Synthesis and biological evaluation of antifungal activities of novel 1,2-trans glycosphingolipids

Article abstract of DOI:10.1002/ardp.201000335

The synthesis and in-vitro biological evaluation of antifungal activities of a series of 1,2-trans glycosphingolipids (GSL) against Candida albicans, Candida parapsilosis, and Candida tropicalis were described. The preliminary study indicated that the sor

Full text of DOI:10.1002/ardp.201000335

786
Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
Full Paper
Synthesis and Biological Evaluation of Antifungal Activities
of Novel 1,2-trans Glycosphingolipids
Ning Ding, Wei Zhang, Guokai Lv, and Yingxia Li
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, P. R. China
The synthesis and in-vitro biological evaluation of antifungal activities of a series of 1,2-trans
glycosphingolipids (GSL) against Candida albicans, Candida parapsilosis, and Candida tropicalis were
described. The preliminary study indicated that the sort of sugar moiety of GSLs affected their
antifungal activities and selectivities towards the three Candida species, and the permeability
might not be the sole parameter affecting their antifungal properties as presumed before, which
would bring new clues to understand the antifungal profile for these types of compounds.
Keywords: Antifungal / Candida / Glycosylation / Glycosphingolipids
Received: November 4, 2010; Revised: February 21, 2011; Accepted: February 25, 2011
DOI 10.1002/ardp.201000335
Introduction
bases in GSLs that contain a C4–C5 double bond in the trans-D-
erythro configuration are called sphingosines (see T2, Fig. 1).
Less frequent are sphinganines (dihydrosphingosine, DHS,
Fig. 1), which lack the double bond, and phytosphingosine
(PHS), which carries an additional hydroxyl group on C4 (see
T4, Fig. 1).
The rising incidence of infections caused by antibiotic-resist-
ant fungi has become a major concern for clinicians and the
public health system. Candida species are among the most
common causes of fungal infections ranging from non-life-
threatening mucocutaneous illnesses to disseminated myco-
ses that affect blood and organs [1]. Studies have shown that
Candida albicans can become transiently resistant to azole
drugs rapidly after exposure to the antifungal drug flucona-
zole [2]. These medical azoles are first-line antifungals for the
treatment of human submucosal and invasive mycoses.
Therefore, the availability of new classes of antifungal agents
enhances opportunities to effectively combat infections that
are notoriously difficult to treat.
The scientific interest in GSLs has increased on account of
their broad biological activities [3]. However, only scattered
data on the antifungal activities of GSLs has been reported in
the literature. To the best of our knowledge, all of these
studies were focused on one specific type of GSL called
‘‘two-headed glycosphingolipids’’ [4]. Among them a few
compounds were discovered to possess moderate antifungal
activities, whereas removal of the O-glycosyl group from the
above compounds enhanced the activities [4]. The presence of
sugar moiety was presumed to reduce the membrane per-
meability and thus depressed the activities. Other studies also
demonstrated that the long-chain sphingoid bases them-
selves (sphingosine, PHS, and DHS) or their ceramide form,
rather than the glycosylated ones, can inhibit the growth of
several fungal species efficiently [5].
Glycosphingolipids (GSLs) are ubiquitous components of
the cellular membranes of all eukaryotic cells. They are
glycolipids composed of a long-chain amino alcohol, known
as a sphingoid base, with a fatty acid residue linked to its
amino group (the resulting amide is called ceramide), and a
carbohydrate chain attached to the primary hydroxyl group
of the ceramide. The most frequently occurring sphingoid
It has been widely known that the sugar is often the critical
determination of the key biological activity in many com-
pounds. With our continuous interest in the effect of sugar
attachments to various types of aglycones on the biological
activities [6] and in order to search for potential new anti-
microorganism agents [6], we decided to investigate the effect
of the sort of sugar moiety of GSL on their fungal growth
Correspondence: Yingxia Li, Department of Medicinal Chemistry, School
of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203,
P. R. China
E-mail: liyx417@fudan.edu.cn
Fax: þ86-21-51980127
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
Evaluation of Antifungal Activities of Novel 1,2-trans Glycosphingolipids
787
N3
C13H27
HO
HO
8
9
T1
R1 = N3, R2 = H, R3 = Bz
R1 = NH2, R2 = H, R3 = Bz
R1 = NH2, R2 = H, R3 = H
c
OBz
O
OR3
O
R1
N3
BzO
R3O
R3O
b
c
a
2
OTBS
d
O
C13H27
O
C13H27
e
BzO
12 R1 = NHCOC25H51, R2 = H, R3 = Bz
T3 R1 = NHCOC25H51, R2 = H, R3 = H
OR3
OBz
OR2
OTBS
d
5
N3
C13H27
OBz
O
BzO
BzO
OR3
O
OBz
O
R3O
R3O
BzO
R1
N3
OBz
3
10
T2
R1 = NH2, R2 = Bz
R1 = NH2, R2 = H
a
d
O
C13H27
O
C13H27
O
BzO
BzO
CCl3
NH
OR3
OBz
OR2
OBz
6
1
N3
OBn
C13H27
HO
a
OBz
O
OR3
O
R3O
R3O
R1 OR2
O C13H27
BzO
BzO
N3 OBn
C13H27
11
13
R1 = NH2, R2 = OBn, R3 = Bz
R1 = NHCOC25H51, R2 = Bn, R3 = Bz
e
c
O
OBn
4
d, f
T4 R1 = NH COC25H51, R2 = H, R3 = H
OR3
OBz
OR2
OBn
7
Conditions: a. donor 1, TMSOTf, CH2Cl2, 92% for 5, 92% for 6, 87% for 7; b. TBAF, THF, 91%; c. PPh3, THF/H2O; d.
NaOCH3, CH3OH/CH2Cl2, 87% for T1 over 2 steps, 73% for T2 over 2 steps, 92% for T3; e. hexacosanoic acid, HOBt,
EDC/HCl, DIPEA, 88% for 12 over 2 steps, 73% for 13 over 2 steps; f. Pd/C-H2, MeOH/EtOAc, 76% over 2 steps.
Scheme 1. Synthesis of T1–T4.
inhibitory activities. Here, we would like to report the syn- Results and discussion
thesis and in-vitro biological evaluation of antifungal activi-
ties of a series of glycosylated sphingolipids against C. albicans,
C. parapsilosis, and C. tropicalis. The results obtained here will
render new clues to the understanding of the antimicroor-
ganism profile for these types of compounds.
Several typical sugar heads, such as D-galactopyranosyl, D-man-
nopyranosyl, D-glucopyranosyl, L-rhamnopyranosyl as well as a
2-O-carbamoyl modified D-galactopyranosyl were introduced to
the primary hydroxyl group of sphingoid bases or ceramides
(T1–T9, Fig. 1). It has been previously demonstrated that the
optimal chain length required for antifungal activities of sphin-
goid bases against C. glabrata lies in the C7–C18 range [4]. In
addition, C18-sphinganine (C18-DHS) was reported to have
excellent fungicidal effect towards C. albicans (MFC 5 mg/mL)
[5]. In this respect, C18-DHS was chosen as the positive control
for comparison in our testing and in all of the designed GSLs 18
is the chain length of selected sphingoid bases.
C15H31
NH
O
NH2
OH
C13H27
HO
OH
O
HO
HO
C
13H27
O
OH
C15H31
NH
OH
OH
T5
DHS
C18-sphinganine(C18-
)
HO
O
OH
O
HO
HO
NH2
O
HO
HO
Compounds T1–T4 were synthesized following an optimized
modular synthetic approach developed in our previous work
(Scheme 1) [7]. With TBS protected azido-sphinganine 2 [8],
benzoyl protected azido-sphingosine 3 [9], and benzyl protected
azido-phytosphingosine 4 [10] in hand, perbenzoylated galac-
topyranosyl trichloroacetimidate 1 [7] was employed as glyco-
syl donor to carry out the glycosylations catalyzed by TMSOTf
efficiently and form azido-GSLs 5, 6 [7] and 7, respectively.
Removal of the protecting TBS group on 5 produced compound
8. Staudinger reductions of the azido groups in 8, 6 and 7 led to
amino intermediates 9, 10 and 11, respectively. 9 and 10 were
then de-esterified to furnish desired compounds T1 and T2. In
the meantime, intermediates 9 and 11 were coupled with
hexacosanoic acid in the presence of HOBt, EDC/HCl to provide
12 and 13, which were followed by global deprotection to give
galactosyl ceramides T3 and T4, respectively.
C
13H27
O
C13H27
T6
15H31
O
OH
OH
NH2
OH
T1
OH
C
O
OH
O
HO
HO
OH
O
NH
C13H27
O
HO
C
13H27
O
HO
OH
T2
OH
OH
15H31
T7
C
O
O
C25H51
NH
HO
HO
OH
O
HO
HO
NH
O
C
13H27
O
C13H27
O
HO
OH
OH
OH
T3
T8
C25H51
O
O
C
25H51
OH
O
HO
HO
NH
OH
O
HO
HO
NH
C
13H27
OH
O
O
C
13H27
O
OH
O
OH
OH
T4
1,2-trans D-Galactopyranosyl, D-mannopyranosyl, D-gluco-
pyranosyl, and L-rhamnopyranosyl ceramides T5–T8, which
all carry a sphingoid base of DHS, were synthesized from
T9
H2N
Figure 1. Structures of glycosphingolipids and sphinganine.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

788
N. Ding et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
O
O
Target compounds T1–T9 were evaluated for their in-vitro
antifungal activities against C. albicans (EU583481),
C. parapsilosis (EF198017), and C. tropicalis (EU651853) using
serial fourfold dilutions technique in triplicate [12]. The
minimum inhibitory concentration (MIC) is defined as the
concentration of the compound required to give 80% inhi-
bition of fungal growth, and MICs of the target compounds
along with C18-DHS for comparison are presented in Table 1.
Compared with the aglycone C18-DHS, galactosyl sphinga-
nine T1 and galactosyl sphingosine T2 showed slightly less
activity against C. albicans and C. tropicalis (MIC 43.1 and
43.3 mM) and retained the moderate inhibitory activities
against C. parasilosis (MIC 173 mM). These results were in
accordance with those previously reported by Nicholas
et al. that glycosyl group could reduce the antifungal
activities [4].
Pd/C, H2
NH
NH
4
R O
R O
5
OH
OH
HO
14-17
T5-T8
OH
O
HO
OH
O
HO
HO
HO
HO
HO
HO
O
O
R =
HO
HO
OH
HO
HO
14, T5
15, T6
16, T7
17, T8
Scheme 2. Synthesis of T5–T8.
their corresponding sphingosine containing forms 14–17
respectively, by hydrogenation of the C4-C5 double bond
(Scheme 2). The synthesis of compounds 14–17 has been
reported by us earlier [7].
The synthesis of 20-O-carbamoyl galactosyl ceramide T9 was
performed as shown in Scheme 3. With the known thioga-
lactoside 18 and monobenzyl protected azido-sphinganine 19
[11] in hand, the coupling reaction was performed under the
promotion of NIS/AgOTf to provide the desired glycoside 20.
The 20-benzoate group of 20 was then transformed to a free
OH, on which the carbamoyl group was introduced by reac-
tion with ClCO2C6H4NO2 in the presence of DMAP in pyri-
dine, and followed by an ammonolysis reaction. After the
deprotection of 24, target T9 was provided.
Interestingly, while T1 was acylated by a C24 or C16 fatty
acid at the sphingoid base (T3 or T5), they were completely
inactive as growth inhibitors. However, replacements of the
D-galactosyl residue of inactive compound T5 by several other
glycosyl residues, like D-mannosyl (T6), D-glucosyl (T7) and
L-rhamnosyl (T8) were all shown to get enhanced inhibitory
activities (MIC 5–80 mg/mL). Within them, the best inhibitory
activity was displayed by T7, which was 2-fold more active
Ph
Ph
O
O
O
N3
O
N3
a
O
C
13H27
O
O
C
13H27
STol HO
+
BnO
BnO
OR
OBz
18
OBn
OBn
20 R = Bz
19
b
21
R = H
Ph
Ph
O
O
N3
O
O
C13H27
O
O
N3
c
BnO
d
O
O
C
13H27
O
O
O
OBn
BnO
H2N
OBn
O
O2N
22
23
O
Ph
C25H51
NH
O
C25H51
NH
O
O
O
OH
O
g
e, f
HO
O
C
13H27
O
C
13H27
O
BnO
H2N
HO
H2N
O
OBn
O
OH
24
O
T9
O
Conditions: a. NIS, AgOTf, CH2Cl2, 86%; b. NaOCH3, CH3OH/CH2Cl2, 92%; c. p-nitrophenyl chloroformate,
DMAP, pyridine; d. NH3, THF, 82% over 2 steps; e. PPh3, THF/H2O; f. hexacosanoyl acid, HOBt, EDC/HCl,
DIPEA, 64% over 2 steps; g. Pd/C-H2, MeOH/EtOAc, 64%.
Scheme 3. Synthesis of T9.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
Evaluation of Antifungal Activities of Novel 1,2-trans Glycosphingolipids
789
Table 1. In-vitro antifungal activities (MIC).
C. albicans
C. parasilosis
C. tropicalis
mg/mL
mM
mg/mL
mM
mg/mL
mM
C18-DHS
T1
T2
T3
T4
T5
T6
T7
T8
5
20
20
>320
20
>320
80
80
20
5
16.5
43.1
43.3
>393
24.1
>456
114
114
29.1
5.8
80
80
80
>320
>320
>320
80
20
80
265
173
173
>393
>385
>456
114
28.5
117
5
20
20
>320
>320
>320
80
5
80
16.5
43.1
43.3
>393
>385
>456
114
7.1
117
5.8
T9
20
23.3
5
than native C18-DHS against C. tropicalis (MIC 7.1 vs. 16.5 mM). Conclusion
The different fungistatic activities between T5 and T6–T8
implied that the permeability might not be the sole
parameter affecting their antifungal properties as presumed
before [4a]. Additionally, some of the GSLs also showed good
selectivities against the selected microbial panel. For
example, glucosyl ceramide T7 was 4-fold more effective
against C. parasilosis than C. albicans (MIC 20 vs. 80 mg/mL),
and 16-fold more effective against C. tropicalis (MIC 5 mg/mL).
Rhamnosyl ceramide T8 showed 4-fold more inhibition
against C. albicans than the other two strains (MIC 20 vs.
80 mg/mL). These results clearly showed the sort of sugar
moiety of GSLs played an important role on their antifungal
activities.
In conclusion, we have conducted the synthesis and in-vitro
biological evaluation of antifungal activities of a series of 1,2-
trans glycosphingolipids against C. albicans, C. parapsilosis, and
C. tropicalis. The preliminary study indicated that the sort of
sugar moiety of the tested GSLs played an important role in
their antifungal activities, and the permeability might not be
the sole parameter affecting their antifungal properties as
presumed before. Additionally, a carbamoyl group modified
GSL (T9) displayed strong antifungal activities against all the
three tested organisms, which would be a potent lead com-
pound to be further optimized. All the results obtained here
would bring new clues to understand the antifungal profile
for these types of compounds.
Another interesting result was observed that when a car-
bamoyl group was introduced to the sugar part of inactive
compound T3, compound T9 displayed strong antifungal
activities against all the three testing organisms with MIC
values of 5.8 mM for C. albicans, 23.3 mM for C. parasilosis, and
5.8 mM for C. tropicalis. Thus, T9 was a potent lead compound
to be further optimized as antifungal agent and the study is
currently on the way. The detailed mechanism and its further
application as fungal growth inhibitors are under
investigation.
Experimental
General
All commercial reagents and solvents were used as received
without further purification unless specified. Reaction solvents
were distilled from CaH2 for dichloromethane and from sodium
metal and benzophenone for tetrahydrofuran. Flash column
chromatography was performed on silica gel (200–300 mesh,
Qingdao, China). 1H-NMR and 13C-NMR spectra were taken on
a JEOL JNM-ECP 600 spectrometer (Jeol, Japan) with tetramethyl-
silane (TMS) as an internal standard at room temperature, except
for the 13C-NMR spectra of compounds T5–T8, which were taken
on a Bruker Avance 400 MHz spectrometer at 608C. Mass spectra
were obtained on a Waters Q-TOF micro mass spectrometer
(Waters).
The investigation also showed that the subtle structure of
sphingoid bases of GSLs affected the antifungal activities. For
example, T4 showed commendable fungistatic activity as com-
pared to T3 against C. albicans (MIC 24.1 vs. >393 mM), indicat-
ing that an additional hydroxyl group at position 4 of the
sphingoid base can increase the antifungal activity, which were
consistent with those reported data [5] that PHS is more effec-
tive than DHS on their antifungal activities in some cases.
Additionally, it has long been known that amino groups could
benefit the antibacterial activity in many antibiotics [13]. In our
study, we observed that GSLs with a primary amine (T1 and T2)
both had better antifungal activities (MIC 20–80 mg/mL) com-
pared to those without free amine in general.
General procedure for the synthesis of glycosides 5, 6,
and 7
To a stirred solution of perbenzoylated glycosyl trichloroaceti-
midate donor 1 (1.3 mmol) and acceptor 2, 3, or 4 (1.0 mmol) in
˚
20 mL of dry CH2Cl2 was added powdered 4-A molecular sieves
(0.2 g). The mixture was stirred for 30 min at ꢀ308C and then
TMSOTf (0.2 mmol) was added to the stirred mixture. The reac-
tion mixture was stirred at ꢀ308C for additional 30 min and
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

790
N. Ding et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
then warmed to room temperature and stirred until TLC
revealed full conversion of acceptor. Et3N was added to quench
the reaction. The solid was then filtered off, and the filtrate was
concentrated under vacuum. The syrupy residue was purified by
silica gel flash chromatography, eluted with EtOAc and hexane
to give glycosides 5 (92%), 6 (92%), or 7 (87%) as a colorless oil.
tant1H-NMR (600 MHz, CDCl3) dH 8.10–7.23 (20H, Ar-H), 6.00 (d,
1H, J ¼ 3.3 Hz, C40-H), 5.81 (dd, 1H, J ¼ 10.4, 7.7 Hz, C20-H), 5.63
(dd, 1H, J ¼ 10.4, 3.3 Hz, C30-H), 4.92 (d, 1H, J ¼ 7.7 Hz, C10-H),
4.69 (dd, 1H, J ¼ 11.0, 6.6 Hz, H-60a), 4.45 (dd, 1H, J ¼ 11.0,
6.1 Hz, C60-Ha), 4.35 (t, 1H, J ¼ 6.6 Hz, C50-H), 4.22 (dd, 1H,
J ¼ 10.4, 5.5 Hz, C1-Ha), 3.90 (dd, 1H, J ¼ 10.4, 3.8 Hz, C2-H),
3.69 (s-like, 1H, C1-Hb), 3.44 (dd, 1H, J ¼ 9.9, 6.0 Hz, C3-H),
1.62 (s-like, 2H, C4-H), 1.46–1.37, 1.26 (m), 0.88 (t, 3H,
J ¼ 7.1 Hz, CH3); 13C-NMR (150 MHz, CDCl3) dC 166.1, 165.6,
165.5, 165.4, 133.7, 133.4, 130.1, 129.9, 129.4, 129.3, 128.8,
128.6, 128.5, 128.4, 101.5, 71.7, 71.6, 71.1, 69.7, 68.6, 68.1,
65.1, 62.1, 32.2, 32.0, 29.8–29.5, 25.7, 22.8, 14.2; HRMS-ESI: m/z
calcd. for C52H63N3NaO11 [MþNa]þ: 928.4360; Found 928.4355.
(2S,3R)-20,30,40,60-Tetra-O-benzoyl-ꢀ-D-
galactopyranosyl-(10!1)-2-azido-3-O-(tert-
butyldimethylsilyl)-octadecan-1,3-diol (5)
1H-NMR (600 MHz, CDCl3) dH 8.09–7.20 (20H, Ar-H), 6.00 (dd, 1H,
J ¼ 3.6, 1.2 Hz, C40-H), 5.82 (dd, 1H, J ¼ 10.2, 7.8 Hz, C20-H), 5.61 (dd,
1H, J ¼ 10.2, 3.6 Hz, C30-H), 4.87 (d, 1H, J ¼ 7.8 Hz, C10-H), 4.67 (dd,
1H, J ¼ 11.4, 7.2 Hz, C60-Ha), 4.43 (dd, 1H, J ¼ 11.4, 5.6 Hz, C60-Hb),
4.34 (dd, 1H, J ¼ 6.6, 1.2 Hz, C50-H), 4.07 (dd, 1H, J ¼ 10.2, 6.0 Hz,
C1-Ha), 3.78 (dd, 1H, J ¼ 9.0, 4.8 Hz, C2-H), 3.68 (dd, 1H, J ¼ 10.2,
4.8 Hz, C1-Hb), 3.57 (dd, 1H, J ¼ 9.0, 5.4 Hz, C3-H), 1.57 (s-like, 2H,
C4-H), 1.31–1.19 (m), 0.88–0.84 (m); 13C-NMR (150 MHz, CDCl3) dC
166.2, 165.8, 165.7, 165.3, 133.8, 133.5, 130.2, 130.0, 128.8, 128.7,
128.6, 128.5, 128.4, 101.6, 71.9, 71.7, 71.6, 69.8, 68.9, 68.2, 64.7, 62.1,
32.2, 32.1, 29.9–29.6, 26.0, 22.9, 14.3; HRMS-ESI: m/z calcd.
for C58H77N3NaO11Si [MþNa]þ: 1042.5220; Found 1042.5237.
General procedure for the synthesis of amino intermediates
9, 10, 11
To a solution of compound 8, 6 or 7 (0.08 mmol) in THF (10 mL)
and water (1.0 mL) co-solvent system was added PPh3
(0.20 mmol). The reaction mixture was heated at 508C for
12 h until TLC indicated the complete transformation of the
starting azide into corresponding amine and then concentrated.
The amine residue was used directly to the next step.
(2S,3R,4E)-20,30,40,60-Tetra-O-benzoyl-ꢀ-D-
galactopyranosyl-(10!1)-2-azido-3-benzoyl-4-
General procedure for the synthesis of glycosyl ceramides
12, 13
octadecene-1,3-diol (6)
To a solution of the above crude amino intermediate 9 or 11 and
hexacosanoic acid (0.10 mmol) in dried CH2Cl2 (10 mL) were
added HOBt (0.10 mmol), EDC ꢂ HCl (0.10 mmol) and DIPEA
(0.12 mmol) at 08C under Ar. The reaction mixture was stirred
at room temperature for 22 h, and then concentrated. The res-
idue was partitioned between CH2Cl2/water. The organic layer
was separated, washed with brine, dried over MgSO4 and con-
centrated. The residue was purified by column chromatography
on silica gel eluted with CH3Cl3 and MeOH (v/v ¼ 20:1) to furnish
12 (88% over 2 steps) or 13 (73% over 2 steps) as a colorless oil.
The physical data are identical with those provided in the liter-
ature: Liu, Y. P.; Ding, N.; Xiao, H. L.; Li, Y. X. J. Carbohydr. Chem.
2006, 25, 471–489.
(2S,3S,4R)-20,30,40,60-Tetra-O-benzoyl-ꢀ-D-
galactopyranosyl-(10!1)-2-azido-3,4-di-O-benzyl-
octadecan-1,3,4-triol (7)
1H-NMR(600 MHz, CDCl3) dH 8.07–7.20 (30H, Ar-H), 5.97 (dd, 1H,
J ¼ 3.6, 1.2 Hz, C40-H), 5.82 (dd, 1H, J ¼ 10.8, 7.8 Hz, C20-H), 5.57
(dd, 1H, J ¼ 10.8, 3.6 Hz, C30-H), 4.79 (d, 1H, J ¼ 7.8 Hz, C10-H),
4.72–4.57 (5H, 2 PhCH2O, C50-H0), 4.40 (dd, 1H, J ¼ 11.4, 7.2 Hz,
C60-Ha), 4.24 (dd, 1H, J ¼ 11.8, 6.6 Hz, C1-Ha), 4.16 (dd, 1H,
J ¼ 11.8, 4.2 Hz, C60-Hb), 3.91 (dd, 1H, J ¼ 10.2, 3.6 Hz, C1-Hb),
3.71–3.69 (m, 1H, C2-H), 3.68–3.61 (m, 2H, C3-H, C4-H), 1.57 (m,
1H, C5-Ha), 1.42 (m, 1H, C5-Hb), 1.31–1.16 (m), 0.88 (t, 3H,
J ¼ 6.6 Hz, CH3); 13C-NMR (150 MHz, CDCl3) dC 166.2, 165.7,
165.6, 165.4, 138.6, 138.2, 133.7, 133.5–133.4, 130.1, 130.0,
129.9, 129.6, 129.2–127.9, 101.4, 79.2, 79.0, 74.1, 72.0, 71.8,
71.4, 69.9, 69.4, 68.2, 63.4, 63.2, 62.4, 62.0, 61.7, 32.1, 30.1–
29.6, 25.4, 22.9, 14.3; HRMS-ESI: m/z calcd. for C66H75N3NaO12
[MþNa]þ: 1124.5248; Found 1124.5227.
(2S,3R)-20,30,40,60-Tetra-O-benzoyl-ꢀ-D-
galactopyranosyl-(10!1)-2-docosanoylamino-
octadecan-1,3-diol (12)
1H-NMR (600 MHz, CDCl3) dH 8.10–7.23 (20H, Ar-H), 6.00 (d, 1H,
J ¼ 3.3 Hz, C40-H), 5.72 (dd, 1H, J ¼ 9.9, 7.7 Hz, C20-H), 5.67 (dd,
1H, J ¼ 9.9, 3.3 Hz, C30-H), 4.71 (d, 1H, J ¼ 7.7 Hz, C10-H), 4.66 (dd,
1H, J ¼ 11.6, 7.1 Hz, C60-Ha), 4.44 (dd, 1H, J ¼ 11.6, 6.6 Hz, C60-
Hb), 4.32 (t, 1H, J ¼ 6.6 Hz, C50-H), 4.30 (dd, 1H, J ¼ 10.4, 2.8 Hz,
C1-Ha), 3.92 (s-like, 1H, C2-H), 3.72–3.71 (m, 1H, C1-Hb), 3.57
(s-like, C3-H), 1.74–1.68 (m, 2H), 1.61–1.25 (m), 0.88 (t, 6H,
J ¼ 6.6 Hz, 2 CH3); HRMS-ESI: m/z calcd. for C78H115NNaO12
[MþNa]þ: 1280.8317; Found 1280.8310.
Preparation of (2S,3R)-20,30,40,60-tetra-O-benzoyl-ꢀ-D-
(2S,3S,4R)-20,30,40,60-Tetra-O-benzoyl-ꢀ-D-
galactopyranosyl-(10!1)-2-azido- octadecan-1,3-diol (8)
To a solution of 5 (400 mg, 0.4 mmol) in dry THF (25 mL), a
solution of TBAF (209 mg, 0.8 mmol) was added at 08C. After
20 min, the solution was allowed to warm to room temperature
and stirred for 90 min. The reaction mixture was treated with
water (50 mL) and extracted with EtOAc (3 ꢁ 100 mL). The
organic phases were combined, dried, and concentrated under
reduced pressure. The crude product was purified by silica gel
flash chromatography eluted with petroleum ether and EtOAc (v/
v ¼ 15:1) to give compound 8 as a colorless oil (323.3 mg, 91.4%).
galactopyranosyl-(10!1)-2-docosanoylamino-3,4-di-O-
benzyl-octadecan-1,3,4-triol (13)
1H-NMR (600 MHz, CDCl3) dH 8.03–7.24 (30H, Ar-H), 6.00 (dd, 1H,
J ¼ 3.3 Hz, C40-H), 5.79 (dd, 1H, J ¼ 10.4, 7.7 Hz, C20-H), 5.66 (dd,
1H, J ¼ 10.4, 3.3 Hz, C30-H), 5.51 (d, 1H, J ¼ 8.8 Hz, NH), 4.86 (d,
1H, J ¼ 11.0 Hz, PhCHHO), 4.81 (d, 1H, J ¼ 7.7 Hz, C10-H), 4.72 (d,
1H, J ¼ 11.0 Hz, PhCHHO), 4.63 (d, 1H, J ¼ 11.5 Hz, PhCHHO),
4.59 (dd, 1H, J ¼ 11.0, 6.6 Hz, H-60a), 4.48 (d, 1H, J ¼ 12.1 Hz,
PhCHHO), 4.44 (dd, 1H, J ¼ 9.9, 3.3 Hz, C1-Ha), 4.41 (dd, 1H,
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
Evaluation of Antifungal Activities of Novel 1,2-trans Glycosphingolipids
791
J ¼ 11.0, 6.6 Hz, C60-Hb), 4.28 (t, 1H, J ¼ 7.1 Hz, C50-H), 4.18 (m,
1H, H-2), 3.85 (dd, 1H, J ¼ 8.8, 2.2 Hz, C3-H), 3.61 (dd, 1H, J ¼ 9.3,
3.3 Hz, C1-Hb), 3.38 (d-like, 1H, J ¼ 8.8, 2.8 Hz, C4-H), 1.69–1.64
(m, 2H), 1.60 (s, 2H), 1.54 (t, 1H, J ¼ 7.1 Hz), 1.31–1.13 (m), 0.88 (t,
6H, J ¼ 6.8 Hz, 2 CH3); 13C-NMR (150 MHz, CDCl3) dC 172.5, 166.2,
165.7, 165.6, 138.9, 133.9, 133.8, 133.5, 130.1–127.8, 101.6, 80.1,
78.4, 74.4, 71.6, 71.5, 71.4, 70.7, 69.0, 68.2, 62.1, 49.4, 36.5, 32.1,
29.9–29.4, 26.3, 25.6, 22.9, 14.3; HRMS-ESI: m/z calcd.
for C92H127NNaO13 [MþNa]þ: 1476.9205; Found 1476.9200.
was 9. After stirring at room temperature for 12 h, the solution
was neutralized with ion-exchange resin (Hþ) and then filtered.
To the filtrate was added silica gel (100 mg), and the solvent was
removed under vacuum. The resulting silica powder was loaded
into a column and purified to form a syrup. To a solution of the
above syrup in MeOH (3 mL) and EtOAc (3 mL) was added Pd/C
(10% wt% Pd dry basis on carbon), and the mixture was hydro-
genated at room temperature for 3 h. The suspension was fil-
tered through Celite pad. The filter cake was rinsed with CHCl3/
MeOH (v/v ¼ 5:1, 30 mL). The combined filtrate and washings
were concentrated in vacuo. The residue was purified by silica gel
flash chromatography eluted with CHCl3 and MeOH (v/v ¼ 10:1)
to give the product T4 (25.9 mg, 76%) as a white powder. 1H-NMR
(600 MHz, pyridine-d5) dH 8.60 (d, 1H, J ¼ 8.7 Hz, NH), 5.18 (bs,
1H, C2-H), 4.91 (d, 1H, J ¼ 7.8 Hz, C10-H), 4.85 (dd, 1H, J ¼ 10.5,
5.5 Hz, C1-Hb), 4.57 (d, 1H, J ¼ 3.2 Hz, C40-H), 4.53–4.50 (m, 1H,
C20-H), 4.47 (dd, 1H, J ¼ 10.5, 4.1 Hz, C1-Ha), 4.45–4.40 (m, 3H, C3-
H, C60-Ha,b), 4.26–4.25 (m, 1H, C4-H), 4.17 (dd, 1H, J ¼ 9.7, 3.7 Hz,
C30-H), 4.06 (t, 1H, J ¼ 5.9 Hz, C50-H), 2.46 (t, 2H, J ¼ 7.7 Hz,
COCH2), 2.20 (s, 1H), 1.97–1.25 (m, 2H, CH2), 0.90–0.88 (m, 6H,
2 CH3); 13C-NMR (150 MHz, pyridine-d5) dC 106.4, 101.1, 77.3, 76.1,
75.6, 73.1, 70.8, 70.4, 62.6, 52.4, 37.3, 33.8, 32.4, 30.8–29.8, 27.0,
26.7, 23.2, 14.5; HRMS-ESI: m/z calcd. for C50H99NNaO9 [MþNa]þ:
880.7218; Found 880.7212.
General procedure for the removal of benzoyl protecting
groups (Preparation of T1–T4)
Compound 9, 10 or 12 was dissolved in MeOH-CH2Cl2 (v/v ¼ 2:1,
6 mL), and then NaOMe in MeOH was added until pH ¼ 9. After
stirring at room temperature for 12 h, the solution was neutral-
ized with ion-exchange resin (Hþ) and then filtered. To the
filtrate was added silica gel (100 mg), and the solvent was
removed under vacuum. The resulting silica powder was loaded
into a silica gel column and purified to form compound T1 (87%
over 2 steps), T2 (73% over 2 steps) or T3 (92%) as a white powder.
(2S,3R)-ꢀ-D-Galactopyranosyl-(10!1)-2-amino-
octadecan-1,3-diol (T1)
1H-NMR (600 MHz, methanol-d4) dH 4.18 (d, 1H, J ¼ 7.3 Hz, C10-H),
3.78–3.76 (m, 3H, C2-H, C1-Ha, C40-H), 3.72 (dd, 1H, J ¼ 11.5,
7.4 Hz, C60-Ha), 3.67 (dd, 1H, J ¼ 11.5, 5.0 Hz, C60-Hb), 3.49–3.48
(m, 1H, C20-H), 3.46–3.45 (m, 2H, C1-Hb, C50-H), 3.42 (dd, 1H,
J ¼ 9.6,3.2 Hz, C30-H), 1.96 (s-like, 1H, C3-H), 1.43–1.41 (m, 2H,
C4-H), 1.31–1.22 (m, CH2), 0.86 (t, 3H, J ¼ 6.9 Hz, CH3); 13C-NMR
(150 MHz, methanol-d4) dC 104.9, 78.1, 76.9, 75.0, 72.6, 70.9, 70.4,
62.7, 49.9, 37.5, 36.1, 34.1, 33.2, 30.9, 30.6, 28.8, 23.8, 14.5; HRMS-
ESI: m/z calcd. for C24H50NO7 [MþH]þ: 464.3587; Found 464.3582.
General procedure for the hydrogenation of the compounds
14–17 (preparation of T5–T8)
To a solution of compound 14, or 15–17 (0.1 mmol) in MeOH (3 mL)
and EtOAc (3 mL) was added Pd/C (100 mg, 10% wt% Pd dry basis on
carbon), and the mixture was hydrogenated at room temperature
for 3 h. The suspension was filtered through Celite pad. The filter
cake was rinsed with CHCl3/MeOH (v/v ¼ 5:1, 30 mL). The com-
bined filtrate and washings were concentrated in vacuo. The residue
was purified by silica gel flash chromatography (CHCl3/MeOH,
v/v ¼ 10:1) to give the product T5, or T6–T8 as a colorless oil.
(2S,3R,4E)-ꢀ-D-Galactopyranosyl-(10!1)-2-amino-4-
octadecene-1,3-diol (T2)
1H-NMR (600 MHz, DMSO-d6) dH 5.54 (dt, 1H, J ¼ 15.2, 7.0 Hz, C5-
H), 5.36 (dd, 1H, J ¼ 15.2, 7.4 Hz, C4-H), 4.03 (d, 1H, J ¼ 7.8 Hz, C10-
H), 3.94–3.90 (m, 2H, C1-Ha, H-3), 3.78–3.76 (m, 1H, H-2), 3.63 (d,
1H, J ¼ 3.2 Hz, C40-H), 3.53 (dd, 1H, J ¼ 11.0, 6.2 Hz, C60-Ha), 3.47
(dd, 1H, J ¼ 11.0, 5.8 Hz, C60-Hb), 3.42 (dd, 1H, J ¼ 11.0, 4.0 Hz, C1-
(2S,3R)-ꢀ-D-Galactopyranosyl (10!1)-2-
(hexadecanoylamido)-octadecane-1,3-diol (T5)
1H-NMR (600 MHz, DMSO-d6): dH 7.60 (d, 1H, J ¼ 9.2 Hz, NH), 4.86
(d, 1H, J ¼ 3.6 Hz, 20-OH), 4.72 (d, 1H, J ¼ 5.5 Hz, 40-OH), 4.57 (t, 1H,
J ¼ 5.5 Hz, 60-OH), 4.56 (d, 1H, J ¼ 6.4 Hz, 3-OH), 4.36 (d, 1H,
J ¼ 4.1 Hz, 30-OH), 4.03 (d, 1H, J ¼ 7.4 Hz, C10-H), 3.96 (dd, 1H,
J ¼ 10.1, 4.6 Hz, C1-Ha), 3.75–3.71 (m, 1H, C2-H), 3.62–3.61 (m, 1H,
C40-H), 3.54–3.50 (m, 1H, C60-Ha), 3.48–3.44 (m, 1H, C60-Hb), 3.42–
3.40 (m, 2H, C1-Hb, C3-H), 3.32–3.25 (m, 3H, C50-H, C20-H, C30-H),
2.11–2.01 (m, 2H, CH2), 1.54–1.41 (m, 4 H, 2 CH2), 1.29–1.18 (m, 50
H, 25 CH2), 0.86–0.84 (m, 6 H, 2 CH3); 13C-NMR (100 MHz, 608C,
DMSO-d6) dC 171.9, 104.2, 75.1, 73.1, 70.6, 69.5, 69.1, 68.0, 60.4,
53.1, 35.5, 33.4, 31.0, 29.0–28.4, 25.2, 24.8, 21.8, 13.6; HRMS-ESI:
m/z calcd. for C40H79NNaO8 [MþNa]þ: 724.5703; Found 724.5698.
Hb), 3.33–3.29 (m, 3H, C20-H, C50-H, C30-H), 1.94–1.92 (m, 2H, CH2
–
–
CH–CH2), 1.45–1.21 (m, CH2), 0.88 (t, 3H, J ¼ 6.8 Hz, CH3); HRMS-
ESI: m/z calcd. for C24H48NO7 [MþH]þ: 462.3431; Found 462.3425.
(2S,3R)-ꢀ-D-Galactopyranosyl-(10!1)-2-
hexacosanoylamino-octadecan-1,3-diol (T3)
1H-NMR (600 MHz, pyridine-d5) d 8.52 (d, 1H, J ¼ 9.1 Hz, NH), 4.88
(d, 1H, J ¼ 7.7 Hz, C10-H), 4.79 (m, 1H, C2-H), 4.60–4.57 (m, 2H,
C40-H, C1-Ha), 4.55–4.52 (m, 1H, C20-H), 4.50–4.44 (m, 2H, C50-H,
C60-Ha), 4.19 (dd, 1H, J ¼ 9.2, 3.0 Hz, C30-H), 4.11–4.09 (m, 1H, C60-
Hb), 4.07 (dd, 1H, J ¼ 11.0, 4.1 Hz, C1-Hb), 3.69–3.66 (m, 1H, C3-
H), 3.45 (s, 3H, OCH3), 2.48 (t, 2H, J ¼ 6.4 Hz, COCH2), 1.91–1.27
(2S,3R)-ꢀ-D-Mannopyranosyl (10!1)-2-
(hexadecanoylamido)-octadecane-1,3-diol (T6)
(CH2), 0.91–0.88 (m, 6H,
2 CH3); HRMS-ESI: m/z calcd.
1H-NMR (600 MHz, DMSO-d6): dH 7.56 (d, 1H, J ¼ 9.2 Hz, NH), 4.67
(d, 1H, J ¼ 5.0 Hz, 20-OH), 4.65 (d, 1H, J ¼ 4.1 Hz, 30-OH), 4.57 (1H,
d, J ¼ 1.0 Hz, C10-H), 4.56 (d, 1H, J ¼ 6.4 Hz, 3-OH), 4.51 (d, 1H,
J ¼ 6.4 Hz, 40-OH), 4.38 (t, 1H, J ¼ 5.9 Hz, 60-OH), 3.75–3.71 (m,
1H, C2-H), 3.69–3.67 (m, 1H, C1-Ha), 3.62–3.59 (m, 1H, C60-Ha),
3.58–3.56 (m, 1H, C30-H), 3.49–3.43 (m, 2H, C40-H, C60-Hb), 3.41–
3.36 (m, 3H, H-1b, C3-H, C20-H), 3.29–3.26 (m, 1H, C50-H), 2.10–2.00
for C50H99NNaO8 [MþH]þ: 864.7263; Found 864.7257.
(2S,3S,4R)-b-D-Galactopyranosyl-(10!1)-2-
hexacosanoylamino-octadecan-1,3,4-triol (T4)
Ceramide 13 (60 mg, 0.041 mmol) was dissolved in MeOH/CH2Cl2
(v/v ¼ 2:1, 6 mL), and then NaOMe in MeOH was added until pH
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

792
N. Ding et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
Preparation of (2S,3R)-30-O-benzyl-40,60-O-benzylidene-
(m, 2H, CH2), 1.44–1.40 (m, 2H, CH2), 1.28–1.18 (m, 52H, 26 CH2),
0.86–0.84 (m, 3H, 2 CH3); 13C-NMR (100 MHz, 608C, DMSO-d6) dC
171.8, 100.5, 73.5, 70.8, 70.2, 69.9, 67.1, 66.5, 61.2, 53.3, 35.4,
33.3, 31.0, 29.0–28.4, 25.2, 24.9, 21.8, 13.6; HRMS-ESI: m/z calcd.
for C40H79NNaO8 [MþNa]þ: 724.5703; Found 724.5698.
ꢀ-D-galactopyranosyl)-(10!1)-2-azido-3-O-benzyl-
octadecan-1,3-diol (21)
Compound 20 (500 mg, 0.6 mmol) was dissolved in 10 mL dry
MeOH and 10 mL dry CH2Cl2. NaOMe (99%, 180 mg) was added to
the mixture and stirred for 20 h. After almost all starting
material was consumed, Amberlyst 15 (Hþ) was added to neutral-
ize the NaOMe, and the mixture was then diluted with MeOH,
and the exchange resin was filtered off. The resin was washed
thoroughly and the filtrate was concentrated. The residue was
purified by silica gel flash chromatography eluted with
petroleum ether and EtOAc (v/v ¼ 7:1) to give the desired prod-
uct 21 (404.4 mg, 92.0%) as a foamy solid.
(2S,3R)-ꢀ-D-Glucopyranosyl (10!1)-2-
(hexadecanoylamido)-octadecane-1,3-diol (T7)
1H-NMR (600 MHz, DMSO-d6): dH 7.59 (d, 1H, J ¼ 9.2 Hz, NH), 4.99
(d, 1H, J ¼ 3.7 Hz, 20-OH), 4.94 (d, 1H, J ¼ 5.0 Hz, 30-OH), 4.91 (d,
1H, J ¼ 5.5 Hz, 40-OH), 4.56 (d, 1H, J ¼ 6.4 Hz, 3-OH), 4.51 (t, 1H,
J ¼ 5.9 Hz, 60-OH), 4.09 (d, 1H, J ¼ 7.8 Hz, C10-H), 3.94 (dd, 1H,
J ¼ 10.1, 5.0 Hz, C1-Ha), 3.75–3.71 (m, 1H, C2-H), 3.68–3.65 (m,
1H, C6-Ha,), 3.90–3.46 (m, 3H, C3-H, C1-Hb, C60-Hb), 3.15–3.11 (m,
1H, C30-H), 3.10–3.07 (m, 1H, C50-H), 3.04–3.00 (m, 1H, C40-H),
2.98–2.94 (m, 1H, C20-H), 2.11–2.01 (m, 2H, CH2), 1.54–1.50 (m,
2H, CH2), 1.45–1.41 (m, 2H, CH2), 1.28–1.20 (m, 50H, 25 CH2),
0.86–0.84 (m, 6H, 2 CH3); 13C-NMR (100 MHz, 608C, DMSO-d6) dC
172.6, 104.2, 77.3, 77.0, 74.0, 70.8, 70.2, 69.8, 61.8, 53.7, 36.1,
34.1, 31.7, 29.6–29.1, 25.8, 25.4, 22.4, 14.2; HRMS-ESI: m/z calcd.
for C40H79NNaO8 [MþNa]þ: 724.5703; Found 724.5698.
1H-NMR (600 MHz, CDCl3) dH 7.52–7.23 (15H, Ar-H), 5.46 (s, 1H,
PhCH), 4.77 (m, 2H, PhCH2O), 4.66 (d, 1H, J ¼ 11.5 Hz, PhCHH),
4.54 (d, 1H, J ¼ 11.5 Hz, PhCHH), 4.37 (d, 1H, J ¼ 7.8 Hz, C10-H),
4.29 (d, 1H, J ¼ 12.1 Hz, C60-Ha), 4.14–4.11 (m, 2H, C40-H0, C1-Ha),
4.04–4.00 (m, 2H, C20-H, C60-Hb), 3.79 (dd, 1H, J ¼ 10.4, 3.8 Hz, C1-
Hb), 3.72 (m, 1H, C2-H), 3.61 (m, 1H, C3-H), 3.50 (dd, 1H, J ¼ 9.4,
3.3 Hz, C30-H), 3.36 (s-like, 1H, C50-H), 1.62–1.53 (m, 2H, H-4), 1.45–
1.44 (m, 2H, H-5), 1.26 (s-like, CH2), 0.88 (t, 3H, J ¼ 7.1 Hz, CH3);
13C-NMR (150 MHz, CDCl3) dC 138.4, 138.3, 138.0, 129.1, 128.6,
128.5, 128.3, 128.2, 128.0, 127.8, 126.6, 103.3, 101.4, 79.2, 78.3,
73.4, 72.7, 71.8, 70.2, 69.3, 68.9, 67.0, 63.5, 32.1, 31.1, 29.9–29.5,
25.3, 22.9, 14.3; HRMS-ESI: m/z calcd. for C45H63N3NaO7 [MþNa]þ:
780.4564; Found 780.4558.
(2S,3R)-ꢁ-D-Rhamnoyranosyl (10!1)-2-
(hexadecanoylamido)-octadecane-1,3-diol (T8)
1H-NMR (600 MHz, DMSO-d6): dH 7.35 (d, 1H, J ¼ 8.7 Hz, NH), 4.51
(brs, 1H, C10-H), 4.47 (d, 1H, J ¼ 4.6 Hz, 40-OH), 4.43 (d, 1H,
J ¼ 4.1 Hz, 20-OH), 4.33 (d, 1H, J ¼ 5.9 Hz, 3-OH), 4.19 (d, 1H,
J ¼ 5.9 Hz, 30-OH), 3.79–3.77 (m, 1H, C2-H), 3.60–3.57 (m, 2H,
C20-H, C1-Ha), 3.48–3.38 (m, 4H, C1-Ha, C20-H, C30-H, C50-H), 3.17–
3.15 (m, 1H, C40-H), 2.12–2.02 (m, 1H, CH2), 1.51–1.46 (m, 2H, CH2),
1.42–1.39 (m, 2 H, CH2), 1.29–1.19 (m, 50 H, 25 CH2), 1.13 (d, 3H,
J ¼ 6.0 Hz, C60-H), 0.87–0.84 (m, 6H, 2 CH3); 13C-NMR (100 MHz,
608C, DMSO-d6) dC 171.7, 99.5, 72.0, 70.7, 70.3, 70.1, 68.1, 66.0, 52.5,
35.4, 33.5, 31.0, 29.0–28.4, 25.2, 24.8, 21.8, 17.6, 13.6; HRMS-ESI: m/z
calcd. for C40H79NNaO7 [MþNa]þ: 724.5754; Found 708.5749.
Preparation of (2S,3R)-3-O-benzyl-4,6-O-benzylidene-2-
O-carbamoyl-ꢀ-D-galactopyranosyl-(10!1)-2-azido-3-O-
benzyl-octadecan-1,3-diol (23)
A solution of compound 21 (76 mg, 0.10 mmol) in Py (10 mL) in
the presence of DMAP (43 mg, 0.30 mmol) was treated with p-
nitrophenyl chloroformate (121.3 mg, 0.60 mmol). The reac-
tion mixture was heated at 408C for 2 h and concentrated.
To a solution of the crude product in dried THF (10 mL) was
added NH3 at 08C. The reaction mixture was stirred for 6 h, and
then concentrated. The residue was partitioned between
CH2Cl2/water. The organic layer was separated, washed with
brine, dried over MgSO4 and concentrated. The residue was
purified by column chromatography on silica gel eluted with
CHCl3 and MeOH (v/v ¼ 40:1) to furnish 23 (65.9 mg, 82.1%) as a
Preparation of (2S,3R)-20-O-benzoyl-30-O-benzyl-40,60-O-
benzylidene-ꢀ-D-galactopyranosyl)-(10!1)-2-azido-3-O-
benzyl-octadecan-1,3-diol (20)
To a mixture of glycosyl donor 18 (800 mg, 1.4 mmol), acceptor 19
˚
(0.5 g, 1.2 mmol) and 4-A MS (200 mg) in CH2Cl2 (30 mL) were
1
white solid. H-NMR (600 MHz, CDCl3) dH 7.52–7.23 (15H, ArH),
5.48 (s, 1H, PhCH), 5.22 (dd, 1H, J ¼ 10.4, 8.3 Hz, C20-H), 4.72–
4.66 (m, 3H, PhCH2), 4.53–4.51 (m, 2H, C10-H, PhCHH), 4.29 (dd,
1H, J ¼ 12.6, 1.6 Hz, C60-Ha), 4.17 (d, 1H, J ¼ 3.3 Hz, C40-H), 4.09
(dd, 1H, J ¼ 10.4, 1.3 Hz, C30-H), 4.02 (dd, 1H, J ¼ 12.1, 1.7 Hz,
C60-Hb), 3.73 (dd, 1H, J ¼ 11.0, 4.4 Hz, C1-Ha), 3.66 (m, 1H, C2-H),
3.61 (m, 2H, H-1b, C3-H), 3.37 (s-like, 1H, C50-H), 1.58 (m, 2H, C4-
H), 1.45–1.44 (m, 2H, C5-H), 1.26 (s-like, CH2), 0.88 (t, 3H,
J ¼ 7.1 Hz, CH3); 13C-NMR (150 MHz, CDCl3) dC 155.6, 138.3,
129.1–126.6, 101.4, 101.1, 78.3, 73.6, 72.9, 71.4, 71.3, 69.2,
67.7, 66.9, 63.4, 32.1, 31.0, 29.9, 29.6, 25.4, 22.9, 14.3; HRMS-
ESI: m/z calcd. for C46H65N4O8 [MþH]þ: 801.4802; Found
801.4797.
added NIS (316.4 mg, 1.4 mmol) and AgOTf (30.8 mg, 0.1 mmol)
at ꢀ208C under Ar. The reaction mixture was stirred for 60 min at
ꢀ208C and then warmed to 08C. After stirring for 30 min at 08C
the reaction mixture was concentrated. The residue was diluted
with CH2Cl2 (50 mL) and washed with 10% Na2S2O3 (50 mL) and
brine (50 mL), dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by silica gel flash chromatog-
raphy eluted with petroleum ether and EtOAc (v/v ¼ 7:1) to give
the desired product 20 as a colorless oil (840 mg, 86.1%).
1H-NMR (600 MHz, CDCl3) dH 8.03–7.20 (20H, Ar-H), 5.72 (dd,
1H, J ¼ 10.1, 8.4 Hz, C20-H), 5.53 (s, 1H, PhCH), 4.71–4.38 (4H, 2
PhCH2O), 4.60 (d, 1H, J ¼ 7.8 Hz, C10-H), 4.26 (dd, 1H, J ¼ 11.8,
1.4 Hz, C60-Ha), 4.21 (d, 1H, J ¼ 3.6 Hz, C40-H), 4.12 (dd, 1H,
J ¼ 10.8, 6.6 Hz, C1-Ha), 4.04 (dd, 1H, J ¼ 11.9, 2.3 Hz, C60-Hb),
3.76 (dd, 1H, J ¼ 9.2, 4.8 Hz, C2-H), 3.72 (dd, 1H, J ¼ 10.8, 4.2 Hz,
C1-Hb), 3.70 (dd, 1H, J ¼ 10.1, 3.2 Hz, C30-H), 3.62 (dd, 2H, J ¼ 9.2,
4.8 Hz, C3-H), 3.26 (s-like, 1H, C50-H), 1.85–1.83 (m, 2H, H-4), 1.32–
1.13 (m), 0.88 (t, 3H, J ¼ 7.4 Hz, CH3); HRMS-ESI: m/z calcd.
for C52H67N3NaO8 [MþNa]þ: 884.4826; Found 884.4820.
Preparation of (2S,3R)-ꢀ-D-galactopyranosyl-(10!1)-
hexacosanoylamino-octadecan-1,3-diol (T9)
PPh3 (0.15 mol) and water (0.01 mol) were added to a stirred
solution of azide compounds 23 (60.0 mg, 0.075 mmol) in THF
(6 mL), and the mixture was then heated at 458C until TLC
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2011, 344, 786–793
Evaluation of Antifungal Activities of Novel 1,2-trans Glycosphingolipids
793
indicated the complete transformation of the starting azide
into corresponding amine. After rotary evaporation, the amine
residue was redissolved in dry CH2Cl2 (6 mL), and
Et3N (0.01 mmol), EDC ꢂ HCl (0.09 mmol), HOBt (0.09 mmol),
and fatty acid (0.083 mmol) were added. The reaction mixture
was stirred at room temperature for 10 h. The solution was
concentrated under vacuum, and the syrupy residue was puri-
fied by column chromatography on silica gel eluted with
petroleum ether and EtOAc (v/v ¼ 5:1). To a solution of the
above purified syrupy residue in MeOH (3 mL) and EtOAc
(3 mL) was added Pd/C (50 mg, 10% wt% Pd dry basis on carbon),
and the mixture was hydrogenated at room temperature for
3 h. The suspension was filtered through Celite pad. The filter
cake was rinsed with CHCl3/MeOH (5:1, 30 mL). The combined
filtrate and washings were concentrated in vacuo. The residue
was purified by silica gel flash chromatography eluted with
CHCl3 and MeOH (v/v ¼ 10:1) to give the product T9 (41.1 mg,
64%) as a light yellow powder.
This work is supported by Research Fund for the Doctoral Program of
Higher Education of China (RFDP, 20070423001) and Startup Foundation
for Young Teachers, School of Pharmacy, Fudan University.
The authors have declared no conflict of interests.
References
[1] M. H. Kossuga, J. B. MacMillan, E. W. Rogers, T. F. Molinski,
G. G. F. Nascimento, R. M. Rocha, R. G. S. Berlinck, J. Nat. Prod.
2004, 67, 1879–1881.
[2] K. A. Marr, C. N. Lyons, T. R. Rustad, R. A. Bowden, T. C. White,
Antimicrob. Agents Chemother. 1998, 42, 2584–2589.
´
[3] J. A. Morales-Serna, O. Boutureira, Y. Dıaz, M. I. Matheu, S.
´
Castillon, Carbohydr. Res. 2007, 342, 1595–1612.
[4] (a) G. M. Nicholas, R. H. Li, J. B. MacMillan, T. F. Molinski,
Bioorg. Med. Chem. Lett. 2002, 12, 2159–2162; (b) B. N. Zhou,
M. P. Mattern, R. K. Johnson, D. G. I. T. Kinston, Tetrahedron
2001, 57, 9549–9554.
[5] (a) K. Thevissen, U. Hillaert, E. M. K. Meert, K. K. Chow, B. P. A.
Cammue, S. V. Calenbergh, I. E. A. Francois, Bioorg. Med. Chem.
Lett. 2008, 18, 3728–3730; (b) D. Mormeneo, A. Manresa, J.
Casas, A. Llebaria, A. Delgado, J. Appl. Microbiol. 2008, 104,
1075–1081.
[6] (a) G. Song, H. Liu, W. Zhang, M. Geng, Y. Li, Bioorg. Med. Chem.
2010, 18, 5183–5193; (b) M. Gui, D. Shi, M. Huang, Y. Zhao, Q.
Sun, J. Zhang, Q. Chen, J. Feng, H. Liu, M. Li, Y. Li, M. Geng, D.
Jian, Invest. New Drug 2010, 28, 1–11; (c) G. Song, S. Yang, W.
Zhang, Y. Cao, P. Wang, N. Ding, Z. Zhang, Y. Guo, Y. Li, J. Med.
Chem. 2009, 52, 7368–7371; (d) Z. Zhang, C. Zong, G. Song, G.
Lv, Y. Chun, P. Wang, N. Ding, Y. Li, Carbohydr. Res. 2010, 345,
750–760.
1H-NMR(600 MHz, pyridine-d5) dH 8.06 (d, 1H, J ¼ 7.3 Hz, NH),
7.87 (m, 2H, NH2), 4.91 (d, 1H, J ¼ 7.8 Hz, C10-H), 4.76 (m, 1H, C2-
H), 4.66 (m, 1H, C1-Ha), 4.58 (m, 1H, C40-H), 4.41–4.40 (m, 2H, C50-
H, C60-Ha), 4.38–4.36 (m, 3H, C20-H, C1-Hb, C3-H), 4.21–4.17 (m,
3H, C60-Hb, C4-H), 4.05 (m, 1H, C30-H), 2.52 (t, 2H, J ¼ 7.3 Hz,
COCH2), 1.97–1.28 (CH2), 0.88 (m, 6H, 3 CH3); 13C-NMR (150 MHz,
pyridine-d5) dC 131.3, 129.2, 102.7, 76.9, 74.2, 71.7, 70.8, 79.9,
65.4, 45.9, 44.3, 36.7, 35.0, 31.9, 31.2, 30.7–29.4, 27.8, 22.7, 14.1;
HRMS-ESI: m/z calcd. for C51H101N2O9 [MþNa]þ: 885.7507; Found
885.7502.
In-vitro antifungal activity evaluation by MIC assay
Compounds C18-DHS and T1–T9 were evaluated for their in-vitro
antifungal activity against Candida albicans (EU583481), Candida
parapsilosis (EF198017), and Candida tropicalis (EU651853). In this
process minimum inhibitory concentration (MIC) of tested com-
pounds were determined by using a modified micro-broth
dilution assay following the guidelines of Clinical and
Laboratory Standard (NCCLS) described elsewhere. The test was
performed by using sterile disposal 96-well polystyrene micro-
titer plates in RPMI 1640 medium buffered with MOPS (3-[N-
morpholino]propane sulfonic acid) (Sigma Chemical Co., USA).
The solutions of tested compounds were prepared by a serial
fourfold dilutions of the product diluted in assay medium to get
the test concentration (320–1.25 mg/mL). Initial inocula of fungal
strains were maintained at 1–5 ꢁ 103 cells/mL. These plates were
incubated in a moist chamber at 358C and absorbance at 630 nm
was recorded on Versa Max micro-plate reader (Molecular devi-
ces, Sunnyvale, USA) after 24, 72 and 168 h. MIC was defined as
the lowest concentration of antifungal agent that inhibits 80%
development of growth reflected by OD value. Experiments were
conducted in triplicate.
[7] Y. P. Liu, N. Ding, H. L. Xiao, Y. X. Li, J. Carbohydr. Chem. 2006, 25,
471–489.
[8] P. Zimmermann, R. Sommer, T. Ba¨r, R. R. Schmidt,
J Carbohydr. Chem. 1988, 7, 435–452.
[9] R. R. Schmidt, P. Zimmermann, Angew. Chem. Int. Ed. 1986, 25,
725–726.
[10] (a) V. Costantino, E. Fattorusso, C. Imperatore, A. Mangoni,
Tetrahedron Lett. 2001, 42, 8185–8187; (b) N. Ding, C. X. Li, Y. P.
Liu, Z. H. Zhang, Y. X. Li, Carbohydr. Res. 2007, 342, 2003–2013.
[11] W. Pilgrim, P. V. Murphy, Org. Lett. 2009, 11, 939–942.
[12] Clinical,Laboratory Standard Institute (Formerly NCCLS),
Methods for determining bactericidal activity of antimicrobial agents;
Approved guidelines: Approved standard M26-A, NCCLS, Wayne,
Pennsylvania, USA 1999.
[13] (a) R. Benveniste, J. Davies, Antimicrob. Agents Chemother. 1973,
4, 402–409; (b) I. G. Smirnova, G. S. Katrukha, A. B. Silaev,
Chem. Natural. Comp. 1974, 10, 641–644.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com