An intermediate compound, a preparation method thereof, and a solid-phase synthesis method for preparing polypeptides using the intermediate compound

Abstract

The present invention relates to an intermediate compound of formula II-1 or formula II-2 or a salt thereof, and a preparation method thereof. The present invention also relates to a process for synthesizing a compound of formula I by a solid-phase synthesis method using the intermediate compound of formula II-2. The synthesis method of the intermediate compound of formula II-1 or formula II-2 of the present invention has simple steps, is easy to operate, and the reaction can be mass-produced in kilograms; the solid-phase synthesis method of the compound of formula I of the present invention has a short synthesis cycle, simple operation steps, and is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and particularly relates to an intermediate compound, a preparation method thereof, and a solid-phase synthesis method for preparing polypeptides using the intermediate compound. Background Art

[0002] Opioid receptors (μ, δ, and κ) are widely present in the central nervous system and the peripheral nervous system. Traditional opioid receptor agonists (such as morphine and its derivatives) are the most effective drugs for treating chronic arthritis, inflammatory neuralgia, postoperative pain, and moderate to severe pain caused by various cancers. However, the first-generation κ-opioid receptor agonists, including spiradoline and enadoline, were discontinued from further development due to side effects such as restlessness and hallucinations caused by these two drugs. The second-generation κ-opioid receptor agonist (such as asimadoline) has poor anesthetic effects at the licensed dose and has been abandoned for development as an opioid anesthetic, but is instead used to treat digestive system diseases, such as irritable bowel syndrome.

[0003] Patent PCT / CN2017 / 103027 synthesized a polypeptide compound by a liquid-phase method. The chemical name is: 4-amino-1-((R)-2-((R)-2-((R)-2-amino-3-phenylpropanamido)-3-phenylpropanamido)-4-methylpentanamido)-6-((2-(2-methoxyethoxy)ethyl)amino)hexanoyl)piperidine-4-carboxylic acid, and its structural formula is as follows:

[0004]

[0005] The compound of formula I has excellent agonist efficacy for the κ-opioid receptor, and while maintaining the peripheral analgesic effect, can effectively reduce the toxic and side effects of the central nervous system. In the above patent, the polypeptide was synthesized by a liquid-phase method, which has a long synthesis cycle, requires real-time monitoring of the reaction process, and after each step of the reaction is completed, the intermediate needs to be purified through a complex process before the next step of the reaction can be carried out. The operation is cumbersome and the cost increases, which is not conducive to large-scale production.

[0006] Therefore, it is necessary to seek a new preparation method for the compound of formula I and the intermediate compound used in this method to solve the problems of long synthesis cycle, complex and cumbersome reaction operation, and difficulty in controlling costs in the prior art. Summary of the Invention

[0007] The object of the present invention is to provide an intermediate compound or its salt represented by formula II-1 or formula II-2, and a method for solid-phase synthesis of the compound of formula I via the intermediate compound. The method for synthesizing the compound of formula I can greatly shorten the reaction cycle, and the reaction operation is simple and convenient, and can be mass-produced industrially on a large scale.

[0008] The present invention provides the following technical solutions to achieve the above object:

[0009] An intermediate compound of formula II-1 or a salt thereof:

[0010]

[0011] Wherein R1 is hydrogen or an amino protecting group, and the amino protecting group is a basic amino protecting group or an acidic amino protecting group.

[0012] In some embodiments, R1 of the intermediate compound II-1 is hydrogen or a basic amino protecting group. In some preferred embodiments, the basic amino protecting group is Fmoc or Tfa, more preferably Fmoc. In some embodiments, R1 is hydrogen, Fmoc or Tfa; preferably Fmoc. In some preferred embodiments, the intermediate compound II-1 is:

[0013]

[0014] An intermediate compound of formula II-2 or a salt thereof:

[0015]

[0016] Wherein R1 is hydrogen or an amino protecting group, and R2 is an amino protecting group, and the amino protecting group is a basic protecting group or an acidic protecting group.

[0017] In some embodiments, R1 of the intermediate compound II-2 is hydrogen or an amino protecting group with the opposite acid-base property to R2. For example, R1 is hydrogen or an acidic amino protecting group, and R2 is a basic amino protecting group; or R1 is hydrogen or a basic amino protecting group, and R2 is an acidic amino protecting group. In some preferred embodiments, R1 is hydrogen or a basic amino protecting group, and R2 is an acidic amino protecting group. In some embodiments, R1 is hydrogen or a basic amino protecting group, and R2 is an acidic amino protecting group. In some embodiments, the basic amino protecting group is preferably Fmoc or Tfa, more preferably Fmoc. In some preferred embodiments, the acidic amino protecting group of the intermediate compound II-2 is Cbz, Boc, Trt, DMB or PMB, more preferably Boc. In some embodiments, R1 is hydrogen, Fmoc or Tfa; R2 is Cbz, Boc, Trt, DMB or PMB. In some embodiments, R1 is Fmoc and R2 is Boc. In some preferred embodiments, the intermediate compound II-2 is:

[0018]

[0019] The salt of intermediate compound II-1 or II-2 of the present invention can be its sodium salt or potassium salt, which can be prepared by methods commonly used in the art.

[0020] Another object of the present invention is to provide a method for preparing intermediate compounds II-1 and II-2. This method has simple steps, is easy to operate, and requires few separation steps, enabling the reaction to be scaled up to kilogram production.

[0021] The present invention achieves this object through the following technical solutions:

[0022] A method for preparing intermediate compound II-1, comprising the following steps:

[0023] Subject the compound represented by formula SM-2-1 to reductive amination reaction with the HX salt of compound III to obtain intermediate compound II-1:

[0024]

[0025] wherein R1 is as defined above; HX is trifluoroacetic acid or hydrochloric acid; preferably, HX is hydrochloric acid.

[0026] In the present invention, the compound of formula III or its HX salt can be synthesized by conventional methods in the art.

[0027] In some embodiments, the reductive amination reaction is carried out in a solvent.

[0028] In some embodiments, the molar ratio of compound SM-2-1 to the HX salt of compound III is (1:1) to (5:1), preferably (1:1) to (2.5:1).

[0029] In some embodiments, the molar amount of the HX salt of compound III to the volume of the solvent in the reductive amination reaction is (1 mol: 5 L) to (1 mol: 10 L), preferably (1 mol: 5 L) to (1 mol: 8 L), such as 1 mol: 5 L, 1 mol: 6 L, 1 mol: 7 L or 1 mol: 8 L.

[0030] In some embodiments, the solvent in the reductive amination reaction is a mixed solvent of an aprotic solvent and an alcohol solvent. In a preferred embodiment, the aprotic solvent is selected from one or more of dichloromethane, tetrahydrofuran and diethyl ether; the alcohol solvent is selected from one or more of methanol, ethanol and isopropanol. In a more preferred embodiment, the solvent is a mixed solvent of dichloromethane and methanol.

[0031] In some embodiments, the volume ratio of the aprotic solvent to the alcohol solvent in the solvent of the reductive amination reaction is (1:5) to (10:1), preferably (1.5:1) to (5:1).

[0032] In some embodiments, the reducing agent used in the reductive amination reaction is sodium borohydride or its derivative, preferably sodium triacetoxyborohydride. In some embodiments, the molar ratio of the HX salt of the compound of formula III to the reducing agent is (1:1) to (1:10), preferably (1:2) to (1:8), such as 1:2.5, 1:5 or 1:7.

[0033] In some embodiments, the reductive amination reaction time is 0.5 - 24 hours, preferably 1 - 3 hours.

[0034] In some embodiments, the reductive amination reaction temperature is -20°C - 25°C, preferably 0°C - 15°C.

[0035] In some embodiments, compound SM-2-1 is prepared by the following oxidation reaction: Diethylene glycol monomethyl ether is oxidized in an oxidation system to the compound shown in formula SM-2-1

[0036]

[0037] In some embodiments, the product obtained from the oxidation reaction is directly used for the preparation of intermediate compound II-1 without separation and purification.

[0038] In some embodiments, the molar ratio of diethylene glycol monomethyl ether to the HX salt of the compound of formula III is (1:1) to (5:1), preferably (1:3) to (3:1), more preferably 2.2:1.

[0039] In some embodiments, the oxidation system in the oxidation reaction includes an oxidizing agent and an organic base.

[0040] In some embodiments, the molar ratio of diethylene glycol monomethyl ether to the oxidizing agent in the oxidation reaction is (1:1) to (1:5), preferably (1:1) to (1:3), more preferably (1:1) to (1:2).

[0041] In some embodiments, the molar ratio of diethylene glycol monomethyl ether to the organic base in the oxidation reaction is (1:2) to (1:10), preferably (1:2) to (1:6).

[0042] In some embodiments, the oxidizing agent in the oxidation reaction is a combination of DMSO and oxalyl chloride, or a combination of DMSO and trifluoroacetic anhydride. In some embodiments, the molar ratio of oxalyl chloride to DMSO, or the molar ratio of trifluoroacetic anhydride to DMSO is (1:1) to (1:5), preferably (1:1) to (1:3), more preferably (1:1) to (1:2), further preferably (1:1) to (1:1.5).

[0043] In some embodiments, the organic base in the oxidation reaction is triethylamine.

[0044] In some embodiments, the oxidation reaction is carried out in an aprotic solvent, which can be selected from one or more of dichloromethane, tetrahydrofuran, and diethyl ether. In a preferred embodiment, the aprotic solvent is dichloromethane.

[0045] In some embodiments, the molar amount of diethylene glycol monomethyl ether to the volume ratio of the aprotic solvent in the oxidation reaction is (1 mol: 1 L) to (1 mol: 2 L).

[0046] In some embodiments, the temperature of the oxidation reaction is less than or equal to -30 °C, preferably less than or equal to -60 °C, more preferably less than or equal to -70 °C.

[0047] In some embodiments, compound SM-2-1 is prepared by the following reaction: Hydrolyzing (2-methoxyethoxy)acetaldehyde dimethyl acetal in an acidic system to obtain the compound shown in formula SM-2-1.

[0048]

[0049] In some embodiments, the acids in the acidic system include, but are not limited to, organic acids and inorganic acids.

[0050] In some embodiments, the organic acids include, but are not limited to, trifluoroacetic acid, acetic acid, formic acid, p-toluenesulfonic acid, fumaric acid, and tartaric acid, with trifluoroacetic acid being particularly preferred.

[0051] In some embodiments, the inorganic acids include, but are not limited to, hydrochloric acid, sulfuric acid, and phosphoric acid, with sulfuric acid being particularly preferred.

[0052] In some embodiments, the reaction system is an aqueous sulfuric acid solution, and the content of the aqueous sulfuric acid solution is 0.1% - 50%, particularly preferably 0.1% - 20%, and even more preferably 1% - 10%.

[0053] In some embodiments, the temperature of the reaction is 0 °C - 50 °C, preferably 0 °C - 25 °C, more preferably 0 °C - 10 °C.

[0054] The present invention further achieves the above object through the following technical solutions:

[0055] A method for preparing intermediate compound II-2, which is obtained by subjecting intermediate compound II-1 to amino protection.

[0056]

[0057] Wherein R1 and R2 are as defined above.

[0058] In some embodiments, intermediate compound II-1 is prepared according to the preparation method described above. In some preferred embodiments, intermediate compound II-1 is directly used for the preparation of intermediate compound II-2 without separation and purification after being prepared.

[0059] In some embodiments, the imino group of intermediate compound II-1 is protected using N,N-diisopropylethylamine and di-tert-butyl dicarbonate. In some embodiments, the molar ratio of intermediate compound II-1 to N,N-diisopropylethylamine is (1:1) to (1:5). In some embodiments, the molar ratio of intermediate compound II-1 to di-tert-butyl dicarbonate is (1:1) to (1:5).

[0060] In some embodiments, the preparation method of intermediate compound II-2 of the present invention further includes the step of separating and purifying the obtained intermediate compound II-2.

[0061] Another object of the present invention is to provide a solid-phase synthesis method of the compound of formula I, which has a short synthesis cycle for the whole process, simple operation steps, and is suitable for large-scale production.

[0062] The present invention achieves this object through the following technical solutions:

[0063] A solid-phase synthesis method of a compound of formula I, comprising the following steps:

[0064] 1) Condensing the compound M-1-1 immobilized on a solid-phase carrier with intermediate compound II-2 to obtain a polypeptide compound M-2 immobilized on a solid-phase carrier, and then removing the R1 protecting group to obtain the compound M-2-1 immobilized on a solid-phase carrier,

[0065]

[0066] 2) Sequentially using amino acid derivatives SM-3, amino acid derivatives SM-4, and amino acid derivatives SM-5 as raw materials to carry out the following reactions, and finally obtaining the compound M-5 immobilized on a solid-phase carrier,

[0067]

[0068] 3) Adding a cleavage solution to the compound M-5 immobilized on a solid-phase carrier obtained in step 2) for deprotection and cleavage to obtain a crude product of compound I,

[0069]

[0070] wherein, R1 and R2 are as defined above; R y, R3, R4, and R5 are basic amino protecting groups or acidic amino protecting groups; and when R3 and R4 are both acidic amino protecting groups, R y and R5 are both basic amino protecting groups; when R3 and R4 are both basic amino protecting groups, R y and R5 are both acidic amino protecting groups.

[0071] In some embodiments, R3 and R4 are basic amino protecting groups, and R y and R5 are acidic amino protecting groups.

[0072] In some embodiments, R3 and R4 are independently selected from one or more of Fmoc and Tfa; R y and R5 are independently selected from one or more of Cbz, Boc, Trt, DMB, and PMB.

[0073] In some embodiments, R3 and R4 are Fmoc; R y and R5 are Boc.

[0074] In some embodiments, R2 and R y , and R5 are both acidic amino protecting groups or both basic amino protecting groups. In some embodiments, when R1 is an amino protecting group, it is the same as R3 and R4 in being both acidic amino protecting groups or both basic amino protecting groups.

[0075] In some embodiments, R3 and R4 are both acidic amino protecting groups, and R2, R y and R5 are both basic amino protecting groups. In some embodiments, R3 and R4 are both basic amino protecting groups, and R2, R y and R5 are both acidic amino protecting groups. In some embodiments, when R1 is an amino protecting group, R1, R3, and R4 are all acidic amino protecting groups, and R2, R y and R5 are both basic amino protecting groups. In some embodiments, when R1 is an amino protecting group, R1, R3, and R4 are all basic amino protecting groups, and R2, R y and R5 are both acidic amino protecting groups. Preferably, R1, R3, and R4 are basic amino protecting groups, and R2, R y and R5 are acidic amino protecting groups.

[0076] In some embodiments, step 4) is optionally included after step 3): The crude compound I obtained in step 3) is separated and purified to obtain the pure compound I.

[0077] In some embodiments, the intermediate compound II-2 is obtained by the preparation method described above.

[0078] In some embodiments, compound M-1-1 of step 1) is prepared by the following method: immobilize compound SM-1 on a solid-phase support, and then remove the protecting group Rx of the piperidine ring imino group to obtain the solid-phase support-immobilized compound M-1-1.

[0079]

[0080] wherein, R x is an amino protecting group with an acid-base property opposite to that of R y ; preferably, R x is a basic amino protecting group; more preferably, R x is Fmoc or Tfa; particularly preferably, R x is Fmoc.

[0081] In some embodiments, the molar ratio of the compound represented by formula SM-1 to the solid-phase support is 1:(2 - 4), preferably 1:3.

[0082] In some embodiments, the solid-phase support is Wang resin or 2-chlorotrityl chloride resin, preferably 2-chlorotrityl chloride resin. Preferably, the substitution degree of Wang resin is 0.3 - 1.0 mmol / g, more preferably 0.4 - 0.7 mmol / g; the substitution degree of 2-chlorotrityl chloride resin is 0.2 - 1.6 mmol / g, preferably 0.7 - 1.2 mmol / g, and preferably 1.0 - 1.2 mmol / g. Using this solid-phase support, the compound has a high yield, high purity of the prepared compound, is easy to purify, and has a low cost.

[0083] In some embodiments, the condensation reactions of steps 1) and 2) are carried out in a solvent selected from one or two of N,N-dimethylformamide and dichloromethane. When the reaction is carried out in a mixed solvent, the volume ratio of N,N-dimethylformamide to dichloromethane is (1 - 5):1, preferably (1 - 3):1.

[0084] In some embodiments, a condensing agent is used for the condensation of the polypeptide in steps 1) and 2), and the condensing agent can be one or more of the following combinations: a) HBTU, Cl-HoBt and DIEA; b) DIC and Cl-HoBt; c) PyBOP, Cl-HoBt and DIEA; d) HBTU, Oxyma, DIEA, wherein the component ratios of group a), group c) and group d) can be 1:1:1.1 respectively, and the component ratio of group b) can be 1:1.1. In a preferred embodiment, a combination of HBTU, Cl-HoBt and DIEA, or DIC and Cl-HoBt is used as the condensing agent.

[0085] In some embodiments, a condensing agent is used for the condensation of the polypeptide in steps 1) and 2), and the condensing agent can be a combination of DIC and HoBt as the condensing agent.

[0086] In the present invention, methods commonly used in the art are used to protect and deprotect amino groups and imino groups with protecting groups. In a preferred embodiment, for the Fmoc protecting group, it can be removed using a 20% piperidine / DMF solution (DBLK); for the Boc protecting group, it can be removed using TFA, HCl, or HF, preferably TFA.

[0087] In some embodiments, when R y , R2, and R5 are acidic amino protecting groups, the cleavage solution in step 3) contains 50%-100% TFA, or further contains one, two, or more of 0%-10% TIS, 0%-10% H2O, and 0%-10% TES, preferably composed of 90% TFA / 5% TIS / 5% H2O (v / v / v) or 95% TFA / 5% H2O (v / v), so as to completely cleave the M-5 resin and improve the purity of the final product.

[0088] In some embodiments, the ratio of the cleavage solution in step 3) to the carrier-fixed polypeptide compound M-5 obtained in step 2) is (6-10) mL:1 g, preferably 8 mL:1 g.

[0089] In some embodiments, the cleavage reaction in step 3) first reacts at a low temperature for 15-30 min, and then is heated to room temperature and reacted until complete.

[0090] In some embodiments, steps before and after the cleavage in step 3) and optional step 4) include a step of precipitating the crude product of the compound of formula I using an ether solvent. In a preferred embodiment, the ether solvent is anhydrous ether or methyl tert-butyl ether.

[0091] In some embodiments, the step of precipitating the crude product of the compound of formula I using the ether solvent is carried out at a low temperature, such as 0 °C, -10 °C, etc.

[0092] In some embodiments, the separation and purification in step 4) is carried out using reverse-phase high performance liquid chromatography.

[0093] The basic amino protecting groups used in this specification and the claims refer to the protecting groups on nitrogen that can be removed under basic conditions, such as Fmoc or Tfa, etc.; the acidic amino protecting groups refer to the protecting groups on nitrogen that can be removed under acidic conditions, such as Cbz, Boc, Trt, DMB or PMB, etc. Those skilled in the art can make appropriate selections and operations with reference to common textbooks in the field, such as Greene's Protective Groups in Organic Synthesis (4th Edition), etc., to selectively or completely remove one or more protecting groups.

[0094] The meanings of the abbreviations used in this specification and the claims are as follows:

[0095]

[0096] Advantages of the present invention:

[0097] 1. Using the intermediate compound II-2 of the present invention to prepare the compound of formula I by the solid-phase method can greatly shorten the reaction cycle, and the reaction operation is simple, the compound yield is high, the production cost is low, and it can be mass-produced on an industrial scale.

[0098] 2. By optimizing the process conditions, the synthesis method of the intermediate compound II-1 or II-2 of the present invention has simple steps and is easy to operate, without too many separation steps, enabling the reaction to be mass-produced in kilograms; in particular, when using diethylene glycol monomethyl ether and the HX salt of compound III as the starting reactants, through the optimization of the reaction process, mild reaction conditions and short reaction times can be adopted, and the intermediate compound II-2 of the present invention can be prepared by the "one-pot method" without any separation and purification steps.

[0099] 3. The solid-phase synthesis method of the compound of formula I of the present invention has a short overall process synthesis cycle, simple operation steps, and is suitable for large-scale production.

[0100] 4. Using the intermediates II-1 and II-2 of the present invention and applying them to the polypeptide compound of formula I prepared by the solid-phase synthesis method, through the optimization of the reaction conditions, the product purity can reach more than 99.5%. Brief Description of the Drawings

[0101] Figure 1 The HPLC chromatogram of the crude product of the compound of formula I obtained in Example 5 of the present invention.

[0102] Figure 2 The HPLC chromatogram of the pure product of the compound of formula I obtained in Example 5 of the present invention. Detailed Description of the Invention

[0103] The raw materials used in the following examples are all commercially available products.

[0104] Instrument and equipment information used in the examples:

[0105]

[0106] Preparation of Compound III-a hydrochloride in Experimental Example 1

[0107]

[0108] Add 150 L of ethyl acetate to a 300 L reaction kettle and cool to -10 - 0 °C. Introduce hydrogen chloride gas into the system and control the system temperature at -10 - 0 °C. Add a mixture of compound Fmoc-D-Lys(Boc)-OH (14 kg, 29.88 mol) and ethyl acetate (50 L) to the above reaction system. Stir and allow it to return to room temperature naturally. After 3 hours, stop the reaction, drain the reaction solution, centrifuge, wash the filter cake with ethyl acetate 3 times, centrifuge again, and dry the filter cake to obtain Compound III-a hydrochloride (11.5 kg, yield 95.1%).

[0109] Preparation of Intermediate Compounds II-1-a and II-2-a in Experimental Example 2

[0110]

[0111] Dissolve oxalyl chloride (1.65 kg, 13.0 mol) in dichloromethane (15 L), cool to <-70 °C under a nitrogen atmosphere, and add a dichloromethane solution (1500 mL) of DMSO (1.47 kg, 18.8 mol), controlling the temperature at <-70 °C. After addition, stir at this temperature for 60 min, then add a dichloromethane solution (1500 mL) of diethylene glycol monomethyl ether (1.5 kg, 12.5 mol), controlling the temperature at <-70 °C. After addition, stir at this temperature for 60 min. Continue to add triethylamine (2.55 kg, 25.2 mol), controlling the temperature at <-70 °C. After addition, slowly raise the temperature to room temperature and stir for 20 min. Obtain a dichloromethane (25 L) solution of 2-(2-methoxyethoxy)acetaldehyde for use.

[0112] A methanol (5 L) solution of compound III-a hydrochloride (2.3 kg, 5.7 mol) was added to the dichloromethane solution (25 L) of 2-(2-methoxyethoxy)acetaldehyde obtained above, and the mixture was stirred at room temperature for 30 min. Sodium triacetoxyborohydride (3.0 kg, 14.2 mol) was added, and the reaction system was reacted at room temperature for 1 h. The reaction was monitored by LC-MS. After the formation of intermediate II-1-a compound, the reaction solution was cooled to 0 °C. N,N-Diisopropylethylamine (2.2 kg, 17.1 mol) and di-tert-butyl dicarbonate (1.36 kg, 6.2 mol) were successively added to the reaction solution. After the addition, the temperature was raised to room temperature and the reaction was carried out for 2 h. Dichloromethane and methanol were removed by distillation under reduced pressure. An aqueous solution of saturated sodium carbonate was added to this crude product, and after stirring for 30 min, it was extracted 3 times with methyl tert-butyl ether. Then the aqueous phase was adjusted to pH 3-4 with 1N hydrochloric acid and extracted twice with ethyl acetate. The ethyl acetate phases were combined, washed 3 times with 1N HCl and saturated brine respectively, dried over anhydrous sodium sulfate, filtered by suction, and silica gel was directly added to the filtrate and concentrated to dryness to obtain 5.0 kg of crude product, which was directly subjected to column chromatography to obtain intermediate compound II-2-a (1.05 kg, yield 32.39%).

[0113] ESI-MS (m / z): 471.2 (M - Boc + H) +

[0114] 1 H NMR (400 MHz, DMSO-d6) δ 12.51 (s, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 7.4 Hz, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 4.30–4.19 (m, 3H), 3.92 (s, 1H), 3.52–3.38 (m, 6H), 3.30–3.21 (m, 5H), 3.17 - 3.12 (m, 2H), 1.78–1.55 (m, 2H), 1.52 - 1.41 (m, 2H), 1.37 (s, 9H), 1.33 - 1.21 (m, 2H).

[0115] Preparation of Intermediate Compounds II-1-a and II-2-a in Experimental Example 3

[0116] Oxalyl chloride (2.39 kg, 18.8 mol) was dissolved in dichloromethane (15 L), cooled to <-70 °C under a nitrogen atmosphere, and a dichloromethane solution (1500 mL) of DMSO (1.95 kg, 25.0 mol) was added, with the temperature controlled at <-70 °C. After addition, the mixture was stirred at this temperature for 60 min, and a dichloromethane solution (1500 mL) of diethylene glycol monomethyl ether (1.5 kg, 12.5 mol) was added, with the temperature controlled at <-70 °C. After addition, the mixture was stirred at this temperature for 60 min. Triethylamine (5.10 kg, 50.4 mol) was continuously added, with the temperature controlled at <-70 °C. After addition, the temperature was slowly raised to room temperature and stirred for 20 min. A dichloromethane (25 L) solution of 2-(2-methoxyethoxy)acetaldehyde was obtained for use.

[0117] A methanol (16.5 L) solution of the compound of formula III-a hydrochloride (2.3 kg, 5.7 mol) was added to the above-obtained dichloromethane solution (25 L) of 2-(2-methoxyethoxy)acetaldehyde, stirred at room temperature for 30 min, sodium triacetoxyborohydride (3.0 kg, 14.2 mol) was added, and the reaction system was reacted at room temperature for 1 h. The reaction was monitored by LC-MS. After the formation of the intermediate II-1-a compound, the reaction solution was cooled to 0 °C. N,N-Diisopropylethylamine (1.5 kg, 11.4 mol) and di-tert-butyl dicarbonate (3.75 kg, 17.1 mol) were successively added to the reaction solution. After addition, the temperature was raised to room temperature and reacted for 2 h. Dichloromethane and methanol were removed by distillation under reduced pressure. An aqueous solution of saturated sodium carbonate was added to this crude product, and after stirring for 30 min, it was extracted 3 times with methyl tert-butyl ether. Then, the aqueous phase was adjusted to pH 3-4 with 1N hydrochloric acid and extracted twice with ethyl acetate. The ethyl acetate phases were combined, washed 3 times with 1N HCl and saturated brine respectively, dried over anhydrous sodium sulfate, filtered by suction, and directly concentrated to dryness with silica gel in the filtrate to obtain 5.0 kg of crude product, which was directly subjected to column chromatography to obtain the intermediate compound II-2-a (1.09 kg, yield 33.74%).

[0118] Preparation of Intermediate Compounds II-1-a and II-2-a in Experimental Example 4

[0119] Oxalyl chloride (3.30 kg, 25.0 mol) was dissolved in dichloromethane (15 L), cooled to <-70 °C under a nitrogen atmosphere, and a dichloromethane solution (1500 ml) of DMSO (2.94 kg, 37.5 mol) was added, with the temperature controlled at <-70 °C. After addition, stirring was carried out at this temperature for 60 min, and a dichloromethane solution (1500 ml) of diethylene glycol monomethyl ether (1.5 kg, 12.5 mol) was added, with the temperature controlled at <-70 °C. After addition, stirring was carried out at this temperature for 60 min. Triethylamine (6.32 kg, 62.5 mol) was continuously added, with the temperature controlled at <-70 °C. After addition, the temperature was slowly raised to room temperature and stirred for 20 min to obtain a dichloromethane (25 L) solution of 2-(2-methoxyethoxy)acetaldehyde for use.

[0120] A methanol (8.5 L) solution of compound formula III-a hydrochloride (2.3 kg, 5.7 mol) was added to the above-obtained dichloromethane solution (25 L) of 2-(2-methoxyethoxy)acetaldehyde, stirred at room temperature for 30 min, sodium triacetoxyborohydride (3.0 kg, 14.2 mol) was added, and the reaction system was reacted at room temperature for 1 h. The reaction was monitored by LC-MS. After the formation of intermediate II-1-a compound, the reaction solution was cooled to 0 °C. N,N-Diisopropylethylamine (3.7 kg, 28.5 mol) and di-tert-butyl dicarbonate (6.25 kg, 28.5 mol) were successively added to the reaction solution. After addition, the temperature was raised to room temperature and reacted for 2 h. Dichloromethane and methanol were removed by distillation under reduced pressure. An aqueous solution of saturated sodium carbonate was added to this crude product, and after stirring for 30 min, it was extracted 3 times with methyl tert-butyl ether. Then the aqueous phase was adjusted to pH 3-4 with 1N hydrochloric acid and extracted twice with ethyl acetate. The ethyl acetate phases were combined, washed 3 times with 1N HCl and saturated brine respectively, dried over anhydrous sodium sulfate, filtered by suction, and directly concentrated to dryness with silica gel in the filtrate to obtain 5.0 Kg of crude product, which was directly subjected to column chromatography to obtain intermediate compound II-2-a (0.95 kg, yield 29.30%).

[0121] Preparation of Compound of Formula I in Example 5

[0122] (1) Preparation of M-1-a Resin

[0123]

[0124] Weigh 1000.0 g of 2-chlorotrityl chloride resin (substitution value: 1.1 mmol / g) and add it to a 20 L polypeptide reactor. At the same time, add 2 L of DCM to wash and swell the resin for 45 min. Weigh compound 4-(tert-butoxycarbonylamino)-1-(9H-fluoren-9-ylmethoxy)piperidine-4-carboxylic acid SM-1-a (1650 mmol, 769.7 g), dissolve it in 7 L of DCM, and add the dissolved reaction solution to the resin. After the resin and the reaction solution are stirred evenly, add DIEA (4950 mmol, 818 mL) to the resin reaction solution and react at 25 °C for 2 h. Continue to add 800 mL of methanol to the reaction solution to block the unreacted active sites and react for 45 min. After the reaction is completed, drain the solution, wash the resin with 4 × 10 L of DMF solution. After the washing is completed, take a part of the resin, carry out deprotection with piperidine, and use ultraviolet spectrophotometry to determine the amount of Fmoc in the piperidine deprotection solution. The substitution degree of M-1-a resin is calculated to be 0.79 mmol / g.

[0125] (2) Preparation of M-1-1-a resin

[0126]

[0127] Treat the M-1-a resin obtained in step (1) with 10 L of 20% piperidine / DMF solution for 5 min, drain the mixture, and then add 10 L of 20% piperidine / DMF solution to continue the treatment for 15 min to remove the Fmoc protecting group. Then wash the resin with 5 × 10 L of DMF. Perform the Kaiser Test. The resin is reddish-brown, and the deprotection is complete, obtaining M-1-1-a resin.

[0128] (3) Preparation of M-2-a resin

[0129]

[0130] Weigh intermediate compound II-2-a (2069 mmol, 1311.7 g), Cl-HoBt (2069 mmol, 351.3 g), and HBTU (2069 mmol, 784.5 g) and dissolve them in 7 L of DMF solution. Under nitrogen protection, ice-bath this solution to 0 - 5 °C, then add DIEA (2275.9 mmol, 376 mL) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step and react at room temperature for 1.5 h. Drain the resin, wash the resin with 3 × 10 L of DMF. Perform the Kaiser Test. The resin is light yellow, and the condensation reaction is complete, obtaining M-2-a resin.

[0131] (4) Preparation of M-2-1-a resin

[0132]

[0133] Treat the M-2-a resin obtained in step (3) with 2×10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5×10 L of DMF. Perform Kaiser Test, the resin is blue-violet, and the deprotection is complete, obtaining M-2-1-a resin.

[0134] (5) Preparation of M-3-a resin

[0135]

[0136] Weigh SM-3-a compound (3270 mmol, 1156.7 g) and Cl-HoBt (3270 mmol, 555.2 g), dissolve them in 7 L of DMF / DCM solution with a volume ratio of 1:1. Under nitrogen protection, ice-bath the solution to 0-5 °C, then add DIC (3597 mmol, 557 ml) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3×10 L of DMF. Perform Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-3-a resin.

[0137] (6) Preparation of M-3-1-a resin

[0138]

[0139] Treat M-3-a resin with 2×10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5×10 L of DMF. Perform Kaiser Test, the resin is blue, and the deprotection is complete, obtaining M-3-1-a resin.

[0140] (7) Preparation of M-4-a resin

[0141]

[0142] Weigh SM-4-a compound (3270 mmol, 1267.8 g) and Cl-HoBt (3270 mmol, 554.9 g), dissolve them in 7 L of DMF / DCM solution with a volume ratio of 1:1. Under nitrogen protection, ice-bath the solution to 0-5 °C, then add DIC (3597 mmol, 557 ml) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3×10 L of DMF. Perform Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-4-a resin.

[0143] Preparation of (8) M-4-1-a resin

[0144]

[0145] The M-4-a resin obtained in step (7) was treated with 2 × 10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, and then the resin was washed with 5 × 10 L of DMF. Kaiser Test showed that the resin was blue-violet, indicating complete deprotection, and M-4-1-a resin was obtained.

[0146] (9) Preparation of M-5-a resin

[0147]

[0148] Weigh SM-5-a compound (3270 mmol, 867.8 g) and Cl-HoBt (3270 mmol, 555.1 g), dissolve them in 7 L of DMF / DCM solution with a volume ratio of 1:1. Under nitrogen protection, the solution was ice-bathed to 0 - 5 °C, and then DIC (3597 mmol, 557 ml) was added and stirred for 5 min. After 5 min, the reaction solution was added to the resin obtained in the previous step, and the reaction was carried out at room temperature for 1.5 h. The resin was drained, washed with 3 × 10 L of DMF. Kaiser Test showed that the resin was light yellow, indicating complete condensation reaction. The resin was washed alternately with 3 × 10 L of DCM and 3 × 10 L of methanol. After washing, the resin was dried in vacuo to a constant weight. Finally, 1967.1 g of M-5-a resin was obtained with a yield of 96.7%.

[0149] (10) Preparation of Compound I

[0150]

[0151] Weigh 1900.1 g of M-5-a resin and add it to a 20 L polypeptide cleavage kettle. Add 15 L of cleavage solution 95% TFA / 5% H2O pre-cooled to about 0 °C, and stir and react at room temperature for 1.5 h. Filter the reaction solution into 150 L of methyl tert-butyl ether pre-cooled to about -10 °C. White solid was produced. Stir the white slurry at -10 °C for 30 min, then centrifuge the white slurry. The centrifuge parameters were set at 3500 r / min for 5 min. After centrifugation, discard the supernatant, collect the white slurry, add 20 L of fresh methyl tert-butyl ether, repeat the above centrifugation process, collect the white slurry, and dry it in vacuo to a constant weight. Finally, 981.9 g of crude Compound I was obtained with a yield of 98.1% and a purity of 92.55%. The HPLC spectrum of the crude Compound I is shown in Figure 1 .

[0152] 99.3 g of the crude compound I was purified by preparative HPLC, and finally 42.7 g of the pure product was obtained with a yield of 43% and a purity of 99.77%. The HPLC spectrum of the purified compound I is shown in Figure 2 .

[0153] ESI-MS (m / z): 782.5 (M + H) +

[0154] Experimental Example 6 Preparation of Compound I

[0155] (1) Preparation of M-1-a resin

[0156] Weigh 1010.8 g of 2-chlorotrityl chloride resin (substitution value: 1.1 mmol / g) and add it to a 20 L polypeptide reactor. At the same time, add 2 L of DCM to wash and swell the resin for 45 min. Weigh 1667 mmol (778.2 g) of SM-1-a compound 4-(tert-butoxycarbonylamino)-1-(9H-fluoren-9-ylmethoxy)piperidine-4-carboxylic acid, dissolve it in 7 L of DCM, and add the dissolved reaction solution to the resin. After the resin and the reaction solution are stirred evenly, add DIEA (5003 mmol, 827 mL) to the resin reaction solution and react at 25 °C for 2 h. Continue to add 800 mL of methanol to the reaction solution to block the unreacted active sites and react for 45 min. After the reaction is completed, drain the solution, wash the resin with 4 × 10 L of DMF solution. After the washing is completed, take a part of the resin, deprotect it with piperidine, and determine the amount of Fmoc in the piperidine deprotection solution by ultraviolet spectrophotometry. The substitution degree of M-1-a resin is calculated to be 0.77 mmol / g.

[0157] (2) Preparation of M-1-1-a resin

[0158] Treat M-1-a resin with 2 × 10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, and then wash the resin with � × 10 L of DMF. Kaiser Test, the resin is reddish-brown, and the deprotection is complete to obtain M-1-1-a resin.

[0159] (3) Preparation of M-2-a resin

[0160] Weigh the intermediate compound II-2-a (1917 mmol, 1093.0 g), Cl-HoBt (1917 mmol, 343.6 g), and HBTU (1917 mmol, 766.3 g), dissolve them in 7 L of DMF solution. Under nitrogen protection, cool this solution to 0 - 5 °C in an ice bath, then add DIEA (2108 mmol, 367 mL) and stir for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 10 L of DMF, perform Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-2-a resin.

[0161] (4) Preparation of M-2-1-a resin

[0162] Treat the M-2-a resin obtained in step (3) with 2 × 10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 10 L of DMF, perform Kaiser Test, the resin is blue, and the deprotection is complete, obtaining M-2-1-a resin.

[0163] (5) Preparation of M-3-a resin

[0164] Weigh the compound SM-3-a (3032.4 mmol, 1071.1 g), Cl-HoBt (3032.4 mmol, 514.6 g), and HBTU (3032.4 mmol, 1149.8 g), dissolve them in 7 L of DMF solution. Under nitrogen protection, cool this solution to 0 - 5 °C in an ice bath, then add DIEA (3322.4 mmol, 550 mL) and stir for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 10 L of DMF, perform Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-3-a resin.

[0165] (6) Preparation of M-3-1-a resin

[0166] Treat the M-3-a resin obtained in step (5) with 2 × 10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 10 L of DMF, perform Kaiser Test, the resin is blue, and the deprotection is complete, obtaining M-3-1-a resin.

[0167] (7) Preparation of M-4-a resin

[0168] Weigh SM-4-a compound (3032.4 mmol, 1175.7 g), Cl-HoBt (3270 mmol, 514.5 g), and HBTU (3032.4 mmol, 1150.3 g) and dissolve them in 7 L of DMF solution. Under nitrogen protection, ice-bath the solution to 0 - 5 °C, then add DIEA (3322.4 mmol, 550 mL) and stir for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 10 L of DMF, perform Kaiser Test, the resin is light yellow, and the condensation reaction is complete to obtain M-4-a resin.

[0169] (8) Preparation of M-4-1-a resin

[0170] Treat the M-4-a resin obtained in step (7) with 2 × 10 L of 20% piperidine / DMF solution for 5 min and 10 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 1 L of DMF, perform Kaiser Test, the resin is blue-violet, and the deprotection is complete to obtain M-4-1-a resin.

[0171] (9) Preparation of M-5-a resin

[0172] Weigh SM-5-a compound (3270 mmol, 867.8 g), Cl-HoBt (3270 mmol, 555.1 g), and HBTU (3032.4 mmol, 1151.7 g) and dissolve them in 7 L of DMF solution. Under nitrogen protection, ice-bath the solution to 0 - 5 °C, then add DIEA (3322.4 mmol, 550 mL) and stir for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step for 1.5 h, drain the resin, wash the resin with 3 × 10 L of DMF, perform Kaiser Test, the resin is light yellow, and the condensation reaction is complete to obtain M-5-a resin. Wash the resin alternately with 3 × 10 L of DCM and 3 × 10 L of methanol. After washing, place the resin in a vacuum dryer and dry it to a constant weight. Finally, 1941.9 g of M-5-a resin is obtained, and the yield is 92.1%.

[0173] (10) Preparation of Compound I

[0174] Weigh 1905.4 g of the obtained M-5-a resin and add it to a 20 L polypeptide cleavage kettle. Add 15 L of a cleavage solution of 95% TFA / 2.5% H2O / 2.5% TIS that has been pre-cooled to about 0 °C. Stir and react at room temperature for 1.5 h. Filter the reaction solution into 150 L of methyl tert-butyl ether that has been pre-cooled to about -10 °C. White solids are produced. Stir the white slurry at -10 °C for 30 min. Then centrifuge the white slurry. Set the centrifuge parameters to 3500 r / min and centrifuge for 5 min. After centrifugation, discard the supernatant, collect the white slurry, add 20 L of fresh methyl tert-butyl ether, repeat the above centrifugation process, collect the white slurry, and vacuum dry to constant weight. Finally, 984.1 g of the crude product of the compound of formula I is obtained, with a yield of 98.1% and a purity of 92.54%.

[0175] Purify 103.2 g of the crude product of the compound of formula I by preparative HPLC, and finally obtain 42.31 g of the pure product, with a yield of 41% and a purity of 99.75%.

[0176] Experimental Example 7 Preparation of the Compound of Formula I

[0177] (1) Preparation of M-1-a resin

[0178] Weigh 1000.1 g of 2-chlorotrityl chloride resin (substitution value: 1.1 mmol / g) and add it to a 20 L polypeptide reactor. At the same time, add 2 L of DCM to wash and swell the resin for 45 min. Weigh 771.0 g (1651 mmol) of SM-1-a compound 4-(tert-butoxycarbonylamino)-1-(9H-fluoren-9-ylmethoxy)piperidine-4-carboxylic acid, dissolve it in 7 L of DCM, and add the dissolved reaction solution to the resin. After the resin and the reaction solution are stirred evenly, add DIEA (4954.8 mmol, 818 mL) to the resin reaction solution and react at 25 °C for 2 h. Add 800 mL of methanol to the reaction solution to block the unreacted active sites and react for 45 min. After the reaction is completed, drain the solution, wash the resin with 4 × 10 L of DMF solution. After washing, take a part of the resin, deprotect it with piperidine, and use ultraviolet spectrophotometry to measure the amount of Fmoc in the piperidine deprotection solution. Calculate that the substitution degree of M-1-a resin is 0.76 mmol / g.

[0179] (2) Preparation of M-1-1-a resin

[0180] Use 2 × 10 L of 20% piperidine / DMF solution to treat the resin obtained in step (1) for 5 min and 15 min respectively to remove the Fmoc protecting group. Then use 5 × 10 L of DMF to wash the resin to remove Fmoc by-products and residual piperidine. Perform Kaiser Test, the resin is red-brown, and the deprotection is complete to obtain M-1-1-a resin.

[0181] (3) Preparation of M-2-a resin

[0182] Weigh II-2-a intermediate compound (1898 mmol, 1230.7 g) and Cl-HoBt (1898 mmol, 322.1 g), dissolve them in 7 L of DMF solution. Under the protection of nitrogen, ice-bath this solution to 0 - 5 °C, then add DIC (2087 mmol, 323 mL) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3×10 L of DMF, Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-2-a resin.

[0183] (4) Preparation of M-2-1-a resin

[0184] Treat the M-2-a resin obtained in step (3) with 2×10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5×10 L of DMF, Kaiser Test, the resin is blue, and the deprotection is complete, obtaining M-2-1-a resin.

[0185] (5) Preparation of M-3-a resin

[0186] Weigh SM-3-a compound (3273 mmol, 1155.7 g) and Cl-HoBt (3032.4 mmol, 514.6 g), dissolve them in 7 L of DMF solution. Under the protection of nitrogen, ice-bath this solution to 0 - 5 °C, then add DIC (2087 mmol, 323 mL) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3×10 L of DMF, Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-3-a resin.

[0187] (6) Preparation of M-3-1-a resin

[0188] Treat the M-3-a resin with 2×10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5×10 L of DMF, Kaiser Test, the resin is blue, and the deprotection is complete, obtaining M-3-1-a resin.

[0189] (7) Preparation of M-4-a resin

[0190] Weigh SM-4-a compound (3273 mmol, 1267.4 g) and Cl-HoBt (3273 mmol, 555.5 g), dissolve them in 7 L of DMF solution. Under nitrogen protection, ice-bath this solution to 0 - 5 °C, then add DIC (2087 mmol, 323 mL) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 10 L of DMF, conduct Kaiser Test, the resin is light yellow, the condensation reaction is complete, and obtain M-4-a resin.

[0191] (8) Preparation of M-4-1-a resin

[0192] Treat the M-4-a resin obtained in step (7) with 2 × 10 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 10 L of DMF, conduct Kaiser Test, the resin is blue-violet, the deprotection is complete, and obtain M-4-1-a resin.

[0193] (9) Preparation of M-5-a resin

[0194] Weigh SM-5-a compound (3273 mmol, 868.6 g) and Cl-HoBt (3273 mmol, 555.2 g), dissolve them in 7 L of DMF solution. Under nitrogen protection, ice-bath this solution to 0 - 5 °C, then add DIC (2087 mmol, 323 mL) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 10 L of DMF, conduct Kaiser Test, the resin is light yellow, the condensation reaction is complete, and obtain M-5-a resin. Wash the resin with 3 × 10 L of DCM and 3 × 10 L of methanol alternately. After the washing is completed, place the resin in vacuum drying and dry it to constant weight. Finally, obtain 1987.4 g of M-5-a resin, and the yield is 98.7%.

[0195] (10) Preparation of the compound of formula I

[0196] Weigh 1911.3 g of the obtained M-5-a resin and add it to a 20 L polypeptide cleavage kettle. Then add 15 L of a cleavage solution of 95% TFA / 5% H2O that has been pre-cooled to around 0 °C, and stir and react at room temperature for 1.5 h. Filter the reaction solution into 150 L of methyl tert-butyl ether that has been pre-cooled to around -10 °C. White solids are produced. Stir the white slurry at -10 °C for 30 min, then centrifuge the white slurry. Set the centrifuge parameters to 3500 r / min and centrifuge for 5 min. After centrifugation is completed, discard the supernatant, collect the white slurry, add 20 L of fresh methyl tert-butyl ether, repeat the above centrifugation process, collect the white slurry, and vacuum dry to constant weight. Finally, 971.2 g of the crude product of the compound of formula I is obtained, with a yield of 97.7% and a purity of 92.71%.

[0197] Purify 105.1 g of the crude product of the compound of formula I by preparative HPLC, and finally obtain 43.1 g of the pure product, with a yield of 41% and a purity of 99.70%.

[0198] Experimental Example 8 Preparation of the Compound of Formula I

[0199] (1) Preparation of M-1-a Resin

[0200] Weigh 100.1 g of Wang resin (substitution value: 0.45 mmol / g) and add it to a 5 L polypeptide reactor. At the same time, add 1 L of DCM to wash and swell the resin for 45 min. Weigh 63.1 g (135 mmol) of SM-1-a compound 4-(tert-butoxycarbonylamino)-1-(9H-fluoren-9-ylmethoxy)piperidine-4-carboxylic acid and 22.9 g (135 mmol) of Cl-HoBt, dissolve them in �00 mL of DMF solution. Under nitrogen protection, ice-bath this solution to 0 - 5 °C, then add 23 mL (148.5 mmol) of DIC, and add the dissolved reaction solution to the resin. After the resin and the reaction solution are stirred evenly, add 2.44 g (27 mmol) of 4-dimethylaminopyridine to the resin reaction solution, and react at 25 °C for 4 h. Add a blocking solution to the reaction solution to block the unreacted active sites, and react for 60 min. After the reaction is completed, drain the solution, wash the resin with 4 × 1 L of DMF solution. After the washing is completed, take a part of the resin, use ultraviolet spectrophotometry to measure the amount of Fmoc in the piperidine deprotection solution, and calculate that the substitution degree of the M-1-a resin is 0.22 mmol / g.

[0201] (2) Preparation of M-1-1-a Resin

[0202] Treat the M-1-a resin with 2 × 100 mL of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 100 mL of DMF. Perform the Kaiser Test. The resin is reddish-brown, and the deprotection is complete, obtaining the M-1-1-a resin.

[0203] (3) Preparation of M-2-a resin

[0204] Weigh the intermediate compound II-2-a (66 mmol, 37.6 g) and Cl-HoBt (66 mmol, 11.2 g), dissolve them in 700 mL of DMF solution. Under nitrogen protection, ice-bath the solution to 0 - 5 °C, then add DIC (72.6 mmol, 12 mL) and stir for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 1 L of DMF. Perform the Kaiser Test. The resin is light yellow, and the condensation reaction is complete, obtaining the M-2-a resin.

[0205] (4) Preparation of M-2-1-a resin

[0206] Treat the M-2-a resin obtained in step (3) with 2 × 1 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 1 L of DMF. Perform the Kaiser Test. The resin is blue, and the deprotection is complete, obtaining the M-2-1-a resin.

[0207] (5) Preparation of M-3-a resin

[0208] Weigh the compound SM-3-a (66 mmol, 23.3 g) and Cl-HoBt (66 mmol, 12.3 g), dissolve them in 700 ml of DMF solution. Under nitrogen protection, ice-bath the solution to 0 - 5 °C, then add DIC (72.6 mmol, 12 mL) and stir for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 1 L of DMF. Perform the Kaiser Test. The resin is light yellow, and the condensation reaction is complete, obtaining the M-3 resin.

[0209] (6) Preparation of M-3-1-a resin

[0210] Treat the M-3 resin obtained in step (5) with 2 × 1 L of 20% piperidine / DMF solution for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 1 L of DMF. Perform the Kaiser Test. The resin is blue, and the deprotection is complete, obtaining the M-3-1-a resin.

[0211] (7) Preparation of M-4-a resin

[0212] Weigh SM-4-a compound (66 mmol, 25.6 g) and Cl-HoBt (66 mmol, 11.9 g), dissolve them in 700 mL of DMF solution. Under the protection of nitrogen, ice-bath this solution to 0 - 5 °C, then add DIC (72.6 mmol, 12 ml) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 1 L of DMF, Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-4-a resin.

[0213] (8) Preparation of M-4-1-a resin

[0214] Use 2 × 1 L of 20% piperidine / DMF solution to treat the M-4-a resin obtained in step (7) for 5 min and 15 min respectively to remove the Fmoc protecting group, then wash the resin with 5 × 1 L of DMF, Kaiser Test, the resin is blue-violet, and the deprotection is complete, obtaining M-4-1-a resin.

[0215] (9) Preparation of M-5-a resin

[0216] Weigh SM-5-a compound (66 mmol, 17.5 g) and Cl-HoBt (66 mmol, 12.0 g), dissolve them in 700 mL of DMF solution. Under the protection of nitrogen, ice-bath this solution to 0 - 5 °C, then add DIC (72.6 mmol, 12 mL) and stir for reaction for 5 min. After 5 min, add the reaction solution to the resin obtained in the previous step, react at room temperature for 1.5 h, drain the resin, wash the resin with 3 × 1 L of DMF, Kaiser Test, the resin is light yellow, and the condensation reaction is complete, obtaining M-5-a resin. Wash the resin with 3 × 1 L of DCM and 3 × 1 L of methanol alternately. After the washing is completed, place the resin in vacuum drying and dry it to a constant weight. Finally, 122.7 g of M-5-a resin is obtained, and the yield is 95.3%.

[0217] (10) Preparation of Compound I

[0218] Weigh 100.1 g of the obtained M-5-a resin and add it to a 5 L polypeptide cleavage kettle. Then add 800 ml of a cleavage solution of 95% TFA / 5% H2O that has been pre-cooled to about 0 °C, and stir and react at room temperature for 1.5 h. Filter the reaction solution into 10 L of methyl tert-butyl ether that has been pre-cooled to about -10 °C. White solids will form. Stir the white slurry at -10 °C for 30 min, then centrifuge the white slurry. Set the centrifuge parameters to 3500 r / min and centrifuge for 5 min. After centrifugation, discard the supernatant, collect the white slurry, add 2 L of fresh methyl tert-butyl ether, repeat the above centrifugation process, collect the white slurry, and vacuum dry it to a constant weight. Finally, 18.7 g of the compound of formula I is obtained, with a yield of 79.1%.

[0219] Purify 10 g of the crude product of the compound of formula I by preparative HPLC to finally obtain 3.51 g of the pure product, with a yield of 35.1% and a purity of 99.50%.

Claims

1. An intermediate compound of formula II-1 or a salt thereof: Among them, R1 is hydrogen or a basic amino protecting group.

2. The intermediate compound or a salt thereof according to claim 1, wherein the basic amino protecting group is Fmoc.

3. An intermediate compound of formula II-2 or a salt thereof: Among them, R1 is hydrogen or a basic amino protecting group, and R2 is an acidic amino protecting group.

4. The intermediate compound or a salt thereof according to claim 3, wherein the basic amino protecting group is Fmoc and the acidic amino protecting group is Boc.

5. A method for preparing the intermediate compound II-1 as described in any one of claims 1-2, characterized in that, Comprising the following steps: Subjecting a compound of formula SM-2-1 to reductive amination with a salt of a compound III of formula III·HX to obtain an intermediate compound II-1: wherein, R1 is as defined in any one of claims 1-2; HX is selected from trifluoroacetic acid and hydrochloric acid; wherein the reductive amination reaction is carried out in a solvent, and the molar amount of the HX salt of the compound III to the volume of the solvent is (1 mol: 4 L) to (1 mol: 8 L); the solvent is a mixed solvent of an aprotic solvent and an alcohol solvent; the aprotic solvent is selected from one or more of dichloromethane, tetrahydrofuran or diethyl ether, and the alcohol solvent is selected from one or more of methanol, ethanol and isopropanol; the volume ratio of the aprotic solvent to the alcohol solvent is (1.5: 1) to (5: 1); sodium triacetoxyborohydride is used as a reducing agent in the reductive amination reaction; the molar ratio of the compound III·HX to the reducing agent is (1: 2) to (1: 8); the compound SM-2-1 is prepared by the following oxidation reaction: oxidizing diethylene glycol monomethyl ether in an oxidation system to a compound of formula SM-2-1, the oxidation system in the oxidation reaction comprises an oxidizing agent and triethylamine, and the oxidation reaction product is directly used for the preparation of the intermediate compound II-1 without separation and purification; the oxidizing agent is a combination of DMSO and oxalyl chloride or a combination of DMSO and trifluoroacetic anhydride; the molar ratio of the diethylene glycol monomethyl ether to the oxidizing agent is (1: 1) to (1: 5); the molar ratio of the diethylene glycol monomethyl ether to the triethylamine is (1: 2) to (1: 10); the molar ratio of oxalyl chloride to DMSO or the molar ratio of trifluoroacetic anhydride to DMSO is (1: 1) to (1: 5).

6. According to the method for preparing the intermediate compound II-1 as claimed in claim 5, wherein: the molar ratio of the diethylene glycol monomethyl ether to the oxidizing agent is (1: 1) to (1: 3); the molar ratio of the diethylene glycol monomethyl ether to the triethylamine is (1: 2) to (1: 6); the molar ratio of oxalyl chloride to DMSO or the molar ratio of trifluoroacetic anhydride to DMSO is (1: 1) to (1: 3).

7. According to the method for preparing the intermediate compound II-1 as claimed in any one of claims 5-6, wherein the oxidation reaction is carried out in an aprotic solvent; the aprotic solvent is selected from one or more of dichloromethane, tetrahydrofuran and diethyl ether; The molar amount of the diethylene glycol monomethyl ether to the volume of the aprotic solvent is (1 mol: 1 L) to (1 mol: 2 L).

8. The preparation method of intermediate compound II-1 according to claim 7, wherein, The aprotic solvent is dichloromethane.

9. The method for preparing the intermediate compound II-1 according to any one of claims 5-6 and 8, wherein the temperature of the oxidation reaction is less than or equal to -30 °C.

10. The method for preparing the intermediate compound II-1 according to any one of claims 5-6 and 8, wherein the temperature of the oxidation reaction is less than or equal to -60 °C.

11. The method for preparing the intermediate compound II-1 according to any one of claims 5-6 and 8, wherein the temperature of the oxidation reaction is less than or equal to -70 °C.

12. A method for preparing the intermediate compound II-2 according to any one of claims 3-4, wherein the intermediate compound II-2 is obtained by subjecting the intermediate compound II-1 according to any one of claims 1-2 to amino protection, Among them, R1 and R2 are as defined in any one of claims 1-2, wherein the intermediate compound II-1 is prepared according to the preparation method described in any one of claims 5-11; the intermediate compound II-1 is directly used for the preparation of the intermediate compound II-2 without separation and purification after being prepared.

13. A solid-phase synthesis method of a compound I represented by the following formula comprising the following steps: 1) Condensing the compound M-1-1 immobilized on a solid-phase carrier with the intermediate compound II-2 to obtain a polypeptide compound M-2 immobilized on a solid-phase carrier, and then removing the R1 protecting group to obtain a compound M-2-1 immobilized on a solid-phase carrier, 2) Using amino acid derivatives SM-3, amino acid derivatives SM-4, and amino acid derivatives SM-5 as raw materials in sequence to carry out the following reactions, and finally obtaining a compound M-5 immobilized on a solid-phase carrier, 3) Adding a cleavage solution to the compound M-5 immobilized on a solid-phase carrier obtained in step 2) for deprotection and cleavage to obtain a crude product of compound I, Among them, R1 and R2 are as defined in claim 3; R3 and R4 are basic amino protecting groups, and R y and R5 are acidic amino protecting groups; Optionally, step 4) is included after step 3): separating and purifying the crude product of compound I obtained in step 3) to obtain a pure product of compound I; The ratio of the cleavage solution in step 3) to the polypeptide compound M-5 immobilized on a carrier obtained in step 2) is (6-10) ml: 1 g; Before the separation and purification in step 4) after the cleavage in step 3), there is also a step of precipitating the crude product of compound I with an ether solvent; The separation and purification in step 4) is carried out in a reverse-phase high-performance liquid chromatography.

14. The solid-phase synthesis method of the compound I according to claim 13, wherein the compound M-1-1 immobilized on a solid-phase carrier in step 1) is prepared by the following method: immobilizing the compound SM-1 on a solid-phase carrier, and then removing the protecting group Rx of the piperidine ring imino group to obtain the compound M-1-1 immobilized on a solid-phase carrier, Among them, R x is an alkaline amino protecting group; the solid-phase carrier is Wang resin or 2-chlorotrityl chloride resin; the substitution degree of the Wang resin is 0.3-1.0 mmol / g, and the substitution degree of the 2-chlorotrityl chloride resin is 0.2-1.6 mmol / g.

15. The solid-phase synthesis method of the compound I according to claim 13 or 14, wherein the intermediate compound II-2 is obtained according to the preparation method described in claim 12.

16. The solid-phase synthesis method of compound I according to any one of claims 13 to 14, wherein a condensing agent is used for peptide condensation in steps 1) and 2); the condensing agent is one or more of the following combinations: a) HBTU, Cl-HoBt and DIEA; b) DIC and Cl-HoBt; c) PyBOP, Cl-HoBt and DIEA; d) HBTU, Oxyma, DIEA; e) DIC and HoBt.

17. The solid-phase synthesis method of compound I according to claim 16, wherein the condensing agent is a combination of HBTU, Cl-HoBt and DIEA, a combination of DIC and Cl-HoBt, or a combination of DIC and HoBt.

18. The solid-phase synthesis method of Compound I according to any one of claims 13 to 14 and 17, wherein when R y , R2 and R5 are acidic amino protecting groups, the cleavage solution in step 3) contains 50%-100% TFA.

19. The solid-phase synthesis method of compound I according to claim 18, wherein the cleavage solution further contains one, two or more of 0%-10% TIS, 0%-10% H2O and 0%-10% TES.

20. The solid-phase synthesis method of compound I according to claim 18, wherein the cleavage solution consists of 90% TFA / 5% TIS / 5% H2O v / v / v or 95% TFA / 5% H2O v / v.

21. The solid-phase synthesis method of compound I according to any one of claims 13 to 14, 17, 19-20, wherein the ether solvent is anhydrous diethyl ether or methyl tert-butyl ether.

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