavonoids and N-substituted
derivatives of 2-oxo-2H-1-benzopyran-3-carboxamides,10 our
structures, inter alia, contained: halogen atoms, methoxyl, hydroxyl
or ester groups, unsaturated bonds and additional aromatic rings.
Methicillin-resistant S. aureus was also susceptible to fourteen
compounds, but the strongest activity was shown by the compounds
and 2, with zone of growth inhibition diameters of up to 23 mm.
E. coli ATCC 25922 was resistant to the majority of tested
1
derivatives, however, this strain displayed susceptibility to five of
them: 1, 2, 4, 13 and 14. The largest growth inhibition zone was
produced by the compound 2, with a diameter of 10 mm.
To sum up, qualitative testing showed that all H. pylori strains were
susceptible to newly synthetized xanthone derivatives, whereas only
few of them were active against other than H. pylori strains.
The strongest anti-H. pylori activity was exhibited by compounds
for which the diameters of the growth inhibition zones were the
largest: 1, 2, 4, 10, 11 and 13.
On the basis of the result of the screening qualitative method (zone
inhibition method) we have chosen the fourteen most active com-
pounds (1, 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 16 and 19; Table 1) and
performed quantitative testing (means determination of minimum
inhibitory concentration (MIC) values).
RESULTS
Antibacterial activity
Results of the qualitative testing. The in vitro antibacterial activity of
the 20 synthesized compounds against 12 representative strains of
bacteria: H. pylori ATCC 43504, H. pylori ATCC 700684, S. aureus
ATCC 25923, E. coli ATCC 25922 and 7 clinical H. pylori strains was
evaluated by disc-diffusion method. Antibacterial activity of the tested
compounds was estimated by measuring the diameters of the
inhibition zones (mm). The obtained results for new compounds
and the reference compounds were presented in Table 1. We used the
interpretative criteria described above, that means zones with
diameters o8 mm indicated no antibacterial activity, zones
with diameters of ⩾ 8 mm indicated antibacterial activity and zones
with diameters of ⩾ 30 mm indicated strong antibacterial activity.
The results presented in Table 1 suggest that all the tested
compounds exhibited an inhibitory effect on the growth of H. pylori
strains.
Results of the quantitative testing. Fourteen samples of xanthone
derivatives were tested quantitatively in order to determine the MIC
value of each compound. These were the following compounds: 1, 2,
, 4, 6, 8, 9, 10, 11, 12, 13, 14, 16, 19.
The compounds were studied with the use of two bacterial strains:
reference H. pylori strain ATCC 43504 (resistant to metronidazole)
and clinical H. pylori strain 132/194 (resistant to clarithromycin).
The obtained results were interpreted according to the following
criteria:
3
Diameters of the growth inhibition zones for the reference
H. pylori strain ATCC 43504, resistant to metronidazole, ranged from
11 mm (compound 5) to 54 mm (compound 2). Strong activity
(
1
inhibition diameter⩾ 30 mm) was displayed by nine compounds: 1,
A, 2, 4, 6, 10, 11, 13 and 19.
-
-
zone of growth inhibitiono8 mm—compound showed no anti-
bacterial activity;
zone of growth inhibition ⩾ 8 mm—compound showed antibac-
terial activity.
Diameters of the growth inhibition zones for the reference
H. pylori strain ATCC 700684, resistant to clarithromycin, ranged
from 19 mm (compound 18) to 38 mm (compound 10). Eleven
compounds: 1, 2, 4, 6, 10, 11, 13, 14, 16 and 19 exhibited strong
activity.
The strongest activity against clinical H. pylori strains resistant to
metronidazole was shown by the compounds 2 and 10, with the
diameters of inhibition zones ranging up to 39 mm or more, whereas
compound 5 presented the weakest activity.
Compounds exhibiting the strongest activity against clinical
H. pylori strains resistant to clarithromycin were 2, 10 and 11 with
diameter of inhibition zone ranging up to 34 mm or more, whereas
compounds 7, 18 showed the weakest activity. It was shown that
twelve compounds had strong antibacterial activity against these
clarithromycin-resistant strains.
The MIC value was considered to be the lowest concentration of the
examined compound that inhibited the growth of bacteria (that is, the
last dilution that exhibited antibacterial activity—zone of growth
inhibition with a diameter⩾ 8 mm).
The highest concentrations of all fourteen compounds exhibited
1
− 1
antimicrobial activity:
I
(5 mg ml− ), II (1 mg ml
)
and
III (500 mg l− ) against both examined H. pylori strains.
1
The results presented in Figure 1 suggest that there were some
differences between the profile of susceptibility of H. pylori strains
expressed as the MIC values of each compound) to the tested
xanthone derivatives. We observed that the MIC values for the
H. pylori strain ATCC 43504 ranged from 5 to 150 mg l− 1, whereas
(
Double-resistant H. pylori strains were susceptible to all tested
xanthones, however, the strongest activity was exhibited by compound
with the inhibition zone diameter of up to 50 mm for strain for the H. pylori strain 132/194 it ranged from 10 to 500 mg l− 1.
43/207, and compound 10 with the diameter size of 56 mm for strain
26/181. The weakest activity against double-resistant strains was H. pylori strain ATCC 43504, that is, the MIC value was the lowest one
displayed by compound 5, with diameter of the growth inhibition (5 mg l− 1). The weakest activity was exhibited by compound 16 with
2
1
1
Compound 3 showed the strongest activity against the reference
zone ranging from 16 to 23 mm.
the MIC value of 150 mg l− 1.
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Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
3
Table 1 Chemical structures of newly synthetized xanthones and the results of qualitative testing: diameter means of the zones of growth
inhibition caused by the tested compounds in 1% concentration
The Journal of Antibiotics

Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
4
Table 1 (Continued)
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Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
5
Table 1 (Continued )
Figure 1 Activity of fourteen newly synthetized xanthone compounds and reference compounds (clarithromycin, metronidazole) expressed by MIC values
against clinical H. pylori strain 132/194 and reference H. pylori strain ATCC 43504.
Three compounds (1, 11, 14) exhibited high MIC values (up to study includes: design, synthesis, physicochemical analysis and
−
1
5
00 mg l ) against the clinical H. pylori strain 132/194. Against the microbiological evaluation of the chosen structures.
same isolate, compound 10 exhibited the strongest activity and
From these results it can be concluded that among xanthone
derivatives new potential anti-H. pylori structures can be found.
Our compounds were much more effective than the reference
compound (metronidazole or clarithromycin), when bacterial strain
was resistant to this drug. Surprisingly, the largest zones of growth
produced the lowest MIC values (10 mg l− 1).
DISCUSSION
The aim of this study was to evaluate anti-H. pylori activity of the new inhibition in qualitative evaluation did not guarantee the lowest MIC
compounds—derivatives of xanthone. In particular, the present values in the quantitative studies. The tested substances manifested
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Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
6
higher antibacterial activity against the reference H. pylori strain t (triplet), qu (quintet) and m (multiplet). Elemental analyses were performed
resistant to metronidazole, than to the clinical H. pylori 132/194 on an Elementar Vario EL III (Elementar Analysansysteme, Hanau, Germany).
For MS analyses, samples were prepared in acetonitrile/water (10/90 v/v)
strain resistant to clarithromycin. This result is consistent with the
mixture. The LC/MS system consisted of a Waters Acquity UPLC, coupled to a
average MIC values of both strains. The average MIC value of the
Waters TQD mass spectrometer (Waters, Milford, MA, USA, electrospray
tested compound for H. pylori ATCC 43504 was lower than the
ionization mode ESI-tandem quadrupole). All the analyses were carried out
average MIC value for H. pylori 132/194 and were determined to be 46
using an Acquity UPLC BEH C18, 1.7 μm, 2.1 × 100 mm column. A flow rate
−
1
and 185 mg l , respectively. Only compound 10 showed higher
activity against H. pylori strains resistant to clarithromycin than
against the metronidazole-resistant one.
− 1
of 0.3 ml min and a gradient of (5–95)% B over 10 min and then 100% B
over 2 min was used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1%
HCO H. LC/MS data were obtained by scanning the first quadrupole in 0.5 s in
2
The lowest MIC value was observed with compound 3 on the a mass range from 50 to 1000 Da; 8 scans were summed up to produce the final
reference H. pylori strain ATCC 43504 and was estimated to be spectrum. The purity of obtained compounds was confirmed by the TLC,
−
1
carried out on pre-coated plates (silica gel, 60F-254, Merck, Darmstadt,
Germany) using the solvent systems mentioned below. The obtained corre-
sponding spots were visualized under UV light.
The synthetic route of the tested compounds and starting materials is
outlined in Scheme 1. The compounds mentioned above were obtained by
5
mg l , whereas the clinical H. pylori strain showed MIC value of
−
1
about 150 mg l . The clinical and reference strains differ by their
profiles and mechanisms of resistance. This can be the cause of
differences between their susceptibility to the same compound.
The analysis of structure–activity relationship revealed that presence
of two hydroxylic groups in the amine moiety strongly contributed to
means of a multistage synthesis. In the first stage, the xanthone skeleton was
24,25
synthesized. This synthesis included Ullmann’s condensation
of appropriate
the reduction in anti-H. pylori activity. Until now, hydroxylic groups chlorobenzoic acid and phenol derivative. The reaction was performed for few
were considered to be a factor enhancing anti-H. pylori activity, but hours—in oil—at 195–200 °C, in the presence of Cu/Cu2O catalyst. After
condensation, intermediate product underwent cyclization in the presence of
concentrated H SO . In case of 2-methoxyphenol derivatives, concomitant
2 4
demethylation occurred. It was noticed that the longer the mixture was heated,
the more effective demethylation was achieved. The optimal refluxing time was
these groups were connected directly to the xanthone scaffold (not to
the side chain). It suggests that activity of the tested compounds is
8
determined more by the structure and spherical configuration than the
hydrophilic character.
We also observed that the compounds containing tert-
butylamine substructure were more active than those containing
iso-propylamine group.
2
days. Then the reacting mixture was poured into crushed ice, filtered and
washed to neutral reaction. Unreacted acid was washed out with NaHCO3
aqueous solution. The detailed methodology and physicochemical properties of
4-hydroxy-9H-xanthen-9-one, 3-chloro-5-hydroxy-9H-xanthen-9-one and
However, we did not observe the structure–activity relationship 3-chloro-5-methyl-9H-xanthen-9-one are described in26–28 3-Chloro-5-
between HCl salts and free-base forms. To ascertain that we methyl-9H-xanthen-9-one underwent methanolysis. Sodium methoxide was
prepared by gradual dissolution of sodium in an excess of methanol (with
performed comparative analysis of the same structure in different
cooling). Then equimolar amount of 3-chloro-5-methyl-9H-xanthen-9-one
forms (base and salt)—compounds
similar zones of growth inhibition for these two forms as shown in
Table 1.
It can be concluded that xanthone derivatives are potential
anti-H. pylori agents. Their activity vary depending on place and type
1 and 1A—we obtained
was added and the mixture was heated for about 48 h. After distillation to
dryness, water and H2SO4 were added to the residue. The precipitate was
filtered and dried. 3-Methoxy-5-methyl-9H-xanthen-9-one was subjected to
demethylation using a tenfold excess of condensed H SO to obtain 3-hydroxy-
2
4
5-methyl-9H-xanthen-9-one. This newly synthetized compound was recrystal-
of substitution in the xanthone core. More-detailed findings lized from ethanol and its physicochemical properties are described below. At
concerning the structure–activity relationship can be obtained from this point the synthesis diverged into two separate pathways, one promoting the
microbiological analysis of a wider group of structures. Currently, we bromopropoxy derivative of xanthone and the other the oxirane products.
5
-(3-Bromopropoxy)-3-chloro-9H-xanthen-9-one was synthesized by the reac-
screen other xanthone derivatives in order to explain their mechanism
of anti-H. pylori action.
tion of 3-chloro-5-hydroxy-9H-xanthen-9-one with 3-chloropropanol in acet-
one, in the presence of a large excess of K CO . Next, the solvent was distilled
2
3
off and the unreacted hydroxyxanthone was elutriated using NaOH solution.
The intermediate product was brominated using PBr3 in CHCl3. Subsequently,
CHCl3 was distilled off and the unreacted intermediate product was washed out
with NaHCO3 aqueous solution. The crude product was recrystallized from
n-hexane/toluene (1:6). The physicochemical properties of 5-(3-bromopro-
MATERIALS AND METHODS
Chemistry
Reagents: 2-amino-2-methylpropan-1,3-diol, 2-amino-2-methylpropan-1-ol,
2
,3-dimethoxyphenethylamine, ethyl 1-piperazinecarboxylate, 2-methoxyben-
zylamine, 2-(methylamino)ethanol, benzylpiperazine, epichlorohydrin and
phenoxyethylpiperazine were purchased from Sigma-Aldrich Chemie
29
poxy)-3-chloro-9H-xanthen-9-one were described formerly. The oxirane
derivatives of xanthone were obtained in the reaction of appropriate hydro-
xyxanthone with epichlorohydrin according to the procedures described
(
Steinheim, Germany), whereas the reagents: allylamine, t-butylamine,
2
1
-chlorobenzoic acid, 3-chloro-1-propanol, 2,4-dichlorobenzoic acid,
-(2-fluorophenyl)piperazine, 2-methoxyphenol, 2-methoxyphenylpiperazine,
30
earlier. Epichlorohydrin was used in double excess. Reacting mixture was
stirred overnight at the room temperature. Next, the precipitate was filtered and
the unreacted substrates were washed out with 10% NaOH solution.
Physicochemical properties of 4-((oxiran-2-yl)methoxy)-9H-xanthen-9-one
N-tert-butylmethylamine, phosphorus tribromide and i-propylamine were
provided by Lancaster Synthesis (Frankfurt am Main, Germany). Solvents were
commercially available and of reagent grade.
26
were described by Marona et al. in 2008, and the characteristic of 3-chloro-
Melting points (m.p.) are uncorrected and were determined using a Büchi
-((oxiran-2-yl)methoxy)-9H-xanthen-9-one was published by Marona et al.27
5
SMP-20 apparatus (Büchi Labortechnik, Flawil, Switzerland). The 1H NMR in 2009. 5-Methyl-3-((oxiran-2-yl)methoxy)-9H-xanthen-9-one was a new
were recorded on a Bruker AMX spectrometer (Brucker, Karlsruhe, Germany) compound, recrystallized from ethanol and its physicochemical properties are
with 500.13 MHz or Varian Mercury-VX 300 NMR spectrometer (Varian, Palo summarized below.
Alto, CA, USA) using signal from DMSO in DMSO-d6 and CHCl3 in CDCl3 as
Compounds 1–19 were obtained by amination of the parent compounds
an internal standard. The results are presented in the following format: with appropriate amines in n-propanol (1–18) or toluene in the presence of
13,16,31
chemical shift δ (p.p.m.), multiplicity, J-values in Hz, number of protons
and proton’s position. Multiplicities are abbreviated as follows: s (singlet), bs distilled off. The resulting amines were dissolved in diluted HCl, cleaned with
broad singlet), d (doublet), dd (doublet of doublets), ddd (double doublet of charcoal and precipitated using NaOH solution. Some of the bases were
doublets), dddd (double doublet of double doublets), td (triplets of doublets), converted into hydrochloride salts using an excess of ethanol saturated with
K CO (19), as described previously.
After aminolysis, the solvents were
2
3
(
The Journal of Antibiotics

Anti-Helicobacter pylori activity of xanthone derivatives
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7
O
COOH
OH
C H ONa
2 5
+
Cu/Cu O
2
R
Cl
HO
1
R
O
R = H, Cl
1
1
R
R
R = CH , OCH
3
3
H SO
2
4
O
O
O
O
O
O
O
H SO
NaOCH3
2
4
H CO
3
O
Cl
R
HO
CH3
CH3
OH
CH3
3
-chloropropan-1-ol/K CO /
2
3
acetone
O
NaOH/
epichlorohydrine
Cl
O
O
OH
PBr /chloroform
3
O
O
O
O
O
R
O
Cl
O
O
O
CH3
O
O
Br
aminolysis/
propan-1-ol
aminolysis/
propan-1-ol
aminolysis/K CO /
2 3
toluene
O
O
O
2
R
O
O
R
O
OH
Cl
O
CH3
CH3
2
OH
CH3
O
R
O
NH
OH
2
2
R = amine
R = amine
Scheme 1 Synthesis of the xanthone derivatives.
HCl. The crude products were recrystallized from acetone/ethanol (1:3). Most 8.01 (dd, J = 7.6, J = 2.7, 1H, H-1), 7.97 (dd, J = 8.0, J = 1.5, 1H, H-8), 7.66
of the synthesized compounds are new and their physicochemical properties are (dd, J = 8.0, J = 1.5, 1H, H-6), 7.31 (t, J = 8.0, 1H, H-7), 6.92-6.87 (m, 2H, H-2,
+
summarized below. Some structures were evaluated previously for their H-4), 2.48 (s, 3H, -CH ). LC-MS: calcd for [M+H] : C H O m/z: 227.06,
3
14 11 3
cardiovascular activity and their physicochemical properties were described found 227.12. R = 0.70 (toluene/acetone (5:3)).
F
elsewhere (compounds 5, 14, 15—Marona et al.,27 compound 8, 10—Marona
5-Methyl-3-((oxiran-2-yl)methoxy)-9H-xanthen-9-one was obtained as a
et al.32 and compounds 17, 18—Szkaradek et al. ). Compounds 3, 4, 11 are white solid (yield 55%), m.p. 153–154 °C. Anal calcd for C H O : C, 72.33;
29
17 14 4
taken under consideration for publication in Bioorganic and Medicinal
1
H, 5.00; found: C, 72.44; H, 5.11. H NMR (DMSO-d ,300 MHz): δ (p.p.m.)
6
Chemistry according to their additional anticancer activity. Compounds 1 and
8
.07 (d, J = 9.0, 1H, H-1), 7.98 (dd, J = 7.6, J = 1.8, 1H, H-8), 7.68 (d, J = 7.6,
H, H-6), 7.33 (t, J = 7.6, 1H, H-7), 7.19 (d, J = 2.3, 1H, H-4), 7.06 (dd, J = 9.0,
1A possessed the same structure but differed by the form: 1 is free base,
1
whereas 1A is a HCl salt. These compounds were evaluated to compare
influence of the form of the compound on its microbiological activity.
Structures of the tested compounds are presented in Table 1.
J = 2.3, 1H, H-2), 4.56 (dd, J = 11.5, J = 2.3, 1H, Ar-O-CHH-), 4.01
dd, J = 11.5, J = 6.9, 1H, Ar-O-CHH-), 3.39 (dddd, J = 6.9, J = 4.7, J = 2.7,
(
J = 2.3, 1H, = CH-), 2.88 (t, J = 4.7, 1H, -CHH-), 2.74 (dd, J = 4.7, J = 2.7, 1H,
3
-Hydroxy-5-methyl-9H-xanthen-9-one was obtained as a white solid (yield
+
-
CHH-), 2.50 (s, 1H, -CH3). LC-MS: calcd for [M+H] : C17H15O4 m/z: 283.29,
5
7
0%), m.p. 288–290 °C. Anal calcd for C H O : C, 74.33; H, 4.46; found: C,
14 10 3
found 283.04. RF = 0.80 (toluene/acetone (5:3)).
1
4.15; H, 4.44. H NMR (DMSO-d , 300 MHz): δ (p.p.m.) 10.93 (s, 1H, -OH),
6
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Anti-Helicobacter pylori activity of xanthone derivatives
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1
3
-(2-Hydroxy-3-(propan-2-ylamino)propoxy)-5-methyl-9H-xanthen-9-one
58.26; H, 5.62; N, 3.40. Found: C, 58.07; H, 6.13; N, 3.41. H NMR (DMSO-d6,
+
(
compound 1) was obtained as a white solid (yield 65%), m.p. 126–127 °C.
300 MHz) δ (p.p.m.): 8.50 (bs, 2H, -NHH ), 8.19 (d, J = 8.4, 1H, H-8), 7.80
Anal calcd for C H NO Cl: C, 63.57; H, 6.40; N, 3.71. Found: C, 63.52; H, (d, J = 1.8, 1H, H-5), 7.76 (dd, J = 8.1, J = 1.5, 1H, H-1), 7.60 (dd, J = 8.0,
2
0
24
4
1
6
.70. N, 3.65. H NMR (CDCl3, 300 MHz): δ (p.p.m.) 8.25 (dd, J = 6.8, J = 2.6,
J = 1.2, 1H, H-3), 7.55 (dd, J = 8.7, J = 2.1, 1H, H-7), 7.42 (t, J = 8.1, 1H, H-2),
.92 (bs, 1H, -OH), 4.26 (bs, 3H, = CH-OH, Ar-O-CH2-), 3.40-3.06 (m, 2H,
CH2-N-), 1.32 (s, 9H, -(CH3)3). RF = 0.16 (methanol/ethyl acetate (1/1)).
-Chloro-5-(2-hydroxy-3-(1-hydroxy-3-methylbutan-2-ylamino)propoxy)-
H-xanthen-9-one (compound 16) was obtained as a white solid (yield 70%),
1
H, H-1), 8.17 (dd, J = 8.0, J = 1.8, 1H, H-8), 7.54 (dd, J = 7.2, J = 1.8, 1H,
5
H-6), 7.29-7.23 (m, 3H, H-4, H-2, H-7), 7.00-6.95 (m, 2H,-OH, -NH-),
.14-4.03 (m, 3H, Ar-O-CH2-, =CH-OH), 2.94 (dd, J = 12.1, J = 3.8, 1H,
-
4
3
-CHH-NH-), 2.85 (qu, J = 6.2, 1H, = CH-), 2.76 (dd, J =12.2, J = 7.6, 1H,
-CHH-NH-), 2.55 (s, 3H, -CH3), 1.11 (d, J = 6.2, 6H, (-CH3)2). RF = 0.16
(methanol).
9
m.p. 133-135 °C. Hydrochloride of compound 16: Anal calcd for
C21H25O5NCl2: C, 57.02; H, 5.70; N, 3.17. Found: C, 56.91; H, 6.06; N,
3
-(2-Hydroxy-3-(prop-2-en-1-ylamino)propoxy)-5-methyl-9H-xanthen-9-
1
+
3
.14. H NMR (DMSO-d , 300 MHz): δ (p.p.m.) 8.77 (bs, 1H, -NHH -), 8.37
6
one (compound 2) was obtained as a white solid (yield 68%), m.p. 94–96 °C.
Anal calcd for C20H21O4N: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.61; H, 6.03;
N, 4.08. H NMR (CDCl , 300 MHz): δ (p.p.m.) 8.25 (dd, J = 6.4, J = 2.8,
+
(
(
(
bs, 1H, -NHH -), 8.18 (d, J =8.5, 1H, H-8), 7.83 (d, J = 2.1, 1H, H-5), 7.75
dd, J = 8.1, J = 1.4, 1H, H-1), 7.59 (dd, J = 8.1, J = 1.4, 1H, H-3), 7.54
dd, J = 8.5, J =2.1, 1H, H-7), 7.41 (t, J = 8.1, 1H, H-2), 5.96 (d, J = 4.4, 1H,
1
3
1
H, H-1), 8.17 (dd, J = 8.0, J = 1.8, 1H, H-8), 7.54 (dd, J = 8.0, J = 1.8, 1H,
4
CH-OH), 5.13 (bs, 1H, -CH -OH), 4.30-4.19 (m, 3H, 4CH-, Ar-O-CH - ),
2
2
H-6), 7.30-7.23 (m, 3H, H-4, H-4, H-7), 7.00-6.95 (m, 2H, -OH, -NH), 5.82-
3.61 (t, J = 6.3, 2H, -CH2-OH), 3.26 (bs, 1H, -CHH-NH-, overlap with signal
6
.00 (m, 1H, -CH= ), 5.22 (ddd, J = 16.9, J = 3.3, J = 1.3, 1H, = CoHH
from H2O in DMSO), 3.07 (bs, 1H, -CHH-NH-), 1.89-1.77 (m, 2H, 4CH-
(trans)), 5.14 (ddd, J = 10.5, J = 3.3, J = 1.3, 1H, =CoHH (cis)), 4.17-4.09
CHo), 1.34 (d, J = 1.3, 6H, (-CH3)2). RF = 0.13 (methanol).
(
m, 3H, Ar-O-CH2-, = CH-OH), 3.35-3.30 (m, 2H, = CH-CH2-NH- ),
3-Chloro-5-(3-(1-hydroxy-2-methylpropan-2-ylamino)propoxy)-9H-
2
.97-2.76 (m, 2H, -CH2-), 2.55 (s, 3H, -CH3). RF = 0.29 (methanol/ethyl
acetate (1/1)).
-(3-(2-Methoxybenzylamino)-2-hydroxypropoxy)-9H-xanthen-9-one
compound 6) was obtained as a white solid (yield 75%), m.p. 138–140 °C. H
NMR (CDCl3, 300 MHz): δ (p.p.m.) 8.33 (dd, J = 8.2, J = 1.5, 1H, H-8), 7.93 (d, J = 8.5 Hz, 1H, H-8), 7.89 (dd, J =7.4, J = 2.1, 1H, H-1), 7.65 (d, J = 2.1,
xanthen-9-one (compound 19) was obtained as a white solid (yield 60%), m.p.
51–153 °C. Anal calcd for C20H23O4NCl2: C, 58.26; H, 5.62; N, 3.40. Found:
C, 58.25; H, 6.25; N, 3.34. 1H NMR (CDCl , 300 MHz): δ (p.p.m.) 8.27
1
4
1
(
3
(
(
7
t, J = 4.6, 1H, H-2), 7.70 (ddd, J = 8.5, J = 7.1, J = 1.5, 1H, H-6), 7.45 1H, H-5), 7.36 (dd, J = 8.5, J = 2.1, 1H, H-7), 7.29 (d, J = 7.4, 1H, H-3), 7.27
dd, J = 8.5, J = 0.7, 1H, H-5), 7.38 (ddd, J = 8.2, J = 7.1, J =0.7, 1H, H-7),
(m, 1H, H-2), 4.26 (t, J = 6.3, 2H, Ar-O-CH -), 3.33 (s, 2H, -CH -OH), 2.82
.28 (d, J =4.6, 2H, H-1, H-7), 7.26-7.22 (m, 2H, H-3’, H-4’), 6.92 (td, J = 7.7,
2
2
2 2 2 2
(t, J = 6.3, 2H, -CH -NH-), 2.08 (qu, J = 6.3, 2H, -CH -CH -CH -), 1.50
J = 1.0, 1H, H-5’), 6.87 (d, J =7.7, 1H, H-6’), 4.25-4.15 (m, 3H, -CH= ,
(bs, 2H, -NH-, -OH), 1.11 (s, 6H, (-CH ) ). R = 0.07 (methanol).
3 2 F
Ar-O-CH -), 3.94-3.83 (m, 2H, -NH-CH -Ar), 3.80 (s, 3H, -O-CH ),
2
2
3
3
.00–2.85 (m, 2H, -CH2-NH-). Hydrochloride of compound 6: Anal calcd
Microbiology
for C24H24O5NCl: C, 65.23; H, 5.47; N, 3.17. Found: C, 64.62; H, 5.58; N, 3.15.
The preliminary assay of antimicrobial activity of the 20 newly synthesized
xanthone derivatives against selected representative bacterial strains were
conducted. Antimicrobial activity was tested in vitro by the Kirby–Bauer
method (agar disc-diffusion method).33,34 This method was used for qualitative
as well as quantitative testing.
The first step of the microbiological examination was the qualitative testing.
The aim of this step was to select the compounds with the strongest
anti-H. pylori activity. The activity of 19 compounds was tested to selected
H. pylori strains. S. aureus and E. coli strains were used as controls.
The second step of the microbiological examination consisted of quantitative
testing that aimed to assess the MIC value of the compound with the strongest
anti-H. pylori activity.
RF =0.65 (methanol:ethyl acetate (1:1)).
4
-(3-(3,4-Methoxyphenyl)ethylamino-2-hydroxypropoxy)-9H-xanthen-9-
one hydrochloride (compound 7) was obtained as a white solid (yield 75%), m.
p. 187–188 °C. Anal calcd for C26H28O6NCl: C, 64.26; H, 5.81; N, 2.88. Found:
1
C, 64.06; H, 6.19; N, 2.88. H NMR (DMSO-d , 300 MHz): δ (p.p.m.) 8.65
6
+
(
bs, 2H, -NHH -), 8.19 (dd, J =8.0, J = 1.5, 1H, H-8), 7.88 (ddd, J = 8.6,
J = 6.9, J = 1.5, 1H, H-6 ), 7.76 (dd, J = 8.0, J = 1.5, 1H, H-1), 7.69 (dd, J = 8.6,
J = 1.0, 1H, H-5), 7.57 (dd, J = 8.0, 1.5 Hz, 1H, H-3), 7.49 (ddd, J =8.0, J = 6.9,
J = 1.0, 1H, H-7), 7.39 (t, J = 8.0, 1H, H-2), 6.86 (d, J = 8.2, 1H, H-5’), 6.84
(
4
d, J = 2.1, 1H, H-2’), 6.75 (dd, J = 8.2, J = 2.1, 1H, H-6’), 5.97 (bs, 1H, -OH),
.36-4.28 (m, 1H, -CH=), 4.26-4.17 (m, 2H, Ar-O-CH2-), 3.72 (s, 3H,
-
O-CH3), 3.69 (s, 3H, -O-CH3), 3.27-3.10 (m, 4H, -CH2-NH-CH2-), 2.86-2.95
Microbiological examination for the antibacterial activity of 20 xanthone
derivatives against representative bacterial strains was carried out in the
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Jagiellonian
University Medical College in Krakow, Poland.
(
m, 2H, -CH -Ar). R = 0.15 (methanol).
2
F
4
-(3-(4-(2-Fluorophenyl)piperazin-1-yl)-2-hydroxypropoxy)-9H-xanthen-9-
one dihydrochloride (compound 9) was obtained as a white solid (yield 70%),
m.p. 224–226 °C. Anal calcd for C26H27O4N2Cl2F: C, 59.89; H, 5.22; N, 5.38.
Found: C, 59.51; H, 5.18; N, 5.17. H NMR (DMSO-d , 300 MHz): δ (p.p.m.)
1
J = 6.9, J = 1.8, 1H, H-6), 7.76 (dd, J = 8.2, J = 1.3, 1H, H-1, H-5), 7.60 (dd,
J = 8.2, J = 1.3, 1H, H-3), 7.50 (ddd, J = 8.0, J = 6.9, J =1.3, 1H, H-7), 7.39
1
6
Bacterial strains. Twelve representative bacterial strains were used in the
microbiological examination. Seven clinical H. pylori strains were included in
the study. Strains were isolated from the endoscopic biopsy specimens of gastric
mucosa from dyspeptic patients who applied to the Falck Medycyna Outpatient
Clinic of Gastroenterology (Krakow, Poland). Isolation of H. pylori strains from
biopsy samples as well as antibacterial susceptibility testing to clarithromycin
and metronidazole with the use of strips impregnated with antimicrobial agents
gradients (E-test, bioMérieux, Lyon, France) were performed as previously
described by Karczewska et al.35
+
0.55 (bs, 1H-NH -), 8.20 (dd, J = 8.0, J = 1.8, 1H, H-8), 7.90 (ddd, J = 8.2,
(t, J = 8.2, 1H, H-2), 7.00-7.21 (m, 4H, H-Ph), 6.02-6.16 (m, 1H, -OH), 4.57
(bs, 1H, -CH= ), 4.23 (d, J = 4.6, 2H Ar-O-CH2-), 3.84-3.12 (m, 10H,
(-N-CH2-)5). RF = 0.86 (methanol/ethyl acetate (1/1)).
3
-Chloro-5-(2-hydroxy-3-(propan-2-ylamino)propoxy)-9H-xanthen-9-one
(
compound 12) was obtained as a white solid (yield 72%), m.p. 169–170 °C.
Anal calcd for C19H20O4NCl: C, 63.07; H, 5.57; N, 3.87. Found: C, 62.70; H,
5
.79; N, 3.51. RF = 0.23 (methanol/ethyl acetate 1/1). Hydrochloride of the
In addition, we used two H. pylori reference strains and single reference
compound 12: H NMR (DMSO-d , 300 MHz) δ (p.p.m.): 8.76 (bs, 2H, -NHH strains of S. aureus and E. coli.
1
6
+
)
, 8.18 (d, J = 8.4, 1H, H-8), 7.85 (d, J = 1.8, 1H, H-5), 7.74 (dd, J = 8.1,
Clinical strains were as follows:
J = 1.5, 1H, H-1), 7.59 (dd, J = 8.1, J =1.5, 1H, H-3), 7.54 (dd, J =8.6, J = 2.1,
H, H-7), 7.41 (t, J = 7.8, 1H, H-2), 5.96 (bs, 1H, -OH), 4.34 (bs, 1H, = CH-
1
- three H. pylori strains resistant to metronidazole: HP 125/180, HP 139/202
and HP 143/207;
OH), 4.24-4.22 (m, 2H, Ar-O-CH -), 3.40-3.06 (m, 3H, -CH -N-, -CH = ),
2
2
1
.28 (dd, J = 6.4, J =2.7, 6H, -(CH3)2).
3
- two H. pylori strains resistant to clarithromycin: HP 115/168 and HP
132/194;
- two double-resistant H. pylori strains (that is, combined resistance to
clarithromycin and metronidazole): HP 106/154 and HP 126/181.
-Chloro-5-(2-hydroxy-3-((2-methylpropan-2-ylamino)propoxy)-9H-
xanthen-9-one (compound 13) was obtained as a white solid (yield 70%), m.p.
54–155 °C. Hydrochloride of compound 13: Anal calcd for C H O NCl : C,
1
20
23
4
2
The Journal of Antibiotics

Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
9
Table 2 Clarithromycin and metronidazole MIC values of twelve
tested strains (clinical and reference H. pylori strains, reference
S. aureus strains, reference E. coli strain)
quantitative testing and to determine the MIC value of each compound. The
dilutions were as follows:
−
1
-
-
-
-
-
-
-
-
-
-
-
-
5000 mg l (0.5%);
−
1
1000 mg l (0.1%);
MIC values of clarithromycin MIC values of metronidazole
−
1
500 mg l (0.05%);
Strains
(mg l− 1)
(mg l− 1
)
− 1
200 mg l (0.02%);
−
1
150 mg l (0.015%);
Strains other than H. pylori
−
1
100 mg l (0.01%);
S. aureus ATCC
0.5
4256
64
4256
4256
4256
−
1
75 mg l (0.0075%);
2
1
2
5923
−
1
50 mg l (0.005%);
S. aureus MRSA
4.002
−
1
10 mg l (0.001%);
−
1
7.5 mg l (0.00075%);
E. coli ATCC
5922
− 1
5 mg l (0.0005%);
− 1
2.5 mg l (0.00025%).
H. pylori strains resistant to clarithromycin
Susceptibility testing of bacterial strains. Qualitative and quantitative assessment
of the susceptibility of H. pylori strains to novel, potential antimicrobial agents
was assessed by the Kirby–Bauer method (agar disk-diffusion method).
H. pylori ATCC
00684
32
0.094
7
HP 132/194
HP 115/168
4256
0.5
Qualitative testing. Qualitative testing was performed with the use of test discs
24
0.094
−
1
impregnated with 1% (10 000 mg l ) solutions of the compounds. In this part
of study all 20 synthesized compounds were tested on all 12 bacterial strains.
The analysis was conducted according to the procedure described below.
Bacterial suspensions were prepared from pure cultures in 0.85% aqueous
NaCl solution (bioMérieux) in order to obtain an equivalent of 3.0 McFarland
units (H. pylori) or 0.5 McFarland units (S. aureus and E. coli).
H. pylori strains resistant to metronidazole
H. pylori ATCC
3504
0.032
96
4
HP 125/180
HP 139/202
HP 143/207
o0.016
0.032
48
4256
4256
The suspensions of H. pylori were inoculated on the Schaedler agar with 5%
sheep blood (bioMérieux) with sterile cotton swab. The suspensions of
S. aureus or E. coli were applied onto Mueller–Hinton Agar. The inoculated
agar was allowed to dry for 15 min. Test discs impregnated with the tested
compounds were applied onto the inoculated agar.
o0.016
Double-resistant H. pylori strains
HP 126/181
4
4256
4256
HP 106/154
4256
The plates with H. pylori were incubated in microaerophilic atmosphere at
37 °C for 72 h, whereas the plates with S. aureus or E. coli were incubated in
aerobic atmosphere at 37 °C for 24 h. After incubation, the diameters of
complete growth inhibition zones were measured. Antibacterial activity was
Abbreviation: MIC, minimal inhibitory concentration. Bold values represent the compounds
revealing significant activity.
The resistance profiles of H. pylori strains were evaluated according to the expressed as the mean of complete inhibition diameters (mm) produced by the
European Committee on Antimicrobial Susceptibility Testing (EUCAST) tested compounds. A growth inhibition zone diameter of 8 mm was defined as
guidelines. The MIC values of clarithromycin and metronidazole were the breakpoint to classify bacteria as susceptible to the tested compounds in
adequate dilutions.
determined with the use of the strips impregnated with the antibiotic gradient
E-test, bioMérieux). The procedure of the examination was performed under
Qualitative testing was performed in order to choose the compounds with
the strongest activity against H. pylori. The interpretative criteria were as
follows:
(
the manufacturer’s instructions. In accordance with the EUCAST guidelines, H.
pylori strains were found as metronidazole or clarithromycin resistant when the
MIC values for metronidazole and clarithromycin were 48 and 40.5 mg l 1,
−
36
- zone of growth inhibitiono8 mm—compound showed no antibacterial
respectively.
activity;
Moreover, the reference strains were used to the examination. We used the
following strains from the American Type Culture Collection (ATCC):
-
-
zone of growth inhibition⩾ 8 mm—compound showed antibacterial
activity;
zone of growth inhibition⩾ 30 mm—compound showed strong antibac-
terial activity.
-
-
-
-
H. pylori strain resistant to metronidazole: ATCC 43504;
H. pylori strain resistant to clarithromycin: ATCC 700684;
S. aureus ATCC 25923;
We also conducted reference qualitative testing with the use of antibiotic. In
order to compare antibacterial activity of the tested compounds to other
antimicrobial agents and to confirm the efficacy of the chosen method,
clarithromycin and metronidazole were used as a reference compounds.
E. coli ATCC 25922.
One methicillin-resistant S. aureus 14.002 strain was also used. This strain
originated from reference collection MIKROBANK of A National Reference
Center for Microbial Susceptibility Testing in Poland.
Qualitative testing. Quantitative testing was conducted with the use of
Susceptibility of the tested strains to clarithromycin and metronidazole fourteen most active compounds (1, 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 16
expressed by MIC values were presented in Table 2.
and 19), for which the stock solutions were diluted to prepare twelve decreasing
solutions tested subsequently against the two selected H. pylori strains (ATCC
Preparation of the compound samples. Qualitative testing was performed with
the use of 1% (10 000 mg l 1) solutions of the tested compounds (synthesized
at the Department of Technology and Biotechnology of Drugs, Jagiellonian
University Medical College) in dimethylsulfoxide (Merck).
4
3504 and 132/194). We also applied the agar disc-diffusion method. Zone of
−
growth inhibitiono8 mm was considered as indicating no antibacterial activity,
whereas an inhibition zone ⩾ 8 mm was considered as indicating antibacterial
activity. The MIC value was considered to be the lowest concentration of the
The most active compounds were classified to the second step of examined compound that inhibited the growth of bacteria. Therefore, the last
microbiological examination, that is, quantitative testing. Twelve decreasing dilution at which antibacterial activity was expressed determined the MIC value
dilutions of the tested compounds in DMSO were prepared in order to perform (zone ⩾ 8 mm).
The Journal of Antibiotics

Anti-Helicobacter pylori activity of xanthone derivatives
K Klesiewicz et al
1
0
We performed qualitative and quantitative tests (Kirby–Bauer method
estimating MIC) to obtain values for clarithromycin and metronidazole against
all clinical strains. Moreover we estimated the MIC values for clarithromycin
and metronidazole using the strips impregnated with the antibiotic gradient
14 IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Schistosomes,
Liver Flukes and Helicobacter pylori/World Health Organization, International Agency for
Research on Cancer 61, 177–242 (WHO: Geneva, Switzerland, 1994).
1
1
1
1
5 Ando, T. et al. Causal role of Helicobacter pylori infection in gastric cancer.
World J. Gastroenterol. 12, 181–186 (2006).
6 Wroblewski, L. E., Peek, R. M. & Wilson, K. T. Helicobacter pylori and gastric cancer:
factors that modulate disease risk. Clin. Microbiol. Rev. 23, 713–739 (2010).
7 Konturek, P. C., Konturek, S. J. & Brzozowski, T. Helicobacter pylori infection in gastric
cancerogenesis. J. Physiol. Pharmacol. 60, 3–21 (2009).
(
E-test, bioMérieux). The procedure of the examination was performed under
the manufacturer’s instructions. MIC values obtained using Kirby–Bauer
method and E-test were the same, so we can conclude that the efficacy and
accuracy of the chosen Kirby–Bauer method was confirmed. Table 2 shows the
MIC values to antimicrobial agents of clinical H. pylori strains.
8 Konturek, S. J., Konturek, J. W., Plonka, M., Brzozowski, T.
& Bielanski, W.
Helicobacter pylori and its involvment in gastritis and peptic ulcer formation. J. Physiol.
Pharmacol. 57, 29–50 (2006).
1
2
2
9 Pellicano, R. et al. Helicobacters and extragastric diseases. Helicobacter 14,
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8–68 (2009).
CONFLICT OF INTEREST
0 Banić, M., Franceschi, F., Babić, Z. & Gasbarrini, A. Extragastric manifestations of
Helicobacter pylori infection. Helicobacter 17, 49–55 (2012).
1 Figura, N. et al. Extragastric manifestations of Helicobacter pylori infection. Helico-
bacter 15, 60–68 (2010).
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The study was supported by research grants from the Ministry of Science and
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