Fungicidal activity of truncated analogues of dihydrosphingosine

Article abstract of DOI:10.1016/j.bmcl.2008.05.067

The minimal fungicidal concentration (MFC) of dihydrosphingosine (DHS), phytosphingosine (PHS), and five short-chain DHS derivatives was determined for Candida albicans and Candida glabrata. In this respect, a C15- and a C17-homologue of DHS showed a 2- to 10-fold decreased MFC as compared to native DHS (i.e. C18-DHS). DHS derivatives that were active, that is, comprising 12, 15, 17, or 18 carbon atoms, induced accumulation of reactive oxygen species (ROS) in C. albicans.

Full text of DOI:10.1016/j.bmcl.2008.05.067

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3728–3730
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fungicidal activity of truncated analogues of dihydrosphingosine
Karin Thevissen a, Ulrik Hillaert b, Els M. K. Meert a, Kuen K. Chow a, Bruno P. A. Cammue a,
,
*
Serge Van Calenbergh b, Isabelle E. J. A. François a
a Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium
b Laboratory for Medicinal Chemistry (FFW), University of Gent, Harelbekestraat 72, 9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The minimal fungicidal concentration (MFC) of dihydrosphingosine (DHS), phytosphingosine (PHS), and
five short-chain DHS derivatives was determined for Candida albicans and Candida glabrata. In this
respect, a C15- and a C17-homologue of DHS showed a 2- to 10-fold decreased MFC as compared to
native DHS (i.e. C18-DHS). DHS derivatives that were active, that is, comprising 12, 15, 17, or 18 carbon
atoms, induced accumulation of reactive oxygen species (ROS) in C. albicans.
Received 11 April 2008
Revised 13 May 2008
Accepted 14 May 2008
Available online 20 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Sphingoid base
Dihydrosphingosine
Fungicidal
Candida albicans
Candida glabrata
Reactive oxygen species
Long-chain sphingoid bases, for example, phytosphingosine
(PHS), sphingosine, and sphinganine (dihydrosphingosine, DHS) in-
hibit the growth of several yeast and fungal species in vitro, includ-
ing Candida albicans,1 Malassezia furfur,1 Aspergillus nidulans,2
Saccharomyces cerevisiae,3 Trichophyton mentagrophytes, and T.
tonsurans.4 Sphingosines also possess antimicrobial activity
in vitro: they are effective against Staphylococcus aureus, Streptococ-
cus pyogenes, Micrococcus luteus, Propionibacterium acnes, and Brev-
ibacterium epidermidis.5 Cheng et al. found that the antifungal
activity of DHS and PHS against A. nidulans acts through the rapid
induction of metacaspase-independent apoptosis, associated with
the rapid accumulation of reactive oxygen species (ROS).2 Regard-
ing the in vivo antifungal activity of sphingoid bases, Bibel et al.
demonstrated that DHS and sphingosine, when topically applied
on human skin, are effective against C. albicans infections and also
prove curative in experimental guinea-pig models for C. albicans
and T. mentagrophytes infections.6 No gross toxicity was observed
among animals or human volunteers,6 which points to the thera-
peutic potential of sphingoid bases against fungal infections.
The aim of this study was to analyse the in vitro antifungal
activity of PHS, DHS, and truncated analogues of DHS. It has been
previously demonstrated that the minimum chain length required
for antifungal activity of sphingoid bases against C. glabrata lies in
the C7–C18 range, based on the fact that three DHS analogues with
fore, a series of truncated DHS analogues with C5 (C5-DHS), C9
(C9-DHS), C12 (C12-DHS), or C15 (C15-DHS) chain lengths were
synthesized and their minimal fungicidal concentration (MFC)
was determined, along with C17-DHS, C18-DHS, and C18-PHS,
against C. albicans strain CAI48 and C. glabrata strain BG2.9 C. glab-
rata is a human pathogen with recognized clinical importance due
to its association with fungemia caused by fluconazole-resistant
yeasts.10 Furthermore, C18-PHS, C18-DHS, and the DHS derivatives
were assessed for ROS accumulation induction in C. albicans.
Compounds tested in this study (Chart 1) were obtained as fol-
lows: C17-DHS, C18-PHS and C18-DHS were purchased from Avan-
ti Polar Lipids (Alabaster, AL, US). C12-DHS was synthesized as
previously described.11 Compounds C5-DHS, C9-DHS, and C15-
DHS12 were synthesized from Garner’s aldehyde (Scheme 1). Treat-
ment of S-enantiomer of Garner’s aldehyde with an appropriate
lithium alkyl acetylide in the presence of HMPA to ensure ery-
thro-selectivity afforded compounds 1a–c13 in reasonable yield
and with excellent stereoselectivity (traces of threo-derivatives).
In case of C5-DHS, deprotection of the intermediate TMS-protected
acetylene was achieved using TBAF in THF.13 Selective deprotec-
tion of the isopropylidene moiety13 afforded synthons 2a–c.
Reduction of the alkyne functionality using Pd/C and subsequent
deprotection of the tert-Boc protecting group under acidic condi-
tions gave access to the envisioned compounds in good overall
yield.
C6 chain displayed no antifungal activity up to 100 l
g/ml.7 There-
The fungicidal activity of each compound against C. albicans and
C. glabrata was determined in PBS19 and the MFC for each com-
pound was calculated as the minimal concentration resulting in
* Corresponding author. Tel.: +32 16 32 96 82; fax: +32 16 32 19 66.
E-mail address: bruno.cammue@biw.kuleuven.be (B.P.A. Cammue).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.067

K. Thevissen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3728–3730
3729
Chart 1. Overview of tested compounds.
Scheme 1. Synthesis of compounds C5-DHS, C9-DHS, and C15-DHS. (See above-mentioned references for further information).
less than 1% survival of the yeast strain relative to the DMSO con-
trol (Table 1).
antifungal activity against C. albicans. Moreover, the fungicidal
activity of C2- and C6-dihydroceramides was tested, with C2 and
C6 being the number of C atoms in the acyl residue (Avanti Polar
Lipids, AL, US,) and these ceramides were completely inactive
C15- and C17-DHS are the most active homologues against both
yeast species: their MFC is 2- to 10-fold lower as compared to na-
tive DHS, and 20-fold lower as compared to C12-DHS. DHS deriva-
tives with shorter chain length, that is, C5-DHS and C9-DHS, are
not active against the tested yeast species. Native PHS is fivefold
more active as compared to native DHS against C. albicans, indicat-
ing that an additional hydroxyl group at position 4 can increase the
(MFC > 100 lg/ml) against both yeast species, indicating that a free
amine at position C2 of the sphingoid base is necessary for fungi-
cidal activity of sphingolipids. These data corroborate with those
of Chung et al. who demonstrated that C2-phytoceramide is not ac-
tive against S. cerevisiae.20 In conclusion, the optimal chain length
for fungicidal activity of DHS derivatives against C. albicans and
C. glabrata lies between C15 and C17.
In the literature, only two other studies report on derivatives of
sphingoid bases with increased antifungal activity. One study de-
scribes a series of new PHS analogues with natural or altered ste-
reochemistry at C3 and/or C4, and OH, NH2, or N3 substituents at
C1, but without alteration of the sphingoid backbone length.21
Table 1
Minimal fungicidal concentration (MFC) for C18-PHS, C18-DHS, and its derivatives in
the absence and presence of 10 mM ascorbic acid against C. albicans and C. glabrata
Compound
MFC (lg/ml)
C. glabrata
C. albicans
The 1-azido derivative, exhibiting the natural D-ribo stereochemis-
0 mM AAa
0 mM AA
10 mM AA
try, showed 10-fold improved antifungal activity against C. albicans
as compared to PHS, based on determination of their minimal
inhibitory concentration (MIC). However, antifungal activity of
the compounds against C. glabrata was not reported.21 Another
study reports on the antifungal activity of dimeric aminoalcohols.7
The most potent derivative was the dimeric aminoalcohol oceanin,
which is characterized by 10-fold improved antifungal activity
against C. glabrata as compared to DHS, based on their MIC. Ocea-
nin is a C28 lipid chain with two polar head groups: one with a
C5–DHS
C9–DHS
C12–DHS
C15–DHS
C17–DHS
DHS
>100
>100
10
0.5
0.5
1
>100
>100
10
0.5
0.5
5
NDb
ND
>25
10
2
5
1
PHS
1
1
a
Ascorbic acid.
Not determined.
b

3730
K. Thevissen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3728–3730
consisting of 15 and 17 carbon atoms, respectively, prove 10-fold
500
400
300
200
100
0
more active against C. albicans and twofold more active against C.
glabrata as compared to native DHS. Since PHS, bearing a hydroxyl
group at position 4, has fivefold increased fungicidal activity
against C. albicans as compared to DHS, the question remains
whether introduction of such hydroxyl group at position 4 of
C15- and C17-DHS can likewise decrease their MFC for C. albicans.
Since it has previously been demonstrated that DHS is non-toxic
upon topical administration and is effective against C. albicans
infections in vivo,6 C15- and C17-DHS hold promising therapeutic
potential as novel antimycotics. Further studies addressing the
mode of action of C15- and/or C17-DHS and their toxicity are
underway.
1
10
100
Acknowledgment
-100
Compound concentration (µg/ml)
Postdoctoral fellowship to K.T. (Industrial Research Fellow)
from K.U. Leuven is gratefully acknowledged.
Figure 1. Accumulation of endogenous ROS in C. albicans upon treatment with
antifungal compounds. Logarithmically growing C. albicans cells were suspended in
PBS, pre-incubated with the compounds for 3 h at 37 °C, washed with PBS, and
incubated with 20,70-dichlorofluorescin diacetate for 3 h at 37 °C. Compounds used
are DHS (open triangles), PHS (open squares), C5-DHS (crosses), C9-DHS (stripes),
C12-DHS (black circles), C15-DHS (black squares), and C17-DHS (black triangles).
Fluorescence emitted by the cells was measured using fluorescence spectrometer
(kex = 485 nm and kem = 525 nm). Experiments have been performed in triplicate.
References and notes
1. Nenoff, P.; Haustein, U. F. Acta Derm. Venereol. 2002, 82, 170.
2. Cheng, J.; Park, T. S.; Chio, L. C.; Fischl, A. S.; Ye, X. S. Mol. Cell. Biol. 2003, 23, 163.
3. Chung, N.; Mao, C.; Heitman, J.; Hannun, Y. A.; Obeid, L. M. J. Biol. Chem. 2001,
276, 35614.
4. Bibel, D. J.; Aly, R.; Shah, S.; Shinefield, H. R. Acta Derm. Venereol. 1993, 73, 407.
5. Bibel, D. J.; Aly, R.; Shinefield, H. R. J. Invest. Dermatol. 1992, 98, 269.
6. Bibel, D. J.; Aly, R.; Shinefield, H. R. Clin. Exp. Dermatol. 1995, 20, 395.
7. Nicholas, G. M.; Li, R.; MacMillan, J. B.; Molinski, T. F. Bioorg. Med. Chem. Lett.
2002, 12, 2159.
(2S,3R)-D-erythro-2-amino-1,3-diol moiety as in natural sphingo-
sine, the other one with a (2R,3R)-2-aminopropan-3-ol group
(threo).7 The MFC of oceanin against C. glabrata is 10
nin is not active against C. albicans.
lg/ml. Ocea-
8. Fonzi, W. A.; Irwin, M. Y. Genetics 1993, 134, 717.
9. The clinical isolate of C. glabrata (strain BG2, URA+) was kindly provided by Dr.
R. Kaur, Department of Molecular Biology and Genetics, Johns Hopkins School
of Medicine, Baltimore, MD, US.
It should be noted that determination of MFC values is preferred
over MIC values, since the former reflects fungicidal activity,
whereas the latter may account for both fungistatic as well as fun-
gicidal activity. Since fungicidal activity pinpoints to inhibition of
targets that are essential for fungal growth22 or induction of an ac-
tive cell death pathway (i.e. apoptosis), these values are more rel-
evant for the design of antifungal drugs. It has previously been
demonstrated that PHS and DHS induce apoptosis in A. nidulans,
concomitant with an accumulation of reactive oxygen species
(ROS).2
In search of the mode of action of PHS, DHS, and its derivatives
against C. albicans, we determined ROS accumulation upon incuba-
tion with various concentrations of the compounds using 20,70-
dichlorofluorescin diacetate staining as previously described.23,24
As can be seen in Figure 1, the inactive C5-DHS and C9-DHS fail
10. (a) Marr, K. A. Oncology 2004, 18, 9; (b) Miyazaki, H.; Miyazaki, Y.; Geber, A.;
Parkinson, T.; Hitchcock, C.; Falconer, D. J.; Ward, D. J.; Marsden, K.; Bennett, J.
E. Antimicrob. Agents Chemother. 1998, 42, 1695.
11. De Jonghe, S.; Van Overmeire, I.; Poulton, S.; Hendrix, C.; Busson, R.; Van
Calenbergh, S.; De Keukeleire, D.; Spiegel, S.; Herdewijn, P. Bioorg. Med. Chem.
Lett. 1999, 9, 3175.
12. C15-DHS has earlier been mentioned in the literature, but to the best of our
knowledge, no synthetic route toward this compound has been described. The
synthesis of rac-C15-DHS has been reported by: Prostenik, M.; Stanavec, N. J.
Org. Chem. 1953, 18, 59.
13. Hillaert, U.; Boldin-Adamsky, S.; Rozenski, J.; Busson, R.; Futerman, A. H.; Van
Calenbergh, S. Bioorg. Med. Chem. Lett. 2006, 14, 5273.
14. Herold, P. Helv. Chim. Acta 1988, 71, 354.
15. Tamotsu, F.; Michiharu, N.; Yoshihiro, K.; Makoto, S. J. Org. Chem. 1994, 59,
5865.
16. Spectroscopic data for C5-DHS: 1H NMR (pyridine-d5) d: 1.08 (t, 3H, J = 7.3 Hz),
1.71–1.86 (m, 2H), 3.60 (ddd, 1H, J = 4.4, 7.0, and 8.8 Hz), 4.14–4.36 (m, 3H);
13C NMR (pyridine-d5) d: 10.86, 26.91, 58.13, 61.68, 73.12; HRMS (ESI)
calculated for C5H14NO2þ: 120.1019, found: 120.1029.
to induce ROS, even at 100 lg/ml, whereas C12-, C15-, and C17-
DHS, and native DHS and PHS induce ROS accumulation in C. albi-
cans. The most active DHS derivatives, that is, C15-DHS and C17-
DHS, induced ROS production to the highest extent. The presence
of 10 mM of the antioxidant ascorbic acid decreased the fungicidal
activity of C12-, C15-, and C17-DHS, whereas the presence of ascor-
bic acid had no effect on the fungicidal activity of native DHS and
PHS (Table 1). These data point to a link between the fungicidal
activity and ROS induction capacity of short-chain DHS derivatives
for C. albicans. In contrast, based on our data, there exists no causal
link between ROS induction and cell death in yeast in case of native
sphingoid bases. In this respect, Cheng et al. demonstrated that
PHS and DHS induce an ROS-independent apoptotic cell death in
A. nidulans.2 Hence, our findings point to a ROS-dependent fungi-
cidal activity of short-chain DHS derivatives on yeast, in contrast
to the ROS-independent fungicidal activity of native sphingoid
bases on yeast.
17. Spectroscopic data for C9-DHS: 1H NMR (CD3OD-d4) d: 0.91 (t, 3H, J = 6.7 Hz),
1.28–1.61 (m, 10H), 3.24 (app. dt, 1H, J = 4.1 and 8.2 Hz), 3.73 (dd, 1H, J = 8.2 Hz
and 11.7 Hz), 3.80–3.86 (m, 1H), 3.86 (dd, 1H, J = 4.1 and 11.7 Hz); 13C NMR
(CD3OD-d4) d: 13.34, 22.52, 25.83, 29.08, 31.77, 33.05, 57.28, 57.77, 69.13;
HRMS (ESI) calculated for C9H22NO2þ: 176,1645, found: 176.1642.
18. Spectroscopic data for C15-DHS: 1H NMR (CD3OD-d4) d: 0.89 (br s, 3H), 1.12–
1.81 (m, 22H), 3.12–3.36 (m, 1H), 3.63–3.94 (m, 3H); 13C NMR (CD3OD-d4) d:
13.33, 22.58, 25.88, 29.32, 29.43, 29.46, 29.61, 31.92, 33.03, 57.28, 57.73,
69.12; HRMS (ESI) calculated for C15H34NO2þ: 260,2584, found: 260,2584.
19. Overnight cultures of C. albicans and C. glabrata were 1/400 diluted in PBS and
treated with the compounds or DMSO in the presence or absence of 10 mM
ascorbic acid for 0 h and 5 h at 37 °C, whereafter colony forming units were
counted on YPD (1% yeast extract, 2% peptone, 2% glucose; 1% agar) plates after
2 days of incubation at 30 °C. MFCs are means of at least three replicates with
standard errors typically below 10%.
20. Chung, N.; Mao, C.; Heitman, J.; Hannun, Y. A.; Obeid, L. M. J. Biol. Chem. 2001,
276, 35614.
21. Mormeneo, D.; Manresa, A.; Casas, J.; Llebaria, A.; Delgado, A. J. Appl. Microbiol.
2008, 104, 1075.
22. Odds, F. C. Rev. Iberoam. Micol. 2005, 22, 229.
23. Aerts, A. M.; François, I. E.; Meert, E. M.; Li, Q. T.; Cammue, B. P.; Thevissen, K. J.
Mol. Microbiol. Biotechnol. 2007, 13, 243.
In conclusion, a series of synthetically easily accessible, trun-
cated DHS analogues have been made and assessed through MFC
measurements. C15- and C17-DHS, that is, DHS homologues
24. François, I. E. J. A.; Cammue, B. P. A.; Borgers, M.; Ausma, J.; Dispersyn, G. D.;
Thevissen, K. Anti-Infect. Agents Med. Chem. 2006, 5, 3.