In vitro inhibitory activity of boropinic acid against Helicobacter pylori

Article abstract of DOI:10.1016/j.bmcl.2006.08.043

In this study, we assessed in vitro minimum inhibitory concentration (MIC) values of some natural geranyloxycoumarins, geranyloxy- and isopentenyloxy acids against growth of Helicobacter pylori. Boropinic acid, active principle isolated from Boronia pinnata (Fam. Rutaceae), was seen to be the most effective compound with a MIC value of 1.62 μg/mL.

Full text of DOI:10.1016/j.bmcl.2006.08.043

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5523–5525
In vitro inhibitory activity of boropinic acid against
Helicobacter pylori
Francesco Epifano,a,* Luigi Menghini,a Rita Pagiotti,b Paola Angelini,b
Salvatore Genovesec and Massimo Curinic
aDipartimento di Scienze del Farmaco, Via dei Vestini 31, 66013 Chieti Scalo (CH), Italy
bDipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Borgo XX Giugno, 74, 06126 Perugia, Italy
cDipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Via del Liceo, 06123 Perugia, Italy
Received 10 July 2006; revised 5 August 2006; accepted 8 August 2006
Available online 30 August 2006
Abstract—In this study, we assessed in vitro minimum inhibitory concentration (MIC) values of some natural geranyloxycoumarins,
geranyloxy- and isopentenyloxy acids against growth of Helicobacter pylori. Boropinic acid, active principle isolated from Boronia
pinnata (Fam. Rutaceae), was seen to be the most effective compound with a MIC value of 1.62 lg/mL.
Ó 2006 Elsevier Ltd. All rights reserved.
Helicobacter pylori is a highly motile, gram-negative
microaerophilic bacterium thought to be an infective
agent of more than 50% of the world population and
can be considered the most common chronic infection
for humans.1 Moreover, H. pylori is now well known
to be the main causal factor in the etiogenesis of chronic
active or type B gastritis, peptic and duodenal ulcer, gas-
tric carcinoma, and mucosa-associated lymphoid tu-
mors.2 Pharmacological treatment of H. pylori
infections includes administration of a proton pump
inhibitor and a combination of two or more antibiotics,
among which the most active ones are amoxicillin, clar-
ithromycin, metronidazole, and tetracyclines.3 However,
bacterial resistance to these antibiotics is a growing
problem: for example, metronidazole is no longer effec-
tive in 10–50% of H. pylori-positive subjects and also
amoxicillin and clarithromycin resistance have increased
in the last few years.4 For these reasons, the search for
alternative therapeutic remedies for H. pylori infections
is a field of current interest.
quite a rare group of natural products. Among these,
coumarins, cinnamic, and benzoic acids have been
recently shown to exert valuable biological properties.5,6
In continuation of our research studies aimed to evalu-
ate pharmacological properties of this novel class of nat-
ural compounds, we wish to report here the activity of
some prenyloxy-phenylpropanoids, namely, 3-(40-gera-
nyloxyphenyl)-2-trans propenoic acid 1, 3-(40-geranyl-
oxy-30-methoxyphenyl)-2-trans propenoic acid 2, both
isolated from Acronychia baueri Schott (Fam. Ruta-
ceae),7 boropinic acid 3, isolated from Boronia pinnata
Sm. (Fam. Rutaceae),8 valencic acid 4, isolated from
Citrus sinensis L. and Aegle marmelos (Fam. Rutaceae),9
4-isopentenyloxy-3-methoxy benzoic acid 5, 4-geranyl-
oxy-3-methoxy benzoic acid 6, both isolated as methyl
esters from the liverwort Trichocolea lanata (Ehrh.)
Dumm. (Fam. Trichocolaceae),10 and finally auraptene,
7, the most common geranyloxycoumarin extracted
from plants belonging to genus Citrus,6 as inhibitors
of growth of H. pylori in vitro.
Secondary metabolites of phenylpropanoid biosynthetic
origin containing a sesquiterpenyl, monoterpenyl, and
isopentenyl chains attached to a phenol group represent
The synthesis of compounds 1, 3, 4, 5, and 6 was accom-
plished following an environment friendly route similar
to that we already reported for the synthesis of com-
pound 2.5 Compound 1 was obtained in 97% overall
yield starting from commercially available p-coumaric
acid that was first converted into its methyl ester by
reaction in refluxing MeOH catalyzed by concd.
H2SO4, then alkylated with geranyl bromide and hydro-
lyzed in a basic medium (Scheme 1).
Keywords: Boropinic acid; Helicobacter pylori; Prenyloxy-
phenylpropanoids.
*
Corresponding author. Tel.: +390 8713555321; fax: +390
8713555315; e-mail: fepifano@unich.it
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.043

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F. Epifano et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5523–5525
Table 1. MIC values for inhibition of growth against Helicobacter
pylori by prenyloxy cinnamic acids 1–6 and auraptene 7
R1
R3
O
MICa (lg/mL)
R2
Compound
1
>200
>200
1.62
100
1 R1 = -CH=CH-COOH, R2 = -H, R3 = geranyl
2 R1 = -CH=CH-COOH, R2 = -OCH3, R3 = geranyl
3 R1 = -CH=CH-COOH, R2 = -OCH3, R3 = isopentenyl
4 R1 = -COOH, R2 = -H, R3 = isopentenyl
2
3
4
5
50
>200
50
5 R1 = -COOH, R2 = -OCH3, R3 = isopentenyl
6 R1 = -COOH, R2 = -OCH3, R3 = geranyl
6
7
Metronidazole
Amoxicillin
Tetracycline
Clarithromycin
>200
0.781
4.00
1.25
O
O O
7
a Values are means of three experiments.
Compounds 3, 5, and 6 were obtained using the same
reaction conditions as above in 96%, 98%, and 99% yield
from ferulic acid and vanillic acid, respectively, while
compound 4 was synthesized in 99% yield by one-pot
alkylation-basic hydrolysis from commercially available
methyl p-hydroxybenzoate and employing in all cases 4-
bromo-2-methyl-2-butene or geranyl bromide as alkylat-
ing agents.11 Auraptene 7 was synthesized in 95% yield
as already described.12
are not significant for a comparison to those obtained
from other secondary metabolites tested as this strain
is clearly resistant to this antibiotic. However, these data
confirm that resistance of H. pylori strains of different
origin to metronidazole has become nowadays quite a
common phenomenon and that boropinic acid could
be claimed as a valuable alternative to bypass this
disadvantage.
From data reported in Table 1 it is also evident that the
presence of a geranyloxy side chain led to a substantial
decrease of activity: in fact, with the only exception of
auraptene (7), geranyloxy acids are virtually ineffective
against growth of H. pylori; on the other hand, the
MIC value recorded for auraptene (7) seems to indicate
that the presence of a coumarin ring could represent
another structural requirement for compounds to be ac-
tive. In fact it has been recently found that differently
substituted coumarins can act as antimicrobial com-
pounds, showing inhibitory activity also against H. pylo-
ri.14 The great difference of activity (from 30 to more
than 120 times) between boropinic acid (3) and all other
secondary metabolites tested seems to indicate that (3)
could have a specific site of action inside H. pylori cells.
Comparing structures of boropinic acid (3) to other
compounds tested it seems that three structural require-
ments may be necessary for the observed activity: an iso-
pentenyloxy side chain, an a,b-unsaturated carboxylic
acid, and an (E) geometry for the conjugated double
bond. In fact it can be seen that increasing the length
of the O-side chain to a geranyl one (compounds 1, 2,
and 6) led to a complete lack of activity, while eliminat-
ing the conjugated double bond (compounds 4, 5, and
6), changing the geometry of the double bond from
(E) to (Z), and introducing a lactone moiety instead of
a carboxylic acid one (compound 7) led to a significative
decrease of activity. These preliminary SAR consider-
ations could be the basis to consequently modify the
structure of boropinic acid (3) in order to obtain more
active compounds against H. pylori.
Compounds 1–7 were assayed using metronidazole,
amoxicillin, tetracycline, and clarithromycin as reference
drugs. The minimum inhibitory concentration (MIC)
tests were performed by the agar dilution method12
according to guidelines provided by NCCLS.13 MIC
was defined as the lowest concentration capable of
inhibiting bacterial colony formation. Results of the test
on inhibition of growth against H. pylori by auraptene
7 and prenyloxy cinnamic acids 1–6 are reported in
Table 1.
As shown in Table 1 only one among the secondary
metabolites tested, namely boropinic acid (3), showed
an appreciable activity against H. pylori. In fact the
inhibitory activity of compound (3) was comparable or
better than that of reference drugs with a MIC value
of 1.62 lg/mL, being by far more active in respect to
all other secondary metabolites, that were only slightly
active (compounds 4, 5, and 7) or totally inactive (com-
pounds 1, 2, and 6). Moreover, the weak activity ob-
served for compounds 4, 5, and 7, because of their
lipophilic nature, is likely due to non-specific binding
or aggregation effects. Data obtained by treating our
H. pylori strain with metronidazole as reference drug
COOH
CO2CH3
a
HO
HO
b,c
COOH
In conclusion, the findings described herein seem to indi-
cate boropinic acid (3) as a potential lead compound of
a novel class of H. pylori inhibitors. Results obtained,
considering also that (3) has been easily synthesized
from widely available and non-toxic starting materials
by a high-yielding, environment friendly, and cheap
O
1
Scheme 1. Reagents and conditions: (a) MeOH, concd H2SO4 (cat.),
reflux, 12 h; (b) geranyl bromide (1.2 equiv), K2CO3 (1.2 equiv),
acetone, reflux, 2 h; (c) NaOH 2N, 70 °C, 1 h.

F. Epifano et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5523–5525
5525
1
1690 cmꢀ1; H NMR: Ref. 7 13C NMR (100 MHz CDCl3
d): 16.1, 17.5, 25.6, 26.2, 39.4, 64.9, 115.3, 117.6, 119.8,
123.8, 128.3, 129.3, 131.4, 141.6, 144.2, 157.7, 168.9; Anal.
Calcd for C19H24O3: C, 75.97; H, 8.05; O, 15.98. Found C,
75.96; H, 8.07, O, 15.99.
synthetic route, prompt us to get further insights into
the mechanism of action of this secondary metabolite,
to perform test in vivo using a suitable animal model
and in vitro and in vivo tests aimed to evaluate the activ-
ity of boropinic acid (3) against strains of H. pylori iso-
lated from clinical patients and finally to synthesize and
test both in vitro and in vivo structural analogues of (3).
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans
propenoic
acid (2). White solid; yield: 96%; analytical data are in
full agreement with those reported in the literature.5
Boropinic acid (3). White solid; yield: 96%; analytical data
are in full agreement with those reported in the literature.8
Valencic acid (4). White solid; yield: 99%; mp: 131–132 °C;
IR: Ref. 15; 1H NMR: Ref. 15; 13C NMR (100 MHz
CDCl3 d): 18.7, 26.2, 66.5, 116.0, 120.9, 122.1, 132.2,
139.1, 162.7, 170.5; Anal. Calcd for C12H14O3: C, 69.89;
H, 6.84; O, 23.27. Found C, 69.88; H, 6.82, O, 23.26.
4-Isopentenyloxy-3-methoxy benzoic acid (5). White solid;
yield: 98%; analytical data are in full agreement with those
reported in the literature.10
Acknowledgments
Authors wish to acknowledge the financial support from
MIUR (Rome, Italy) National Project ‘Sviluppo di pro-
cessi sintetici ecocompatibili nella sintesi organica’
COFIN 2004 and Regione Abruzzo (L.R. 35/97) Project
`
‘Tutela della Biodiversita’.
4-Geranyloxy-3-methoxy benzoic acid (6). White solid;
yield: 98%; analytical data are in full agreement with those
reported in the literature.10
Supplementary data
Auraptene (7). White solid; yield: 95%; analytical data are
in full agreement with those reported in the literature.12
Microbiological tests. Strain of H. pylori (DSMZ 4867,
originated from human gastric samples) was cultured on
Columbia agar (Difco, Italy) with 4% horse blood (Difco,
Italy). Before tests, H. pylori plates were prepared by
subculturing onto Mueller–Hinton Agar supplemented
with 5% defibrinated horse blood and incubated for 48 h
microaerobically. An evaluation of the activity of each
compound with reference drugs was made by comparison
of bacterial growth degree in each plate of H. pylori.
Auraptene 7 and acids 1–6 were dissolved in dimethyl-
sulfoxide (DMSO). By serial double diluitions of
antimicrobial agents, they were diluted in melted
Mueller–Hinton Agar supplemented with 5% defibrinated
horse blood to give concentrations ranging from 200 to
0.781 lg/mL. They were inoculated with 5 lL of bacterial
suspension (107 CFU/mL) and incubated at 37 °C for 3
days under microaerobic conditions. An antimicrobic-free
plate and plates with corresponding dilutions of DMSO
were used as negative controls to ensure bacterial viability
and no contaminants in inoculums.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.08.043.
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