Antifungal activity of bifunctional sphingolipids. Intramolecular synergism within long-chain alpha,omega-bis-aminoalcohols.

Article abstract of DOI:10.1016/S0960-894X(02)00367-0

The in vitro antifungal activity of a series of alpha,omega-bifunctionalized aminoalcohols against Candida glabrata was measured. The dimeric bi-functionalized lipids exhibited activity about approximately 10-fold higher higher than D-sphingosine, which is a larger factor than expected from the simple additive effects of vicinal aminoalcohols groups.

Full text of DOI:10.1016/S0960-894X(02)00367-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2159–2162
Antifungal Activity of Bifunctional Sphingolipids.
Intramolecular Synergism within Long-Chain
ꢀ
,!-bis-Aminoalcohols
Gillian M. Nicholas, Ronghua Li, John B. MacMillan and Tadeusz F. Molinski*
Department of Chemistry, University of California, Davis, CA 95616, USA
Received 6 February 2002; accepted 30 April 2002
Abstract—The in vitro antifungal activity of a series of a,o-bifunctionalized aminoalcohols against Candida glabrata was measured.
The dimeric bi-functionalized lipids exhibited activity about ꢀ10-fold higher higher than d-sphingosine, which is a larger factor
than expected from the simple additive effects of vicinal aminoalcohols groups. # 2002 Elsevier Science Ltd. All rights reserved.
1
We recently reported the structure of a novel sphingo-
lipid, the a,o-bis-amino alcohol oceanapiside (1), which
was isolated from the temperate marine sponge Ocean-
apia phillipensis. The structure of 1—a C28 lipid chain
terminated at each end by a vicinal amino alcohol—is
differentially functionalized. The glycosidic terminus of
non-natural dimeric aminoalcohols have not been
described previously. We report here that dimeric
sphingolipids have ꢀ10-fold improved antifungal activity
against C. glabrata compared to 3 or sphinganine.
C. glabrata is a human pathogen with recognized clin-
ical importance due its association with fungemia
1
bears a conventional (2S,3R)-d-erythro-2-amino-1,3-
7
diol, identical to that of d-sphingosine (3), while the
opposing terminus lacks the primary OH group and
displays an enantiomeric configuration 2R,3R, of oppo-
site relative stereochemistry (threo). Compound 1 and
its aglycone 2 exhibited potent selective antifungal
activity against the fluconazole-resistant strain Candida
glabrata (MIC 10 and 3 mg/mL, respectively), but not
against other tested strains (Candida albicans ATCC
caused by fluconazole-resistant yeasts. The widespread
use of fluconazole and the rise of resistant strains has
lead to a shifting pathology of clinical patient isolates
from C. albicans to fluconazole-resistant, non-albicans
7
c
species. The selective activity of 1 against C. glabrata
prompted us to investigate the structure–activity rela-
tionship of 1, 2, and several synthetic analogues with the
goal of defining the contributions to activity as from the
lipid-chain length and the bipolar sphingolipid-like head
groups. We found a significant intramolecular syner-
gism with long-chain a,o-bis-aminoalcohols that leads
to a higher than expected antifungal activity compared
with simple mono-functionalized vicinal aminoalcohols
such as 3. These observations may have broader
implications for the mode of action of bifunctional
sphingolipids and their potential use in antifungal
therapy.
14503, Candida krusei or Candida albicans 96–489).
2
Related compounds also exhibit biological activity.
For example, the isomeric calyxoside (4), from an
3
undescribed Calyx sp., exhibited modest activity
against two yeast indicator strains for DNA-damaging
agents (RS321 and RS322, 62 and 36 mg/mL, respec-
tively) while rhizochalin (5) from Rhizochalia incrustata
4
showed modest antibacterial activity.
In humans, the sphingolipid pool of the stratum corneum
forms an important barrier to cutaneous infection by
Compounds tested in this study (Scheme 1) were
obtained as follows. Oceanapiside (1) was isolated from
extracts of O. phillipensis, as previously described. The
5
fungi and bacteria. Sphingosine is known to inhibit the
6
1a
growth of C. albicans, but the antifungal properties of
algycone 2 was obtained by methanolysis of 1 followed
by separation on silica chromatography. The dimeric
bis-aminoalcohol 6 was synthesised from the terminal
*
8
Corresponding author. Tel.: +1-530-752-6358; fax: +1-530-752-
995; e-mail: tfmolinski@ucdavis.edu
1
b
olefin 7. Olefin metathesis of 7 (Grubbs’ catalyst) gave
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00367-0

2160
G. M. Nicholas et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2159–2162
Scheme 1. Structures of naturally occurring a,o-aminoalcohol lipids and synthetic analogues.
a tetrabenzyl dimer which was subjected to hydrogenation–
hydrogenolysis to provide 6 and, subsequently, 8 (BzCl,
The long lipid chain was found to be a requirement for
activity—all the short chain analogues 9–12 were inactive
(MIC >100 mg/mL). d-Sphingosine (3) and dihydro-
sphingosine (12) both inhibited C. glabrata, albeit with
modest activity (MIC 30 mg/mL). Long-chain bis-ana-
logue 6 also exhibited activity comparable to the natural
product 1. The corresponding per-benzoyl derivative 8,
and also short-chain analogue 9 were essentially inactive,
which showed that antifungal activity required free primary
1
b
pyr). Short-chain analogues 9, 10, and 11 were syn-
1
b
thesized as described previously. d-Sphinganine (12)
8
was obtained by hydrogenation of d-sphingosine (3).
Finally, the bis-quaternary ammounium compound 13
was obtained by exhaustive methylation of 1,12-diamino-
9
dodecane (14).
Bioassay of each compound for antifungal activity
against C. glabrata was carried out using modifications
of a published microbroth dilution assay.10 The results
are shown in Table 1. The most potent of the com-
pounds tested was the algycone, oceanin (2). Removal
of the O-glucosyl group enhanced activity, possibly by
rendering the compound more membrane-permeable.
NH and OH groups.
2
Significant differences in activity were observed between
the simple symmetrical C26 bipolar lipid 6 (MIC 10 mg/
mL) and the C28 aglycone 2 (3 mg/mL). A difference of
two CH groups in the carbon chains seems unlikely to
2
account for the differential activity, however, the pre-
sence of the 11-keto group in 1 and 2 is expected to
predispose a ‘hairpin’ bend into the long alkyl chains.
Consequently, the molecule suffers a reduction in the
degrees of freedom of chain movement and closer aver-
age distance between the two polar head groups. Alter-
natively, the presence of the extra primary OH may play
a role in the activity of 2.
Table 1. Antifungal minimum inhibitory concentrations (MIC’s) for
aminoalcohols 1, 2 and synthetic analogues against C. glabrataa,b
Compd
MIC (mg/mL)
MIC (mM)
1
2
3
6
8
9
10
3
30
10
15
6.2
100
24
—
—
—
—
100
—
We tested the possibility that the antifungal activity
against C. glabrata was manifested through nonspecific
amphiphilic properties of long-chain aminolipids. The
The MIC of the antiseptic cetyltrimethylammonium
bromide (15) was 5 mg/mL, however, 1,6-diaminohexane
(16), 1,12-diaminododecane (13), the corresponding
N,N’-hexamethyl-bis-ammonium dichloride salt, 13,
were all inactive at up to 100 mg/mL. From these data
we can draw the conclusion that the minimal require-
ments for antifungal activity in this series are the pre-
sence of a long lipid chain and a terminal vicinal 2,3-
aminoalcohol. The minimum chain length required for
activity in monofunctionalized aminoalcohols, as
judged from antifungal activity of 3 and 9–12, is
between C6 and C18. No activity (MIC >100 mg/mL) is
>100
>100
>100
>100
30
>100
>100
5
10
11
12
13
14
15
16
—
14
—
>100
aMinimum inhibitory concentrations (MIC’s) are from duplicate
experiments.
b
Dimeric compounds exhibited no significant activity against C. albi-
cans ATCC 14503, the fluconazole-resistant strains C. albicans 96–489
(a patient isolate, MIC fluconazole >64 mg/mL) and C. krusei (up to
100 mg/mL).

G. M. Nicholas et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2159–2162
2161
Table 2. Fungicidal versus fungistatic activity of bifunctional sphingolipids against C. glabrataa
Compd
Compd concentration before dilution-replating (mg/mL)
0
.3
1.0
3.0
5.0
6.0
10
1230
Amphotericin Bc
FC À
+
+
+
+
FC À
FS À
+
+
+
À
FS À
+
+
+
À
À
À
À
À
À
À
À
d
d
d
d
Fluconazolec
1
2
3
+
FS À
FS À
+
FC À
FC À
+
À
FS À
À
b
b
+
+
+
FS À
À
6
+
+
+
+
FS À
À
a
ꢁ
C. glabrata was grown in the presence of compound and Saboraub media overnight (37 C) in 96-well plates. Innocula from those wells which were
near the MIC (10 mL) were then diluted and replated onto Saboraub agar plates before overnight reincubation. Codes: +, growth in media; À, no
growth in media; FS, fungistatic (growth on plate); FC, fungicidal (no growth on plate). Compounds 11 and threo-11 (structure not shown) were
inactive in this assay (>100 mg/mL).
b
Indistinct fungistatic activity.
c
Amphotericin B and fluconazole were used as positive controls for fungicidal and fungistatic activity, respectively.
Not tested.
d
seen in 10–11 where the correct functionality is present
but the chain length is truncated (C6).
We briefly examined the fungistatic or fungicidal prop-
erties of the bifunctional sphingolipids in comparison to
fungistatic fluconazole and fungicidal amphotericin B
(
Table 2). Fungistatic activity was estimated as that
concentration which inhibited growth in liquid media,
but produced viable colonies after dilution-replating.
Oceanin (2) was fungistatic as low as 5.0 mg/mL, but at
1
0 mg/mL became fungicidal. Oceanapiside (1) was also
fungicidal at 10 mg/mL, however in constrast, the syn-
thetic dimer 6 was only fungistatic at the highest con-
centration tested (12 mg/mL). Under these conditions,
sphingosine (3) exhibited only indistinct fungistatic
activity at high concentration (30 mg/mL). It thus
appears that the bipolarity of a,o-bis-aminoalcohols
and the presence of a keto group within the long chain
lipid promotes fungicidal activity of sphingolipids,
although additional analogues must be tested before we
may draw more firm conclusions.
Figure 1. Hypothetical binding of dimeric sphingolipid to dimeric
receptor or two units of monomeric receptor.
The most remarkable feature of the antifungal profile
was the potent activity exhibited by the natural product
receptors may, in turn, translate to improved efficacy in
antifungal drug–receptor interactions.
1
and its algycone 2 compared to d-sphingosine 3.
When compared on a molar basis, the MIC’s for 1 and
appear to be an order of magnitude lower than those
Oceanapiside (1) and related compounds exhibit pro-
nounced synergism in their antifungal activity against
C. glabrata. The activity of 1 and analogues appear to
be specific to the bis-aminoalcohol functionality and not
a simple function of the amphiphilic nature of these
long chain polar lipids. While other natural product
aminoalcohols have been reported with modest activity
2
of 3. The increase in activity (ꢀ10-fold) is much larger
than expected from simple molar additivity of two vic-
inal aminoalcohol groups in each of the bifunctional
compounds and is consistent with a synergistic effect.
1
2
The synergism may involve binding and inhibition of
multiple receptor sites by 2 in a manner observed with
multivalent drug-receptor interactions (Fig. 1). An
obvious entropic advantage for binding is gained
through dimerism as illustrated in a hypothetical ligand–
receptor Scheme (Fig. 1). Binding of the first sphingo-
against C. albicans, the dimeric functionality confers
upon compounds 1 and 2 a unique selectivity and par-
ticularly high potency as a result of intramolecular
1
3
synergism. Sphingofungin and related compounds
bear structural similarity to 1 and 3 and are known
inhibitors of palmitoyl transferase, a key enzyme
involved in the committed step of d-sphingosine bio-
synthesis. We are currently exploring the possibility that
the natural products are specific inhibitors of one or
more key steps in the biosynthesis of yeast sphingosine
or sphingolipid metabolism. These results will be reported
in due course.
lipid polar group (K 1) promotes cooperativity in the
D
second binding event (K 2). For example, Whitesides et
D
al. showed a synthetic dimer of the antibiotic vancomycin
exhibited a 1000-fold increase in binding to a receptor
peptide analogue (diacetyl-l-lysd-ala-d-ala) compared to
1
1
the monomeric parent. Improved binding to biological

2162
G. M. Nicholas et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2159–2162
7. (a) Miyazaki, H.; Miyazaki, Y.; Geber, A.; Parkinson, T.;
Hitchcock, C.; Falconer, D. J.; Ward, D. J.; Marsden, K.;
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4
00 MHz NMR spectrometer was partially funded by
NSF (CHE 9808183). This work was supported by a
grant from NIH (AI 39987).
1
00, 617.
. All compounds were purified (>95% pure, TLC or HPLC)
8
and gave satisfactory H NMR (400 MHz),
1
13
C NMR
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9
. The bis-ammonium dichloride salt of 13 was prepared by
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