Synthesis of isosteric triterpenoid derivatives and antifungal activity

Article abstract of DOI:10.1111/cbdd.12251

Dermatomycoses are among the most widespread and common superficial and cutaneous fungal infections in humans. There is an urgent need to develop efficient and non-toxic antimycotic agents with a specific spectrum of activity. Triterpenes have been demonstrated to exhibit a wide range of biological activities, including antifungal activities. In this study, through hemisynthesis, we aimed to obtain triterpene-isosteric molecules from betulinic and ursolic acids to improve the antifungal activity and spectrum of action of these compounds. Six compounds were resynthesized and tested against eleven mucocutaneous and cutaneous mycotic agents. The results of the susceptibility assays were expressed as the minimal inhibitory concentration (MIC). The MIC values of the piperazinyl derivatives of ursolic and betulinic acids that were active against pathogenic yeasts were in the range of 16-32 μg/mL and 4-16 μg/mL, respectively, whereas fungicidal effects were observed at concentrations ranging from 16 to 128 μg/mL and 8 to 128 μg/mL, respectively. The piperazinyl derivative of betulinic acid exhibited an antifungal profile similar to that of terbinafine and was the most effective derivative against dermatophytes. This strategy led to a promising candidate for the development of a new antifungal agent.

Full text of DOI:10.1111/cbdd.12251

Chem Biol Drug Des 2014; 83: 344–349
Research Article
Synthesis of Isosteric Triterpenoid Derivatives and
Antifungal Activity
Adrine Innocente1, Bruna B. Casanova1,
Received 5 June 2013, revised 4 September 2013 and
accepted for publication 15 October 2013
Fernanda Klein2, Aline D. Lana2, Dariane
Pereira2, Mauro N. Muniz1, Pascal Sonnet3,
Grace Gosmann1, Alexandre M. Fuentefria2 and
The risk of fungal infections has increased over the last
Simone C. B. Gnoatto1,*
decades, predominantly among individuals who are immu-
nocompromised due to malignancies, AIDS, and the use
1
ꢀ
ꢀ
ꢀ
ˆ
Laboratorio de Fitoquımica e Sıntese Organica, Programa
ꢀ
of intensive chemotherapy and immunosuppressive drugs
(1–3). Mycoses are not always successfully treated
because the available antifungal drugs may be ineffective,
produce drug-related toxicity, or lead to the development
of resistance (4). The development of resistant fungal
strains in response to the extensive use of antifungal drugs
in the past decades is likely to cause serious problems in
the future. Consequently, there is an urgent need to
develop efficient and non-toxic antimycotic agents with a
specific spectrum of activity (5).
^
˜
ˆ
de Pos-graduacßao em Ciencias Farmaceuticas,
Universidade Federal do Rio Grande do Sul, Av. Ipiranga,
2752, Porto Alegre, 90610-000 RS, Brazil
2
´
Laboratorio de Micologia Aplicada, Departamento de
ꢀ
´
Analises, Faculdade de Farmacia, Universidade Federal do
Rio Grande do Sul, Av. Ipiranga, 2752, Porto Alegre,
90610-000 RS, Brazil
3Laboratoire des Glucides, FRE CNRS 3517, UFR de
ꢀ
Pharmacie Universite de Picardie Jules Verne1, Rue des
Louvels, 80037 Amiens Cedex 1, France
*Corresponding author: Simone C. B. Gnoatto,
simone.gnoatto@ufrgs.br
Cutaneous mycoses are among the most common fungal
infections and are typically caused by dermatophytes, fila-
mentous keratinophilic fungi that use keratin as a nutrient
when they infect skin, hair, and nails (6). Mucocutaneous
mycosis is characterized by recurrent or persistent infec-
tions of the nails or oral and genital mucosae by Candida
albicans, non-albicans Candida, and other emerging yeast
species (7). The azole group of antifungal drugs has been
used for the treatment of various fungal infections, espe-
cially dermatomycosis and candidiasis (8); however, the
widespread use of these drugs has resulted in the devel-
opment of clinically resistant strains of Candida spp.,
Microsporum canis, M. gypseum, Epidermophyton flocco-
sum, Trichophyton rubrum, and T. mentagrophytes (9,10).
Dermatomycoses are among the most widespread
and common superficial and cutaneous fungal infec-
tions in humans. There is an urgent need to develop
efficient and non-toxic antimycotic agents with
a
specific spectrum of activity. Triterpenes have been
demonstrated to exhibit a wide range of biological
activities, including antifungal activities. In this study,
through hemisynthesis, we aimed to obtain triterpene-
isosteric molecules from betulinic and ursolic acids to
improve the antifungal activity and spectrum of action
of these compounds. Six compounds were resynthe-
sized and tested against eleven mucocutaneous and
cutaneous mycotic agents. The results of the suscepti-
bility assays were expressed as the minimal inhibitory
concentration (MIC). The MIC values of the piperazinyl
derivatives of ursolic and betulinic acids that were
active against pathogenic yeasts were in the range of
16–32 lg/mL and 4–16 lg/mL, respectively, whereas
fungicidal effects were observed at concentrations
ranging from 16 to 128 lg/mL and 8 to 128 lg/mL,
respectively. The piperazinyl derivative of betulinic
acid exhibited an antifungal profile similar to that of
terbinafine and was the most effective derivative
against dermatophytes. This strategy led to a promis-
ing candidate for the development of a new antifungal
agent.
Triterpenes, including the pentacyclic triterpenes ursolic
acid (1) and betulinic acid (2), are natural products that
have been demonstrated to exhibit a wide range of bio-
logical activities (11,12). Ursolic acid was identified as
one of the active components in Rosmarinus officinalis
(13), which inhibits the growth of some food-associated
bacteria and yeasts (14). Furthermore, ursolic acid was
tested against seven species of fungi by Shai et al.,
yielding minimal inhibitory concentration (MIC) values of
8–63 lg/mL. Additionally, Shai et al. (15) reported the
excellent antifungal activity of betulinic acid against Cryp-
tococcus neoformans, Sporothrix schenckii, Aspergillus
fumigatus, M. canis, and C. albicans, with MIC values of
12–250 lg/mL.
Key words: antifungal activity, betulinic acid, mucocutaneous
and cutaneous mycosis agents, synthesis, triterpenes, ursolic
acid
344
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12251

Antifungal Activity of Triterpenoid
Our research group have been looking for semisynthetic
compounds derived from natural sources, which have
potential to be use in the treatment of the protozoal and
fungal diseases through a simple and cheap strategy. This
approach is important considering a large of protozoal and
fungal disease like a neglected tropical diseases (NTDs). In
this sense, we aim to obtain triterpene-derived molecules
from ursolic and betulinic acids to improve the previously
reported antifungal activity of these aglycones. Moreover,
we expect to propose a preliminary structure–activity rela-
tionship that could be used in the development of new
drug candidates. In this work, we continue the pharmaco-
logical investigation on new piperazinyl derivatives at C-28
of acetyl betulinic acid and acetyl ursolic acid previously
synthesized by us (16,18) focusing herein to the antifungal
activity considering the reported potential activity of this
class of compounds.
tetramethylsilane (TMS) as a reference. HR-EI-MS spectra
~
were recorded with an UltrOTOF (Bruker Daltonics, Sao
Paulo, Brazil) mass spectrometer. Melting points were
determined using a Koffler instrument.
Synthesis
The synthetic route is presented in Scheme 1. The full
details of the chemical and structural elucidation of the
ursolic acid and betulinic acid derivatives have been
described previously (16–18).
Antifungal activity
Isolates
The set of isolates included mucocutaneous and cutane-
ous mycosis agents: Candida albicans ATCC 90028, Can-
dida krusei ATCC 6258, Candida glabrata HCCGL01,
Candida tropicalis ATCC 750, Candida parapsilosis ATCC
22019, Epidermophyton floccosum EPF32, Trichophyton
mentagrophytes TME22, Trichophyton rubrum TRU31,
Microsporum canis MCA02, Microsporum gypseum
MGY09, and Scytalidium dimidiatum SCY04. These iso-
lates were obtained from the culture collections of the
Laboratory of Applied Mycological Research, Universidade
Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Methods and Materials
Plant materials
The bark of Platanus acerifolia found on the ground was
collected, and a voucher specimen, No. 182537, was
deposited in the Botany Department Herbarium of Univer-
sidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Peels of Malus domestica (apple) were obtained as a
byproduct from the juice industry. Bark was dried in an
air-circulating stove at 40 °C for 1 week and crushed,
resulting in a fine powder that was submitted to extraction.
Antifungal activity screening assay
Antifungal activity screening was performed using the disk
diffusion agar method and confirmed with the broth micro-
dilution technique. Disk diffusion testing was performed
essentially as described by the Clinical Laboratory and
Standards Institute (CLSI) document M44-A (19,20) using
Sabouraud dextrose agar (SDA; Difco, Detroit, MI, USA).
Blank disks were pretreated with 10 lL of a 512 lg/mL
solution and incubated at 35 °C for 24 h. Confirmatory
screening using the broth microdilution technique was per-
formed in 96-well flat-bottom microplates, with each well
containing appropriate test samples: approximately 104
spores or 103 yeast cells in the test compound at a range
of concentrations (0.5–256 lg/mL) in Sabouraud broth
(Difco). The plates were incubated at 35 °C for 24 h for
yeast and 48–72 h for dermatophytes.
Powdered peel materials of M. domestica (150 g) were
submitted to extraction with ethanol by reflux for 2 h. Most
of the ethanol was evaporated to dryness under vacuum,
and the resulting precipitate was submitted to crystalliza-
tion using AcOEt:MeOH (70:30), resulting in 2.6 g of urso-
lic acid 1, which was identified by TLC and spectroscopic
methods.
Powdered bark material (150 g) of P. acerifolia was sub-
jected to reflux with dichloromethane for 2 h. The extract
was filtered and evaporated to dryness under vacuum.
The corresponding residue was submitted to crystallization
using MeOH, resulting in 2.3 g of betulinic acid 2, which
was identified by TLC and spectroscopic methods.
Chemistry
Minimal inhibitory concentration
All of the solvents were dried and distilled prior to use.
Column chromatography (CC) was carried out on silica gel
Inocula were prepared according to the documents M27-
A3 (yeast) and M38-A2 (filamentous fungi) of the Clinical
Laboratory and Standards Institute (CLSI) (21,22). Minimal
inhibitory concentration (MIC) values were determined by
broth microdilution using the twofold dilution method
according to the CLSI guidelines (21,22) with RPMI-MOPS
(RPMI 1640 medium containing L-glutamine and without
sodium bicarbonate (Sigma-Aldrich Co., St Louis, MO, USA)
buffered to pH 7.0 with 0.165 M MOPS buffer). The sample
concentrations tested ranged from 0.5 to 256 lg/mL.
~
(Merck, Sao Paulo, Brazil, 60–230 mesh) using dichlorom-
ethane/methanol gradient eluent mixtures (CH2Cl2/MeOH).
IR analysis was performed with a PerkinElmer FT-IR
1
System Spectrum BX (Sao Paulo, Brazil). H and 13C NMR
~
spectra were recorded with a Varian Inova 300 NMR
spectrometer and a Varian VNMRS 300 spectrometer.
Chemical shifts are shown in parts per million (d) with
Chem Biol Drug Des 2014; 83: 344–349
345

Innocente et al.
The MIC was defined as the lowest concentration of
sample at which the micro-organism tested did not dem-
onstrate visible growth. A total of 64 lg/mL fluconazole
(Sigma-Aldrich Co.) was used as a positive inhibition
control.
betulinic acid were previously reported (16–18) as 3-acety-
lursolic acid 3, N-{3-[4-(3-aminopropyl)piperazinopropyl]
terbutylcarbamate}-3-acetylursolamide 7, and N-{3-[4-(3-
aminopropyl)piperazinyl]propyl}-3-acetylursolamide 9. The
derivatives of betulinic acid were 3-acetylbetulinic acid 4,
N-{3-[4-(3-aminopropyl)
piperazinopropyl]terbutylcarba-
mate}-3-acetylbetulinamide 8, and N-{3-[4-(3-aminopro-
pyl)piperazinyl]propyl}-3-acetylbetulinamide 10 (18). All of
the compounds were tested for antifungal activity against
eleven mucocutaneous and cutaneous mycosis agents.
Minimal fungicidal concentration
The minimal fungicidal concentration (MFC) was deter-
mined by subculturing 10-lL aliquots of all wells without
visible growth in SDA with chloramphenicol (Difco) and
incubating at 35 °C for 48 h for yeast and 7 days for
dermatophytes. MFC values represent the lowest concen-
tration of a compound that produces a 99.9% end-point
reduction.
The results of the susceptibility assays are expressed as
MICs (Table 1) and showed that 10 had a broad spectrum
of activity and was active against both yeast and filamen-
tous fungi. However, 9 showed antifungal activity against
only yeast. Neither the aglycones alone (1 and 2) nor their
derivatives (3, 4, 7 and 8) showed antifungal activity in the
range of 5.5–256 lg/mL. Because derivatives 9 and 10
were the most active among the tested compounds, the
piperazinyl substituent C-28 appears to be important for
the antifungal activity of these compounds. To exclude the
possibility that the activity could be due to the piperazinyl
group alone, 5 was tested and showed no antifungal
properties.
Results and Discussion
In our previous studies, we produced a series of new ur-
solic acid derivatives with a piperazinyl side chain that
showed good antimalarial and aromatase inhibition activi-
ties (16–18). Because of the antifungal activity reported for
ursolic and betulinic acids (13–15), they were also selected
for a medicinal chemistry research program with the aim
of identifying new triterpene series with antifungal activity.
The rational design was based on the physicochemical
properties and structural similarities between ursolic (1)
and betulinic (2) acids (Scheme 1), considering that ursolic
acid has an ursane skeleton and betulinic acid has a
lupane skeleton. In the present investigation, the lupane
skeleton was used as an isostere of the lead structure to
develop the compound 10.
The MICs of 9 and 10 for pathogenic yeasts ranged from
16 to 32 lg/mL and from 4 to 16 lg/mL, respectively.
Fungicidal effects were observed at concentrations ranging
from 16 to 128 lg/mL and 8 to 128 lg/mL, respectively.
Based on both the MIC and minimal fungicidal concentra-
tion (MFC) parameters, C. glabrata and C. tropicalis were
more susceptible than other tested species. Systemic
terbinafine is the treatment of choice for onychomycosis,
not topical treatment (19). In vitro tests have shown that its
MIC is much lower than those of azoles, and it is not only
fungistatic but fungicidal (19). Our results showed that
compound 10 presented a profile response than terbina-
In this work, six pentacyclic derivatives were synthesised
using ursolic acid 1 and betulinic acid 2, as shown in
Scheme 1. The derivatives obtained from ursolic acid and
5
NH2
H2N
N
N
N
Boc O, MeOH
0ºC, 48 h
3
1
COOH
COOH
6
H2N
NHBOC
Acetic anhydride,
pyridine, 24 h, rt.
N
AcO
HO
1. ClCOCOCl, 0ºC, 3 h.
2. TEA, BAPP, rt, 24 h.
COOH
COOH
2
4
HO
AcO
NH
N
N
NH2
NH
N
N
N
N
NHBoc
O
O
9
O
O
TFA 10%, CH Cl
2
2
AcO
AcO
7
3 h, rt.
AcO
NH
N
N
NH2
NHBoc
NH
8
Scheme 1: Synthesis of ursolic
acid and betulinic acid derivatives.
10
AcO
346
Chem Biol Drug Des 2014; 83: 344–349

Antifungal Activity of Triterpenoid
Table 1: MIC and MFC values of compounds 9 (ursolic acid derivative) and 10 (betulinic acid derivative) against yeast and dermatophytes
9 (lg/mL)
10 (lg/mL)
Antifungal drugs MIC (lg/mL)
MIC
MFC
MIC
MFC
AmpBa
Mzlb
Tbnc
Candida albicans
Candida krusei
16
32
16
16
16
>256
>256
>256
>256
>256
>256
128
32
16
128
32
NT
NT
NT
NT
NT
NT
4
16
4
4
4
32
16
64
64
32
64
32
128
8
8
32
128
128
256
256
256
256
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.5
0.5
0.25
2
0.25
8
0.5
0.5
0.5
0.5
2
1
8
16
16
4
0.25
4
0.25
0.25
4
Candida glabrata
Candida tropicalis
Candida parapsilosis
Epidermophyton floccosum
Trichophyton mentagrophytes
Trichophyton rubrum
Microsporum canis
Microsporum gypseum
Scytalidium dimidiatum
0.25
NT, not tested.
Compounds 1 to 8 did not exhibit activity against any of the tested species in the range of 0.5–256 lg/mL.
aAmphotericin.
bMiconazole.
cTerbinafine.
fine, as 10’s MIC of 4 lg/mL against C. albicans, C. glab-
rata, C. tropicalis, and C. parapsilosis was better than the
MIC of terbinafine against the three latter strains.
These results are important because the number of cases
of sepsis involving fungi has grown in recent years, with
Candida spp. being the most common in hospital infec-
tions.
2
1
Cutaneous mycoses are also an important cause of mor-
bidity, especially in immunocompromised patients. Minor
effectiveness of 9 against dermatophytes was observed,
but compound 10 showed good activity against dermato-
phytes, with MICs ranging from 16 to 64 lg/mL and MFCs
ranging from 64 to 256 lg/mL.
Figure 1: 3D steric images of ursolic acid (1) and betulinic acid
(2). (MM2/ChemBio3D Ultra 11.0 – ChemBioOffice2008).
Finally, to determine the mode of action of the compound
10 on the integrity of the fungal cell wall, the Sorbitol Pro-
tection Assay was performed (23,24,26). In this assay,
MIC determinations were conducted in parallel with and
without 0.8 M sorbitol. Sorbitol causes a slight stress to
cells, and in the presence of some weak non-specific cell
wall inhibitors, cell growth can be inhibited. Results
showed that MIC of the 10 did not vary in the presence of
sorbitol after 7 days of incubation suggesting that 10
would not act by inhibiting the mechanisms controlling cell
wall synthesis. In contrast, a 125-fold increase in MIC was
observed for the positive control drug anidulafungin. Cellu-
lar Leakage Assay was performed to assess whether com-
pound 10 produces fungal membrane damage (23–25). In
this assay, cellular components that absorb at 260 nm
represent on class of leakage components, especially nu-
cleotides. SDS was used as reference compound, which
produces 100% cellular leakage, compound 10 caused
much less leakage, about 20%. To determine whether
damages the fungal membrane are due binding to the
ergosterol, the Ergosterol Effect Assay was performed.
A preliminary structure–activity relationship (SAR) could be
proposed:
●
The piperazine chain might be a pharmacophore
involved in the antifungal activities of these series of com-
pounds.
● Through rational design based on the isosteric replace-
ment of ursane with lupane, the importance of the lupane
skeleton for antifungal activity was demonstrated.
● Compound 9 and 10 differ with respect to topological
space (Figure 1). Betulinic acid and compound 10 have a
cis junction between the A and B rings and lack a double
bond in ring C compared with compound 9, which is
derived of ursolic acid. Compound 10 was effective
against dermatophytes, suggesting that topological param-
eters could be responsible for the activity of this
compound.
Chem Biol Drug Des 2014; 83: 344–349
347

Innocente et al.
This test is based on offering exogen ergosterol to a com-
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form a complex, thus preventing the complexation with
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a
consequence, an
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Odds F.C., Rex J.H. (2010) An insight into the antifun-
gal pipeline: selected new molecules and beyond. Nat
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enhancement of MIC is observed (23,24). Ours results
showed that MIC of 10 against C. albicans cells remains
unchanged in the presence of different concentrations
(50–250 lg/mL) of exogenous ergosterol, therefore sug-
gesting that this compound did not act by binding to
ergosterol. In contrast, a fourfold increase in MIC was
observed for the positive control drug amphotericin B,
whose interaction with ergosterol is well known.
€
€
6. Turel O. (2011) Newer antifungal agents. Expert Rev
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Conclusion and Future Directions
This is the first report of the in vitro antifungal activity of
piperazinyl derivatives of betulinic and ursolic acids against
human mycosis agents. Through rational design based on
the isosteric replacement of ursane with a lupane skeleton,
we demonstrated that this strategy led to a substantial
increase in antifungal activity. Compound 10 exhibited
growth inhibitory and fungicidal activities in the range of
4–64 lg/mL against clinical yeast and dermatophyte
strains. Regarding the mechanism of action, this triterpene
derivative produces a weak damage in fungal membrane
and did not act on the ergosterol assay. Further assays
are required to elucidate the pathways, whereby this
antifungal compound exerts their effects. This study may
contribute to the development of new pharmaceutical
prototypes derived from natural sources for treating these
diseases.
Acknowledgments
This work was supported by grants and financial support
^
from Instituto Nacional de Ciencia e Tecnologia para
~
^
Inovacßao Farmaceutica (INCT-if), Conselho Nacional de
ꢀ
ꢀ
Desenvolvimento Cientıfico e Tecnologico (CNPQ-Brazil),
~
and Coordenacßao de Aperfeicßoamento de Pessoal de
ꢀ
Nıvel Superior (CAPES-Brazil). We also thank Prof. Dr. N.
P. Lopes (USP-RP, Brazil) for the HRMS analysis, Prof. Dr.
J. Duppont (UFRGS, Brazil) for the NMR analysis, and
Tecnovin do Brasil for the apple material.
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