CN113527050A

CN113527050A - Intermediate of vaccine adjuvant MPLA, synthesis and application

Info

Publication number: CN113527050A
Application number: CN202010305744.XA
Authority: CN
Inventors: 高祺; 隋强; 李�根; 郑致伟; 韩子怡; 薛俊娣
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2021-10-22
Anticipated expiration: 2040-04-17
Also published as: CN113527050B

Abstract

The invention discloses an intermediate of vaccine adjuvant MPLA, synthesis and application. The intermediate provided by the invention takes Nap as a protecting group, and can be conveniently removed in subsequent operations; the synthesis route is short, and the total yield is obviously increased. Provides a basis for the synthesis and amplification of MPLA.

Description

Intermediate of vaccine adjuvant MPLA, synthesis and application

Technical Field

The invention relates to an intermediate of vaccine adjuvant MPLA, synthesis and application.

Background

MPLA is the innermost liposome (Lipid a) derived from endotoxin (LPS) in the cell wall of gram-negative bacteria, Monophosphoryl Lipid a (hereinafter abbreviated as MPL). Lipid A is an amphiphilic structure, is also a key structure of gram-negative bacteria with toxicity and immunogenicity, and can cause immune response of organisms. It can therefore be added to vaccines as an adjuvant to increase the immunogenicity of the vaccine; generally, although the attenuated live vaccine has strong immunogenicity, the attenuated live vaccine has high toxicity and may have pathogenic risks; and the non-toxic inactivated vaccine has weaker immunogenicity. The addition of the adjuvant can enhance the immunogenicity of the inactivated vaccine and does not bring pathogenic risk to the weak. Meanwhile, the use amount of the antigen is reduced, and the supply amount of the vaccine is increased. MPL is the first FDA-approved human novel immunoadjuvant substance other than aluminum salts. MPL acts on toll-like receptor 4(TLR4), and has the advantages of strong immunogenicity, clear mechanism and low toxicity. However, the existing aluminum salt adjuvant has an undefined mechanism, generates red swelling at an injection site, has a higher pain probability than MPL, and cannot generate specific cellular immunity. A plurality of new vaccine varieties adopting the MPL adjuvant system are sold in the market at home and abroad, such as hepatitis B vaccine Fendrix, cervical cancer vaccine Cerivix, herpes zoster vaccine Shingrix and malaria vaccine Mosquirix. And more than twenty are in clinical stage.

Because MPL derived from different bacteria or different serotypes of the same bacteria has different structures, the basic difference is the number/connection position of fatty chains and the carbon chain length of the fatty chains, for example, the synthesized structure of the patent is shown as the following formula;

n1 and n3 are independently 10, 12 or 14, and n2 and n4 are independently 8, 10 or 12.

The existing MPL source depends on biological fermentation extraction, the cost is high, the problem of heterogeneity of lipopolysaccharide is difficult to solve in the extraction process, the purity problem is easily caused, and the potential safety hazard is brought. Based on special physicochemical properties and complex structures, chemical methods are very difficult to synthesize MPL, and biological fermentation is difficult to replace. The total synthesis of MPL and its Lipid A analogues has been reported to be limited to milligram levels, and the ligands that normally provide phosphate groups are typically benzyl pyrophosphate (N, N-diazopyro) phosphine) or O-xylylene N, N-diethylphosphoramidite (N, N-diethyl-1,5-dihydro-3H-2,4, 3-benzodioxaphospho-3-amine) as the source of phosphate groups, with Bn being a permanent protecting group. These protecting groups must eventually be removed using hydrogenation conditions. For example, when the prior literature is used for preparing similar lipid A and derivatives thereof, the benzyl is used, Pd/C is required to react for 10-20 h under the atmospheric pressure or hydrogen pressurization condition, the impurities are more, and the yield is 45-68%. And the purification mode is complicated, and the method needs to use regenerated cellulose for filtration and remove the catalyst by ultrasound; then DEAE-cellulose ion exchange resin is used for separation, and a plurality of mixed solvents with different components are used as mobile phases to flush out the product. And repeatedly separating and concentrating to obtain the final product. (see Ref 1: Alla Zamyyana, Harald Sekljic, Helmut Brade. Synthesis and purity assessment of tetra-and pentaacyl Lipid A of Chlamydiacontaining (R) -3-hydroxyicosanoic acid. Tetrahedron 60(2004) 12113-12137. Ref 2: Kaustabh K.Maiti, Michael Decasto, Abu-Baker M.Abdel-Aal El-Sayed.chemical Synthesis and ProInflammatory reactions of Monophoroyl Lipid A Adjuvantation Candidata. Eur.J.Org.Chem.2010, 80-91).

The synthesis routes reported by the total synthesis of the MPL analogue at present are all longer; except that the benzyl protecting group used is difficult to remove completely at later deprotection; in order to avoid side reactions in the reaction, some temporary protecting groups are usually needed, so that a plurality of protecting groups are involved, and repeated protection and deprotection are needed; in the reported literature, the linking and connecting order of four fatty chains is different, or four fatty chains (0+4) or 3+1 are connected after glycosylation, so that the fatty chain linking and connecting selectivity is not strong, or the steric hindrance is large, the selectivity is low during glycosylation, and the like. ([1] J.AM.CHEM.SOC.2007,129, 5200-5216; [2] WO 2013072768; [3] The chemical record, Vol 6,333-343(2006))

Therefore, in the current literature reports, the hydrogenation result is not ideal, the yield is low, impurities are more, the reaction time is long (generally more than 20 hours), the purification conditions are complicated, and the large-scale production is difficult to meet the commercial requirements.

Therefore, a new preparation method with short route and high total yield is urgently needed so as to synthesize MPLA in large quantity.

Disclosure of Invention

The invention aims to overcome the defect of lack of the preparation method of the traditional vaccine adjuvant MPLA, and provides an intermediate, synthesis and application of the vaccine adjuvant MPLA. The intermediate synthesized by the invention has short route and obviously increased total yield. The key intermediate of MPLA is adopted, and MPLA can be obtained after deprotection, thus providing a foundation for the synthesis and amplification of MPLA.

The present invention solves the above-mentioned problems by the following technical means.

The invention provides a preparation method of a compound shown as a formula 20, which comprises the following steps: in a mixed solvent of an organic solvent and water, in the presence of alkali, carrying out hydrolysis reaction on a compound shown as a formula 24 as shown in the specification to obtain a compound shown as a formula 20;

the organic solvent may be a cyclic ether solvent (e.g., tetrahydrofuran, THF).

The volume ratio of the organic solvent to water can be from 1:1 to 1:2 (e.g., from 1:1.2 to 1:1.5, and further e.g., from 1: 1.33).

The amount of the mixed solvent may not be particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 24 to the mixed solvent may be 10g/L to 200g/L (e.g., 50g/L to 100g/L, and for example, 80 g/L).

The base may be an alkali metal hydroxide (e.g., one or more of lithium hydroxide, sodium hydroxide, and potassium hydroxide, again, for example, lithium hydroxide). The base may be in the form of an aqueous solution.

The molar ratio of the base to the compound of formula 24 can be 3:1 to 10:1 (e.g., 5:1 to 8: 1).

The temperature of the hydrolysis reaction may be 50 ℃ to 100 ℃ (e.g., the azeotropic temperature of the mixed solvent, further e.g., 60 ℃ to 65 ℃).

The progress of the hydrolysis reaction can be monitored by means conventional in the art (e.g., TLC, HPLC or LCMS), and is generally at the end of the reaction when the compound of formula 24 is absent or no longer reduced; the hydrolysis reaction time is preferably 0.5 to 12 hours (e.g., 1 hour to 5 hours).

The workup of the hydrolysis reaction may be a workup conventional in the art, for example comprising the steps of: after the hydrolysis reaction is finished, quenching until the pH value is 7, diluting with an organic solvent, washing, drying an organic phase, filtering, concentrating, separating and purifying to obtain the product. The quenching may be with a saturated sodium bicarbonate solution. The quenching to pH 7 may be carried out using aqueous HCl (e.g., 1.5M). The organic solvent diluted by the organic solvent can be halogenated alkane solvent (such as CH)₂Cl₂). The washing may be with a saturated sodium bicarbonate solution. The separation and purification is preferably column chromatography, the packing for column chromatography separation can be silica gel column, and the eluent for column chromatography separation can be petroleum ether and ethyl acetate (for example, petroleum ether: ethyl acetate: 5: 1).

The preparation method of the compound shown in the formula 20 can further comprise the following steps: in an organic solvent, in the presence of a silicon reagent, a reducing agent and a Lewis acid, a compound shown as a formula 23 and 2-naphthaldehyde are mixed

Carrying out Nap protection reaction shown as follows to obtain the compound shown as the formula 24;

the operation and reaction conditions of the Nap protection reaction can be the operation and reaction conditions which are conventional in the similar Nap protection reaction in the field; in the present invention, the following are preferred:

in the Nap protection reaction, the organic solvent may be a cyclic ether solvent, and the cyclic ether solvent may be Tetrahydrofuran (THF).

The amount of the organic solvent to be used is not particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 23 to the organic solvent may be 5g/L to 100g/L (e.g., 10g/L to 50g/L, and for example, 33 g/L).

The silicon reagent may be a silicon reagent conventional in this type of reaction in the art, which is silylation protected for the hydroxyl group of compound 23; for example hexamethyldisiloxane (silyl ether, (TMS)₂O) and/or chlorotrimethylsilane, for example hexamethyldisiloxane.

The reducing agent can be triethylsilane (Et)₃SiH)。

The lewis acid may be a triflate, such as trimethylsilyltrifluoromethane sulfonate (TMSOTf).

The molar ratio of the silicon reagent to the compound of formula 23 can be 1:1 to 10:1 (e.g., 4:1 to 8:1, and further e.g., 6: 1).

The molar ratio of the reducing agent to the lewis acid may be 3:1 to 10:1 (e.g., 4:1 to 6:1, such as 4.4: 1).

The molar ratio of the reducing agent to the compound of formula 23 can be 1:1 to 5:1 (e.g., 3:1 to 4:1, and further e.g., 3.5: 1).

The molar ratio of the 2-naphthaldehyde to the compound of formula 23 can be 0.5:1 to 2:1 (e.g., 0.7:1 to 1:1, and further e.g., 0.8: 1).

The Nap protection reaction can be at a temperature of-20 ℃ to 30 ℃ (e.g., -10 ℃ to 10 ℃).

In the Nap protection reaction, the following steps can be adopted: and adding the reducing agent and the Lewis acid into a mixed system of the compound shown as the formula 23, the 2-naphthaldehyde and the organic solvent to carry out the Nap protection reaction.

The progress of the Nap protection reaction can be monitored by means conventional in the art (e.g., TLC, HPLC or LCMS), and is generally the end point of the reaction when the compound represented by formula 23 is absent or no longer reduced; the Nap protection reaction time is preferably 0.5 to 12 hours (e.g., 1 to 5 hours).

The workup of the Nap protection reaction can be a workup conventional in the art, for example comprising the steps of: after the Nap protection reaction is finished, diluting with an organic solvent, washing, concentrating, separating and purifying to obtain the Nap-based catalyst. The organic solvent diluted by the organic solvent can be halogenated alkane solvent (such as CH)₂Cl₂). The washing may be with a saturated sodium bicarbonate solution. The separation and purification is preferably column chromatography separation or crystallization, and the solvent of the crystallization can be petroleum ether and ethyl acetate (for example, petroleum ether: ethyl acetate ═ 5: 1).

The invention provides a compound shown as the following formula 20 and 24,

the invention also provides a preparation method of the compound shown as the formula 24, which comprises the following steps: in an organic solvent, in the presence of a silicon reagent, a reducing agent and a Lewis acid, a compound shown as a formula 23 and 2-naphthaldehyde are mixed

in the Nap protection reaction, the organic solvent may be a cyclic ether solvent, and the cyclic ether solvent may be tetrahydrofuran THF. The amount of the organic solvent to be used is not particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 23 to the organic solvent may be 5g/L to 100g/L (e.g., 10g/L to 50g/L, and for example, 33 g/L).

The silicon reagent may be a silicon reagent conventional in this type of reaction in the art, which is silylation protected for the hydroxyl group of compound 23; for example hexamethyldisiloxane (silyl ether, (TMS)₂O) and/or chlorotrimethylsilane.

The reducing agent can be triethylsilane (Et)₃SiH)。

The lewis acid may be a triflate (e.g., trimethylsilyltrifluoromethanesulfonate TMSOTf).

The workup of the Nap protection reaction can be a workup conventional in the art, for example comprising the steps of: after the Nap protection reaction is finished, diluting with an organic solvent, washing, concentrating, separating and purifying to obtain the Nap-based catalyst. The organic solvent diluted by the organic solvent can be halogenated alkane solvent (such as CH)₂Cl₂). The washing may be with a saturated sodium bicarbonate solution. The separation and purification is preferably column chromatography separation or crystallization, and the solvent of the crystallization can be petroleum ether and ethyl acetate (for example, petroleum ether: ethyl acetate: 5:1 by volume).

The invention also provides an application of the compound shown in the formula 20 in preparing the compound shown in the formula 18; it may comprise the steps of:

the method comprises the following steps of (1) carrying out amidation reaction on a compound shown as a formula 14 and a compound shown as a formula 20 in an organic solvent in the presence of a condensing agent to obtain the compound shown as the formula 15; wherein n1 and n3 are independently 10, 12 or 14, and n2 and n4 are independently 8, 10 or 12; and when n2 and n4 are 10, n1 and n3 are not simultaneously 12;

step (2), in an organic solvent, in the presence of a TBS removing protective agent, carrying out TBS (TBDMS, tert-butyl dimethyl) removing protecting group reaction on a compound shown as a formula 15 to obtain the compound shown as a formula 16;

step (3), in an organic solvent, in the presence of alkali, HCOOH, a Pd catalyst and a phosphine ligand, carrying out an allyl (allyl) protecting group removing reaction on a compound shown as a formula 16 to obtain the compound shown as a formula 17;

step (4), in an organic solvent, carrying out a Nap protecting group removing reaction on a compound shown as a formula 17 and DDQ (2, 3-dichloro-5, 6-dicyan-p-benzoquinone) as shown in the specification to obtain an MPLA compound shown as a formula 18;

in the present invention, in the general formula

Is conventional in the art and denotes the alpha configuration, beta configuration, or a mixture of alpha and beta configurations.

In a certain aspect, n1 and n3 are independently 10, 12, or 14, and n2 and n4 are independently 10; and n1 and n3 are not both 12 at the same time.

In a certain embodiment, n1 and n3 are 10 and n2 and n4 are 10.

In a certain aspect, n1 is 10, n3 is 14, and n2 and n4 are 10.

In a certain aspect, n1 is 12, n3 is 10, and n2 and n4 are 10.

In step (1), the operation and reaction conditions of the amidation reaction may be those conventional in the art for such amidation reactions; in the present invention, the following are preferred:

in the amidation reaction, the organic solvent may be a halogenated alkane solvent, and the halogenated alkane solvent may be dichloromethane and/or trichloromethane. The amount of the organic solvent to be used is not particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 14 to the organic solvent may be 50g/L to 200g/L (e.g., 80g/L to 100 g/L).

The condensing agent may be one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), Dicyclohexylcarbodiimide (DCC) and N, N' -Diisopropylcarbodiimide (DIC), for example 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.

The molar ratio of the condensing agent to the compound of formula 14 can be 1:1 to 10:1 (e.g., 4:1 to 8:1, such as 6: 1).

The molar ratio of the compound shown in the formula 14 to the compound shown in the formula 20 can be 1:1-1:4 (for example, 1:2-1: 3).

The temperature of the amidation reaction may be from 0 ℃ to 50 ℃ (e.g., 10 ℃ to 30 ℃).

The amidation reaction may be carried out under the protection of inert gas, and the inert gas may be nitrogen or argon.

The progress of the amidation reaction can be monitored by means conventional in the art (e.g., TLC, HPLC or LCMS), and is generally at the end of the reaction when the compound of formula 14 is absent or no longer reduced; the time for the amidation reaction is preferably 5 to 24h (e.g., 10h to 15 h).

The work-up of the amidation reaction may be a work-up conventional in the art, for example comprising the following steps: and after the amidation reaction is finished, concentrating, separating and purifying to obtain the product. The separation and purification is preferably a column chromatography separation, and the eluent for the column chromatography separation can be a mixed solution of toluene and ethyl acetate (for example, toluene/ethyl acetate is 10:1 by volume).

In the step (2), the operation and reaction conditions of the TBS protecting group removing reaction can be those conventional in the TBS protecting group removing reaction in the field; in the present invention, the following are preferred:

in the TBS protecting group removing reaction, the organic solvent can be a cyclic ether solvent, and the cyclic ether solvent can be tetrahydrofuran THF. The amount of the organic solvent to be used is not particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 15 to the organic solvent may be 5g/L to 100g/L (e.g., 10g/L to 50g/L, and for example, 33 g/L).

The TBS removing protective agent can be a hydrofluoric acid pyridine complex (HF/pyridine, also called Olah reagent, wherein the mass percentage concentration of HF is 65-70%); preferably a pyridine solution of pyridine hydrofluoric acid complex (e.g., a 3-6.5 fold pyridine diluted solution of pyridine hydrofluoric acid complex).

The volume-to-mass ratio of the TBS removal protective agent to the compound shown in the formula 15 can be 1mL/g-20mL/g (for example, 3 mL/g-10 mL/g).

The temperature of the TBS deprotection reaction may be from-80 ℃ to 50 ℃ (e.g., -40 ℃ to 30 ℃).

In the TBS protecting group removing reaction, the following steps can be adopted: and adding the TBS removing protective agent into the mixed system of the compound shown as the formula 15 and the solvent to carry out the TBS removing protective group reaction. The addition temperature may be from-70 ℃ to-30 ℃ (e.g., -40 ℃). After the addition is finished, the temperature of the TBS deprotection reaction can be 0-30 ℃.

The progress of the TBS deprotection reaction can be monitored by means conventional in the art (e.g., TLC, HPLC or LCMS), and is generally determined as the end point of the reaction when the compound represented by formula 15 disappears or the content thereof is not reduced; the TBS deprotection reaction time is preferably 1 to 24 hours (e.g., 2 to 12 hours).

The post-treatment of the TBS deprotection reaction may be a post-treatment conventional in the art, for example, comprising the steps of: and after the TBS protecting group removing reaction is finished, quenching, extracting by using an organic solvent, drying, filtering, concentrating, separating and purifying to obtain the TBS. The quenching may be with a saturated sodium bicarbonate solution. The extracted organic solvent may be a haloalkane solvent (e.g., chloroform and/or dichloromethane). The separation and purification is preferably column chromatography separation, the filler for column chromatography separation can be C18 filler, and the eluent for column chromatography separation can be CH in sequence₃CN, MeOH/EA (e.g. M)eOH/EA 8:1 by volume) and MeOH.

In the step (3), the operation and reaction conditions of the deallyl protecting group reaction can be the operation and reaction conditions which are conventional in the deallyl protecting group reaction in the field; in the present invention, the following are preferred:

in the deallyl protecting group reaction, the organic solvent may be a cyclic ether solvent, and the cyclic ether solvent may be Tetrahydrofuran (THF). The amount of the organic solvent to be used is not particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 16 to the organic solvent may be 1g/L to 50g/L (e.g., 10g/L to 25 g/L).

In the deallyl protecting group reaction, the base can be an organic base, and the organic base can be Triethylamine (TEA) and/or n-butylamine (n-bunH)₂)。

The Pd catalyst can be Pd (PPh)₃)₄。

The phosphine ligand may be PPh₃。

The amount of the Pd catalyst may be a conventional amount, for example, the molar ratio of the Pd catalyst to the compound represented by formula 16 may be 0.1:1 to 0.5:1 (e.g., 0.2:1 to 0.4: 1).

The molar ratio of the phosphine ligand to the Pd catalyst can be 2:1 to 5:1 (e.g., 4.4:1 to 4.75: 1).

The molar ratio of the base to the Pd catalyst can be 20:1 to 50:1 (e.g., 42:1 to 45: 1).

The molar ratio of the HCOOH to the Pd catalyst can be 20:1 to 50:1 (e.g., 42:1 to 45: 1).

The temperature at which the deallyl protecting group is reacted can be from 0 ℃ to 50 ℃ (e.g., 10 ℃ to 30 ℃).

The progress of the reaction for removing the allyl protecting group can be monitored by means conventional in the art (such as TLC, HPLC or LCMS), and is generally the end point of the reaction when the compound shown in formula 16 disappears or the content is not reduced any more; the reaction time for the deallyl protecting group is preferably 0.5 to 24 hours (e.g., 1.5 to 12 hours).

Said deallyl protecting groupThe work-up of the reaction may be a work-up as conventional in the art, for example comprising the steps of: and after the reaction of the deallyl protecting group is finished, concentrating, separating and purifying to obtain the allyl-protecting group-removing catalyst. The separation and purification is preferably column chromatography separation, and the packing of the column chromatography can be C18 packing; the column chromatography separation can comprise the following steps: using CH with 0.1% TEA₃CN, MeOH containing 0.1% TEA, CH containing 0.1% TEA₂Cl₂The elution was carried out sequentially with MeOH as eluent.

In the step (4), the operation and reaction conditions of the reaction for removing the Nap protecting group can be the operation and reaction conditions which are conventional in the reaction for removing the Nap protecting group in the field; in the present invention, the following are preferred:

the organic solvent can be halogenated alkane solvent, and the halogenated alkane solvent can be dichloromethane and/or trichloromethane. The amount of the organic solvent to be used is not particularly limited so as not to affect the reaction; for example, the mass-to-volume ratio of the compound represented by formula 17 to the organic solvent may be 1g/L to 50g/L (e.g., 10g/L to 20 g/L).

The molar ratio of the compound of formula 17 to DDQ can be 1:2 to 1:220 (e.g., 1:4 to 1:20, e.g., 1:6 to 1: 8). Since 4 equivalents of Nap are present per molecule, compound 17 is obtained in high yield when DDQ is used in conventional amounts (e.g., a molar ratio of DDQ to Nap of about 1:1 to 2.25: 1).

The temperature of the reaction for removing the Nap protecting group can be 10 ℃ to 50 ℃ (for example, 20 ℃ to 30 ℃).

The Nap deprotection reaction can be carried out under the protection of inert gas, and the inert gas can be nitrogen or argon.

The reaction for removing the Nap protecting group is preferably carried out under ultrasonic conditions to accelerate the reaction process.

The process of the reaction for removing the Nap protecting group can be monitored by means conventional in the art (such as TLC, HPLC or LCMS), and is generally the end point of the reaction when the compound shown in the formula 17 disappears or the content is not reduced any more; the reaction time for removing the Nap protecting group is preferably 0.5 to 6 hours (e.g., 1 hour to 1.5 hours).

The post-treatment for the reaction for removing the Nap protecting group may be a post-treatment conventional in the art, and may include, for example, the following steps: after the reaction for removing the Nap protecting group is finished, quenching, concentrating, separating and purifying to obtain the Nap protecting group. The quenched solvent can be an amine organic base (e.g., triethylamine). The separation and purification is preferably column chromatography separation, and the packing of the column chromatography can be C18 packing; the column chromatography separation can comprise the following steps: removing excess DDQ by using a nitrile solvent (such as acetonitrile), and eluting by using a mixed solvent of a haloalkane solvent (such as dichloromethane) and an alcohol solvent (such as methanol) as an eluent; the volume ratio of the halogenated alkane solvent to the alcohol solvent can be 2: 1-4:1 (for example, 3: 1).

In the present invention, room temperature means 10 to 30 ℃ unless otherwise specified; "h" means hours; "overnight reaction" means reaction for 8-16 hours.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the invention takes Nap as a protecting group, and can be conveniently removed in subsequent operations; the synthesis route is short, and the total yield is obviously increased. Provides a basis for the synthesis and amplification of MPLA.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Compound 18-a series of examples

Preparation of starting Compound 1

After a reaction flask added with 2-deoxy-1-oxo- (1, 1-dimethylethyl) dimethylsilyl-2- [ (2,2, 2-trichloroethoxy) carbonyl ] amino-3, 4, 6-triacetyl-beta-D-glucose (10g, 16.8mmol) is slowly added with a guanidinidine hydrochloride buffer solution (100mL, pH 8), stirred at room temperature for 3.5h, TLC detection raw material is consumed, the reaction solution is neutralized with a cationic resin, filtered and concentrated, the product is extracted with dichloromethane and a saturated sodium bicarbonate solution, the organic layer is collected and concentrated to obtain 2-deoxy-1-oxo- (1, 1-dimethylethyl) dimethylsilyl-2- [ (2,2, 2-trichloroethoxy) carbonyl ] amino-beta-D-glucose (1-1,8.23 g).

In a reaction flask, 1-1 and 2- (dimethoxymethyl) -naphthalene (5.1g, 25mmol, 1.5eq) were dissolved in 50mL acetonitrile, camphorsulfonic acid (0.39g, 1.69mmol, 0.1eq) was added, the reaction was stirred at room temperature for 4h, triethylamine was added to neutrality, the reaction solution was extracted with dichloromethane and saturated sodium bicarbonate solution, and the solution was separated. The organic phase was dried and dried to give a yellow solid. The crude product was passed through a silica gel frit funnel (PE: EA ═ 5:1) to afford product 1 (pale yellow solid, 6.97g) in 68.3% yield over two steps.

Compound 1:¹H NMR(400MHz,CDCl₃)δ7.96–7.50(m,7H),5.72(s,1H),5.17(d,J＝6.3Hz,1H), 4.88(d,J＝7.7Hz,1H),4.73(q,J＝12.0Hz,2H),4.36(dd,J＝10.5,5.0Hz,1H),4.13–4.01(m,1H),3.86 (t,J＝10.3Hz,1H),3.70–3.58(m,1H),3.52(td,J＝9.7,5.0Hz,1H),3.47–3.35(m,1H),2.96(s,1H),0.94 (d,J＝8.2Hz,9H),0.20–0.08(m,6H).

¹³C NMR(101MHz,CDCl₃)δ154.54,134.52,133.78,132.87,128.41,128.29,127.75,126.69,126.41, 126.07,123.93,101.97,96.33,95.30,81.52,74.85,70.71,68.68,66.20,60.73,26.94,25.59,17.90,-4.14,- 5.26.

preparation of starting Compound 23

Compound 23 was prepared by reference to the following reference and ee was determined using the same method: belma Hasdemir, Hu lya

Onar,

Asymmetry synthesis of long chain β -hydroxy fatty acid esters as new elastase inhibitors tetrahedron: Asymmetry (23)2012, 1100-1105. The document title is not normative, has been modified

Step (1) preparation of Compound 22

Meldrum's acid (64.8g, 0.45mol) and pyridine (48mL) were dissolved in CH₂Cl₂To (100mL) was added lauroyl chloride (21, 65.6g, 0.3mol) at 0 ℃. Stir at room temperature for 2.5 hours. After complete consumption of starting material, wash with 1M HCl (100mL) and water (100 mL). The organic layer was dried, filtered and concentrated. It was dissolved in methanol (250mL) and refluxed overnight. After the intermediate is consumed, the intermediate is concentrated and passes through Al₂O₃Purification by column chromatography (toluene: ethyl acetate 2: 1) gave 22 as a pale yellow solid (59.3g, 77%).

¹H NMR(400MHz,CDCl₃)δ3.70(3H,d,J＝1.5),3.42(2H,s),2.50(2H,t,J＝7.4),1.28(18H,s), 0.90(3H,t,J＝6.6).

¹³C NMR(101MHz,CDCl₃)δ202.51,167.58,137.67,128.96,128.15,125.25,52.02,48.84,42.90, 31.92,29.62,29.47,29.38,29.35,29.01,23.43,22.68,21.31,14.04.

Step (2) preparation of Compound 23

Preparation of ruthenium catalyst: 180mg of (R) -Ru (OAc)₂(BINAP) (diacetate [ (R) - (+) -2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl group)]Ruthenium (II), cas number: 325146-81-4) in CH₂Cl₂(5mL), 1.42N HCl (0.35mL) was added, and the mixture was stirred at room temperature for 1 hour and then spin-dried.

Compound 22(15g) was dissolved in methanol (50mL) with the prepared ruthenium catalyst. H₂Stirring for 6h at 65 ℃ under the condition of 1.5 MPa. After the starting material was completely completed, the reaction mixture was concentrated and purified by silica gel column chromatography (petroleum ether)Ethyl acetate 5:1) to give compound 23 (white solid, 15g, 98%, ee 98.7%).

¹H NMR(400MHz,CDCl₃)δ4.01(1H,dq,J＝11.8,4.0),3.71(3H,s),2.90(1H,d,J＝4.0),2.46(2 H,ddd,J＝25.4,16.4,6.1),1.26(18H,s),0.88(3H,t,J＝6.8).

¹³C NMR(101MHz,CDCl₃)δ173.41,67.99,51.63,41.18,36.57,31.89,29.60,29.56,29.51,29.32, 25.47,22.65,14.05.

Reaction condition screening for the preparation of compound 23:

relationship of substrate to catalyst ratio (S/C) to ee value: the reaction is completed in about 4-6h at 50 ℃ with the amount of S/C-300 catalyst, and the yield reaches over 95%. To reduce the use of catalyst, a reaction of 180mg of catalyst per 20g of substrate was attempted (S/C >300) with a yield of only 71%.

Compound 22(20g) was dissolved in methanol (50mL) with 180mg of the prepared ruthenium catalyst. H₂Stirring for 6h at 65 ℃ under the condition of 1.5 MPa. After the starting material was completely removed, the reaction mixture was concentrated and purified by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) to obtain compound 3 (white solid, 10.7g, 71%, ee 92.3%).

Analysis of the ee values of the products under the different conditions showed that the catalyst was small and the yield decreased and the ee value was only 92%. The ee value of several batches of S/C300 products is above 98%.

EXAMPLE 1 preparation of Compound 24

Compound 23(10g, 39mmol,1eq) and 2-naphthaldehyde (18.14g, 116mmol,3eq) were dissolved in THF (100mL) and TMSOTf (6.88g, 31mmol,0.8eq), (TMS) was added under ice bath₂O (37.68g, 232mmol,6eq) and Et₃SiH (15.7 g, 135mmol,3.5 eq). Reacting at 0 deg.C for 1.5 hr, and reacting with CH₂Cl₂Diluted (150mL) and saturated NaHCO₃And (6) washing. The organic layer was spin dried and recrystallized (to give methylnaphthalene,white solid at room temperature) (petroleum ether: ethyl acetate ═ 5:1) and filtered to remove impurities (methylnaphthalene). The filtrate (containing compound 24) was collected and spin-dried to give crude 24 as a pale yellow oily liquid, which was used in the next step without purification.¹H NMR(400MHz,CDCl₃)0.89(3H,t，J6.7),1.27–1.64(18H,m),2.35(2H,dd,J 8.4)2.49 (1H,dd,J15.0,5.3),2.62(1H,dd,J15.0,7.3),3.68(3H,s),3.88(1H,m),4.54(2H,m),7.25–7.35(7H,m).

EXAMPLE 2 preparation of Compound 20

Compound 24 prepared in example 1 was dissolved in THF-H₂O solution (5: 1, 100mL), aqueous lithium hydroxide (9.41g, 224mmol, 94mL) was added and refluxed for 12 h. After disappearance of the starting material, it was cooled to room temperature and quenched by addition of 1.5M aqueous HCl to pH 7. Subjecting the mixture to CH₂Cl₂(150mL) diluted with saturated NaHCO₃And (6) washing. The organic phase was dried and spin dried. Purification by silica gel column chromatography (5: 1 petroleum ether/ethyl acetate) gave compound 20(11.6g, 77.9% yield in two steps, colorless syrup).

¹H NMR(400MHz,CDCl₃)δ10.00(1H,s,OH),7.85(4H,dd,J＝14.5,10.8),7.53(3H,d,J＝4.2), 4.85–4.71(2H,m,NapCH₂O),4.08–3.94(1H,m,H-3),2.76(1H,dd,J＝15.2,6.9,H-2),2.64(1H,dd,J ＝15.2,4.3,H-2),1.84–1.58(2H,m,H-4),1.57–1.31(18H,m),1.00(3H,t,J＝6.0).

¹³C NMR(101MHz,CDCl₃)δ177.86,135.86,133.38,133.10,128.20,128.00,127.76,126.57,126.10, 125.99,125.89,75.94,71.71,39.81,34.37,32.05,29.77,29.71,29.48,25.27,22.82,14.26.

EXAMPLE 3 preparation of Compound 2

Reacting compound 19-A(5g,11.7mmol) and EDC & HCl (2.25g,11.7mmol, 1.2eq) in CH₂Cl₂(50mL), stirred at room temperature for 15min, then added with Compound 1(5.93g,9.8mmol) and DMAP (0.06g,0.5mmol, 0.05eq), stirred at room temperature for 10h, and reacted with CH in turn₂Cl₂(100mL) and saturated NaHCO₃(60mL) washing. The fractions were dried, concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to give compound 2-a (8.6g, 86.7%, colorless syrup). TOF-MS M/z 1036.46[ M + Na ]]⁺。

δ_H(400MHz,CDCl₃)7.91(1H,s),7.82(3H,dt,J 9.3,4.9),7.54(1H,d,J 8.5),7.50–7.44(2H,m), 5.65(1H,s,NapCH),5.40–5.29(2H,m,H-3,NH),5.17(1H,dd,J 12.2,6.2,lipid-H-3),4.88(1H,d,J 7.8, H-1),4.69(2H,dd,J 48.4,12.0,Troc),4.34(1H,dd,J 10.5,4.9,H-6),3.84(1H,t,J 10.3,H-6),3.75(1H,t, J 9.4,H-4),3.62(1H,dd,J 16.8,7.7,H-2),3.54(1H,td,J 9.7,5.0,H-5),2.57(2H,ddd,J 20.5,15.2,6.3), 2.12(2H,t,J 7.4),1.56–1.41(4H,m),1.32–1.13(34H,m),0.92–0.85(15H,m),0.11(6H,d,J 8.6).

δ_C(101MHz,CDCl₃)173.41,170.19,154.12,134.29,133.71,132.85,128.38,128.11,127.67,126.47, 126.16,125.81,123.73,101.76,96.82(C-1),95.31,78.95(C-4),74.66,71.14(C-3),69.98,68.72(C-6), 66.47(C-5),59.11(C-2),39.26,34.31,33.83,31.93,29.63,29.50,29.35,29.29,29.06,25.53,25.07,24.93, 22.69,17.88,14.13,-4.21,-5.29.

EXAMPLE 4 preparation of Compound 3-a

Compound 2-a (2g,1.97mmol) was dissolved in THF (20mL) and BH was added sequentially under ice bath conditions₃·Me₃N(0.57g,7.69 mmol，3.9eq)，AlCl₃(1.52g,11.4mmol)，H₂O (69mg,3.83mmol, 1.9eq) the reaction was stirred at room temperature for 1.5 h. Water (20mL) and 1M HCl solution (20mL) were added to quench, and the reaction solution was quenched with CH₂Cl₂(30mL), drying, concentrating, and purifying by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to obtainTo compound 3-a (1.72g, 86%, colorless oily liquid). TOF-MS M/z 1038.52[ M + Na ]]⁺.

δ_H(400MHz,CDCl₃)7.85–7.75(4H,m),7.46(3H,dd,J 7.7,4.0),5.37(1H,d,J 9.2,NH),5.15(1H, s,lipid-H-3),5.03(1H,t,J 9.7,H-3),4.81–4.71(4H,m,H-1,NapCH₂,Troc),4.63(1H,d,J 11.9,Troc), 3.81(2H,qd,J 10.5,3.8,H-6),3.69(1H,t,J 9.8,H-4),3.65–3.54(2H,m,H-2,H-5),3.47(1H,s,OH), 2.62–2.48(2H,m,lipid-H-2),2.28(2H,t,J 7.3,lipid’-H-2),1.57(4H,s,lipid’-H-3,lipid-H-4),1.25(34H, s),0.88(15H,s),0.12(6H,d,J 17.5).

δ_C(101MHz,CDCl₃)174.35,171.77,154.27,135.54,133.29,133.02,128.20,127.89,127.71,126.29, 126.11,125.88,125.56,96.50(C-1),75.91(C-3),74.72(C-5),74.61,73.73,70.96,70.19(C-4),70.01(C-6), 60.43,57.85(C-2),40.10,34.58,34.50,31.94,29.65,29.56,29.52,29.37,29.30,29.14,25.61,25.16,24.99, 22.71,21.06,17.93,14.21,14.14,-4.06,-5.28.

EXAMPLE 5 preparation of Compound 4

(1)

The compound 3-a (0.5g,0.489mmol) and tetrazole (102mg,1.45mmol,3eq) were dissolved in extra dry acetonitrile (10mL), allyl ligand (hexadiene-N, N-diisopropylphosphoramidite, 0.24g,0.978mmol,2eq) was added, and the reaction was carried out at room temperature for 40min, after which the starting material was consumed.

mCPBA (211mg,1.22mmol,2.5eq) was dissolved in super dry dichloromethane (15mL) at-40 ℃, the reaction system was added, slowly warmed to-10 ℃, quenched with saturated sodium thiosulfate solution (20mL) after 40min of reaction, washed with saturated sodium bicarbonate (40mL), and purified by dry spin-dry silica gel column chromatography (petroleum ether: ethyl acetate ═ 8:1) to give compound 4-a (0.53g, 92%). TOF-MS M/z 1198.67[ M + Na ]]⁺.

δ_H(400MHz,CDCl₃)7.85–7.77(4H,m),7.50–7.44(3H,m),5.90–5.73(2H,m,CH₂＝CH-CH₂O-), 5.41–5.16(6H,m,NH,lipid-H-3,CH₂＝CH-CH₂O-),4.97(1H,d,J 7.9,H-1),4.79-4.63(4H,td,J 12.3,12.4,12.0,Troc,Nap-CH₂),4.49–4.38(5H,m,H-4,CH₂＝CH-CH₂O-),3.85(1H,d,J 9.6,H-6),3.75(1 H,d,J 5.4,H-6),3.72–3.65(1H,m,H-5),3.50–3.40(1H,m,H-2),2.64–2.60(2H,m),2.28(2H,t,J 7.4),1.58(4H,br,J 7.3),1.25(34H,s),0.91–0.86(15H,m),0.13(6H,d,J 19.2).

δ_C(101MHz,CDCl₃)173.57,170.37,153.95,135.65,133.27,132.98,132.27,132.21,132.14,128.09, 127.87,127.68,126.23,126.05,125.81,125.65,118.56,118.40,95.63(C-1),95.32,74.59,74.12(C-5), 74.06(C-4),73.61,72.42(C-3),70.06,68.70(C-6),68.62,68.56,68.46,68.41,58.67(C-2),39.59,34.51,34.24, 31.92,29.66,29.64,29.57,29.52,29.35,29.20,25.61,25.17,25.07,22.69,22.57,17.93,14.12,-4.15,-5.25.

(2)

Dissolving the compound 3-a (0.5g,0.489mmol) and tetrazole (102mg,1.45mmol,3eq) in ultra-dry acetonitrile (10mL), adding allyl ligand (0.18g,0.73mmol,1.5eq), reacting at room temperature for 2h until the raw material is not consumed, supplementing 0.5eq of allyl ligand, and consuming the raw material after 30 min. mCPBA (211mg,1.22mmol,2.5eq) was dissolved in super dry dichloromethane (15mL) at-40 ℃, added to the reaction system, slowly raised to-10 ℃, quenched after 30min reaction with saturated sodium thiosulfate solution (20mL), washed with saturated sodium bicarbonate (40mL), and purified by dry spin-dry silica gel column chromatography (petroleum ether: ethyl acetate ═ 8:1) to give compound 4-a (0.49g, 84.7%).

(3)

Dissolving the compound 3-a (0.5g,0.489mmol) and tetrazole (171mg,2.45mmol,5eq) in ultra-dry acetonitrile (10mL), adding allyl ligand (0.24g,0.978mmol,2eq), reacting at room temperature for 40min until the raw material is consumed, dissolving mCPBA (211mg,1.22mmol,2.5eq) in ultra-dry dichloromethane (15mL) at-40 ℃, adding the reaction system, slowly raising the temperature to-10 ℃, quenching after 40min, adding saturated sodium thiosulfate solution (20mL), washing with saturated sodium bicarbonate (40mL), and purifying by dry spin-drying silica gel column chromatography (petroleum ether: ethyl acetate 8:1) to obtain the compound 4-a (0.474g, 82.3%).

EXAMPLE 6 preparation of Compound 5-a

Compound 4-a (0.8g,0.68mmol) was dissolved in THF (24mL), HF/pyridine (2.4mL, 65-70%) diluted in 15mL pyridine was added at-40 ℃. Slowly heating to room temperature for reaction for 12h, and using NaHCO to react reaction liquid₃(40mL) quench and add CH₂Cl₂(50mL) was separated, dried and purified by spin-dried silica gel column chromatography (petroleum ether: ethyl acetate 4:1) to give compound 5-a (0.657g, 91%, white solid). TOF-MS M/z 1084.48[ M + Na ]]⁺。

δ_H(400MHz,CDCl₃)7.85–7.77(4H,m),7.51–7.44(3H,m),5.91–5.65(2H,m,CH₂＝CH-CH₂O-), 5.61(1H,d,J 9.4,NH),5.42–5.26(3H,m,H-1,CH₂＝CH-CH₂O-),5.23–5.09(4H,m,H-3,CH₂＝CH- CH₂O-,lipid-H-3),4.80-4.69(4H,m,Troc,Nap-CH₂),4.45–4.40(3H,m,H-4,CH₂＝CH-CH₂O-),4.36– 4.27(2H,m,CH₂＝CH-CH₂O-),4.25–4.19(1H,m,H-5),4.01–3.94(1H,m,H-2),3.84–3.75(2H,m,H- 6),2.60(2H,ddd,J 21.4,16.2,6.3),2.24(2H,t,J 7.6),1.56(4H,d,J 7.0),1.25(34H,s),0.88(6H,t,J 6.8).

δ_C(101MHz,CDCl₃)173.38,170.76,154.31,135.19,133.23,133.04,132.28,132.22,132.09,132.02, 128.23,127.92,127.70,126.67,126.15,125.95,125.81,118.66,118.38,95.37,91.50(C-1),74.65,73.63(C- 4),70.82(C-3),69.90,69.57,69.52(C-5),68.67,68.61,68.47,68.40,68.35(C-6),54.27(C-2),39.10,34.46, 34.15,31.92,29.64,29.57,29.53,29.36,29.32,29.18,25.18,25.01,22.69,14.12.

EXAMPLE 7 preparation of Compound 6-a

Compound 5-a (1.3g,1.22mmol) was dissolved in ultra dry (dry) CH₂Cl₂(20mL)，N₂2,2, 2-trifluoro-N-phenylacetylimine chloride (1.52g,7.32mmol, 6eq) and DBU (371mg,2.44mmol, 2eq) were added under protection and reacted at room temperature for 30 mins. The reaction was purified by column chromatography (petroleum ether: ethyl acetate 15:1with 0.1% Et)₃N) gave compound 6-a (1.11g, 74%, colorless syrup). TOF-MS M/z 1255.48[ M + Na ]]⁺。

EXAMPLE 8 preparation of Compound 7

(1)

Compound 1(3.39g,5.58mmol) and compound 20(4.29g,11.16mmol,2eq) were added to a reaction flask, after addition of 40mL of dichloromethane, EDC · HCl (2.14g,11.16mmol,2eq) and DMAP (135mg,1.1mmol,0.2eq) were added under ice-bath, and compound 1 was consumed after stirring at room temperature for 3 h. Saturated sodium bicarbonate solution (25mL) was added to wash the separated liquid, dried, filtered, dried and purified by recrystallization (dichloromethane: methanol ═ 1: 12) to obtain compound 7(5.08g, 93.5%). TOF-MS M/z 994.41[ M + Na ]]⁺。

δ_H(400MHz,CDCl₃)7.81(1H,s),7.76–7.61(7H,m),7.48–7.35(5H,m),7.30(1H,d,J 8.4),5.50 (1H,s,NapCH),5.39(1H,t,J 9.9,H-3),5.24(1H,d,J 9.1,NH),4.84(1H,d,J 7.8,H-1),4.67–4.62(3H, m,NapCH_2,-OCH₂CCl₃),4.52(1H,d,J 11.8,-OCH₂CCl₃),4.31(1H,dd,J 10.4,4.7,H-6),3.85–3.81(1H, m,Lipid-H-3),3.78(1H,d,J 10.3,H-6),3.71(1H,t,J 9.5,H-4),3.67–3.60(1H,m,H-2),3.53(1H,td,J 9.5,5.1,H-5),2.74(1H,dd,J 14.9,6.0,Lipid-H-2),2.55(1H,dd,J 14.8,5.6,Lipid-H-2),1.54(2H,m, Lipid-H-4),1.27(18H,s),0.91(12H,s),0.13(6H,d,J 12.0).

δ_C(101MHz,CDCl₃)171.82,154.36,136.11,134.41,133.85,133.41,133.13,132.99,128.53,128.28, 128.23,128.11,127.88,127.85,126.60,126.51,126.31,126.20,126.04,125.96,125.91,123.85,101.88,97.14 (C-1),79.18(C-4),75.73(C-6),74.88,71.39(C-3),71.28,68.91(C-6),66.76(C-5),60.64,59.29(C-2),39.77, 34.71,32.15,29.87,29.86,29.80,29.77,29.58,25.74,25.45,22.92,21.28,18.08,14.43,14.37,-3.98,-5.08.

(2)

EDC & HCl (1.087g,5.67mmol,1.2eq), compound 20(2.18g,5.67mmol,1.2eq) was added to a reaction flask, 25mL of dichloromethane was added, and after stirring at room temperature for 15min, compound 1(2.87g,4.72mmol), DMAP (29mg,0.237mmol, 0.05eq) were added and the reaction was continued for 12h with compound 1 being unconsumed. Saturated sodium bicarbonate solution (25mL) was added to wash the separated liquid, dried, filtered, dried and purified by column chromatography (petroleum ether: ethyl acetate: 7: 1) to obtain compound 7(2.72g, 59.23%).

(3)

EDC & HCl (1.81g,9.44mmol,2eq), compound 20(2.54g,6.6mmol,1.4eq) were added to a reaction flask, 25mL of dichloromethane was added, and after stirring at room temperature for 15min, compound 1(2.87g,4.72mmol), DMAP (29mg,0.237mmol, 0.05eq) were added and the reaction was continued for 12h with compound 1 unconsumed. Saturated sodium bicarbonate solution (25mL) was added to wash the separated liquid, dried, filtered, dried and purified by column chromatography (petroleum ether: ethyl acetate: 7: 1) to obtain compound 7(3.27g, 71.2%).

(4)

EDC & HCl (2.65g,13.8mmol,2eq) and compound 20(5.33g,13.8mmol,2eq) were added to a reaction flask, 50mL of dichloromethane was added, and after stirring at room temperature for 15min, compound 1(4.12g,6.78mmol), DMAP (170mg,1.39mmol,0.2 eq) were added and the reaction was run for 12h with compound 1 being consumed. Saturated sodium bicarbonate solution (50mL) was added to wash the separated liquid, dried, filtered, dried and purified by column chromatography (petroleum ether: ethyl acetate: 7: 1) to obtain compound 7(5.22g, 79.1%).

(5)

Compound 1(3.39g,5.58mmol) and compound 20(4.29g,11.16mmol,2eq) were added to a reaction flask, after addition of 40mL of dichloromethane, EDC · HCl (2.14g,11.16mmol,2eq) and DMAP (135mg,1.1mmol,0.2eq) were added under ice-bath, and compound 1 was consumed after stirring at room temperature for 3 h. Saturated sodium bicarbonate solution (25mL) was added to wash the separated liquid, dried, filtered, dried and purified by column chromatography (petroleum ether: ethyl acetate: 7: 1) to obtain compound 7(4.71g, 86.7%).

(6)

Compound 1(3.39g,5.58mmol) and compound 20(4.29g,11.16mmol,2eq) were added to a reaction flask, after addition of 40mL of dichloromethane, EDC · HCl (2.14g,11.16mmol,2eq) and DMAP (135mg,1.1mmol,0.2eq) were added under ice-bath, and compound 1 was consumed after stirring at room temperature for 3 h. Saturated sodium bicarbonate solution (25mL) was added to wash the separated liquid, dried, filtered, dried and purified by recrystallization (dichloromethane: methanol ═ 1:8) to obtain compound 7(3.93g, 72.4%).

EXAMPLE 9 preparation of Compound 8

Compound 7(6g,6.16mmol) was dissolved in methylene chloride (60mL), and acetic acid (12mL,2V) and zinc powder (6g,1V) (V means mass equivalent) were added thereto, and the mixture was stirred vigorously at room temperature for 2 hours to complete the reaction, followed by washing with saturated sodium bicarbonate (60mL) and saturated brine (60 mL). Separation, drying and concentration gave compound 8(5.3 g). TOF-MS M/z 821.134[ M + Na ]]⁺. Directly used for the next reaction.

EXAMPLE 10 preparation of Compound 9

(1)

Compound 8 prepared in example 9 was dissolved in dichloromethane (100mL), FmocCl (3.2g,12.36mmol,2eq) and DIPEA (1.6g,12.36mmol,2eq) were added under ice bath, the reaction was completed by stirring at room temperature for 2h, and the mixture was washed with saturated brine (100mL), separated, dried and recrystallized (dichloromethane: methanol 1: 10) to obtain compound 9(5.6g, 89.1%). TOF-MS M/z 1042.7[ M + Na ]]⁺。

δ_H(400MHz,CDCl₃)7.83(1H,s),7.78–7.65(7H,m),7.64–7.44(5H,m),7.40(6H,ddd,J 15.3, 9.7,5.3),7.32–7.24(3H,m),5.53(1H,s,NapCH),5.44(1H,t,J 9.4,H-3),4.97(1H,d,J 8.7,NH),4.91 (1H,d,J 7.2,H-1),4.63(1H,d,J 11.8,Fmoc-CH₂),4.51(1H,d,J 11.8,,Fmoc-CH₂),4.32(1H,br,H-6), 4.28(2H,d,J 6.5,NapCH₂),4.17(1H,d,J 6.4,,Fmoc-CH),3.82(2H,s,H-6,Lipid-H-3),3.78–3.71(1H, m,H-4),3.67(1H,d,J 8.9,H-2),3.58(1H,br,H-5),2.70(1H,dd,J 14.8,6.3),2.50(1H,dd,J 14.8,5.5), 1.55–1.37(2H,m),1.29–0.96(18H,m),0.91–0.81(12H,m),0.09(6H,d,J 13.5).

δ_C(101MHz,CDCl₃)155.78,143.79,141.23,135.92,134.24,133.63,133.17,132.87,132.78,128.31, 128.05,127.92,127.86,127.63,127.04,126.34,126.26,126.06,125.93,125.81,125.69,125.15,123.64, 119.96,101.65,97.14(H-1),79.00(H-4),75.62(H-6),71.25(H-3),71.19,68.76(H-6),67.19,66.60(H-5), 58.98(H-2),47.04,39.74,34.53,31.92,29.61,29.53,29.34,25.49,25.16,22.69,17.86,14.14,-4.21,-5.36.

(2)

Compound 7(6g,6.16mmol) was dissolved in methylene chloride (60mL), acetic acid (12mL,2V) and zinc powder (6g,1V) were added, the mixture was stirred vigorously at room temperature for 2 hours, and the reaction was completed, followed by saturated sodium bicarbonate (60mL) and saturated brine (60 mL). Separation, drying and concentration gave compound 8(5.3 g). This was dissolved in dichloromethane (100mL), FmocCl (3.2g,12.36mmol,2eq), DIPEA (1.6g,12.36mmol,2eq) were added under ice-bath, the reaction was completed by stirring at room temperature for 2h, and the mixture was washed with saturated brine (100mL), dried by liquid separation and then recrystallized (methanol, 5V) to obtain compound 9(4.91g, 78.1%).

EXAMPLE 11 preparation of Compound 10

Compound 9(1.5g,1.47mmol) is reacted with molecular sieves (b), (d)

1.5g) in ultra-dry CH₂Cl₂(60mL)，N₂Triethylsilane (0.55mL,3.67mmol,2.5eq) and PhBCl were added at-78 ℃ under protection₂(0.76mL,5.88mmol) and the reaction was stirred at-78 deg.C for 1 h. After the reaction is finished, methanol (6mL) is added for quenching, and the pH value is adjusted to 8 by triethylamine. The reaction solution was filtered and then saturated NaHCO₃(10mL) washing. Dry spin-dry silica gel column chromatography (petroleum ether: ethyl acetate ═ 6:1) afforded compound 10(1.2g, 80.1%, colorless oily liquid). TOF-MS m/z:1044.45[M+Na]⁺。

δ_H(400MHz,CDCl₃)7.75–7.73(1H,m),7.70(7H,dd,J 14.7,5.4),7.66(2H,d,J 5.3),7.62(2H,d, J 5.8),7.53(1H,d,J 3.9),7.44–7.38(6H,m),7.30–7.25(3H,m),5.32(1H,t,J 9.8,H-3),4.93(1H,d,J 9.3,NH),4.79(1H,d,J 6.6,H-1),4.73(2H,q,J 11.7,NapCH₂),4.57(2H,dd,J 24.5,11.7,Fmoc-CH₂), 4.24(2H,d,J 7.1,Lipid-NapCH₂),4.17–4.10(1H,m,Fmoc-CH),3.85-3.80(2H,s,H-6,Lipid-H-3),3.74 (1H,d,J 3.9,H-4),3.73–3.69(1H,m,H-6),3.60(1H,dd,J 18.4,9.4,H-2),3.49(1H,d,J 8.3,H-5),2.55 (1H,dd,J 15.6,7.1),2.39(1H,dd,J 15.5,4.9),1.55–1.39(2H,m),1.22–1.06(18H,m),0.87–0.82(12 H,m),0.08(6H,d,J 13.4).

δ_C(101MHz,CDCl₃)172.00,155.84,143.86,141.23,135.99,135.05,133.23,133.15,132.98,132.92, 128.26,128.04,127.95,127.87,127.69,127.65,127.04,126.67,126.29,126.14,126.00,125.97,125.87, 125.79,125.74,125.22,119.96,96.66(H-1),75.72(H-4),75.28(H-5),74.64(H-3),71.47(Fmoc-CH₂),67.24 (Lipid-NapCH₂),62.00(H-6),58.48(H-2),53.46,47.06(Fmoc-CH),39.74,34.19,31.96,29.67,29.65,29.62, 29.60,29.38,25.52,25.15,22.73,17.90,14.17,-4.07,-5.25.

EXAMPLE 12 preparation of Compound 11

(1)

The reaction flask was charged with acceptor compound 10(606mg,0.593mmol), donor compound 6-a (1.1g,0.89mmol,1.5eq) molecular sieve (S) ((R))

600mg)，N₂Adding ultra-dry CH under the condition₂Cl₂(10mL), 100-fold dilution of TfOH (10. mu.L, 0.12mmol, 0.2eq) with dichloromethane at-20 ℃ and stirring for 40min at-20 ℃; quench with methanol (5mL) and triethylamine to pH 8. CH for reaction solution₂Cl₂(35mL) and saturated NaHCO₃(10mL) washing. The organic phase was concentrated by drying and purified by column chromatography (PE: EA ═ 10:1) to give the glycosidated product 11-a (1.03g, 84%). TOF-MS M/z 2088.14[ M + Na ]]⁺。

δ_H(400MHz,CDCl₃)7.80–7.21(29H,m),5.89–5.68(2H,m,diallyl),5.48(1H,d,J 7.0,NH’),5.37 (1H,t,J 9.6,H-3’),5.32–5.10(6H,m,H-3,lipid-H-3,diallyl),4.87(1H,d,J 9.1,NH),4.80(1H,d,J 7.7, H-1’),4.74(1H,d,J 3.3,H-1),4.70(4H,d,J 12.8,Troc,Nap),4.64(2H,d,J 3.3,Nap-CH₂),4.53(2H,dd, J 29.1,11.9,Fmoc),4.45(1H,s,H-4’),4.43–4.31(5H,m,H-4,diallyl),4.24(2H,d,J 6.8,acceptor-lipid- Nap),4.16–4.11(1H,m,Fmoc-CH),4.07(1H,d,J 10.3,H-6),3.83(2H,d,J 10.2,H-6,acceptor-lipid-H- 3’),3.72(2H,dd,J 10.3,6.0,H-6’),3.65–3.54(3H,m,H-2,H-5,H-5’),3.41(1H,dd,J 17.7,8.4,H-2’), 2.61(2H,t,J 6.6),2.50(1H,dd,J 15.6,7.1),2.35(1H,dd,J 15.8,5.1),2.28(2H,t,J 7.4),1.59(6H,d,J 7.7),1.42(4H,ddd,J 14.5,10.8,6.1),1.32–1.21(52H,m),0.86(18H,dd,J 14.9,7.8),0.15–0.05(6H, m).

δ_C(101MHz,CDCl₃)δ172.65,170.83,169.04,154.72,152.94,142.82,140.16,134.94,134.60,134.29, 132.19,132.15,132.08,131.91,131.84,131.82,131.29,131.22,131.13,131.06,129.85,127.80,127.12, 127.06,126.93,126.85,126.79,126.62,126.60,126.55,125.97,125.32,125.17,125.10,125.02,124.90, 124.85,124.78,124.70,124.67,124.61,124.18,118.86,117.44,117.27,99.00(C-1’),95.53(C-1),94.38, 75.08(C-5’),74.58(C-5)73.71(C-3),73.43(C-4’),73.31(C-4),73.11,73.05,72.96,72.91,72.60,71.37(C-3’), 70.37,69.10,67.55(C-6),67.49(C-6’),67.34,67.28,66.14,64.51,57.32(C-2),55.78(C-2’),46.00,38.78, 38.64,33.50,33.41,33.13,30.89,28.67,28.63,28.61,28.59,28.54,28.51,28.31,28.17,24.54,24.15,24.07, 24.03,21.66,18.16,16.82,13.12,13.10,-4.91,-6.31.

(2)

Compound 6-a (50mg,0.04mmol) and compound 10(51mg,0.049mmol,1.2eq) were charged to a reaction flask, 1mL of ultra dry dichloromethane was added under nitrogen protection, and 100 fold diluted TfOH (0.25mg,0.00167mmol, 0.05eq) was added at-20 ℃. After 1h compound 6-a (donor) was consumed and compound 10 (acceptor) was not consumed. Methanol is added to quench triethylamine and adjust to be neutral. Purification by column chromatography (toluene: ethyl acetate ═ 12: 1) gave compound 11-a (49mg, 58.5%). Since the product 11-a is close in polarity to the acceptor 10, it is more polar than the donor 6-a. Therefore, when the acceptor 10 is excessive, the isolated yield is low although the reaction conversion rate is substantially equivalent.

(3)

Compound 6-a (100mg,0.081mmol, 1.2eq) and compound 10(69mg,0.067mmol) were charged into a reaction flask, 1mL of ultra dry dichloromethane was added under nitrogen protection, and 100-fold diluted TfOH (0.5mg,0.0033mmol, 0.05eq) was added at-20 ℃. After 1h, the donor acceptor is not consumed, and the reaction time is not changed after the reaction is prolonged. Methanol is added to quench triethylamine and adjust to be neutral. Purification by column chromatography (toluene: ethyl acetate ═ 12: 1) gave compound 11-a (58mg,0.0278mmol, 41.7%).

(4)

Compound 6-a (200mg,0.162mmol, 1.5eq) and compound 10(110mg,0.107mmol) were charged to a reaction flask, 1mL of ultra dry dichloromethane was added under nitrogen protection, and TfOH (0.8mg,0.0053mmol, 0.05eq) diluted 100-fold was added at-20 ℃. After 1h, the donor acceptor is not consumed, and the reaction time is not changed after the reaction is prolonged. Methanol is added to quench triethylamine and adjust to be neutral. Purification by column chromatography (toluene: ethyl acetate ═ 12: 1) gave compound 11-a (107mg, 48.2%).

(5)

Compound 6-a (200mg,0.162mmol, 1.5eq) and compound 10(110mg,0.107mmol) were charged to a reaction flask, 1mL of ultra dry dichloromethane was added under nitrogen protection, and TfOH (2.43mg,0.0162mmol, 0.15eq) diluted 100-fold was added at-20 ℃. The donor was consumed after 1 h. Methanol is added to quench triethylamine and adjust to be neutral. Purification by column chromatography (toluene: ethyl acetate ═ 12: 1) gave compound 11-a (192mg, 86.48%). TOF-MS M/z 2088.14[ M + Na ]]⁺。

EXAMPLE 13 preparation of Compound 12-a

In a reaction flask11-a (860mg,0.029mmol) in CH₂Cl₂(10mL)，N₂Under the conditions, zinc powder (2.7g, 40mmol) and acetic acid (2.7mL, 45mmol) were added and stirred at room temperature for 2h, after completion of the reaction, the reaction mixture was filtered, and the filtrate was azeotropically dried with toluene and subjected to column chromatography (PE: EA ═ 6:1) to give compound 12-a. TOF-MS M/z 1912.06[ M + Na ]]⁺。

EXAMPLE 14 preparation of Compound 13-a (MPL-8)

Compound 12-a, prepared in example 13, was dissolved in ultra dry DCM (10mL), N₂EDC & HCl (2g,10.43mmol) and aliphatic chain 19-A (1.48g,3.47mmol) were added at-10 deg.C, the reaction stirred for 12h and purified by spin-dry column chromatography (toluene/ethyl acetate 10:1) to give compound 13-a (680mg, 71%, colorless clear syrup). TOF-MS M/z 2323.15[ M + Na ]]⁺。

EXAMPLE 15 preparation of Compound 14-a

Compound 13-a (580mg,0.252mmol) was dissolved in DMF (12mL), N in a reaction flask₂Triethylamine (12mL, 86mmol) was added under these conditions, the mixture was stirred at room temperature overnight, and the reaction mixture was purified by column chromatography (PE: EA ═ 6:1) by spin drying to give compound 14-a. TOF-MS M/z 2030.2[ M + Na ]]⁺。

EXAMPLE 16 preparation of Compound 15-a

Compound 14-a, prepared in example 15, is dissolved in ultra dry CH₂Cl₂(5mL)，N₂EDC & HCl (295mg,1.53mmol) and aliphatic chain 20(296mg,0.77mmol) were added at room temperature, the reaction mixture was stirred at room temperature for 12h, and purification was performed by spin-dry column chromatography (toluene/ethyl acetate ═ 5:1) to give the compound15-a (480mg, yield 78%, colorless clear syrup). TOF-MS M/z 2468.4[ M + Na ]]⁺。

EXAMPLE 17 preparation of Compound 16-a

Compound 15-a (300mg, 0.123mmol) was dissolved in THF (10mL), and a solution of HF/Py (3mL, 65-70%) in pyridine (9mL) was added at-40 ℃. Warm to room temperature and stir overnight. After the reaction, saturated aqueous sodium bicarbonate solution was added to quench, and chloroform was added to extract the solution several times. The organic layer was dried, filtered and concentrated and purified by a C18 packing (CH)₃CN, MeOH/EA ═ 8:1, MeOH eluting sequentially) gave 16-a (0.214g, 75%, white solid).

δ_H(600MHz,CDCl₃)7.77–7.24(28H,m),6.28(1H,d,J 9.5,NH),6.22(1H,d,J 7.4,NH’),5.77– 5.70(1H,m,allyl),5.67–5.59(1H,m,allyl),5.44–5.31(3H,m,H-3,H-1’,H-3’),5.22–5.03(6H,m, allyl,H-1,lipid-H-3),4.99(1H,t,J 5.8,lipid-H-3),4.67–4.57(5H,m,Nap),4.52–4.42(3H,m,Nap), 4.35–4.21(5H,m,H-4’,diallyl),4.17(1H,td,H-2),4.05(1H,d,J 7.2,H-5),3.87(1H,d,J 11.7,H-6’), 3.80–3.74(3H,m,lipid-H-3*2,H-6’),3.67–3.56(4H,m,H-4’,H-6*2,H-5’),3.32–3.28(1H,m,H-2’), 3.27(1H,t,J 11.6,7.7,H-4),2.55–2.43(3H,m),2.28–2.11(9H,m),1.45–1.37(8H,m),1.22–1.10 (108H,m),0.80(18H,dt,J 7.0,5.1).

δ_C(151MHz,CDCl₃)173.28,172.55,170.87,170.29,169.85,168.98,135.06,134.48,133.92,132.31, 132.23,132.19,132.10,131.96,131.90,131.86,131.22,131.17,131.11,131.06,127.15,127.09,127.05, 126.91,126.83,126.66,126.57,125.46,125.41,125.36,125.24,125.06,124.99,124.93,124.88,124.85, 124.82,124.73,124.64,124.61,117.45,117.30,98.02(C-1’),90.43(C-1),75.46(C-4),74.52,73.50,73.23, 73.09,72.86(C-5’),72.51(C-3),71.64(C-3’),70.60(C-5),70.36,69.75,69.33,67.55(C-4’),67.51,67.28(C- 6’),67.24,66.43(C-6),59.37,55.37(C-2’),51.48(C-2),41.01,40.61,38.95,38.86,33.63,33.54,33.40,33.34, 30.92,28.64,28.56,28.35,28.21,24.17,24.06,23.89,21.67,13.09.

EXAMPLE 18 preparation of Compound 17-a

(1)

The compound 16-a (240mg, 0.103mmol), PPh₃(25mg,0.095mmol) was charged into a reaction flask, and after nitrogen blanketing, THF (10mL), TEA (125. mu.L, 0.9mmol), HCOOH (75. mu.L, 1.957mmol), Pd (Ph) were added₃)₄(25mg,0.02mmol, 0.2 eq). Reacting at 25 deg.C for 5 hr, reacting for 12 hr, removing allyl, and spin-drying on C18 column CH₃CN with 0.1％TEA,MeOH with 0.1％TEA,CH₂Cl₂MeOH eluting sequentially with 0.1% TEA afforded compound 17-a (184mg, 79.65%).

(2)

The compound 16-a (240mg, 0.103mmol), PPh₃(50mg,0.190mmol) was added to the reaction flask, and after nitrogen blanketing, THF (10mL), TEA (250. mu.L, 1.8mmol), HCOOH (150. mu.L, 3.914mmol), Pd (Ph) were added₃)₄(50mg,0.043mmol, 0.4 eq). After the reaction is completed for 1.5h at 25 ℃, the reaction solution is spun through a C18 column CH₃CN with 0.1％TEA,MeOH with 0.1％TEA, CH₂Cl₂MeOH with 0.1% TEA afforded compound 17-a (225mg, 97.4%). TOF-M: M/z:2248.76[ M-H]⁺。

When PPh contains two equivalents of allyl per molecule₃And other agents in conventional amounts (e.g., PPh)₃A molar ratio to allyl of about 0.9:1 to 1.1: 1), compound 17 can be obtained in high yield.

EXAMPLE 19 preparation of Compound 18-a

(1)

Compound 17-a (50mg,0.022mmol) was mixed with DDQ (1.1g,4.84mmol, 22)0eq) was added to the reaction flask, and dry CHCl was added under nitrogen protection₃(5 mL). And (3) finishing the reaction after ultrasonic treatment at 30 ℃ for 60 min. Quench with a drop of triethylamine and spin dry the reaction. Acetonitrile was added to the spin-dried reaction flask, and the mixture was poured into a column chromatography packed with C18 to decolorize and purify the mixture. When the excess DDQ is completely removed, CH₂Cl₂MeOH ═ 3:1 gave compound 18-a (25mg, 67.6%) as the final product. (since DDQ is greatly beyond the conventional amount, in the case of a small feed scale, although the reaction yield can reach the theoretical value, a large amount of residual DDQ affects the yield of the post-treatment purification)

(2)

The compound 17-a (50mg,0.022mmol) and DDQ (10mg,0.044mmol,2eq) were added to a reaction flask, and dried CHCl was added under nitrogen protection₃(5 mL). And (3) finishing the reaction after ultrasonic treatment at 30 ℃ for 60 min. Quench with a drop of triethylamine and spin dry the reaction. Acetonitrile was added to the spin-dried reaction flask, and the mixture was poured into a column chromatography packed with C18 to decolorize and purify the mixture. When the excess DDQ is completely removed, CH₂Cl₂MeOH ═ 3:1 gave compound 18-a (17mg, 45.9%) as the final product. As in (4), the yield can be greatly improved by prolonging the reaction time.

(3)

The compound 17-a (50mg,0.022mmol) and DDQ (40mg,0.177mmol, 8eq) were added to a reaction flask, and dried CHCl was added under nitrogen protection₃(5 mL). The reaction is completed after 30 ℃ ultrasonic treatment for 60 min. Quench with a drop of triethylamine and spin dry the reaction. Acetonitrile was added to the spin-dried reaction flask, and the mixture was poured into a column chromatography packed with C18 to decolorize and purify the mixture. When the excess DDQ is completely removed, CH₂Cl₂MeOH ═ 3:1 gave compound 18-a (29mg, 78.4%) as the final product. As in (4), the yield can be greatly improved by prolonging the reaction time.

(4)

The compound 17-a (50mg,0.022mmol) and DDQ (40mg,0.177mmol, 8eq) were added to a reaction flask, and dried CHCl was added under nitrogen protection₃(5 mL). The reaction is completed after 30 ℃ ultrasonic treatment for 80 min. Quench with a drop of triethylamine and spin dry the reaction. Acetonitrile was added to the spin-dried reaction flask, and the mixture was poured into a column chromatography packed with C18 to decolorize and purify the mixture. When the excess DDQ is completely removed, CH₂Cl₂MeOH ═ 3:1 gave compound 18-a (35.8mg, 95.26%, HPLC purity: 98.5%) as a final product. TOF-MS M/z 1689.36[ M-H ]]⁺。

Examples of Compound 18-b series

EXAMPLE 20 preparation of Compound 13-b

11-a (400mg,0.194mmol) was dissolved in CH in a reaction flask₂Cl₂(5mL)，N₂Adding zinc powder 1.2g) and acetic acid (1.2 mL) under the condition, stirring at room temperature for 2h, filtering after the reaction is finished, performing azeotropic spin-drying on the filtrate by using toluene and performing column chromatography (PE: EA is 6:1) to obtain a compound 12-a (TOF-MS: M/z 1915.49[ M + Na & lt/EN & gt verified by MS)]⁺)

Compound 12-a was dissolved in ultra dry DCM (5mL), N₂EDC & HCl (0.892g,4.656mmol) and fatty chain 19-C (749mg,1.552mmol) (cas:93390-37-5) were added at-10 deg.C, the reaction was stirred for 12h and then purified by spin-column chromatography (toluene/ethyl acetate ═ 10:1) to give compound 13-b (722.7mg, 63.4%, colorless clear syrup). TOF-MS M/z 2380.26[ M + Na ]]⁺。

EXAMPLE 21 preparation of Compound 14-b

Compound 13-b (600mg,0.254mmol) was dissolved in DMF (12mL), N in a reaction flask₂Triethylamine (12mL) was added under these conditions, stirred at room temperature overnight, and the reaction was purified by column chromatography (PE: EA ═ 6:1) to give compound 14-b (verified by MS). TOF-MS M/z 2158.02[ M + Na ]]⁺。

EXAMPLE 22 preparation of Compound 15-b

Dissolving Compound 14-b in SuperDry CH₂Cl₂(5mL)，N₂EDC · HCl (390.35mg,2.036mmol) and fatty chain 20(390.7mg,1.016mmol) were added at room temperature, the reaction mixture was stirred at room temperature for 12h, and purified by spin-dry column chromatography (toluene/ethyl acetate ═ 5:1) to give compound 15-b (504mg, 79.3% colorless clear syrup). TOF-MS M/z 2524.56[ M + Na ]]⁺。

EXAMPLE 23 preparation of Compound 16-b

Compound 15-b (500mg,0.2 mmol) was dissolved in THF (18mL), and a solution of HF/Py (5mL, 65-70%) in pyridine (15mL) was added at-40 ℃. Warm to room temperature and stir overnight. After the reaction, saturated aqueous sodium bicarbonate solution was added to quench, and chloroform was added to extract the solution several times. The organic layer was dried, filtered and concentrated and purified by a C18 packing (CH)₃CN, MeOH/EA ═ 8:1, MeOH) gave 16-b (0.351g, 73.5%, white solid). TOF-MS M/z 2410.34[ M + Na ]]⁺。

EXAMPLE 24 preparation of Compound 17-b

The compound 16-b (470mg, 0.196mmol), PPh₃(90mg,0.370mmol) was added to the reaction flask, and after nitrogen blanketing, THF (20mL), TEA (490. mu.L, 3.5mmol), HCOOH (290. mu.L, 7.8mmol), Pd (Ph) were added₃)₄(90mg,0.08 mmol). After the reaction is completed for 1.5h at 25 ℃, the reaction solution is spun through a C18 column CH₃CN with 0.1％TEA,MeOH with 0.1％TEA,CH₂Cl₂MeOH with 0.1% TEA afforded compound 17-b (428mg, 94.3%). TOF-M: M/z:2330.16[ M + Na ]]⁺。

EXAMPLE 25 preparation of Compound 18-b

Will combine withThe compound 17-b (90mg,0.039mmol) and DDQ (71mg,0.312mmol, 8eq) were charged to a reaction flask and dried CHCl was added under nitrogen₃(10 mL). The reaction is completed after 30 ℃ ultrasonic treatment for 80 min. Quench with a drop of triethylamine and spin dry the reaction. Acetonitrile was added to the spin-dried reaction flask, and the mixture was poured into a column chromatography packed with C18 to decolorize and purify the mixture. When the excess DDQ is completely removed, CH₂Cl₂MeOH ═ 3:1 gave compound 18-b (65mg, 95.26%) as a final product. TOF-MS M/z 1745.47[ M-H ]]⁺. HPLC purity: 97.5 percent.

Examples of the series of Compounds 18-c

EXAMPLE 26 preparation of Compound 2-c

Compound 19-B (cas:163310-36-9) (2.613g,5.93mmol) and EDC & HCl (1.136g,5.93mmol, 1.2eq) were dissolved in CH₂Cl₂(25mL), after stirring at room temperature for 15min, compound 1(3g,4.94mmol) and DMAP (0.03g,0.247mmol, 0.05eq) were added, and the mixture was stirred at room temperature for 10h to complete the reaction, and the reaction solution was successively reacted with CH₂Cl₂(50mL) and saturated NaHCO₃(30mL) washed. The fractions were dried, concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to give compound 2-c (4.29g, 84.3%, colorless syrup). TOF-MS M/z 1052.7[ M + Na ]]⁺。

EXAMPLE 27 preparation of Compound 3-c

Compound 2-c (1g,0.97mmol) was dissolved in THF (10mL) and BH was added sequentially under ice bath conditions₃·Me₃N(0.28g,3.783 mmol，3.9eq)，AlCl₃(0.76g,5.7mmol)，H₂O (35mg,1.92mmol, 1.9eq) the reaction was stirred at room temperature for 1.5 h. Water (10mL) and 1M HCl solution (10mL) were added to quench, and the reaction solution was quenched with CH₂Cl₂(15mL), followed by drying, concentration and purification by silica gel column chromatography (petroleum ether/ethyl acetate 10:1) to give a compound3-c (0.882g, 87.6%, colorless oily liquid). TOF-MS M/z 1059.42[ M + Na ]]⁺.

EXAMPLE 28 preparation of Compound 4-c

The compound 3-c (0.5g,0.482mmol) and tetrazole (102mg,1.45mmol,3eq) were dissolved in extra dry acetonitrile (10mL), allyl ligand (hexadiene-N, N-diisopropylphosphoramidite, 0.236g,0.964mmol,2eq) was added, and the reaction was carried out at room temperature for 40min, after which the starting material was consumed. mCPBA (207.9mg,1.205mmol,2.5eq) was dissolved in ultra-dry dichloromethane (15mL) at-40 ℃, the reaction system was added, the temperature was slowly raised to-10 ℃, the reaction was quenched by addition of saturated sodium thiosulfate solution (20mL) after 40min, washed with saturated sodium bicarbonate (40mL), and purified by dry spin-dry silica gel column chromatography (petroleum ether: ethyl acetate ═ 8:1) to give compound 4-c (0.537g, 93.3%). TOF-MS M/z 1214.8[ M + Na ]]⁺.

EXAMPLE 29 preparation of Compound 5-c

Compound 4-c (1.5g,1.26mmol) was dissolved in THF (45mL), HF/pyridine (4.8 mL, 65-70%) diluted in 30mL pyridine was added at-40 ℃. Slowly heating to room temperature for reaction for 12h, and using NaHCO to react reaction liquid₃(80mL) quench and add CH₂Cl₂(100mL) and dried by spin-dry silica gel column chromatography (petroleum ether: ethyl acetate 4:1) to give compound 5-c (1.26g, 93.4%, white solid). TOF-MS M/z 1100.53[ M + Na ]]⁺。

EXAMPLE 30 preparation of Compound 6-c

Compound 5-c (1.3g,1.21mmol) was dissolved in ultra-dry CH₂Cl₂(20mL)，N₂Adding 2,2, 2-trifluoro under protectionN-phenylacetylimine chloride (1.50g,7.24mmol, 6eq) was reacted with DBU (368mg,2.42mmol, 2eq) at room temperature for 30 mins. The reaction was purified by column chromatography (petroleum ether: ethyl acetate 15:1with 0.1% Et)₃N) gave compound 6-c (1.19g, 79%, colorless syrup). TOF-MS M/z 1271.65[ M + Na ]]⁺。

EXAMPLE 31 preparation of Compound 11-c

The reaction flask was charged with acceptor compound 10(700mg,0.684mmol), donor compound 6-c (1.28g,1.026mmol,1.5eq) molecular sieve (1

600mg)，N₂Adding ultra-dry CH under the condition₂Cl₂(10mL), 100-fold dilution of TfOH (20. mu.L, 0.137mmol, 0.2eq) with dichloromethane at-20 ℃ and stirring for 40min at-20 ℃; quench with methanol (5mL) and triethylamine to pH 8. CH for reaction solution₂Cl₂(35mL) and saturated NaHCO₃(10mL) washing. The organic phase was concentrated by drying and purified by column chromatography (PE: EA ═ 10:1) to give the glycosidated product 11-c (1.27g, 89%). TOF-MS M/z 2104.9[ M + Na ]]⁺。

EXAMPLE 32 preparation of Compound 12-c

11-c (500mg,0.24mmol) was dissolved in CH in a reaction flask₂Cl₂(5mL)，N₂Adding zinc powder 1.5g) and acetic acid (15mL) under the condition, stirring at room temperature for 2h, filtering after the reaction is finished, performing azeotropic spin-drying on the filtrate by using toluene and performing column chromatography (PE: EA is 6:1) to obtain a compound 12-c (TOF-MS: M/z 1929.5[ M + Na & lt/EN & gt verified by MS)]⁺)。

EXAMPLE 33 preparation of Compound 13-c

Compound 12-c was dissolved in ultra dry DCM (5mL), N₂EDC & HCl (1.1g,5,76mmol) and fatty chain 19-A (819mg,1.92mmol) were added at-10 ℃ for displacement, the reaction was stirred for 12h, and then purification was performed by spin-dry column chromatography (toluene/ethyl acetate 10:1) to obtain compound 13-c (441mg, 79.4%, colorless clear syrup). TOF-MS M/z 2338.23[ M + Na ]]⁺。

EXAMPLE 34 preparation of Compound 14-c

Compound 13-c (600mg,0.259mmol) was dissolved in DMF (12mL), N in a reaction flask₂Triethylamine (12mL) was added under these conditions, stirred at room temperature overnight, and the reaction was purified by column chromatography (PE: EA ═ 6:1) to give compound 14-c (verified by MS). TOF-MS M/z 2115.9[ M + Na ]]⁺。

EXAMPLE 35 preparation of Compound 15-c

Dissolving Compound 14-c in SuperDry CH₂Cl₂(5mL)，N₂EDC · HCl (397.2mg,2.072mmol) and fatty chain 20(398.4mg,1.036mmol) were added at room temperature, the reaction mixture was stirred at room temperature for 12h, and purified by spin-column chromatography (toluene/ethyl acetate ═ 5:1) to give compound 15-c (518.8mg, 81.4%, colorless clear syrup). TOF-MS M/z 2482.47[ M + Na ]]⁺。

EXAMPLE 36 preparation of Compound 16-c

Compound 15-c (400mg, 0.162mmol) was dissolved in THF (18mL), and a solution of HF/Py (4mL, 65-70%) in pyridine (12mL) was added at-40 ℃. Warm to room temperature and stir overnight.After the reaction, saturated aqueous sodium bicarbonate solution was added to quench, and chloroform was added to extract the solution several times. The organic layer was dried, filtered and concentrated and purified by a C18 packing (CH)₃CN, MeOH/EA ═ 8:1, MeOH) gave 16-c (0.293g, 76.9%, white solid). TOF-MS M/z 2368.26[ M + Na ]]⁺。

EXAMPLE 37 preparation of Compound 17-c

The compound 16-c (459mg, 0.196mmol), PPh₃(90mg,0.370mmol) was added to the reaction flask, and after nitrogen blanketing, THF (20mL), TEA (490. mu.L, 3.5mmol), HCOOH (290. mu.L, 7.8mmol), Pd (Ph) were added₃)₄(90mg,0.08 mmol). After the reaction is completed for 1.5h at 25 ℃, the reaction solution is spun through a C18 column CH₃CN with 0.1％TEA,MeOH with 0.1％TEA,CH₂Cl₂MeOH with 0.1% TEA afforded compound 17-c (405mg, 91.5%). TOF-MS M/z 2288.05[ M + Na ]]⁺。

EXAMPLE 38 preparation of Compound 18-c

The compound 17-c (100mg,0.044mmol) and DDQ (80mg,0.353mmol, 8eq) were added to a reaction flask, and dried CHCl was added under nitrogen protection₃(10 mL). The reaction is completed after 30 ℃ ultrasonic treatment for 80 min. Quench with a drop of triethylamine and spin dry the reaction. Acetonitrile was added to the spin-dried reaction flask, and the mixture was poured into a column chromatography packed with C18 to decolorize and purify the mixture. When the excess DDQ is completely removed, CH₂Cl₂MeOH ═ 3:1 gave compound 18-c (68mg, 91.5%) as a final product. TOF-MS M/z 1703.4[ M-H ]]⁺. HPLC purity: 96.5 percent

Comparative example 1

Compound 17(50mg,0.022mmol) and Pd (400mg) were added to a hydrogenation kettle, dissolved in THF: h₂O4: 1(20mL), 1MPa,30 ℃ for 24 h. Quench with a drop of triethylamine and filter the reaction and spin dry. Use ofPurification by column chromatography on C18 packing was performed to afford the final product, Compound 18(12mg, 32.4%). The TLC plate layer shows that the raw material is remained, the impurity points are more, and the raw material is not reduced for a prolonged time. The yield and purity are clearly inferior to the above examples.

As can be seen from the above, the allyl ligand used in the invention avoids the subsequent hydrogenation reaction, and the reaction can be completed under the condition of using the tetratriphenylphosphine for 1.5 h. The final product can be obtained after simple decolorization by using a C18 packed column. The problems of more impurities, lower yield and complex purification mode caused by hydrogenation debenzylation protecting group in the prior art are solved. Has obviously better effect.

(2) Compared with the prior art which needs hydrogenation reaction of palladium on carbon for more than 20h and then repeated filtration and ion column chromatography, the yield is lower (only can reach about 50%), the method using the Nap protecting group simplifies the operation, the yield of the deprotection step can reach more than 91.5% after optimization, and the purity reaches 97% (the determination method refers to that the content of MPL in the BLP25 liposome vaccine is determined by an HPLC-ELSD method, and Chinese medicines are listed as Juanxuan, Wang and Chang at No. 26 and No. 5 in 2012).

Claims

1. A method for preparing a compound shown as a formula 20 is characterized by comprising the following steps: in a mixed solvent of an organic solvent and water, in the presence of alkali, carrying out hydrolysis reaction on a compound shown as a formula 24 as shown in the specification to obtain a compound shown as a formula 20;

2. the process of claim 1 for the preparation of a compound of formula 20,

the organic solvent is a cyclic ether solvent;

and/or the volume ratio of the organic solvent to the water is 1:1-1: 2;

and/or the mass volume ratio of the compound shown in the formula 24 to the mixed solvent is 10g/L-200 g/L;

and/or, the base is an alkali metal hydroxide;

and/or the molar ratio of the alkali to the compound shown in the formula 24 is 3:1-10: 1;

and/or the temperature of the hydrolysis reaction is 50-100 ℃;

and/or the post-treatment of the hydrolysis reaction comprises the following steps: after the hydrolysis reaction is finished, quenching until the pH value is 7, diluting with an organic solvent, washing, drying an organic phase, filtering, concentrating, separating and purifying to obtain the product.

3. The process of claim 2 for the preparation of a compound of formula 20,

the organic solvent is tetrahydrofuran;

and/or the volume ratio of the organic solvent to the water is 1:1.2-1: 1.5;

and/or the mass volume ratio of the compound shown in the formula 24 to the mixed solvent is 50g/L-100 g/L;

and/or, the alkali is one or more of lithium hydroxide, sodium hydroxide and potassium hydroxide;

and/or the molar ratio of the alkali to the compound shown in the formula 24 is 5:1-8: 1;

and/or the temperature of the hydrolysis reaction is 60-65 ℃;

and/or, in the post-treatment of the hydrolysis reaction, the HCl aqueous solution is adopted for quenching to the pH value of 7;

and/or in the post-treatment of the hydrolysis reaction, the organic solvent diluted by the organic solvent is a halogenated alkane solvent;

and/or, in the post-treatment of the hydrolysis reaction, saturated sodium bicarbonate solution is adopted for washing;

and/or in the post-treatment of the hydrolysis reaction, the separation and purification is column chromatography separation, the filler of the column chromatography separation is a silica gel column, and the eluent of the column chromatography separation is petroleum ether and ethyl acetate.

4. The method of claim 1, further comprising the steps of: in an organic solvent, in the presence of a silicon reagent, a reducing agent and Lewis acid, carrying out Nap protection reaction on a compound shown as a formula 23 and 2-naphthaldehyde to obtain a compound shown as a formula 24;

5. the process of claim 4, wherein the compound of formula 20,

in the Nap protection reaction, the organic solvent is a cyclic ether solvent;

and/or the mass volume ratio of the compound shown in the formula 23 to the organic solvent is 5g/L-100 g/L;

and/or the silicon reagent is hexamethyldisiloxane and/or trimethylchlorosilane;

and/or the reducing agent is triethylsilane;

and/or the Lewis acid is trimethyl silicon triflate;

and/or the molar ratio of the silicon reagent to the compound shown in the formula 23 is 1:1-10: 1;

and/or the molar ratio of the reducing agent to the Lewis acid is 3:1-10: 1;

and/or the molar ratio of the reducing agent to the compound shown in the formula 23 is 1:1-5: 1;

and/or the molar ratio of the 2-naphthaldehyde to the compound shown in the formula 23 is 0.5:1-2: 1;

and/or the temperature of the Nap protection reaction is-20 ℃ to 30 ℃;

and/or the Nap protection reaction comprises the following steps: adding the reducing agent and Lewis acid into a mixed system of the compound shown as the formula 23, 2-naphthaldehyde and the organic solvent to carry out Nap protection reaction;

and/or the post-treatment of the Nap protection reaction comprises the following steps: after the Nap protection reaction is finished, diluting with an organic solvent, washing, concentrating, separating and purifying to obtain the Nap-based catalyst.

6. The process of claim 5 for the preparation of a compound of formula 20,

in the Nap protection reaction, the organic solvent is tetrahydrofuran;

and/or the mass volume ratio of the compound shown in the formula 23 to the organic solvent is 10g/L-50 g/L;

and/or, the silicon reagent is hexamethyldisiloxane;

and/or the molar ratio of the silicon reagent to the compound shown in the formula 23 is 4:1-8: 1;

and/or the molar ratio of the reducing agent to the Lewis acid is 4:1-6: 1;

and/or the molar ratio of the reducing agent to the compound shown in the formula 23 is 3:1-4: 1;

and/or the molar ratio of the 2-naphthaldehyde to the compound shown in the formula 23 is 0.7:1-1: 1;

and/or the temperature of the Nap protection reaction is-10 ℃ to 10 ℃;

and/or in the post-treatment step of the Nap protection reaction, the organic solvent diluted by the organic solvent is a halogenated alkane solvent;

and/or, in the post-treatment step of the Nap protection reaction, saturated sodium bicarbonate solution is adopted for washing;

and/or in the post-treatment step of the Nap protection reaction, the separation and purification is crystallization, and the solvents of the crystallization are petroleum ether and ethyl acetate.

7. The process of claim 6 for the preparation of a compound of formula 20,

in the post-treatment step of the Nap protection reaction, the organic solvent diluted by the organic solvent is CH₂Cl₂；

And/or, when the separation and purification are crystallization in the post-treatment step of the Nap protection reaction, the solvent of the crystallization is petroleum ether and ethyl acetate with the volume ratio of 5: 1.

8. A compound shown as a formula 20 and a formula 24;

9. a method for preparing a compound shown as a formula 24 is characterized by comprising the following steps: in an organic solvent, in the presence of a silicon reagent, a reducing agent and Lewis acid, carrying out Nap protection reaction on a compound shown as a formula 23 and 2-naphthaldehyde to obtain a compound shown as a formula 24;

the reaction conditions and operations in the process for preparing the compound represented by the formula 24 are as described in any one of claims 5 to 7.

10. The application of the compound shown as the formula 20 in preparing the compound shown as the formula 18 is characterized by comprising the following steps:

step (2), in an organic solvent, in the presence of a TBS removing protective agent, carrying out TBS removing protecting group reaction on a compound shown as a formula 15 to obtain the compound shown as a formula 16;

step (3), in an organic solvent, in the presence of alkali, HCOOH, a Pd catalyst and a phosphine ligand, carrying out an allyl protecting group removing reaction on a compound shown as a formula 16 as shown in the specification to obtain a compound shown as a formula 17;

step (4), in an organic solvent, carrying out a Nap protecting group removing reaction on the compound shown in the formula 17 and DDQ as shown in the specification to obtain an MPLA compound shown in the formula 18;