CN1324798A - Synthesis of mannitose-glucose tetrasaccharide repeated unit with anti-tumor activity - Google Patents

Abstract

The synthesis of tetrasaccharide repeating unit of manno-glucosan which possesses biological activity and can be used as anti-cancer medicine includes the following steps: firstly, coupling the benzoylated mannose trichloroacetyliminoester with glucose whose 3-position is free hydroxyl group to obtain disaccharide, then making acid hydrolysis and selectively removing 4,6-benzal group or 5,6 isopropylidene group, and selectively coupling with another benzoylated mannose trichloroacetyliminoester at 6-position to obtain trisaccharide, making acid hydrolysis and removing 1,2-position ethylidene group or isopropylidene group; after activation, coupling with monosaccharide receptor whose 4-position is free hydroxyl group, and making deprotection so as to obtain the tetrasaccharide repeating unit.

Description

Synthesis of Tetrasaccharide Repeating Units of Manno-glucosan with Antitumor Activity

The present invention relates to the synthesis of tetrasaccharide repeating units of manno-glucosan which are biologically active, in particular useful as antineoplastic agents.

A manna-glucose polysaccharide was isolated from the microorganism Microellobosporia grisea. This polysaccharide has strong antitumor activity and is a polysaccharide composed of a tetrasaccharide repeating unit (see K.Inoue et al. CarbohydrateResearch 114(1983) 245-256; ) 199-208; 123 (1983) 305-314). This tetrasaccharide is composed of two mannose and two glucose, and its structure is:

This tetrasaccharide has not been synthesized so far. We believe that the synthesis of this tetrasaccharide is not only important for the study of the relationship between the structure and activity of oligosaccharides, but also that this tetrasaccharide may be used as a new antitumor drug.

The purpose of the present invention is to adopt a new idea to provide a simple step, time-saving and labor-saving synthesis method of the tetrasaccharide repeating unit of mannose-glucose polysaccharide that can be used as an antitumor drug.

The object of the present invention is achieved in this way: the tetrasaccharide is divided into two parts, namely the trisaccharide donor on the left in the above figure and the monosaccharide acceptor on the right. As shown below: R=acyl or alkyl protecting group X=leaving group R'=hydrogen atom or alkyl

The synthesis method of the present invention is: 1. Benzoylated mannose trichloroacetimidate 1 as the glycosyl donor and 4,6-O-benzylidene-1,2-O-ethylidene-protected glucose 2 as the glycosyl acceptor , dissolving the glycosyl donor and the glycosyl acceptor in dichloromethane respectively, and then mixing the two, adding a catalytic amount of Lewis acid, stirring and reacting at room temperature for 2-4 hours, to prepare disaccharide 3; The benzylidene group of disaccharide 3 was removed by acid hydrolysis to obtain disaccharide 4; then benzoylmannose trichloroacetimidate 1 was used as the sugar donor, and disaccharide 4 in equimolar ratio was used as the sugar acceptor. Dissolving the glycosyl donor and the glycosyl acceptor in dichloromethane respectively, then mixing the two, adding a catalytic amount of Lewis acid, stirring and reacting at room temperature for 2-4 hours, to prepare trisaccharide 5; Acetylation of trisaccharide 5, removal of ethylidene group by acid hydrolysis, and reacetylation of trisaccharide 7; selective removal of the 1-position acetyl group of trisaccharide 7, followed by standard methods to prepare trichloroacetylide of the trisaccharide Urethane 8;

以上图中  Bz=苯甲酰基  Ac=乙酰基2．2,3,6酰基保护的单糖烷基苷受体由4,6苄叉化的葡萄糖烷基苷12经2,3位苯甲酰化得到全保护的葡萄糖苷13，酸水解移除苄叉基得到14，然后选择性6位苯甲酰化得到单糖受体15；式中   R=烷基或芳基   R’=酰基3．将三糖供体8与单糖受体15溶于二氯甲烷中，在路易斯酸催化下进行偶联反应，得到保护的四糖16，按常规方法脱掉保护，得到四糖17；式中    R=氢原子或烷基或芳基  R’=酰基  Bz=苯甲酰基  Ac=乙酰基Or use another method to synthesize the trichloroacetimide ester 8 of trisaccharides, that is, starting with 1,2:5,6-di-O-isopropylidene glucose, first with benzoylmannose three Chloroacetimide ester coupling of 1 affords the disaccharide, followed by selective hydrolysis to remove the 5,6-isopropylidene group to yield 10, coupled with 1 to yield the trisaccharide 11, followed by acid hydrolysis to remove the 1,2-isopropylidene Propylene, then acetylated to give the trisaccharide, and the trichloroacetimidate 8 of the trisaccharide was prepared according to a standard method;

In the above figure, Bz = benzoyl Ac = acetyl 2.2, 3, 6 acyl protected monosaccharide alkyl glycoside acceptor by 4, 6 benzylated glucosyl glucoside 12 through 2, 3 benzoyl The fully protected glucoside 13 was obtained by oxidation, the benzylidene group was removed by acid hydrolysis to obtain 14, and then the selective 6-position benzoylation was obtained to obtain the monosaccharide acceptor 15; Where R = alkyl or aryl R' = acyl 3. The trisaccharide donor 8 and the monosaccharide acceptor 15 were dissolved in dichloromethane, and the coupling reaction was carried out under the catalysis of Lewis acid to obtain the protected tetrasaccharide 16, which was deprotected according to the conventional method to obtain the tetrasaccharide 17; In the formula, R=hydrogen atom or alkyl group or aryl group R'=acyl group Bz=benzoyl group Ac=acetyl group

The Lewis acid is trimethylsilyl trifluoromethanesulfonate (TMSOTf) or boron trifluoride-diethyl ether complex (BF ₃ .Et ₂ O).

Describe below in conjunction with embodiment.

Example: Preparation Method 1 of Trisaccharide Donor 8

1. Disaccharide 3 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-4,6-O-benzylidene-1,2-O-(R,S)- Preparation of ethylidene-α-D-glucopyranose):

Benzoylated mannose trichloroacetimidate 1 (741 mg, 1 mmol) was dissolved in 20 mL of dichloromethane to give solution A, 4,6-O-benzylidene-1,2-O -Ethylidene glucose 2 (293 mg, 1 mmol) was dissolved in 10 ml of dichloromethane to obtain solution B, B was mixed with A to obtain solution C, and TMSOTf (20 μl, 0.23 mmol) was added to C , after two hours of reaction at room temperature, thin-layer chromatographic analysis showed that the reaction was complete. The reaction solution was diluted with 10 ml of dichloromethane, neutralized with triethylamine, washed with water, the aqueous phase was discarded, and the organic phase was dried under vacuum. The obtained crude product was purified by silica gel column chromatography and purified with ethyl acetate Ester/petroleum ether (1/3) was used as eluent to wash, and the corresponding components were collected to obtain pure disaccharide 3 785 mg, yield: 90%; melting point: 140-145 ° C, specific rotation [α] _D +13°;

2. Disaccharide 4 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-1,2-O-(R,S)-ethylene-α-D- glucopyranose) preparation:

3 (872 mg, 1 mmol) was dissolved in 10 ml of acetic acid, 1 ml of 80% dichloroacetic acid was added, and after stirring at room temperature for six hours, TLC analysis showed that the reaction was complete. The reaction solution was diluted with water, extracted with 30 ml of dichloromethane, the organic phase was washed successively with saturated sodium bicarbonate and water, and then dried under vacuum to obtain 731 mg of a crystalline crude product, yield: 91%; melting point: 150-152°C, specific rotation [α] _D -24°;

3. Trisaccharide 5(2,3,4,6-Tetra-O-henzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D -Mannopyranosyl-(1→6)]-1,2-O-(R,S)-ethylene-α-D-glucopyranose):

Benzoylated mannose trichloroacetimidate 1 (741 mg, 1 mmol) was dissolved in 10 mL of dichloromethane to give solution A, disaccharide 4 (784 mg, 1 mmol) was dissolved in 10 mL In dichloromethane, solution B was obtained, B and A were mixed to obtain solution C, TMSOTf (20 microliters, 0.23 mmol) was added to C, and after three hours of reaction at room temperature, thin-layer chromatography analysis showed that the reaction was complete. The reaction solution was diluted with 10 ml of dichloromethane, neutralized with triethylamine, washed with water, the aqueous phase was discarded, and the organic phase was dried under vacuum. The obtained crude product was purified by silica gel column chromatography and purified with ethyl acetate Ester/petroleum ether (1/3) was used as eluent to wash, and the corresponding components were collected to obtain 1158 mg of pure trisaccharide 5, yield: 85%; melting point: 131-134 ° C, specific rotation [α] _D -1.5°;

4. Trisaccharide 6(2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D Preparation of -mannopyranosyl-(1→6)]-4-O-acetyl-1,2-O-(R,S)-ethylidene-α-D-glucopyranose):

Trisaccharide 5 (1362 mg, 1 mmol) was quantitatively acetylated with acetic anhydride-pyridine according to a conventional method to obtain fully protected trisaccharide 6, 1376 mg, with a yield of 98%; melting point: 131-134 ° C, specific rotation [α] _D -2.5°;

5. Trisaccharide 7 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D -mannopyranosyl-(1→6)]-1,2,4-tri-O-acetyl-α,β-D-glucopyranose):

Dissolve trisaccharide 6 (1404 mg, 1 mmol) in 10 ml of 90% trifluoroacetic acid, react at room temperature for one hour under stirring, and drain the solution under vacuum. Quantitative acetylation of anhydride-pyridine afforded 7,1023 mg of trisaccharide, mainly composed of α-isomer, yield: 70%. Melting point: 137-140°C, specific rotation [α] _D -5.1°;

6. Trisaccharide donor 8 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α -D-mannopyranosyl-(1→6)]-2,4-di-O-acetyl-α-D-glucopyranosyltrichloroacetimidate):

Trisaccharide 7 (731 mg, 0.5 mmol) and potassium carbonate (69 mg, 0.5 mmol) were dissolved in 10 ml of DMF, reacted at room temperature for twelve hours under stirring, the reactant was diluted with water, and then dichloromethane Extract, wash the organic phase with 1N hydrochloric acid, water, sodium bicarbonate solution successively, dry and concentrate the organic phase, the obtained slurry is dissolved in 20 ml of dichloromethane, then add trichloroacetonitrile (0.1 ml, 1 mmol) , and then DBU 14 microliters, the reaction mixture was stirred at room temperature for two hours, the reaction mixture was dried under vacuum, purified by silica gel column chromatography, and ethyl acetate/petroleum ether (1/2) was used as The eluent was washed, and the corresponding components were collected to obtain 8,669 mg of trisaccharides, with a yield of 90%. Melting point: 123-126, specific rotation [α] _D +1.5°;

Example: Preparation Method 2 of Trisaccharide Donor 8

1. Disaccharide 10 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose) Preparation of:

Benzoylated mannose trichloroacetimidate 1 (741 mg, 1 mmol) was dissolved in 10 mL of dichloromethane to give solution A,1,2:5,6-di-O-isopropylidene Glucose 9 (260 mg, 1 mmol) was dissolved in 10 ml of dichloromethane to obtain solution B, B was mixed with A to obtain solution C, and BF ₃ .OEt ₂ (20 microliters, 0.16 mmol ), after reacting at room temperature for four hours, thin-layer chromatographic analysis showed that the reaction was complete. The reaction solution was diluted with 10 ml of dichloromethane, washed with 1N hydrochloric acid, the aqueous phase was discarded, and the organic phase was dried under vacuum. The obtained crude product was purified by silica gel column chromatography and washed with ethyl acetate/petroleum ether (1 /3) Rinse as an eluent, collect the corresponding components, and obtain 10720 mg of pure disaccharide, yield: 90%; melting point: 110-113° C., specific rotation [α] _D + 18°;

2. Trisaccharide 11 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl -(1→6)]-1,2-O-isopropylidene-α-D-glucofuranose) preparation:

Benzoylated mannose trichloroacetimidate 1 (741 mg, 1 mmol) was dissolved in 10 mL of dichloromethane to give solution A, disaccharide 10 (798 mg, 1 mmol) was dissolved in 10 mL In dichloromethane, solution B was obtained, B was mixed with A to obtain solution C, and BF ₃ .OEt ₂ (20 microliters, 0.16 mmol) was added to C, and after reacting at room temperature for four hours, thin-layer chromatographic analysis showed that the reaction Finish. The reactant was neutralized to neutral with triethylamine, and then dried under vacuum, and the obtained crude product was purified by silica gel column chromatography, and washed with ethyl acetate/petroleum ether (1/3) as eluent , collected the corresponding components to obtain 970 mg of pure trisaccharide 11, yield: 80%; melting point: 120-123°C, specific rotation [α] _D +28°;

3. Trisaccharide 7 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl -(1→6)]-1,2,4-tri-O-acetyl-α,β-D-glucopyranose):

Dissolve trisaccharide 11 (1216 mg, 1 mmol) in 10 ml of 90% trifluoroacetic acid, react at room temperature for one hour under stirring, and drain the solution under vacuum. Quantitative acetylation of anhydride-pyridine afforded 7,1042 mg of trisaccharide, mainly composed of α-isomer, yield: 71%. The product property is identical with the property described in preparation method one, 5;

Example: Preparation of monosaccharide receptor 15 (Allyl 2,3,6-tri-O-benzoyl-α-D-glucopyranoside):

4,6-O-benzylidene-α-D-glucopyranosyl allylside 12 (313 mg, 1 mmol) was dissolved in 10 ml of pyridine, and benzoyl chloride ( 0.34 ml, 3 mmol), the reaction was carried out at room temperature under stirring for three hours, and the disaccharide 13 was quantitatively obtained, and the reaction solution was processed by a conventional method, and the obtained crude product was dissolved in tetraoxyfuran (20 ml), and added to the solution Add 3 ml of 1M sulfuric acid dropwise, and then heat the reactant to 70°C with stirring. After two hours, the reaction is complete. After cooling to room temperature, neutralize with sodium bicarbonate solution, extract with dichloromethane, and dry the organic phase under vacuum. , the obtained crude product was purified by silica gel column chromatography, washed with ethyl acetate/petroleum ether (1/1) as eluent, and the corresponding components were collected to obtain 381 mg of disaccharide 14 with a yield of 89%. The obtained 14 was dissolved in 15 ml of pyridine, and benzoyl chloride (0.125 ml, 1 mmol) was added dropwise under stirring at room temperature, and the reaction was completed after three hours. Refined by chromatography, washed with ethyl acetate/petroleum ether (1/2) as eluent, collected the corresponding components, and obtained pure monosaccharide acceptor 9 464 mg, yield: 98%; melting point: 148- 151°C, specific rotation [α] _D +28°;

Example: Tetrasaccharide 16 (Allyl 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-α -D-mannopyranosyl-(1→6)]-2,4-di-O-acetyl-β-D-glucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D- glucopyranoside) preparation:

Use 8 as a glycosyl donor (782 mg, 0.5 mmol) and 15 as a glycosyl acceptor (266 mg, 0.5 mmol), dissolve them in 10 ml of dichloromethane, and add to this solution under stirring TMSOTf (10 μL, 0.11 mmol) was added and reacted at room temperature for four hours. TLC analysis showed that the reaction was complete. The reactant was neutralized to neutral with triethylamine, and then dried under vacuum, and the obtained crude product was purified by silica gel column chromatography, and washed with ethyl acetate/petroleum ether (1/3) as eluent , collected the corresponding components to obtain 870 mg of pure tetrasaccharide 16, yield: 90%; melting point: 133-136°C, specific rotation [α] _D +38°;

Example: tetrasaccharide allyl glycoside 17a (Allyl α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→6)--β-D-glucopyranosyl-(1→4)-α -D-glucopyranoside) preparation:

Tetrasaccharide 16 (154 mg, 0.08 mmol) was suspended in 10 ml of methanol, 2M 0.1 ml of solution was added to this solution, and the solution was stirred overnight at room temperature, and the resulting solution was neutralized with a cation exchange resin, After filtration and concentration, 48 mg of tetrasaccharide allyl glycoside 17a was obtained, yield: 85%; melting point: 154-156°C, specific rotation [α] _D -7°;

Example: Free tetrasaccharide 17b (α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→6)--β-D-glucopyranosyl-(1→4)-α,β-D -glucopyranose) preparation:

Tetrasaccharide 16 (307 mg, 0.16 mmol) was dissolved in 10 ml of dichloromethane, 10 ml of methanol was added, palladium dichloride (30 mg, 0.17 mmol) was added under stirring, and the reaction was carried out at room temperature after 6 hours Complete, filter and wash the filter cake with dichloromethane, combine the washings with the reaction solution, dry it under vacuum, then suspend it in 10 milliliters of methanol, add 0.18 milliliters of 2M solution to this solution, this solution is at room temperature, After stirring overnight, the resulting solution was neutralized with a cation exchange resin, filtered and concentrated to obtain 90 mg of free tetrasaccharide 17b, yield: 82%; specific rotation [α] _D -2°.

Claims (4)

1. A kind of synthetic method that can be used as antineoplastic, the tetrasaccharide repeating unit of mannose-glucose polysaccharide, it is characterized in that: take benzoylated mannose trichloroacetimidate 1 as sugar group donor, use 4 , 6-O-benzylidene-1,2-O-ethylidene-protected glucose 2 is the glycosyl acceptor, the glycosyl donor and the glycosyl acceptor are dissolved in dichloromethane respectively, and then the two Mix, add a catalytic amount of Lewis acid, stir and react at room temperature for 2-4 hours to prepare disaccharide 3; remove the benzylidene group of disaccharide 3 by acid hydrolysis to obtain disaccharide 4; then use benzoyl Mannose trichloroacetimidate 1 is used as a sugar donor, and disaccharide 4 in an equimolar ratio is used as a sugar acceptor. The sugar donor and the sugar acceptor are respectively dissolved in dichloromethane, and then the two Mix them together, add a catalyst amount of Lewis acid, stir and react at room temperature for 2-4 hours to prepare trisaccharide 5; acetylate trisaccharide 5, remove the ethylidene group by acid hydrolysis, and then acetylate to obtain trisaccharide 7; The 1-position acetyl group of trisaccharide 7 was selectively removed, and then the trichloroacetimidate 8 of the trisaccharide was prepared according to standard methods; Or use another method to synthesize the trichloroacetimide ester 8 of trisaccharides, that is, starting with 1,2:5,6-di-O-isopropylidene glucose, first with benzoylmannose three Chloroacetimide ester coupling of 1 affords the disaccharide, followed by selective hydrolysis to remove the 5,6-isopropylidene group to yield 10, coupled with 1 to yield the trisaccharide 11, followed by acid hydrolysis to remove the 1,2-isopropylidene Propylene, then acetylated to give the trisaccharide 7; selective removal of the acetyl group at position 1 followed by standard procedures to produce the trichloroacetimidate 8 of the trisaccharide; In the above figure Bz=benzoyl Ac=acetyl 2. A method for synthesizing the tetrasaccharide repeating unit of manno-glucosan, which can be used as an antineoplastic agent, is characterized in that: 2,3,6 acyl-protected monosaccharide alkyl glycoside acceptors are composed of 4,6 benzylidene Glucoside 12 was benzoylated at the 2 and 3 positions to obtain the fully protected glucoside 13, and the benzylidene group was removed by acid hydrolysis to obtain 14, and then selectively benzoylated at the 6 position to obtain the monosaccharide acceptor 15: Where R=alkyl or aryl R'=acyl 3. A kind of synthetic method that can be used as antineoplastic, the tetrasaccharide repeating unit of mannose-glucose polysaccharide, it is characterized in that: trisaccharide donor 8 and monosaccharide acceptor 15 are dissolved in methylene chloride, in Lewis acid catalysis The coupling reaction is carried out under the following conditions to obtain the protected tetrasaccharide 16, and the protection is removed according to the conventional method to obtain the tetrasaccharide 17;

In the formula, R=hydrogen atom or alkyl group or aryl group R'=acyl group Bz=benzoyl group Ac=acetyl group 4. The Lewis acid described in claims 1 and 3 is trimethylsilyl trifluoromethanesulfonate (TMSOTf) or boron trifluoride-diethyl ether complex (BF ₃ .Et ₂ O).