Novel disaccharide inhibitors of human glioma cell division

Article abstract of DOI:10.1021/jm980365i

Several α-L-Fuc-(1→3)-α-D-GlcNAcOC₈H₁₇ disaccharide derivatives bearing different hydroxylated alkyl chains, with or without sulfate groups at C-4 and/or C-6 positions of the GlcNAc unit, have been synthesized and tested as inhibitor

Full text of DOI:10.1021/jm980365i

J . Med. Chem. 1998, 41, 4599-4606
4599
Novel Disa cch a r id e In h ibitor s of Hu m a n Gliom a Cell Division
Begon˜a Aguilera,† Lorenzo Romero-Ram´ırez,‡ J ose´ Abad-Rodr´ıguez,‡ Guillermo Corrales,†
Manuel Nieto-Sampedro,*,‡ and Alfonso Ferna´ndez-Mayoralas*,†
Instituto de Quı´mica Orga´nica General, CSIC, J uan de la Cierva 3, 28006 Madrid, Spain, and Neural Plasticity Group,
Instituto Cajal, CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain
Received J une 15, 1998
Several R-L-Fuc-(1f3)-R-D-GlcNAcOC8H17 disaccharide derivatives bearing different hydrox-
ylated alkyl chains, with or without sulfate groups at C-4 and/or C-6 positions of the GlcNAc
unit, have been synthesized and tested as inhibitors of human astrocytoma lines U-373 and
U-118. The antimitotic activity was dependent on the structure and position of the hydroxylated
chain linked to the disaccharide. The compounds with a pentaerythritol or L-glyceryl chain at
the C-6 position showed the best inhibitory properties, with an ID50 value of ca. 200 µM. On
the contrary, sulfated disaccharide derivatives were inactive. The antimitotic activities of the
compounds tested were essentially independent of the mitogen used to stimulate cell division.
Ch a r t 1
In tr od u ction
Astrocyte number is maintained constant in the
mammalian central nervous system (CNS),1 during
adulthood and old age under normal circumstances, as
a result of the balance of division promoters and division
inhibitors. Mitogenic polypeptides are abundant in
brain, and their structure has been known for many
years. In contrast, evidence for the existence in mam-
malian brain of antimitotic molecules was presented
only 10 years ago;2 their purification has been achieved
only recently,3 and their precise structure remains
unknown. The mitogen inhibitors are immunologically
related to blood group oligosaccharides and to some
glycidic epitopes of the epidermal growth factor recep-
tor.2,4 On the basis of these data, we synthesized a
family of oligosaccharides with a common Lewis X-type
structure and tested their activity as inhibitors of the
proliferation of normal and transformed neural cells.5-8
The tetrasaccharide R-D-GalNac(1f3)-â-D-Gal-(1f4)-
[R-L-Fuc-(1f3)]-â-D-GlcOMe inhibited the proliferation
of astrocytes and astrocytoma in culture, showing ID50
(50% inhibition) values in the micromolar range.6 As-
trocytes and neurons were viable at tetrasaccharide
concentrations 10-fold higher than the ID50, indicating
that growth inhibition did not involve cytotoxicity. The
effect of the tetrasaccharide may be related to the
increased expression of connexin 43 observed in glioma
cell cultures treated with the oligosaccharide.7 How-
ever, the tetrasaccharide caused not only the inhibition
but also the destruction of a malignant rat brain glioma
after transplantation of C6 cells. This effect appeared
to be caused indirectly, either by activation of natural
killers cells, cytotoxic lymphocytes, or by inhibition of
tumor vascularization.7 From results obtained with the
oligosaccharides tested in culture, some structure-
activity relationships could be drawn: (i) an R-fucosyl
residue linked to the C-3 position of a glucose moiety
was important for the antimitotic activity and (ii) a
negatively charged group at the nonreducing end of the
oligosaccharide seemed beneficial for the inhibitory
activity against tumoral cells. Thus, a sulfated trisac-
charide (compound 1 in Chart 1) showed higher activity
than the nonsulfated trisaccharide (ID50 ) 79 and 4300
µM, respectively).8 A more flexible and straightforward
synthesis of these molecules is required, both to get a
deeper insight into their structure-activity relationship
and to facilitate the large-scale preparation required for
their possible clinical use.
The practical syntheses of a second generation of
inhibitory compounds was carried out based on previous
data. The compounds were tested as inhibitors of
human astrocytoma grades 3 and 4. The new oligosac-
charides have as common novel structural feature: a
disaccharide fragment with the beneficial R-fucosyl
residue linked to the C-3 position of the N-acetylglu-
cosamine (structure I in Chart 1). The disaccharide was
glycosylated with n-octanol giving the molecule an
amphiphilic glycolipid character.9 The different prod-
ucts originated from a variable hydroxylated alkyl
chain, intended to mimic a glycoside unit linked to the
C-4 and/or C-6 positions of the N-acetylglucosamine.
Some derivatives with a sulfate group at the end of the
hydroxylated chain will be useful for learning about the
importance of a negatively charged group for antimitotic
* To whom correspondence should be addressed.
† Instituto de Qu´ımica Orga´nica General.
‡ Instituto Cajal.
10.1021/jm980365i CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/10/1998

4600 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Aguilera et al.
Sch em e 1a
3). Thus, treatment of diol 6 with NaH followed by
reaction with 1.5 equiv of cyclic sulfate 9 at 60 °C gave
a mixture of mono- and dialkylated compounds, 10-
12. After column chromatography 10, 11, and 12 were
isolated in 30%, 21%, and 30% yields, respectively.
Separated hydrogenolysis of 10-12 provided target
sulfates 13, 15, and 17. The nonsulfated compounds
4P (14), 6P (16), and 4,6P (18) were obtained by
treatment of 13, 15, and 17, respectively, with sulfuric
acid in dioxane.
The structures of 13-18 were unequivocally deter-
mined by their NMR spectra. Thus, the number of
1
protons and carbons in the H and 13C NMR spectra of
13-18 was in agreement with the presence of one or
two pentaerythritol chains. In the case of monoalkyla-
ted derivatives 13-16, the position of the chain was
determined by analyzing the 13C NMR spectra on the
basis of the downfield R-effect caused by alkylation.11
The assignment of the signals was previously carried
out using DEPT, HMQC, and HMBC experiments. The
C-6 carbon is 7.0-8.0 ppm more deshielded in 15 and
16 than in 13 and 14, indicating that the former
compounds are bearing the hydroxylated alkyl chain at
the C-6 position. Finally, the presence of the sulfate
groups was characterized by a sharp band at 1200 cm-1
in the IR spectra of compounds 13, 15, and 17.
a
Reagents and conditions: (a) octanol, BF3‚Et2O, CH3CN,
reflux, 35%; (b) R,R′-dimethoxytoluene, p-TsOH, CH3CN, rt, 73%;
(c) 1. Br2, CH2Cl2, 0 ˚C, 2. Bu4NBr, DMF, rt, 87%; (d) camphor-
sulfonic acid, MeOH, 50 ˚C, 74%; (e) H2, Pd/C, MeOH, rt, 97%.
Sch em e 2a
We next synthesized another series of compounds
containing a linear hydroxylated chain derived from
glycerol (G series). Partial alkylations on diol 6 were
carried out using commercially available tosylates 19
and 20 derived from D- and L-glycerol (Scheme 4). The
reaction of 6 with 1.5 equiv of tosylate 19 in the presence
of NaH gave a mixture of 21, 23, and 25 in 52%, 22%,
and 16% yields. Under similar conditions the L-glyceryl
tosylate 20 afforded a mixture of 22, 24, and 26 in 56%,
24%, and 14% yields. Hydrogenation of 21-26 sepa-
rately in the presence of trifluoroacetic acid led to the
cleavage of benzyl and isopropylidene groups providing
target products 27-32. The structure of 27-32 was
a
Reagents and conditions: (a) p-anisaldehyde, HCl, H2O, rt,
84%; (b) NaH, 1,1′-sulfonyldiimidazole, THF, rt, 81%.
activity. In this paper we report the synthesis of the
novel inhibitors and their antimitotic activity against
human astrocytoma cells U-373 and U-118.
Resu lts a n d Discu ssion
1
again determined from their H and 13C NMR spectra.
Ch em istr y. To minimize protecting group manipu-
lations, the target compounds were obtained by carrying
out partial alkylations on a common diol intermediate,
6. The synthesis of the disaccharide derivative 6 started
from inexpensive N-acetyl-D-glucosamine (2) (Scheme
1). Fischer-type glycosidation of 2 with n-octanol gave
the R-glycoside 3, which was subsequently benzylide-
nated to afford alcohol 4. Glycosidation10 of 4 with
freshly prepared tri-O-benzyl-R-L-fucopyranosyl bromide
gave protected disaccharide 5. The R-configuration of
the newly formed glycosidic bond was determined from
It is interesting to note that in both cases a selective
alkylation on the secondary hydroxyl HO-4 over the
primary HO-6 took place. A similar preference, al-
though to a lesser extent, was also observed in the
above-described alkylation using the cyclic sulfate.
Schmidt has reported12 a selective allylation on the
secondary C-3 position in the presence of two primary
hydroxyls of an azidolactose derivative using allyl
bromide and NaH. The surprising result was explained
by intramolecular complexation of the sodium ion from
the O-3 alkoxide. Similarly, in diol 6 a preferential
formation of O-4 alkoxide could take place due to Na+
chelation with O-3 and the ring oxygen atom of the
fucose moiety (Chart 2). As evidence to support this
hypothesis, the alkylation of diol lacking the fucose
residue (33) with tosylate 20 led to a complete reverse
of the regioselectivity, with a predominant formation of
the 6-O-alkylated derivative.
1
the H NMR spectrum of 5 by a doublet signal of J )
3.3 Hz at 5.23 ppm assigned to the H-1 of the R-fucosyl
residue. Treatment of 5 with camphorsulfonic acid in
MeOH gave the desired intermediate 6. Hydrogenolysis
of 6 afforded the unsubstituted disaccharide 7.
From intermediate 6 we first prepared a series of
compounds containing a sulfated pentaerythritol chain
(P series). A straightforward synthesis of sulfates 4PSu
(13), 6PSu (15), and 4,6PSu (17) (Scheme 3) was per-
formed by using the cyclic sulfate derivative 9 as
alkylating agent, easily prepared from pentaerythritol
(Scheme 2). The nucleophilic ring opening of 9 allowed
to introduce the pentaerythritol chain generating, at the
same time, the negatively charged sulfate group (Scheme
For comparative purposes the 6-O-galactosyl trisac-
charide derivative 36 was prepared (Scheme 5). From
intermediate 6 a highly regio- and stereoselective ga-
lactosylation was achieved using the trichloroacetimi-
date 34 in the presence of TMSOTf as promoter. The
6-O-â-glycosylated product 35 was obtained in 82%

Disaccharide Inhibitors of Glioma Cell Division
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4601
Sch em e 3a
a
Reagents and conditions: (a) NaH, 9, THF-DMF, 60 °C; (b) H2, Pd/C, rt, 13 (96%), 15 (96%), 17 (quant); (c) H2SO4 (1 M), dioxane-
MeOH, rt, 14 (64%), 16 (71%), 18 (38%).
yield, which was transformed, by removal of the benzoyl
and benzyl groups, into the desired product 36.
activity was observed in 6GD (29), substituted at C-6
with a D-glyceryl chain, with respect to its diastereo-
isomer 6GL(30). As in the pentaerythritol series, wherea
second alkyl chain at C-4 of 6P (16) led to the less
inhibitory compound 4,6P (18), the disubstituted deriva-
tive 4,6GL (32) showed a decrease of activity as com-
pared to the monosubstituted disaccharide 6GL (30).
Biologica l Activity. The antimitotic activity of
compounds containing a pentaerythritol chain (13-18,
P series) as well as that of the nonsubstituted disac-
charide 7 was tested on rat (C6) and human glioma (U-
373 and U-118) cell cultures, stimulated with different
mitogens. The results are summarized in Table 1. In
contrast to our previous results using the sulfated
trisaccharide 1 (Chart 1) on rat C6 glioma,8 sulfated
compounds 4PSu (13), 6PSu (15), and 4,6PSu (17) were
inactive in all the cell types. However, the nonsulfated
derivatives 4P (14), 6P (16), and 4,6P (18) showed
moderate inhibitory activity on thymidine incorporation
promoted by EGF, the ID50 value of which depended on
the position of the pentaerythritol chain. The substitu-
tion at the C-4 position (compound 4P, 14) led to a slight
increase in activity compared with the nonsubstituted
disaccharide 7, while 6P (16), containing the pen-
taerythritol chain at C-6, showed the best antimitotic
activity of this family.
Taken together, the results indicate that a hydrox-
ylated alkyl chain at the C-6 position of the glucosamine
enhanced the inhibitory activity, although the effect of
the substitution was dependent on the relative orienta-
tion of the hydroxyl groups. Thus, a small modification
such as the chirality of the glyceryl chain in 6GL (30)
or 6GD (29) produced a change in the activity. Steric
factors could also influence the inhibitory properties of
these compounds since a second alkyl chain at C-4 gave
less active inhibitors. Besides, the trisaccharide deriva-
tive 36 with a bulky galactosyl residue at C-6 was
inactive against U-373 glioma cells (Table 2).
Human glioma U-373 cells produced molecules which
would allow them to proliferate, when cultured in the
absence of mitogens (DMEM medium). Several authors
have reported the expression of growth factor13 and
cytokine13,14 receptors by U-373 cells, although the
astrocytoma proliferated much better in the presence
of exogenous growth factors, such as EGF and bFGF.
6GL (30) and 6GD (29) were the most active molecules
of the two series studied here, with small differences
depending on whether division was driven by EGF,
bFGF, or autocrine (DMEM only). Therefore, antimi-
totic activity was substantially independent of the
All the compounds of the G series, substituted with a
glyceryl chain, showed ID50 values below 1 mM (Table
2) improving the inhibitory activity with respect to the
P series. Some appreciable differences were observed
depending on both the position and the configuration
of the glyceryl chain. The 6-L-glyceryl derivative 6GL
(30) was the best inhibitor with ID50 values ranging
between 105 and 234 µM. Its 4-L-glyceryl regioisomer
4GL (28) showed a 2-4-fold higher ID50 value depending
on the mitogen used. A similar, though smaller, loss of

4602 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Aguilera et al.
Sch em e 4a
a
Reagents and conditions: (a) NaH, 19 or 20, DMF, rt; (b) H2, Pd/C, TFA, rt, 27 (quant), 28 (quant), 29 (97%), 30 (96%), 31 (quant),
32 (98%).
Ta ble 1. Inhibition of Human (U-373 and U-118) and Rat
(C6) Glioma Cell Cultures by Compounds of the Pentaerythritol
(P) Series
Ch a r t 2
ID50 (mM)
U-373
U-373
EGFa
U-118
C6
compd
1% FCSa
1% FCSa
10% FCSa
7
>1000
>1000
>1000
>1000
>1000
202
>1000
>1000
>1000
>1000
710
993
>1000
>1000
>1000
785
756
ndb
4PSu (13)
6PSu (15)
4,6PSu (17)
4P (14)
ndb
Sch em e 5a
ndb
>1000
6P (16)
210
425
ndb
4,6P (18)
>1000
433
>1000
>1000
a
Cell cultures were stimulated with different mitogens: FCS,
fetal bovine serum; EFG, epidermal growth factor. For details of
b
the assay, see the Experimental Section. nd, not detectable.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. Melting points are not
corrected. TLC was performed using TLC plates GF254 with
detection by charring with 5% H2SO4 in EtOH. Column
chromatography was performed on silica gel (230-400 mesh).
The eluent used is indicated, and solvent ratios refer to
volume. Solvents were distilled over drying agents: dimeth-
ylformamide, BaO; dichloromethane, CaH2; tetrahydrofuran,
sodium/benzophenone ketyl; acetonitrile, CaH2; and pyridine,
BaO. 1H NMR spectra were registered at 500, 400, 300, or
200 MHz. 13C NMR spectra were obtained at 125, 75, or 50
MHz. Elemental analyses were determined in a Perkin-Elmer
240 analyzer.
Octyl 2-Aceta m id o-2-d eoxy-r-D-glu cop yr a n osid e (3).
To a suspension of N-acetyl-D-glucosamine (4.42 g, 20 mmol)
and octanol (9.45 mL, 60 mmol) in dry acetonitrile (160 mL)
was added under Ar boron trifluoride diethyl etherate (406
µL, 3.3 mmol), and the reaction mixture was stirred under
reflux for 18 h. After cooling, the unreacted starting material
a
Reagents and conditions: (a) TMSOTf, CH2Cl2, rt, 82%; (b) 1.
NaOMe, MeOH, rt, 2. H2, Pd/C, rt, 93%.
mitogen, and the inhibitor must be acting at a common
point downstream from the different kinases induced
by the various mitogens. This is advantageous for the
possible therapeutic use of these molecules because,
under physiological conditions, tumor growth is pro-
moted by many different mitogens. New compounds,
based on the structures and synthetic strategies pre-
sented here, will provide valuable information on struc-
ture-function relationships and permit the production
of compounds with very low ID50 values.

Disaccharide Inhibitors of Glioma Cell Division
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4603
(c 0.6, MeOH). 1H NMR (300 MHz, CD3OD): δ 5.16 (d, 1H, J
) 2.4 Hz), 4.97 (d, 1H, J ) 3.6 Hz), 4.45 (m, 1H), 4.24 (dd, 1H,
J ) 3.6 Hz, J ) 10.5 Hz), 4.03-3.60 (m, 10H), 2.16 (s, 3H),
1.81 (m, 2H), 1.53 (m, 10H), 1.38 (d, 1H, J ) 6.6 Hz), 1.09 (m,
3H). 13C NMR (50 MHz, CD3OD): δ 174.03, 101.69, 98.54,
81.57, 73.82, 73.61, 71.64, 70.82, 70.12, 69.01, 68.45, 62.60,
54.56, 32.98, 30.51, 30.39, 27.27, 23.67, 22.77, 16.56, 14.37.
Anal. (C22H41NO10) C, H, N.
Mon o-p-m eth oxyben zilid en e-p en ta er yth r itol (8). In a
flask were placed pentaerythritol (10 g, 73.4 mmol) and water
(72 mL). The mixture in the flask was heated with stirring
until all the solid was dissolved and then was allowed to cool
undisturbed. When the solution had cooled to room temper-
ature, stirring was started and concentrated hydrochloric acid
(370 µL) was added, followed by p-anisaldehyde (1 mL, 8.2
mmol). When the precipitate of mono-p-methoxybenzilidene-
pentaerythritol started forming, dropwise addition of p-anis-
aldehide (8.32 mL, 68.4 mmol) was begun. After the addition
of p-anisaldehide was complete (ca 2 h), the mixture was
stirred for an additional 3 h. The precipitate was collected on
a Bu¨chner funnel and washed with ice-cold water which had
been made slightly alkaline by addition of sodium carbonate.
The solid was dried over phosphorus pentoxide to give 8 as a
white solid (15.7 g, 84%). Rf: 0.47 (CH2Cl2-MeOH, 10:1).
Mp: 163-166 °C. Anal. (C13H18O5) C, H.
3,3-Dioxo-9-(p -m e t h oxyp h e n yl)-2,4,8,10-t e t r a oxo-3-
th ia sp ir o[5.5]d eca n e (9). To a suspension of 8 (2 g, 7.84
mmol) in dry tetrahydrofuran (40 mL) was added sodium
hydride (423 mg, 17.6 mmol) and 1,1′-sulfonyldiimidazole (2.3
g, 11.7 mmol) dissolved in dry tetrahydrofuran (30 mL). The
reaction mixture was stirred at room temperature for 2 h.
Then, methanol (1 mL) was added, and the mixture was
evaporated to dryness. The residue was purified by column
chromatography (hexane-CH2Cl2, 5:1) to give 9 as a solid (2
g, 81%). Rf: 0.77 (CH2Cl2-MeOH 10:1). Mp: 181-184 °C.
IR (KBr): 1410, 1210 cm-1. Anal. (C13H16O7S) C, H, S.
Alk yla tion Rea ction of Diol 6 w ith Cyclic Su lfa te 9.
To a solution of 6 (1 g, 1.33 mmol) in a mixture of dry
tetrahydrofuran-dimethylformamide (7:1, 48 mL) were added
sodium hydride (128 mg, 5.33 mmol) and then 9 (634 mg, 2
mmol), and the reaction mixture was heated at 60 °C for 1.5
h. After cooling, methanol (5 mL) was added and the mixture
was evaporated to dryness. The residue was purified by
column chromatography (CH2Cl2-MeOH, 20:1) to give 10 (386
mg, 30%), 11 (283 mg, 21%), and 12 (570 mg, 30%). 10: Rf
0.28 (CH2Cl2-MeOH, 8:1). 11: Rf 0.57 (CH2Cl2-MeOH, 8:1).
12: Rf 0.16 (CH2Cl2-MeOH, 8:1).
Ta ble 2. Inhibition of Human U-373 Glioma Cell Cultures by
Compounds of the Glycerol (G) Series
ID50 (mM), U-373
compd
DMEMa
EGFa
bFGFa
4GD (27)
4GL (28)
6GD (29)
6GL (30)
4,6GD (31)
4,6GL (32)
36
>1000
430
788
490
291
205
500
604
710
443
234
529
195
105
900
363
500
363
>1000
>1000
>1000
a
Human U-373 glioma cell cultures were stimulated with the
appropriate mitogen and treated with the different molecules at
various concentrations as indicated in the Experimental Section:
DMEM, Dulbecco’s modified Eagle’s medium; EGF, epidermal
growth factor; bFGF, basic fibroblast growth factor.
was filtered off, and the filtrate was evaporated to dryness.
The residue was purified by column chromatography (CH2Cl2-
MeOH 20:1f6:1) to give 3 as a solid (2.34 g, 35%). Rf: 0.54
(CH2Cl2-MeOH, 10:1). Mp: 157-159 °C. [R]D: +129.5° (c 1,
MeOH). Anal. (C16H31NO6) C, H, N.
Octyl 2-Aceta m id o-4,6-O-ben zylid en e-2-d eoxy-r-D-glu -
cop yr a n osid e (4). To a suspension of 3 (2.34 g, 7.02 mmol)
in dry acetonitrile (50 mL) under Ar were added p-toluene-
sulfonic acid (66 mg, 0.34 mmol) and then R,R′-dimethoxytolu-
ene (5.2 mL, 35.09 mmol), and the reaction mixture was stirred
at room temperature for 3 h. After this time, the mixture was
diluted with dichloromethane, neutralized with triethylamine
and concentrated. Purification of the residue by column
chromatography (CH2Cl2-MeOH, 40:1f20:1) gave 4 as a solid
(2.17 g, 73%). Rf: 0.61 (CH2Cl2-MeOH, 10:1). Mp: 187-189
°C. [R]D: +49.7° (c 1, CHCl3). Anal. (C23H35NO6) C, H, N.
Octyl O-(2,3,4-Tr i-O-ben zyl-r-L-fu cop yr a n osyl)-(1f3)-
2-a cet a m id o-4,6-O-b en zylid en e-2-d eoxy-r-D-glu cop yr a -
n osid e (5). Ethyl 2,3,4-tri-O-benzyl-1-thio-â-L-fucopyrano-
side18 (2.82 g, 5.89 mmol) was dissolved in dry dichloromethane
(45 mL) under argon. At 0 °C, bromine (0.31 mL, 6 mmol)
was added, and the mixture was stirred for 30 min. The
mixture was concentrated in vacuo, and residual bromine was
removed by evaporation with toluene (3 × 20 mL). The crude
bromide was used without purification in the next step.
Acceptor 4 (1.64 g, 3.91 mmol) was dissolved in dry DMF
(36 mL), and 4 Å molecular sieves (5 g) and Bu4NBr (1.28 g,
3.95 mmol) were added. The mixture was kept under argon
atmosphere, and the crude bromide, obtained as shown before
(ca 5.89 mmol), dissolved in dichloromethane (16 mL) was
added. After stirring for 18 h, the reaction was quenched with
ethanol. After 30 min, the mixture was diluted with dichlo-
romethane (300 mL) and filtered through a pad of Celite. The
filtrate was washed with saturated aqueous NaHCO3 (200 mL)
and brine (200 mL), dried (Na2SO4), and concentrated in vacuo.
The residue was purified by column chromatography (hexane-
acetate, 4:1) to give 5 as a solid (2.86 g, 87%). Rf: 0.34
(hexane-acetone, 2:1). Mp: 93-96 °C. [R]D: -28.7° (c 1,
CHCl3). Anal. (C50H63NO10) C, H, N.
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-4-O-[2,2-bis(h yd r oxym eth yl)-3-h yd r oxy-3-O-(oxo-
su lfon yl)p r op yl]-r-D-glu cop yr a n osid e (4P Su (13)). To a
solution of 10 (200 mg, 0.184 mmol) in methanol (15 mL) was
added 10% Pd/C (100 mg), and the reaction mixture was
stirred under H2 atmosphere for 5 h. After this time, the
mixture was filtered through Celite and evaporated to dryness
to give 13 as a solid (124 mg, 96%). Rf: 0.10 (CH2Cl2-MeOH,
3:1). Mp: 149-153 °C. [R]D: +12.4° (c 0.7, MeOH). 1H NMR
(300 MHz, CD3OD): δ 5.34 (d, 1H, J ) 2.7 Hz), 5.16 (d, 1H, J
) 3.4 Hz), 4.37 (m, 1H), 4.27-3.59 (m, 19H), 2.18 (s, 3H), 1.75
(m, 2H), 1.50 (m, 10H), 1.44 (d, 3H, J ) 6.5 Hz), 1.09 (m, 3H).
13C NMR (50 MHz, CD3OD): δ 173.47 (CO), 101.87 (C-1 Fuc),
97.76 (C-1 GlcNAc), 80.04, 78.61, 73.49, 73.29, 72.17 (C-1′),
71.06, 70.86, 69.25, 69.01 (CH2 (octyl)), 67.88 (C-3′), 62.39 (CH2-
OH), 62.27 (CH2OH), 61.90 (C-6), 55.77 (C-2 GlcNAc), 46.98
(C-2′), 32.98 (CH2), 30.43 (CH2), 27.23 (CH2), 23.68 (CH2), 22.79
Octyl O-(2,3,4-Tr i-O-ben zyl-r-L-fu cop yr a n osyl)-(1f3)-
2-a ceta m id o-2-d eoxy-r-D-glu cop yr a n osid e (6). A mixture
of 5 (2.61 g, 3.11 mmol) and camphorsulfonic acid (180 mg,
0.77 mmol) in methanol (60 mL) was stirred for 90 min at 50
°C. After cooling, the mixture was neutralized with triethy-
lamine and concentrated. The residue was purified by column
chromatography(hexane-AcOEt (1:1)fAcOEt) to give 6 as a
solid (1.73 g, 74%). Rf: 0.11 (hexane-acetone, 2:1). Mp: 156-
159 °C. [R]D: +9.4° (c 1, CHCl3). Anal. (C43H59NO10) C, H,
N.
(NHAc), 16.70, 14.41. IR (KBr): 1240, 1080 cm-1
Oct yl O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
.
d eoxy-6-O-[2,2-bis(h yd r oxym eth yl)-3-h yd r oxy-3-O-(oxo-
su lfon yl)p r op yl]-r-D-glu cop yr a n osid e (6P Su (15)). 10 (200
mg, 0.184 mmol) was reacted by the procedure used to
synthesize compound 13 and afforded the title compound as a
solid (124 mg, 96%). Rf: 0.10 (CH2Cl2-MeOH, 3:1). Mp:
121-124 °C. [R]435Hg: +3.0° (c 0.5, MeOH). 1H NMR (300
MHz, CD3OD): δ 5.16 (s, 1H), 4.93 (d, 1H, J ) 3.6 Hz), 4.50
(m, 1H), 4.30-3.54 (m, 19H), 2.17 (s, 3H), 1.81 (m, 2H), 1.58
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-r-D-glu cop yr a n osid e (7). To a solution of 6 (150.9
mg, 0.2 mmol) in methanol (15 mL) was added Pd/C 10% (100
mg), and the mixture was stirred under H2 atmosphere for 5
h. After this time, the mixture was filtered through Celite
and evaporated to dryness to give 7 as a solid (93.6 mg, 97.4%).
Rf: 0.65 (CH2Cl2-MeOH, 3:1). Mp: 214-217 °C. [R]D: +4.4°

4604 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Aguilera et al.
(m, 10H), 1.38 (d, 3H, J ) 6.6 Hz), 1.09 (m, 3H). 13C NMR (50
MHz, CD3OD): δ 173.97 (CO), 101.42 (C-1 Fuc), 98.69 (C-1
GlcNAc), 81.07, 73.67, 72.97, 72.14 (C-1′), 71.73, 71.68 (C-6),
70.73, 70.17, 69.23 (OCH2 (octyl)), 68.35, 68.21 (C-3′), 62.99
(CH2OH), 62.92 (CH2OH), 54.65 (C-2), 46.66 (C-2′), 32.92
(CH2), 30.50 (CH2), 30.47 (CH2), 30.32 (CH2), 27.27 (CH2), 23.61
23.69 (CH2), 22.74 (NHAc), 16.70 (CH3-Fuc), 14.43 (CH3).
Anal. (C32H61NO16‚H2O) C, H, N.
Alk yla tion Rea ction of Diol 6 w ith Tosyla te 19. To a
solution of 6 (330 mg, 0.44 mmol) in dry dimethylformamide
(4.8 mL) at room temperature under Ar was added sodium
hydride (27 mg, 1.1 mmol), and the mixture was stirred for 5
min. Then, 19 (188 mg, 0.66 mmol) dissolved in dimethylfor-
mamide (2 mL) was added dropwise, and the reaction mixture
was heated at 40 °C for 8 h. After cooling, the reaction was
quenched with methanol (1 mL) and the solvent was evapo-
rated under reduced pressure. The residue was purified by
column chromatography (hexane-acetone, 5:1f1:1) to give 21
(194 mg, 52%), 23 (82 mg, 22%), and 25 (66 mg, 16%). 21: Rf
0.34 (hexane-acetone, 1:1). 23: Rf 0.51 (hexane-acetone, 1:1).
25: Rf 0.54 (hexane-acetone, 1:1).
(CH2), 22.82 (NHAc), 16.51, 14.30. IR (KBr): 1250, 1060 cm-1
Oct yl O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
.
d eoxy-4,6-bis-O-[2,2-bis(h yd r oxym eth yl)-3-h yd r oxy-3-O-
(oxosu lfon yl)p r op yl]-r-D-glu cop yr a n osid e (4,6P Su (17)).
12 (206 mg, 0.144 mmol) was reacted by the procedure used
to synthesize compound 13 and afforded the title compound
as a solid (133 mg, quantitative). Rf: 0.25 (CH2Cl2-MeOH,
3:2). Mp: 204-208 °C. [R]D: +12.3° (c 0.5, MeOH). 1H NMR
(300 MHz, CD3OD): δ 5.15 (d, 1H, J 1,2 ) 2.2 Hz), 4.93 (d, 1H,
J ) 3.4 Hz), 4.69-3.55 (m, 28H), 1.99 (s, 3H), 1.52 (m, 10H),
1.56 (m, 2H), 1.25 (d, 3H, J ) 6.6 Hz), 0.89 (m, 3H). 13C NMR
(50 MHz, CD3OD): δ 173.56 (CO), 101.65 (C-1 Fuc), 97.90 (C-1
GlcNAc), 79.77, 78.39, 73.50, 72.37 (C-1′), 72.31, 71.59 (C-1′),
71.11, 70.89, 70.59 (C-6), 69.25, 69.19 (CH2 (octyl)), 68.45 (C-
3′), 68.15 (C-3′), 62.87 (CH2OH), 62.40 (CH2OH), 55.75 (C-2
GlcNAc), 46.86 (C-2′), 46.51 (C-2′), 33.02 (CH2), 30.46 (CH2),
27.27 (CH2), 23.72 (CH2), 22.83 (NHAc), 16.73, 14.45. IR
Octyl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a ceta m id o-2-
d eoxy-4-O-(2(R),3-d ih yd r oxyp r op yl)-r-D-glu cop yr a n o-
sid e (4GD (27)). To a solution of 21 (100 mg, 0.116 mmol) in
methanol (9 mL) were added trifluoroacetic acid (2.6 µL, 0.035
mmol) and then Pd/C (132 mg), and the mixture was stirred
under H2 atmosphere for 4 h. After this time, the reaction
mixture was filtered through Celite and evaporated to give
27 as a solid (65 mg, quantitative). Rf: 0.24 (CH2Cl2-MeOH,
5:1). Mp: 96-99 °C. [R]D: +16.7° (c 1, MeOH). 1H NMR (300
MHz, CD3OD): δ 5.31 (d, 1H, J ) 2.2 Hz), 5.11 (d, 1H, J ) 3.0
Hz), 4.38 (m, 1H), 4.18-3.54 (m, 16H), 2.17 (s, 3H), 1.75 (m,
2H), 1.41 (m, 10H), 1.09 (m, 3H). 13C NMR (50 MHz, CD3-
OD): δ 101.77 (C-1 Fuc), 98.10 (C-1 GlcNAc), 79.97, 78.18,
75.59 (C-1′), 73.50 (C-1′), 73.23, 72.73, 71.25, 70.68, 69.00 (CH2
(octyl)), 68.75, 64.29 (C-3′), 61.87 (C-6), 55.69, 32.99 (CH2),
30.47 (CH2), 30.43 (CH2), 27.27 (CH2), 23.69 (CH2), 22.76
(NHAc), 16.79 (Me-Fuc), 14.41 (CH3). Anal. (C25H47NO12) C,
H, N.
(KBr): 1250, 1045 cm-1
Oct yl O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
.
d eoxy-4-O-[2,2-bis(h yd r oxym eth yl)-3-h yd r oxyp r op yl]-r-
D-glu cop yr a n osid e (4P (14)). To a solution of 13 (190 mg,
0.27 mmol) in dioxane (7.8 mL) and methanol (2.5 mL) was
added 1 M sulfuric acid (0.012 mL, 0.012 mmol), and the
reaction mixture was stirred at room temperature for 24 h.
After this time, the mixture was neutralized with saturated
NaHCO3 solution and concentrated in vacuo. The residue was
purified by column chromatography (EtOAc-MeOH, 4:1) to
give 14 as a solid (104 mg, 64%). Rf: 0.44 (CH2Cl2-MeOH,
3:1). Mp: 61-65 °C. [R]D: +11.0° (c 1, MeOH). 1H NMR (300
MHz, CD3OD): δ 5.31 (d, 1H, J ) 3.2 Hz), 5.22 (d, 1H, J ) 3.3
Hz), 4.39 (m, 1H), 4.21-3.51 (m, 19H), 2.18 (s, 3H), 1.75 (m,
2H), 1.64 (m, 10H), 1.43 (d, 3H, J ) 6.6 Hz), 1.09 (m, 3H). 13C
NMR (50 MHz, CD3OD): δ 173.52 (CO), 102.75 (C-1 Fuc),
95.66 (C-1 GlcNAc), 80.38, 79.39, 73.43, 72.49 (C-1′), 71.08,
70.97, 69.60, 68.99 (CH2 (octyl)), 62.95 (CH2OH), 61.93 (C-6),
55.89 (C-2 GlcNAc), 40.52 (C-2′), 33.02 (CH2), 30.46 (CH2),
27.28 (CH2), 23.72 (CH2), 22.79 (NHAc), 16.72 (Me-Fuc), 14.45
(CH3). Anal. (C27H51NO13‚H2O) C, H, N.
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-6-O-(2(R),3-d ih yd r oxyp r op yl)-r-D-glu cop yr a n o-
sid e (4GD (29)). 23 (64 mg, 0.074 mmol) was reacted by the
procedure used to synthesize compound 27 and afforded the
title compound (40 mg, 97%). Rf: 0.24 (CH2Cl2-MeOH, 5:1);
[R]D: +6.2° (c 0.8, MeOH). 1H NMR (300 MHz, CD3OD): δ
5.16 (s, 1H), 4.94 (d, 1H, J ) 3.5 Hz), 4.50 (m, 1H), 4.23 (dd,
1H, J ) 3.5 Hz, J ) 10.6 Hz), 4.00-3.55 (m, 15H), 2.16 (s,
3H), 1.81 (m, 2H), 1.50 (m, 10H), 1.38 (d, 3H, J ) 6.5 Hz),
1.11 (m, 3H). 13C NMR (50 MHz, CD3OD): δ 101.61 (C-1 Fuc),
98.66(C-1 GlcNAc), 80.94, 74.14 (C-1′), 73.64, 72.85, 72.22,
71.66, 71.63 (C-6), 70.69, 70.09, 69.15 (CH2 (octyl)), 68.27, 64.42
(C-3′), 54.60, 33.00 (CH2), 30.53 (CH2), 30.45 (CH2), 30.43
(CH2), 27.29 (CH2), 23.71 (CH2), 22.74 (NHAc), 16.57 (Me-Fuc),
14.41 (CH3). Anal. (C25H47NO12) C, H, N.
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-6-O-[2,2-bis(h yd r oxym eth yl)-3-h yd r oxyp r op yl]-r-
D-glu cop yr a n osid e (6P (16)). Compound 15 (180 mg, 0.26
mmol) was reacted by the procedure used to synthesize
compound 14 and afforded the title compound as a solid (110
mg, 71%). Rf: 0.36 (CH2Cl2-MeOH, 3:1). Mp: 55-59 °C. [R]D:
+4.8° (c 1, MeOH). 1H NMR (300 MHz, CD3OD): δ 5.16 (s,
1H), 4.94 (d, 1H, J ) 3.5 Hz), 4.48 (m, 1H), 4.25 (dd, 1H, J )
3.5 Hz, J ) 10.6 Hz), 3.96-3.50 (m, 18H), 2.16 (s, 3H), 1.82
(m, 2H), 1.61-1.43 (m, 10H), 1.38 (d, 3H, J ) 6.6 Hz), 1.1 (m,
3H). 13C NMR (50 MHz, CD3OD): δ 174.05 (CO), 101.66 (C-1
Fuc), 98.67 (C-1 GlcNAc), 80.94, 73.66, 72.73, 72.47 (C-1′),
71.89 (C-6), 71.66, 70.95, 70.09, 69.15 (CH2 (octyl)), 68.29, 63.20
(CH2OH), 54.62 (C-2 GlcNAc), 46.96 (C-2′), 33.04 (CH2), 30.57
(CH2), 30.48 (CH2), 27.35 (CH2), 23.74 (CH2), 22.75 (NHAc),
16.60 (Me-Fuc), 14.45 (CH3). Anal. (C27H51NO13) C, H, N.
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-4,6-bis-O-(2(R),3-d ih yd r oxyp r op yl)-r-D-glu cop yr a -
n osid e (4,6GD (31)). 25 (46 mg, 0.047 mmol) was reacted by
the procedure used to synthesize compound 27 and afforded
the title compound (30 mg, quantitative). Rf: 0.17 (CH2Cl2-
MeOH, 3:1); [R]D: +15.2° (c 0.3, MeOH). 1H NMR (300 MHz,
CD3OD): δ 5.31 (d, 1H, J ) 2.7 Hz), 5.01 (d, 1H, J ) 3.3 Hz),
4.38 (m, 1H), 4.20-3.51 (m, 21H), 2.17 (s, 3H), 1.75 (m, 2H),
1.49 (m, 10H), 1.41 (d, 3H, J ) 6.5 Hz), 1.09 (m, 3H). 13C NMR
(50 MHz, CD3OD): δ 101.79 (C-1 Fuc), 98.19 (C-1 GlcNAc),
79.80, 78.12, 75.58 (C-1′), 73.93 (C-1′), 73.50, 72.60, 72.34,
72.25, 71.26, 70.76 (C-6), 70.67, 69.17 (CH2 (octyl)), 68.74, 64.41
(C-3′), 64.28 (C-3′), 55.65, 32.99 (CH2), 30.47 (CH2), 30.43
(CH2), 27.27 (CH2), 23.67 (CH2), 22.74 (NHAc), 16.79 (Me-Fuc),
14.41 (CH3). Anal. (C28H53NO14) C, H, N.
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-4,6-bis-O-[2,2-bis(h yd r oxym eth yl)-3-h yd r oxyp r o-
p yl]-r-D-glu cop yr a n osid e (4,6P (18)). Compound 17 (38.7
mg, 0.04 mmol) was reacted by the procedure used to synthe-
size compound 14. The yield after column chromatography
was 38% (11.5 mg). Rf: 0.32 (CH2Cl2-MeOH, 3:1); [R]D: +24.3°
(c 1, MeOH). 1H NMR (300 MHz, CD3OD): δ 5.30 (d, 1H, J )
3.2 Hz), 5.22 (d, 1H, J ) 3.4 Hz), 4.38 (m, 1H), 4.18-3.51 (m,
27H), 2.18 (s, 3H), 1.77 (m, 2H), 1.49 (m, 10H), 1.43 (d, 3H, J
) 6.6 Hz), 1.09 (m, 3H). 13C NMR (50 MHz, CD3OD): δ 173.52
(CO), 102.79 (C-1 Fuc), 97.68 (C-1 GlcNAc), 80.24, 79.28, 73.36,
72.40 (C-1′), 72.21, 71.75 (C-1′), 71.03, 70.89, 70.56 (C-6), 69.61,
69.11 (CH2 (octyl)), 63.10 (CH2OH), 62.79 (CH2OH), 55.83 (C-2
GlcNAc), 32.97 (CH2), 30.44 (CH2), 30.41 (CH2), 27.24 (CH2),
Alk yla tion Rea ction of Diol 6 w ith Tosyla te 20. 6 (425
mg, 0.56 mmol) was treated with 20 (243 mg, 0.85 mmol),
following the procedure shown above, to give after column
chromatography (hexane-acetone, 5:1f1:1) 22 (278 mg, 56%),
24 (114 mg, 24%), and 26 (75 mg,14%). 22 Rf: 0.33 (hexane-
acetone, 1:1). 24: Rf 0.51 (hexane-acetone, 1:1). 26: Rf 0.54
(hexane-acetone, 1:1).
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-4-O-(2(S),3-d ih yd r oxyp r op yl)-r-D-glu cop yr a n o-
sid e (4GL (28)). 22 (145 mg, 0.168 mmol) was reacted by the
procedure used to synthesize compound 27 and afforded the

Disaccharide Inhibitors of Glioma Cell Division
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4605
title compound (93 mg, quantitative). Rf: 0.34 (CH2Cl2-
MeOH, 3:1). Mp: 96-99 °C. [R]D: +19.0° (c 1, MeOH). 1H
NMR (300 MHz, CD3OD): δ 5.32 (d, 1H, J ) 2.8 Hz), 5.11 (d,
1H, J ) 3.3 Hz), 4.40 (m, 1H), 4.18 (dd, 1H, J ) 8.7 Hz, J )
10.7 Hz), 4.05-3.52 (m, 14H), 2.17 (s, 3H), 1.75 (m, 2H), 1.49
(m, 10H), 1.41 (d, 3H, J ) 6.5 Hz), 1.09 (m, 3H). 13C NMR (50
MHz, CD3OD): δ 102.84 (C-1 Fuc), 98.94(C-1 GlcNAc), 81.00,
79.33, 75.84 (C-1′), 74.42, 74.11, 73.36, 72.15, 71.64, 69.92 (CH2
(octyl)), 69.79, 65.35 (C-3), 62.86 (C-6), 56.62, 33.87 (CH2),
31.36 (CH2), 28.16 (CH2), 24.59 (CH2), 23.70 (NHAc), 17.69,
15.33. Anal. (C25H47NO12) C, H, N.
71,72, 70.57, 70.34, 70.18, 69.16, 68.32, 62.54, 54.61, 32.79,
30.60, 30.54, 30.47, 27.30, 23.73, 22.75, 16.61, 14.42. Anal.
(C28H51NO15‚2H2O) C, H, N.
Cell Cu ltu r e. C6 rat glioma cells15 and U-373 MG and
U-118 MG human glioma cells16 were seeded on plastic flasks
and maintained in DMEM culture medium, supplemented
with 10% fetal bovine serum (FCS), 2 mM glutamine, 50 IU/
mL penicillin, and 50 µg/mL streptomycin (DM-10S) and
maintained in a humidified atmosphere of 5% CO2, at 37 °C.
All the plastic material was obtained from Costar (Cambrigde,
MA). Culture media, supplements, and antibiotics were from
Sigma Spain (Madrid, Spain).
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
In h ibition of Cell P r olifer a tion . Proliferation assays
were performed on 96-well plates, using a modification of the
method described by Nieto-Sampedro,2,17 as follows. All cells
lines were seeded in DMEM supplemented with 10% FCS, at
5 × 103 cells/well, and allowed to attach for 6 h. For human
glioma culture, after the cells were attached to the substrate,
the medium was changed to serum-free DMEM and the cells
were incubated for 48 h. By comparison, C6 cells were
incubated in DMEM supplemented with 0.5% FCS, overnight.
After either 48 h (human lines) or 24 h (rat glioma C6), the
medium was replaced by DMEM medium containing the test
inhibitors (from 1 mM to 62 µM, in triplicate) and mitogen.
The mitogens were 10% FCS for C6 cells and 1% FCS for
U-118. Astrocytoma U-373 proliferation was driven by 1%
FCS, by human EGF (10 ng/mL), or by human bFGF (100 ng/
mL), as indicated for each experiment. The cultures were
further incubated for 48 h and washed with serum-free DMEM
medium, and [3H]thymidine incorporation was measured
following a 6-h pulse (0.5 µCi/well). Nonincorporated radio-
activity was washed, and the cells were collected on glass fiber
filters, using a cell harvester(Skatron Inc., Norway). Radio-
activity was measured on a Pharmacia-LKB liquid scintillation
counter. Inhibition (%) was calculated as follows:
d eoxy-6-O-(2(S),3-d ih yd r oxyp r op yl)-r-D-glu cop yr a n o-
sid e (6GL (30)). 24 (65 mg, 0.075 mmol) was reacted by the
procedure used to synthesize compound 27 and afforded the
title compound (40 mg, 96%). Rf: 0.34 (CH2Cl2-MeOH, 3:1);
[R]D: +9.9° (c 0.9, MeOH). 1H NMR (300 MHz, CD3OD): δ
5.16 (s, 1H), 4.95 (d, 1H, J ) 3.2 Hz), 4.48 (m, 1H), 4.23 (dd,
1H, J ) 3.5 Hz, J ) 10.6 Hz), 4.00-3.55 (m, 15H), 2.16 (s,
3H), 1.83 (m, 2H), 1.50 (m, 10H), 1.38 (d, 3H, J ) 6.5 Hz),
1.11 (m, 3H). 13C NMR (50 MHz, CD3OD): δ 101.59 (C-1 Fuc),
98.66 (C-1 GlcNAc), 80.88, 74.05 (C-1′), 73.64, 72.87, 72.12,
71.64, 71.55 (C-6), 70.66, 70.07, 69.14 (CH2 (octyl)), 68.24, 64.40
(C-3′), 54.59, 33.01 (CH2), 30.56 (CH2), 30.54 (CH2), 30.44
(CH2), 27.29 (CH2), 23.71 (CH2), 22.73 (NHAc), 16.57, 14.42.
Anal. (C25H47NO12) C, H, N.
Oct yl
O-(r-L-F u cop yr a n osyl)-(1f3)-2-a cet a m id o-2-
d eoxy-4,6-bis-O-(2(S),3-d ih yd r oxyp r op yl)-r-D-glu cop yr a -
n osid e (4,6GL (32)). 26 (52 mg, 0.053 mmol) was reacted by
the procedure used to synthesize compound 27 and afforded
the title compound (33 mg, 98%). Rf: 0.16 (CH2Cl2-MeOH,
3:1); [R]D: +18.0° (c 0.5, MeOH). 1H NMR (300 MHz, CD3-
OD): δ 5.32 (d, 1H, J ) 2.5 Hz), 5.12 (d, 1H, J ) 3.3 Hz), 4.40
(m, 1H), 4.16 (dd, 1H, J ) 8.9 Hz, J ) 11.8 Hz), 4.06-3.50 (m,
20H), 2.17 (s, 3H), 1.77 (m, 2H), 1.49 (m, 10H), 1.41 (d, 3H, J
) 6.5 Hz), 1.10 (m, 3H). 13C NMR (50 MHz, CD3OD): δ 102.03
(C-1 Fuc), 98.11 (C-1 GlcNAc), 79.93, 78.39, 74.98 (C-1′), 73.51,
72.45, 72.33, 72.26, 72.14, 71.22 (C-6), 69.15 (CH2 (octyl)),
68.93, 64.42 (C-3′), 64.37 (C-6), 55.70, 32.99 (CH2), 30.44 (CH2),
27.26 (CH2), 23.69 (CH2), 22.73 (NHAc), 16.76 (Me-Fuc), 14.41
(CH3). Anal. (C28H53NO14) C, H, N.
% inhibition ) 100-100[(X - B)/(A - B)]
where A is the [3H]thymidine dpm incorporated by cells
maintained in 10% FCS, 1% FCS, or growth factors (high
mitosis control), B is the dpm incorporated by cells in mitogen-
free medium (low mitosis control), and X represents the dpm
incorporated by cells treated with test inhibitors. Dose-
response plots of percent inhibition versus concentration were
adjusted to sigmoidal curves from which ID50 values were
calculated.
Octyl O-(2,3,4-Tr i-O-ben zyl-r-L-fu cop yr a n osyl)-(1f3)-
O-[(2,3,4,6-tetr a -O-ben zoyl-â-D-ga la ctop yr a n osyl-(1f6)]-
2-a ceta m id o-2-d eoxy-r-D-glu cop yr a n osid e (35). A mix-
ture of 2,3,4,6-tetra-O-benzoyl-R/â-D-galactopyranosyl trichloro-
acetimidate19 (178 mg, 0.24 mmol), 6 (150 mg, 0.2 mmol), and
molecular sieves (4 Å) (ca 60 mg) in dry dichloromethane (2
mL) was stirred at room temperature under Ar for 30 min.
Then, a solution of TMSOTf in dichloromethane (0.1 M, 0.4
mL) was added dropwise, and the reaction mixture was stirred
at room temperature under Ar for 3 h. After this time, the
mixture was neutralized with triethylamine, filtered through
Celite, and evaporated to dryness. The residue was purified
by column chromatography (hexane-acetone, 4:1) to give 35
(217 mg, 82%). Rf: 0.44 (hexane-AcOEt, 1:1); [R]D: +45.5°.
Anal. (C77H85NO19) C, H, N.
Ack n ow led gm en t. We are very grateful to Prof.
Manuel Bernabe´ for his interest and helpful discussions.
The financial support of the Ministerio de Educacio´n y
Cultura (to B.A.), the Gobierno Auto´nomo Vasco (to
L.R.-R., BF195.121), and the Comunidad de Madrid
(Grants 07/115/96 and 8.1/0011/1997) is greatly appreci-
ated.
Su p p or tin g In for m a tion Ava ila ble: NMR spectral data
for compounds 3-6, 8-12, 21-26, and 35 (4 pages). Ordering
information is given on any current masthead page.
Refer en ces
Octyl O-(r-L-F u cop yr a n osyl)-(1f3)-O-[(â-D-ga la ctop y-
r a n osyl-(1f6)]-2-a cet a m id o-2-d eoxy-r-D-glu cop yr a n o-
sid e (36). 35 (86 mg, 0.065 mmol) was treated with a solution
of sodium methoxide in methanol (0.1 M, 2.6 mL, 0.26 mmol)
for 2 h at room temperature. After this time, the reaction
mixture was neutralized with Amberlite IR-120 (H+), filtered,
and concentrated. The residue was dissolved in methanol (5
mL), Pd/C (74 mg) was added, and the reaction mixture was
stirred under H2 atmosphere for 3 h. Then, the mixture was
filtered through Celite and evaporated to dryness to give 36
(38 mg, 93%). Rf: 0.22 (CH2Cl2-MeOH, 2:1). Mp: 223-226
°C. [R]D: +2.0° (c 0.7, MeOH). 1H NMR (300 MHz, CDCl3):
δ 5.16 (d, 1H, J ) 1.7 Hz), 4.93 (d, 1H, J ) 3.6 Hz), 4.48 (d,
1H, J ) 7.7 Hz), 4.48 (m, 1H), 4.33 (dd, 1H, J ) 1.7 Hz, J )
10.8 Hz), 4.25 (dd, 1H, J ) 10.7 Hz, J ) 3.5 Hz), 4.03-3.51
(m, 15H), 2.16 (s, 3H), 1.81 (m, 2H), 1.59 (m, 10H), 1.38 (d,
1H, J ) 6.5 Hz), 1.09 (m, 3H). 13C NMR (50 MHz, CD3OD):
δ 105.45, 101.64, 99.66, 80.95, 76.77, 74.99, 73.70, 72.89, 72.62,
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