Effects of 3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans propenoic acid and its ester derivatives on biofilm formation by two oral pathogens, Porphyromonas gingivalis and Streptococcus mutans

Article abstract of DOI:10.1016/j.ejmech.2007.11.001

The aim of this study was to investigate the effect of 3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans propenoic acid, active principle isolated from Acronychia baueri Schott, and its ester derivatives on biofilm formation by two important oral pathogens, Porphyromonas gingivalis and Streptococcus mutans. The parent acid and conjugates with vanillic acid, 2-hydroxynaphthoquinone and guaiacol caused a significant and reproducible inhibition of P. gingivalis biofilm formation. This effect could be related to the ability of the compounds to inhibit bacterial growth. These compounds also efficiently caused a reduction of biofilm formation by S. mutans, a phenomenon not related to growth inhibition. These data suggest that 3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans propenoic acid and some of its ester derivatives may have a therapeutic/preventive potential for oral infections.

Full text of DOI:10.1016/j.ejmech.2007.11.001

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1612e1620
http://www.elsevier.com/locate/ejmech
Original article
Effects of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid
and its ester derivatives on biofilm formation by two oral pathogens,
Porphyromonas gingivalis and Streptococcus mutans
Charles Bodet a, Francesco Epifano b, Salvatore Genovese c, Massimo Curini c, Daniel Grenier a,
*
a
´
Groupe de Recherche en Ecologie Buccale, Faculte´ de Me´decine Dentaire, Universite´ Laval, Quebec City, Quebec G1K 7P4, Canada
b Dipartimento di Scienze del Farmaco, Universita` ‘‘G. D’Annunzio’’ di Chieti-Pescara, Via dei Vestini, 31, 66013 Chieti Scalo, Chieti, Italy
c Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, Universita` degli Studi di Perugia, Via del Liceo, 06123 Perugia, Italy
Received 22 June 2007; received in revised form 10 October 2007; accepted 1 November 2007
Available online 21 February 2008
Abstract
The aim of this study was to investigate the effect of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid, active principle isolated
from Acronychia baueri Schott, and its ester derivatives on biofilm formation by two important oral pathogens, Porphyromonas gingivalis and
Streptococcus mutans. The parent acid and conjugates with vanillic acid, 2-hydroxynaphthoquinone and guaiacol caused a significant and re-
producible inhibition of P. gingivalis biofilm formation. This effect could be related to the ability of the compounds to inhibit bacterial growth.
These compounds also efficiently caused a reduction of biofilm formation by S. mutans, a phenomenon not related to growth inhibition. These
data suggest that 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid and some of its ester derivatives may have a therapeutic/preventive
potential for oral infections.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Oral infection; Bacteria; Biofilm; Ferulic acid
1. Introduction
enamel dissolution by acid-producing bacteria inhabiting den-
tal biofilm, especially when the biofilm reaches a critical mass
due to poor oral hygiene. Streptococcus mutans, known as
a major cariogenic bacteria, produces extracellular polysac-
charide that contributes to the formation of the dental biofilm
[4].
Periodontitis is a destructive inflammatory disorder that
leads to the loss of tooth-support. It is initiated by a specific
group of Gram-negative anaerobic bacteria, which modulate
periodontal tissue destruction through complex interactions
with mucosal and immune cells [1,2]. Much evidence points
to Porphyromonas gingivalis as the key pathogen in chronic
periodontitis [1]. The ability of this bacterium to form biofilm
and to colonize subgingival sites is a critical step in the initi-
ation of periodontitis. P. gingivalis is well known to express
several adhesins, associated with either the outer membrane
or fimbriae, that promote its adhesion to tooth surfaces, gingi-
val epithelial cells, basement membrane components, erythro-
cytes and oral bacteria [3]. Dental caries is the direct result of
3-(40-Geranyloxy-30-methoxyphenyl)-2-trans
propenoic
acid 1 (Fig. 1) is a secondary metabolite biosynthetically re-
lated to ferulic acid in which a geranyl chain is attached to
the phenolic group. It has been isolated in 1966 from the
bark of Acronychia baueri Schott, an Australian small tree be-
longing to the family of Rutaceae [5]. Although known for
four decades, only in the last decade this natural compound
showed valuable pharmacological properties that have been
recently reviewed [6]. Both 1 and its some structurally simple
ester derivatives (e.g. ethyl ester) are considered potential
drugs for chemoprevention of different types of cancer, espe-
cially those of gastrointestinal tract [6].
* Corresponding author. Tel.: þ1 418 656 7341; fax: þ1 418 656 2861.
E-mail address: daniel.grenier@greb.ulaval.ca (D. Grenier).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.001

C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
COOH
1613
3. Biological results and discussion
The effect of 1, alcohols 2e11 and esters 12e21 were first
tested for their capacity to inhibit biofilm formation by the pe-
riodontopathogenic bacterium P. gingivalis (Fig. 2). Com-
pound 1 at 31.3 mg/ml (78.1 mM) caused an inhibition of
biofilm formation by P. gingivalis of approximately 80%.
Among alcohols tested, only 2-hydroxynaphthoquinone 3
and hydroquinone 10 reduced the formation of biofilm by P.
gingivalis. Even at the lowest concentration tested (3.9 mg/ml;
35.5 mM) 10 caused a reduction (>80%) of biofilm formation.
Three ester derivatives of 1 showed a capacity to interfere with
biofilm formation by P. gingivalis: 2-hydroxynaphthoquinone
13, vanillic acid 17 and guaiacol 18 derivatives. The inhibition
obtained with 13 and 17 was not better than that obtained with
compound 1. However, the guaiacol derivative 18 was much
more potent than the parent molecule and was able to signif-
icantly (P ꢀ 0.05) inhibit biofilm formation at the lowest con-
centration tested (3.9 mg/ml; 8.6 mM).
We then investigated whether the effects of the above cited
compounds on biofilm formation by P. gingivalis were related
to an inhibition of bacterial growth. As reported in Table 2, all
compounds showed a capacity to reduce growth of P. gingivalis
that correlates with their ability to inhibit biofilm formation. Pre-
formed biofilms (48 h) of P. gingivalis were incubated with the
compounds for 4 h prior to determination of cell viability using
an XTT assay. None of the compounds caused a significant de-
crease in viability of P. gingivalis suggesting that the antibacte-
rial effect is bacteriostatic rather than bactericidal.
In the second part of the study, the compounds that showed
activity on P. gingivalis were tested for their effect on biofilm
formation by S. mutans (Fig. 3). Compound 1 at the lowest
concentration tested (3.9 mg/ml; 9.8 mM) still caused a signifi-
cant and reproducible inhibition (P ꢀ 0.05) of biofilm forma-
tion. Compounds 3 and 10 were effective in reducing
biofilm formation by S. mutans at a concentration down to
31.3 mg/ml (180 and 284.4 mM, respectively). The three deriv-
atives of compound 1 (13, 17 and 18), although they could re-
duce biofilm formation to some extent, were less effective that
the parent molecule. On the contrary to what was observed
with P. gingivalis, none of the compounds showed a capacity
to inhibit growth of S. mutans (data not shown). The com-
pounds thus have a specific anti-biofilm effect on S. mutans.
To better evaluate the significance of the data obtained, the
effect of chlorhexidine, an antimicrobial agent largely used to
treat oral infections, on P. gingivalis and S. mutans was tested.
The chlorhexidine minimal inhibitory concentrations on
P. gingivalis and S. mutans were 4 mg/ml (6 mM) and 2 mg/ml
(3 mM), respectively. The reduction in biofilm formation
observed in the presence of sub-optimal inhibitory concentra-
tions of chlorhexidine correlated with partial growth inhibition
of both bacterial species. Therefore, no specific effect on
biofilm formation could be associated with chlorhexidine, as
previously reported [15].
O
1
OMe
Fig. 1. 3-(40-Geranyloxy-30-methoxyphenyl)-2-trans propenoic acid.
In continuation of our research studies aimed to demon-
strate the beneficial properties of 1 for human health, we
wish to report herein the preparation of some ester derivatives
of the parent acid using the following natural compounds
possessing an alcohol or phenol moiety: umbelliferone 2 [7],
2-hydroxynaphthoquinone 3 [8], salicylic 4, ferulic 5, caffeic
6, and vanillic 7 acids [9], guaiacol 8 [10], naringenin 9
[11], hydroquinone 10 [12], and anthraflavin 11 [13] which
are known to have antimicrobial activity. Thereafter, 1 and
its derivatives were tested for their ability to inhibit biofilm
formation by two important oral pathogens: P. gingivalis and
S. mutans. It is thought that such a chemical modification
may allow synergism of biological action, once the two por-
tions of the ester are cleaved by bacterial lipases.
2. Chemistry
Compound 1 was synthesized in 98% yield as already re-
ported [14]. Esters of 1 were synthesized in two steps by con-
version of the acid into the corresponding acyl chloride by
reaction with oxalyl chloride in dry diethyl ether at room tem-
perature, followed by its reaction with the alcohol or phenol
and triethylamine in dry diethyl ether at room temperature
(Scheme 1). Yields of ester derivatives are reported in Table 1.
Salicylic, ferulic, caffeic and vanillic acid were conjugated to 1
in form of methyl esters.
Crucial for the success of this synthesis is the time of the
reaction of conversion of compound 1 into the corresponding
chloride that had to be rigorously of 5 min, while for longer
reaction times the acyl chloride went rapidly through decom-
position. Moreover each attempt to synthesize esters by direct
condensation of the acid with alcohol or phenol in the pres-
ence of different reagents like dicyclohexyl carbodiimide or
1,1-carbonyl diimidazole were unsuccessful.
COOH
COCl
a
GerO
GerO
OMe
OMe
b
COOR(Ar)
GerO
OMe
Colonization and subsequent biofilm formation by oral
pathogens such as P. gingivalis and S. mutans is the initial
step in the pathogenesis of oral infections. Biofilms, which
Scheme 1. Reagents and conditions: (a) (COCl)2, dry Et2O, room temperature
for 5 min; (b) ROH (ArOH), Et3N, dry Et2O, room temperature for 40 min.

1614
C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
Table 1
Synthesis of ester derivatives of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid
Substrate
Product
Yield (%)a
65
O
O
O
HO
O
O
O
2
GerO
OMe
12
95
O
O
O
HO
O
O
O
3
GerO
OMe
13
81
OH
O
COOMe
O
COOMe
GerO
4
OMe
14
63
COOMe
COOMe
O
O
HO
OMe
OMe
GerO
5
OMe
15
80
COOMe
COOMe
O
OGer
O
HO
O
OH
OMe
GerO
6
O
OMe
16
87
COOMe
COOMe
O
O
HO
OMe
OMe
7
GerO
OMe
17

C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
1615
Table 1 (continued)
Substrate
Product
Yield (%)a
31
OH
O
OMe
O
OMe
GerO
8
OMe
18
69
OH
OGer
OMe
HO
O
GerO
MeO
O
O
O
O
GerO
MeO
OH
O
O
9
O
O
19
O
44
OH
OGer
OMe
O
HO
O
O
10
O
GerO
OMe
20
36
O
OGer
O
OH
O
O
OMe
O
HO
O
O
O
GerO
11
21
OMe
Yields of pure isolated product fully characterized by IR, GCeMS, 1H NMR and 13C NMR.
a
are defined as structured microbial communities attached to
surfaces, play an important role in most bacterial infections
of the human body. In the oral cavity, biofilms allow bacteria
to evade immune defenses and to better resist to mechanical
removal and chemotherapeutic agents. Only in the last five
years it has been observed that the presence of a terpenyl hy-
drocarbon chain, either farnesyl, geranyl or isopentenyl,
greatly enhance the antibacterial properties of a natural or
semisynthetic compounds [16]. In particular natural prenyloxy
secondary metabolites of phenylpropanoid biosynthetic origin
very recently were shown to exert valuable antimicrobial prop-
erties against several bacteria such as Klebsiella pneumoniae,
Mycobacterium smegmatis, Pseudomonas aeruginosa, Salmo-
nella galinnarum, Staphylococcus aureus and Helicobacter
pylori [17e19]. Among this latter class of natural products
3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid 1
was seen to be pharmacologically active against several bio-
logical targets [6,14,20], while other structurally similar mol-
ecules (e.g. the acid lacking the methoxy group in position 3)
do not share the same pharmacological properties. This al-
lowed us to exclude a non-specific effects on the lipid matrix
of cell membranes, acting like a surfactant, in considering the
biological action of compound 1. Although the underlying
mechanism is not known at the moment, although it is cur-
rently under investigation, the same hypothesis could be
made in explaining the observed effect on prevention of bio-
film formation by 3-(40-geranyloxy-30-methoxyphenyl)-2-
trans propenoic acid. It is noteworthy that, although

1616
C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
B 1,6
A
1,6
1,4
1,2
1
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0,8
0,6
0,4
0,2
0
*
*
*
*
*
*
*
*
control 250
625
125
62,5
31,3
15,6
39,1
7,8
19,5
3,9 µg/ml
9,8 µM
control 250
1440
125
720
62,5
360
31,3
180
15,6
90
7,8
45
3,9 µg/ml
22,5 µM
312,5 156,3 78,1
Concentration of 3-(4’-geranyloxy-3’-methoxyphenyl)-2-trans
Concentration of 2-hydroxynaphthoquinone (3)
propenoic acid (1)
C
1,6
D
1,4
1,2
1
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0,8
0,6
0,4
0,2
0
*
*
*
*
*
*
*
*
**
*
control 250
125
62,5
31,3
15,6
7,8
3,9 µg/ml
35,5 µM
control 250
500
125
250
62,5
125
31,3
62,5
15,6
31,3
7,8
15,6
3,9 µg/ml
7,8 µM
2275 1137,5 568,8 284,4 142,2 71,1
Concentration of hydroquinone (10)
Concentration of vanillic acid derivative of 3-(4’-geranyloxy-
3’-methoxyphenyl)-2-trans propenoic acid (17)
F
E
1,6
1,4
1,2
3
2,5
2
*
1
*
0,8
1,5
1
0,6
**
*
**
0,4
0,2
0
*
*
*
*
0,5
0
*
control
250
550
125
62,5
31,3
15,6
34,4
7,8
17,2
3,9 µg/ml
8,6 µM
control 250
500
125
250
62,5
125
31,3
62,5
15,6
31,3
7,8
15,6
3,9 µg/ml
7,8 µM
275 137,5 68,8
Concentration of gaiacol derivative of 3-(4’-geranyloxy-
Concentration of 2-hydroxynaphthoquinone derivative of 3-
3’-methoxyphenyl)-2-trans propenoic acid (18)
(4’-geranyloxy-3’-methoxyphenyl)-2-trans propenoic acid (13)
Fig. 2. Effect of 1 (A), 3 (B), 10 (C), 17 (D), 13 (E), and 18 (F) on biofilm formation by P. gingivalis.
esterification of acid 1 with other phenolic natural compounds
greatly improve the lipophilicity of the resulting adduct, lead-
ing in theory to a better capacity to permeate biofilm surface
and thus to a better antimicrobial activity, only in one case
the activity of the ester was superior to that of the parent
acid. Moreover, compound 1 in many cases performed better
than other parent natural products. The preliminary data about
the capacity of compound 1 to inhibit biofilm formation herein
described will prompt us to use 3-(40-geranyloxy-30-methoxy-
phenyl)-2-trans propenoic acid as a lead compound to draw
and synthesize properly structured isosters (e.g. those deriving
from substation of the methoxy group in position 3 with an
halogen atom) in order to improve the title pharmacological
profile. In conclusion, overall, our data suggest that 3-(40-
geranyloxy-30-methoxyphenyl)-2-trans propenoic acid and
some of its ester derivatives (vanillic acid, 2-hydroxynaphtho-
quinone and guaiacol) may have a therapeutic potential for
pharmacological treatment of oral infections by reducing the
capacity of P. gingivalis and S. mutans to form biofilm.
4. Experimental protocols
4.1. Synthesis of esters of 3-(40-geranyloxy-30-
methoxyphenyl)-2-trans propenoic acid e
general procedure
To a solution of 3-(40-geranyloxy-30-methoxyphenyl)-2-
trans propenoic acid 1 (0.55 mmol) in anhydrous Et2O
(2.5 ml), oxalyl chloride (1.11 mmol) was added and the re-
sulting solution was stirred under N2 for 5 min at room

C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
1617
Table 2
Effect of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid, 2-hydroxynaphthoquinone, hydroquinone, vanillic acid derivative of 3-(40-geranyloxy-30-
methoxyphenyl)-2-trans propenoic acid, 2-hydroxynaphthoquinone derivative of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid, and guaiacol deriv-
ative of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans propenoic acid on growth of P. gingivalis
Compound
P. gingivalis relative growth (%)
Control
250a
125a
62.5a
31.2a
15.6a
7.8a
3.9a
1
100 ꢃ 12
100 ꢃ 12
100 ꢃ 12
100 ꢃ 12
100 ꢃ 12
100 ꢃ 12
30 ꢃ 8*
27 ꢃ 9*
15 ꢃ 11*
21 ꢃ 11*
39 ꢃ 7*
39 ꢃ 6*
13 ꢃ 5*
49 ꢃ 15*
14 ꢃ 9*
17 ꢃ 7*
40 ꢃ 11*
32 ꢃ 10*
9 ꢃ 5*
79 ꢃ 16
13 ꢃ 12*
16 ꢃ 3*
49 ꢃ 14*
25 ꢃ 14*
15 ꢃ 6*
86 ꢃ 14
17 ꢃ 6*
34 ꢃ 10*
72 ꢃ 16
28 ꢃ 15*
34 ꢃ 12*
92 ꢃ 15
68 ꢃ 23
81 ꢃ 26
3
100 ꢃ 14
27 ꢃ 15*
124 ꢃ 4
87 ꢃ 18
25 ꢃ 11*
103 ꢃ 10
45 ꢃ 8*
110 ꢃ 17
98 ꢃ 14
61 ꢃ 9*
10
13
17
18
15 ꢃ 5*
105 ꢃ 15
76 ꢃ 18
22 ꢃ 10*
*P < 0.05 in Student’s t-test.
a
Dose expressed in mg/ml.
temperature. The solvent was evaporated under vacuum, the
resulting syrup was dissolved in anhydrous Et2O (3 ml) and
to this solution was added dropwise over a period of 30 min
a solution of phenol derivative (0.48 mmol) and Et3N
(1.2 mmol) in anhydrous Et2O (3 ml). The resulting mixture
was stirred for 10 min and the white precipitate formed was
filtered under vacuum and washed twice with Et2O (5 ml).
The filtrate was then extracted twice with a 1% solution of cit-
ric acid (10 ml), the organic phase washed twice with a 1% so-
lution of NaHCO3 (5 ml), dried over anhydrous Na2SO4 and
the solvent evaporated under vacuum to yield the desired ester.
4.1.3. Methyl 2-{[(2E)-3-(4-{[(2E)-2,7-dimethylocta-2,6-die-
nyl]oxy}-3-methoxyphenyl)prop-2-enoyl]oxy}benzoate (14)
Yellow solid; yield 81%; mp: 182e183 ꢁC; IR (KBr):
1
1698, 1695 cmꢂ1; H NMR (200 MHz, CDCl3 d): 1.67 (s,
3H), 1.69 (s, 3H), 1.75 (s, 3H), 2.10e2.18 (m, 4H), 3.98
(s, 3H), 3.99 (s, 3H), 4.68e4.72 (m, 2H), 5.06e5.09 (m,
1H), 5.50e5.53 (m, 1H), 6.54e8.08 (m, 9H); 13C NMR
(50 MHz, CDCl3 d) 16.4, 17.7, 25.7, 26.6, 27.5, 39.6, 51.4,
56.1, 67.9, 111.7, 114.2, 116.7, 119.4, 121.0, 121.9, 123.3,
123.9, 127.8, 128.6, 131.9, 132.1, 133.2, 135.2, 144.8,
146.2, 150.9, 155.0, 168.2, 168.5. Anal. Calcd for
C28H32O6: C, 72.39; H, 6.94; O, 20.66. Found: C, 72.40;
H, 6.93; O, 20.68.
4.1.1. Umbelliferyl (2E )-3-(4-{[(2E )-2,7-dimethylocta-2,6-
dienyl]oxy}-3-methoxyphenyl)prop-2-enoate (12)
White solid; yield 65%; mp: 186e188 ꢁC; IR (KBr): 1687,
1684 cmꢂ1; 1H NMR (200 MHz, CDCl3 d): 1.62 (s, 3H), 1.69
(s, 3H), 1.75 (s, 3H), 2.09e2.20 (m, 4H), 3.95 (s, 3H), 4.69e
4.72 (m, 2H), 4.97e5.13 (m, 2H), 5.47e5.56 (m, 1H), 6.48 (d,
1H, J ¼ 9.9 Hz), 6.74 (d, 1H, J ¼ 12.4 Hz), 6.92 (d, 1H,
J ¼ 8.1 Hz) 7.15e7.29 (m, 4H), 7.54 (d, 1H, J ¼ 8.1 Hz),
7.73 (d, 1H, J ¼ 9.9 Hz), 7.86 (d, 1H, J ¼ 12.4 Hz); 13C
NMR (50 MHz, CDCl3 d) 16.5, 17.6, 25.4, 26.0, 39.4, 55.9,
65.9, 110.1, 111.6, 114.3, 115.3, 115.9, 116.7, 118.0, 119.4,
121.9, 123.6, 128.4, 130.1, 131.8, 141.6, 144.1, 144.4,
146.2, 150.7, 156.1, 158.0, 161.7, 165.3. Anal. Calcd for
C29H30O6: C, 73.40; H, 6.37; O, 20.23. Found: C, 73.39; H,
6.34; O, 20.24.
4.1.4. 2-Methoxy-4-[(1E )-3-methoxy-3-oxoprop-1-
enyl]phenyl (2E )-3-(4-{[(2E )-2,7-dimethylocta-2,6-
dienyl]oxy}-3-methoxyphenyl)prop-2-enoate (15)
Yellowish solid; yield 63%; mp: 159e161 ꢁC; IR (KBr):
1
1695, 1692 cmꢂ1; H NMR (200 MHz, CDCl3 d): 1.62 (s,
3H), 1.69 (s, 3H), 1.75 (s, 3H), 2.11e2.20 (m, 4H), 3.92 (s,
3H), 3.94 (s, 3H), 3.97 (s, 3H), 4.68e4.71 (m, 2H), 5.09e
5.11 (m, 1H), 5.49e5.53 (m, 1H), 6.51e7.89 (m, 7H); 13C
NMR (50 MHz, CDCl3 d) 16.2, 17.6, 25.8, 26.9, 27.2, 39.4,
55.7, 55.9, 67.9, 111.7, 112.9, 114.0, 117.7, 118.4, 121.5,
123.6, 123.8, 123.9, 127.8, 128.5, 131.5, 132.1, 132.6,
139.2, 144.2, 144.7, 146.4, 150.5, 152.9, 164.9, 169.8. Anal.
Calcd for C31H36O7: C, 71.52; H, 6.97; O, 21.51. Found: C,
71.50; H, 6.98; O, 21.50.
4.1.2. 1,4-Dioxo-1,4-dihydronaphthalen-2-yl (2E )-3-
(4-{[(2E)-2,7-dimethylocta-2,6-dienyl]oxy}-3-
methoxyphenyl)prop-2-enoate (13)
4.1.5. Methyl 4-{[(2E )-3-(4-{[(2E )-2,7-dimethylocta-2,6-
dienyl]oxy}-3-methoxyphenyl)prop-2-enoyl]oxy}-3-
methoxybenzoate (16)
Yellowish solid; yield 95%; mp: 208e209 ꢁC; IR (KBr):
1705, 1695, 1685 cmꢂ1 1H NMR (200 MHz, CDCl3 d):
;
1.62 (s, 3H), 1.69 (s, 3H), 1.73 (s, 3H), 2.08e2.21 (m, 4H),
3.97 (s, 3H), 4.71e4.73 (m, 2H), 4.98e5.11 (m, 2H), 5.51e
5.59 (m, 1H), 6.67 (d, 1H, J ¼ 12.3 Hz) 6.94e8.17 (m, 4H);
13C NMR (50 MHz, CDCl3 d) 16.2, 17.4, 25.1, 25.6, 26.2,
39.5, 55.9, 65.9, 107.5, 111.6, 114.4, 119.8, 121.9, 123.9,
127.1, 127.3, 129.6, 131.2, 131.3, 132.8, 133.5, 134.1,
134.9, 141.6, 144.3, 147.4, 146.9, 150.7, 152.2, 161.8,
176.7, 183.8. Anal. Calcd for C30H30O6: C, 74.06; H, 6.21;
O, 19.73. Found: C, 74.03; H, 6.19; O, 19.70.
Yellowish solid; yield 80%; mp: 162e163 ꢁC; IR (KBr):
1
1698, 1695 cmꢂ1; H NMR (200 MHz, CDCl3 d): 1.62 (s,
3H), 1.69 (s, 3H), 1.74 (s, 3H), 2.11e2.19 (m, 4H), 3.94 (s,
3H), 3.95 (s, 3H), 3.97 (s, 3H), 4.68e4.71 (m, 2H), 5.06e
5.11 (m, 1H), 5.49e5.53 (m, 1H), 6.51e7.89 (m, 8H); 13C
NMR (50 MHz, CDCl3 d) 16.5, 17.6, 25.6, 26.9, 27.3, 39.6,
51.2, 55.9, 56.0, 67.9, 111.7, 112.8, 114.0, 117.7, 121.9,
122.3, 123.6, 126.0, 127.7, 128.8, 131.5, 132.1, 144.8,
146.0, 146.2, 149.2, 150.4, 164.8, 166.2. Anal. Calcd for

1618
C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
0,35
0,35
A
B
0,3
0,3
0,25
0,2
0,25
0,2
*
*
*
*
0,15
0,1
0,15
0,1
0,05
0
*
*
*
*
*
*
*
0,05
0
250
1440
125
720
62,5
360
31,3
180
15,6
90
7,8
45
3,9 µg/ml
22,5 µM
control 250
625
125
62,5
31,3
15,6
39,1
7,8
19,5
3,9 µg/ml
9,8 µM
control
312,5 156,3 78,1
Concentration of 2-hydroxynaphthoquinone (3)
Concentration of 3-(4’-geranyloxy-3’-methoxyphenyl)-2-trans
propenoic acid (1)
0,35
0,35
0,3
C
D
0,3
0,25
0,2
0,25
0,2
*
*
*
*
0,15
0,1
0,15
0,1
*
*
*
*
0,05
0
0,05
0
control 250
500
125
250
62,5
125
31,3
62,5
15,6
31,3
7,8
15,6
3,9 µg/ml
7,8 µM
control 250
125
62,5
31,3
15,6
7,8
3,9 µg/ml
35,5 µM
2275 1137,5 568,8 284,4 142,2 71,1
Concentration of hydroquinone (10)
Concentration of vanillic acid derivative of 3-(4’-geranyloxy-
3’-methoxyphenyl)-2-trans propenoic acid (17)
0,35
0,35
0,3
F
E
0,3
0,25
0,2
0,25
0,2
*
0,15
0,1
0,15
0,1
*
*
*
*
*
0,05
0
0,05
0
control 250
550
125
275
62,5
31,3
15,6
34,4
7,8
17,2
control 250
500
125
250
62,5
125
31,3
62,5
15,6
31,3
7,8
15,6
3,9 µg/ml
7,8 µM
3,9 µg/ml
8,6 µM
137,5 68,8
Concentration of gaiacol derivative of 3-(4’-geranyloxy-
Concentration of 2-hydroxynaphthoquinone derivative of 3-
3’-methoxyphenyl)-2-trans propenoic acid (18)
(4’-geranyloxy-3’-methoxyphenyl)-2-trans propenoic acid (13)
Fig. 3. Effect of 1 (A), 3 (B), 10 (C), 17 (D), 13 (E), and 18 (F) on biofilm formation by S. mutans.
C29H34O7: C, 70.43; H, 6.93; O, 22.64. Found: C, 70.41; H,
6.92; O, 22.65.
4.1.7. 4-(5,7-Bis{[(2E )-3-(4-{[(2E )-3,7-dimethylocta-2,6-
dienyl]oxy}-3-methoxyphenyl)prop-2-enoyl]oxy}-4-oxo-3,
4-dihydro-2H-chromen-2-yl)phenyl (2E )-3-(4-{[(2E )-3,
7-dimethylocta-2,6-dienyl]oxy}-3-methoxyphenyl)prop-2-
enoate (18)
4.1.6. 2-Methoxyphenyl (2E )-3-(4-{[(2E )-3,7-dimethylocta-
2,6-dienyl]oxy}-3-methoxyphenyl)prop-2-enoate (17)
Orange solid; yield 87%; mp: 145e147 ꢁC; IR (KBr):
Yellowish solid; yield 31%; mp: 262e264 ꢁC (d); IR
(KBr): 1695, 1693 cmꢂ1 1H NMR (200 MHz, CDCl3 d):
;
1696 cmꢂ1
;
1H NMR (200 MHz, CDCl3 d): 1.62 (s, 3H),
1.64 (s, 9H), 1.68 (s, 9H), 1.75 (s, 9H), 2.09e2.26 (m, 14H),
3.95 (s, 9H), 4.69e4.75 (m, 6H), 5.08e5.56 (m, 7H), 6.53e
7.82 (m, 20H); 13C NMR (50 MHz, CDCl3 d) 16.3, 17.5,
25.6, 26.4, 39.3, 55.6, 65.9, 78.5, 106.1 108.3, 111.7, 113.1,
114.5, 116.6, 116.7, 119.8, 121.6, 121.8, 123.9, 128.7,
130.0, 131.3, 135.9, 142.0, 144.4, 146.2, 150.1, 150.8,
153.5, 158.4, 165.2, 165.7, 169.3, 189.8. Anal. Calcd for
C75H84O14: C, 74.48; H, 7.00; O, 18.52. Found: C, 74.49; H,
7.02; O, 18.51.
1.69 (s, 3H), 1.72 (s, 3H), 2.11e2.20 (m, 4H), 3.91 (s, 3H),
3.94 (s, 3H), 4.68e4.71 (m, 2H), 5.07e5.12 (m, 1H), 5.50e
5.53 (m, 1H), 6.53e7.86 (m, 9H); 13C NMR (50 MHz,
CDCl3 d) 16.2, 17.5, 25.4, 26.2, 39.4, 55.8, 55.9, 65.9,
111.6, 114.4, 114.8, 117.7, 119.9, 121.9, 123.8, 123.9,
125.0, 126.6, 128.8, 131.3, 141.7, 142.5, 144.4, 146.2,
150.8, 151.2, 165.0. Anal. Calcd for C27H32O5: C, 74.29; H,
7.39; O, 18.33. Found: C, 74.28; H, 7.37; O, 18.35.

C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
1619
4.1.8. 4-{[(2E )-3-(4-{[(2E )-3,7-Dimethylocta-2,6-
dienyl]oxy}-3-methoxyphenyl)prop-2-enoyl]oxy}phenyl
(2E )-3-(4-{[(2E )-3,7-dimethylocta-2,6-dienyl]oxy}-3-
ethylphenyl)prop-2-enoate (19)
on growth and biofilm formation by P. gingivalis was deter-
mined in a microplate. A 24 h culture of P. gingivalis in THB-
HK was diluted in fresh broth medium to obtain an optical
density at 660 nm (OD660) of 0.07. Samples (100 ml) were added
to the wells of a 96-well tissue culture plate (Sarstedt, Newton,
NC) containing 100 ml of serial dilutions (500e7.8 mg/ml) of
sterile compounds in THB-HK. Control wells with no com-
pounds were also inoculated. After incubation for 48 h at
37 ꢁC under anaerobic conditions, the absorbance at 660 nm
(A660) was recorded. Thereafter, spent media and free-floating
bacteria were removed by aspiration using a 26 G needle and
the wells were gently washed three times with distilled water.
The bacterial biofilms were stained with 0.04% crystal violet
(100 ml) for 15 min. The wells were washed four times with dis-
tilled water to remove unbound crystal violet dye and dried for
2 h at 37 ꢁC. After adding 100 ml of 95% (v/v) ethanol to each
well, the plate was shaken for 10 min to release the stain from
the biofilms and the absorbance at 550 nm (A550) was recorded.
A preliminary assay revealed that the compounds tested, even at
the highest concentration, did not affect the reading of the A550
values. Assays were run in triplicate and the means ꢃ standard
deviations of two independent experiments were calculated.
Compounds that showed an inhibitory effect on biofilm forma-
tion by P. gingivalis were then tested for their capacity to inter-
fere with biofilm formation by S. mutans. Lastly, to compare the
effect of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans prope-
noic acid 1, alcohols 2e11 and ester derivatives 12e21 with
a known antimicrobial agent used in dentistry, chlorhexidine
was tested.
Bright yellow solid; yield 69%; mp: 232e233 ꢁC (d); IR
1
(KBr): 1693 cmꢂ1; H NMR (200 MHz, CDCl3 d): 1.61 (s,
6H), 1.67 (s, 6H), 1.70 (s, 6H), 2.09e2.17 (m, 8H), 3.93 (s,
6H), 4.67e4.72 (m, 4H), 5.08e5.12 (m, 4H), 5.52e5.56 (m,
2H), 6.53e7.67 (m, 14H); 13C NMR (50 MHz, CDCl3 d)
16.1, 17.4, 25.7, 26.3, 39.4, 55.8, 65.7, 111.8, 114.3, 116.8,
119.7, 119.8, 121.8, 123.8, 128.5, 131.5, 141.4, 144.2,
145.4, 146.2, 150.9, 165.8. Anal. Calcd for C46H54O9: C,
77.02; H, 7.70; O, 15.28. Found: C, 77.01; H, 7.68; O, 15.30.
4.1.9. 6-{[(2E )-3-(4-{[(2E )-2,7-Dimethylocta-2,6-
dienyl]oxy}-3-methoxyphenyl)-1-methyleneprop-2-
enyl]oxy}-9,10-dimethylene-9,10-dihydroanthracen-2-yl
(2E )-3-(4-{[(2E )-2,7-dimethylocta-2,6-dienyl]oxy}-3-
methoxyphenyl)prop-2-enoate (20)
Yellowish solid; yield 44%; mp: 252e255 ꢁC (d); IR
1
(KBr): 1695 cmꢂ1; H NMR (200 MHz, CDCl3 d): 1.63 (s,
6H), 1.67 (s, 6H), 1.70 (s, 6H), 2.07e2.20 (m, 8H), 3.97 (s,
6H), 4.67e4.71 (m, 4H), 5.08e5.11 (m, 2H), 5.51e5.54 (m,
2H), 6.50e8.43 (m, 16H); 13C NMR (50 MHz, CDCl3 d)
16.2, 17.6, 25.6, 26.1, 39.4, 55.9, 65.8, 111.6, 114.7, 116.7,
119.8, 120.3, 121.9, 123.8, 127.6, 128.4, 129.2, 131.3,
132.0, 137.6, 141.8, 144.4, 146.9, 150.7, 157.0, 165.7,
182.9. Anal. Calcd for C54H56O10: C, 74.98; H, 6.53; O,
18.50. Found: C, 74.95; H, 6.51; O, 18.46.
4.1.10. Methyl (2E )-3-(3,4-bis{[(2E )-3-(4-{[(2E )-3,7-
dimethylocta-2,6-dienyl]oxy}-3-methoxyphenyl)prop-2-
enoyl]oxy}phenyl)prop-2-enoate (21)
4.4. Effect on bacterial viability
Compounds showing a capacity to inhibit biofilm formation
by P. gingivalis were selected and tested for their effect on
bacterial viability using the tetrazolium sodium 30-{1-[(phe-
nylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)-
benzene sulfonic acid hydrate (XTT; SigmaeAldrich Canada
Ltd., Oakville, Ontario) reduction assay. Briefly, XTT was dis-
solved in PBS at 1 mg/ml and menadione was prepared in
acetone at 1 mM. The XTT/menadione reagent was prepared
fresh and contained 12.5 parts XTT/1 part menadione. Bacte-
rial biofilms were prepared as above and treated with com-
pounds (250e3.9 mg/ml) for 4 h (anaerobiosis, 37 ꢁC) prior
to adding 25 ml XTT/menadione. After 1 h at 37 ꢁC, the absor-
bance at 490 nm (A490) was read using a microplate reader.
Yellowish solid; yield 36%; mp: 208e210 ꢁC; IR (KBr):
1
1699, 1695 cmꢂ1; H NMR (200 MHz, CDCl3 d): 1.64 (s,
6H), 1.68 (s, 6H), 1.72 (s, 6H), 2.11e2.20 (m, 8H), 3.97 (s,
6H), 3.99 (s, 3H), 4.65e4.72 (m, 4H), 5.05e5.11 (m, 2H),
5.50e5.56 (m, 2H), 6.40e7.82 (m, 16H); 13C NMR (50 MHz,
CDCl3 d) 16.5, 17.9, 25.4, 26.0, 39.3, 51.4, 55.9, 65.9, 111.6,
114.3, 116.7, 117.8, 119.4, 121.9, 122.0, 123.7, 126.6, 128.5,
129.5, 131.8, 134.8, 136.5, 141.3, 144.4, 144.6, 146.1, 146.4,
150.7, 164.8, 164.9, 168.2. Anal. Calcd for C50H58O10: C,
73.33; H, 7.14; O, 19.54. Found: C, 73.35; H, 7.11; O, 19.56.
4.2. Bacteria and culture conditions
P. gingivalis ATCC 33277 and S. mutans ATCC 25175
were used throughout the study. Bacteria were routinely grown
in Todd Hewitt Broth (BBL Microbiology Systems, Cockeys-
ville, MD) supplemented with hemin (10 mg/ml) and vitamin
K (1 mg/ml) (THB-HK) and incubated for 24 h in an anaerobic
chamber (N2/H2/CO2 75/10/15) at 37 ꢁC.
4.5. Statistical analyses
Differences between means were analyzed for statistical
significance using Student’s t-test. Differences were consid-
ered significant at the 0.05 level (P value).
4.3. Effect on growth and biofilm formation
Acknowledgements
The effect of 3-(40-geranyloxy-30-methoxyphenyl)-2-trans
propenoic acid 1, alcohols 2e11 and ester derivatives 12e21
Authors wish to thank the Canadian Institutes of Health Re-
`
search and the Ministero dell’Universita e della Ricerca,

1620
C. Bodet et al. / European Journal of Medicinal Chemistry 43 (2008) 1612e1620
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Rome, Italy. We also thank Louis Grignon for excellent tech-
nical assistance.
[12] J. Cudlin, M. Blumaerova, N. Steinorova, J. Mateju, J. Zalabak, Folia Mi-
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