Exploring the anti-biofilm activity of cinnamic acid derivatives in Candida albicans

Article abstract of DOI:10.1016/j.bmcl.2016.10.091

Some compounds, characterized by phenylethenyl moiety, such as methyl cinnamate and caffeic acid phenethyl ester, are able to inhibit C. albicans biofilm formation. On these bases, and as a consequence of our previous work, we synthesized a series of cinnamoyl ester and amide derivatives in order to evaluate them for the activity against C. albicans biofilm and planktonically grown cells. The most active compounds 7 and 8 showed ?50% biofilm inhibition concentrations (BMIC₅₀) of 2 μg/mL and 4 μg/mL respectively, against C. albicans biofilm formation; otherwise, 7 showed an interesting activity also against mature biofilm, with BMIC₅₀of 8 μg/mL.

Full text of DOI:10.1016/j.bmcl.2016.10.091

Accepted Manuscript
Exploring the anti-biofilm activity of cinnamic acid derivatives in Candida al-
bicans
Daniela De Vita, Giovanna Simonetti, Fabiana Pandolfi, Roberta Costi, Roberto
Di Santo, Felicia Diodata D’Auria, Luigi Scipione
PII:
S0960-894X(16)31139-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.091
BMCL 24394
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 September 2016
29 October 2016
31 October 2016
Please cite this article as: De Vita, D., Simonetti, G., Pandolfi, F., Costi, R., Di Santo, R., Diodata D’Auria, F.,
Scipione, L., Exploring the anti-biofilm activity of cinnamic acid derivatives in Candida albicans, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.091
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Exploring the anti-biofilm activity of cinnamic acid derivatives in Candida
albicans
Daniela De Vita a #, Giovanna Simonetti c #, Fabiana Pandolfi a, Roberta Costi a,b, Roberto Di Santo
a,b, Felicia Diodata D'Auria c, Luigi Scipione b*
a Dept. Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro, 5, I-00185,
Rome, Italy
b “Istituto Pasteur-Fondazione Cenci Bolognetti”, Dept. Chimica e Tecnologie del Farmaco,
Sapienza University of Rome, P.le A. Moro, 5, I-00185, Rome, Italy
c Department of Public Health and Infectious Diseases “Sapienza” University of Rome, Piazzale
Aldo Moro 5, 00185 Rome, Italy
# The authors Daniela De Vita and Giovanna Simonetti equally contributed to the work.
* Corresponding author: luigi.scipione@uniroma1.it
1

Abstract
Some compounds, characterized by phenylethenyl moiety, such as methyl cinnamate and caffeic
acid phenethyl ester, are able to inhibit C. albicans biofilm formation. On these bases, and as a
consequence of our previous work, we synthesized a series of cinnamoyl ester and amide
derivatives in order to evaluate them for the activity against C. albicans biofilm and planktonically
grown cells.
The most active compounds 7 and 8 showed ≥ 50 % biofilm inhibition concentrations (BMIC50) of
2µg/mL and 4µg/mL respectively, against C. albicans biofilm formation; otherwise, 7 showed an
interesting activity also against mature biofilm, with BMIC50 of 8 µg/mL.
Keywords
Biofilm; Candida albicans; Cinnamic esters; Cinnamoyl amides.
2

Candida albicans (C. albicans) is the most common fungal pathogen in clinical settings and it is
responsible of infections that can affect the skin and the mucosa or cause life-threatening systemic
disease.1,2 The mortality among patients with invasive candidiasis is as higher than 40%, even when
patients receive antifungal therapy.3
One of the major problem related to the treatment of C. albicans infections is therapeutic failure,
especially due to the onset of therapeutic resistance phenomena, which are very often associated
with the biofilm formation.4
Biofilms are defined as complex microbial communities encased in a matrix of extracellular
polymeric substances, that develops when a community of microorganisms irreversibly adheres to
an inert or living surface. Contact with a solid surface triggers the expression of a panel of enzymes,
which catalyze the formation of sticky polysaccharides that promote the surface colonization and
the microbial cells protection. This adherent community is considered an important virulence factor
because it is difficult to eradicate and often responsible for treatment failures.5 Indeed, the biofilm
represents a physical barrier that prevents drugs from entering and expressing their activity.
In C. albicans the morphogenesis and the biofilm formation are controlled by a complex
mechanism of communication termed quorum sensing (QS), a process that is sensitive to the cell
density in the biofilm population. The QS is based on the exchange and sensing of low molecular
weight signal compounds,6 in particular, three substances have been identified as QS molecules:
farnesol, phenylethyl alcohol and tryptophol.7 However, the antifungal resistance of C. albicans
biofilm is complex and it involves not only the physical barrier due to the polysaccharide layer, but
a number of different mechanisms, such as over-expression of efflux pumps, genetic changes of
drug targets, persister cells, biofilm-host immune system interaction.8
Azoles currently used for the treatment of systemic infections (e.g. fluconazole, itraconazole) have
little effect against Candida biofilms, even at high doses and in combination with caspofungin.9 For
these reasons, new therapies are urgently needed to treat the wide variety of Candida biofilm
infections in the medical setting.
3

In literature there are few compounds able to inhibit C. albicans biofilm formation,10 among them
there are methyl cinnamate and caffeic acid phenethyl ester (CAPE), characterized by
phenylethenyl moiety.11 Based on these evidences, in our previous work we tested, against C.
albicans planktonic and biofilm cells, a series of caffeic and cinnamic acid derivatives (Chart 1).
O
O
CH3
HO
O
O
HO
CAPE
Methyl cinnamate
O
O
HO
HO
OH
OH
Cinnamic acid
Caffeic acid
O
O
O
NO2
Lead compound (1)
Chart 1. Compounds characterized by a phenylethenyl moiety and our lead compound 1.
12,13,14
By referring to the structure of CAPE and its biological properties,
we synthesized several
ester and amide compounds with simple chains or several more complex groups. Furthermore, we
have removed the two hydroxylic groups, typical of caffeic acid derivatives, saturated or removed
the ethenyl double bond of the acylic moiety. As a result of that work, we identified a new
cinnamate derivative ((5-nitrofuran-2-yl)methyl-(2E)-3-phenylprop-2-enoate (1) showing a good
inhibition activity against C. albicans biofilm. It was able to inhibit the biofilm formation and to
reduce the metabolic activity of preformed biofilm, better than the reference drug fluconazole.
Our basic SAR study has showed that the conjugation of the carbonyl group with an unsaturated
system seems to be relevant for the anti-biofilm activity; furthermore, the presence of the
hydroxylic groups appears to be not necessary.15
4

In this paper we report the synthesis and the activity evaluation, on C. albicans biofilm, of a new
series of cinnamoyl ester and amide derivatives that have been designed with the idea of preserving
the cinnamoyl moiety and removing the hydroxyl groups of caffeic acid, in order to obtain an
improved chemical stability and a simplified reaction work up.
We also investigated the importance of the nitro group, present in our reference compound (1),
forasmuch as, it is known from the literature that compounds able to induce the production of
reactive oxygen species (ROS) possess a potential antibiofilm activity.16, 17 The ROS are usually
generated by in vivo electron transfer processes that involve specific chemical functionalities
including aromatic nitro compounds.18 In this context, we have synthesized several ester and amide
derivatives combining the carboxylic function of cinnamic acid with different groups, including
nitro heterocyclic groups, azole and indole rings (Chart 2).
O
R
Compound
1
R
Compound
6
R
O
O
O
O
O
NO
NO
2
Cl
2
O
N
Cl
2
7
N
N
N
H C
3
F
N
N
O
N
F
N
N
NO
2
3
4
5
8
9
N
N
N
O N
2
NH
N
N
N
N
N
H
N
N
S
NH
10
NH
NO
2
Chart 2. Studied cinnamic acid derivatives 1-10.
5

We also synthesized some molecular hybrids between cinnamic acid and other compound endowed
with own antifungal activity. In particular, we have chosen molecular fragment of anti-biofilm
compounds as miconazole (7) and molecules that are active against planktonic cells as fluconazole
(8).19
Furthermore, we synthesized the derivative 10, a hybrid between cinnamic acid and tryptamine,
because it is demonstrated that compounds, such as indole and 3-indolylacetonitrile, are able to
reduce biofilm formation and virulence, by the regulation of NRG1, a transcriptional factor that
influences filamentation and biofilm formation in C. albicans.20
The synthetic procedures of the studied compounds are illustrated in the schemes 1 and 2; briefly,
the cinnamic acid was activated to cinnamoyl chloride by the treatment with SOCl2, then the
opportune alcohol or amine were added to synthesize the corresponding ester (2, 6) or amide (3-5,
9, 10).
O
O
O
b
a
Cl
OH
R
(2-6, 9-10)
Scheme 1: General procedure for the synthesis of 2-6, 9-10. a) SOCl2, 2 hrs, reflux; b) opportune alcohol or amine,
TEA, DCM, 12 hrs, reflux (2-6, 9) or r.t. (10).
The compounds 2, 5 and 10 were synthesized modifying the procedures reported in literature.21, 22, 23
For the synthesis of the compound 2, the metronidazole was obtained by extraction with
dichloromethane after shattering commercial tablets in warm water;24 whereas, the 1-(3-
nitropyridin-2-yl)piperazine, necessary to synthesize compound 4, was prepared as we have
previously reported.25
Attempts to synthesize esters 7 and 8 using the above illustrated scheme were unsuccessful, then it
was necessary to preventively activate the alcoholic function by NaH before the reaction with
cinnamoyl chloride.
6

N
N
N
X
O
N
R3
X
a, b
R3
HO
O
R2
R1
R2
(7-8)
R1
7
8
. R1=R2=Cl; R3=H; X=CH
. R1=R2=F; R3=1H-1,2,4-triazole; X=N
Scheme 2: General procedure for the synthesis of 7, 8. a) NaH, CH3CN, 2 hrs, r. t. (7) or 50°C (8); b) cinnamoyl
chloride, 12 hrs, r. t. (7) or 50°C (8).
The detailed synthetic procedures, the analytical and spectroscopic data of new synthesized
compounds are reported in the supplementary material and are in agreement with the proposed
structures.
Cinnamic acid, its derivatives 2–10 and parent alcohol or amine compounds, have been screened to
evaluate the activity against planktonically growing C. albicans cells and against C. albicans
biofilm formation and preformed. For the in vitro antifungal and anti-biofilm experiments,
described in detail in the supplementary material, C. albicans ATCC 10231 was used. This strain is
sensitive to fluconazole on planktonic cells (MIC50 0.5 µg/mL) and it is resistant in the different
phases of biofilm formation (BMIC50 128 µg/mL and >128 µg/mL respectively on biofilm
formation and mature biofilm). The minimal inhibitory concentration (MIC) was calculated and
expressed as the lowest drug concentration at which a significant decrease in turbidity (>50%) was
detected in comparison with the control in the absence of drug. The 50% inhibitory concentration
(BMIC50) were defined as the concentrations causing 50% inhibition of either biofilm formation or
preformed biofilms due to drug treatment.
The results of the microbiological assays are summarized in table 1, where the MIC50 and BMIC50
values of the synthesized cinnamic derivatives 2-10 are compared to the earlier published
compound 1, cinnamic acid and parent alcohol and amine compounds.
7

Table 1. Antifungal activity of compounds 1–10, cinnamic acid and the parent compounds against C. albicans ATCC
10231 biofilm and planktonic cells.
MIC50
BMIC50 (µg/mL)
(µg/mL)
Ester or
amide
compound
Parent
alcohol or
amine
Mature
biofilm
Biofilm
formation
Planktonic
cells
1
64
64
16
64
32
2
16
O
HO
NO2
2
>128
128
NO2
HO
N
>128
>128
>128
N
H3C
3
4
>32
>128
>128
>128
>128
>128
32
>128
>128
HN
N
NO2
O2N
HN
N
>128
>128
64
>128
>128
64
>128
>128
>128
N
5
N
H2
N
S
NO2
6
7
128
>128
8
16
>128
2
64
>128
2
O
HO
HO
N
N
Cl
64
64
4
16
64
Cl
8
>32
N
N
N
N
N
N
>128
128
0.5
F
HO
F
9
>128
>128
>128
>128
>128
8
>128
>128
>128
H2N
N
N
10
H2N
>128
128
>128
NH
Cinnamic
acid
>128
>128
>128
The inhibition of biofilm formation and destruction of pre-formed biofilm by different compounds were evaluated by
measuring the metabolic activity of cells within the biofilm (XTT assay). The BMIC end point for biofilm is based on
the lowest drug concentration producing a decrease of 50% metabolic activity relative the untreated growth control.
8

MIC end point for planktonic cells is based on lowest drug concentration that prevented 50% of growth with respect to
the untreated control. At least two experiments were performed on two separate dates for each compound tested in
triplicate. The results were expressed as median.
In most cases the presence of the cinnamic moiety produces an increase of the antibiofilm activity,
particularly for the biofilm formation, as can be observed for compounds 2, 6, 7, 8 and 10 with
respect of the corresponding amines or alcohols. The only exception is presented by the compound
5, for which was observed an increase of the BMIC50 values compared to the corresponding amine.
The activities of compounds 2-5 on mature biofilm, on biofilm formation, as well on planktonic
cells, range from 32 to >128 µg/mL. These data indicate that the replacement of the nitrofuran
moiety of the reference compound 1 with other nitroheterocyclic groups, as nitroimidazole (2),
nitropyridine and (4) nitrobenzothiazole (5), or nitrophenyl group (3), resulted in a significant
reduction of the antibiofilm activity. Furthermore, the removal of the nitro group from the
compound 1 to obtain the furan derivative 6 did not produce significant changes against biofilm in
formation but, it reduced the activity on preformed biofilm, as the BMIC50 increased from 64 to 128
µg/mL, and more markedly on planktonic cells, as the MIC50 increased from 2 to 64 µg/mL.
Taken together, the activity data for compound 2-5 compared to 1 indicate that the presence of
nitrofuran moiety appears to be critical to obtain the anti-biofilm activity. These data also suggest
that the antibiofilm activity of the nitrofuran moiety could be due to a combined action on
planktonic cells and on the biofilm, especially in the mature phases.
It is noteworthy that in this study we have identified other new interesting anti biofilm compounds.
Cinnamic acid not showed antibiofilm activity, as reported in table 1, but combining it with
molecular fragments that are present in antifungal drugs, new compounds have been obtained with
antibiofilm activities.
In particular, the most active derivative 7, containing a molecular fragment of miconazole, has
shown inhibition of biofilm in formation at the concentration of 2 µg/mL and reduction in metabolic
9

activity of preformed biofilm at 8 µg/mL, lower than the lead compound 1 (16 and 64 µg/mL,
respectively) and the reference drug fluconazole (128 and >128 µg/mL, respectively).
Finally, the esterification of fluconazole with cinnamic acid, to produce the hybrid compound (8), it
has led to a significant increase in the activity on biofilm formation (BMIC50 4 µg/mL vs 128
µg/mL) and in minor extent on preformed biofilm. On the other hand, the hybrid of cinnamic acid
with tryptamine (10) have produced an enhancement of the anti-biofilm activity, selectively against
biofilm in formation, with the reduction of BMIC50 value from 128 µg/mL to 8 µg/mL.
In conclusion, the synthesis of amide and ester derivatives of cinnamic acid has allowed to identify
new compounds endowed of antibiofilm activity, particularly effective in the early phases of
biofilm formation. The cinnamic moiety does not seem to act as a simple lipophilic carrier but
instead appears to be responsible for antibiofilm activities; in fact, in most cases, quite active esters
or amides were obtained from amines or alcohols which do not have activity on biofilm
(compounds 1, 2, 3 and 6 vs parent alcohols or amides). Furthermore, the cinnamic group enhances
the weak antibiofilm activity of the 1-(2,5-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol, the parent
alcohol of ester 7, and confers good antibiofilm activity to fluconazole, the parent alcohol of ester 8.
The activity data obtained for these cinnamic derivatives also indicate that the approach of
combining into a hybrid, two molecular fragments characterized by their own activities, could be
advantageously exploited to develop new compounds with anti-biofilm activity.
AKNOWLEDGEMENT
This work was supported by “Progetto di Ateneo 2014” funding from Sapienza University of Rome.
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Chart 1. Compounds characterized by a phenylethenyl moiety and our lead compound 1.
Chart 2. Studied cinnamic acid derivatives 1-10.
Scheme 1: General procedure for the synthesis of 2-6, 9-10. a) SOCl2, 2 hrs, reflux; b) opportune alcohol or amine,
TEA, DCM, 12 hrs, reflux (2-6, 9) or r.t. (10).
Scheme 2: General procedure for the synthesis of 7, 8. a) NaH, CH3CN, 2 hrs, r. t. (7) or 50°C (8); b) cinnamoyl
chloride, 12 hrs, r. t. (7) or 50°C (8).
13

14