M. Bortolami et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128087
concentration of ligand. The variation of the UV spectra of the ligand in
presence of metal ions is indicative of the complex formation. These
spectra showed that benzylated derivatives 3a and 9a can chelate only
Fe3+, while debenzylated compounds 3b and 9b chelate both Fe3+ and
Cu2+ (Fig. S1–S8 supporting information).
μ
g/mL and 64
μ
g/mL respectively, but not towards planktonic cells. The
corresponding deprotected compounds 9b and 10b show a lower or a
similar potency against biofilm formation, with BMIC50 values of 128
μ
g/mL and 64 μg/mL respectively. These data suggest that their activity
on biofilm could be due not only to chelation proprieties, but also to
other inhibition mechanisms, probably related to the NSAIDs moiety. On
the contrary, the debenzylated derivatives 9b and 10b show a very
interesting activity against C. albicans planktonic cells, with MIC50 of 4
The synthesized compounds 2a,b-13a,b have been screened to
evaluate the activity against C. albicans planktonic cells and biofilm,
both in formation and mature (48 h), using C. albicans ATCC 10231, a
strain sensitive to fluconazole on planktonic cells (0.5 µg/mL) and
resistant in the different phases of biofilm formation (BMIC50 128 µg/mL
on biofilm formation and > 128 µg/mL on mature biofilm).45 The results
are reported in Table 1.
μg/mL and 8 μg/mL respectively.
Finally, none of the compounds obtained by molecular duplication of
DFP group 11a,b-13a,b were found active against biofilm and plank-
tonic cells up to a concentration of 128 g/mL.
μ
Among the amine derivatives 2a,b-5a,b, the compounds 2b and 3b
are the most interesting, both for their activity against biofilm formation
In order to support the hypothesis that a chelation mechanism could
be involved in the activity of the best inhibitor of biofilm formation 3b,
other experiments were carried out using culture medium enriched with
increasing concentration of Fe3+ and Cu2+. The obtained data are re-
ported in the Fig. 1. As expected, a dose/related decrease of the activity
against biofilm formation was observed in presence of increasing con-
centration of the metallic ions. Recently, the relationship between the
inhibition of biofilm formation and the reduction of available iron in
C. albicans has also been demonstrated by Hsu et al. These authors
showed the antifungal activity of a compound, which inhibits yeast-to-
hyphal transition and biofilm formation of C. albicans by interfering
with iron ion homeostasis.22 Moreover, Sumant Puri et al. have
demonstrated that C. albicans cells treated with the iron chelator
deferasirox have a significantly reduced adhesion ability.19 Adhesion is
the first step in the formation of the biofilm. This could explain the ac-
tivity of 3b in the formation of the biofilm and not in the dispersion of
the preformed biofilm.
(BMIC50 32 μg/mL and 16 μg/mL respectively), comparable or superior
to that of the parent compound DFP (BMIC50 32
activity against planktonic cells (MIC50 32 g/mL and 16
respectively), much higher than that of DFP (MIC50 128 g/mL). These
μ
g/mL), and for their
μ
μ
g/mL
μ
compounds contain a phenyl alkyl moiety connected with DFP; it can be
noted that passing from three (compound 2b) to four (compound 3b)
methylene units the biofilm inhibition activity probably increases due to
the higher lipophilicity of 3b (MLogP = 1.83) compared to 2b (MLogP =
1.58).46,47
It is noteworthy that the corresponding benzylated molecules 2a and
3a are not active up to a concentration of 128 μg/mL, indicating that the
free hydroxyl function in position 3 is of great importance for obtaining
antibiofilm activity and this could be related to the depletion of metals
from the cellular environment by a chelation mechanism; indeed, the
presence of the benzyl protecting group on the oxygen atom in position 3
of DFP makes the latter less available for the chelation of metals.
Regarding the amide derivatives 7a,b-10a,b, the most potent in-
hibitors are compounds 9a,b and 10a,b which derive from the
connection between DFP with NSAIDs, in particular ibuprofen and
ibufenac respectively. In particular, the benzylated DFP derivatives 9a
and 10a are active against biofilm formation, with BMIC50 values of 32
The stoichiometry of 3b complexed with Fe3+ and Cu2+ was deter-
mined by the method of continuous variations of Job,48 which is
described in detailed in supporting information. In Fig. 2 the UV titra-
tion spectra of ligand 3b with Fe3+ and the related Job’s plot obtained at
302 nm are depicted. Both for Fe3+ and Cu2+ complexes a 1:1 stoichi-
ometry among the ligand and the metal ion was observed. Known the
stoichiometry, it was possible to calculate the stability constants (Kstab
)
of these complexes using UV–Vis spectroscopy method, as described in
the supporting information.49 The corresponding logKstab of 3b-Fe3+
and 3b-Cu2+ complexes were 5.16 ± 0.27 and 4.63 ± 0.09 respectively.
Finally, we evaluated the in vivo toxicity of the most active anti-
biofilm compound 3b on larvae of Galleria mellonella. This is a simple
and low-cost validated model widely used, because its results correlate
with those observed in mammals.50 The detailed procedure is reported
in the supplementary materials. The lethal dose that reduces the number
of G. mellonella larvae by 50% (LD50) of 3b has not been found even
when testing a dose more of 10 times higher (512 µg/mL) than the active
against C. albicans biofilm (BMIC50 16 µg/mL). The results, reported in
Table 2, showed that G. mellonella larvae treated with 3b at the con-
centration of 512 µg/mL displayed a 100%, 96% and 86% of survival
rate respectively at 24 h, 48 h and 72 h; no toxic effects were observed at
lower concentration of 3b (Table 2).
Table 1
Antifungal activity of the DFP derivatives 2a,b-13a,b against C. albicans ATCC
10,231 biofilms and planktonic cells.
BMIC50
(μ
g/mL)
MIC50 (μg/mL)
Compound
Mature biofilm
Biofilm formation
Planktonic cells
2a
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
32
>128
32
2b
3a
>128
16
>128
16
3b
4a
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
4b
5a
5b
7a
7b
8a
In conclusion, in this work we describe the synthesis of a new series
of DFP derivatives with the aim of obtaining compounds able to chelate
the metal cations Fe3+ and Cu2+ and endowed with antibiofilm activity.
Two representative compounds of the benzylated series on the oxygen 3
of DFP (3a, 9a) showed the ability to chelate exclusively the Fe3+ ion,
while the corresponding debenzylated compounds with free hydroxyl
8b
9a
32
9b
128
10a
10b
11a
11b
12a
12b
13a
13b
DFP
64
>128
8
64
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
128
group (3b, 9b) resulted chelators of both Fe3+ and Cu2+
.
The data obtained by antifungal and antibiofilm activity tests in
cultured ATCC 10,231C. albicans indicate that none of the synthesized
compound resulted active on mature biofilm. Otherwise, among the
amine derivatives 2a,b-5a,b, only 2b and 3b resulted moderately active
both on biofilm formation and on planktonic cells, with 3b being the
128
>128
32
BMIC50: the lowest drug concentration producing a 50% decrease of biofilm
relative to the untreated growth control. MIC50: the lowest drug concentration
producing 50% growth inhibition. The antifungal activities are the result of
three independent experiments performed in triplicate. The data were presented
as median.
most active antibiofilm compound identified (BMIC50 = 16 μg/mL). The
reduction in the inhibitory effect of compound 3b on biofilm formation
in the presence of increasing amounts of Fe3+ and Cu2+ suggests that this
4