CA2055381A1

CA2055381A1 - Oligosaccharide derivatives

Info

Publication number: CA2055381A1
Application number: CA 2055381
Authority: CA
Inventors: Markus Hosang; Niggi Iberg; Michel Trumtel; Thomas B. Tschopp; Hans P. Wessel
Original assignee: Individual
Current assignee: F Hoffmann La Roche AG
Priority date: 1991-07-30
Filing date: 1991-11-13
Publication date: 1993-01-31

Abstract

Abstract Sulphated oligosaccharide derivatives of formula I

I

wherein R, R' and R have the significance given in the description, can be used as medicaments, especially for the treatment of arteriosclerotic disorders.

Description

2 ~
RAN 407 l /30 The present invention is concerned with novel sulphated oligosaccharides of the formula CH2 OR H O.;~
0 ~ H~ H
,~ 1 O~ O~R~oR
H O~ H

wherein R is hydrogen or a residue -SO3M; M is a cation; R is a an equatorially or quasiequatorially linked sulphated mono-, di- or tri- saccharide residue; or an axially linked sulphated trisaccharide residue; and R is hydrogen or an equatorially or quasiequatorially linked sulphated mono- or disaccharide residue;
whereby the molecule contains a ma~imum of 6monosaccharide units and on average at least one -SO3M group is present per monosaccharide unit.

2s The invention is also concerned with a process for the manufacture of the compounds of formula I, their use as medicaments or as active ingredients for the manufacture of pharmaceutical preparations, pharmaceutical preparations based on compounds of formula I and a method of treating or 30 preventing arteriosclerotic disorders in man.

As the cation M there come into consideration all physio-logically compatible cations, e.g. alkali metal cations such as ~a+
and K+; ammonium ions and substituted ammonium ions which 35 are derived from tertiary amines such as triethylamine, or pyridine or imida;~ole; or quaternary ammonium ions such as dodecyltri- methylammonium, ethylpyridinium and Grn/7 . 8 . 9 l ... . . . . . . . . . . . . .. . . . . . . .. . . .. . .. . . . . . . .

. ~ .

2 ~5~
~enzethonium: as well as alkaline earth metal cations such as Ca++.
Compounds in which M is Na~ are prefe~ed.

The degree of sulphation means the number of -SO3M
s residues per monosaccharide unit w}lich are present in the molecule on average. The degree of sulphation 1 therefore exists e.g. when a hexasaccharide of formula I contains 6-SO3M
residues in the molecule. The degree of sulphation in the compounds of formula I preferably amounts to 2-3.

Equatorially linked sulphated mono-, di- or trisaccharide residues R' are preferably ,~-glycosidically linked residues; but an equatorial linlcing, e.g. in the case of arabinose glycosides, can accompany an a-glycosidically bonding of the residue R' to the 5 trehalose residue. The term "~uasiequatorial" relates to the conformation of furanosides.

The compounds of formula I can be manufactured in accordance with the invention by treating a corresponding tri-, 20 tetra-, penta- or hexasaccharide with a sulphating agent and converting the reaction product into a salt or isolating it as such.
Examples of monosaccharide residues are glucopyranosyl, manno-pyranosyl, arabinopyranosyl, galactofuranosyl, ar~binofuranosyl, ribofuranosyl and rhamnofuranosyl. Examples of disaccharide 2s residues are maltosyl, cellobiosyl, lactosyl, melibiosyl, gentio-biosyl and galactopyranosidoarabinopyranosyl. A trisaccharide residue is, for example, maltotriosyl. The aforementioned residues are sulphated as substituents R or R" in the scope of the definition of formula I. Examples of compounds of formula I are 30 the compounds of formulae Ia and Ib in which R has the signifi-cance given above and on average a Ieast one residue R per mono-saccharide unit is -SO3M. Ill the compound Ia R is ,~-D-malto-triosyl and R is hydrogen, in the compound Ib R' and R' are ,~-D-maltosyl.

. .. _ . . . . . . . _ . _ . _ . . _ _ . . . . . . .

? ~ ~L

ClkOR CIIzOR CHZORCIIzOR 11 OR

R i\lo~ Il/Lo~\ol~ I~/l o,)\OR I~f ~J\OR 1~oJ\QIl2c/lo~ I~
H OR 11 01~ 1 I OR 11 OR 11 Cl120R CIIzOR CIIzOR 1~ 0~ CIIzOR CIIzOR
o ~ o ~0~ /OR 11\~ J I~ l Ib R l~ ()J\oR ll/l ol~RO112l,~ OR
I 1 01~ 11 o~ 11 OR

The sulphation in accordance with the present invention can be carried out using methods which are known per se for the sulphation of hydroxy groups.

Examples of sulphating agents which can be used for the 20 manufacture of the compounds of formula I are S03-complexes such as S03-pyridine, SO30trimethylamine, SO30dioxan and S03-dimethylformamide. Other examples of sulphating agents are chlorosulphonic acid, mixtures of chlorosulphonic acid and sulphuric acid; and piperidine N-sulphate.
2~
The reaction is conYeniently effected in a suitable solvent, especially a polar solvent, e.g. dimethylformamide, dimethyl sulphoxide or hexamethylphosphortriamide. The reaction can be carried out at room temperature or a higher temperature, e.g. at 30 20-70C, whereby the degree of sulphation can be influenced by varying the reaction duration and reaction temperature. The degree of sulphation achieved in each case can be assessed by HPLC. The working-up of the reaction mixture and, respectively, the isolation of the reaction product of formula I from the reaction 35 mixture can be effected according to methods known per se, e.g.
by gel filtration or ultrafiltration.

....... , .. , .. _ . . ..... .. . . .. _ _ . _ ~ _ _ _ ~ .. _ _ _ _ ~ _ . . . .. ...

2 ~
The free saccharides which are used as starting materials are known or are accessible according to methods which are known in principle. Enzymatic or synthetic chemical procedures come into consideration for the preparation. The oligosaccha-rides 5 can be synthesized principally using sequential synthesis or block synthesis. In this case glycosidic bonds are formed by reacting a glycosyl acceptor with a glycosyl donor in the presence of a suitable catalyst. Derivatized glycosyl compounds which are activated at the anomeric centre, such as e.g. chlorides, bromides, 0 fluorides, acetates, trichloroacetimidates, alkylthio derivatives, etc, are suitable as glycosyl donors.

Those saccharide derivatives in which the O~I groups to be glycosylated are free and the remaining OH groups are completely 5 or partially protected are suitable as glycosyl acceptors. When the remaining OH groups are only partially protected the glyco-sylidation can be effected selectively or can be directed in a particular direction by virtue of the hydroxyl groups having a different environment.
The compounds of formula I inhibit the migration and proliferation of cells of the vascular smooth musculature and prevent proliferative arteriosclerotic lesions. Their blood coagulation-inhibiting activity is lower than that of heparin. In 2s particular, the compounds have no in vitro anticoagulant activity, i.e. they have no effect or only a very slight effect on the coagu-lation factors thrombin (F.IIa) and F.Xa. The compounds of formula I can therefore be used for the prophyla~cis of arterio-sclerotic disorders, in man especially after bypass operations or 30 angioplasty, as well as for the treatment of patients having progressive arteriosclerosis.

The blood coagulation-inhibiting activity was determined as follows:
3s aPTT factivated partial thromboplastin time! Test (see Walenga et al., CRC Critical Reviews in Laboratory Sciences 22 (4) 361-389 (1986)): 100 ~,11 of citrated human plasma, which contains various .. . _ _ _ , _ . . , .. . .. . _ _ _ . ~ ~ ~ _ _ _ . . _ _ _ _ _ _ . . ~ ~ _ j _ s concentrations of test compound, is incubated at 37C for 8 minutes with 100 ~LI of Activated Thrombofax (Ortho Diagnostics, Raritan, N.J., U.S.A.). 100 ~LI of pre-warmed 25 mM calcium chloride solution are then added and the coagulation time is 5 measured in a Fibrometer Coagulation Timer (Becton, Diskinson B asle).

anti-Xa Clotting Assay- 25 ~Ll of citrated plasma having various concentrations of test compound are mixed with 75 ~,11 of 0 Factor Xa (Diagnostic Reagents, Thame, Oxon, Great Britain) diluted 1:100 in 0.63% citrate buffer (pH 7.3~ which contains 41 mM
imidazole, 82mM NaCl and 0.1% albumin. After warming to 37C
for 2 minutes 200 ~,11 of a 1:1 mixture of Factor X Deficient Plasma (Diagnostic Reagents) and Platelet Substitute (Diagnostic Reagents) 5 are added and the mixture is incubated at 37C for 20 seconds.
After the addition of 100 ~LI of pre-warmed 50 mM calcium chloride solution the coagulation time is measured in a Fibrometer.

The activity of the test compound is given as the ICso, which is that concentration [~lg/ml] which leads to a coagulation time which is double the control value.

Inhibition of Thrombin or Factor Xa in the Chromogenic 2s Substrate Assav (Teien et al., Thrombosis Research 10, 399-410 - (1977)): The determination was effect in a Cobas-Bio centrifugal automatic spectrophotometer. The buffer solution used consisted of 50mM Tris buffer, 18û mM NaCl, 7.~ mM EDTA Na2, 1% PEG
6000 and 0.02% Tween 80, pH 8.4. The test solution consisted of 50 ~Ll of buffer, 30111 of anti-thrombin III (1 U/ml), Kabi Diagnostica) and 20 ~1 of plasma which contained vanous con-centrations of test compounds. 30 ~11 of sample solution and 20 ~Ll of water with 18û 111 of thrombin were added to the test cuvette in the automatic analyzer. After incubation at 37C for 3s 240 seconds 60 ~,11 of S-2238 (H-D-Phe-Pip-Arg-NH.pNA, Kabi Diagnostica, Mondal, Sweden, 0.75 mM in water) and 20 111 of water were added. The liberation of pNA (p-nitroaniline) was followed during 60 seconds at 405 nm in 10 second intervals in ... .... , . . . . . .. . .. . ~ _ .

comparison to water as the blank. The inhibitory activity is given as the ICso, which is the concentration [~,lg/ml] at which the amidolytic activity of thrombin is reduced by 50% in comparison to the plasma control value.
s The inhibition of ~actor Xa was measured in the same manner using a solution of Factor ~a (2.8 nkat/ml and 2nM S-222 (Bz-CO-Ile-Glu-Arg-NH.pNA, Kabi Diagnostica) in water in place of thrombin and, respectively, S-2238.

The anti-proliferative activity of the substances was determined in cell cultures as follows: smooth muscle cells of rats (cultivated in DMEM with 10% FCS at 37C and 5% CO2) were applied to cell culture pla~es with a density of 8 x 103 cells/well.
15 After 4 hours the number of adhered cells was determined and the substances to be tested (100 mg/mi) were added. Cells to which test substance was not added served as a comparison and heparin (100 mg/ml) served as a positive control. The cells were incubated for 7 days and then the cell number was determined.
20 The antiproliferative activity of the individual substances was calculated as the % inhibition in comparison to non-inhibited growth:

cell number ( ) - cell number (inhib) 25 % Inhibition = x 100 cell number ( ) - cell number (d=o) wherein cell number(d=o) = cell number after 4 h cell number( ) = cell number to which test substances was not added, a~ter 7 days.
cell number(inhib) = cell number with 100 ~LI/ml of the test substance The results obtained in the experimental procedures 35 described above with compounds of formula I are listed in Table 1. Heparin served as the reference compound.

6~

Table I

Anti- Anti-coagulation activity IC50 [llg/ml]
Compoun proliferative Coagulation Amidolytic d of activity % inhibitior activity ~xample inhibition aPI~ ~a Thrombin F.Xa lG. 5 7 11 31 >1000960 2C 38 40 1 ]l~ >1000>1000 3C 5 9 1 2 33.5 >1000>1000 4C 64 1 1 32 >1000>1000 5C 39 33 69 >1000>1000 6D 6 7 14 . 5 28.5 > 1000900 7C 49 13.5 34 >1000~1000 Heparin 4 7 1.2 0.6 1.9_ 2.7 The test results show that the compounds in accordance s with the invention have an anti-proliferative activity which, in contrast to the likewise anti-proliferatively active heparin, is not accompanied by or is accompanied to a very insignificant extent by an anticoagu]ant activity.

o The medicaments based on the compounds in accordance with the invention can be administered enterally, e.g orally in the form of tablets, coated tablets, dragées, hard and soft gelatine capsules, solutions, emulsions or suspensions, or rectally, e.g. in the form of suppositories. However, the administration is 5 preferably effected parenterally, e.g. in the form of injection solutions.

For the manufacture of tablets, coated tablets, dragées and hard gelatine capsules the active ingredient can be mixed with 20 pharmaceutically inert, inorganic or organic excipients. As such excipients for tablets, dragées and hard gelatine capsules there can be used e.g. lactose, maize starch or derivatives thereof, talc, stearic acid or its salts. Suitable excipients for soft gelatine capsules are e.g. vegetable oils, waxes, fats, semi-solid and liquid 25 polyols; depending on the nature of the active ingredient no excipients are, ho~ivever, usually required in the case of soft ... . . ... . . _ _ .. _ _ ... .. .. _ , ~.. _. _ . __ __ ._ ____ .. __ .. __ ._ . ... ,_ ....... .. ~ . , .

2 ~

gelatine capsules. Suitable excipients for the manufacture of solutions and syrups are e.g. water, polyols, saccharose, invert sugar and glwcose, suitable excipients for injection solutions are e.g. water, alcohols, polyols, glycerine and vegetable oils and 5 s~itable excipients for s~ppositories are e.g. natural or hardened oils, waxes, fats and semi-liquid or li4uid polyols.

The pharmaceutical preparations can contain, in addition, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, o sweeteners, colorants, flavorants, salts for varying ~he osmotic pressure, buffers, coating agents or antioxidants. In the case of enteral administration the resorption of the active ingredient can be increased with the aid of liposomes.

The dosage of the active ingredient can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of parenteral administration a dosage of about 0.1 to 100 mg/lcg, preferrably of about 1.5 to 15 mg/kg, per day should be appropriate for adults, 20 although the upper limit just given can also be exceeded when this is shown to be indicated.

Example 1 25 A. A solution of 28.5 g of 2,2',3,3'-tetTa-O-benzyl-4,6-O-benzylidene-~,oc-D-trehalose (Carbohydr. Res. ~, 51 (1978)) and 27 ml of bistributyltin oxide in 2.21 of toluene was heated under reflex for 4.5 hours on a water separator and reduced to a volume of 800 ml. Subsequently, 3.84 g of tetrabutylammonium bromide 30 and 42.8 ml of benzyl bromide were added and the mixture was stirred at 100C. After 16 hours the solution was cooled and worked-up by extraction in a sodium hydrogen carbonate/methylene chloride system. The combined organic phases were dried (MgSO4) and chromatographed on silica gel 3s with acetone/hexane 1:2 (containingl %o of triethylamine) as the eluent. Product *actions were crystallized and gave 27.5 g (~6%) of 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalose, m.p. 122C.

. ... .. . ... . ... . . . . . _ . _ _ . .. _ . _ . _ _ _ _ _ _ _ ~ . _ _ 2 ~ 3 ,j 3 ~ ~, B. A solution of 32.7 g of deca-O-acetyl-a-D-maltotriosyl bromide (K. Tabeo, K. Mine and T. Kuge, Carbohydr. Res 48, 197 (1976)), in 85 ml of allyl alcohol was stirred at 50-60~ for 5 90 minutes in the presence of 8.5 g of mercury-II cyanide, concentrated and extracted in ethereal solution successively with 1 molar potassium iodide solution, sodium bicarbonate solution and water. Chromatography of the crude product on silica gel with ethyl acetate/hexane 1:1 as the eluent gave 25.3 g (79.6%) 10 of pure allyl deca-O-acetyl-,B-D-maltotrioside, [CC]2D=7S.OO (c= 0.5, dioxan).

C A solution of 24.8 g of allyl deca-O-acetyl-,B-D-malto-trioside in 150 ml of methanol was stirred at room temperature 5 for 18 hours in the presence of catalytic amounts of anhydrous sodium carbonate, then filtered and neutralized with acidic ion exchanger. After removing the solvent the residue was taken up in 3~0 ml of dimethylformamide and treated at room temper-ature with 8.0 g of sodium hydride (80% in Fefined oil). After 20 stirIing for 90 minutes the mixture was cooled to 1C, treated with 32 ml of benzyl bromide and stirred while cooling with ice for 30 minutes and at room temperature for 1 hour. Thereafter, the mixture was added dropwise to 100 ml of methanol and stirred for 1 hour. The reaction mixture was concentrated, taken 2s up in ethyl acetate and extracted with aqueous sodium bicarbonate solution and with water. The organic phases were dried and concentrated (38.5 g). 5 g of benzylated crude product were suspended in 40 ml of acetic acidlwater 9:1 and treated in an ul~asound bath for 3 hours in the presence of 2.46 g of 30 palladium chloride and 2.46 g of sodium acetate. Thereafter, the mixture was suction filtered, washed, evaporated, the residue was taken up in ethyl acetate and the solution was washed in succession with aqueous sodium bicarbonate solution and water.
The ethyl acetate phases were dried (Na2~O4), concentrated and 35 chroma~ographed on silica gel with toluene/ethyl acetate 7:1 as the eluent. There were obtained 3.99 g (90%) of syrupy deca-O-benzyl-D-maltotriose.

. . .. .. _ _ . _ ____.__ ., _ .. .. _ _. , . . ._. .. .. _ __ _ .. _ ._ . . ..... __ _ _ _ 2 ~
D. A solution of 0.25 ml of oxalyl chloride in 7 ml of absolute dichlorome~hane was added dropwise at 3C during 45 minutes to a solwtion of 3.69 g of deca-O-benzyl-D-maltotriose in 25 ml of absolute dichloromethane and 0.1 ml of absolute dimethyl-5 formamide. After stirring at room tempera~ure for 6 hours themixture was evaporated. The dried crude deca-O-benzyl-a- D -maltotriosyl chloride was dissolved in 10 ml of absolute aceto-nitrile and stirred at room temperatl~re for 3 hours in the presence of 0.97 g of dry silver fluori~de. The reaction mixture o was filtered and the precipitate was washed with ether. The organic solutions were treated with aqueous saturated sodium chloride solution and stirred vigorously for 15 minutes. After suction filtration of the separated precipitate the filtrate was concentrated, diluted with ether and washed in succession with 15 sodium chloride solution and water. The ethereal solutions were evaporated. Chromatography on silica gel with ethyl acetate/
hexane 1:6 as the eluent gave 2.19 g (66%) of pure deca-O-benzyl-~-D-maltotriosyl fluoride, [a]2D = 58.9 (c = 0.9, CHCl3).

20 E A solution of l l O ml of trifluoromethanesulphonic anhydride in 5 ml of dry ether was added dropwise at -20C
within 1 hour in the presence of molecular sieve (4A) to a solution of 1.69 g of well dried deca-~-benzyl-,~-D-maltotriosyl fluoride and 53~ mg of 2,2',3,3',6'-penta-O-benzyl-4,6-O-2s benzylidene-o~,a-D-trehalose in 20 ml of dry ether. The mixture was warmed to room temperature and stirred for 48 hours. After filtration over a filter aid, washing with ether and concentrating the residue was chromatographed on silica gel using toluene/
ethyl acetate 14:1 as the eluent. There were obtained 414 mg 30 (30%) of 2,2',3,3',6'-penta-O-benzyl-4'-O-(deca-O-benzyl-oc-D-maltotriosyl)-4,6-O-benzylidene-~,a-D-trehalose as a syrupy product, [a]2D = +44 (c = 0.0846, Etr)H) F. A solution of 295 mg of 2,2',3,3',6'-penta-O-benzyl-4'-O-(deca-3s O-benzyl-a-D-matlotriosyl)-4,6-O-benzylidene-a~a-D-trehalose in 10 ml of ethanol and 5 ml of water was hydrogen- ated at room temperature for 2 hours in the presence of 300 mg of 10%
palladium-on-charcoal. The reaction solution was filtered over a .. . . ~

~ ~ r3 ~ .rJ3 '~.

filter aid, rinsed and freeze-dried. The crude product was gel-chromatographed with water (on Sephadex6~ L1120) and the product was lyophilized. There were obtained 65 mg o~ ~-O-(a-D-maltotriosyl)-a,a-D-trehalose, [a]2D = +179.5 (c = 0.2, ~I2O).

C~ A solution of 30 mg of 4-O-(a-D-maltotriosyl)-oc,a-D-trehalose in 1 ml of absolute dimethylformamide was stirred at 50C for 20 hours in the presence of 170 mg of sulphur trioxide-trimethylamine complex, whereby a viscous syrup separated. The 0 solvent was decanted off, the residue was washed with methanol, dissolved in 1.5 ml 10% sodium acetate solution and concen-trated. The residue was taken up several times in water and evaporated in order to remove trimethylamine. The residue was gel-chromatographed (Sephadex~' LH 20) in order to remove salts.
s After ~reeze-drying there were obtained 67 mg of sulphated 4-O-(a-D-maltotriosyl)-a,a-D-trehalose, S = 18.66%, AS (average degree of sulphation) about 2.4.

Example 2 A. A solution of 4.2 g of a-D-acetobromoglucose in absolute dichloromethane was added dropwise at -30C to a solution of 3.0 g of dry 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,oc-D-trehalose in 30 ml of absolute dichloromethane and 2.5 ml of 2s absolute tetramethylurea in the presence of 2.61 g of dry silver trifluoromethanesulphonate. After stirring at room temperature for 20hours a further addition of 3.44g of a-D-acetobromo-glucose in 5 ml of dichloromethane, 2 ml of tetramethylurea and 1.75 g of silver trifluoromethanesulphonate was carried out at -30 30~C. After stirring at room temperature for 18 hours themixture was filtered and washed with dichloromethane. The organic solutions were washed with sodium bicarbonate solution and water and dried over magnesium sulphate. Chromatography on silica gel with toluene/ether 6:1 as the eluent gave 64% o~ pure 35 4'-0-(2,3,4,6-tetra-O-acetyl-~-D-glucopyranosyl)-2,2',3,3',6-penta-O-benzylidene-a,a-D-trehalose as a syrup, [a]2D = +53.0 (c = 0.2, dioxan~.

.. .... , . . .. . .. , . .. ... _ , . , .. . . . .. . _ _ . _ . .. _ . _ .. _ _ . _ _ _ ... . _ _ _ _ . _ . _ _ _ 12 2~$~
B. A solution of 1.14 g of 4'-0-(2,3,4,6-tetra-O-acetyl-~-D-glucopyranosyl)-2,2',3 ,3 ',6'-penta-O-ben~yl-4,6-O-ben~ylidene-a,a-D-trehalose in 60 ml of absolute methanol and 20 ml of absolute cycloh~xane (20 ml) was treated with 1.4 ml of 2%
5 sodium methylate solution. After 4 hours at room temperature the mixture was neutralized with acidic ion exchanger, filtered, concentrated and chromatographed on silica gel with e~hyl acetate/methanol/water 95:1:1 as the eluent, whereby 840 mg of product were obtained. A 370 mg a]liquot was hydrogenated at 10 room temperature in 60 ml ethanol/water 5:1 in the presence of 10% palladium-on-charcoal. After 90 minutes the mixture was filtered, washed with ethanol and water and concentrated. There was obtained a quantitative yield of 4-O-(~-D-glucopyranosyl)-a,a-D-trehalose, [a]2D = +121.9 ~c = 0.2, H20).
C A solution of 840 mg of well-dried 4-O-(,~-D-gluco-pyranosyl)-a,a-D-trehalose in 30 ml absolute dimethyl-formamide was stirred at 50C for 20 hours in the presence of 5.08 g of sulphur trioxide-trimethylamine complex, whereby a 20 viscous syrup separated. The solvent was decanted off, the residue was washed with methanol, dissolved in 30 ml of 10%
sodium acetate solution and concentrated. The residue was taken up several times in water and evaporated in order to remove trimethylamine. The residue was purified by gel chromatography 2s (Sephadex~' LH 20) in order to remove salts. After freeze-drying there were obtained 2.0 g of sulphated 4-O-(,I~-D-glucopyranosyl) -a,a-D-trehalose, S = 20.40%, AS about 3Ø
.

Example 3 A. A solution of 1.19 g of hepta-O-acetyl-a-D-maltosyl bromide (J. Chem. Soc. 1962, 2823) in 5ml of absolute dichloro-methane was added dropwise at 0C to a solution of 1.0 g of well-dried 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-3s trehalose in 6 ml of absolute dichloromethane and 0.22 ml oftetramethylurea in the presence of 0.43 g of silver trifluoro-methanesulphonate. After 8 hours at room temperature 109 mg of silver triflate, 0.05 ml of tetramethylurea and 2g8 mg of .. . .. . . .... . . _ _ _ _ 2 ~ ~ ~ T3 ~3 ~

maltosyl bromide were added. After stiTring at room temperature for 18 hours the mixture was filtered and washed with dichloro-methane. The combined organic solutions were washed with sodium bicarbonate solution and water and dried over magnesium 5 sulphate. Chromatography on silica gel with ethyl acetate/hexane 1:1 as the eluent gave 1.49 g (88%) of pure 4'-O-(hepta-O-acetyl-~-D-maltosyl)-2,2',3,3 ',6'-penta-O-benzyl-4,6-O-beslzylidene-O-a,a-D-trehalose as a syrup, [a]20= +84.0 (c = 0.2, dioxan).

o B. A solution of 1.43 g of 4'-(hepta-O-acetyl-~-D-maltosyl)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,oc-D-trehalose in 15 ml of absolute methanol and 4 ml of absolute cyclohexane was treated with 6 ml of 2% sodium methylate solution. After 1 minutes at room temperature the mixture was neutralized with acidic ion exchanger, filtered and concentrated. The crude product was dissolved in 20 ml of ethanol/water (3:1) and hydrogenated at room temperature for 2 hours with 275 mg of 10% palladium-on-charcoal. Filtration over a filter aid and rinsing gave pure 4-O-(~-D-maltosyl)-a,oc-D-trehalose, (635 mg), [a]20= +150.0 (c = 0.2, ~0 H20)-C A solution of 1.0 g of well-dried 4-O-(~-D-maltosyl)-a,a-D-trehalose in 2~ ml of absolute dimethylformamide was stirred at 50C for 18 hours in the presence of 5.845 g of sulphur trioxide-25 trimethylamine complex, whereby a viscous syrup separated.
Working-up as described in Example IG. gave 2.63 g of sulphated 4-O-~,B-D-maltosyl)-a,a-D-trehalose, S = 19.91%, AS about 2.8.

Example 4 A. A solution of 6.67 g of deca-O-acetyl-a-D-maltotriosyl bromide (Carbohydr. Res 48, 197 (1976)) in 40ml of absolute of dichloromethane was added dropwise at -30C to a solution of 4.0 g of well-dried 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzyl-35 idene-a,a-D-trehalose in 3~ ml of absolute dichloromethane and 3.1 ml of tetramethylurea in the presence of 1.74 g of silver trifluoromethanesu lphonate. After 3 hours at room temperature 3.4 g of deca-O-acetyl-a-D-maltotriosyl bromide in 20 ml of 2 ~ 8 ~

absolute di~hloromethane and 1 g of 4A molecular sieve were added at -30OC. The mixture was worked-up after 90 hours.
Chromatography on silica gel with ethyl acetate/hexane 3 :7 as the eluent gave 4.03 g (50%) of 4'-(deca-O-acetyl-~-D-maltotriosyl)-5 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalos~ as a syrup, [a]20= +100.5 (c = 0.2, dioxan).

B. A solution of 3.46 g of 4'-deca-O-acetyl-,B-D-maltotriosyl)-2,2',3,3',6'-penta-O-~enzyl-4,6-O-benzylidene-a,a-D-trehalose in 10 45 ml absolute methanol was stirred at room tempera~ure for 24 hours in the presence of a catalytic arnount of anhydrous sodium carbonate. After filtration and neutralization with acidic ion exchanger the mixture was concentrated and chromatographed on silica gel with ethyl acetate/methanol/ water ~5:10:5 as the 15 eluent. The product fraction (2.29 g, 86%) was dissolved in 24 ml of ethanol/water (5:1) and hydrogenated at room temperature for 3 hours in the presence of 10% pallidium-on-charcoal. Filtration over a fil~er aid and rinsing gave pure 4-O-(,~-~-maltotriosyl)-a,a-D-trehalose as a foam (1.21 g, 100%), [a]2D=
20 +172.5 (c=0.2, H2O).

C A solution of 500 mg of well-dried 4-O-(,~-D-maltotriosyl~-a,a-D-trehalose in 10 ml of absolute dimethylformamide was stirred at 50C for 18 hours in the presence of 2.85 g of sulphur 2s trioxide-trimethylamine complex, whereby a viscous syrup separated. Working-up as described in Example lG. gave 1.32 g of sulphated 4-O-(,I~-D-maltotriosyl)-a,a-D-trehalose, S =19.5%, AS
about 2.6.
Example 5 A. A solution of 1.19 g of dry hepta-O-acetyl-a-D-cellobiosyl bromide (Ann. 435~ 1 (1923)) in 5 ml of absolute dichloromethane was added dropwise at -30C to a solution of 1 g of well-dried 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-3s trehalose in 1 ml of absolute dichloromethane and 0.3 mltetramethylurea in the presence of 442 mg of silver trifluoromethanesulphonate. After stirring at room temperature for 60 hours the mixture was filtered over a filter aid, rinsed with , . _ ... .. . _ _ . _ __ __. _ . _. .. . __ _ _ ~_ .. _ . .. __ ~ ,_ _ , . ..... ~ ~

2~fi~3~.'1.

dichloromethane, evaporated and chromatographed on silica gel with dichloromethane/acetone 24: 1 as the eluent. There were obtained 1.51 g (g8%) of pure 4'-O-(hepta-O-acetyl-~-D-cellobiosyl)-2,2',3.3 ',6'-penta-O-benzyl-4~6-O-benzylidene-o~,oc-D -s trehalose, [O~]2D= +35.0 (c = 0.3, CHC13) B. A solution of 1.0 g of 4'-O-(hepta-O-acetyl-,B-D-cello-biosyl)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-o~,a-D -trehalose in 30 ml absolute methanol was stirred at room o temperature in the presence of a catalytic amount of sodium (a few mg). After 2 hours the mixture was neutralized with acidic ion exchanger, evaporated and filtered over silica gel in chloro-form/methanol 9:1. Evaporation gave a deacetylated crude product (764 mg) of which 168 mg were hydrogenated at room 15 temperature in 10 ml of ethanol/water (4:1) in the presence of 10% palladium-on-charcoal. After 2.5 hours the mixture was filtered over a filter aid, rinsed an evaporated. There were obtained 93 mg (100%) of 4'-O-(,B-D-cellobiosyl)-oc,~-D-trehalose, [a]2D= +97 (c = 0.3, H2O).
C A solution or 72 mg of 4'-O-(,B-D-cello~iosyl)-c~,a-D-trehalose in 5 ml of anhydrous dimethylformamide was stirred at 5C for 20 hours in a presence of sulphur trioxide-trimethylamine complex, whereby a viscous syrup separated. Working-up as 2s described in Example lG. gave 176 mg of sulphated 4-O-(,I~-D-cellobiosyl)-(x,a-D-trehalose, S = 18.2%, AS about 2.7.

Example 6 30 A. 2.19 ml of bis-tributyltin oxide were added to a suspension of 1.0 g of 2,2',3,3'-tetra-O-ben~yl-a,o~-D-trehalose (Chem. Pharm.
Bull. 3 0, 1169 (1982)) in 80 ml of toluene and the mixture was heated under reflux on a water separator for 3.5 hours. Thereby, the volume was reduced by 40 ml. Then, 157 mg of 3~ tetrabutylammonium bromide and 0.84 ml of benzyl bromide were added and the mixture was stirred at 80C for 30 hours.
After the addition of the same amounts of tetrabutylammonium bromide and benzyl bromide the mixture was stirred at 80C for a , . . . . _ . .. .. .. . ..... .. . . .. _ _ _ _ _ . _ .. .. _ . .. _ _ . _ . .. _ _ _ _ . _ . _ . .

further 72 hours. The reaction mixture was poured into ice-water/ methylene chloride and extracted with ~,vater and sodium bicarbonate solution. After evaporating the organic phase the residue was chromatographed on silica gel with ethyl acetate/
5 hexane 1 :2 as the eluent. There was obtained pure 2,2'3,3',6'-hexa-O-benzyl-a,a-D-trehalose (1120 mg, 89%) as a syrup, [a]20 =
+111.5 (c = 0.5, dioxan).

B. A solution of 2.38 g of hepta-O-acetyl-a-maltosyl bromide 10 in 12 ml of anhydrous dichloromethane was added at -30C to a suspension of 1.0 g of 2,2',3,3',6,6'-hexa-O-benzyl-oc,a-D-trehalose and 0.88 g of silver triflate in 3 ml of anhydrous dichloromethane and 0.61 ml of tetramethylurea. ~fter 10 days at room temperature the mixture was filtered over a filter aid, 5 washed with dichloromethane, evaporated and the crude product was chromatographed on silica gel with acetone/hexane 2:3 as the eluent. There were obtained 1.96 g of pure 4,4'-bis-O-(hepta-O-acetyl-~-D-maltosyl)-2,2',3,3',6,6'-hexa-O-benzyl-a,a-D-trehalose (82%), melting point 85C, [a]2D= +84.5 (c = 0.2, chloroform).
C A catalytic amount of sodium methylate was added at room temperature to a solution of 1.0 g of 4,4'-bis-O-(hepta-O-acetyl-~-D-maltosyl)-2,2',3,3',6,6'-hexa-O-benzyl-a,a-D-trehalose in 40 ml of methanol/dioxan (1:1). After 4 hours the mixture was 2s neutralized with acidic ion exchanger, filtered and evaporated.
The deacetylated compound was dissolved in 50 ml of ethanol/
water (1:1) and hydrogenated at room temperature in the presence of 300 mg of 10% palladium-on-charcoal. After 1 hcur the mixture was filtered over a filter aid, washed with ethanol/
30 water (1:1) and evaporated. There were obtained 465 mg of pure 4,4'-bis-O-(~-D-maltosyl)-a,oc-D-trehalose, [a]20= +148 (c = 1.3, H20).
D. A solution of ~.3 g of 4,4'-bis-O-(~-D-maltosyl)-a,a-D- ;
35 trehalose in 15 ml of anhydrous dimethylformamide was stirred at 5~C for 20 hours in the presence of 1.685 g of sulphur trioxide-trimethylamine complex, whereby a viscous syrup separated.
Working-up as described in Example lG. gave 652 mg of .. . . , . . . . . . .. . .. .. _ . _ . . . .. . .. , _ .... , . , . , _ . . , . . , _ 2~5~-~3 sulphated 4,4'-bis-O-(,B-D-maltosyl)-a,a-D-trehalose, S - 18.93%, AS = 2.5.

Example 7 A. 0.21 ml of trifluoromethanesulphonic anhydride was added under argon to a cooled solution (-80C) of 618 mg of 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl trichloroacetimidate and 553 mg of 2,2',3,3',6,6'-hexa-O-benzyl-a,a-D-trehalose in 5 ml of absolute o dichloromethane. After 2.5 hours at -10C a further 123 mg of 2,3,4,6-tetra-O-acetyl-D-glucopyranosyl trichloro- acetimidate and 41 ml of trifluoromethanesulphonic anhydride were added at -80C. The mixture was warmed to -10C, triethylamine was added and the solvent was evaporated. After co-distillation with 15 toluene the residue was chromatographed on silica gel with toluene/ethyl acetate 7:3 as the eluent and there were obtained 832 mg (86%) of pure 4,4'-bis-0-(2,3,4,6-tetra-O-acetyl-~-D-glucopyranosyl)-2,2',3 ,3 ',6,6'-hexa-O-benzyl-a,a-D-trehalose [a]2D =
+ 42 (c = 0.3, chloroform).
~0 B. A solution of 696 mg of 4,4'-bis-0-(2,3,4,6-tetra-O-acetyl-,B-D-glucopyranosyl)-2,2',3,3',6,6'-hexa-O-benzyl-a,oc-D-trehalose in 40 ml of anhydrous methanol was treated with a catalytic amount of sodium. After 20 minutes at room temperature the 2s mixture was neutralized with acidic ion exchanger, filtered and evaporated. Filtration over silica gel with chloroform/methanol 9:1 as the eluent gave 480 mg of deacetyl-ated compound which was hydrogena~ed at room temperature in 50 ml of ethanol/water (4:1) in the presence of 200 mg of 10% palladium-30 on-charcoal. After 2 hours the mixture was filtered over a filter aid, washed with ethanol/water (1:1) and evaporated. There were obtained 250 mg of 4,4'-bis-O-(~B-D-glucopyranosyl)-a,a-D-trehalose [a]20= ~108 (c = 0.2, H2O).

35 C A solution of 208 mg of 4~4'-bis-O-(,B-D-glucopyranosyl)-a,a-D-trehalose in 5 ml absolute dimethylformamide was stirred at 50C for 20 hours in the presence of 1.22 g of sulphur trioxide-trimethylamine cornplex, whereby a viscous syrup separated.

.. ., . . . . ... .. .. . . .. . .. ... ~
.

2~'33~1 Working-up as described as in Example lG. gave 520 mg of sulphated 4,4'-bis-O-(~-D-glucopyranosyl)-a,a-D-trehalose, S =
19.52%, AS about 2.7.

Example ~, A. 0.52 g of silver trifluoromethanesulphonate and im~nedi-ately thereafter 0.6 ml of absolute tetramethylurea were added at 0-~C to a solution of 2.64 g of high vacuum-dried 2,2',3,3',6'-0 penta-O-benzyl-4,6-O-benzylidene-a,lx-D-trehalose and 1.29 g of 2,3,4-tri-O-acetyl-6-0-(2,3,4-tri-O-acetyl-6-desoxy-a-L -mannopyranosyl~-a-D-glucopyranosyl bromide [Ber. 70, 1098-1101 (1938)] in 20 ml of absolute dichloromethane. After stirring at 0-5C for a further 75 minutes the reaction mixture 5 was suction filtered over Dicalite and rinsed with dichloro-methane, and the filtrate was washed twice with saturated sodium bicarbonate solution, dried over magnesium sulphate and concentrated. The residue was chromatographed on 85 g of silica gel (70-230 mesh) with ethyl acetate/hexane 1 :4, 1 :2 and 1: 1 as 20 the eluent and gave 830 mg (29%) of 0-(2,3,~-tri-O-acetyl-6-desoxy-a-~-mannopyranosyl)-( l ~ 6)-0-(2,3,4-tri-O-acetyl-~-D-glucopyranosyl)-(l ~4)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalose as a foam, [a]20= + 32.2 (c = 0.5, (~HC13).
2s B. 1.5 ml of 1% Na in methanol were added at room temper-ature to a solutiDn of 750 mg of 0-(2,3,4-~ri-O-acetyl-6-desoxy-a-L-mannopyranosyl)-( l ~ 6)-0-(2,3,4-tri-O-acetyl-,~-D-gluco-pyranosyl)-(l ~4)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene- ' 30 oc,a-D-trehalose in 1.5 ml of diethyl ether and 7.5 ml of methanol and the mixture was stirred for a further 3 hours. The reaction solution was then neutralized with acidic ion exchanger (Amberlite IR-120), stirred for 30 minutes, filtered off, rinsed with methanol and the filtrate was evaporated. The residue was 3s chromatographed on 27 g of silica gel (70-230 mesh) with ethyl acetate/methanol/water 98:1:1 as the eluent. 380 mg (62%) of deacylated product were obtained, [a]2D= +7.0 (c =0.1, CHC13).
360 mg (0.303 mmol) of this in 27 ml of ethanol/water 2:1 were .. ... _ . _ _ 2~ ;3 I ~
hydrogenated at room temperature in the presence of 180 mg of 10% palladium-on charcoal. After 14 hours the reaction mixture was suction filtered over Dicalite, rinsed with ethanol/water and the filtrate was concentrated and dried in a high vacuum. There were obtained 205 mg (100%) of 0-(6-desoxy-a-L-mannopyranosyl)-(1~6)-O-~-D-glucopyranosyl-(1~4)-a,oc-D-trehalose as an amorphous powder, [OC]ZD= + g.3 (c = 0.1, ~I2O).

C A solution of 200 mg of 0-(6-d~esoxy-a-L-mannopyranosyl)-0 (1~6)-O-~-D-glucopyranosyl-(l~4)-a,a-D-trehalose in 7 ml of absolute dimethylformamide was treated with 1.72 g of sulphur trioxide-trimethylamine complex and stirred under argon at 60-65C for 20 hours. A resinous precipitate separated during this time. Tbe solvent was evaporated and the residue was 5 dissolved in 11 ml of 10% sodium acetate solution and concentrated in a waterjet vacuum. The residlle was tal~en up several times (lOx) in 50 ml of water each time and evaporated in order to remove triethylamine and was then gel-chroma-tographed (Sephadex~' LH20, 150 g) in order to remove salts. The 20 fractions containing the sulphated tetrasaccharide were evaporated and Iyophilized. There were obtained 320 mg (54%) of sulphated 0-(6-desoxy-1~c-L-mannopyranosyl)-(1~6)-O-,B-D-glucopyranosyl)-(1~4)-a,a-D-trehalose as an amorphous powder, S = 19.62%, AS = about 2.7.
2s Example 9 A. 0.52 g of silver trifluoromethanesulphonate, 1.40 g of 2,3,4-tri-O-acetyl-6-0-(2,3,4,6-tetrta-O-acetyl-a-D-glucopyranosyl)-a-30 D-glucopyranosyl bromide [Wolfram et al, J. Am. Chem. Soc., 71, 125-127, ~1949)] and 0.6 ml of absolute tetramethylurea (5.0 mmol) were added in rapid sequence at 0-5C to a solution of 2.64 g of high vacuum-dried 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalose in 20 ml of absolute 3s dichloromethane and the mixture was stirred at 0-5C for a further 1 hour. The reaction mixture was suction filtered over Dicalite, rinsed with dichloromethane, the filtrate was washed twice with saturated sodium bicarbonate solution, dried over .. .. . .. ... . . . . _ _ _ _ _ ... . .. .. .. .. _ . _. . .. .. ._ ~ _ . ~; ... .. _ . ~ _ _ _ _. .
. , _. _ _ 2 i~

magnesium sulphate, filtered off and concentrated. The residue was chromatographed on 85 g of silica gel (70-230 mesh) with ethyl acetate/hexane 1:4, 1:2 and 1:1 as the eluent. 740 mg (25%) of 0-(2,3,4,6-tetra-O-acetyl-a-D-gluco- pyranosyl)-(1~6)-s 0-(2,3,4-tri-O-acetyl-,B-D-glucopyranosyl)-(1~4~-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-o~,a-D-trehalose were obtained as a foam, [a]2D= + 78.2 (c = O.S, CHC13).

B. 1.32 ml of 1% Na in methanol were added at room temper-o ature to a solution of 660 mg of 0-(2,3,4,6-tetra-O-acetyl-oc - D -glucopyranosyl)-( l ~ 6)-0-(2,3,4-tri-O-acetyl-~-D-gluco-pyranosyl)-(1 ~ 4)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalose in 1.5 ml of diethyl ether and 7.5 ml of methanol and the mixture was stirred at room temperature for a further 15 3 hours. The reaction solution was then stirred with acidic ion exchanger (Amberlite IR-120) for 10 minutes, filtered off and rinsed with methanol. The filtrate was evaporated and chroma-tographed on 27 g of silica gel (70-23û mesh) with ethyl acetate/methanol/water 96:2:2 as the eluent. 420 mg (79%) of 20 deacylated product were obtained, [a]20= +103.0 (c = 0.1, CHC13).
400 mg (0.33 mmol) of this in 30 ml of ethanol/wa~er 2:1 were hydrogenated at room temperature in the presence of 200 mg of 10% palladium-on charcoal. After 14 hours the mixture was ~' suction filtered over Dicalite, rinsed with ethanol/ water and the `
2s filtrate was concentrated and dried in a high vacuum. There were obtained 230 mg (100%) of O-(~-D-glucopyranosyl~-(1~6)-O~ -D-glucopyranosyl)-(1~4)-o~,oc-D-trehalose as an amorphous powder, [a]20= + 133.0 (c = 0.1, H2O).

30 C A solution ~f 213 mg of O-(a-D-glucopyranosyl)-(1~6)-O-(,B-D-glucopyranosyl)-(1~4)-o~,a-D-trehalose in 8 ml of dry dimethylformamide was stirred with 1.80 g of sulphur trioxide-trimethylamine complex at 60-65C for 20 hours, whereby a glassy, resinous precipitate separated. After decanting off the 35 solvent the residue was dissolved in 11 ml of 10% sodium acetate solution and concentrated in a waterjet vacuum. The residue was dissolved several times (12 x) in S0 ml of water each time and evaporated in order to remove triethylamine. In order to remove .

21 20.~3 ~ri~)$ I
salts, the residue was gel-chromatographed (Sephadex~ LH20, 150 g). Product fractions were evaporated and lyophilized. There were obtained 390 mg (~58%) of sulphated O-a-D-glucopyranosyl-(1~6)-0-,B-D-glucopyranosyl-(1~4)-oc,a-D-trehalose as an amorphous powder, S = 20.36%, AS = about 3Ø

Example_ 10 A. 1.52 g of silver trifluoromethanlesulphonate, 4.13 g of 0 hepta-O-acetyl-oc-D-melibiosyl bromide [Jeanes at al, J. Am. Chem.
Soc., Z~, 3667-3672, (1953)] in 15 ml of absolute dichloromethane and 0.76 ml of absolute tetramethylurea were added successiYely and rapidly at 0-5C to a solution of 3.84 g of 2,2',3,3',4,6,6' -hepta-0-benzyl-4,S-O-benzylidene-a,a-D -trehalose 5 [S.Kato, K.Yogo, Bull. Chem. Soc. Jpn., 59, 411-14 (1986)] in 25 ml of absolute dichloromethane. After stirring at 0-5C for a further 2 hours the reaction mixture was suction filtered over Dicalite and rinsed with dichloromethane. The filtrate was washed twice with saturated sodium bicarbonate solution, dried over magnesium 20 sulphate, filtered off, concentrated and chromatographed on 350 g of silica gel (70-230 mesh) with ethyl acetate/hexane 1 :4, 1:3 and 1:2 as the eluent. 2.~3 g (47%~ of pure 4-0-(hepta-0-acetyl-,B-D-melibiosyl)-2,2',3,3',4,6,6'-hepta-0-benzyl-a,a-D -trehalose were obtained as a foam, [a]2=+93.4(c=0.5,CHCl3).
B. A solution of 2.75 g of 4-0-(hepta-0-acetyl-,B-D-melibiosyl)-2,2',3,3',4,6,6'-hepta-0-benzyl-~,oc-D-trehalose in 5.5 ml of diethyl ether and 27 ml of methanol was treated at room temperature with 5.5 ml of 1% sodium in methanol and stirred 30 for 2 1/2 hours. The reaction solution was then stirred with acidic ion exchanger (Amberlite IR-12û) for 10 minutes, filtered off under suction and rinsed with methanol. The filtrate was evaporated and the residue was chromatographed on 75 g of silica gel (70-230 mesh) with ethyl acetate/methanol/water 35 96:2:2 as the eluent. 1.53 g (68%) of deacylated product were obtained, [CC]20= +115 (c = 0.2, CHC13). 1.296g (lOmmol) of this in 40 ml of ethanol/H20 3:1 were hydrogenated at room temperature in the presence of 0.7 g of 10% palladium-on-2 ~22 charcoal. After 8 hours the reaction mixture was suction filtered over Dicalite and rinsed with ethanol/water. The filtrate was concentrated and dried in a high vacuum and gave 0.679 g (100%) of 4-O-(~-D-melibiosyl)-a,oc-D-trehalose as an amorphous s powder, [a]2D= + 143.2 (c = ~.5, H2O).

C A solution of 660 mg of 4-O-(,B-D-melibiosyl)-a,a-D-trehalose in 15 ml of dry dimethylformamide was treated with 4.17 g (30 mmol) of sulphur trioxidle-trimethylamine complex o and stirred at 60-65C for 20 hours, whereby a viscous syrup separated after 3 hours. The solvent was decanted off a~d the residue was dissolved in 25 ml of 10% sodium ace~ate solution and concentrated in a waterjet vacuum. The residue was dissolved several times (12 x) in 50 ml of water each time and 5 concentrated in order to remove triethylamine. In order to remove salts, the residue was gel-chromatographed (Sephadex~' LH20, 150 g). Fractions containing sulphated tetrasaccharide were evaporated and lyophilized. There were obtained 1.59 g (about 80%) of sulphated 4-O-(~-D-melibiosyl)-a,a--D-trehalose, [OC]20 = ,`~
20 +135.2 (c = 1.0, H2O), S = 20.58%, AS = about 3Ø

Example 1 1 A. 0.37 g of absolute N,N,N,N-tetramethylurea and 0.76 g of 25 dry silver trifluoromethanesulphonate were added at 0-5C under argon and with the exclusion of light to a solution of 1.74 g of dry 2,2',3 ,3',6'-penta-O-benzyl-4,6-O-benzylidene-o~,a-trehalose (Carbohydr. Res. 63, 51, (1978)) in 12.5 ml of absolute dichloro-methane. A solution of 1.92 g of acetobromo-D-lactose (J. Am.
30 Chem. Soc. 37, 1270 (1915)) in 7.5 ml of dichloromethane was added dropwise. After stirring at 0-5C for 1.5 hours the reaction solution was filtered over silica gel and washed with dichloromethane. The organic solutions were washed with sodium bicarbonate solution and water and dried over magnesium 3s sulphate. Chromatography on silica gel with ethyl acetate/hexane 1:2 as the eluent gave 1.54 g of pure 4'-O-(hepta-O-acetyl-~-D-lactosyl)-2,2',3,3 ',6'-penta-O-benzyl-4,6-O-benzylidene-a,cc-D -trehalose as a white resin, FAB-MS (1521.2 (M+~a)+).

2~

B. A solution of 1.22 g of 4'-O-(hepta-O-acetyl-~-D-lactosyl)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-oc,a-D-trehalose in 12.2 ml of absolute methanol and 2.5 ml of absolute ether was s treated with 3.1 ml of 1 % sodium methylate solution. After 5.5 hours ar room temperature the mixture was neutralized with acidic ion exchanger, filtered and concentra~ed. Chromatography on silica gel with ethyl acetate/methanol/water 85:10:5 as the eluent gave 0.91 g of pure 4'-O-(~-D-lactosyl)-2,2',3,3',6'-penta-O-0 benzyl-4,6-O-benzylidene-a,a-D-trehalose as a white resin, FAB-~IS (1227.3 (M+Na)+).

C A solution of 0.90 g of 4'-O-(,~-D-lactosyl)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,cc-D-trehalose in 28 ml of ethanol/water 3:1 was hydrogenated at room temperature for 6 hours with 0.5 g of 10% palladium-on-charcoal. Filtration over a filter aid and rinsing gave, after drying in a high vacuum, 0.51 g of pure 4'-O-(~-D-lactosyl)-a,a-D-trehalose as a white resin, FAB-MS (689.0 (M+Na)+).
D. A solution of 0.49 g of well-dried 4'-O-(,B-D-lactosyl)-a,a-D-trehalose in 11.2 ml of absolute dimethylformamide was stirred at 65C for 22 hours in the presence of 3.094 g of sulphur trioxide-trimethylamine complex, whereby a viscous syrup 2s separated. Working-up as described in Example lG gave ].1 g of sulphated 4'-O-(,B-D-lactosyl)-a,a-D-trehalose, S = 19.93%, AS
about 2.9.

Example 12 A. 0.30 g of absolute N,N,N,N-tetramethylurea and 0.625 g of dry silver trifluoromethanesulphonate were added at 0-5C under argon and with the exclusion of light to a solution of 1.43 g of dry 2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalose 3s (Carbohydr. Res. 6~, 51, (1978)) in lO.Oml of absolute dichloromethane. A solution of 1.70 g of acetobromo-D-gentiobiose (J. Am. Chem. Soc. 49, 3170 (1927)) in 6.0ml of methylene chloride was added dropwise. After stirring at room ,, . ... . . . . .. . . . . .. . . ....... . . . ... .. . .. ... .. . . _ . . . . . . .. ..... . _ _ _ 2 ~ l3 ~r~

temperature for 20 hours the reactis)n solution was filtered over silica gel and washed ~vith dichloromethane. The organic solutions were washed with sodium bicarbonate solution and wate~ and dried over magnesium sulphate. Chromatography on silica gel s with ethyl acetate/toluene 1:2 as the eluent gave 1.22 g of pure 4'-O-(hepta-O-acetyl-~-D-gentiobiosyl)-2,2',3,3',- 6'-penta-O-benzyl-4,6-O-benzylidene-a,a-trehalose as a white resin, ~AB-MS
(1521 .2 (M+Na)+).

B. A solution of 1.22 g of 4'-O-(hepta-O-acetyl-,~-D-gentio-biosyl)-2,2',3 ,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D -trehalose in 12.0 ml of absolute methanol and 2.5 ml of absolute ether was treated with 2.44 ml of 1% sodium methylate solution.
After 5.5 hours ar room temperature the mixture was neutralized l 5 with acidic ion exchanger, filtered and concentrated. Chroma-tography on silica gel with ethyl acetate/methanol/water 85:10:5 as the eluent gave O.50g of pure 4'-O-(,B-D-gen~iobiosyl)-2,2',3,-3',6'-penta-O-benzyl-4,6-O-benzylidene-oc,a-D-trehalose as a white resin, FAB-MS (1227.4 (M+Na)+).
C A solution of 0.48 g of 4'-O-(~-D-gentiobiosyl)-2,2',3,3',6'-penta-O-benzyl-4,6-O-benzylidene-a,a-D-trehalose in 11 ml of ethanol and 3.7 ml of water 3:1 was hydrogenated at room temperature for 6 hours with 0.27 g of 10% palladium-on-25 charcoal. ~iltration over a filter aid and rinsing gave, after dryingin a high vacuum, 0.26 g of pure 4'-O-(,B-D-gentiobiosyl)-a,a-D-trehalose, as a white resin, FAB-MS (689.0 ((M+Na)+).

D. A solution of 0.20 g of well-dried 4'-O-(~-D-gentiobiosyl)-30 c~,a-D-trehalose in 5.0 ml of absolute dimethylformamide was stiIred at 65C for 22 hours in the presence vf 1.26 g of sulphur trioxide-trimethylamine complex, whereby a viscous syrup separated. Working-up as described in Example lG gave 0.50 g of sulphated 4'-O-(,B-D-gentiobiosyl)-a,a-D-trehalose, S = 20.25%, 3s AS about 3.3.

, . . .. . . . . .. . .... . . . . . .. .... .. . . .. . ~ . . . , . . . ~ . . . .

2 '') ~ ~ ~ ? 1 E~xample 13 A. 50 ml of acetic anhydride were added to a suspension of S g of 3-O-~B-D-galactopyranosyl-D-arabinose in 75 ml of 5 pyridine and the mixture was stirred at room temperature for 6 honrs. The reaction solution was concentrated in a waterjet vacuu n at 140C bath temperature. The syrupy residue was treated with 200 ml of ice-wa~er andl extracted twice with 200 ml of e~hyl acetate each time. The extracts were washed twice with 0 cold 5% H2SO4, twice with saturated sodium bicarbonate solution and once with saturated sodium chloride solution, dried over magnesium sulphate, filtered of~ and evaporated and dried in a high vacuum overnight. There were obtained 8.84 g (91~o) of 1 ,2,4-tri-O-acetyl-3 -0-(2,3,4,6-tetra-O-acetyl-,B-D -5 galactopyranosyl)-D-arabinose (according to NMR the mixture also contained furanose derivative). 7.6 g thereof were dissolved in 15 ml of dichloromethane, cooled to 0C and treated within lS' - with 45 ml of 33% hydrobromic acid in acetic acid and stiTred at 0C for a further 2hours. The reaction mixture was poured on to 20 250 ml of ice-water and extracted three times with 100 ml of dichloromethane each time. The extracts were washed twice with 100 ml of ice-water each time and twice with 100 ml of cold saturated sodium bicarbonate solution each time, dried over magnesium sulphate, filtered off and evaporated at <25C. The 2s residue was chromatographed on 200 g of silica gel (70-230 mesh) with dichloromethane/diethyl ether 9:1 and 4:1 as the eluent. There were obtained 4.15 g (53%) of 2,4-di-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-,i~-D-galacto- pyranosyl)-,l~-D-arabinopyranosyl bromide in the form of a foam, [a]20= -148.8 (c 30 = 0.5, CHC13).

B. 2.0 ml of absolute tetramethylurea and 1.55 g of silver trifluoromethanesulphonate were added in succession at 0-5C to a solution of 3.52 g of high vacuum-dried 2,2',3,3',6'-penta-O-35 benzyl-4,6-O-benzylidene-a,a-D-trehalose and 3.76 g of 2,4-di-O
acetyl-3 -0-(2,3,4,6-tetra-O-acetyl-~-D-galactopyranosyl)-,B-D -arabinopyranosyl bromide in 30 ml of absolute dichloro-methane. After ome hour at 0-~C the reaction mixture was 3 ~

sucti~n filtered over Celite, rinsed with dichloromethane, the f;ltrate was washed twice with saturated sodium bicarbonate solution, dried over magnesium sulphate, filtered off and evapor-ated. The residue was chromatographed on 190 g of silica gel (70-s 230 mesh) with ethyl acetate/hexane 1 :2 and 1: ] as the eluent.There were obtained 3.33 g (58%) of 0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl)-(1 ~ 3)-0-(2,4-di-0-acetyl-a-D -arabinopyran osyl)- (1 ~ 4) -2,2' ,3,3 ' ,6'-penta-0- ben~yl-4,6- 0 -benzylidene-a,a-D-trehalose as a foam, [a]20= +42.0 (c = 0.5, I o CHC13).

C 3.4ml of 1% Na in methanol were added to a solution of 3.4 g of 0-(2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl)-(1~3)-0-(2,4-di-0-acetyl-a-D-arabinopyranosyl)-(1 ~4)-2,2',3,3',6'-penta-S 0-benzyl-4,6-0-benzylidene-a,a-D-trehalose in 7 ml of diethyl ether and 35 ml of methanol and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was neutralized with ion exchanger (Amberlite IR-120), stirred for 15 minutes, filtered 03ff, rinsed with methanol and the filtra~e was evaporated.
20 The residue was chromatographed on 90 g of silica gel (70-230 mesh) with ethyl acetate/methanol/ water 93 :5 :2 as the eluent.
1.93 g (69%) of deacylated product were obtained as a foam, ~a]23 = +52.0 (c = 0.5, CHC13). 1.80 g (1.53 mmol) of this in 60 ml of ethanol/water 3: 1 were hydrogenated at room temperature in the 2s presence of 1.0 g of 10% palladium-on charcoal. After 16 hours the reaction mixture was suction filtered over 1:3icalite, rinsed with ethanol/water and ~he filtrate was evaporated and dried. There were obtained 1.12 g of a product which, for ~urther purification, was acetylated using pyridine (22 ml) and acetic anhydride (11 30 ml). After 22 hours at room temperatule the reaction mixture was evaporated and chromatographed on 85 g of silica gel (70-230 mesh) with ethyl acetate/hexane 1: 1 and 2: 1 as the eluent. There were obtained 1.06 g (59%) of 0-(2,3,4,6-tetra-0-acetyl-,l~-D-galactopyranosyl)-(1 ~3)-0-(2,4-di-0-acetyl-a-D-35 arabinopyranosyl)-(1~4)-hepta-0-acetyl-a,a-trehalose as a foam, [a] D = + 68.00 (c = 0.4, CHC13). 1.02 g (0.86 m3mol) of this were dissolved in 30 ml of methanol/ dimethoxyethane/water l:l:1 and treated dropw3ise with 1 % sodium methylate solution (pH

. ... ., . . .... . . .. . _ . _ .. . . . .. ... _ _ _ _ _ _ _ _ .

2 ~ 3 :1 1~-13) until saponification was complete. After 6 hours the reaction solution was neutralized with ion exchanger (Amberlite IR-120), stirred for 15 minutes, suction filtered, rinsed with methanol/water and the filtrate was evaporated and dried in a s high vacuum. There were obtained 480 mg (88%) of O-~-D-galactopyranosyl-(1~3)-O-a-D-arabino- pyranosyl-(1~4)-a,a-trehalose as an amorphous powder, [a]20= + 9.4O (c = 0.1, H2O).

D. A solution of 440 mg (0.69 mmol) of O-,B-D-galacto-o pyranos yl- ( 1 ~ 3 ) - O-a -D- arabinopyran osyl -(1~ 4)-a, a-trehalose in 15 ml of dry dimethylformamide was treated with 2.87 g (20.7 mmol) of sulphur trioxide-trimethylamine complex and stirred at 60-65C under argon for 20 hours. During this time a viscous precipitate separated. Working-up as described in 5 Example lG gave 960 mg (about 75%) of sulphated O-~-D-galactopyranosyl-(1~3)-O-a-D-arabinopyranosyl-(1~4)-a,a-trehalose as an amorphous powder, [a]2D= +158.8 (c = 0.5, H2O), S
= 19.24%, AS about 3Ø

Example 14 For the production of an injection solution, 5 mg of a compound of formula I and 9 mg of sodium chloride are dissolved in water ad 1 ml. The solution is treated with ascorbic acid 2s (0.5 mg/ml) and benzyl alcohol (0.1 mg/ml) and then filtered s~erile.

Claims

1. Compounds of the formula I

wherein R is hydrogen or a residue -SO3M; M is a cation; R is a an equatorially or quasiequatorially linked sulphated mono-, di- or trisaccharide residue; or an axially linked sulphated trisaccharide residue; and R'' is hydrogen or an equatorially or quasiequatorially linked sulphated mono or disaccharide residue;
whereby the molecule contains a maximum of 6 monosaccharide units and on average at least one -SO3M group is present per monosaccharide unit.

2. Compounds of formula I in accordance with claim 1, wherein R is hydrogen or a residue -SO3M; M is a cation; R' is a .beta.-glycosidically linked sulphated mono-, di- or trisaccharide residue; or an a-glycosidically linked sulphated trisaccharide residue; and R" is hydrogen or a .beta.-glycosidically linked sulphated mono- or disaccharide residue; whereby the molecule contains a maximum of 6 monosaccharide units and on average at least one -SO3M group is present per monosaccharide unit.

3. Compounds according to claim 1, wherein R' is a sulphated .beta.-glucosyl, .beta.-maltosyl, .beta.-cellobiosyl or .alpha.- or ,.beta.-maltotriosyl residue.

4. Sulphated 4-O-(.alpha.-D-maltotriosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4-O-(.beta.-D-glucopyranosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4-O-(.beta.-D-maltosyl)-.alpha.,.alpha.-D-trehalose;

sulphated 4-O-(.beta.-D-maltotriosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4-O-(.beta.-D-cellobiosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4,4'-bis-O-(.beta.-D-maltosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4,4'-bis-O-(.beta.-D-glucopyranosyl)-.alpha.,.alpha.-D-trehalose.

5. Sulphated O-(6-desoxy-.alpha.-L-mannopyranosyl)-(1?6)-O-.beta.-D-glucopyranosyl)-(1?4)-.alpha.,.alpha.-D-trehalose;
sulphated O-.alpha.-D-glucopyranosyl-(1?6)-O-.beta.-D-glueopyranosyl-(1?4)-.alpha.,.alpha.-D-trehalose;
sulphated 4-O-(.beta.-D-melibiosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4'-O-((.beta.-D-lactosyl)-.alpha.,.alpha.-D-trehalose;
sulphated 4'-O-((.beta.-D-gentiobiosyl)-.alpha.,.alpha.-D-trehalose;
sulphated O-.beta.-D-galactopyranosyl-(1?3)-O-.alpha.-D-arabinopyranosyl-(1?4)-.alpha.,.alpha.-trehalose.

6. Compounds of formula I as in claim 1 for use as medicaments, especially for the treatment and prevention of arteriosclerotic disorders.

7. A process for the manufacture of compounds of formula I, as in claim 1 which process comprises treating a corresponding tri-, tetra-, penta- or hexasaccharide with a sulphating agent and isolating the reaction product as the salt.

8. Pharmaceutical preparations, containing a compound of formula I as in claim 1 and usual pharmaceutical adjuvants.

9. The use of compounds of formula I as in claim 1 as active ingredients for the manufacture of pharmaceutical preparations for the treatment of arteriosclerotic disorders.

10. Compounds of formula I as in claim 1 whenever prepared by the process of claim 7 or by an obvious chemical equivalent thereof.

11. The invention as hereinbefore described, especially with reference to the Examples.

12. A method of treating or preventing arteriosclerotic disorders in man which comprises administering an effective amount of a compound of formula I to a patient in need of such treatment.