Design and synthesis of a heparanase inhibitor with pseudodisaccharide structure

Article abstract of DOI:10.1016/S0040-4020(01)00642-1

Aza-analogue of the basic disaccharide unit of heparane sulfate was designed as a potent inhibitor against heparanase produced by solid tumors cell, and synthesized via a coupling reaction of phenyl 2-azide-1-thio-D-glucopyranoside derivatives with a partially protected 1-deoxynojirimycin derived from D-glucose. The azapseudodisaccharide inhibited tumor cell heparanase with IC₅₀ value of 58-63 μM.

Full text of DOI:10.1016/S0040-4020(01)00642-1

TETRAHEDRON
Pergamon
Tetrahedron 57 72001) 6915±6926
Design and synthesis of a heparanase inhibitor with
pseudodisaccharide structure
Shunya Takahashi,a,p Hiroyoshi Kuzuharaa and Motowo Nakajimab
aRIKEN ꢀThe Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan
bDiscovery Research, Takarazuka Research Institute, Novartis Pharma K. K., 10-66 Miyuki-cho, Takarazuka 665-8666, Japan
Received 9 February 2001; accepted 15 June 2001
AbstractÐAza-analogue of the basic disaccharide unit of heparane sulfate was designed as a potent inhibitor against heparanase produced
by solid tumors cell, and synthesized via a coupling reaction of phenyl 2-azide-1-thio-d-glucopyranoside derivatives with a partially
protected 1-deoxynojirimycin derived from d-glucose. The azapseudodisaccharide inhibited tumor cell heparanase with IC50 value of
58±63 mM. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
gulonic acid 72). The latter is known as a naturally occurring
powerful enzyme inhibitor against human liver b-d-glu-
curonidase.9 Although 1-deoxynojirimycin 73)10 and its
stereoisomers have been widely utilized for designing and
synthesizing new glycosidase inhibitors,11 such utilizations
of 2 have not been reported.12 Here we describe the syn-
thesis of 1 and its enzyme inhibitory activities 7Fig. 1).13
It has been recognized that tumor metastasis occurs via
complex multistage process which involves tumor cell
adhesion to various basement membrane components and
degradation of extracellular matrix and basement
membranes.1 Glycosaminoglycans such as heparan sulfate
7HS) are the important constituents in these structures. In
1983, HS degradative activities of murine B 16 melanoma
sublines were found to correlate with their metastatic lung
colonization and invasive abilities, and this activity was
proven to be caused by HS speci®c endo-b-glucronidase
7heparanase) cleaving the linkage between glucuronic acid
and N-acetyl-glucosamine in HS.2 These results suggest that
heparanase plays an important role in cell invasion of some
malignant solid tumors through basement membranes.
Consequently, molecules that are able to inhibit heparanase
might possibily prevent tumor metastasis. Such con-
sideration stimulated researchers to develop heparanase
inhibitors. Heparin, modi®ed heparin derivatives,3 and
suramine4 have been reported to inhibit heparanase and
suppress tumor metastatis. However, the diverse function-
ality and higher-molecular weight nature of these sub-
stances have also led to search of low-molecular weight
heparanase inhibitors with a potentialy higher speci®-
city.5±7 In connection with our chemical studies on endo-
glycosidase inhibitors,8 we were interested in heparanase
inhibitor, and designed a new compound 1, a mimic of the
basic repeating disaccharide unit of heparane sulfate, as
a potential heparanase inhibitor. The compound 1 is
composed of 2-acetamido-2-deoxy-6-O-sulfonyl-a-d-
glucopyranosyl moiety and 2,6-dideoxy-2,6-imino-l-
2. Results and discussions
Our synthetic process directed towards 1 involved chemical
conversion14 of 1,2:5,6-di-O-isopropylidene-a-d-gluco-
furanose 74) into 2,3-di-O-benzyl-N-benzyloxycarbonyl-6-
O-t-butyl-diphenylsilyl 7TBDPS)-1,5-dideoxy-1,5-imino-d-
glucitol 714) and benzyl N-benzyloxycarbonyl-4,5-di-O-
benzyl-2,6-dideoxy-2,6-imino-l-gulonate 728) as glycosyl
acceptors, their coupling with phenyl 2-azido-3,4-di-O-
benzyl-2-deoxy-6-O-p-methoxybenzyl-1-thio-d-gluco-
pyranosides 717) as a glycosyl donor, and regioselective
manipulations such as imino acid formation and O-sul-
fation.
Keywords: aza compounds; carbohydrate mimetics; enzyme inhibitors;
thioglycosides.
Corresponding author. E-mail: shunyat@riken.go.jp
p
Figure 1.
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020701)00 642-1

6916
S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
Scheme 1. 7a) NaH, allyl bromide, 08C; 7b) 80% AcOH, 608C; 7c) BzCl, and then MsCl, pyridine±CH2Cl2, 215±08C; 7d) NaOMe, MeOH, rt; 7e) t-BuOK,
THF±DMF, 08C±rt; 7f) NaN3, NH4Cl, aq. DMF, 808C; 7g) BnBr, NaH, n-Bu4N1I2, DMF, 08C; 7h) 3% HCl in MeOH, rt; 7i) Tf2O, pyridine, CH2Cl2, 2608C;
7j) Ph3P, CH2Cl2, rt±458C; 7k) K2CO3, H2O, dioxane, MeOH, THF, rt; and then ZCl, 08C; 7l) TFA, H2O±dioxane, rt; 7m) NaBH4, 95% EtOH, 08C;
7n) TBDPSCl, imidazole, DMF, rt; 7o) PdCl2, NaOAc, aq. AcOH, 508C; 7p) TBAF, AcOH, THF, rt; 7q) 10% Pd±C, H2, AcOH±EtOH±H2O, rt.
Initially, 1,2:5,6-di-O-isopropylidene-a-d-glucofuranose
74) was converted into the 6-O-benzoyl-5-O-mesyl deriva-
tive 5 in 70% overall yield by the three-sequence which
included 7a) 3-O-allylation with sodium hydride 7NaH)
and allyl bromide in N,N-dimethylformamide 7DMF),
7b) selective hydrolysis of the 5,6-O-isopropylidene group
with 90% acetic acid, 7c) one-pot esteri®cation with benzoyl
chloride followed by methanesulfonyl chloride in pyridine
7Scheme 1). The compound 5 was treated with methanolic
sodium methoxide to afford an epoxide 6 in 60% yield,
while successive treatments of 5 with sodium methoxide
and potassium t-butoxide in tetrahydrofuran afforded 6 in
76% yield. The oxirane in 6 was opened by the action of
sodium azide in aqueous DMF in the presence of NH4Cl,
and subsequent benzylation {benzyl bromide 7BnBr), NaH,
n-tetrabutylammonium iodide 7n-Bu4NI)} gave a benzyl
ether 7 in 85% yield. This was treated with 3%HCl
in methanol to afford an anomeric mixture of methyl glyco-
sides, which was readily separated into each isomer 8 750%
yield) and 9 747% yield) by chromatography on silica gel. In
order to attain the effective construction of the piperidine
ring system 7vide infra), the b-isomer 9 was again equili-
brated under the same conditions to the anomeric mixture,
from which 8 was isolated in 52% yield. By repetition of this
procedure, 80% yield of 8 was attained. Treatment of 8 with
tri¯ic anhydride in pyridine±dichloromethane at 2608C
gave an unstable 2-O-tri¯ate derivative, from which the
piperidine ring system was formed via reduction of the
azide group to amine with triphenylphosphine, followed
by cyclization through an intramolecular displacement of
the tri¯uoromethanesulfonyl group under basic condition.
The bicyclic amine thus formed was isolated, upon treat-
ment with benzyloxycarbonyl chloride, as a carbamate 10
²
in 69% yield from 8. On the other hand, the stereoisomer 11
was obtained from 9 in only 8% yield under the same con-
ditions. The formation of the bicyclic amine from 9 as
monitored by TLC analysis was sluggish to decompose
the intermediary tri¯ate derivative under these conditions.
This low reactivity would be explained by the steric
hindrance arising from the b-methoxy group. Acidic
hydrolysis of 10, followed by sodium borohydride reduction
gave a diol 12 in 71% yield. In the same way, 11 was also
converted into 12 in 70% yield. The primary hydroxyl group
in 12 was protected as the TBDPS ether, and the resulting
secondary hydroxyl group was benzylated to afford a fully
protected nojrimycin derivative 13 in 88% overall yield.
Cleavage of the allyl group with palladium 7II) chloride
and sodium acetate in aqueous acetic acid provided the
³
glycosyl acceptor 14 in 72% yield.
²
All 1H NMR 7400 or 500 MHz, 238C) spectra of 10±14, 18±30 appears as
broadened and complicated signals probably due to the presence of some
Scheme 2. 7a) PhSTMS, ZnI2, 7CH2Cl)2, rt; 7b) K2CO3, aq. MeOH, rt;
7c) MPMCl, NaH, n-Bu4N1I2, DMF, 08C.
1
rotamers. This made dif®cult the H NMR analyses. However, the dif®-
culty was circumvented by measuring such spectra in d6-dimethyl sulf-
oxide at the elevated temperature 780±1208C). In addition, the preferred
conformation of a series of deoxynojirimycin derivatives having the
benzyloxycarbonyl group seems to be near 1C4 rather than 4C1 form
7vide supra, see also Ref. 11).
³
In order to con®rm the piperidine ring system, 14 was also transformed
into 3 7see Section 3).

S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
6917
The glycosyl donor 17 was prepared from 1,6-anhydro-2-
azido-3,4-di-O-benzyl-2-deoxy-b-d-glucopyranose 1515 as
follows 7Scheme 2). The anhydro sugar 15 was treated
with 7phenylthio)trimethylsilane in the presence of zinc
iodide,16 and subsequently with potassium carbonate in
methanol to afford thioglycosides 16. The hydroxyl group
in 16 was protected as p-methoxybenzyl ether, giving the
donor 17 7a/bˆ2/1) in 72% yield from 15. Although each
anomer was readily separated by chromatography on silica
gel, these isomers were employed to the next glycosidation
step without separation.
High a-selectivity 719/18ˆ92/8, 59% yield) was observed
when methyl tri¯ate19 was used as a promoter in ether. The
stereochemistry of newly created glycosidic linkage in
18 and 19 was con®rmed by the H NMR analyses in
d6-dimethylsulfoxide at the elevated temperature 7110±
1208C). Thus, the spectrum of 19 showed the H-10 reso-
1
0
0
nance at d 5.17 as a doublet with J1 ,2 ˆ3.7 Hz, whereas
one of 18 exhibited a signal, due to H-10, at d 4.52 as a
doublet with 7.8 Hz.
Constructing a desired glycosidic linkage between 14 and
17, we turned our attention next to functionalization of this
pseudodisaccharide 19. Initially, the azide 19 was reduced
with triphenylphosphine, after N-acetylation, giving a
20-acetamido derivative 20 in 93% yield. Treatment of 19
with thioacetic acid in pyridine afforded 20 in a single step
785% yield). This compound 20 underwent de-silylation
with tetrabutylammonium ¯uoride 7TBAF) in the presence
of acetic acid to give 21 in 99% yield. Deprotection in the
absence of acetic acid resulted in the product contaminated
with an N,O-cyclic carbonate produced by an attack of
6-O-alkoxide anion to benzyloxycarbonyl group. Con-
version of the hydroxyl group in 21 into the carboxylic
function was achieved by Jones oxidation at room tempera-
ture in a short time. The resulting carboxylic acid was,
after methylation, subject to de-O-p-methoxybenzylation
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone 7DDQ) to
afford an alcohol 22 in 65% overall yield. Attempts to
synthesize 1 from 22, however, gave unsatisfactory results
because of contamination of side-products encountered in
alkaline hydrolysis at the later stage of total synthesis.
This problem was ®nally overcome by the replacement of
protection for the carboxyl group. Thus, the carboxylic acid
obtained from 21 reacted with BnBr in the presence of
cesium carbonate, giving the corresponding benzyl ester
23 in 72% yield. Upon treatment with DDQ, 23 afforded
Coupling reaction of 14 and 17 was examined under several
conditions17±19 as shown in Table 1 7Scheme 3). Best result
was obtained by the action of N-iodosuccinimide 7NIS) in
the presence of tri¯ic acid in ether±dichloromethane 75:1)18
at 2508C to afford an a-glycoside 19 and the corresponding
b-isomer 18, in 77 and 15% yield, respectively 7entry 5).
Table 1. Glycosidation reactions
Entry Donor 17 7equiv.)
Conditions
Yield 7%)a
18
19
1
2
3
1.2
1.2
1.2
NBS, CH2Cl2, MS4A, rt, 16 h
NBS, CH3CN, MS4A, rt, 16 h
NBS, LiClO4, Et2O, MS4A,
215±08C, 5 h
6
18
3
28
12
27
4
5
6
7
1.3
1.1
1.1
1.3
MeOTf, Et2O, MS4A, 08C, rt,
30h
5
15
8
54
77
67
62
NIS, TfOH, Et2O±CH2Cl2
75:1), MS4A, 2508C, 50min
NIS, TESOTf, Et2O±7CH2Cl)2
75:1), MS4A, 2108C, 30min
DMTST, CH2Cl2, MS3A,
2108C, 2 h
21
a
The calculation was based on the acceptor 14.
Scheme 3. 7a) NIS, TfOH, MS4A, ether±CH2Cl2, 2508C; 7b) Ph3P, THF, 608C, and then H2O and Ac2O, rt; 7c) TBAF, AcOH, THF, rt; 7d) Jones reagent, rt;
7e) diazomethane in Et2O, rt; 7f) DDQ, aq. CH2Cl2, 08C; 7g) BnBr, CsCO3, DMF, rt; 7h) SO3´Me3N, DMF, 08C±rt; 7i) 10% Pd±C, H2, AcOH±MeOH±H2O, rt.

6918
S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
Scheme 4. 7a) TBAF, AcOH, THF, rt; 7b) Jones reagent, rt; 7c) BnBr, CsCO3, DMF, rt; 7d) PdCl2, NaOAc, aq. AcOH, 508C; 7e) 17, NIS, TESOTf, MS4A,
ether± 7CH2Cl)2, 2788C; 7f) AcSH, pyridine, 08C±rt.
an alcohol 24 in 77% yield.§ Finally, the alcohol 24 was
suffered to O-sulfation with sulfur trioxide trimethylamine
complex 7Me3N´SO3) to provide a 60-O-sulfated derivative
25, in which all O-benzyl groups were removed by hydro-
genolysis with 10% palladium carbon under hydrogen
atmosphere in aqueous methanol±acetic acid to give 1 in
high yield.
of [3H] HS was monitored by HPLC. In the case of B16-F10
melanoma heparanase, this degradation was completely
inhibited by 2.1 mM of 1, but it was almost unaffected by
2.1 mM concentration. The concentration of 1 required for
50% inhibition of the heparanase was 140 mM. In addition,
compound 1 also showed more potent activity 7IC50ˆ
58±63 mM) against the heparanase derived from colon
26N-17 cells. The activity was comparable to those of
siastatin B analogs previously reported as a heparanase
inhibitor.5,6
The structure of 1 was unambiguously elucidated on the
basis of NMR analyses. The vicinal coupling constants,
1
J2,3ˆJ3,4ˆJ4,5ˆJ5,6aˆca. 7±9 Hz, in the H NMR spectra
strongly supported the4 C1 conformation of the imino acid
moiety in 1, which was the same as for glucronic acid unit in
heparin oligosaccharides.20
In conclusion, we have developed a new type of heparanase
inhibitor by taking advantage of glycosidation reaction
between a partially protected nojrimycin derivatives 14 or
28 and phenyl 2-azido-2-deoxy-1-thio-d-glucopyranosides
17.
Furthermore we have also developed an improved route to
1 7Scheme 4). The ®rst step was de-silylation of 13, giving
an alcohol 26 in 98% yield. Jones oxidation of 26 and
subsequent benzylation afforded a benzyl ester 27 in
81% yield. This was subject to de-allylation to give an
imino acid derivative 28 in 74% yield. The imino acid 28
was allowed to react with 17 in the presence of NIS-
triethylsilyl tri¯ate in ether-1,2-dichloroethane 75:1) at
2788C to give a desired pseudodisaccharide 30 in 72%
yield along with the corresponding b-anomer 29 79%).
Nonetheless the two anomers were easily distinguished
3. Experimental
3.1. General procedures
Melting points were determined in a capillary with an Ishii
melting-point apparatus and are uncorrected. Optical
rotations were determined with a JASCO DIP-370polari-
meter. IR spectra were recorded with a Shimazu-FT-IR-
8100M spectrophotometer. 1H NMR spectra were recorded
at 400 or 500 MHz with JEOL JNM-a400 or GX 500
spectrometers. Column chromatography was performed on
silica gel 60723±0400mesh; E. Merck, Darmstadt,
1
by comparisons of the anomeric protons in the H NMR
0
0
0
0
spectra; d 5.22 7J1 ,2 ˆ3.9 Hz) for 30 vs 4.69 7J1 ,2
ˆ
8.3 Hz) for 29. The azide residue in 30 was cleanly reduced
with thioacetic acid to produce the known acetamide 23
almost quantitatively; the overall yield from 13 attained to
41% 7cf. 36% in the previous route). Transformation of 23
into 1 has already established. This quite convergent
strategy would make it feasible to attach a variety of
sugar residues to the C-3 position of 2, and therefore
could be of interest as a concise and ¯exible method for
preparation of new pseudooligosaccharides.
Germany). Merk precoated silica gel 60F
plates, 0.25
254
or 0.5 mm thickness, were used for analytical or preparative
thin layer chromatography, respectively. Organic solutions
obtained after extractive work-up were dried over MgSO4,
®ltered through a pad of Celite, and evaporated under
reduced pressure.
3.1.1. 3-O-Allyl-6-O-benzoyl-1,2-O-isopropylidene-5-O-
methanesulfonyl-a-d-glucofuranose -5). To a stirred
solution of 4 7150g, 0.58 mol) in N,N-dimethylformamide
71000 mL) was added sodium hydride 760% mineral oil
dispersion, 30g, 0.75 mol) by portions at 0 8C, and then
the mixture was stirred for 0.5 h. Allyl bromide 775 ml,
0.87 mol) was added dropwise, and stirring was continued
for 3.5 h at 08C. After quenching with sat. NH4Cl solution,
Heparanse inhibition of 1 was examined according to the
method previously reported by Nakajima et al.4 Thus, [3H]
HS was incubated at 428C with partially puri®ed heparanase
in the presence or absence of 1, and the degradation course
§
Transesteri®cation of 22 with titanium7iv) isopropoxide-benzyl alcohol
into 24 failed.

S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
6919
the resulting mixture was extracted with ether. The extracts
were washed with water and brine, dried, and evaporated.
The residual syrup 7188 g) was dissolved in acetic acid±
water 74:1, 1000 ml). The mixture was heated at 608C for
16 h with stirring, cooled, evaporated, and then co-evapo-
rated with toluene to dryness. To a stirred solution of the
residual syrup 7175 g) in dichloromethane±pyridine 73:5;
800 ml) was added dropwise a solution of benzoyl chloride
767 mL, 0.58 mol) in dichloromethane 770 ml) at 2158C.
After 1 h, methanesulfonyl chloride 790ml, 1.17 mol) was
added and the mixture was stirred at 2158C±rt for 12 h.
More methanesulfonyl chloride 790ml, 1.17 mol) was
added and the mixture was stirred for 8 h, poured into
ice±water and allowed to stand overnight. The resulting
mixture was extracted with ether. The extracts were washed
with dil. HCl solution, sat. NaHCO3 solution, water and
brine, dried, and evaporated. The residue was treated with
ether±hexane to give 210g of crystalline mass, which was
3.1.3. 3-O-Allyl-6-azido-5-O-benzyl-6-deoxy-1,2-O-iso-
propylidene-b-l-idofuranose -7). To a stirred mixture of
6 73.2 g, 13.2 mmol) and NH4Cl 75.66 g, 0.11 mol) in
N,N-dimethylformamide±water 710:1, 55 ml) was added
sodium azide 72.58 g, 39.7 mmol). The mixture was stirred
at 808C for 2 h, cooled, poured into ice±water and extracted
with ether. The extracts were washed with water and brine,
dried, evaporated and then co-evaporated with toluene to
dryness. To a stirred mixture of the residual syrup
73.70g), benzyl bromide 72.36 ml, 19.8 mmol) and n-tetra-
butylammonium iodide 74.88 g, 13.2 mmol) in N,N-di-
methylformamide 730ml) was added sodium hydride
760% mineral oil dispersion, 793 mg, 19.8 mmol) at 08C
under Ar and the mixture was stirred for 2.5 h. Sat. NH4Cl
solution was added and the resulting mixture was extracted
with ether. The extracts were washed with water and brine,
dried, and evaporated. The residual syrup was chroma-
tographed on silica gel with hexane±ethyl acetate 710:1)
27
recrystallized from ether±hexane to give 179 g of 5 770%
ˆ
to give 7 73.84 g, 78%): [a]D ˆ257.48 7c 1.10, CHCl3);
IR nmax7CHCl3): 2103, 1650 cm21 1H NMR 7CDCl3)
;
26
yield from 4): mp 132±1338C 7ether±hexane); [a]D
210.98 7c 1.25, CHCl3); IR nmax7KBr): 1730, 1350, 1270,
dˆ1.32, 1.49, 76H, each s, acetonide), 3.3071H, dd,
1
1170, 1070 cm21; H NMR 7CDCl3) dˆ1.34, 1.5076H,
J5,6aˆ6.2 Hz, J6a,6bˆ13 Hz, H-6a), 3.39 71H, dd, J5,6b
3.3 Hz, H-6b), 3.84 71H, br.d, J3,4ˆ3.4 Hz, H-3), 3.90
71H, br.dd, Jˆ13, 3.4 Hz, allyl), 3.92 71H, ddd, J4,5ˆ
7.5 Hz, H-5), 4.14 71H, br.dd, Jˆ5.4 Hz, allyl), 4.31 71H,
dd, H-4), 4.56 71H, br.d, J1,2ˆ4.0Hz, H-2), 4.68, 4.87 72H,
each d, Jˆ12 Hz, PhCH2), 5.22 71H, br.dd, Jˆ11, 1.5 Hz,
ole®n), 5.28 71H, br.dd, Jˆ17 Hz, ole®n), 5.85 71H, m,
ole®n), 5.97 71H, d, H-1). 7.25±7.41 75H, m, Ph).
ˆ
each s, acetonide), 3.03 73H, s, Ms), 4.03 71H, d, J3,4ˆ
3.0Hz, H-3), 4.09±4.18 72H, m, allyl), 4.45 71H, dd,
J4,5ˆ7.8 Hz, H-4), 4.49 71H, dd, J5,6aˆ6.4 Hz, J6a,6b
ˆ
13 Hz, H-6a), 4.62 71H, d, J1,2ˆ3.9 Hz, H-2), 4.93 71H,
dd, J5,6bˆ2.5 Hz, H-6b), 5.22 71H, br.qd, Jˆ10Hz, ole®n),
5.32 71H, qd, Jˆ17, 1.5 Hz, ole®n), 5.38 71H, ddd, H-5),
5.93 71H, d, H-1), 5.97 71H, m, ole®n), 7.44 72H, br.t,
Jˆ7.8 Hz, Ph), 7.57 71H, td, Jˆ7.4, 1.5 Hz, Ph), 8.07 72H,
dd, Jˆ8.8 Hz, Ph).
Anal. Found: C, 60.79; H, 6.70; N, 11.15. Calcd for
C19H25O5N3 requires C, 60.79; H, 6.71; N, 11.19%.
Anal. found: C, 54.17; H, 5.94; S, 7.42. Calcd for C20H26O9S
requires C, 54.29; H, 5.92; S, 7.25%.
3.1.4. Methyl 3-O-allyl-6-azido-5-O-benzyl-6-deoxy-a-l-
idofuranoside -8) and methyl 3-O-allyl-6-azido-5-O-
benzyl-6-deoxy-b-l-idofuranoside -9). A mixture of 7
7163 mg, 0.43 mmol) in 3% HCl in methanol 75 ml) was
stirred at rt for 18 h, made neutral anhydrous Na2CO3, the
mixture ®ltered, and the ®ltrate evaporated. The residual
syrup was chromatographed on silica gel with hexane±
ethyl acetate 74:1) to give 8 775 mg, 50%) and 9 771 mg,
47%). The b-anomer 9 742 mg, 0.12 mmol) was treated with
3% HCl in methanol 75 ml) at rt for 8 h and treated as
described above to give 8 722 mg, 52%) and 9 719 mg,
45%). By repetition of this procedure once again, total
3.1.2. 3-O-Allyl-5,6-anhydro-1,2-O-isopropylidene-b-l-
idofuranose -6). To a stirred solution of 5 77.5 g,
16.9 mmol) in methanol±tetrahydrofuran±dichloromethane
75:2:2, 90ml) was added sodium methoxide 7150mg,
2.8 mmol). The mixture was stirred at rt for 3 h, treated
with Dowex-50W X-8 7H1) resin, and then ®ltered. The
®ltrate was evaporated in high vacuum, and then co-
evaporated with toluene to give a syrup 76.8 g). To a stirred
solution of the syrup 76.8 g) in N,N-dimethylformamide±
tetrahydrofuran 72:1; 60ml) was added potassium
t-butoxide 72.9 g, 25.4 mmol) at 08C, and the mixture was
stirred at 08C±rt for 6 h, poured into ice±water, and
extracted with ether. The extracts were washed with water
and brine, dried, and evaporated. The residual syrup was
chromatographed on silica gel with hexane±ethyl acetate
27
121 mg 780%) of 8 was obtained. 8: [a]D ˆ188.88 7c
1.21, CHCl3); IR nmax7CHCl3): 3500, 2103 cm21
;
1H
NMR 7CDCl3) dˆ2.75 71H, d, Jˆ7.8 Hz, OH), 3.37 71H,
dd, J5,6aˆ5.9 Hz, J6a,6bˆ13 Hz, H-6a), 3.39 71H, dd, J5,6b
ˆ
5.4 Hz, H-6b), 3.49 73H, s, OMe), 3.81 71H, ddd, J4,5ˆ
7.5 Hz, H-5), 3.91 71H, dd, J2,3ˆ3.9 Hz, J3,4ˆ5.9 Hz,
H-3), 4.01 71H, br.dd, Jˆ12, 5.7 Hz, allyl), 4.25 71H, dd,
H-4), 4.26 71H, m, allyl), 4.28 71H, ddd, J1,2ˆ4.9 Hz, H-2),
4.67, 4.82 72H, each d, Jˆ11 Hz, PhCH2), 4.99 71H, d, H-1),
5.21 71H, br.d, Jˆ10, 1.5 Hz, ole®n), 5.30 71H, br.dd,
Jˆ17 Hz, ole®n), 5.9071H, m, ole®n), 7.28±7.4075H, m,
Ph).
26
78:1!4:1) to give 6 73.2 g, 78%): [a]D ˆ252.48 7c 0.28,
1
CHCl3); IR nmax7®lm): 1647 cm21; H NMR 7CDCl3) dˆ
1.32, 1.46, 76H, each s, acetonide), 2.65 71H, dd, J5,6a
ˆ
2.8 Hz, J6a,6bˆ5.8 Hz, H-6a), 2.84 71H, dd, J5,6bˆ4.3 Hz,
H-6b), 3.25 71H, ddd, J4,5ˆ6.1 Hz, H-5), 3.82 71H, dd,
J3,4ˆ3.4 Hz, H-4), 3.95 71H, br.d, H-3), 4.01 71H, dd,
Jˆ13, 5.9 Hz, allyl), 4.18 71H, dd, Jˆ4.9 Hz, allyl), 4.58
71H, br.d, J1,2ˆ3.7 Hz, H-2), 5.22 71H, br.d, Jˆ 10Hz,
ole®n), 5.3071H, br.d, Jˆ17 Hz, ole®n), 5.86 71H, m,
ole®n), 5.98 71H, d, H-1).
Anal. Found: C, 58.14; H, 6.70; N, 11.98. Calcd for
C17H23O5N3 requires C, 58.44; H, 6.64; N, 12.03%.
27
9: [a]D ˆ247.38 7c 0.55, CHCl3); IR nmax7CHCl3): 3490,
Anal. Found: C, 59.39; H, 7.45. Calcd for C12H18O5 requires
C, 59.49; H, 7.49%.
1
2103 cm21; H NMR 7CDCl3) dˆ1.87 71H, d, Jˆ4.4 Hz,

6920
S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
OH), 3.33 71H, dd, J5,6aˆ5.9 Hz, J6a,6bˆ13 Hz, H-6a), 3.45
73H, s, OMe), 3.47 71H, dd, J5,6bˆ3.4 Hz, H-6b), 3.83 71H,
dd, J2,3ˆ2.4 Hz, J3,4ˆ5.4 Hz, H-3), 3.91 71H, ddd, J4,5ˆ
7.3 Hz, H-5), 3.95 71H, br.dd, Jˆ13, 6.5 Hz, allyl), 4.15
71H, br.dd, Jˆ5.4 Hz, allyl), 4.25 71H, dd, H-2), 4.37 71H,
dd, H-4), 4.72, 4.86 72H, each d, Jˆ11 Hz, PhCH2), 4.85
71H, br.s, H-1), 5.22 71H, br.dd, Jˆ10, 1.5 Hz, ole®n),
5.28 71H, br.dd, Jˆ17 Hz, ole®n), 5.88 71H, m, ole®n),
7.27±7.43 75H, m, Ph).
7880mg). A mixture of the crude tri¯ate 7880mg) and
triphenylphospine 7473 mg, 1.81 mmol) in dichloromethane
76 ml) was stirred at rt for 0.5 h and then at 458C for 2 h. The
reaction mixture was cooled and stirred vigorously with
50% potassium carbonate solution 75 ml) in tetrahydro-
furan±methanol±water 73:1:1; 11 ml) for 2 d. To the
resulting suspension was added benzyloxycarbonyl chloride
70.31 g, 1.82 mmol) at 08C, and then the mixture was stirred
for 1 hr, diluted with dichloromethane. The organic layer
was separated and the aqueous phase was extracted with
dichloromethane. The combined organic phase was
washed with water and brine, dried and evaporated. The
residue was chromatographed on silica gel with benzene±
ethyl acetate 715:1) and then puri®ed by preparative TLC
with carbontetrachloride±ethyl acetate 72:1) to give 11
Anal. Found: C, 58.03; H, 6.78; N, 11.91. Calcd for
C17H23O5N3 requires C, 58.44; H, 6.64; N, 12.03%.
3.1.5. Methyl 3-O-allyl-5-O-benzyl-N-benzyloxycarbo-
nyl-2,6-dideoxy-2,6-imino-a-l-gulofuranoside -10). To a
stirred mixture of 8 731.5 g, 90mmol) and pyridine
719.8 ml, 0.25 mol) in dichloromethane 7300 ml) was
added dropwise tri¯ic anhydride 733.3 g, 0.12 mol) at
2608C under Ar and then the mixture was stirred at
2608C for 1.6 h. After quenching with methanol 75.0ml),
the resulting mixture was warmed to rt, diluted with ether,
washed with oxalic acid solution, satureted NaHCO3
solution, water, and brine, dried, and evaporated to give a
syrup 744.1 g), which was employed to the next step without
further puri®cation. A mixture of the tri¯ate 744.1 g) and
triphenylphospine 726.2 g, 0.10 mol) in dichloromethane
7263 ml) was stirred at rt for 1.5 h and at 458C for 2.5 h,
cooled and stirred vigorously with 50% potassium carbonate
solution 7263 ml) in tetrahydrofuran±methanol±water
73:1:1; 550ml) for 4 d. To the resulting suspension was
added benzyloxycarbonyl chloride 730.8 g, 0.18 mol) at
08C, and then the mixture was stirred for 7 h, diluted with
dichloromethane. The organic layer was separated and the
aqueous phase was extracted with dichloromethane. The
combined organic phase was washed with water and
brine, dried, and evaporated. The residue was treated with
hexane±ether to remove phosphine oxide as semicrystalline
solids. The mother liquid was puri®ed by chromatography
on silica gel with hexane±ethyl acetate 710:1!5:1) to give
25
764 mg, 8%); [a]D ˆ240.08 7c 0.43, CHCl3); IR
n
max7CHCl3): 1700, 1650 cm21 1H NMR 7d6-DMSO,
;
1208C) dˆ3.34 73H, s, OMe), 3.64 71H, m, H-6a), 3.76
71H, m, H-5), 3.84 71H, dd, J5,6bˆ7.1 Hz, J6a,6bˆ13 Hz, H-
6b), 3.99±4.09 73H, m, H-3, allyl), 4.29 71H, m, H-4), 4.56
72H, br.s, PhCH2), 4.65 71H, m, H-2), 5.02 71H, d,
J1,2ˆ2.3 Hz, H-1), 5.07±5.15 73H, m, PhCH2, ole®n), 5.22
71H, dd, Jˆ17, 1.5 Hz, ole®n), 5.85 71H, m, ole®n), 7.25±
7.36 710H, m, Ph).
Anal. Found: C, 67.96; H, 6.66; N, 3.09. Calcd for
C25H29O6N requires C, 68.32; H, 6.65; N, 3.19%.
3.1.7. 4-O-Allyl-2-O-benzyl-N-benzyloxycarbonyl-1,5-di-
deoxy-1,5-imino-d-gulcitol -12). 7i) From 10. A mixture of
10 73.5 g, 7.97 mmol) in dioxane±tri¯uoroacetic acid±
water 72:1:1; 20ml) was stirred at rt for 11 h, and poured
into ice±sodium acetate solution. The resulting mixture was
extracted with chloroform. The extracts were washed with
sat. NaHCO3 solution, water and brine, dried, and evapo-
rated. The residual syrup was dissolved in 95% ethanol
740ml) and treated with sodium borohydride 71 g,
26.4 mmol) at 08C. After 2 h, the reaction mixture was
quenched with excess acetic acid, and then evaporated.
The residue was diluted with dichloromethane±water, and
extracted with dichloromethane. The extracts were washed
with water and brine, dried, and evaporated. The residual
syrup was chromatographed on silica gel with benzene±
ethyl acetate 71:1) to give 12 72.42 g, 71%).
27
10 727.3 g, 69%): [a]D ˆ128.58 7c 1.20, CHCl3); IR
n
max7CHCl3): 1700, 1649 cm21 1H NMR 7d6-DMSO,
;
1108C) dˆ3.26 73H, s, OMe), 3.63 71H, dd, J5,6aˆ4.9 Hz,
J6a,6bˆ13 Hz, H-6a), 3.74 71H, ddd, J4,5ˆ2.0Hz, J5,6b
ˆ
6.8 Hz, H-5), 3.82 71H, dd, H-6b), 4.00-4.12 72H, m,
allyl), 4.26±4.3072H, br.s, H-2,3), 4.36 71H, ddd, J3,4ˆ
3.9 Hz, J2,4ˆ2.0Hz, H-4), 4.53, 4.58 72H, each d, Jˆ
12 Hz, PhCH2), 4.76 71H, br.s, H-1), 5.10, 5.13 72H, each
d, Jˆ12 Hz, PhCH2), 5.07±5.25 72H, m, ole®n), 5.84 71H,
m, ole®n), 7.24±7.35 710H, m, Ph).
7ii) From 11. A mixture of 11 760mg, 0.14 mmol) in di-
oxane±tri¯uoroacetic acid±water 72:1:1; 1.2 ml) was stirred
at rt for 1 h, and treated with the procedure as described
above to give a crude hemiacetal, which was reduced with
sodium borohydride 710mg, 0.26 mmol) in 95% ethnaol
75 ml) and puri®ed by prepartive TLC with carbon tetra-
chloride±ethyl acetate 72:1) to give 12 741 mg, 70%):
Anal. Found: C, 68.22; H, 6.63; N, 3.17. Calcd for
C25H29O6N requires C, 68.32; H, 6.65; N, 3.19%.
24
[a]D ˆ17.78 7c 0.49, CHCl3); IR nmax7CHCl3): 3400,
1
3.1.6. Methyl 3-O-allyl-5-O-benzyl-N-benzyloxycarbonyl-
2,6-dideoxy-2,6-imino-b-l-gulofuranoside -11). To
1690cm 21; H NMR 7d6-DMSO, 808C) dˆ3.32 71H, dd,
a
J1a,1bˆ14 Hz, J1a,2ˆ3.4 Hz, H-1a), 3.49 71H, q, J2,3ˆ
4.2 Hz, H-2), 3.53 71H, dd, J3,4ˆ5.2 Hz, J4,5ˆ4.9 Hz, H-
stirred mixture of 9 7630mg, 1.81 mmol) and pyridine
70.44 ml, 5.44 mmol) in dichloromethane 75 ml) was
added dropwise tri¯ic anhydride 70.76 g, 2.69 mol) at
2608C under Ar and the mixture was stirred at 2608C for
45 min. After quenching with methanol 70.8 ml), the result-
ing mixture was warmed to rt, diluted with ether, washed
with oxalic acid solution, sat. NaHCO3 solution, water, and
brine, dried, and evaporated to give a tri¯ate as a syrup
4), 3.61 71H, dd, J5,6aˆ5.5 Hz, J6a,OHˆ5.5 Hz, J6a,6b
ˆ
11 Hz, H-6a), 3.63 71H, dd, J5,6bˆ5.5 Hz, J6b,OHˆ5.5 Hz,
H-6b), 3.74 71H, q, J2,3ˆ4.2 Hz, J3,OHˆ5.2 Hz, J3,4ˆ
5.2 Hz, H-3), 3.92 71H, dd, J1b,2ˆ3.7 Hz, H-1b), 4.00 71H,
q, H-5), 4.06 71H, ddt, Jˆ13, 5.5, 1.5 Hz, allyl), 4.14 71H,
ddt, Jˆ5.2, 1.5 Hz, allyl), 4.53, 4.59 72H, each d, Jˆ12 Hz,
PhCH2), 4.56 71H, t, 6-OH), 5.05±5.14 74H, m, 3-OH,

S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
6921
PhCH2, ole®n), 5.9071H, m, ole®n), 7.25±7.34 710H, m,
Ph).
3.1.10. 1,5-Dideoxy-1,5-imino-d-glucitol -3). To a stirred
mixture of 14 7440mg, 0.61 mmol) and acetic acid 748 mg,
0.80 mmol) in tetrahydrofuran 73 ml) was added a 1.0 M
solution of tetrabutylammonium ¯uoride 70.68 mL,
0.68 mmol) at rt under Ar. The mixture was stirred at rt
for 8 h, diluted with ethyl acetate, successively washed
with water and brine, dried and evaporated. The residual
syrup was chromatographed on silica gel with hexane±
ethyl acetate 72:1!1:1) to give 2,3-di-O-benzyl-N-benzyl-
oxycarbonyl-1,5-dideoxy-1,5-imino-d-glucitol21 7255 mg,
Anal. Found: C, 67.61; H, 6.60; N, 3.16. Calcd for
C24H29O6N requires C, 67.43 H, 6.84; N, 3.28%.
3.1.8. 4-O-Allyl-N-benzyloxycarbonyl-2,3-di-O-benzyl-6-
O-tert-butyldiphenyl-silyl-1,5-dideoxy-1,5-imino-d-gul-
citol -13). To a stirred solution of 12 72.42 g, 5.67 mmol)
and imidazole 71.24 g, 18.2 mmol) in N,N-dimethyl-
formamide 720ml) was added tert-butylchlorodiphenyl-
silane 72.39 g, 8.70mmol) at rt and the mixture was
stirred at rt for 1 h, poured into ice±water. The resulting
mixture was extracted with ether. The extracts were washed
with water and brine, dried and evaporated. To a stirred
mixture of the residual syrup, benzyl bromide 74.26 g,
24.9 mmol) and tetrabutylammonium iodide 73.07 g,
8.31 mmol) in N,N-dimethylformamide 750ml) was added
sodium hydride 7a 60% mineral oil dispersion, 1 g,
25 mmol) at 08C. After 3 h, the reaction mixture was
quenched with methanol, and poured into ice±water. The
resulting mixture was extracted with ether. The extracts
were washed with water and brine, dried and evaporated.
The residual syrup was chromatographed on silica gel with
hexane±ethyl acetate 710:1) to give 13 73.76 g, 88%);
87%), [a]D ˆ123.28 7c 0.57, CHCl3); 1H NMR
23
7d6-DMSO, 1008C) dˆ3.3071H, dd, J1a,1bˆ14 Hz, J1a,2
ˆ
3.1 Hz, H-1a), 3.55 71H, dd, J2,3ˆ4.3 Hz, J3,4ˆ5.5 Hz,
H-3), 3.66-3.69 72H, m, H-6), 3.7071H, q, H-2), 3.88±
3.92 72H, m, H-4, 5), 4.07 71H, dd, J1b,2ˆ3.1 Hz, H-1b),
4.37 71H, br.t, Jˆ5.1 Hz, 6-OH), 4.44 71H, d, Jˆ6.4 Hz,
4-OH), 4.48 71H, d, Jˆ12 Hz, PhCH2), 4.59±4.73 73H, m,
PhCH2), 5.1072H, s, PhCH ), 7.26±7.37 715H, m, Ph). A
2
mixture of the above glucitol 7174 mg, 0.36 mmol) and 10%
Pd±C 730mg) in acetic acid±ethanol±water 71:1:1; v/v/v,
3 ml) was stirred at rt under a hydrogen atmosphere for
45 hr. The catalyst was then ®ltered off and washed with
aq. methanol. The ®ltrate and washings were combined,
concentrated in vacuo and then treated with dil. hydro-
chloric acid solution, concentrated in vacuo. The residue
was crystallized from ethanol±methanol to give 3 731 mg,
21
[a]D ˆ114.98 7c 0.95, CHCl3); IR nmax7®lm): 1701,
1425, 1111 cm21
;
1H NMR 7d6-DMSO, 808C) dˆ0.99
53%) as the hydrochloride. mp 207±2098C 7dec., aq. EtOH)
23
79H, s, t-butyl), 3.24 71H, dd, J1a,1bˆ14 Hz, J1a,2ˆ2.8 Hz,
H-1a), 3.66±3.68 72H, m, H-2,3), 3.81±3.88 73H, m, H-4,6),
3.98 71H, dd, J1b,2ˆ3.7 Hz, H-1b), 4.00 71H, m, allyl), 4.12
71H, ddt, Jˆ13, 4.9, 1.5 Hz, allyl), 4.15 71H, q, H-5), 4.47,
4.57 72H, each d, Jˆ12 Hz, PhCH2), 4.61 72H, br.s, PhCH2),
4.98 71H, d, Jˆ13 Hz, PhCH2), 5.06±5.09 72H, m, PhCH2,
ole®n), 5.17 71H, qd, Jˆ17, 5.8, 1.8 Hz, ole®n), 5.84 71H,
m, ole®n), 7.24±7.60725H, m, Ph).
[lit.22 mp 196±1988C, lit.14 mp 204±2058C]; [a]D
ˆ
135.08 7c 0.45, H2O), [lit.22 [a]D ˆ138.08]; H NMR
7D2O) dˆ2.99 71H, t, J1a,1bˆJ1a,2ˆ12 Hz, H-1a), 3.22 71H,
ddd, H-5), 3.52 71H, dd, J1b,2ˆ4.9 Hz, H-1b), 3.53 71H, dd,
J2,3ˆ9.3 Hz, J3,4ˆ9.7 Hz, H-3), 3.62 71H, dd, J4,5ˆ9.3 Hz,
H-4), 3.8071H, ddd, H-2), 3.89 71H, dd, J5,6aˆ5.4 Hz,
J6a,6bˆ13 Hz, H-6a), 3.96 71H, dd, J5,6bˆ2.9 Hz, H-6b);
13C NMR 7D2O) dˆ46.3, 58.1, 60.4, 67.4, 68.2, 76.7.
22
1
Anal. Found: C, 74.57; H, 6.91; N, 1.94. Calcd for
C47H53O6NSi requires C, 74.67; H, 7.07; N, 1.85%.
3.1.11. Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-p-
methyoxybenzyl-1-thio-d-glucopyranosides -17). To a
stirred solution of 15 71.28 g, 3.48 mmol) and 7phenylthio)-
trimethylsilane 71.90g, 10.4 mmol) in 1,2-dichloroethane
730ml) was added zinc iodide 74.0g, 12.5 mmol) at rt.
The mixture was stirred at rt for 12 h, diluted with chloro-
form and washed with dil. HCl solution, sat. NaHCO3
solution, water, and brine, dried and evaporated. The
residual syrup 72.6 g) was dissolved in methanol±water
710:1; 20 ml) and treated with potassium carbonate
7964 mg, 6.97 mmol) at rt for 0.5 hr. The resulting mixture
was concentrated, and diluted with chloroform, washed with
water, and brine, dried, and evaporated to give 16 72.0g). To
a stirred mixture of 16 72.0g), p-methoxybenzyl chloride
71.50g, 9.58 mmol) and tetrabutylammonium iodide
71.29 g, 3.48 mmol) in N,N-dimethylformamide 720ml)
was added sodium hydride 7a 60% oil suspension, 420 mg,
10.5 mmol) at 08C. After 4 h, the reaction mixture was
quenched with methanol, and poured into sat. NH4Cl
solution. The resulting mixture was extracted with ether.
The extracts were washed with water and brine, and evapo-
rated. The residual syrup was chromatographed on silica gel
with hexane±ethyl acetate 720:3) to give 17 71.49 g, 72%
from 15) as an anomeric mixture 7a/bˆ2:1, 1H NMR analy-
sis). A small quantity of the anomeric mixture was separated
by chromatography on silica gel with hexane±ethyl acetate
710:1).
3.1.9.
N-Benzyloxycarbonyl-2,3-di-O-benzyl-6-O-tert-
butyldiphenylsilyl-1,5-dideoxy-1,5-imino-d-gulcitol -14).
A mixture of 13 73.10g, 4.10mmol), sodium acetate
71.72 g, 21.0mmol) and palladium chloride 7931 mg,
5.25 mmol) in 90% acetic acid was heated at 508C with
stirring for 3.5 h, cooled and concentrated. The residue
was diluted with chloroform and washed with water, sat.
NaHCO3 solution, water, and brine, dried and evaporated.
The residual syrup was chromatographed on silica gel with
hexane±ethyl acetate 77:1) to give 14 72.10g, 72%);
26
[a]D ˆ127.48 7c 0.59, CHCl3); IR nmax7CHCl3): 3500,
1
1700 cm21; H NMR 7d6-DMSO, 808C) dˆ0.98 79H, s,
t-butyl), 3.21 71H, dd, J1a,1bˆ14 Hz, J1a,2ˆ2.9 Hz, H-1a),
3.54 71H, dd, J2,3ˆ3.9 Hz, J3,4ˆ5.8 Hz, H-3), 3.7071H,
q, H-2), 3.86 71H, dd, J5,6aˆ4.9 Hz, J6a,6bˆ10Hz, H-6a),
3.92 71H, dd, J5,6bˆ5.4 Hz, H-6b), 3.98 71H, m, H-4),
4.05 71H, q, J4,5ˆ6.3 Hz, H-5), 4.12 71H, dd, J1b,2
ˆ
3.0Hz, H-1b), 4.46, 4.59 72H, each d, Jˆ12 Hz,
PhCH2), 4.60, 4.64 72H, each d, Jˆ12 Hz, PhCH2), 4.67
71H, br.s, OH), 4.99, 5.09 71H, d, Jˆ13 Hz, PhCH2),
7.23±7.61 725H, m, Ph).
Anal. Found: C, 73.67; H, 6.92; N, 2.01. Calcd for
C44H49O6NSi requires C, 73.81; H, 6.90; N, 1.96%.

6922
S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
22
a-anomer: mp 63±648C 7diisopropylether); [a]D ˆ11288
7c 1.01, CHCl3); 1H NMR 7CDCl3) dˆ3.59 71H, dd,
J5,6aˆ2.0Hz, J6a,6bˆ11 Hz, H-6a), 3.72±3.79 73H, m, H-4,
5, 6b), 3.76 73H, s, OMe), 3.81 71H, dd, J2,3ˆ10Hz, J3,4ˆ
8.9 Hz, H-3), 3.94 71H, dd, J1,2ˆ5.5 Hz, H-2), 4.34 71H, m,
H-5), 4.36, 4.56 72H, each d, Jˆ11 Hz, PhCH2), 4.48, 4.78
72H, each d, Jˆ11 Hz, PhCH2), 4.88, 4.91 72H, each d,
Jˆ11 Hz, PhCH2), 5.61 71H, d, H-1), 6.82 72H, d, Jˆ
8.5 Hz, Ph), 7.13±7.40715H, m, Ph), 7.5072H, dd, Jˆ
1.8 and 7.5 Hz, Ph).
Anal. Found: C, 71.59; H, 6.62; N, 4.69. Calcd for
C72H78O11N4Si requires C, 71.86; H, 6.53; N, 4.66%.
26
19: [a]D ˆ141.98 7c 0.88, CHCl3); IR nmax7CHCl3): 2103,
1
1700 cm21; H NMR 7d6-DMSO, 1208C) dˆ0.99 79H, s,
t-butyl), 3.28 71H, dd, J1a,1bˆ14 Hz, J1a,2ˆ3.1 Hz, H-1a),
0
0
0
0
0
3.45 71H, dd, J1 ,2 ˆ3.7 Hz, J2 ,3 ˆ10Hz, H-2 ), 3.52 71H,
br.d, J6 a,6 bˆ14 Hz, H-60a), 3.58 71H, dd, J5 ,6 bˆ3.4 Hz,
0
0
0
0
H-60b), 3.66 71H, dd, J3 ,4 ˆ8.8 Hz, J4 ,5 ˆ9.8 Hz, H-4 ),
0
0
0
0
0
3.68 71H, m, H-2), 3.72 73H, s, OMe), 3.79 71H, dd,
H-30), 3.8071H, m, H-5 ), 3.86 71H, t, J2,3ˆ2.4 Hz, J3,4ˆ
0
Anal. Found: C, 68.16; H, 5.87; N, 6.86; S, 5.37. Calcd
for C34H35O5N3S requires C, 68.32; H, 5.90; N, 7.03; S,
5.36%.
2.4 Hz, H-3), 3.89 72H, br.d, H-6), 3.9071H, dd, J1b,2
ˆ
5.2 Hz, H-1b), 4.13 71H, t, J4,5ˆ2.4 Hz, H-4), 4.30, 4.39
72H, each d, Jˆ12 Hz, PhCH2), 4.54±4.69 710H, m,
PhCH2), 5.03 72H, br.s, PhCH2), 5.17 71H, d, H-10), 6.83
72H, dd, Jˆ6.7, 2.1 Hz, Ph), 7.6±7.41 733H, m, Ph), 7.60
74H, br.t, Jˆ6.2 Hz).
22
b-isomer: mp 86±878C 7diisopropylether); [a]D ˆ243.98
7c 1.03, CHCl3); 1H NMR 7CDCl3) dˆ3.34 71H, dd,
J1,2ˆ10Hz, J2,3ˆ9.4 Hz, H-2), 3.45 71H, ddd, H-5), 3.49
71H, dd, J3,4ˆ9.2 Hz, H-3), 3.58 71H, dd, J4,5ˆ9.5 Hz,
H-4), 3.7071H, dd, J5,6aˆ4.1 Hz, J6a,6bˆ11 Hz, H-6a),
3.73 71H, dd, J5,6bˆ2.1 Hz, H-6b), 3.79 73H, s, OMe),
4.4071H, d, H-1), 4.46, 4.54 72H, each d, Jˆ11 Hz,
PhCH2), 4.54, 4.76 72H, each d, Jˆ11 Hz, PhCH2), 4.81,
4.84 72H, each d, Jˆ11 Hz, PhCH2), 6.85 72H, d, Jˆ
8.6 Hz, Ph), 7.17±7.32 715H, m, Ph), 7.59 72H, dd, Jˆ1.2
and 8.2 Hz, Ph).
Anal. Found: C, 71.52; H, 6.59; N, 4.72. Calcd for
C72H78O11N4Si requires C, 71.86; H, 6.53; N, 4.66%.
3.1.13. 4-O--2-Acetamido-3,4-di-O-benzyl-2-deoxy-6-O-
p-methoxybenzyl-a-d-glucopyranosyl)-N-benzyloxycar-
bonyl-2,3-di-O-benzyl-6-O-tert-butyldiphenylsilyl-1,5-
dideoxy-1,5-imino-d-gulcitol -20). 7a) A mixture of 19
71.15 g, 0.96 mmol) and triphenylphosphine 7276 mg,
1.05 mmol) in tetrahydrofuran 738 ml) was heated at 608C
for 16 h with stirring, cooled to rt. Water 74.0ml) was added
and stirring was continued for further 20h. Acetic
anhydride 70.3 ml) was added to the resulting mixture and
the solution was stirred at rt for 3 h, and evaporated. The
residual syrup was chromatographed on silica gel with
hexane±ethyl acetate 72:1) to give 20 71.09 g,93%).
Anal. Found: C, 68.16; H, 5.87; N, 6.96; S, 5.36. Calcd for
C34H35O5N3S requires C, 68.32; H, 5.90; N, 7.03; S, 5.36%.
3.1.12. 4-O--2-Azido-3,4-di-O-benzyl-2-deoxy-6-O-p-meth-
oxybenzyl-b-d-glucopyranosyl)-N-benzyloxycarbonyl-
2,3-di-O-benzyl-6-O-tert-butyldiphenylsilyl-1,5-dideoxy-
1,5-imino-d-gulcitol -18) and 4-O--2-azido-3,4-di-O-
benzyl-2-deoxy-6-O-p-methoxybenzyl-a-d-glucopyrano-
syl)-N-benzyloxycarbonyl-2,3-di-O-benzyl-6-O-tert-butyl-
diphenylsilyl-1,5-dideoxy-1,5-imino-d-gulcitol -19). To a
stirred suspension of 14 71.01 g, 1.41 mmol), 17 7942 mg,
7b) To a stirred mixture of 19 77.4 g, 6.15 mmol) in pyridine
710ml) was added dropwise thioacetic acid 720ml) at 5 8C
and then the mixture was stirred at rt for 18 h, evaporated.
The residual syrup was co-evaporated with toluene in
3 times, and then chromatographed on silica gel with
hexane±ethyl acetate 74:1!1:1) to give 20 76.40g, 85%);
Ê
1.58 mmol) and pulverized activated 4 A molecular sieves
72.40g) in ether±dichloromethane 732:5, 37 ml) was added
N-iodosuccinimide 7887 mg, 3.94 mmol) at 2508C under
Ar. A sat. solution of tri¯uoromethanesulfonic acid in
dichloromethane 71.2 ml) was then added dropwise. After
50min, the reaction mixture was poured into sat. NaHCO 3
and extracted with ether. The extract was successively
washed with sat. Na2S2O3, sat. NaHCO3, water and brine,
dried, and evaporated. The residual syrup was chromato-
graphed on silica gel with benzene±ethyl acetate 7200:1)
to give 19 71.30g, 77%) and 18 7257 mg, 15%).
23
[a]D ˆ130.28 7c 0.42, CHCl3); IR nmax7CHCl3): 1699,
1
1684, 1110cm 21; H NMR 7d6-DMSO, 1108C) dˆ0.97
79H, s, t-butyl), 1.58 73H, s, NAc), 3.21 71H, br.d, J1a,1b
0
ˆ
14 Hz, H-1a), 3.5071H, br.d, J6 a,6 bˆ11 Hz, H-60a), 3.55±
0
0
4.1076H, m, H-2, 3, 6, 3 , 40), 3.6071H, dd, J5 ,6bˆ3.4 Hz,
0
H-60b), 3.71 73H, s, OMe), 3.85 71H, br.d, H-50), 3.98 71H,
m, H-20), 4.03 71H, br.s, H-4), 4.05 71H, br.d, H-1b), 4.30,
4.4072H, each d, Jˆ12 Hz, PhCH2), 4.55 71H, br.s, H-5),
0
0
4.45±4.71 78H, m, PhCH2), 4.87 71H, d, J1 ,2 ˆ3.0Hz,
H-10), 5.04, 5.07 72H, each d, Jˆ13 Hz, PhCH2), 6.63
26
18: [a]D ˆ114.78 7c 0.28, CHCl3); IR nmax7CHCl3): 2103,
0
Ph), 7.16±7.59 737H, m, Ph).
0
0
71H, d, J2 NH ˆ7.3 Hz, NH ), 6.82 72H, br.d, Jˆ8.5 Hz,
1700 cm21; 1H NMR 7d6-DMSO, 1108C) dˆ0.99 79H, s, t-
0
0
0
0
0
butyl), 3.29 71H, dd, J1 ,2 ˆ7.8 Hz, J2 ,3 ˆ9.3 Hz, H-2 ), 3.30
71H, dd, J1a,1bˆ14 Hz, J1a,2ˆ3.4 Hz, H-1a), 3.47 71H, m, H-
Anal. Found: C, 72.63 H, 6.67 N, 2.31. Calcd for
C74H82O12N2Si requires C, 72.88; H, 6.78; N, 2.30%.
50), 3.48 71H, dd, J3 ,4 ˆ7.8 Hz, H-3 ), 3.53 71H, dd,
0
0
0
0
0
0
0
J4 ,5 ˆ9.5 Hz, H-4 ), 3.6072H, br.d, H-6 ), 3.64 71H, br.d,
H-2), 3.68 73H, s, OMe), 3.77 71H, dd, J1b,2ˆ5.9 Hz, H-1b),
3.86 72H, br.d, H-6), 3.92 71H, dd, J2,3ˆ4.4 Hz, J3,4ˆ2.4 Hz,
H-3), 4.31 71H, J4,5ˆ2.0Hz, H-4), 4.35, 4.4072H, each d,
Jˆ12 Hz, PhCH2), 4.51 71H, m, H-5), 4.52 71H, d, H-10),
4.50±4.80 78H, m, PhCH2), 5.00, 5.04 72H, each d,
Jˆ13 Hz, PhCH2), 6.76 72H, dd, Jˆ6.4, 2.3 Hz, Ph),
7.13±7.61 737H, m, Ph).
3.1.14. 4-O--2-Acetamido-3,4-di-O-benzyl-2-deoxy-6-O-
p-methoxybenzyl-a-d-glucopyranosyl)-N-benzyloxy-
carbonyl-2,3-di-O-benzyl-1,5-dideoxy-1,5-imino-d-gulci-
tol -21). To a stirred mixture of 20 76.0g, 4.92 mmol) and
acetic acid 70.50 g, 8.33 mmol) in tetrahydrofuran 7100 ml)
was added dropwise a 1.0M solution of tetrabutylammo-
nium ¯uoride 77.87 ml, 7.87 mmol) at rt. The mixture was

S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
6923
stirred at rt for 22 h, diluted with chloroform and washed
with water and brine, dried, and evaporated. The residual
syrup was chromatographed on silica gel with hexane±ethyl
residual syrup and benzyl bromide 713 ml, 0.11 mmol) in
N,N-dimethylformamide 71.0ml) was added cesium car-
bonate 727 mg, 0.06 mmol) at rt. The mixture was stirred
for 6 h, and poured into water, extracted with chloroform.
The extracts were successively washed with water and
brine, dried, and evaporated. The residual syrup was
chromatographed on silica gel with hexane±ethyl acetate
25
acetate 71:1) to give 21 74.80g, 99%). [ a]D ˆ120.18 7c
0.67, CHCl3); IR
nmax7CHCl3): 3400, 1698, 1680,
1
1612 cm21; H NMR 7d6-DMSO, 1208C) dˆ1.58 73H, s,
0
0
0
0
0
NAc), 3.54 71H, dd, J3 ,4 ˆ9.3 Hz, J4 ,5 ˆ9.2 Hz, H-4 ),
3.35 71H, dd, J1a,1bˆ14 Hz, J1a,2ˆ2.9 Hz, H-1a), 3.59±
3.67 75H, m, H-6, 30, 60), 3.69±3.75 72H, m, H-2,3), 3.74
25
72:1) to give 23 743 mg, 72%); [a]D ˆ153.18 7c 0.56,
CHCl3); IR nmax7CHCl3): 1748, 1703, 1684 cm21 1H
;
0
0
0
0
73H, s, OMe), 3.87 71H, ddd, J5 ,6 aˆ3.5 Hz, J5 ,6 bˆ2.5 Hz,
H-50), 3.98 71H, m, H-20), 4.00 71H, br.t, J3,4ˆ3.0Hz,
J4,5ˆ3.0Hz, H-4), 4.06 71H, dd, J1b,2ˆ3.9 Hz, H-1b), 4.34
NMR 7d6-DMSO, 1208C) dˆ1.62 73H, s, NAc), 3.50±
3.6072H, m, H-6a, 4 0), 3.49 71H, dd, J5 ,6 aˆ8.8 Hz,
0 0
J6 a,6 bˆ11 Hz, H-60a), 3.54 71H, t, J2 ,3 ˆ9.3 Hz, J3 ,4
ˆ
0
0
0
0
0
0
9.3 Hz, H-30), 3.59 71H, dd, J5 ,6 bˆ3.9 Hz, H-60b), 3.72
73H, s, OMe), 3.74±3.84 73H, m, H-4, 5, 50), 3.98 71H, m,
H-20), 4.08 71H, dd, J6a,6bˆ13 Hz, H-6b), 4.32 71H, d,
Jˆ12 Hz, PhCH2), 4.39 71H, br.s, H-3), 4.39±4.67 79H,
0
0
71H, dt, J5,6aˆ6.3 Hz, J5,6bˆ6.3 Hz, H-5), 4.36±4.71 710H,
0
0
0
m, PhCH2), 4.88 71H, d, J1 ,2 ˆ3.4 Hz, H-1 ), 5.07, 5.12 72H,
0
0
0
each d, Jˆ13 Hz, PhCH2), 6.62 71H, d, J2 NH ˆ7.3 Hz, NH ),
6.85 72H, br.d, Jˆ8.2 Hz, Ph), 7.17±7.32 712H, m, Ph).
m, PhCH2), 4.93 71H, d, Jˆ13 Hz, PhCH2), 4.94 71H, d,
0
0
0
Anal. Found: C, 70.89 H, 6.60 N, 2.86. Calcd for
C58H64O12N2 requires C, 71.00; H, 6.58; N, 2.86%.
J1 ,2 ˆ3.9 Hz, H-1 ), 4.99 71H, br.s, H-2), 5.05±5.14 73H,
0
0
0
m, PhCH2), 6.68 71H, d, J2 NH ˆ8.3 Hz, NH ), 6.82 72H,
br.d, Jˆ8.8 Hz, Ph), 7.17±7.32 732H, m, Ph).
3.1.15. Methyl 3-O--2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-d-glucopyranosyl)-N-benzyloxycarbonyl-4,5-di-
O-benzyl-2,6-dideoxy-2,6-imino-l-gulonate -22). To a
stirred solution of 21 7427 mg, 0.44 mmol) in acetone
715 ml) was added dropwise Jones reagent 7ca. 0.5 ml) at
rt. After 10min, 2-propanol was added, and the resulting
mixture was poured into water, extracted with chloroform.
The extracts were washed successively with water and
brine, dried, and evaporated, co-evaporated with toluene.
The residue was treated with diazomethane in ether and
the solution evaporated. The residue was passed through a
short colomn of silica gel {chloroform±ethyl acetate 76:1)}
to give a syrup 7420mg), which was dissolved in dichloro-
methane±water 719:1, 20ml). To this solution was
added 2,3-dichloro-5,6-dicyano-p-benzoquinone 7283 mg,
1.25 mmol) at 08C. The mixture was stirred at 08C for 2 h,
diluted with dichloromethane, and washed successively
with sat. Na2S2O3, sat. NaHCO3, water and brine, dried,
and evaporated. The residual syrup was chromatographed
on silica gel with hexane±ethyl acetate 71:2) to give 22
Anal. Found: C, 71.90H, 6.38 N, 2.52. Calcd for
C65H68O13N2 requires C, 71.94; H, 6.32; N, 2.58%.
3.1.17. Benzyl 3-O--2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-d-glucopyranosyl)-N-benzyloxycarbonyl-4,5-di-
O-benzyl-2,6-dideoxy-2,6-imino-l-gulonate -24). To a
stirred solution of 23 71.30g, 1.20mmol) in dichloro-
methane±water 719:1, 20ml) was added 2,3-dichloro-5,6-
dicyano-p-benzoquinone 7544 mg, 2.40mmol) at 0 8C. The
mixture was stirred at 08C for 2 h, diluted with dichloro-
methane, and washed successively with sat. Na2S2O3, sat.
NaHCO3, water and brine, dried, and evaporated. The
residual syrup was chromatographed on silica gel with
hexane±ethyl acetate 71:1) to give 24 70.89 g, 77%),
25
[a]D ˆ144.48 7c 0.12, CHCl3); IR nmax7CHCl3): 3350,
1
1745, 1710, 1680 cm21; H NMR 7d6-DMSO, 1108C) dˆ
1.65 73H, s, NAc), 3.55±3.69 76H, m, H-6a, 30, 40, 50, 60),
3.78±3.84 72H, m, H-4, 5), 4.00 71H, br.t, H-20), 4.05 71H,
dd, J5,6bˆ4.0Hz, J6a,6bˆ14 Hz, H-6b), 4.21 71H, br.s, OH),
4.38 71H, br.t, H-3), 4.43±4.71 78H, m, PhCH2), 4.96071H, d,
24
7252 mg, 65%), [a]D ˆ150.28 7c 0.67, CHCl3); IR
max7CHCl3): 3360, 1755, 1710, 1690 cm21
;
1H NMR
Jˆ12 Hz, PhCH2), 4.97 71H, d, J1 ,2 ˆ3.4 Hz, H-1 ), 4.99
0
0
n
7d6-DMSO, 808C) dˆ1.64 73H, s, NAc), 3.48±3.69 76H,
m, H-6a, 30, 40, 50, 60), 3.54 73H, s, Me), 3.78±3.84 72H,
m, H-4, 5), 3.98 71H, br.t, H-20), 4.07 71H, dd, J5,6bˆ3.9 Hz,
J6a,6bˆ14 Hz, H-6b), 4.34 71H, br.s, H-3), 4.38±4.73 79H,
71H, br.s, H-2), 5.009-5.15 73H, m, PhCH2), 6.74 71H, d,
0
0
J2 NH ˆ8.5 Hz, NH ), 7.16±7.33 730H, m, Ph).
Anal. Found: C, 70.68 H, 6.19; N, 2.86. Calcd for
C57H60O12N2 requires C, 70.94; H, 6.27; N, 2.90%.
0
0
m, OH, PhCH2), 4.95 71H, br.s, H-2), 4.96 71H, d, J1 ,2
ˆ
3.4 Hz, H-10), 5.07, 5.16 72H, each d, Jˆ13 Hz, PhCH2),
0
0
0
6.87 71H, d, J2 NH ˆ9.3 Hz, NH ), 7.16±7.33 725H, m, Ph).
3.1.18. Benzyl 3-O--2-acetamido-3,4-di-O-benzyl-2-
deoxy-6-O-sulfo-a-d-glucopyranosyl)-N-benzyloxycar-
bonyl-4,5-di-O-benzyl-2,6-dideoxy-2,6-imino-l-gulonate
sodium salt -25). Sulfur trioxide±trimethylamine complex
7177 mg, 1.27 mmol) was added to a solution of 24 7612 mg,
0.64 mmol) in N,N-dimethylformamide 73 ml) at 08C, and
the mixture was stirred for 12 h at rt, diluted with methanol
70.5 ml), placed on a column of Sephadex LH-20 pre-
equilibrated with 1:1 7v/v) chloroform±methanol, and
eluted with the same solvent. The product was chromato-
graphed on a column of Dowex 50W X-8 7Na 1) resin,
preequilibrated with 80% methanol by using the same
solvent to give a crude product, which was then chromato-
graphed on silica gel with chloroform±methanol 710:1) to
Anal. Found: C, 68.57; H, 6.27; N, 3.03. Calcd for
C51H56O12N2 requires C, 68.90; H, 6.35; N, 3.15%.
3.1.16. Benzyl 3-O--2-acetamido-3,4-di-O-benzyl-2-
deoxy-6-O-p-methoxybenzyl-a-d-glucopyranosyl)-N-
benzyloxycarbonyl-4,5-di-O-benzyl-2,6-dideoxy-2,6-
imino-l-gulonate -23). To a stirred solution of 21 754 mg,
0.06 mmol) in acetone 72 ml) was added dropwise Jones
reagent 7ca. 50 ml) at rt. After 20min, 2-propanol was
added, and the resulting mixture was poured into water,
extracted with chloroform. The extracts were washed
successively with water and brine, dried, and evaporated,
co-evaporated with toluene. To a stirred solution of the
26
give 25 7665 mg, 98%): [a]D ˆ118.78 7c 0.52, CHCl3); IR

6924
S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
1
n
max7®lm): 1740, 1703, 1653, 1431, 1215 cm21; H NMR
each d, Jˆ12 Hz, PhCH2), 5.05±5.25 74H, m, ole®n and
PhCH2), 5.82 71H, m, ole®n), 7.20±7.33 720H, m, Ph).
HRFABMS: Found; 644.2612, Calcd for C38H39O7NNa:
644.2624 7M1Na)1.
7d6-DMSO, 808C) dˆ1.62 73H, s, NAc), 3.51±3.58 73H, m,
H-6a, 30, 40), 3.75 71H, m, H-50), 3.77 71H, br.t, H-5), 3.79
71H, br.t, H-4), 3.94 71H, m, H-20), 3.95 71H, dd, J5 ,6 a
ˆ
0
0
1.7 Hz, J6 a,6 bˆ11 Hz, H-60a), 4.04 71H, dd, J5,6bˆ4.7 Hz,
0
0
J6a,6bˆ14 Hz, H-6b), 4.13 71H, dd, J5 ,6 bˆ2.9 Hz, H-60b),
3.1.21. Benzyl N-benzyloxycarbonyl-4,5-di-O-benzyl-
2,6-dideoxy-2,6-imino-l-gulonate -28). A mixture of 27
785 mg, 0.14 mmol), sodium acetate 747 mg, 0.57 mmol)
and palladium chloride 725 mg, 0.14 mmol) in 90% acetic
acid 71 ml) was heated 508C with stirring for 1.5 h, cooled,
diluted with ethylacetate, and then ®ltered through a pad of
Celite. The ®ltrate was evaporated, and then co-evaportaed
with toluene. The residue was chromatographed on silica
gel with hexane±ethyl acetate 710:1!4:1!2:1) to give 28
0
0
4.38 71H, br.s, H-3), 4.36±4.65 77H, m, PhCH2), 4.83
0
0
71H, d, Jˆ11 Hz, PhCH2), 4.96 71H, d, J1 ,2 ˆ3.4 Hz, H-
10), 4.94±4.99 72H, m, H-2, PhCH2), 5.07±5.15 73H, m,
0
0
0
PhCH2), 6.83 71H, d, J2 NH ˆ8.6 Hz, NH ), 7.12±7.36
730H, m, Ph).
Anal. Found: C, 63.43 H, 5.60; N, 2.56; S, 2.55. Calcd for
C57H59O15N2SNa´H2O requires C, 63.09; H, 5.66; N,2.59; S,
2.95%.
26
759 mg, 74%); [a]D ˆ136.68 7c 0.39, CHCl3); IR nmax
7®lm): 3487, 1744, 1705 cm21 1H NMR 7d6-DMSO,
;
3.1.19. 4-O-Allyl-N-benzyloxycarbonyl-2,3-di-O-benzyl-
1,5-dideoxy-1,5-imino-d-gulcitol -26). To a stirred mixture
of 13 7890mg, 1.18 mmol) and acetic acid 70.11 ml,
2.00 mmol) in tetrahydrofuran 715 ml) was added dropwise
a 1.0M solution of tetrabutylammonium ¯uoride 71.88 ml,
1.88 mmol) at rt. The mixture was stirred at rt for 22 h,
diluted with ethyl acetate and washed with water and
brine, dried, and evaporated. The residual syrup was
chromatographed on silica gel with hexane±ethyl acetate
1008C) dˆ3.52 71H, dd, J6a,6bˆ14 Hz, J6a,5ˆ3.0Hz,
H-6a), 3.67 71H, t, Jˆ4.0Hz, H-4), 3.76 71H, m, H-5),
4.03 71H, dd, J5,6bˆ3.5 Hz, H-6b), 4.28 71H, m, H-3),
4.51, 4.59 72H, each d, Jˆ12 Hz, PhCH2), 4.52 72H, s,
PhCH2), 4.7071H, d, J2,3ˆ3.9 Hz, H-2), 4.93, 5.03 72H,
each d, Jˆ12 Hz, PhCH2), 5.1072H, s, PhCH ), 7.20±7.32
2
720H, m, Ph). HRFABMS: Found; 604.2308, Calcd for
C35H35O7NNa: 604.2311 7M1Na)1.
26
74:1) to give 26 7599 mg, 98%); [a]D ˆ115.18 7c 0.85,
3.1.22. Benzyl 3-O--2-azido-3,4-di-O-benzyl-2-deoxy-6-
O-p-methoxybenzyl-b-d-glucopyranosyl)-N-benzyloxy-
carbonyl-4,5-di-O-benzyl-2,6-dideoxy-2,6-imino-l-gulo-
nate -29) and benzyl 3-O--2-azido-3,4-di-O-benzyl-2-
deoxy-6-O-p-methoxybenzyl-a-d-glucopyranosyl)-N-
benzyloxycarbonyl-4,5-di-O-benzyl-2,6-dideoxy-2,6-imino-
l-gulonate -30). To a stirred suspension of 28 744 mg,
77 mmol), 17 760mg, 100 mmol), N-iodosuccinimide 743
1
CHCl3); IR nmax7®lm): 3447, 1699, 1428, 1072 cm21; H
NMR 7d6-DMSO, 1008C) dˆ3.34 71H, dd, J1a,1bˆ14 Hz,
J1a,2ˆ4.2 Hz, H-1a), 3.58±3.68 74H, m, H-2,3, 6a, 6b),
3.75 71H, dd, J3,4ˆ5.4 Hz, J4,5ˆ4.9 Hz, H-4), 3.92 71H,
dd, J1b,2ˆ4.4 Hz, H-1b), 4.01±4.15 73H, m, H-5, allyl),
4.47 71H, t, Jˆ5.5 Hz, OH), 4.49, 4.58 72H, each d, Jˆ
12 Hz, PhCH2), 4.63, 4.67 72H, each d, Jˆ12 Hz, PhCH2),
5.06±5.24 74H, m, PhCH2, ole®n), 5.84 71H, m, ole®n),
7.24±7.60715H, m, Ph).
Ê
mg, 190 mmol) and pulverized activated 4 A molecular
sieves 7100 mg) in ether±1,2-dichloroethane 75:1, 0.6 ml)
was added dropwise triethylsilytri¯ate 723 ml, 100 mmol)
at 2788C under Ar. After 2.5 h, the reaction mixture was
poured into sat. NaHCO3 and extracted with ether. The
extracts were washed successively with sat. Na2S2O3, sat.
NaHCO3, water and brine, dried, and evaporated. The
residual syrup was chromatographed on silica gel with
hexane±ethyl acetate 710:1!4:1) and then puri®ed by
preparative TLC {chloroform±ethyl acetate 7200:1)} to
give 29 77 mg, 9%) and 30 758 mg, 72%).
Anal. Found: C, 71.53; H, 6.84; N, 2.51. Calcd for
C31H35O6N requires C, 71.93; H, 6.82; N, 2.71%.
3.1.20. Benzyl 3-O-allyl-N-benzyloxycarbonyl-4,5-di-O-
a
benzyl-2,6-dideoxy-2,6-imino-l-gulonate -27). To
stirred solution of 26 7165 mg, 0.33 mmol) in acetone
77 ml) was added dropwise Jones reagent 7ca. 0.4 ml) at rt.
After 5 min, 2-propanol was added, and the resulting
mixture was poured into water, extracted with dichloro-
methane. The extracts were washed successively with
water and brine, dried, and evaporated, co-evaporated
with toluene. To a stirred solution of the residual syrup
7179 mg) and benzyl bromide 70.11 ml, 0.93 mmol) in
N,N-dimethylformamide 72.0ml) was added cesium carbo-
nate 7195 mg, 0.40 mmol) at rt. The mixture was stirred for
3 h, and poured into water, extracted with ether. The
extracts were washed successively with water and brine,
dried, and evaporated. The residual syrup was chromato-
graphed on silica gel with hexane±ethyl acetate 710:1!
26
29: [a]D ˆ118.68 7c 0.30, CHCl3); IR nmax7®lm): 2113,
1748, 1705, 1455, 1249 cm21; 1H NMR 7d6-DMSO, 1108C)
0
0
0
0
0
dˆ3.26 71H, dd, J1 ,2 ˆ8.3 Hz, J2 ,3 ˆ9.3 Hz, H-2 ), 3.45±
3.72 77H, m, H-5, 6a, 30, 40, 50, 60), 3.68 73H, s, OMe), 3.82
71H, J5,6ˆ5.4 Hz, J6a,6bˆ14 Hz, H-6b), 3.96 71H, dd,
J3,4ˆ3.4 Hz, J4,5ˆ4.4 Hz,, H-4), 4.36, 4.4072H, each d,
Jˆ12 Hz, PhCH2), 4.44±4.56 76H, m, PhCH2), 4.56 71H,
dd, J2,3ˆ2.0Hz, H-3), 4.67±4.75 73H, m, PhCH 2), 4.69
71H, d, H-10), 4.94±5.1073H, m, PhCH ), 5.01 71H, d,
2
H-2), 6.78 72H, d, Jˆ8.4 Hz, Ph), 7.25±7.31 732H, m,
Ph); HRFABMS: Found; 1091.4386, Calcd for
C63H64O12N4Na: 1091.4418 7M1Na)1.
25
5:1) to give 27 7160mg, 81%); [ a]D ˆ125.18 7c 0.56,
1
CHCl3); IR nmax7®lm): 1747, 1705, 1455, 1132 cm21; H
NMR 7d6-DMSO, 1008C) dˆ3.57 71H, dd, J6a,6bˆ14 Hz,
26
30: [a]D ˆ164.48 7c 0.10, CHCl3); IR nmax7®lm): 2103,
J6a,5ˆ3.2 Hz, H-6a), 3.7071H, m, H-5), 3.77 71H, dd,
J3,4ˆ3.4 Hz, J4,5ˆ3.9 Hz, H-4), 3.9071H, dd,
J5,6b
ˆ
1748, 1705, 1455, 1124 cm21; 1H NMR 7d6-DMSO, 1108C)
0
0
0
4.4 Hz, H-6b), 4.04±4.10 72H, m, allyl), 4.11 71H, J2,3ˆ
3.0Hz, H-3), 4.51, 4.59 72H, each d, Jˆ14 Hz, PhCH2),
4.53 72H, s, PhCH2), 4.82 71H, d, H-2), 4.97, 5.07 72H,
dˆ3.44 71H, dd, J1 ,2 ˆ3.9 Hz, J2,3ˆ9.8 Hz, H-2 ), 3.48±
3.78 710H, m, H-5, 6a, 30, 40, 50, 60), 3.72 73H, s, MeO),
3.43 71H, br.t, Jˆ2.5 Hz, H-4), 3.98 71H, J5,6ˆ3.9 Hz,

S. Takahashi et al. / Tetrahedron 57 ꢀ2001) 6915±6926
6925
Poste, G.; Fidler, I. J. Nature ꢀLondon) 1980, 283, 139±146.
Liotta, L. A. Cancer Res. 1986, 46, 1±7. Nicolson, G. L. Curr.
Opin, Cell. Biol. 1989, 1, 1009±1019.
J6a,6bˆ14 Hz, H-6b), 4.29, 4.38 72H, each d, Jˆ12 Hz,
PhCH2), 4.46 71H, t, Jˆ2.1 Hz, H-3), 4.47±4.67 78H, m,
PhCH2), 4.93±5.14 74H, m, PhCH2), 5.06 71H, d, H-2),
5.22 71H, d, H-10), 6.82 72H, d, Jˆ8.4 Hz), 7.25±7.31
732H, m, Ph); HRFABMS: Found; 1091.4436, Calcd for
C63H64O12N4Na: 1091.4418 7M1Na)1.
2. Irimura, T.; Nakajima, M.; Di Ferrante, N.; Nicolson, G. L.
Anal. Biochem. 1983, 130, 461±468. Nakajima, M.; Irimura,
T.; Di Ferrante, N.; Nicolson, G. L. J. Biol. Chem. 1984 , 259,
2283±2290.
3.1.23. Chemical transformation of 30 into 23. To a
stirred mixture of 30 714 mg, 0.01 mmol) in pyridine
70.2 ml) was added dropwise thioacetic acid 70.4 ml) at
08C and then the mixture was stirred at 08C!rt for 18 h,
evaporated. The residual syrup was co-evaporated with
toluene in 3 times, and then chromatographed on silica gel
with hexane±ethyl acetate 74:1!1:1) to give 23 714 mg,
98%).
3. Irimura, T.; Nakajima, M.; Nicolson, G. L. Biochemistry 1986,
25, 5322±5328. Saiki, I.; Murata, J.; Nakajima, M.; Tokura,
S.; Azuma, I. Cancer Res. 1990, 50, 3631±3637.
4. Nakajima, M.; DeChavigny, A.; Johnson, C. E.; Hamada, J.;
Stein, C. A.; Nicolson, G. L. J. Biol. Chem. 1991, 266, 9661±
9666.
5. Takatsu, T.; Takahashi, M.; Kawase, Y.; Enokita, R.; Okazaki,
T.; Matsukawa, H.; Ogawa, K.; Sakaida, Y.; Kagasaki, T.;
Kinoshita, T.; Nakajima, M.; Tanzawa, K. J. Antibiot. 1996,
49, 54±60. Kawase, Y.; Takahashi, M.; Takatsu, T. M.; Arai,
M.; Nakajima, M.; Tanzawa, K. J. Antibiot. 1996, 49, 61±64.
6. Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeuchi, T.;
Azetaka, M. H.; Fukuyasu, H.; Iizuka, Y J. Am. Chem. Soc.
1996, 118, 3051±3052. Nishimura, Y.; Satoh, T.; Adachi, H.;
Kondo, S.; Takeuchi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka,
Y. J. Med. Chem. 1997, 40, 2626±2633. Nishimura, Y.;
Shitara, E.; Adachi, H.; Toyoshima, M.; Nakajima, M.;
Okami, Y.; Takeuchi, T. J. Org. Chem. 2000, 65, 2±11 and
references cited therein.
3.1.24. 3-O--2-Acetamido-2-deoxy-6-O-sulfo-a-d-gluco-
pyranosyl)-2,6-dideoxy-2,6-imino-l-gulonic acid sodium
salt -1). A mixture of 25 7524 mg, 0.49 mmol) and 10% Pd±
C 7400 mg) in methanol±acetic acid±water 74:1:1; 18 ml)
was stirred under a hydrogen atmosphere at rt for 2 d. The
mixture was ®ltered and the ®ltrate was evaporated. The
residue was chromatographed on a gel-permeation column
7Sephadex G-10), with water as the eluent, to give 1
ˆ
25
7229 mg, 96%) as a hygroscopic white powder: [a]D
184.48 7c 0.44, H2O); IR nmax7KBr): 3480, 1650, 1560,
1
1250, 1230, 1060 cm21; H NMR 7D2O) dˆ2.07 73H, s,
7. Tsuruoka, T.; Fukuyasu, H.; Ishii, M.; Usui, T.; Shibahara, S.;
Inouye, S. J. Antibiot. 1996, 49, 155±161.
NAc), 2.98 71H, dd, J5,6aˆ8.9 Hz, J6a,6bˆ13 Hz, H-6a),
0
0
3.56 71H, dd, J5,6bˆ4.0Hz, H-6b), 3.61 71H, dd, J3 ,4
ˆ
8. Takahashi, S.; Terayama, H.; Kuzuhara, H. Tetrahedron Lett.
1992, 33, 7565±7568. Terayama, H.; Kuzuhara, H.;
Takahashi, S.; Sakuda, S.; Yamada, Y. Biosci. Biotechnol.
Biochem. 1993, 57, 2067±2069. Takahashi, S.; Terayama,
H.; Kuzuhara, H. Tetrahedron Lett. 1994, 35, 4149±4152.
Takahashi, S.; Terayama, H.; Kuzuhara, H.; Sakuda, S.;
Yamada, Y. Biosci. Biotechnol. Biochem. 1994, 58, 2301±
2302.
0
0
0
9.4 Hz, J4 ,5 ˆ10Hz, H-4 ), 3.65 71H, d, J2,3ˆ7.6 Hz,
0
0
0
H-2), 3.76 71H, dd, J2 ,3 ˆ10Hz, H-3 ), 3.81 71H, dd,
J3,4ˆ7.3 Hz, J4,5ˆ7.6 Hz, H-4), 3.89 71H, ddd, H-5), 3.96
71H, br.d, H-50), 3.98 71H, dd, J1 ,2 ˆ3.7 Hz, H-2 ), 4.08
0
0
0
71H, dd, H-3), 4.26 71H, br.d, J6 a,6 bˆ11 Hz, H-60a), 4.34
0
0
71H, dd, J5 ,6 bˆ2.9 Hz, H-60b), 5.42 71H, d, H-10). 13C NMR
7D2O) dˆ22.8 7Me), 44.9 7C-6), 54.1 7C-20), 61.3 7C-2),
67.4 7C-60), 67.5 7C-5), 69.9 7C-40), 71.3 7C-50), 71.6
7C-30), 72.9 7C-4), 74.3 7C-3), 97.7 7C-10), 171.4 7COOH),
175.2 7NHCO). HRFABMS: Found; 459.0941, Calcd for
C14H23O13N2SNa: 459.0921 7M2Na)2.
0
0
9. Manning, K. S.; Lynn, D. G.; Shabanowitz, J.; Fellows, L. E.;
Singh, M.; Schrire, B. D. J. Chem. Soc., Chem. Commun.
1985, 127±129.
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802±815. Inouye, S.; Tsuruoka, T.; Ito, T.; Niida, T.
Tetrahedron 1968, 24, 2125±2144. Yagi, M.; Kouno, T.;
Aoyagi, Y.; Murai, H Nippon Nogei Kagaku Kaishi 1976,
50, 571±572. Schmidt, D. D.; Frommer, W.; Muller, L.;
Truscheit, E. Naturwissenschaften 1979, 66, 584±585.
Daigo, K.; Inamori, Y.; Takemoto, T. Chem. Pharm. Bull.
1986, 34, 2243±2246.
3.1.25. Heparanase assay. The assay was performed
according to the method previously reported by Nakajima
et al.4 In the presence or absence of 1, N-3H-acetylated
heparan sulfate 7bovine lung) was incubated at 428C for
4 h with heparanase partially purifed from colon 26 N-17
or B16-F10 melanoma cells in 0.1% Triton X-100 and
0.1 M sodium acetate 7pH 5.0). Incubation products were
analyzed by size exclusion chromatography using a Bio-Gel
TSK-30XL column. Percent inhibition was determined by
measuring the decrease in the area of the ®rst one-half of the
peak of intact [3H] heparan sulfate on the chromatogram.
11. Kiso, M.; Katagiri, H.; Furui, H.; Hasegawa, A. J. Carbohydr.
Chem. 1992, 11, 627±644 and references cited therein.
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B. Tetrahedron Lett. 1985, 26, 4981±4982. Bashyal, B. P.;
Chow, H.-F.; Fleet, G. W. J. Tetrahedron Lett. 1986, 27,
3205±3208.
13. Preliminary communication, Takahashi, S.; Kuzuhara, H.
Chem. Lett. 1994, 2119±2122.
Acknowledgements
14. Fleet, G. W. J.; Carpenter, N. M.; Petursson, S.; Ramsden,
N. G. Tetrahedron Lett. 1990, 31, 409±412.
We are grateful to Ms M. Yoshida and her collaborators in
RIKEN for the elemental analyses.
15. Dasgupta, F.; Garegg, P. J. Synthesis 1988, 626±628.
16. Wang, L-X.; Sakairi, N.; Kuzuhara, H. J. Chem. Soc. Perkin
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