A short and efficient transformation of rhamnose into activated daunosamine, acosamine, ristosamine and epi-daunosamine derivatives, and synthesis of an anthracycline antibiotic acosaminyl-ε-iso-rhodomycinone

Article abstract of DOI:10.1016/S0008-6215(00)00257-3

3-Amino-2,3,6-trideoxyhexopyranoses are essential constituents of most anthracycline antitumour antibiotics. For an investigation of structure-activity relationships, the four diastereomeric amino sugars daunosamine, acosamine, ristosamine, and epidaunosamine were synthesised in short and efficient routes starting from commercially available rhamnose. Several glycosyl donors were provided and their use was exemplified in the synthesis of acosaminyl-ε-iso-rhodomycinone.

Full text of DOI:10.1016/S0008-6215(00)00257-3

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Carbohydrate Research 329 (2000) 861–872
Note
A short and efficient transformation of rhamnose into
activated daunosamine, acosamine, ristosamine and
epi-daunosamine derivatives, and synthesis of an
anthracycline antibiotic acosaminyl-o-iso-rhodomycinone
Bernd Renneberg a, Yue-Ming Li a, Hartmut Laatsch a,*, Heinz-Herbert Fiebig b
aInstitute of Organic Chemistry, Uni6ersity of Go¨ttingen, Tammannstrasse 2, D-37077 Go¨ttingen, Germany
bOncotest GmbH, Am Flughafen 8-10, D-79110 Freiburg, Germany
Received 1 June 2000; accepted 22 August 2000
Abstract
3-Amino-2,3,6-trideoxyhexopyranoses are essential constituents of most anthracycline antitumour antibiotics. For
an investigation of structure–activity relationships, the four diastereomeric amino sugars daunosamine, acosamine,
ristosamine, and epi-daunosamine were synthesised in short and efficient routes starting from commercially available
rhamnose. Several glycosyl donors were provided and their use was exemplified in the synthesis of acosaminyl-o-iso-
rhodomycinone. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Acosamine; Daunosamine; Ristosamine; Anthracycline; Acosaminyl-o-iso-rhodomycinone
Many anthracycline antibiotics show strong
antitumour activity [1,2]: the highest intracel-
lular concentration of the drug is found in the
nucleus, where it intercalates into the DNA
double helix forming a ternary complex with
DNA topoisomerase II [3], with consequent
inhibition of replication and transcription. Be-
cause of the small difference between normal
DNA and DNA from cancer cells, anthracy-
clines are generally very toxic also to hosts,
and administration of the drug may cause
serious side-effects such as nausea, vomiting,
gastrointestinal toxicity, and most of all, cu-
mulative cardiotoxicity (Scheme 1).
Anthracycline antibiotics like daunomycin
or the o-iso-rhodomycinone derivative -788-6
(1a) consist of a tetracyclic chromophore and
one or more sugar residues; the first moiety is
usually an amino sugar, which may be the
only sugar residue, or be part of an oligosac-
charide chain. The configuration of the amino
D
* Corresponding author.
E-mail address: hlaatsc@gwdg.de (H. Laatsch).
Scheme 1.
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00257-3

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B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
have been synthesised from commercially
available cheap -monosaccharides, however,
with rather long reaction sequences or low
yields [5]. The -amino hexose 5a bearing xylo
configuration is the C-3 epimer of
D
L
L
-
daunosamine and has often been referred as
3-epi-daunosamine. It is not a naturally occur-
ring isomer, and has been obtained most usu-
ally as a minor by-product during the
synthesis of other amino sugars [6] (Scheme
2).
The first total syntheses of 5a were reported
by Cheung [7] and Boivin [8], starting from
a-
D
-glucopyranoside. In our synthesis of
-di-O-acetyl-
amino sugar derivatives 2a–5a,
L
rhamnal (7) was used as the starting material.
The latter was easily obtained in a 100 g scale,
when a literature procedure for the synthesis
of acetobromo sugars [9] and the reductive
dehalogenation were optimised and performed
Scheme 2.
in a one-pot-reaction.
L
-(+)-Rhamnose (6a)
was first peracetylated and transformed into
acetobromorhamnose (6b) using PBr3–H2O as
the brominating agent and then reduced by a
zinc–copper alloy giving the expensive -(+)-
L
di-O-acetyl-rhamnal (7) in more than 85%
yield (Scheme 3).
Heating the aqueous suspension of 7 at
80 °C [10] delivered the hydrolysis product 8a
which was used for the synthesis of acosamine
(3a) and ristosamine (4a) derivatives. In con-
trast to the literature [11], addition of azide on
8a occurred with only moderate stereoselectiv-
ity, and both 3b and 4b were obtained in a
ratio of merely 2.1:1. Silylation of the crude
product with tert-butyl(dimethyl)silyl chlo-
ride–imidazole1 using DMF as the solvent
afforded a mixture of a and b anomers [12]; in
dichloromethane or 1,2-dichloroethane, how-
ever, both 3c and 4c (84%) were obtained as
pure b anomers [13]. After removal of the
acetyl groups using K2CO3 –MeOH, the ob-
Scheme 3.
sugar influences strongly the bioactivity of the
drug: replacement of -daunosamine (2a) with
-acosamine (3a) or -ristosamine (4a) in an-
thracyclines produces analogues having simi-
lar antitumour activity but reduced
cardiotoxicity [4]. In this paper we describe an
optimised short synthesis for the -amino sug-
L
L
L
L
1 The condensation of o-rhodomycinone (1b) with an a-1-
O-trimethylsilylated daunosamine donor, catalyzed by TMS–
triflate, gave a complex mixture of products, from which a,a-
and a,b-disaccharide as well as an o-rhodomycin with b-gly-
cosidic linkage have been isolated. As these side reactions
could be avoided in the case of daunosamine donor 2f by
using the more stable b-1-O-tert-butyldimethylsilyl glycoside,
we selected TBDMS as the protective group in the syntheses
of other glycosyl donors as well.
ars 2a–5a in an activated form useful for the
glycosylation of anthracyclinones, as is exem-
plified in the coupling reaction of the
acosamine derivative 3f with 1b affording
acosaminyl-o-iso-rhodomycinone (1c).
The amino sugars 2a–4a occur naturally as
constituents of anthracycline antibiotics and

B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
863
TLC analysis showed that again only one
product was formed, which was further con-
firmed as the b anomer 5i.
tained diastereomeric mixture of azides could
be separated easily by column chromatogra-
phy to yield pure 3d and 4d.
1
From the H NMR spectrum of the final
The result that azide addition on 8a gave
both 3b and 4b, allowed a divergent synthesis
product it was not clear whether the com-
pound had the lyxo or the xylo configuration.
Removal of the benzoyl group with K2CO3–
of the diastereomeric -amino sugars 2b and
L
5b in the same way. Thus, compound 7 was
first converted in a Ferrier reaction to 8b (a
and b anomer in a ratio of ꢀ4:1), using zinc
chloride in methanol as a catalyst [14], fol-
lowed by deacetylation to afford compound
8c. Epimerisation of the 4-OH group by the
Mitsunobu reaction furnished compound 9b
with an axial 4-OH group. In the presence of
hydrochloric acid in acetic acid and THF, the
acetal group in 9b was cleaved, the resulting
9c was then subjected to azide addition. The
resulting mixture was again protected with
TBDMS without further purification and
characterisation. After column chromatogra-
phy, a colourless viscous oil was obtained.
The 1H NMR spectrum showed that 2c and 5c
were obtained in a ratio of about 1:2.4, very
similar to the synthesis of 3c and 4c. Removal
of the acetate groups delivered 2d and 5d.
Another facile access to the daunosamine
series was possible starting from the
acosamine derivative 3d: first, this was con-
verted to the triflate 3h, followed by epimeri-
sation at C-4 using AcOCs and sodium
acetate. Although the reaction failed to give a
high yield of 2c (43% overall yield from 3d), it
is still more efficient than results reported
previously [15].
1
CH3OH, however, gave a compound, the H
NMR spectrum of which was not identical
with that of the authentic lyxo-compound 2d,
identifying the acetal as being the xylo deriva-
tive 5d with an axial azido group. This stereo-
chemistry was further confirmed by NMR
experiments with the derivative 5f: significant
nuclear Overhauser effects (NOE) of NH with
1-H and 5-H allowed the assignment of the
NHCOCF3 group as being axial, and a strong
NOE between one 2-H2 signal and a weak
NOE with the other allowed the assignment of
chemical shifts of 2-He and 2-Ha. The 1-pro-
ton was assigned as being axial according to
the coupling constant and also the strong
NOE with the NH-group.
Reduction of all azides was done in the
same way: when catalytic hydrogenation of
3d–4d was carried out in methanol in the
presence of two equivalents of ethyl trifluoro-
acetate and triethyl amine, trifluoro-acetyla-
tion of the amino group occurred simulta-
neously to give 3e and 4e, and the yield was
12% higher than using an improved literature
procedure [17]. Protection of the 4-hydroxyl
group as acetate or p-nitrobenzoate gave the
acosamine derivatives 3f–3g and the ris-
tosamine derivative 4g in good yields (\
90%). With similar results, compound 2d was
finally converted into the glycosyl donor 2f,
and from 5d, 5f was obtained. All four pro-
The low stereoselectivity during azide addi-
tion on 8a or 9c is due to the small steric
shielding of their 4-O-acetyl group, and reac-
tion of benzoate 9a with azide should give
much better yields of the 3-epi-daunosamine
configuration, therefore. The hexenopyran-
oside 9a was obtained, however, by mesyla-
tion of 8c and subsequent nucleophilic substi-
tution of the mesylate with caesium benzoate
in only 39% yield; better results (80–90%)
were obtained using the Mitsunobu reaction
[16]: when the acetal group in 9a was cleaved
with 1 N HCl in HOAc–THF and subse-
quently azide was added, only a single product
was detectable by TLC. The 1-hydroxy group
of this intermediate was finally protected as
tert-butyl(dimethyl)silyl (TBDMS) derivative.
tected -amino sugars are versatile intermedi-
L
ates for the synthesis of anthracycline type
anticancer antibiotics.
The glycosylation of o-iso-rhodomycinone
(1b) with the acosamine donor 3f proceeded
smoothly in 1:1 dichloromethane–acetone in
the presence of 1.6 equivalents of Me3Si–tri-
,
flate and powdered molecular sieve (4 A),
when a temperature of about −35 °C was
maintained. At lower temperature (−45 to
−50 °C), the sparingly soluble 1b precipi-
tated, and prolonged reaction time lead to side
reactions, such as enhanced bisglycosylation,
and decomposition of the glycosyl donor 3f.

864
B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
cinon (1c) showed a very similar IC70-pattern
like doxorubicin (see Table 2). The colon can-
cer model (CCL HT29) and the lung cancer
(LXF 529) were the most sensitive in contrast
to the gastric cancer (GXF 251), lung cancer
(LXF 629) and the melanoma (MEXF 462).
1. Experimental
General.—IR spectra (KBr pellet or neat)
were recorded on Perkin–Elmer 297 spec-
1
trometer, H NMR spectra were recorded on
Scheme 4.
Varian VXR 200 (200 MHz), Bruker AM 300
(300 MHz); 13C NMR spectra were recorded
on Varian VXR 200 (50.3 MHz), Bruker AM
300 (75.5 MHz), coupling constants J in
Hertz, chemical shifts in l related to tetra-
methylsilane as internal standard. Mass spec-
tra were obtained on Varian MAT 73 and
Varian 311A at 70 eV by electron impact.
TLC plates (Silica Gel PF254, E. Merck) and
silica gel for column chromatography (0.05–
0.2 mm, 70–270 mesh) was purchased from
Macherey–Nagel & Co (Du¨ren, Germany).
By column chromatography on silica gel, 10
was obtained in 70% yield along with about
15% of the bisglycosylated product as an in-
separable mixture. Removal of the tri-
fluoroacetyl and p-nitrobenzoyl groups with 1
N sodium hydroxide [18] gave the pure an-
thracycline analogue 1c in 68% yield (Scheme
4).
As expected, the in vitro cytotoxicity of 1c
is lower than that of doxorubicin and of o-iso-
rhodomycin (1b) (see Tables 1 and 2). In an in
vitro-cytotoxicity study with six human tu-
mour cell lines, acosaminyl-o-iso-rhocomy-
L
-(+)-rhamnose monohydrate (6a, [h]D20 = +
8.20°, c=10, water, 1 h) was purchased from
Acros (Geel, Belgium) and used without fur-
ther purification.
Table 1
In-vitro-cytotoxicity (IC50, mg/mL) of doxorubicin, o-iso-rhodomycin and acosaminyl-o-iso-rhodomycinone (1c) against L1210
leukemia cells in short time and long-termed exposure tests
Time of exposure
Doxorubicin
o-iso-Rhodomycin
Acosaminyl-o-iso-rhodomycinone (1c)
1 h
\1
\1
\1
7 days
0.01
0.6
0.8
Table 2
In-vitro-cytotoxicity (IC70, mg/mL) of doxorubicin, o-iso-rhodomycinone and acosaminyl-o-iso-rhodomycinone (1c) against six
human tumour cell lines in a modified propidium iodide assay [19]
Compound
Colon cancer Gastric cancer Melanoma xenograft Non-small cell lung cancer
HT29
GXF 251
MEXF 462NL
LXF 529L LXF 629L LXF 66NL
Doxorubicin
1c
o-iso-Rhodomycinone (1b) \3.00
b-Rhodomycinone \3.00
B0.30
0.71
3.81
2.21
\3.00
\3.00
1.45
2.19
\3.00
\3.00
B0.30
0.33
\3.00
\3.00
1.47
0.75
1.58
\3.00
1.88
\3.00
\3.00
3.14

B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
865
tert-Butyldimethylsilyl 4-O-acetyl-3-azido-
2,3,6-trideoxy-i- -lyxo-hexopyranoside (2c)
and tert-butyldimethylsilyl 3-azido-4-O-acetyl-
2,3,6-trideoxy-i- -xylo-hexopyranoside (5c)
vacuum dried at 150 °C/0.05 Torr) was added
to a solution of crude triflate 3h (8.1 g, ca.
19.3 mmol) dissolved in dry DMF (50 mL).
After stirring for 18 h at 20 °C, the reaction
mixture was diluted with CH2Cl2 (200 mL)
and washed three times with water (50 mL
each time). The organic layer was dried with
Na2SO4, concentrated in vacuo, and the crude
product was purified by column chromatogra-
phy (1:30 EtOAc–cyclohexane) to give 2c
(3.55 g) as a colourless oil, along with 20%
unidentified impurity which could not be
separated.
L
L
Method A. A solution of 9b (3.70 g, ca. 20
mmol) in 1 N HCl (20 mL), AcOH (8 mL)
and THF (30 mL) was stirred for 15 h at
20 °C. TLC analysis showed complete cleav-
age of the acetal group. Sodium azide (2.30 g,
ca. 35 mmol) was added and the reaction
mixture was stirred for 10 h. The organic layer
was separated and the water phase was ex-
tracted with CH2Cl2. The combined organic
layer was washed with satd NaHCO3 and
dried with Na2SO4. The solvent was removed
and the crude product was redissolved in dry
CH2Cl2 (100 mL), together with tert-
butyl(dimethyl)silyl chloride (6.00 g, ca. 40
mmol) and imidazole (2.72 g, ca. 40 mmol).
After stirring for 2 h, the mixture was washed
with water and dried with Na2SO4. The crude
product was purified by column chromatogra-
phy (silica gel: 80 g, 20:1 cyclohexane–EtOAc)
to give a colourless viscous oil (3.30 g, 50%) of
both 2c and 5c in a ratio of about 1:2.4.
tert - Butyldimethylsilyl 3 - azido - 2,3,6 - tri-
deoxy-i- -lyxo-hexopyranoside (2d).—Deace-
L
tylation of 2c (3.40 g, ca. 8 mmol, containing
ca. 20% impurity) carried out as described in
the synthesis of 3d gave, after column chro-
matography (1:15 EtOAc–cyclohexane), 2d
(2.02 g, 88%) as a colourless solid with mp
69–70 °C (Lit.: 72 °C [17]). [h]2D0= −3.6° (c=
1
0.80, CHCl3). H NMR (200 MHz, CDCl3):
l=4.74 (dd, J1,2ax=8.9, J1,2eq =2.8, 1 H, 1-
H), 3.60 (br s, 1 H, 4-H), 3.46 (dq, J5,4 =1.0,
J5.6=6.5, 1 H, 5-H) 3.30 (ddd, J3,4 =2.7,
1
Compound 2c: H NMR (300 MHz, CDCl3):
J
J
3,2ax=12.5, J3,2eq =5.1, 1 H, 3-H), 1.99 (ddd,
2ax,2eq=12.7, 1 H, 2eq-H), 1.97 (br s, 1 H,
l=5.04 (dd, J4,3 =3.2, J4,5=1, 1 H, 4-H),
4.77 (dd, J1,2ax =8.5, J1,2eq =3.0, 1 H, 1-H),
3.58 (dq, J5,6 =6.5, 1 H, 5-H), 3.40 (ddd,
4-OH), 1.84 (ddd, 1 H, 2ax-H), 1.29 (d, 3 H,
6-H3), 0.91 (br s, 9 H, SiCMe3), 0.13 and 0.11
(2 s, 2 H, SiMe2).
J
3,2ax =12.0, J3,2eq =5.0, 1 H, 3-H), 2.17 (s, 3
H, MeCO), 2.00 (ddd, 1 H, 2eq-H), 1.93 (ddd,
2ax,2eq =12.0, 1 H, 2ax-H), 1.16 (d, 3 H, 6-H3),
1-O-tert-Butyldimethylsilyl 2,3,6-trideoxy-3-
trifluoroacetamido - i - - lyxo - hexopyranoside
L
J
(2e).—Catalytic hydrogenation of 2d (1.87 g,
6.5 mmol) carried out as described in the
synthesis of 3e gave, after column chromatog-
raphy (1:5 EtOAc–cyclohexane), 2e (2.11 g,
91%) as a colourless solid with mp 54 °C (Lit.:
55 °C [18]). [h]D25= +16.2° (c=0.92, CHCl3).
1H NMR (300 MHz, CDCl3): l=6.82 (br d,
0.90 (br s, 9 H, SiCMe3), 0.12, 0.10 (2s, 6 H,
SiMe2). 13C NMR (CDCl3, 75 MHz): l=
170.64 (MeCO), 94.93 (C-1), 70.33 (C-5),
69.19 (C-4), 57.50 (C-3), 33.66 (C-2), 25.77
(Me3C), 20.83 (MeCO), 18.17 (Me3C), 16.78
(C-6), −4.13, −5.07 (Me2Si). Compound 5c:
1H NMR (300 MHz, CHCl3): l=4.95 (dd,
JNH,3=8, 1 H, NH), 4.77 (dd, J1,2ax =9.5,
J1,2eq=2.5, 1 H, 1-H), 4.10 (dddd, J3,2ax =13,
J3,2eq=5.0, J3,4=2.3, 1 H, 3-H), 3.62 (qd,
J
1,2eq =3.9, J1,2ax =7.3, 1 H, 1-H), 4.54 (dd,
J4,3 =3.2, J4,5=1.5, 1 H, 4-H), 3.95 (m, 2 H,
3-, 5-H), 2.10 (s, 3 H, MeCO), 1.82 (m, 2 H,
2-H2), 1.15 (d, J6,5 =6.5, 3 H, 6-H3), 0.89 (s, 9
J5,4=1.0, J5,6 =6.5, 1 H, 5-H), 3.48 (dd,
4,OH=10, 1 H, 4-H), 2.12 (d, 1 H, 4-OH),
13
J
H, SiC(Me)3), 0.17, 0.16 (2 s, 6 H, SiMe2). C
2.04 (ddd, J2eq,2ax=13, 1 H, 2eq-H), 1.54 (ddd,
1 H, 2ax-H), 1.28 (d, 3 H, 6-H3), 0.88 (s, 9 H,
SiCMe3), 0.11 and 0.10 (2 s, 6 H, SiMe2).
1-O-tert-Butyldimethylsilyl 4-O-p-nitroben-
NMR (75 MHz, CHCl3: l=170.39 (MeCO),
92.49 (C-1), 68.88 (C-4), 68.00 (C-5), 58.10
(C-3), 33.55 (C-2), 25.81 (Me3C), 20.92
(MeCO), 18.19 (Me3C-), 16.63 (C-6), −4.20,
−5.12 (Me2Si).
zoyl-2,3,6-trideoxy-3-trifluoroacetamido-i- -
L
Method B. Anhydrous AcONa (1.64 g, 20
mmol) and anhyd AcOCs (1.92 g, 10 mmol,
lyxo-hexopyranoside (2f).—p-Nitrobenzoyl-
ation of 2e (1.97 g, 5.5 mmol) carried out as

866
B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
(2 s, 6 H, SiMe2). Compound 4c: l=4.99 (dd,
1,2ax=8.8, J1,2eq =2.2, 1 H, 1-H), 4.63 (dd,
J4,3=3.5, J4,5=9.4, 1 H, 4-H), 4.16 (ddd,
3,2ax=3.5, J3,2eq =3.5, 1 H, 3-H), 3.94 (qd,
J5,6=6.2, 1 H, 5-H), 2.12 (s, 3 H, MeCO),
2.00 (ddd, J2eq,2ax =13.0, 1 H, 2eq-H), 1.82
(ddd, 1 H, 2ax-H), 1.20 (d, 3 H, 6-H3), 0.90 (s,
9 H, SiCMe3), 0.12 and 0.11 (2 s, 6 H, SiMe2).
tert - Butyldimethylsilyl 3 - azido - 2,3,6 - tri-
described in the synthesis of 3f gave, after
column chromatography (1:6 EtOAc–cyclo-
hexane) of the crude product, 2f (2.59 g, 93%)
as a colourless solid, mp 73 °C (Lit.: 75 °C
J
J
1
[18]). [h]2D5= −90.1° (c=0.935, CHCl3). H
NMR (300 MHz, CDCl3): l=8.32, 8.28
(A2B2, J=9, 4 H, ArꢀH), 6.35 (d br, JNH,3
=
7.5, 1 H, NH), 5.34 (d br, J4,3=3, 1 H, 4-H),
4.94 (dd, J1,2ax =8.7, J1,2eq =2.0, 1 H, 1-H),
4.37 (m, 1 H, 3-H), 3.85 (qd, J5,4=1.0, J5,6=
6.5, 1 H, 5-H), 2.08 (ddd, J2eq,3 =4.5,
deoxy-i-
tert-butyldimethylsilyl 3-azido-2,3,6-trideoxy-
i- -ribo-hexopyranoside (4d).—A solution of
L
-arabino-hexopyranoside (3d) and
J
2eq,2ax =12.3, 1 H, 2eq-H), 1.83 (ddd, J2ax,3=
L
12.5, 1 H, 2ax-H), 1.24 (d, 3 H, 6-H3), 0.94 (s,
9 H, SiCMe3), 0.18 and 0.16 (2 s, 6 H, SiMe2).
4-O-Acetyl-3-azido-2,3,6-trideoxy-
bino-hexopyranose (3b) and 4-O-acetyl-3-
azido - 2,3,6 - trideoxy - - ribo - hexopyranose
the previously obtained 3c–4c mixture (19.8 g,
60 mmol) and K2CO3 (0.5 g) in dry MeOH
(200 mL) was stirred for 15 h. The solvent was
removed in vacuo to give a viscous oil (17.4 g)
which was purified by column chromatogra-
phy (silica gel: 400 g, 15:1 cyclohexane–
EtOAc). Two fractions were obtained.
From the faster moving fraction, compound
3d (10.2 g, 59%) was obtained as a colourless
viscous oil. [h]D25= +27.9° (c=1.87, CHCl3).
1H NMR (200 MHz, CDCl3): l=4.80 (dd,
L
-ara-
L
(4b).—A suspension of 7 (17.14 g, 80 mmol)
in water (100 mL) was stirred for 2 h at 80 °C.
At 20 °C, AcOH (16 mL) and NaN3 (8.1 g,
0.12 mol) was added. After stirring for 24 h at
20 °C the reaction mixture was poured into a
satd aq NaHCO3 solution and extracted three
times with EtOAc (100 mL each time). The
organic layer was dried with Na2SO4 and
evaporated to dryness. The crude mixture of
3b and 4b (16.5 g) was used without further
purification and characterisation.
J
J
1,2ax=9.4, J1,2eq =2.4, 1 H, 1-H), 3.38 (ddd,
2ax,3=12.5, J3,2eq =5.0, J3,4 =9.5, 1 H, 3-H),
3.32 (dq, J5,4=9.2, J5,6 =6.5, 1 H, 5-H), 3.15
(ddd, J4,OH=3.0, 1 H, 4-H), 2.17 (ddd,
J2eq,2ax=12.5, 1 H, 2eq-H), 2.14 (d, 1 H, 4-
tert-Butyldimethylsilyl 4-O-acetyl-3-azido-
OH), 1.65 (ddd, 1 H, 2ax-H), 1.32 (d, 3 H,
6-H3), 0.90 (s, 9 H, SiCMe3), 0.13 and 0.12 (2
s, 6 H, SiMe2). Anal. Calcd for C12H25N3O3Si:
C, 50.14; H, 8.77; N, 14.62. Found C, 49.97;
H, 9.00; N, 14.55.
2,3,6-trideoxy-i- -arabino-hexopyranosid (3c)
L
and tert-butyldimethylsilyl 4-O-acetyl-3-azido-
2,3,6-trideoxy-i- -ribo-hexopranoside (4c).—
L
tert-Butyldimethylsilyl chloride (13.9 g, 92
mmol) in dry 1,2-dichloroethane (50 mL) was
added dropwise to a solution of a crude 3b–
4b mixture (16.5 g) and imidazole (10.5 g, 154
mmol) in dry CH2Cl (200 mL) at 0 °C. After
stirring for 24 h, the reaction mixture was
washed with water, satd NaHCO3, and again
water. After drying with Na2SO4, the crude
product was purified by column chromatogra-
phy (silica gel: 200 g, 30:1 cyclohexane–
EtOAc) to give 3c–4c (21.7 g 2.1:1 mixture,
From the slower moving fraction, com-
pound 4d (4.9 g, 28%) was obtained as a
colourless viscous oil. [h]2D5 = −36.6° (c=
1
0.90, CHCl3). H NMR (200 MHz, CDCl3):
l=5.00 (dd, J1,2ax=9.0, J1,2eq =2.3, 1 H, 1-
H), 4.07 (ddd, J3,2eq =J3,2ax=J3,4 =3.5, 1 H,
3-H), 3.63 (dq, J5,4 =9.2, J5,6=6.2, 1 H, 5-H),
3.40 (m, 1 H, 4-H; after D2O-exchange: m“
dd), 2.12 (ddd, J2eq,2ax =14, 1 H, 2eq-H), 1.83
(s br, 1 H, 4-OH), 1.82 (ddd, 1 H, 2ax-H), 1.29
(d, 3 H, 6-H3), 0.91 (s, 9 H, SiCMe3), 0.13 and
0.12 (2 s, 6 H, SiMe2). Anal. Calcd for
C12H25N3O3Si: C, 50.14; H, 8.77; N, 14.62.
Found C, 50.34; H, 8.66; N, 14.68.
1
83% from 7) as a colourless oil. H NMR (200
MHz, CDCl3), 3c: l=4.81 (dd, J1,2ax=9.2,
J
1,2eq =2.0, 1 H, 1-H), 4.66 (dd, J4,3 =J4,5=
9.5, 1 H, 4-H), 3.50 (ddd, J3,2ax=12.8, J3,2eq
=
5.0, 1 H, 3-H), 3.44 (qd, J5,6=6.2, 1 H, 5-H),
2.20 (ddd, J2eq,2ax =13.0, 1 H, 2eq-H), 2.12 (s, 3
H, MeCO), 1.68 (ddd, 1 H, 2ax-H), 1.20 (d, 3
H, 6-H3), 0.90 (s, 9 H, SiCMe3), 0.12 and 0.11
1-O-tert-Butyldimethylsilyl 2,3,6-trideoxy-3-
trifluoroacetamido-i-
L
-arabino-hexopyranoside
(3e).—Triethylamine (1 mL) and ethyl tri-

B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
867
dropwise to a solution of 3e (4.29 g, 12 mmol)
and pyridine (5 mL) in CH2Cl2 (40 mL) at
0 °C. After stirring for 12 h at 20 °C, CH2Cl2
(50 mL) and water (50 mL) was added and the
mixture was stirred for 30 min. The organic
layer was separated, washed with water and
dried with Na2SO4. After concentrating in
vacuo, the residue was co-evaporated three
times with toluene (30 mL each time), and the
crude product was purified on silica gel (1:10
EtOAc–cyclohexane) to give 3g (4.45 g, 92%)
as a colourless solid, mp 76 °C (Lit. 78 °C
fluoroacetate (5.11 g, 36 mmol) was added to
a solution of 3d (5.17 g, 18 mmol) in MeOH
(50 mL), and the reaction mixture was shaken
for 4 h in a hydrogen atmosphere in the
presence of Pd–C (10% Pd, 400 mg). After
filtering off the catalyst, the solvent was re-
moved in vacuo and the green–brown oil was
purified on silica gel (1:5 EtOAc–cyclohexane)
to give 3e (5.28 g, 82%) as a colourless solid,
1
mp 140–141 °C (Lit.: 142 °C [18]). H NMR
(200 MHz, CDCl3): l=6.46 (d br, JNH,3=7.5
Hz, 1 H, NH), 4.87 (dd, J1,2ax =8.7, J1,2eq
=
1
[18]). [h]2D5= −23.9° (c=1.99, CHCl3). H
2.1 Hz, 1 H, 1-H), 3.96 (m, 1 H, 3-H), 3.37
(qd, J5,4=9.1, J5,6 =6.2 Hz, 1 H, 5-H), 3.17 (t
br, J4,3=10.0, 1 H, 4-H), 2.47 (s br, 1 H,
4-OH), 2.23 (ddd, J2eq,3 =4.8, J2eq,2ax =12.7, 1
H, 2eq-H), 1.62 (ddd, J2ax,3 =12.7, 1 H, 2ax-H),
1.33 (d, 3 H, 6-H3), 0.89 (s, 9 H, SiCMe3), 0.12
and 0.11 (2 s, 6 H, SiMe2).
NMR (200 MHz, CDCl3): l=6.74 (d br,
J
NH,3=7.5, 1 H, NH), 4.87 (dd, J1,2ax =9.2,
1,2eq=2.1, 1 H, 1-H), 4.54 (dd, J4,3=J4,5 =
J
9.5, 4-H), 4.12 (m, 1 H, 3-H), 3.58 (qd, 1 H,
J5,6=6.4, 5-H), 2.33 (ddd, 1 H, J2eq,3 =4.8,
J
2ax,2eq=13.0, 2eq-H), 2.08 (s, 3 H, MeCO),
1.62 (ddd, J2ax,3=13, 1 H, 2ax-H), 1.24 (d, 3
H, 6-H3), 0.90 (s, 9 H, SiCMe3), 0.12 and 0.11
(2 s, 6 H, SiMe2).
1-O-tert-Butyldimethylsilyl 4-O-p-nitroben-
zoyl-2,3,6-trideoxy-3-trifluoro-acetamido-i- -
L
arabino-hexopyranoside (3f).—p-Nitrobenzoyl
chloride (2.26 g, 12.2 mmol) was added to a
solution of 3e (3.57 g, 10 mmol) and 4-
dimethylaminopyridine (DMAP) in CH2Cl2
and pyridine (6:1, 70 mL) at 0 °C. After stir-
ring for 24 h at 20 °C, the reaction mixture
was washed with water, satd NaHCO3, and
again water. After drying with Na2SO4, the
solvent was removed in vacuo and the residue
was co-evaporated three times with toluene
(30 mL each time) to remove pyridine.
Column chromatography (1:6 EtOAc–cyclo-
hexane) of the crude product gave 3f (4.60 g,
91%) as a colourless solid, mp 88–90 °C.
[h]2D5= −5.5° (c=1.96, CHCl3). 1H NMR
(300 MHz, CDCl3): l=8.29, 8.14 (A2B2, J=
9, 4 H, ArꢀH), 6.74 (d br, JNH,3=8.3, 1 H,
NH), 4.97 (dd, J1,2ax =9.0, J1,2eq =2.2, 1 H,
1-H), 4.84 (dd, J4,5=J4,3=9.7, 1 H, 4-H), 4.38
(m, 1 H, 3-H), 3.79 (qd, J5,6=6.2, 1 H, 5-H),
2.37 (ddd, J2eq,3 =4.7, J2eq,2ax=12.5, 1 H, 2eq-
H), 1.76 (ddd, J2ax,3 =12.5, 1 H, 2ax-H), 1.31
(d, 3 H, 6-H3), 0.92 (s, 9 H, SiCMe3), 0.16 and
0.14 (2 s, 6 H, SiMe2). Anal. Calcd for
C21H29F3N2O7Si: C, 49.79; H, 5.77; N, 5.53.
Found C, 50.03; H, 5.76; N, 5.47.
tert - Butyldimethylsilyl 3 - azido - 2,3,6 - tri-
deoxy-4-O-trifluoromethanesulfonyl-i- -ara-
L
bino-hexopyranoside (3h).—Dry pyridine
(3.96 g, 50 mmol) and DMAP (100 mg) was
added to 3d (5.75 g, 20 mmol) in dry CH2Cl2
(150 mL). At −40 °C, a solution of trifluoro-
methanesulfonic acid anhydride (3.4 mL, 6.77
g, 24 mmol) in dry CH2Cl2 (50 mL, containing
1% pyridine) was added dropwise to the reac-
tion mixture. After stirring for 4 h at −20 °C
(reaction was monitored by thin layer chro-
matography (TLC), 1:5 EtOAc–cyclohexane),
the reaction mixture was washed twice with
10% aq AcONa, 5% NaHCO3, and finally
with water. The solvent was removed in
vacuo, and residue was co-evaporated three
times with toluene (30 mL each time) to re-
move pyridine. The crude product 3h was used
directly for the next step (inversion) without
further purification. For NMR measurement,
a small amount was purified by column chro-
matography (1:5 EtOAc–cyclohexane) to give
3h as a colourless unstable oil which decom-
posed visibly within some hours by going
1
dark. H NMR (200 MHz, CDCl3): l=4.86
(dd, J1,2ax=9.2, J1,2eq =2.2, 1 H, 1-H), 4.34
(dd, J3,4 =J4,5=9.5, 1 H, 4-H), 3.64 (ddd,
tert-Butyldimethylsilyl
4-O-acetyl-2,3,6-
- arabino-
trideoxy - 3 - trifluoroacetamido - i -
L
J
3,2ax=13, J3,2eq =5.0, 1 H, 3-H), 3.60 (qd,
hexopyranoside (3g).—Acetic anhydride (4.8
mL, 48 mmol) in CH2Cl2 (20 mL) was added
J5,6=6.2, 1 H, 5-H), 2.38 (ddd, J2eq,2ax =13.0,
1 H, 2eq-H), 1.83 (ddd, 1 H, 2ax-H), 1.38 (d, 3

868
B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
2.8, J2ax,2eq =13.0, 2eq-H), 2.08 (s, 3 H,
MeCO), 1.90 (ddd, J2ax,3 =4.5, 1 H, 2ax-H),
1.33 (d, 3 H, 6-H3), 0.90 (s, 9 H, SiCMe3), 0.13
and 0.12 (2 s, 6 H, SiMe2). Anal. Calcd for
C16H28F3NO5Si: C, 48.11; H, 7.06; N, 3.51.
Found: C, 48.33; H, 7.19; N, 3.64.
H, 6-H3), 0.90 (s, 9 H, SiCMe3), 0.13 and 0.12
(2 s, 6 H, SiMe2).
tert-Butyldimethylsilyl 2,3,6-trideoxy-3-trifl-
uoroacetamido-i- -ribo-hexopyranoside
L
(4e).—Catalytic hydrogenation of 4d (4.00 g,
13.9 mmol) carried out as described in the
synthesis of 3e gave, after column chromatog-
raphy (1:5 EtOAc–cyclohexane), 4e (4.32 g,
tert - Butyldimethylsilyl 3 - azido - 2,3,6 - tri-
deoxy-i- -xylo-hexopyranoside (5d)
L
1
Method A. A solution of 5i (6.00 g, 15.3
mmol) and K2CO3 (0.5 g) in dry MeOH (50
mL) was stirred overnight and solvent was
removed in vacuo. Column chromatography
(80 g of silica gel, 5:1 cyclohexane–EtOAc) of
the crude product yielded 5d (4.00 g, 91%) as
87%) as colourless viscous oil. H NMR (200
MHz, CDCl3): l=6.46 (d br, 1 H, NH), 5.07
(dd, J1,2ax =6.0, J1,2eq =3, 1 H, 1-H), 4.45 (m,
1 H, 3-H), 3.79 (qd, J5,4=J5,6=6.5, 1 H,
5-H), 3.65 (s br, 1 H, 4-H), 2.20 (ddd, J2eq,3
=
7.5, J2eq,2ax =13.5, 1 H, 2eq-H), 2.10 (s br, 1 H,
4-OH), 1.81 (ddd, J2ax,3 =4, 1 H, 2ax-H), 1.39
(d, 3 H, 6-H3), 0.89 (s, 9 H, SiCMe3), 0.12 and
0.12 (2 s, 6 H, SiMe). Anal. Calcd for
C14H26F3NO4Si: C, 47.04; H, 7.33; N, 3.92.
Found: C, 47.46; H, 7.46; N, 3.93.
1
a white solid, mp 85–86 °C. H NMR (200
MHz, CDCl3): l=4.93 (dd,
J1,2eq =4.3
J
1,2ax=7.3, 1 H, 1-H), 3.98 (dd, J3,4 =3.2,
3,2ax=6.6, 1 H, 3-H), 3.91 (dq, J5,6 =6.6,
J
J5,4=1, 1 H, 5-H), 3.32 (ddd, J4-OH =10,
3–4=3.2,J4–5=1, 1 H,4-H),2.24(d,1 H, OH),
J
tert-Butyldimethylsilyl 4-O-p-nitrobenzoyl-
1.85 (m, 2 H, 2eq-, 2ax-H), 1.25 (d, 3 H, 6-H3)
0.90 (s, 9 H, SiC(CH3)3), 0.13, 0.12 (2 s, 2×3
H, SiMe2). 13C NMR (75.5 MHz, CDCl3):
l= −4.24, −5.21 (ꢀSiCH3), 16.59 (C-6),
18.13 [(CH3)3Cꢀ], 25.75 [ꢀ(CH3)3], 32.91 (C-2),
60.47 (C-3), 69.22/68.31(C-4, C-5), 93.12 (C-1).
Method B. Deacetylation of a mixture of
2c–5c was carried out as depicted in the
deacetylation of 3c–4c. Thus, when 2.50 g
(7.59 mmol) of the mixture was deacetylated
in anhyd MeOH (50 mL), using K2CO3 (0.5 g)
as the base, a white solid (1.90 g) was ob-
tained. Further separation of the mixture by
column chromatography (silica gel: 200 g, 15:1
cyclohexane–EtOAc) afforded two fractions:
from the faster moving fraction, compound 2d
(0.50 g, 24%) was obtained as a colourless
solid with mp 68–70 °C. From the slower
moving fraction, compound 5d (1.20 g, 56%)
was obtained as a colourless solid, mp 85–
86 °C. Anal. Calcd for C12H25N3O3Si: C,
50.14; H, 8.77. Found: C, 50.11; H, 8.50.
tert-Butyldimethylsilyl 3-trifluoroacetamido-
2,3,6-trideoxy-3-trifluoroacetamido-i- -ribo-
L
hexopyranoside (4f).—p-Nitrobenzoylation of
4e (5.36 g, 15 mmol) carried out as described
in the synthesis of 3f gave, after column chro-
matography (1:6 EtOAc–cyclohexane), 4f
(6.26 g, 82%) as a colourless solid with mp
1
106 °C. [h]2D5= +46.2° (c=2.09, CHCl3). H
NMR (300 MHz, CDCl3): l=8.30, 8.14 (m,
4H, A2B2, J=9, 4 H, ArꢀH), 6.47 (d br,
J
J
NH,3=7.5, 1 H, NH), 5.20 (dd, J1,2ax=6.5,
1,2eq =2.7, 1 H, 1-H), 5.09 (dd, J4,3=3.8,
J4,5 =6.7, 1 H, 4-H), 4.85 (m, 1 H, 3-H), 4.06
(qd, J5,6=6.7, 1 H, 5-H), 2.19 (ddd, J2eq,3
6.8, J2eq,2ax =13.5, 1 H, 2eq-H), 2.04 (ddd,
2ax,3 =4.5, 1 H, 2ax-H), 1.42 (d, 3 H, 6-H3),
=
J
0.92 (s, 9 H, SiCMe3), 0.16 and 0.15 (2 s, 6 H,
SiMe2). Anal. Calcd for C21H29F3N2O7Si: C,
49.79; H, 5.77; N, 5.53. Found C, 50.07; H,
5.85; N, 5.59.
tert-Butyldimethylsilyl 4-O-acetyl-2,3,6-tri-
deoxy-3-trifluoroacetamido-i- -ribo-hexopyran-
L
oside (4g).—Acetylation of 4e (1.79 g, 5
mmol) carried out as described in the synthe-
sis of 3g gave, after column chromatography,
4g (1.77 g, 89%) as a colourless solid with mp
2,3,6-trideoxy-i- -xylo-hexopyranoside (5e).
L
—Hydrogenation of 5d (2.40 g, 8.35 mmol)
carried out as described in the synthesis of 3e
gave, after column chromatography (silica gel:
60 g, 5:1 cyclohexane–EtOAc), 5e (2.65 g,
92%) as a colourless solid, mp 125–126 °C.
122 °C. [h]2D5= +21.4° (c=0.94, CHCl3). H
1
NMR (200 MHz, CDCl3): l=6.38 (d br,
J
J
NH,3=7.5, 1 H, NH), 5.09 (dd, J1,2ax=6.5,
1,2eq =2.9, 1 H, 1-H), 4.75 (dd, J4,3=4.0,
1
[h]2D5= +16.4° (c=1.97, CHCl3). H NMR
J4,5 =6.7, 4-H), 4.66 (m, 1 H, 3-H), 3.88 (qd,
1 H, J5,6=6.7, 5-H), 2.13 (ddd, 1 H, J2eq,3
(200 MHz, CDCl3): l=6.26 (br, 1 H, NH),
4.92 (dd, J1,2e =2.4, J1–2a=8.5, 1 H, 1-H),
=

B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
869
for 15 h at 20 °C until TLC analysis showed
complete cleavage of the acetal group. Sodium
azide (2.30 g, ca. 35 mmol) was added and the
reaction mixture was stirred for 10 h. The
organic layer was separated and the water
phase was extracted with CH2Cl2. The com-
bined organic layers were washed with satd
NaHCO3 and dried with Na2SO4. The solvent
was removed and the crude product was redis-
solved in dry CH2Cl2 (100 mL), together with
of tert-butyl(dimethyl)silyl chloride (3.00 g,
ca. 20 mmol) and imidazole (2.72 g, ca. 40
mmol). After stirring for 2 h, the mixture was
washed with water and dried with Na2SO4.
The crude product was purified by column
chromatography (80 g of silica gel, 20:1 cyclo-
hexane–EtOAc) to give 5i (7.0 g, 88%) as a
colourless viscous oil. [h]2D5 = −60.4° (c=
4.29 (m, 1 H, 3-H), 3.80 (dq, J5–4 =1.7, J5–6
6.6, 1 H, 5-H), 3.50 (m, J4–3 =3.6, 1 H, 4-H),
2.30 (d, J=9.1, 1 H, OH), 2.07 (ddd, J2a,2e
=
=
13.6, J2a–1 =8.5, J2a,3=4.7, 1 H, 2a-H), 1.79
(ddd, J2a,2e=13.6, J2e,3 =2.7, J2e,1 =2.4, 1 H,
2e-H), 1.31 (d, J=6.6, 3 H, 6-H), 0.90 (s, 9 H,
tert-Bu), 0.14 and 0.12 (2 s, 2×3 H, ꢀSiꢀMe).
13C NMR (75.5 MHz, CDCl3): l= −4.05,
−5.17 (ꢀSiMe), 16.73 (C-6), 18.01 [(Me)3Cꢀ],
25.70 [ꢀC(Me)3], 33.25 (C-2), 49.92 (C-3),
69.65/68.10 (C-4, C-5), 92.97 (C-1), 115.60
(JCF=287.9 Hz, CF3), 156.88 (JCF=37.4 Hz,
ꢀCOCF3). MS (70 eV): m/z (%)=356.2 ([M+
−1], 0.5), 119 (100), 101 (20), 75 (36), 73 (26).
Anal. Calcd for C14H26F3NO4Si: C, 47.04; H,
7.34. Found: C, 46.95; H, 7.20.
tert-Butyldimethylsilyl 3-trifluoracetamido-
4-O-(p-nitrobenzoyl)-2,3,6-trideoxy- -xylo-
L
1
0.37, CHCl3). H NMR (200 MHz, CDCl3):
hexopyranoside (5f).—p-Nitrobenzoylation of
5e (1.55 g, 4.34 mmol) carried out as described
in the synthesis of 3f gave, after column chro-
matography (silica gel: 60 g, 20:1 cyclohex-
ane–EtOAc), 5f (2.0 g, 91%) as a pale yellow
l=8.09 (m, 2 H) and 7.65–7.42 (m, 3 H,
phenylꢀH), 5.06 (dd, J1,2eq=3.9, J1,2ax =7.3, 1
H, 1-H), 4.81 (dd, J4,3 =3.2, J4,5=1.5, 1 H,
4-H), 4.10 (m, 2 H, 3-, 5-H), 1.93 (m, 2 H,
2-H2), 1.25 (d, J6,5=6.5, 3 H, 6-H3), 0.94 (s, 9
H, SiC(CH3)3), 0.17, 0.16 (2 s, 2×3 H,
SiMe2).
1
foam. [h]2D5= −12.1° (c=2.05, CHCl3). H
NMR (200 MHz, CDCl3): l=8.33, 8.24
(A2B2, J=9, 4 H, ArꢀH), 6.45 (d, J=7.3,
NH), 5.13 (m, 2 H, 1-, 4-H), 4.61 (m, 1 H,
3-H), 4.18 (dq, J5–6 =6.6, J4–5 =3.4, 1 H,
5-H), 2.15 (ddd, J2a–2e =13.7, J1,2a=6.4,
3,4-Di-O-acetyl-1,5-anhydro-2,6-dideoxy-
L
-
arabino-hex-1-enol (di-O-acetyl- -(+)-rham-
L
nal) (7).—Rhamnose (6a) (100 g, 0.55 mol)
was added to a mixture of Ac2O (340 mL) and
70% perchloric acid (2 mL) in a 1-L three-
necked flask in such a way that the reaction
temperature was maintained between 30 and
40 °C. After stirring for 2 h, phosphorus tri-
bromide (70 mL) was added dropwise below
10 °C. Finally water (33 mL) was added drop-
wise below 15 °C, and the reaction mixture
was then stirred for another 2 h at 20 °C.
Sodium acetate (226 g), water (700 mL) and
AcOH (295 mL) were added to a 4-L three-
necked round bottom flask equipped with me-
chanic stirrer, thermometer and dropping
funnel. The mixture was cooled to −10 °C
using MeOH–dry ice bath. Zinc powder (200
g) and finally aq CuSO4 solution (16.5 g
CuSO4×5 H2O in 65 mL water) were added.
After disappearance of the blue colour and
development of hydrogen, the crude bro-
moaceto–rhamnose solution prepared above
was added dropwise to this mixture and the
reaction temperature was maintained between
J3,2a =4.7, 1 H, 2a-H), 1.93 (ddd, 1 H, J2a–
2e=13.7, J3–2e =6.4, J1–2e =2.7, 2e-H), 1.34
(d, 3H, 6-H3), 0.94 (s, 9 H, tert-Bu), 0.18 and
0.16 (2 s, 2×3 H, ꢀSiꢀMe). 13C NMR (75.5
MHz, CDCl3): l= −4.08, −5.08 (SiMe),
17.34 (C-6), 18.08 [(Me)3Cꢀ], 25.75 [ꢀC(Me)3],
35.64 (C-2), 46.36 (C-3), 69.17–71.16 (C-4,
C-5), 92.47 (C-1), 115.52 (q, J1CF=288 Hz,
CF3), 156.86 (q, J2CF=38 Hz, ꢀCOCF3),
123.74 (C-3%, C-5%), 131.04 (C-2%, C-6%), 134.46
(C-1%), 150.89 (C-4%), 164.19 (ꢀCOO). MS (70
eV): m/z (%)=505.1 ([M+−1], 491 (32), 449
(36), 336 (48), 238 (50), 224 (36), 208 (20), 150
(100),
101
(16).
Anal.
Calcd
for
C21H29N2O7F3Si: C, 49.79; H, 5.77; Found: C,
49.79; H, 5.71.
tert-Butyldimethylsilyl 3-azido-4-O-benzoyl-
2,3,6 - trideoxy - i - - xylo - pyranoside (5i).—
L
Compound 9a (5.00 g, ca. 20 mmol) was
dissolved in a mixture of 1 N HCl (20 mL),
HOAc (8 mL) and THF (30 mL), and stirred

870
B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
CH2Cl2. The crude product was dried with
Na2SO4, and redissolved in DMF (50 mL).
Caesium benzoate (3.81 g) was added and the
suspension was stirred for 5 h at 80 °C. At
20 °C, water (50 mL) was then added and the
reaction mixture was extracted four times with
diethyl ether (50 mL of each time). The or-
ganic phase was washed with water, dried
with Na2SO4, and the crude product was
purified by column chromatography to give 9a
(0.95 g, 39%) as a colourless oil. [h]2D5 = +
214.0° (c=0.86, CHCl3).
−5 and −10 °C. After stirring for 6 h at
−10 °C, 1 L of ice–water was added and the
reaction mixture was stirred for another 5
min. Unreacted zinc powder and Cu were
filtered off, and the filtrate was extracted five
times with CH2Cl2 (300 mL each time). The
combined organic layer was washed with ice–
water, satd NaHCO3, again ice–water, and
dried with Na2SO4. The solvent was removed
in vacuo and the crude product was vacuum-
distilled at 78–79 °C/0.1 Torr to give 7 (100 g,
85%) as a colourless liquid (Lit.: 68–69 °C/
1
0.06 Torr [9]). H NMR (200 MHz, CDCl3):
Method B (Mitsunobu reaction). To a solu-
tion of 8c (9.6 g, 66.6 mmol), benzoic acid
(17.0 g, 0.14 mol) and triphenylphosphine
(36.5 g, 0.14 mol) in anhyd THF (100 mL),
DEAD (22.0 g, ca. 0.13 mol) dissolved in
THF (30 mL) was added dropwise at 0 °C.
The reaction mixture was stirred for 15 h and
the solvent was removed in vacuo, 100 mL of
ether was added to the residue and the insolu-
ble part was filtered off. After removal of the
solvent, the crude product was purified by
column chromatography (200 g of silica gel,
10:1 cyclohexane–EtOAc) to give 9a (15.00 g,
l=6.43 (dd, 1 H, J1,2=6.1, 1-H), 5.34 (m, 1
H, 2-H), 5.03 (dd, 1 H, J4,5 =8.1, J3,4=6.1,
4-H), 4.78 (dd, 1 H, J3,4=6.1, J2,3 =3.0, 3-H),
4.11 (dq, 1 H, J4,5=6.1, J5,6 =6.6, 5-H), 2.09,
2.05 (2 s, 6 H, 2 AcO), 1.32 (d, 3 H, J5,6=6.6,
6-H).
Methyl
4-O-acetyl-2,3,6-trideoxy-h,i-
L
-
erythro-hex-2-enopyranoside
(8b).—Anhy-
drous ZnCl (14.0 g, c.a. 0.10 mol) was added
to a solution of 7 (43 g, 0.20 mol) in dry
CH2Cl2 (330 mL) and dry MeOH (20 mL).
After stirring overnight, satd NaHCO3 (100
mL) was added to the reaction mixture and
the organic layer was washed with water. Af-
ter drying with Na2SO4, the crude product was
vacuum-distilled at 55–56 °C/0.1 Torr to give
colourless oily 8b (30.0 g, 81%) as mixture of
1
90%) as a colourless viscous liquid. H NMR
(200 MHz, CDCl3): a-9a: l=8.09 (m, 2 H,
ArꢀH), 7.61–7.39 (m, 3 H, ArꢀH), 6.21 (dd,
J3,4=5, J3,2 =10, 1 H, 3-H), 6.06 (dd, J2,1=3,
1 H, 2-H), 5.15 (dd, J4,5 =5.2, 1 H, 4-H), 4.98
(d, 1 H, 1-H), 4.33 (dq, J5,6=6.5, 1 H, 5-H),
3.47 (s 3 H, OMe), 1.32 (d, 3 H, 6-H3). b-9a:
l=8.09 (m, 2 H, ArꢀH), 7.61–7.39 (m, 3 H,
ArꢀH), 6.18 (ddd, J3,1 =1.5, J3,4 =4.8, J3,2 =
10, 1 H, 3-H), 6.06 (ddd, J2,1=J2,4 =1, 1 H,
2-H), 5.26 (m, 1 H, 4-H), 5.09 (br q, 1 H,
1-H), 4.01 (dq, J5,4 =2.8, J5,6=6.5, 1 H, 5-H),
3.53 (s 3 H, OMe), 1.34 (d, 3 H, 6-H3).
1
a and b anomer (a:b=4:1). H NMR (200
MHz, CDCl3), a-8b: l=5.83 (m, 2 H, 2-,
3-H), 5.05 (m, 1 H, 4-H), 4.83 (dd, J1,2=1.5,
J1,3 =0.9, 1 H, 1-H), 3.94 (dq, J5,6=6.5,
J5,4 =9.5, 1 H, 5-H), 3.44 (s, 3 H, OMe), 2.09
(s, 3 H, MeCO), 1.24 (d, 3 H, 6-H3). b-8b:
l=5.90 (m, 2 H, 2-, 3-H), 5.05 (m, 1 H, 4-H),
4.78 (d, J1,2 =4, 1 H, 1-H), 3.85 (m, 1 H, 5-H),
3.47 (s, 1 H, OMe), 2.10 (s, 3 H, MeCO), 1.32
(d, J6,5 =6.5, 3 H, 6-H3).
Methyl 4 - O - acetyl - 2,3,6 - trideoxy - h,i -
threo-hex-2-enopyranoside (9b)
L
-
Methyl 4-O-benzoyl-2,3,6-trideoxy-h,i-
L
-
Deacetylation reaction. A mixture of anhyd
K2CO3 (0.1 g) and 8b (6.50 g, ca. 35 mmol) in
dry MeOH (30 mL) was stirred for 15 h at
20 °C and solvent was removed in vacuo. The
resulting yellowish oil was purified on silica
gel (column, 10:1 cyclohexane–EtOAc) to give
8c (4.70 g) as a colourless oily mixture of both
anomers.
threo-hex-2-enopyranoside (9a)
Method A. Compound 8b (8.00 g, ca. 10
mmol) was dissolved in dry MeOH (30 mL)
and anhyd K2CO3 (0.1 g) was added. After
stirring overnight, the solvent was removed in
vacuo. The residue was dissolved in dry pyri-
dine (10 mL) and mesyl chloride (2.06 g, ca.
18 mmol) was added dropwise at 0 °C. After
stirring for 15 h at 20 °C, water (30 mL) was
added and the mixture was extracted with
Mitsunobu reaction.—Diethyl azodicar-
boxylate (11.40 g, ca. 65 mmol) in dry THF
(15 mL) was added dropwise to the solution

B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
871
of 8c (4.7 g, 65 mmol) obtained above, AcOH
(4.0 g, ca. 65 mmol) and triphenylphosphine
(17.10 g, 65 mmol) in dry THF (100 mL) at
0 °C. The reaction mixture was stirred 15 h
and solvent was removed in vacuo, diethyl
ether (50 mL) was added to the residue and
the insoluble part was filtered off. After re-
moval of solvent, the crude product was
purified by column chromatography (200 g
silica, 10:1 cyclohexane–EtOAc) to give 9b
(4.85 g, 85%) as a colourless viscous liquid.
7-O-(2,3,6-Trideoxy-4-O-p-nitrobenzoyl-3-
concentrated in vacuo, redissolved in 2:1
CH2Cl2–butanol (200 mL), washed with satd
NaHCO3 and with water (50 mL). The or-
ganic phase was concentrated in vacuo and
the residue was purified by column chro-
matography (60 g of silica gel, 100:5:0.5
CH2Cl2–MeOH–concentrated NH3) giving 1c
(1.04 g, 67%) as a red sparingly soluble pow-
der, mp 169–171 °C (Lit. 173–175 °C).
Biological testing.—The samples were
solved in DMSO and stored at −20 °C. For
the in vitro study the compounds were further
diluted with culture medium under consider-
ation that the final DMSO concentration
never exceeds 0.3%. The supplied drugs were
tested in a final concentration of 0.3 and 3
mg/mL.
trifluoroacetamido-h-L-arabino-hexopyranosyl)-
m-iso-rhodomycinone
(7-O-acosaminyl-m-iso-
rhodomycinone) (10).—Me3Si–triflate (1.50 g,
6.72 mmol) was added dropwise at −35 °C to
a suspension of 1b (1.80 g, 4.05 mmol), 2f
(2.60 g, 5.13 mmol) and powdered molecular
Human tumor cell lines.—For the in vitro
cytotoxicity study the following six human
tumor cell lines were used:
,
sieves (4 A, 4.5 g) in 10:1 CH2Cl2–acetone
(210 mL). After stirring for 6 h at this temper-
ature, the reaction was quenched by addition
of triethylamine (2.8 mL). The molecular
sieves were filtered off, and the filtrate was
washed twice with water (100 mL each time).
After drying with Na2SO4, the crude product
was purified by column chromatography (170
g of silica gel, 200:10:1 chloroform–EtOAc–
formic acid) to give 10 (2.32 g, 70%) as a red
powder, mp 268–271 °C (Lit. 279–281 °C
[11]) which contained ca. 12% bis-glycoside.
1H NMR (300 MHz, CDCl3): l=13.02,
12.84, 12.30, 12.29 (4 s, 4 H, 1-, 4-, 6-, 11-
OH), 8.32, 8.18 (A2B2, 4 H, nitrobenzoylꢀH),
7.30 (s, 2 H, 2-, 3-H), 6.60 (d, J3%,NH=8, 1 H,
NH), 5.52 (d, J1%,2ax =4, 1 H, 1%-H), 5.26 (dd,
Colon cancer:
Gastric cancer:
Melanoma:
HT 29
GXF 251
MEXF 462NL
Non-small cell lung LXFL 529L, LXF
cancer: 629, LXF 66
Cell culture.—Human tumor cells were
grown at 37 °C in a humidified atmosphere
(95% air, 5% CO2) in monolayer cultures in
RPMI 1640 medium with phenol red (Life
Technologies, Karlsruhe, Germany) supple-
mented with 10% of fetal calf serum. Cells
were trypsinised and maintained weekly.
Cytotoxicity assay.—A modified propidium
iodide assay was used to examine the antipro-
liferative activity of the studied compounds
[19]. Briefly, cells were harvested from expo-
nential phase cultures growing in RPMI 1640
medium supplemented with 10% of fetal calf
serum by trypsination, counted and plated in
96 well flat-bottomed microtitre plates (100 ml
cell suspension, 1×105 and 5×104 cells/mL).
After a 24 h recovery, to allow cells to resume
exponential growth, 50 ml culture medium (six
control wells per plate) or culture medium
containing the test drug were added to the
wells. Each drug concentration was plated in
triplicate. After 3–7 days of incubation, de-
pending on cell doubling time, culture medium
J7,8a =10, J7,8b =2, 1 H, 7-H), 4.91 (dd, J3%,4%=
J4%,5%=10, 1 H, 4%-H), 4.48–4.38 (m, 2 H, 5%-,
3%-H), 4.34 (s, 1 H, 10-H), 4.09 (s, 1 H, 9-OH),
3.74 (s, 3 H, COOMe), 2.44 (m, 2 H, 2%eq-,
8a-H), 2.30 (dd, 1 H, 8b-H), 1.92 (m, 2 H, 2%ax-,
13b-H), 1.34 (d, J5%,6%=6, 6%-H3), 1.18 (t,
J
14,13 =7.3, 3 H, 14-H3).
7-O-(3-Amino-2,3,6-trideoxy-h- -arabino-
L
hexopyranosyl)-m-iso-rhodomycinone (7-acosa-
minyl-m-iso-rhodomycinone) (1c).—To a solu-
tion of 10 (2.21 g, 2.7 mmol) in CH2Cl2–
MeOH (2: 1, 25 mL), 1 N NaOH (9.5 mL) of
was added and the reaction mixture was ho-
mogenised by adding MeOH. After stirring
20 °C for 30 min, the reaction mixture was
neutralised by adding 1 N HCl (9.5 mL),

872
B. Renneberg et al. / Carbohydrate Research 329 (2000) 861–872
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zlowski, J.A. Rice, Proc. Natl. Acad. Sci. USA, 78 (1981)
3333–3337.
was replaced by fresh medium containing pro-
pidium iodide (6 mg/mL). Microplates were
then kept at −18 C for 24 h, resulting in a
total cell kill. After thawing of the plates,
fluorescence was measured using a Millipore
Cytofluor 2350-microplate reader (excitation
530 nm, emission 620 nm) in order to quantify
the total cell number. The assay included un-
treated and positive controls (Doxorubicin).
Growth inhibition was expressed as treated/
control×100 (T/C%). IC50 and IC70 values
were determined by plotting compound con-
centration versus cell number. Mean IC50 and
IC70 values were calculated according to the
[2] (a) J.W. Lown, S.-K Sim, K.C. Majumdar, R.-Y. Chang,
Biochem. Biophys. Res. Commun., 76 (1977) 705–710. (b)
N.R. Bachur, S.L. Gordon, M.V. Gee, Cancer Res., 38
(1978) 1745–1750.
[3] (a) W.E. Ross, D.L. Glaubiger, K.W. Kohn, Biochim.
Biophys. Acta, 519 (1978) 23–30. (b) K.M. Tewey, G.L.
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9182–9187.
[4] (a) F. Arcamone, in B.W. Fox (Ed.), Ad6ances in Medical
Oncology, Research and Education, Basis of Cancer Ther-
apy, Vol. 5, Pergamon, Oxford, 1979, pp. 21–32. (b) U.
Bonfante, G. Bonadonna, F. Villani, G. Di Fronzo, A.
Martini, A.M. Casazza, Cancer Treat. Rep., 63 (1979)
915–918. (c) A. Suarato, S. Penco, A. Vigevani, F.
Arcamone, Carbohydr. Res., 98 (1981) C1–C3.
[5] D. Socha, M. Jurczak, M. Chmielewski, Tetrahedron, 53
(1997) 739–746.
formula:
n
[6] F.M. Hauser, S.R. Ellenberger, Chem. Re6., 86 (1986)
35–67 and the literature cited herein.
%
log(IC )
50,70 x
x=1
[7] T.M. Cheung, D. Horton, R.J. Sorensen, W. Weckerle,
Carbohydr. Res., 63 (1978) 77–89.
n
mean IC50,70 =10
[8] J. Boivin, M. Pa¨ıs, C. Monneret, Carbohydr. Res., 64
(1978) 271–278.
[9] B. Iselin, T. Reichstein, Hel6. Chim. Acta, 27 (1944)
1146–1149.
[10] K.H. Hollenbeak, M.E. Kuehne, Tetrahedron, 30 (1974)
2307–2316.
[11] J.-C. Florent, C. Monneret, J. Chem. Soc., Chem. Com-
mun., (1987) 1171–1172.
With x=specific tumor cell line and n=to-
tal number of cell lines studied. If IC50 or IC70
could not be determined within the examined
dose range, the lowest or highest concentra-
tion studied was used for the calculation.
[12] B. Kraska, A. Klemer, H. Hagedorn, Carbohydr. Res., 36
(1974) 398–403.
[13] C. Kolar, G. Kneißl, Angew. Chem., Int. Ed. Engl., 102
(1990) 827–828.
Acknowledgements
[14] (a) R.J. Ferrier, N. Prasad J. Chem. Soc. C, (1969)
570–575. (b) B. Fraser-Reid, D.R. Kelly, D.B. Tulshian,
P.S. Ravi, J. Carbohydr. Chem., 2 (1983) 105–114.
[15] P.G. Sammes, D. Thetford, J. Chem. Soc. Perkin Trans.
1, (1988) 111–123.
[16] O. Mitsunobu, Synthesis, (1981) 1–28.
[17] C. Kolar, K. Dehmel, H. Moldenhauer, M. Gerken, J.
Carbohydr. Chem., 9 (1990) 873–890.
We thank Dr G. Remberg for the mass
spectra, Mr R. Machinek for the NMR mea-
surements and the Fonds der Chemischen
Industrie for financial support.
[18] C. Kolar, K. Dehmel, U. Kno¨dler, M. Paal, P. Her-
mentin, M. Gerken, J. Carbohydr. Chem., 8 (1989) 295–
305.
[19] W.A. Dengler, J. Schulte, D.P. Berger, R. Mertelsmann,
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