Preparation method of β-D-(1,4)-mannuronic acid oligosaccharide and its intermediate

Abstract

The present invention discloses a method for economically and efficiently preparing β-D-(1,4)-mannuronic acid oligosaccharide shown in Formula IX. In particular, the present invention uses readily available 1,2,3,4,6-penta-O-acetyl-D-mannopyranose (Formula X) as a raw material to prepare key intermediate compounds I, II and III; couples intermediate compounds II and III into an oligosaccharide receptor compound V, and couples intermediate compounds I and II into an oligosaccharide donor compound VII; couples compound V and compound VII into an oligosaccharide compound VIII, and removes the protecting groups from the oligosaccharide compound VIII to obtain β-D-(1,4)-mannuronic acid oligosaccharide IX.

Description

Technical Field

[0001] The present invention belongs to the field of organic chemical synthesis and relates to a preparation method of β-D-(1,4)-mannuronic acid oligosaccharides (disaccharides to eicosasaccharides). Background Art

[0002] β-D-(1,4)-Mannuronic acid oligosaccharides widely exist in natural products and have the effects of promoting plant root growth, antibacterial, promoting the generation of human keratinocytes, etc. They can bind to Toll-like receptors 2 and 4 and show immunomodulatory effects (CN103275133A). β-D-(1,4)-Mannuronic acid oligosaccharides also have good curative effects in the treatment of vascular dementia (CN106344593A). On November 2, 2019, the National Medical Products Administration approved the listing of Mannaten capsules for the treatment of mild to moderate Alzheimer's disease, and its main ingredient is β-D-(1,4)-mannuronic acid oligosaccharides and their derivatives.

[0003] At present, β-D-(1,4)-mannuronic acid oligosaccharides are mainly obtained by degrading sodium alginate (CN100508985C). The degradation preparation process poses great challenges to the control of its purity and impurities. There is an urgent need to develop a preparation process that is efficient, simple to operate, and can be precisely controlled. There are literature reports on using solid-phase synthesis (WO2012138698) or liquid-phase synthesis (Codée, Jeroen D.C, van den Bos, Leendert J, de Jong, Ana-Rae, et al. The Stereodirecting Effect of the Glycosyl C5-Carboxylate Ester: Stereoselective Synthesis of β-Mannuronic Acid Alginates[J]. Journal of Organic Chemistry, 74(1):38-47) to prepare derivatives of β-D-(1,4)-mannuronic acid oligosaccharides. Among them, the solid-phase synthesis method has high costs and high requirements for equipment, and is not easy to be industrially produced. In addition, the existing liquid-phase synthesis method has problems such as low synthesis efficiency and cumbersome removal of protecting groups, and is not easy to be industrially produced. Therefore, developing a new, efficient, and process-controllable preparation method of β-D-(1,4)-mannuronic acid oligosaccharides has important application value and economic value. Summary of the Invention

[0004] In the first aspect of the present invention, a method for preparing β-D-(1,4)-mannuronic acid oligosaccharide is provided, which can economically and efficiently prepare β-D-(1,4)-mannuronic acid oligosaccharide with high purity. The method includes coupling compound V and compound VII and selectively removing the protecting group R 3 to generate compound VIII;

[0005]

[0006] and optionally, removing the protecting group R from compound VIII in one step 2 to generate β-D-(1,4)-mannuronic acid oligosaccharide shown in formula IX;

[0007]

[0008] wherein, m is an integer selected from 2 - 18; n and n' are each independently an integer selected from 0 - 8; R 1 is selected from C 1-8 alkyl, C 1-8 aryl optionally substituted by C 6-14 alkyl; R 2 is a hydroxyl protecting group that can be removed by a palladium-carbon catalyzed hydrogenation reaction or a palladium-carbon catalyzed oxidation reaction; R 3 is a hydroxyl protecting group that cannot be removed by a palladium-carbon catalyzed hydrogenation reaction or a palladium-carbon catalyzed oxidation reaction.

[0009] In particular, the present invention can use readily available 1,2,3,4,6-penta-O-acetyl-D-mannopyranose (formula X) as a raw material to prepare intermediate compounds I, II and III; then couple intermediate compounds II and III into an oligosaccharide receptor compound V, couple intermediate compounds I and II into an oligosaccharide donor compound VII; then couple compound V and compound VII into an oligosaccharide compound VIII, and remove the protecting group to obtain the final product compound IX, namely β-D-(1,4)-mannuronic acid oligosaccharide. The present invention provides an economical and efficient solution for synthesizing β-D-(1,4)-mannuronic acid oligosaccharide compounds with a degree of polymerization of 2 to 20. Compared with the process of extracting and preparing oligosaccharide mixtures by degrading sodium alginate and the like, this solution can obtain oligosaccharides with a single degree of polymerization and high purity, laying a solid foundation for further studying the pharmacological and biological activities of oligosaccharides with a single degree of polymerization.

[0010] In the second aspect of the present invention, key intermediate compounds I, II, III, V, VII and VIII for synthesizing β-D-(1,4)-mannuronic acid oligosaccharide are provided, and the structural formulas are shown as follows:

[0011]

[0012] Among them,

[0013] R 1 is selected from C 1-8 alkyl, optionally C 1-8 alkyl-substituted C 6-14 aryl; preferably, R 1 is selected from phenyl, o-tolyl, p-tolyl, 4-tert-butyl-2-methylphenyl, 2,4-di-tert-butylphenyl, methyl or ethyl;

[0014] R 2 is a hydroxyl protecting group that can be removed by a palladium-carbon-catalyzed hydrogenation reaction; preferably, R 2 is selected from C 6-14 arylmethyl or allyl, and the C 6-14 arylmethyl is optionally substituted with C 1-8 alkyl, C 1-8 alkoxy, halogen; more preferably, R 2 is selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl; most preferably, R 2 is selected from benzyl;

[0015] R 3 is a hydroxyl protecting group that cannot be removed by a palladium-carbon-catalyzed hydrogenation reaction; preferably, R 3 is selected from C 1-8 alkylacyl, C 1-8 alkoxyacyl, C 6-14 arylacyl, tris(C 1-8 alkyl)silyl, 9-fluorenylmethoxycarbonyl, tris(C 6-14 aryl)methyl; wherein any carbon atom in C 1-8 alkylacyl and C 1-8 alkoxyacyl can be optionally oxo; more preferably, R 1-8 3 is selected from acetyl, acetylpropionyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, 9-fluorenylmethoxycarbonyl, or triphenylmethyl; most preferably, R 3 is selected from acetylpropionyl;

[0016] R 4 is selected from H, C 1-8 alkyl, C 6-14 aryl;

[0017] X is selected from fluorine, chlorine, bromine, iodine;

[0018] m is an integer from 2 to 18; preferably, m is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;​

[0019] n and n' are each independently selected from integers from 0 to 8; preferably, n and n' are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8. Description of the Drawings

[0020] Attached Figure 1 is the general synthetic route of Compound VIII and Compound IX.

[0021] Attached Figure 2 is the synthetic route of Compound IX-1 and Compound IX-2.

[0022] Attached Figure 3 is the synthetic route of Compound IX-3 and Compound IX-4.

[0023] Attached Figure 4 is the synthetic route of Compound IX-5. Detailed Description of the Invention

[0024] Unless otherwise specified, the term "alkyl" herein may be a straight-chain or branched-chain saturated hydrocarbon group, such as methyl, ethyl, propyl, butyl, octyl, isopropyl, tert-butyl, sec-pentyl and similar groups. The alkyl may be unsubstituted or substituted by one or more substituents (such as, halogen, alkoxy, aryl, aralkyl, aralkyloxy and similar groups). C 1-n Cn alkyl (where n is an integer) refers to an alkyl group containing 1 - n carbon atoms, for example, 1 - 18 carbon atoms, 1 - 12 carbon atoms, 1 - 10 carbon atoms, 1 - 8 carbon atoms, 1 - 6 carbon atoms, 1 - 4 carbon atoms, and so on.

[0025] The term "aryl" refers to a monocyclic (such as phenyl) or polycyclic (such as naphthyl or anthracenyl) monovalent unsaturated aromatic group, optionally substituted by substituents such as halogen (including fluorine, chlorine, bromine, iodine), alkyl, aralkyl, alkoxy, aralkyloxy and similar groups. C 6-n Cn aryl (where n is an integer) refers to an aryl group having 6 to n carbon atoms, such as phenyl, naphthyl, anthracenyl, or its optionally substituted group.

[0026] The first aspect of the present invention relates to a method for preparing a β-D-(1,4)-mannuronic acid oligosaccharide represented by formula (VIII) or formula (IX),

[0027]

[0028] comprising:

[0029] coupling Compound V and Compound VII and selectively removing the protecting group R 3 to produce Compound VIII;

[0030]

[0031] and optionally, removing the protecting group R from compound VIII in one step 2 to produce a β-D-(1,4)-mannuronic acid oligosaccharide as shown in formula IX;

[0032] wherein, m is an integer selected from 2 - 18; n and n' are each independently an integer selected from 0 - 8; R 1 is selected from C 1-8 alkyl, C 1-8 alkyl optionally substituted with C 6-14 aryl; R 2 is a hydroxyl protecting group that can be removed by a hydrogenation reaction catalyzed by palladium on carbon; R 3 is a hydroxyl protecting group that cannot be removed by a hydrogenation reaction catalyzed by palladium on carbon.

[0033] The above coupling reaction is carried out in the presence of a sterically hindered organic base, diphenyl sulfoxide and a sulfonic anhydride catalyst; then the hydroxyl protecting group R at the 4-position and the hydroxyl protecting groups R at other positions are removed in sequence 3 to obtain a β-D-(1,4)-mannuronic acid oligosaccharide compound as shown in IX. Preferably, the sulfonic anhydride catalyst is selected from methanesulfonic anhydride, trifluoromethanesulfonic anhydride or p-toluenesulfonic anhydride. 2

[0034] In one embodiment of the present invention, a method for synthesizing compound V is provided, comprising:

[0035]

[0036] Dissolving intermediate compound II and intermediate compound III in a suitable organic solvent, adding dry molecular sieve, protecting with nitrogen or argon, adding a sulfonic acid catalyst at an appropriate temperature, carrying out a coupling reaction to form a 1,4-glycosidic bond, and then selectively removing the hydroxyl protecting group at the 4-position to obtain compound IV; repeating the above coupling reaction and the step of selectively removing the hydroxyl protecting group R at the 4-position for compound IV and compound II 3 optionally repeating the above coupling reaction and deprotection step for the obtained compound until compound V is obtained.

[0037] ​In a preferred embodiment of the present invention, the organic solvents in the synthesis of compound V are selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethyl sulfoxide, preferably anhydrous dichloromethane; the sulfonic acid catalysts are selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate, preferably trimethylsilyl trifluoromethanesulfonate; the reagent for removing the hydroxyl protecting group at the 4-position is preferably hydrazine acetate, and the molar equivalent of hydrazine acetate is 3-8eq, preferably 3-5.5eq, such as 4.3eq.

[0038] In one embodiment of the present invention, a method for synthesizing compound VII is provided, including:

[0039]

[0040] Dissolve compound I and compound II in a suitable organic solvent, add dry molecular sieve, protect with nitrogen or argon, and at an appropriate temperature, add a sulfonic acid catalyst to carry out a coupling reaction to form a 1,4-glycosidic bond, and then selectively remove the hydroxyl protecting group R at the 4-position 3 , to obtain compound VI; repeat the above coupling reaction and selective removal of the 4-position protecting group R 3 for compound VI and compound II; optionally, continue to repeat the above coupling reaction and deprotection steps for the obtained compound until compound VII is obtained.

[0041] In a preferred embodiment of the present invention, the organic solvents in the synthesis of compound VII are selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethyl sulfoxide, preferably anhydrous dichloromethane; the sulfonic acid catalysts are selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate; the sulfonic acid catalyst is preferably trimethylsilyl trifluoromethanesulfonate; the reagent for removing the hydroxyl protecting group at the 4-position is preferably hydrazine acetate.

[0042] In one embodiment of the present invention, a method for synthesizing compound IX is provided, including:

[0043]

[0044] Dissolve compound V and compound VII in a suitable organic solvent, add dry molecular sieve, protect with nitrogen or argon, and at an appropriate temperature, add a bulky organic base, diphenyl sulfoxide and a sulfonic anhydride catalyst to carry out a coupling reaction to form a 1,4-glycosidic bond, and then selectively remove the protecting group R of the hydroxyl group at the 4-position 3Compound VIII was obtained; palladium on carbon (palladium content 5%-10%) was added to Compound VIII, and all R protecting groups of Compound VIII were removed by hydrogenation to obtain the β-D-(1,4)-mannuronic acid oligosaccharide compound IX. 2 The protecting groups were removed to obtain the β-D-(1,4)-mannuronic acid oligosaccharide compound IX.

[0045] In a preferred embodiment of the present invention, in the synthesis of compound IX, the molar ratio of compound V to compound VII is 1:0.9; the solvent for the coupling reaction is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous diethyl ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethyl sulfoxide, preferably anhydrous dichloromethane; the bulky organic base is selected from: 1,8-diazabicycloundec-7-ene, 2,6-di-tert-butylpyridine or 2,4,6-tri-tert-butylpyrimidine, preferably 2,6-di-tert-butylpyridine; the sulfonic anhydride catalyst is selected from: methanesulfonic anhydride, trifluoromethanesulfonic anhydride or p-toluenesulfonic anhydride, preferably trifluoromethanesulfonic anhydride; the molar equivalent of the sulfonic anhydride is preferably 0.05 eq; the reaction temperature is preferably -60 °C; the reagent for removing the 4-position hydroxyl protecting group is preferably hydrazine acetate, and its molar equivalent is preferably 4.3 eq; the temperature for removing the protecting group is preferably 25 °C.

[0046] The starting compounds of formula I and formula II used above can be obtained from the compound of formula X respectively.

[0047] The compound of formula X is 1-5 substituted pyranomannose, and the substituents can be C1-C6 acyl groups, such as formyl, acetyl, propionyl. An example of the compound of formula X is 1,2,3,4,6-penta-O-acetyl-D-pyranomannose.

[0048] In a preferred embodiment of the present invention, taking 1,2,3,4,6-penta-O-acetyl-D-pyranomannose (the compound shown in formula X), which is a simple and readily available raw material, as an example, the overall reaction route is as follows:

[0049]

[0050] wherein R 5 is an acyl group of C 1-8 and is preferably acetyl.

[0051] In an embodiment of the present invention, a method for synthesizing the intermediate compound I is provided, including:

[0052]

[0053] Step 1: 1,2,3,4,6-penta-O-acetyl-D-pyranomannose and the anomeric carbon protecting agent R 1The SH reaction is carried out, followed by alkaline hydrolysis to obtain compound A;

[0054] Step 2: Selectively protect the hydroxyl groups at the 2- and 3-positions of compound A to obtain compound B;

[0055] Step 3: Oxidize the hydroxyl group at the 6-position of compound B to a carboxyl group, and then esterify the carboxyl group to obtain compound I.

[0056] In a preferred embodiment, the method includes:

[0057] Step 1. In a dry organic solvent, using 1,2,3,4,6-penta-O-acetyl-D-mannopyranose as a raw material, stirring in the presence of an acidic catalyst at an appropriate temperature, protected by nitrogen or argon, adding an appropriate anomeric carbon protecting agent R 1 React with SH, and then carry out base hydrolysis to obtain compound A;

[0058] Step 2. Under the catalysis of a Lewis acid, select an appropriate hydroxyl protecting group to selectively protect the hydroxyl groups at the 4- and 6-positions of compound A, then selectively protect the hydroxyl groups at the 2- and 3-positions, and finally selectively remove the protecting groups at the 4- and 6-positions to obtain compound B;

[0059] Step 3. In an organic solvent, add an oxidizing agent to compound B to selectively oxidize the hydroxyl group at the 6-position of compound B to a carboxyl group, and then react with an alkylating agent to form an ester in a basic condition at an appropriate temperature and solvent to obtain compound I.

[0060] In a more preferred embodiment, the synthesis of the intermediate compound I adopts a one-pot two-step reaction, and the intermediate does not need to be separated and purified. Among them, the organic solvent described in Step 1 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide; the appropriate temperature is 0-25 °C, preferably 0-5 °C; the acidic catalyst is selected from: boron trifluoride diethyl ether, acetyl chloride or hydrogen chloride gas, preferably boron trifluoride diethyl ether; the molar equivalent of boron trifluoride diethyl ether is preferably 0.1eq; the base is selected from: sodium ethoxide, potassium ethoxide, magnesium ethoxide, sodium methoxide, potassium methoxide or magnesium methoxide, preferably sodium methoxide; the molar equivalent of sodium methoxide is preferably 0.1eq; the solvent for base hydrolysis is selected from: methanol, ethanol or tetrahydrofuran, preferably methanol; the anomeric carbon protecting agent added in the reaction is selected from: benzenethiol, o-toluenethiol, p-methylbenzenethiol, 4-tert-butyl-2-methylbenzenethiol, 2,4-di-tert-butylbenzenethiol, methanethiol or ethanethiol, preferably p-methylbenzenethiol; the molar equivalent of the anomeric carbon protecting agent is preferably 1.05eq.

[0061] Among them, the protecting agents for protecting the 4-position and 6-position of compound A in step 2 are selected from: benzaldehyde dimethyl acetal, p-methoxybenzaldehyde dimethyl acetal or 2,2-dimethoxypropane (acetone acetal), preferably benzaldehyde dimethyl acetal; the molar equivalent of benzaldehyde dimethyl acetal is preferably 0.9 eq; the Lewis acid catalyst is selected from: p-toluenesulfonic acid, trifluoromethanesulfonic acid, aluminum trichloride and ferric trichloride, preferably ferric trichloride; the protecting agents for protecting the 2-position and 3-position hydroxyl groups of compound A are selected from benzyl bromide, benzyl chloride, p-methoxybenzyl bromide, p-methoxybenzyl chloride or allyl bromide, preferably benzyl bromide; the equivalent of benzyl bromide is preferably 1.9 eq.

[0062] Among them, the oxidants described in step 3 are selected from: 2,2,6,6-tetramethylpiperidine 1-oxyl, sodium hypochlorite, potassium bromide, hydrogen peroxide, tert-butyl hydroperoxide or iodobenzene diacetate, preferably 2,2,6,6-tetramethylpiperidine 1-oxyl or iodobenzene diacetate; the molar equivalent of 2,2,6,6-tetramethylpiperidine 1-oxyl is preferably 0.2 eq, or the molar equivalent of iodobenzene diacetate is preferably 2.5 eq; the organic solvents are selected from any one or more of dichloromethane, acetonitrile, water, tetrahydrofuran, N,N-dimethylformamide or N,N-dimethylacetamide, preferably selected from: acetonitrile-water mixed solvent (the volume ratio of acetonitrile to water is about 5:1 - 1:5), tetrahydrofuran-water mixed solvent (the volume ratio of tetrahydrofuran to water is about 5:1 - 1:5) or dichloromethane - water mixed solvent (the volume ratio of dichloromethane to water is about 5:1 - 1:5), more preferably dichloromethane-water mixed solvent (the volume ratio of dichloromethane to water is about 1:2); the temperature of the oxidation reaction is 0 °C to 25 °C, and the preferred temperature is 25 °C; the bases are selected from: potassium carbonate, cesium carbonate, sodium carbonate, calcium carbonate, silver carbonate, triethylamine or diisopropylethylamine, preferably potassium carbonate or triethylamine; the molar equivalent of the base is preferably 0.5 eq to 3 eq, more preferably potassium carbonate with a molar equivalent of 1.5 eq or triethylamine with a molar equivalent of 1 eq; the alkylating agent is selected from: benzyl bromide, benzyl chloride, p-methoxybenzyl bromide, p-methoxybenzyl chloride or allyl bromide, preferably benzyl bromide; the solvent for the alkylation reaction is selected from: tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, acetone or acetonitrile, preferably acetone; the temperature of the alkylation reaction is 25 °C to 100 °C, and the preferred temperature is 30 °C.

[0063] In one embodiment of the present invention, a method for synthesizing intermediate compound II is provided, including:

[0064]

[0065] Step 1: Protect the 4-position hydroxyl group of compound I to obtain compound D;

[0066] Step 2: Selectively remove the anomeric carbon protecting group -SR of compound D 1 , to obtain compound E;

[0067] Step 3: Under alkaline conditions, react compound E with CX3CN or CX3C(=NR 4 )X to obtain compound II.

[0068] In a preferred embodiment, the method comprises:

[0069] Step 1: Under alkaline conditions in a dry organic solvent, use a suitable protecting agent to protect the 4-hydroxy group of compound I to obtain compound D;

[0070] Step 2: Selectively remove the anomeric carbon protecting group -SR of compound D 1 , to obtain compound E;

[0071] Step 3: Under alkaline conditions in a dry organic solvent, react compound E with CX3CN or CX3C(=NR 4 )X (for example, trichloroacetonitrile) to obtain compound II.

[0072] In a more preferred embodiment, the synthesis of intermediate compound II adopts a one-pot three-step reaction, and the intermediate does not need to be separated and purified. Among them, the suitable protecting agent described in Step 1 is selected from acetyl chloride, acetic anhydride, trimethylchlorosilane, tert-butyldimethylchlorosilane, benzoyl chloride, 9-fluorenylmethyl chloroformate, triphenylchloromethane, acetylpropionyl chloride or levulinic acid, preferably levulinic acid; the molar equivalent of the protecting agent is preferably 1.5eq; the organic solvent is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide, preferably anhydrous dichloromethane; the preferred reaction temperature is 25°C; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, N,N-dimethylaminopyridine or pyridine, preferably N,N-dimethylaminopyridine; the molar equivalent of the base is preferably 1.5eq.

[0073] Among them, the reagent used to remove the anomeric carbon protecting group in Step 2 is selected from N-chlorosuccinimide or N-bromosuccinimide, preferably N-bromosuccinimide; the reaction temperature is preferably 25°C.

[0074] Among them, the organic solvent described in step 3 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide, preferably anhydrous dichloromethane; the reaction temperature is preferably from 0 °C to 10 °C; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N-dimethylaminopyridine or pyridine, preferably 1,8-diazabicyclo[5.4.0]undec-7-ene; the molar equivalent of the base is preferably 0.5 eq.

[0075] In one embodiment of the present invention, a method for synthesizing intermediate compound III is provided, including:

[0076]

[0077] Step 1: Protect the 4-hydroxy group of compound I to obtain compound D;

[0078] Step 2: Under alkaline conditions, react compound D with R 2 OH to obtain compound F;

[0079] Step 3: Selectively remove the 4-hydroxy protecting group R 3 of compound F to obtain compound III.

[0080] In a preferred embodiment, the method includes:

[0081] Step 1. In a dry organic solvent, under alkaline conditions, use a suitable protecting agent to protect the 4-hydroxy group of compound I to obtain compound D;

[0082] Step 2. In a dry organic solvent, in the presence of a bulky base, add R 2 OH, diphenylthiol and a sulfonic anhydride catalyst to compound D, and react to obtain compound F;

[0083] Step 3. Selectively remove the 4-hydroxy protecting group of compound F in an organic solvent to obtain compound III.

[0084] In a more preferred embodiment of the present invention, the synthesis of the intermediate compound III adopts a one-pot three-step method. Wherein, the suitable protective agent in step 1 is selected from: acetyl chloride, acetic anhydride, trimethylsilyl chloride, tert-butyldimethylsilyl chloride, 9-fluorenylmethyl chloroformate, benzoyl chloride, triphenylmethane chloride, levulinic acid chloride or levulinic acid, preferably levulinic acid; the molar equivalent of the protective agent is preferably 1.5eq; the organic solvent is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ethyl acetate, anhydrous acetonitrile, anhydrous toluene, anhydrous N,N-dimethylformamide or anhydrous N,N-dimethylacetamide, preferably anhydrous dichloromethane; the reaction temperature is preferably 25°C; the base is selected from: anhydrous potassium carbonate, anhydrous sodium carbonate, triethylamine, N,N-dimethylaminopyridine or pyridine, preferably N,N-dimethylaminopyridine; the molar equivalent of the base is preferably 1.5eq.

[0085] Wherein, the organic solvent in step 2 is selected from: anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous ether, anhydrous N,N-dimethylformamide, anhydrous N,N-dimethylacetamide, anhydrous toluene or anhydrous dimethyl sulfoxide, preferably anhydrous dichloromethane; the bulky hindered organic base is selected from: 1,8-diazabicycloundec-7-ene, 2,6-di-tert-butylpyridine or 2,4,6-tri-tert-butylpyridine, preferably 2,6-di-tert-butylpyridine; the molar equivalent of the base is preferably 2.2eq; the sulfonic anhydride catalyst is selected from: methanesulfonic anhydride, trichloromethanesulfonic anhydride, tribromomethanesulfonic anhydride, trifluoromethanesulfonic anhydride or toluenesulfonic anhydride, preferably trifluoromethanesulfonic anhydride; the molar equivalent of trifluoromethanesulfonic anhydride is preferably 0.05eq; the reaction temperature is preferably -60°C.

[0086] Wherein, the organic solvent in step 3 is dichloromethane and / or pyridine, preferably dichloromethane:pyridine=5:1 (V / V); the reagent used to selectively remove the 4-hydroxyl protecting group of compound F is preferably hydrazine acetate, and its equivalent is preferably 4.3eq.

[0087] In a preferred embodiment of the present invention, the compound I, compound II and compound III are compounds I-1, II-1 and III-1 shown in the following structural formulas respectively:

[0088]

[0089] The advantages of the present invention are:

[0090] The method for preparing β-D-(1,4)-mannuronic acid oligosaccharide (Compound IX) described in this article adopts a convergent synthesis strategy, through the new oligosaccharide intermediates Compound I, Compound II and Compound III, the oligosaccharide donor (Compound VII) and the oligosaccharide acceptor (Compound V) are assembled into an oligosaccharide compound VIII with a higher sugar base number, and the protecting group R in Compound VIII is removed at one time.2 , synthesize β-D-(1,4)-mannuronic acid oligosaccharide (Compound IX). Compared with the prior art, the preparation method of the present invention is more concise, economical and efficient.

[0091] Example

[0092] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0093] The following examples further illustrate the present invention by providing the total synthesis schemes of disaccharide (Compound IX-1), tetrasaccharide (Compound IX-2), trisaccharide (Compound IX-3), pentasaccharide (Compound IX-4) and heptasaccharide (Compound IX-5) of β-D-(1,4)-mannuronic acid, but the present invention is not limited thereto.

[0094] The raw materials or reagents in the examples are commercially available unless otherwise specified.

[0095] Room temperature in the examples refers to 20 - 30 °C.

[0096] Atmospheric pressure in the examples refers to 1 atmosphere.

[0097] Water in the examples refers to deionized water.

[0098] Meaning of abbreviations

[0099] Abbreviations Meaning TLC Thin Layer Chromatography EA Ethyl Acetate Hex Hexane DCM Dichloromethane TEMPO 2,2,6,6 - Tetramethylpiperidin - 1 - yloxy DIC N,N - Diisopropylcarbodiimide DMAP 4 - (Dimethylamino)pyridine NBS N - Bromosuccinimide DBU 1,8 - Diazabicyclo[5.4.0]undec - 7 - ene TTBP 2,4,6 - Tri - tert - butylpyridine <![CDATA[Tf2O]]> Trifluoromethanesulfonic Anhydride LevOH Levulinic Acid TMSOTf Trimethylsilyl Trifluoromethanesulfonate <![CDATA[CNCCl3]]> Trichloroacetonitrile min Minute mL Milliliter mmol Millimole

[0100] Example 1: Synthesis of Compound I-1

[0101] Step 1: Weigh 1,2,3,4,6-penta-O-acetyl-D-mannopyranose (19.5 g, 50.0 mmol) and place it in a 500 mL eggplant-shaped flask. Add anhydrous dichloromethane (150 mL). The solution is colorless and transparent. Under an ice bath, add p-toluenethiol (6.2 g, 50.0 mmol) and boron trifluoride diethyl etherate solution (12.7 mL, 100 mmol). Stir for half an hour under the ice bath and then continue to stir at room temperature for 24 hours. The solution changes from light yellow to pink. When TLC (EA / Hex = 1 / 2) shows that the raw material has disappeared, quench the reaction with saturated NaHCO3 solution. Separate the dichloromethane layer with a separatory funnel, evaporate the solvent under reduced pressure, then redissolve it in 200 mL of anhydrous methanol. Add sodium methoxide (256 mg, 4.7 mmol) and stir at room temperature for 12 hours. When TLC (EA / Hex = 1 / 2) shows that the raw material has completely reacted, adjust the pH to neutral with dilute hydrochloric acid, evaporate the solvent under reduced pressure, then add dichloromethane (100 mL) and water (100 mL), stir at room temperature for half an hour, separate the aqueous layer with a separatory funnel, and evaporate the water under reduced pressure to obtain crude compound A-1, which is directly used for the next step without purification.

[0102] Step 2: Dissolve the crude compound A-1 (about 47.3 mmol) in 200 mL of anhydrous DMF, add PhCH(OMe)2 (7.9 g, 52.03 mmol, 1.1 eq), and p-toluenesulfonic acid (899 mg, 4.7 mmol, 0.1 eq). Control the reaction temperature at 50 °C and react under reduced pressure for 2 hours. TLC (CH3OH / DCM = 1 / 9) shows that the raw material has completely reacted. Cool the reaction to room temperature, add NaH (5.68 g, 141.9 mmol, 3.0 eq) under an ice bath and react for 20 min, then add BnBr (20.2 g, 118.3 mmol, 2.5 eq). Remove the ice bath after 20 minutes and stir at room temperature for 12 hours. TLC (EA / Hex = 1 / 4) shows that the raw material has been completely converted. Quench NaH with methanol (10 mL) under an ice bath, add ethyl acetate (200 mL) to dilute the reaction solution, wash the reaction solution three times with saturated brine (200 mL), concentrate and remove the solvent under reduced pressure, then redissolve it in 300 mL of methanol, add p-toluenesulfonic acid (899 mg, 10%), and react at room temperature for 12 hours. TLC (EA / Hex = 1 / 9) shows that the raw material has completely reacted. Then adjust the pH of the reaction solution to 8 with saturated sodium bicarbonate solution, separate the organic layer with a separatory funnel, remove the solvent under reduced pressure, separate and purify by column chromatography. The eluent ratio is (EA / Hex = 1 / 12 - CH3OH / DCM = 1 / 9). Rotate dry the organic phase and evacuate to constant weight to obtain white solid compound B-1 (11.8 g, 25.5 mmol), with a yield of 51% (yield calculated based on acetylmannose). The NMR data are as follows:

[0103] 1 1H NMR (600 MHz, CDCl3) δ 7.37–7.21 (m, 12H), 7.06 (d, J = 7.9 Hz, 2H), 5.45 (d, J = 1.3 Hz, 1H), 4.60 (d, J = 12.2 Hz, 1H), 4.57–4.46 (m, 3H), 4.17–4.00 (m, 2H), 3.94 (dd, J = 3.0, 1.5 Hz, 1H), 3.85–3.73 (m, 2H), 3.68 (dd, J = 9.1, 3.1 Hz, 1H), 3.35 (s, 1H), 2.70 (d, J = 3.7 Hz, 1H), 2.28 (s, 3H).

[0104] Step 3: Weigh compound B-1 (6.3 g, 13.6 mmol) and place it in a 250 mL eggplant-shaped flask. Add 87 mL of a mixed solution of water and dichloromethane (v / v = 1 / 2), which is colorless and transparent. Add TEMPO (424 mg, 2.7 mmol, 0.2 eq), and the solution turns reddish-brown. Then add PhI(OAc)2 (10.9 g, 33.9 mmol, 2.5 eq), and stir vigorously at room temperature for 3 hours. The solution turns yellowish-brown. TLC (EA / Hex = 1 / 1) indicates that the raw material has completely reacted. Add 10 mL of saturated aqueous Na2S2O3 solution to the reaction solution, adjust the pH to 3 with dilute hydrochloric acid, stir for 10 minutes, add 100 mL of dichloromethane for extraction and separation, remove the solvent under reduced pressure, then redissolve in 180 mL of acetone. Add BnBr (4.6 g, 27.2 mmol, 2 eq) and K2CO3 (2.8 g, 20.4 mmol, 1.5 eq). Under nitrogen protection, stir at room temperature for 3 hours. The solution is reddish-brown. TLC (CH3OH / DCM = 1 / 9) indicates that the raw material has completely disappeared. Adjust the pH of the reaction solution to neutral with dilute hydrochloric acid, extract and separate with dichloromethane (100 mL), concentrate the organic phase under reduced pressure, and purify by column chromatography (EA / Hex = 1 / 10 - 1 / 1) to obtain a reddish-brown oily compound I-1 (4.3 g, 7.5 mmol), with a yield of 55% (calculated based on compound B-1). The NMR spectrum data is as follows:

[0105] 11H NMR (600 MHz, CDCl3) δ 7.70–7.28 (m, 17H), 6.97 (d, J = 7.9 Hz, 2H), 5.23 (dd, J = 44.0, 12.3 Hz, 2H), 5.00 (d, J = 11.4 Hz, 1H), 4.86 (d, J = 11.4 Hz, 1H), 4.75 (q, J = 12.0 Hz, 2H), 4.68 (d, J = 0.9 Hz, 1H), 4.45 (td, J = 9.5, 2.5 Hz, 1H), 4.10 (t, J = 6.4 Hz, 1H), 3.76 (d, J = 9.6 Hz, 1H), 3.58–3.41 (m, 1H), 3.08 (d, J = 2.5 Hz, 1H), 2.30 (s, 3H).

[0106] Example 2: Synthesis of Compound II-1

[0107] Step 1: Weigh Compound I-1 (15.0 g, 26.3 mmol) and place it in a 1000 mL eggplant-shaped flask. Add 400 mL of anhydrous dichloromethane. The solution turns reddish-brown. Add LevOH (7.6 g, 65.8 mmol, 2.5 eq), DIC (8.3 g, 10.2 mL, 65.8 mmol, 2.5 eq), and DMAP (8.1 g, 65.8 mmol, 2.5 eq). Stir at room temperature for 3 hours. The solution becomes yellowish-brown and turbid. TLC (EA / Hex = 1 / 2) indicates complete conversion of the starting material. Add saturated brine (500 mL), extract and separate the layers, and concentrate the organic phase under reduced pressure to obtain the crude product Compound D-1, which is directly used in the next step.

[0108] Step 2: Dissolve the crude Compound D-1 in 320 mL of acetone / water (v / v = 15 / 1). The solution is light yellow. Add NBS (16.8 g, 4 eq). After a few minutes, it turns reddish-brown. Stir at room temperature for 1 hour. The solution turns light yellow. TLC (EA / Hex = 1 / 2) indicates complete conversion of the starting material. Quench the reaction with saturated Na2S2O3 solution (10 mL), then add dichloromethane (500 mL) for extraction. Wash the organic layer with saturated brine once, and concentrate under reduced pressure to remove the solvent to obtain the yellow oil Compound E-1, which is directly used in the next reaction.

[0109] Step 3: Dissolve the crude product compound E-1 obtained in the previous step in 90 mL of anhydrous dichloromethane, add CNCCl3 (6.3 g, 4.3 mL, 5 eq) and DBU (663 mg, 0.7 mL, 4.4 mmol, 0.5 eq). The solution turned dark brown and was stirred for 3 hours in an ice bath. TLC (EA / Hex = 1 / 2) indicated complete conversion of the starting material. The reaction solution was concentrated under reduced pressure and purified by column chromatography with the eluent ratio (EA / Hex = 1 / 6 - 1 / 1) to obtain the light yellow oily compound II-1 (13.5 g, 19.2 mmol) with a yield of 73% (calculated based on compound I-1). The NMR data are as follows:

[0110] 1 H NMR(400MHz,CDCl3)δ7.43–7.16(m,15H),6.53(d,J=3.3 Hz,1H),5.60(t,J=7.5Hz,1H),5.12(t,J=12.9Hz,1H),5.03(d,J=12.2Hz,1H),4.88(d,J=46.7Hz,2H),4.65(ddd,J=34.3,16.0,6.9Hz,1H), 4.51(d,J=7.6Hz,1H),4.49–4.38(m,1H),3.89(dd,J=7.8,2.9Hz,1H),3.85–3.76(m,1H),2.72–2.44(m,3H),2.39(d,J=7.2Hz,1H),2.15(d,J =11.7Hz,3H).

[0111] Example 3: Synthesis of compound III-1

[0112] Weigh compound D-1 (300 mg, 0.45 mmol) and dissolve it in 20 mL of anhydrous DCM. Under nitrogen protection, add TTBP (334.8 mg, 1.35 mmol, 3 eq), diphenyl sulfoxide (109 mg, 0.54 mmol, 1.2 eq), and dry 4 Å molecular sieve (500 mg). Stir the solution at -78 °C for 10 minutes. Add Tf2O (141 mg, 0.5 mmol, 1.1 eq). Stir the solution for 10 minutes, then add benzyl alcohol (54 mg, 0.5 mmol, 1.1 eq). React at -78 °C for 1 hour. TLC (EA / Hex = 1 / 2) indicates complete conversion of the starting material. Adjust the pH to neutral with saturated sodium bicarbonate, remove the molecular sieve, and concentrate the organic phase under reduced pressure to obtain the crude product of compound F-1. Redissolve it in 20 mL of DCM / pyridine (v / v = 4 / 1), add hydrazine acetate (180 mg, 1.95 mmol, 4.33 eq), and stir at room temperature for 2 hours. TLC (EA / Hex = 1 / 2) indicates the disappearance of the starting material. Quench the reaction with acetone, dilute with dichloromethane, then adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase over anhydrous sodium sulfate, filter, concentrate the organic phase, and purify by column chromatography with an eluent ratio of (EA / Hex = 1 / 4 - 1 / 1) to obtain the colorless oil compound III-1 (224 mg, 0.41 mmol) with a yield of 90% (calculated based on compound D-1). The NMR data is as follows:

[0113] 1 H NMR (600 MHz, CDCl3) δ 7.49–7.06 (m, 20H), 5.24 (q, J = 12.3 Hz, 2H), 5.03 (d, J = 1.6 Hz, 1H), 4.74 (d, J = 11.9 Hz, 1H), 4.70–4.54 (m, 3H), 4.50 (d, J = 11.9 Hz, 1H), 4.35 (td, J = 9.2, 2.4 Hz, 1H), 4.20 (d, J = 9.3 Hz, 1H), 3.80 (dd, J = 9.2, 3.0 Hz, 1H), 3.78–3.71 (m, 1H), 2.82 (d, J = 2.4 Hz, 1H).

[0114] Example 4: Synthesis of Compound IV-1

[0115] Weigh compound II-1 (2.0 g, 2.84 mmol) and compound III-1 (1.7 g, 3.12 mmol, 1.1 eq) and dissolve them in 80 mL of anhydrous DCM. The solution is reddish-brown. Add dry 4 Å molecular sieves (1.8 g). After stirring at -40 °C for 10 min, add TMSOTf (126 mg, 0.57 mmol, 0.2 eq). Stir the reaction under N2 protection for 1 hour. TLC (EA / Hex = 1 / 2) indicates the disappearance of compound II-1. Adjust the pH to neutral with triethylamine, remove the molecular sieves, concentrate the organic phase under reduced pressure, redissolve it in 20 mL of DCM / pyridine (v / v = 4 / 1), add hydrazine acetate (1130 mg, 12.3 mmol, 4.33 eq), and stir at room temperature for 5 hours. TLC (EA / Hex = 1 / 2) indicates the disappearance of the starting materials. Quench the reaction with acetone, dilute with dichloromethane, then adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the organic phase, and purify by column chromatography with an eluent ratio of (EA / Hex = 1 / 8 - 1 / 1) to obtain a pale yellow oily compound IV-1 (1813 mg, 1.8 mmol) with a yield of 64%. The NMR data are as follows:

[0116] 1 H NMR (600 MHz, CDCl3) δ 7.35–6.92 (m, 35H), 5.19 (s, 1H), 5.08 (d, J = 12.2 Hz, 1H), 5.00 (d, J = 12.2 Hz, 1H), 4.94 (d, J = 12.2 Hz, 1H), 4.80 (dd, J = 27.2, 12.1 Hz, 2H), 4.71 (d, J = 12.2 Hz, 1H), 4.57 (dd, J = 12.1, 4.8 Hz, 2H), 4.52–4.35 (m, 8H), 4.35–4.28 (m, 1H), 4.18 (t, J = 9.5 Hz, 1H), 4.03–3.93 (m, 1H), 3.72 (t, J = 4.2 Hz, 1H), 3.64–3.56 (m, 2H), 3.15 (dd, J = 9.5, 2.8 Hz, 1H), 2.94 (s, 1H).

[0117] Example 5: Synthesis of β-D-(1,4)-mannuronic acid disaccharide (Compound IX-1)

[0118] Compound IV-1 (500 mg, 0.5 mmol) was dissolved in 22 mL of THF / H2O / t-BuOH (v / v / v = 1 / 1 / 0.2), and palladium on carbon (palladium content 10%) (50 mg) was added. The reaction was stirred at 25 °C under hydrogen for 48 hours. TLC (EA / Hex = 1 / 2) indicated the disappearance of the starting material. The palladium on carbon was filtered off, and the palladium on carbon layer was washed three times with water (50 mL * 3). The aqueous phases were combined, extracted once with EA (100 mL), concentrated under reduced pressure to 10 mL, and freeze-dried to obtain white solid compound IX-1 (160 mg, 0.4 mmol), with a yield of 86%. The NMR data are as follows:

[0119] 1 H NMR (600 MHz, D2O) δ 5.23–5.12 (m, 1H), 4.75 (d, J = 9.4 Hz, 1H), 4.67–4.57 (m, 1H), 4.40 (t, J = 6.8 Hz, 1H), 4.08 (dt, J = 45.1, 20.4 Hz, 1H), 4.02–3.85 (m, 2H), 3.85–3.67 (m, 2H), 3.67–3.48 (m, 1H).

[0120] 13 C NMR (151 MHz, D2O) δ 172.85, 172.73, 101.35, 101.06, 99.82, 95.47, 94.08, 93.28, 92.97, 79.52, 77.87, 76.14, 75.36, 75.22, 72.32, 72.27, 71.82, 71.16, 70.11, 69.96, 69.84, 69.80, 69.75, 69.64, 69.59, 69.53, 69.43, 69.21, 69.04, 67.91, 67.79, 67.75, 67.66.

[0121] Example 6: Synthesis of Compound VI-1

[0122] Weigh compound II-1 (2.0 g, 2.84 mmol) and compound I-1 (1.78 g, 3.13 mmol, 1.1 eq) and dissolve them in 80 mL of anhydrous DCM, which shows a reddish-brown color. Add dry 4 Å molecular sieves (2.0 g). After stirring at -40 °C for 10 min, add TMSOTf (126 mg, 0.57 mmol, 0.2 eq). Stir for 1 hour under N2 protection. TLC (EA / Hex = 1 / 2) indicates that the reaction of compound II-1 is complete. Add saturated sodium bicarbonate to adjust the pH to neutral, filter off the molecular sieves, extract, dry, and concentrate. Purify by column chromatography with the eluent ratio (EA / Hex = 1 / 8 - 1 / 1) to obtain the pale yellow solid compound VI-1 (2.7 g, 2.42 mmol) with a yield of 86%. The NMR data are as follows:

[0123] 1 H NMR (600 MHz, CDCl3) δ 7.39–7.15 (m, 34H), 6.90 (d, J = 8.0 Hz, 2H), 5.68 (d, J = 6.8 Hz, 1H), 5.56–5.46 (m, 1H), 5.13–5.02 (m, 2H), 4.92 (dd, J = 36.4, 12.2 Hz, 2H), 4.75–4.53 (m, 3H), 4.53–4.38 (m, 7H), 4.38–4.26 (m, 1H), 4.16 (s, 1H), 3.83 (dt, J = 9.6, 3.2 Hz, 2H), 3.73 (dd, J = 8.1, 2.7 Hz, 1H), 3.40 (dd, J = 9.6, 2.8 Hz, 1H), 2.57 (dt, J = 19.8, 7.8 Hz, 1H), 2.52–2.37 (m, 2H), 2.36–2.25 (m, 1H), 2.22 (s, 3H), 2.15–2.05 (m, 3H)

[0124] Example 7: Synthesis of β-D-(1,4)-mannuronic acid tetrasaccharide (Compound IX-2)

[0125] Step 1: Weigh VI-1 (500 mg, 0.45 mmol) and dissolve it in 20 mL of anhydrous DCM. Under nitrogen protection, add TTBP (334.8 mg, 1.35 mmol, 3 eq), diphenyl sulfoxide (109 mg, 0.54 mmol, 1.2 eq), and 500 mg of dry 4 Å molecular sieve. Stir the solution at -78 °C for 10 minutes. Add Tf2O (141 mg, 0.5 mmol, 1.1 eq). Stir the solution for 10 minutes, then add compound IV-1 (500 mg, 0.5 mmol, 1.1 eq). React at -78 °C for 1 hour. TLC (EA / Hex = 1 / 2) indicates complete conversion of the starting materials. Adjust the pH to neutral with saturated sodium bicarbonate, remove the molecular sieve, concentrate the organic phase under reduced pressure, redissolve it in 20 mL of DCM / pyridine (v / v = 4 / 1), add hydrazine acetate (180 mg, 1.95 mmol, 4.33 eq), and stir at room temperature for 2 hours. TLC (EA / Hex = 1 / 2) shows the disappearance of the starting materials. Quench the reaction with acetone, dilute with dichloromethane, then adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase over anhydrous sodium sulfate, filter, concentrate the organic phase, and purify by column chromatography with an eluent ratio of (EA / Hex = 1 / 4 - 1 / 1) to obtain a pale yellow solid, compound VIII-1 (766 mg, 0.405 mmol), with a yield of 90% (calculated based on compound VI-1).

[0126] Step 2: Dissolve compound VIII-1 (766 mg, 0.405 mmol) in 22 mL of THF / H2O / t-BuOH (v / v / v = 1 / 1 / 0.2), add 10% palladium on carbon (300 mg), and stir the reaction at 25 °C under hydrogen for 48 hours. TLC (EA / Hex = 1 / 2) shows the disappearance of the starting materials. Filter off the palladium on carbon, wash the palladium on carbon layer three times with water (50 mL * 3), combine the aqueous phases, extract once with EA (100 mL), concentrate the water under reduced pressure to 10 mL, and freeze-dry to obtain a white solid, compound IX-2 (263 mg, 0.365 mmol), with a yield of 90%.

[0127] 1 1H NMR (600 MHz, D2O) δ 5.22 (d, J = 4.4 Hz, 1H), 4.98–4.80 (m, 2H), 4.82–4.76 (m, 2H), 4.46 (d, J = 6.6 Hz, 1H), 4.17 (t, J = 6.9 Hz, 1H), 4.13–3.87 (m, 8H), 3.87–3.65 (m, 3H), 3.65–3.48 (m, 2H).

[0128] 1313C NMR (150 MHz, D2O) δ 172.68, 172.11, 171.66, 100.40, 100.37, 99.86, 95.47, 93.99, 92.93, 77.94, 77.84, 77.66, 76.54, 75.23, 74.38, 73.03, 72.93, 72.31, 71.81, 71.16, 70.97, 69.97, 69.60, 69.53, 69.46, 69.19, 69.04, 67.63。

[0129] Example 8: Synthesis of β-D-(1,4)-mannuronic acid trisaccharide (Compound IX-3)

[0130] Step 1: Weigh Compound II-1 (2.0 g, 2.84 mmol) and Compound IV-1 (3.12 g, 3.13 mmol, 1.1 eq) and dissolve them in 100 mL of anhydrous DCM. The solution is reddish-brown. Add dry 4 Å molecular sieves (1.8 g). After stirring at -40 °C for 10 min, add TMSOTf (126 mg, 0.57 mmol, 0.2 eq), and stir the reaction for 1 hour under N2 protection. TLC (EA / Hex = 1 / 2) shows the disappearance of Compound II-1. Adjust the pH to neutral with triethylamine, remove the molecular sieves, concentrate the organic phase under reduced pressure, redissolve it in 50 mL of DCM / pyridine (v / v = 4 / 1), add hydrazine acetate (1130 mg, 12.3 mmol, 4.33 eq), and stir at room temperature for 5 hours. TLC (EA / Hex = 1 / 2) shows the disappearance of the starting materials. Quench the reaction with acetone, dilute with dichloromethane, then adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the organic phase, and purify by column chromatography. The eluent ratio is (EA / Hex = 1 / 8 - 1 / 1), to obtain a pale yellow oily Compound V-1 (2547 mg, 1.76 mmol) with a yield of 62%. The NMR data are as follows:

[0131] 11H NMR (600 MHz, CDCl3) δ 7.26 (ddd, J = 48.8, 27.9, 19.6 Hz, 50H), 5.25 (s, 1H), 5.14–4.97 (m, 4H), 4.88 (dd, J = 27.9, 12.0 Hz, 3H), 4.73 (t, J = 11.9 Hz, 2H), 4.62 (ddd, J = 33.3, 22.0, 11.5 Hz, 6H), 4.54–4.39 (m, 8H), 4.39–4.27 (m, 2H), 4.17 (t, J = 8.7 Hz, 1H), 4.04 (s, 1H), 3.79 (d, J = 8.3 Hz, 1H), 3.70 (d, J = 14.7 Hz, 3H), 3.48 (d, J = 9.3 Hz, 1H), 3.45–3.34 (m, 1H), 3.11 (d, J = 8.3 Hz, 1H), 2.82 (s, 1H).

[0132] Step 2: Dissolve compound V-1 (500 mg, 0.34 mmol) in 20 mL of THF / H2O / t-BuOH (v / v / v = 1 / 1 / 0.2), add palladium on carbon (palladium content 10%) (50 mg), stir and react under hydrogen at 25 °C for 48 hours. TLC (EA / Hex = 1 / 2) indicates that the raw material has disappeared. Filter off the palladium on carbon, wash the palladium on carbon layer three times with water (50 mL * 3), combine the aqueous phases, extract once with EA (100 mL), concentrate the water under reduced pressure to 10 mL, and freeze-dry to obtain white solid compound IX-3 (167 mg, 0.31 mmol), with a yield of 90%. The NMR data are as follows:

[0133] 1 1H NMR (600 MHz, D2O) δ 5.17 (d, J = 4.3 Hz, 1H), 4.77 (s, 1H), 4.41 (d, J = 6.6 Hz, 1H), 4.11 (d, J = 6.7 Hz, 1H), 4.05–3.94 (m, 3H), 3.94–3.89 (m, 2H), 3.88 (d, J = 9.9 Hz, 1H), 3.82–3.77 (m, 1H), 3.77–3.65 (m, 3H), 3.58 (dd, J = 9.5, 3.0 Hz, 1H).

[0134] 1313C NMR (151 MHz, D2O) δ 185.70, 172.66, 172.09, 171.67, 100.34, 99.85, 93.97, 92.93, 77.93, 77.84, 77.73, 77.64, 74.36, 73.03, 72.30, 71.79, 71.12, 70.95, 70.56, 70.08, 69.98, 69.55, 69.48, 69.18, 69.02, 67.72, 67.61。

[0135] Example 9: Synthesis of β-D-(1,4)-mannuronic acid pentasaccharide (Compound IX-4)

[0136] Step 1: Weigh VI-1 (500 mg, 0.45 mmol) and dissolve it in 20 mL of anhydrous DCM. Under nitrogen protection, add TTBP (334.8 mg, 1.35 mmol, 3 eq), diphenyl sulfoxide (109 mg, 0.54 mmol, 1.2 eq), and 500 mg of dry 4 Å molecular sieve. Stir the solution at -78 °C for 10 minutes, add Tf2O (141 mg, 0.5 mmol, 1.1 eq), stir the solution for 10 minutes, add Compound V-1 (723 mg, 0.5 mmol, 1.1 eq), and react at -78 °C for 1 hour. TLC (EA / Hex = 1 / 2) indicates complete conversion of the starting materials. Adjust the pH to neutral with saturated sodium bicarbonate, remove the molecular sieve, concentrate the organic phase under reduced pressure, redissolve it in 20 mL of DCM / pyridine (v / v = 4 / 1), add hydrazine acetate (180 mg, 1.95 mmol, 4.33 eq), and stir at room temperature for 2 hours. TLC (EA / Hex = 1 / 2) indicates the disappearance of the starting materials. Quench the reaction with acetone, dilute with dichloromethane, then adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase over anhydrous sodium sulfate, filter, concentrate the organic phase, and purify by column chromatography with an eluent ratio of (EA / Hex = 1 / 4 - 1 / 1) to obtain a pale yellow solid, Compound VIII-2 (935 mg, 0.40 mmol), with a yield of 88% (calculated based on Compound VI-1).

[0137] Step 2: Dissolve compound VIII-2 (935 mg, 0.40 mmol) in 22 mL of THF / H2O / t-BuOH (v / v / v = 1 / 1 / 0.2), add palladium on carbon (palladium content 10%) (300 mg), stir and react at 25 °C under hydrogen for 48 hours. TLC (EA / Hex = 1 / 2) indicates the disappearance of the raw materials. Filter out the palladium on carbon, wash the palladium on carbon layer three times with water (50 mL * 3), combine the aqueous phases, extract once with EA (100 mL), concentrate the water under reduced pressure to 10 mL, and freeze-dry to obtain white solid compound IX-4 (323 mg, 0.36 mmol) with a yield of 90%. The NMR data are as follows:

[0138] 1 H NMR(600MHz,D2O)δ5.17(s,1H),4.86(s,1H),4.63–4.51(m, 3H),4.29(d,J=5.8Hz,1H),4.05(s,1H),3.97(s,3H),3.91(t,J=10.4Hz,2H),3.84(d,J=9.3Hz,6H),3.76(dd,J=14.9,7.5Hz,2H),3.72(d,J=18.2Hz,3H),3.58(d,J=7.0Hz,1H),3.53(s,1H).

[0139] 13C NMR(151MHz,D2O)δ174.35,174.12,173.65,173.37,172.69, 100.23,100.15,99.78,97.86,93.81,93.18,78.07,78.00,77.86,77.79,74.50,72.39,71.24,70.38,70.15,69.80,69.71,69.51,69.01,67.99,67.45,61.48.

[0140] Example 10: Synthesis of β-D-(1,4)-mannuronic acid heptasaccharide (Compound IX-5)

[0141] Step 1: Weigh compound VI-1 (500 mg, 0.45 mmol) and dissolve it in 20 mL of DCM / pyridine (v / v = 4 / 1). Add hydrazine acetate (180 mg, 1.95 mmol, 4.33 eq), and stir at room temperature for 2 hours. TLC (EA / Hex = 1 / 2) indicates that the raw material has disappeared. Quench the reaction with acetone, dilute with dichloromethane, adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the organic phase, add dry 4 Å molecular sieve (2.0 g), redissolve in 20 mL of anhydrous DCM, then add compound II-1 (317 mg, 0.45 mmol). The solution turns reddish-brown. Under N2 protection, control the temperature below -40 °C, stir for 10 min, then add TMSOTf (20 mg, 0.09 mmol, 0.2 eq), and stir for 1 hour. TLC (EA / Hex = 1 / 2) indicates that compound II-1 has completely reacted. Add saturated sodium bicarbonate to adjust the pH to neutral, filter off the molecular sieve, extract, dry, and concentrate. Stir with silica gel and separate by column chromatography. The eluent ratio is (EA / Hex = 1 / 8 - 1 / 1) to obtain a pale yellow solid compound VII-1 (560 mg, 0.36 mmol), with a yield of 80% (calculated based on compound VI-1).

[0142] Step 2: Weigh VII-1 (500 mg, 0.32 mmol) and dissolve it in 20 mL of anhydrous DCM. Under nitrogen protection, add TTBP (238 mg, 0.96 mmol, 3 eq), diphenyl sulfoxide (78 mg, 0.38 mmol, 1.2 eq), and dry 4 Å molecular sieve (500 mg). Stir the solution at -78 °C for 10 minutes, add Tf2O (100 mg, 0.35 mmol, 1.1 eq), stir the solution for 10 minutes, then add compound VIII-1 (663 mg, 0.35 mmol, 1.1 eq). React at -78 °C for 1 hour. TLC (EA / Hex = 1 / 2) indicates that the raw material has been completely converted. Add saturated sodium bicarbonate to adjust the pH to neutral, remove the molecular sieve, concentrate the organic phase under reduced pressure, redissolve in 20 mL of DCM / pyridine (v / v = 4 / 1), add hydrazine acetate (128 mg, 1.39 mmol, 4.33 eq), and stir at room temperature for 2 hours. TLC (EA / Hex = 1 / 2) indicates that the raw material has disappeared. Quench the reaction with acetone, dilute with dichloromethane, adjust the pH to neutral with dilute hydrochloric acid, wash once with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the organic phase, and purify by column chromatography. The eluent ratio is (EA / Hex = 1 / 4 - 1 / 1) to obtain a pale yellow solid compound VIII-3 (880 mg, 0.27 mmol), with a yield of 85% (calculated based on compound VII-1).

[0143] Step 3: Dissolve compound VIII-3 (880 mg, 0.27 mmol) in 22 mL of THF / H2O / t-BuOH (v / v / v = 1 / 1 / 0.2), add palladium on carbon (palladium content 10%) (300 mg), stir and react at 25 °C under hydrogen for 48 hours. TLC (EA / Hex = 1 / 2) indicates that the raw material has disappeared. Filter out the palladium on carbon, wash the palladium on carbon layer three times with water (50 mL * 3), combine the aqueous phases, extract once with EA (100 mL), concentrate the water under reduced pressure to 10 mL, and freeze-dry to obtain white solid compound IX-5 (300 mg, 0.24 mmol), with a yield of 89%. The NMR data are as follows:

[0144] 1 H NMR (600 MHz, D2O) δ 5.96 (d, J = 3.9 Hz, 1H), 5.15 (s, 1H), 4.84 (s, 2H), 4.65 (d, J = 3.9 Hz, 3H), 4.60 (s, 3H), 4.16 (s, 1H), 4.10–4.01 (m, 1H), 3.96 (s, 3H), 3.91 (s, 2H), 3.84 (d, J = 16.4 Hz, 5H), 3.70 (s, 6H), 3.66 (d, J = 6.7 Hz, 4H), 3.56 (dd, J = 12.2, 6.7 Hz, 3H).

[0145] 13 C NMR (151 MHz, D2O) δ 174.37, 174.05, 174.02, 174.01, 173.94, 173.84, 173.60, 100.23, 100.15, 99.78, 97.86, 93.81, 93.19, 78.08, 78.00, 77.87, 77.79, 74.64, 72.39, 71.25, 70.38, 70.16, 69.80, 69.71, 69.51, 69.01, 68.00, 67.45, 61.49.

Claims

1. A method for preparing a compound represented by formula VIII or a β-D-(1,4)-mannuronic acid oligosaccharide represented by formula IX, comprising: Couple compound II and compound III to form a 1,4-glycosidic bond, and then selectively remove the hydroxyl protecting group R at the 4-position 3 to obtain compound IV; Afterwards, compound IV is coupled with compound II to form a 1,4-glycosidic bond, and then the hydroxyl protecting group R at the 4-position is selectively removed. 3 Optionally, the obtained compound is repeatedly subjected to the above-mentioned coupling reaction and deprotection of protecting group R 3 until compound V is obtained. The compound I and the compound II are subjected to a coupling reaction to form a 1,4-glycosidic bond, and then the hydroxyl protecting group R at the 4-position is selectively removed 3 to obtain the compound VI; Couple compound VI with compound II to form a 1,4-glycosidic bond, and then selectively remove the hydroxyl protecting group R at the 4-position. 3 Optionally, continue to repeat the above-mentioned coupling reaction and deprotection of protecting group R for the resulting compound. 3 until compound VII is obtained. Couple compound V and compound VII and selectively remove the protecting group R 3 Compound VIII is formed, wherein the coupling reaction in this step is carried out in the presence of a bulky organic base, diphenyl sulfoxide and a sulfonic anhydride catalyst; Optionally, the protecting group R of compound VIII is removed in one step 2 to produce a β-D-(1,4)-mannuronic acid oligosaccharide as shown in formula IX; Among them, m is selected from integers of 2 - 18; n and n' are each independently selected from integers of 0 - 8; R 1 is an optionally C 1-8 alkyl-substituted C 6-14 aryl; R 2 is a hydroxyl protecting group that can be removed by a palladium-carbon-catalyzed hydrogenation reaction or a palladium-carbon-catalyzed oxidation reaction; R 3 is a hydroxyl protecting group that cannot be removed by a palladium-carbon-catalyzed hydrogenation reaction or a palladium-carbon-catalyzed oxidation reaction; R 4 is H; X is selected from fluorine, chlorine, bromine, iodine.

2. The method according to claim 1, wherein the sulfonic anhydride catalyst is selected from methanesulfonic anhydride, trifluoromethanesulfonic anhydride or p-toluenesulfonic anhydride.

3. The method according to claim 1, wherein the coupling reaction of compound II and compound III to form a 1,4-glycosidic bond is carried out in the presence of a sulfonic acid catalyst; the sulfonic acid catalyst is selected from: methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid or trimethylsilyl trifluoromethanesulfonate.

4. The method according to claim 1, wherein compound II is obtained as follows: Step 1: Protect the 4-hydroxy group of compound I to obtain compound D; Step 2: Selectively remove the anomeric carbon protecting group -SR 1 , to obtain compound E; Step 3: Under alkaline conditions, react compound E with CX3C(=NR 4 )X or CX3CN to obtain compound II; wherein R 1 , R 2 and R 3 are as defined in claim 1; R 4 is H; X is selected from fluorine, chlorine, bromine, iodine.

5. The method according to claim 1, wherein compound III is obtained as follows: Step 1: Protect the 4-hydroxy group of compound I to obtain compound D; Step 2: Under alkaline conditions, react compound D with R 2 OH to obtain compound F; Step 3: Selectively remove the hydroxyl protecting group R at the 4-position of compound F 3 to obtain compound III; wherein R 1 , R 2 and R 3 are as defined in claim 1.

6. The method according to claim 1, further comprising: Step 1: React 1,2,3,4,6-penta-O-acetyl-D-mannopyranose with an anomeric carbon protecting agent R 1 in an SH reaction, followed by a hydrolysis reaction to obtain compound A; Step 2: Selectively protect the hydroxyl groups at the 2- and 3-positions of compound A to obtain compound B; Step 3: Oxidize the hydroxyl group at the 6-position of compound B to a carboxyl group, and then esterify the carboxyl group to obtain compound I; wherein R 1 and R 2 are as defined in claim 1.

7. The method according to any one of claims 1-6, wherein, R 2 selected from C 6-14 arylmethyl or allyl, wherein said C 6-14 arylmethyl is optionally substituted by C1-8 alkyl, C 1-8 alkoxy, or halogen; R 3 selected from C 1-8 alkylcarbonyl, C 1-8 alkoxycarbonyl, C 6-14 arylcarbonyl, tris(C 1-8 alkyl)silyl, 9-fluorenylmethoxycarbonyl, tris(C 6-14 aryl)methyl; wherein any carbon atom in the C 1-8 alkylcarbonyl and C 1-8 alkoxycarbonyl is optionally oxo-substituted. 1-8 ​ 8. The method according to claim 7, wherein, R 2 selected from benzyl, p-methoxybenzyl, naphthylmethyl, allyl; R 3 Selected from acetyl, acetylpropionyl, trimethylsilyl, tert-butyldimethylsilyl, benzoyl, 9-fluorenylmethoxycarbonyl, trityl.

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