Hydrophobic derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate: A new type of D-glucosamine-6-phosphate synthase inhibitors with antifungal action

Article abstract of DOI:10.1016/S0968-0896(03)00049-X

Several N-acyl and ester derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) have been synthesised and tested as inhibitors of fungal enzymes involved in early steps of chitin biosynthesis and for antifungal activity. All the tested derivatives w

Full text of DOI:10.1016/S0968-0896(03)00049-X

Bioorganic & Medicinal Chemistry 11 (2003) 1653–1662
Hydrophobic Derivatives of 2-Amino-2-deoxy-D-glucitol-6-phosphate:
A New Type of D-Glucosamine-6-phosphate Synthase Inhibitors
with Antifungal Action
Agnieszka M. Janiak,a Maria Hoffmann,b Maria J. Milewskab and S•awomir Milewskia,*
a
´
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk,
´
11/12 Narutowicza St., 80-952 Gdansk, Poland
´ ´
Department of Organic Chemistry, Technical University of Gdansk, 80-952 Gdansk, Poland
b
Received 9 August 2002; accepted 17 January 2003
Abstract—Several N-acyl and ester derivatives of 2-amino-2-deoxy-d-glucitol-6-phosphate (ADGP) have been synthesised and tes-
ted as inhibitors of fungal enzymes involved in early steps of chitin biosynthesis and for antifungal activity. All the tested derivatives
were found to be much poorer inhibitors of the enzyme, d-glucosamine-6-phosphate (GlcN-6-P) synthase, than the parent com-
pound but some of them exhibited much better antifungal activity. MIC values for the investigated compounds ranged between 10
mg mLꢀ1, found for ADGP and 0.3 mg mLꢀ1 for the most active derivative, namely ADGP dimethyl ester. Increased affinity of
ADGP derivatives to the artificial immobilised cell membrane was correlated with their enhanced ability to be taken up by fungal
cells by free diffusion. It was found that some of the examined derivatives behaved as ‘pro-drugs’ and after internalisation were
converted into ADGP in the cell-free extract. This conversion was relatively rapid for ADGP esters but very slow for N-acyl deri-
vatives. Results of our studies demonstrate a possibility of design and preparation of GlcN-6-P synthase inhibitors exhibiting
antifungal activity.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
cell wall biosynthesis. One of them is GlcN-6-P syn-
thase, enzyme catalysing the first committed step in
chitin biosynthesis pathway, that is transformation of
d-fructose-6-phosphate (Fru-6-P) to d-glucosamine-6-
phosphate. Although the enzyme is also present in
mammalian systems, substantial difference in physio-
logical consequences of GlcN-6-P synthase inhibition in
fungi and in mammals, constitute a firm molecular basis
for the selective toxicity of specific enzyme inhibitors.4
Disseminated infections caused by human pathogenic
fungi remain one of the most difficult problems in
modern chemotherapy. Our ability to treat such infec-
tions with established agents do not satisfy medical
needs.1 The situation becomes more complicated
because of the rapidly increasing number of immuno-
compromised patients and the emerging challenge of
multidrug resistance. Only a very limited number of
antifungal chemotherapeutics are in clinical use. The
high toxicity of Amphotericin B for mammalian cells
and fungistatic but not fungicidal action of azole anti-
fungals limit their clinical applications. Therapeutic
potential of the latter is also severely limited due to
the substantial development of resistance, especially of the
multidrug character.2 One of the possible solutions to
this problem is to consider an exploitation of new anti-
fungal targets,3 including enzymes involved in fungal
Known specific inhibitors of GlcN-6-P synthase belong
to two different structural groups: l-glutamine mimics
and analogues of the putative transition state inter-
mediates. Among a number of known glutamine ana-
logues, some are selective inhibitors of GlcN-6-P
synthase, not interacting with other enzymes utilising
l-glutamine as a substrate. One of them, N3-(4-methoxy-
fumaroyl)-l-2,3-diaminopropanoic acid (FMDP), gave
rise to oligopeptidic compounds demonstrating
remarkable antifungal activity.5 Incorporation of
FMDP into peptide structure allowed effective inter-
nalisation of the enzyme inhibitor by the way of peptide
permeases, but on the other hand was a reason for
*Corresponding author. Tel.: +48-58-347-2107; fax: +48-58-347-
1144; e-mail: milewski@altis.chem.pg.gda.pl
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00049-X

1654
A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
substantial specific resistance,6 since peptides permeases
are not essential for fungal cells.7
were obtained upon the action of diazomethane on
N-benzyloxycarbonyl-ADGP or N-acetyl-ADGP (2). In
the former case, hydrogenolytic de-protection of the
amino function afforded the required compound 7.
Synthesis of ethyl ester derivatives of ADGP (9, 10, and 11)
was accomplished by multi-step procedures. d-Glucos-
amine was first N-protected upon acylation with ben-
zylchloroformate or acetic anhydride and the obtained
compounds were converted into methyl or benzyl
glycosides 9a or 10a, respectively. The protected gluco-
samine derivatives were selectively phosphorylated at
6-OH with diethylchlorophosphate in the presence of
AgCN, thus giving rise to derivatives 9b and 10b.
Hydrogenolysis of 10b afforded diethyl ester of N-ace-
tyl-d-glucosamine-6-phosphate 10c, while treatment of
9b with traces of hydrochloric acid gave the diethyl ester
of N-benzyloxycarbonyl-d-glucosamine-6-phosphate 9c.
Both compounds (9c and 10c) were reduced with
sodium borohydride to give products 9d, 10 or 11.
Product 9d was isolated and treated with H2/Pd/C to
give finally the required diethyl ester of ADGP 9.
Reduction of 10c for a short time (2 h) afforded the
diethyl ester of AcADGP 10, but prolonged treatment
of 10c with NaBH4 (overnight) led to the partial
hydrolysis of the diethyl ester and eventual formation
of the monoethyl ester of AcADGP 11. Graphical
representation of synthetic strategies is shown in
Scheme 1. All the final ester products: 7, 8, 9, 10 and
11 were obtained as the oily substances. All final com-
pounds 1–11 were at least 99% pure, as judged by
HPLC testing (see below).
The second group of compounds comprises derivatives
of phosphorylated aminosugars, including: 2-amino-2-
deoxy-d-glucitol-6-phosphate (ADGP),8 arabinose-5-
phosphate oxime9 and 5-methylenephosphono-d-arabino
hydroximolactone,10 as the most powerful GlcN-6-P
synthase inhibitors. These compounds exhibit very
poor, if any, antifungal activity. We have recently
demonstrated that the antifungal activity of ADGP is
limited due to the highly inefficient uptake of this com-
pound by an unidentified active transport system and
apparent inability to cross the membrane by free diffu-
sion.11 In the present paper, we describe a method of
improving an antifungal activity of ADGP by modifi-
cations of its structure, giving rise to derivatives of
enhanced lipophilicity, able to diffuse through the fun-
gal cytoplasmic membrane.
Results and Discussion
Rationale for modification
In the 2-amino-2-deoxy-d-glucitol-6-phosphate (1)
molecule there are two sites charged at physiological
conditions: the amino group and the phosphate group.
These are also possible sites of modifications, resulting
in increase of a general lipophilicity of the molecule,
supposed to be advantageous for better penetration of
the derivatives through the cytoplasmic cell membrane.
We have therefore decided to prepare and test several
N-acyl and ester derivatives of ADGP of general for-
mula showed in Figure 1.
Comparison of apparent lipophilicity of the obtained
compounds
The general idea of our approach was to convert the
good GlcN-6-P synthase inhibitor ADGP into its deri-
vatives demonstrating higher apparent lipophilicity than
the parent compound and, in consequence, an increased
potential to cross the cytoplasmic membrane by free
diffusion. In order to compare the relative lipophilicity
of ADGP and its derivatives, we determined their affi-
nity to the artificial biological membrane immobilised
on the bed of the IAM PC DD2 column. The column
bed is especially designed for investigation of low and
moderately lipophilic compounds.13 It is composed of
phosphatidylcholine residues attached to the silica
Synthesis
Compound 1 was prepared from d-glucosamine-6-
phosphate upon reduction with sodium borohydride.12
N-Acyl derivatives of ADGP 2–6 were obtained by
acylation of the 2-amino group with respective acyl
anhydrides under alkaline conditions. Compound 2 was
also alternatively synthesised by acetylation of d-gluco-
samine-6-phosphate, followed by the selective reduction
of the aldehyde group. All the compounds were obtained
as white solids. Dimethyl ester derivatives of ADGP (7, 8)
Figure 1. Structures of the investigated compounds.

A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
1655
Scheme 1. Synthetic strategies applied in this work. Cbz, benyloxycarbonyl; Bnz, benzyl.
support, thus forming a lipidic surface mimicking the
monolayer of the fluidic cell membrane. The retention
time measured in this system for a given compound, is
not only a measure of lipophilicity but reflects also
possible interactions with the artificial membrane, like
hydrogen or electrostatic bond formation. We have
determined the retention parameters, k0IAM, for ADGP
and its derivatives. It should be noted that all examined
compounds gave single peaks, with noise level not
exceeding 1%, thus confirming their at least 99% purity.
The obtained data are0 presented in Table 1. The
extreme values of log k AM were found for reference
compounds: ꢀ0.7583 for GlcN-6-P and +0.8893 for
benzene. Apparent lipophilicity of ADGP was inter-
mediate, surprisingly enough much higher than that of
the closely related reference compound, that is, GlcN-6-
P. Acylation of the amino group or conversion of the
phosphate group into dimethyl or diethyl esters caused
further increase of apparent lipophilicity, as well as the
elongation of the carbon chain in the series of N-acyl

1656
A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
Table 1. Data characterising affinity of the investigated compounds
to the IAM.PC.DD2 column and their TLC mobility. ND=not
determined
Badet-Denisot et al., who investigated the inhibitory
potential of compounds 1, 2, 5 and 6 towards Escher-
ichia coli GlcN-6-P synthase.15 This discrepancy may
suggest differences between the active centres of eukary-
otic and prokaryotic GlcN-6-P synthases. Esterification
of the phosphate group also reduced the inhibitory
potential of ADGP, but the effect was less pronounced
than in the case of N-acylation. Since Teplyakov et al.16
previously demonstrated that interactions of the ADGP
phosphate oxygen atoms with amino acid side chains at
the GlcN-6-P synthase active centre involved exclusively
hydrogen bonds, one may assume that such interactions
are still possible for ADGP esters, provided no steric
hindrance is implemented. Moreover, the IC50 values of
ADGP esters were generally lower when determined for
the enzyme present in the crude extract, in comparison
to those found for the homogenous enzyme. One of the
likely explanations for this finding is the possibility of
relatively rapid conversion of ADGP esters into ADGP,
catalysed by esterases present on the cell-free extract.
Compound
log k0IAM
Rf
GlcN-6-P
Benzene
1
2
3
5
6
7
8
ꢀ0.7583
0.8893
ꢀ0.0437
0.2408
0.3170
0.3942
0.4407
0.2493
0.4074
0.2802
0.4238
0.3602
0.34 (B)
ND
0.28 (B)
0.67 (B)
0.65 (B)
0.75 (B)
0.80 (B)
0.81 (A)
0.95 (A)
0.89 (A)
0.85 (A)
0.67 (A)
9
10
11
derivatives 2–6. The log k0IAM value found for the most
lipophilic compound in the series, that is N-hexanoil-
ADGP (6) was slightly higher than those of the N-acetyl
esters 8 and 10. The compounds 2–11 could be therefore
considered as moderately lipophilic and thus potentially
able to cross the cell membrane by free diffusion.
Both acylation of the amino group and esterification of
the phosphate group in ADGP molecule had no influ-
ence on the inhibition manner. In all cases inhibition
was competitive with regard to d-fructose-6-phosphate
and non-competitive with regard to l-Gln (data not
shown). It is therefore clear that the investigated com-
pounds interact with GlcN-6-P synthase at the d-fructose-
6-phosphate binding site.
Enzyme inhibitory activity
Inhibitory activity of all the synthesised compounds
towards Candida albicans GlcN-6-P synthase was deter-
mined. Some of these compounds were also tested as
potential inhibitors of two other enzymes, catalysing
respective: preceding and following steps in the chitin
biosynthesis pathway, namely phosphoglucose iso-
merase and GlcN-6-P N-acetylase. The obtained data
have been summarised in Table 2. All the derivatives
appeared to be poorer inhibitors of GlcN-6-P synthase
than ADGP. The enzyme inhibitory activity was espe-
cially reduced upon N-acylation of ADGP and the
resulting loss of the positive charge (compare com-
pounds 1 and 2), whereas the elongation of the acyl
chain was of the secondary importance (compounds 2, 5
and 6). The former observation is consistent with the
earlier suggestion of Bearne and Blouin,14 based on the
results of analysis of the other GlcN-6-P synthase inhi-
bitors. Surprisingly enough, enzyme inhibitory activity
of N-haloacetyl ADGP derivatives 3 and 4 was very
low. This is in contrast with the observations made by
Phosphoglucose isomerase catalyses a reaction preced-
ing the GlcN-6-P synthase-catalysed step in the chitin
biosynthesis pathway. On the other hand, the isolated
Fru-6-P-binding domain of GlcN-6-P synthase exhibits
the phosphoglucose isomerase-like activity.17 It seemed
thus reasonable to check whether the tested compounds
demonstrated phosphoglucose isomerase-inhibiting
activity. We found that ADGP (1), at 5 mM, reduced
the yeast enzyme activity by only 10%. Surprisingly,
N-acetyl-ADGP (2) appeared a relatively good inhibitor
of phosphoglucose isomerase, inhibiting 50% of the
enzyme activity at 76ꢁ2 mM. Actually, the phospho-
glucose isomerase inhibitory activity of 2 was much higher
than that against GlcN-6-P synthase. Other derivatives
did not affect the phosphoglucose isomerase activity.
GlcN-6-P acetylase follows GlcN-6-P synthase in the
chitin biosynthesis pathway. Amino sugar phosphates
and their derivatives can be potential inhibitors of
GlcN-6-P acetylase as potential analogues of its sub-
strate. However, none of the tested compounds inhib-
ited enzyme activity up to 20mM. Thus, with the
exception of compound 2, investigated compounds were
shown to be specific inhibitors of GlcN-6-P synthase
and did not significantly inhibit activity of enzymes cat-
alysing step adjacent to formation of GlcN-6-P by
GlcN-6-P synthase.
Table 2. Inhibition of C. albicans GlcN-6-P synthase by ADGP and
its derivatives
Compd
IC50 (mM)
Pure enzyme
In cell-free extract
0.24
1
2
3
4
5
6
7
8
0.23
6.05.5
5.2
5.2
>20
10.1
17.5
1.7
>20
11
19
4.75
12
4.7
Antifungal activity of ADGP and its derivatives
5.2
3.8
9
Antifungal activity of compounds 1–3 and 5–11 was
determined by a serial dilution microplate method.
N-iodoacetyl-ADGP (4) was not tested, as this compound
10
11
19
13
12
6.5

A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
Table 3. Antifungal in vitro activity of ADGP and its derivatives
1657
Compd
MIC (mg mLꢀ1
)
C. albicans
ATCC 10261
C. albicans
ATCC 26278
C. famata
C. humicola
C. glabrata
S. cerevisiae
ATCC 9763
1
2
3
5
6
7
8
9
5
5
5
5
5
>105
>10
2.5
5
>102.5
2.5.75
0
>10
5
>10
2.5
10105
0.3
5
0.6
105
2.5
>10
2.5
11005
0.3
>10
5
>10
2.5
0.6
>10ND
1.25
5
105
ND
2.5
0.6
0.6
10
5
1.25
0.6
ND10
2.5
1.25
1.25
1.25
2.5
10
10
11
ND, not determined.
did not show any GlcN-6-P synthase inhibitory activity.
Minimal inhibitory concentrations (MICs) were deter-
mined in YNBG minimal medium with glucose as car-
bon source. Obtained results are summarised in Table 3.
Practically identical results were obtained when the
antifungal activity was determined in buffered RPMI
1640medium (exact data not shown).
compounds, thus indicating that GlcN-6-P synthase is
their primary target in fungal cells.
Uptake of ADGP, AcADGP and dimethyl ester of
ADGP
The initial uptake rates of compounds 1, 2 and 7 to
C. albicans cells were determined in the presence of
d-glucose, d-glucose and NaN3, and in the absence of
the carbon source. Results are summarised in Table 4.
Most of the investigated compounds exhibited rather
weak antifungal activity (MICs ranging from 2.5 to 10
mg mLꢀ1). Notable exceptions are the dimethyl and
diethyl esters of ADGP (7 and 9), demonstrating growth
inhibitory effect at much lower concentrations. On the
other hand, it is worth noticing that N-acetyl-ADGP (2)
inhibited fungal growth at concentrations similar to that
of ADGP or even lower, despite its significantly poorer
GlcN-6-P synthase inhibitory activity (see Table 2).
Actually, the MIC values for this compound, as well as
those for other N-acyl derivatives of ADGP (5 and 6)
were quite comparable to their IC50 values determined
in respect to C. albicans GlcN-6-P synthase. On the
other hand, the MICs for ADGP esters 7 and 9 were
even lower than their IC50s against the enzyme. Anti-
fungal activity of the monoethyl ester of AcADGP (11)
was very similar or slightly better than that of N-acetyl-
ADGP (2), while that of the dimethyl and diethyl esters
of N-acetyl-ADGP (8 and 10) was poorer.
Presence of d-glucose reduced N-acetyl-ADGP (2)
uptake rates, but had no influence on ADGP (1) and
ADGP dimethyl ester (7) uptake. In the presence of
sodium azide, uptake of ADGP was completely inhib-
ited but uptake of compounds 2 and 7 was only slowed
down. The initial uptake rate of 2, determined in the
presence of sodium azide, was linearly dependent on the
initial concentration of the former, while in the absence of
NaN3 was hyperbolic (Fig. 2). Similar phenomenon was
observed for the compound 7 (details not shown). Maxi-
mal uptake rates were: 6.6 nmol (min mg dry weight)ꢀ1, 20
nmol (min mg dry weight)ꢀ1 and 15.5 nmol (min mg dry
weight)ꢀ1, for compounds 1, 2 and 7, respectively.
One may therefore conclude that N-acetyl-ADGP and
ADGP ester are transported into C. albicans cells both
by an active transport system and by free diffusion. We
have not studied the uptake of other ADGP derivatives
but it is very likely that their accumulation in fungal
cells is also in part possible by free diffusion. On the
other hand, ADGP is transported exclusively by an
unidentified active transport system.
The microscopic examination of cells treated with
ADGP, N-acetyl-ADGP or ADGP dimethyl ester
revealed morphological changes characteristic for yeast
cells affected by compounds inhibiting chitin and/or
mannoprotein biosynthesis. These alterations included
cell agglutination, visible destruction of cell integrity,
cell swelling and inhibition of septum formation (data
not shown). Similar morphological changes were pre-
viously described as a result of the action of oligopeptides
containing an inhibitor of GlcN-6-P synthase.18 On the
other hand, known inhibitors of chitin synthase, nikko-
mycins and polyoxins, inhibit septum formation and
cause cell swelling and lysis.19, 20 while tunicamycin, a
selective inhibitor of mannoprotein biosynthesis, causes
mainly cell agglutination.21 It should be noted that the
presence of 10mM d-glucosamine in the medium com-
pletely abolished the antifungal activity of investigated
Table 4. Initial rates of uptake of ADGP, AcADGP and ADGP
diethyl ester by C. albicans cells
Buffer/additives
Initial uptake ratea
(nmol/min/mg dry weight)
(1)
(2)
(7)
Tris/glucose
Tris
Tris/glucose, NaN3
1.1ꢁ0.2
1.0ꢁ0.2
<0.01
3.1ꢁ0.2
5.0ꢁ0.3
0.5ꢁ0.06
2.8ꢁ0.3
2.7ꢁ0.2
1.6ꢁ0.1
aInitial compound concentration 5 mg mLꢀ1
.

1658
A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
Conclusion
We have designed and synthesised a number of
2-amino-2-deoxy-d-glucitol-6-phosphate (ADGP) deri-
vatives, modified at 2-amino and/or 6-phosphate
groups. All the obtained derivatives demonstrated
higher apparent affinity to the artificial immobilised cell
membrane IAM PC DD2 than the parent compound
and therefore a higher potential to cross the real cell
membrane by free diffusion. The log k0IAM values
determined for these compounds were in the 0.2–0.5
range, thus indicating their moderately lipophilic
character. In general opinion, log k0IAM values ranging
between 0and 2 are considered advantageous since
compounds demonstrating negative log k0IAM are unli-
kely to diffuse through the biological membranes, while
those showing log k0IAM>2 may be easily retained in
the membrane environment.13
Figure 2. Kinetics of N-acetyl-ADGP uptake by C. albicans cells.
Fungal cells harvested from the exponentially growing culture were
washed, suspended in an appropriate buffer system and the cell sus-
pension was incubated at 30 ꢂC. N-Acetyl-ADGP was added and
samples of the cell culture were collected at time intervals. Cells were
harvested by filtration and concentration of the remaining ADGP
derivative was determined in filtrates. The initial uptake velocities,
determined from the plots of remaining AcADGP concentration ver-
sus time were re-plotted against the initial compound concentration.
Values are the means of three independent determinations ꢁS.D.
Buffer composition: (&) 50mM Tris/HCl, pH 6.5, containing 1%
glucose; (*) 50mM Tris/HCl, pH 6.5, containing 1% glucose and 100
mM NaN3.
ADGP, a relatively strong inhibitor of GlcN-6-P syn-
thase, was previously found to be a very poor antifungal
agent, transported into C. albicans cells by unidentified,
energy-dependent transport system.11 We have been
able to improve the antifungal activity of ADGP by its
conversion into more lipophilic N-acyl and ester deri-
vatives, able to cross the cell membrane by free diffu-
sion. Although the obtained derivatives demonstrated
lower inhibitory potential against the target enzyme,
GlcN-6-P synthase, their intracellular metabolism,
demonstrated for N-Acetyl-ADGP and ADGP dimethyl
ester and suspected for other derivatives, gives rise to
formation of the more active enzyme inhibitor. This
mode of action is therefore consistent with the ‘pro-
drug’ concept.22,23 However, intracellular deacetylation
of N-acetyl-ADGP is very slow and that of other N-acyl
derivatives is possibly even slower. On the other hand,
the ADGP dimethyl ester is much faster metabolised to
ADGP, thus giving rise to intracellular accumulation of
the more efficient enzyme inhibitor. We can therefore
conclude that esterification is a more promising way of
preparation of potential antifungal agents based on
amino sugar phosphate analogues. Further studies,
aimed at design and synthesis of novel GlcN-6-P syn-
thase inhibitors, more effective than ADGP, should
result in development of their ester derivatives as
potential antifungals. These studies are in progress in
our laboratory.
Metabolism of ADGP, N-acetyl-ADGP and ADGP
dimethyl ester
Composition of the cell-free extracts prepared from the
C. albicans cells grown in the presence of ADGP,
N-acetyl-ADGP or ADGP dimethyl ester was investi-
gated by thin layer chromatography (TLC) analysis.
These studies showed that ADGP (1) was not meta-
bolised to any detectable derivative. On the other hand,
N-acetyl-ADGP (2) and ADGP ester (7) were meta-
bolised to compound chromatographically identical
with ADGP. The conversion rate of 2 was very low,
since the presence of ADGP was detected only in the
cell-free extract prepared from the cells harvested after
8-h incubation with N-acetyl-ADGP. The ADGP ester
(7) was metabolised much faster, since a spot corres-
ponding to ADGP was detected as early as after 1 h
incubation.
Similar results were obtained when compositions of the
C. albicans cell-free extracts, incubated with 1, 2 or 7,
were analysed by TLC. However, in that case it was
found that the rate of conversion of 2 into 1 sub-
stantially increased when the cell free extract was addi-
tionally supplemented with ATP, 10mM.
Experimental
Chemistry
Low rate of intracellular deacetylation of N-acetyl-
ADGP suggests that this compound, as a good inhibitor
of phosphoglucose isomerase, may target this enzyme
upon its action on fungal cells. Such inhibition should,
in principle, result in growth inhibitory effect. However,
we believe that this possibility seems rather unlikely,
since the lowest MIC value of this compound (0.6 mg
mLꢀ1 against Saccharomyces cerevisiae) is almost three
orders of magnitude higher than its IC50 against pure
yeast phosphoglucose isomerase.
Butanoic acid anhydride, hexanoic acid anhydride,
chloroacetyl chloride, 2-amino-2-deoxy-d-glucose-6-
phosphate and sodium borohydride were purchased
from Sigma-Aldrich Chemical Co. All other chemicals
were of the highest purity commercially available.
1H and 31P NMR spectra were obtained with Varian
Unity Plus 500 at 200 and 500 MHz, respectively. The
deuterated solvents were used as an internal lock for
1H NMR and external aq 85% H3PO4 for 31P NMR.

A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
1659
Specific rotations were measured on a Rudolph Autopol
II digital polarimeter. TLC was carried out on Kieselgel
60F 254 plates (Merck) in solvent systems: A (methanol–
25% aq ammonia–water, 6:2:1 v/v/v), B (n-butanol–acetic
acid–water 4:1:1 v/v/v) and C (chloroform–methanol
5:1, v/v). The spots were visualised with ninhydrin, cer-
ium sulphate or Hanes’ reagent.24
2H), 3.9 (m, 1H); Rf (A) 0.56; Rf (B) 0.67. Compound 3:
1H NMR (D2O) d: 3.58–3.64 (m, 1H); 3.65–3.78 (m,
2H), 3.84–4.02 (m, 3H), 4.09 (s, 2H, CH2ClCO); 4.18–
4.28 (m, 2H); Rf (B) 0.65. Compound 4: 1H NMR
(D2O) d: 3.48–3.64 (m, 4H); 3.63 (s, 2H, CH2ICO);
3.72–3.85 (m, 3H); 3.87–3.93 (m, 1H); Rf (B) 0.62.
Compound 5: 1H NMR (D2O) d: 0.96 (t, 3H, J=7.3 Hz,
CH3CH2); 1.66 (m, 2H, J=7.3 Hz, CH3CH2CH2CO);
2.23 (t, 2H, J=7.9 Hz, –CH2CO); 3.35 (m, 2H, J=1.7
Hz); 3.68 (m, 2H); 3.76 (m, 1H); 4.07 (m, 3H); Rf (B)
0.75. Compound 6: 1H NMR (D2O) d: 0.9 (t, 2H,
J=6.67 Hz, CH3CH2); 1.3 (m, 4H, CH3(CH2)2), 1.6 (m,
2H, J=7.5 Hz, CH3(CH2)2CH2CH2); 2.4 (t, 2H, J=7.3
Hz, CH2CH2CO); 3.31 (m, 2H, J=1.74 Hz); 3.64 (m,
2H); 3.74 (m, 1H); 4.07 (m, 3H); Rf (B) 0.80.
2-Amino-2-deoxy-D-glucitol-6-phosphate (1). This com-
pound was synthesised according to the method descri-
bed by Bearne12 with 80% yield (literature yield12 60%).
N-Acetyl-2-amino-2-deoxy-D-glucitol-6-phosphate (2),
N-chloroacetyl-2-amino-2-deoxy-D-glucitol-6-phosphate
(3), N-iodoacetyl-2-amino-2-deoxy-D-glucitol-6-phosphate
(4), N-n-butanoil-2-amino-2-deoxy-D-glucitol-6-phosphate
(5) and N-n-hexanoil-2-amino-2-deoxy-D-glucitol-6-phos-
phate (6). To the solution of 1 (100 mg, 0.38 mmol)
and a base (1.78 mmol) in an appropriate solvent system
(5 mL), an acylating agent was added for 20–60 min and
then the reaction mixture was stirred for another 20–60
min. A course of the reaction was followed with TLC
(solvent system A or B). Further details are shown in
2-Amino-2-deoxy-d-glucitol-6-phosphate dimethyl ester
(7). 2-Amino-2-deoxy-d-glucitol-6-phosphate (281 mg,
1.1 mmol) was dissolved in 1 M NaOH solution (1 mL)
and benzylchloroformate (0.21 mL) was added. The
reaction mixture was stirred at room temperature for 24
h, maintaining pH at 9–10with 1 M NaOH water solu-
tion. The resulting solution was extracted 3ꢃ with ethyl
ether. The remaining water layer was chromatographed
over Dowex 50WX8 (H) column. Fractions containing
2-(N-benzyloxycarbonylamino)-2-deoxy-d-glucose-6-
phosphate were evaporated in vacuo. Product (200 mg)
was dissolved in water (5 mL) and the diazomethane
ethyl ether solution was added to permanent yellow tint.
Solvent was distilled off and the product was purified by
silicagel column chromatography in solvent system
chloroform/methanol 5:1, v/v, affording after evapora-
tion 90mg of 2-( N-benzyloxycarbonylamino)-2-deoxy-
d-glucose-6-phosphate dimethyl ester (yield 41%). This
product was dissolved in methanol (6 mL), NaBH4 (30
mg, 0.78 mmol) was added and the mixture was kept for
4 h at room temperature. The resulting solution was
purified on a Dowex 50WX4 (H +) ion-exchange resin
developed with methanol and then the methanolic solu-
tion of the product was subjected to catalytic hydro-
genation for 4 h. Final overall yield: 25%.1H NMR
(D2O) d: 3.32 (m, 1H); 3.41 (m, 1H); 3.70(2d, 6H,
J=10.7 Hz, 2ꢃCH3OP); 3.78 (m, 2H); 3.86 (m, 1H);
3.91 (m, 1H); 4.06 (m, 1H); 4.11 (m, 1H); Rf (A) 0.9.
Table 5. Products 2, 3 and 4 were purified by ion-
+
exchange chromatography on Dowex 50WX4 (H
)
column and obtained finally from the lyophilised fil-
trate. Compounds 5 and 6 were purified by consecutive
adsorption (Silicagel) and ion-exchange (Dowex 50
WX4/H+/) column chromatography Fractions con-
taining product were pooled and lyophilised.
N-Acetyl-2-amino-2-deoxy-d-glucitol-6-phosphate (2)
Method 2. To the solution of 2-amino-2-deoxy-d-glu-
cose-6-phosphate (100 mg, 0.36 mmol) and NaHCO3
(150mg, 1.78 mmol) in water (5 mL), acetic acid anhy-
dride (10% solution in acetone, 0.6 mL) was added
during 10min. After another 10min of stirring at room
temperature, the reaction mixture was diluted with
2-propanol (5 mL) and then the solvent was evaporated.
The oily residue was dissolved in water (3 mL) and cooled
to 0 ꢂC. Then NaBH4 (150mg, 4 mmol) was added por-
tionwise for 2 h. The reaction mixture was stirred at room
temperature for another 1 h and then the solvent was
evaporated. The residue was dissolved in a solvent system
A and chromatographed over Silicagel column in the same
solvent system. Fractions containing the product were
evaporated, re-dissolved in water and chromatographed
over Dowex 50 WX4 (H+) column. Fractions containing
the product were pooled and lyophilised. Compound 2 was
obtained with 86% yield (94 mg, 0.31 mmol).
2-N-Acetylamino-2-deoxy-D-glucitol-6-phosphate dimetyl
ester (8). To the solution of d-glucosamine-6-phosphate
(140 mg, 0.5 mmol) in 0.5 M NaOH (1 mL), acetic
anhydride (95 mL, 1 mmol) was added. The reaction
mixture was stirred for 2 h at room temperature and
then chromatographed over Dovex 50WX8 (H +) col-
umn. Fractions containing reaction product were
pooled and evaporated in vacuo, affording 150mg of
crude 2-N-acetylamino-2-deoxy-d-glucose-6-phosphate.
Compound 2. 1H NMR (D2O) d: 1.84 (s, 3H,
CH3CO); 3.48–3.55 (m, 4H); 3.6 (m, 1H); 3.74–3.82 (m,
Table 5. Acylation of ADGP — details
Compd
Solvent
Base
Acylating agent
Reaction conditions
Yield (%)
2
3
4
5
6
Water
Water
Water
NaHCO3
NaHCO3
NaHCO3
Triethylamine
Triethylamine
10% Acetic anhydride in acetone, 0.6 mL
10% Chloroacetic anhydride in acetone, 0.8–1 mL
Iodoacetic anhydride, 0.2 mL
40 min, room temp
1 h, room temp
2 h, 0 ꢂC
2 h, room temp
2 h, 60 ꢂC
96
66
49
60
70
Water/methanol
Water/methanol
Butanoic anhydride, 0.2 mL
Hexanoic anhydride, 0.5 mL

1660
A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
This product was dissolved in methanol (5 mL) and an
ethyl ether solution of diazomethane was added drop-
wise to permanent yellow tint. Solvent was distilled off
in vacuo affording 165 mg of the crude 2-N-acetyl-
amino-2-deoxy-d-glucose-6-phosphate dimethyl ester.
This product was purified by silicagel column chroma-
tography in solvent system chloroform–methanol 5:1
(v/v). After evaporation of the solvent, the purified pro-
duct was dissolved in methanol (2 mL) and NaBH4 (20mg,
0.52 mmol) was added. The reaction mixture was stirred
for 4 h at room temperature and then chromatographed
over Dowex 50WX4 (H +) ion-exchange resin. Product
was eluted with methanol. Solvent was distilled off in
vacuo, affording compound 8 (cummulative yield 63%).
1H NMR (MeOD) d: 1.99 (s, 3H, CH3CO); 3.52 (d, 1H,
J=7.3 Hz); 3.65 (d, 2H, J=4.88 Hz); 3.80(d, 6H, J=11.2
Hz, 2ꢃCH3OP); 3.84 (m, 1H); 4.02 (m, 1H); 4.04 (t, 1H,
J=4.88 Hz); 4.14 (m, 1H); 4.27 (m, 1H); Rf (C) 0.2.
phate (418 mg, 2.4 mmol) and AgCN (5 mg, 0.04 mmol)
in anhydrous pyridine (6 mL) was stirred at room tem-
perature. After 30min benzyl 2-deoxy-2-( N-acetyl-
amino)-d-glucopyranoside 10a (750mg, 2.4 mmol) or
methyl 2-deoxy-2-(N-benzyloxycarbonylamino)-d-gluco-
pyranoside 9a (370mg, 1.13 mmol) was added and stir-
ring was continued. After 72 h, an excess of pyridine
was distilled off in vacuo and the residue was dissolved
in water (10mL) and extracted with chloroform (3 ꢃ5
mL). The organic layer was washed with water (7 mL),
hydrochloric acid (2% in water), and water (7 mL)
again, and dried over anhydrous MgSO4. After solvent
evaporation the residue was purified on the Silicagel
column (Merck 70–270 mesh) with chloroform as
mobile phase.
Compound 9b. Yield 40% (200 mg), colourless oil,
[a]2D0ꢀ20( c 0.8, CHCl3), [a]D20ꢀ135 (c 1, MeOH). 1H
NMR (CDCl3) d: 1.31 (dt, 6H, J=7.3 and 10.3 Hz,
2ꢃCH3CH2OP); 3.45 (s, 3H, CH3O); 3.47 (d, 1H,
J=8.4 Hz, H4); 3.67 (t, 1H, J=9.3 Hz, H3); 3.88 (m,
2-Deoxy-2-(N-benzyloxycarbonylamino)-D-glucopyranose.
This compound was prepared as white solid, mp 205–
207 ꢂC; [a]D20+73 (c 1, Pyr) [lit. 214 ꢂC; [a]2D0+75.4 (c
3.42, Pyr)], using the described procedure.25
3H, H2+2ꢃOH); 4.10(qv, 4H,
J=7.3 Hz,
2ꢃCH3CH2OP); 4.24 (ddd, 1H, J=4.4, 9.3 and 11.2 Hz,
H6); 4.3 (m, 2H, H5+H60); 5.08 and 5.10 (AB+m, 3H,
H1+CH2C6H5); 5.7 (d, 1H, NH); 7.29 (m, 5H, C6H5).
Methyl 2-deoxy-2-(N-benzyloxycarbonylamino)-D-gluco-
pyranoside (9a). 2-Deoxy-2-(N-benzyloxycarbonyl-
amino)-d-glucopyranose (626 mg,
suspended in methanol (12 mL) saturated with dry HCl.
2
mmol) was
The reaction mixture was stirred for 3 h at 40 ꢂC. Sus-
pension containing 50mg PbCO in 2 mL of water was
Compound 10b. Yield 35% (375 mg), colourless oil, [a]D20
+63 (c 2.25, EtOH), 1H NMR (MeOD) d: 1.35 (dt, 6H,
3
added. The resulting precipitate was filtered off. The fil-
trate was concentrated in vacuo, and the residue was
crystallised from methanol/diethyl ether mixture. The
product 9a was obtained with 75% yield (490mg), mp.
3.0and
J=7.1 Hz, 2ꢃCH3CH2OP); 1.95 (s, 3H,
CH3CO); 3.39 (t, 1H, J=9.2 Hz, H4); 3.71 (t, 1H,
J=9.8 Hz, H3); 3.80( dd, 1H, J=4.8 and 9.8 Hz, H2);
3.89 (m, 1H, H5); 4.14 (qv, 4H, J=7.1 Hz,
2ꢃCH3CH2OP); 4.21 (m, 1H, H6); 4.28 (m, 1H, H60);
4.51 and 4.73 (AB, 2H, CH2C6H5); 7.09–7.38 (m, 5H,
C6H5); 8.06 (d, J=8.3 Hz, NH).
138–40 ꢂC, [a]D22ꢀ4 (c 1, MeOH). H NMR (DMSO-d6)
1
d: 3.08 (m, 2H, 2ꢃOH); 3.16 (t, 1H, J=9.1 Hz, H4);
3.22 (m, 2H, H3+OH); 3.36 (s, 3H, CH3O); 3.44 (m,
1H, J=6.1 Hz, H6); 3.66 (dd, 1H, 6.1 and J=11.3 Hz,
H60); 4.12 (d, 1H, J=8.2 Hz, H2); 4.52 (t, 1H, J=6.1
Hz, H5); 4.97 (AB+m, 3H, H1+CH2C6H5); 7.12 (d,
1H, J=9.1 Hz, NH); 7.34 (m, 5H, C6H5).
6-[2-Deoxy-2-(N-benzyloxycarbonylamino)-D-glucopyrano-
sidyl]diethyl phosphate (9c). Compound 9a (87 mg, 0.2
mmol) was suspended in methanol (5 mL) and one drop
of hydrochloric acid was added. The reaction mixture
was stirred for 4–5 h at 70 ꢂC, then the suspension of
PbCO3 (50mg) in water (2 mL) was added. The resulting
precipitate was filtered off. The filtrate was concentrated
in vacuo and the residue was purified by ion-exchange
chromatography on Dowex 50X W[H+] column. Com-
pound 9c was obtained with 60% yield (50 mg),
Benzyl 2-deoxy-2-(N-acetylamino)-D -glucopyranoside
(10a). To the solution containing 884 mg (4 mmol) of
N-acetyl-d-glucosamine in dry benzyl alcohol (10mL),
diethyl ether saturated with dry HCl was added (2 mL).
The reaction mixture was vigorously stirred for 3 h at
50–60 ꢂC. The crude product was precipitated by addi-
tion of benzene (10mL) and diethyl ether (40mL). The
product was filtered off and crystallised twice from
anhydrous ethanol. Product 10a wasꢂ obtained as a
white solid (930mg, yield 75%), mp. 183 C, [a] 2D0+104 (c
1, 94% EtOH); [a] 2D0+112 (c 1, Pyr). 1H NMR (MeOD)
d: 1.95 (s, 3H, CH3CO); 3.36 (t, 1H, J=9.1 Hz, H4); 3.65–
3.72 (m, 3H, H5, H3, H6); 3.81 (dd, 1H, J=10.6 Hz, H60);
3.88 (dt, 1H, J=10.6 and 3.3 Hz, H2); 4.50 and 4.75 (AB
2d, 2H, CH2C6H5); 4.86 (d, 1H, J=3.6 Hz, H1); 7.27–7.39
(m, 5H, C6H5); 8.03 (d, J=8.2 Hz, NH)
22
1
[a]D +20( c 1.5, H2O). H NMR (MeOD) d: 1.18 (dt,
6H, J=7.3 and 10.3 Hz, 2ꢃCH3CH2OP); 3.21–3.95 (m,
7H); 4.05 (qv, 4H, J=7.3 Hz, 2ꢃCH3CH2OP); 4.18 (m,
2H); 5.08 and 5.10 (AB+m, 3H, H1+CH2C6H5); 7.28
(m, 5H, C6H5).
6-[2-Deoxy-2-(N-acetylamino)-d-glucopyranosyl]diethyl
phosphate (10c). Suspension of compound 10b (112 mg,
0.254 mmol) and 10% Pd/C (60 mg) in methanol (5 mL)
was saturated with H2 for 6 h at room temperature. The
catalyst was then filtered off and the solvent was dis-
tilled off in vacuo. Compound 10c was obtained as a
colourless oil with 90% yield (80 mg), [a]2D0+22.9 (c 1,
EtOH). 1H NMR (MeOD) d: 1.35 (t, 6H, J=7 Hz,
2ꢃCH3CH2OP); 2.03 (s, 3H, CH3CO); 3.38 (t, 1H,
6-[Methyl-2-deoxy-2-(N-benzyloxycarbonylamino)-D-gluco-
pyranosidyl]diethyl phosphate (9b) and 6-[benzyl-2-
deoxy-2-(N-acetylamino)-D-glucopyranosidyl]diethyl phos-
phate (10b). Suspension containing diethyl chlorophos-

A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
1661
J=9.2 Hz, H4); 3.73 (t, 1H, J=9.6 Hz, H3); 3.85 (dd,
1H, 2.9 and J=10.8 Hz, H2); 3.97 (m, 1H, H5); 4.14
(qv, 4H, J=7.2 Hz, 2ꢃCH3CH2OP); 4.21 (m, 1H, H6);
4.27 (m, 1H, H60); 5.10(d, 1H, J=2.9 Hz, H1).
imidazolide) bonded to silica-propylamine, with the
unreacted propylamine moieties end-capped with C10
and C3 alkyl chains. The column dimensions were 3
cmꢃ4.6 mm, particle diameter 12 mm and pore diameter
300 A. For all studies the injection volume was ca. 10
mL of a solute aqueous solution. The standard solvent
system was acetonitrile/50mM potassium-phosphate
buffer pH 7.2 (50:50, 30:70, 20:80, 10:90, 5:95 v/v) con-
taining 0.1% acetone. The flow rate was 1 mL minꢀ1
and solute detection was at 495 nm. The chromato-
graphic system consisted of a Model l-6200 A pump, a
Model l-4250UV/VIS detector and a Model d-2500
chromatointegrator (Merck-Hitachi, Vienna, Austria).
The dead volume of the column was determined by the
t0 value, i.e., retention time of citric acid (applied as an
aqueous solution, 50mg mL ꢀ1). Retention times of the
studied compounds (tR) were determined for different
compositions of the mobile phase and used to calculate
the capacity parameters, k0IAM=(trꢀt0)/t0. Values of log
k0IAM were plotted against the acetonitryle content in
the mobile phase and the resulting linear plots were
extrapolated to give the log k0IAM value for the hypo-
thetical purely aqueous mobile phase.
2-Amino-2-deoxy-D-glucitol-6-phosphate diethyl ester (9),
2-(N-acetylamino)-2-deoxy-D-glucitol-6-phosphate diethyl
ester (10) and 2-(N-acetylamino)-2-deoxy-D-glucitol-6-
phosphate ethyl ester (11). Solution of compound 9c (50
mg, 0.12 mmol) or 10c (53 mg, 0.15 mmol) in ethanol (4
mL) was cooled to 0 ꢂC and NaBH4 (35 mg) was added.
The reaction mixtures were stirred for 2 h at room tem-
perature (for preparation of compounds 9d or 10, respec-
tively) or overnight for preparation of compound 11 from
10c. The resulting solutions were chromatographed over
the ion-exchange resin Amberlit IR-120[H +]. Fractions
containing either of the products were pooled, combined
and evaporated in vacuo with methanol (2ꢃ10mL).
1
Compound 9d. Yield 65% (34 mg), colourless oil, H
NMR (D2O) d: 1.12 (qv, 3H, J=7.3 Hz, CH3CH2OP);
1.20(qv, 3H, J=7.3 Hz, CH3CH2OP); 3.48 (m, 1H); 3.52
(m, 1H); 3.59 (m, 2H); 3.7 (m, 2H); 3.8 (m, 3H,
CH3CH2OP+Hglucitol); 3.92 (m, 1H); 4.2 (m, 2H,
CH3CH2OP); 5.0(AB, 2H, C H2C6H5); 7.25 (s, 5H, C6H5).
Determination of enzyme activity
GlcN-6-P synthase activity was determined according to
the modified Elson-Morgan method described pre-
viously.26 GlcN-6-P N-acetylase activity was assayed in
solution containing 5 mM phosphate buffer pH 6.9, 8
mM glucosamine-6-phosphate, 2 mM acetyl-coenzyme
A, appropriately diluted C. albicans cell-free extract and
enzyme inhibitor in appropriate concentration. The
reaction was carried out at 30 ꢂC for 30min and stop-
ped by heating. Concentration of the N-acetyl-d-gluco-
samine-6-phosphate formed was determined as
described previously.26 Phosphoglucose isomerase
activity was measured according to the procedure
described by Noltmann et al.27,28 using the yeast enzyme
preparation (Sigma-Aldrich Chemical Co).
1
Compound 10. Yield 60% (32 mg), colourless oil, H
NMR (D2O) d: 1.12 (qv, 3H, CH3CH2OP); d: 1.18 (qv,
3H, CH3CH2OP); 1.87 (s, 3H, CH3CO); 3.48 (m, 2H);
3.57 (m, 1H); 3.79 (m, 1H); 3.81 (m, 4H,
CH3CH2OP+2Hglucitol); 3.91 (m, 1H); 4.04 (m, 3H,
CH3CH2OP+Hglucitol).
Compound 11. Yield 40% (13 mg), colourless oil,
[a]2D0ꢀ5 (c 0.4, EtOH), 1H NMR (MeOD) d: 1.1 (dt, 3H,
2.9 and J=6.8 Hz, CH3CH2OP); 1.87 (s, 3H, CH3CO);
3.48 (m, 2H); 3.57 (dd, 1H, J=11.3 and 4.8 Hz); 3.79
(m, 1H); 3.81 (m, 4H, CH3CH2OP+2Hglucitol); 3.91 (dd,
1H, J=4.8 and 6.3 Hz); 4.04 (t, 1H, J=7.3 Hz).
Determination of antifungal in vitro activity
Compound 9d was dissolved in ethanol (5 mL) and 10%
Pd/C (30mg) was added. The suspension was saturated
with H2 for 2 h at room temperature. Then the catalyst
was filtered off, and the solvent was evaporated in vacuo
Clinical strains of different Candida species were from
the collection of the Department of Clinical Micro-
biology, Medical Academy of Gdansk. Candida clinical
and reference strains and S. cerevisiae ATCC 9763
(American Type Culture Collection, Manassas, Va,
USA) were stored on Sabouraud Agar slants. Fungal
inocula were grown in Sabouraud broth at 30C with
shaking. MICs were determined by a serial dilution
microtitrer plate method in Yeast Nitrogen Base med-
ium containing 1% glucose as a carbon source. Wells
containing serially diluted compound and control wells
were inoculated with 105 cells mLꢀ1 of an overnight
culture of fungal cells and incubated for 24 h at 30 ꢂC.
MIC was defined as the lowest antifungal agent con-
centration preventing visible growth. Alternatively,
MICs were determined in RPMI 1640medium buffered
with 3-[N-morpholino]propanesulfonic acid (MOPS) to
pH 7.0, under conditions recommended by NCCLS.29
In all cases, reproducible sharp end points were
obtained and trailing effects were not observed.
1
to give 9 as a colourless oil with 55% yield (19 mg). H
NMR (D2O) d: 1.12 (qv, 3H, J=7.3 Hz, CH3CH2OP);
1.20(qv, 3H, J=7.3 Hz, CH3CH2OP); 3.48 (m, 1H);
3.52 (m, 1H); 3.59 (m, 2H); 3.7 (m, 2H); 3.8 (m, 3H,
CH3CH2OP+Hglucitol); 3.92 (m, 1H); 4.2 (m, 2H,
CH3CH2OP).
Determination of the affinity to the artificial biological
membrane
Interactions between examined compounds and immo-
bilised artificial biological membrane were investigated
with the use of a HPLC column IAM PC DD2 (Regis
Techologies, Inc., Morton Grove, IL, USA), containing
as a stationary phase 1-myristoyl-2-[13-carbonylimid-
azolide-tridecanoyl]-sn-3-glycerophosphocholine (lecithin-

1662
A. M. Janiak et al. / Bioorg. Med. Chem. 11 (2003) 1653–1662
Uptake studies
References and Notes
C. albicans ATCC 10261 cells, grown exponentially in
YNB medium at 30C, were harvested, washed with sal-
ine and re-suspended in 50mM Tris/HCl buffer, pH 6.5
containing 1% glucose to the final cell density of 108
cells mLꢀ1 (about 1.0mg dry weight cells mL ꢀ1). The
suspension was pre-incubated at 30 ꢂC for 10min,
investigated compounds (1, 2 or 7) dissolved in a mini-
mal buffer aliquot were added to the final concentration
of 5 mg mLꢀ1 and the incubation was continued. At
that moment and after 5, 10, 15, 30, 60 and 90 min, 100-
mL samples were withdrawn and diluted 1:100. The
diluted suspensions were immediately filtered through
GF/C glass fibre filters (Whatman International Ltd.,
Maidstone, UK) under suction and filtrates were col-
lected. Concentration of all investigated compounds
was measured by determination of phosphate (V) ester
concentration with Fiske Subba Row reagent.30 Data
were plotted as nanomoles of ADGP or its derivative
taken up by 1 mg (dry weight) of cells versus time. The
initial uptake velocities were determined from the slopes
of the linear part of the curves originating at 0.0 point
and then re-plotted against the initial compound con-
centration.
1. Neely, M. N.; Ghannoum, M. Eur. J. Microbiol. Infect.
Dis. 2000, 19, 897.
2. Georgopapadakou, N. H. Curr. Opin. Microbiol. 1998, 1,
547.
3. Walsh, T. J.; Vivani, M. A.; Arathoon, E.; Chiou, C.;
Ghannoum, M.; Groll, A. H.; Odds, F. C. Med. Mycol. 2000,
38, 335.
4. Borowski, E. Il Farmaco 2000, 55, 206.
5. Milewski, S.; Chmara, H.; Andruszkiewicz, R.; Borowski,
E.; Zaremba, M.; Borowski, J. Drugs Exptl. Clin. Res. 1988,
14, 461.
6. Milewski, S.; Andruszkiewicz, R.; Borowski, E. FEMS
Microbiol. Lett. 1988, 50, 73.
7. Horak, J. Biochim. Biophys. Acta 1997, 1331, 41.
8. Badet-Denisot, M.-A.; Leriche, C.; Masserie, F.; Badet, B.
Bioorg. Med. Chem. Lett. 1995, 5, 815.
9. Le Camus, C.; Badet-Denisot, M.-A.; Badet, B. Tetra-
hedron Lett. 1998, 39, 2571.
10. Le Camus, C.; Chassange, A.; Badet-Denisot, M.-A.;
Badet, B. Tetrahedron Lett. 1998, 39, 287.
11. Janiak, A.; Cybulska, B.; Szlinder-Richert, J.; Borowski,
E.; Milewski, S. Acta Biochim. Polon. 2002, 49, 77.
12. Bearne, S. L. J. Biol. Chem. 1996, 271, 3052.
13. Ong, S.; Liu, H.; Pidgeon, C. J. Chrom. A 1996, 728, 113.
14. Bearne, S. L.; Blouin, C. J. Biol. Chem. 2000, 275, 135.
15. Badet-Denisot, M.-A.; Leriche, C.; Badet, B. In Chitin
Enzymology; Muzzarelli, R.A.A., Ed., European Chitin
Society: Ancona, 1993, p 161.
Investigation of metabolism of ADGP derivatives in a
cell-free extract or by C. albicans cells
16. Teplyakov, A.; Obmolova, G.; Badet-Denisot, M.-A.;
Badet, B. Protein Sci. 1999, 8, 596.
A concentrated solution of compound 1, 2 or 7 was
added to the cell-free extract, prepared from C. albicans
ATCC 10261 cells as described previously, to give a
final concentration of 5 mg mLꢀ1. The mixture was
incubated at 30 ꢂC. Samples of 1 mL were collected at
hourly intervals and de-proteinised by addition of 1 mL
of ethanol. The protein precipitate was removed by fil-
tration and the composition of the filtrate was deter-
mined by TLC analysis of the filtrates (solvent system
A). Alternatively, C. albicans ATCC 10261 cells were
grown in YNB liquid medium at 30 ꢂC in the presence
17. Leriche, C.; Badet-Denisot, M.-A.; Badet, B. J. Am.
Chem. Soc. 1996, 118, 1797.
18. Milewski, S.; Andruszkiewicz, R.; Kasprzak, L.; Mignini,
F.; Mazerski, J.; Borowski, E. Antimicrob. Agents Chemother.
1988, 35, 36.
19. Becker, J. M.; Covert, N. L.; Shenbagamurthi, P.; Stein-
feld, A. S.; Naider, F. Antimicrob. Agents Chemother. 1983, 23,
926.
20. Hector, R. F.; Brown, P. C. Antimicrob. Agents Che-
mother. 1986, 29, 389.
21. Vespa, M. N.; Lebecq, J. C. Mycoses 1996, 39, 271.
22. Connors, T. A. Xenobiotica 1989, 16, 975.
23. Manoharan, M.; Johnson, L. K.; McGee, G. P.; Gui-
nosso, C. J.; Ramasamy, K.; Springer, R. H.; Bennett, C. F.;
Ecker, D. J.; Vickers, T.; Cowsert, L. Ann. New York Acad.
Sci. 1992, 660, 306.
or absence of an investigated compound, 1 mg mLꢀ1
.
The compound was added when the density of the cell
suspension measured at l=660nm reached .06. At
hourly time intervals samples of the cell suspension were
collected, cells were harvested by centrifugation and
broken by the small-scale glass beads procedure.31
Crude extracts were analysed TLC (solvent system A,
spots visualised with ninhydrin or Hanes reagent).
24. Merck, E. In Anfarbereagenzien fur Dunnschicht- und
¨
¨
¨
Papier-Chromatographie, Darmstadt, 1970, p 4–5.
25. Chargaff, E.; Bovarnick, M. J. Biol. Chem. 1937, 118, 421.
26. Kenig, M.; Vandamme, E.; Abraham, E. P. J. Gen.
Microbiol. 1976, 94, 46.
27. Noltmann, E. A. J. Biol. Chem. 1963, 239, 1545.
28. Chirgwin, J. M.; Noltmann, E. A. J. Biol. Chem. 1975,
250, 7272.
Acknowledgements
29. Document M27-A. National Committee for Clinical
Laboratory Standards: Wayne, PA, 1997.
30. Bartlett, G. J. Biol. Chem. 1959, 234, 466.
31. Bollag, D. M.; Edelstein, J. S. In Protein Methods. Wiley-
Liss: New York, 1991.
The authors acknowledge the financial support of these
studies by the State Committee for Scientific Research
(KBN), Warsaw (grant No. 4 P05F 00216) and in part by
the Chemical Faculty, Technical University of Gdansk.