Activity of caffeic acid derivatives against Candida albicans biofilm

Article abstract of DOI:10.1016/j.bmcl.2014.02.005

The aim of this study was to evaluate the caffeic acid (1) and ester derivatives (2-10) against Candida albicans biofilm and to investigate whether these compounds are able to inhibit the biofilm formation or destroy pre-formed biofilm. Caffeic acid ester 7, cinnamic acid ester 8 and 3,4-dihydroxybenzoic acid ester 10 are more active than fluconazole, used as reference drug, both on biofilm in formation with MIC₅₀ values of 32, 32 and 16 μg/mL, respectively, and in the early stage of biofilm formation (4 h) with MIC ₅₀ values of 64, 32 and 64 μg/mL, respectively. These esters result also more active than fluconazole on mature biofilm (24 h), especially 8 and 10 with MIC₅₀ values of 64 μg/mL.

Full text of DOI:10.1016/j.bmcl.2014.02.005

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1502–1505
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Activity of caffeic acid derivatives against Candida albicans biofilm
Daniela De Vita a, , Laura Friggeri a, , Felicia Diodata D’Auria c, Fabiana Pandolfi a, Francesco Piccoli a,
a,
b
Simona Panella c, Anna Teresa Palamara c, Giovanna Simonetti c, , Luigi Scipione , Roberto Di Santo ,
⇑
⇑
Roberta Costi b, Silvano Tortorella a
a Department of ‘Chimica e Tecnologie del Farmaco’, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
b ‘Istituto Pasteur-Fondazione Cenci Bolognetti’, Department of ‘Chimica e Tecnologie del Farmaco’, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
c Department of ‘Sanità Pubblica e Malattie Infettive’, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was to evaluate the caffeic acid (1) and ester derivatives (2–10) against Candida albi-
cans biofilm and to investigate whether these compounds are able to inhibit the biofilm formation or
destroy pre-formed biofilm.
Received 5 December 2013
Revised 30 January 2014
Accepted 3 February 2014
Available online 13 February 2014
Caffeic acid ester 7, cinnamic acid ester 8 and 3,4-dihydroxybenzoic acid ester 10 are more active than
fluconazole, used as reference drug, both on biofilm in formation with MIC50 values of 32, 32 and 16
mL, respectively, and in the early stage of biofilm formation (4 h) with MIC50 values of 64, 32 and 64
l
l
g/
g/
Keywords:
Candida albicans biofilm
Caffeic acid
Anti-biofilm agents
Biofilm formation
mL, respectively. These esters result also more active than fluconazole on mature biofilm (24 h), espe-
cially 8 and 10 with MIC50 values of 64 g/mL.
l
Ó 2014 Elsevier Ltd. All rights reserved.
Candida species are opportunistic pathogens that can cause a
wide variety of infections and they are the main agents responsible
for nosocomial fungal infections. Candida spp. can form readily bio-
film on indwelling medical devices and mucosal tissues. This bio-
film turns out to be an infectious reservoir difficult to eradicate,
and it causes device-associated infections with high mortality.1,2
The most prevalent fungal biofilm-forming pathogen is Candida
albicans, which cause both superficial and systemic infections. C.
albicans is able to colonize and form biofilms on devices such as
shunts, stents, endotracheal tubes and various types of catheters.3
Indeed, it has been reported that infections caused by biofilm-
forming C. albicans were significantly correlated with increased
mortality.4
The early stage of C. albicans biofilm formation is characterized
by the adhesion of single cells to the substratum, that was followed
by the formation of an intricate network of hyphae and the begin-
ning of a dense structure. Changes in the transcriptome begin
within 30 min of contact with the substrate. Some of these changes
are initiated early and maintained throughout the process; other
changes are typical of the earliest stages of biofilm formation.5
Among the phenotypic alterations displayed by cells in biofilms,
the most relevant from the clinical point of view is the increased
resistance to antifungal treatments.6,7 Compared to their plank-
tonic counterparts, biofilm cells exhibit up to a 1000-fold increased
resistance.
C. albicans biofilms have been reported to be resistant to a
variety of clinical antifungal agents, including fluconazole,8
a
well-tolerated antifungal drug, commonly used in the treatment
of candidiasis. Among the available antifungal agents, only
echinocandins and amphotericin B lipid formulations have shown
consistent activity against Candida biofilms. Unfortunately,
amphotericin B can cause severe nephrotoxicity to the host and
echinocandins are highly expensive to be used routinely.9 There-
fore, in this scenario, there is an urgent need for low-cost
compounds with a better efficacy and low side effects.
Several molecules were reported in literature to inhibit or
to prevent the C. albicans biofilm formation. Many of these
compounds are characterized by phenylethenyl moieties, such as
4-hydroxycordoin and methyl cinnamate;10,11 among these
molecules, caffeic acid phenethyl ester (CAPE) is able to inhibit
both Candida filamentation and biofilm formation (Chart 1).12
We have decided to test caffeic acid (1) and the ester derivatives
(2–10) against C. albicans planktonic and biofilm cells, in order to
evaluate the effects on anti-biofilm activity of structural modifica-
tion on the alcoholic and carboxylic moieties. The assessment of
anti-biofilm activity of these compounds may allow to understand
⇑
Corresponding authors. Tel.: +39 0649970115; fax: +39 064468625 (G.S.); tel.:
+39 0649913737; fax: +39 0649913133 (L.S.).
E-mail addresses: giovanna.simonetti@uniroma1.it (G. Simonetti), luigi.scipio-
ne@uniroma1.it (L. Scipione).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.02.005
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

D. De Vita et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1502–1505
1503
Chart 1. Phenylethenyl derivatives active on C. albicans biofilm.
Scheme 1. Synthetic procedures. (a) (Compounds 1–5): H2SO4, R3-OH, reflux;
(compounds 6 and 7): (i) NaOH, DMF, rt, 20 min; (ii) 4-(bromomethyl)-2-fluoro-1-
methoxybenzene (for 6), 2-(bromomethyl)-5-nitrofuran (for 7), rt, 50 h. (Compound
8) (i) Cs2CO3, DMF, rt, 20 min; (ii) 2-(bromomethyl)-5-nitrofuran, 12 h, rt. (b) (i)
NaOH, DMF, rt, 20 min, (ii) 2-(bromomethyl)-5-nitrofuran, 20 h, rt; (iii) 2 h, 50 °C.
the real importance of phenylethenyl group trying to delineate a
basic SAR rules.
We have synthesized different esters modifying the alcoholic
function with simple small alkyl chains (2–5), with small hydro-
phobic aromatic moiety (6) and nitro-furan group (7) that was cho-
sen on the basis of its known antibacterial properties.13
Furthermore, we have also modified the acylic moiety removing
the hydroxyl groups (8), saturating (9) or removing the ethenyl
double bond (10) (Chart 2).
The synthetic procedures were described in Scheme 1. Caffeic
acid esters (2–5) have been synthesized by Fisher esterification
starting from commercially available caffeic acid (1) as described
in the literature.14 The derivatives 6 and 7 were prepared via
the direct carboxylate oxygen alkylation of the caffeic acid,
respectively, by 2-(bromomethyl)-5-nitrofuran and 4-(bromo-
methyl)-2-fluoro-1-methoxybenzene,15 according to the method-
ology reported by Prasad et al.16
Similarly, compound 8 was prepared by O-alkylation of cin-
namic acid with 2-(bromomethyl)-5-nitrofuran using Cs2CO3 as
basic catalyst,17 following the procedure described by Parrish
et al.18 and the esters 9 and 10 have been synthesized by direct
O-alkylation of 3,4-dihydroxybenzylacetic acid and 3,4-dihydroxy-
benzoic acid with 2-(bromomethyl)-5-nitrofuran in presence of
aqueous 0.4 M NaOH,19 as described by Brewster et al.20
The analytical and spectroscopic data of new synthesized com-
pounds 6–10 are reported in Table S.1 in the Supplementary mate-
rial and are in agreement with the proposed structures.
Caffeic acid (1) and the ester derivatives 2–10 have been tested,
as described below, against planktonically grown C. albicans cells
and against C. albicans in formation biofilm, and early stage (4 h)
and mature (24 h) C. albicans biofilms.
For the in vitro antifungal experiments C. albicans ATCC 10231
purchased by the American Type Culture Collection (ATCC, Rock-
ville, MD, USA) were used. This strain is sensible to fluconazole
on planktonic cells (0.5 lg/mL) and it is resistant in the different
phases of biofilm formation. Antifungal activity of 1–10 and fluco-
nazole (Table 1) against C. albicans was evaluated in accordance
with the CLSI M27–A3 broth microdilution method.21 The minimal
inhibitory concentration (MIC) was calculated and expressed as the
lowest drug concentration at which a significant decrease in tur-
bidity (>50%) was detected in comparison with the control in the
absence of drug.
The effect of 1–10 and fluconazole on biofilm was evaluated as
described by Pierce et al. The anti-biofilm activity was evaluated
and expressed as the lowest drug concentration at which a 50% de-
crease in absorbance was detected in comparison with the level for
the biofilms formed in the absence of drug.22
The cytotoxicity of compounds 7–10 (Table 1) was evaluated
on human pulmonary mucoepidermoid carcinoma cell line
(NCI-H292) obtained from the ATCC (Rockville, MD, USA) using a
Chart 2. Synthesized compounds.

1504
D. De Vita et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1502–1505
Table 1
Antifungal activity of 1–10 against Candida albicans ATCC 10231 biofilms and planktonic cells and effect of compounds 7–10 on the human pulmonary mucoepidermoid
carcinoma cell line (NCI-H292) growth
Candida albicans ATCC10231
MIC50 g/mL
NCI-H292 CC50 lg/mL
l
Compd
(a) Biofilms in formation
(b) Biofilms at 4 h
(b) Biofilms at 24 h
(c) Planktonically grown cells
1
2
3
4
5
6
7
8
256
256
256
256
256
256
32
32
128
16
256
nd
nd
256
nd
nd
64
32
128
64
256
256
256
256
256
256
256
128
64
128
128
128
128
128
128
16
2
64
16
0.5
nd
nd
nd
nd
nd
nd
191.5 4.0
174.4 8.4
256.7 22.3
132.2 45.3
nd
9
10
128
64
256
Fluconazole
128
Candida albicans: The inhibition of biofilm formation (a) and destruction of pre-formed biofilm (b) by different compounds were examined by measuring the metabolic
activity of cells within the biofilm (XTT assay). The MIC end point for biofilms is based on the lowest drug concentration producing a decrease of 50% metabolic activity
relative to metabolic activity of the untreated growth control (XTT reduction assay). (c) MIC end point for planktonic cells is based on lowest drug concentration that
prevented P50% of growth with respect to the untreated control determined according to CLSI guidelines (CLSI document M27–A3.2008). At least two experiments were
performed on two separate dates for each compound tested in triplicate. The results were expressed as median.
NCI-H292 CC50: concentration required to reduce cell viability of the human pulmonary mucoepidermoid carcinoma cell line (NCI-H292) by 50%. Data represent
means standard deviation from two independent experiments, each performed in triplicate.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reduction assay.23 The cells were exposed to the compounds at the
final concentration ranging from 32 to 512 lg/mL. Each concentra-
The esters 7, 8 and 10 showed a better activity on biofilm in for-
mation with MIC values of 32, 32 and 16 g/mL, respectively, with
respect to mature biofilm at 4 and 24 h. Ester 8 also resulted quite
active at early stage biofilm (4 h) with MIC value of 32 g/mL.
Preliminary toxicity study on the esters 7–10 on human pul-
monary mucoepidermoid carcinoma cells (NCI-H292) shows no
cytotoxicity at anti-biofilm concentration (Table 1). Further studies
are needed for the evaluation of therapeutic index.
In conclusion, promising anti-biofilm activity was displayed by
7, 8 and 10, which could be considered as lead compounds to de-
velop new agents against Candida biofilm infections. Moreover
the combination of these compounds with established antifungal
agents could have additive or synergistic action that should be
studied further.
l
tion and control was assayed in three replicates with at least five
concentrations. The cytotoxicity of the compounds was calculated
as percentage reduction in viable cells with respect to the control
culture. The 50% cytotoxic concentration (CC50) was evaluated as
the drug concentration required to reduce human cell viability
by 50% compared to the drug-free control.
The results of the microbiological tests, reported in Table 1,
show that esters 7–10 are more active than the reference drug
fluconazole on biofilm in formation, on early formed biofilm (4 h)
and on mature biofilm (24 h). In particular, the anti-biofilm activity
of compound 8 and 10 was higher than fluconazole on biofilm in
formation and mature biofilms (P < 0.05 and P < 0.01, respectively).
The ester 10 has showed an activity eightfold higher than the ref-
erence drug fluconazole against the biofilm in formation. Ester 8
resulted also active at early stage of formation (4 h) with MIC value
l
Acknowledgments
We thank Miss Valentina Lilla and Professor Maria Elisa Cre-
stoni (Department of ‘Chimica e Tecnologie del Farmaco’ Sapienza
University of Rome, Italy) for obtaining the Mass spectra.
This work was supported by the Project PON 01_01802.
of 32 lg/mL. However, the tests clearly show that esters 7–10 are
also active on planktonic cells albeit less than fluconazole. These
data suggest that these compounds may inhibit the adhesion and
the aggregation of cells in the biofilm, with the consequent elimi-
nation of sessile cells.
Supplementary data
The 3,4-dihydroxybenzoic acid ester 10, the caffeic acid ester 7
and the cinnamic esters 8 are active versus biofilm in formation as
well as in the early stage and mature biofilm. In all these molecules
the ester function is conjugated, directly or via double bond, with
an aromatic ring. Differently, the reduction of the 2,3 double bond
of the ester 7, to obtain the saturated ester 9, has produced a reduc-
tion of the anti-biofilm activity compared to 7, 8 and 10. These data
suggest that the conjugation of the ester function with an unsatu-
rated system could be relevant for the anti-biofilm activity.
The presence of the two hydroxylic groups appears to be not
necessary for the inhibition of the biofilm in formation, as can be
evidenced by the comparison of the activity data of caffeic ester
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
02.005. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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7 and cinnamic ester 8 (32 lg/mL).
Data in Table 1 also show that all the compounds that contain
the (5-nitrofuran-2-yl)methyl moiety (7–10) retain the anti-
biofilm activity both on biofilm formation, on biofilm at the
early stage of formation (4 h) and on mature biofilm (24 h). The
(5-nitrofuran-2-yl)methyl appear to be fundamental for the anti-
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15. Synthesis of 6 and 7: In an ice bath, to 1.61 mmol of caffeic acid, dissolved in
2 mL of DMF, 0.4 mL of aqueous 0.4 M NaOH were added. After 20 min, at rt,
1.65 mmol of the opportune bromide were added in small portions over a
period of 60 min. The mixture was raised to room temperature and stirred for
50 h. The reaction mixture was poured in 10 mL of water and extracted with
ethyl acetate (3 Â 20 mL). The combined organic layers were washed with
20 mL of 1 M HCl and water, drying over Na2SO4 and evaporated under
reduced pressure. For 6, the crude residue was subjected to silica gel column
chromatography using Et2O/n-hexane (80:20 v/v) until the output of the
bromide and after Et2O to give 6 as white solid crystallized from AcOEt/
petroleum ether (25% yield). For 7 the residue was subjected to silica gel
column chromatography using AcOEt–MeOH (95:5 v/v) to give a yellow solid
(20% yield).
formed precipitated was filtered off and the separated organic layers were
washed with water, dried over Na2SO4 and evaporated under reduced pressure.
The obtained crude residue was purified by column chromatography on silica
gel using CH2Cl2/n-hexane (80:20 v/v) as eluent to give 8 as yellowish solid
(yield 40%).
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dissolved in DMF (2 mL) then 0.4 mL of aqueous 0.4 M sodium hydroxyde were
added. The mixture was stirred for 20 min and then 2-(bromomethyl)-5-
nitrofuran (1.6 mmol) was slowly added. After the addition was complete, the
mixture was allowed to reach ambient temperature for 20 h and afterward it
was stirred for 2 h at 50 °C. The reaction mixture was treated with H2O and
AcOEt (3:1). The ethyl acetate layer was separated and the aqueous layer
extracted with ethyl acetate (20 mL). The combined ethyl acetate extracts were
washed with water (30 mL), saturated sodium hydrogen carbonate solution
(30 mL), water (30 mL) and with saturated sodium chloride solution (30 mL)
and dried over Na2SO4. The solvent was removed under vacuum and the
residue was purified with column chromatography using silica gel and AcOEt/
n-hexane as eluent to give a yellow solid which was crystallized from toluene
(yield 40–50%).
20. Brewster, A. G.; Caulkett, P. W. R.; Faull, A. W.; Pearce, R. J.; Shute, R. E. US
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17. Synthesis of 8: Under an atmosphere of nitrogen, 1.35 mmol of trans-cinnamic
acid were dissolved in DMF. Then cesium carbonate (1.35 mmol) was added.
After 10 min, 2-(bromomethyl)-5-nitrofuran (1.35 mmol) was added. The
corresponding brownish suspension was stirred for 1 h at room temperature
under N2. Next, the reaction mixture was quenched by adding 1 M HCl (15 mL),
then the resulting solution was treated with n-hexane/EtOAc (30:10 v/v). The
21. Clinical and Laboratory Standards Institute Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard
Document M27–A3, 3rd ed.; CLSI: Wayne, PA, 2008.
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