Preventing Candida albicans biofilm formation using aromatic-rich piperazines

Article abstract of DOI:10.1016/j.bmc.2020.115810

The global increase in microbial resistance is an imminent threat to public health. Effective treatment of infectious diseases now requires new antimicrobial therapies. We report herein the discovery of aromatic-rich piperazines that inhibit biofilm forma

Full text of DOI:10.1016/j.bmc.2020.115810

Bioorg. Med. Chem. 28 (2020) 115810
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preventing Candida albicans biofilm formation using
aromatic-rich piperazines
a,
b
,
1
,
a,
1
b
¨
´
´
ˆ ´ a
Gaelle Simon
, Christopher Berube , Pierre-Alexandre Paquet-Cote , Daniel Grenier ,
*
Normand Voyera
a
´
´
´
´
Departement de Chimie and PROTEO, Universite Laval, 1045 Avenue de la Medecine, Quebec, QC G1V 0A6, Canada
b
´
´
´
´
´
Groupe de Recherche en Ecologie Buccale, Faculte de Medecine Dentaire, Universite Laval, 2420 Rue de la Terrasse, Quebec, QC G1V 0A6, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords:
The global increase in microbial resistance is an imminent threat to public health. Effective treatment of in-
fectious diseases now requires new antimicrobial therapies. We report herein the discovery of aromatic-rich
piperazines that inhibit biofilm formation by C. albicans. 22 piperazines, including 16 novel ones, were pre-
pared efficiently using a combination of solid- and solution phase synthesis. The most potent compound prevents
morphological switching under several hypha-inducing conditions and reduces C. albicans’ ability to adhere to
epithelial cells. These processes are essential to the development of Candida biofilms, which are associated with
its increased resistance to immune defenses and antifungal agents.
Piperazines
Candida albicans
Antibiofilm
Morphological switching
Oxime resin
1. Introduction
pathogenesis.13
On the other hand, the biofilm life cycle is initiated by the adherence
Candida albicans, a commensal microorganism that colonizes the
human mucosa, is an opportunistic pathogen that can cause various
infections1 ranging from superficial dermal or mucosal infections, such
as candidiasis2,3 and denture stomatitis,4 to often-deadly bloodstream
infections.5 This fungus represents a serious threat, especially for
immunocompromised individuals6 and for healthy patients with
implanted medical devices,7,8 including heart valves and pacemakers,
prostheses, dentures and catheters.9 In fact, C. albicans is the main
fungus responsible for medical-device-associated infections, and
Candida species are the third-most frequent causal agents of sepsis, ac-
counting for 15% of all nosocomial bloodstream infections.1,10 Coloni-
zation of medical devices, mucosa, and tooth enamel by C. albicans is
closely associated with its ability to adhere to, and form biofilms on,
these surfaces.
of yeast to a surface, followed by proliferation until a basal layer is
formed. Then, the biofilm matures through the elongation of hyphae and
secretion of extracellular matrix components.1 Finally, the yeast is
released, which disperses the biofilm to colonize new areas.14
Considering the socioeconomic burden of Candida medical-device-
associated infections15–18 and the resistance of fungal biofilms to
drugs,19–22 the development of novel therapies based on new modes of
action is imperative.6,23-25 Small molecules that interfere with
morphogenesis could be useful adjuvants for antifungal therapies.
The piperazine ring is the fourth most frequent heterocyclic scaffold
found in drugs.26 Piperazines are well known for their wide range of
bioactivities27 and can be synthesized by reduction of 1,4-diketopipera-
zines, a class of cyclic dipeptides (CDPs) often reported for anti-biofilm
properties.28,29 Herein, we report the synthesis of a series of 22 piper-
azines, including 16 novel ones (Figure 1) and their anti-biofilm for-
mation against C. albicans.
Indeed, it forms highly structured biofilms that harbor multiple cell
types, including budded round cells (yeast) and elongated joint cells
such as hyphae and intermediate pseudohyphae.11 It has been shown
that C. albicans can switch from one morphotype to another in response
to specific environmental conditions, a process called morphogenesis.12
Each morphology is implicated in a different step of biofilm develop-
ment, which all play critical roles in C. albicans virulence and
* Corresponding author.
E-mail address: normand.voyer@chm.ulaval.ca (N. Voyer).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmc.2020.115810
Received 19 July 2020; Received in revised form 4 October 2020; Accepted 5 October 2020
Available online 9 October 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
2. Results
6-chlorohydroxybenzotriazole (6-Cl-HOBt) and (2-(6-Chloro-1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate
2.1. Synthesis of piperazine analogs
(HCTU). After deprotection, the linear dipeptide was simultaneously
cyclized and cleaved from the resin in the presence of diisopropyle-
thylamine (DIEA; 2.5 equiv) and acetic acid (AcOH; 5 equiv) in
dichloromethane, leading to high-purity CDPs. Using this synthetic
procedure, we successfully synthesized CDPs with a variety of chiral cis
and trans side chains. A total of 9 CDPs were prepared and characterized
by 1H NMR, 13C NMR and mass spectrometry analyses.31,32 Then,
piperazine analogs were readily produced by the reduction of the CDPs
using an excess of lithium aluminum hydride in THF at reflux for 24–72
h. The key to a successful reduction here is the treatment of the crude
reduction mixture with sodium sulfate decahydrate for 30 min
(Figure 1).37,38 Aromatic piperazines 1, 3, 5 were obtained with 80% to
87% yields. Following the reduction step, a part of the cis piperazines
bearing protected phenol side chains (1a, 1b, 3a and 3b) were imme-
diately subjected to hydrogenolysis using H2 and Pd on charcoal as
Based on the possibility of synthesizing rapidly aromatic CDPs,30
a
series of piperazines was prepared using L- or D-phenylalanine and
tyrosine following the general procedure illustrated in Figure 1. A
noteworthy feature of our synthetic approach is the efficient solid-phase
preparation of chiral CDPs from readily available N-Boc protected amino
acids,31–34 which can be converted to the corresponding piperazines.
A set of 22 piperazines (1–5) and N,N’-dimethyl-piperazines (1′-5′)
with various stereochemistries and aromatic substituents was synthe-
sized to allow structure–activity investigations. Briefly, the synthesis
started by coupling the first amino acid on the oxime resin35,36 for three
hours using diisopropylcarbodiimide (DIC) as coupling reagent. The N-
Boc protecting group was removed using a mixture of 1:1 trifluoroacetic
acid (TFA)/dichloromethane. The second amino acid was coupled using
R1
R2
O
1. 50% TFA
CH2Cl2, 30 min
O
DIC, 6-Cl-HOBt
H
Boc-HN
HO
COOH
Boc-HN
N
N
N
O
2. Boc-AA2-COOH
HBTU, 6-Cl-HOBt
DIEA, CH2Cl2/DMF, 3h, r.t.
Boc-HN
O
+
DMAP, DIEA
CH2Cl2/DMF, 3h, r.t.
O
N
R1
1. 50% TFA
CH2Cl2, 30 min
R1
Oxime resin
2. DIEA, AcOH
CH2Cl2, 24h, r.t.
O
O
O
O
O
R2
R1
R2
R2
R1
NH
NH
NH
HN
NH
R2
HN
HN
HN
R1
R1
O
O
O
cis-(L,L)
cis-(D,D)
trans-(L,D)
CDPs
R1 and R2 = H, OBn or OH
1. LiAlH4,THF
reflux, 24-72h.
2. Na2SO4 10H2O
reflux, 30 min.
R2
R1
R2
R2
NH
NH
NH
HN
2,5-piperazines (1-5)
HN
HN
R1
R1
cis-(L,L)
cis-(D,D)
trans-(L,D)
1a: R1 = H, R2 = OBn : 84%
2a: R1 = H, R2 = OH : 64% (2 steps)
3a: R1 = R2 = OBn : 80%
1b: R1 = H, R2 = OBn : 82%
1c: R1 = H, R2 = OBn : 85%
1d: R1 = OBn, R2 = H : 87%
3c: R1 = R2 = OBn : 83%
4c: R1 = R2 = OH : 64% (2 steps)
5c: R1 = R2 = H : 86%
2b: R1 = H, R2 = OH : 60% (2 steps)
3b: R1 = R2 = OBn : 81%
4b: R1 = R2 = OH : 58% (2 steps)
5b: R1 = R2 = H : 82%
4a: R1 = R2 = OH : 60% (2 steps)
5a: R1 = R2 = H : 86%
HCOOH / HCOH
70 oC, 30 min.
R2
R1
R2
R2
N
N
N
N,N'-2,5-dimethylpiperazines (1'-5')
N
N
N
R1
R1
cis-(L,L)
cis-(D,D)
5b': R1 = R2 = H : 78%
trans-(L,D)
5c': R1 = R2 = H : 80%
1a': R1 = H, R2 = OBn : 74%
2a': R1 = H, R2 = OH : 58% (2 steps)
3a': R1 = R2 = OBn : 75%
4a': R1 = R2 = OH : 55% (2 steps)
5a': R1 = R2 = H : 80%
Figure 1. Synthesis of piperazines (1–5), and their N,N-dimethylated analogs (1′-5′) under investigations.
2

G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
catalyst, leading to compounds 2 and 4 with 58% to 64% yields for the
two steps. N-methylation of piperazines using the Eschweiler–Clarke
reaction led to the N,N’-dimethyl-piperazines 1′, 3′ and 5′ with 74% to
80% yields.39 To obtain N,N’-dimethylated compounds with unpro-
tected phenol 2a’ and 4a’, the OBn-protected precursors 1a and 3a were
subjected to double N-methylation followed by a final hydrogenolysis.
Their respective overall yields are 58% and 55% after the double N-
methylation and hydrogenolysis steps respectively. Characterization of
the 2,5-piperazines and their N,N’-dimethylated analogs is available in
the Supplementary data (Figure S1–S41).
Figure 3. Biofilm formation by C. albicans ATCC 28,366 in the presence of 1a-
1d. Data are represented as mean ± standard deviation. (* p < 0.01).
2.2. Antibiofilm activity
formation (data not shown). Therefore, the large hydrophobic benzyl
groups are required for bioactivity.
The antibiofilm activity was evaluated against C. albicans ATCC
28,366 at piperazine concentrations up to 96 µg/mL. The piperazines
made only from phenylalanine residues (5a-c, 5a’-c’) were inactive and
therefore will not be discussed. Overall, compounds 1a-1c and 3a-3c
were the most effective inhibitors of C. albicans biofilm formation.
Piperazine 1a significantly inhibited biofilm formation (p < 0.01) at
a concentration as low as 1 µg/mL (Figure 2A). Additional experiments
were performed using the clinical isolate LAM-1, a wild strain from a
sepsis patient. There was significant inhibition of C. albicans LAM-1
biofilm formation in the presence of 4 µg/mL of 1a (Figure 2B). Alto-
gether, the antibiofilm activity of piperazine 1a against C. albicans was
observed at concentrations 25- to 100-fold lower than the minimal
inhibitory concentration (MIC = 96 µg/mL) that was obtained under the
same experimental conditions. Piperazines 1a-d did not impede yeast
growth and viability at concentrations up to 64 µg/mL, as shown by the
measurement of optical density at 660 nm (OD660) and by the plate
count determination.
Compared to piperazines 1, compounds with two benzyl ether moi-
eties (3) showed a slightly lower antibiofilm properties (Figure 4a). In-
hibition of biofilm formation was significantly measured at 2 µg/mL for
compounds 3a-3b (Figure 4A), making their antibiofilm activity effec-
tive at concentrations about 12-fold less than the MIC-. Indeed, com-
pounds with a cis configuration 3a-b have better antifungal activities,
with MIC and minimum fungicidal concentrations (MFC) of 24 µg/mL
under the experimental biofilm conditions.
The trans isomer 3c was less potent than its analogs and did not
impede fungal growth at the concentration range tested (MIC > 128 µg/
mL), but impeded biofilm formation at concentration of 48 µg/mL
(Figure 4B). Removal of the benzyl group (4a, 4b) and/or N-methylation
(3a’, 4a’) of these compounds suppressed all activity, therefore con-
–
firming the proposed involvement of the N H groups and importance of
the benzyl ether for activity.
Surprisingly, N-methylated compound 2a’ with only one free hy-
droxyl led to a weak antibiofilm activity at concentrations of 24 to 96
µg/mL (Figure 4C). Considering the distant structural analogy that re-
lates compound 2a’ with the other antibiofilm piperazines, its activity is
thought to use a different mechanism. As the MIC of 2a’ was signifi-
cantly higher than those of the other compounds, no specific investi-
gation was conducted to shed light on its possible mode of action.
Although eight compounds that prevent C. albicans biofilm devel-
opment were identified, no piperazine was effective in disrupting a
preformed biofilm (data not shown). These findings suggest that the
molecules interfere with the normal life cycle of the biofilm, but do not
have the ability to affect the mature biofilm and its polymeric matrix.
Although similar concentration ranges of all four stereoisomers of
piperazine 1 inhibited fungal biofilm formation (Figure 3), compound
1d exhibited the weakest antibiofilm and antifungal activities (MIC >
128 µg/mL). Piperazine 1d is also less soluble than compounds 1a-c,
which may contribute to the lower activity observed. The N-methylated
–
analog 1a’ was inactive (data not shown). This indicates that the N
H
groups are involved in the antibiofilm mechanism of action. In addition,
free hydroxyl analogs (2a-b) had no effect on fungal growth and biofilm
Figure 4. Fungal growth and biofilm formation by C. albicans ATCC 28,366 in
the presence of (A) cis piperazines 3a and 3b; (B) trans piperazine 3c;(C) N,N’-
dimethyl-piperazine 2a’. Data are represented as mean ± standard deviation. (*
p < 0.01).
Figure 2. Fungal growth and biofilm formation by C. albicans ATCC 28,366 (A)
and LAM-1 (B) in the presence of piperazine 1a. Data are represented as mean
± standard deviation. (* p < 0.01).
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G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
2.3. Antibiofilm mechanisms of action of piperazines
To investigate the mechanism of action of the antibiofilm activity of
piperazine 1a on C. albicans, we studied its impact on morphogenesis
and cell adherence, two on the main processes involved in the life cycle
of the fungus and in the biofilm formation.40,41
2.4. Inhibition of C. Albicans morphogenesis
C. albicans remains in the budded morphotype when grown at 30 ◦C
in Yeast Extract-Peptone-Dextrose broth (YPD). Its morphogenesis can
be initiated by using appropriate hyphae-inducing culture media.12,42–44
The impact of piperazine 1a on C. albicans’ ability to form hyphae
was studied by microscopy after 4 h of growth in Spider medium
(Figure 5), a carbon deprivation environment which stimulates forma-
tion of hyphae. No real hyphae cells were present after incubation with
4 µg/mL of piperazine 1a (Figure 5 D). The first real hyphae cells to be
observed were short and represented only a small subpopulation of the
culture grown with 2 µg/mL of piperazine 1a (Figure 5C). Even though
filamentation effectively led to the elongation of joint hypha cells in the
presence of 1 µg/mL of 1a in Spider medium (Figure 5B), their
appearance was still remarkably distinct from the positive control
(Figure 5A) and an important proportion of pseudohyphae and inter-
mediate morphologies were present.
Figure 6. Proportion of C. albicans LAM-1 (white) and ATCC 28,366 (color)
cells in the hypha morphology after 4 h of incubation in Spider medium in the
presence of piperazine 1 analogs. Data are represented as mean ± standard
deviation. (* p < 0.01).
Lee’s medium, an amino acid deprived medium.45 In addition, pipera-
zine 1a at 1 µg/mL inhibited morphological switching in YPD despite
supplementation with 10% fetal bovine serum (FBS), which strongly
induces morphogenesis. Finally, piperazine 1a had no effect on
morphological switching induced by N-acetylglucosamine as the only
carbon source (GlcNAc 0.5%) or by nitrogen deprivation (SLAD).
Methylation of the amine functions of 1a (1a’) resulted in the loss of
activity for Lee’s media and YDP + 10% FBS or no activity for the
GlcNAc 0.5% and SLAD conditions.
The cells were counted as hypha or yeast to determine the percentage
of individuals that underwent morphological changes. This count
confirmed that the morphogenesis of C. albicans ATCC 28,366 was
significantly inhibited in Spider medium (p < 0.01) in the presence of
compounds 1a and 1b at the lowest antibiofilm concentrations
(Figure 6). A comparable activity level was observed using the clinical
strain LAM-1. With both strains there was no activity using twice as
These results were obtained within 4 h of incubation of a diluted
culture and demonstrate an inhibition of filamentation in the early
exponential growth phase. Experiments do not prove that filamentation
impairment occurs under the biofilm conditions or under dense popu-
lation. It should be noted that the rise in temperature from 30 to 37 ◦C
alone can initiate the filamentation of yeast cultured in YPD. This
experimental factor was reproduced in each experiment above and its
potential contribution cannot be quantified. The characterization of
grown biofilms by scanning electron microscopy would provide evi-
dence of this effect under biofilm-forming conditions and would help
determine whether the inhibition of morphogenesis contributes to
antibiofilm activity.
–
much of the N- methylated piperazine 1a’, suggesting that the N
H
groups also play a key role in morphogenesis inhibition. The measure of
OD660 and a further plate count after incubation confirmed that the
growth and viability of C. albicans were not affected by the piperazines
in this experiment. This indicates that the inhibition of filamentation by
these compounds does not result from their fungicidal activity.
The ability of compound 1a to inhibit morphogenesis was investi-
gated further in other hyphae-inducing media (Figure 7).42,43 Inhibition
of filamentation was significantly observed starting from 2 µg/mL in
2.5. Adherence to epithelial cells
Adherence to a surface is the initial step of biofilm development and
a key process for pathogenesis and tissue infection. Consequently, genes
and regulators involved in adhesion often contribute to biofilm forma-
tion among other vital elements, such as cell wall integrity.46 To
determine if the antibiofilm piperazine 1a could also inhibit the first step
of biofilm establishment, adherence of C. albicans ATCC 28,366 on oral
epithelial cells GMSM-K was evaluated by fluorescence microscopy
using microorganisms labeled with fluorescein-isothiocyanate (FITC)
after 4 h of incubation (Figure 8). Clearly, the adherence of C. albicans
decreases significantly in the presence of piperazine 1a. A dose-
dependent reduction of fungal adherence to epithelial cells was
observed in the antibiofilm concentration range, starting from 6 µg/mL
of 1a (p < 0.01) (Figure 9).
2.6. In vitro biocompatibility with oral epithelial cells
In view of a potential therapeutic use of piperazine 1a, its biocom-
patibility was assessed using an oral epithelial cell model. Human oral
epithelial cells (cell line B11) were treated with various concentrations
of piperazine 1a for 2 and 24 h prior to monitor cell viability using a
MTT (3-[4, 5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay. Results showed that following a 2-h treatment, only the highest
concentration (96 µg/mL) of piperazine 1a significantly decreases the
cell viability. When the treatment was extended to 24 h, a significant
Figure 5. Morphology of C. albicans ATCC 28,366 after incubation in Spider
medium with piperazine 1a at concentrations of (A) 0 µg/mL;(B) 1 µg/mL; (C)
2 µg/mL; (D) 4 µg/mL.
4

G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
Figure 7. Morphological switching in C. albicans ATCC 28,366 yeast cells induced using different hypha-inducing culture media in the presence of piperazines 1a
and 1a’. Data are represented as mean ± standard deviation. (* p < 0.01).
Figure 8. Adherence of FITC-labeled C. albicans to GMSM-K epithelial cells with piperazine 1a at concentrations of (A) 0 µg/mL; (B) 12 µg/mL; (C) 24 µg/mL; (D) 48
µg/mL.
reduction in cell viability was observed at concentrations ≥ 24 µg/mL
biofilm dispersion, degradation enzymes secretion, and other virulence
factors.41,51 These processes are influenced, and therefore can be
controlled, by environmental cues and small molecules.12–49,52 On this
basis, we have studied the effect of piperazine 1a on C. albicans
morphological switching and adherence.
(Figure 10).
3. Discussion
Over 2000 genes47 and 50 transcriptional regulators48 are implicated
in biofilm formation by C. albicans through the different stages of biofilm
development, in which the cells adopt differentiated gene expression. A
core transcriptional network of nine regulators was identified among the
complex set of interconnected metabolic pathways required for biofilm
development.47,49,50 Most of them are also involved in the regulation of
adherence, morphological switching,40 extracellular matrix production,
In this study, we have synthesized 22 piperazines, including 16 new
piperazines and identified eight that inhibit biofilm formation by
C. albicans without affecting planktonic growth. Structure-activity
relationship studies reveal the importance of the benzyl ether moiety
present in antibiofilm piperazines 1 and 3. Analogs with free hydroxyls
(2, 4) were inactive. N-methylation of the antibiofilm compounds also
–
resulted in a loss of activity, suggesting the participation of the N
H
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G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
transcription factor Cph1 via N-acetylglucosamine transporter Ngt160 or
through the MAPK pathway,51,61 although other regulators are
63,64
involved.50,62,
Thus, many targets might be suggested for the
mechanisms through which piperazine 1a inhibits C. albicans fila-
mentation. Altogether, the results suggest that the piperazine interferes
with the Ras-cAMP-PKA pathway and the upstream regulators of Tec1
and Efg1, core regulators of biofilm development.40,49 Overall, inhibi-
tion of morphogenesis is likely to contribute to the antibiofilm activity of
piperazines 1a-d. Using mutant strains in the morphogenesis inhibition
assay to systematically investigate the effect of gene depletion could
lead to a better understanding of the precise sites where the piperazines
interfere.
Figure 9. Relative fluorescence of FITC-labeled C. albicans adhered to GMSM-K
epithelial cells in the presence of piperazine 1a. Data are represented as mean
± standard deviation. (* p < 0.01).
Compound 1a was effective in reducing the ability of C. albicans to
form a biofilm and to adhere to oral epithelial cells at relatively low
concentrations. In addition, we showed that at concentrations required
for anti-biofilm and anti-adhesion effects, compound 1a did not affect
the human cell viability following a 2-h exposure. These results are
interesting in view of a potential therapeutic use of compound 1a for
treating/preventing C. albicans infections, even though toxicity at higher
concentrations (≥24 µg/mL) after a 24-h exposure may limit its efficacy.
4. Conclusions
A set of 22 piperazines were synthesized efficiently and studied for
their ability to inhibit biofilm formation by C. albicans. Some compounds
alter biofilm-related processes far below their fungicidal concentration.
Most importantly, piperazine 1a inhibits the morphological switching of
C. albicans and its adherence to epithelial cells at concentrations that
does not reduce the cells viability.
Overall, considering the inhibition of the C. albicans biofilm-related
processes and the highly interconnected mechanisms that regulate bio-
film development, the piperazines may act as perturbators of metabolic
or signaling pathways of the fungus. The action of 1a is most probably
through interferences with the Ras-cAMP-PKA pathway and the up-
stream regulators of Tec1 and Efg1, although more work is needed to
pinpoint the exact mechanism of action.
Figure 10. Cell viability of human oral epithelial cell line B11 in the presence
of piperazine 1a after 2 h and 24 h. Data are represented as mean ± standard
deviation. (θ p between 0.01 and 0.001) (* p < 0.001).
groups in the mechanism of action. These results are in line with ob-
servations made during other antibiofilm activity studies of cyclic di-
peptides, analogs of piperazines under investigation.30 Notably,
piperazines have better solubilities than their cyclic dipeptide analogs.
Microbial adherence is ensured by adhesion proteins and by non-
specific physicochemical interactions as well. Environmental factors
such as quorum-sensing molecules, colonization by other microorgan-
isms, and even host hormones can influence the adhesion process,53–56
which in vitro seems to mostly depend on the nature of the surface. The
results reported show a significant reduction of adherence on epithelial
cells by 1a. However, it does not identify the reduction of adherence as a
causal factor for the antibiofilm activity of compound 1a at the condi-
tions of the biofilm assay, as the biofilm adhered to a different material.
The inhibition of fungal adherence to epithelial cells by 1a demonstrates
a supplementary and complementary mechanism of action by which the
compound may inhibit biofilm development under biological
conditions.
It is believed that therapeutic strategies that do not result in the
direct killing of pathogens would prevent the rise of resistance and
reduce side effects of antimicrobial drugs.65,66 Therefore, the results
reported herein targeting metabolic pathways that regulate biofilm
development and virulence expression with small aromatic-rich piper-
azines constitute an interesting avenue towards novel antifungal
therapies.67–70
5. Experimental section
5.1. General information
Oxime resin, coupling reagents and N-Boc-protected amino acids
´
were purchased from Matrix Innovation (Quebec City, QC, Canada).
Unless indicated otherwise, all chemicals were purchased from com-
mercial sources and used directly. NMR spectra were recorded using
Varian Inova 400 MHz and NMR Agilent DD2 500 MHz spectrometers.
The coupling constants are reported in hertz (Hz). Chemical shifts are
reported in parts per million (ppm) downfield from tetramethylsilane.
Splitting patterns are designated as s (singlet), d (doublet), dd (doublet
of doublets), t (triplet), q (quartet), br (broad) and m (multiplet). Mass
spectra were obtained on an Agilent 6210 LC Time of Flight Mass
Spectrometer in direct injection mode. Optical density and absorbance
values for biofilm formation assays were obtained using a BIO-RAD
xMark Microplate spectrophotometer. Adherence of C. albicans on
epithelial cells was measured by fluorescence spectrometry using a
Synergy 2 spectrophotometer (BioTek Instruments, USA) and pictures
were taken with a FSX100 Olympus camera.
On the other hand, piperazine 1a inhibits the morphological
switching of C. albicans under two hyphae-inducing conditions. In Spi-
der medium, the transcriptional regulator Efg1 induces hypha formation
in response to carbon deprivation through Gpr1 in the cAMP-PKA
pathway. Additionally, 1a inhibits morphological switching in Lee’s
medium (Figure 7), in which the amino acid limitation activates the
transcription factors Cph2 and Tec1 to upregulate filamentation genes.43
Likewise, 1a is also active in YPD with 10% of FBS (Figure 7). The FBS
strongly induces morphogenesis through both MAPK and cAMP-PKA in
response to activation of Ras GTPases.57–59 However, piperazine 1a had
no effect on morphological switching induced by N-acetylglucosamine
as the only carbon source (0.5% GlcNAc) or by nitrogen deprivation
(SLAD), which respectively induce morphogenesis by activation of the
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G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
5.2. Synthesis
157.0. HRMS (ESI-TOF, m/z) calcd for C32H35N2O2 (M + H)+
=
479.2693, found 479.2688. HPLC (retention time, purity): 30.69 min,
5.2.1. General procedures for the synthesis of 1,4-piperazines
99%.
All 1,4-piperazine (1-5a-c) were prepared from the corresponding
cyclic dipeptides (CDPs). The latter were prepared by solid-phase pep-
tide synthesis with oxime resin using the appropriate phenylalanine or
tyrosine derivatives.31,32 1,4-Piperazines were readily produced by the
reduction of CDPs using LiAlH4 (1 M in THF) for 48 h in dry THF under
an inert atmosphere. Once the reaction was completed, Na2SO4⋅10H2O
was added to neutralize residual LiAlH4 and other active species, and the
mixture was refluxed for 30 min.37,38 Residual solids were eliminated by
filtration which led to the desired crude aromatic 1,4-piperazines with
80% to 87% yields. Crude products were purified by flash chromatog-
raphy using CH2Cl2/MeOH as eluent.
5.2.1.6. (2R,5R)-2,5-bis(4-(benzyloxy)benzyl)piperazine
(3b). White
powder, 81% yield. mp = 135 ◦C. 1H NMR (400 MHz, DMSO‑d6): δ 1.80
(s, 2H), 2.50–2.57 (m, 4H), 2.58–2.63 (m, 6H), 5.01 (s, 4H), 6.87 (d, J =
8.3 Hz, 4H), 7.06 (d, J = 8.3 Hz, 4H), 7.27 (t, J = 7.1 Hz, 2H), 7.34 (t, J
= 7.4 Hz, 4H), 7.39 (d, J = 7.4 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6):
δ 47.6, 55.9, 61.2, 69.6, 115.0, 128.1, 128.2, 128.9, 130.5, 132.6, 137.8,
157.0. HRMS (ESI-TOF, m/z) calcd for C32H35N2O2 (M + H)+
=
479.2693, found 479.2700. HPLC (retention time, purity): 30.69 min,
95%.
5.2.1.7. (2S,5R)-2,5-bis(4-(benzyloxy)benzyl)piperazine
(3c). White
powder, 83% yield. mp = 135 ◦C. 1H NMR (400 MHz, DMSO‑d6): δ 1.80
(s, 2H), 2.50–2.57 (m, 4H), 2.58–2.63 (m, 6H), 5.01 (s, 4H), 6.87 (d, J =
8.3 Hz, 4H), 7.06 (d, J = 8.3 Hz, 4H), 7.27 (t, J = 7.1 Hz, 2H), 7.34 (t, J
= 7.4 Hz, 4H), 7.39 (d, J = 7.4 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6):
δ 47.6, 55.9, 61.2, 69.6, 115.0, 128.1, 128.2, 128.9, 130.5, 132.6, 137.8,
5.2.1.1. (2S,5S)-2-benzyl-5-(4-(benzyloxy)benzyl)piperazine (1a). Pale
yellow powder, 84% yield. mp = 170 ◦C. 1H NMR (500 MHz, CDCl3): δ
1.74 (s, 2H), 2.75 (dd, J = 13.4, 5.6 Hz, 1H), 2.81 (dd, J = 13.4, 5.6 Hz,
1H), 2.84–2.89 (m, 4H), 2.91–3.03 (m, 4H), 5.06 (s, 2H), 6.92–6.95 (m,
2H), 7.13–7.16 (m, 2H), 7.21–7.24 (m, 3H), 7.30–7.35 (m, 3H),
7.38–7.41 (m, 2H), 7.44–7.46 (m, 2H). 13C NMR (126 MHz, CDCl3) δ
37.6, 38.5, 47.9, 48.0, 55.6, 55.7, 70.1, 114.9, 126.2, 127.5, 127.9,
128.5, 128.6, 129.2, 130.1, 131.2, 137.1, 139.3, 157.3. HRMS (ESI-TOF,
m/z): calcd for C25H29N2O (M + H)+ = 373.2274, found 373.2269.
HPLC (retention time, purity): 23.45 min, 99%.
157.0. HRMS (ESI-TOF, m/z) calcd for C32H35N2O2 (M + H)+
=
479.2693, found 479.2680. HPLC (retention time, purity): 30.69 min,
96%.
5.2.1.8. (2S,5S)-2,5-dibenzylpiperazine (5a)71–76. Yellow powder, 75%
yield. mp = 162 ◦C. 1H NMR (500 MHz, DMSO‑d6): δ 3.02–3.15 (m, 6H),
3.36–3.47 (m, 4H), 7.29–7.33 (m, 6H), 7.36–7.39 (m, 4H). 13C NMR
(126 MHz, CDCl3) δ 36.4, 44.0, 53.7, 127.0, 128.8, 129.3, 136.5. HRMS
(ESI-TOF, m/z): calcd for C18H23N2 (M + H)+ = 267.1856, found
267.2196. HPLC (retention time, purity): 13.47 min, 99%.
5.2.1.2. (2R,5R)-2-benzyl-5-(4-(benzyloxy)benzyl)piperazine
(1b).
White powder, 82% yield. mp = 170 ◦C. 1H NMR (500 MHz, CDCl3): δ
1.74 (s, 2H), 2.75 (dd, J = 13.4, 5.6 Hz, 1H), 2.81 (dd, J = 13.4, 5.6 Hz,
1H), 2.84–2.89 (m, 4H), 2.91–3.03 (m, 4H), 5.06 (s, 2H), 6.92–6.95 (m,
2H), 7.13–7.16 (m, 2H), 7.21–7.24 (m, 3H), 7.30–7.35 (m, 3H),
7.38–7.41 (m, 2H), 7.44–7.46 (m, 2H). 13C NMR (126 MHz, CDCl3) δ
37.6, 38.5, 47.9, 48.0, 55.6, 55.7, 70.1, 114.9, 126.2, 127.5, 127.9,
128.5, 128.6, 129.2, 130.1, 131.2, 137.1, 139.3, 157.3. HRMS (ESI-TOF,
m/z): calcd for C25H29N2O (M + H)+ = 373.2274, found 373.2259.
HPLC (retention time, purity): 23.45 min, 99%.
5.2.1.9. (2R,5R)-2,5-dibenzylpiperazine (5b). Yellow powder, 71%
yield. mp = 162 ◦C. 1H NMR (500 MHz, DMSO‑d6): δ 3.02–3.15 (m, 6H),
3.36–3.47 (m, 4H), 7.28–7.33 (m, 6H), 7.36–7.39 (m, 4H). 13C NMR
(126 MHz, DMSO‑d6) δ 34.5, 41.5, 52.4, 127.6, 129.2, 129.8, 136.1.
HRMS (ESI-TOF, m/z): calcd for C18H23N2 (M + H)+ = 267.1856, found
267.2460. HPLC (retention time, purity): 13.57 min, 99%.
5.2.1.3. (2S,5R)-2-benzyl-5-(4-(benzyloxy)benzyl)piperazine (1c). Pale
yellow powder, 85% yield. mp = 171 ◦C. 1H NMR (500 MHz, CDCl3): δ
2.43–2.48 (m, 3H), 2.53 (dd, J = 13.4, 8.6 Hz, 1H), 2.66 (ddd, J = 28.8,
13.5, 5.1 Hz, 2H), 2.82–2.92 (m, 2H), 2.96 (ddd, J = 11.4, 5.5, 2,7 Hz,
2H), 5.04 (s, 2H), 6.91 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H),
7.18–7.23 (m, 3H), 7.28–7.34 (m, 3H), 7.37–7.44 (m, 4H). 13C NMR
(126 MHz, CDCl3) δ 39.9, 40.8, 52.6, 52.7, 62.6, 70.0, 114.9, 126.4,
127.4, 127.9, 128.5, 129.2, 130.1, 137.0, 138.4, 157.4. HRMS (ESI-TOF,
m/z): calcd for C25H29N2O (M + H)+ = 373.2278, found 373.2269.
HPLC (retention time, purity): 23.12 min, 97%.
5.2.1.10. (2S,5R)-2,5-dibenzylpiperazine (5c). Pale yellow powder,
80% yield. mp = 163 ◦C. 1H NMR (400 MHz, DMSO‑d6): δ = 1.72 (s,
1H), 2.49 (ddd, J = 24.4, 12.3, 9.3 Hz, 4H), 2.68 (dd, J = 13.4, 5.1 Hz,
2H), 2.85–2.92 (m, 2H), 2.95 (dd, J = 11.3, 2.7 Hz, 2H), 7.15–7.23 (m,
6H), 7.25–7.30 (m, 4H). 13C NMR (101 MHz, CDCl3): δ = 40.8, 52.6,
57.0, 126.4, 128.5, 129.2, 138.4. HRMS (ESI-TOF, m/z): calcd for
C18H23N2O2 (M + H)+ = 267.1856 , found 267.1865. HPLC (retention
time, purity): 12.74 min, 96%.
5.2.2. General procedure for the synthesis of N,N’-dimethyl-1,4-piperazines
A 1,4-piperazines was dissolved in formic acid (50 equiv.) in a reg-
ular round bottom flask and stirred for a few minutes. Formaldehyde (33
equiv, 37% v/v in water) was added and the mixture was stirred at 70 %
C for 30 min. EtOAc was used to dilute the reaction mixture and a
saturated NaHCO3 solution was added dropwise until no further gas
formation was observed. The reaction mixture was extracted three times
with EtOAc. The organic layers were combined, dried with Na2SO4,
filtered, and concentrated in vacuo, giving N,N’-dimethyl-1,4-pipera-
zines with 74% to 80% yields . Crude products were purified by flash
chromatography using (90:5:5) CH2Cl2/MeOH/AcOH).
5.2.1.4. (2R,5S)-2-benzyl-5-(4-(benzyloxy)benzyl)piperazine (1d). Pale
yellow powder, 87% yield. mp = 171 ◦C. 1H NMR (500 MHz, CDCl3): δ
2.43–2.48 (m, 3H), 2.53 (dd, J = 13.4, 8.6 Hz, 1H), 2.66 (ddd, J = 28.8,
13.5, 5.1 Hz, 2H), 2.82–2.92 (m, 2H), 2.96 (ddd, J = 11.4, 5.5, 2,7 Hz,
2H), 5.04 (s, 2H), 6.91 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H),
7.18–7.23 (m, 3H), 7.28–7.34 (m, 3H), 7.37–7.44 (m, 4H). 13C NMR
(126 MHz, CDCl3) δ 39.9, 40.8, 52.6, 52.7, 62.6, 70.0, 114.9, 126.4,
127.4, 127.9, 128.5, 129.2, 130.1, 137.0, 138.4, 157.4. HRMS (ESI-TOF,
m/z): calcd for C25H29N2O (M + H)+ = 373.2272, found 373.2269.
HPLC (retention time, purity): 23.12 min, 97%.
5.2.1.5. (2S,5S)-2,5-bis(4-(benzyloxy)benzyl)piperazine
(3a). White
5.2.2.1. (2S,5S)-2-benzyl-5-(ρ-(benzyloxy)benzyl)-1,4-dimethylpiper-
powder, 80% yield. mp = 135 ◦C. 1H NMR (400 MHz, DMSO‑d6): δ 1.80
(s, 2H), 2.50–2.57 (m, 4H), 2.58–2.63 (m, 6H), 5.01 (s, 4H), 6.87 (d, J =
8.3 Hz, 4H), 7.06 (d, J = 8.3 Hz, 4H), 7.27 (t, J = 7.1 Hz, 2H), 7.34 (t, J
= 7.4 Hz, 4H), 7.39 (d, J = 7.4 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6):
δ 47.6, 55.9, 61.2, 69.6, 115.0, 128.1, 128.2, 128.9, 130.5, 132.6, 137.8,
azine (1a’)31. White powder, 74% yield. mp = 111 ◦C. 1H NMR (500
MHz, CDCl3): δ = 2.23–2.28 (m, 2H), 2.37 (s, 3H), 2.38 (s, 3H),
2.47–2.56 (m, 3H), 2.58–2.63 (m, 2H), 2.71 (dd, J = 13.2, 9.6 Hz, 2H),
2.78 (dd, J = 13.0, 9.9 Hz, 2H), 2.95 (dd, J = 13.2, 3.5 Hz, 2H), 2.99 (dd,
J = 13.0, 3.7 Hz, 2H), 5.07 (s, 2H), 6.92–6.95 (m, 2H), 7.12–7.16 (d,
7

G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
2H), 7.22–7.24 (m, 3H), 7.29–7.36 (m, 3H), 7.39–7.42 (m, 2H),
7.45–7.47 (m, 2H); 13C NMR (125 MHz, CDCl3): δ = 32.6, 42.8, 42.9,
55.3, 55.7, 62.8, 63.0, 70.0, 114.7, 125.8, 127.5, 127.9, 128.3, 128.6,
129.4, 130.3, 132.4, 137.2, 140.3, 157.1; HRMS (ESI-TOF, m/z) calcd
for C27H33N2O (M + H)+ = 401.2587, found 401.2593. HPLC (retention
time, purity): 29.26 min, 95%.
3.05–3.09 (m, 1H), 3.11–3.16 (m, 1H), 6.67 (d, J = 8.4 Hz, 2H), 6. 99 (d,
J = 8.4 Hz, 2H), 7.21–7.25 (m, 3H), 7.30–7.33 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 37.8, 46.5, 46.9, 50.8, 54.8, 55.4, 115.8, 126.6, 128.7,
129.2, 129.3, 130.2, 138.3, 155.2. HRMS (ESI-TOF, m/z) calcd for
C
18H23N2O (M + H)+ = 283.1805, found 283.1815. HPLC (retention
time, purity): 12.40 min, 95%.
5.2.2.2. (2S,5S)-2,5-bis(
ρ
-(benzyloxy)benzyl)-1,4-dimethylpiperazine
5.2.3.2. 4-(((2R,5R)-5-benzylpiperazin-2-yl)methyl)phenol (2b). White
powder, 60% yield (2 steps). mp = 45 ◦C. 1H NMR (500 MHz, CDCl3): δ
1H NMR (500 MHz, CDCl3): δ 2.76 (dd, J = 13.6, 6.7 Hz, 1H), 2.82–2.91
(m, 5H), 2.94–2.98 (m, 2H), 3.05–3.09 (m, 1H), 3.11–3.16 (m, 1H), 6.67
(d, J = 8.4 Hz, 2H), 6. 99 (d, J = 8.4 Hz, 2H), 7.21–7.25 (m, 3H),
7.30–7.33 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 37.8, 46.5, 46.9, 50.8,
54.8, 55.4, 115.8, 126.6, 128.7, 129.2, 129.3, 130.2, 138.3, 155.2.
HRMS (ESI-TOF, m/z) calcd for C18H23N2O (M + H)+ = 283.1805,
found 283.1810. HPLC (retention time, purity): 12.40 min, 95%.
(3a’)31. White powder, 75% yield. mp = 172 ◦C. 1H NMR (500 MHz,
CDCl3): δ = 2.24 (dd, J = 11.4, 3.0 Hz, 2H), 2.36 (s, 6H), 2.49 (dd, J =
11.4, 6.2 Hz, 2H), 2.52–2.57 (m, 2H), 2.70 (dd, J = 13.2, 9.7 Hz, 2H),
2.91 (dd, J = 13.2, 3.5 Hz, 2H), 5.06 (s, 4H), 6.92 (d, J = 8.6 Hz, 4H),
7.13 (d, J = 8.6 Hz, 4H), 7.32–7.37 (m, 2H), 7.39–7.41 (m, 4H),
7.44–7.46 (m, 4H); 13C NMR (125 MHz, CDCl3): δ = 32.0, 42.8, 55.5,
63.9, 114.7, 127.5, 127.9, 128.6, 130.3, 132.4, 137.2, 157.1. HRMS
(ESI-TOF, m/z) calcd for C34H39N2O2 (M + H)+ = 507.3006, found
507.3011. HPLC (retention time, purity): 35.32 min, 96%.
5.2.3.3. 4,4′-(((2S,5S)-piperazine-2,5-diyl)bis(methylene))diphenol
(4a)74,77. Beige powder, 60% yield (2 steps). mp = 90 ◦C. 1H NMR (500
MHz, DMSO‑d6): δ 1.72 (s, 2H), 2.55 (dd, J = 17.1, 3.6 Hz, 2H),
2.56–2.62 (m, 4H), 3.35–3.38 (m, 4H), 6.64 (d, J = 8.4 Hz, 4H), 6.95 (d,
J = 8.4 Hz, 4H). 13C NMR (126 MHz, DMSO‑d6): δ 47.7, 56.1, 61.2,
115.5, 130.1, 130.3, 156.2. HRMS (ESI-TOF, m/z) calcd for C18H23N2O2
(M + H)+ = 299.1754, found 299.1764. HPLC (retention time, purity):
9.44 min, 95%.
5.2.2.3. (2S,5S)-2,5-dibenzyl-1,4-dimethylpiperazine
(5a’)72. Yellow
powder, 80% yield. mp = 120 ◦C. 1H NMR (500 MHz, CDCl3): δ 2.24
(dd, J = 11.6, 3.2 Hz, 2H), 2.37 (s, 6H), 2.50 (dd, J = 11.6, 6.2 Hz, 2H),
2.57–2.61 (m, 2H), 2.76 (dd, J = 13.0, 9.9 Hz, 2H), 2.99 (dd, J = 13.0,
3.7 Hz, 2H), 7.19–7.22 (m, 6H), 7.27–7.31 (m, 4H). 13C NMR (100 MHz,
DMSO‑d6): δ 33.1, 43.0, 55.8, 63.1, 128.1, 128.6, 129.6, 129.7, 140.5.
HRMS (ESI-TOF, m/z) calcd for C20H27N2 (M + H)+ = 295.2169, found
295.2173. HPLC (retention time, purity): 19.95 min, 96%.
5.2.3.4. 4,4′-(((2R,5R)-piperazine-2,5-diyl)bis(methylene))diphenol
(4b). White powder, 58% yield (2 steps). mp = 90 ◦C. 1H NMR (500
MHz, DMSO‑d6): δ 1.72 (s, 2H), 2.55 (dd, J = 17.1, 3.6 Hz, 2H),
2.56–2.62 (m, 4H), 3.35–3.38 (m, 4H), 6.64 (d, J = 8.4 Hz, 4H), 6.95 (d,
J = 8.4 Hz, 4H). 13C NMR (126 MHz, DMSO‑d6): δ 47.7, 56.1, 61.2,
115.5, 130.1, 130.3, 156.2. HRMS (ESI-TOF, m/z) calcd for C18H23N2O2
(M + H)+ = 299.1754, found 299.1762. HPLC (retention time, purity):
9.45 min, 96%.
5.2.2.4. (2R,5R)-2,5-dibenzyl-1,4-dimethylpiperazine (5b’). Pale yellow
powder, 78% yield. mp = 120 ◦C. 1H NMR (400 MHz, CDCl3): δ 2.29
(dd, J = 11.8, 3.3 Hz, 2H), 2.38 (s, 6H), 2.52 (dd, J = 11.7, 6.2 Hz, 2H),
2.61–2.67 (m, 2H), 2.78 (dd, J = 13.0, 9.9 Hz, 2H), 2.99 (dd, J = 13.0,
3.7 Hz, 2H), 7.19–7.22 (m, 6H), 7.26–7.31 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ 33.1, 42.8, 55.2, 62.7, 126.0, 128.4, 129.4, 129.7, 139.8.
HRMS (ESI-TOF, m/z) calcd for C20H27N2 (M + H)+ = 295.2169, found
295.2177. HPLC (retention time, purity): 19.94 min, 95%.
5.2.3.5. 4-(((2S,5S)-5-benzyl-1,4-dimethylpiperazin-2-yl)methyl)phenol
(2a’). Brown oil, 58% yield (2 steps). mp = 148 ◦C. 1H NMR (500 MHz,
CDCl3): δ 2.40 (dd, J = 11.2, 5.8 Hz, 1H), 2.44 (s, 3H), 2.47 (s, 3H),
2.52–2.54 (m, 2H), 2.61 (dd, J = 12.9, 6.0 Hz, 2H), 2.86–2.90 (m, 2H),
2.96–3.03 (m, 3H), 6.73 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H),
7.20–7.25 (m, 3H), 7.30–7.33 (m, 2H). 13C NMR (125 MHz, CDCl3): δ
18.4, 22.2, 41.7, 42.0, 53.4, 58.5, 61.0, 61.6, 115.7, 126.5, 128.5, 128.6,
129.3, 130.2, 155.7, 176.6. HRMS (ESI-TOF, m/z) calcd for C20H27N2O
(M + H)+ = 311.2118, found 311.2128. HPLC (retention time, purity):
16.60 min, 95%.
5.2.2.5. (2R,5S)-2,5-dibenzyl-1,4-dimethylpiperazine (5c’). White pow-
der, 80% yield. mp = 123 ◦C. 1H NMR (400 MHz, CDCl3): δ 2.26 (dd, J
= 11.8, 3.0 Hz, 2H), 2.41 (s, 3H), 2.43 (s, 3H), 2.42–2.51 (m, 2H),
2.54–2.59 (m, 2H), 2.83 (dd, J = 13.2, 9.8 Hz, 2H), 3.01 (dd, J = 13.1,
6.9 Hz, 2H), 7.18–7.24 (m, 6H), 7.23–7.33 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ 32.1, 43.2, 56.8, 63.3, 128.1, 128.5, 129.6, 129.7, 140.5.
HRMS (ESI-TOF, m/z) calcd for C20H27N2 (M + H)+ = 295.2169, found
295.2181. HPLC (retention time, purity): 16.00 min, 99%.
5.2.3. General procedure for benzyl hydrogenolysis
5.2.3.6. 4,4′-(((2S,5S)-1,4-dimethylpiperazine-2,5-diyl)bis(methylene))
diphenol (4a’). White powder, 55% yield (2 steps). mp = 155 ◦C. 1H
NMR (400 MHz, DMSO‑d6): δ 2.17 (s, 6H), 2.29 (d, J = 8.3 Hz, 4H),
2.44–2.50 (m, 4H), 2.70–2.73 (m, 2H), 6.62 (d, J = 8.1 Hz, 4H), 6.91 (d,
J = 8.1 Hz, 4H), 9.10 (s, 2H). HRMS (ESI-TOF, m/z) calcd for
The 1,4-piperazines and N,N’-dimethyl-1,4-piperazines containing
benzylether side chains have been subjected to hydrogenolysis using
classical debenzylation procedure using palladium/H2 chemistry. The
benzyl containing piperazine was dissolved in AcOH and placed in a
high-pressure vessel. The vessel was installed on an hydrogenator and,
after four purges, the H2 pressure was set at 50 psi at room temperature.
After 3 h, the reaction was stopped. The mixture was filtered on Celite®
pad. The filtrate was concentrated in vacuo. The crude product, if
necessary, was purified column chromatography using CH2Cl2/MeOH
mixture as eluent. The 1,4-piperazines 2a-2b and 4a-4b were directly
hydrogenated after the reduction of the CDP without purification
therefore the yield is reported for those two steps. The N,N’-dimethyl-
1,4-piperazines 2a’ and 4a’ were directly hydrogenated after the double
N-methylation of 1a and 3a respectively without purification therefore
the yield is reported for those two steps.
C
20H27N2O2 (M + H)+ = 327.2027, found 327.2040. HPLC (retention
time, purity): 12.60 min, 96%.
5.3. Antifungal and antibiofilm assays
Inhibition of biofilm formation and the minimal inhibitory concen-
tration (MIC) were assessed by absorbance spectrometry using the
crystal violet staining method in a culture-treated flat-bottom 96-well
microplate as previously described.78 Candida albicans ATCC 28,366
and clinical isolate LAM-1 were cultivated overnight at 37 ◦C in aerobic
conditions using Sabouraud media pH 7.0. Optical density at 660 nm
(OD660) of the overnight culture was adjusted to 0.2. Then, equal vol-
umes (100 µL) of the microbial suspension and two-fold serial dilutions
of the piperazine solutions in culture medium were incubated at 37 ◦C
5.2.3.1. 4-(((2S,5S)-5-benzylpiperazin-2-yl)methyl)phenol (2a). White
powder, 64% yield (2 steps). mp = 45 ◦C. 1H NMR (500 MHz, CDCl3): δ
2.76 (dd, J = 13.6, 6.7 Hz, 1H), 2.82–2.91 (m, 5H), 2.94–2.98 (m, 2H),
8

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Bioorganic & Medicinal Chemistry 28 (2020) 115810
for 24 to 48 h in a 96-well plate to allow biofilm formation. After
recording the OD660 to monitor microbial growth, the culture medium
was removed from the wells which were then rinsed with water before
staining the biofilm by adding 100 µL of an aqueous solution of crystal
violet (0.001%) for 15 min. The wells were rinsed twice with water for
dye removal and allowed to dry. The dye was released from biofilm by
adding 75% ethanol (100 µL per well) and the biofilm was quantified by
measuring the absorbance at 550 nm (A550). Control wells containing no
piperazine were included as a reference of maximal planktonic and
biofilm growth (100%). Blanks containing no microorganisms were
included for each concentration of piperazine as well as controls con-
taining only culture medium. Relative biofilm growth was calculated for
each concentration by reporting its A550 as a percentage of the A550 of
wells containing only microorganisms in culture broth (100%) after
subtraction of their respective blanks.
monitor fungal growth, and the viability of C. albicans was confirmed by
plate count.
5.5. Reduction of C. Albicans adherence
The adherence of C. albicans on epithelial cells was evaluated as
described by Ben Marquis et al. (2012)79 using fluorescein isothiocya-
nate (FITC)-labeled cultures with slight modifications. The GMSM-K
human epithelial cells80 were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat-inactivated FBS and
100 μ
g/mL penicillin G/streptomycin and incubated at 37 ◦C in an at-
mosphere of 5% CO2. The cells were seeded in 96-well clear bottom
black microplates (100 µL at 1.5 × 106 cells/ml) and incubated over-
night to reach confluence.
A compound possesses antibiofilm properties when a significant
reduction in biofilm formation is observed at concentrations not
affecting microbial growth. The MIC is the lowest concentration where
no microbial growth is visually observed after 24 h. The minimal
fungicidal concentration (MFC) is the lowest concentration (≥MIC) that
prevents colony growth after inoculation (5 µL per well) on nutritive
agar and further incubation (24 h, 37 ◦C). In the case where the MIC and
MFC vary between replicates, the highest value prevails. The results are
a mean of at least two independent experiments performed in triplicates.
The significance of the results was determined according to p values
calculated with the t Test.
The C. albicans cells were labeled by using an overnight culture (10
mL) of C. albicans that was centrifuged at 7000 × g for 10 min and
suspended in 12 mL of fresh 0.5 M NaHCO3 (pH 8) containing 0.03 mg/
mL of FITC. The suspensions were incubated in the dark at 37 ◦C for 30
min with constant shaking. Then, the FITC-labeled yeasts were washed
three times by centrifugation (7000 × g for 5 min) and suspended in
DMEM.
The confluent monolayers of the immortalized human epithelial cell
line GMSM-K seeded in 96-well clear-bottom black microplates were
pre-incubated with two-fold serial dilutions of piperazines in DMEM for
30 min (37 ◦C, 5% CO2). A suspension of FITC-labeled C. albicans in
DMEM was added to the wells at a multiplicity of infection (MOI) of 103
before further incubation for 4 h in the same conditions. The wells were
washed twice with PBS to remove unbound microorganisms. Relative
fluorescence units (RFU; excitation wavelength 495 nm; emission
wavelength 525 nm) corresponding to the level of Candida adherence
were determined using a Synergy 2 microplate reader. Control wells
without piperazines were used to determine 100% adherence values,
while wells without microorganisms were used as controls to measure
basal autofluorescence. The results were reported as a percentage
compared to the control wells containing no piperazine (100% adher-
ence). They are shown as the mean ± standard deviation of the relative
fluorescence obtained from two independent experiments produced in
triplicate. Pictures were taken with a FSX100 Olympus camera.
In a separate experiment performed in black-bottomed 96-well
microplate, the fluorescence of 100 µL solutions of FITC (0.5 µg/mL in
PBS) in the absence and presence of piperazines were compared as a
control experiment to detect possible interference or fluorescence
quenching by the piperazines. Such interference was not observed. FITC
concentration was adjusted to 0.5 µg/mL to obtain a fluorescence signal
in the same range as in the adherence assay.
The disruption of pre-established biofilms was assessed by growing
C. albicans biofilm in Sabouraud pH 7.0 for 36 to 48 h in a culture-
treated flat-bottom 96-well microplate in the absence of piperazines.
The culture medium and free-floating microorganisms were removed,
and the biofilm was rinsed once with 50 mM phosphate-buffered saline
(PBS, pH 7.0). Two-fold serial dilutions of piperazines in PBS were added
to the preformed mature biofilms. Microplates were further incubated
for 12 h, then wells were washed, and residual biofilm stained with
crystal violet as described above. Biofilms treated with PBS were used as
controls.
5.4. Inhibition of C. Albicans morphogenesis
Fresh cultures of Candida albicans were grown overnight in YPD (1%
yeast extract, 2% peptone, 2% dextrose) at 30 ◦C to favor the yeast
morphology. Morphogenesis was induced by incubation at 37 ◦C
following yeast suspension in hyphae-inducing media, including:
carbon-limiting Spider media (1% nutrient broth, 1% mannitol, 0.2%
K2HPO4, pH 7.2); minimal amino-acid Lee’s media (0.05% L-Ala, 0.13%
L-Leu, 0.1% L-Lys, 0.01% L-Met, 0.05% L-Phe, 0.05% L-Pro, 0.05% L-Thr,
0.007% L-ornithine, 1 mg/mL Biotin, 0.5% NaCl, 0.25% K2HPO4, 0.5%
ammonium sulfate, 1% dextrose); FBS-enriched YPD (1% yeast extract,
2% peptone, 2% dextrose, 10% fetal bovine serum); GlcNAc media
(0.5% N-acetylglucosamine, 0.5% peptone, 0.3% KH2PO4); low-
nitrogen SLAD media (YNB without amino acids and ammonium sul-
fate, 2% raffinose, 7 mg/mL ammonium sulfate);
5.6. In vitro biocompatibility assay with oral epithelial cells
The human oral epithelial cell line B11, that has been previously
characterized81, was kindly provided by S. Groeger (Justus Liebig Uni-
versity Giessen, Germany). Cells were cultured in keratinocyte serum-
free medium (K-SFM; Life Technologies Inc.) supplemented with
growth factors (50 µg/mL of bovine pituitary extract and 5 ng/mL of
human epidermal growth factor) and 100 µg/mL of penicillin G-strep-
tomycin. The cultures were incubated at 37 ◦C in a 5% CO2. To evaluate
the effect of compound 1a on cell viability, cells were seeded (1 × 105
cells in 100 µL) into wells of a 96-well tissue culture plate and incubated
at 37 ◦C in a 5% CO2 atmosphere until they reached confluence. Cells
were treated with two-fold serial dilutions of the compound (from 96 to
1.5 µg/mL) for 2 and 24 h. Thereafter, an MTT (3-[4, 5-diethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide) assay was performed according to
the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany)
to assess cell viability. The assays were performed in triplicate and the
means ± standard deviations were calculated.
Two-fold dilutions of piperazines were made in chosen culture me-
dium in a final volume of 5 mL per sample. Each tube was inoculated
with 50 µL of overnight cultures of C. albicans yeast (YPD, 30 ◦C) and
then incubated at 37 ◦C for 4 h to induce morphogenesis. The percentage
of cells in the hypha and yeast morphotypes was determined by mi-
croscopy, counting at least 100 cells for each replicate. Hypha cells were
counted as one cell unless a septum was clearly visible. Intermediate
shapes such as linear cells and pseudohyphae were counted as hypha
cells on the rational basis that their morphology had changed. Budding
yeast cells were counted as one cell if the daughter cell was significantly
smaller than the mother. A control sample without piperazine was
included to indicate maximal conversion. The results are presented as
mean ± standard deviation of at least two independent experiments
produced in triplicate. After incubation, the OD660 was measured to
9

G. Simon et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115810
Quantitative Review of Randomized Trials. Clin Infect Dis. 2012;54:1110–1122.
https://doi.org/10.1093/cid/cis021.
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C.B. synthesized and characterized the piperazines. P.A.P.C
contributed to the synthesis of the piperazines. G.S. performed and
analyzed the biological experiments. G.S. and C.B. co-wrote the first
draft of the manuscript. P.A.P.C, D.G and N.V revised and edited the
manuscript. D.G and N.V. designed and supervised the project and
contributed equally to the work. All authors approved the final version
of the manuscript.
18 Pappas PG, Kauffman C, Andes D, et al. Clinical Practice Guidelines for the
Management of Candidiasis: 2009 Update by the Infectious Diseases Society of
America. Clin Infect Dis. 2009;48:503–535. https://doi.org/10.1086/596757.
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10.1128/AAC.01638-09.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
21 Nett J, Lincoln L, Marchillo K, et al. Putative Role of {beta}-1,3 Glucans in Candida
albicans Biofilm Resistance. Antimicrob Agents Chemother. 2007;51:510. https://doi.
org/10.1128/AAC.01056-06.
Acknowledgements
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This work was supported by Natural Sciences and Engineering
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