Synthesis of galactosyl and lactosyl derivatives as potential anti-metastasis compounds

Article abstract of DOI:10.1016/S0008-6215(00)00137-3

Based on the known anti-metastasis activities of lactosides and galactosides, a galactosyl and a lactosyl trimannoside were prepared via the conventional Koenigs-Knorr and trichloroacetimidate methods, respectively. Through typical deblocking procedures,

Full text of DOI:10.1016/S0008-6215(00)00137-3

www.elsevier.nl/locate/carres
Carbohydrate Research 328 (2000) 611–615
Note
Synthesis of galactosyl and lactosyl derivatives as
potential anti-metastasis compounds
Hui Li, Qing Li, Meng-Shen Cai, Zhong-Jun Li *
Department of Bioorganic Chemistry, School of Pharmaceutical Sciences, Beijing Medical Uni6ersity,
Beijing 100083, PR China
Received 16 November 1999; received in revised form 18 April 2000; accepted 21 April 2000
Abstract
Based on the known anti-metastasis activities of lactosides and galactosides, a galactosyl and a lactosyl
trimannoside were prepared via the conventional Koenigs–Knorr and trichloroacetimidate methods, respectively.
Through typical deblocking procedures, a tetrasaccharide a-
-ManpOCH3 and a pentasaccharide b- -Galp-(1“4)-b- -Glcp-(1“2)-a-
ManpOCH3 were obtained. © 2000 Elsevier Science Ltd. All rights reserved.
D
-Galp-(1“2)-a-
D
-Manp-(1“2)-a-
D
-Manp-(1“6)-a-
D
D
D
D
-Manp-(1“2)-a-
D
-Manp-(1“6)-a-D-
Keywords: a-Galactosyl trimannoside; b-Lactosyl trimannoside; Glycosylation; Synthesis
cells from agglomerating in the lungs. Further
study of the lactosides indicated that conju-
gates having a lactosyl group linked to a
lysine–lysine peptide or other polylysine pep-
tides through a carbon chain spacer arm have
different activities in regard to inhibiting
metastasis. Interestingly some of the glycocon-
jugates have strong anti-metastasis activity
while others show no such activity or even
enhance metastasis, a factor apparently re-
lated to the difference in length of the spacer
arms [4]. In addition, such ramified mannans
as hexamannoside Man6 and nonamannoside
Man9 are also able to induce cancer cell adhe-
sion, whereas trimannoside Man3 has not
[5,6].
1. Introduction
Raz and Lotan [1,2] discovered that lactose,
-galactose,
D
D
-glucosamine, and N-acetyl- -
D
galactosamine can inhibit the adhesion of
some cancer cells, such as human melanoma
A375, SH4, Hs294, Hs852, human adenoma
Hela-S3, murine melanoma B16-F1, and
murine fibrosarcoma UV-2237P. Among the
aforementioned glycoses, lactose has the
strongest inhibition activity, whereas other
glycoses such as
D-mannose,
L
-fucose, N-ace-
tyl- -glucosamine have none. The adhesion of
D
cancer cells to other normal cells is the basis
of cancer metastasis and proliferation. Oguchi
et al. [3] reported that methyl b-lactoside can
significantly inhibit murine melanoma B-16
We therefore postulated that a combina-
tion of a galactosyl or lactosyl group and a
trimannoside might lead to new anti-metasta-
sis compounds. For this purpose, a tetra-
* Corresponding author. Tel.: +86-10-62091504; fax: +
86-10-62015584.
E-mail address: zjli@mail.bjmu.edu.cn (Z.-J. Li).
saccharide a-
D
-Galp-(1“2)-a- -Manp-(1“2)
D
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00137-3

612
H. Li et al. / Carbohydrate Research 328 (2000) 611–615
-a-
D
-Manp-(1“6)-a-
D
-ManpOCH3 (8), and a
-Galp-(1“4)-b- -Glcp-
-Manp-(1“2)-a- -Manp-(1“6)-
version of the corresponding ethyl 1-thiogly-
pentasaccharide b-
D
D
coside 5 [8] reacting with bromine in
dichloromethane [9], was coupled to 4 by the
conventional Koenigs–Knorr method with
Hg(CN)2 as promoter to give in 54% yield of
an exclusive a-galacosylated tetrasaccharide 7,
which was then debenzylated by catalytic hy-
drogenolysis to afford the deblocked tetra-
saccharide 8. Compound 5 could be coupled
directly to glycosyl acceptor 4 using methyl
triflate as promoter to afford tetrasaccharide
7, but the yield was relatively low (20–30%).
The configuration of 8 was confirmed by the
(1“2)-a-
a-
D
D
D
-ManpOCH3 (13) have been synthesized,
using 1+3 and 2+3 coupling modes (see
Scheme 1).
2. Results and discussion
The trimannoside 3 was prepared using 2-
O-acetyl-3,4,6-tri-O-benzyl-a- -mannopyran-
D
osyl trichloroacetimidate (1) as glycosyl donor
and the dimannoside 2 [7] as acceptor. The
condensation was carried out in a solution of
dichloromethane with trimethylsilyl triflate
(Me3SiOTf) as promoter and the yield was
good (72.7%). Zemple´n deacetylation gave the
trisaccharide acceptor 4 in 83.7% yield.
3
galactosyl anomeric JH-1,2, value of 3.68 Hz.
To prepare pentasaccharide 11, hepta-O-
acetyllactosyl trichloroacetimidate 10 [9], ob-
tained by reaction of lactoseheptaacetate 9
and trichloroacetonitrile in the presence of
DBU as catalyst, was chosen as glycosyl
donor to couple with 4 in the presence of
Me3SiOTf as promoter. The resultant pen-
tasaccharide 11 was obtained in moderate
yield (45%). Deacetylation of 11 in NaOMe–
Me3OH gave compound 12 in 81.7% yield.
Subsequent catalytic hydrogenolysis gave the
deblocked pentasaccharide 13 in 82.6% yield.
1H NMR spectroscopy indicated that the non-
reducing terminal galactosyl and glucosyl
2,3,4,6-Tetra-O-benzyl-a- -galactopyranosyl
bromide (6), obtained readily by in situ con-
D
3
anomeric JH-1,2 values are 7.97 and 7.99 Hz,
respectively, and thus the b configuration of
the lactosylation was thus confirmed.
Compounds 8 and 13, along with several
other related oligosaccharides synthesized in
our laboratory are being tested for their anti-
metastasis activity, and the results will be re-
ported later.
3. Experimental
General methods.—Spectra were recorded
1
with the following instruments: H and 13C
1
NMR, Bruker ARX-400; the H NMR spec-
tra were recorded with Me4Si as the internal
standard and 13C NMR with CDCl3 as solvent
and internal standard; Mass spectra, VG
ZAB-Hs; IR, Perkin–Elmer 983. Elemental
analyses were performed on a Perkin–Elmer
240C instrument. Optical rotations were mea-
sured at 25 °C with a Optical Activity AA-
10R polarimeter. The progress of reactions
Scheme 1.

H. Li et al. / Carbohydrate Research 328 (2000) 611–615
613
the residue on silica gel, eluting with 4:1 cyclo-
hexane–acetone gave compound 4 (yield 1.08
g, 83.7%) as a colorless syrup. Rf 0.37 (4:1
cyclohexane–acetone); [h]D +19.6° (c, 0.12,
was monitored by thin-layer chromatography
(TLC) on Silica Gel GF254 (Hai Yang Chemi-
cal Factory, Qingdao, Shandong, PR China).
Detection was effected by examination under
UV light and by charring with 5% phospho-
molybdic acid hydrate in EtOH or 20% concd
H2SO4 in EtOH and heating. PTLC was per-
formed on Silica Gel GF254 and column chro-
matography on silica gel H (Hai Yang
Chemical Factory, Qingdao, Shandong, PR
China). The solvent systems indicated are vol-
ume volume ratios, and the petroleum ether
used in the experiment has the boiling range
of 60–90 °C.
1
CHCl3); IR: 3460 cm−1 (OH); H NMR: l
2.60 (br, 1 H, OH), 3.22 (s, 3 H, OCH3),
3.67–4.70 (m, 49 H, H-1, 2, 3, 4, 5, 6, H-1%, 2%,
3%, 4%, 5%, 6%, H-1¦, 2¦, 3¦, 4¦, 5¦, 6¦, 9×
PhCH2), 7.20–7.32 (m, 45 H, aromatic H);
13CNMR: l 54.61 (OCH3), 65.27–77.36 (C-2,
3, 4, 5, 6, C-2%, 3%, 4%, 5%, 6%, C-1¦, 2¦, 3¦, 4¦, 5¦,
6¦, 9×PhCH2), 98.82, 99.10, 101.13 (C-1, C-
1%, C-1¦), 127.27–128.41 (aromatic carbon),
138.09, 138.12, 138.25, 138.29, 138.47, 138.55
(2 C), 138.68, 138.72 (aromatic quaternary
carbon).
Methyl 2,3,4-tri-O-benzyl-6-O-[3,4,6-tri-O-
benzyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-h-
mannopyranosyl)-h- -mannopyranosyl]-h-
D
-
-
D
D
Methyl 2,3,4-tri-O-benzyl-6-O-{3,4,6-tri-O-
benzyl-2-O-[3,4,6-tri-O-benzyl-2-O-(2,3,4,6-
mannopyranoside (3).—2-O-Acetyl-3,4,6-tri-
-mannopyranosyl trichloroacet-
O-benzyl-a-
D
tetra - O - benzyl - h -
D
- galactopyranosyl) - h -
-mannopyranosyl}-h-
D
-
-
imidate (1) (1.04 g, 1.63 mmol) and methyl
2,3,4-tri-O-benzyl-6-O-(3,4,6-tri-O-benzyl-2-
mannopyranosyl]-h-
D
D
mannopyranoside (7).—To a solution of com-
pound 4 (550 mg, 0.40 mmol) in 25 mL of dry
O - a -
D
- mannopyranosyl) - a -
D
- mannopy-
,
ranoside (2) (1.32 g, 1.44 mmol) were dis-
CH2Cl2, Hg(CN)2 (0.33 g) and powdered 4A
solved in dry CH2Cl2 (25 ml). To the solution
was added powdered 4A molecular sieve (1.0
molecular sieve (1.0 g) were added and the
mixture was stirred for 1 h at rt. Meanwhile,
to a solution of compound 5 (780 mg, 1.33
mmol) in 15 mL of dry CH2Cl2 was added
liquid bromine dropwise (0.25 mL) with stir-
ring for 45 min at rt. The solvent was evapo-
rated in vacuo and toluene (2×25 mL) was
co-distilled from the residue. The resulting
galactosyl bromide 6 was dissolved in dry
CH2Cl2 (10 mL) and the solution added drop-
wise to the aforementioned mixture. After stir-
ring for 24 h at rt, the mixture was filtered and
the filtrate was washed successively with satd
brine, satd NaHCO3 solution, and water, re-
spectively, and dried (MgSO4). Evaporation of
the solvent in vacuo gave a yellowish residue,
which was subjected to column chromatogra-
phy on silica gel eluting with 5:1 petroleum
ether–EtOAc to give tetrasaccharide 7 (yield
420 mg, 54.0%) as a colorless syrup; Rf 0.33
(5:1 petroleum ether–EtOAc); [h]D +37.1° (c
,
g) and the mixture was stirred at room tem-
perature (rt) for 1 h and then cooled to
−10 °C for another 20 min. Trimethylsilyl
triflate (ten drops) was added and the mixture
was stirred for 5 h, during which time the
mixture attained to rt. The mixture was
filtered through a layer of Celite and rinsed
with CH2Cl2. The combined filtrate was
washed with water, satd aq NaHCO3, and
water, and then dried (MgSO4). The residue
obtained by evaporation of the solvent was
subjected to silica gel column chromatography
eluting with 6:1 petroleum—acetone to give
compound 3 (yield 1.47 g, 72.7%) as a color-
less syrup. Rf 0.50 (4:1 cyclohexane–acetone);
[h]D +10.5° (c 0.39, CHCl3). Anal. Calcd. for
C84H90O17: C, 73.58; H, 6.57. Found: C, 73.37;
H, 6.28. IR: 1732 cm−1 (CꢀO).
Methyl 2,3,4-tri-O-benzyl-6-O-[3,4,6-tri-O-
1
benzyl-2-O-(3,4,6-tri-O-benzyl-h-
ranosyl) - h - - mannopyranosyl] - h -
D
-mannopy-
- manno-
0.35, CHCl3). H NMR: l 3.17 (s, 3 H,
D
D
OCH3), 3.49–5.20 (54 H, sugar ring H and
13×PhCH2), 7.17–7.45 (65 H, aromatic H).
13C NMR: l 53.48 (OCH3), 60.42–80.50 (C-2,
3, 4, 5, 6, C-2%, 3%, 4%, 5%, 6¦, C-2¦, 3¦, 4¦, 5¦, 6¦,
C-2§, 3§, 4§, 5§, 6§, 13×PhCH2), 97.96,
98.91, 99.05, 101.12 (C-1, C-1%, C-1¦, C-1§),
pyranoside (4).—To a solution of compound 3
(1.33 g, 0.95 mmol) in abs MeOH (30 ml),
metal sodium (30 mg) was added. The solu-
tion was stirred at rt overnight, decationized
and evaporated. Column chromatography of

614
H. Li et al. / Carbohydrate Research 328 (2000) 611–615
h -
benzyl-h-
O-benzyl-h-
D
- mannopyranosyl) - (1“2) - (3,4,6 - tri - O-
-mannopyranosyl)-(1“6)-2,3,4-tri-
-mannopyranoside (11).—A mix-
127.00–128.53 (aromatic carbon), 138.01,
138.25, 138.36, 138.53, 138.62, 138.70, 138.73,
138.79, 138.85, 138.90, 138.95, 139.17, 139.21
(13C, aromatic quaternary carbon).
D
D
ture of compound 10 (270 mg, 0.35 mmol),
compound 4 (290 mg, 0.22 mmol) and pow-
Methyl
6-O-[2-O-(2-O-h-
D
-galactopyran-
,
ered 4 A molecular sieve (1 g) in dry CH2Cl2
osyl-h-
D
-mannopyranosyl)-h-
-mannopyranoside (8).—Compound
D
-mannopyran-
(20 mL) was stirred for 2 h at rt and then
cooled to −15 °C. Trimethylsilyl triflate (10
drops) was added and the mixture was stirred
for 5 h. The mixture was filtered through a
layer of Celite, rinsed with CH2Cl2, and then
dried (MgSO4). The residue resulting from
evaporation of the solvent was subjected to
silica gel column chromatography eluting with
3:1 cyclohexane–acetone to give compound 11
(yield 190 mg, 45%) as a colorless syrup; [h]D
+11.5° (c 0.17, CHCl3); IR: 1748 cm−1
(CꢀO). 1H NMR: l 1.97, 2.04, 2.06, 2.08, 2.10,
2.15, 2.17 (7 s, 21 H, 7×CH3CO), 3.23 (s, 3
H, OCH3), 3.68–5.22 (m, 53 H, sugar ring H,
9×PhCH2), 7.20–7.30 (45 H, aromatic H).
13C NMR: l 20.49, 20.62 (2 C), 20.71, 20.76,
20.80, 20.85 (7×CH3CO), 54.65 (OCH3),
60.95–77.40 (39 C, sugar ring carbon, 9×
PhCH2), 98.90, 99.0 (2 C) (C-1, C-1%, C-1¦),
99.35, 101.14 (C-1§, C-1¨), 127.41–128.88
(aromatic carbon), 138.04, 138.30, 138.33,
138.37(2 C), 138.49(2 C), 138.53, 138.62 (aro-
matic quaternary carbon), 168.99, 169.46,
169.85, 170.03, 170.15, 170.25, 170.36 (7 C,
CH3CO). Anal. Calcd. for C108H122O17: C,
66.60; H, 6.27. Found: C, 67.03; H, 6.20.
osyl]-h-
D
7 (320 mg, 0.17 mmol) was dissolved in 4:1
MeOH–EtOAc (65 mL) and the solution was
hydrogenlyzed with palladium on charcoal
(200 mg) in a hydrogen stream under a pres-
sure of 330 KPa for 24 h. The mixture was
filtered and the resulting solution was evapo-
rated to give compound 8 a colorless syrup,
which was then dissolved in distilled water
and lyophilized to a colorless foam (98 mg,
83.3%), FABMS: (m/z) 681 [M+1]+; [h]D
1
+54.0° (c 0.63, water). H NMR (400 MHz,
D2O): l 3.43 (s, 3 H, OCH3), 3.37–4.25 (m, 37
H, sugar ring H, 13×OH), 4.77 (d, 1 H, J1,2
1.48 Hz, mannosyl H-1), 5.17, 5.42 (2 s, 2 H,
mannosyl H-1), 5.18 (d, 1 H, J1,2 3.68 Hz,
galactosyl H-1); 13C NMR: l 57.33 (OCH3),
63.46, 63.60, 63.82 (mannosyl C-6), 68.35
(galactosyl C-6), 69.05, 69.47, 69.68, 71.37,
71.82 (2 C), 72.41, 72.74, 72.88, 73.80 (2 C),
73.93, 75.31, 75.66, 81.48, 82.09 (C-2, 3, 4, 5,
C-2%, 3%, 4%, 5%, C-2¦, 3¦, 4¦, 5¦, C-2§, 3§, 4§,
5§), 100.51 (galactosyl C-1), 103.20, 103.57,
103.74 (mannosyl C-1). Anal. Calcd.
C25H44O21: C, 44.05; H, 6.47. Found: C, 44.36;
H, 6.61.
2,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-
Methyl (i-
-glucopyranosyl)-(1“2)-(3,4,6-tri-O-benzyl-
h- -mannopyranosyl)-(1“2)-(3,4,6-tri-O-ben-
zyl-h- -mannopyranosyl)-(1“6)-(2,3,4-tri-O-
benzyl-h- -mannopyranoside) (12).—To a so-
D
-galactopyranosyl)-(1“4)-(i-
acetyl-i-
D
-galactopyranosyl)-
D
-glucopyrano-
D
syl trichloroacetimidate (10).—To a solution
of 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-ace-
D
D
tyl-b-D-galactopyranosyl)-D-glucopyranose [9]
D
(9, 1.13 g, 1.78 mmol) in dry CH2Cl2 (20 mL),
trichloroacetonitrile (1 mL) was added and the
mixture was stirred in an ice-water bath for 10
min. DBU (ten drops) was then added and the
mixture was stirred for 2 h during which time
the bath reached rt. Evaporation of the sol-
vent in vacuo gave a dark-brown oil, which
was purified by column chromatography elut-
ing with 1:1 petroleum ether–EtOAc to give
compound 10 (1.1 g, 79.7%), as a yellow
syrup. Rf 0.31 (1:1 petroleum ether–EtOAc).
lution of compound 11 (180 mg, 0.09 mmol)
in abs MeOH (20 mL) CH3ONa (10 mg) was
added and the mixture was stirred for 2 h at
rt. TLC showed that the reaction was com-
plete. The solution was decationized, evapo-
rated, and was purified by PTLC (3:1
CHCl3–MeOH) to afford compound 12 (yield
125 mg, 81.7%) as a colorless syrup. Rf 0.33
(3:1 CHCl3–MeOH).
Methyl (i-
-glucopyranosyl)-(1“2)-(h-
osyl) - (1“2) - (h - - mannopyranosyl) - (1“6)-
(h- -mannopyranoside) (13).—To a solution
of compound 12 (114 mg, 0.07 mmol) in
D
-galactopyranosyl)-(1“4)-(i-
D
D
-mannopyran-
Methyl (2,3,4,6-tetra-O-acetyl-i-
topyranosyl)-(1“4)-(2,3,6-tri-O-acetyl-i-
glucopyranosyl) - (1“2) - (3,4,6 - tri - O - benzyl-
D-galac-
D
D-
D

H. Li et al. / Carbohydrate Research 328 (2000) 611–615
615
MeOH (50 mL) was added Pd–C (10%) and
the mixture was hydrogenolyzed in a hydro-
gen stream under a pressure of 33 KPa for 24
h. The mixture was filtered and the catalyst
was rinsed with MeOH and a small amount of
distilled water. Evaporation and lyophilization
of the filtrate gave compound 13 (yield 48 mg,
82.6%) as a white amorphous solid; FABMS:
(m/z) 843 [M+1]+, 865 [M+Na]+; [h]D
for C31H56O26: C, 44.18; H, 6.65. Found: C,
43.96; H, 6.88.
Acknowledgements
The project was supported by National
Natural Sciences Foundation of China and a
grant from the Ministry of Science and Tech-
nology of PR China.
1
+35.6° (c 0.51, water). H NMR (400 MHz,
D2O): l 3.44 (s, 3 H, OCH3), 3.35–3.93 (m,
sugar ring H), 4.05 (dd, 1 H, mannosyl H-2),
4.27 (dd, 1 H, mannosyl H-2), 4.48 (d, 1 H,
J1,2 7.97 Hz, glucosyl H-1), 4.59 (d, 1 H, J1,2
7.99 Hz, galactosyl H-1), 4.76 (d, 1 H, J1,2 1.48
Hz, mannosyl H-1), 5.14 (d, 1 H, J1,2 1.44 Hz,
mannosyl H-1), 5.16 (d, 1 H, J1,2 1.28 Hz,
mannosyl H-1). 13C NMR: l 57.37 (OCH3),
62.58, 63.10, 63.48, 63.52, 68.29 (C-6, C-6%,
C-6¦, C-6§, C-6¨), 69.09, 69.46, 71.13 (2 C),
72.08, 72.45, 72.75, 73.21, 73.32, 73.53, 74.98,
75.09, 75.36, 75.73, 76.66, 77.34, 77.95 (2 C),
79. 86, 80.82, 81.38 (C-2, 3, 4, 5, C-2%, 3%, 4%, 5%,
C-2¦, 3¦, 4¦, 5¦, C-2§, 3§, 4§, 5§, C-2¨, 3¨,
4¨, 5¨), 100.50, 102.87, 103.62, 104.01, 105.54
(C-1, C-1%, C-1¦, C-1§, C-1¨). Anal. Calcd.
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