Simplified sialyl Lewisx analogues with improved E-selectin inhibition

Article abstract of DOI:10.1002/hlca.200390085

The simplified sialyl Lewis^x mimic 5 containing a D-arabinose, a 3-cyclohexyl-2-hydroxypropanoate, and a tetrahydropyran building block instead of L-fucose, sialic acid, and N-acetylglucosamine, respectively was synthesized. Compound 5 was 10-f

Full text of DOI:10.1002/hlca.200390085

Helvetica Chimica Acta Vol. 86 (2003)
855
Simplified Sialyl Lewisx Analogues with Improved E-Selectin Inhibition
by Gebhard Thoma* and Franz Schwarzenbach
Novartis Pharma AG, PO Box, WSJ-507.4.12, CH-4002 Basel
(tel. 0613243342, fax 0613246735; e-mail: gebhard.thoma@pharma.novartis.com)
The simplified sialyl Lewisx mimic 5 containing a d-arabinose, a 3-cyclohexyl-2-hydroxypropanoate, and a
tetrahydropyran building block instead of l-fucose, sialic acid, and N-acetylglucosamine, respectively was
synthesized. Compound 5 was 10-fold more potent than sLex in a static E-selectin binding assay and showed at
50 mm 75% inhibition in a dynamic-flow assay in which sLex did not inhibit neutrophil rolling at up to 1000 mm.
Compound 7 with a lactic acid instead ofsialic acid building block showed threeofld improved potency
compared to sLex.
Introduction. The initial interaction (tethering and rolling) ofleukocytes with the
endothelium is mediated by the selectin family of adhesion molecules (E-, P-, and L-
selectin) [1]. Although their physiological ligands are emerging as a diverse group of
glycosylated proteins recognizing one or more ofthe selectins [2], it has been shown
that the minimal carbohydrate epitope recognized by all three selectins is the
tetrasaccharide sialyl Lewisx (sLex) 1 (Fig.) [1].
The carbohydrate/selectin-mediated rolling is a prerequisite for leukocyte extrav-
asation, and thus, inhibition of this process is a possible therapy for adverse effects
caused by accumulating leukocytes [3]. Evidence stems from experiments with
knockout mice, antibodies, and soluble carbohydrates recognized by the selectins [4].
Patients with the LAD-2 syndrome (leukocyte adhesion deficiency 2) indicate the
importance ofselectins in humans [5]. They have an impaired ability to synthesize the
physiological ligands ofthe selectins, show markedly reduced leukocyte recruitment to
sites of inflammation, and consequently suffer from frequent bacterial infections.
The sialyl Lewisx tetrasaccharide 1, which is a weak E-selectin inhibitor (KD ˆ
1060 mm)1) [6] became a lead structure to identify simplified but more-potent selectin
antagonists. Extensive work elucidated that the essential groups required for binding to
E-selectin are the three OH groups ofthe fucose, the 4- and the 6-OH groups ofthe
galactose, and the carboxylic acid function [7]. Based on this knowledge, a multitude of
E-selectin antagonists have been prepared [7].
Earlier, we reported the simplified sLex analogue 2, which is 30-fold more potent
than sLex in an E-selectin binding assay and can be prepared from commercially
available starting materials in ten linear steps in an overall yield of18% [8a] (ofr an
alternative synthesis of 2, see [8b]. Compound 2 was designed from sLex by replacing
sialic acid and N-acetylglucosamine by 3-cyclohexyl-2-hydroxypropanoate and a
tetrahydropyran building block derived from d-glucal, respectively. We also described
Individual protein-carbohydrate interactions are generally weak (KD ˆ 10À3 10À4 mÀ1), see [6].
1
)

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Helvetica Chimica Acta Vol. 86 (2003)
Figure. Sialyl Lewisx 1 and analogs 2
7
compound 3, which is similar to 2 but contains a benzoate at the 2-position ofthe
galactose [9]. It was found to be 100-fold more potent than sLex 1 in the binding assay
and showed an IC50 value of1 2 mm in a cell-based in vitro flow assay. The high
potencies ofsLe x analogues 2 and 3 cannot be explained by additional interactions of
the methyl substituent or the benzoyl group with E-selectin but are most probably due
to improved preorganization ofthe bioactive conof rmation in solution compared to
sLex 1 [10]. Both compounds 2 and 3 contain costly residues such as l-fucose and 3-
cyclohexyl-2-hydroxypropanoate. It would be desirable to replace these groups by
inexpensive ones such as arabinose and readily available carboxylic acids.

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857
Sialyl Lewisx analogues with d-arabinose instead of l-fucose2)3) or (S)-lactic acid
instead ofsialic acid 4) have been described but showed unsatisfying E-selectin
inhibition (four- and fivefold less potent than sLex 1, resp.). We reasoned that performing
similar modifications on highly potent inhibitors 2 and 3 could although a drop of
activity has to be expected lead to derivatives with good potency compared to sLex 1.
Here we report on the synthesis and biological evaluation ofthe novel E-selectin
antagonists 4 7 (see Fig.) which are based on highly potent inhibitors 2 and 3 but
contain d-arabinose instead of l-fucose (see 4 and 5) or glycolic acid instead of3-
cyclohexyl-2-hydroxypropanoate (see 6 and 7). Compounds 4 and 5 were less potent
than 2 and 3 but still showed 3- and 10-fold improved inhibition of E-selectin binding
compared to sLex 1. Furthermore antagonist 5 gave 75% inhibition in the flow assay at
50 mm. Compound 6 did not inhibit E-selectin but its galactose-2-benzoate derivative 7
was found to be three times more potent than sLex 1.
Syntheses. Compounds 4 and 5 were prepared from advanced intermediate 8, the
synthesis ofwhich we reported earlier ( Scheme 1) [14]. Selective tritylation ofthe
primary OH group gave 9. d-Arabinose was stereoselectively introduced by means of
donor 10, which was transformed into the corresponding glycosyl bromide by slow
addition ofa small excess ofBr 2. Hydrolysis ofthe reaction mixture led to the cleavage
ofthe trityl group, and the modiifed trisaccharide 11 was isolated in 61% yield. The
corresponding a-d-glycoside could not be isolated. Hydrogenolysis followed by mild
transesterification at room temperature gave compound 5, which still contains the
benzoate at the 2-position ofthe d-galactose unit. To obtain compound 4, we removed
the benzoate by applying harsher conditions and elevated temperatures. With related
compounds, we had observed this selective deprotection earlier [9]. The sodium salt 4
‡
was obtained by passing the lithium salt through an ion-exchange-resin (Na ) column5)
[15].
Thioarabinoside 10 was prepared from glycosyl bromide 12, which was reacted with
thiourea in acetone and, subsequently, with bromoethane to give acetylated thioar-
abinoside 13 (Scheme 1). Removal ofthe acetate groups and introduction ofbenzyl
protecting groups afforded the required arabinose donor 10.
Compounds 6 and 7 were prepared starting from thiogalactoside 14 [16] by tin-
mediated regioselective alkylation6) with ethyl iodoacetate (Scheme 2). Intermediate
15 was benzoylated ( ! 16) and, subsequently, treated with tetrahydropyran building
block 17 [8] to give the desired b-d-linked disaccharide 18 (76%) along with small
2
)
)
Replacement offucose by arabinose in a sLe x pentasaccharide resulted in a fivefold less potent E-selectin
inhibitor [11].
Very recently, a complex glycopeptide from E-selectin ligand 1 (ESL-1) with the arabino sLex structure
was reported that showed ninefold improved E-selectin inhibition compared to sLex [12]; synthesis and
3
x
biological evaluation ofthe corresponding glycopeptide with the fuco sLe was not included.
4
5
)
The corresponding (R)-lactic acid derivative was inactive up to 10 mm [13]. An sLex mimetic containing
x
glycolic acid instead ofsialic acid was 4.5-fold less potent than sLe 1 [13].
)
It has been reported that compounds contaminated with trace amounts ofpolyanions released from ion-
exchange resins can give false positive test results in selectin assays [15]. To exclude such effects, we tested
several compounds that were not treated with ion-exchange resin as sodium salts and as free acids. The
biological results in our assays were not affected.
6
)
For regioselective manipulations ofOH groups via organotin derivatives, see [17].

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 1
quantities ofits a-d-anomer 19 (7%). This was unexpected since neighboring-group
participation ofthe benzoate at the 2-position ofdonor 16 should selectively lead to the
b-d-glycosidic linkage. In related transformations of acceptor 17 and galactosyl donors
having an ether group at the 3-position and a benzoate at the 2-position, we have
observed incomplete stereo-induction before [14]. The stereoisomers were separated
by chromatography. Reductive opening of the benzylidene acetal afforded regioisom-
ers 20 and 21 as a separable 10 :1 mixture (86%). Compound 20 was reacted with
fucosyl donor 22 [8] applying similar conditions as described for the arabinosylation of
9. The fully protected trisaccharide 23 was obtained in 52% yield. Hydrogenolytic
removal ofthe benzyl protecting groups gave pentol 24, which was treated with NaOH
(3 equiv.) in MeOH/H2O 9 :1 at 08 for 2 h. The reaction mixture was quenched with
AcOH, and the products 6 (80%) and 7 (15%) were separated by reversed-phase
chromatography. The 2-benzoate ofthe galactose unit in 24 was clearly less stable
under basic conditions than the 2-benzoate in 11, most probably due to shielding by the
adjacent bulky 3-cyclohexyl-2-hydroxypropanoate in 11.
Biological Evaluation. Compounds 4 7 were tested in a static, cell-free ligand-
binding assay that measures E-selectin inhibition under equilibrium conditions [18]. To
compare the data for different compounds obtained on different test plates, sLex 1
(IC50 ˆ 1000 1500 mm) was assayed on each plate as a reference. This allows the
determination of IC50 values relative to sLex 1, which are defined as relative IC50 ˆ IC50
(test compound)/IC50(sLex 1). All compounds were tested at least twice. Compounds 4,

Helvetica Chimica Acta Vol. 86 (2003)
859
Scheme 2
5, and 7 were found to be significantly more active than sLex 1, showing relative IC50
values of0.31, 0.09, and 0.29, respectively. Compound 6 did not inhibit E-selectin
binding at concentrations up to 5000 mm. The most potent antagonist, compound 5, was
also profiled in a dynamic in vitro assay which allows monitoring ofE-selectin-
dependent rolling ofneutrophils on activated endothelial cells and, hence, mimics the
non-equilibrium in vivo conditions [19]. It inhibited rolling by 75% at 50 mm, whereas
sLex 1 did not show any inhibition in this more relevant assay at up to 1000 mm.
Conclusions. Based on our highly active E-selectin antagonist 3, we designed
compounds 5 and 7 with inexpensive replacements for the costly l-fucose and 3-
cyclohexyl-2-hydroxypropanoate, which were less potent than 3 but 10- and 3-fold more
potent than sLex 1.

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Helvetica Chimica Acta Vol. 86 (2003)
Experimental Part
General. All reactions were carried out under dry Ar. Commercially available abs. solvents were used.
CC ˆ column chromatography, FC ˆ flash chromatography. NMR Spectra: Bruker Avance-DPX-400 spec-
trometer; assignments by 2D 1H,1H correlation (COSY) and 1H,13C correlation (HSQC); d in ppm rel. to SiMe4,
J in Hz; Gal ˆ b-d-galactopyranosyl, Ara ˆ b-d-arabinopyranosyl, Fuc ˆ 6-deoxy-a-l-galactopyranosyl, Hex ˆ
d-arabino-hexitol), cC6H11 ˆ cyclohexyl. MS: Finnigan MAT-90 mass spectrometer.
O-3-O-[(1S)-1-Carboxy-2-cyclohexylethyl]-b-d-galactopyranosyl-(1 ! 3)-O-[b-d-arabinopyranosyl-(1 !
4)]-1,5-anhydro-2-deoxy-d-arabino-hexitol Sodium Salt (4). A mixture of 5 (12 mg, 0.021 mmol), LiOH (13 mg,
0.312 mmol), MeOH (1.5 ml), and H2O (0.5 ml) was stirred at 258 for 24 h. AcOH (0.02 ml) was added, and the
‡
solvents were evaporated. The residue was passed through an ion-exchange column (Na ) and then subjected to
CC (RP-18, H2O/MeOH 5 :1 ! 2 :1): 4 (11 mg, quant.). 1H-NMR (400 MHz, D2O): 0.75 1.72 (m, cC6H11CH2,
HaxÀC(2) (Hex)); 2.15 (br. dd, J ˆ 13.0, 5.9, HeqÀC(2) (Hex)); 3.31 (dd, J ˆ 9.5, 3.5, HÀC(3) (Gal)); 3.32
(m, HÀC(5) (Hex)); 3.40 (br. t, J ˆ 12.0, HaxÀC(1) (Hex)); 3.46 (t, J ˆ 9.5, HÀC(4) (Hex)); 3.49 (d, J ˆ 12.0,
HaxÀC(5) (Ara)); 3.52 (t, J ˆ 9.0, HÀC(2) (Gal)); 3.54 (t, J ˆ 6.0, HÀC(5) (Gal)); 3.65 (m, CH2(6) (Gal)); 3.74
(dd, J ˆ 8.0, 3.5, HÀC(2) (Ara)); 3.75 (m, CH2(6) (Hex)); 3.82 (dd, J ˆ 9.0, 3.0, HÀC(3) (Ara)); 3.83
(m, HÀC(4) (Gal)); 3.85 3.95 (m, CHCO2Na, HeqÀC(1) (Hex), HÀC(4) (Ara)); 4.00 (m, HÀC(3) (Hex));
4.44 (d, J ˆ 8.0, HÀC(1) (Gal)); 4.47 (d, J ˆ 12.0, HeqÀC(5) (Ara)); 4.93 (d, J ˆ 3.5, HÀC(1) (Ara)). HR-MS:
595.2604 (C26H43O15À, [M À Na]À; calc. 595.2602).
O-2-O-Benzoyl-3-O-[(1S)-1-carboxy-2-cyclohexylethyl]-b-d-galactopyranosyl-(1 ! 3)-O-[b-d-arabinopy-
ranosyl)-(1 ! 4)]-1,5-anhydro-2-deoxy-d-arabino-hexitol Sodium Salt (5).
A mixture of 11 (200 mg,
0.158 mmol), 10% Pd/C (400 mg) and MeOH (15 ml) was hydrogenated at 258 for 16 h at 1 atm. The catalyst
was filtered off and the solvent evaporated. The residue was dissolved in MeOH (10 ml). NaOMe (0.169 mmol)
was added, and the mixture was stirred at 258 for 16 h. AcOH (0.02 ml) was added, and the solvent was
‡
evaporated. The residue was passed through an ion-exchange column (Na ) and then subjected to CC (RP-18,
H2O/MeOH 3 :1 ! 1:2): 5 (81 mg, 71%). 1H-NMR (400 MHz, D2O): 0.27 1.47 (m, cC6H11CH2, HaxÀC(2)
(Hex)); 2.08 (m, HeqÀC(2) (Hex)); 3.27 (m, HÀC(5) (Hex)); 3.34 (m, HaxÀC(1) (Hex)); 3.35 (t, J ˆ 9.0,
HÀC(4) (Hex)); 3.55 (dd, J ˆ 13.0, 2.0, HaxÀC(5) (Ara)); 3.64 3.82 (m, 8 H); 3.92 (br. d, J ˆ 3.0, HÀC(4)
(Gal)); 3.95 (br. d, J ˆ 2.0, HÀC(4) (Ara)); 3.96 (m, HÀC(3) (Hex)); 4.49 (d, J ˆ 13.0, HeqÀC(5) (Ara)); 4.86
(d, J ˆ 8.0, HÀC(1) (Gal)); 4.87 (d, J ˆ 3.0, HÀC(1) (Ara)); 5.17 (br. t, J ˆ 9.0, HÀC(2) (Gal)); 7.48 (t, J ˆ 8.0,
À
2 arom. H); 7.62 (br. t, J ˆ 8.0, 1 arom. H); 8.05 (dd, J ˆ 8.0, 1.0, 2 arom. H). HR-MS: 699.2865 (C33H47O16
[M À Na]À; calc. 699.2864).
,
O-3-O-(Carboxymethyl)-b-d-galactopyranosyl-(1 ! 3)-O-[6-deoxy-a-l-galactopyranosyl-(1 ! 4)]-1,5-an-
hydro-2-deoxy-d-arabino-hexitol (6) and O-2-O-Benzoyl-3-O-(carboxymethyl)-b-d-galactopyranosyl-(1 ! 3)-
O-[6-deoxy-a-l-galactopyranosyl-(1 ! 4)]-1,5-anhydro-2-deoxy-d-arabino-hexitol (7). A mixture of 24 (98 mg,
0.131 mmol), NaOH (0.39 mmol), MeOH (25 ml), and H2O (0.1 ml) was stirred at 08 for 2 h. AcOH (0.1 ml)
was added, and the solvents were evaporated. The residue was subjected to CC (RP-18, H2O/MeOH 9 :1 !
1:1): 6 (13 mg, 19%) followed by 7 (65 mg, 80%).
Data of 6: 1H-NMR (400 MHz, D2O) 1.10 (J ˆ 6.5, Me(6) (Fuc)); 1.54 (qd, J ˆ 12.5, 5.0, HaxÀC(2)); 2.13
(dd, J ˆ 12.5, 5.0, HeqÀC(2) (Hex)); 3.29 (m, HÀC(5) (Hex)); 3.37 (dd, J ˆ 9.5, 3.0, HÀC(3) (Gal)); 3.40
(br. t, J ˆ 12.0, HexÀC(1) (Hex)); 3.49 (t, J ˆ 9.0, HÀC(4) (Hex)); 3.52 (dd, J ˆ 7.0, 4.5, HÀC(5) (Gal)); 3.65
(m, CH2(6) (Gal)); 3.68 (dd, J ˆ 10.5, 4.0, HÀC(2) (Fuc)); 3.71 (d, J ˆ 3.5, HÀC(4) (Fuc)); 3.73 (dd, J ˆ 12.0,
5.0, HÀC(6) (Hex)); 3.79 (dd, J ˆ 10.5, 3.5, HÀC(3) (Fuc)); 3.83 (dd, J ˆ 12.0, 2.0, 1 HÀC(6) (Hex)); 3.91
(br. dd, J ˆ 12.0, 5.0, HeqÀC(1) (Hex)); 3.96 (m, HÀC(3) (Hex)); 3.97 (s, CH2CO2H); 3.98 (d, J ˆ 3.0, HÀC(4)
(Gal)); 4.45 (d, J ˆ 8.0, HÀC(1) (Gal)); 4.67 (q, J ˆ 6.5, HÀC(5) (Fuc)); 4.87 (d, J ˆ 4.0, HÀC(1) (Fuc)). HR-
MS: 513.1816 (C20H33O15À, [M À H]À; calc. 513.1819).
Data of 7: 1H-NMR (400 MHz, D2O): 1.21 (m, HaxÀC(2) (Hex)); 1.23 (d, J ˆ 6.5, Me(6) (Fuc)); 2.03
(m, HeqÀC(2) (Hex)); 3.26 (m, HÀC(5) (Hex)); 3.33 (td, J ˆ 12.0, 1.5, HaxÀC(1) (Hex)); 3.37 (t, J ˆ 9.0,
HÀC(4) (Hex)); 3.65 (br. t, J ˆ 6.0, HÀC(5) (Gal)); 3.66 3.79 (m, 8 H); 3.82 (dd, J ˆ 10.0, 3.0, HÀC(3)
(Gal)); 3.88 (m, HÀC(3) (Hex)); 3.89 (d, J ˆ 16, 1 H, CH2CO2H); 3.97 (d, J ˆ 16, 1 H, CH2CO2H); 4.10 (d, J ˆ
3.0, HÀC(4) (Gal)); 4.76 (br. q, J ˆ 6.5, HÀC(5) (Fuc)); 4.81 (d, J ˆ 8.0, HÀC(1) (Gal)); 4.83 (d, J ˆ 4.0,
HÀC(1) (Fuc)); 5.24 (dd, J ˆ 10.0, 8.0, HÀC(2) (Gal)); 7.49 (t, J ˆ 8.0, 2 arom. H); 7.64 (t, J ˆ 8.0, 1 arom. H);
8.03 (t, J ˆ 8.0, 2 arom. H). HR-MS: 617.2091 (C27H37O16À, [M À H]À; calc. 617.2082).
1,5-Anhydro-2-deoxy-3-O-{2,4,6-tri-O-benzoyl-3-O-[(1S)-1-(cyclohexylmethyl)-2-oxo-2-(phenylmethoxy)-
ethyl]-b-d-galactopyranosyl}-6-O-(triphenylmethyl)-d-arabino-hexitol (9). A soln. of 8 [14] (177 mg, 0.204 mol)
and chlorotriphenylmethane (114 mg, 0.408 mmol) in pyridine (5 ml) was heated at 708 for 16 h. The solvent

Helvetica Chimica Acta Vol. 86 (2003)
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was evaporated and the residue subjected to FC (SiO2, hexane/AcOEt 3 :1 ! 2 :1): 9 (226 mg, quant.).
Colorless oil. 1H-NMR (400 MHz, CDCl3): 0.45 1.70 (m, cC6H11CH2, HaxÀC(2) (Hex), HeqÀC(2) (Hex)); 3.20
(m, HÀC(5) (Hex), 1 HÀC(6) (Hex)); 3.26 (td, J ˆ 11.5, 2.0, HaxÀC(1) (Hex)); 3.41 (m, HÀC(3) (Hex),
1 HÀC(6), Hex)); 3.49 (br. t, J ˆ 9.0, HÀC(4) (Hex)); 3.85 (d, J ˆ 1.0, OHÀC(4) (Hex)); 3.87 (m, HÀC(1)
(Hex)); 3.88 (dd, J ˆ 10.0, 3.5, HÀC(3) (Gal)); 3.96 (ddd, J ˆ 8.5, 3.5, 1.0, HÀC(5) (Gal)); 4.17 (dd, J ˆ 8.0, 4.5,
CHCO2Bn); 4.24 (dd, J ˆ 12.0, 8.5, 1 HÀC(6) (Gal)); 4.61 (d, J ˆ 8.0, HÀC(1) (Gal)); 4.63 (dd, J ˆ 12.0, 3.5,
1 HÀC(6) (Gal)); 5.07 (d, J ˆ 12.0, 1 H, PhCH2); 5.21 (d, J ˆ 12.0, 1 H, PhCH2); 5.63 (dd, J ˆ 10.0, 8.0, HÀC(2)
‡
(Gal)); 5.88 (dd, J ˆ 3.5, 1.0, HÀC(4) (Gal)); 7.13 8.14 (m, 35 arom. H). ESI-MS: 1126 ([M ‡ NH4] ).
S-Ethyl 2,3,4-Tris-O-(phenylmethyl)-1-thio-a-d-arabinopyranoside (10).
A mixture of 13 (13.2 g,
41.3 mmol) and NaOCH3 (4.13 mmol) in MeOH (150 ml) was stirred for 16 h at 258. The mixture was
‡
neutralized with Amberlyst 15 (H form) and filtered and the solvent evaporated to give the corresponding triol
as a colorless oil (7.34 g, quant.), which was used without further purification. 1H-NMR (400 MHz, D2O): 1.04
(t, J ˆ 7.0, MeCH2S); 2.48 (m, MeCH2S); 3.31 (m, 2 HÀC(5)); 3.37 (t, J ˆ 9.5, HÀC(2)); 3.64 (m, HÀC(4)); 3.70
(dd, J ˆ 10.0, 3.0, HÀC(3)); 4.10 (d, J ˆ 9.0, HÀC(1)).
To a soln. ofthis triol (1.00 g, 5.15 mmol) in DMF (15 ml) was added NaH (0.50 g, 20.8 mmol) in small
portions. Benzyl bromide (2.82 g, 16.5 mmol) was added within 45 min, and the mixture was stirred at 258 for
additional 30 min. MeOH (0.25 ml) and 2m NaOMe/MeOH (1 ml) and then H2O (30 ml) were added. The
mixture was extracted with Et2O, the org. phase dried (Na2SO4) and evaporated, and the residue subjected to FC
1
(SiO2, hexane/AcOEt 7:1 ! 4 :1): 10 (1.96 g, 82%). Colorless foam. H-NMR (400 MHz, CDCl3): 1.31 (t, J ˆ
7.0, MeCH2S); 2.72 (m, MeCH2S); 3.38 (d, J ˆ 12.0, 1.5, 1 HÀC(5)); 3.62 (m, HÀC(4)); 3.82 (m, HÀC(2),
HÀC(3)); 4.20 (dd, J ˆ 12.0, 3.0, 1 HÀC(5)); 4.52 (d, J ˆ 9.0, HÀC(1)); 4.60 4.82 (m, HÀC(1), 3 PhCH2);
‡
7.28 7.41 (m, 15 arom. H). ESI-MS: 482 ([M ‡ NH4] ).
O-2,4,6-Tri-O-benzyl-3-O-[(1S)-1-(cyclohexylmethyl)-2-oxo-2-(phenylmethoxy)ethyl]-b-d-galactopyrano-
syl-(1 ! 3)-O-[2,3,4-tris-O-(phenylmethyl)-b-d-arabinopyranosyl-(1 ! 4)]-1,5-anhydro-2-deoxy-d-arabino-
hexitol (11). A soln. ofBr (332 mg, 2.08 mmol) in CH2Cl2 (1.5 ml) was added dropwise at 08 to a soln. of 10
2
(840 mg, 1.81 mmol) in CH2Cl2 (1.5 ml). After stirring for 30 min at 08, cyclohexene (0.25 ml) was added to
consume excessive Br2. The soln. was added within 20 min to a mixture of 9 (1000 mg, 0.90 mmol), Et4NBr
(377 mg, 1.81 mmol; dried for 2 h at 2008), and molecular sieves (3 ä, 1.0 g; dried for 24 h at 3008) in DMF/
CH2Cl2 1:1 (9 ml). The mixture was stirred for 40 h at 258, diluted with AcOEt and filtered. The resulting soln.
was washed with H2O and brine and dried (Na2SO4). The solvent was evaporated and the residue dissolved in
Et2O (40 ml). Formic acid (15 ml) was added, and the mixture was stirred at 258 for 2 h, washed with 1n NaOH,
H2O, and brine. The org. phase was dried (Na2SO4) and evaporated and the residue subjected to FC (SiO2,
toluene/AcOEt 4 :1 ! 3 :1): 11 (690 mg, 61%). Colorless foam. 1H-NMR (400 MHz, CDCl3): 0.43 1.42
(m, cC6H11CH2, HaxÀC(2) (Hex)); 1.90 (br. dd, J ˆ 13.0, 5.0, HeqÀC(2) (Hex)); 2.35 (br. s, OHÀC(6) (Hex));
3.03 (dt, J ˆ 9.0, 3.5, HÀC(5) (Hex)); 3.16 (t, J ˆ 12.0, HaxÀC(1) (Hex)); 3.49 (t, J ˆ 9.0, 1 HÀC(6) (Hex)); 3.64
(br. s, HÀC(4) (Ara)); 3.66 3.80 (m, HeqÀC(1) (Hex), HÀC(3) (Hex), HÀC(4) (Hex), 1 HÀC(6) (Hex),
1 HÀC(6) (Gal)); 3.82 (br. d, J ˆ 12.5, 1HÀC(5) (Ara)); 3.87 (dd, J ˆ 10.0, 3.0, HÀC(3) (Gal)); 3.93 (t, J ˆ 6.5,
HÀC(5) (Gal)); 3.95 (dd, J ˆ 10.0, 3.0, HÀC(3) (Ara)); 4.08 (dd, J ˆ 10.0, 3.5, HÀC(2) (Ara)); 4.13 (dd, J ˆ
8.0, 4.5, OCHCO2Bn); 4.34 (d, J ˆ 6.5, 1 HÀC(6) (Gal), 4.38 (br. d, J ˆ 12.5, 1 HÀC(5) (Ara)); 4.40 (d, J ˆ 12.0,
1 H, PhCH2); 4.45 (d, J ˆ 12.0, 1 H, PhCH2); 4.50 (d, J ˆ 12.0, 1 H, PhCH2); 4.57 (d, J ˆ 12.0, 1 H, PhCH2); 4.67
(d, J ˆ 8.0, HÀC(1) (Gal)); 4.69 (d, J ˆ 11.5, 1 H, PhCH2); 4.86 (d, J ˆ 11.5, 1 H, PhCH2); 5.04 (d, J ˆ 12.0, 1 H,
CO2CH2Ph); 5.09 (d, J ˆ 3.5, HÀC(1) (Ara)); 5.13 (d, J ˆ 12.0, 1 H, CO2CH2Ph); 5.62 (br. t, J ˆ 9.0, HÀC(2)
‡
(Gal)); 5.87 (br. d, J ˆ 3.0, HÀC(4) (Gal)); 7.14 8.13 (m, 35 arom. H). ESI-MS: 1286 ([M ‡ NH4] ).
S-Ethyl 2,3,4-Tri-O-acetyl-1-thio-a-d-arabinopyranoside (13). A soln. of 12 (24.0 g, 70.8 mmol) and
thiourea (6.30 g, 83.3 mmol) in acetone (96 ml) was stirred and heated under reflux for 1 h. To the grey
suspension, acetone (500 ml), H2O (600 ml), bromoethane (8.54 g, 78.5 mmol), K2CO3 (10.1 g, 73.1 mmol), and
NaHCO3 (6.72 mg, 64.7 mmol) were added, and the mixture was stirred at 258 for 16 h. The mixture was
extracted with CH2Cl2, the org. phase dried (Na2SO4) and evaporated, and the residue subjected to FC (SiO2,
hexane/AcOEt 4 :1 ! 1:1): 13 (13.2 g, 58%). Colorless oil. 1H-NMR (400 MHz, CDCl3): 1.29 (t, J ˆ 7.0,
MeCH2S); 2.05 (s, 1 AcO); 2.09 (s, 1 AcO); 2.04 (s, 1 AcO); 2.71 (m, MeCH2S); 3.67 (dd, J ˆ 12.0, 1.5,
1 HÀC(5)); 4.11 (dd, J ˆ 12.0, 3.0, 1 HÀC(5)); 4.52 (d, J ˆ 9.0, HÀC(1)); 5.08 (dd, J ˆ 10.0, 3.0, HÀC(3)); 5.24
‡
(t, J ˆ 9.5, HÀC(2)); 5.20 (m, HÀC(4)). ESI-MS: 343 ([M ‡ Na] ).
S-Ethyl 3-O-(2-Ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-1-thio-b-d-galactopyranoside (15). A suspension
of 14 [16] (2.54 g, 8.06 mmol) and Bu2SnO (2.21 g, 8.87 mmol) in MeOH (100 ml) was heated under reflux for
2 h. The solvent was removed and the resulting colorless foam dried in vacuo for 16 h. The residue was dissolved
in DMF (40 ml). At 08, iodoacetic acid ethyl ester (8.83 g, 40.3 mmol) and CsF (2.45 g, 16.1 mmol) were added.

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The resulting suspension was stirred at 08 for 20 h. The mixture was diluted with AcOEt, washed with H2O and
brine, dried (Na2SO4), and evaporated and the crude 15 used without further purification in the next step.
S-Ethyl 2,4-Di-O-benzoyl-3-O-(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-1-thio-b-d-galactopyranoside
(16). At 08, benzoyl chloride (4.54 g, 32.2 mmol) was slowly added to a soln. ofcrude 15 (8.06 mmol) and
N,N-dimethylpyridin-4-amine (DMAP; 0.39 g, 3.22 mmol) in pyridine (30 ml). The mixture was stirred at 258
for 16 h. The mixture was diluted with AcOEt and washed with 0.2n, HCl, NaHCO3 soln., and brine, dried
(Na2SO4), and evaporated and the residue subjected to FC (SiO2, toluene/AcOEt 20 :1): 16 (2.44 g, 50%).
Colorless oil. 1H-NMR (400 MHz, CDCl3): 1.11 (t, J ˆ 7.0, MeCH2O); 1.31 (t, J ˆ 7.5, MeCH2S); 2.74 2.90
(m, MeCH2S); 3.61 (dd, J ˆ 9.5, 7.0, 1 HÀC(6)); 3.70 (dd, J ˆ 9.5, 6.0, 1 HÀC(6)); 3.99 (br. t, J ˆ 6.5, HÀC(5));
4.00 4.09 (m, MeCH2O); 4.10 (dd, J ˆ 10.0, 3.5, HÀC(3)); 4.15 (d, J ˆ 17, 1 H, OCCH2CO2Et); 4.26 (d, J ˆ 17,
1 H, OCH2CO2Et); 4.46 (d, J ˆ 11.5, 1 H, PhCH2); 4.54 (d, J ˆ 11.5,1 H, PhCH2); 4.71 (d, J ˆ 10.0, HÀC(1));
‡
5.58 (t, J ˆ 10.0, HÀC(2)); 5.98 (d, J ˆ 3.5, HÀC(4)); 7.20 8.14 (m, 15 arom. H). ESI-MS: 631 ([M ‡ Na] ).
1,5-Anhydro-2-deoxy-3-O-[2,4-di-O-benzoyl-3-O-(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-b-d-galacto-
pyranosyl]-4,6-O-(phenylmethylene)-d-arabino-hexitol (18) and 1,5-Anhydro-2-deoxy-3-O-[2,4-di-O-benzoyl-
3-O-(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-a-d-galactopyranosyl]-4,6-O-(phenylmethylene)-d-arabino-
hexitol (19). At 08, trifluoromethanesulfonic acid (3 drops) was added to a soln. of 16 (1.40 g, 2.30 mmol), 17 [8]
(0.81 g, 3.45 mmol), and N-iodosuccinimide (NIS; 0.65 g, 2.88 mmol) in CH2Cl2 (10 ml). The mixture was
stirred for 30 min. Solid NaHCO3 (0.2 g) was added. The mixture was diluted with CH2Cl2 and extracted with
NaHCO3 soln. and brine. The solvent was evaporated and the residue subjected to FC (SiO2, toluene/AcOEt
9 :1 ! 5 :1): 19 (0.13 g, 7%) followed by 18 (1.36 g, 76%).
Data of 19: Colorless oil. 1H-NMR (400 MHz, CDCl3): 1.15 (t, J ˆ 7.0, MeCH2O); 1.90 (qd, J ˆ 12.0, 5.0,
HaxÀC(2) (Hex)); 2.08 (dd, J ˆ 12.0, 5.0, HeqÀC(2) (Hex)); 3.27 (td, J ˆ 9.5, 5.0, HÀC(5) (Hex)); 3.45 3.65
(m, HaxÀC(1) (Hex), HÀC(4) (Hex), 1 HÀC(6) (Hex), CH2(6) (Gal)); 3.92 (dd, J ˆ 12.0,5.0, HeqÀC(1)
(Hex)); 3.98 (m, HÀC(3) (Hex)); 4.08 (q, J ˆ 7.0, MeCH2O); 4.17 (dd, J ˆ 10.5, 5.0, 1 HÀC(6) (Hex)); 4.21
(br. s, CH2CO2Et); 4.30 (dd, J ˆ 10.5, 3.5, HÀC(3) (Gal)); 4.39 (br. t, J ˆ 6.0, HÀC(5) (Gal)); 4.42 (d, J ˆ 11.5,
1 H, PhCH2); 4.51 (d, J ˆ 11.5, 1 H, PhCH2); 5.04 (s, PhCH(O)2); 5.47 (dd, J ˆ 10.5, 3.5, HÀC(2) (Gal)); 5.64
(d, J ˆ 3.5, HÀC(1) (Gal)); 5.97 (d, J ˆ 3.5, HÀC(4) (Gal)); 7.10 8.12 (m, 20 arom. H). ESI-MS: 805 ([M ‡
‡
Na] ).
Data of 18: Colorless solid. 1H-NMR (400 MHz, CDCl3): 1.09 (3 H, t, J ˆ 7.0, MeCH2O); 1.80 (m, HaxÀC(2)
(Hex), HeqÀC(2) (Hex)); 3.28 (td, J ˆ 9.5, 5.0, HÀC(5) (Hex)); 3.38 (td, J ˆ 12.0, 3.0, HaxÀC(1) (Hex)); 3.48
(dd, J ˆ 9.0, 5.5, 1 HÀC(6) (Gal)); 3.56 (br. t, J ˆ 8.5, 1 HÀC(6) (Gal)); 3.67 (t, J ˆ 9.0, HÀC(4) (Hex)); 3.70
(t, J ˆ 10.0, 1 HÀC(6) (Hex)); 3.78 (br. t, J ˆ 7.0, HÀC(5) (Gal)); 3.86 (m, HeqÀC(1) (Hex)); 3.90 (m, HÀC(3)
(Hex)); 3.94 4.09 (m, HÀC(3) (Gal), MeCH2O); 4.12 (d, J ˆ 17.0, 1 H, CH2CO2Et); 4.23 (d, J ˆ 17.0, 1 H,
CH2CO2Et); 4.26 (dd, J ˆ 10.0, 5.0, 1 HÀC(6) (Hex)); 4.31 (d, J ˆ 11.5, 1 H, PhCH2); 4.40 (d, J ˆ 11.5, 1 H,
PhCH2); 4.89 (d, J ˆ 8.0, HÀC(1) (Gal)); 5.48 (dd, J ˆ 10.0, 8.0, HÀC(2) (Gal)); 5.58 (s, PhCH(O)2); 5.85
‡
(br. d, J ˆ 3.0, HÀC(4) (Gal)); 7.16 8.14 (m, 20 arom. H). ESI-MS: 805 ([M ‡ Na] ).
1,5-Anhydro-2-deoxy-3-O-[2,4-di-O-benzoyl-3-O-(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-b-d-galacto-
pyranosyl]-6-O-(phenylmethyl)-d-arabino-hexitol (20) and 1,5-Anhydro-2-deoxy-3-O-[2,4-di-O-benzoyl-3-O-
(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-b-d-galactopyranosyl]-4-O-(phenylmethyl)-d-arabino-hexitol (21).
At 08, a sat. HCl soln. in Et2O (freshly prepared) was slowly added in small portions to a mixture of 18
(1.36 g, 1.74 mmol) and NaCNBH3 (1.09 g, 17.4 mmol) in THF (30 ml). The addition was stopped (11 ml) when
TLC (toluene/AcOEt 4 :1) indicated complete consumption of 18. NaHCO3 (solid, 0.75 g) and AcOEt were
added. The mixture was washed with NaHCO3 soln. and brine dried (Na2SO4), and evaporated, and the residue
subjected to FC (SiO2, toluene/acetone 9 :1 ! 5 :1): 20 (1.06 g, 78%) followed by 21 (0.12 g, 8%).
Data of 20: Colorless foam. 1H-NMR (400 MHz, CDCl3): 1.11 (t, J ˆ 7.0, MeCH2O); 1.63 1.74 (m, CH2(2)
(Hex)); 3.29 3.37 (m, HaxÀC(1) (Hex), HÀC(5) (Hex)); 3.45 (td, J ˆ 9.0, 1.5, HÀC(4) (Hex)); 3.52
(m, HÀC(3) (Hex)); 3.59 3.67 (m, HÀC(6) (Hex), CH2(6) (Gal)); 3.82 (dd, J ˆ 10.5, 2.0, 1 HÀC(6)
(Hex)); 3.90 (m, HeqÀC(1) (Hex)); 3.98 4.10 (m, MeCH2O, OHÀC(4), HÀC(3) (Gal), HÀC(5) (Gal));
4.12 (d, J ˆ 17.0, 1 H, CH2CO2Et); 4.21 (d, J ˆ 17.0, 1 H, CH2CO2Et); 4.43 (d, J ˆ 11.5, 1 H, PhCH2); 4.52 (d, J ˆ
11.5, 1 H, PhCH2); 4.59 (m, PhCH2); 4.74 (d, J ˆ 8.0, HÀC(1) (Gal)); 5.53 (dd, J ˆ 10.0, 8.0, HÀC(2) (Gal));
‡
5.87 (d, J ˆ 3.0, HÀC(4) (Gal)); 7.20 8.11 (m, 20 arom. H). ESI-MS: 807 ([M ‡ Na] ).
Data of 21: Colorless foam. 1H-NMR (400 MHz, CDCl3): 1.11 (t, J ˆ 7.0, MeCH2O); 1.46 (qd, J ˆ 12.0, 5.0,
HÀC(2) (Hex)); 1.85 (m, OHÀC(6)); 1.95 (dd, J ˆ 12.0, 5.0, HeqÀC(2) (Hex)); 3.23 (ddd, J ˆ 9.5, 5.0, 3.0,
HÀC(5) (Hex)); 3.33 (td, J ˆ 12.0, 1.5, HaxÀC(1) (Hex)); 3.37 (t, J ˆ 9.0, HÀC(4) (Hex)); 3.60 (m, CH2(6)
(Gal)); 3.68 (m, 1 HÀC(6) (Hex)); 3.81 3.89 (m, HeqÀC(1) (Hex), 1 HÀC(6) (Hex)); 3.94 (br. t, J ˆ 6.5,
HÀC(5) (Gal)); 3.97 4.09 (m, HÀC(3) (Hex), MeCH2O); 4.11 (dd, J ˆ 10.0, 3.0, HÀC(3) (Gal)); 4.17 (d, J ˆ

Helvetica Chimica Acta Vol. 86 (2003)
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17.0, 1 H, CH2CO2Et); 4.27 (d, J ˆ 17.0, 1 H, CH2CO2Et); 4.39 (d, J ˆ 11.5, 1 H, PhCH2); 4.49 (d, J ˆ 11.5, 1 H,
PhCH2); 4.66 (d, J ˆ 11.5, 1 H, PhCH2); 4.85 (d, J ˆ 8.0, HÀC(1) (Gal)); 5.16 (d, J ˆ 11.5, 1 H, PhCH2); 5.54
(dd, J ˆ 10.0, 8.0, HÀC(2) (Gal)); 5.93 (br. d, J ˆ 3.0, HÀC(4) (Gal)); 7.20 8.15 (m, 20 arom. H). ESI-MS: 807
‡
([M ‡ Na] ).
O-2,4-Di-O-benzoyl-3-O-(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-b-d-galactopyranosyl-(1 ! 3)-O-[6-
deoxy-2,3,4-tris-O-(phenylmethyl)-a-l-galactopyranosyl-(1 ! 4)]-1,5-anhydro-2-deoxy-6-O-(phenylmethyl)-d-
arabino-hexitol (23). A soln. ofBr 2 (307 mg, 1.92 mmol) in CH2Cl2 (2 ml) was added dropwise at 08 to a soln. of
22 [8] (840 mg, 1.81 mmol) in CH2Cl2 (2 ml). After stirring for 30 min at 08, cyclohexene (0.20 ml) was added to
consume excessive Br2. The soln. was added within 10 min to a mixture of 20 (1050 mg, 1.34 mmol) and Et4NBr
(335 mg, 1.61 mmol; dried for 2 h at 2008) in DMF/CH2Cl2 1:1 (10 ml). The mixture was stirred for 70 h at 258,
diluted with AcOEt and filtered. The resulting soln. was washed with H2O and brine, dried (Na2SO4), and
evaporated and the residue subjected to FC (SiO2, hexane/AcOEt 1:1 ! 1 :2): 23 (838 mg, 52%). Colorless
foam. 1H-NMR (400 MHz, CDCl3): 1.10 (t, J ˆ 7.0, MeCH2O); 1.41 (d, J ˆ 6.5, Me(6) (Fuc)); 1.49 (qd, J ˆ 12.0,
5.0, HaxÀC(2) (Hex)); 2.03 (br. dd, J ˆ 12.0, 5.0, HeqÀC(2) (Hex)); 3.29 3.37 (m, HaxÀC(1) (Hex), HÀC(5)
(Hex)); 3.56 3.65 (m, 1 HÀC(6) (Hex), CH2(6) (Gal)); 3.66 (br. d, J ˆ 2.5, HÀC(4) (Fuc)); 3.76 (dd, J ˆ 10.5,
4.0, 1 HÀC(6) (Hex)); 3.79 (t, J ˆ 9.0, HÀC(4) (Hex)); 3.86 3.97 (m, HeqÀC(1) (Hex), HÀC(3) (Hex),
HÀC(3) (Fuc), HÀC(5) (Gal)); 3.97 4.09 (m, MeCH2O, HÀC(2) (Fuc), HÀC(3) (Gal)); 4.13 (d, J ˆ 17.0,
1 H, CH2CO2Et); 4.23 (d, J ˆ 17.0, 1 H, CH2CO2Et); 4.41 (d, J ˆ 11.5, 1 H, PhCH2); 4.42 (s, PhCH2); 4.49 (d, J ˆ
11.5, 1 H, PhCH2); 4.52 (d, J ˆ 11.5, 1 H, PhCH2); 4.57 (d, J ˆ 11.5, 1 H, PhCH2); 4.58 (d, J ˆ 11.5, 1 H, PhCH2);
4.63 (d, J ˆ 11.5, PhCH2); 4.73 (br. q, J ˆ 6.5, HÀC(5) (Fuc)); 4.75 (d, J ˆ 11.5, 1 H, PhCH2); 4.78 (d, J ˆ 8.0,
HÀC(1) (Gal)); 4.93 (d, J ˆ 11.5, 1 H, PhCH2); 5.06 (d, J ˆ 3.5, HÀC(1) (Fuc)); 5.49 (dd, J ˆ 10.0, 8.0, HÀC(2)
‡
(Gal)); 5.93 (br. d, J ˆ 3.0, HÀC(4) (Gal)); 7.16 8.12 (m, 35 arom. H). ESI-MS: 1223 ([M ‡ Na] ).
O-2,4-Di-O-benzoyl-3-O-(2-ethoxy-2-oxoethyl)-6-O-(phenylmethyl)-b-d-galactopyranosyl-(1 ! 3)-O-[6-
deoxy-a-l-galactopyranosyl)-(1 ! 4)]-1,5-anhydro-2-deoxy-d-arabino-hexitol (24). A mixture of 23 (300 mg,
0.250 mmol), 10% Pd/C (150 mg), dioxan (10 ml), and H2O (2 ml) was hydrogenated at 258 for 16 h at 1 atm.
The catalyst was filtered off and the solvent evaporated. The residue was subjected to CC (SiO2, iPrOH/AcOEt/
H2O 1:1:0.05 ! 1:1 :0.1): 24 (127 mg, 68%). Colorless solid. 1H-NMR (400 MHz, CD3OD):1.07 (t, J ˆ 7.0,
MeCH2O); 1.30 (m, HaxÀC(2) (Hex)); 1.38 (d, J ˆ 6.5, Me(6) (Fuc)); 2.13 (m, HeqÀC(2) (Hex)); 3.19
(br. d, J ˆ 9.0, HÀC(5) (Hex)); 3.38 (t, J ˆ 12.0, HaxÀC(1) (Hex)); 3.58 (t, J ˆ 9.0, HÀC(4) (Hex)); 3.63 3.72
(m, 1 HÀC(6) (Hex), CH2(6) (Gal)); 3.47 (dd, J ˆ 10.0, 3.0, HÀC(2) (Fuc)); 3.76 3.86 (m, HeqÀC(1) (Hex),
1 HÀC(6) (Hex), HÀC(3) (Fuc), HÀC(4) (Fuc)); 3.86 4.03 (m, MeCH2O, HÀC(3) (Hex), HÀC(5) (Gal));
4.13 4.25 (m, CH2CO2Et, HÀC(3) (Gal)); 4.89 (m, HÀC(5) (Fuc), HÀC(1) (Gal)); 4.97 (d, J ˆ 3.0, HÀC(1)
(Fuc)); 5.44 (br. t, J ˆ 9.0, HÀC(2) (Gal)); 5.85 (br. d, J ˆ 2.5, HÀC(4) (Gal)); 7.47 8.13 (m, 10 arom. H). ESI-
‡
MS: 773 ([M ‡ Na] ).
We would like to thank Drs. J. L. Magnani and J. T. Patton for developing and performing the biological
7
assays ). We also would like to thank Mrs. Karin Krayer for technical assistance.
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Received September 21, 2002