Synthesis and antimicrobial activity of geranyloxy- and farnesyloxy-acetophenone derivatives against oral pathogens

Article abstract of DOI:10.1016/j.fitote.2012.06.003

The compounds 2′,6′-dihydroxy-4′-geranyloxyacetophenone (1) and 2′,6′-dihydroxy-4′-farnesyloxy-acetophenone (2) are oxyprenylated secondary metabolites extracted from plants belonging to the Rutaceae family. In this study, 1 and 2 were synthesized and tested for their antimicrobial activity toward major oral pathogens. Compounds 1 and 2 were synthesized by selective prenylation of 2,4,6-trihydroxyacetophenone at the 4′ position with geranyl and farnesyl bromide, respectively. Compound 1 showed stronger antimicrobial activity than 2 against major oral pathogens, including Gram positive bacteria (Streptococcus mutans, Streptococcus sobrinus), Gram negative bacteria (Prevotella intermedia, Porphyromonas gingivalis) and Candida albicans. Evidences were obtained that the mode of action of 1 and 2 may be related to their iron-chelating property. This study suggests that 1 and 2 may represent potential natural molecules for the prevention/treatment of common oral infections, including dental caries, periodontal disease, and candidiasis.

Full text of DOI:10.1016/j.fitote.2012.06.003

Fitoterapia 83 (2012) 996–999
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Synthesis and antimicrobial activity of geranyloxy- and
farnesyloxy-acetophenone derivatives against oral pathogens
a
a
b
b
a,
Laetitia Bonifait , Annie Marquis , Salvatore Genovese , Francesco Epifano , Daniel Grenier ⁎
a
Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, 2420 Rue de la Terrasse, Québec City, QC, Canada, G1V 0A6
Dipartimento di Science del Farmaco, Università G. D'Annunzio, Via Dei Vestini 31, 66013 Chieti Scalo, Chieti, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The compounds 2′,6′-dihydroxy-4′-geranyloxyacetophenone (1) and 2′,6′-dihydroxy-4′-
farnesyloxy‐acetophenone (2) are oxyprenylated secondary metabolites extracted from plants
belonging to the Rutaceae family. In this study, 1 and 2 were synthesized and tested for their
antimicrobial activity toward major oral pathogens. Compounds 1 and 2 were synthesized by
selective prenylation of 2,4,6-trihydroxyacetophenone at the 4′ position with geranyl and farnesyl
bromide, respectively. Compound 1 showed stronger antimicrobial activity than 2 against major
oral pathogens, including Gram positive bacteria (Streptococcus mutans, Streptococcus sobrinus),
Gram negative bacteria (Prevotella intermedia, Porphyromonas gingivalis) and Candida albicans.
Evidences were obtained that the mode of action of 1 and 2 may be related to their iron-chelating
property. This study suggests that 1 and 2 may represent potential natural molecules for the
prevention/treatment of common oral infections, including dental caries, periodontal disease, and
candidiasis.
Received 28 February 2012
Received in revised form 30 May 2012
Accepted 2 June 2012
Available online 11 June 2012
Keywords:
Oxyprenylated secondary metabolites
Antimicrobial agents
Oral infections
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
the Rutaceae family. Since these secondary metabolites are
found in relatively low amounts in nature, the development of
an efficient chemical synthesis procedure is required to better
characterize their pharmacological properties.
Oxyprenylated secondary metabolites have been consid-
ered for decades merely as biosynthetic intermediates of
C-prenylated compounds and only in the last ten years
have been characterized as phytochemicals exerting valuable
biological activities. Oxyprenylated acetophenones represent a
rare class of natural products in which a terpenyl chain, more
frequently a geranyl (C10) or a farnesyl (C15) one, is attached to
the aromatic ring by a carbon–carbon or a carbon–oxygen link.
In this context the compounds 2′,6′-dihydroxy-4′-geranylox-
yacetophenone (1) and 2′,6′-dihydroxy-4′-farnesyloxyaceto-
phenone (2) have been previously extracted from the fruits of
Evodia merrillii Kanehira & Sasaki [1] from the aerial parts of
Boronia ramosa Benth. [2], from the fruits and aerial parts of
Melicope obscura (Coode) T.G. Hartley, Melicope obtusifolia sp.
obtusifolia var. arborea (Coode) T.G. Hartley [3], and Melicope
semecarpifolia (Merr.) T. G. Hartley [4], all plants belonging to
Oral diseases, including dental caries, periodontal disease
and candidiasis, are major public health problems because of
their high prevalence and incidence in all regions of the world.
The etiology of both dental caries and periodontal disease has
long been known to be related to colonization of oral sites by
specific bacteria. Streptococcus mutans and Streptococcus sobrinus,
the primary acidogenic components of dental biofilm, are able to
metabolize exogenous dietary carbohydrates and to generate
lactic acid, resulting in demineralization of tooth enamel [5]. As
dental biofilm matures, colonization shifts toward facultative
and anaerobic bacterial species, leading to supra-gingival plaque
accumulation and gingival inflammation which are the first
clinical signs of periodontal disease. While gingivitis is a mild and
reversible form of periodontal disease, periodontitis causes
permanent damages to tooth-supporting tissues and may lead
to tooth loss [6]. A group of about ten bacterial species, mostly
Gram negative anaerobic bacteria, have been associated to
⁎
Corresponding author. Tel.: +1 418 656 7341; fax: +1 418 656 2861.
E-mail address: Daniel.Grenier@greb.ulaval.ca (D. Grenier).
0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2012.06.003

L. Bonifait et al. / Fitoterapia 83 (2012) 996–999
997
periodontal disease [7]. Oral candidiasis is an opportunistic
infection of the oral cavity caused by an overgrowth of Candida
species, the most common being Candida albicans [8]. The most
prevalent forms of oral candidiasis are pseudomembranous
candidiasis (also called thrush) and erythematous candidiasis,
which includes denture stomatitis [8].
grown aerobically, all at 37 °C. C. albicans was cultivated in
Yeast Nitrogen Base (YNB; BBL Microbiology Systems) medium
supplemented with 0.5% glucose and incubated aerobically at
37 °C.
2.3. Determination of minimal inhibitory concentrations and
Controlling the above pathogens is the primary action for
maintaining oral health. Active compounds endowed with a
capacity to exert antimicrobial activity toward oral pathogens
have received considerable attention as they may represent
potential new therapeutic agents for the prevention/treatment
of oral infections. In this study, we synthesized compounds 1
and 2 and evaluated the antimicrobial properties toward major
pathogens associated with dental caries, periodontal disease,
and candidiasis.
minimal microbicidal concentrations
Cultures of microorganisms were diluted in fresh broth
medium to obtain an optical density at 660 nm (OD660) of 0.2.
Samples (100 μl) were added to the wells of a 96-well tissue
−
1
culture plate containing 100 μl of serial dilutions (100 μg ml
−
1
to 6.25 μg ml ) of 1 or 2. Control wells with no substance
but with substance vehicle were also inoculated. Penicillin G
(
(
Sigma-Aldrich Canada, Oakville, ON, CANADA) and Nystatin
EMD Biosciences Inc., San Diego, CA, USA) were used as
reference controls for growth inhibition. After incubation for
8 h at 37 °C (aerobic or anaerobic according to the microor-
2
. Materials and methods
4
ganism), the OD660 was recorded using a microplate reader.
The concentration of compounds that caused complete growth
inhibition was recorded as the minimal inhibitory concentra-
tion (MIC). Ten-μl samples obtained from the wells showing
no visible growth was spread on agar plates to determine the
minimal microbicidal concentration (MMC) of the compounds.
All the above assays were run in triplicate.
2
.1. Chemical synthesis
Compounds
1 and 2 were synthesized by selective
prenylation of 2,4,6-trihydroxyacetophenone at the 4′ posi-
tion with geranyl and farnesyl bromide, respectively, in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the
base in acetone. Briefly, to a solution of 2,4,6-trihydroxyace-
tophenone (1.0 mM) in acetone (5.0 ml), DBU (1.0 mM) and
geranyl/farnesyl bromide (1.02 mM) were added in sequence
and the resulting mixture was left to react for 2 h at room
temperature. The solution was then diluted with water (50 ml)
and extracted with n-hexane (3×10 ml). The collected organic
2.4. Influence of iron content on growth inhibition of P. gingivalis
The effect of 1 and 2 on growth of P. gingivalis was further
investigated under iron-limiting conditions. Mycoplasma Broth
Base (MBB; BBL Microbiology Systems) (iron content≤8 nM)
medium supplemented with 10 μM hemin was used as a
medium with iron-restricted conditions. The growth inhibitory
effect was determined as above.
phases were dried over anhydrous Na
dryness. The desired compound was obtained by crystallization
2 4
SO and evaporated to
(
n-hexane). The resulting compounds were characterized by
1
13
H-nuclear magnetic resonance (NMR), C NMR, infrared
(
IR) and mass spectroscopy, as routinely performed in our
laboratory.
2.5. Analysis of complex formation between iron and
compounds 1 and 2
2
.2. Microorganisms
UV/Vis spectroscopic analysis was performed as previously
reported [9] to monitor the formation of complexes between
iron and compounds 1 and 2.
Aggregatibacter actinomycetemcomitans ATCC 29522 (sero-
type 2), Porphyromonas gingivalis ATCC 33277, Prevotella inter-
media 5W2, S. mutans ATCC 25175, S. sobrinus ATCC 33478, and
C. albicans ATCC 28366 were used. Bacteria were grown in
Todd–Hewitt broth (BBL Microbiology Systems, Cockeysville,
MD, USA) supplemented with 0.001% hemin and 0.0001%
vitamin K (THB-HK). A. actinomycetemcomitans, P. gingivalis,
and P. intermedia were incubated under anaerobic conditions
3. Results and discussion
Compounds
1 and 2 were synthesized by selective
prenylation of 2,4,6-trihydroxyacetophenone at the 4′ posi-
tion with geranyl and farnesyl bromide, respectively, in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the
2 2 2
(N \H \CO /80:10:10), while S. mutans and S. sobrinus were
Fig. 1. Synthesis of compounds 1 and 2.

998
L. Bonifait et al. / Fitoterapia 83 (2012) 996–999
Table 1
Minimal inhibitory concentrations (MICs) and minimal microbicidal concentrations (MMCs) of compounds
1
and
2
toward S. sobrinus, S. mutans,
A. actinomycetemcomitans, P. intermedia, P. gingivalis, and C. albicans.
Compound 1 (μg ml−1)
Compound 2 (μg ml−1
)
Reference control(μgml )
−1 a
Microorganism
MIC
100
100
>100
25
MMC
MIC
MMC
MIC
MMC
S. sobrinus
S. mutans
A. actinomycetemcomitans
P. intermedia
P. gingivalis
100
100
>100
50
12.5
50
>100
>100
>100
>100
12.5
>100
>100
>100
>100
>100
12.5
>100
0.098
0.049
1.56
0.392
0.098
3.125
1.56
1.56
6.25
3.125
0.78
6.25
12.5
25
C. albicans
a
Penicillin G was used for bacteria and Nystatin for C. albicans.
base in acetone (Fig. 1). The yields of 1 and 2 were 55% and 62%,
respectively. The selectivity in geranylation/farnesylation of
the parent acetophenone may be explained by a preferential
hydrogen abscission by the bulky amine DBU from the more
sterically accessible 4-OH. Both products were characterized
by IR, 1H NMR, 13C NMR and GC–MS. The recorded data were
in full agreement with those already reported for the same
compounds [1–4].
the lowest concentration tested (6.25 μg ml−1) that also
corresponded to the MMC. The structural features of 1 and 2
(e.g. the presence of a double OH function in ortho position
to an acetyl group) could allow an efficient metal chelation.
To validate this hypothesis we performed UV/Vis spectroscopic
analysis to monitor the formation of complexes between iron
and compounds 1 and 2. This analysis showed the formation
of iron complexes with 1 and 2 (data not shown). Since iron
is an essential nutrient to support microbial growth, iron
chelation by 1 and 2 likely contributes to the antimicrobial
properties of the compounds.
Very few studies have investigated the pharmacological
properties of 1 and 2. In a recent study, Bruyère et al. [12]
reported the capacity of these two oxyprenylated acetophe-
nones to inhibit proliferation of cancer cell lines. On the basis
of the data reported in this study, compounds 1 and 2 may
represent potential natural molecules for the prevention/
treatment of common oral infections, including dental caries,
periodontal disease, and candidiasis.
MICs and MMCs of compounds 1 and 2 as well as of
reference controls (penicillin G and Nystatin) are reported
in Table 1. The MIC of compound 1 for both Gram positive
−
1
bacteria (S. mutans, S. sobrinus) tested was 100 μg ml . This
concentration also represented the MMC. At this concentration,
compound 2 only caused a 40% and 36% growth inhibition
of S. sobrinus and S. mutans, respectively (data not shown).
The effect of 1 and 2 on growth of three Gram negative
oral pathogens was also investigated. While 1 and 2 had no
significant effect on growth of A. actinomycetemcomitans,
compound 1 was highly active on P. intermedia and P. gingivalis
−
1
showing MIC of 25 and 12.5 μg ml
1
and MMC of 50 and
2.5 μg ml , respectively. Although compound 2 only caused
a 32% inhibition of growth of P. intermedia at the highest
−
1
Acknowledgments
−
1
concentration tested (100 μg ml ), it showed MIC and MMC
This study was financially supported by an International
Association for Dental Research/GlaxoSmithKline Innovation
in Oral Care Award.
−
1
of 12.5 μg ml
on P. gingivalis. Lastly, the effect of 1 and 2 on
growth of the pathogenic yeast C. albicans was tested. As it
was the case for bacteria, compound 1 was more effective than
−
1
−1
2
with a MIC of 25 μg ml
the highest concentration tested (100 μg ml ), compound
caused a 33% inhibition of the growth of C. albicans. The
and a MMC of 50 μg ml . At
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