WO2017071152A1 - 磷酰甘露五糖及其衍生物，及其制备方法和应用 - Google Patents

磷酰甘露五糖及其衍生物，及其制备方法和应用 Download PDF

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WO2017071152A1
WO2017071152A1 PCT/CN2016/077370 CN2016077370W WO2017071152A1 WO 2017071152 A1 WO2017071152 A1 WO 2017071152A1 CN 2016077370 W CN2016077370 W CN 2016077370W WO 2017071152 A1 WO2017071152 A1 WO 2017071152A1
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formula
compound
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mannose
pentasaccharide
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胡文浩
周俊
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如东瑞恩医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C07H11/04—Phosphates; Phosphites; Polyphosphates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical fields of medicinal chemistry, organic chemistry, natural product chemistry and the like. Specifically, it relates to phosphoryl mannose pentasaccharide and its derivatives, a preparation method thereof and application thereof in preparing an antitumor drug.
  • Angiogenesis is a key process in tumor growth and metastasis. Therefore, inhibition of the angiogenesis process is recognized as one of the strategies for treating cancer.
  • approved anti-angiogenic treatment methods such as tyrosine kinase inhibitors, have obvious clinical anti-tumor effects, but still have problems such as low survival rate and toxic side effects on the heart. Therefore, the development of angiogenesis inhibitors with low side effects and targeting various angiogenic factors is the current research direction.
  • Heparan Sulfate which is distributed on the cell surface and extracellular matrix, is a complex type of sulfated polysaccharide that is ubiquitous in nature and plays a dominant role in cancer biology, including: inhibiting angiogenesis, regulating tumor evolution and metastasis. Wait.
  • heparin sulfate mimics can block the process of HS-related angiogenic factor signaling; it also inhibits heparanase, effectively inhibits heparinase degradation of HS and promotes tumor metastasis.
  • the Heparan Sulfate Simulator PI-88 (structure shown in Figure 1) developed by Vigen Ferro Group, Progen, Australia, is a complex mixture of highly sulfated mannooligosaccharides with anti-angiogenesis and anti-tumor metastasis.
  • PI-88 can simultaneously inhibit angiogenesis and tumor metastasis by two mechanisms: 1) direct inhibition of angiogenesis (Angiogenesis): interference with HS and angiogenic growth factors FGF-1, FGF-2 The interaction between VEGF, thereby avoiding the cascade of cellular signaling that triggers angiogenesis.
  • Blocking tumor metastasis By strongly inhibiting heparanase, it prevents the degradation of the extracellular matrix and the proteoglycan HS side chain on the basement membrane, thereby preventing the metastatic tumor cells from escaping into the blood circulation.
  • heparinase also plays an important role in angiogenesis, so PI-88 also inhibits angiogenesis indirectly by inhibiting heparanase.
  • PI-88 is a mixture of low molecular weight phosphorylmannose oligosaccharides degraded by the extracellular phosphopolysaccharide of yeast Pichia holstii NRRL Y-2448, and the highly sulfated mannooligosaccharide complex obtained by sulfation modification Mixture (see Figure 1).
  • the Danish Parolis Group and the Australian Vito Ferro Group reported in the Bobohydr.Res. 1998, 309, 77-87 and Carbohydr. Res.
  • yeast Pichia holstii NRRL Y-2448 extracellular phosphate mannose in 1 Mild acidic hydrolysis is carried out in % acetic acid to give a mixture of phosphorylmannose oligosaccharides.
  • the Australian Vito Ferro team also reported in Carbohydr. Res. 2002, 337, 139–146 the use of capillary electrophoresis to analyze the content of each component in the mixture.
  • the main component of the phosphoryl mannose pentasaccharide is 59% and the tetrasaccharide is 28.5%.
  • trisaccharides 5.5%), disaccharides (3%) and hexoses (1%) (see Figure 1).
  • a single component of the phosphoryl mannose pentasaccharide of the formula (I) is obtained by chemical synthesis, and then modified by sulfation to obtain a sulfated glucomannan pentasaccharide of the formula (III), which is advantageous for the development of a new component.
  • Tumor anti-angiogenic pharmaceutical molecules with a view to optimizing biological activity and associated quality control.
  • the main problems are: 1) the imidate donor is unstable and unsuitable for storage; therefore, it is not suitable for large-scale preparation and modular combination synthesis; 2) The activity of the imidate donor is relatively high, and a series of side reactions easily occur in the glycosylation reaction, thereby reducing the yield; in order to solve this problem, a larger amount of the imidate donor is generally required, but at the same time increasing The cost.
  • glucosinolate Another common glycosylation donor, glucosinolate, has better stability and moderate activity; however, the glycosylation reaction with mannose glucoside as donor is in the preparation of ⁇ -(1-2) -/ ⁇ -(1-3)-linked mannooligosaccharide chains are rarely reported, the main reasons may include: 1) inherent low activity of mannose donors; 2) inherent low activity of glucosinolate donors; Together, the factors result in the inability of the mannose glucoside donor to be effectively activated under glycosylation conditions.
  • the phosphate ester for the non-reducing end of the sugar chain is mainly realized by an "early phosphorylation strategy", namely: installing a protected phosphate ester (O, O'-diphenyl group) in a monosaccharide building block. Phosphoryl), followed by glycosylation assembly to construct oligosaccharide chains, and removal of protecting groups on phosphates in the late deprotection phase, eg Vito Ferro Group in Bioorg. Med. Chem. 2004, 12, 6063–6075 Synthesis of phosphorylmannose disaccharide reported in .
  • PI-88 is a highly sulfated mixture of phosphorylmannose oligosaccharides: 1) the length of the sugar chain is from disaccharide to hexose, the main component is pentasaccharide ( ⁇ 60%) and four Sugar ( ⁇ 30%); 2) Due to incomplete sulfation of the oligosaccharide mixture, the degree of sulfation and the site are uncertain, as in the Vito Ferro group at J. Med. Chem. 2005, 48, 8229-8236 [3] The oligosaccharide chain is composed of an ⁇ -(1-2)-/ ⁇ -(1-3)-glycosidic bond.
  • Progen mainly dephosphorylates the phosphoryl mannose oligosaccharide mixture and then SEC-purified to separate components with a single sugar chain length (such as pentasaccharide); For example, peracetylation, reduction end activation, etc., a series of aglycon analogs with different fatty chains at the reducing end were prepared, and their in vitro activities and pharmacokinetics were studied, such as the Vito Ferro group. Reported in J. Med. Chem. 2005, 48, 8229-8236, J. Med. Chem. 2010, 53, 1686-1699.
  • the PI-88 analog is mainly limited to the modification of the reducing end of the oligosaccharide chain, and has not been studied for the following aspects: 1) reduction ring-opening derivatization of the reducing terminal hemiacetal; 2) determination of incomplete sulfuric acid at the site 3) different glycosidic linkages.
  • the present invention overcomes the problems of the prior art, and proposes a novel phosphoryl mannose oligosaccharide and a derivative thereof, as well as a preparation method and application thereof.
  • the invention provides a phosphoryl mannose pentasaccharide and a derivative thereof, wherein the phosphoryl mannose pentasaccharide is the highest content of the yeast Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide mixture (disaccharide to hexasaccharide).
  • the components are present in the form of a single component of pentasaccharide, a compound that does not contain other sugar chain lengths.
  • the present invention provides a single component of the phosphoric acid mannose pentasaccharide of the formula (I) in a yeast Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide mixture, the derivative of which is: a phosphoryl mannose as shown in formula (II) a pentasaccharitol, a sulfated phosphoryl mannose pentasaccharide represented by formula (III), a sulfated phosphoryl mannitol pentasaccharide represented by formula (IV), and an incompletely sulfated mannooligosaccharide at a defined position Compounds represented by (V), (VI), (VII), (VIII), (IX), (X) and (XI).
  • the phosphorylated mannose pentasaccharide represented by the formula (I) and the sulfated phosphoryl mannose pentasaccharide represented by the formula (III) in the pure form means that the yeast Pichia holstii NRRL Y-2448 includes two cells. a single form component of a mixture of sugar to hexasaccharide phosphoryl mannose oligosaccharides.
  • the determination site is not determined by the formula (V)-(XI) Finish
  • Fully sulfated mannooligosaccharides means that the sulfated group is located at several specific positions on the oligosaccharide chain.
  • the present invention provides for the first time a pure form of phosphoryl mannose pentasaccharide of formula (I).
  • the present invention provides, for the first time, a pure form of the phosphoryl mannose pentasaccharide of formula (I) associated with the yeast Pichia holstii NRRL Y-2448.
  • the phosphoryl mannose pentasaccharide of the formula (I) in pure form has not been obtained, and is only present in the form of the yeast Pichia holstii NRRL Y-2448 extracellular phosphorylmannose oligosaccharide mixture (disaccharide to hexasaccharide).
  • a single component of the phosphoryl mannose pentasaccharide of the formula (I) is obtained for the first time by chemical synthesis, which is remarkably superior to the phosphorylmannose oligosaccharide mixture of the prior art.
  • the phosphoryl mannose pentasaccharide of the formula (I) can be further modified by sulfation to obtain a single component of the sulfated phosphoryl mannose pentasaccharide of the formula (III).
  • the present invention also proposes for the first time a phosphoryl mannitol pentasaccharide represented by the formula (II).
  • the present invention provides, for the first time, a phosphoryl mannose pentasaccharide of the formula (II) which is reduced by a reducing end of the yeast Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide.
  • the phosphoryl mannitol pentasaccharide represented by the formula (II) in the present invention is a reduced ring-opening derivative of the first reducing terminal hemiacetal of the yeast Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide.
  • the present invention also provides for the first time a sulfated phosphoryl mannose pentasaccharide of the formula (III) in the form of a fully sulfated octadecyl salt.
  • the fully sulfated phosphorylmannose pentasaccharide represented by formula (III) in the present invention is the first obtained yeast form Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide-related pure form of fully sulfated phosphoryl mannose five sugar.
  • a single component of the phosphoryl mannose pentasaccharide of the formula (I) obtained by chemical synthesis is subjected to sulfation to obtain a single component of the sulfated phosphoryl mannose pentasaccharide represented by the formula (III), which is remarkably It is superior to the highly sulfated phosphorylmannose oligosaccharide mixture (PI-88) of the prior art. Conducive to the development of new high-purity tumor anti-angiogenic pharmaceutical compounds with a view to optimizing biological activity and associated quality control.
  • the present invention also provides for the first time a sulfated phosphoryl mannitol pentasaccharide of the formula (IV) in the form of a fully sulfated nineteen sodium salt.
  • the sulfated phosphoryl mannitol pentasaccharide represented by the formula (IV) in the present invention is the first reduction-opening-sulfation of the reducing end hemiacetal of the yeast Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide derivative.
  • the present invention also proposes for the first time an incompletely sulfated mannose pentasaccharide at a defined site represented by formula (V).
  • the incompletely sulfated mannooligosaccharide derivative represented by the formula (V) in the present invention is the first indeterminate site incompletely sulfated derivative of the yeast Pichia holstii NRRL Y-2448 extracellular phosphorylmannose oligosaccharide. In terms of quality control, it is significantly superior to the highly sulfated mannooligosaccharide mixture with a degree of sulfation and site uncertainty in the prior art.
  • the present invention also proposes for the first time a incompletely sulfated mannotriose at a defined site as shown in formula (VIII).
  • the incompletely sulfated mannotriose of the defined site represented by the formula (VIII) in the present invention is the first incomplete sulfation of a certain site containing an ⁇ -(1-4)-/ ⁇ -(1-2)-glucosidic bond. Mannan oligosaccharide derivatives.
  • R is a C 1 -C 5 alkyl group
  • the present invention also proposes for the first time an incompletely sulfated branched chain phosphoryl mannose triose as defined in formula (X).
  • the present invention firstly proposes an incompletely sulfated phosphoryl mannose branched-chain oligo with a defined site containing an ⁇ -(1-3)-/ ⁇ -(1-6)-glucosidic bond.
  • the indeterminate site of formula (X) in the present invention is not fully sulfated branched-chain phosphorylmannose trisaccharide is the first case containing a defined position containing an ⁇ -(1-3)-/ ⁇ -(1-6)-glycosidic bond. The point is not fully sulfated branched-chain phosphorylmannose oligosaccharide derivative.
  • the sulfated oligosaccharide derivatives of the above formulae (V) to (XI) are mainly modified for the following aspects: 1) sulfated mannooligosaccharide derivatives; 2) incompletely sulfated mannooligos at defined positions a sugar derivative; and 3) a sulfated mannooligosaccharide derivative containing an ⁇ -(1-4)-/ ⁇ -(1-6)-glycosidic linkage; providing a novel medicinal molecule for anti-tumor angiogenesis space.
  • the invention also provides a preparation method of the phosphoryl mannose pentasaccharide of the formula (I), wherein three monosaccharide synthesis blocks are prepared by using D-mannose as raw materials through a plurality of reaction steps: formula (A), formula (B) And formula (C), which is obtained by glycosylation of glucosinolate as a donor, linear assembly starting from the reducing end C, to obtain a protected pentasaccharide intermediate represented by formula (D), and then subjected to a later stage. Deprotection of the non-reducing end, phosphorylation-oxidation, and complete deprotection to obtain the phosphoryl mannose pentasaccharide of the formula (I).
  • the significant progress of the preparation method of the present invention from the prior art is as follows: 1) by rationally designing the protecting group of the hydroxy group in the mannose glucoside donor to regulate its reactivity, and realizing the glycosylation of glycosylation as a donor. -(1-2)-/ ⁇ -(1-3)-linked mannooligosaccharide chains; 2) Phosphorylation by a "late phosphorylation strategy" with a phosphate protecting group that is easily removed Efficient and economical.
  • the preparation method of the invention comprises the following steps and reaction formula:
  • Step (1) using D-mannose as a raw material, respectively preparing different three monosaccharide synthesis blocks through a plurality of reaction steps (ie, through routes a, b, c respectively): formula (A), formula (B) ), formula (C), whose chemical structure is as follows:
  • the protecting groups R 1 to R 10 and R E are each independently selected from the group consisting of: acetyl (Ac), benzoyl (Bz), benzyl (Bn), tert-butyldimethylsilyl (TBS), Triisopropylsilyl (TIPS);
  • the groups R A and R C are each independently selected from the group consisting of phenyl (Ph), 4-methylphenyl (Lev), methyl (Me), ethyl (Et) And propyl (Pr);
  • the temporary protecting groups R B and R D are each independently selected from the group consisting of: 3-levulinyl (Lev), 4-methoxybenzyl (PMB), allyl (All), alkene Propoxycarbonyl (Alloc), 9-fluorenylmethoxycarbonyl (Fmoc).
  • TBS tert-butyldimethylsilyl
  • TIPS triisopropylsilyl
  • PMB 4-methoxybenzyl
  • Step (2) reacting the monosaccharide synthesis block (C) and formula (B) under glycosylation conditions to obtain a disaccharide intermediate formula (E); removing the protective group R D to obtain a compound Formula (F); compound formula (F) and monosaccharide synthesis block (B) are reacted under glycosylation conditions to obtain a trisaccharide intermediate formula (G); and the protective group R D is removed to obtain a compound formula ( H); compound formula (H) and monosaccharide synthesis block (B) are reacted under glycosylation conditions to obtain a tetrasaccharide intermediate formula (I); the protective group R D is removed to obtain a compound formula (J) Compound (J) and monosaccharide synthesis block (A) are reacted under glycosylation conditions to give pentasaccharide intermediate formula (D).
  • glycosylation conditions are a halogen-containing oxidizing agent, a Lewis acid or a protic acid, a solvent, a combination of temperatures, or a diphenyl sulfoxide, a trifluoromethanesulfonic anhydride, a 2,4,6-tri-tert-butylpyrimidine, a solvent , the combination of temperatures.
  • the halogen-containing oxidizing agent is: N-iodosuccinimide, N-bromosuccinimide;
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate, tert-butyldimethyl a silyl trifluoromethanesulfonate, a triethylsilyl trifluoromethanesulfonate, a silver trifluoromethanesulfonate;
  • the protonic acid is trifluoromethanesulfonic acid, trifluoroacetic acid;
  • the solvent is diethyl ether, two Methyl chloride, 1,4-dioxane, tetrahydrofuran or a binary mixture thereof; the temperature is -70 ° C to 0 ° C.
  • Step (3) using the compound of the formula (D) as a raw material, first removing the protecting group R B to obtain a compound of the formula (K); and sequentially obtaining a phosphate compound under the conditions of phosphorylation and oxidation. (L); Finally, the protecting groups R 1 to R 10 and R E are removed to give a phosphoryl mannose pentasaccharide of the formula (I).
  • the preparation method of the phosphoryl mannose pentasaccharide of the formula (I) of the present invention comprises the following steps and a reaction formula:
  • Step (1) using D-mannose as a raw material, obtaining a compound of formula (1) by peracetylation; obtaining a glucosyl group by thiolation of 4-methylthiophenol; (2); 4,6-O-benzylidene protection gives the diol formula (3); the compound (4) is obtained by 3-O-benzylation, 2-O-acetylation; and then, under reducing ring-opening conditions, Compound (5); Finally, by 3-acetylpropionylation conditions, a monosaccharide synthesis block (6) is obtained.
  • Step (2) using the compound of the formula (3) as a starting material, through 3-O-(4-methoxybenzyl) to give the compound formula (7);
  • Step (3) D-mannose is used as a raw material, and the compound (11) is obtained by acylation of benzyl alcohol Fischer, and acetylation is carried out; the acetyl group is removed, and the 4,6-O-benzylidene group is protected to obtain a diol type. (12); obtaining a compound of the formula (13) by 3-O-benzylation; obtaining a compound of the formula (14) by 2-O-(4-methoxybenzyl); and then, under acidic conditions, 4,6 Removal of -O-benzylidene protecting group, 4,6-O-acetylation to give compound (15); finally, under the condition of removing 2-O-(4-methoxybenzyl), Monosaccharide synthesis block type (16).
  • Step (4) reacting the monosaccharide synthesis block (16) and formula (10) under glycosylation conditions to obtain a disaccharide intermediate formula (17; in removing 3-O-(4-methoxy Under the conditions of the benzyl group), the compound of the formula (18) is obtained; the compound of the formula (18) and the monosaccharide synthetic block (10) are reacted under glycosylation conditions to obtain a trisaccharide intermediate formula (19); Under the conditions of 3-O-(4-methoxybenzyl), the compound of formula (20) is obtained; the compound of formula (20) and the monosaccharide synthetic block (10) are reacted under glycosylation conditions to obtain a tetrasaccharide.
  • the glycosylation conditions are a halogen-containing oxidizing agent, a Lewis acid or a protic acid, a solvent, a combination of temperatures, or diphenyl sulfoxide, trifluoromethanesulfonic anhydride, 2,4,6-tri-tert-butylpyrimidine, a combination of a solvent and a temperature;
  • the halogen-containing oxidizing agent is: N-iodosuccinimide, N-bromosuccinimide
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate, Tert-butyldimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulfonate
  • the protic acid is trifluoromethanesulfonic acid, trifluoroacetic acid
  • the solvent is diethyl ether, dichloromethane, 1,4-d
  • Step (5) treating the compound of the formula (23) under conditions of removing levulinyl group to obtain a compound of the formula (24); and sequentially obtaining a phosphate compound formula under the conditions of phosphorylation and oxidation.
  • the compound of the formula (26) is obtained; under the condition of removing the silicon group, the compound of the formula (27) is obtained; and under the condition of removing the benzyl group, the phosphoryl group of the formula (I) is obtained.
  • the benzyl removal condition is a combination of a catalyst and an amount thereof, a solvent, and hydrogen (1 atm); wherein the catalyst is palladium/carbon, palladium hydroxide/carbon, palladium black, platinum/carbon, Lanney nickel; the amount of the catalyst is 0.1 to 5 times the mass of the compound of the formula (27); the solvent is methanol, ethanol, water or a binary mixture thereof.
  • the present invention also proposes a phosphoryl mannose pentasaccharide represented by the formula (I) synthesized by the above production method.
  • the invention also proposes for the first time a preparation method of a phosphoryl mannose pentasaccharide of the formula (II), which is obtained by using the compound of the formula (27) as a raw material under the condition of removing benzyl-reductive ring-opening.
  • the benzyl-reduction ring-opening condition is a combination of a catalyst and an amount thereof, a solvent, a hydrogen gas and a pressure thereof; wherein the catalyst is palladium/carbon, palladium hydroxide/carbon, palladium black, platinum/carbon , Raney nickel; the amount of the catalyst is 5 to 40 times the mass of the compound (27); the solvent is methanol, ethanol, water or a binary mixture thereof; and the hydrogen pressure is 1 to 5 atm.
  • the invention also provides a preparation method of a phosphoryl mannitol pentasaccharide of the formula (II), wherein the compound of the formula (I) is used as a raw material to obtain a phosphoryl mannose pentasaccharide of the formula (II) under reducing conditions.
  • the preparation method includes the following steps and a reaction formula.
  • the reducing condition is a combination of a reducing agent and a solvent; wherein the reducing agent is sodium borohydride, potassium borohydride, lithium borohydride, sodium triacetoxyborohydride, sodium cyanoborohydride; the solvent It is methanol, ethanol, dichloromethane, 1,2-dichloromethane or a binary mixture thereof.
  • the present invention also proposes a phosphoryl mannitol pentasaccharitol represented by the formula (II) which is synthesized according to any of the above-described methods for preparing a phosphoryl mannitol pentasaccharide.
  • the invention also provides a preparation method of the sulfated phosphoryl mannose pentasaccharide of the formula (III), which is obtained by using the phosphoryl mannose pentasaccharide of the formula (I) as a raw material to obtain the sulfation of the formula (III) under O-sulfation conditions.
  • Phosphorylmannose pentasaccharide the preparation method comprises the following steps and a reaction formula.
  • the present invention also proposes a sulfated phosphoryl mannose pentasaccharide represented by the formula (III) synthesized by the above production method.
  • the invention also provides a method for preparing a sulfated mannose pentasuitol of the formula (IV), which is characterized in that the phosphoryl mannitol pentasaccharide of the formula (II) is used as a raw material under the condition of O-sulfation.
  • the (IV) sulfated phosphoryl mannose pentasaccharide; the preparation method comprises the following steps and a reaction formula.
  • the present invention also proposes a sulfated phosphorylmannitol pentasuitol as shown in the formula (IV) synthesized by the above production method.
  • the invention also provides a preparation method of the formula (VI) for determining the incompletely sulfated mannose pentasaccharide (determining the site partial sulfated mannose pentasaccharide), wherein the three monosaccharide synthesis blocks are first synthesized separately.
  • the protected pentasaccharide intermediate is obtained by linear assembly starting from the reducing end, and then deprotected, sulfated, and deprotected in the latter stage to obtain a partially sulfated mannose pentasaccharide at the site of formula (VI);
  • the preparation method includes The following steps and reaction formula:
  • Step (1) using the compound of the formula (6) as a raw material, the compound of the formula (28) is obtained under oxidative hydrolysis conditions; and the monosaccharide synthesis block type (29) is obtained by trichloroacetonitrile or alkali treatment.
  • Step (2) using the compound of the formula (7) as a raw material, which is protected by 2-O-benzylation to obtain a compound of the formula (30); and further subjected to a 4,6-O-benzylidene protecting group under acidic conditions. Removal, 6-O-silylation, to give the compound of formula (31); finally, after 4-O-benzylation protection, to obtain monosaccharide synthesis block type (32);
  • Step (3) using compound formula (33) as a raw material, and protecting with trimethylsilyl group to obtain compound formula (34); further passing 4,6-O-benzylidene protection, 2,3-O-silicon Base removal, to obtain the compound formula (35); finally, by 3-O-benzylation protection, to obtain a monosaccharide synthesis block type (36);
  • Step (4) reacting the monosaccharide synthesis block type (36) and formula (32) under the first glycosylation condition to obtain a disaccharide intermediate formula (37); and removing 3-O-(4) -Methoxybenzyl), the compound of formula (38) is obtained; the compound formula (38) and the monosaccharide synthesis block (32) are reacted under glycosylation conditions to obtain a trisaccharide intermediate formula (39); Under the condition of removing 3-O-(4-methoxybenzyl), the compound of formula (40) is obtained; the compound of formula (40) and the monosaccharide synthetic block (32) are reacted under glycosylation conditions, The tetrasaccharide intermediate formula (41) is obtained; under the condition of removing 3-O-(4-methoxybenzyl), the compound of formula (42) is obtained; compound 42 and monosaccharide synthetic block type (29) are in the first The reaction is carried out under disaccharification conditions to obtain a pentasaccharide intermediate formula (43).
  • the glycosylation condition 1 is a halogen-containing oxidizing agent, a Lewis acid or a protic acid, a solvent, a combination of temperatures, or diphenyl sulfoxide, trifluoromethanesulfonic anhydride, 2,4,6-tri-tert-butylpyrimidine a combination of a solvent and a temperature;
  • the halogen-containing oxidizing agent is: N-iodosuccinimide, N-bromosuccinimide;
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate , tert-butyldimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulfonate;
  • the protic acid is trifluoromethanesulfonic acid, trifluoroacetic acid;
  • the solvent is diethyl ether, dichloromethane
  • the glycosylation condition 2 is a combination of a Lewis acid or a protic acid, a solvent, and a temperature; wherein the Lewis acid is trimethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyltrifluoride Mesylate, triethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulfonate; the protic acid is trifluoromethanesulfonic acid, trifluoroacetic acid; the solvent is diethyl ether, dichloromethane, 1 , 4-dioxane, Tetrahydrofuran or a binary mixture thereof; the temperature is -40 ° C to 0 ° C.
  • Step (5) treating the compound of the formula (43) under the conditions of removing an acyl group and removing a silicon group to obtain a compound of the formula (44); and further obtaining a compound 45 under O-sulfation conditions; Under the conditions of removing the benzyl group, the sulfated pentose pentasaccharide is partially obtained at the site of the formula (VI).
  • the conditions for removing the benzyl group are a combination of a catalyst and an amount thereof, a solvent, and hydrogen (1 atm); wherein the catalyst is palladium/carbon, palladium hydroxide/carbon, palladium black, platinum/carbon , Raney nickel; the amount of the catalyst is 0.1 to 5 times the mass of the compound 44; the solvent is methanol, ethanol, water or a binary mixture thereof.
  • the present invention also proposes an incompletely sulfated mannose pentasaccharide at a defined site represented by the formula (VI) synthesized by the above preparation method.
  • the invention provides a preparation method of the formula (VII) for determining the incompletely sulfated mannose pentasaccharide, which is characterized in that firstly, the monosaccharide synthesis block type (48) is synthesized, and then the monosaccharide is synthesized with the monosaccharide.
  • Block (10), formula (6) obtain a protected pentasaccharide intermediate by linear assembly starting from the reducing end, and obtain desulfurization, sulfation, and deprotection in the latter stage to obtain a partial sulfuric acid in the formula (VII).
  • the mannose pentasaccharide; the preparation method comprises the following steps and a reaction formula:
  • Step (1) using the compound 36 as a raw material, and protecting by 2-O-(4-methoxybenzyl), the compound of the formula (46) is obtained; and the 4,6-O-benzylidene under acidic conditions is sequentially passed. Removal of the base protecting group, 4,6-O-acetylation, to obtain the compound of the formula (47); finally, under the condition of removing 3-O-(4-methoxybenzyl), the monosaccharide synthesis is obtained.
  • Block type (48)
  • Step (2) reacting the monosaccharide synthesis block type (48) and formula (10) under glycosylation conditions to obtain a disaccharide intermediate formula (49); and removing 3-O-(4-A) Under the conditions of oxybenzyl), the compound of formula (50) is obtained; the compound of formula (50) and the monosaccharide synthetic block (10) are reacted under glycosylation conditions to obtain a trisaccharide intermediate formula (51); In addition to 3-O-(4-methoxy Under the conditions of benzyl), the compound of formula (52) is obtained; the compound formula (52 and the monosaccharide synthesis block (10) are reacted under glycosylation conditions to obtain a tetrasaccharide intermediate formula (53); Under the conditions of O-(4-methoxybenzyl), the compound of formula (54) is obtained; the compound of formula (54) and the monosaccharide synthetic block (6) are reacted under glycosylation conditions to obtain a pentasaccharide intermediate. (55
  • the glycosylation conditions are a halogen-containing oxidizing agent, a Lewis acid or a protic acid, a solvent, a combination of temperatures, or a diphenyl sulfoxide, a trifluoromethanesulfonic anhydride, a 2,4,6-tri-tert-butylpyrimidine, a solvent a combination of temperatures;
  • the halogen-containing oxidizing agent is: N-iodosuccinimide, N-bromosuccinimide
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate, uncle Butyldimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, silver triflate
  • the protic acid is trifluoromethanesulfonic acid, trifluoroacetic acid
  • the solvent is diethyl ether, dichloromethane, 1,4-dioxane,
  • Step (3) the compound of the formula (55) is sequentially treated under conditions of removing an acyl group and removing a silicon group to obtain a compound
  • the compound of the formula (56) is further obtained under O-sulfation conditions to obtain the compound of the formula (57); finally, under the condition of removing the benzyl group, the sulfated pentose pentasaccharide is partially obtained at the site of the formula (VII).
  • the benzyl removal condition is a combination of a catalyst and an amount thereof, a solvent, and hydrogen (1 atm); wherein the catalyst is palladium/carbon, palladium hydroxide/carbon, palladium black, platinum/carbon, blue Nickel nickel; the amount of the catalyst is 0.1 to 5 times the mass of the compound 56; the solvent is methanol, ethanol, water or a binary mixture thereof.
  • the present invention also proposes an incompletely sulfated mannose pentasaccharide having a defined position as shown in the formula (VII) synthesized by the above production method.
  • the invention also provides a preparation method of the incompletely sulfated mannotriose of the formula (IX) (determining the site partially sulfated mannotriose), first synthesizing the monosaccharide synthesis block type (59), and then combining the same
  • the monosaccharide synthesis block type (36), formula (29), obtains a protected trisaccharide intermediate by linear assembly starting from a non-reducing end, and obtains a formula (IX) by post-stage deprotection, sulfation, and deprotection. Determining a portion of the sulfated mannotriose; the preparation method comprises the following steps and a reaction formula:
  • Step (1) using the compound of the formula (3) as a raw material, after 2,3-O-benzylation, the compound of the formula (58) is obtained; and then the 4,6-O-benzylidene group is protected under acidic conditions.
  • Step (2) reacting the monosaccharide synthesis block type (59) and formula (29) under the first glycosylation condition to obtain a disaccharide intermediate formula (60); then, compound 60 and monosaccharide synthesis Block (36) is reacted under a second glycosylation condition to obtain a trisaccharide intermediate formula (61);
  • the first glycosylation condition is a combination of a Lewis acid or a protic acid, a solvent, and a temperature; wherein the Lewis acid is trimethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl a trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulfonate; the protic acid is trifluoromethanesulfonic acid, trifluoroacetic acid; the solvent is diethyl ether, dichloromethane , 1,4-dioxane, tetrahydrofuran or a binary mixture thereof; the temperature is -40 ° C to 0 ° C.
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl a trifluoromethanesulfonate, triethylsily
  • the second glycosylation condition is a halogen-containing oxidizing agent, a Lewis acid or a protic acid, a solvent, a combination of temperatures, or diphenyl sulfoxide, trifluoromethanesulfonic anhydride, 2,4,6-tri-tert-butylpyrimidine, a solvent a combination of temperatures;
  • the halogen-containing oxidizing agent is: N-iodosuccinimide, N-bromosuccinimide
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate, uncle Butyldimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, silver triflate
  • the protic acid is trifluoromethanesulfonic acid, trifluoroacetic acid
  • the solvent is diethyl ether, dichloromethane, 1,4-dioxane, tetrahydro
  • Step (3) the compound of the formula (61) is subjected to treatment for removing an acyl group and removing a silicon group in order to obtain a compound of the formula (62); 4,6-O-benzylidene protection under acidic conditions The removal of the group gives the compound of the formula (63); then, under the O-sulfation condition, the compound of the formula (64) is obtained; finally, under the condition of removing the benzyl group, the sulfuric acid of the formula (XI) is obtained.
  • the mannose trisaccharide wherein the benzyl removal condition is a combination of a catalyst and an amount thereof, a solvent, and hydrogen (1 atm); wherein the catalyst is palladium/carbon, palladium hydroxide/carbon, Palladium black, platinum/carbon, Raney nickel; the amount of the catalyst is 0.1 to 5 times the mass of the compound 63; the solvent is methanol, ethanol, water or a binary mixture thereof.
  • the present invention also proposes a defined site incompletely sulfated mannotriose represented by the formula (IX) synthesized by the above preparation method.
  • the invention also provides a preparation method of the formula (XI) for determining the incompletely sulfated branched chain phosphoryl mannose triose (determining the site partial sulfated branched phosphonomannosyl trisaccharide), first synthesizing the monosaccharide synthesis building block Formula (67), which is further combined with the monosaccharide building block (6) to obtain a protected branched trisaccharide intermediate formula (68) by glycosylation, and then deprotected by a non-reducing end of the latter stage, Phosphorylation-oxidation, deprotection, sulfation, debenzylation, to obtain a partially sulfated branched chain phosphoryl mannose trisaccharide of formula (XI);
  • the preparation method comprises the following steps and a reaction formula:
  • Step (1) using D-mannose as a raw material, followed by acetylation and phenolylation to obtain a compound of formula (65); and then passing through deacetylated, 2,3:4,6-O-bisbenzylidene Protection, to obtain the compound of formula (65); then, under reducing ring-opening conditions, to obtain monosaccharide synthesis block type (67);
  • Step (2) reacting the monosaccharide synthesis block (67) and formula (6) under glycosylation conditions to obtain a branched trisaccharide intermediate formula (68); wherein the glycosylation conditions are a combination of a halogen-containing oxidizing agent, a Lewis acid or a protic acid, a solvent, a temperature, or a combination of diphenyl sulfoxide, trifluoromethanesulfonic anhydride, 2,4,6-tri-tert-butylpyrimidine, a solvent, and a temperature;
  • the halogen-containing oxidizing agent is: N-iodosuccinimide, N-bromosuccinimide;
  • the Lewis acid is trimethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl III Fluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, silver trifluoromethanesulf
  • Step (3) treating the compound of the formula (68) by removing the levulinyl group to obtain a diol formula (69); and sequentially obtaining a bisphosphonate compound under phosphorous acylation and oxidation conditions. (70); then, under the condition of removing the acetyl group, the compound of the formula (71) is obtained; finally, the treatment of the condition of removing the benzyl group by O-sulfation conditions in order to obtain the site of the formula (XI) Sulfating branched-chain phosphorylmannose trisaccharide; wherein the benzyl removal condition is a combination of a catalyst and an amount thereof, a solvent, and hydrogen (1 atm); wherein the catalyst is palladium/carbon, hydrogen Palladium oxide / carbon, palladium black, platinum / carbon, Raney nickel; the amount of the catalyst is 0.1 to 5 times the mass of the compound 71; the solvent is methanol, ethanol, water or a binary mixture thereof.
  • the present invention also proposes an incompletely sulfated branched chain phosphoryl mannose trisaccharide having a defined position as shown in the formula (XI) synthesized by the above production method.
  • the present invention also provides the use of a phosphorylmannopenta pentasaccharide represented by the formula (I) and a derivative of phosphorylmannopentaose represented by the formula (II)-(XI) for the preparation of an antitumor drug.
  • the invention also provides the use of the compound of the phosphoryl mannose pentasaccharide formula (I) and the compound of the phosphoryl mannitol pentasaccharide formula (II) for preparing an antitumor medicament.
  • the present invention also provides the sulfated oligosaccharide formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X)
  • sulfated oligosaccharide formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) The use of a compound of formula (XI) for the preparation of an antitumor drug.
  • the present invention also proposes the use of a phosphorylmannopenta pentasaccharide represented by the formula (I) and a derivative of phosphorylmannopentaose represented by the formulae (II)-(XI) for the preparation of a medicament for inhibiting angiogenesis.
  • the formula (III), the formula (IV), the formula (V), the formula (VI), the formula The compound of (VII), formula (VIII), formula (IX), formula (X), and formula (XI) has an activity of inhibiting angiogenesis.
  • the present invention also provides the use of the phosphorylmannose pentasaccharide represented by the formula (I) for the preparation of a sulfated phosphorylmannose pentasaccharide compound represented by the formula (III) having an activity of inhibiting angiogenesis.
  • the present invention also proposes the use of the phosphorylmannitol pentasaccharide represented by the formula (II) for the preparation of a sulfated phosphorylmanna pentasaccharitol compound represented by the formula (IV) having an activity of inhibiting angiogenesis.
  • the compounds of the formula (I) and the formula (II) are the key raw materials for preparing the compound of the formula (III) and the formula (IV), respectively, which have an activity of inhibiting angiogenesis.
  • the present invention provides, for the first time, a pure form of the phosphoryl mannose pentasaccharide of formula (I) associated with the yeast Pichia holstii NRRL Y-2448.
  • the single component of the phosphorylmannose pentasaccharide of the formula (I), which is obtained for the first time by chemical synthesis, is significantly superior to the phosphorylmannose oligosaccharide mixture of the prior art.
  • the phosphoryl mannose pentasaccharide of the formula (I) can be further modified by sulfation to obtain a single component of the fully sulfated phosphoryl mannose pentasaccharide of the formula (III) for the first time, which is remarkably superior to the prior art.
  • Sulfated phosphoryl mannose oligosaccharide mixtures (PI-88) facilitate the development of new high purity tumor anti-angiogenic pharmaceutical compounds with a view to optimizing biological activity and associated quality control.
  • the phosphoryl mannitol pentasaccharide represented by the formula (II) in the present invention is a reduced ring-opening derivative of the first reducing terminal hemiacetal of the yeast Pichia holstii NRRL Y-2448 extracellular phosphoryl mannose oligosaccharide.
  • the sulfated phosphoryl mannose pentasaccharide represented by the formula (IV) is obtained by the sulfation modification of the phosphoryl mannitol pentasaccharide of the formula (II), which is the yeast Pichia holstii NRRL Y-2448.
  • the first reduction of the extracellular phosphorylmannose oligosaccharide is a reduced ring-sulfated derivative of the hemiacetal.
  • the incompletely sulfated mannooligosaccharide derivative represented by the formula (V) in the present invention is the first indeterminate site incompletely sulfated derivative of the yeast Pichia holstii NRRL Y-2448 extracellular phosphorylmannose oligosaccharide.
  • a highly sulfated mannooligosaccharide mixture that is significantly superior to the prior art in terms of degree of sulfation and site uncertainty.
  • the incompletely sulfated mannotriose of the defined site represented by the formula (VIII) in the present invention is the first incomplete sulfation of a certain site containing an ⁇ -(1-4)-/ ⁇ -(1-2)-glucosidic bond.
  • Mannan oligosaccharide derivatives The indeterminate site of formula (X) in the present invention does not completely sulfate the branched phosphonomannosin trisaccharide as the first defined site containing the ⁇ -(1-3)-/ ⁇ -(1-6)-glycosidic bond. Incompletely sulfated branched chain phosphorylmannose oligosaccharide derivatives.
  • the sulfated oligosaccharide derivatives of the above formulae (V) to (XI) are mainly modified for the following aspects: 1) sulfated mannooligosaccharide derivatives; 2) incompletely sulfated mannooligos at defined positions a sugar derivative; and 3) a sulfated mannooligosaccharide derivative containing an ⁇ -(1-4)-/ ⁇ -(1-6)-glycosidic linkage, which provides a novel drug molecule for anti-tumor angiogenesis space.
  • the significant progress of the preparation method of the present invention from the prior art is as follows: 1) glycosylation with glucosinolate as a donor by rationally designing a protecting group of a hydroxyl group in the mannose glucoside donor to control its reactivity Construction of ⁇ -(1-2)-/ ⁇ -(1-3)-linked mannooligosaccharide chains; 2) Phosphorus is achieved by a "late phosphorylation strategy" and a phosphate protecting group that is easily removed The acylation is highly efficient and economical.
  • Fig. 1 is a schematic view showing the preparation state of phosphoryl mannose Wutang and its derivatives in the prior art.
  • FIG 2 is an experimental result of inhibition of angiogenesis by a sulfated oligosaccharide compound of the formula (III), (IV), (VI), (VII), (IX), (XI), wherein PI-88 is used as a comparative compound.
  • Figure 2-1 is a bar graph of the experimental results of inhibition of HMEC-1 microtubule formation by each compound.
  • Figure 2-2 is an image diagram of the inhibition of HMEC-1 microtubule production by a compound of formula (III).
  • the compound of the formula (I) (16.1 mg, 0.0177 mmol) and SO 3 ⁇ Py (216 mg, 1.357 mmol) prepared in Example 5 were dissolved in DMF (1.5 mL) and stirred at 60 ° C for 6 h. It was then cooled to room temperature and stirring was continued for 2 d. The supernatant was aspirated and the residue was dissolved in purified water, adjusted to pH 9-10 using 1M NaOH (aq.) and concentrated to dryness.
  • DEPT135 (100MHz, D 2 O, 300K, HSQC) ⁇ 101.88, 99.12, 98.93, 77.57, 77.07, 75.04, 73.86, 71.39, 70.63,70.45,69.88,69.10,68.98,68.70,67.58,67.38,66.52,65.73,55.10.
  • HRMS(ESI-TOF)m/z Calcd for C 19 H 29 O 31 Na 3 S 5 [M+3Na] 2- 490.9495, found 490.9453.
  • HMEC-1 Human microvascular endothelial cells
  • MCDB131 commercial endothelial cell serum-free medium
  • MCDB131 commercial endothelial cell serum-free medium
  • a cell density of 5 ⁇ 10 4 /mL Take a 200 ⁇ L of the above HMEC-1 cells, respectively, using a volume of sulfated oligosaccharide compounds and PI-88 compounds of formula (III), (IV), (VI), (VII), (IX), (XI)
  • the standard solution was treated to give a final solution having a compound concentration of 10, 50, 100 ⁇ M, respectively, and a final volume of 300 ⁇ L.
  • HMEC-1 cells 100 ⁇ L of the treated HMEC-1 cells were dispensed onto a 96-well plate plated with a low growth factor artificial basement membrane, followed by incubation at 37 ° C for 18 h.
  • Microtubule generation was detected by a phase contrast microscope and images were acquired using a digital camera attached to an Olympus IX50 inverted phase contrast microscope.
  • the inhibition rate of each sulfated oligosaccharide compound on microtubule formation is obtained by manual quantification of an image, specifically, the total number of branch nodes connecting three or more microtubules. The results are expressed as a percentage inhibition rate relative to the blank control, and HMEC cells treated with the sulfated oligosaccharide compound were used as a standard reference for normal cell growth and microtubule production on the artificial basement membrane.
  • FIG. 2 Experimental results of inhibition of angiogenesis by the sulfated oligosaccharide compounds of the formulae (III), (IV), (VI), (VII), (IX), (XI) are shown in Fig. 2, wherein PI-88 is used as a comparative compound.
  • Figure 2-1 is a bar graph showing the results of experiments in which each compound inhibits angiogenesis.
  • Figure 2-2 is an image diagram of the inhibition of angiogenesis by a compound of formula (III).

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Abstract

本发明提出了如式（I）所示的磷酰甘露五糖及如式（II）-（XI）所示的磷酰甘露五糖的衍生物，以及其制备方法。本发明制备方法中，通过设计甘露糖硫苷给体中羟基的保护基来调控反应活性，且在糖基化反应中副反应少，反应收率高，适宜大规模及模块化组合合成。本发明中的磷酰甘露五糖及其衍生物具有抑制血管生成的活性，具有作为抗肿瘤药物的应用前景。

Description

磷酰甘露五糖及其衍生物，及其制备方法和应用 技术领域
本发明属于药物化学、有机化学、天然产物化学等技术领域。具体涉及磷酰甘露五糖及其衍生物、及其制备方法和在制备抗肿瘤药物中的应用。
背景技术
血管生成(Angiogenesis)是肿瘤生长和转移的关键过程。因此，抑制血管生成过程是公认的治疗癌症的策略之一。近年获批的抗血管生成的治疗方法，如：酪氨酸激酶抑制剂等，虽然具有明显临床抗肿瘤效果，但仍然存在存活率低、对心脏有毒副作用等问题。因此，开发低副作用、且对多种血管生成因子具有靶向性的血管生成抑制剂是目前研究的方向。分布在细胞表面和细胞外基质的硫酸肝素(Heparan Sulfate)是一类在自然界普遍存在的复杂硫酸化多糖，在癌症生物学中发挥着支配性作用，包括：抑制血管生成、调节肿瘤演化和转移等。采用硫酸肝素模拟物(HS mimetics)调节这些过程是一种具有前景的癌症治疗方法。研究发现，使用硫酸肝素模拟物能阻碍HS参与的血管生长因子信号传递的过程；同时也能抑制肝素酶，对肝素酶降解HS、促进肿瘤转移的过程进行有效抑制。
澳大利亚Progen的Vito Ferro小组开发的硫酸肝素模拟物PI-88(结构见图1)，是一个具有抗血管生成、抗肿瘤转移的高度硫酸化甘露寡糖复杂混合物。目前，正在进行III期临床试验，用于切除术后肝细胞癌的治疗。PI-88作为硫酸肝素模拟物，能同时抑制血管生成和肿瘤转移，通过以下两种机制发挥作用：1)直接抑制血管生成(Angiogenesis)：干扰HS和血管生成生长因子FGF-1、FGF-2、VEGF之间的相互作用，从而避免了引发血管生成的细胞信号级联放大。2)阻碍肿瘤转移：通过强烈抑制肝素酶(heparanase)，阻止其降解细胞外基质和基膜上的蛋白聚糖HS侧链，从而阻碍转移瘤细胞逃离进入血液循环。另外，肝素酶在血管生成中也发挥重要作用，所以PI-88通过抑制肝素酶也是间接抑制了血管生成。
从制备方法上看，PI-88是由酵母菌Pichia holstii NRRL Y-2448细胞外磷酸多糖降解成的低分子量磷酰甘露寡糖的混合物，经硫酸化修饰而得的高度硫酸化甘露寡糖复杂混合物(见图1)。丹麦Parolis小组和澳大利亚Vito Ferro小组分别在Carbohydr.Res.1998,309,77-87和Carbohydr.Res.2001,332,183–189中报道了将酵母菌Pichia holstii NRRL Y-2448细胞外磷酸甘露多糖在1％乙酸中进行温和酸性水解，得到磷酰甘露寡糖混合物。澳大利亚Vito Ferro小组还在Carbohydr.Res.2002,337,139–146中报道了采用毛细管电泳对混合物中各组分含量进行分析，主要组分磷酰甘露五糖的含量是59％，四糖是28.5％；此外，还有少量的三糖(5.5％)、二糖(3％)和六糖(1％)(见图1)。在前述的现有技术中(Carbohydr. Res.1998,309,77-87)，此磷酰甘露寡糖混合物的各个组分通过凝胶排阻色谱(SEC)尚无法进行有效的分离纯化(见图1)；因此，作为此混合物的主要组分，单一组分的式(I)磷酰甘露五糖的纯净样品尚未获得，而其化学合成也未见文献报道。未经分离的磷酰甘露寡糖混合物经硫酸化修饰，即得到PI-88，为高度硫酸化的磷酰甘露寡糖混合物，制备方法如Robert J.Linhardt小组在Eur.J.Med.Chem.2002,37,783-791中所报道的。同样由于凝胶排阻色谱(SEC)有限的纯化效率，式(III)硫酸化磷酰甘露五糖及其不完全硫酸化衍生物的纯净样品也未能获得，如Robert J.Linhardt小组在Eur.J.Med.Chem.2002,37,783-791中所报道的，以及Vito Ferro小组在J.Med.Chem.2003,46,4601-4608中所描述的。因此，通过化学合成的方式获得单一组分的式(I)磷酰甘露五糖，再经硫酸化修饰，获得式(III)硫酸化磷酰甘露五糖，有利于开发新的组分单一的肿瘤抗血管生成的药用分子，以期优化生物活性和相关的质量控制。
据前所述，单一组分的式(I)磷酰甘露五糖的纯净样品尚未获得，而其化学合成也未见文献报道。对于构成式(I)磷酰甘露五糖的主要糖链骨架：α-(1-2)-/α-(1-3)-连接的甘露寡糖，在现有技术中，其构建方法主要是通过以亚胺酸酯为给体的糖基化反应来合成，如：Vito Ferro小组在Bioorg.Med.Chem.2004,12,6063–6075中报道的磷酰甘露二糖的合成；在Bioorg.Med.Chem.2008,16,699–709中报道的甘露五糖的合成；Yuguo Du小组在Carbohydr.Res.2004,339,1155–1162中报道的6V-O-硫酸化甘露五糖的合成；以及Alfonso Iadonisi小组在J.Org.Chem.2008,73,4496–4503中报道的甘露五糖的合成等等。以亚胺酸酯为给体的糖基化反应中，主要存在的问题是：1)亚胺酸酯给体不稳定，不宜保存；因此，不适宜大规模制备以及模块化组合合成；2)亚胺酸酯给体的活性较高，在糖基化反应中很容易发生一系列副反应，从而降低收率；为了解决此问题，一般需要较多量的亚胺酸酯给体，但同时增加了成本。另一类常见的糖基化给体——硫苷具有较好的稳定性和较适中的活性；然而，以甘露糖硫苷为给体的糖基化反应在制备α-(1-2)-/α-(1-3)-连接的甘露寡糖链中少见报道，主要原因可能包括：1)甘露糖给体固有的低活性；2)硫苷给体固有的低活性；两方面的因素共同导致了甘露糖硫苷给体在糖基化条件下无法进行有效的活化。因此，合理设计甘露糖硫苷给体中羟基的保护基是调控其反应活性的主要途径，也是实现以甘露糖硫苷为给体的糖基化反应在制备α-(1-2)-/α-(1-3)-连接的甘露寡糖链中的应用的重要方法。
在现有技术中，对于构建糖链非还原端的磷酸酯，主要是通过“早期磷酰化策略”实现，即：在单糖砌块中安装保护的磷酸酯(O,O’-二苯基磷酰基)，随后进行糖基化组装构建寡糖链，在后期的脱保护阶段将磷酸酯上的保护基脱除，如：Vito Ferro小组在Bioorg.Med.Chem.2004,12,6063–6075中报道的磷酰甘露二糖的合成。在上述现有技术中，存在若干问 题，例如：1)O,O’-二苯基磷酰基中苯基的脱除需要高压等较剧烈的条件(PtO2/H2/100psi)，并且剧烈的条件可能导致其他的副反应；2)“早期磷酰化策略”中，单糖砌块中的O,O’-二苯基磷酰基，由于其较强的吸电子作用，导致糖基化给体的活性降低；3)在大规模制备中，“早期磷酰化策略”可能增加磷酰化试剂的用量，从而提高成本。因此，设计采用“后期磷酰化策略”，并采用容易脱除的磷酸酯保护基，有望解决上述问题。
另外，从化学组成上看，PI-88是高度硫酸化的磷酰甘露寡糖混合物：1)糖链的长度从二糖到六糖分布，主要组分为五糖(～60％)和四糖(～30％)；2)由于寡糖混合物的硫酸化进行不完全，其硫酸化程度、位点具有不确定性，如Vito Ferro小组在J.Med.Chem.2005,48,8229-8236中所报道的；3)寡糖链由α-(1-2)-/α-(1-3)-糖苷键构成。在PI-88类似物的研究方面，Progen主要对磷酰甘露寡糖混合物进行去磷酰化，再经SEC纯化，分离出糖链长度单一的组分(如五糖)；再经过化学修饰(如：全乙酰基化、还原端活化等)，制备了一系列还原端具有不同脂肪链的苷元类似物，并对其体外活性、药物代谢动力学等方面进行了研究，如Vito Ferro小组在J.Med.Chem.2005,48,8229-8236、J.Med.Chem.2010,53,1686–1699中所报道的。在现有技术中，PI-88类似物主要限于寡糖链还原端的修饰，未针对以下方面进行研究：1)还原端半缩醛的还原开环衍生化；2)确定位点的不完全硫酸化；3)不同的糖苷键连接方式。因此，通过化学合成方式获得：1)硫酸化甘露寡糖醇衍生物；2)确定位点的不完全硫酸化甘露寡糖衍生物；以及3)含有α-(1-4)-/α-(1-6)-糖苷键的硫酸化甘露寡糖衍生物，为我们提供了发现新的抗肿瘤血管生成的药用分子的机会。
发明内容
本发明克服现有技术存在的问题，提出了一种新的磷酰甘露寡糖及其衍生物，以及其制备方法和应用。
本发明提出了一种磷酰甘露五糖及其衍生物，所述磷酰甘露五糖是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖混合物(二糖～六糖)中含量最高的组分，且存在形式是单一组分的五糖，不含其他糖链长度的化合物。本发明提供酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖混合物中单一组分的式(I)磷酰甘露五糖，其衍生物为：如式(II)所示的磷酰甘露五糖醇、如式(III)所示的硫酸化磷酰甘露五糖、如式(IV)所示的硫酸化磷酰甘露五糖醇，确定位点的不完全硫酸化甘露寡糖如式(V)、(VI)、(VII)、(VIII)、(IX)、(X)和(XI)所示的化合物。本发明中，所述纯净形式的式(I)所示的磷酰甘露五糖和式(III)所示的硫酸化磷酰甘露五糖是指酵母菌Pichia holstii NRRL Y-2448细胞外包括二糖～六糖的磷酰甘露寡糖混合物中单一形式的组分。相对于现有技术中的硫酸化程度、位置不确定的微观不均一性的混合物来说，本发明中，所述确定位点的式(V)-(XI)所示的确定位点的不完 全硫酸化甘露寡糖是指硫酸化基团是位于寡糖链上已知的若干特定位置。
本发明首次提供了一种式(I)所示的纯净形式的磷酰甘露五糖。本发明首次提供了酵母菌Pichia holstii NRRL Y-2448相关的纯净形式的式(I)磷酰甘露五糖。在现有技术中，纯净形式的式(I)磷酰甘露五糖尚未获得，仅以酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖混合物(二糖～六糖)的形式存在。本发明中，通过化学合成的方式首次获得了单一组分的式(I)磷酰甘露五糖，显著地优于现有技术中的磷酰甘露寡糖混合物。该式(I)磷酰甘露五糖可再经硫酸化修饰，获得单一组分的式(III)硫酸化磷酰甘露五糖。
Figure PCTCN2016077370-appb-000001
本发明还首次提出了一种式(II)所示的磷酰甘露五糖醇。本发明首次提供了酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖相关的还原端还原开环的式(II)磷酰甘露五糖醇。本发明中的式(II)所示的磷酰甘露五糖醇是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖的首例还原端半缩醛的还原开环衍生物。
Figure PCTCN2016077370-appb-000002
本发明还首次提出了一种式(III)所示的硫酸化磷酰甘露五糖，其存在形式是全硫酸化十八钠盐。本发明中的式(III)所示的全硫酸化磷酰甘露五糖是首次获得的酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖相关的纯净形式的全硫酸化磷酰甘露五糖。本发明中，将化学合成获得的单一组分的式(I)磷酰甘露五糖，经硫酸化修饰，获得单一组分的式(III)所示的硫酸化磷酰甘露五糖，显著地优于现有技术中的高度硫酸化磷酰甘露寡糖混合物(PI-88)。有利于开发新的高纯度的肿瘤抗血管生成的药用化合物，以期优化生物活性和相关的质量控制。
Figure PCTCN2016077370-appb-000003
本发明还首次提出了一种式(IV)所示的硫酸化磷酰甘露五糖醇，其存在形式是全硫酸化十九钠盐。本发明中的式(IV)所示的硫酸化磷酰甘露五糖醇是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖的首例还原端半缩醛的还原开环-硫酸化衍生物。
Figure PCTCN2016077370-appb-000004
本发明还首次提出了一种式(V)所示的确定位点不完全硫酸化甘露五糖。本发明中式(V)所示的确定位点不完全硫酸化甘露寡糖衍生物是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖的首例确定位点不完全硫酸化衍生物，在质量控制方面显著地优于现有技术中的硫酸化程度、位点不确定的高度硫酸化甘露寡糖混合物。
Figure PCTCN2016077370-appb-000005
其中，式(V)中，R选自C1～C5的烷基；R1～R16各自独立地选自SO3 -和H，其中，SO3 -的个数不小于5，不大于15，其余为H；钠离子的数目n＝SO3 -的数目。
所述式(V)中，当R＝CH3，R1＝R2＝R3＝R4＝R6＝R7＝R8＝R10＝R11＝R14＝R15＝H，R6＝R9＝R12＝R13＝R16＝SO3 -，n＝5时，即为式(VI)所示的确定位点不完全硫酸化甘露五糖；
Figure PCTCN2016077370-appb-000006
所述式(V)中，当R＝CH3，R2＝R3＝R4＝R6＝R7＝R9＝R10＝R12＝R13＝R16＝SO3 -，R1＝R5＝R8＝R11＝R14＝R15＝H，n＝10时，即为式(VII)所示的确定位点不完全硫酸化甘露五糖：
Figure PCTCN2016077370-appb-000007
本发明还首次提出了一种式(VIII)所示的确定位点不完全硫酸化甘露三糖。本发明中式(VIII)所示的确定位点不完全硫酸化甘露三糖是首例含有α-(1-4)-/α-(1-2)-糖苷键的确定位点不完全硫酸化甘露寡糖衍生物。
Figure PCTCN2016077370-appb-000008
其中，式(VIII)中，R为C1～C5的烷基；R1～R10选自SO3 -和H，其中，SO3 -的个数大于0，H的个数大于0，即1～9个为SO3 -，其余均为H；钠离子的数目n＝SO3 -的数目。
本发明还首次提出了一种如式(IX)所示的确定位点不完全硫酸化甘露三糖，即，当所述式(VIII)中，R为CH3，R2＝R3＝R6＝R7＝R10＝SO3-，R1＝R4＝R5＝R8＝R9＝H，n＝5时，其结构如式(IX)所示：
Figure PCTCN2016077370-appb-000009
本发明还首次提出了一种如式(X)所示的确定位点不完全硫酸化支链磷酰甘露三糖。
本发明首次提出了一种含有α-(1-3)-/α-(1-6)-糖苷键的确定位点不完全硫酸化磷酰甘露支链寡 糖衍生物。本发明中式(X)所示的确定位点不完全硫酸化支链磷酰甘露三糖是首例含有含有α-(1-3)-/α-(1-6)-糖苷键的确定位点不完全硫酸化支链磷酰甘露寡糖衍生物。
Figure PCTCN2016077370-appb-000010
其中，式(X)中，R为芳基；R1～R8选自SO3 -和H，其中，SO3 -的个数大于0，H的个数大于0，即1～7个为SO3 -，其余均为H；钠离子的数目n＝SO3 -的数目+4；
所述式(X)中，优选地，当R＝p-CH3OC6H4，R4＝R6＝SO3 -，R1＝R2＝R3＝R5＝R7＝R8＝H，n＝6时，其结构如式(XI)所示：
Figure PCTCN2016077370-appb-000011
上述式(V)～式(XI)硫酸化寡糖衍生物，主要是针对如下方面进行的结构修饰：1)硫酸化甘露寡糖醇衍生物；2)确定位点的不完全硫酸化甘露寡糖衍生物；以及3)含有α-(1-4)-/α-(1-6)-糖苷键的硫酸化甘露寡糖衍生物；为发现新的抗肿瘤血管生成的药用分子提供了空间。
本发明还提出了一种式(I)磷酰甘露五糖的制备方法，以D-甘露糖为原料分别通过多个反应步骤制备三个单糖合成砌块：式(A)、式(B)、式(C)，再通过以硫苷为给体的糖基化、从还原端C起始进行线性组装，获得保护的式(D)所示的五糖中间体，然后经后阶段的非还原端脱保护、亚磷酰化-氧化，再经全脱保护得到式(I)磷酰甘露五糖。本发明制备方法区别于现有技术的显著进步在于：1)通过合理设计甘露糖硫苷给体中羟基的保护基以调控其反应活性，实现了以硫苷为给体的糖基化构建α-(1-2)-/α-(1-3)-连接的甘露寡糖链；2)通过“后期磷酰化策略”，并采用容易脱除的磷酸酯保护基，实现了磷酰化的高效性、经济性。
本发明制备方法包括以下步骤及反应式：
步骤(1)：以D-甘露糖为原料，分别通过多个反应步骤(即，分别通过路线a，b，c)制备不同的三个单糖合成砌块：式(A)，式(B)，式(C)，其化学结构如下所示：
Figure PCTCN2016077370-appb-000012
其中，保护基团R1～R10以及RE各自独立地选自：乙酰基(Ac)、苯甲酰基(Bz)、苄基(Bn)、叔丁基二甲基硅基(TBS)、三异丙基硅基(TIPS)；基团RA和RC各自独立地选自：苯基(Ph)、4-甲基苯基(Lev)、甲基(Me)、乙基(Et)、丙基(Pr)；临时保护基团RB和RD各自独立地选自：3-乙酰丙酰基(Lev)、4-甲氧基苄基(PMB)、烯丙基(All)、烯丙氧羰基(Alloc)、9-芴甲氧羰基(Fmoc)。上述保护基中的：苄基(Bn)、叔丁基二甲基硅基(TBS)、三异丙基硅基(TIPS)、4-甲氧基苄基(PMB)等，均可通过给电子的效应，有效地提高甘露糖硫苷给体的反应活性，从而实现以甘露糖硫苷为给体的糖基化在制备α-(1-2)-/α-(1-3)-连接的甘露寡糖链中的应用。
步骤(2)：将所述单糖合成砌块式(C)和式(B)在糖基化条件下反应，得到二糖中间体式(E)；将保护基团RD脱除，得到化合物式(F)；化合物式(F)和单糖合成砌块式(B)在糖基化条件下反应，得到三糖中间体式(G)；将保护基团RD脱除，得到化合物式(H)；化合物式(H)和单糖合成砌块式(B)在糖基化条件下反应，得到四糖中间体式(I)；将保护基团RD脱除，得到化合物式(J)；化合物式(J)和单糖合成砌块式(A)在糖基化条件下反应，得到五糖中间体式(D)。
其中，所述糖基化条件为含卤素氧化剂、路易斯酸或质子酸、溶剂、温度的组合，或者二苯亚砜、三氟甲烷磺酸酐、2,4,6-三叔丁基嘧啶、溶剂、温度的组合。其中，所述含卤素氧化剂为：N-碘代琥珀酰亚胺、N-溴代琥珀酰亚胺；所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-70℃～0℃。
Figure PCTCN2016077370-appb-000013
步骤(3)：以所述化合物式(D)为原料，先将保护基团RB脱除，得到化合物式(K)；再依次在亚磷酰化、氧化条件下，得到磷酸酯化合物式(L)；最后，将保护基团R1～R10以及RE脱除，得到式(I)磷酰甘露五糖。
Figure PCTCN2016077370-appb-000014
优选地，当所述保护基团R1＝R4＝R8＝R9＝乙酰基(Ac)，保护基团R2＝R3＝R5＝R7＝RE＝苄基(Bn)，R6＝三异丙基硅基(TIPS)，RD＝4-甲氧基苄基(PMB)，RB＝3-乙酰丙酰基(Lev)，RA＝RC＝4-甲基苯基(Tol)时，本发明式(I)磷酰甘露五糖的制备方法包括以下步骤及反应式：
步骤(1)：以D-甘露糖为原料，经全乙酰化得到化合物式(1)；通过4-甲基苯硫酚进行硫苷化得到硫苷式(2)；经过乙酰基脱除、4,6-O-亚苄基保护得到二醇式(3)；通过3-O-苄基化、2-O-乙酰化得到化合物式(4)；然后，经还原性开环条件，得到化合物式(5)；最后，经3-乙酰丙酰化条件，得到单糖合成砌块式(6)。
Figure PCTCN2016077370-appb-000015
式中：a_全乙酰化；b_硫苷化；c_全脱乙酰基；d_4,6-O-亚苄基保护；e_3-O-苄基保护；f_2-O-乙酰基保护；g_还原性开环；h_3-乙酰丙酰化；
步骤(2)：以所述化合物式(3)为原料，经过3-O-(4-甲氧基苄基)化得到化合物式(7)；
经过2-O-乙酰化得到化合物式(8)；然后，经还原性开环条件，得到化合物式(9)；最 后，经硅基化得到单糖合成砌块式(10)。
Figure PCTCN2016077370-appb-000016
式中：a_3-O-PMB保护；b_2-O-乙酰化；c_还原性开环；d_6-O-硅基化；
步骤(3)：以D-甘露糖为原料，经苄醇Fischer糖苷化、全乙酰化得到化合物式(11)；经过乙酰基脱除、4,6-O-亚苄基保护得到二醇式(12)；通过3-O-苄基化得到化合物式(13)；经2-O-(4-甲氧基苄基)化得到化合物式(14)；然后，经酸性条件下4,6-O-亚苄基保护基的脱除、4,6-O-乙酰化得到化合物式(15)；最后，在脱除2-O-(4-甲氧基苄基)的条件下，得到单糖合成砌块式(16)。
Figure PCTCN2016077370-appb-000017
式中：a_苄苷化；b_全乙酰化；c_全脱乙酰基化；d_4,6-O亚苄基保护；e_3-O-苄基保护；f_2-O-PMB保护；g_酸性条件；h_4,6-O-乙酰化；i_脱除PMB；
步骤(4)：将所述单糖合成砌块式(16)和式(10)在糖基化条件下反应，得到二糖中间体式(17；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(18)；化合物式(18)和单糖合成砌块式(10)在糖基化条件下反应，得到三糖中间体式(19)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(20)；化合物式(20)和单糖合成砌块式(10)在糖基化条件下反应，得到四糖中间体式(21)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(22)；化合物式(22)和单糖合成砌块式(6)在糖基化条件下反应，得到五糖中间体式(23)。
其中，所述的糖基化条件为含卤素氧化剂、路易斯酸或质子酸、溶剂、温度的组合，或者二苯亚砜、三氟甲烷磺酸酐、2,4,6-三叔丁基嘧啶、溶剂、温度的组合；其中，所述含卤素氧化剂为：N-碘代琥珀酰亚胺、N-溴代琥珀酰亚胺；所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-70℃～0℃。
Figure PCTCN2016077370-appb-000018
步骤(5)：将所述化合物式(23)在脱除乙酰丙酰基的条件下处理，得到化合物式(24)；再依次在亚磷酰化、氧化条件下，得到磷酸酯化合物式(25)；在脱除乙酰基的条件下，得到化合物式(26)；在脱除硅基的条件下，得到化合物式(27)；在脱除苄基的条件下，得到式(I)磷酰甘露五糖。
所述的脱除苄基的条件为催化剂及其用量、溶剂、氢气(1个大气压)的组合；其中，所述的催化剂为钯/炭、氢氧化钯/炭、钯黑、铂/炭、兰尼镍；催化剂用量为化合物式(27)质量的0.1～5倍；所述溶剂为甲醇、乙醇、水或其二元混合物。
Figure PCTCN2016077370-appb-000019
本发明还提出了由上述制备方法合成的如式(I)所示磷酰甘露五糖。
本发明还首次提出了一种式(II)一种磷酰甘露五糖醇的制备方法，以所述化合物式(27)为原料，在脱除苄基-还原开环的条件下，获得式(II)磷酰甘露五糖醇；所述制备方法包括以下步骤及反应式。
所述脱除苄基-还原开环的条件为催化剂及其用量、溶剂、氢气及其压力的组合；其中，所述的催化剂为钯/炭、氢氧化钯/炭、钯黑、铂/炭、兰尼镍；催化剂用量为化合物式(27)质量的5～40倍；所述溶剂为甲醇、乙醇、水或其二元混合物；氢气压力为1～5atm。
Figure PCTCN2016077370-appb-000020
本发明还提出了一种式(II)一种磷酰甘露五糖醇的制备方法，以所述式(I)化合物为原料，在还原条件下，获得式(II)磷酰甘露五糖醇；所述制备方法包括以下步骤及反应式。所述的还原条件为还原剂、溶剂的组合；其中，所述的还原剂为硼氢化钠、硼氢化钾、硼氢化锂、三乙酰氧基硼氢化钠、氰基硼氢化钠；所述溶剂为甲醇、乙醇、二氯甲烷、1,2-二氯甲烷或其二元混合物。
Figure PCTCN2016077370-appb-000021
本发明还提出了按上述任一磷酰甘露五糖醇制备方法合成的如式(II)所示的磷酰甘露五糖醇。
本发明还提出了一种式(III)硫酸化磷酰甘露五糖的制备方法，以式(I)磷酰甘露五糖为原料，在O-硫酸化条件下，获得式(III)硫酸化磷酰甘露五糖；所述制备方法包括以下步骤及反应式。
Figure PCTCN2016077370-appb-000022
本发明还提出了一种由上述制备方法合成的如式(III)所示的硫酸化磷酰甘露五糖。
本发明还提出了一种式(IV)硫酸化磷酰甘露五糖醇的制备方法，其特征在于，以式(II)磷酰基甘露五糖醇为原料，在O-硫酸化条件下，获得式(IV)硫酸化磷酰甘露五糖醇；所述制备方法包括以下步骤及反应式。
Figure PCTCN2016077370-appb-000023
本发明还提出了由上述制备方法合成的如式(IV)所示的硫酸化磷酰甘露五糖醇。
本发明还提供一种式(VI)确定位点不完全硫酸化甘露五糖(确定位点部分硫酸化甘露五糖)的制备方法，其特征在于，首先分别合成三个单糖合成砌块，通过从还原端起始的线性组装获得保护的五糖中间体，然后经后阶段的脱保护、硫酸化、脱保护得到式(VI)确定位点部分硫酸化甘露五糖；所述制备方法包括以下步骤及反应式：
步骤(1)：以所述化合物式(6)为原料，在氧化水解条件下，得到化合物式(28)；再通过三氯乙腈、碱处理，得到单糖合成砌块式(29)。
Figure PCTCN2016077370-appb-000024
式中：a_氧化水解条件；b_制备三氯乙酰亚胺酸酯；
步骤(2)：以所述化合物式(7)为原料，经过2-O-苄基化保护，得到化合物式(30)；再经过酸性条件下4,6-O-亚苄基保护基的脱除、6-O-硅基化，得到化合物式(31)；最后，经过4-O-苄基化保护，得到单糖合成砌块式(32)；
Figure PCTCN2016077370-appb-000025
式中：a_苄基化；b_酸性条件；c_6-O-硅基化；d_苄基化；
步骤(3)：以化合物式(33)为原料，经过全三甲基硅基保护，得到化合物式(34)；再经过4,6-O-亚苄基保护、2,3-O-硅基脱除，得到化合物式(35)；最后，通过3-O-苄基化保护，得到单糖合成砌块式(36)；
Figure PCTCN2016077370-appb-000026
式中：a_全三甲基硅基化；b_4,6-O-亚苄基保护、2,3-O-硅基脱除；c_3-O-苄基化；
步骤(4)：将所述单糖合成砌块式(36)和式(32)在第一糖基化条件下反应，得到二糖中间体式(37)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(38)；化合物式(38)和单糖合成砌块式(32)在糖基化条件下反应，得到三糖中间体式(39)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(40)；化合物式(40)和单糖合成砌块式(32)在糖基化条件下反应，得到四糖中间体式(41)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(42)；化合物42和单糖合成砌块式(29)在第二糖基化条件下反应，得到五糖中间体式(43)。
其中，所述的糖基化条件1为含卤素氧化剂、路易斯酸或质子酸、溶剂、温度的组合，或者二苯亚砜、三氟甲烷磺酸酐、2,4,6-三叔丁基嘧啶、溶剂、温度的组合；其中，所述含卤素氧化剂为：N-碘代琥珀酰亚胺、N-溴代琥珀酰亚胺；所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-70℃～0℃。
所述的糖基化条件2为路易斯酸或质子酸、溶剂、温度的组合；其中，所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、 四氢呋喃或其二元混合物；所述温度为-40℃～0℃。
Figure PCTCN2016077370-appb-000027
步骤(5)：将所述化合物式(43)，依次在脱除酰基、脱除硅基的条件下处理，得到化合物式(44)；再在O-硫酸化条件下，得到化合物45；最后，在脱除苄基的条件下，得到式(VI)确定位点部分硫酸化甘露五糖。
其中，所述脱除苄基的条件为催化剂及其用量、溶剂、氢气(1个大气压)的组合；其中，所述的催化剂为钯/炭、氢氧化钯/炭、钯黑、铂/炭、兰尼镍；催化剂用量为化合物44质量的0.1～5倍；所述溶剂为甲醇、乙醇、水或其二元混合物。
Figure PCTCN2016077370-appb-000028
本发明还提出了一种由上述制备方法合成的式(VI)所示的确定位点不完全硫酸化甘露五糖。
本发明提供一种式(VII)确定位点不完全硫酸化甘露五糖的制备方法，其特征在于，首先合成单糖合成砌块式(48)，再将其与所述的单糖合成砌块式(10)、式(6)，通过从还原端起始的线性组装获得保护的五糖中间体，经后阶段的脱保护、硫酸化、脱保护得到式(VII)确定位点部分硫酸化甘露五糖；所述制备方法包括以下步骤及反应式：
步骤(1)：以所述化合物36为原料，经过2-O-(4-甲氧基苄基)保护，得到化合物式(46)；再依次经过酸性条件下4,6-O-亚苄基保护基的脱除、4,6-O-乙酰化，得到化合物式(47)；最后，在脱除3-O-(4-甲氧基苄基)的条件下，得到单糖合成砌块式(48)；
Figure PCTCN2016077370-appb-000029
式中：a_2-O-PMB保护；b_酸性条件；c_4,6-O-乙酰基保护；d_脱除PMB；
步骤(2)：将所述单糖合成砌块式(48)和式(10)在糖基化条件下反应，得到二糖中间体式(49)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(50)；化合物式(50)和单糖合成砌块式(10)在糖基化条件下反应，得到三糖中间体式(51)；在脱除3-O-(4-甲氧基 苄基)的条件下，得到化合物式(52)；化合物式(52和单糖合成砌块式(10)在糖基化条件下反应，得到四糖中间体式(53)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(54)；化合物式(54)和单糖合成砌块式(6)在糖基化条件下反应，得到五糖中间体式(55)。
其中，所述糖基化条件为含卤素氧化剂、路易斯酸或质子酸、溶剂、温度的组合，或者二苯亚砜、三氟甲烷磺酸酐、2,4,6-三叔丁基嘧啶、溶剂、温度的组合；其中，所述含卤素氧化剂为：N-碘代琥珀酰亚胺、N-溴代琥珀酰亚胺；所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-70℃～0℃。
Figure PCTCN2016077370-appb-000030
步骤(3)：将所述化合物式(55)，依次在脱除酰基、脱除硅基的条件下处理，得到化合 物式(56)；再在O-硫酸化条件下，得到化合物式(57)；最后，在脱除苄基的条件下，得到式(VII)确定位点部分硫酸化甘露五糖。
所述脱除苄基的条件为催化剂及其用量、溶剂、氢气(1个大气压)的组合；其中，所述的催化剂为钯/炭、氢氧化钯/炭、钯黑、铂/炭、兰尼镍；催化剂用量为化合物56质量的0.1～5倍；所述溶剂为甲醇、乙醇、水或其二元混合物。
Figure PCTCN2016077370-appb-000031
本发明还提出了一种由上述制备方法合成的如式(VII)所示确定位点不完全硫酸化甘露五糖。
本发明还提供一种式(IX)不完全硫酸化甘露三糖(确定位点部分硫酸化甘露三糖)的制备方法，首先合成单糖合成砌块式(59)，再将其与所述的单糖合成砌块式(36)、式(29)，通过从非还原端起始的线性组装获得保护的三糖中间体，经后阶段的脱保护、硫酸化、脱保护得到式(IX)确定位点部分硫酸化甘露三糖；所述制备方法包括以下步骤及反应式：
步骤(1)：以所述化合物式(3)为原料，经过2,3-O-苄基化，得到化合物式(58)；再依次经过酸性条件下4,6-O-亚苄基保护基的脱除、6-O-硅基化，得到单糖合成砌块式(59)；
Figure PCTCN2016077370-appb-000032
式中：a_2,3-O-苄基保护；b_酸性条件；c_6-O-硅基化；
步骤(2)：将所述单糖合成砌块式(59)和式(29)在第一糖基化条件下反应，得到二糖中间体式(60)；然后，化合物60和单糖合成砌块式(36)在第二糖基化条件下反应，得到三糖中间体式(61)；
其中，所述第一糖基化条件为路易斯酸或质子酸、溶剂、温度的组合；其中，所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-40℃～0℃。
所述第二糖基化条件为含卤素氧化剂、路易斯酸或质子酸、溶剂、温度的组合，或者二苯亚砜、三氟甲烷磺酸酐、2,4,6-三叔丁基嘧啶、溶剂、温度的组合；其中，所述含卤素氧化剂为：N-碘代琥珀酰亚胺、N-溴代琥珀酰亚胺；所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-70℃～0℃。
Figure PCTCN2016077370-appb-000033
步骤(3)：将所述化合物式(61)，依次经过脱除酰基、脱除硅基的条件的处理，得到化合物式(62)；经酸性条件下4,6-O-亚苄基保护基的脱除，得到化合物式(63)；然后，在O-硫酸化条件下，得到化合物式(64)；最后，在脱除苄基的条件下，得到式(XI)确定位点部分硫酸化甘露三糖；其中，所述的脱除苄基的条件为催化剂及其用量、溶剂、氢气(1个大气压)的组合；其中，所述的催化剂为钯/炭、氢氧化钯/炭、钯黑、铂/炭、兰尼镍；催化剂用量为化合物63质量的0.1～5倍；所述溶剂为甲醇、乙醇、水或其二元混合物。
Figure PCTCN2016077370-appb-000034
本发明还提出了一种由上述制备方法合成的式(IX)所示的确定位点不完全硫酸化甘露三糖。
本发明还提供一种式(XI)确定位点不完全硫酸化支链磷酰甘露三糖(确定位点部分硫酸化支链磷酰甘露三糖)的制备方法，首先合成单糖合成砌块式(67)，再将其与所述的单糖合成砌块式(6)，通过糖基化获得保护的支链三糖中间体式(68)，然后经后阶段的非还原端脱保护、亚磷酰化-氧化、脱保护、硫酸化、脱苄基，得到式(XI)确定位点部分硫酸化支链磷酰甘露三糖；所述制备方法包括以下步骤及反应式：
步骤(1)：以D-甘露糖为原料，依次经过乙酰化、酚苷化，得到化合物式(65)；再依次经过脱乙酰基、2,3:4,6-O-双亚苄基保护，得到化合物式(65)；然后，在还原开环条件下，得到单糖合成砌块式(67)；
Figure PCTCN2016077370-appb-000035
式中：a_全乙酰化；b_酚苷化；c_脱乙酰基化；d_2,3:4,6-O-双亚苄基保护；e_还原开环；
步骤(2)：将所述单糖合成砌块式(67)和式(6)在糖基化条件下反应，得到支链三糖中间体式(68)；其中，所述的糖基化条件为含卤素氧化剂、路易斯酸或质子酸、溶剂、温度的组合，或者二苯亚砜、三氟甲烷磺酸酐、2,4,6-三叔丁基嘧啶、溶剂、温度的组合；其中，所述含卤素氧化剂为：N-碘代琥珀酰亚胺、N-溴代琥珀酰亚胺；所述路易斯酸为三甲基硅基三氟甲磺酸酯、叔丁基二甲基硅基三氟甲磺酸酯、三乙基硅基三氟甲磺酸酯、三氟甲磺酸银；所述质子酸为三氟甲磺酸、三氟乙酸；所述溶剂为乙醚、二氯甲烷、1,4-二氧六环、四氢呋喃或其二元混合物；所述温度为-70℃～0℃。
Figure PCTCN2016077370-appb-000036
步骤(3)：将所述化合物式(68)，经脱除乙酰丙酰基的条件处理，得到二醇式(69)；再依次在亚磷酰化、氧化条件下，得到双磷酸酯化合物式(70)；然后，在脱除乙酰基的条件下，得到化合物式(71)；最后，依次经O-硫酸化条件、脱除苄基的条件的处理，得到式(XI)确定位点部分硫酸化支链磷酰甘露三糖；其中，所述的脱除苄基的条件为催化剂及其用量、溶剂、氢气(1个大气压)的组合；其中，所述的催化剂为钯/炭、氢氧化钯/炭、钯黑、铂/炭、兰尼镍；催化剂用量为化合物71质量的0.1～5倍；所述溶剂为甲醇、乙醇、水或其二元混合物。
Figure PCTCN2016077370-appb-000037
本发明还提出了一种由上述制备方法合成的如式(XI)所示确定位点不完全硫酸化支链磷酰甘露三糖。
本发明还提出了式(I)所示的磷酰甘露五糖及如式(II)-(XI)所示的磷酰甘露五糖的衍生物在制备抗肿瘤药物中的应用。本发明还提供了所述磷酰甘露五糖式(I)化合物、磷酰甘露五糖醇式(II)化合物在制备抗肿瘤药物中的应用。本发明还提供了所述硫酸化寡糖式(III)、式(IV)、式(V)、式(VI)、式(VII)、式(VIII)、式(IX)、式(X)、式(XI)化合物在制备抗肿瘤药物中的应用。
本发明还提出了式(I)所示的磷酰甘露五糖及如式(II)-(XI)所示的磷酰甘露五糖的衍生物在制备抑制血管生成药物中的应用。所述式(III)、式(IV)、式(V)、式(VI)、式 (VII)、式(VIII)、式(IX)、式(X)、式(XI)化合物具有抑制血管生成的活性。
本发明还提出了式(I)所示的磷酰甘露五糖在制备具有抑制血管生成活性的式(III)所示的硫酸化磷酰甘露五糖化合物中的应用。本发明还提出了式(II)所示的磷酰甘露五糖醇在制备具有抑制血管生成活性的式(IV)所示的硫酸化磷酰甘露五糖醇化合物中的应用。所述式(I)、式(II)化合物分别是制备具有抑制血管生成活性的化合物式(III)、式(IV)的关键原料。
本发明首次提供了酵母菌Pichia holstii NRRL Y-2448相关的纯净形式的式(I)磷酰甘露五糖。本发明通过化学合成的方式首次获得的单一组分的式(I)所示的磷酰甘露五糖，显著地优于现有技术中的磷酰甘露寡糖混合物。本发明中，该式(I)磷酰甘露五糖可再经硫酸化修饰，首次获得单一组分的式(III)全硫酸化磷酰甘露五糖，显著地优于现有技术中的高度硫酸化磷酰甘露寡糖混合物(PI-88)，有利于开发新的高纯度的肿瘤抗血管生成的药用化合物，以期优化生物活性和相关的质量控制。
本发明中的式(II)所示的磷酰甘露五糖醇是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖的首例还原端半缩醛的还原开环衍生物。本发明中，由式(II)磷酰甘露五糖醇经硫酸化修饰，首次获得了式(IV)所示的硫酸化磷酰甘露五糖醇，该化合物是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖的首例还原端半缩醛的还原开环-硫酸化衍生物。
本发明中式(V)所示的确定位点不完全硫酸化甘露寡糖衍生物是酵母菌Pichia holstii NRRL Y-2448细胞外磷酰甘露寡糖的首例确定位点不完全硫酸化衍生物，显著地优于现有技术中的硫酸化程度、位点不确定的高度硫酸化甘露寡糖混合物。本发明中式(VIII)所示的确定位点不完全硫酸化甘露三糖是首例含有α-(1-4)-/α-(1-2)-糖苷键的确定位点不完全硫酸化甘露寡糖衍生物。本发明中式(X)所示的确定位点不完全硫酸化支链磷酰甘露三糖是首例含有α-(1-3)-/α-(1-6)-糖苷键的确定位点不完全硫酸化支链磷酰甘露寡糖衍生物。上述式(V)～式(XI)硫酸化寡糖衍生物，主要是针对如下方面进行的结构修饰：1)硫酸化甘露寡糖醇衍生物；2)确定位点的不完全硫酸化甘露寡糖衍生物；以及3)含有α-(1-4)-/α-(1-6)-糖苷键的硫酸化甘露寡糖衍生物，为发现新的抗肿瘤血管生成的药用分子提供了空间。
本发明中的制备方法区别于现有技术的显著进步在于：1)通过合理设计甘露糖硫苷给体中羟基的保护基以调控其反应活性，实现了以硫苷为给体的糖基化构建α-(1-2)-/α-(1-3)-连接的甘露寡糖链；2)通过“后期磷酰化策略”，并采用容易脱除的磷酸酯保护基，实现了磷酰化的高效性、经济性。
附图说明
图1为现有技术中磷酰甘露五塘及其衍生物的制备现状示意图。
图2是式(III)、(IV)、(VI)、(VII)、(IX)、(XI)硫酸化寡糖化合物抑制血管生成的实验结果，其中，PI-88作为对比化合物。图2-1是各化合物抑制HMEC-1微管生成的实验结果柱状图。图2-2是式(III)化合物抑制HMEC-1微管生成的成像图。实验表明，式(III)、(IV)、(VI)、(VII)、(IX)、(XI)硫酸化寡糖化合物对HMEC-1微管生成具有显著的抑制作用，其中，化合物(III)和(IV)表现出优于PI-88的活性。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明，本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1 化合物6的制备
Figure PCTCN2016077370-appb-000038
化合物式(1)的制备：室温下，将D-甘露糖(10.0g)悬浮于Ac2O(70mL)中，并加入NaOAc(10g)。将温度升至120℃并搅拌2h。将反应液倒入400g冰中，搅拌1h，然后用乙酸乙酯萃取(200×3)萃取。有机相合并，并依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，Na2SO4干燥，浓缩即得化合物式(1)(100％)。
化合物式(2)的制备：0℃，将BF3·OEt2(50mL)滴加到化合物式(1)(52.0g，133.2mmol)和4-甲苯硫酚(22g,177mmol)的CH2Cl2(200mL)溶液中。升至室温，继续搅拌，TLC检测反应完成。用DCM稀释，有机相依次用5％NaOH(aq)和饱和食盐水洗涤,MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE＝1:3)得到化合物式(2)(64.3g,100％)。1H NMR(400MHz,CDCl3)δ7.40–7.36(m,2H),7.12(d,J＝7.9Hz,2H),5.55–5.45(m,1H),5.41(d,J＝1.2Hz,1H),5.37–5.27(m,2H),4.60–4.51(m,1H),4.30(dd,J＝12.2,5.9Hz,1H),4.11(dd,J＝12.2,2.4Hz,1H),2.33(s,3H),2.14(s,3H),2.07(s,3H),2.06(s,2H),2.01(s,3H).
化合物式(3)的制备：化合物式(2)(15.9g，55.4mmol)溶解在MeOH(300mL)中，加入MeONa(0.29g,5.54mmol)。室温下搅拌4h，经Dowex50WX8酸性树脂中和、过滤和 蒸干后得粗产物。将粗产物与甲苯共沸，然后悬浮于100mL CH3CN中。加入苯甲醛二甲缩醛(5.8mL,1.1eq)和DL-樟脑磺酸(811mg,0.1eq)，室温下搅拌15min，升至50℃，继续搅拌30min。然后加入三乙胺(5mL)淬灭，蒸去溶剂。粗产物经EtOAc重结晶得到化合物式(3)(11.02g，84％，2步)。1H NMR(400MHz,DMSO)δ7.49–7.44(m,2H),7.42–7.32(m,5H),7.18(d,J＝8.0Hz,2H),5.63(s,1H),5.52(d,J＝4.1Hz,1H),5.36(d,J＝0.9Hz,1H),5.20(d,J＝6.1Hz,1H),4.13–4.01(m,2H),4.01–3.97(m,1H),3.94(dd,J＝9.3,9.3Hz,1H),3.82–3.67(m,2H),2.29(s,3H).
化合物式(4)的制备：化合物式(3)(6.4g,17.1mmol)和Bu2SnO(4.68g,18.8mmol)溶解在甲苯(100mL)中，加热至140℃，搅拌4小时，并不断蒸出溶剂至原体积的1/4。冷却至60℃，依次加入BnBr(3.0mL,25.6mmol)和TBAI(1.26g,3.42mmol)，并继续在60℃加热12小时。用水终止反应，并用甲苯共沸除去溶剂得到粗产物。将此粗产物溶于DCM(100mL)，依次加入吡啶(17.3mL)、DMAP(100mg)和乙酸酐(8.46mL)，室温下搅拌直至TLC监测反应完全。NaHCO3(sat.aq.)淬灭，水相用DCM萃取。有机相用饱和食盐水洗涤,MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE＝1:30～1:5)得到化合物式(4)(6.1g,70％，2步)。1H NMR(400MHz,CDCl3)δ7.45(dd,J＝7.6,1.9Hz,2H),7.36–7.20(m,10H),7.04(d,J＝7.9Hz,2H),5.57(s,1H),5.54(dd,J＝3.4,1.4Hz,1H),5.31(d,J＝1.2Hz,1H),4.65(d,J＝12.1Hz,1H),4.61(d,J＝12.1Hz,1H),4.30(ddd,J＝9.8,9.8,4.8Hz,1H),4.16(dd,J＝10.3,4.9Hz,1H),4.06(dd,J＝9.6,9.6Hz,1H),3.94(dd,J＝9.9,3.4Hz,1H),3.78(dd,J＝10.3,10.3Hz,1H),2.25(s,3H),2.07(s,3H).
化合物式(5)的制备方法：将化合物式(4)(482.2mg,0.95mmol)溶解于乙腈(10mL)，并冷却至0℃，然后加入NaBH4(360mg,9.5mmol)。搅拌5min，加入三聚氯氰(1.4g,7.6mmol)，并继续在0℃搅拌3h。升至室温后继续搅拌6h至TLC监测反应完全。反应液用乙酸乙酯稀释，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:4:1)得到化合物式(5)(416mg,86％)。1H NMR(400MHz,CDCl3)δ7.38–7.27(m,12H),7.11(d,J＝7.9Hz,2H),5.59(dd,J＝3.2,1.7Hz,1H),5.37(d,J＝1.4Hz,1H),4.93(d,J＝10.9Hz,1H),4.73(d,J＝11.3Hz,1H),4.64(d,J＝10.9Hz,1H),4.57(d,J＝11.3Hz,1H),4.0(ddd,J＝7.0,3.8,3.1Hz,1H),3.96(dd,J＝9.4,3.2Hz,1H),3.87(dd,J＝9.5,9.5Hz,1H),3.84–3.74(m,2H),2.32(s,3H),2.13(s,3H),1.74(s,1H).HRMS(ESI-TOF)m/z Calcd for C29H32O6NaS[M+Na]+531.1817,found 531.1818.
化合物式(6)的制备方法：将5(403mg,0.79mmol)溶解在DCM(10mL)中，室温下加入
Figure PCTCN2016077370-appb-000039
(500mg)、EDCI(304mg,1.58mmol)、DMAP(97mg,0.79mmol)和LevOH(184mg,1.58mmol)，搅拌过夜，TLC监测反应完全。过滤，用乙酸乙酯洗涤滤饼，然后依 次用饱和食盐水洗、水洗，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:5:1～1:3:1)得到化合物式(6)(431mg,90％)。1H NMR(400MHz,CDCl3)δ7.38–7.27(m,12H),7.10(d,J＝7.9Hz,2H),5.59(dd,J＝3.2,1.7Hz,1H),5.40(d,J＝1.6Hz,1H),4.92 and 4.58(ABq,JAB＝10.8Hz,2H,CH2Ar),4.72 and 4.56(ABq,JAB＝11.2Hz,2H,CH2Ar),4.42–4.35(m,2H),4.30(dd,J＝14.2,4.9Hz,1H),3.95(dd,J＝9.2,3.2Hz,1H),3.78(dd,J＝9.2,9.2Hz,1H),2.78–2.67(m,2H),2.59–2.52(m,2H),2.32(s,3H),2.16(s,3H),2.14(s,3H).HRMS(ESI-TOF)m/z Calcd for C34H38O8NaS[M+Na]+629.2185,found 629.2162.
实施例2 化合物式(10)的制备
Figure PCTCN2016077370-appb-000040
化合物式(7)的制备：将化合物式(3)(2.0g,5.34mmol)和Bu2SnO(1.46g,5.86mmol)溶解在甲苯(80mL)中，加热至130℃，4h内将溶剂体积浓缩至约20mL。然后冷至60℃，依次加入DMF(15mL),PMBCl(1.09mL,8.0mmol),CsF(1.62g,10.7mmol)和TBAI(395mg,1.07mmol)，并继续在70℃下搅拌4小时(TLC监测)。冷却至室温，用水终止反应，并用乙酸乙酯萃取。有机相依次用水、饱和食盐水洗涤，MgSO4干燥,过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:4:1)得到化合物式(7)(2.85g,100％)。1H NMR(400MHz,CDCl3)δ7.55–7.48(m,2H),7.43–7.27(m,7H),7.12(d,J＝7.9Hz,2H),6.93–6.84(m,2H),5.61(s,1H),5.51(d,J＝0.8Hz,1H),4.81(d,J＝11.4Hz,1H),4.66(d,J＝11.4Hz,1H),4.34(ddd,J＝9.8,9.8,4.9Hz,1H),4.24–4.22(m,1H),4.20(dd,J＝10.5,5.0Hz,1H),4.14(J＝9.5,9.5Hz,1H),3.94(dd,J＝9.6,3.4Hz,1H),3.85(J＝10.3,10.3Hz,1H),3.81(s,3H),2.85(d,J＝1.3Hz,1H),2.33(s,3H).
化合物式(8)的制备：将化合物式(7)(1.314g,2.66mmol)溶于DCM(15mL)中，室温下加入三乙胺(4.4mL,12eq)、DMAP(32mg，0.1eq)后，逐滴加入Ac2O(1.26mL，5eq)，搅拌过夜。在0℃下，用NaHCO3(sat.aq.)淬灭。有机相用水、饱和食盐水洗涤，MgSO4干燥，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(8)(1.24g,87％)。1H NMR(400MHz,CDCl3)δ7.55–7.47(m,2H),7.43–7.36(m,3H),7.36–7.32(m,2H),7.31–7.26(m,2H),7.12(d,J＝7.9Hz,2H),6.91–6.78(m,2H),5.63(s,1H),5.59(dd,J＝3.4,1.4Hz,1H),5.37(d,J＝1.2Hz,1H),4.66 and 4.60(ABq,JAB＝11.6Hz,2H,CH2Ar),4.36(ddd,J＝9.8,9.8,4.8Hz,1H),4.22(dd,J＝10.3,4.8Hz,1H),4.11(dd,J＝9.6,9.6Hz,1H),3.99(dd,J＝9.9,3.4 Hz,1H),3.84(dd,J＝10.3,10.3Hz,1H),3.80(s,3H),2.33(s,3H),2.14(s,3H).HRMS(ESI-TOF)m/z Calcd for C30H32O7NaS[M+Na]+559.1766,found 559.1738.
化合物式(9)的制备：将化合物式(8)(1.22mg,2.27mmol)溶于CH3CN(22mL)，冷至0℃，再加入NaBH4(859mg,13.4mmol)。搅拌10min，加入三聚氯氰(3.35g,18.2mmol)，继续在0℃搅拌30min。升至室温后继续搅拌，TLC监测反应完全(约9h)。乙酸乙酯稀释、硅藻土过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:3:1)得到化合物式(9)(1.03g,84％)。1H NMR(400MHz,CDCl3)δ7.39–7.25(m,9H),7.11(d,J＝7.9Hz,2H),6.90–6.82(m,2H),5.58(dd,J＝3.1,1.6Hz,1H),5.37(d,J＝1.4Hz,1H),4.92 and 4.62(ABq,JAB＝10.9Hz,2H,CH2Ar),4.66 and 4.49(ABq,JAB＝10.9Hz,2H,CH2Ar),4.19(ddd,J＝7.2,4.0,3.2Hz,1H),3.94(dd,J＝9.4,3.2Hz,1H),3.88–3.77(m,6H),2.32(s,3H),2.13(s,3H),1.74(t,J＝6.5Hz,1H,OH).HRMS(ESI-TOF)m/z Calcd for C30H34O7NaS[M+Na]+561.1923,found 561.1915.
化合物式(10)的制备：将化合物式(9)(496mg,0.92mmol)溶解在DCM(10mL)中，室温下加入咪唑(188mg,3eq)。搅拌下加入TIPSCl(335μL,1.7eq)。TLC监测反应完全后(5h)，用DCM稀释，有机相依次用水、NaHCO3(sat.aq.)和饱和食盐水洗涤。Na2SO4干燥，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:20:1)得到化合物式(10)(559mg，87％)。1H NMR(400MHz,CDCl3)δ7.37–7.25(m,9H),7.07(d,J＝8.0Hz,2H),6.88–6.82(m,2H),5.54(dd,J＝3.0,1.7Hz,1H),5.39(d,J＝1.5Hz,1H),4.90 and 4.65(ABq,JAB＝10.8Hz,2H,CH2Ar),4.65 and 4.50(ABq,JAB＝10.9Hz,2H,CH2Ar),4.11(dd,J＝9.4,2.5Hz,1H),4.04(dd,J＝11.2,3.9Hz,1H),3.99(dd,J＝9.5,9.5Hz,1H),3.95–3.87(m,2H),3.80(s,3H),2.31(s,3H),2.09(s,3H),1.15–0.97(m,21H).HRMS(ESI-TOF)m/z calcd for C39H54O7NaSiS[M+Na]+717.3257,found 717.3247.
实施例3 化合物式(16)的制备
Figure PCTCN2016077370-appb-000041
化合物式(11)的制备：将2.0g D-甘露糖溶解于12mL苄醇中，加热至50℃后缓慢滴加乙酰氯(2.0mL)，并在此温度下保持2h。冷至室温，继续搅拌15h。将反应液倒入100mL水中，用乙酸乙酯萃取除去苄醇。水相浓缩，残余物溶于20mL DCM，再依次加入吡啶(36 mL,40eq)和乙酸酐(21mL,20eq)，室温下搅拌过夜。NaHCO3(sat.aq.)淬灭，DCM萃取，有机相用饱和食盐水洗涤，MgSO4干燥，过滤，蒸干。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:4:1)得到化合物式(11)(2.52g，52％，2步)。1H NMR(400MHz,CDCl3)δ7.42–7.28(m,5H),5.38(dd,J＝10.0,3.4Hz,1H),5.295(dd,J＝10.0,10.0Hz,1H),5.288(dd,J＝3.5,1.8Hz,1H),4.89(d,J＝1.6Hz,1H),4.72(d,J＝11.9Hz,1H),4.57(d,J＝11.9Hz,1H),4.28(dd,J＝12.1,5.1Hz,1H),4.05(dd,J＝12.2,2.5Hz,1H),4.01(ddd,J＝9.9,5.1,2.5Hz,1H),2.14(s,3H),2.11(s,3H),2.03(s,3H),1.99(s,3H).
化合物式(12)的制备：将化合物式(11)(775mg,1.77mmol,1.0eq)溶解在MeOH(15mL)中，加入MeONa(10mg,0.18mmol)。室温下搅拌12h，经Dowex50WX8酸性树脂中和，过滤，蒸干。将粗产物与甲苯共沸，然后溶于7mL乙腈中，加入苯甲醛二甲缩醛(0.29mL,1.1eq)和DL-樟脑磺酸(41mg,0.1eq)，室温下搅拌15min，升温至50℃搅拌15min。三乙胺(5mL)淬灭，蒸干。硅胶柱层析纯化(EA/PE/DCM＝1:5:1～1:2:1)得到化合物式(12)(287mg，45％，2步)。1H NMR(400MHz,CDCl3)δ7.52–7.45(m,2H),7.41–7.28(m,8H),5.56(s,1H),4.95(d,J＝1.3Hz,1H),4.74(d,J＝11.9Hz,1H),4.53(d,J＝11.9Hz,1H),4.25(dd,J＝9.2,3.5Hz,1H),4.16–4.09(m,1H),4.08–4.04(m,1H),3.94(dd,J1＝J2＝8.9Hz,1H),3.91–3.85(m,1H),3.85–3.79(m,1H),2.70(d,J＝3.2Hz,1H),2.68(d,J＝2.3Hz,1H).
化合物式(13)的制备：将12(278mg,0.78mmol)和Bu2SnO(212mg,3.07mmol)溶于甲苯(30mL)，加热至140℃，搅拌4h，浓缩至起始体积的1/4。冷至60℃，加入DMF(5mL),BnBr(0.14mL,1.5eq),CsF(236mg,2.0eq)和TBAI(57mg,0.2eq)，并继续在60℃加热12h。用水终止反应，EtOAc萃取。有机相用饱和食盐水洗涤，MgSO4干燥,过滤，蒸干。硅胶柱层析纯化(EA/PE/CH2Cl2＝1:10:1～1:3:1)得到13(365mg,100％)。1H NMR(400MHz,CDCl3)δ7.49(s,2H),7.42–7.28(m,13H),5.62(s,1H),4.97(d,J＝1.4Hz,1H),4.86(d,J＝11.8Hz,1H),4.73(d,J＝4.5Hz,1H),4.70(d,J＝4.5Hz,1H),4.52(d,J＝11.8Hz,1H),4.26(ddd,J＝4.0,3.9,1.5Hz,1H),4.17–4.08(m,2H),3.98(dd,J＝9.6,3.5Hz,1H),3.95–3.89(m,1H),3.89–3.82(m,1H),2.67(d,J＝1.1Hz,1H).
化合物式(14)的制备：将化合物式(13)(365mg)溶于DMF(5mL)，0℃下，分批加入NaH(60％,62mg,1.6mmol)。30min后,加入PMBCl(0.21mL,1.6mmol)。升至室温，继续搅拌12h。反应完成后，加入水淬灭，用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:10:1)得到化合物式(14)(403mg，91％)。1H NMR(400MHz,CDCl3)δ7.55–7.45(m,2H),7.41–7.18(m,15H),6.88–6.78(m,2H),5.64(s,1H),4.84(s,1H),4.82(d,J＝10.4Hz,1H),4.72(d,J＝11.9Hz,1H),4.68(d,J＝12.0Hz,1H),4.63(2 overlapping d,J＝12.1Hz,2H),4.44(d,J＝12.0Hz,1H),4.31–4.17(m,2H),4.06–3.96(m,1H),3.92–3.83(m,3H),3.80(s,3H).
化合物式(15)的制备：将化合物式(14)(380mg,0.67mmol)溶解在AcOH/H2O(4:1,10mL)中，50℃下搅拌4h。冷至室温后加入乙酸乙酯稀释，用NaHCO3(sat.aq.)中和，有机相用饱和食盐水洗涤，MgSO4干燥，浓缩。所得粗产物溶于DCM(5mL)中，再加入三乙胺(4.4mL,48eq)、DMAP(16mg，0.2eq)，并逐滴加入Ac2O(1.26mL,20eq)，室温下搅拌过夜。用NaHCO3(sat.aq.)中和，并以乙酸乙酯稀释。有机相用饱和食盐水洗涤，MgSO4干燥，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(15)(369mg,98％,2步)。1H NMR(400MHz,CDCl3)δ7.39–7.26(m,10H),7.24–7.18(m,2H),6.85–6.74(m,2H),5.42(dd,J＝9.9,9.9Hz,1H),4.91(d,J＝1.8Hz,1H),4.68(d,J＝11.9Hz,1H),4.65(d,J＝12.0Hz,1H),4.58(d,J＝12.1Hz,1H),4.55(d,J＝12.1Hz,1H),4.47(d,J＝12.0Hz,2H),4.45(d,J＝12.1Hz,1H),4.22(dd,J＝12.1,5.5Hz,1H),4.09(dd,J＝12.1,2.4Hz,1H),3.86(dd,J＝9.7,3.1Hz,1H),3.85(ddd,J＝9.7,5.6,2.5Hz,1H),3.82–3.78(m,1H),3.78(s,3H),2.09(s,3H),2.00(s,3H).HRMS(ESI-TOF):m/z Calcd for C32H36O9Na[M+Na]+587.2257,found 587.2243.
化合物式(16)的制备：0℃下，化合物式(15)(361mg,0.64mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)的溶液中，并加入DDQ(174mg,0.77mmol,1.2eq)。0℃，搅拌1h。升至室温，继续搅拌3h。NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:2:1)得到化合物式(16)(253mg,89％)。1H NMR(400MHz,CDCl3)δ7.57–7.19(m,10H),5.29(t,J＝9.8Hz,1H),5.00(d,J＝1.5Hz,1H),4.71(d,J＝11.8Hz,1H),4.66(d,J＝11.9Hz,1H),4.54(d,J＝11.9Hz,1H),4.53(d,J＝11.8Hz,1H),4.24(dd,J＝12.2,5.2Hz,1H),4.09–4.03(m,2H),3.90(ddd,J＝10.2,5.1,2.3Hz,1H),3.84(dd,J＝9.5,3.4Hz,1H),2.55(d,J＝1.5Hz,1H),2.09(s,3H),1.99(s,3H).HRMS(ESI-TOF):m/z Calcd for C24H28O8Na[M+Na]+467.1682,found 467.1675.
实施例4 化合物式(23)的制备。
Figure PCTCN2016077370-appb-000042
化合物式(17)的制备:将给体式(10)(469mg,0.675mmol)和受体式(16)(250mg,0.562mmol)溶于无水CH2Cl2(9mL)中，并加入
Figure PCTCN2016077370-appb-000043
(1.1g)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(152mg,0.675mmol)。搅拌10min后，加入TMSOTf(12.5μL，0.068mmol)。在-20℃下搅拌约40min，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到二糖式(17)(482mg,84％)。1H NMR(400MHz,CDCl3)δ7.38–7.25(m,17H),6.90–6.81(m,2H),5.43(dd,J＝3.0,2.0Hz,1H),5.29(dd,J＝9.8,9.8Hz,1H),4.96(d,J＝1.7Hz,1H),4.94(d,J＝1.8Hz,1H),4.87(d,J＝10.8Hz,1H),4.67(d,J＝10.6Hz,1H),4.66(d,J＝11.8Hz,1H),4.65(d,J＝10.8Hz,1H),4.57(d,J＝11.1Hz,1H),4.54(d,J＝12.2Hz,1H),4.47(d,J＝11.5Hz,1H),4.44(d,J＝12.2Hz,1H),4.09(d,J＝3.9Hz,2H),4.03–3.97(m,2H),3.94–3.85(m,3H),3.84–3.78(m,1H),3.77(s,1H), 3.74(dd,J＝11.2,1.3Hz,1H),3.70–3.62(m,1H),2.06(s,3H),1.99(s,3H),1.97(s,3H),1.13–0.99(m,21H).HRMS(ESI-TOF)m/z Calcd for C56H74O15NaSi[M+Na]+1037.4695,found 1037.4685.
化合物式(18)的制备：0℃下，化合物式(17)(479mg,0.475mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,6mL)中，并加入DDQ(151mg,0.665mmol)。0℃搅拌30min，升至室温，继续搅拌3h，TLC检测反应完全。用NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:2:1)得到化合物式(18)(329mg,78％)。1H NMR(400MHz,CDCl3)δ7.39–7.26(m,15H),5.26(dd,J＝9.8,9.8Hz,1H),5.20(dd,J＝3.3,1.8Hz,1H),4.97(d,J＝1.7Hz,1H),4.96(d,J＝1.9Hz,1H),4.83(d,J＝11.1Hz,1H),4.70(d,J＝11.1Hz,1H),4.66(d,J＝11.8Hz,1H),4.64(d,J＝12.0Hz,1H),4.52(d,J＝12.1Hz,1H),4.45(d,J＝11.9Hz,1H),4.22(ddd,J＝9.3,4.4,3.5Hz,1H),4.13–4.04(m,2H),3.97(dd,J＝2.9,2.0Hz,1H),3.94(dd,J＝11.4,3.7Hz,1H),3.91–3.83(m,2H),3.82–3.78(m,1H),3.76(dd,J＝11.3,1.1Hz,1H),3.67(ddd,J＝9.7,3.3,1.3Hz,1H),2.16(d,J＝4.7Hz,1H),2.07(s,3H),2.02(s,3H),1.97(s,3H),1.14–1.01(m,21H).HRMS(ESI-TOF)m/z Calcd for C48H66O14NaSi[M+Na]+917.4120,found 917.4074.
化合物式(19)的制备：将10(332mg,0.478mmol)和18(329mg,0.368mmol)溶解在无水CH2Cl2(8mL)中，并加入
Figure PCTCN2016077370-appb-000044
(900mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(108mg,0.478mmol)。搅拌10min后，加入TMSOTf(6.7μL，0.037mmol)。在-20℃下搅拌约40min，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:30:1～1:6:1)得到19(419mg,78％)。1H NMR(400MHz,CDCl3)δ7.37–7.24(m,20H),7.22–7.16(m,2H),6.80–6.74(m,2H),5.43(dd,J＝3.0,1.9Hz,1H),5.24–5.17(m,2H),5.09(d,J＝1.4Hz,1H),4.97(d,J＝1.8Hz,1H),4.94(d,J＝1.9Hz,1H),4.86(d,J＝10.7Hz,1H),4.77(d,J＝10.8Hz,1H),4.71(d,J＝10.7Hz,1H),4.64(d,J＝11.9Hz,1H),4.63(d,J＝12.3Hz,1H),4.58(d,J＝10.8Hz,1H),4.55(d,J＝10.9Hz,1H),4.51(d,J＝12.3Hz,1H),4.42(d,J＝11.9Hz,1H),4.36(d,J＝10.8Hz,1H),4.19–4.12(m,2H),4.10–4.05(m,3H),3.96–3.76(m,7H),3.72(s,3H),3.71–3.62(m,3H),2.08(s,3H),2.00(s,3H),1.98(s,3H),1.96(s,3H),1.12–0.99(m,42H).HRMS(ESI-TOF)m/z Calcd for C80H112O21NaSi2[M+Na]+1487.7132,found 1487.7081.
化合物式(20)的制备：0℃下，化合物式(19)(149mg,0.101mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(28mg,0.122mmol)。0℃搅拌30min，升至室温，继续搅拌3h。TLC检测反应完全，用NaHCO3(sat.aq.)和CH2Cl2 稀释。有机相依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(20)(106mg,74％)。1H NMR(400MHz,CDCl3)δ7.39–7.24(m,20H),5.24–5.15(m,3H),5.10(d,J＝1.3Hz,1H),4.95(d,J＝1.9Hz,1H),4.93(d,J＝1.9Hz,1H),4.83(d,J＝11.2Hz,1H),4.80(d,J＝10.9Hz,1H),4.76(d,J＝11.3Hz,1H),4.65(d,J＝11.4Hz,1H),4.62(d,J＝11.5Hz,1H),4.60(d,J＝10.7Hz,1H),4.52(d,J＝12.2Hz,1H),4.42(d,J＝11.8Hz,1H),4.15(dd,J＝9.3,3.2Hz,1H),4.12–4.01(m,5H),3.97–3.84(m,5H),3.80(ddd,J＝9.7,5.1,2.9Hz,1H),3.72–3.61(m,3H),2.05(s,3H),2.03(s,3H),1.99(s,3H),1.97(s,3H),1.15–0.97(m,42H).HRMS(ESI-TOF)m/z Calcd for C72H104O20NaSi2[M+Na]+1367.6557,found 1367.6591.
化合物式(21)的制备：将化合物式(10)(71mg,0.102mmol)和化合物式(20)(106mg,0.079mmol)溶解在无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000045
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(23mg,0.102mmol)。搅拌10min后，加入TMSOTf(2μL，0.0118mmol)。在-20℃下搅拌约40min，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:30:1～1:8:1)得到化合物式(21)(139mg,92％)。1H NMR(400MHz,CDCl3)δ7.40–7.20(m,27H),6.83–6.74(m,2H),5.42(dd,J＝2.9,1.9Hz,1H),5.26(dd,J＝2.6,1.8Hz,1H),5.21–5.14(m,2H),5.09(d,J＝1.1Hz,1H),5.08(d,J＝1.4Hz,1H),4.94(d,J＝1.5Hz,1H),4.91(d,J＝1.8Hz,1H),4.84(d,J＝10.1Hz,1H),4.83(d,J＝10.8Hz,1H),4.77(d,J＝11.4Hz,1H),4.74(d,J＝11.6Hz,1H),4.632(d,J＝10.8Hz,1H),4.628(d,J＝12.3Hz,1H),4.61(d,J＝12.2Hz,1H),4.59(d,J＝10.7Hz,1H),4.54–4.49(m,2H),4.41(d,J＝10.8Hz,1H),4.40(d,J＝11.9Hz,1H),4.22–4.09(m,4H),4.08–4.00(m,3H),3.98–3.80(m,7H),3.80–3.75(m,1H),3.74(s,3H),3.67–3.61(m,2H),3.61–3.54(m,3H),2.04(s,3H),2.03(s,3H),2.03(s,3H),1.96(s,3H),1.93(s,3H),1.15–1.01(m,42H),0.99–0.88(m,21H).HRMS(ESI-TOF)m/z Calcd for C104H150O27NaSi3[M+Na]+1937.9570,found 1937.9478.
化合物式(22)的制备：0℃下，化合物式(21)(314mg,0.164mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(49mg,0.213mmol)。0℃搅拌30min，升至室温，继续搅拌3h，TLC检测反应完全。NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:15:1～1:3:1)得到式(22)化合物(238mg,81％)。1H NMR(400MHz,CDCl3)δ7.37–7.24(m,20H),5.27(dd,J＝3.0,1.8Hz,1H),5.23–5.13(m,3H),5.09(d,J＝1.6Hz,1H),5.08(d,J＝1.3Hz,1H),4.94(d,J＝1.6Hz,1H),4.91(d,J＝1.8Hz,1H),4.83(d,J＝10.0Hz,1H),4.79(d,J＝10.7Hz,1H),4.75(d,J＝11.2Hz,1H),4.74(d,J＝10.8Hz,1H),4.70(d,J＝11.4Hz,1H),4.62(d,J＝11.9Hz,1H),4.61(d,J＝12.3Hz,1H),4.52(d,J＝12.4Hz,1H), 4.51(d,J＝10.0Hz,1H),4.40(d,J＝11.9Hz,1H),4.23–4.08(m,3H),4.09–3.91(m,6H),3.92–3.81(m,4H),3.81–3.72(m,2H),3.70–3.50(m,5H),2.07(s,3H),2.03(s,3H),2.00(s,3H),1.96(s,3H),1.92(s,3H),1.16–1.00(m,42H),1.00–0.88(m,21H).HRMS(ESI-TOF)m/z Calcd for C96H142O26NaSi3[M+Na]+1817.8995,found 1817.8987.
化合物式(23)的制备：将化合物式(6)(121mg,0.199mmol)和化合物式(22)(238mg,0.133mmol)溶解在无水CH2Cl2(6mL)中，并加入
Figure PCTCN2016077370-appb-000046
(650mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(46mg,0.205mmol)。搅拌10min后，加入TMSOTf(6μL，0.033mmol)。在-20℃下搅拌约1h，逐渐升至0℃，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭。硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:15:1～1:3:1)得到化合物式(23)(218mg,72％)。1H NMR(400MHz,CDCl3)δ7.38–7.22(m,35H),5.43(dd,J＝3.0,1.9Hz,1H),5.28–5.22(m,2H),5.20–5.14(m,2H),5.12(d,J＝1.4Hz,1H),5.11(d,J＝1.6Hz,1H),5.08(d,J＝0.8Hz,1H),4.93(d,J＝1.6Hz,1H),4.90(d,J＝1.8Hz,1H),4.88–4.79(m,3H),4.74–4.66(m,3H),4.66–4.58(m,3H),4.56–4.47(m,3H),4.45–4.35(m,3H),4.21–4.10(m,5H),4.09–4.00(m,4H),3.97–3.91(m,2H),3.90–3.71(m,8H),3.66–3.59(m,2H),3.60–3.51(m,3H),2.73–2.57(m,2H),2.57–2.49(m,2H),2.11(s,3H),2.09(s,3H),2.08(s,3H),2.01(s,3H),1.98(s,3H),1.96(s,3H),1.92(s,3H),1.13–1.02(m,42H),1.00–0.90(m,21H).HRMS(ESI-TOF)m/z Calcd for C123H172O34NaSi3[M+Na]+2300.0936,found 2300.0989.
实施例5 式(I)化合物的制备
Figure PCTCN2016077370-appb-000047
化合物式(24)的制备：0℃，将化合物式(23)(107mg,0.051mmol)溶解在CH2Cl2(5mL)和MeOH(0.4mL)中，加入NH2-NH2·HOAc(1M in MeOH，0.33mL，0.33mmol)，并搅拌10min。升至室温，继续搅拌10h，TLC显示反应完全，用丙酮淬灭，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:6:1～1:3:1)得到化合物式(24)(92mg,90％)。1H NMR(400MHz,CDCl3)δ7.44–7.18(m,35H),5.43(dd,J＝3.1,1.9Hz,1H),5.25(dd,J＝2.8,1.7Hz,1H),5.24–5.21(m,1H),5.20–5.12(m,2H),5.09–5.07(m,2H),5.06(d,J＝1.6Hz,1H),4.94(d,J＝1.6Hz,1H),4.91(d,J＝1.6Hz,1H),4.88–4.79(m,3H),4.74–4.65(m,3H),4.65–4.56(m,4H),4.55–4.48(m,2H),4.43(d,J＝11.2Hz,1H),4.40(d,J＝11.9Hz,1H),4.21–4.09(m,5H),4.08–3.99(m,3H),3.97–3.73(m,9H),3.71–3.52(m,8H),2.08(s,3H),2.07(s,3H),2.02(s,3H),1.99(s,3H),1.96(s,3H),1.92(s,3H),1.11–1.01(m,42H),0.99–0.90(m,21H).HRMS(ESI-TOF)m/z  Calcd for C118H166O32NaSi3[M+Na]+2202.0568,found 2202.0613.
化合物式(25)的制备：将式(24)(88mg,0.0407mmol)溶解在无水CH2Cl2(4mL)中，并加入1H-四氮唑(15mg，0.203mmol)和
Figure PCTCN2016077370-appb-000048
(400mg)，在室温下搅拌30min。加入(BnO)2PN(iPr)2(54μL，0.163mmol)，搅拌2h。待TLC显示反应完全后，将温度降至-20℃。加入mCPBA(36mg,77％，0.163mmol)，搅拌1h。反应用三乙胺淬灭。加入NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)，用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:3:1)得到式(25)(99mg,100％，2步)。1H NMR(400MHz,CDCl3)δ7.39–7.22(m,45H),5.41(s,1H),5.29–5.26(m,1H),5.26(dd,J＝2.8,1.6Hz,1H),5.20–5.13(m,2H),5.12(d,J＝1.3Hz,1H),5.10–5.05(m,2H),5.03–4.89(m,6H),4.86–4.78(m,3H),4.72(d,J＝10.6Hz,1H),4.69–4.65(m,2H),4.65–4.57(m,4H),4.55–4.46(m,2H),4.40(2 overlapping d,J＝11.1Hz,2H),4.32(ddd,J＝10.8,5.8,1.7Hz,1H),4.22–4.01(m,9H),3.96–3.81(m,7H),3.81–3.73(m,3H),3.63(d,J＝10.1Hz,2H),3.60–3.50(m,3H),2.08(s,3H),2.00(s,3H),1.96(s,3H),1.95(s,3H),1.93(s,3H),1.92(s,3H),1.12–1.01(m,42H),0.99–0.92(m,21H).31P NMR(162MHz,CDCl3)δ-0.93.HRMS(ESI-TOF)m/z Calcd for C132H179O35NaPSi3[M+Na]+2462.1170,found 2462.1118.
化合物式(26)的制备：将化合物式(25)(99mg，0.041mmol)溶于MeOH(5mL)中，室温下加入MeONa(11.6mg，0.205mmol)，搅拌12h。反应完成后，用Dowex50WX8酸性树脂中和、过滤和浓缩后得化合物式(26)，未经纯化直接用于下一步。HRMS(ESI-TOF)m/z Calcd for C120H167O29NaPSi3[M+Na]+2210.0536,found 2210.0562.
化合物式(27)的制备：将上述所得化合物式(26)(0.041mmol)溶解在THF(3mL)中，室温下加入TBAF(1M in THF,0.24mL,0.24mmol),AcOH(2.4mmol，14μL)，继续搅拌20h，HR-MS监测反应完成。蒸干溶剂。硅胶柱层析纯化(EA/PE/MeOH＝1:10:0～1:0:0.03)得到化合物式(27)(70mg，100％)。1H NMR(400MHz,CDCl3)δ7.35–7.12(m,45H),5.15–5.11(m,2H),5.09(s,1H),5.03(s,1H),4.92–4.84(m,4H),4.83–4.77(m,2H),4.71(d,J＝10.9Hz,1H),4.67–4.34(m,12H),4.24–4.01(m,9H),4.00–3.84(m,7H),3.82–3.73(m,3H),3.71–3.51(m,10H),3.46(d,J＝9.6Hz,1H),3.29(brs,1H),2.70(brs,5H).31P NMR(162MHz,CDCl3)δ-1.35.HRMS(ESI-TOF)m/z Calcd for C93H107O29NaP[M+Na]+1741.6533,found 1741.6511。
式(I)化合物的制备：将化合物式(27)(30.4mg，0.0177mmol)溶解在混合溶剂MeOH/H2O(v/v＝4/1，5mL)中，室温下加入10％Pd/C(43mg)，并在H2气氛下搅拌20h。HR-MS显示反应完成，过滤、浓缩，粗产品经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到式(I)化合物(16.1mg，100％)。1H NMR(400MHz,D2O)δ5.38(s,1H),5.14–5.10(m,2H),5.10(s,1H),5.04(s,1H),4.33(s,1H),4.25–4.20(m,3H),4.09(s,1H),4.05–3.64(m,25H).13C-APT  NMR(100MHz,D2O,300K,HSQC)δ102.78,102.48,102.02(2C),92.52,79.24,79.05,78.34,77.90,73.56,73.48,73.35,72.51,72.42(d,J＝6.6Hz),70.27,70.02,69.98,69.79,69.57,69.47,67.04,66.67,66.18,65.92(2C),64.41,61.10,60.97(2C),60.94(2C).31P NMR(162MHz,D2O,pH 7.0,300K)δ1.31.HRMS(ESI-TOF)m/z Calcd for C30H52O29P[M-H]-907.2332,found 907.2347.
实施例6 式(II)化合物的制备
Figure PCTCN2016077370-appb-000049
将化合物式(27)(23mg，0.0135mmol)溶解在混合溶剂MeOH/H2O(v/v＝4/1，5mL)中，加入20％Pd(OH)2/C(250mg)，并在H2气氛(2atm)下搅拌36h。HR-MS显示反应完全，过滤，浓缩，粗产品经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到式(II)化合物(12.3mg，100％)。1H NMR(400MHz,D2O)δ5.14(d,J＝1.1Hz,1H),5.10(d,J＝1.5Hz,1H),5.08(d,J＝1.1Hz,1H),4.98(d,J＝1.3Hz,1H),4.38(dd,J＝3.0,1.8Hz,1H),4.21(dd,J＝3.0,1.8Hz,1H),4.14(dd,J＝2.9,1.9Hz,1H),4.13–4.09(m,1H),4.08(dd,J＝3.4,1.5Hz,1H),4.03(dd,J＝8.7,0.8Hz,1H),4.01–3.70(m,24H),3.70–3.64(m,2H).13C-APT NMR(100MHz,D2O,300K,HSQC)δ102.85,102.57,102.18,101.36,79.50,79.40,78.26,78.16,73.72,73.44,73.36,72.73(d,J＝6.2Hz),70.88,70.25,70.11,69.97,69.79,69.32,69.25,67.26,66.82,66.04,65.86,65.84,63.36(d,J＝3.0Hz),63.17,61.12(2C),60.99,60.82.HRMS(ESI-TOF)m/z Calcd for C30H54O29P[M-H]-909.2488,found 909.2443.
实施例7 式(II)化合物的制备
Figure PCTCN2016077370-appb-000050
将实施例5制备的如式(I)所示的化合物(23mg，0.0253mmol)溶解在混合溶剂MeOH/H2O(v/v＝1/1，5mL)中，加入NaBH4(10mg)，并室温下搅拌12h。HR-MS显示反应完全，过滤，浓缩，粗产品经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到如式(II)所示的化合物(23mg，100％)。
实施例8 式(III)化合物的制备
Figure PCTCN2016077370-appb-000051
将实施例5制备的如式(I)所示的化合物(16.1mg,0.0177mmol)和SO3·Py(216mg,1.357mmol)溶解在DMF(1.5mL)中，在60℃下搅拌6h。随后冷至室温，继续搅拌2d。将上清液吸去，残余物用纯水溶解，用1M NaOH(aq.)调节至pH 9～10，浓缩至干。粗产物经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到硫酸化衍生物式(III)化合物(37.5mg，82％)。1H NMR(400MHz,D2O,300K,HSQC)δ5.78(s,1H),5.53(s,1H),5.47(s,1H),5.46–5.38(m,2H),5.20(s,1H),4.99(s,1H),4.86–4.79(m,2H),4.79–4.73(m,2H),4.69–4.45(m,8H),4.43–4.15(m,14H),4.11(d,J＝9.8Hz,1H).13C NMR and DEPT135(100MHz,D2O, 300K,HSQC)δ100.34,99.94,99.80,98.26(missing),96.11,76.94,76.43,76.28,76.14,75.98,75.36,75.09,74.82(missing),74.52(missing),74.06,72.65,72.35,72.12,71.70,71.55,71.31,70.19(d,J＝8.8Hz),69.59,69.25,69.15,66.86,66.45,66.01,65.87,62.88.31P NMR(162MHz,D2O,pH 7.0,300K)δ0.68.HRMS(ESI-TOF)m/z Calcd for C30H36O77Na11PS16[M+H+11Na]6-403.8841,found 403.2216；Calcd for C30H38O71Na10PS14[M+3H–2SO3+11Na]5-448.4833,found 448.2916.
实施例9 式(IV)化合物的制备
Figure PCTCN2016077370-appb-000052
将实施例6或7制备的如式(II)所示的化合物(12.3mg,0.0135mmol)和SO3·Me3N络合物(150mg,1.08mmol)溶解在DMF(1.5mL)中。50℃搅拌3d。冷至室温，加入Na2CO3(1.08mmol)和水淬灭，再搅拌1h，浓缩。残余物用水溶解，经Dowex50WX8(Na+)树脂柱交换盐型，浓缩。经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到式(IV)化合物(24.3mg，67％)。1H NMR(400MHz,D2O,300K,HSQC)δ5.51(s,1H),5.50–5.47(m,2H),5.46(s,1H),5.27(s,1H),5.12(dd,J＝2.0,2.0,1H),5.06(dd,J＝2.0,2.0,1H),5.04(dd,J＝2.0,2.0,1H),4.99(ddd,J＝8.0,2.0,2.0,1H),4.95(dd,J＝2.0,2.0,1H),4.90–4.84(m,2H),4.83–4.66(m,4H),4.62–4.54(m,2H),4.51–4.21(m,18H).DEPT135(100MHz,D2O,300K,HSQC)δ100.07,99.85,99.78,95.69,78.65,78.26(2C),76.76,76.38,76.32,76.13,75.95(2C),75.31,75.09,74.03,72.64,72.38,72.30,71.65,70.15(d,J＝8.0Hz),69.23,69.09,69.00,67.47,66.87,66.27,65.96,65.87,62.93.HRMS(ESI-TOF)m/z Calcd for C30H37O80Na12PS17[M+H+12Na]6-421.2098,found 421.2110；Calcd for C30H37O77Na14PS16[M+H–SO3+14Na]4-623.3204,found 623.0717；Calcd for C30H38O74Na13PS15[M+2H–2SO3+13Na]4-597.8357,found 598.9766。
实施例10 化合物式(29)的制备
Figure PCTCN2016077370-appb-000053
化合物式(28)的制备：将化合物式(6)(325mg,0.54mmol)溶解在湿DCM(7mL)中，0℃下加入TTBP(266mg,1.07mmol)。搅拌30min后，加入NIS(241mg,1.07mmol)和AgOTf(27.5mg,0.11mmol)并继续在0℃搅拌1h，TLC监测反应完全。依次用NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，并用DCM萃取。有机相用饱和食盐水洗涤、Na2SO4干燥，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:4:1～1:1:1)得到化合物式(28)α和(28)β的混合物(190mg,6/1,71％)。Data for(28)α:1H NMR(400MHz,CDCl3)δ7.38–7.28(m,10H),5.40(dd,J＝3.3,1.8Hz,1H),5.20(dd,J＝2.6,2.0Hz,1H),4.91 and 4.57(ABq,JAB＝10.9Hz,2H,CH2Ar),4.71 and 4.54(ABq,JAB＝11.0Hz,2H,CH2Ar),4.43(dd,J＝11.6,2.1Hz,1H),4.25(dd,J＝11.7,6.4Hz,1H),4.10(ddd,J＝9.8,6.4,2.0Hz,1H),4.06(dd,J＝9.3,3.4Hz,1H),3.69(dd,J＝9.6,9.6Hz,1H),3.43(d,J＝3.3Hz,1H),2.79–2.74(m,2H),2.63–2.55(m,2H),2.18(s,3H),2.15(s,3H).HRMS(ESI-TOF)m/z Calcd for C27H32O9Na[M+Na]+523.1944,found 523.1945.
化合物式(29)的制备：将化合物式(28)(181mg,0.54mmol)溶解在DCM(5mL)中，室温下加入CCl3CN(109μL,3.0eq)和K2CO3(100mg,2.0eq)。TLC监测反应完全后(约20h)，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:5:1～1:3:1)得到化合物式(29)(181mg,78％)。1H NMR(400MHz,CDCl3)δ8.70(s,1H),7.38–7.27(m,10H),6.24(d,J＝1.9Hz,1H),5.49(dd,J＝3.2,2.1Hz,1H),4.92 and 4.59(ABq,JAB＝10.6Hz,2H,CH2Ar),4.75 and 4.59(ABq,JAB＝11.2Hz,2H,CH2Ar),4.40–4.29(m,2H),4.04(dd,J＝9.4,3.2Hz,1H),4.01(dd,J＝4.1,2.5Hz,1H),3.85(dd,J＝9.7,9.7Hz,1H),2.79–2.71(m,2H),2.62–2.56(m,2H),2.21(s,3H),2.18(s,3H).HRMS(ESI-TOF)m/z Calcd for C29H32Cl3NO9Na[M+Na]+666.1040,found 666.1014.
实施例11 化合物式(32)的制备。
Figure PCTCN2016077370-appb-000054
化合物式(30)的制备：将化合物7(2.556g,5.168mmol)和TBAI(191mg,0.517mmol)溶于THF(30mL)，0℃下，分批加入NaH(60％,270mg,6.718mmol)。30min后,加入BnBr(0.92mL,7.752mmol)。升至室温，继续搅拌12h。反应完成后，加入水淬灭，用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝ 1:40:1～1:10:1)得到化合物式(30)(2.23g,74％)。1H NMR(400MHz,CDCl3)δ7.54–7.49(m,2H),7.41–7.24(m,12H),7.10(d,J＝7.9Hz,2H),6.89–6.83(m,2H),5.64(s,1H),5.42(d,J＝1.3Hz,1H),4.73(d,J＝11.8Hz,1H),4.70(s,2H),4.58(d,J＝11.8Hz,1H),4.33–4.24(m,2H),4.24–4.18(m,1H),3.99(dd,J＝3.2,1.4Hz,1H),3.97–3.93(m,1H),3.87(m,1H),3.81(s,3H),2.33(s,3H).
化合物式(31)的制备：将化合物式(30)(2.23g,3.81mmol)溶于MeOH(25mL)，加入TsOH·H2O(73mg,0.381mmol)。室温搅拌12h，用三乙胺(5mL)淬灭，浓缩。所得粗产物溶于DMF(15mL)，室温下加入咪唑(600mg,8.77mmol)。冷却至0℃，加入TIPSCl(0.9mL,4.20mmol)，然后逐渐升至室温，搅拌5h。加入水淬灭反应，再用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:10:1)得到化合物式(31)(2.47g,99％,2步)。HRMS(ESI-TOF):m/z Calcd for C37H52O6NaSSi[M+Na]+675.3152,found 675.3162.
化合物式(32)的制备：将化合物式(31)(2.47g,3.77mmol)和TBAI(141mg,0.382mmol)溶于THF(30mL)，0℃下，分批加入NaH(60％,198mg,4.95mmol)。30min后,加入BnBr(0.91mL,7.66mmol)。升至室温，继续搅拌12h。反应完成后，加入水淬灭，用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:50:1～1:30:1)得到化合物式(32)(2.73g,96％)。1H NMR(400MHz,CDCl3)δ7.36–7.26(m,14H),7.06(d,J＝8.0Hz,2H),6.88–6.83(m,2H),5.48(d,J＝1.5Hz,1H),4.92(A of ABq,JAB＝10.9Hz,1H),4.69–4.59(m,3H,benzylic),4.56(s,2H,CH2Ar),4.09(ddd,J＝9.7,4.4,1.8Hz,1H),4.03–3.90(m,4H),3.84(dd,J＝9.2,3.1Hz,1H),3.81(s,3H),2.31(s,3H),1.09–1.00(m,21H).HRMS(ESI-TOF):m/z Calcd for C44H58O6NaSiS[M+Na]+765.3621,found 765.3643.
实施例12 化合物式(36)的制备。
Figure PCTCN2016077370-appb-000055
化合物式(34)的制备：将-D-甘露糖甲苷(500mg，2.57mmol)悬浮于DCM(5mL)中，再加入HMDS(914mg,2.2eq)。搅拌下加入TMSOTf(45μL)，TLC监测反应完全后，浓缩，加入正己烷溶解，依次用水、饱和食盐水洗涤，MgSO4干燥，浓缩，得到化合物式(34)(1.185g,99％).1H NMR(400MHz,CDCl3)δ4.46(d,J＝1.8Hz,1H),3.83–3.78(m,1H),3.78–3.76(m,1H),3.76–3.73(m,1H),3.73–3.67(m,2H),3.46–3.39(m,1H),3.31(s,3H),0.15(s,9H),0.13(s,9H),0.11(s,9H),0.11(s,9H).
化合物式(35)的制备：将化合物式(34)(1.185g,2.45mmol)溶于DCM中，加入苯 甲醛(0.28mL，2.52mmol)，溶液冷却至-78℃,加入TMSOTf(44μL)。TLC监测反应完全后，加入TBAF(7.4mL,1M in THF),逐渐升至室温。反应完成后，用NaHCO3(sat.aq.)淬灭。反应液用乙酸乙酯萃取。合并有机相，用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～2:1:2)得到单苄叉保护的产物35(440mg,64％)：1H NMR(400MHz,CDCl3)δ7.52–7.45(m,2H),7.41–7.34(m,3H),5.56(s,1H),4.73(d,J＝1.3Hz,1H),4.35–4.20(m,1H),4.04(dd,J＝9.4,3.5Hz,1H),4.00(dd,J＝3.5,1.3Hz,1H),3.94–3.87(m,1H),3.86–3.76(m,2H),3.39(s,3H),2.88(s,1H),2.84(s,1H).HRMS(ESI-TOF)m/z Calcd for C14H18O6Na[M+Na]+305.1001,Found 305.1014.
化合物式(36)的制备：将化合物式(35)(708mg,2.51mmol)和Bu2SnO(687mg,1.1eq)悬浮于甲苯(30mL)中，加热至140℃，搅拌4小时，不断蒸出甲苯和水。冷至60℃，依次加入10mL DMF、BnBr(0.45mL,1.5eq)、CsF(762mg,2.0eq)和TBAI(185mg,0.2eq)。在70℃下搅拌12h。加水终止反应，用EtOAc稀释。有机相用饱和食盐水洗涤,MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:3:1)得到化合物式(36)(897mg,96％，2步)。1H NMR(400MHz,CDCl3)δ7.52–7.45(m,2H),7.42–7.26(m,8H),5.61(s,1H),4.85(d,J＝11.9Hz,1H),4.76(d,J＝1.3Hz,1H),4.71(d,J＝11.9Hz,1H),4.31–4.25(m,1H),4.09(dd,J＝9.3,9.3Hz,1H),4.05(dd,J＝3.0,1.3Hz,1H),3.90(dd,J＝8.9,2.8Hz,1H),3.86(dd,J＝9.0,9.0Hz,1H),3.81(ddd,J＝12.6,7.4,2.7Hz,1H),3.37(s,3H),2.68(d,J＝1.3Hz,1H).HRMS(ESI-TOF)m/z Calcd forC21H24O6Na[M+Na]+395.1471,found 395.1482.
实施例13 化合物式(43)的制备。
Figure PCTCN2016077370-appb-000056
化合物式(37)的制备：将化合物式(32)(188mg,0.253mmol)和化合物36(72mg,0.194mmol)溶解在无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000057
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(53mg,0.233mmol)。搅拌10min后，加入TMSOTf(4μL，0.1eq)。在-20℃下搅拌约30min，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:70:1～1:5:1)得到化合物式(37)(110mg,57％)：1H NMR(400MHz,CDCl3)δ7.52(dd,J＝7.7,1.8Hz,2H),7.42–7.24(m,20H),6.89–6.81(m,2H),5.61(s,1H),5.25(d,J＝1.5Hz,1H),4.91(d,J＝10.8Hz,1H),4.76(d,J＝11.7Hz,1H),4.68(d,J＝1.5Hz,1H),4.66–4.54(m,4H),4.47(s,2H),4.22(dd,J＝9.2,3.7Hz,1H),4.14–4.08(m,1H),3.99–3.82(m,6H),3.82–3.64(m,7H),3.30(s,3H),1.15–0.99(m,21H).HRMS(ESI-TOF)m/z  Calcd for C58H74O12NaSi[M+Na]+1013.4847,found 1013.4866.
化合物式(38)的制备：0℃下，化合物37(92mg,0.092mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(27mg,0.120mmol)。0℃搅拌30min，升至室温，继续搅拌3h(TLC检测)。NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:4:1)得到化合物式(38)(69mg,86％)。1H NMR(400MHz,CDCl3)δ7.54–7.48(m,2H),7.41–7.16(m,18H),5.64(s,1H),5.34(d,J＝1.2Hz,1H),4.91(d,J＝11.0Hz,1H),4.83(d,J＝11.4Hz,1H),4.67(d,J＝1.4Hz,1H),4.63(d,J＝11.4Hz,2H),4.62(d,J＝11.0Hz,1H),4.40(d,J＝11.8Hz,1H),4.28–4.18(m,2H),4.16(dd,J＝3.0,1.6Hz,1H),4.12–4.01(m,2H),4.00–3.94(m,2H),3.91(dd,J＝10.9,4.9Hz,1H),3.86–3.79(m,2H),3.78–3.71(m,1H),3.70–3.58(m,2H),3.32(s,3H),2.34(d,J＝9.8Hz,1H,OH),1.18–1.00(m,21H).HRMS(ESI-TOF)m/z Calcd for C50H66O11NaSi[M+Na]+961.4382,found 961.4388.
化合物式(39)的制备：将给体式(32)(71mg,0.095mmol)和二糖受体式(38)(64mg,0.073mmol)溶解在无水CH2Cl2(5mL)中，加入
Figure PCTCN2016077370-appb-000058
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(20mg,0.088mmol)。搅拌10min后，加入TMSOTf(2μL，0.1eq)。在-20℃下搅拌约1h，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:7:1)得到-构型三糖式(39)(94mg,85％):1H NMR(400MHz,CDCl3)δ7.53–7.47(m,2H),7.39–7.16(m,30H),6.78–6.71(m,2H),5.61(s,1H),5.34(d,J＝1.5Hz,1H),5.18(s,1H),4.92(d,J＝11.1Hz,1H),4.80(d,J＝11.7Hz,1H),4.71–4.65(m,2H),4.64–4.49(m,6H),4.46–4.36(m,3H),4.25(dd,J＝9.8,4.4Hz,1H),4.20–4.13(m,2H),4.10(dd,J＝9.3,3.1Hz,1H),4.03(dd,J＝9.8,4.4Hz,1H),3.98–3.91(m,3H),3.87–3.70(m,9H),3.69(s,3H),3.66–3.60(m,1H),3.32(s,3H),1.09–0.96(m,42H).HRMS(ESI-TOF)m/z Calcd for C87H116O17NaSi2[M+Na]+1511.7649,found 1511.7619.
化合物式(40)的制备：0℃下，化合物式(39)(81mg,0.054mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(35mg,0.159mmol)。0℃搅拌1h，升至室温，继续搅拌3h(TLC检测)。NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:5:1)得到化合物式(40)(54mg,73％)。1H NMR(400MHz,CDCl3)δ7.50(dd,J＝7.6,1.9Hz,2H),7.41–7.35(m,3H),7.34–7.16(m,25H),5.60(s,1H),5.34(d,J＝1.2Hz,1H),5.24(s,1H),4.87(d,J＝11.2Hz,1H),4.80(d,J＝11.7Hz,1H),4.76(d,J＝11.5Hz,1H),4.71(d,J＝1.1Hz,1H),4.67(d,J＝11.3Hz,2H),4.63(d,J＝11.7Hz,1H),4.46–4.34(m,3H),4.29–4.20(m,2H),4.18(dd,J＝2.9,1.5Hz,1H),4.13(dd,J＝9.4,3.1Hz,1H),4.09–3.98(m,2H),3.98– 3.84(m,6H),3.84–3.71(m,5H),3.69–3.62(m,2H),3.33(s,3H),2.31(d,J＝9.3Hz,1H),1.12–0.95(m,42H).HRMS(ESI-TOF)m/z Calcd for C79H108O16NaSi2[M+Na]+1391.7074,found 1391.7096.
化合物式(41)的制备：将化合物式(32)(38mg,0.051mmol)和化合物式(40)(116mg,0.039mmol)溶解在无水CH2Cl2(5mL)中，加入
Figure PCTCN2016077370-appb-000059
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(12mg,0.051mmol)。搅拌10min后，加入TMSOTf(1μL，0.1eq)。在-20℃下搅拌约1h，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:14:1)得到化合物式(41)(52mg,68％)。1H NMR(400MHz,CDCl3)δ7.53–7.46(m,2H),7.37–7.13(m,40H),6.79–6.70(m,2H),5.59(s,1H),5.36(d,J＝1.2Hz,1H),5.24(s,1H),5.17(s,1H),4.89(d,J＝11.1Hz,1H),4.80(d,J＝11.6Hz,1H),4.75–4.58(m,6H),4.58–4.42(m,5H),4.39–4.20(m,5H),4.20–4.07(m,5H),4.05–3.73(m,13H),3.71(s,3H),3.68–3.58(m,5H),3.31(s,3H),1.11–0.86(m,63H).HRMS(ESI-TOF)m/z Calcd for C116H158O22NaSi3[M+Na]+2010.0450,found 2010.0350.
化合物式(42)的制备：0℃下，化合物式(41)(52mg,0.026mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(8mg,0.034mmol)。0℃搅拌1h，升至室温，继续搅拌2h(TLC检测)。用NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:50:1～1:10:1)得到化合物式(42)(37mg,77％)。1H NMR(400MHz,CDCl3)δ7.52–7.47(m,2H),7.39–7.17(m,35H),7.15–7.08(m,3H),5.60(s,1H),5.37(s,1H),5.24(s,1H),5.22(s,1H),4.85(d,J＝11.5Hz,1H),4.82(d,J＝12.8Hz,1H),4.76(s,2H),4.70(d,J＝10.7Hz,1H),4.68(s,1H),4.65(d,J＝11.2Hz,2H),4.63(d,J＝11.6Hz,1H),4.55(d,J＝11.0Hz,1H),4.52–4.48(m,2H),4.45(d,J＝12.4Hz,1H),4.31(d,J＝12.1Hz,1H),4.28–4.15(m,5H),4.12(dd,J＝9.5,3.0Hz,1H),4.07–3.98(m,3H),3.97–3.73(m,12H),3.68(d,J＝8.6Hz,1H),3.66–3.57(m,4H),3.32(s,3H),1.12–0.81(m,63H).HRMS(ESI-TOF)m/z Calcd for C108H150O21NaSi3[M+Na]+1889.9875,found 1889.9867.
化合物式(43)的制备：将化合物式(29)(29mg,0.045mmol)和化合物式(42)(33mg,0.018mmol)溶解在无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000060
(500mg)，在室温下搅拌1h。溶液冷至-20℃，搅拌10min后，加入TMSOTf(0.3μL，0.1eq)。在-20℃下搅拌约30min，逐渐升至0℃，TLC显示反应完全。用Et3N中和，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:30:1～1:4:1)得到化合物式(43)(25mg,63％)。1H NMR(400MHz,CDCl3)δ7.53–7.47(m,2H),7.42–7.07(m,48H),5.60(s,1H),5.47–5.44(m,1H),5.38(s,1H),5.24(s,1H),5.23(s,1H),5.15(s,1H),4.84(d,J＝10.5Hz,1H),4.81(d,J＝11.2Hz,1H),4.75–4.29(m,15H),4.29–4.16 (m,6H),4.15–4.00(m,5H),3.99–3.59(m,18H),3.59–3.52(m,2H),3.31(s,3H),2.61–2.55(m,2H),2.54–2.47(m,2H),2.10(s,3H),2.09(s,3H),1.13–0.81(m,63H).HRMS(ESI-TOF)m/z Calcd for C135H180O29NaSi3[M+Na]+2372.1816,found 2372.1731.
实施例14 式(VI)化合物的制备。
Figure PCTCN2016077370-appb-000061
化合物式(44)的制备：将化合物式(43)(23.5mg，0.010mmol)溶于MeOH(3mL)中，室温下加入MeONa(5.4mg，0.010mmol)，搅拌24h。TLC及HR-MS显示反应完成后，用Dowex50WX8酸性树脂中和，过滤，浓缩，粗产品未经纯化直接用于下一步。将粗产物溶于THF(3mL)，加入TBAF(1M in THF，0.15mL)，50℃下搅拌36h。HR-MS显示反应完成，浓缩。硅胶柱层析纯化(EA/DCM＝1:3:1～1:1:1)得到化合物式(44)(12.7mg，72％)。1H NMR(400MHz,CDCl3)δ7.50(dd,J＝7.6,1.9Hz,2H),7.40–7.13(m,48H),5.66(s,1H),5.26(s,1H),5.24(s,1H),5.18(s,1H),5.13(s,1H),4.84(d,J＝11.6Hz,1H),4.83(d,J＝11.1Hz,1H),4.73–4.52(m,11H),4.47–4.26(m,6H),4.25–4.16(m,2H),4.13–4.03(m,4H),3.98–3.82(m,7H),3.82–3.52(m,17H),3.34(s,3H).HRMS(ESI-TOF)m/z Calcd for C101H112O26Na[M+Na]+1763.7340,found 1763.7283.
式(VI)化合物的制备：将化合物式(44)(12.7mg,0.0073mmol)和SO3·Me3N络合物(25.4mg,0.182mmol)溶解在DMF(1mL)中，加热至50℃，搅拌3d。冷却至0℃，加入Et3N和H2O淬灭，浓缩。残余物用水溶解，经Dowex50WX8(Na+)柱交换盐型，浓缩得 式(45)粗产品，不经纯化直接用于下一步。将上一步所得的式(45)粗产物溶于混合溶剂MeOH/H2O(v/v＝2/1,3mL)，加入20％Pd(OH)2/C(70mg)，并将体系内置换成H2氛围，搅拌3d。用硅藻土过滤，浓缩，再经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到式(VI)化合物(6.7mg，68％，2步)。1H NMR(400MHz,D2O,300K)δ5.39(d,J＝1.2Hz,1H),5.07(d,J＝1.7Hz,1H),5.06(d,J＝1.4Hz,1H),4.97(d,J＝1.2Hz,1H),4.95(d,J＝1.3Hz,1H),4.63(dd,J＝3.2,1.6Hz,1H),4.39–4.27(m,4H),4.26–4.08(m,7H),4.07–3.92(m,7H),3.92–3.81(m,3H),3.81–3.61(m,7H),3.58–3.50(m,1H),3.38(s,1H).13C NMR(100MHz,D2O,300K,HSQC)δ102.56,102.54,102.34,100.01,99.27,78.96,78.85(2C),78.82,76.76,72.65,71.39,71.35(3C),70.33,69.60,69.54,69.47,68.84,67.80,67.74,67.39(2C),67.01,66.47,65.84,65.72,65.60,61.09,54.96.HRMS(ESI-TOF)m/z Calcd for C31H49O41Na3S5[M+3Na]2-653.0023,found 652.7643；Calcd for C31H49O41Na2S5[M+2Na]3-427.6716,found 427.5134.
实施例15 化合物式(48)的制备。
Figure PCTCN2016077370-appb-000062
化合物式(46)的制备：将化合物式(36)溶于DMF(897mg,2.41mmol)，0℃下分批加入NaH(60％,193mg,4.8mmol)，再搅拌5min,加入PMBCl(0.65mL,4.8mmol)。升至室温，继续搅拌12h。0℃，加入水淬灭反应，然后用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:10:1)得到46(703mg，59％)。1H NMR(400MHz,CDCl3)δ7.53–7.46(m,2H),7.40–7.25(m,10H),6.90–6.83(m,2H),5.63(s,1H),4.80(d,J＝12.3Hz,1H),4.75(d,J＝11.9Hz,1H),4.67(d,J＝12.1Hz,1H),4.65(s,1H),4.63(d,J＝10.2Hz,1H),4.28–4.19(m,2H),3.93(dd,J＝10.0,3.3Hz,1H),3.88(t,J＝10.3Hz,1H),3.83–3.80(m,4H),3.79–3.72(m,1H),3.31(s,3H).HRMS(ESI-TOF)m/z Calcd for C29H32O7Na[M+Na]+515.2046,found 515.2037.
化合物式(47)的制备：将化合物式(46)(692mg,1.41mmol)溶解在甲醇(10mL)中，并加入对甲苯磺酸单水合物(27mg,0.1eq)。室温搅拌12h,用三乙胺(5mL)淬灭，浓缩，得粗产品。未经纯化直接用于下一步。所得粗产物溶于DCM(30mL)中，依次加入DMAP(17mg,0.1eq)和乙酸酐(1.33mL,10eq)，室温下搅拌过夜。NaHCO3(sat.aq.)淬灭，水相用DCM萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，蒸干。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:5:1)得到化合物式(47)(658mg,96％,2步)。1H NMR(400MHz,CDCl3)δ7.37–7.24(m,7H),6.86–6.78(m,2H),5.43–5.35(m,1H),4.71(d,J＝1.5Hz,1H),4.68(d,J＝12.1Hz,1H),4.62(d,J＝12.1Hz,1H),4.55(d,J＝12.2Hz,1H),4.44(d,J＝12.2Hz, 1H),4.22(dd,J＝12.1,5.7Hz,1H),4.12(dd,J＝12.1,2.5Hz,1H),3.85–3.72(m,6H),3.33(s,3H),2.08(s,3H),2.00(s,3H).HRMS(ESI-TOF)m/z Calcd for C26H32O9Na[M+Na]+511.1944,found 511.1919.
化合物式(48)的制备：0℃下，化合物式(47)(642mg,1.32mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,10mL)的溶液中，并加入DDQ(358mg,1.2eq)。0℃搅拌1h，升至室温，继续搅拌3h。用NaHCO3饱和溶液淬灭，CH2Cl2萃取。有机相依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:2:1)得到化合物式(48)(437mg,90％)。1H NMR(400MHz,CDCl3)δ7.38–7.27(m,5H),5.26(t,J＝9.8Hz,1H),4.81(d,J＝1.6Hz,1H),4.66(d,J＝12.0Hz,1H),4.53(d,J＝12.0Hz,1H),4.23(dd,J＝12.2,5.3Hz,1H),4.10(dd,J＝12.2,2.4Hz,1H),4.03(ddd,J＝3.4,3.4,1.8Hz,1H),3.83(ddd,J＝10.1,5.3,2.4Hz,1H),3.77(dd,J＝9.4,3.4Hz,1H),3.38(s,3H),2.56(d,J＝1.8Hz,1H),2.08(s,3H),1.99(s,3H).HRMS(ESI-TOF)m/z Calcd for C18H24O8Na[M+Na]+391.1369,found 391.1379.
实施例16 化合物式(55)的制备
Figure PCTCN2016077370-appb-000063
化合物式(49)的制备：将化合物式(10)(235mg,0.338mmol)和化合物式(48)(104mg,0.282mmol)溶于无水CH2Cl2(5mL)中，加入
Figure PCTCN2016077370-appb-000064
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(76mg,0.338mmol)。搅拌10min后，加入TMSOTf(5μL,0.1eq)。在-20℃下搅拌约1h，TLC显示反应完全。用NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(49)(181mg,80％)。1H NMR(400MHz,CDCl3)δ7.36–7.26(m,12H),6.89–6.81(m,2H),5.45(dd,J＝3.1,2.0Hz,1H),5.26(dd,J＝9.9,9.9Hz,1H),5.01(d,J＝1.8Hz,1H),4.87(d,J＝10.8Hz,1H),4.76(d,J＝1.7Hz,1H),4.68(d,J＝11.0Hz,1H),4.65(d,J＝12.5Hz,1H),4.57(d,J＝10.8Hz,1H),4.52(d,J＝12.3Hz,1H),4.47(d,J＝10.8Hz,1H),4.16–4.06(m,2H),4.04–3.98(m,2H),3.94(dd,J＝11.1,4.3Hz,1H),3.89(dd,J＝11.0,1.6Hz,1H),3.82(ddd,J＝9.5,6.3,3.2Hz,2H),3.77(s,3H), 3.76–3.68(m,2H),3.31(s,3H),2.06(s,3H),1.99(s,3H),1.97(s,3H),1.13–1.00(m,21H).HRMS(ESI-TOF)m/z Calcd for C50H70O15NaSi[M+Na]+961.4382,found 961.4388.
化合物式(50)的制备：0℃下，将化合物式(49)(205mg,0.219mmol)溶于混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(60mg,0.262mmol)。0℃搅拌1h，升至室温，继续搅拌3h(TLC检测)。反应用NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:2:1)得到化合物式(50)(137mg,76％)。1H NMR(400MHz,CDCl3)δ7.36–7.25(m,10H),5.28–5.19(m,2H),5.02(d,J＝1.7Hz,1H),4.84(d,J＝11.1Hz,1H),4.78(d,J＝1.7Hz,1H),4.70(d,J＝11.1Hz,1H),4.64(d,J＝12.2Hz,1H),4.50(d,J＝12.2Hz,1H),4.28–4.19(m,1H),4.13(d,J＝12.8Hz,1H),4.08(d,J＝12.5Hz,1H),4.02–3.95(m,2H),3.92(dd,J＝11.2,1.5Hz,1H),3.86–3.78(m,2H),3.77–3.70(m,2H),3.31(s,3H),2.13(s,1H),2.08(s,3H),2.02(s,3H),1.97(s,3H),1.16–1.00(m,21H).HRMS(ESI-TOF)m/z Calcd for C42H62O14NaSi[M+Na]+841.3807,found 841.3796.
化合物式(51)的制备：将化合物式(10)(144mg,0.207mmol)和化合物式(50)(131mg,0.159mmol)溶于无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000065
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(47mg,0.207mmol)。搅拌10min后，加入TMSOTf(3μL，0.1eq)。在-20℃下搅拌约1h，TLC显示反应完全。用NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:30:1～1:5:1)得到化合物式(51)(185mg,84％)。1H NMR(400MHz,CDCl3)δ7.37–7.26(m,15H),7.22–7.16(m,2H),6.81–6.74(m,2H),5.43(dd,J＝3.1,1.9Hz,1H),5.21(dd,J＝3.1,2.2Hz,1H),5.18(dd,J＝9.8Hz,1H),5.09(d,J＝1.5Hz,1H),5.02(d,J＝1.9Hz,1H),4.86(d,J＝10.7Hz,1H),4.81–4.75(m,2H),4.71(d,J＝10.8Hz,1H),4.63(d,J＝12.4Hz,1H),4.58(d,J＝10.8Hz,1H),4.56(d,J＝10.8Hz,1H),4.49(d,J＝12.4Hz,1H),4.37(d,J＝10.9Hz,1H),4.21–4.13(m,2H),4.13–4.04(m,3H),3.97–3.88(m,4H),3.87–3.77(m,3H),3.77–3.70(m,5H),3.68–3.63(m,1H),3.29(s,3H),2.08(s,3H),2.00(s,3H),1.98(s,3H),1.96(s,3H),1.10–0.99(m,42H).HRMS(ESI-TOF)m/z Calcd for C74H108O21NaSi2[M+Na]+1411.6819,found 1411.6771.
化合物式(52)的制备：0℃下，将化合物式(51)(181mg,0.130mmol)溶解在混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(35mg,0.159mmol)。0℃搅拌1h，升至室温，继续搅拌3h(TLC检测)。用NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(52)(123mg,82％)。1H NMR(400MHz,CDCl3)δ7.44–7.15(m,15H),5.22–5.13(m,3H),5.10(d,J＝1.4Hz,1H),5.00(d,J＝1.9Hz,1H),4.83 (d,J＝11.3Hz,1H),4.82(d,J＝10.8Hz,1H),4.76(d,J＝11.4Hz,1H),4.75(d,J＝1.8Hz,1H),4.62(d,J＝11.9Hz,1H),4.60(d,J＝10.4Hz,1H),4.49(d,J＝12.3Hz,1H),4.20–4.10(m,2H),4.10–4.00(m,4H),3.98–3.82(m,5H),3.79(dd,J＝9.5,3.1Hz,1H),3.76–3.68(m,2H),3.68–3.60(m,1H),3.29(s,3H),2.05(s,3H),2.03(s,3H),1.99(s,3H),1.97(s,3H),1.16–0.97(m,42H).HRMS(ESI-TOF)m/z Calcd for C66H100O20NaSi2[M+Na]+1291.6244,found 1291.6193.
化合物式(53)的制备：将化合物式(10)(83mg,0.119mmol)和化合物式(52)(116mg,0.091mmol)溶于无水CH2Cl2(5mL)中，加入
Figure PCTCN2016077370-appb-000066
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(27mg,0.119mmol)。搅拌10min后，加入TMSOTf(2μL，0.1eq)。在-20℃下搅拌约1h，TLC显示反应完全。用NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:30:1～1:6:1)得到化合物式(53)(136mg,81％)。1H NMR(400MHz,CDCl3)δ7.39–7.19(m,22H),6.84–6.76(m,2H),5.42(dd,J＝3.0,1.9Hz,1H),5.25(dd,J＝2.7,1.8Hz,1H),5.20–5.12(m,2H),5.09(d,J＝1.2Hz,1H),5.08(d,J＝1.4Hz,1H),5.00(d,J＝1.6Hz,1H),4.86(d,J＝12.4Hz,1H),4.83(d,J＝13.2Hz,1H),4.79–4.74(m,2H),4.74(d,J＝1.6Hz,1H),4.63(d,J＝10.9Hz,1H),4.61(d,J＝12.3Hz,1H),4.59(d,J＝10.6Hz,1H),4.53–4.47(m,2H),4.41(d,J＝10.8Hz,1H),4.21–4.00(m,7H),3.94–3.76(m,8H),3.74(s,3H),3.73–3.66(m,2H),3.63–3.54(m,3H),3.27(s,3H),2.04–2.03(m,6H),2.03(s,3H),1.96(s,3H),1.93(s,3H),1.12–1.03(m,42H),1.00–0.91(m,21H).HRMS(ESI-TOF)m/z Calcd for C98H146O27NaSi3[M+Na]+1861.9257,found 1861.9171.
化合物式(54)的制备：0℃下，将化合物式(53)(120mg,0.065mmol)溶于混合溶剂(CH2Cl2/pH7.0磷酸盐缓冲溶液＝20/1,5mL)中，并加入DDQ(19mg,0.084mmol)。0℃搅拌1h，升至室温，继续搅拌3h(TLC检测)。NaHCO3(sat.aq.)淬灭，CH2Cl2萃取。有机相用NaHCO3(sat.aq.)和饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(54)(90mg,80％)。1H NMR(400MHz,CDCl3)δ7.43–7.19(m,20H),5.26(dd,J＝3.0,1.8Hz,1H),5.20–5.11(m,3H),5.10–5.06(m,2H),4.99(d,J＝1.7Hz,1H),4.85(d,J＝10.1Hz,1H),4.79(d,J＝10.7Hz,1H),4.76–4.72(m,3H),4.70(d,J＝11.4Hz,1H),4.61(d,J＝12.5Hz,1H),4.51(d,J＝10.1Hz,1H),4.49(d,J＝12.4Hz,1H),4.22–4.08(m,4H),4.07–3.96(m,4H),3.95–3.83(m,4H),3.83–3.75(m,3H),3.74–3.66(m,2H),3.64–3.53(m,3H),3.27(s,3H),2.07(s,3H),2.03(s,3H),2.01(s,3H),1.96(s,3H),1.91(s,3H),1.17–1.02(m,42H),1.01–0.91(m,21H).HRMS(ESI-TOF)m/z Calcd for C90H138O26NaSi3[M+Na]+1741.8682,found 1741.8627.
化合物式(55)的制备：将化合物式(6)(41mg,0.067mmol)和化合物式(54)(82mg,0.048mmol)溶于无水CH2Cl2(5mL)中，加入
Figure PCTCN2016077370-appb-000067
(500mg)，在室温下搅拌1h。溶液 冷至-20℃，加入NIS(15mg,0.067mmol)。搅拌10min后，加入TMSOTf(2μL，0.2eq)。在-20℃下搅拌约1h，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:3:1)得到化合物式(55)(80mg,76％)。1H NMR(400MHz,CDCl3)δ7.39–7.20(m,30H),5.43(dd,J＝3.0,1.9Hz,1H),5.27–5.22(m,2H),5.20–5.09(m,4H),5.08(d,J＝1.0Hz,1H),4.98(d,J＝1.6Hz,1H),4.89–4.80(m,3H),4.76–4.64(m,4H),4.64–4.57(m,2H),4.56–4.46(m,3H),4.42(d,J＝11.1Hz,1H),4.37(dd,J＝12.0,3.5Hz,1H),4.21–4.01(m,9H),3.97–3.74(m,10H),3.73–3.66(m,2H),3.62–3.51(m,3H),3.27(s,3H),2.11(s,3H),2.09(s,3H),2.08(s,3H),2.02(s,3H),1.98(s,3H),1.96(s,3H),1.92(s,3H),1.15–1.02(m,42H),1.00–0.91(m,21H).HRMS(ESI-TOF)m/z Calcd for C117H168O34NaSi3[M+Na]+2224.0623,found 2224.0684.
实施例17 式(VII)化合物的制备
Figure PCTCN2016077370-appb-000068
化合物式(56)的制备：将化合物式(55)(40mg，0.018mmol)溶于MeOH(2mL)中，室温下加入MeONa(20mg，0.363mmol)。将温度升至50℃，搅拌24h。反应完成后，冷却至室温，用Dowex50WX8酸性树脂中和、过滤和浓缩后的粗产物，未经纯化直接用于下一步。将所得粗产物溶解在THF(2mL)中，室温下加入TBAF(1M in THF,0.18mL)，搅拌 3d。使用HR-MS检测显示反应完成后，蒸干。硅胶柱层析纯化(EA/MeOH＝40:1～10:1)得到式(56)(23.6mg，94％，2步)。1H NMR(400MHz,CDCl3)δ7.39–7.05(m,30H),5.10(s,1H),5.05(s,1H),5.04(s,1H),5.01(s,1H),4.80–4.69(m,2H),4.68–4.42(m,11H),4.16–3.73(m,17H),3.72–3.54(m,10H),3.51–3.36(m,3H),3.26(s,3H).HRMS(ESI-TOF)m/z Calcd for C73H90O26Na[M+Na]+1405.5618,found 1405.5598.
式(VII)化合物的制备：将化合物式(56)(23.6mg,0.0171mmol)和SO3·Me3N络合物(166mg,1.194mmol)溶解在DMF(1mL)中，并加热至50℃，搅拌3d。冷却至0℃，加入三乙胺和水淬灭，浓缩。残余物用水溶解，经Dowex50WX8(Na+)柱交换盐型，浓缩得化合物式(57)粗产品，不经纯化直接用于下一步。将上一步所得的化合物式(57)粗产物溶于混合溶剂MeOH/H2O(v/v＝4/1，5mL)中，加入20％Pd(OH)2/C(45mg)，并将体系内置换成H2氛围，搅拌3d。硅藻土过滤，浓缩，再经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到式(VII)化合物(26.3mg，83％，2步)。1H NMR(400MHz,D2O,300K,HSQC)δ5.47(d,J＝1.4Hz,1H),5.46–5.45(m,2H),5.36(d,1H),4.97(d,J＝1.6Hz,1H),4.77(dd,J＝3.1,1.7Hz,1H),4.74–4.69(2H,overlapped by HOD),4.63(dd,J＝3.3,1.7Hz,1H),4.50(dd,J＝11.3,1.6Hz,1H),4.38–4.21(m,9H),4.21–3.95(m,12H),3.89(dd,J＝9.9,9.9Hz,1H),3.85(dd,J＝9.9,9.9Hz,1H),3.77(2overlapping dd,J＝10.0,10.0Hz,2H),3.41(s,3H).13C-APT NMR(100MHz,D2O,300K,HSQC)δ100.14,100.06,99.93,99.30,98.83,78.55,77.43,76.88,76.67,76.48,76.42,76.21,76.00,75.06,71.17,70.90,70.84,70.74,69.12,68.69(2C),67.61,67.35,66.67(2C),66.57,65.90(2C),65.78,65.36,55.23.HRMS(ESI-TOF)m/z Calcd for C31H44O56Na7S10[M+7Na]3-597.5695,Found 597.3513；Calcd for C31H44O56Na6S10[M+6Na]4-442.4297,found 442.2662.
实施例18 化合物式(59)的制备
Figure PCTCN2016077370-appb-000069
化合物式(58)的制备：将化合物3(2.0g,5.34mmol)和TBAI(0.4g,1.07mmol)溶于THF(20mL)，0℃下，分批加入NaH(60％,855mg,21.36mmol)。30min后,加入BnBr(2.5mL,21.36mmol)。升至室温，继续搅拌12h。反应完成后，加入水淬灭，用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:10:1)得到化合物式(58)(2.96g,100％)。1H NMR(400MHz,CDCl3)δ7.52(dd,J＝7.6,1.8Hz,2H),7.41–7.23(m,15H),7.10(d,J＝8.0Hz,2H),5.64(s,1H),5.43(d,J＝1.4Hz,1H),4.82(d,J＝12.2Hz,1H),4.71(s,2H),4.65(d,J＝12.2Hz,1H),4.35–4.25(m,2H),4.22(dd,J＝9.9,4.0Hz, 1H),4.03(dd,J＝3.2,1.4Hz,1H),4.00–3.94(m,1H),3.87(ddd,J＝5.8,5.0,2.7Hz,1H),2.33(s,3H).HRMS(ESI-TOF):m/z Calcd for C34H34O5NaS[M+Na]+577.2025,found 577.2013.
化合物式(59)的制备：将化合物式(58)(2.96g,5.34mmol)溶于MeOH(40mL)，加入TsOH·H2O(102mg,0.534mmol)。室温搅拌12h，用三乙胺(5mL)淬灭，浓缩。所得粗产物溶于DMF(15mL)，室温下加入咪唑(836mg,12.28mmol)。冷却至0℃，加入TIPSCl(1.26mL,5.87mmol)，然后逐渐升至室温，搅拌5h。加入水淬灭反应，再用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:40:1～1:15:1)得到化合物式(59)(3.22g,97％)。1H NMR(400MHz,CDCl3)δ7.37–7.25(m,12H),7.08(d,J＝7.9Hz,2H),5.48(d,J＝1.6Hz,1H),4.64 and 4.55(ABq,JAB＝12.2Hz,2H,CH2Ar),4.63and 4.60(ABq,JAB＝11.9Hz,2H,CH2Ar),4.18–4.07(m,2H),4.04–3.89(m,3H),3.75–3.68(m,1H),2.97(brs,1H,OH),2.32(d,J＝5.0Hz,3H),1.13–1.01(m,21H).HRMS(ESI-TOF):m/z Calcd for C36H50O5NaSiS[M+Na]+645.3046,found 645.3015.
实施例19 化合物式(61)的制备。
Figure PCTCN2016077370-appb-000070
化合物式(60)的制备：将化合物式(29)(68mg,0.106mmol)和化合物式(59)(60mg,0.096mmol)溶解在无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000071
(500mg)，在室温下搅拌1h。溶液冷至-20℃，搅拌10min后，加入TMSOTf(2μL，0.1eq)。在-20℃下搅拌约30min，逐渐升至0℃，TLC显示反应完全。用Et3N淬灭，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:15:1～1:5:1)得到化合物式(60)(90mg,72％)。1H NMR(400MHz,CDCl3)δ7.36(dd,J＝9.3,4.8Hz,2H),7.34–7.23(m,20H),7.13–7.04(m,2H),5.54–5.42(m,3H),4.88(Aof ABq,JAB＝11.0Hz,1H),4.70 and 4.46(ABq,JAB＝11.0Hz,2H,CH2Ar),4.60 and 4.47(ABq,JAB＝12.1Hz,2H,CH2Ar),4.56–4.51(m,3H,benzylic),4.41(dd,J＝11.8,4.3Hz,1H),4.23(dd,J＝11.8,1.9Hz,1H),4.14–4.08(m,2H),4.02–3.87(m,5H),3.84–3.78(m,1H),3.77–3.69(m,1H),2.78–2.62(m,2H),2.62–2.52(m,2H),2.32(s,3H),2.14(s,3H),2.05(s,3H),1.11–0.93(m,21H).HRMS(ESI-TOF)m/z Calcd for C63H80O13NaSiS[M+Na]+1127.4987,found  1127.5011.
化合物式(61)的制备：将化合物式(60)(44mg,0.039mmol)和化合物式(36)(15mg,0.041mmol)溶解在无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000072
(500mg)，在室温下搅拌1h。溶液冷至-20℃，加入NIS(9mg,0.038mmol)。搅拌10min后，加入TMSOTf(1μL，0.1eq)。在-20℃下搅拌约1h后，逐渐升至0℃，TLC显示反应完全。NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:20:1～1:5:1)得到化合物式(61)(40mg，72％)。1H NMR(400MHz,CDCl3)δ7.53(dd,J＝7.7,1.8Hz,2H),7.43–7.36(m,3H),7.34–7.25(m,18H),7.25–7.15(m,7H),5.67(s,1H),5.49(dd,J＝3.1,1.9Hz,1H),5.46(d,J＝1.7Hz,1H),5.31(d,J＝1.6Hz,1H),4.88(Aof ABq,JAB＝11.0Hz,1H),4.81 and 4.64(ABq,JAB＝11.5Hz,2H,CH2Ar),4.73(d,J＝1.5Hz,1H),4.72 and 4.47(ABq,JAB＝10.9Hz,2H,CH2Ar),4.57–4.50(m,3H,benzylic),4.40(dd,J＝12.1,4.1Hz,1H),4.38(d,J＝12.4Hz,1H),4.30(d,J＝12.1Hz,1H),4.28(dd,J＝9.7,4.2Hz,1H),4.21(dd,J＝11.8,1.9Hz,1H),4.18(dd,J＝2.7,1.7Hz,1H),4.04–3.94(m,4H),3.92–3.78(m,7H),3.74–3.70(m,1H),3.69–3.61(m,1H),3.34(s,3H),2.75–2.65(m,2H),2.60–2.53(m,2H),2.13(s,3H),2.03(s,3H),1.09–0.97(m,21H).HRMS(ESI-TOF)m/z Calcd for C77H96O19NaSi[M+Na]+1375.6213,found 1375.6171.
实施例20 化合物式(IX)的制备
Figure PCTCN2016077370-appb-000073
化合物式(63)的制备：将化合物式(61)(25.8mg，0.019mmol)溶于MeOH(3mL)中，室温下加入MeONa(5.2mg，0.095mmol)，搅拌24h。TLC及HR-MS显示反应完成，用Dowex50WX8酸性树脂中和，过滤，浓缩，未经纯化直接用于下一步。将所得粗产物溶解在THF(3mL)中，加入TBAF(1M in THF，0.1mL)，50℃下搅拌24h，TLC检测显示反应完成。蒸干溶剂，得粗产物式(62)，未经纯化直接用于下一步。将化合物式(62)粗产物溶 解在MeOH(3mL)中，加入TsOH·H2O(10mg)。室温搅拌12h，用Et3N(2mL)淬灭，浓缩。硅胶柱层析纯化(EA/DCM/MeOH＝1:1:0～1:1:0.02)得到化合物式(63)(14mg,76％，3步)。1H NMR(400MHz,CDCl3)δ7.38–7.26(m,20H),7.25–7.17(m,5H),5.34(s,1H),5.02(s,1H),4.82(d,J＝11.0Hz,1H),4.77(s,1H),4.66–4.53(m,5H),4.52–4.46(m,3H),4.41(d,J＝11.7Hz,1H),4.12(dd,J＝9.1Hz,1H),4.00–3.89(m,3H),3.87–3.68(m,12H),3.67–3.61(m,1H),3.59(ddd,J＝9.3,3.7Hz,1H),3.35(s,3H),2.53(br,5H,OH).HRMS(ESI-TOF)m/z Calcd for C54H64O16Na[M+Na]+991.4092,found 991.4122.
式(IX)化合物的制备：将化合物式(63)(13.4mg,0.0144mmol)和SO3·Me3N络合物(50mg,0.361mmol)溶解在无水DMF(1mL)中，在50℃搅拌2d。冷却至室温，加入Na2CO3(38mg,0.361mmol)和水，搅拌2h，蒸干，得化合物式(64)的粗产品，不经纯化直接用于下一步。将粗产物式(64)(0.0144mmol)溶解在混合溶剂MeOH/H2O(v/v＝1/2，6mL)中，加入20％Pd(OH)2/C(35mg)，并将体系内置换成H2氛围，搅拌4d。1H NMR监测反应完全，硅藻土过滤，浓缩，Sephadex G-25凝胶排阻色谱纯化，再经冷冻干燥得到式(IX)化合物(10mg，67％,2步)。1H NMR(400MHz,D2O,300K,HSQC)δ5.63(d,J＝1.6Hz,1H),5.10(d,J＝1.6Hz,1H),4.96(d,J＝1.3Hz,1H),4.70(dd,J＝3.1,2.0Hz,1H),4.52(dd,J＝11.4,2.0Hz,1H),4.43–4.36(m,2H),4.34(dd,J＝11.4,1.9Hz,1H),4.30(dd,J＝11.3,1.8Hz,1H),4.23(dd,J＝11.3,6.0Hz,1H),4.17(dd,J＝11.4,7.5Hz,1H),4.12–4.00(m,7H),3.94–3.80(m,4H),3.46(s,3H).DEPT135(100MHz,D2O,300K,HSQC)δ101.88,99.12,98.93,77.57,77.07,75.04,73.86,71.39,70.63,70.45,69.88,69.10,68.98,68.70,67.58,67.38,66.52,65.73,55.10.HRMS(ESI-TOF)m/z Calcd for C19H29O31Na3S5[M+3Na]2-490.9495,found 490.9453.
实施例21 化合物式(67)的制备。
Figure PCTCN2016077370-appb-000074
化合物式(65)的制备：室温下，将D-甘露糖(5.0g)悬浮于Ac2O(50mL)中，并加入NaOAc(7.5g)。将温度升至120℃并搅拌2h。将反应液倒入200g冰中，搅拌1h，然后用乙酸乙酯萃取(100×3)萃取。有机相合并，并依次用NaHCO3(sat.aq.)和饱和食盐水洗涤，Na2SO4干燥，浓缩得全乙酰化粗产品(12.3g)。将全乙酰化粗产品和PMBOH(5.17g,0.042mmol)溶于DCM(50mL)。0℃，加入BF3·Et2O(7mL)，然后升至室温，搅拌过夜。用DCM稀释，有机相依次用5％NaOH(aq)和饱和食盐水洗涤,MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE＝1:3)得到化合物式(65)(9.86g,77％，2步)。1H NMR(400MHz,CDCl3)δ7.05– 6.99(m,2H),6.86–6.79(m,2H),5.55(dd,J＝10.0,3.5Hz,1H),5.44(dd,J＝3.5,1.9Hz,1H),5.41(d,J＝1.8Hz,1H),5.35(t,J＝10.0Hz,1H),4.28(dd,J＝12.0,5.3Hz,1H),4.17–4.12(m,1H),4.09(dd,J＝12.0,2.3Hz,1H),3.77(s,3H),2.19(s,3H),2.06(s,3H),2.05(s,3H),2.03(s,3H).HRMS(ESI-TOF)m/z Calcd forC21H26O11Na[M+Na]+477.1373,found 477.1362.
化合物式(66)的制备：化合物式(65)(1.26g，2.8mmol)溶解在MeOH(30mL)中，加入MeONa(0.29g,5.54mmol)。室温搅拌4h，经Dowex 50WX8酸性树脂中和，过滤，蒸干。将粗产物与甲苯共沸，然后溶于15mL CH3CN。加入苯甲醛二甲缩醛(0.46mL,3.1mmol)和DL-樟脑磺酸(33mg,0.14mmol)，室温下搅拌15min，升温至50℃，继续搅拌30min。然后加入Et3N(5mL)淬灭，蒸干。EtOAc重结晶得化合物式(66)(942mg，73％，2步)。exo-66:1H NMR(400MHz,CDCl3)δ7.58–7.47(m,4H),7.43–7.33(m,6H),7.01–6.94(m,2H),6.87–6.80(m,2H),6.34(s,1H),5.74(s,1H),5.65(s,1H),4.80(dd,J＝7.6,5.5Hz,1H),4.38(d,J＝5.4Hz,1H),4.27(dd,J＝10.4,4.8Hz,1H),4.06–3.93(m,2H),3.80(dd,J＝10.0Hz,1H),3.77(s,3H).HRMS(ESI-TOF)m/z Calcd forC27H26O7Na[M+Na]+485.1576,found 485.1581.
化合物式(67)的制备：0℃，将BH3·THF(5mL,1M in THF,5mmol)滴加到化合物式(66)(349mg，0.753mmol)的无水CH2Cl2(6mL)溶液中。0℃下搅拌30min，再升至室温，TLC检测反应完成。用Et3N和MeOH淬灭，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:3:1)得到化合物式(67)(147mg,45％)。1H NMR(400MHz,CDCl3)δ7.42–7.26(m,10H),6.96–6.88(m,2H),6.86–6.77(m,2H),5.45(d,J＝1.5Hz,1H),4.92 and 4.70(ABq,JAB＝11.1Hz,2H,CH2Ar),4.76 and 4.67(ABq,JAB＝11.6Hz,2H,CH2Ar),4.19(ddd,J＝8.5,8.5,3.8Hz,1H),3.93(dd,J＝3.7,1.7Hz,1H),3.87–3.67(m,7H),2.39(d,J＝8.9Hz,1H,OH),1.91(brs,1H,OH).HRMS(ESI-TOF)m/z Calcd for C50H66O11NaSi[M+Na]+893.4272,found 893.4246.
实施例22 化合物式(68)的制备。
Figure PCTCN2016077370-appb-000075
化合物式(68)的制备：将化合物式(6)(150mg,0.247mmol)和化合物式(67)(110mg,0.235mmol)溶解在无水CH2Cl2(5mL)中，并加入
Figure PCTCN2016077370-appb-000076
(500mg)，室温下搅拌1h。溶液冷至-20℃，加入NIS(61mg,0.270mmol)。搅拌10min后，加入TMSOTf(4.5μL，0.1eq)。在-20℃下搅拌约30min,逐渐升至0℃，TLC显示反应完全。用NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，硅藻土过滤，CH2Cl2洗涤滤饼。有机相用饱和食盐水洗涤，MgSO4干燥， 过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:1:1)得到化合物式(68)(50mg,15％):1H NMR(400MHz,CDCl3)δ7.40–7.22(m,30H),6.96–6.87(m,2H),6.82–6.74(m,2H),5.53(dd,J＝3.1,1.8Hz,1H),5.46(d,J＝1.7Hz,1H),5.38(dd,J＝3.1,1.9Hz,1H),5.22(d,J＝1.4Hz,1H),4.92(d,J＝10.9Hz,1H),4.90(d,J＝10.9Hz,1H),4.84(d,J＝1.7Hz,1H),4.78(d,J＝11.3Hz,1H),4.77(d,J＝12.1Hz,1H),4.70(d,J＝12.0Hz,1H),4.68(d,J＝11.2Hz,1H),4.59(d,J＝11.4Hz,1H),4.56(d,J＝12.1Hz,2H),4.49(d,J＝11.2Hz,1H),4.48(d,J＝11.3Hz,1H),4.38(d,J＝11.1Hz,1H),4.34–4.22(m,5H),4.03(dd,J＝9.2,3.3Hz,1H),3.99–3.89(m,3H),3.89–3.75(m,4H),3.74–3.67(m,2H),3.60(s,3H),3.58(d,J＝7.0Hz,1H),2.73–2.58(m,4H),2.58–2.47(m,4H),2.13(s,3H),2.13(s,3H),2.09(s,3H),2.09(s,3H).HRMS(ESI-TOF)m/z Calcd for C81H90O23Na[M+Na]+1453.5771,found 1453.5735.
实施例23 式(XI)化合物的制备
Figure PCTCN2016077370-appb-000077
化合物式(69)的制备：将化合物式(68)(30mg,0.021mmol)溶解在CH2Cl2(5mL)中，室温下加入NH2-NH2·HOAc(1M in MeOH，0.08mL，0.08mmol)。待TLC显示反应完全后，用丙酮淬灭，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:10:1～1:1:1)得到化合物式(69)(20mg,77％)。1H NMR(400MHz,CDCl3)δ7.38–7.16(m,30H),6.95–6.88(m,2H),6.80–6.74(m,2H),5.54(dd,J＝3.2,1.8Hz,1H),5.41(d,J＝1.9Hz,1H),5.40(dd,J＝3.2,2.0Hz,1H),5.21(d,J＝1.5Hz,1H),4.94(d,J＝11.0Hz,1H),4.91(d,J＝10.9Hz,1H),4.83(d,J＝1.7Hz,1H),4.77(d,J＝11.2Hz,1H),4.73(d,J＝12.1Hz,1H),4.69(d,J＝11.3Hz,1H),4.68(d,J＝12.0Hz,1H),4.62(d,J＝11.1Hz,1H),4.61(d,J＝11.1Hz,1H),4.60(d,J＝11.2Hz,1H),4.51(d,J＝11.3Hz,1H),4.48(d,J＝11.3Hz,1H),4.39(d,J＝11.2Hz,1H),4.30(dd,J＝9.3,3.0Hz,1H),4.07–3.99(m,1H),3.98–3.87(m,3H),3.86–3.66(m,10H),3.62(s,3H),3.61–3.56(m,1H),2.12(s,3H),2.09(s,3H),1.87(t,J＝6.0Hz,1H,OH),1.79(t,J＝6.4Hz,1H,OH).HRMS (ESI-TOF)m/z Calcd for C71H78O19Na[M+Na]+1257.5035,found 1257.4972.
化合物式(70)的制备：将化合物式(69)(20mg,0.0162mmol)溶解在无水CH2Cl2(2mL)中，并加入1H-四氮唑(12mg，0.162mmol)和
Figure PCTCN2016077370-appb-000078
(100mg)，在室温下搅拌30min。加入(BnO)2PN(iPr)2(43μL，0.130mmol)，搅拌3h，TLC显示反应完全。将温度降至0℃。加入mCPBA(22mg,85％，0.130mmol)，搅拌1h。加入NaHCO3(sat.aq.)和Na2S2O3(sat.aq.)淬灭，用乙酸乙酯萃取。有机相用饱和食盐水洗涤，MgSO4干燥，过滤，浓缩。硅胶柱层析纯化(EA/PE/DCM＝1:3:1～1:1:1)得到化合物式(70)(27.8mg,98％，2步)。1H NMR(400MHz,CDCl3)δ7.33–7.17(m,50H),6.92–6.83(m,2H),6.76–6.67(m,2H),5.50(dd,J＝3.1,1.8Hz,1H),5.37(dd,J＝3.0,2.0Hz,1H),5.35(d,J＝1.7Hz,1H),5.18(d,J＝1.2Hz,1H),5.07–4.97(m,8H),4.89(d,J＝10.9Hz,1H),4.88(d,J＝10.8Hz,2H),4.82(d,J＝1.6Hz,1H),4.72(d,J＝11.3Hz,1H),4.65(d,J＝11.4Hz,2H),4.61–4.54(m,4H),4.47(d,J＝11.2Hz,1H),4.41(d,J＝11.3Hz,1H),4.36(d,J＝11.1Hz,1H),4.30–4.18(m,5H),4.03(dd,J＝9.2,3.3Hz,1H),3.95–3.84(m,4H),3.83–3.70(m,6H),3.59(s,3H),3.53–3.47(m,1H),2.02(s,3H),2.01(s,3H).HRMS(ESI-TOF)m/z Calcd for C99H104O25NaP2[M+Na]+1777.6240,found 1777.6300.
化合物式(71)的制备：将化合物式(70)(27.8mg，0.016mmol)溶于MeOH(3mL)中，室温下加入MeONa(3mg，0.056mmol)，搅拌5h。TLC及HR-MS显示反应完成后，用Dowex50WX8酸性树脂中和，过滤，浓缩。硅胶柱层析纯化(EA/DCM＝1:4～1:2)得到化合物式(71)(18.2mg,69％)。1H NMR(400MHz,CDCl3)δ7.35–7.17(m,50H),6.91–6.85(m,2H),6.76–6.68(m,2H),5.34(d,J＝1.8Hz,1H),5.21(d,J＝1.2Hz,1H),5.08–4.97(m,8H),4.91(d,J＝1.5Hz,1H),4.85(d,J＝11.0Hz,1H),4.85(d,J＝10.9Hz,1H),4.65(d,J＝11.3Hz,1H),4.63–4.53(m,7H),4.50(d,J＝11.5Hz,1H),4.43(d,J＝11.3Hz,1H),4.29–4.13(m,5H),4.03–3.95(m,2H),3.94–3.80(m,6H),3.79–3.69(m,4H),3.61(s,3H),3.56(d,J＝10.2Hz,1H),2.60(d,J＝3.2Hz,1H),2.59(d,J＝3.6Hz,1H).HRMS(ESI-TOF)m/z Calcd for C95H100O23NaP2[M+Na]+1693.6028,found 1693.6068.
式(XI)化合物的制备：将化合物式(71)(18.2mg,0.0111mmol)和SO3·Me3N络合物(16mg,0.111mmol)溶解在DMF(1mL)中，并加热至50℃，搅拌2d。冷却至室温，加入Na2CO3(12mg,0.111mmol)和水淬灭，搅拌2h，浓缩，不经纯化直接用于下一步。将所得的粗产物溶解在混合溶剂MeOH/H2O(v/v＝1/1,6mL)中，加入20％Pd(OH)2/C(35mg)，在H2气氛下搅拌2d。1H NMR监测反应完全，硅藻土过滤，浓缩。残余物用水溶解，经Dowex50WX8(Na+)柱交换盐型，再经Sephadex G-25凝胶排阻色谱纯化，冷冻干燥得到式(XI)化合物(11.0mg，97％，2步)。1H NMR(400MHz,D2O,300K,HSQC)δ7.19–7.13(m,2H),7.05–6.99(m,2H),5.49(d,J＝1.4Hz,1H),5.46(d,J＝1.2Hz,1H),5.12(d,J＝1.4Hz,1H),4.72(dd,J＝3.4,1.5Hz,1H),4.49(dd,J＝3.3,1.6Hz,1H),4.40(dd,J＝3.0,1.7Hz,1H), 4.18–3.98(m,7H),3.99–3.85(m,4H),3.83(s,3H),3.80(dd,J＝9.8,2.8Hz,1H),3.78(dd,J＝9.7,2.9Hz,1H),3.76–3.70(m,2H).13C NMR(100MHz,D2O,300K,HSQC)δ154.67,149.38,118.89,115.09,100.35,99.14,97.39,80.02,76.92,76.75,72.62(d,J＝6.5Hz),71.85(d,J＝7.7Hz),71.82,69.25,68.86,68.79,66.50,66.12,65.98,65.34,63.56(d,J＝3.3Hz),63.22(d,J＝3.2Hz),55.84.31P NMR(162MHz,D2O,pH 7.0,300K)δ2.60.HRMS(ESI-TOF)m/z Calcd for C25H36O29Na3P2S2[M+2H+3Na]-994.9952,found 994.9931；Calcd for C25H36O29Na2P2S2[M+2H+2Na]2-486.0027,found 486.0003.
实施例24 式(III)、(IV)、(VI)、(VII)、(IX)、(XI)化合物抑制血管生成的活性测试(人工基底膜微管生成实验)
将人微血管内皮细胞(HMEC-1)以5×104/mL的细胞密度分散在商品化的内皮细胞无血清培养基(MCDB131)中。取一份200μL的上述HMEC-1细胞，分别采用一定体积的式(III)、(IV)、(VI)、(VII)、(IX)、(XI)硫酸化寡糖化合物以及PI-88化合物的标准溶液进行处理，得到最终溶液的化合物浓度分别是10,50,100μM，最终体积均为300μL。分取经处理的HMEC-1细胞100μL涂抹到镀有低生长因子的人工基底膜的96-孔板上，然后在37℃下培养18h。微管生成是通过相差显微镜进行检测，图像采用连接在Olympus IX50倒置相差显微镜上的数字摄像机进行采集。各硫酸化寡糖化合物对微管生成的抑制率是通过对图像的人工定量获得的，具体是记录连接3个及以上微管的分支节点的总数。结果表述为相对于空白对照的百分比抑制率，未经硫酸化寡糖化合物处理的HMEC细胞被作为人工基底膜上正常细胞生长、微管生成的标准参照。
式(III)、(IV)、(VI)、(VII)、(IX)、(XI)硫酸化寡糖化合物抑制血管生成的实验结果见图2，其中，PI-88作为对比化合物。。图2-1是各化合物抑制血管生成的实验结果柱状图。图2-2是式(III)化合物抑制血管生成的成像图。实验表明，式(III)、(IV)、(VI)、(VII)、(IX)、(XI)硫酸化寡糖化合物对HMEC-1微管生成具有显著的抑制作用，其中，化合物(III)和(IV)表现出优于PI-88的活性。

Claims (32)

  1. 一种纯净形式的磷酰甘露五糖，其特征在于，其化学结构如式(I)所示：
    Figure PCTCN2016077370-appb-100001
    所述纯净形式的磷酰甘露五糖是酵母菌Pichia holstii NRRL Y-2448细胞外包括二糖～六糖的磷酰甘露寡糖混合物中含量最高的组分，且存在形式是单一组分。
  2. 一种磷酰甘露五糖醇，其特征在于，其化学结构如式(II)所示：
    Figure PCTCN2016077370-appb-100002
  3. 一种纯净形式的硫酸化磷酰甘露五糖，其特征在于，其为全硫酸化十八钠盐，其化学结构如式(III)所示：
    Figure PCTCN2016077370-appb-100003
  4. 一种硫酸化磷酰甘露五糖醇，其特征在于，其为全硫酸化十九钠盐，其化学结构如式(IV)所示：
    Figure PCTCN2016077370-appb-100004
  5. 一种确定位点不完全硫酸化甘露五糖，其特征在于，其化学结构如式(V)所示：
    Figure PCTCN2016077370-appb-100005
    其中，R为C1～C5的烷基；R1～R16各自独立地选自SO3 -和H，其中，SO3 -的个数不小于5，不大于15，其余为H；钠离子的个数n＝SO3 -的个数。
  6. 如权利要求5所述的确定位点不完全硫酸化甘露五糖，其特征在于，当所述R＝CH3，R6＝R9＝R12＝R13＝R16＝SO3 -，R1＝R2＝R3＝R4＝R6＝R7＝R8＝R10＝R11＝R14＝R15＝H，n＝5时，其结构如式(VI)所示；
    Figure PCTCN2016077370-appb-100006
    当所述R＝CH3，R2＝R3＝R4＝R6＝R7＝R9＝R10＝R12＝R13＝R16＝SO3 -，R1＝R5＝R8＝R11＝R14＝R15＝H，n＝10，其结构如式(VII)所示：
    Figure PCTCN2016077370-appb-100007
  7. 一种确定位点不完全硫酸化甘露三糖，其特征在于，其化学结构如式(VIII)所示，其中，R为C1～C5的烷基；R1～R10各自独立地选自SO3 -和H，其中，SO3 -的个数大于0，H的个数大于0；钠离子的个数n＝SO3 -的个数：
    Figure PCTCN2016077370-appb-100008
  8. 如权利要求7所述的确定位点不完全硫酸化甘露三糖，其特征在于，当所述R2＝R3＝R6＝R7＝R10＝SO3 -，R1＝R4＝R5＝R8＝R9＝H，n＝5时，其结构如式(IX)所示：
    Figure PCTCN2016077370-appb-100009
  9. 一种确定位点的不完全硫酸化支链磷酰甘露三糖，其特征在于，其结构如式(X)所示，其中，R为芳基，R1～R8各自独立地选自SO3 -和H，其中，SO3 -的个数大于0，H的个数大于0；钠离子的个数n＝SO3 -的个数+4：
    Figure PCTCN2016077370-appb-100010
  10. 如权利要求9所述的确定位点不完全硫酸化支链磷酰甘露三糖，其特征在于，当所述R为p-CH3OC6H4，R4＝R6＝SO3 -，R1＝R2＝R3＝R5＝R7＝R8＝H，n＝6时，其结构如式(XI)所示：
    Figure PCTCN2016077370-appb-100011
  11. 如权利要求1所述的式(I)磷酰甘露五糖的制备方法，其特征在于，以D-甘露糖为原料分别制备得到式(A)、式(B)、式(C)三个单糖合成砌块，再通过从还原端C起始的线性组装获得保护的五糖中间体式(D)，然后经后阶段的非还原端脱保护、亚磷酰化-氧化，再经全脱保护得到式(I)磷酰甘露五糖；所述制备方法包括以下步骤及反应式：步骤(1)：以D-甘露糖为原料分别制备式(A)、式(B)、式(C)，如路线(1)所示；
    Figure PCTCN2016077370-appb-100012
    其中，基团R1～R10、RE各自独立地选自乙酰基、苯甲酰基、苄基、叔丁基二甲基硅基或三异丙基硅基；基团RA和RC分别为苯基、4-甲基苯基、甲基、乙基或丙基；基团RB和RD各自独立地选自3-乙酰丙酰基、4-甲氧基苄基、烯丙基、烯丙氧羰基或9-芴甲氧羰基；步骤(2)：将所述式(B)和式(C)在糖基化条件下反应，得到二糖中间体式(E)；将所述中间体式(E)的基团RD脱除，得到化合物式(F)；将化合物式(F)和式(B)在糖基化条件下反应，得到三糖中间体式(G)；将所述三糖中间体式(G)的保护基团RD脱除，得到化合物式(H)；将所述化合物式(H)和式(B)在糖基化条件下反应，得到四糖中间体式(I)；将所述四糖中间体(I)的保护基团RD脱除，得到化合物式(J)；将所述化合物式(J)和式(A)在糖基化条件下反应，得到五糖中间体式(D)，如路线(2)所示；
    Figure PCTCN2016077370-appb-100013
    步骤(3)：以所述五糖中间体式(D)为原料，将其保护基团RB脱除，得到化合物式(K)；再依次在亚磷酰化、氧化条件下反应，得到磷酸酯化合物式(L)；将所述化合物式(L)的基团R1～R10以及RE脱除，得到如式(I)所示的化合物磷酰甘露五糖，如路线(3)所示；
    Figure PCTCN2016077370-appb-100014
  12. 如权利要求11所述的制备方法，其特征在于，当所述基团R1＝R4＝R8＝R9＝乙酰基，R2＝R3＝R5＝R7＝RE＝苄基，R6＝三异丙基硅基，RD＝4-甲氧基苄基，RB＝3-乙酰丙酰基，RA＝RC＝4-甲基苯基，所述制备方法包括：
    步骤(1)：以D-甘露糖为原料，经全乙酰化得到化合物式(1)；通过4-甲基苯硫酚进行硫苷化得到硫苷式(2)；经过乙酰基脱除、4,6-O-亚苄基保护得到二醇式(3)；通过3-O-苄基化、2-O-乙酰化得到化合物式(4)；然后，经还原性开环条件，得到化合物式(5)；最后，经3-乙酰丙酰化条件，得到单糖合成砌块式(6)，如路线(4)所示；
    Figure PCTCN2016077370-appb-100015
    路线(4)中：a_全乙酰化；b_硫苷化；c_全脱乙酰基；d_4,6-O-亚苄基保护；e_3-O-苄基保护；f_2-O-乙酰基保护；g_还原性开环；h_3-乙酰丙酰化；
    步骤(2)：以所述化合物式(3)为原料，经过3-O-(4-甲氧基苄基)化得到化合物式(7)； 经过2-O-乙酰化得到化合物式(8)；然后，经还原性开环条件，得到化合物式(9)；最后，经硅基化得到单糖合成砌块式(10)，如路线(5)所示；
    Figure PCTCN2016077370-appb-100016
    路线(5)中：a_3-O-PMB保护；b_2-O-乙酰化；c_还原性开环；d_6-O-硅基化；
    步骤(3)：以D-甘露糖为原料，经苄醇Fischer糖苷化、全乙酰化得到化合物式(11)；经过乙酰基脱除、4,6-O-亚苄基保护得到二醇式(12)；通过3-O-苄基化得到化合物式(13)；经2-O-(4-甲氧基苄基)化得到化合物式(14)；然后，经酸性条件下脱除4,6-O-亚苄基保护基、4,6-O-乙酰化得到化合物式(15)；最后，在脱除2-O-(4-甲氧基苄基)的条件下，得到单糖合成砌块式(16)，如路线(6)所示；
    Figure PCTCN2016077370-appb-100017
    路线(6)中：a_苄苷化；b_全乙酰化；c_全脱乙酰基化；d_4,6-O亚苄基保护；e_3-O-苄基保护；f_2-O-PMB保护；g_酸性条件；h_4,6-O-乙酰化；i_脱除PMB；
    步骤(4)：将所述单糖合成砌块式(10)和式(16)在糖基化条件下反应，得到二糖中间体式(17)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(18)；将所述化合物式(18)和所述单糖合成砌块式(10)在糖基化条件下反应，得到三糖中间体式(19)；在脱除所述三糖中间体式(19)的3-O-(4-甲氧基苄基)的条件下，得到化合物式(20)；将所述化合物式(20)和单糖合成砌块式(10)在糖基化条件下反应，得到四糖中间体式(21)；在脱除所述四糖中间体21的3-O-(4-甲氧基苄基)的条件下，得到化合物式(22)；将所述化合物式(22)和所述单糖合成砌块式(6)在糖基化条件下反应，得到五糖中间体式(23)，如路线(7)所示；
    Figure PCTCN2016077370-appb-100018
    步骤(5)：将所述化合物式(23)在脱除乙酰丙酰基的条件下处理，得到化合物式(24)；再依次进行亚磷酰化、氧化，得到磷酸酯化合物式(25)；在脱除乙酰基的条件下，得到化合物式(26)；在脱除硅基的条件下，得到化合物式(27)；在脱除苄基的条件下，得到式(I)磷酰甘露五糖，如路线(8)所示；
    Figure PCTCN2016077370-appb-100019
  13. 一种按权利要求11或12所述方法合成的式(I)磷酰甘露五糖。
  14. 如权利要求2所述的式(II)磷酰甘露五糖醇的制备方法，其特征在于，以化合物式(27)为原料，在脱除苄基-还原开环的条件下，获得如式(II)所示的磷酰甘露五糖醇，其反应如路线(9)所示：
    Figure PCTCN2016077370-appb-100020
  15. 如权利要求2所述的式(II)磷酰甘露五糖醇的制备方法，其特征在于，以式(I)磷酰甘露五糖化合物为原料，在还原条件下，获得所述如式(II)所示的磷酰甘露五糖醇，其反应如路线(10)所示：
    Figure PCTCN2016077370-appb-100021
  16. 一种按权利要求14或15所述方法合成的式(II)磷酰甘露五糖醇。
  17. 如权利要求3所述的式(III)硫酸化磷酰甘露五糖的制备方法，其特征在于，以式(I)磷酰甘露五糖为原料，在O-硫酸化条件下，获得如式(III)所示的硫酸化磷酰甘露五糖；其反应如路线(11)所示：
    Figure PCTCN2016077370-appb-100022
  18. 一种按权利要求17所述方法合成的式(III)硫酸化磷酰甘露五糖。
  19. 如权利要求4所述的式(IV)硫酸化磷酰甘露五糖醇的制备方法，其特征在于，以式(II)磷酰基甘露五糖醇为原料，在O-硫酸化条件下，获得如式(IV)所示的硫酸化磷酰甘露五糖醇；其反应如以下路线(12)所示：
    Figure PCTCN2016077370-appb-100023
  20. 一种按权利要求19所述方法合成的式(IV)硫酸化磷酰甘露五糖醇。
  21. 如权利要求6所述的式(VI)确定位点不完全硫酸化甘露五糖的制备方法，其特征在于，首先分别合成三个单糖合成砌块，通过从还原端起始的线性组装获得保护的五糖中间体，然后经后阶段的脱保护、硫酸化、脱保护得到式(VI)确定位点不完全硫酸化甘露五糖；所述制备方法包括：
    步骤(1)：以化合物式(6)为原料，在氧化水解条件下，得到化合物式(28)；再通过三氯乙腈、碱处理，得到单糖合成砌块式(29)，其反应如路线(13)所示；
    Figure PCTCN2016077370-appb-100024
    路线(13)中：a_氧化水解条件；b_制备三氯乙酰亚胺酸酯；
    步骤(2)：以化合物式(7)为原料，经过2-O-苄基化保护，得到化合物式(30)；再经过酸性条件下脱除4,6-O-亚苄基保护基、6-O-硅基化，得到化合物式(31)；最后，经过4-O-苄基化保护，得到单糖合成砌块式(32)，其反应如路线(14)所示；
    Figure PCTCN2016077370-appb-100025
    路线(14)中：a_苄基化；b_酸性条件；c_6-O-硅基化；d_苄基化；
    步骤(3)：以化合物式(33)为原料，经过全三甲基硅基保护，得到化合物式(34)；再经过4,6-O-亚苄基保护、2,3-O-硅基脱除，得到化合物式(35)；最后，通过3-O-苄基化保护，得到单糖合成砌块式(36)，其反应如路线(15)所示；
    Figure PCTCN2016077370-appb-100026
    路线(15)中：a_全三甲基硅基化；b_4,6-O-亚苄基保护、2,3-O-硅基脱除；c_3-O-苄基化；
    步骤(4)：将所述单糖合成砌块式(36)和式(32)在第一糖基化条件下反应，得到二糖中间体式(37)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(38)；化合物式(38)和单糖合成砌块式(32)在糖基化条件下反应，得到三糖中间体式(39)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(40)；化合物式(40)和单糖合成砌块式(32)在糖基化条件下反应，得到四糖中间体式(41)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(42)；化合物式(42)和单糖合成砌块式(29)在第二糖基化条件下反应，得到五糖中间体式(43)，其反应如路线(16)所示；
    Figure PCTCN2016077370-appb-100027
    步骤(5)：将所述化合物式(43)，依次在脱除酰基、脱除硅基的条件下处理，得到化合物式(44)；再在O-硫酸化条件下，得到化合物式(45)；最后，在脱除苄基的条件下，得到如式(VI)所示的确定位点不完全硫酸化甘露五糖，其反应如路线(17)所示；
    Figure PCTCN2016077370-appb-100028
  22. 一种按权利要求21所述方法合成的式(VI)确定位点不完全硫酸化甘露五糖。
  23. 如权利要求6所述的式(VII)确定位点不完全硫酸化甘露五糖的制备方法，其特征在于，所述制备方法包括：
    步骤(1)：以所述化合物式(36)为原料，经过2-O-(4-甲氧基苄基)保护，得到化合物式(46)；再依次经过酸性条件下4,6-O-亚苄基保护基的脱除、4,6-O-乙酰化，得到化合物式(47)；最后，在脱除3-O-(4-甲氧基苄基)的条件下，得到单糖合成砌块式(48)，其反应如路线(18)所示；
    Figure PCTCN2016077370-appb-100029
    路线(18)中：a_2-O-PMB保护；b_酸性条件；c_4,6-O-乙酰基保护；d_脱除PMB；
    步骤(2)：将所述单糖合成砌块式(48)和式(10)在糖基化条件下反应，得到二糖中间体式(49)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(50)；化合物式(50)和单糖合成砌块式(10)在糖基化条件下反应，得到三糖中间体式(51)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(52)；化合物式(52)和单糖合成砌块式(10)在 糖基化条件下反应，得到四糖中间体式(53)；在脱除3-O-(4-甲氧基苄基)的条件下，得到化合物式(54)；化合物式(54)和单糖合成砌块式(6)在糖基化条件下反应，得到五糖中间体式(55)，其反应如路线(19)所示；
    Figure PCTCN2016077370-appb-100030
    步骤(3)：将所述化合物式(55)，依次在脱除酰基、脱除硅基的条件下处理，得到化合物式(56)；再在O-硫酸化条件下，得到化合物式(57)；最后，在脱除苄基的条件下，得到式(VII)不完全硫酸化甘露五糖，其反应如路线(20)所示；
    Figure PCTCN2016077370-appb-100031
  24. 一种按权利要求23所述方法合成的式(VII)确定位点不完全硫酸化甘露五糖。
  25. 如权利要求8所述的式(IX)确定位点不完全硫酸化甘露三糖的制备方法，其特征在于，首先合成单糖合成砌块式(59)，再将其与所述的单糖合成砌块式(36)、式(29)，通过从非还原端起始的线性组装获得保护的三糖中间体，经后阶段的脱保护、硫酸化、脱保护得到所述式(IX)确定位点不完全硫酸化甘露三糖；
    所述制备方法包括：
    步骤(1)：以化合物式(3)为原料，经过2,3-O-苄基化，得到化合物式(58)；再依次经过酸性条件下4,6-O-亚苄基保护基的脱除、6-O-硅基化，得到单糖合成砌块式(59)，其反应如路线(21)所示；
    Figure PCTCN2016077370-appb-100032
    路线(21)中：a_2,3-O-苄基保护；b_酸性条件；c_6-O-硅基化；
    步骤(2)：将所述单糖合成砌块式(59)和式(29)在第一糖基化条件下反应，得到二糖中间体式(60)；然后，化合物式(60)和单糖合成砌块式(36)在第二糖基化条件下 反应，得到三糖中间体式(61)，其反应如路线(22)所示；
    Figure PCTCN2016077370-appb-100033
    步骤(3)：将所述化合物式(61)，依次经过脱除酰基、脱除硅基的条件的处理，得到化合物式(62)；经酸性条件下4,6-O-亚苄基保护基的脱除，得到化合物式(63)；然后，在O-硫酸化条件下，得到化合物式(64)；最后，在脱除苄基的条件下，得到式(IX)不完全硫酸化甘露三糖，其反应如路线(23)所示；
    Figure PCTCN2016077370-appb-100034
  26. 一种按权利要求25所述方法合成的式(IX)确定位点不完全硫酸化甘露三糖。
  27. 如权利要求10所述的式(XI)确定位点不完全硫酸化支链磷酰甘露三糖的制备方法，其特征在于，首先合成单糖合成砌块式(67)，再将其与所述的单糖合成砌块式(6)，通过糖基化获得保护的支链三糖中间体式(68)，然后经后阶段的非还原端脱保护、亚磷酰化-氧化、脱保护、硫酸化、脱苄基，得到式(XI)确定位点不完全硫酸化支链磷酰甘露三糖；所述制备方法包括：
    步骤(1)：以D-甘露糖为原料，依次经过乙酰化、酚苷化，得到化合物式(65)；再依次经过脱乙酰基、2,3:4,6-O-双亚苄基保护，得到化合物式(65)；然后，在还原开环条件下， 得到单糖合成砌块式(67)，其反应如路线(24)所示；
    Figure PCTCN2016077370-appb-100035
    路线(24)中：a_全乙酰化；b_酚苷化；c_脱乙酰基化；d_2,3:4,6-O-双亚苄基保护；e_还原开环；
    步骤(2)：将所述单糖合成砌块式(67)和式(6)在糖基化条件下反应，得到支链三糖中间体式(68)，其反应如路线(25)所示；
    Figure PCTCN2016077370-appb-100036
    步骤(3)：将所述化合物式(68)，经脱除乙酰丙酰基的条件处理，得到二醇式(69)；再依次在亚磷酰化、氧化条件下，得到双磷酸酯化合物式(70)；然后，在脱除乙酰基的条件下，得到化合物式(71)；最后，依次经O-硫酸化条件、脱除苄基的条件的处理，得到式(XI)确定位点不完全硫酸化支链磷酰甘露三糖，其反应如路线(26)所示；
    Figure PCTCN2016077370-appb-100037
  28. 一种按权利要求27所述方法合成的式(XI)确定位点不完全硫酸化支链磷酰甘露三糖。
  29. 如权利要求1-10之任一项所述的化合物在制备抗肿瘤药物中的应用。
  30. 如权利要求1-10之任一项所述的化合物在制备抑制血管生成药物中的应用。
  31. 根据权利要求1所述的化合物在制备具有抑制血管生成活性的如式(III)所示的化合物 中的应用。
  32. 根据权利要求2所述的化合物在制备具有抑制血管生成活性的如式(IV)所示的化合物中的应用。
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