Rational design of N-alkyl derivatives of 2-amino-2-deoxy-d-glucitol-6P as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2007.09.072

N-Alkyl and N,N-dialkyl derivatives of 2-amino-2-deoxy-d-glucitol-6P (ADGP) were synthesized and found to inhibit growth of human pathogenic fungi (MICs in the 0.08-0.625 mg mL^-1 range for the most active compounds). It was thus shown that N-alkylation of ADGP provides novel inhibitors of a fungal enzyme, glucosamine-6P synthase, exhibiting higher antifungal activity than the parent compound, due to the increased lipophilicity and better uptake by fungal cells.

Full text of DOI:10.1016/j.bmcl.2007.09.072

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6602–6606
Rational design of N-alkyl derivatives of
2-amino-2-deoxy-D-glucitol-6P as antifungal agents
Anna Melcer,a Izabela Ła˛cka,b Iwona Gabriel,b Marek Wojciechowski,b
a
a
b,
*
´
Beata Liberek, Andrzej Wisniewski and Sławomir Milewski
aDepartment of Chemistry, University of Gdan´sk, 18 Sobieskiego Street, 80-952 Gdan´sk, Poland
bDepartment of Pharmaceutical Technology & Biochemistry, Gdan´sk University of Technology,
11/12 Narutowicza Street, 80-952 Gdan´sk, Poland
Received 14 August 2007; revised 13 September 2007; accepted 18 September 2007
Available online 25 September 2007
Abstract—N-Alkyl and N,N-dialkyl derivatives of 2-amino-2-deoxy-D-glucitol-6P (ADGP) were synthesized and found to inhibit
growth of human pathogenic fungi (MICs in the 0.08–0.625 mg mLꢀ1 range for the most active compounds). It was thus shown
that N-alkylation of ADGP provides novel inhibitors of a fungal enzyme, glucosamine-6P synthase, exhibiting higher antifungal
activity than the parent compound, due to the increased lipophilicity and better uptake by fungal cells.
Ó 2007 Elsevier Ltd. All rights reserved.
Emerging challenge of systemic fungal infections, espe-
cially in immunocompromised patients, and a limited
repertoire of effective antifungals stimulate a quest for
novel targets and drug candidates. Enzymes involved
in biosynthesis of the fungal cell wall components are
of a special interest in this respect. Glucosamine-6P
(GlcN-6P) synthase catalyzes the first committed step
in the biosynthetic pathway leading to the formation
of UDP-GlcNAc, a sugar nucleotide precursor provid-
ing D-glucosamine for the formation of chitin and man-
noproteins.1 The enzyme was proposed as a target for
antifungal chemotherapy2 and a search for its selective
inhibitors as potential antifungals has been continued.
So far, two main groups of such compounds were iden-
tified: L-glutamine analogs and mimics of a putative cis-
enolamine transition state intermediate but none of
them demonstrated high antifungal activity, due to the
inefficient uptake by the fungal cells. On the other hand,
some of the L-glutamine analogs, namely N-acyl deriva-
tives of L-2,3-diaminopropanoic acid, gave rise to oligo-
peptides exhibiting high antifungal in vitro and in vivo
activity3 but poor serum stability and unfavorable phar-
macokinetic properties.4
2-Amino-2-deoxy-D-glucitol-6P (ADGP) and 2-amino-
2-deoxy-D-mannitol-6P (ADMP) are known as the
strongest inhibitors of fungal GlcN-6P synthase, belong-
ing to the second group, with inhibitory constants at
micromolar level.5,6 Our previous studies showed that
N-acylation of the 2-amino group and/or esterification
of the phosphate increased lipophilicity of the ADGP
molecule and facilitated uptake of the inhibitor into fun-
gal cells.7 Unfortunately, N-acylation of ADGP
strongly decreased GlcN-6P synthase-inhibitory poten-
tial of this molecule, thus suggesting that presence of
the 2-amino group, positively charged at physiological
conditions, may be of crucial importance for high en-
zyme-inhibitory activity.6 On the other hand, dimethyl
and diethyl esters of ADGP demonstrated improved
antifungal in vitro activity,7 but were unstable in human
serum, one may therefore predict their poor in vivo
efficiency.
Taking into account the above-mentioned results we
reasoned that N-alkylation of ADGP should afford
derivatives demonstrating substantially higher anti-
fungal in vitro activity than the parent molecule and
high stability in serum, provided their GlcN-6P syn-
thase-inhibitory potential is not diminished, relative to
ADGP. In the first stage of our project we performed
in silico docking simulations. Structures of N-mono-
alkyl- (C2–C6) and N,N-dialkyl- (C2–C6) derivatives of
ADGP were docked into an active site of the hexose-
Keywords: Antifungals; Aminoglucitol derivatives; Glucosamine-6-p-
hosphate synthase.
*
Corresponding author. Tel.: +48 58 3472107; fax: +48 58 3471144;
e-mail: milewski@chem.pg.gda.pl
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.072

A. Melcer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6602–6606
6603
phosphate isomerase domain (ISOM) of Candida albi-
cans GlcN-6P synthase.8 Inspection of the structures
of resulting enzyme: ligand complexes clearly showed
that the ligands can easily accommodate at the active
site, and all favorable ligand: active site residues interac-
tions—noted in the known crystal structure of the bac-
terial GlcN-6P synthase:ADGP complex9—can be
preserved. Figure 1 shows that presence of alkyl substit-
uents does not disturb ligand binding, as the alkyl
chain(s) can either fit near the hydrophobic entrance
to the ISOM binding pocket—shielded by Val709 and
Val501 residues—or even protrude out of the pocket.
Unfortunately, a three-dimensional structure of the
whole fungal GlcN-6P synthase is not known, so that
one cannot predict how the N-alkyl chains may interact
with the remaining part of the enzyme, namely the glu-
tamine amide hydrolyzing domain (GAH) and the puta-
tive interdomain linker. However, taking into account
the known structure of bacterial GlcN-6P synthase10
and some sequence homology between the prokaryotic
and eukaryotic version of the enzyme monomer sub-
units, it seems possible that the N-alkyl substituents
may also participate in the favorable interactions with
residues constituting the hydrophobic intramolecular
channel connecting active centers of ISOM and GAH.
Evidence supporting presence of such channel in C. albi-
cans enzyme has been shown recently.11
Scheme 1. Synthesis of N,N-dialkyl derivatives of ADGP.
our previous studies we showed that reductive alkyl-
ation of D-glucosamine under such conditions leads to
the formation of N-alkyl and N,N-dialkyl derivatives
of D-glucosamine.13 Now we found that the reductive
alkylation of ADGP 2, obtained from GlcN-6P 1 by
the method of Bearne,14 afforded unequivocally the
respective N,N-dialkyl derivatives, provided at least a
threefold molar excess of aldehyde with respect to 2
was used. Thus, the procedure shown in Scheme 1 was
applied for the preparation of N,N-dialkyl ADGP deriv-
atives 3–5.
N-Monoalkyl derivatives of ADGP were synthesized by
the two-step procedure15 shown in Scheme 2, starting
from 1, N-acylated in the first step with a respective car-
boxylic acid anhydride. Subsequently, the resulting N-
acyl-GlcN-6P was treated with LiBH4 in THF. Under
these conditions both an amide and an aldehyde groups
were reduced, thus affording N-monoalkyl ADGP deriv-
atives 6–8. Identity of compounds 3–8 was confirmed by
Results of the docking simulations suggested that N-al-
kyl derivatives of ADGP should at least preserve the en-
zyme-inhibitory potential of ADGP and encouraged us
to elaborate synthetic procedures for unequivocal syn-
thesis of N-alkyl and N,N-dialkyl derivatives of ADGP.
N,N-Dialkyl derivatives of ADGP were synthesized by
reductive alkylation involving the reaction of ADGP 2
with a suitable aldehyde in the presence of NaCNBH3
in the mixture of CH3CN–H2O ꢁ 3:1 (Scheme 1).12 In
1
IR, H NMR, and MS.16
ADGP 2 and its N-alkyl and N,N-dialkyl derivatives 3–8
were tested for physicochemical and biological proper-
ties, including apparent lipophilicity, enzyme-inhibitory
potential, uptake by fungal cells, and antifungal
in vitro activity. Affinity of the examined compounds
to the artificial biological membrane immobilized on
the bed of the IAM PC DD2 column, especially de-
signed for investigation of low and moderately lipophilic
compounds, was quantified and retention parameters
k0IAM were determined as a measure of apparent lipophil-
icity.17 Benzene and GlcN-6P, taken as reference com-
pounds, demonstrated the k0IAM values 0.8893 and
ꢀ0.7583, respectively. Data shown in Table 1 indicate
that ADGP exhibited the k0IAM value close to 0.0 and
all the mono and dialkyl derivatives of ADGP demon-
strated higher k0IAM values than the parent compound.
As expected, the affinity to the immobilized artificial
membrane depended on length and number of N-alkyl
Figure 1. N,N-Dipropyl- (top) and N-hexyl-ADGP (bottom) docked
into an active site of the ISOM domain of C. albicans GlcN-6P
synthase. Red wire model represents a reference ADGP conformation,
based on the geometry of its complex with a bacterial enzyme (1 mos).
Scheme 2. Synthesis of N-monoalkyl derivatives of ADGP.

6604
A. Melcer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6602–6606
Table 1. Data characterizing lipophilicity, uptake by C. albicans cells,
and inhibition of C. albicans GlcN-6P synthase by ADGP and its N-
alkyl derivatives
der of inhibitory potential reflected by Ki values deter-
mined from kinetic data was similar to that found for
the IC50 data. Interestingly, Floquet et al. reported re-
cently that Ki values of ADGP, N-acetyl-ADGP and
N-ethyl-ADGP toward Escherichia coli GlcN-6P syn-
thase were 25, 28, and 500 lM, respectively, so that in
the case of the bacterial enzyme, N-acetyl-ADGP was
as effective as ADGP, while N-ethyl-ADGP was much
poorer.19 It seems therefore possible that despite the
close sequence homology between E. coli and C. albicans
ISOM domains and conservation of all residues impor-
tant for catalytic activity, there could be a difference in
spatial arrangements of catalytic residues of bacterial
and fungal enzyme versions, especially in the region con-
taining residues interacting with the 2-amino group of
ADGP and its derivatives. None of the studied com-
pounds at concentrations up to 20 mM inhibited activity
of yeast phosphoglucose isomerase, thus confirming the
previously suggested structural requirements for effec-
tive transition state analog inhibitors of this enzyme.6
Compound Lipophilicity Initial
log k0IAM
Inhibition of
GlcN-6P synthasea
uptake ratea
(nmol/min/mg
dry weight)
IC50 (lM) Ki (lM)
2
3
4
5
6
7
8
ꢀ0.0437
0.3895
0.4625
0.5190
0.2283
0.3567
0.4222
1.1 ( 0.2)
3.9 ( 0.6)
4.7 ( 0.5)
5.0 ( 0.8)
2.9 ( 0.4)
3.4 ( 0.2)
4.8 ( 0.6)
230 ( 15) 35 ( 4)
40 ( 8)
52 ( 6)
82 ( 8)
180 ( 9)
115 ( 7)
8 ( 1)
10 ( 2)
12 ( 3)
26 ( 5)
20 ( 3)
290 ( 21) 49 ( 6)
a Values are means of three experiments, standard deviation is given in
parentheses.
substituents. However, all the compounds 3–8 could be
considered as moderately lipophilic and thus potentially
able to cross the real cell membrane by free diffusion.
This assumption was confirmed by the results of deter-
mination of initial uptake rates of compounds 2–8 by
C. albicans cells, shown in Table 1. The rates, deter-
mined under conditions described previously,7 were
much higher for N-alkyl ADGP derivatives, in compar-
ison to that of ADGP. Kinetic studies on the uptake of
N-alkyl derivatives of ADGP provided evidence for its
passive character, including a linear dependence of up-
take rates on initial compound concentration and lack
of any effect of metabolic inhibitors, such as NaN3 (de-
tailed results not shown). It is worth mentioning there-
fore that N-acyl ADGP derivatives were effectively
taken up by C. albicans cells by a passive transport
mechanism, while the ADGP molecule was slowly accu-
mulated by the way of an unidentified active transport
system.7 The novel N-alkyl ADGP derivatives 3–8 thus
closely resemble their N-acyl counterparts in terms of
lipophilicity and uptake properties.
Compounds 2–8 were tested for their antifungal in vitro
activity against 3 strains of human pathogenic yeast
from the genus Candida and 2 recombinant strains of
Saccharomyces cerevisiae: AD and ADCDR1. The AD
cells are sensitive to antifungals, due to the deletion of
the seven genes encoding main drug exporting mem-
brane proteins of S. cerevisiae,20 while the ADCDR1
cells were constructed by transformation of AD with a
plasmid containing a gene encoding Cdr1p, the main
drug exporter of C. albicans.21 Minimum inhibitory con-
centration (MIC) values determined by a serial dilution
microplate method22 for compounds 2–8 and a well-
known antifungal drug fluconazole as a reference are
summarized in Table 2.
Antifungal activity of ADGP (2) is very poor, since a
growth inhibitory effect was observed at concentrations
as high as 5–10 mg mLꢀ1. MIC values determined for N-
alkyl ADGP derivatives 3–8 were generally an order or
even nearly two orders of magnitude lower and the
lowest values 0.08–0.625 mg mLꢀ1 were observed for
N,N-dialkyl compounds 3 and 4. Presence of 10 mM
N-acetyl-D-glucosamine in the growth medium com-
pletely abolished the antifungal activity of investigated
compounds, thus indicating that GlcN-6P synthase is
their primary target in fungal cells. Comparison of the
MIC values determined against the recombinant S. cere-
visiae cells AD and ADCDR1 clearly indicates that
presence of the Cdr1p, mediating multidrug resistance,
does not affect antifungal activity of both ADGP and
its N-alkyl derivatives, while strongly decreases that of
fluconazole. This result suggests that ADGP and its
derivatives are probably not extruded from yeast cells
by the Cdr1p drug transporter.
Compounds 2–8 were tested for their GlcN-6P synthase-
and phosphoglucose isomerase-inhibitory activities.18
Both these enzymes utilize D-fructose-6-phosphate as a
substrate and catalyze ketose–aldose isomerization,
according to the very similar mechanisms involving,
respectively, cis-enolamine or cis-endiol intermediate.
N-alkyl ADGP derivatives 3–8 appeared relatively
strong inhibitors of C. albicans GlcN-6P synthase, as
under in vitro conditions, 50% inhibition of enzyme
activity (IC50) was observed in the 40–290 lM range.
Data shown in Table 1 reveal that the inhibitory poten-
tial in terms of the IC50 values of nearly all the N-alkyl
derivatives, except the N-hexyl-ADGP 8, was even high-
er than that of ADGP. Especially low IC50 values were
found for N,N-dialkyl compounds 3–5. This is in con-
trast to the inhibitory potential of N-acyl ADGP deriv-
atives, for which we previously found the IC50 values in
the 6–19 mM range.6 Good inhibitory potential of com-
pounds 3–8 thus confirmed the suggestion resulting
from the docking simulations described above. Inhibi-
tion of C. albicans GlcN-6P synthase by ADGP and
its N-alkyl derivatives was competitive in respect to D-
Fru-6P and non-competitive in respect to L-Gln. The or-
Compounds 2–8 were stable in human serum, as no
products of their possible degradation were detected
by HPLC23 upon their incubation for 72 h at 37 °C in
5% human serum (details not shown).
According to our best knowledge, N-alkyl derivatives of
ADGP constitute the first reported example of N-alkyl-

A. Melcer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6602–6606
Table 2. Antifungal in vitro activity of ADGP and its N-alkyl derivatives
6605
Compound
MIC (mg mLꢀ1
)
Candida albicans Candida glabrata Candida tropicalis Saccharomyces cerevisiae AD Saccharomyces cerevisiae ADCDR1
2
5
10
0.625
10
0.625
5
5
3
0.31
0.16
0.312
1.25
0.625
0.625
0.008
0.31
0.08
1.25
2.5
0.31
0.16
1.25
2.5
4
0.625
1.25
2.5
0.625
2.5
2.5
5
6
7
1.25
0.625
0.016
1.25
0.31
0.016
1.25
0.31
0.004
1.25
0.31
0.032
8
FLUCa
a FLUC, fluconazole.
11. Olchowy, J.; Gabriel, I.; Milewski, S. Biochem. J. 2007,
404, 121.
ated aminohexitol phosphates demonstrating antimicro-
bial activity. In the literature, there are also very few
examples of structurally related N-alkylated amino sug-
ars exhibiting any type of biological activity.24,25 Struc-
tures of our novel compounds have been rationally
designed, taking into account results of in silico docking
simulations and predictions concerning expected
improvement of the uptake parameters upon introduc-
tion of alkyl substituents at the 2-amino group of
ADGP. This approach has appeared successful,
although the antifungal activity of the obtained com-
pounds is not high enough to consider them true drug
candidates.
12. General procedure for N,N-dialkylation of ADGP:
ADGP 2 (200 mg, 0.76 mmol) and appropriate aldehyde
(2.28 mmol) were stirred in the mixture of acetonitrile and
water (3:1, 14 mL) at rt for 30 min. Then NaCNBH3
(132 mg, 2.1 mmol) was added and stirring was continued
for 1 h at rt. The end of reaction was detected by TLC
(MeOH/NH3/H2O, 6:2:1). The reaction mixture was
concentrated and chromatographed on silica gel (MeOH/
NH3/H2O, 6:2:1).
´
13. Liberek, B.; Melcer, A.; Osuch, A.; Wakiec, R.; Milewski,
´
S.; Wisniewski, A. Carbohydr. Res. 2005, 340, 1876.
14. Bearne, S. L. J. Biol. Chem. 1996, 271, 3052.
15. General procedure for N-monoalkylation of ADGP:
GlcN-6P 1 (200 mg, 0.76 mmol) and NaHCO3 (300 mg,
3.6 mM) were dissolved in a smallest possible amount of
water (ꢁ1.5 mL) and 25% acetone solution of appropriate
anhydride (1.5 mM) was added dropwise. The reaction
mixture was stirred at rt for 24 h. Then isopropanol
(20 mL) was added and stirring was continued for a few
min. The reaction mixture was concentrated and purified
on a Dowex 50 WX4 (H+) ion-exchange resign. Obtained
N-acyl derivative of GlcN-6P (0.66 mmol) was dissolved in
dry THF (5 mL) and LiBH4 (216 mg, 9.9 mmol) was
added. The reaction mixture was stirred at rt for 2 h.
When the end of reduction was detected by TLC (MeOH/
CHCl3, 1:1), methanol (5 mL) was added and the reaction
mixture was kept at rt for a few hours. Then it was
concentrated and purified on a Dowex 50 WX4 (H+) ion-
exchange resign.
References and notes
1. Herscovics, A.; Orlean, P. FASEB J. 1993, 7, 540.
2. Borowski, E. Il Farmaco 2000, 55, 206.
3. Andruszkiewicz, R.; Milewski, S.; Zieniawa, T.; Borowski,
E. J. Med. Chem. 1990, 33, 132.
4. Kasprzak, L.; Gumieniak, J.; Milewski, S.; Borowski, E.
J. Chemother. 1992, 4, 88.
5. Badet-Denisot, M.-A.; Leriche, C.; Masserie, F.; Badet, B.
Bioorg. Med. Chem. Lett. 1995, 5, 815.
6. Milewski, S.; Janiak, A.; Wojciechowski, M. Arch.
Biochem. Biophys. 2006, 450, 39.
7. Janiak, A. M.; Hoffmann, M.; Milewska, M. J.; Milewski,
S. Bioorg. Med. Chem. 2003, 11, 1653.
8. The structure of the receptor (ISOM domain of C. albicans
GlcN-6P synthase) was homology modeled on the basis of
the available crystal structure of E. coli enzyme (1jxa)
which was used as a template. The structures of ligands
studied were built by means of InsightII molecular
modeling package from Accelrys,26 as described earlier.6
The putative geometries of the ligand–enzyme complexes
were then obtained by docking each of the ligands to the
modeled receptor using the AutoDock program.27
Lamarckian genetic algorithm (LGA) was used as the
search method and ligands were flexible during the
calculations with all single bonds marked for rotation.
The crystal geometry of ADGP complexed with E. coli
GlcN-6P synthase ISOM domain (1 mos) was used as the
reference. For each ligand studied, geometry of the ligand
best resembling the reference was, in all cases, found either
as the best scored solution or within the top five solutions
found by AutoDock.
16. Compound 3: Yield 86%, colorless syrup. IR: m 3460, 3140
(O–H), 3040, 2806 (C–H), 2349 (R3N–H+) cmꢀ1 1H
;
NMR (400 MHz, D2O): d 4.33 (d, J = 8.8 Hz, 1H), 4.04
(m, 4H), 3.80–3.85 (m, 3H), 3.59 (m, J = 6.8 Hz, 2H,
CH2), 3.43 (m, 2H, CH2), 1.41 (t, J = 7.2 Hz, 6H, 2CH3).
MALDITOF: m/e 317.1 (M+Å), 318.1 (M+Å+1), 339.0
(M+Åꢀ1+Na), 361.0 (M+Åꢀ2+2 Na).
Compound 4: Yield 78%, colorless syrup. IR: m 3500, 3235
(O–H), 3043, 2816 (C–H), 2349 (R3N–H+) cmꢀ1 1H
;
NMR (400 MHz, D2O): d 4.21 (d, 1H), 3.96–4.02 (m, 3H),
3.78–3.86 (m, 2H), 3.64 (dd, J = 10.8, 6.4 Hz, 1H), 3.51
(dd, J = 10.8, 7.2 Hz, 1H), 3.26 (m, 2H, CH2), 3.14 (m, 2H,
CH2), 1.78 (m, 4H, 2CH2), 1.01 (t, J = 7.3 Hz, 6H, 2CH3).
MALDITOF: m/e 345.2 (M+Å), 346.2 (M+Å+1).
Compound 5: Yield 90%, colorless syrup. IR: m 3234 (O–
H), 3070, 2964, 2936 (C–H), 2347 (R3N–H+) cmꢀ1 1H
;
NMR (500 MHz, D2O): d 4.14 (d, J = 8.79 Hz, 1H), 3.80–
3.89 (m, 4H), 3.64–3.68 (m, 3H), 3.30 (m, 2H, CH2), 3.13
(m, 2H, CH2), 1.66 (m, 2H, CH2), 1.57 (m, 2H, CH2), 1.25
(m, J = 7.32 Hz, 4H, 2CH2), 0.80 (t, J = 7.32 Hz, 6H,
2CH3). MALDITOF: m/e 371.5 (M+Åꢀ2), 394.5
(M+Åꢀ2+Na).
9. Teplyakov, A.; Obmolova, G.; Badet-Denisot, M.-A.;
Badet, B. Protein Sci. 1999, 8, 596.
10. Teplyakov, A.; Obmolova, G.; Badet, B.; Badet-Denisot,
M.-A. J. Mol. Biol. 2001, 313, 1093.

6606
A. Melcer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6602–6606
Compound 6: Yield 88%, colorless syrup. IR: m 3296 (O–
concentration. For determination of IC50 values concen-
1
H), 2943, 2883 (C–H), 2348 (R2HN–H+) cmꢀ1; H NMR
trations of both substrates (D-Fru-6P and L-Gln) were
10 mM. Data were plotted as Michaelis–Menten graphs
and kinetic data were subjected to the non-linear regres-
sion analysis. The IC50 and Ki were determined in
triplicate, to give the mean values SD. Activity of yeast
phosphoglucose isomerase (Sigma) was determined using
the Glc-6P dehydrogenase-coupled assay.30
(400 MHz, D2O): d 4.18 (m, 1H), 4.12 (m, J = 6.8 Hz, 2H),
3.91 (dt, J = 12.4, 3.6 Hz, 1H), 3.80 (dd, 1H), 3.74 (dt,
1H), 3.68 (m, 1H), 3.61 (m, 2H, CH2), 3.55 (m, 1H), 1.58
(t, J = 6.4 Hz, 3H, CH3). MALDITOF: m/e 288.2 (M+Å).
Compound 7: Yield 77%, colorless syrup. 3200 (O–H),
2934 (C–H), 2397 (R2HN–H+) cmꢀ1; 1H NMR(400 MHz,
D2O): d 4.04–4.18 (m, 3H), 3.96 (m, 1H), 3.86 (m, 1H),
3.73 (m, 1H), 3.64 (m, 1H), 3.57 (m, 2H, CH2), 3.55 (m,
1H), 1.55 (m, 4H, 2CH2), 0.86 (t, 3H, CH3). MALDITOF:
m/e 317.0 (M+Å), 339.0 (M+Åꢀ1+Na).
19. Floquet, N.; Richez, C.; Durand, P.; Maigret, B.; Badet,
B.; Badet-Denisot, M.-A. Bioorg. Med. Chem. Lett. 2007,
17, 1966.
20. Decottignies, A.; Grant, A. M.; Nichols, J. W. J. Biol.
Chem. 1998, 274, 12612.
21. Smriti; Krishnamurthy, S.; Dixit, B. L.; Gupta, C. M.;
Milewski, S.; Prasad, R. Yeast 2002, 19, 303.
22. Document M27-A2. National Committee for Clinical
Laboratory Standards. Wayne, PA, 2002.
Compound 8: Yield 94%, colorless syrup. IR: m 3334 (O–
1
H), 2957, 2931, 2860 (C–H), 2336 (R2HN–H+) cmꢀ1; H
NMR (400 MHz, D2O): d 4.08–4.20 (m, 3H), 3.90 (dt, 1H,
J = 10.4 Hz, J = 3.2 Hz), 3.82 (m, 1H), 3.75 (m, 1H), 3.64
(m, 1H), 3.60 (m, 2H, CH2), 3.55 (m, 1H), 1.57 (m, 4H, 2
CH2), 1.28 (m, 4H, 2CH2) 0.86 (t, 3H, CH3). MALDI-
TOF: m/e 344.1 (M+Åꢀ1), 367.1 (M+Åꢀ1+Na).
23. Examined compounds were dissolved in a solution of 5%
human serum and the mixtures were incubated for 72 h at
37 °C. Samples withdrawn at 12 h intervals were deprote-
inized with 1.5 M HClO4, neutralized with 2 M K2CO3,
centrifuged, and supernatants were analyzed by HPLC on
a Phenomenex Luna amino column developed with
acetonitrile-phosphate buffer (75:25, v/v, pH 7.5), flow
17. Interactions between the examined compounds and the
IAM PC DD2 column (Regis Technologies, Inc., Morton
Grove, IL, USA) were studied under conditions described
previously.7 Briefly, the column loaded with a sample was
developed with the solvent system acetonitrile/50 mM
potassium phosphate buffer, pH 7.2 (50:50, 30:70, 20:80,
10:90, 5:95 v/v) containing 0.1% acetone, flow rate
1 mL minꢀ1. Retention times of the studied compounds
(tR) were determined for different compositions of the
mobile phase and used to calculate the capacity parameters
rate 1.5 mL minꢀ1
.
24. Liu, H.-M.; Zhang, F.; Wang, S. Org. Biomol. Chem.
2003, 1, 1641.
25. Ogawa, S.; Sakata, Y.; Ito, N.; Watanabe, M.; Kabayama,
K.; Itoh, M.; Korenaga, T. Bioorg. Med. Chem. 2004, 12,
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26. InsightII, Accelrys, San Diego, CA, 1997. Available from
<http://www.accelrys.com>.
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k0IAM = (tR ꢀ t0)/t0. Values of logk0
were plotted
IAM
against the acetonitrile content in the mobile phase and
the resulting linear plots were extrapolated to give the kI0AM
value for the hypothetical purely aqueous mobile phase.
18. Candida albicans GlcN-6P synthase was purified to near
homogeneity, as described previously.28 Enzyme activity
was determined by the modified Elson–Morgan method.29
Reactions were carried out in 25 mM phosphate buffer,
pH 6.9, containing 1 mM EDTA, 1 mM DTT, D-Fru-6P
(0.5–7.5 mM), L-glutamine (0.625–10 mM), GlcN-6P syn-
thase (0.1–0.2 lM), and a given inhibitor in appropriate
28. Sachadyn, P.; Je˛drzejczak, R.; Milewski, S.; Kur, J.;
Borowski, E. Protein Expr. Purif. 2000, 19, 343.
29. Kenig, M.; Vandamme, E.; Abraham, E. P. J. Gen.
Microbiol. 1976, 94, 46.
30. Noltman, E. A. J. Biol. Chem. 1963, 239, 1545.