Synthesis and preliminary antifungal evaluation of a library of phytosphingolipid analogues

Article abstract of DOI:10.1039/b709421c

A library of 64 phytosphingolipid analogues resulting from the systematic variation of the C1, C3, C4, and the N-acyl moiety of phytosphingosine (PHS) has been prepared from common scaffolds derived from the chiral pool and Sharpless asymmetric dihydroxylation reactions. Library members have been evaluated as growth inhibitors of the yeast Saccaromyces cerevisiae. In addition, 1-amino-N-pivaloyl PHS analogues were also tested as IPC synthase inhibitors, in comparison with the natural product khafrefungin. The Royal Society of Chemistry 2007.

Full text of DOI:10.1039/b709421c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and preliminary antifungal evaluation of a library of
phytosphingolipid analogues†
David Mormeneo,a,b Josefina Casas,a Amadeu Llebariaa and Antonio Delgado*a,b
Received 21st June 2007, Accepted 23rd July 2007
First published as an Advance Article on the web 8th October 2007
DOI: 10.1039/b709421c
A library of 64 phytosphingolipid analogues resulting from the systematic variation of the C1, C3, C4,
and the N-acyl moiety of phytosphingosine (PHS) has been prepared from common scaffolds derived
from the chiral pool and Sharpless asymmetric dihydroxylation reactions. Library members have been
evaluated as growth inhibitors of the yeast Saccaromyces cerevisiae. In addition, 1-amino-N-pivaloyl
PHS analogues were also tested as IPC synthase inhibitors, in comparison with the natural product
khafrefungin.
action of inositolphosphoryl ceramide synthase (IPC synthase)
Introduction
(Scheme 1). It has been reported that the reaction catalyzed by IPC
synthase plays a role in the transition from the G1 to the S phase
of the cell cycle16 and is essential for sphingolipid biosynthesis
in fungi. Therefore specific IPC synthase inhibitors, such as the
natural product khafrefungin (Scheme 1), are attractive targets
for the design of new, nontoxic antifungal agents.14,17,18
In this context, we are currently interested in the search of
new, selective, modulators of fungal SL metabolism based on
the design and synthesis of SL analogues. In particular, we
wish to report on a series of new phytosphingolipid (PHSL)
analogues shown in Fig. 1. They have been designed according
to three different structural criteria: a) Modification of the C3–C4
dihydroxy framework present in natural PHC; b) replacement of
the C1-OH group with amino and azido functionalities, and c)
variations on the N-acyl substitution.
Fungi are eukaryotic organisms that present a rigid cell wall
together with an ergosterol-rich cytoplasmatic membrane. They
grow as yeasts or moulds, and species such as Candida, Aspergillus
and Cryptococcus are among the most common human pathogens.
However, new pathogenic fungal species are emerging that can give
rise to a wide variety of fungal infections, from superficial to deeply
invasive and disseminated systemic ones.1
In recent years, the number of serious fungal opportunistic
infections has risen dramatically due to the increased number
of immune-suppressed patients, either by HIV,2,3 cancer chemo-
therapy4,5 or organ allograft transplantation.6–8
The increase of fungal resistance to standard treatments9,10 has
stimulated the search for new therapeutic agents in this field. One
of the current strategies relies on the discovery of new pharma-
cological targets.11 In this context, exploitation of the metabolic
differences between fungal and mammalian sphingolipid (SL)
metabolism represents an attractive approach. SLs are essential
components in eukaryotic cells that are also involved in cell growth
regulation and communication processes.12,13 Since they present
significant differences in mammalian and fungal cells, they are
attractive new targets for fungal intervention, as evidenced by
several natural products that have been discovered in recent years
as selective inhibitors of fungal sphingolipid biosynthesis.14 One
of the most significant differences in SL biosynthesis between
fungi and mammals is the hydroxylation of dihydrosphingosine
(DHS) to phytosphingosine (PHS) by means of a C4 hydroxylase.15
Moreover, PHS can be acylated to phytoceramide (PHC), which,
in turn, can be transformed into complex fungal SLs via its
initial conversion into inositolphosphoryl ceramide (IPC) by the
Preliminary results on antifungal activity as Saccharomyces
cerevisiae growth inhibitors is reported in this work, as well as
the inhibitory properties against IPC synthase for the most active
analogues.
Results and discussion
Chemistry
Synthesis of PHSL analogues started from N-Boc olefins (E)- and
(Z)-13b (Fig. 1), obtained from Garner aldehyde through slight
modifications of reported protocols.19–21 Azido olefins (E)- and
(Z)-14b were obtained from (E)- and (Z)-13b following standard
mesylation to (E)- and (Z)-16b and azide displacement. Sharpless
asymmetric dihydroxylation (AD) of olefins (E)- and (Z)-13b
with AD-mix-a or -b catalysts was carried out as described in
the literature20 to give the expected triols 1b–4b. Application of
the same protocol to azido olefins (E)- and (Z)-14b afforded the
corresponding azido diols 5b–8b in good yields and diastereose-
lectivities (Table 1).
As expected from the Kishi and Sharpless empirical rules for
the AD reaction,22,23 the stereochemical course from olefins 13
and 14 was not affected by the presence of the azide group,
as evidenced by the close similarities found in the 1HNMR
spectra for each equivalent isomeric pair (1b/5b, 2b/6b, 3b/7b,
aResearch Unit on BioActive Molecules (RUBAM); Departament de
Qu´ımica Orga`nica Biolo`gica, Institut d’Investigacions Qu´ımiques i Ambien-
tals de Barcelona (IIQAB-C.S.I.C), Jordi Girona 18-26, 08034, Barcelona,
Spain. E-mail: adelgado@cid.csic.es; Fax: +34 932 045 904; Tel: +34 .934
006 108
bUniversitat de Barcelona, Facultat de Farma`cia, Unitat de Qu´ımica
Farmace`utica (Unitat Associada al CSIC), Avgda. Juan XXIII, s/n, 08028,
Barcelona, Spain
† Electronic supplementary information (ESI) available: Additional exper-
imental data. See DOI: 10.1039/b709421c
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Scheme 1 Divergent biosynthetic pathways in mammals and fungi.
Fig. 1 General structures of the library of PHSL analogues described in this study.
4b/8b). In addition, we further confirmed the above assumption
by chemical correlation. Thus, 1b and 5b afforded the same bis-
OTBS derivative 18 by simple functional group manipulations, as
shown in Scheme 2.
Despite the identical stereochemical course observed for azido
and hydroxy olefins in the AD reaction, some reactivity differences
are remarkable. Thus, a higher stereoselectivity arose from azido
olefin (Z)-14b on reaction with AD-mix-b, compared with the
corresponding hydroxy counterpart (Z)-13b (entries 1 and 5,
Table 1). Interestingly, an opposite trend was observed on reaction
with AD-mix-a (entries 2 and 6). In addition, dihydroxylation
of azido olefins in the presence of AD-mix-a led to incomplete
conversions and lower yields than the corresponding hydroxy
olefins (compare entries 2 and 4 with entries 6 and 8). This trend
can be attributed to a presumed unfavourable interaction of the
azido group with the chiral ligand.
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Scheme 2 Reagents and conditions: a) TBSOTf, 2,6-lutidine, DCM, 0 → 25 ◦C; b) HF·pyr, THF, 0 → 25 ◦C; c) MsCl, TEA, DCM, 0 → 25 ◦C; d) NaN3,
DMF, 65 ◦C.
Table 1 Sharpless asymmetric dihydroxylation (AD) of olefins (Z)- and
(E)-13b and (Z)- and (E)-14b
Entry Olefina
AD-mix Yield (%) Major isomera Isomeric ratio
1
2
3
4
5
6
7
8
(Z)-13b
(Z)-13b
(E)-13b
(E)-13b
(Z)-14b
(Z)-14b
(E)-14b
(E)-14b
b
a
b
a
b
a
b
a
96
82
94
93
94
49b
96
62c
1b (D-ribo)
87 : 13 (1b/2b)
2b (L-arabino) 13 : 87 (1b/2b)
3b (D-xylo)
4b (L-lyxo)
5b (D-ribo)
6b (L-arabino) 29 : 71 (5b/6b)
7b (D-xylo)
8b (L-lyxo)
85 : 15 (3b/4b)
15 : 85 (3b/4b)
92 : 8 (5b/6b)
88 : 12 (7b/8b)
12 : 88 (7b/8b)
a See Fig. 1. b 39% recovered starting material. c 33% recovered starting
material.
N-Boc removal of 1b–8b and 13b, 14b (E and Z isomers) under
classical acidic conditions19,20 set the stage for amide formation to
gain access to PHC analogues. This was carried out by selective
coupling the resulting amino derivatives 1a–8a and 13a, 14a (E
and Z isomers) with some of the acyl derivatives c–k shown in
Fig. 2.
Fig. 2 Acylating agents used for the synthesis of PHC analogues.
the corresponding amides shown in Scheme 3. Carboxylic acid 1k
was coupled in the presence of HOBt–EDC in THF.25
Acylating agents were chosen in order to compare the biological
activities of the resulting PHC analogues with those of related
ceramide analogues currently under evaluation in our group.24
Acyl chlorides c–j reacted in good to moderate yields under
Schotten–Baumann conditions (THF, 50% aq. NaOAc) to afford
1-Amino PHSL analogues were obtained by reduction of the
corresponding azide precursors (Scheme 4). Catalytic hydrogena-
tion was the method of choice for 9–12, whereas azide reduction via
Staudinger reaction was required for azido olefins 14 to preserve
the double bond integrity of the final analogues.
Scheme 3 PHC analogues synthesized in this study. a: Rꢀ = H; b: Rꢀ = Boc; c–k: see Fig. 2.
Scheme 4 Synthesis of 1-amino PHC analogues; a: Rꢀ = H; b: Rꢀ = Boc; c,d: see Fig. 2. Reagents and conditions: (a) H2, Pd/C, THF, 20 h, rt; (b) Ph3P,
THF–H2O (9 : 1), 60–72 h, 25 ◦C.
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Table 2 Inhibition of yeast growth by PHSL analogues at 400 lM
Yeast growth inhibition
S. cerevisiae, whose SL metabolism and signalling functions are
well known,26,27 has also been considered as a pathogen model for
antifungal activity.28 In this context, PHS has been shown to inhibit
S. cerevisiae growth through ubiquitin-dependent proteolysis of
different nutrient permeases.29 Along this line, the PHS analogues
described in this work (1a–12a) were evaluated as S. cerevisiae
growth inhibitors in order to determine the influence of the
sphingoid chain configuration at the C3–C4 positions and the
nature of the functional group at C1 (Table 2 and Fig. 3).
Compound
Inhibition (%) (mean SD)
IC50/lM
1a
77
84
92
2
3
1
280
110
98
2a
3a
4a
100
98
85
2
1
2
90
5a
150
135
—
6a
7a
40
4
n/a
3
4
6
5
5
8a
—
(Z)-13a
(E)-13a
(Z)-15a
(E)-15a
1b
79
83
80
82
23
145
128
102
98
—
—
—
—
—
176
—
—
—
72
98
71
—
—
—
All four PHS diastereomers (R = OH, 1a–4a, Fig. 1) were good
growth inhibitors, especially the unnatural isomers 2a–4a (IC50
90–110 lM). Analogues resulting from the replacement of C1-OH
with a primary amine (R = NH2, 9a–12a, Fig. 1) were devoid
of activity, an indication of the importance of C1-OH in natural
PHS. On the other hand, striking differences on growth inhibitory
properties were found among azide derivatives (R = N3, 5a–8a,
Fig. 1), where the naturally configured D-ribo analogue 5a and
L-arabino analogue 6a (IC50 150 and 135 lM, respectively) were
the most active ones. It is worth noting that the stereoselectivity
observed in the azide analogues was not found in the amino
(R = NH2) or hydroxy (R = OH) counterparts. Since the simple
aliphatic alcohol (1-octadecanol), amine (1-octadecylamine) or
azide (1-azidooctadecane) were totally devoid of activity in this
assay, and unsaturated alcohols ((E)- and (Z)-13a and (E)- and
(Z)-15a) showed a similar inhibition profile (IC50 98–145 lM),
it is reasonable to assume that the observed effects for our PHS
analogues are the result of specific, albeit yet undisclosed, cellular
effects rather than unspecific membrane-disrupting mechanisms.
It is accepted that effective incorporation of D-ribo PHS into phy-
tosphingolipids requires both phosphorylation and subsequent
dephosphorylation. These reactions serve to properly localize the
sphingoid base and to allow for efficient action of downstream
enzymes, since yeasts with a deleted phosphatase gene (LCB3)
are more resistant to PHS treatment.30 Although additional
experiments are required along this line, targets of natural PHS,
such as AGC-type protein kinases31 and/or nutrient permeases29,32
seem reasonable candidate targets for PHS stereoisomers 1a–4a.
2b
n/a
3
6
3b
64
6
4b
5b
6
7
7
9b
90
43
59
43
100
83
92
50
70
4
(Z)-13b
1c
6
5
5
5
5
3
5
6
9c
10c
11c
12c
(Z)-15c
(E)-15c
(Z)-15d
1e
2
n/a
4
—
—
—
—
—
—
—
1f
55
44
1g
3
1h
n/a
8
1i
1j
3
n/a
n/a
1k
Preliminary experiments on PHC analogues were first carried
out from N-acyl derivatives of natural D-ribo stereochemistry (1c–
k, Table 2, Fig. 1 and 2). Branched a- or b-N-acyl substituents
led to the most potent growth inhibitors (compounds 1c, 1f,
and 1g).33 These promising data led us to explore other N-
pivaloyl derivatives arising from the systematic variation of the
C1 functionality and/or the stereochemistry around the C3–C4
Fig. 3 Yeast growth inhibition (%) in the presence of PHSL analogues at 400 lM.
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Table 3 Inhibition of IPC synthase by PHSL analogues
moiety of the sphingoid backbone. Thus, replacement of C1-
OH with an azido group (5c–8c, Fig. 1 and 2), as well as the
presence of a non-natural stereochemistry on the sphingoid base
(2c–4c) was detrimental for growth inhibition. However, this trend
was not observed for the C1-NH2 analogues (9c–12c, Fig. 1 and
2), where the non-naturally configured 10c–12c were the most
potent growth inhibitors (IC50 71–98 lM, Table 2 and Fig. 3).
Unsaturated analogues ((E)- and (Z)-15c), bearing the C1-NH2
and N-pivaloyl moieties, showed growth inhibitory properties
comparable to those of the corresponding D-ribo analogue 9c, this
stressing the importance of an unnatural stereochemistry at C3–
C4 for the activity of these kinds of PHC analogues.34 Finally,
although N-octanoyl analogue 1d was inactive, the inhibitory
activity elicited by the C1-NH2 analogues 9c–12c and (E)- and (Z)-
15c prompted us to test the corresponding C1-NH2 N-octanoyl
derivatives 9d and (Z)-15d. However, none of them led to yeast
growth inhibition.35
Different C1-functionalized N-Boc PHS (1b–5b and 9b, Fig. 1
and 2) and olefin isomers ((Z)- and (E)-13b, 14b, and 15b) were also
tested (Table 2 and Fig. 3). As a general trend, the activity of C1-
OH analogues (1b–4b) was lower than that of the corresponding
free sphingoid base (1a–4a). However, some of them exhibited
higher inhibitory activity than the corresponding N-pivaloyl
analogues (compare, for example 3b/3c and (Z)-13b/(Z)-13c,
Fig. 3). Among the N-Boc C1-NH2 analogues tested, the D-ribo
compound 9b was the most active one, exhibiting an IC50 value
of 176 lM (Table 2). With the exception of olefin (Z)-13b, the
remaining olefin isomers were inactive as yeast growth inhibitors.
Compound
Inhibition (%) (mean SD)
1c
35.4
31.7
29.2
48.1
29.3 0.5
24.9 0.6
14.3
28.2
45.3
25.4
24.1
22.0
34.0
31.2 0.4
26.9
5.3
10.6
18.5
8.0
3
3
6
9
2c
3c
4c
5c
6c
7c
2
2
9
3
5
2
2
8c
9c
10c
11c
12c
(Z)-13c
(E)-13c
(Z)-15c
(E)-15c
9b
5
3
1
4
5
2
(Z)-15b
(E)-15b
1d
33.3
11.8 0.4
9d
moderate inhibitors, the L-lyxo compound 4c being the most
potent (around 48% inhibition, see Table 3). Both the D-ribo
analogue 1c, and the corresponding N-octanoyl analogue 1d,
having the natural configuration of the sphingoid backbone, as
well as the non-natural 2c and 3c analogues, afforded similar
results (30–35% inhibition). These data seem to indicate that the
stereochemistry of the C3–C4 moiety in this series is irrelevant
for the inhibitory activity, since the inhibition shown by both
(Z)- and (E)-13c was comparable to that of the above analogues.
Replacement of the C1-OH with an amino group led to more
striking differences. Thus, D-ribo analogue 9c was the most potent
inhibitor of this series (45% inhibition), while stereochemical
variations around the C3–C4 moiety led to the markedly less
potent inhibitors 10c–12c (around 20–25% inhibition). Likewise,
replacement of the N-pivaloyl group with the N-octanoyl (9d)
or N-Boc (9b) groups led to an even more pronounced loss of
activity (around 10% inhibition). In contrast to the C1-OH series,
replacement of the C3–C4 dihydroxy moiety with a double bond
led to a substantial loss of activity, as found in (Z)-15b, (E)-15b,
and (E)-15c.
IPC synthase inhibition
As mentioned above, specific IPC synthase inhibitors are attractive
targets for the design of new, nontoxic antifungal agents. However,
only some structurally complex natural products have been
reported so far.14 Attempts to obtain simplified analogues have
found limited success so far, as reported for galbonolide36 and
khafrefungin (Scheme 1).37,38 The complex chemical structures of
the above compounds and the lack of significant activity found
for simplified analogues indicate the striking difficulty of finding
new IPC synthase inhibitors. Along this line, several of our PHC
analogues were assayed against IPC synthase using khafrefungin
as a reference inhibitor.
Using a membrane preparation from S. cerevisiae obtained as
described by Aeed et al.39 and an HPLC-fluorimetric assay to
assess the formation of fluorescent inositol phosphoryl C6-NBD
ceramide (C6-NBD-Cer),40 we were able to determine a Km of
4.3 lM for C6-NBD-Cer, a similar value to that reported for IPC
synthase from Candida albicans.39 This assay was optimized and
routinely used, as described in the Experimental section.
For the most active compounds (4c and 9c), incubations at
different concentrations were carried out in comparison with
khafrefungin (Fig. 4). Calculated IC50 values of 33, 35 and
Based on the above analytical protocol, N-pivaloyl PHC
analogues 1c–12c were evaluated in vitro as IPC synthase inhibitors
in comparison with khafrefungin.17,37,41 For comparative purposes,
the D-ribo N-octanoyl analogues 1d and 9d, as well as N-Boc
analogues 9b, (Z)-15b and (E)-15b, were studied. Compounds
were evaluated at 30 lM against the fluorescent substrate C6-
NBD-Cer at 10 lM. The results are shown in Table 3.
As a general trend, C1-azido analogues (5c–8c) were weaker
IPC synthase inhibitors than their corresponding C1-OH (1c–
4c) or C1-NH2 (9c–12c) counterparts. All C1-OH analogues were
Fig. 4 Inhibition of IPC synthase by khafrefungin (ꢀ) and phytoceramide
analogues 4c (᭹) and 9c (ꢁ).
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0.19 lM, respectively, were obtained. Although the inhibitory
activity of our PHC analogues was two orders of magnitude lower
than that of khafrefungin, their structural simplicity will make
possible the design of focused libraries for further screening in the
search of more potent IPC synthase inhibitors.
didates were tested at 30 lM whereas the fluorescent substrate
was used at 10 lM. The IC50 values were determined by plotting
percent activity versus the inhibitor concentration, expressed as
log [I], using at least five different inhibitor concentrations.
Chemistry
Conclusions
General methods. Garner aldehyde was obtained by oxidation
of the corresponding primary alcohol,42 prepared according to
reported methods.43,44 Solvents were distilled prior to use and
dried by standard methods.45 FT-IR spectra are reported in cm−1.
1H and 13C NMR spectra were obtained in CDCl3 solutions at
A library of 64 PHSL analogues resulting from the systematic
variation of the C1, C3, C4, and the N-acyl moiety of phytosphin-
gosine has been prepared. The antifungal activity of the library has
been evaluated based mainly on its growth inhibitory properties
against the yeast S. cerevisiae. PHS analogues (Rꢀ = H, Fig. 1)
bearing the natural C1-OH functional group were potent growth
inhibitors, irrespective of the nature or the stereochemistry of the
C3–C4 moiety of the sphingoid backbone. In contrast, only C1-
NH2 PHS analogues with a C3–C4 double bond showed significant
growth inhibition. Among PHC analogues (Rꢀ = acyl, Fig. 1),
C1-NH2 N-pivaloyl derivatives were good growth inhibitors, once
again irrespective of the nature of the C3–C4 moiety. Attempts
to correlate growth inhibition with IPC synthase inhibition in N-
pivaloyl analogues were unsuccessful, since the most active enzyme
inhibitors (4c and 9c) were inactive or weakly active, respectively, in
the growth inhibition assay. Nevertheless, the promising results on
IPC synthase inhibition obtained with some of our PHC analogues
will help to define new structural modifications for future research
along this line.
1
300 MHz (for H) and 75 MHz (for 13C), respectively. Chemical
shifts are reported in d units, in parts per million (ppm) relative to
the singlet at 7.24 ppm of CDCl3 for 1H, and in ppm relative to the
center line of a triplet at 77.0 ppm of CDCl3 for 13C. [a]D values
are given in 10−1deg cm2 g−1. HPLC-MS analyses were obtained
on a Hewlett Packard MSD system. ESI/HRMS spectra were
recorded on a Waters LCT Premier Mass spectrometer at CID–
CSIC (Barcelona) and IQAC (Tarragona).
Synthesis of mesylates (E)- and (Z)-16b from alcohols (E)- and
(Z)-13b. To 1.3 mmol of the corresponding alcohol (E)- or
(Z)-13b were added 13 mL of anhydrous DCM under argon
atmosphere, and the reaction was cooled to 0 ◦C. The reaction
mixture was next treated with 0.36 mL (2.6 mmol) of freshly
distilled TEA and, after stirring for 10 min at 0 ◦C, 0.12 mL
(1.6 mmol) of freshly distilled MsCl was also added. The reaction
mixture was stirred at 0 ◦C for 2 h until no starting material
was observed by TLC. The mixture was then diluted with DCM
(15 mL) and sequentially washed with 3 × 15 mL of 1 N HCl,
sat. aq. NaHCO3, and brine. The organic phase was dried over
MgSO4, filtered, and the solvent evaporated in vacuo to give the
required mesylate.
Experimental
Inhibition assays
Biological assays. The isogenic S. cerevisiae strain W303a
(leu2, ura3, trp1, ade2, his3) was grown in YPD medium, composed
of 1% yeast extract, 2% peptone and 2% glucose. The yeasts
were grown with aeration at 30 ◦C and growth was followed
turbidimetrically at 600 nm. Yeast suspension on YPD medium to
a final concentration of 0.15 OD in 0.1 mL was prepared. Tested
compounds were dispensed into the wells of a 96-well polystyrene
microtiter plate to a final concentration of 0.4 mM and inoculated
with 0.1 mL of yeast suspension. Growth in liquid YPD with or
without 0.5% tergitol in the presence of synthesized compounds
was monitored over a 16 h period. All experiments were repeated
at least three times. The IC50 values were determined by plotting
percent activity versus log [I], using at least five different inhibitor
concentrations.
(2ꢀR,3ꢀZ)-Methanesulfonic acid 2-(tert-butoxycarbonylamino)-
octadec-3-enyl ester ((Z)-16b). Obtainedas awhite solid(524 mg,
1.1 mmol, 89% from (Z)-13b), which was used in the next step
◦
without further purification. Mp 58–59 C; [a]2D5 +6.5 (c 1.10,
CHCl3); IR (film): 3368, 2926, 2855, 1708, 1513, 1463, 1361, 1245,
1174, 1054, 963, 829. 1H NMR (CDCl3, 500 MHz): 5.64 (dt, J =
10.8, 7.5 Hz, 1H), 5.32 (dd, J = 11, 9 Hz,1H), 4.67 (br s, 2H), 4.26
(m, 1H), 4.16 (dd, J = 10, 5 Hz, 1H), 3.02 (s, 3H), 2.11 (m, 2H),
1.44 (s, 9H), 1.36 (m, 2H), 1.25 (m, 22H), 0.88 (t, J = 7 Hz, 3H).
13C NMR (CDCl3, 75 MHz): 155.1, 136.0, 124.5, 80.1, 71.3, 47.4,
37.6, 37.4, 32.0, 30.8, 29.8, 29.6, 29.5, 29.4, 28.5, 28.4, 28.1, 27.3,
22.8, 14.3, 14.1 (rotamers observed). ESI-MS m/z 484 [M + Na],
500 [M + K].
IPC synthase assay. IPC synthase activity was determined
using detergent-treated microsomal membranes from S. cerevisiae,
prepared as described by Aeed et al.39 The assay was based on an
HPLC analysis of the conversion of the fluorescent C6-NBD-
Cer to inositol phosphoryl C6-NBD-Cer.40 Chromatographic
conditions: column: Kromasil 100, C18, 5 lm, 15 × 0.40 cm;
flow rate 1 mL min−1, CH3CN–H2O, gradient from 50 : 50 to 70 :
30 in 20 min; detection at 530 nm (excitation at 450 nm); retention
times: inositol phosphoryl C6-NBD-Cer (7.1 min); C6-NBD-Cer
(21.3 min). The protein was incubated with different concentra-
tions of C6-NBD-Cer, and the Km value was calculated by a
Lineweaver–Burk plot. Yeast microsomes were incubated in the
presence or absence of potential inhibitors for 1 h at 37 ◦C. Can-
(2ꢀR,3ꢀE)-Methanesulfonic acid 2-(tert-butoxycarbonylamino)-
octadec-3-enyl ester ((E)-16b). Obtained from 125 mg
(0.33 mmol) of alcohol (E)-13b as a white solid (126 mg,
0.27 mmol, 81%), which◦was used in the next step without further
purification. Mp 64–65 C; [a]2D5 +3.0 (c 1.15, CHCl3); IR (film):
3344, 2917, 2849, 1685, 1525, 1463, 1353, 1280, 1247, 1168, 1054,
1
965, 836. H NMR (CDCl3, 500 MHz): 5.73 (dtd, J = 15.5, 6.5,
1 Hz, 1H), 5.38 (dd, J = 15.5, 6 Hz,1H), 4.72 (br s, 2H), 4.38 (m,
1H), 4.27 (dd, J = 9.5, 4 Hz, 1H), 4.18 (dd, J = 10, 5 Hz, 1H),
3.02 (s, 3H), 2.03 (m, 2H), 1.44 (s, 9H), 1.36 (m, 2H), 1.23–1.32
(m, 22H), 0.87 (t, J = 7 Hz, 3H). 13C NMR (CDCl3, 75 MHz):
155.1, 135.1, 125.0, 80.1, 71.3, 51.5, 37.5, 32.4, 32.0, 29.8, 29.7,
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29.6, 29.5, 29.2, 29.0, 28.4, 22.8, 14.2. ESI-MS m/z 484 [M + Na],
946 [2M + Na].
26.5, 26.3, 26.2, 26.0, 26.0, 25.9, 25.6, 22.9, 18.5, 18.4, 18.3, 18.3,
14.3, −2.8, −3.5, −3.7, −4.6, −5.0, −5.1, −5.4. ESI-MS m/z
783 [M + Na].
General procedure for the synthesis of azido olefins 14b.
A
A solution of the above tris-OTBS derivative (95 mg, 0.12 mmol)
solution of the required starting mesylate 16b (346 mg, 0.75 mmol)
in DMF (12 mL) was treated with NaN3 (122 mg, 1.87 mmol).
The reaction mixture was heated at 65 ◦C and stirred for 20 h.
The mixture was then diluted with 40 mL of H2O and 40 mL of
Et2O. The organic phase was washed with 1 N HCl (3 × 25 mL),
dried over MgSO4, filtered and the solvent removed under reduced
pressure. The resulting crude was purified by flash chromatography
(hexane–EtOAc 9 : 1) to give the final azide.
◦
in THF (2 mL) under N2 at 0 C was treated with HF·pyridine
(0.41 mL of a ∼70% solution, 15.8 mmol) in 2.5 mL of a 65 :
35 mixture of THF–pyridine. The reaction mixture was stirred for
30 min at 0 ◦C and allowed to warm to rt. After 1 h, the mixture was
quenched by addition of sat. aq. NaHCO3 (15 mL) and stirred for
10 min. The reaction mixture was extracted with EtOAc and the
organic phases were washed with brine, filtered and evaporated to
dryness. The residue was flash chromatographed to afford 57 mg
(0.09 mmol, 70%) of 17 as a colourless oil. [a]2D5 −11.6 (c 1.93,
CHCl3); IR (film): 3446, 2929, 2857, 1699, 1501, 1467, 1387, 1368,
(2ꢀR,3ꢀZ)-[1-(Azidomethyl)heptadec-2-enyl]carbamic acid tert-
butyl ester ((Z)-14b). Obtained in 94% yield (288 mg, 0.70 mmol)
as a white solid from 346 mg (0.75 mmol) of (Z)-16b. Mp 55–56 ◦C;
[a]2D5 +3.5 (c 0.80, CHCl3); IR (film): 3359, 2921, 2853, 2102, 1756,
1
1253, 1171, 1054, 936, 836, 776. H NMR (CDCl3, 500 MHz):
5.22 (d, J = 8.5 Hz, 1H), 4.10 (m, 1H), 3.87 (m, 1H), 3.76 (dt, J =
6.3, 3 Hz, 1H), 3.72 (dd, J = 7.5, 3.8 Hz, 1H), 3.62 (ddd, J = 8.5,
8.1, 3.3, 1H), 2.97 (br d, J = 11.5 Hz, 1H), 1.53 (m, 2H), 1.44 (s,
9H), 1.34 (m, 2H), 1.25–1.30 (m, 22H), 0.92 (s, 9H), 0.90 (s, 9H),
1
1687, 1525, 1462, 1372, 1301, 1241, 1168, 1109, 852. H NMR
(CDCl3, 500 MHz): 5.59 (dt, J = 10, 7.5 Hz,1H), 5.31 (dd, J = 10,
9 Hz, 1H), 4.61 (br s, 1H), 4.55 (br s, 1H), 3.41 (m, 1H), 3.34 (dd,
J = 12, 4.5 Hz, 1H), 2.11 (m, 2H), 1.44 (s, 9H), 1.36 (m, 2H), 1.25
(m, 22H), 0.88 (t, J = 7 Hz, 3H). 13C NMR (CDCl3, 75 MHz):
155.0, 135.2, 126.3, 79.9, 55.4, 48.1, 32.0, 29.8, 29.8, 29.7, 29.6,
29.5, 29.5, 29.4, 28.5, 28.1, 22.8, 14.2. ESI-MS m/z 431 [M + Na],
840 [2M + Na].
0.88 (t, J = 7 Hz, 3H), 0.10 (s, 3H), 0.10 (s, 6H), 0.08 (s, 3H). 13
C
NMR (CDCl3, 75 MHz): 155.5, 79.3, 77.6, 76.1, 63.6, 52.2, 34.2,
32.1, 29.8, 29.7, 29.7, 29.5, 28.6, 26.2, 26.1, 25.9, 22.8, 18.3, 18.3,
14.3, −3.6, −3.9, −4.4, −4.7. ESI-MS m/z 547 [M − Boc + H],
669 [M + Na].
(2ꢀR,3ꢀE)-[1-(Azidomethyl)heptadec-2-enyl]carbamic acid tert-
butyl ester ((E)-14b). Obtained in 77% yield (557 mg, 1.36 mmol)
as a white solid from 815 mg (1.77 mmol) of (E)-16b. Mp 49–
(2ꢀS,3ꢀS,4ꢀR)-[1-Azidomethyl-2,3-bis-(tert-butyldimethylsilanyl-
oxy)heptadecyl]carbamic acid tert-butyl ester (18). Method A:
From silylation of 5b: A solution of azide 5b (64 mg, 0.14 mmol) in
DCM (2 mL) under argon was cooled to 0 ◦C and treated with neat
TBSOTf (111 lL, 0.48 mmol). After stirring for 10 min at 0 ◦C, 2,6-
lutidine (168 lL, 1.45 mmol) was added to the mixture. Stirring was
maintained for 1 h at 0 ◦C and for 2 h at 25 ◦C. The reaction mixture
was quenched with CH3OH (1 mL) and the solvent was removed
under reduced pressure. The residue was flash chromatographed
(hexane–EtOAc 98 : 2) to afford 60 mg (0.10 mmol) of 18 as a
colourless oil.
◦
50 C; [a]2D5 +8.1 (c 1.05, CHCl3); IR (film): 3341, 2920, 2851,
1
2100, 1683, 1525, 1463, 1392, 1364, 1293, 1246, 1169. H NMR
(CDCl3, 500 MHz): 5.59 (dtd, J = 15.5, 6.5, 1.5 Hz, 1H), 5.38
(dd, J = 15.5, 6.5 Hz, 1H), 4.67 (br s, 1H), 4.27 (m, 1H), 3.42 (dd,
J = 12, 5 Hz, 1H), 3.34 (dd, J = 12, 4.5 Hz, 1H), 2.03 (m, 2H),
1.45 (s, 9H), 1.36 (m, 2H), 1.23–1.32 (m, 22H), 0.88 (t, J = 7 Hz,
3H). 13C NMR (CDCl3, 75 MHz): 155,1, 134.2, 126,8, 79.9, 55,2,
52,3, 32,4, 32,1, 29.8, 29.7, 29.6, 29.5, 29,3, 29,1, 28.5, 22.8, 14.2.
ESI-MS m/z 431 [M + Na], 841 [2M + Na].
Method B: From alcohol 17: Following the mesylation–
azidation protocol described above for the synthesis of azides 14b
from alcohols 13b, alcohol 17 afforded azide 18 in 45% combined
yield. [a]2D5 +1.7 (c 0.87, CHCl3); IR (film): 2928, 2857, 2102, 1710,
1498, 1466, 1371, 1254, 1169, 835. 1H NMR (CDCl3, 500 MHz):
4.78 (d, J = 9.5 Hz, 1H), 3.80 (m, 1H), 3.67 (m, 2H), 3.58 (m, 2H),
1.57 (m, 1H), 1.47 (m, 2H), 1.44 (s, 9H), 1.37 (m, 1H), 1.22–1.32
(m, 22H), 0.91 (s, 9H), 0.90 (s, 9H), 0.88 (t, J = 7 Hz, 3H), 0.12
(s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.05 (s, 3H). 13C NMR (CDCl3,
100 MHz): 155.2, 79.7, 76.5, 75.5, 52.1, 51.9, 33.1, 32.1, 29.9, 29.8,
29.8, 29.8, 29.7, 29.5, 28.5, 26.2, 26.2, 26.1, 24.9, 22.8, 18.5, 18.3,
14.3, −3.5, −3.8, −4.5, −5.1. ESI-MS m/z 572 [M − Boc + H]+,
694 [M + Na].
(1ꢀS,2ꢀS,3ꢀR)-[2,3-Bis-(tert-butyldimethylsilanyloxy)-1-(hydroxy-
methyl)heptadecyl]carbamic acid tert-butyl ester (17). A solution
of 1b (152 mg, 0.36 mmol) in DCM (10 mL) at 0 ◦C was
sequentially treated at 0 ◦C with neat TBSOTf (418 lL,
1.82 mmol) and 2,6-lutidine (0.64 mL). The reaction mixture was
stirred at 0 ◦C for 30 min, and then warmed to 25 ◦C and stirred
at this temperature for 20 h. The reaction was quenched with
CH3OH (5 mL) and stirred for 10 min. The solvent was removed
at reduced pressure and the residue taken up in Et2O and washed
with H2O, aq. NaHCO3, and brine. The organic phase was dried
over MgSO4, filtered and evaporated to give a residue, which was
purified by flash chromatography to afford 178 mg (0.23 mmol,
65%) of the tris-OTBS derivative as a colourless oil. [a]2D5 + 6.3
(c 0.50, CHCl3); IR (film): 3453, 2960, 2930, 2858, 1723, 1495,
1468, 1386, 1365, 1254, 1172, 1063, 1008, 941, 838, 778. 1H NMR
(CDCl3, 500 MHz): 4.91 (d, J = 9 Hz, 1H), 3.79 (dd, J = 10.2,
4.2 Hz, 1H), 3.74 (m, 2H), 3.65 (dd, J = 10, 4.5 Hz, 1H), 3.58 (m,
1H), 1.57 (m, 1H), 1.48 (m, 1H), 1.42 (s, 9H), 1.24–1.32 (m, 24H),
0.90 (br s, 6H), 0.89 (m, 6H), 0.89 (m, 6H), 0.86 (m, 12H), 0.12
(s, 2H), 0.07 (s, 2H), 0.06 (s, 2H), 0.05 (s, 2H), 0.04 (s, 4H), 0.01
(d, J = 0.5 Hz, 6H). 13C NMR (CDCl3, 75 MHz): 155.5, 78.9,
75.7, 75.6, 62.0, 54.3, 32.4, 32.1, 30.0, 29.9, 29.8, 29.8, 29.5, 28.6,
General method for N-Boc removal. A solution of 0.50 mmol
of the starting N-Boc-protected amine in 5 mL of a 95 : 5 TFA–
H2O mixture was stirred for 20 min at rt. The reaction mixture was
evaporated to dryness under a stream of N2 and the residual solid
was washed with aq. NaHCO3, filtered and thoroughly washed
with H2O. The white residue was purified by flash chromatography
(92 : 8 : 1 DCM–CH3OH–NH4OH) to afford analytically pure
samples of the target amino alcohols. Compounds 1a–8a and
13–15a (E and Z isomers) from 1b–8b and 13–15b (E and Z
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isomers) were obtained following this protocol (see Electronic
Supplementary Information†).
identification by ninhydrin staining on the TLC. Compounds
(Z)-15b–d, (E)-15b,c were obtained following this protocol (see
Electronic Supplementary Information†).
General method for acylation reactions with acyl chlorides c–
j. A solution of the starting amine (0.1 mmol) in 5 mL of
a 1 : 1 mixture of THF and 50% aq. NaOAc was cooled to
Acknowledgements
0
◦C and stirred vigorously at this temperature for 15 min. The
corresponding acyl chloride (0.19 mmol) was added dropwise
to the reaction mixture, which was allowed to warm to 25 ◦C
and stirred for an additional 20 h. The mixture was extracted
with EtOAc (3 × 10 mL) and the combined organic phases
were washed with 1 N NaOH and brine, dried over MgSO4,
filtered and evaporated. The resulting solid was purified by flash
chromatography to give the final amide. Compounds 1c–j, 2c–
8c, (E) and (Z)–13c, (E) and (Z)–14c, 5d, and (Z)-13d were
obtained following this protocol (see Electronic Supplementary
Information†).
Financial support from Ministerio de Educacio´n y Ciencia,
Spain (Projects MCYT CTQ2005-00175/BQU and AGL2004–
05252), Fondos Feder (EU), and Generalitat de Catalunya
(2005SGR01063) is acknowledged. D.M. is acknowledged to
Ministerio de Educacio´n y Ciencia, Spain, for a predoctoral
fellowship. We also thank Mrs. Marta Mart´ın and Mrs. Victoria
Framis for experimental contributions. Finally, we are grateful to
Prof. Kobayashi (Graduate School of Pharmaceutical Sciences,
The University of Tokyo, Japan) for a sample of khafrefungin.
(1ꢀS,2ꢀS,3ꢀR)-N-[2,3-Dihydroxy-1-(hydroxymethyl)heptadecyl]-
2-oxoctanamide (1k). A solution of 37 mg (0.23 mmol) of
2-oxooctanoic acid, 45 mg (0.23 mmol) of EDC and 32 mg
(0.23 mmol) of HOBt in THF (5 mL) under Ar was stirred for
40 min. A solution of 1a (56 mg, 0.16 mmol) in THF (5 mL)
was added dropwise and the reaction mixture was stirred at rt
for 24 ◦C. The resulting viscous liquid was evaporated to dryness
and taken up with 10 mL of a 1 : 1 H2O–DCM mixture. The
organic phase was separated and washed with aq. NaHCO3,
filtered and evaporated to dryness. The residue was purified by
flash chromatography to afford 16 mg (0.03 mmol, 15%) of 1k as a
white waxy solid. [a]2D5 +5.4 (c 1.14, CHCl3); IR (film): 3308, 2919,
2851, 1707, 1669, 1541, 1468, 1212, 1184, 1067. 1H NMR (CDCl3,
500 MHz): 7.66 (d, J = 8 Hz, 1H, NH), 4.13 (m, 1H, H2), 3.95 (dd,
J = 11.5, 3 Hz, 1H, H1a), 3.89 (dd, J = 11.5, 5.5 Hz, 1H, H1b),
3.66–3.70 (m, 2H, H3, H4), 2.91 (t, J = 7.4 Hz, 2H, H2ꢀ), 1.69 (m,
1H), 1.60 (m, 2H), 1.49 (m, 2H), 1.23–1.36 (m, 29H), 0.88 (t, J =
7 Hz, 3H), 0.87 (t, J = 7 Hz, 3H). 13C NMR (CDCl3, 100 MHz):
198.9, 160.6, 75.9, 75.4, 73.0, 73.0, 61.5, 61.2, 52.3, 52.1, 37.0, 33.2,
32.1, 31.6, 29.8, 29.8, 28.8, 29.7, 29.7, 29.5, 28.7, 25.8, 23.2, 22.8,
14.3, 14.2. ESI-MS m/z 458 [M + H]+, 938 [2M + Na]; HPLC:
>95% pure (H2O–CH3CN 20 : 80, tR = 8.96 min).
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