Synthesis of β-D-galactofuranosyl nucleoside analogues. A new type of β-D-galactofuranosidase inhibitor

Article abstract of DOI:10.1016/S0008-6215(01)00146-X

The development of β-D-galactofuranosidase inhibitors provides a good chemotherapeutic target for treatment of major human diseases, because β-D-galactofuranose is a constituent of important pathogen microorganisms but is absent in mammals. With this purpose we have prepared β-D-galactofuranosyl nucleoside analogues, derived by the addition of nucleophiles to perbenzoylated β-D-galactofuranosyl isothiocyanate, a compound previously prepared in this laboratory. N-β-D-Galactofuranosyl-O-ethylthiourethane, N-β-D-galactofuranosyl-4-oxoimidazolidine-2-thione, N-β-D-galactofuranosyl-4-imidazoline-2-thione, and N-β-D-galactofuranosyl-4-methoxyimidazolidine-2-thione, were prepared. The biological assays showed that imidazoline and imidazolidine-2-thione derivatives act as a new type of exo β-D-galactofuranosidase inhibitor.

Full text of DOI:10.1016/S0008-6215(01)00146-X

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Carbohydrate Research 333 (2001) 123–128
Synthesis of b-
D
-galactofuranosyl nucleoside analogues.
A new type of b-
D-galactofuranosidase inhibitor
Carla Marino,a,* Pal Herczegh,b Rosa M. de Lederkremera
aCIHIDECAR, Departamento de Qu´ımica Orga´nica, Facultad de Ciencias Exactas y Naturales,
Uni6ersidad de Buenos Aires, Pabello´n II, Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina
bResearch Group for Chemistry of Antibiotics of the Hungarian Academy of Sciences,
and Faculty of Pharmaceutical Chemistry, Uni6ersity of Debrecen, H-4010 Debrecen, Hungary
Received 8 January 2001; accepted 11 May 2001
Abstract
The development of b-
major human diseases, because b-
absent in mammals. With this purpose we have prepared b-
addition of nucleophiles to perbenzoylated b- -galactofuranosyl isothiocyanate, a compound previously prepared in
this laboratory. N-b- -Galactofuranosyl-O-ethylthiourethane, N-b- -galactofuranosyl-4-oxoimidazolidine-2-thione,
N-b- -galactofuranosyl-4-imidazoline-2-thione, and N-b- -galactofuranosyl-4-methoxyimidazolidine-2-thione, were
prepared. The biological assays showed that imidazoline and imidazolidine-2-thione derivatives act as a new type of
exo b- -galactofuranosidase inhibitor. © 2001 Elsevier Science Ltd. All rights reserved.
D
-galactofuranosidase inhibitors provides a good chemotherapeutic target for treatment of
-galactofuranose is a constituent of important pathogen microorganisms but is
-galactofuranosyl nucleoside analogues, derived by the
D
D
D
D
D
D
D
D
Keywords: Galactofuranosidase inhibitors; Isothiocyanate; Thiohydantoin; 4-Imidazoline; Imidazolidine-2-thion derivatives
found in the lipopeptidophosphoglycan
(LPPG) of Trypanosoma cruzi.1 Variations in
the galactofuranosyl content of these glyco-
conjugates could be due to the action of an
1. Introduction
The development of specific inhibitors of
glycosidases is one of the primary objectives
of glycobiology. b- -Galactofuranose is a
exo b-
We have previously developed the synthesis
of 1-thio-b- -galactofuranosides, and they
D
-galactofuranosidase.
D
constituent of important microorganism gly-
coconjugates. The absence of galactofuranose
in mammal glycoconjugates suggests that the
enzymes involved in the metabolism of this
sugar in bacteria, fungi and protozoa would
be a good target for the design of drugs.1 An
D
have been evaluated as inhibitors of the en-
zyme from P. fellutanum.5 Taking into ac-
count their biological activity, we have
developed an affinity chromatographic system
for the isolation of the enzyme6 and studied
the influence of the inhibitors in cultures of
the fungi (unpublished).
exo b- -galactofuranosidase has been detected
D
in only a few species: Penicillium fellutanum,2
Helminthosporium sacchari,3 and Aspergillius.4
Terminal nonreducing residues of Galf are
We also found that
D
-galactono-1,4-lactone
inhibits the enzyme,5 and in general, this char-
acteristic of the aldonolactones has been at-
tributed to their structural similarity with the
glycosyloxocarbonium ion intermediate of the
* Corresponding author. Tel./fax: +54-11-45763352.
E-mail address: cmarino@qo.fcen.uba.ar (C. Marino).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00146-X

124
C. Marino et al. / Carbohydrate Research 333 (2001) 123–128
enzymatic glycoside cleavage.7 The N-thiocar-
bonyl glycosyl derivatives also provide planar
resonance structures, similar to the hydrolytic
reaction intermediate. Thus, these type of
compounds, usually obtained from the corre-
sponding isothiocyanates, are convenient pre-
cursors for the design and synthesis of
glycosidase inhibitors.8,9 Furthermore, thiohy-
dantoin derivatives have previously been
shown to act as inhibitors of carbohydrate
processing enzymes.10,11
1H and 13C NMR spectra. The 13C NMR
spectrum of 5 showed signals at 179.1 and
168.2 ppm, which were assigned to the thio-
carbonyl and the carbonyl groups of the thio-
hydantoin moiety, respectively, and a signal at
l 46.8 for C-5 of the same unit. Signals for the
sugar carbon atoms were similar to those ob-
served for other b- -galactofuranosyl deriva-
D
tives, with a higher field signal for C-1% (l
88.5), because of the higher shielding effect of
nitrogen, in comparison with the oxygenated
analogues (C-1, l 105).14
The synthesis of 2,3,5,6-tetra-benzoyl-b- -
D
galactofuranosyl isothiocyanate (1) was previ-
ously described as a simple and efficient
one-step procedure from 1,2,3,5,6-penta-O-
Nucleophilic addition of aminoacetaldehyde
dimethyl acetal to 1 led almost quantitatively,
after 2 h of reaction, to the thiourea derivative
benzoyl-b-
-galactofuranose.12,13 We also de-
D
1
6 (92%, Scheme 2). The H NMR spectrum of
scribed its reaction with several alcohols,
amines, and amino acids, which led to a vari-
6 showed the characteristic galactofuranosyl
pattern, with a signal at 6.08 ppm for the
anomeric proton as observed for analogous
ety of N-glycosyl derivatives of b- -
D
b-
-galactofuranosyl nucleosides.12 The 13C
D
galactofuranose.
We now describe the preparation of 1,3-di-
NMR spectrum showed the resonance for the
thiocarbonyl group at 183.8 ppm and signals
at 46.8 (C-4), 102.2 (C-5) and 54.4 ppm
(OCH3), as expected for the aglycon moiety.
aza-1-b- -galactofuranosyl-2-thione heterocy-
D
cles, starting from compound 1, and their
evaluation as inhibitors of exo b-
D
-galacto-
furanosidase from P. fellutanum.
For the b- -galactofuranosyl unit, the ex-
D
pected signals were observed.
Compound 6 was treated with 0.1 N HCl in
DMF in order to hydrolyze the acetal group
and lead to cyclization by condensation of the
generated aldehyde with the glycosylic nitro-
gen. A similar reaction was earlier communi-
2. Results and discussion
Debenzoylation of compound 2, derived
from the addition of ethanol to 1,13 led to
fully unprotected compound 3 (Scheme 1).
Similar treatment of 4, obtained by condensa-
cated for the
D
-ribofuranose analogue of 6,
which led to the imidazole-2-thione derivative
in 29% yield. This structure was assigned on
tion of 1 and
L
-glycine ethyl ester12 led, by
1
the basis of the H NMR spectrum. The reac-
cyclization, to the glycosyl thiohydantoin 5.
This structure was assigned on the basis of the
tion time was not specified and no other
Scheme 1. (i) EtOH, 60 °C; (ii) NaOMe–MeOH; (iii) H2NCH2CO2Et·HCl, toluene, EDPA.

C. Marino et al. / Carbohydrate Research 333 (2001) 123–128
125
Scheme 2. (i) Dimethylaminoacetaldehyde, toluene; (ii) HCl 0.1 M–DMF; (iii) NaOMe–MeOH.
product was isolated.15 In our case, we ob-
served that, at room temperature, the reaction
proceeded very slowly and after 30 h, two
products (7 and 8) with similar chromato-
graphic behavior were formed, although start-
ing compound 6 was still present. Isolation by
column chromatography (4:1 toluene–ethyl
acetate) afforded 7 (52%), 8 (35%), along with
some unreacted 6 (8%). When the reaction
was performed by heating in a boiling-water
bath, the starting material was completely
transformed into compound 7 (69%) in 2 h,
and no compound 8 was detected. The iso-
lated compound 8 could also be transformed
into 7 by heating under reflux with 0.1 N HCl
in DMF.
No vinylic carbons were detected, and signals
at l 64.9 and 93.5 were observed instead. The
1H NMR spectrum of 8 showed the presence
of two epimeric compounds in similar
amounts, displaying the pattern of a methyl-
ene group with no equivalent hydrogens as
two double doublets centered at 3.96 and 4.14
ppm (H-5a,5b), with a large Jgem (14.2 Hz)
coupling, each of them coupled with a depro-
tected proton (H-4). This pattern indicated the
imidazolidine structure 8. Both epimers
showed distinguishable signals for the aglycon
moiety and the anomeric proton, but the
sugar signals were overlapped.
Compounds 7 and 8 were debenzoylated
(NaOMe–MeOH), affording compounds 9
and 10. For compound 9, the1H NMR spec-
trum showed vinylic signals at 7.21 and 6.88
ppm with J4,5 2.7 Hz, and the galactofuranosyl
signals with J1%,2% 5.0 Hz, larger than for 7,
suggesting that a change in the conformation
occurred, as previously observed for
analogous compounds.8,12 In the 13C NMR
spectrum of 9 the vinylic carbons were ob-
served at 115.6 and 115.2 ppm, and the signal
at l 162 showed that compound 9 was in the
thioenolic form.
1
The H NMR spectrum of 7 showed a
doublet at 6.70 ppm for the anomeric proton
with J1,2ꢀ2.5 Hz, characteristic of the b- -
D
Galf configuration. For the 4-imidazoline-2-
thione ring, the vinylic protons showed
overlapped signals at 6.98 ppm with J4,5 4.8
Hz, in agreement with the values reported for
the ribofuranosyl analogue.15 The 13C NMR
spectrum showed a signal at 162.9 ppm for
C-2, indicating that the tautomeric equi-
librium of the imidazole moiety was mainly
displaced to the thioenol form, and vinylic
signals (C-4,5) were observed at l 114.6 and
114.9.
Compound 10 showed similar signals to 9
for the sugar unit, and to 8 for the aglycon
moiety, indicating unequivocally the presence
of the methoxyl group, and the thioenolic
form. On being kept, the epimeric mixture of
10 partly decomposed to 9, and only one of
the epimers was evident by NMR. NOE ex-
The 13C NMR spectrum of 8 clearly showed
that one of the methoxyl groups was still
present in this compound, and the 2-thione
function was present as the thioenolic form.

126
C. Marino et al. / Carbohydrate Research 333 (2001) 123–128
periments did not afford information on the
configuration of C-4, due to the conforma-
tional flexibility of the molecule. Attempts to
crystallize compounds 8 and 10 for the deter-
mination of the absolute configuration were
not successful.
3. Experimental
General methods.—Optical rotations were
measured with a Perkin–Elmer 343 polarime-
ter. NMR spectra were recorded with Bruker
AC 200 or AM 500 spectrometers. TLC exam-
ination was performed on precoated plates (E.
Merck, Silica 60 F254). Column chromatogra-
phy was performed on Silica Gel 60 (200–400
mesh, E. Merck). Compounds 213 and 412 were
prepared as previously described.
With the purpose of evaluating compounds
3, 5, 9, and 10 as inhibitors of exo b- -galac-
D
tofuranosidase, we performed typical experi-
ments for the measurement of the enzymatic
activity, in the presence of different amounts
of these compounds (Fig. 1). 4-Nitrophenyl
N-(2,3,5,6-Tetra-O-benzoyl-i-
furanosyl)-N%-(2,2-dimethoxyethyl)
D
-galacto-
b-
D
-galactofuranoside16 was used as substrate
thiourea
(Fig. 1), and
D
-galactono-1,4-lactone was
(6).—To a solution of 2,3,5,6-tetra-O-ben-
-galactofuranosyl isothiocyanate (1,
tested as a reference inhibitor (IC50 0.03
zoyl-b-
D
mM).6
0.70 g, 1.01 mmol)12,13 in anhyd toluene (15.0
mL), aminoacetaldehyde dimethyl acetal (0.16
mL, 1.5 mmol) was added. The solution was
kept at rt until complete reaction occurred (2
h). TLC examination (2:1 toluene–EtOAc)
showed a slower-moving component (Rf 0.35)
than 1 (Rf 0.80). The solution was poured over
5% HCl, extracted (CH2Cl2), and dried
(MgSO4). After evaporation of the solvent,
compound 6 (0.69 g, 92%) was obtained as a
We observed that 3 and 5 were not biologi-
cally active (data not shown). Compound 9
was found to be a weak inhibitor, with IC50
1.05 mM. Compound 10 was ten times more
active (IC50 0.10 mM), suggesting that a
change in the flexibility of the imidazolidine
ring facilitates the interaction with the active
site of the enzyme.
In summary, we found a new type of b- -
D
galactofuranosidase inhibitor, the imidazole-2-
thione derivatives, and observed that the
activity is dependent on the structure of the
imidazole ring. This fact opens the possibility
of continuing with the design of more potent
inhibitors by modifications of this structure.
Furthermore, the comparative activity of these
compounds should provide information about
the chemical topography of the active site of
the enzyme.
1
syrup having [h]D −25° (c 1, CHCl3); H
NMR (200 MHz, CDCl3), l 7.00 (s, 1 H,
NH), 6.08 (m, 2 H, H-1,5), 5.71, (m, 2 H,
H-2,3), 4.77 (m, 3 H, H-4,6a,b), 4.52 (t, 1 H,
J4%,5% 5.1 Hz, H-5%), 3.73 (d, 2 H, H-4%), 3.36 (2
13
s, 6 H, OCH3); C NMR (25.3 MHz, CDCl3),
l 183.8 (C-2%), 102.2 (C-5%), 88.3 (C-1), 81.6
(C-4), 80.7 (C-2), 78.1 (C-3), 71.1 (C-5), 63.3
(C-6), 46.8 (C-4%). Anal. Calcd for
C39H38N2O11S: C, 63.06; H, 5.16. Found: C,
63.19, H, 5.10.
N-(2,3,5,6-Tetra-O-benzoyl-i-
furanosyl)-4-imidazoline-2-thione (7) and N-
(2,3,5,6-tetra-O-benzoyl-i- -galactofuranosyl)-
D
-galacto-
D
4-methoxyimidazolidine-2-thione (8).—Com-
pound 6 was dissolved in 0.1 N HCl–DMF
and stirred during 48 h at rt. TLC analysis
showed two products (Rf 0.26 and 0.18) and
some starting material. Complete reaction did
not occurred, even after several days. Column
chromatography (5:1 toluene–EtOAc) of the
mixture gave compound 7 (0.28 g, 52%), 8
(0.20 g, 35%), and 6 (0.04 g, 8%).
Fig. 1. Effect of concentration of inhibitors on the enzymatic
activity of b-D-galactofuranosidase from P. fellutanum. The
When the reaction was performed in a boil-
ing-water bath, the starting material was com-
pletely transformed after 1 h, and only
amount of 4-nitrophenol released from 4-nitrophenyl b-D-
galactofuranoside was determined as a measure of galacto-
furanosidase activity.

C. Marino et al. / Carbohydrate Research 333 (2001) 123–128
127
H-5); 13C NMR (D2O, 25.3 MHz), l 179.1
(C-2), 168.2 (C-4), 88.5 (C-1%), 84.8 (C-4%), 80.3
(C-2%), 75.1 (C-3%), 71.8 (C-5%), 62.9 (C-6%), 49.7
(C-5). Anal. Calcd for C9H14N2O6S: C, 38.84,
H, 5.07. Found: C, 39.02, H, 5.10.
compound 7 (0.37, 69%) was obtained. Com-
1
pound 7 had [h]D −19° (c 1, CHCl3); H
NMR (200 MHz, CDCl3), l 6.98 (d, 2 H, J4,5
4.8 Hz, H-4,5), 6.70 (d, 1 H, J1%,2% 2.5 Hz,
H-1%), 6.09 (m, 1 H, J5%,6%a 3.5, J5%,6%b 4.6 Hz,
H-5%), 5.98 (dd, 1 H, J2%,3% 2.5, J3%,4% 3.3 Hz,
H-3%), 5.73, (dd, 1 H, H-2%), 4.88 (dd, 1 H, J4%,5%
3.3 Hz, H-4%), 4.82 (dd, 1 H, J6%a,6%b 11.9 Hz,
N-i- -Galactofuranosyl-4-imidazoline-2-
D
thione (9).—Yield 92%, [h]D −22° (c 1, wa-
1
ter); H NMR (500 MHz, Me2SO-d6), l 7.21
13
H-6%a), 4.72 (dd, 1 H, H-6%b); C NMR (25.3
(d, 1 H, J4,5 2.7 Hz, H-4), 6.88 (d, 1 H, H-5),
6.06 (d, 1 H, J1,2 5.0 Hz, H-1%), 4.07 (m, 2 H,
J2%,3% 2.5, J3%,4% 3.3 Hz, H-2%,3%), 4.04 (dd, 1 H,
J4%,5% 3.3 Hz, H-4%), 3.39 (m, 2 H, H-6%a,b), 3.03
MHz, CDCl3), l 162.9 (C-2), 114.9, 114.6
(C-5,4), 89.8 (C-1%), 84.0 (C-4%), 80.6 (C-2%),
78.9 (C-3%), 71.7 (C-5%), 63.2 (C-6%). Anal.
Calcd for C37H30N2O9S: C, 65.48; H, 4.45.
Found: C, 65.53, H, 4.59.
13
(m, 1 H, H-5%); C NMR (25.3 MHz, Me2SO-
d6), l 162.0 (C-2), 115.6, 115.2 (C-5,4), 89.2
(C-1%), 83.5 (C-4%), 80.7 (C-2%), 75.3 (C-3%), 70.5
(C-5%), 62.3 (C-6%). Anal. Calcd for
C9H14N2O5S: C, 41.22; H, 5.38. Found: C,
41.35, H, 5.26.
Compound 8 had [h]D −7° (c 1, CHCl3);
1H NMR (200 MHz, CDCl3), selected data: l
5.84 (d, 1 H, J1%,2% 2.0 Hz, H-1%), 5.55 (dd, 1 H,
J4,5a 5.1, J4,5bꢀ1.0 Hz, H-4), 4.15, 3.96 (2 dd,
2 H, J5a,5b 14.2 Hz, H-5a,b). For the other
epimer, l 5.89 (d, 1 H, J1%,2% 2.0 Hz, H-1%), 5.48
(dd, 1 H, J4,5a 4.8, J4,5b ꢀ1.0 Hz, H-4), 4.10,
3.87 (2 dd, 2 H, J5a,5b 14.2 Hz, H-5a,b). 13C
NMR (25.3 MHz, CDCl3), l 160.2 (C-2),
93.5, 93.1 (C-4 both epimers), 90.7 (C-1%), 81.1
(C-2%,4%), 78.3 (C-3%), 71.3 (C-5%), 64.9, 64.6
(C-5 both epimers), 63.6 (C-6%), 55.9 (OCH3).
Anal. Calcd for C38H34N2O10S: C, 64.22; H,
4.82. Found: C, 64.00, H, 4.69.
N-i- -Galactofuranosyl-4-methoxyimidazo-
D
lidine-2-thione (10).—Yield 93%, [h]D −64°
1
(c 1, water); H NMR (500 MHz, Me2SO-d6),
selected data: l 5.63 (d, 1 H, J1%,2% 5.2 Hz, H-1%,
both epimers), 5.23, 5.18 (2 d, 2 H, J4,5 7.0 Hz,
H-4 both epimers), 4.21 (dd, 1 H, H-5 one
epimer), 4.03 (m, 2 H, H-2%,3%), 3.95 (dd, 1 H,
H-4%), 3.10 (m, 1 H, H-5%), 3.45 (m, 2 H,
13
H-6%a,b); C NMR (25.3 MHz, Me2SO-d6), l
167.5 (C-2), 93.6 (C-4), 88.3 (C-1%), 81.5 (C-4%),
79.7 (C-2%), 75.6 (C-3%), 71.5 (C-5%), 65.1 (C-5),
63.4 (C-6%), 56.4 (OCH3). Anal. Calcd for
C10H18N2O6S: C, 40.80; H, 6.16. Found: C,
40.63, H, 6.24.
Debenzoylation: general procedure.—Com-
pounds 2,13 4,12 7 and 8 were suspended in 0.5
N NaOMe–MeOH, and stirred at rt during 2
h. The solution was passed through a column
(1.5×5.0 cm) containing Dowex 50 W(H+)
resin. The solvent was removed under vacuum
and the remaining MeOBz was eliminated by
several coevaporations with water. The fol-
lowing compounds were obtained.
Alternatively the mixture of 7 and 8 was
debenzoylated and compounds 9 and 10 were
separated by column chromatography with 4:1
AcOEt–iPrOH.
Assays of i- -galactofuranosidase inhibi-
D
N-i-
D
-Galactofuranosyl-O-ethylthioureth-
tion.—The filtered medium of a stationary
culture of P. fellutanum was used as the en-
zyme source.5 The standard assay (100% of
activity) was performed with 100 mm of 66
mM NaOAc buffer (pH 4.0), 62 mL of a 5
mM solution of the substrate (4-nitrophenyl
ane (3).—Yield 95%, [h]D −15° (c 1, water);
1H NMR (200 MHz, D2O), selected data: l
13
5.58 (d, 1 H, J1,2 4.2 Hz, H-1); C NMR (25.3
MHz, D2O), l 183.8 (CꢀS), 88.9 (C-1), 81.7
(C-4), 79.9 (C-2), 75.5 (C-3), 71.7 (C-5), 63.1
(C-6), 61.6 (OCH3). Anal. Calcd for
C9H17NO6S: C, 40.44; H, 6.41. Found: C,
40.29, H, 6.33.
b- -galactofuranosidase) and 100 mL (20 mg
D
protein) of the enzyme medium, in a final
volume of 500 mL. The inhibitors were incor-
porated in various amounts to give a final
concentration range of 0.05–1.30 mM. The
enzymatic reaction was started with the addi-
tion of the enzyme, and after 1.5 h at 37 °C it
was stopped with 1 mL of 0.1 M Na2CO3
N - i -
D
- Galactofuranosyl - 5 - oxo - imidazo-
lidine-2-thione (5).—Yield 83%, [h]D −25° (c
1
1, CHCl3); H NMR (D2O, 200 MHz), se-
lected data, l 5.98 (d, 1 H, J1%,2% 6.7 Hz, H-1%),
5.06 (dd, 1 H, J2%,3% 5.1 Hz, H-2%), 4.15 (s, 2 H,

128
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buffer (pH 9). The 4-nitrophenol released was
measured spectrophotometrically at 410 nm.
Acknowledgements
We are indebted to Agencia Nacional de
Promocio´n Cient´ıfica (ANCYT, BID 802/OC-
AR Pict 06-3036), University of Buenos Aires,
CONICET (Consejo Nacional de Investiga-
ciones Cient´ıficas y Tecnolo´gicas) and SEP-
CYT (Argentina)/OMFB (Hungary) for
financial support (HU/A 98/06–EVI/06).
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