Synthesis of C2-symmetric guanidino-sugars as potent inhibitors of glycosidases

Article abstract of DOI:10.1016/S0968-0896(99)00294-1

A series of enantiomerically pure C₂-Symmetric guanidino-sugars was synthesized from D-mannitol. The first method described involves direct opening of a bis-epoxide by guanidine, whereas the second one deals with a mercury-catalyzed transformation of a cyclic thiourea into a N,N',N'- trisubstituted guanidine as a key step. The biological activity of these compounds towards several glycosidases has been evaluated. One of them (5) was found to selectively inhibit α-L-fucosidase of bovin kidney (2.8 μM). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00294-1

Bioorganic & Medicinal Chemistry 8 (2000) 307±320
Synthesis of C2-Symmetric Guanidino-Sugars as
Potent Inhibitors of Glycosidasesy
Yves Le Merrer,* Laurence Gauzy, Christine Gravier-Pelletier
and Jean-Claude Depezay
 Â
Universite Rene Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
Á
UMR 8601 CNRS, 45, rue des Saints-Peres, 75270 Paris Cedex 06, France
Received 2 August 1999; accepted 20 September 1999
AbstractÐA series of enantiomerically pure C2-Symmetric guanidino-sugars was synthesized from d-mannitol. The ®rst method
described involves direct opening of a bis-epoxide by guanidine, whereas the second one deals with a mercury-catalyzed transfor-
mation of a cyclic thiourea into a N,N0,N00-trisubstituted guanidine as a key step. The biological activity of these compounds
towards several glycosidases has been evaluated. One of them (5) was found to selectively inhibit a-l-fucosidase of bovin kidney
(2.8 mM). # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Here we report full results on both the synthesis of C2-
Symmetric guanidino-sugars 1, 2 and 3, 431 from d-
mannitol (Scheme 1), and the introduction on com-
pound 4 of a n-butyl or glucopyranosyl group as an
aglycon part (respectively, 5 and 6). Results on the bio-
logical evaluation of those compounds as potent inhibi-
tors of glycosidases will be presented.
Glycosidases are enzymes which are responsible for the
hydrolysis of the glycosyl bond; they play a key role in
glycoprotein trimming, catabolism of glycoconjugates
and degradation of polysaccharides. As a consequence,
speci®c reversible inhibitors of those enzymes can have
major therapeutic utility in the treatment of diabetes,1
cancer2 and viral infections.3
Compounds 1, 2 and 3, 4 are respectively analogues of
the azepanes 7, 8 and of the pyrrolidines 9, 10, we pre-
cedently described and that inhibit glycosidases in low
micromolar range.32 Two retrosynthetic pathways
(Scheme 2) have been imagined as a solution to this
challenging synthesis. The ®rst one involves the hetero-
cyclization of bis-electrophiles derived from d-mannitol
by guanidine. The use of bis-epoxydes will lead to cyclic
guanidine 1 or 2 , while an activation at positions 2 and
5 of d-mannitol will lead to 3 or 4. The second one
involves the hetero-cyclization of a diamine as a bis-
nucleophile.
The strategy commonly encountered to design such
inhibitors is to synthesize compounds that are able to
mimick the oxocarbenium-type transition state,4 which
is supposed to arise during glycosyl bond hydrolysis. In
most cases, azasugars5±13 or amidine14±21 derivatives
have been chosen in order to resemble the transient
oxonium. More recently, guanidino-sugars22±24 have
been proposed as a new class of glycosyl cation mimics.
Moreover, in the aim of synthesizing compounds cap-
able of interacting with glycosidases, it has been shown
that the aglycon part of the glycoside also plays an
important role in the interaction. In particular, pseudo-
disaccharides25±29 or pseudo-sugars containing an alkyl
aglycone part30 deserve more consideration.
Results and Discussion
The commercially available guanidine in salt form (hy-
drochloride, carbonate, nitrate or sulfate) has already
been used as a nucleophile in reactions with a-dike-
tones,33 a,b-ethylenic esters34 and alkyl halides,35 and
more recently in the opening of epoxides. For example,
guanidine reacts with benzene trioxide36 to lead cyclic
guanidine, and with the 1,2:5,6-dianhydro-3,4-di-O-
Keywords: d-mannitol; guanidine; a-l-fucosidase inhibitor; mercuric
chloride; thiourea.
*Corresponding author. Tel: 33-1-4286-2181; fax: 33-1-4286-8387;
e-mail: yves.le-merrer@biomedicale.univ-paris5.fr
yWarmly dedicated to Professeur Pierre Sinay on the occasion of his
62nd birthday.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00294-1

308
Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
Scheme 1.
Scheme 2.
benzyl-l-iditol37 to give the corresponding guanidino-
methyl tetrahydrofuran by subsequent O-cyclization. In
the latter case, considering that the protective groups
play a decisive role in the selectivity of ring opening of
the bis-epoxide, we chose to protect the 3,4-diol as an
acetonide to prevent O-cyclization; the corresponding
bis-epoxides 11 and 12 (Scheme 3) being easily prepared
from d-mannitol.38,39
Thus, treatment of 1,2:5,6-dianhydro-3,4-O-methyl-
ethylidene-d-mannitol 11 (or l-iditol 12) by 1 equivalent
of guanidine (generated by ethanolic elution of its hy-
drochloride salt on Amberlite1 IRA 400 (OH )40) in
re¯uxing ethanol a€orded the corresponding C2-Sym-
metric cyclic guanidine 13 (or 14) in 97% yield.
Removal of the acetonide group was easily achieved
with hydrogen chloride in methanol or with aqueous
tri¯uoroacetic acid.
Scheme 3. (a) Guanidine, EtOH Á, 97%; (b) HClg, MeOH, 70%;
(c) TFA, H2O, 70%.

Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
309
According to the same strategy, the synthesis of cyclic
guanidines 3±6 supposed the nucleophilic substitution
of alcohols in positions 2 and 5 of d-mannitol or l-idi-
tol, after protection of all the other alcohol functions.
Thus, remaining secondary alcohols could be activated
as mesylates or by the Mitsunobu reaction in order to
react with guanidine.41,42 Both approaches have been
tried on di€erent protected derivatives 15, 16, 17, or 18
(Scheme 4), o€ering di€erent spatial restraints. Diols 1643
and 1744,45 were prepared as already described, whereas
the diol 15 was easily obtained from the commercially
available 3,4-O-methylethylidene-d-mannitol 19 by selec-
tive benzylation of both primary hydroxyl groups through
stannylidene activation with Bu2SnO46,47 (Scheme 5), and
subsequent treatment with benzyl bromide (86%).
Lastly, the diol 18 was prepared from the dicarbonate
2048,49 by protection of the alcohol functions in posi-
tions 3 and 4 as trioxepane50 according to a method
developed by Beck.51 Reaction of dicarbonate 20 with
paraformaldehyde and BF3.Et2O led to an unseparable
mixture of 21 and 22 (71% yield in 3:1 ratio according
1
to H NMR). Hydrolysis of carbonates in presence of
pyridine yielded a mixture of the corresponding tetrols
23 and 24. Final protection of both primary alcohols
with Bu2SnO and benzyl bromide, as described above,
a€orded the corresponding mixture of 18 and 25 which
could be separated by ¯ash chromatography (30 and
8% overall yield from 20, respectively).
On one hand, the dimesylate 26, 27 or 28, issued,
respectively, from diol 15, 16 or 17 after treatment with
methanesulfonyl chloride, failed to react with di€erent
sources of guanidine (free guanidine, acetyl-guanidine,
N,N0-di-benzyloxy-carbonyl-guanidine), and on the other
hand, Mitsunobu reaction involving N,N0-di-benzyloxy-
carbonyl-guanidine on diol 16 or 18 led to the corre-
sponding tetrahydrofuran 29 or 30 (Scheme 6). In the
absence of the guanidine derivative, the yield of 29 or 30
could even be improved up to 91 or 68%, respectively.
Thus, this ®rst strategy involving the N,N0-dialkylation
of guanidine revealed to only be ecient with a 1,6-bis
activated derivative of d-mannitol. In order to obtain
the cyclic guanidines 3±6 we turned our attention to the
second strategy dealing with the cyclization of a 2,5-
diamine derived from d-mannitol or l-iditol into
thiourea and subsequent transformation into guanidine.
Synthesis of the readily available cyclic thiourea 33 via
the l-ido-diamine 32 is depicted in Scheme 7. Nucleo-
philic substitution of the 2,5-dimesylate 26 by sodium
azide a€orded the diazido derivative 31 (57% overall
yield from 15) with inversion of con®guration at C2 and
C5, then subsequent heterogeneous catalytic reduction
led to the corresponding diamine 32 (87%). Cyclization
of 32 into thiourea 33 smoothly occurred by treatment
with carbon disul®de52,53 in pyridine (86%).
In order to transform the thiourea 33 into guanidine, the
well-known method of thiourea alkylation into inter-
Scheme 4.
Scheme 5.

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Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
mediary methylisothiourea 3454±57 was ®rst tried
(Scheme 8). Although methylation at the exocyclic sulfur
atom of 33 with methyl para-toluene-sulfonate58 in
re¯uxing methanol was easily achieved (88%), all
attempts to substitute the thiomethyl group either with
ammonia59 or benzylamine60 failed. We therefore
turned to a more direct method using HgCl2.61,62 Such
an approach had already successfully been used on N-
tert-butyloxycarbonyl thioureas in DMF but was repor-
ted to be inecient on unactivated thioureas.63 Indeed,
this method involving the thiourea 33 in the presence of
benzylamine and CH2Cl2 as solvent,64 revealed to e-
ciently a€ord the N-benzyl cyclic guanidine 35 (91%). The
last step was the deprotection of cyclic guanidine 35.
Concerning reduction of benzyl ethers and N-benzyl gua-
nidine, di€erent attempts involving hydrogeneous cataly-
tic reduction failed, while the use of sodium in liquid
ammonia was successful. After 6 h, the reaction was
quenched by addition of solid ammonium chloride. Then,
an extraction with absolute ethanol was followed by dis-
carding ammonium chloride in excess by an ethanolic
elution on Amberlite1 IRA 400 (OH ). Final hydrolysis
of the acetonide group was achieved in aqueous tri-
¯uoroacetic acid to yield the guanidino-sugar 4.
Scheme 6.
This strategy could also be applied to the synthesis of
cyclic guanidines 3, 2 and 1 from corresponding dia-
mines 40, 43 and 47 (Scheme 9). Interestingly, this sec-
ond way of synthesizing cyclic guanidines 1 and 2
o€ered a justi®cation for their structures. In that pur-
pose, d-manno diamine 40 and l-ido diamine 43 were
obtained from l-ido bis-epoxide 12. On one hand,
opening of bis-epoxide 12 with sodium benzylate cleanly
occurred to give the 1,6-di-O-benzyl derivative 37 (57%)
which was then routinely converted into the 2,5-diamine
40 as described above for diamine 32 from diol 15. On
the other hand, opening of bis-epoxide 12 by sodium
azide led to the diol 41 (83%) which was protected as
dibenzyl ether 42, by successive treatment with sodium
hydride and benzyl bromide (77%). Finally, hetero-
geneous catalytic reduction in ethanol led to the diamine
43 (70%). The last diamine 47 was obtained from tetrol 19
as follows: selective tosylation of both primary hydroxyl
groups with para-toluenesulfonyl chloride in pyridine39
(86%), then nucleophilic substitution with sodium azide
(75%) and heterogeneous catalytic reduction (82%).
Cyclization of diamines 40, 43 and 47 into thioureas 48, 51
and 53 and subsequent transformations into the corre-
sponding guanidino-sugars are summarized in Table 1.
Scheme 7.
Furthermore, this strategy which involves transforma-
tion of a cyclic thiourea into guanidine with benzylamine
could be extended to other primary amines such as n-
butylamine or methyl 6-amino-6-deoxy-2,3,4-tri-O-ben-
zyl-d-gluco-pyranoside65 (Table 2). Thus, n-butylamine
treatment of thiourea 33 in CH2Cl2 in the presence of
mercuric chloride at 20 ꢀC led to the cyclic N-butyl
guanidine 55 (65%), while reaction with the 6-amino-6-
deoxy derivative of d-glucose in 1,2-dichloroethane at
80ꢀC a€orded the protected pseudo-disaccharide 57
(86%). By sodium reduction and subsequent acidic
hydrolysis, as above, 55 and 57 yielded the corresponding
deprotected guanidino-sugars 5 and 6.
Scheme 8. (a) MeOTs, MeOH Á, 88%; (b) BnNH2, HgCl2, Et3N,
CH2Cl2, 91%; (c) Na, NH3, 74%; (d) TFA, H2O, quant.

Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
311
Scheme 9.
Biological activity of synthesized guanidino-sugars
towards di€erent glycosidases (a- and b-d-glucosidases, a-
d-mannosidase and a-l-fucosidase) was evaluated (Table
3). The results show that guanidino-sugars are weak inhi-
bitors of glycosidases. For example, 3 is a less potent
inhibitor of a- and b-d-glucosidases than its parent aza-
sugar 9 (DMDP). The best results observed concerned the
N-butyl and the N-glucosyl derivatives 5 and 6 which
competitively inhibit a-l-fucosidase, Ki=2.8 and 500 mM,
respectively. Selective strong inhibition of a-l-fucosidase
by compound 5, and more weakly by compound 6 con-
®rms that the aglycon part plays a critical role in recogni-
tion. In particular, an alkyl aglycon moiety may be
decisive in order to inhibit a-l-fucosidase.
Paris, France. Mass spectra were performed by the `Service
de Spectrometrie de Masse de l'Ecole Normale Superieure',
Paris, France. All reactions were monitored by thin-layer
chromatography with Merck 60F 254 precoated silica
(0.2 mM) on glass. Flash chromatography was performed
with Merck Kieselgel 60H (5±40 mM). Prior to use, THF
and Et2O were distilled from benzophenone-sodium, and
CH2Cl2 from CaH2. Spectroscopic (1H and 13C NMR,
MS) and/or analytical data were obtained using chroma-
tographically homogeneous samples.
(5R,6R,7R,8R)-5,6,7,8-Tetrahydroxy-1,3-diazonan-2-imi-
nium chloride (1). A solution of the guanidine 13 (50 mg,
21 mmol) in gazeous hydrogen chloride saturated
methanol (1 mL) was heated at 60 ꢀC during 1 h. After
cooling to 20ꢀC, the solid was ®ltered and washed with
ethanol to yield the guanidinium chloride 1 (35mg, 70%)
Experimental66
1
as a white hygroscopic solid. [a] +5 (c 0.8, H2O); H
General methods
NMR (D2O): 3.35±3.50 (m, 2H, H5,8), 3.60±3.75 (m, 2H,
H6,7), 3.75±4.05 (m, 4H, H4,9); 13C NMR (D2O): 47.9
(C4,9), 71.6, 72.2 (C5,6,7,8), 160.4 (C2); HRMS (FAB+) for
C7H16O4N3: (MH+) calcd 206.1141, found: 206,1118.
1H NMR (250MHz) and 13C NMR (63 MHz) spectra were
recorded in CDCl3 (unless otherwise indicated) on a Bru-
ker ARX 250 spectrometer. Optical rotations were mea-
sured on a Perkin±Elmer 241C polarimeter with sodium
lamp (589nm) at 20 ꢀC. IR spectra were recorded on a
Perkin±Elmer 783 spectrophotometer and wave-numbers
of characteristic absorption bands are given in cm 1. Ele-
mental analyses were performed by the `Service Regional
de Microanalyse de l'Universite Pierre et Marie Curie',
(5S,6R,7R,8S)-5,6,7,8-Tetrahydroxy-1,3-diazonan-2-imi-
nium tri¯uoroacetate (2). A solution of the guanidine 14
(180 mg, 76 mmol) in 10% aq tri¯uoroacetic acid (4 mL)
was heated at 60 ꢀC during 1 h. After concentration in
vacuo, the resulting oil was triturated with diethylether to
leave a white solid collected by ®ltration and washed with

312
Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
Table 1.
Diamine
Thiourea
Protected guanidine
Guanidino-sugar
40
48 (81%)
49: P=Bn (78%)
50: P=H (75%)
3 (100%)
43
51 (100%)
52: P=Bn (94%)
14: P=H (80%)
2 (70%)
47
53 (100%)
54: P=Bn (94%)
13: P=H (77%)
1 (70%)
1
diethylether to yield the guanidinium tri¯uoroacetate 2
(177mg, 70%) as a white hygroscopic solid. [a] +19 (c
0.3, H2O); 1H NMR (D2O): 3.25±3.40 (m, 2H, H6,7), 3.45±
4.10 (m, 6H, H4,5,8,9); 13C NMR (D2O): 54.7 (C4,9), 74.2,
76.1 (C5,6,7,8), 159.6 (C2).
1.0, H2O); H NMR (D2O): 3.65±4.10 (m, 8H, H4,5,6,7
,
CH2OH); 13C NMR (D2O): 59.6 (C4,7), 63.9 (CH2OH),
72.3 (C5,6), 160.6 (C2); HRMS (FAB+) for C7H16N3O4:
(MH+) calcd 206.1141, found 206.1165.
(4S,5R,6R,7S)-5,6-Dihydroxy-4,7-dihydroxy methyl-1,3-
diazepan-2-(N-butyl)-iminium tri¯uoroacetate (5). The
guanidino-sugar5 (120 mg, 96%) was obtained as a whitish
hygroscopic solid from the guanidine 58 on a 0.33 mmol
scale, according to the procedure described above for the
guanidino-sugar 2. [a] +2 (c 1.0, MeOH); 1H NMR
(4R,5R,6R,7R)-5,6-Dihydroxy-4,7-dihydroxy methyl-1,
3-diazepan-2-iminium tri¯uoroacetate (3). The guanidi-
nium tri¯uoroacetate
3 (40 mg) was quantitatively
obtained as a whitish hygroscopic solid from the guani-
dine 50 on a 0.12mmol scale, according to the procedure
described above for the guanidino-sugar 2. [a] +8 (c 1.3,
H2O); 1H NMR (D2O): 3.40±3.50 (m, 2H, H4,7), 3.60±3.70
0
0
0
(CD3OD): 0.97 (t, 3H, J3 ,4 =7.3 Hz, H4 ), 1.37 (m, 2H,
0
H3 ), 1.64 (m, 2H, H2 ), 3.65±4.00 (m, 8H, H4,7,1 , CH2OH),
0
0
13
0
4.17 (m, 2H, H5,6); C NMR (CD3OD): 15.8 (C4 ), 22.8
(m, 2H, H5,6), 3.93 (dd, 2H, J4,CHOH=6.4, JCH OH
=
2
0
0
0
0
12.0 Hz, CH2OH), 4.06 (dd, 2H, J4,CH OH=3.6, JCH2
OH=12.0 Hz, CH2OH); 13C NMR (D2O): 60.7 (C4,7), 63.2
(CH2OH), 75.3 (C5,6); HRMS (FAB+) for C7H16 N3O4:
(MH+) calcd 206.1141, found 206.1095.
(C3 ), 33.7 (C2 ), 45.2 (C1 ), 59.4 (C4,7), 65.8 (CH2OH), 80.0
(C5,6), 160.6 (C2); MS (NH3): 262 (MH+).
Methyl (40S,50R,60R,70S)-6-deoxy-D-gluco pyranoside-6-
(40,70-dihydroxy methyl-50,60-dihydroxy-10,30-diazepan-20-
yl) - iminium tri¯uoroacetate (6). The guanidino-sugar 6
(160mg, 98%) was obtained as a whitish hygroscopic
solid from the guanidine 60 on a 0.33mmol scale accord-
ing to the procedure described above for guanidino-sugar
(4S,5R,6R,7S)-5,6-Dihydroxy-4,7-dihydroxy methyl-1,3-
diazepan - 2 - iminium tri¯uoroacetate (4). The guanidi-
nium tri¯uoroacetate
4 (27 mg) was quantitatively
obtained as a whitish hygroscopic solid from the guanidine
36 on a 0.08 mmol scale, according to the procedure
described above for the guanidino-sugar 2. [a] +12 (c
1
2. [a] +25 (c 0.9, CH3OH); H NMR (CD3OD): 3.35±
3.43 (m, 11H, H4,7, CH2N, OCH3, CH2OH), 3.69±3.77

Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
313
Table 2.
Reactants
Protected guanidine
Guanidino-sugars
33+nBuNH2, HgCl2,CH2Cl2, 20 ꢀC
33+nBuNH2, HgCl2,CH2Cl2, 20 ꢀC
55: P=Bn (65%)
56: P=H (73%)
5 (96%)
33+
HgCl2,ClCH2CH2Cl, 80 ꢀC
57: P=Bn (86%)
58: P=H (79%)
6 (98%)
Table 3.
CH2Cl2 were added. The organic layer was washed with
water and the combined aqueous layers were con-
centrated in vacuo to yield the guanidine 13 (208 mg,
97%) as an hygroscopic solid.
Enzyme
Inhibitors (%)a
1
2
3
4
5
6
a-d-glucosidaseb
b-d-glucosidasec
a-d-mannosidased
a-l-fucosidasee
24
7
10
21
36
34
41
31
14
10
4
37
16
4
41
NI
18
94
2.8 mMf
14
6
12
96
500 mMf
From (5R,6R,7R,8R)-5,8-dibenzyloxy-7,8-dihydroxy-7,
8-di-O-methylethylidene-2-benzylimino-1,3-diazonane (54).
A solution of the benzylated guanidine 54 (50 mg,
97 mmol) in a minimum of anhydrous tetrahydrofuran
was added into liquid ammonia (5 mL). After addition
of sodium until persistance of a dark blue color, the
reaction mixture was stirred under re¯uxing ammonia
during 6 h. Then, careful addition of solid ammonium
chloride resulted in decoloration and was followed by
ammonia evaporation. The resulting solid was washed
with CH2Cl2 and extracted with absolute ethanol. The
combined alcoholic layers were concentrated in vacuo,
neutralized over Amberlite1 IRA 400 (OH ), eluted
with ethanol and concentrated in vacuo to yield the gua-
nidine 13 (23 mg, 77%) as an hygroscopic solid. [a] +24
(c 1.0, MeOH); 1H NMR (D2O): 1.47 (s, 6H, CH3), 3.38±
3.58 (m, 4H, H4,9), 3.68±4.00 (m, 4H, H5,6,7,8); 13C NMR
(CD3OD): 27.3 (CH3), 58.3 (C4,9), 73.4 (C5,8), 82.5 (C6,7),
110.5 (CMe2), 161.2 (C2); MS (NH3): 246 (MH+), 263
(M+NH+4 ).
12
32
a% Inhibition determined at 1 mM concentration of inhibitor; NI for
no inhibition.
bBacillus stearothermophilus, pH 6.8.
cAlmond, pH 5.0.
dJack beans, pH 4.5.
eBovine kidney, pH 5.5.
fInhibition constants (Ki in mM) determined by the Lineweaver±Burk
method.
(m, 6H, H5,6,2 ,3 ,4 ,5 ), 4.77 (m, 1H, H1 ); 13C NMR (CD3
OD): 46.0 (CH2N), 52.5, 58.7 (C4,7, OCH3), 66.0 (CH2
0
0
0
0
0
0
OH), 71.8, 74.3, 74.9, 75.8, 77.1 (C5,6,2 ,3 ,4 ,5 ), 103.8 (C1 );
0
0
0
0
MS (FAB+): 382 (MH+).
(5R,6R,7R,8R)-5,6,7,8-Tetrahydroxy-6,7-O-methylethy-
lidene-2-imino-1,3-diazonane (13)
From 1,2:5,6-dianhydro-3,4-O-methylethylidene-D-man-
nitol (11). A solution of guanidinium chloride (83 mg,
87mmol, 0.95 equiv) in a minimum of 95% ethanol:water
was neutralized on Amberlite1 IRA 400 (OH ), then
eluted with absolute ethanol. After concentration in
vacuo, free guanidine was dissolved in absolute ethanol
(1 mL), the bis-epoxide 1139 (170 mg, 92 mmol) was
added and the resulting mixture was heated under re¯ux
during 1 h. After concentration in vacuo, water and
(5S,6R,7R,8S)-5,6,7,8-Tetrahydroxy-6,7-O-methylethyli-
dene-2-imino-1,3-diazonane (14). As described above for
13, the protected guanidino-sugar 14 was obtained as an
hygroscopic solid on one hand, from the bis-epoxide 12
(92 mmol scale, 97%), and on the other hand by N,O-
debenzylation of 52 (97 mmol scale, 80%). [a] +27 (c

314
Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
1
0.9, MeOH); H NMR (D2O): 1.49 (s, 6H, CH3), 3.42
0
chromatographied (acetone:dichloromethane, 5:95) to
yield a mixture of the trioxepane 21 and of the methyl-
ene acetal 22 (75:25 according to 1H NMR, 137 mg,
ꢁ71%) as a solid.
(dd, 2H, J4,5=5.8, J4,4 =15.6 Hz, H4,9), 3.64 (m, 2H,
H4 ,9 ), 3.75±4.10 (m, 4H, H5,6,7,8); 13C NMR (CD3OD):
27.3 (CH3), 55.5 (C4,9), 72.4 (C5,8), 82.3 (C6,7), 110.6
(CMe2), 161.7 (C2); MS (NH3): 246 (MH+).
0
0
1
0
21. H NMR: 3.78 (m, 2H, H4,5), 4.43 (dd, 2H, J4 ,5
0
1,6-Di-O-benzyl-3,4-O-methylethylidene-D-mannitol (15).
In a ¯ask equipped with a Dean±Stark, a suspension of
3,4-O-methylethylidene-d-mannitol (500 mg, 2.25 mmol)
and dibutyl-tin oxide (1.15 g, 4.7 mmol, 2.1 equiv) in
toluene (23 mL) was heated under re¯ux during 15 h.
After concentration in vacuo, to a solution of the resi-
due in toluene (11 mL), benzyl bromide (1.1 mL,
9 mmol, 4 equiv) and tetrabutylammonium iodide
(805 mg, 2.25 mmol, 1 equiv) were added and the mix-
ture was stirred at 70 ꢀC for 15 h. Concentration in
vacuo and subsequent chromatography of the resulting
residue (EtOAc:cyclohexane, 3:7) a€orded the diol 15
0
=6.3, J5 ,5 =9.0 Hz, H5 ), 4.55 (dd, 2H, J5 ,5 =9.0,
0
J4 ,5 =8.2 Hz, H5 ), 4.78±4.86 (m, 2H, H4 ), 4.90 (d, 2H,
J2,2 =6 Hz, H2,7), 5.07 (d, 2H, J2,2 =6.0 Hz, H2,7); MS
(NH3): 277 (MH+), 294 (M+NH4+).
00
0
0
00
0
00
00
0
0
1
22. H NMR: 4.11 (m, 2H, H3,4), 4.42 (dd, 2H, J1,2
0
0
0
=5.3, J1,1 =8.7 Hz, H1,6), 4.58 (dd, 2H, J1 ,2=9.5, J1,1
0
0
=8.7 Hz, H1 ,6 ), 4.70±4.75 (m, 2H, H2,5), 5.10 (s, 2H,
OCH2O); MS (NH3): 264 (M+NH4+).
(4R,5R,10R,100R) - 4,5 - Di - (10,20 - dihydroxyethyl) - 1,3,6 -
trioxepan (23) and 3,4-O-methylene-D-mannitol (24). A
solution of the mixture of carbonates 21 and 22 (137 mg,
ꢁ0.5 mmol) in a 1:1 pyridine:water solution (26 mL) was
heated under re¯ux during 1 h to yield a quantitative
mixture of tetrols 23 and 24 (110 mg, ꢁ75:25), after
concentration in vacuo.
1
(777 mg, 86%) as an oil. [a] +21 (c 1.2, CHCl3); H
NMR: 1.36 (s, 6H, CH3), 3.57 (dd, 2H, J1,2=6.2,
0
0
0
J1,1 =9.7 Hz, H1,6), 3.71±3.85 (m, 4H, H1 ,2,5,6 ), 3.85±
3.95 (m, 2H, H3,4), 4.57 (s, 4H, OCH2Ph), 7,32 (s, 10H,
CHPh); 13C NMR: 26.9 (CH3), 71.7 (C1,6), 72.0 (C2,5),
73.5 (OCH2Ph), 79.9 (C3,4), 109.4 (CMe2), 127.8, 128.4
(CHPh), 138.1 (CPh). Anal. calcd for C23H30O6: C,
68.62; H, 7.52. Found: C, 68.77; H, 7.41.
(4R,5R,10R,100R)-4,5-Di-(20 -benzyloxy-10 -hydroxyethyl)-
1,3,6-trioxepan (18) and 1,6-di-O-benzyl-3,4-O-methylene-
D-mannitol (25). The diols 18 and 25 were obtained from
the mixture of the tetrols 23 and 24 on a 0.22 mmol scale,
as described above for the diol 15. Flash chromatography
of the residue (cyclohexane:EtOAc, 6:4) respectively
a€orded 18 (40 mg, 30%) and 25 (10 mg, 8%).
1
0
23. H NMR (D2O): 3.64±3.81 (m, 4H, H2 ,2 ), 3.86
00
0
(m, 2H, H1 ,1 ), 3.99 (m, 2H, H4,5), 5.01 (d, 2H,
0
J2,2 =8.0 Hz, H2,7), 5.18 (d, 2H, J2,2 =8.0 Hz, H2 ,7 ).
00
0
0
0
24. 1H NMR (D2O): 3.64±3.81 (m, 4H, H1,6), 3.86 (m,
2H, H2,5), 4.18 (m, 2H, H3,4), 5.08 (s, 2H, OCH2O).
1,6-Di-O-benzyl-2,5-di-O-methane sulfonyl-3,4-O-methyl-
ethylidene-D-mannitol (26). Methane sulfonyl chloride
(150mL, 1.9 mmol, 3 equiv) was dropwise added to a solu-
tion of the diol 15 (250mg, 0.62mmol) and triethylamine
(350mL, 2.5 mmol, 4 equiv) in dichloromethane (1 mL)
at 0 ꢀC. After stirring at 20ꢀC for 5 min, water was added
and the mixture was extracted with dichloromethane. The
combined organic layers were dried (MgSO4) and con-
centrated in vacuo to quantitatively yield the dimesylate 26
(345mg). 1H NMR 1.38 (s, 6H, CH3), 3.03 (s, 6H,
1
18. [a] 2 (c 0.7, CHCl3); H NMR: 3.60±3.67 (m,
6H, H1,3,4,6), 4.01 (m, 2H, H2,5), 4.50, 4.55 (AB, 4H,
JAB=11.0 Hz, OCH2Ph), 4.78, 4.95 (2d, 4H, J=6.0 Hz,
OCH2O), 4.95 (d, 2H, J2,2=6.0 Hz, OCH2O), 7.30 (s,
10H, CHPh); 13C NMR: 69.4 (C2,5), 70.7 (C1,6), 73.4
(OCH2Ph), 83.0 (C3,4), 91.9 (OCH2O), 127.8, 128.5
(CHPh), 137.9 (CPh); MS (NH3) 422 (M+NH4+).
1
0
SO3Me), 3.70 (dd, 2H, J1,2=6.9, J1,1 =11.3 Hz, H1,6), 3.85
25. [a] +11 (c 0.4, CHCl3); H NMR: 3.57 (dd, 2H,
0
J1,2=6.0, J1,1 =10.0 Hz, H1,6), 3.71 (dd, 2H, J1 ,2=3.2,
0
0
0
0
0
(dd, 2H, J1 ,2=3.2, J1,1 =11.3 Hz, H1 ,6 ), 4.33 (m, 2H,
H3,4), 4.51, 4.57 (AB, 4H, JAB=11.7 Hz, OCH2Ph), 4.86
(m, 2H, H2,5), 7.30 (m, 10H, CHPh).
0
0
0
J1,1 =10.0 Hz, H1 ,6 ), 3.80 (m, 2H, H2,5), 3.97 (m, 2H,
H3,4), 4.56 (s, 4H, OCH2Ph), 4.93 (s, 2H, OCH2O), 7.32
(s, 10H, CHPh); MS (NH3): 392 (M+NH4+).
1,3,4,6-Tetra-O-benzyl-2,5-di-O-methane sulfonyl-D-man-
nitol (27). The dimesylate 27 (130 mg) was quantitatively
obtained from the diol 16 as described above for the
dimesylate 26 on a 0.18 mmol scale. H NMR 2.96 (s,
0
6H, SO2CH3), 3.80 (dd, 2H, J1,2=7.0, J1,1 =11.0 Hz,
(4S,5S,40R,400R)-4,5-Di-(20-oxo-10,30-dioxol-40-yl)-1,3,6-
trioxepan (21) and 1,2:5,6-di-O-carbonyl-3,4-O-methyl-
ene-D-mannitol (22). Boron tri¯uoride etherate (215 mL)
was dropwise added to a suspension of both the diol 20
(163 mg, 0.69 mmol) and paraformaldehyde (62 mg) in
ethyl acetate (1.6 mL) at 0 ꢀC. Then, the temperature
was allowed to warm up to 20 ꢀC and stirring was con-
tinued for 15 h. After addition of a saturated aqueous
solution of NaHCO3, the pH was adjusted to 8 by
addition of solid NaHCO3. Then the mixture was
extracted with ethyl acetate and the combined organic
layers were dried (MgSO4), concentrated in vacuo and
1
0
0
0
0
H1,6), 3.92 (dd, 2H, J1 ,2=3.0, J1,1 =11.0 Hz, H1 ,6 ),
4.03 (m, 2H, H3,4), 4.45, 4.52 (AB, 4H, JAB=12.0 Hz,
OCH2Ph), 4.62, 4.68 (AB, 4H, JAB=11.0 Hz, OCH2Ph),
4.93 (m, 2H, H2,5), 7.32 (m, 20H, CHPh).
1,3:4,6-Di-O-benzylidene-2,5-di-O-methanesulfonyl-D-
mannitol (28). The dimesylate 28 (157 mg, 79%) was
obtained from the diol 17 as described above for the

Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
315
dimesylate 26 on a 0.28mmol scale. 1H NMR: 3.03 (s, 6H,
0
SO2CH3), 3.84 (dd, 2H, J1,2=10.5, J1,1 =10.6 Hz, H1,6),
0
4.13 (d, 1H, J2,3=9.0 Hz, H3,4), 4.55 (dd, 2H, J1 ,2=5.5,
(MH+ N2), 470 (M + NH4+). Anal. calcd for
C23H28O4N6: C, 61.03; H, 6.24; N, 18.58. Found: C,
61.14; H, 6.18; N, 18.56.
0
J1 ,1=10.6 Hz, H1 ,6 ), 5.05 (m, 2H, H2,5), 5.50 (s, 2H,
0
0
OCHPh), 7.35 (m, 6H, CHPh), 7.47 (m, 4H, CHPh).
2,5-Diamino-1,6-di-O-benzyl-2,5-dideoxy-3,4-O-methyl-
ethylidene-L -iditol (32). Palladium (10%) on charcoal
(125 mg) in ethanol (3 mL) was completely hydro-
genated under 1 atm of H2 at 20 ꢀC prior to the addition
of the diazido derivative 31 (189 mg, 0.42 mmol) in eth-
anol (2 mL). After 2 h, the catalyst was removed through
a celite pad and the ®ltrate was concentrated in vacuo
and puri®ed by ¯ash chromatography (CH2Cl2:MeO-
H:ammonium hydroxide, 94:3:3) to yield the diamine 32
1,3,4,6-Tetra-O-benzyl-2,5-anhydro-D-glucitol (29).
Diisopropyl azodicarboxylate (55 mL, 0.27 mmol, 3
equiv) was dropwise added to a solution of both the diol
16 (50 mg, 0.09mmol) and triphenylphosphine (55 mg,
0.27mmol, 3 equiv) in toluene (1mL) at 0 ꢀC. After stir-
ring at 0 ꢀC for 1 h, the reaction mixture was concentrated
in vacuo and the residue was chromatographied
(EtOAc:cyclohexane, 1:9) to yield the tetrahydrofuran 29
(44 mg, 91%). [a] +21 (c 1.2, CHCl3) {lit.67 [a]d20 +24.9
(c 4.28, CHCl3)}; 1H NMR: 3.54 (dd, 1H, J1,2=6.8,
1
(146 mg, 87%) as an oil. [a] +14 (c 0.8, CHCl3); H
NMR: 1.38 (s, 6H, CH3), 2.93 (m, 2H, H2,5), 3.80 (dd,
0
2H, J1,2=7.3, J1,1 =9.2 Hz, H1,6), 3.49 (dd, 2H, J1 ,2=5,
0
0
0
0
J1,1 =9.2 Hz, H1 ,6 ), 4.06 (br.s, 2H, H3,4), 4.49 (s, 4H,
OCH2Ph), 7.30 (s, 10H, CHPh); 13C NMR (CDCl3):
19.7 (CH3), 51.9 (C2,5), 73.3, 73.8 (C1,6, OCH2Ph), 78.2
(C3,4), 108.8 (CMe2), 127.7, 128.4 (CHPh), 138.1 (CPh).
0
J1,1 =9.9Hz, H1), 3.65 (dd, 1H, J1 ,2=5.7, J1,1 =9.9Hz,
0
0
0
H1 ), 3.73 (dd, 1H, J5,6=6.0, J6,6 =11.3 Hz, H6), 3.79 (dd,
0
0
0
0
1H, J5,6 =5.3, J6,6 =11.3 Hz, H6 ), 3.96 (br.d, 1H,
J2,3=3.0 Hz, H3), 3.98 (br.d, 1H, J4,5=3.9 Hz, H4), 4.12
0
(ddd, 1H, J2,3=3.0, J1 ,2=5.7, J1,2=6.8 Hz, H2), 4,26
(4S,5R,6R,7S)-4,7-Dibenzyloxymethyl-5,6-dihydroxy-5,6-
O-methylethylidene-1,3-diazepan-2-thione (33). Carbon
disul®de (118 mL, 1.96 mmol, 2 equiv) was added to a
solution of the diamine 32 (391 mg, 0.98 mmol) in pyri-
dine (800 mL). After stirring at 60 ꢀC for 15 h, the pH
was adjusted to 2±3 by the addition of a 1 M aq HCl
solution. The mixture was extracted with dichloro-
methane and the combined organic layers were washed
with a 1 M aq NaOH solution, dried (MgSO4) and
concentrated in vacuo to yield the thiourea 33 (372 mg,
(ddd, 1H, J4,5=3.9, J5,60=5.3, J5,6=6.0 Hz, H5), 4.39,
4.62 (AB, 2H, JAB=12.0 Hz, OCH2Ph), 4.61±4.47 (m, 6H,
OCH2Ph), 7.29 (br.s, 20H, CHPh); 13C NMR: 68.3, 70.5
(C1,6), 71.5, 73.4 (OCH2Ph), 80.1, 82.8, 83.8 (C2,3,4,5),
127.6, 128.4, 129.7, 133.5 (CHPh), 137.9 (CPh); MS (NH3):
542 (M+NH4+).
(1R,7R,8S,10R)-8,10-Dibenzyloxy methyl-2,4,6,9-tetra-
oxa - bicyclo[5.3.0] decane (30). The tetrahydrofuran 30
was obtained from the diol 18 as described above for the
tetrahydrofuran 29 on a 0.1 mmol scale. After ¯ash chro-
matography (cyclohexane:EtOAc, 8:2), the compound 30
(26 mg, 68%) was obtained as an oil. [a] 4 (c 1.0,
CHCl3); 1H NMR: 3.53 (dd, 1H, J10,CHOBn=4.8,
1
86%) as a yellow foam. [a] +21 (c 1.0, CHCl3); H
NMR: 1.35 (s, 6H, CH3), 3.30±3.80 (m, 4H, CH2OBn),
3.80±4.20 (m, 4H, H4,5,6,7), 4.20±4.60 (m, 4H, OCH2Ph),
7.24 (m, 10H, CHPh); 13C NMR: 27.0 (CH3), 53.0 (C4,7),
69.5 (C5,6), 73.1 (CH2OBn, OCH2Ph), 110.2 (CMe2),
127.8, 128.4 (CHPh), 136.3 (CPh), 184.0 (C2); IR (neat):
1230 (nCˆS); HRMS (NH3): for C24H31N2O4S (MH+)
calcd 443.2004, found 443.1983.
JCH OBn=10.6 Hz, CH2OBn), 3.60±3.69 (m, 3H,
2
CH2OBn), 4.03 (m, 1H, H10), 4.25±4.36 (m, 2H, H1,8),
4.45 (dd, 1H, J7,8=8.3, J1,7=8.1 Hz, H7), 4.53 (s, 2H,
OCH2Ph), 4.58 (s, 2H, OCH2Ph), 4.82 (d, 1H,
0
0
J3,3 =6.2Hz, H3), 4.95 (d, 1H, J5,5 =6.2 Hz, H5), 5.11 (d,
(4R,5R,6R,7R)-4,7-Dibenzyloxymethyl-5,6-dihydroxy-2-
methylthio-4,5,6,7-tetrahydro-1,3-diazepinium para-tolu-
ene sulfonate (34). A solution of the thiourea 33 (40 mg,
0.09 mmol) and methyl para-toluenesulfonate (24 mL,
0.16 mmol, 1.7 equiv) in methanol (200 mL) was heated
under re¯ux during 30 min. After concentration in
vacuo, the resulting oil was triturated with diethylether
to yield the isothiourea 34 (47 mg, 88%) as an hygro-
2H, J3,3 =J5,5 =6.2 Hz, H3,5), 7.31 (s, 10H, CHPh); 13C
NMR: 69.7, 69.9 (CH2OBn), 73.5 (OCH2Ph), 76.4, 77.5,
80.1, 82.3 (C1,7,8,10), 95.5, 95.9 (C3,5), 127.5, 127.6, 129.7,
133.0 (CHPh), 138.3 (CPh); MS (NH3): 404 (M+NH4+).
0
0
2,5-Diazido-1,6-di-O-benzyl-2,5-dideoxy-3,4-O-methyl-
ethylidene-L-iditol (31). A solution of the dimesylate 26
(345 mg, 0.62 mmol) and sodium azide (405 mg,
6.2 mmol, 10 equiv) in DMF (7.5 mL) was heated at
120 ꢀC during 15 h. After concentration in vacuo and
addition of water, the mixture was extracted with
dichloromethane. The combined organic layers were
dried (MgSO4), concentrated in vacuo and chromato-
graphied (cyclohexane:dichloromethane, 2:8) to yield
the diazido compound 31 (160 mg, 57%) as a colorless
oil. [a] +67 (c 1.7, CHCl3); 1H NMR: 1.41 (s, 6H,
1
scopic solid. [a] +109 (c 0.4, CHCl3); H NMR: 2.29
(br.s, 3H, SCH3), 2.44 (br.s, 3H, ArCH3), 3.80 (m, 6H,
H4,7, CH2OBn), 4.40 (m, 6H, H5,6, OCH2Ph), 7.09 (br.s,
2H, CHAr), 7.24 (br.s, 10H, CHAr), 7.72 (br.s, 2H,
CHAr); 13C NMR: 15.4 (SCH3), 21.3 (ArCH3), 60.6
(C4,7), 69.6 (CH2OBn), 71.6 (C5,6), 73.4 (OCH2Ph),
126.0, 127.8, 128.0, 128.5, 128.9, 129.9 (CHAr), 137.5,
140.3, 141.9 (CAr); MS (FAB+): 417 (MH+).
0
CH3), 3.67 (dd, 2H, J1,2=4.7, J1,1 =10 Hz, H1,6), 3.75
(4S,5R,6R,7S)-4,7-Dibenzyloxymethyl-5,6-dihydroxy-5,6
-O-methylethylidene-2-(N-benzyl)-imino-1,3-diazepane
(35). Benzylamine (57 mL, 0.52 mmol, 1.1 equiv) and
triethylamine (63 mL, 0.94 mmol, 2 equiv) were succes-
sively added to a solution of both the thiourea 33
(208 mg, 0.47 mmol) and mercuric chloride (160 mg,
0
0
0
0
(dd, 2H, J1 2=7.8, J1,1 =10 Hz, H1 ,6 ), 4.13 (br.s, 2H,
H3,4), 4.46 (m, 2H, H2,5), 4.55 (s, 4H, OCH2Ph), 7.34 (s,
10H, CHPh); 13C NMR: 26.9 (CH3), 60.1 (C2,5), 69.9,
73.6 (C1,6, OCH2Ph), 77.1 (C3,4), 110.5 (CMe2), 127.8,
127.9, 128.5 (CHPh), 137.5 (CPh); MS (NH3): 425

316
Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
0.61 mmol, 1.3 equiv) in dichloromethane (6.5 mL).
After stirring at 20 ꢀC during 15 h, mercuric salts were
removed by ®ltration through a celite pad and washed
with dichloromethane. The combined organic layers
were successively washed with a 1 M aq HCl solution
and a 1 M aq NaOH solution then dried (MgSO4) and
concentrated in vacuo to yield the guanidine 35 (220 mg,
4.54 (AB, 4H, JAB=11.5 Hz, OCH2Ph), 4.92 (m, 2H,
H2,5), 7.33 (m, 10H, CHPh).
2,5-Diazido-1,6-di-O-benzyl-2,5-dideoxy-3,4-O-methyl-
ethylidene-D -mannitol (39). The diazido compound 39
was obtained from the dimesylate 38 as described above
for the diazide 31 on a 4.6 mmol scale. After ¯ash chro-
matography (dichloromethane:cyclohexane, 8:2), the
product 39 (1.3 g, 63% yield) was obtained as an oil. [a]
1
91%). [a] +32 (c 0.5, CHCl3); H NMR: 1.25 (br.s,
6H, CH3), 3.30±3.70 (m, 4H, CH2OBn), 3.70±4.00 (m,
6H, H4,5,6,7, NCH2Ph), 4.00±4.38 (m, 4H, OCH2Ph),
7,24 (br.s, 15H, CHPh); 13C NMR: 27.1 (CH3), 53.4
(C4,7), 56.3 (C5,6), 71.5, 73.0 (NCH2Ph, CH2OBn,
OCH2Ph), 109.5 (CMe2), 127.7, 128.4 (CHPh), 138.0
(CPh); IR (neat): 1625 (nCˆN); HRMS (NH3) for
C31H38N3O4: (MH+) calcd 516.2862, found 516.2845.
1
+8 (c 0.8, CHCl3); H NMR: 1.35 (s, 6H, CH3), 3.61
0
(dd, 2H, J1,1 =8.3, J1,2=9.4 Hz, H1,6), 3.69±3.75 (m,
0
0
0
0
2H, H2,5), 3.81 (dd, 2H, J1 ,2=3.0, J1,1 =8.3 Hz, H1 ,6 ),
4.01 (m, 2H, H3,4), 4.57 (s, 4H, OCH2Ph), 7.33 (s, 10H,
CHPh); 13C NMR: 27.4 (CH3), 63.1 (C2,5), 70.0, 73.6
(C1,6, OCH2Ph), 78.0 (C3,4), 110.7 (CMe2), 126.5, 127.8,
128.5 (CHPh), 137.6 (CPh); IR (neat): 2100 (nN ). Anal.
calcd for C23H28O4N6: C, 61.05; H, 6.24; N, 18.57.
Found: C, 61.02; H, 6.35; N, 18.47.
3
(4S,5R,6R,7S)-5,6-Dihydroxy-4,7-dihydroxy methyl-5,6-
O-methylethylidene-2-imino-1,3-diazepane (36). A solu-
tion of the benzylated guanidine 35 (191 mg, 0.42 mmol)
in a minimum of anhydrous THF was poured into
liquid ammonia (10 mL). Then, sodium was added until
persistance of a dark blue color and the mixture was
stirred in re¯uxing ammonia during 6 h. After careful
addition of solid ammonium chloride until decolora-
tion, ammonia was evaporated and the solid residue was
washed with dichloromethane and extracted with abso-
lute ethanol. The combined alcoholic layers were con-
centrated in vacuo. Neutralization of the resulting residue
by ethanolic elution through an Amberlite1 IRA 400
(OH ) packed column followed by concentration in
vacuo a€orded the guanidine 36 (67 mg, 74%) as an
2,5-Diamino-1,6-di-O-benzyl-2,5-dideoxy-3,4-O-methyl-
ethylidene-D-mannitol (40). The diamine 40 was obtained
from the diazido compound 39 as described above for the
diamine 32 on a 0.89mmol scale. After ¯ash chromato-
graphy (CH2Cl2:MeOH:ammonium hydroxide, 94:3:3),
the compound 40 (280mg, 79%) was obtained as an oil.
[a] +7 (c 0.7, CHCl3); 1H NMR: 1.33 (s, 6H, CH3), 3.07
0
(m, 2H, H2,5), 3.46 (dd, 2H, J1,2=7.1, J1,1 =9.1 Hz, H1,6),
0
0
0
0
3.69 (dd, 2H, J1 ,2=3.5, J1,1 =9.1 Hz, H1 ,6 ), 3.85 (m, 2H,
H3,4), 4.52 (s, 4H, OCH2Ph), 7.32 (s, 10H, CHPh); 13C
NMR: 27.3 (CH3), 54.0 (C2,5), 72.6, 73.3 (C1,6, OCH2Ph),
81.1 (C3,4), 108.9 (CMe2), 127.7, 128.4 (CHPh), 138.3
(CPh).
1
hygroscopic solid. H NMR (D2O): 1.60 (m, 6H, CH3),
3.70±4.30 (m, 8H, H4,5,6,7, CH2OH); 13C NMR (D2O):
28.9 (CH3), 57.0 (C4,7), 64.6 (CH2OH), 79.3 (C5,6), 113.4
(CMe2), 160.2 (C2).
1,6-Diazido-1,6-dideoxy-3,4-O-methyl ethylidene-L-iditol
(41). A solution of the bis-epoxide 1239 (688 mg,
3.7 mmol), ammonium chloride (870 mg, 16.3 mmol, 4.4
equiv) and sodium azide (2.4 g, 36.9 mmol, 10 equiv) in
methanol:water 8:1 (15 mL) was stirred at 20 ꢀC during
15 h. After concentration in vacuo, water was added and
the mixture was extracted with dichloromethane. The
combined organic layers were dried (MgSO4), con-
centrated in vacuo and chromatographied (dichlor-
omethane:ether, 80:20) to yield the diazido-diol 41
(835mg, 83%) as an oil. [a] 4 (c 1.0, CH2Cl2); 1H
1,6-Di-O-benzyl-3,4-O-methylethylidene-L-iditol (37).
Benzylic alcohol (4.2 mL, 40.3 mmol, 2.5 equiv) was
added to a suspension of sodium hydride (851 mg,
35.5 mmol, 2.2 equiv) in DMF (32 mL) and the reaction
mixture was stirred at 20 ꢀC during 3 h. Then, a solution
of the bis-epoxide 1239 (3 g, 16.11 mmol) in a minimum
of DMF was dropwise added at 0 ꢀC. After stirring at
20 ꢀC during 20 h, methanol was added and the reaction
mixture was concentrated in vacuo prior to the addition
of water. The mixture was then extracted with
dichloromethane and the combined organic layers were
dried (MgSO4), concentrated in vacuo and chromato-
graphied (cyclohexane:AcOEt, 70:30) to yield the diol 37
(3.7 g, 57%) as an oil. [a] 19 (c 5.1, CH2Cl2); 1H NMR:
0
NMR: 1.43 (s, 6H, CH3), 3.35 (dd, 2H, J1,2=5.2, J1,1
=
0
0
12.6Hz, H1,6), 3.45 (dd, 2H, J1 ,2=6.9, J1,1 =12.6 Hz,
0
H1 ,6 ), 3.73 (m, 2H, H2,5), 4.04 (s, 2H, H3,4); 13C NMR:
27.0 (CH3), 54.2 (C1,6), 68.9 (C2,5), 77.0 (C3,4), 110.3
0
(CMe2); IR (neat): 2100 (nN ). Anal. calcd for C9H16N6O4:
C, 39.70; H, 5.92; N, 30.87. Found: C, 39.83; H, 3.17; N,
30.11.
3
0
1.43 (s, 6H, CH3), 3.51 (dd, 2H, J1,2=5.5, J1,1 =10.0 Hz,
0
0
0
0
H1,6), 3.56 (dd, 2H, J1 ,2=6.0, J1,1 =10.0 Hz, H1 ,6 ), 3.78
(m, 2H, H2,5), 4.11 (m, 2H, H3,4), 4.52 (s, 4H, OCH2Ph),
7.33 (s, 10H, CHPh). Anal. calcd for C23H30O6: C, 68.64;
H, 7.51. Found: C, 68.54; H, 7.50.
1,6-Diazido-2,5-di-O-benzyl-1,6-dideoxy-3,4-O-methyl-
ethylidene-L-iditol (42). A solution of the diol 41
(748 mg, 2.75 mmol) in THF (4.5 mL) was dropwise
added to a suspension of sodium hydride (195 mg,
8.1 mmol, 3 equiv) in THF (4.5 mL) at 0 ꢀC. After stir-
ring at 20 ꢀC for 3 h, benzyl bromide (4 mL, 8.1 mmol, 3
equiv) was added and the mixture was stirred at 20 ꢀC
for an additional 15 h. Then, methanol was added and
the reaction mixture was concentrated in vacuo. After
addition of water and extraction with dichloromethane,
the combined organic layers were washed with brine,
1,6-Di-O-benzyl-2,5-di-O-methane sulfonyl-3,4-O-methyl-
ethylidene-L-iditol (38). The dimesylate 38 (2.57 g) was
quantitatively obtained from the diol 37 as described
above for the dimesylate 26 on a 4.59 mmol scale. H
NMR: 1.40 (s, 6H, CH3), 3.02 (s, 6H, SO3CH3), 3.70
1
0
(dd, 2H, J1,2=3.3, J1,1 =11.0 Hz, H1,6), 3.87 (dd, 2H,
0
J1,2=8.5, J1,1 =11.0 Hz, H1 ,6 ), 4.13 (m, 2H, H3,4), 4.48,
0
0

Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
317
dried (MgSO4), and concentrated in vacuo. Flash chro-
matograpy (dichloromethane:cyclohexane, 8:2) a€orded
the compound 42 (957 mg, 77%) as an oil. [a] +34 (c
the compound 47 (94 mg, 82%) was obtained as an oil.
[a] +16 (c 1.0, CHCl3); 1H NMR: 1.36 (s, 6H, CH3), 2.93
(m, 4H, H1,6), 3.51 (m, 2H, H2,5), 4.14 (m, 2H, H3,4), 4.56
(s, 4H, OCH2Ph), 7.27 (br.s, 10H, CHPh); 13C NMR: 27.3
(CH3), 41.2 (C1,6); 72.1 (OCH2Ph), 78.7, 81.4 (C2,3,4,5),
109.7 (CMe2), 127.9, 128.4 (CHPh), 138.2 (CPh).
1
1.0, CHCl3); H NMR: 1.37 (s, 6H, CH3), 3.41 (br.s,
6H, H1,2,5,6), 4.04 (s, 2H, H3,4), 4.51, 4. 71 (AB, 4H,
JAB=11.7 Hz, OCH2Ph), 7.32 (br.s, 10H, CHPh); 13C
NMR: 26.9 (CH3), 51.8 (C1,6), 73.4 (OCH2Ph), 76.0,
76.2 (C2,3,4,5), 109.5 (CMe2), 128.1, 128.2, 128.5 (CHPh),
(4R,5R,6R,7R)-4,7-Dibenzyloxymethyl-5,6-dihydroxy-
5,6-O-methylethylidene-1,3-diazepan-2-thione (48). The
thiourea 48 (125mg, 81%) was obtained as a yellow foam
from the diamine 40 on a 0.35 mmol scale, according to
the procedure described above for the thiourea 33. [a]
137.5 (CPh); IR (neat) 2100 (nN ). Anal. calcd for
C23H28N3O4: C,61.0; H, 6.24; N, 18.57. Found: C,
61.00; H, 6.25; N, 18.43.
3
1
1,6-Diamino-2,5-di-O-benzyl-1,6-dideoxy-3,4-O-methyl-
ethylidene - L - iditol (43). The diamine 43 was obtained
from the diazido compound 42 as described above for the
diamine 32 on a 0.85mmol scale. After ¯ash chromato-
graphy (CH2Cl2:MeOH:ammonium hydroxide, 90:7:3),
the compound 43 (240mg, 70%) was obtained as an oil.
[a] +12 (c 0.4, CHCl3); 1H NMR: 1.40 (s, 6H, CH3), 2.77
+40 (c 0.4, CHCl3); H NMR: 1.34 (s, 6H, CH3), 3.44
(m, 6H, H4,7, CH2OBn), 3.74 (m, 2H, H5,6), 4.55 (s, 4H,
OCH2Ph), 7.33 (s, 10H, CHPh); 13C NMR: 26.8 (CH3),
56.7 (C4,7), 69.0, 73.4 (CH2OBn, OCH2Ph), 78.9 (C5,6),
111.6 (CMe2), 127.0, 128.6 (CHPh), 137.2 (CPh), 187.3
(C2); IR (neat) 1230 (nCˆS); HRMS (NH3) for C24H31
N2O4S: (MH+) calcd 443.2004, found 443.2003.
0
(dd, 2H, J1,2=5.9, J1,1 =13.3 Hz, H1,6), 2.89 (dd, 2H,
0
0
0
0
J1 ,2=5.0, J1,1 =13.3 Hz, H1 ,6 ), 3.34 (m, 2H, H2,5), 4.14
(br.s, 2H, H3,4), 4.57, 4.64 (AB, 4H, JAB=11.8 Hz,
OCH2Ph), 7.30 (br.s, 10H, CHPh); 13C NMR: 27.1 (CH3),
42.3 (C1,6), 72.7 (OCH2Ph), 77.7, 78.8 (C2,3,4,5), 109.1
(CMe2), 127.8, 128.0, 128.4 (CHPh), 138.2 (CPh).
(4R,5R,6R,7R)-4,7-Dibenzyloxymethyl-5,6-dihydroxy-
5,6-O-methylethylidene-2-(N-benzyl)-imino-1,3-diazepane
(49). The guanidine 49 (45 mg, 78%) was obtained as a
yellow foam from the thiourea 48 on a 0.11 mmol scale,
according to the procedure described above for the
guanidine 35. [a] +37 (c 0.4, CHCl3); 1H NMR: 1.33 (s,
6H, CH3), 3.42±3.72 (m, 6H, H4,7, CH2OBn), 3.80 (m,
2H, H5,6), 4.18±4.41 (m, 2H, NCH2Ph), 4.54 (m, 4H,
OCH2Ph), 7.14±7.25 (m, 15H, CHPh); 13C NMR: 26.7
(CH3), 55.5 (C4,7), 68.4, 73.5 (NCH2Ph, CH2OBn,
OCH2Ph), 78.2 (C5,6), 111.6 (CMe2), 127.0, 128.0, 128.3,
128.5, 129.0, 129.1 (CHPh), 135.2, 137.4 (CPh), 159.8
(C2); IR (neat): 1635 (nCN); HRMS (NH3) for
C31H38N3O4: (MH+) calcd 516.2862, found 516.2872.
1,6-Diazido-1,6-dideoxy-3,4-O-methyl ethylidene-D-man-
nitol (45). A solution of the ditosylate 4439 (5 g,
9.5 mmol) and sodium azide (2.5 g, 38 mmol, 4 equiv) in
DMF (38 mL) was heated at 70 ꢀC during 3 h. After
concentration in vacuo and addition of water, the mix-
ture was extracted with dichloromethane. The combined
organic layers were dried (MgSO4), concentrated in
vacuo and chromatographied (dichloromethane:ether,
90:10) to yield the diazido-diol 45 (2 g, 75%) as an oil.
1
[a] +45 (c 2.0, CH2Cl2); H NMR: 1.35 (s, 6H, CH3),
3.44 (dd, 2H, J1,2=4.6, J1,1 =12.2 Hz, H1,6), 3.64 (d,
(4R,5R,6R,7R)-5,6-Dihydroxy-4,7-dihydroxy methyl-5,6-
O-methylethylidene-2-imino-1,3-diazepane (50). The
guanidino-sugar 50 (21 mg, 75%) was obtained as an
hygroscopic solid from the guanidine 49 on a 0.13 mmol
scale, according to the procedure described above for
0
2H, J1,1 =12.2 Hz, H1 ,6 ), 3.78 (m, 4H, H2,3,4,5); 13C
NMR: 26.7 (CH3), 54.2 (C1,6), 72.2 (C2,5), 80.1 (C3,4),
109.9 (CMe2); IR (neat) 2100 (nN3). Anal. calcd for
C9H16N6O4: C, 39.70; H, 5.92; N, 30.87. Found: C,
39.72; H, 6.23; N, 29.93.
0
0
0
1
the guanidino-sugar 36. H NMR (D2O): 1.52 (s, 6H,
CH3), 3.55 (m, 2H, H4,7), 3.79 (dd, 2H, J4,CHOH=5.6,
JCH OH=12.1 Hz, CH2OH), 3.91±3.99 (m, 4H, H5,6
,
2
1,6-Diazido-2,5-di-O-benzyl-1,6-dideoxy-3,4-O-methyl-
ethylidene - D - mannitol (46). The dibenzyl-ether 46 was
obtained from the diol 45 as described above for the
dibenzyl-ether 42 on a 1.78mmol scale. After ¯ash chro-
matography (dichloromethane:cyclohexane, 8:2), the
CH0OH); 13C NMR (D2O): 28.7 (CH3), 59.2 (C4,7), 63.4
(CH2OH), 80.4 (C5,6), 115.4 (CMe2), 162.8 (C2).
(5S,6R,7R,8S)-5,8-Dibenzyloxy-6,7-dihydroxy-6,7-O-me-
thylethylidene-1,3-diazonan-2-thione (51). The thiourea
51 (335 mg) was quantitatively obtained as a yellow
foam from the diamine 43 on a 0.77 mmol scale
according to the procedure described above for thiourea
1
compound 46 (223mg, 81%) was obtained as an oil. H
0
NMR: 1.34 (s, 6H, CH3), 3.39 (dd, 2H, J1,2=5.8, J1,1
0
=
13.2 Hz, H1,6), 3.49 (dd, 2H, J1 ,2=3.3, J1,1 =13.2 Hz,
0
1
0
0
H1 ,6 ), 3.61 (m, 2H, H2,5), 4.06 (m, 2H, H3,4), 4.53, 4.66
(AB, 4H, JAB=11.5 Hz, OCH2Ph), 7.29 (m, 10H, CHPh);
13C NMR: 27.2 (CH3), 51.1 (C1,6), 72.8 (OCH2Ph), 78.3,
79.3 (C2,3,4,5), 110.2 (CMe2), 128.0, 128.5 (CHPh), 137.5
(CPh). Anal. calcd for C23H28N6O4: C, 61.05; H, 6.24; N,
18.57. Found: C, 61.21; H, 6.16; N, 18.51.
33. [a] +21 (c 1.1, CHCl3); H NMR: 1.36 (br.s, 6H,
CH3), 3.43 (m, 4H, H4,9), 3.65 (m, 2H, H5,8), 4.01 (br.s,
2H, H6,7), 4.43, 4.58 (AB, 4H, JAB=11.8 Hz, OCH2Ph),
7.25 (s, 10H, CHPh); 13C NMR: 27.0 (CH3), 44.9 (C4,9),
72.3 (OCH2Ph), 73.8, 77.4 (C5,6,7,8), 109.5 (CMe2), 128.3
(CHPh), 137.5 (CPh), 182.6 (C2); IR (neat): 1215 (nCˆS);
HRMS (NH3) for C24H31N2O4S: (MH+) calcd
443.2004, found 443.2003.
1,6-Diamino-2,5-di-O-benzyl-1,6-dideoxy-3,4-O-methyl-
ethylidene-D-mannitol (47). The diamine 47 was obtained
from the diazido compound 46 as described above for the
diamine 32 on a 0.29 mmol scale. After ¯ash chromato-
graphy (CH2Cl2:MeOH:ammonium hydroxide, 90:7:3),
(5S,6R,7R,8S)-5,8-Dibenzyloxy-6,7-dihydroxy-6,7-O-me-
thylethylidene-2-(N-benzyl)-imino-1,3-diazonane (52).
The guanidine 52 (55 mg, 94%) was obtained as a yellow

318
Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
0
(CH3), 35.3 (C2 ), 45.9 (C1 ), 58.6 (C4,7), 67.7 (CH2OH),
80.9 (C5,6), 112.1 (CMe2), 159.5 (C2); MS (NH3): 302
(MH+).
0
foam from the thiourea 51 on a 0.11 mmol scale
according to the procedure described above for the
guanidine 35. [a] +21 c 1.1, CHCl3); H NMR: 1.25
1
(br.s, 6H, CH3), 3.15±3.82 (m, 8H, H4,5,8,9, NCH2Ph),
3.8±4.30 (m, 4H, H6,7), 4.30±4.80 (m, 4H, OCH2Ph),
7.20 (br.s, 15H, CHPh); 13C NMR: 27.0 (CH3), 43.6
(C4,9, NCH2Ph), 73.5 (OCH2Ph), 77.7 (C5,6,7,8), 109.9
(CMe2), 128.1, 128.5 (CHPh), 137.6 (CPh), 156 (C2); IR
(neat): 1630 (nCˆN); HRMS (NH3) for C31H38N3O4:
(MH+) calcd 516.2862, found 516.2872.
Methyl (40S,50R,60R,70S)-6-deoxy-6-(40,70 -dibenzyloxy-
methyl-50,60-dihydroxy-50,60-O-methylethylidene-10,30-dia-
zepan-20-yl)-imino-2,3,4-tri-O-benzyl-D-glucopyranoside
(57). The guanidine 57 (327 mg, 86%) was obtained as
a yellow foam from the thiourea 33 on a 0.79mmol scale,
according to the procedure described above for the gua-
nidine 35 using methyl 6-amino-6-deoxy-2,3,4-tri-O-ben-
zyl-d-glucopyranoside. [a] +36 (c 0.5, CHCl3); 1H NMR:
(5R,6R,7R,8R)-5,8-Dibenzyloxy-6,7-dihydroxy-6,7-O-me-
thylethylidene-1,3-diazonan-2-thione (53). The thiourea
53 (145 mg) was quantitatively obtained as a yellow
foam from the diamine 47 on a 335 mmol scale, accord-
ing to the procedure described above for the thiourea
33. [a] +20 (c 0.4, CHCl3); 1H NMR: 1.34 (s, 6H,
CH3), 3.65 (m, 6H, H4,5,8,9), 4.06 (m, 2H, H6,7), 4.54 (m,
4H, OCH2Ph), 7.26 (br.s, 10H, CHPh); 13C NMR: 27.0
(CH3), 44.3 (C4,9), 72.3 (OCH2Ph), 77.9, 78.4 (C5,6,7,8),
110.1 (CMe2), 127.9, 128.2, 129.4 (CHPh), 137.7 (CPh),
182.7 (C2); IR (neat): 1210 (nCˆS); HRMS (NH3) for
C24H31N2O4S: (MH+) calcd 443.2004, found 443.2009.
0
0
0
0
1.37 (m, 6H, CH3), 3.25±4.10 (m, 17H, H4,5,6,7,2 ,3 ,4 ,5 ,
CH2N, OCH3, CH2OBn), 4.39±5.00 (m, 11H, OCH2Ph,
H1 ), 7.24 (m, 25H, CHPh); 13C NMR: 27.0 (CH3),
0
46.0 (CH2N), 54.0, 55.3 (C4,7, OCH3), 69.5 (C5,6), 73.3,
74.9, 75.7 (CH2OBn, OCH2Ph), 77.3, 78.8, 80.1, 81.3
0
0
0
0
0
(C2 ,3 ,4 ,5 ), 98.0 (C1 ), 110.0 (CMe2), 127.7, 128.0, 128.4
(CHPh), 138.1, 138.7 (CPh), 158.0 (C2); IR (neat): 1635
(nCˆN); HRMS (NH3) for C52H62O9N3: (MH+) calcd
872.4486, found 872.4501.
Methyl (40S,50R,60R,70S)-6-deoxy-6-(40,70-dihydroxyme-
thyl-50,60-dihydroxy-50,60-O-methylethylidene-10,30-diaze-
pan-20 -yl)-imino-D-glucopyranoside (58). The guanidine
58 (209mg, 79%) was obtained as an hygroscopic solid
from the guanidine 57 on a 0.62mmol scale according to
the procedure described above for the guanidino-sugar 36.
(5R,6R,7R,8R)-5,8-Dibenzyloxy-6,7-dihydroxy-6,7-O-
methylethylidene-2-(N-benzyl)-imino-1,3-diazonane (54).
The guanidine 54 (139 mg, 94%) was obtained as a yel-
low foam from the thiourea 53 on a 287 mmol scale,
according to the procedure described above for the
guanidine 35. [a] 8 (c 0.5, CHCl3); 1H NMR: 1.26
(br.s, 6H, CH3), 3.30±3.56 (m, 4H, H4,9), 3.75 (m, 2H,
NCH2Ph), 4.07±4.58 (m, 8H, H5,6,7,8, OCH2Ph), 7.22
(br.s, 15H, CHPh); 13C NMR: 26.8 (CH3), 43.1, 46.0
(C4,9, NCH2Ph), 72.2 (OCH2Ph), 79.0 (C5,6,7,8), 109.8
(CMe2), 128.0, 128.5 (CHPh), 137.3 (CPh); IR (neat):
1630 (nCˆN); HRMS (NH3) for C31H38O4N3: (MH+)
calcd 516.2862, found 516.2877.
1
[a] +55 (c 0.75, CH3OH); H NMR (CD3OD): 1.38 (s,
3H, CH3), 1.46 (s, 3H, CH3), 3.42 (m, 5H, CH2N, OCH3),
3.55±3.85 (m, 8H, H4,5,6,7, CH2OH), 3.90±4.40 (m, 4H,
H2 ,3 ,4 ,5 ), 4.68 (m, 1H, H1 ); 13C NMR (CD3OD): 30.0
(CH3), 47.0 (CH2N), 51.8, 58.3 (C4,7, OCH3), 66.0
0
0
0
0
0
0
(CH2OH), 76.1, 76.9, 77.4, 79.8, 81.7 (C5,6,2 ,3 ,4 ,5 ), 103.8
0
0
0
0
(C1 ), 112.0 (CMe2), 159.9 (C2).
Inhibition analysis of compounds 1, 2, 3, 4, 5 and 6
against various glycosidases. a-d-glucosidase from bacillus
3
(4S,5R,6R,7S)-4,7-Dibenzyloxymethyl-5,6-dihydroxy-5,6
-O-methylethylidene-2-(N-butyl)-imino-1,3-diazepane
(55). The guanidine 55 (318mg, 65%) was obtained as a
yellow foam from the thiourea 33 on a 1.02 mmol scale,
according to the procedure described above for the gua-
nidine 35 using n-butylamine. [a] +42 (c 0.7, CHCl3); 1H
stearothermophilus (11 Â 10 unit, EC 3.2.1.20), b-d-
glucosidase from almonds (15 Â 10 3 unit, EC 3.2.1.21),
3
a-d-mannosidase from Jack beans (10Â10 unit, EC
3
3.2.1.24) or a-l-fucosidase from bovine kidney (4Â 10
unit, EC 3.2.1.111) were purchased from Sigma-Aldrich
Chimie. Incubation mixtures (1 mL) contained 0.05 M
citrate±phosphate bu€er at a pH of 6.8, 5.0, 4.5 and 5.5,
respectively, according to the enzyme, 4-nitrophenyl-a-d-
glucopyranoside (2 mM), or 4-nitrophenyl-b-d-glucopyr-
anoside (2 mM), or 4-nitrophenyl-a-d-mannopyranoside
(2 mM) or 4-nitrophenyl a-l-fucopyranoside (0.25mM),
the potential inhibitor 1, 2, 3, 4, 5, or 6 at a ®nal con-
centration of 1 mM and ®nally, the enzyme a-d-gluco-
0
0
0
NMR: 0.84 (m, 3H, H4 ), 1.24±1.40 (m, 10H, H2 ,3 , CH3),
0
3.20±4.20 (m, 10H, H4,5,6,7,1 , CH2OBn), 4.19 (m, 4H,
13
0
OCH2Ph), 7.23 (m, 10H, CHPh); C NMR: 13.6 (C4 ),
0
0
0
19.9 (C3 ), 27.1 (CH3), 30.7 (C2 ), 41.0 (C1 ), 52.8 (C4,7),
73.1 (CH2OBn, OCH2Ph), 77.5 (C5,6), 109.9 (CMe2),
127.6, 128.3 (CHPh), 138.0 (CPh), 159.0 (C2); IR (neat):
1625 (nCˆN); HRMS (NH3) for C28H40O4N3: (MH+)
calcd 482.3019, found 482.2986.
3
3
sidase, 11 Â 10 unit; b-d-glucosidase, 15 Â 10 unit;
3
a-d-mannosidase, 10 Â 10
unit; or a-l-fucosidase,
3
(4S,5R,6R,7S)-5,6-Dihydroxy-4,7-hydroxy methyl-5,6-O
-methylethylidene-2-(N-butyl)-imino-1,3-diazepane (56).
The guanidine 56 (140 mg, 73%) was obtained as an
hygroscopic solid from the guanidine 55 on a 0.64 mmol
scale, according to the procedure described above for
the guanidino-sugar 36. [a] +45 (c 0.7, MeOH); 1H
4 Â 10 unit per sample). After a 10 min incubation
period at 37 ꢀC, the reaction was quenched by the addi-
tion of a 0.2 M glycine±sodium hydroxide bu€er at
pH 10 (1 mL). The optical absorbance at 400 nm was
measured to determine the amount of liberated 4-nitro-
phenol and the percentage of inhibition was calculated.
0
0
0
NMR (CD3OD): 0.93 (t, 3H, J3 ,4 =7.1 Hz, H4 ), 1.39±
0
1.45 (m, 8H, H3 , CH3), 1,55 (m, 2H, H2 ), 3.14 (m, 2H,
0
When the percentage of inhibition was greater than
50%, as it was the case for the compounds 5 and 6 against
a-l-fucosidase, the assay was performed in presence of
0
H1 ), 3.55±3.80 (m, 6H, H4,7, CH2OH), 4.05 (br.s, 2H,
13
0
0
H5,6); C NMR (CD3OD): 16.2 (C4 ), 23.2 (C3 ), 29.3

Y. Le Merrer et al. / Bioorg. Med. Chem. 8 (2000) 307±320
319
variable amounts of the 4-nitrophenyl-a-l-fucopyrano-
side (in the range of 0.05 to 0.5 mM) and in the absence
or in the presence of the inhibitor (at a constant ®nal con-
centration of 1 mM) in 0.05M citrate±phosphate bu€er at
pH 5.5. Lineweaver±Burk plots were then constructed for
both reactions (with and without inhibitor) and the Ki was
calculated from the two Michaelis±Menten constants
(Km) thus obtained.
27. Bleriot, Y.; Dintinger, T.; Genre-Grandpierre, A.; Padrines,
M.; Tellier, C. Bioorg. Med. Chem. Lett. 1995, 5, 2655.
28. Lehmann, J.; Rob, B. Tetrahedron: Asymmetry 1994, 5,
2255.
29. Kobayashi, Y.; Miyazaki, H.; Shiozaki, M. J. Am. Chem.
Soc. 1992, 114, 10065.
30. Asano, N.; Kizu, H.; Oseki, K.; Tomioka, E.; Matsui, K.;
Okamoto, M.; Baba, M. J. Med. Chem. 1995, 38, 2349.
31. Preliminary results: Gauzy, L., Le Merrer, Y., Depezay,
J.C. Synlett 1998, 402.
32. Le Merrer, Y.; Poitout, L.; Depezay, J. C.; Dosbaa, I.;
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Acknowledgements
We wish to thank Rhone±Poulenc Rorer for ®nancial
support (LG) and Drs D. Damour-Barbalat and S.
Mignani for helpful discussions. We are grateful to I.
Dosbaa of Laboratoire de Biochimie et de Glycobio-
logie (Pr. M.J. Foglietti) for her help in assaying guan-
idino-sugars as potent inhibitors of glycosidases.
37. Gravier-Pelletier, C., Bourissou, D., Le Merrer, Y., Depe-
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are attributed. For example, in the molecule 14, H4 and H9
0
0
have the same chemical shift as well as H4 and H9 . The cor-
responding notation for these protons is: 3.42 (dd, 2H,
0
J4,5=5.8, J4,4 =15.6 Hz, H4,9), 3.64 (m, 2H, H4 ,9 ).
0
0
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