SU1694065A3 - Method of obtainine pentasaccharide - Google Patents

Description

sulfating agent of the type indicated above at pH 9.5 in an aqueous medium, followed by saponification of group A and the isolation of the target product in the form of a sodium salt on an ion exchange resin in sodium form. Priority featured:

01.02.82 during the synthesis of PI disaccharide), 28.05.82 during the condensation of monosaccharide IV with glucuronic acid,

08/06/82 in the synthesis of disaccharide (I), the interaction of disaccharides (II) and (III), polypentasaccharide (I).

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 i C, t Ie-bretne btn is related to a method for producing oligosaccharides, fragments of mucopolysaccharide acids, in particular to a method for producing pentasaccharide of the general formula OR COO OR

NHR

NHR

OR j-CU

: 00 uch / ik Uchon

 OR -NHR

where OR1 is the OH group or

R is a hydrogen atom or a group, in the form of a sodium salt.

The aim of the invention is to obtain natural fragments of mucopolysaccharide acids using chemical reagents of a given structure containing active groups capable of glycosylation reaction, as well as blocking groups allowing the introduction of functional groups

Example 1. Preparation of the disaccharide 20 of the general formula (II) or 1-bromo-3,6-dm-0-acetyl-2-azido-4-0-methyl- (2,3-di-0-benzyl-4 - 0-chloroacetyl - / 3 -D-glucopyranosyl) uroate} -a-D-glucopyranoside.

a) Preparation of allyl-a-D-glucopyranoside (compound 1).

A solution of gaseous hydrogen chloride (18 g) in allyl alcohol (600 ml) is heated to 70 ° C. Anhydrous glucose (300 g) is then added and kept at this temperature for 3 hours.

The reaction was monitored by thin layer chromatography (TLC) in methanol / chloroform (1/4 by volume). The solution obtained after 3 hours of brown color is concentrated to dryness under vacuum, neutralized with concentrated ammonia solution (50 ml), then concentrated again to dryness. Acetone (500 ml) is added to the obtained residue, brought to a boil and allowed to dissolve. After cooling, the liquid is decanted. The residue is again subjected to the same treatment until

TLC of the extract will not show the depletion of the residue composition by derivative 1 or too high contamination of the extract with impurities. Part of the extracted first fraction

(12 g) chromatographic on silica. The derivative 1 is recovered, which can be crystallized from an acetone / ether mixture (6.5 g, mp 95-99 ° C). The residue of the product can be purified in the same way.

b) Blocking of the positions 4 and 6 of the allyl derivative, resulting in allyl-4.6-0-benzylidene-a-D-glucopyranoside (compound 2).

Compound 1 (37 g) was dissolved in dimethylformamide (200 ml). Dimethoxytoluene (41 g) was added, followed by hydrated p-toluenesulfonic acid (130 mg). After heating in a water bath under vacuum and in the presence of phlegm

the reaction ends. TLC in methanol / chloroform (2/25 by volume). The solvent is evaporated. The syrup is dissolved in methanol (the minimum amount, this solution is poured dropwise into an aqueous solution of sodium bicarbonate (6.3 g in 320 ml of water). The resulting precipitate is recrystallized from ethanol (21 g, mp. 120- 121 ° C.) 8 stock solutions give another product 2. Total yield 37 g (71.4%).

c) Introduction of a benzyl group, leading to allyl-2,3-di-0-5enzyl-4,6-0-benzidene-α-D-glucopyranoside (compound 3).

Compound 2 (45 g) was dissolved in non-aodic dimethylformamide (500 ml). Sodium hydride (18 g of a 50% dispersion in oil) is added. After 30 minutes, the mixture was cooled to 0 ° C and benzyl bromide (52 ml) was added dropwise. The reaction is monitored by TLC (ether / hexane, 1/1 by volume). Methanol (150 ml) is then slowly added, evaporated to dryness and taken up in chloroform. The chloroform phase is washed with water, dried over sodium sulfate. After evaporation of the solvent, the residue is crystallized from ether / hexane (36.5 g, mp. 83-84 ° C). This product is slightly contaminated with impurity migrating upward in TLC (ether / hexane, 1/1 by volume).

0

d) Removal of the benzylidene group, resulting in allyl-2,3-di-0-benzyl-a-D-glucopyranoside (compound 4).

Water (450 ml) is added to a solution of compound 3 (56 g) in methanol (1 l), then hydrated p-toluenesulfonic acid (17 g). After 2 hours at 80 ° C, the mixture was allowed to cool, the solvent was evaporated and the residue was dissolved in chloroform (1 L). The chloroform solution is washed with water until neutral pH, then dried over sodium sulfate. Thus, a light yellow color syrup (48 g) is obtained, which is used to synthesize compound 5.

e) Introduction of the triphenylmethyl group at position 6 followed by acetylation of position 4, which leads successively to allyl-2,3-di-0-benzyl-b-0-triphenylmethyl-a-D-glucopyranoside (compound 5) and its 4- 0-acetylated analogue (compound 6a).

The resulting derivative 4 (48 g) is dissolved in pyridine (250 ml) and triphenylmethyl chloride (38.5 g) is added. After 1 h at 100 ° C, the reaction is complete (TLC, ether / hexane, 1/1 by volume ). Acetic anhydride (200 ml) is added to the previous solution. After overnight, the reaction is ended (TLC, ether / hexane, 1/2 in volume). It is evaporated to dryness, the residue is dissolved in chloroform (500 ml), the chloroform phase is washed with a 10% potassium bisulfate solution, water and dried over sodium sulfate. The chloroform is evaporated. Compound 6a is obtained, which is used to prepare compound 7a.

(e) Removal of the triphenylmethyl group, resulting in allyl 4-0-acetyl-2,3-di-0-benzyl-a-D-glucopyranoside (compound 7a).

The resulting derivative 6a is dissolved in chloroform (500 ml). In this solution, cooled to 0 ° C with stirring, a solution of boron threefurture in methanol (20%, 120 ml) is added dropwise. The reaction is monitored by TLC (toluene / acetone, 10/2 by volume). The reaction mixture is poured into a separatory funnel. The chloroform phase is washed with water (2x100 ml), saturated sodium bicarbonate solution, then with water until neutral pH. After drying and evaporation, the residue obtained is introduced into a column of silica gel (500 g) equilibrated in toluene. After most impurities are eluted with pure toluene, the product is eluted with a toluene / acetone mixture (10/2 by volume). Thus, 48 g of compound 7a is obtained, which is directly introduced into the synthesis of compound 8a. Connection Part 7a

get a pure form, a D + -11 ° (chloroform). Its PC and NMR spectra, as well as elemental analysis, confirm the structure.

g) Oxidation of the alcohol primary 5 group in position 6, resulting in allyl 4-0-acetyl-2, 3-di-0-benzyl-a-D-glucopyranosypuronic acid (compound 8a).

A solution of compound 7a (48 g) in acetone 0 (300 ml) is cooled to -50 ° C. Then a solution of chromium trioxide (30 g) in sulfuric acid (3.4 M, 125 ml) is added dropwise. The mixture was allowed to rise to room temperature. The reaction is monitored by TLC (methanol / chloroform, 1/10 by volume). Upon completion of the reaction, the reaction mixture was poured into water (500 ml). The product is extracted with chloroform (3x250 ml). The chloroform phase is washed with water until the pH is neutral, dried over sodium sulfate and concentrated to dryness. The resulting syrup (83 g) is used to prepare compound 9a.

h) Methylation of the carboxyl group 5 in position 6, resulting in methyl- (allyl-40-acetyl-2, 3-di-0-benzyl-a-D-glucopyranoyl) -uronate (compound 9a).

The syrup obtained in the preparation of compound 8a is dissolved in ether (300 ml).

0 Then an ether solution of diazomethia is added until compound 8e disappears (TLC, ether / hexane, 1/1 by volume). After acidification with acetic acid, the solvents are evaporated. The resulting residue (53 g)

5 is dissolved in hot ethanol. Derivative 9a crystallizes upon cooling. After recrystallization, compound 9a (13.4 g) is obtained, m.p. 85-86 ° C a. (, 2, chloroform). This

0 product is characterized by IR, NMR spectra and elemental analysis.

An additional 7.6 g of compound 9a is obtained from the filtrate after crystallization. The overall yield of compound 9a from compound 2 of co5 is 38%.

i) Incorporation of the propenyl group into position 1, resulting in methyl- (prop-1-en11l-4-0-acetyl-2,8-di-0-benzyl-a-0-glucopyranosyl) -uronate (com. 0. 10a).

Derivative 9a (4g) is dissolved in a mixture of ethanol (119 ml) and benzene (52 ml) and water (17 ml). Then diazabicyclo-5 tan (170 mg) is added and brought to the boil under reflux. Tris- (triphenylphosphine) - rhodium -1 (550 mg) chloride is added to the boiling solution. Boiling is maintained for 4 hours (TLC. Ether / hexane, 1/1 by volume).

At the end of the reaction, the solution is filtered and the solvents are removed. The residue is chromatographed on silica gel (150 g) in ethyl acetate / chloroform (1/50 by volume). Compound 10a (3.25 g, 81%) is obtained, which is crystallized from ethanol. G (, chloroform); m.p. 90 ° C. The structure is confirmed by elemental analysis and NMR and IR spectra.

j) Removal of the acetyl group in position 4, resulting in methyl- {prop-1-yl-2,3-di-0-benzyl-a-0-glucopyranosyl) -uronate (compound 13).

The Yua derivative (350 mg) is dissolved in methanol (5 ml). Sodium methoxide (0.2 ml, 2M) is added. After keeping for 1 hour at room temperature, the reaction is stopped by adding Dowex 50 resin in the H + form. After filtration, product 13 is obtained, which is contaminated with a small amount of product resulting from a-ft-cleavage.

According to a variant of step d, instead of an acetylation reaction, a benzoylation reaction is carried out which leads to allyl-4-0-benyl-2,3-di-0-benzyl-4-0-benzoyl-BTO-triphenylmethane-a-D-glucopyrano - zid (compound 66), from which the triphenylmethyl group is then removed, which allows to obtain an allyl-4-0-benzoyl-2,3-di-O-benzyl-a-O-glucopyranoside (compound 76). These reactions are carried out as follows.

Preparation of compounds 66 and 76. Benzoyl chloride (1.5 eq.) Is added to the pyridine solution of compound 5 and the reaction is monitored by TLC (ethyl acetate / benzene, 1/20 by volume). Excess benzoyl chloride is destroyed by adding excess methanol. After evaporation to dryness in chloroform, the residue is washed with 10% potassium bisulfate solution, water, dried and concentrated to dryness. The resulting syrup is used to synthesize compound 76, This syrup (105 g, obtained from 80 g of compound 3) is dissolved in chloroform (300 ml) P-Toluene sulphonic acid (76 g of monohydrate in 100 ml of methanol) is added. After overnight, the reaction is complete (TLC, ethyl acetate / chloroform, 1/20 by volume), the chloroform phase is washed with water to neutral pH, dried and concentrated to dryness. The resulting syrup (98 g) is chromatographed on a silica gel column (1.2 g), eluted with chloroform (0.6 l), then with a mixture of ethyl acetate / chloroform (1/20 by volume). A pure derivative 76 (30 g) is thus obtained, which is used in the preparation of compound 86.

Starting from compound 76, it is possible to oxidize the CHAOH group in position 6, then introduce a methyl group into the resulting carboxyl group, obtaining the allyl-4-0-benzoyl-2,3-di-0-benzyl-aD-glucopyranosyluronic acid acid (compound 86) and the corresponding methyl ester (compound 96).

These derivatives are obtained as follows

in a way.

Compound 76 (27 g) is treated as indicated1 for 7a in preparation of 8a. The resulting syrup / 1 treatment syrup contains compound 86, which is methylated

diazomethane (as for compound 8a). The residue from the methylation stage is purified on silica gel (200 g, ether / hexane, 1/1) and compound 95 is obtained (21 g, 77.5%). Its IR and NMR spectra confirm its structure.

Starting from derivative 96, the corresponding propenyl derivative 106 is obtained, working as in the preparation of Yu.

Derivative 13 is prepared starting from 106 according to the reaction given for the preparation of Yu.

In another embodiment, allyl-2,3-di-O-benzyl-o: -D-glucopyranosyluronic acid and methyl- (allyl-2,3-di-0-benzyl - # - O-glucopyracosyl) -uronate ( compounds 11 and 12).

Compound 86 (1.9 g) was dissolved in methanol (40 ml). Then 5 n is added. a solution of sodium hydroxide in an amount sufficient to have a 1M concentration of sodium δ peroxide. Over the course of the reaction after washing with TLC (methanol / chloroform, 1/4 in volume). When it is finished, water (100 ml) is added. The mixture is washed with ether, acidified, and the product is extracted with ether. The acidic ether phase is washed.

water to neutral pH. One does not allocate 11. It is methylated with an ether solution of diazomethane to give compound 12 (900 mg, 56%), which is then purified on a silica gel column.

(sir / hexane, 1/1 by volume). , 20 (, 3, chloroform). Its PC and NMR spectra and elemental analysis data confirm its structure.

In the same way, derivatives 11 and 12

can be obtained from 9a or 96,

Compound 13 can be obtained from compound 12 as follows.

Derivative 12 is treated with a rhodi complex, as indicated for 9a. Compound 5 nor 13 is obtained with a yield of 90%. It is characterized by IR and NMR spectra. Moreover, when treated with acetic anhydride (1 ml per 180 mg 9a), it gives the compound Yua.

Alternatively, a derivative 13 can be obtained from 10a or 106, following the procedure given for the preparation of compounds 17 of 9a or 96.

l) Chloroacetylation of compound 13, leading to compound 14, namely methyl (prop-1 (-yl-2,3-di-0-benzyl-4-0-chloroacetyl-a-0-glucopyranosyl) -uronate.

2.8 g of compound 13 is dissolved in 30 ml of pyridine (6.56 mmol). After cooling to 0 ° C, a solution of 2 ml of chloroacetyl chloride in 20 ml of dichloromethane is added dropwise (10 ml). After 30 minutes, evaporated to dryness, the residue is treated with 200 ml of chloroform, washed with 10% potassium bicarbonate solution, then with water, dried and concentrated. The syrup obtained is chromatographed on silica gel (200 g, ethyl acetate / hexane, 1/3 by volume). Thus, 2.7 g (80%) of pure compound 14 are obtained in the form of a syrup, - + 2 ° (, 5; chloroform). Elemental analysis and NMR spectrum confirm the structure of the compound.

m) Release of anomeric carbon leading to compound 15 or methyl- (2.3-di-0-benzyl-4-0-chloroacetyl-a-0-glucopyranosyl) -uronate.

2.7 g (5.3 mmol) of derivative 14 are dissolved in 80 ml of a mixture of acetone / water (5/1 by volume). Mercury oxide (3.1 g) was added, followed by a solution of mercury chloride (3.9 g) in acetone (27 ml). After 5 minutes salt is filtered off. After concentration to dryness, the residue is treated with chloroform. The chloroform phase is washed with a 10% solution of potassium chloride, then with water. After evaporation, the product is crystallized from ethyl acetate / hexane. Get 2 g (80%) of a solid substance, so pl. 105-107 ° C .; oi about -4.7 ° (chloroform). Elemental analysis and NMR spectrum confirm the structure of the substance.

n) Bromination of eunomolar carbon, leading to compound 16 or methyl- (1-bromo-2,3-di-0-benzyl-4-0-chloroacetyl-a - 0-glucopyranosyl) uronate.

2 g (4.30 mol) of compound 15 is dissolved in 50 ml of dichloromethane. 4.8 ml (34.4 mmol) of simm-collidine are added at 0 ° C, followed by dimethylammonium bromide (17 mmol).

After 4 hours, the mixture was diluted with 100 ml of dichloromethane, then poured into ice water, washed with ice water and the solvent was evaporated. After chromatography on silica gel (20 g, hexane / ethyl acetate, 2/1 by volume), 2.06 g (90%) of compound 16 is obtained in the form of syrup, + 82.5 ° (, 5; chloroform). Elemental analysis and NMR spectrum confirm the structure of the compound.

o) Preparation of a disaccharide 18 or 3-0-acetyl-1,6-zngydro-2-azido-4-0-methyl- (2,3-di 5 -O-benzyl-4-O-chloroacetyl- / - D-glucopyranosyl) -uronate} - / -0-glucopyranose.

This synthesis is based on the condensation of monosacrides 16 and 17.

To a solution of 870 mg (3.8 mmol) of compound 17 in dichloromethane, add 1 gels, 0.6 g molecular sieves 4 A in powder form and 0.525 g freshly prepared silver carbonate. After stirring for 2 hours, 670 mg (1.3 mg) are added dropwise at 0 ° C.

5 mmol) of compound 16. After 6 days, the solids are filtered. The syrup obtained after concentration is chromatographed on silica gel (50 g, chloroform / ethyl acetate, 4/1 by volume). Disaccharide 18 is obtained as a foam (421 mg, 50%). about. D -17 ° (chloroform). Elemental analysis confirms the structure. The study of the NMR spectrum confirms the configuration of the interglycosidic bond.

5p) Preparation of disaccharide structure 19

or 1,3,6-tri-0-acetyl-2-azido-4-0-methyl- (2,3-di-0-benzyl-4-0-chloroacetyl- -0-gluco-pyrenosyl) -uronate -0-glucopyrous by acetolysis of the disaccharide.

0 Disaccharides get 19 acetolysis

disaccharide 18.

300 mg of compound 18 are dissolved in a mixture of 4 ml of acetic anhydride and 0.5 ml of freshly distilled trifluoroacetic acid.

5 The reaction mixture is stirred 10 h at 18 ° C, then evaporated to dryness and evaporated with toluene. The residue is chromatographed on a silica gel column (15 g). Elution with dichloromethane / ethyl acetate (19/1 by volume) gave 282 mg (86%) of anomeric acetates of structure 19 as a colorless syrup. The a-and / -form ratio determined by NMR analysis is 4/1.

5 The NMR spectrum confirms the expected structure.

p) Preparation of the disaccharide 20 of general formula II by brominating disaccharides 19. F solution of 140 mg of acetates 19 in a mixture of 3 ml

0 dichloromethane and 0.3 ml of ethyl acetate are stirred at 17-18 ° C under dry argon atmosphere in the presence of 140 mg of titanium tetrabromide (approximately 2 eq.) For 20 hours. After cooling to 0 ° C and dilution 30

With 5 ml of dichloromethane, the mixture is washed with ice water, then with a 5% aqueous solution of potassium bromide, with water and dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on a column.

Silkkagel (10 g). Elution with a dichloromethane / ethyl acetate mixture (19/1 by volume) was isolated in the order of elution:

Bromide 20 of formula (II) (74 mg, 50%) as a colorless unstable syrup (immediately added to the next solution); The NMR spectrum confirms the expected structure;

the fraction (28 mg, 20%) corresponding to the initial unreacted product;

sedentary fraction corresponding to products of partial 0-debenzylation.

Example 2. Synthesis of monosaccharide 22 or benzyl-6-0-acetyl-3-0-benzyl-2-benzyloxycarbonylamino-2-deoxy-a-0-gluco-pyroside.

A suspension of benzyl-3-O-benzyl-2-benzyloxycarbonylamino-2-desoxy-cr-0-glucopyranoside (derivative 21) (987 mg, 2 mM) in anhydrous 1,2-dichloroethane (15 ml) is stirred at reflux temperature for 30 h in the presence of N-acetylamidazole (2.5 mM, freshly prepared). After cooling and diluting with chloroform (50 ml), the organic phase is washed with ice-cold 1 M hydrochloric acid, water, saturated aqueous sodium bicarbonate solution, water, dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on a sipica gel column (50 g). Elution with a mixture of dichloromethane / acetone (15/1 by volume) Zet derivative 22 in the form of a syrup crystallized from a mixture of ethyl acetate / hexes) (759 mg, 71%), m.p. 114-115 ° C; a o20 + 88 ° (chloroform).

Example 3. Synthesis of monosaccharide 33 or 3-O-benzyl-O-methyldiopyranuronate,

a) 63 g of 3-0-benzyl-1,2-0-isopropylidene-β-D-glucofrenoside (compound 23) are dissolved in 500 ml of anhydrous pyridine. 85 g of triphenylmethyl chloride are added and heated at 80 ° C for 1 hour. In this way, trityl protection is introduced in position 6 of compound 23 and compound 24 is obtained. A -34.7 ° (chloroform).

The structure of this compound is confirmed by IR and NMR spectra, elemental analysis is correct.

b) The mixture is cooled to 0 ° C and 45 ml of benzoyl chloride is added. An overnight reagent is decomposed by adding 300 ml of methanol, the mixture is evaporated to dryness and treated with chloroform. The chloroform phase is washed with water, dried over sodium sulfate and concentrated. Thus, in addition

the benzene group is introduced into position 5 and compound 25 is obtained.

c) To remove the trityl protection, the resulting syrup is dissolved in 400 ml of chloroform. After adding 100 ml of a 5 M solution of p-toluenesulfonic acid in methanol, the solution was left overnight at A ° C. After washing the organic phase with water, 215 g of mixture is obtained. By chromium chromatography on silica gel (zfir / hexane, 2/1 by volume), 36 g of compound 26, .3 ° (chloroform) are obtained. The structure of compound 26 is confirmed by IR and NMR spectra.

d) Methylation of the carboxyl function at position 6 of compound 26.

Compound 26 (1.88 g) was dissolved in acetone (20 ml). At -50 ° C, 3.5 ml of a solution of chromium trioxide (13 g) in 3.5 M are added dropwise.

H2 $ Cm (29 ml). The temperature is allowed to rise and is kept for 1 hour under these conditions. Then the reaction mixture is poured on ice and the product is extracted with chloroform. After washing with water and drying, evaporated to dryness. Get connection 27.

The mixture was dissolved in methanol (20 ml), then 10 ml of 1N was added. sodium hydroxide solution and left overnight at room temperature. The sharp mixture is then passed through a column (25 ml) with Dowex 50 resin in the AND + form previously washed with methanol. By concentrating the eluate, compound 28 is obtained.

 This compound is dissolved in ether and is methylated in a classical manner with diazomethane. After evaporation, compound 29 is obtained (1.08 g, 70.4%), and O-27 (chloroform). Elemental

Compound 29 is valid. Its structure, in addition, is confirmed by the IR and I | u1P spectra.

e) Isomerization of the OH group at position 5 of compound 29.

To a solution of trifluoromethanesulfonate anhydride (0.8 ml) in dichloromethane (16 ml) cooled to -20 ° C, pyridine solution (0.8 ml) in dichloromethane (8 ml) is added dropwise. Then at -10 ° C, 80 mg are added dropwise.

Compound 29 dissolved in dichloromethane (8 ml). After 1 hour at 50 ° C, the reaction mixture was poured into ice-water (8 ml) containing 160 mg of sodium bicarbonate. Stir until the organic and aqueous phases are separated. The organic phase is washed with 30% hydrochloric acid, water, saturated sodium chloride solution, dried, and concentrated to give compound 30.

The syrup is treated with dimethylformamide (10 ml). Sodium trifluoroacetate (1.6 g) is added and heated at 80 ° C for

3h Compound 31 is thus obtained. After evaporation, the mixture is treated with dichloromethane, the resulting solution is washed with water and dried. The residue is treated with methanol, then the solvent after 1

4 steamed. After column chromatography (ether / hexane, 2/1), compound 32 (450 mg, 56.2%) is obtained; ofo -3 ° (chloroform). The structure of the compound was confirmed by IR and NMR spectra. Elemental analysis is correct.

e) Formation of the pyran cycle of compound 33.

Compound 32 (200 mg) is dissolved in a mixture of trifluoroacetic acid and water (9/1). After 15 minutes, the solvents are evaporated. The residue is crystallized from ethyl acetate / hexane to give 110 mg of compound 33, mp. 125-126 ° C.

IR (chloroform) v, cm: 3450 (OH), 3080, 3060, 3080, (CH2: benzyl) and 1740 (COOCH3).

NMR spectrum (d, TMS), ppm: 3.75 (singlet, ЗН +, СООМе), 4.98 (1 Н4), 7.30 (singlet, 5Н +, CeHs). and about 20 + 130 ° (methanol).

Calculated,%: C 56.37; H 6.08

Cmhiao

Found,%: C 56.17; H 5.85.

Example 4. Synthesis of a derivative 38 or 3-0-benzyl-4-0-chloro-acetyl-1,2-0-tert-butoxyethylidene- / -L-methyldiopyranuronate.

a) Acetylation reaction of compound 33, resulting in 1,2,4-tri-O-acetyl-3-O-benzyl-a-P-L-methyldiopyranuronates (derivatives 34 and 35). .

A solution of compound 33 (3 g) in a mixture of anhydrous pyridine (20 ml) and acetic anhydride (10 ml) is stirred at 0 ° C in the presence of moisture for 5 hours. The reaction mixture is evaporated to dryness, evaporated with toluene (4x20 ml) and dried under vacuum. The residue is chromatographed on a silica gel column (150 g). Elution with toluene / ethyl acetate (4/1; v / v) gives, in order of elution:

fraction of the head compound of furan derivatives,

Compound 34 (anomer a), syrup (1-70 mg, a (chloroform), NMR spectrum () 5: 6.23 (singlet, 1H, H-1);

Compound 35 (anomer / 3), crystallized from ether / hexane (2.688 g, 63%), m.p. 112-113 ° C, a (chloroform). Nuclear Magnetic Resonance Spectrum (CDCIs): 6.08 (doublet, 1H, H-1), Ji.2 1.5 Hz).

Anomers a and 3 34 and 35 are not separated if a continuous series of the syntheses described is carried out. Their mixture is used directly as a syrup for 5 subsequent reactions.

b) Bromination reaction leading to compound 36 or 2,4-di-O-acetyl-3-O-5 benzyl-o1-methyl di-pyranuronyl bromide. A mixture of acetates 34 and 35 (212 mg; 0.5 mM) was dissolved in anhydrous dichloromethane (5 ml) and anhydrous ethyl acetate (0.5 ml). Titanium tetrabromide (250 mg, 0.7 mmol) is added at one time and the reaction mixture is stirred for 24 hours at room temperature.

5 lack of moisture. After cooling to 0 ° C and diluting with dichloromethane, the organic phase is washed with ice water (3 times), dried over sodium sulfate, filtered and evaporated to obtain

0 derivative 36 in the form of slightly colored syrup (217mg, 96%). NMR spectrum () b: 6.41 (singlet, 1H, H-1). This compound is very unstable; it is immediately introduced into the next reaction.

5c) Preparation of ortho ester 37 or 4-0-acetyl-3-0-benzyl-1, 2,0-tert-butoxyethylidene- / 3-1 -methyl isopyranuronate.

A solution of bromide 36 (freshly prepared from 2,122 g, 5 mmol of a mixture of acetates

34 and 35 in anhydrous dichloromethane) (20 ml) is stirred at room temperature under a dry argon atmosphere. Simm-collidine (2.65 ml, 20 mmol) and anhydrous tert-butanol (3 ml, 30

5 mmol) and the reaction mixture is stirred for 15 hours under these conditions. After dilution with dichloromethane (50 ml), the organic phase is washed with aqueous saturated sodium bicarbonate solution, water, dried .. over sodium sulfate, filtered and evaporated. The residue is chromatographed on a silica gel column (120 g). Elution with a mixture of hexane / ethyl acetate (2/1 by volume) containing 0.5% triethylamine gave 37 as a pure syrup (1.54 g, 70% of 34 and 35).

a (chloroform), NMR spectrum (d): 5.48 (doublet, 1H, H-1), Ji..5ru).

0 g) Monochloro acetylation of the orthoester 37.

A solution of orthoester 37 (220 mg, 0.5 mmol) in anhydrous methanol (10 ml) is cooled to -20 ° C with stirring under a atmosphere of dry argon, anhydrous potassium carbonate (40 mg) is added and the reaction mixture is stirred for 5 h in these conditions. The solids are filtered off with suction, the filtrate is evaporated and the residue is treated with chloroform (50 ml). The organic phase is quickly washed three times with ice water, dried over sodium sulfate, filtered and evaporated. The residue was immediately dissolved in anhydrous pyridine (4 ml) and anhydrous dichloromethane (2 ml). After cooling to -20 ° C under dry argon atmosphere, a solution of chloroacetyl chloride (0.1 ml, 1.24 mmol, freshly distilled) in anhydrous dichloromethane (1 ml) was added dropwise, stirred for 30 minutes and poured into a mixture of ice-water (100 ml). After stirring for 15 minutes, the mixture is extracted with chloroform (3 x 20 ml). The organic phases are washed with ice-water, aqueous 2% sodium bicarbonate solution, water, dried over sodium sulfate, filtered and evaporated. The residue is quickly chromatographed on a silica gel column (12 g). Elution with a hexane / ethyl acetate mixture (5/2 by volume) containing 0.2% triethylamine gives, in order of elution:

saturated compound 39 (15 mg, 8%, no further use),

orthoester 38, syrup (145 mg, 61% of 12); a o - + 19 ° (chloroform), NMR () 6: 5.45 (doublet, H N, H-v |, Ji, 5 Hz); 5.24 (doublet-doublet, 1H, H-4, Lz ,, 5 Hz; J4., 5 Hz); 4.00 (singlet, 2H, CI-CH2-COO-), which is used in the next step.

Example 5. Synthesis of disaccharide 41 of general formula (I) or benzyl-5-O-acetyl-3-0-benzyl-2-benzyloxycarbonylamino-2-deoxy-4-0- (2-0-acetyl) -3-0- benzi-a-L-methylidopyramuronyl) -a-D-glucopyrano-zoid,

a) Preparation of disaccharide 40 or benzi. - i - 6-0-acetyl-3-0-benzyl-2-benzyloxycarbonyl-amino-2-deoxy-4-0- (2-0-acetyl) -3-С-benzyl -4-O-chloroacetyl-aL-methylidopyranuronyl) - "-D-glucopyranoside,

A solution of orthoester 38 (284 mg, 0.6 mmol) and compound 22 (214 mg, 0.4 mmol) in anhydrous chlorobenzene (12 ml) is heated at 140 ° C with stirring and a light stream of dry argon. After a slow distillation, 10 ml of solvent for A solution of 2,6-dimethylpyridine perchlorate (0.006 mmol, freshly prepared) in chlorobenzene (4 ml) was added dropwise over 30 minutes while the solvent was distilled off (4 ml). The reaction mixture is stirred for 1 h with the addition of fresh solvent (10 ml) and simultaneous distillation so that the reaction volume remains constant and equals 4 ml. After cooling and diluting with chloroform, the organic phase is washed with saturated sodium bicarbonate solution, water, and dried over sulfate

sodium, filtered and evaporated. The residue is chromatographed on a silica gel column (40 g). Elution with a hexane / ethyl acetate mixture (4/3 by volume) gives, in order

elution:

product 22 (120mg, 65%); disaccharide 40 crystallized from ether / hexane (112 mg, 30%), m.p. 144-145 ° C, + 35 ° (chloroform); NMR spectrum () confirms the expected structure.

b) Removal of monochrome cetyl group.

The mixture of the disaccharide 40 (56 mg, 0.06 mmol)

and thiourea (7 mg, 0 1 mmol) in pyridine (2.5 ml) and absolute ethanol (0.5 ml) were stirred at 100 ° C for 30 minutes. After cooling and evaporation to dryness, the residue is treated with a mixture of water / chloroform (1/1 by volume) (40 ml). The organic phase is washed with water, dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on a silica gel column (2 g), eluted with ethyl acetate / hexane (2/1 by volume) gives a disaccharide 41 of formula (II) crystallizing from ether (46 mg, 30%), m.p. 146-147 C; “L –44 ° C (chloroform). NMR spectrum () confirms the expected structure.

RU.

Example 6: Synthesis of tegrasaccharide 43. a) A condensation reaction of disaccharides of the formula (I) and (ill) (compounds 20 and 41). Smag 64 mg (80 mM) of freshly prepared briide 20, 51 mg (60 µm / M) of compound 41 and 80 mg of molecular sieve 4 A in the form of a powder in 1.5 ml of anhydrous dichloroethane are stirred for 0.5 h at room temperature in dry argon atmosphere, then

cool tso -20 ° C. Sequentially add 20 ml (150 µm) of symmm collidine and 31 mg (120 µmol of silver trifluoroacetate). The reaction mixture is stirred for 1 h at -20 ° C, then left to rise

after dilution with 50 ml of dichloromethane, the solids are sucked off and the filtrate is washed with ice-water and 1M hydrochloric acid, then with water (twice).

0, the filtrate is dried over sodium sulfate, filtered and evaporated,

The residue is chromatographed on a silica gel column (8 g, 230-400 mesh gel). Elution with a mixture of hexane / ethyl acetate (4/3 on

5 vol) allows you to isolate 37 mg of tetrasaccharide 42 (yield 39%) as a colorless glass, and 56 ° (.6: chloroform); The NMR spectrum confirms the expected structure.

Elution with ethyl acetate / hexane (2/1 by volume) allows 23 mg (44%) of starting product 41 to be isolated.

b) 0-dichloro-acetylation reaction.

A solution of 36 mg (23 µmol) of tetrasaccharide 42 in a mixture of 1.25 ml of pyridine and 0.25 ml of absolute ethanol is heated for 20 minutes at 100 ° C in the presence of 7 mg (100 µmol) of thiourea. After cooling and evaporation to dryness treated with 20 ml of water and extracted with chloroform (5x5 ml), the organic phases are washed with 10% aqueous solution of sodium bisulfate, water, dried over sodium sulfate, filtered and evaporated. The residue is chromatographed on a column of silica gel (3 g). Eluted with ethyl acetate / hexane (3/2 by volume), get 27 mg (80%) of derivative 43 as colorless glass, ap20 + 61 ° (, 8; chloroform): The NMR spectrum confirms the expected structure.

Example 7. Synthesis of pentasaccharide 50 of general formula (I).

a) Conduct a condensation reaction between tetrasaccharide 43 and a monosaccharide.

44 of general formula (IV).

A mixture of 27 mg (54 µmol) of bromide 44 of formula (IV), 26 mg (18 µmol) of tetrasaccharide 43 and 50 mg of a molecular sieve 4 A is in the form of a powder in 0.8 ml of dichloroethane and stirred for 0.5 h at room temperature in dry argon atmosphere, then cooled to -20 ° C. 16 ml (120 µmol) of simm-collidine and 26 mg (100 µmol) of silver trifluoroacetate are sequentially added and the reaction mixture is stirred for 18 hours while the temperature rises to room temperature.

After dilution with 50 ml of dichloromethane, the solids are filtered off with suction and the filtrate is washed with chilled 1M aqueous hydrochloric acid, then twice with water. Dry over sodium sulfate, filter, and evaporate.

The residue is chromatographed on a silica gel column (5 g, 230-400 mesh gel). Elute with a mixture of hexane / egylacetate (4/3 by volume), remove 30 mg (90%) of pentasaccharide

45 in the form of colorless glass, "67 ° (chloroform). The NMR spectrum confirms the expected structure. In particular, for anomeric protons of glucosamine units, shifts (d, TMS) are 5.36 and 5.52 ppm. respectively.

b) Removal of acetyl groups from derivative 45.

A solution of 28 mg of pentasachrid 45 in a mixture of 2.5 ml of 1,2-dimethoxyethane and 0.8 ml of methanol is cooled to 0 ° C with stirring. Then 1 M sodium hydroxide solution is added dropwise over 10 minutes.

The reaction mixture is stirred for 1 hour at then 12 hours at room temperature. After cooling to 0 ° C, 3 ml of 1 M hydrochloric acid and milky color mixture 5 are immediately extracted with chloroform (5 x 5 ml). The organic phases are washed with water, dried over sodium sulfate, filtered and evaporated. The residue is dissolved in 2 ml of methanol and treated with ether-10% solution of diazomethane (in excess up to a stable yellow color) for 0.5 h.

After evaporation to dryness, the residue is chromatographed on a column of silica gel (2.5 g, 220-400 mesh). Elution with a mixture of dichloromethane / methanol (15/1 by volume) afforded 18 mg (72%) of pentasaccharide 46 as a colorless glass, + 57 ° (chloroform). The expected structure is consistent with the NMR spectrum.

c) Sulfation of the OH groups of compound 46.

To a solution of compound 46 (22 mg) in dimethylspamide (0.5 ml) is added a complex of trimethylamine with CHF (22 mg, 2.5 eq. (OH). The reaction mixture is heated at 50 ° C for about 14 hours. Then the trimethylamine complex with the FOS (10 mg) is added again and the mixture is left for 24 hours. Methanol (0.5 ml) and chloroform (0.5 ml) are added to the reaction mixture. The solution is introduced into the upper part of the column. with Sephadex LH-20 equilibrated in chloroform / methanol (1/1 by volume). The fractions containing the sulfated product 5 are combined and the solvent is evaporated. The residue is in the form of a glass (30 mg) by chromatography rut on silica gel (10 g) in a solvent consisting of 3 parts of an ethyl ts.tat / pyridine / acetic acid / water 0 mixture (6/2/0, 6/1 by volume) in 2 hours of an ethyl acetate mixture acetic acid / water (5/5/1 / 3 by volume).

The fractions containing the desired product are combined and concentrated. After 5 evaporations of the solvents, the resulting residue is dissolved in methanol, to which water is added, then passed through a column of Dowex 50 wx 4, in a N a + form, equilibrated in methanol / water (50/50 by volume). Thus, a sodium salt is obtained (compound 47).

g) Hydrogenation of Compound 47. The product obtained above is dissolved in methanol (3.7 ml), to which water 5 is added (0.8 ml). The catalyst is added to this solution (Pd / C, 6%. 40 mg) and stirred under a hydrogen atmosphere for 5 days. After filtering the catalyst, the UV spectrum of the solution obtained shows the complete disappearance of

absorption caused by benzyl groups. Then the solvent is evaporated and compound 48 is obtained in the residue.

e) Sulfation of 1MH2-groups, then saponification of carboxyl.

Compound 48 was dissolved in water (4 ml), the pH was adjusted to 9.5, after which the trimethylamine complex was added with 50h (54 mg). The pH is maintained at 9.5 throughout the process by adding 0.1 n. sodium hydroxide solution.

After a night, the sulfating agent (27 mg) is added again. The last addition is made after 24 hours.

After 48 hours, a solution of sodium hydroxide (PQ, 34 ml) is added to the resulting compound 49, then the solution is stirred for 3 hours at room temperature in order to hydrolyze the methyl esters

links like uronic acid. The reaction mixture is neutralized, then concentrated to a volume of about 2 ml. The resulting solution was introduced into the top of a Sephadex G 25 (Pa +) column (100 ml), eluting with water. The collected fractions were analyzed by UV absorption (206 nm) and polymetry (265 nm). The fractions possessing optical activity are grouped, the solvent is removed, the residue is dissolved in about 2 ml of water and lyophilized.

The derivative 50 of the general formula (I) is obtained in the form of a white powder (5.6 mg, 25% with respect to the product 45).

The NMR spectrum confirms the expected structure. In particular, for the anomeric protons of the glucoamine units there are shifts (d, TMS) of 5.36; 5.45 and 5.52 ppm

Claims (1)

METHOD FOR PRODUCING PENTASACCHARIDE of the general formula C00 ’. OR '. NHR where OR is a group of OH or-OBOZ; R is a hydrogen atom or an SO3 group, in the form of a sodium salt, characterized in that a condensation reaction of a disaccharide of the general formula </ BR> where Op and A have the indicated meanings; Z - benzyloxycarbonylamino group, in a medium of an organic solvent such as dichloroethane in the presence of silver trifluoroacetate and sim-collidine at a temperature of -, 20 ° С followed by a gradual increase in temperature and termination of the process at room temperature with the release of the formed tetrasaccharide in which the T group is removed by the action thiourea in ethanol at a temperature of 100 ° C, and the modified tetrasaccharide is condensed with a derivative of the general formula C00A where Op is acyl for the positions intended for sulfation or benzyl for the positions in which the OH group is to be released; Z is H3-azido group; A is lower alkyl: X is a halogen atom; T is monochloroacetyl, With a saccharide of the general formula where Op, X and Z have the indicated meanings, under the conditions of the above condensation, a pentasaccharide is isolated in which the acyl groups are removed by saponification with an alkali solution, followed by the re-introduction of group A, removed by saponification, by the action of an alkylating agent such as diazomethane, then sulfate liberation of the hydroxyl groups by the trimethylamine / BOS complex in dimethylformamide at a temperature of 50 ° C, the benzyl groups and the Z and Z ^y groups are hydrogenated, respectively, to the OH and NH2 groups in the presence of palladium catalysis ora in a hydrogen atmosphere, sulfate amino groups by action 1694065 AZ sulphating agent of the type indicated above at pH 9.5 in an aqueous medium, followed by saponification of group A and the isolation of the target product in the form of sodium salt on an ion-exchange resin in sodium form. Priority by signs: 02/01/82 in the synthesis of disaccharide (III), 05/28/82 in the condensation of monosaccharide IV with glucuronic acid, 08/06/82 during the synthesis of disaccharide (II), the interaction of disaccharides (II) and (III), obtaining pentasaccharide (I).