CN118271395A

CN118271395A - Preparation method of intermediate of antibody-coupled drug

Info

Publication number: CN118271395A
Application number: CN202311836261.2A
Authority: CN
Inventors: 李振东; 王东生; 杨周; 沈磊; 刘宁; 邱雪飞; 范丹
Original assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Current assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Priority date: 2022-12-30
Filing date: 2023-12-28
Publication date: 2024-07-02

Abstract

The invention discloses a preparation method of an intermediate of an antibody coupled drug, and particularly provides a preparation method of a compound I. The preparation method provided by the invention comprises the following steps of separating a mixture containing a compound I and main control impurities under a high-pressure preparation chromatographic condition, wherein a chromatographic column is a dynamic axial compression column; the stationary phase is unbound silicon hydroxyl; the mobile phase is a mixed solution of an alcohol solvent and a halogenated hydrocarbon solvent. The preparation method can realize the effective separation of the compound I and the main control impurity, thereby improving the purity of the compound I and better controlling the quality of the subsequently synthesized antibody coupling drug.

Description

Preparation method of intermediate of antibody-coupled drug

Technical Field

The invention belongs to the field of drug synthesis and purification, and mainly relates to a preparation method of an intermediate of an antibody coupled drug.

Background

Antibody coupled drugs (ADCs) become drug lines which are in the arrangement of medical enterprises at home and abroad in recent years, and are one of the hot spots of attention in the pharmaceutical industry in recent years. The ADC medicine consists of three basic modules, including antibody, connector and effector molecule. The ADC medicine utilizes the specific targeting effect of the antibody to transmit effector molecules to tumor sites for enrichment, so that the aim of killing tumor cells is fulfilled. Among effector molecules, a class of camptothecin analogs is widely used in the ADC field. In recent years, immunomedics company applies the camptothecin as an effector molecule to an ADC drug IMMU-132 (ZL 200980156218) thereof, which shows better anti-tumor effect, and the first three uses another camptothecin analogue as an ADC drug DS-8201a (ZL 201380053256) of the effector molecule together, which also shows better anti-tumor effect.

Patent WO2020259258A1 discloses an ADC compound with a camptothecin derivative DXd as effector molecule and provides a method for the preparation and purification of intermediates (compound I) of two linker drug conjugates, and patent WO2022204947A1, CN115215921a discloses improved routes 1 and 2, according to which both routes 1 and 2 compound I can be prepared with a purity of 98% and an impurity content of < 1%. The specific route is as follows.

Route 1:

route 2:

Patent WO2022204947A1, i.e. route 1, further discloses an HPLC detection method for a compound I finished product, by which no obvious single impurity peak is found in the spectrum with relative retention time between rrt=1.0 and 1.1, no impurity with consistent relative retention time is found, and the purity of the product reaches more than 98%, but the purity of an antibody coupling drug obtained by coupling a compound I with an antibody is not high, and potential toxic and side effects are contained, so that different HPLC conditions are screened, and when detecting a raw material compound I, it is found that an obvious impurity peak exists at a position with a relative retention time of 1.04 under the new HPLC condition. In the subsequent ADC medicine preparation process, main control impurities are introduced into the antibody coupling medicine after the patent medicine due to the characteristics of similar structure and the like of the main product, and the main control impurities have potential safety toxicity to the antibody coupling medicine, so that the compound I is required to be further purified, the main control impurities with potential safety toxicity are separated, and the product purity of the compound I is improved.

In view of the product yield and the operation convenience of the intermediate of the antibody-coupled drug during the amplified production, a reasonable and effective preparation and purification method is needed to solve the problem.

Disclosure of Invention

Aiming at the problem that the existing main control impurities with the properties and structures similar to those of the compound I are difficult to detect and separate in the preparation route of the antibody coupling drug intermediate, the invention provides a preparation method of the antibody coupling drug intermediate, wherein the main control impurities are introduced during chloromethylation reaction in the synthesis route of the compound I, and finally the main control impurities in the compound I are generated through subsequent reaction steps, and the mass spectrum is ESI-MS m/z:1171; the structure of the compound I and the chloromethylation reaction step are as follows:

The invention mainly solves the technical problems through the following technical scheme.

The invention provides a purification method of a compound I, which comprises the following steps: separating the mixture containing the compound I and the main control impurity under the high-pressure chromatography condition;

Wherein, the high-pressure chromatography conditions are as follows:

The chromatographic column is a dynamic axial compression column;

the stationary phase is unbound silicon hydroxyl;

The mobile phase is a mixed solution of an alcohol solvent and a halogenated hydrocarbon solvent.

In some embodiments, the master impurity has the structure:

In some embodiments, the purification process collects a fraction having a compound I content of greater than 90.0%, preferably greater than 95%, and more preferably greater than 98%.

In some embodiments, the dynamic axial compression column inner diameter is 50-200mm, preferably 50mm or 200mm.

In some embodiments, the dynamic axial compression column length is 650-1000mm, preferably 650mm or 1000mm.

In some embodiments, the particle size of the stationary phase is 5-50 μm, preferably 8-20 μm, further preferably 10-20 μm, most preferably 20 μm.

In some embodiments, the stationary phase is8-120、10-120 Or20-120, Preferably10-120 Or20-120, Further preferred20-120。

In some embodiments, the stationary phase has a pore size of

In some embodiments, the volume ratio of the alcoholic solvent to the halogenated hydrocarbon solvent is from 35:65 to 2:98, more preferably from 35:65 to 3:97, for example, 35:65, 30:70, 25:75, 20:80, 10:90, or 3:97, still more preferably from 10:90 to 3:97, and most preferably 10:90.

In some embodiments, the alcoholic solvent is methanol, ethanol, or isopropanol; methanol is preferred.

In some embodiments, the haloalkane solvent is a C _1-2 saturated or unsaturated chloroalkane, such as methylene chloride, chloroform, carbon tetrachloride, 1-dichloroethane, 1, 2-dichloroethane, 1-trichloroethane, 1, 2-trichloroethane 1, 2-tetrachloroethane, 1, 2-tetrachloroethane, pentachloroethane, 1-dichloroethylene, 1, 2-dichloroethylene, trichloroethylene, or tetrachloroethylene; preferably dichloromethane, trichloromethane, 1-trichloroethane, trichloroethylene or tetrachloroethylene; further preferred is dichloromethane, chloroform; most preferred is methylene chloride.

In some embodiments, the flow rate of the mobile phase is selected as usual in the art, preferably 50 to 1000ml/min, further preferably 50ml/min or 1000ml/min.

In some embodiments, the dynamic axial compression column pressure is 1.0-2.0 Mpa, preferably 1.1-1.5Mpa, more preferably 1.1Mpa, 1.2Mpa, 1.25Mpa, 1.3Mpa or 1.5Mpa.

In some embodiments, the dynamic axial compression column is model ZDAC50 0 x 650, and the mobile phase flow rate is 50-70 ml/min, preferably 50ml/min.

In some embodiments, the dynamic axial compression column is ZDAC200 x 1000, and the mobile phase has a flow rate of 800 to 1000ml/min; preferably 1000ml/min.

In some embodiments, the purification method may further comprise the steps of: and (3) dissolving the mixture in a chlorinated alkane solvent, separating under the high-pressure chromatographic condition, and collecting a main peak product to obtain the compound I. Preferably, the mass to volume ratio of the mixture to the chlorinated alkane solvent is 0.1-1g/mL.

In some embodiments, in the purification method, when the stationary phase is8-120, Wherein the volume ratio of the alcohol solvent to the chlorinated alkane solvent is 35:65-10:90.

In some embodiments, in the purification method, when the stationary phase is10-120, Wherein the volume ratio of the alcohol solvent to the chlorinated alkane solvent is 30:70-3:97.

In some embodiments, in the purification method, when the stationary phase is20-120, The volume ratio of the alcohol solvent to the chlorinated alkane solvent is 30:70-3:97, preferably 10:90-3:97, and most preferably 10:90.

In some embodiments, the purification method can be any of the following schemes:

Scheme 1: in the purification method, the stationary phase is 8-120; The volume ratio of the methanol to the dichloromethane is 35:65; preferably, the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

scheme 2: in the purification method, the stationary phase is 8-120; The volume ratio of the methanol to the dichloromethane is 25:75; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC50 0x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

scheme 3: in the purification method, the stationary phase is 8-120; The volume ratio of the methanol to the dichloromethane is 20:80; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC50 0x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

Scheme 4: in the purification method, the pressure is 1.2Mpa; the stationary phase is 8-120; The volume ratio of the methanol to the dichloromethane is 10:90; preferably, the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

scheme 5: in the purification method, the stationary phase is 10-120; The volume ratio of the methanol to the dichloromethane is 3:97; preferably, the pressure is 1.1Mpa; the model of the dynamic axial compression column is ZDAC50 0x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

Scheme 6: in the purification method, the stationary phase is 10-120; The volume ratio of the methanol to the dichloromethane is 30:70; preferably, the pressure is 1.5Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

Scheme 7: in the purification method, the stationary phase is 20-120; The volume ratio of the methanol to the dichloromethane is 3:97; preferably, the pressure is 1.25Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

scheme 8: in the purification method, the stationary phase is 20-120; The volume ratio of the methanol to the dichloromethane is 30:70; preferably, the pressure is 1.3Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

scheme 9: in the purification method, the stationary phase is 20-120; The volume ratio of the methanol to the dichloromethane is 10:90; preferably, the pressure is 1.5Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength is 254nm;

Scheme 10: in the purification method, the stationary phase is 20-120; The volume ratio of the methanol to the dichloromethane is 10:90; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC ×1000; the flow rate of the mobile phase is 1000ml/min; the detection wavelength is 254nm.

In some embodiments, the mass content of compound I in the mixture is at least 65%, preferably 65.0% to 95.0%, and more preferably 70.0% to 85.0%.

In some embodiments, the amount of the host impurity in the mixture is up to 10.0%, preferably 1.0% to 10.0%, and more preferably 3.0% to 5.5%.

In some embodiments, the master impurity mass spectrum is ESI-MS m/z:1171.

In some embodiments, the relative (compound I) retention time of the master impurity under any of the HPLC conditions described below is 1.03-1.05, preferably 1.04;

HPLC conditions 2-1: chromatographic column model: agilent AdvanceBio PEPTIDE MAP,3.5 μm, 2.1X1250 mm; mobile phase: phase a is 0.01mol/L potassium dihydrogen phosphate aqueous solution (ph=5.0), phase B is 10% methanolic acetonitrile solution (volume ratio of methanol to acetonitrile 10:90); sample injection amount: 5uL; column temperature: 45 ℃; detection wavelength: 254nm, flow rate: 0.25mL/min;

HPLC conditions 2-2: chromatographic column model: agilent AdvanceBio PEPTIDE MAP,3.5 μm, 2.1X1250 mm; mobile phase: phase a is 0.01mol/L potassium dihydrogen phosphate aqueous solution (ph=5.0), phase B is 10% methanolic acetonitrile solution (volume ratio of methanol to acetonitrile 10:90); sample injection amount: 5uL; column temperature: 45 ℃; detection wavelength: 254nm, flow rate: 0.25mL/min; high performance liquid chromatograph: agilent 187260.

In some embodiments, the master impurity has a retention time of 29.8min to 33.0min, preferably 32.578min, under any of the HPLC conditions described below;

In some embodiments, the retention time of compound I under any one of the following HPLC conditions is 29.5min to 32.5min, preferably 31.321;

In some embodiments, the master impurity is produced by a chloromethylation reaction step in the process for the preparation of compound I, followed by subsequent reactions to produce the master impurity; the preparation method of the compound I can comprise a route 1, a route 2, a route 3 or a route 4; the chloromethylation reaction step has the following reaction formula:

in some embodiments, the route 1 comprises the steps of:

Step 1: reacting Sup>A compound of the formulSup>A V-A with paraformaldehyde and trimethylchlorosilane in Sup>A solvent to obtain Sup>A compound of the formulSup>A IV-A;

step 2: carrying out substitution reaction on Sup>A compound of formulSup>A IV-A and DXd or DXd derivatives in Sup>A solvent in the presence of alkali to obtain Sup>A compound of formulSup>A III-A;

Step 3: carrying out reduction reaction on a compound of the formula III-A and a reducing agent in an organic solvent and in the presence of an acid buffer solution to obtain a compound of the formula II-A;

Step 4: coupling the compound of the formula II-A and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain a compound I;

Wherein R ¹ is C ₁～C₆ alkyl, one or more N (R ^1-1)(R^1-2) -substituted C ₁～C₆ alkyl, or one or more R ^1-3S(O)₂ -substituted C ₁～C₆ alkyl;

R ² and R ³ are each independently C ₁～C₆ alkyl, one or more halogen substituted C ₁～C₆ alkyl, or halogen;

R ^1-1、R^1-2 and R ^1-3 are each independently C ₁～C₄ alkyl.

In some embodiments, the route 2 comprises the steps of:

Step 1: reacting a compound of the formula VI-B with paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of the formula V-B;

Step 2: carrying out substitution reaction on a compound of the formula V-B and DXd derivatives in a solvent in the presence of alkali to obtain a compound of the formula IV-B;

Step 3: carrying out reduction reaction on a compound of the formula IV-B and a reducing agent in an organic solvent and in the presence of an acid buffer solution to obtain the compound of the formula III-B;

Step 4: coupling the compound of the formula III-B with 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain the compound of the formula II-B;

Step 5: removing the R ⁴ protecting group of the compound of the formula II to obtain a compound of the formula I;

R ^1-1、R^1-2 and R ^1-3 are each independently C ₁～C₄ alkyl;

R ⁴ is a hydroxyl protecting group.

In some embodiments, the route 3 comprises the steps of:

Step 1: carrying out substitution reaction on a compound of the formula IX-C, paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of the formula VI II-C;

step 2: etherification reaction is carried out on a compound of the formula VIII-C and a reagent VII-C in a solvent in the presence of alkali to obtain a compound of the formula VI-C;

step 3: deprotection of a compound of formula VI-C in the presence of a deprotection agent to give a compound of formula V-C;

Step 4: carrying out amide condensation reaction on the intermediate compound of the formula V-C and the compound of the formula III or the mesylate thereof in the presence of a condensing agent, alkali and a solvent to obtain a compound of the formula IV-C;

step 5: carrying out reduction reaction on a compound of the formula IV-C and a reducing agent in a solvent and in the presence of an acid buffer solution to obtain a compound of the formula II-C;

step 6: coupling the compound of the formula II-C and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain a compound I;

wherein R is-Si (C ₁～C₆)₃ substituted C ₁～C₆ alkyl).

In some embodiments, the route 4 comprises the steps of:

step 1: carrying out substitution reaction on the compound of the formula IX-D, paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of the formula VIII-D;

step 2: etherification reaction is carried out on the compound of the formula VIII-D and the reagent VII-D in a solvent in the presence of alkali to obtain the compound of the formula VI-D;

step 3: carrying out reduction reaction on a compound of the formula VI-D and a reducing agent in a solvent and in the presence of an acid buffer solution to obtain a compound of the formula V-D;

Step 4: coupling the compound of the formula V-D and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain a corresponding compound of the formula IV-D;

step 5: deprotection reaction is carried out in the presence of a deprotection agent of the formula IV-D in a solvent to obtain a compound of the formula II-D;

Step 6: performing amide condensation reaction on the compound of the formula II-D and the compound of the formula III or the mesylate thereof in the presence of a condensing agent, alkali and a solvent to obtain a compound I;

Wherein R is-Si (C ₁～C₆)₃ substituted C ₁-C₆ alkyl).

The invention also provides a separation and detection method of the compound I, which comprises the following steps: separating and detecting the mixture containing the compound I under the condition of high performance liquid chromatography;

wherein, the high performance liquid chromatography conditions are as follows:

The chromatographic column is Agilent AdvanceBio PEPTIDE MAP,3.5 μm, 2.1X1250 mm;

the mobile phase is a mixed solution of 0.01mol/L potassium dihydrogen phosphate aqueous solution (PH=5.0) and 10 percent methanol acetonitrile solution (the volume ratio of methanol to acetonitrile is 10:90);

The detection wavelength is 254nm;

The elution conditions were as follows:

In some embodiments, the methods of preparing compound I are as set forth in any one of schemes 1-4 above.

As used herein, "room temperature" refers to 10-30deg.C.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

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

The invention has the positive progress effects that:

The preparation and purification method provided by the invention has a better purification effect on the intermediate crude product of the antibody coupling drug and can better remove main control impurities, and the preparation and purification method adopts a high-pressure normal phase preparation and purification technology, optimizes the proportion of silica gel filler, eluent and eluent, can purify to obtain a compound I product with the main control impurity content less than or equal to 0.2%, and can obtain a compound I product with the main control impurity content less than or equal to 0.05% under a better condition, so that the purity of the compound I is further improved, the requirement of subsequent antibody coupling is met, and the antibody coupling drug with higher purity is prepared.

Specifically, the invention also has the following characteristics:

Compared with the traditional glass column chromatography, the purification method disclosed by the invention is simple and efficient to operate, short in time consumption, less in solvent quantity, capable of carrying out process amplification, and capable of separating and obtaining a large number of antibody coupling drug intermediates with high purity, low impurities and high yield.

Drawings

FIG. 1 is an HPLC plot of crude compound I obtained in scheme 3 of example 3 (compound I RT 31.321min, master impurity RT 32.578 min).

FIG. 2 is an HPLC plot of the compound I product obtained in example 14 (compound I RT 31.321min, master impurity RT 32.578 min).

FIG. 3 is a mass spectrum of Compound I.

Fig. 4 is a mass spectrum of a master impurity.

Fig. 5 is a nuclear magnetic resonance hydrogen spectrum of a host impurity.

Fig. 6 is a mass spectrum of an initial source compound of a host impurity.

FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of an initial source compound of a host impurity.

Detailed Description

The present invention will be further described with reference to the following examples, which are given by way of illustration only and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will now occur to those skilled in the art in light of the foregoing disclosure.

Unless otherwise specified, the proportions of the substances referred to in the following examples are all volume ratios.

The parameters of the column chromatography packed stationary phase used in the following examples are shown in table 1.

TABLE 1 packing stationary phase parameters

The preparation separation apparatus parameters used in the following examples are shown in table 2.

TABLE 2 preparation of separation plant parameters

In the following comparative examples and examples, the HPLC analysis conditions for the content of Compound I in the final compound were as follows:

HPLC condition 1:

HPLC conditions 1 are liquid chromatography conditions published by WO2022204947A1, CN115215921 a: phase A is 0.1% formic acid aqueous solution (volume ratio of formic acid to water is 0.1:99.9), phase B is 0.1% formic acid acetonitrile solution (volume ratio of formic acid to acetonitrile is 0.1:99.9), detection wavelength is 370nm, instrument is Agilent 1260, chromatographic column is ZORBAX Eclipse Plus C, 3.5 μm, 4.6X1150 mm. The gradient settings are as follows in table 3.

TABLE 3 gradient setup of mobile phases

Time (min)	Mobile phase A%	Mobile phase B%
			0.00	80.0	20.0
10.00	60.0	40.0
			25.00	60.0	40.0
35.00	30.0	70.0
			41.00	30.0	70.0
41.10	80.0	20.0
			43.00	80.0	20.0

HPLC condition 2:

Phase a was 0.01mol/L potassium dihydrogen phosphate aqueous solution (ph=5.0), phase B was 10% methanolic acetonitrile solution (volume ratio of methanol to acetonitrile 10:90), detection wavelength was 254nm, instrument was agilent 187260, column chromatography was Agilent AdvanceBio PEPTIDE MAP,3.5 μm,2.1×250mm, gradient set as in table 4 below.

TABLE 4 HPLC elution gradient

Time (min)	Mobile phase A%	Mobile phase B%
			0.00	80.0	20.0
5.00	60.0	40.0
			35.00	60.0	40.0
40.00	30.0	70.0
			46.00	30.0	70.0
46.10	80.0	20.0
			60.00	80.0	20.0

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

EXAMPLE 1 preparation of crude and finished Compounds I

Route 1 refers to the entire disclosure of publication No. WO2022204947 A1; route 2 refers to the entire disclosure of publication number CN115215921 a.

The preparation of the finished compounds I is described in patent WO2022204947A1 (scheme 1), CN115215921A (scheme 2), and further developed by the inventors in scheme 3 and scheme 4.

Route 1:

step 1: synthesis of Compound 4

Compound 5 (150 g,0.32 mol) synthesized according to the method of step 5 in patent WO2022204947A1 and paraformaldehyde (29 g, 1.00mol in terms of formaldehyde, 3.1 eq) were added to 3L of ultra-dry tetrahydrofuran, trimethylchlorosilane (122 mL,1.24mmol,3.9 eq) was added dropwise to the resulting mixture, after the addition was completed, the resulting mixture was stirred at room temperature overnight, the reaction solution was then directly filtered, the filtrate was distilled off under reduced pressure to remove the solvent and vacuum was applied on a high vacuum pump for 2 hours to give a solid compound 4 (directly used in the next step) as bubbles.

Step 2: synthesis of Compound 3

The crude product of compound 4 obtained according to the method of step 6 in patent WO2022204947A1 was dissolved with 2L of ultra-dry 1, 4-dioxane, then Dxd (50 g,0.10mol, commercially available) and panpiridine (87 mL,0.48 mol) were added thereto, and the resulting mixture was heated to 60℃and stirred for 2 hours. The solvent was distilled off from the resultant reaction solution under reduced pressure, and the crude product was dissolved in 1.5L of methylene chloride, and then the crude solution was washed with 0.1mol/L of dilute hydrochloric acid, water and saturated brine in this order, and the resultant organic phase was dried over anhydrous sodium sulfate overnight. The dried solution was distilled off to remove the solvent and then subjected to silica gel column chromatography eluting with methylene chloride/methanol=60:1 to 10:1 to give compound 3 (95 g, which was used directly in the next step).

Step 3: synthesis of Compound 2

Compound 3 obtained according to the procedure of step 7 of patent WO2022204947A1 was dissolved in 600mL of tetrahydrofuran, then 400mL of tetrahydrofuran and acetic acid buffer (1000 mL,100 mM) at pH 5.0 were added, followed by 1M solution of trimethylphosphine in tetrahydrofuran (290 mL,290 mmol). The resulting mixture was stirred at 0-5℃for 2 hours, and after completion of the reaction, 1000mL of saturated brine was added to the reaction mixture, followed by extraction with 2.5L of methylene chloride. The resulting organic phase was dried over anhydrous sodium sulfate overnight, the solvent was distilled off under reduced pressure, and the crude product was chromatographed on a silica gel column, eluting with dichloromethane/methanol 20:1-10:1 to give compound 2 (50 g, 97% purity, 53% yield based on Dxd).

Step 4: synthesis of Compound 1

Compound 2 (50 g,53.0 mmol) was mixed with compound EMCS (commercially available, 32g,0.104 mmol) and dissolved in 2L dichloromethane and the resulting mixture was stirred overnight at 40 ℃. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography, eluting with methylene chloride/methanol=50:1 to 15:1 to give compound 1 (39 g, purity 98%, yield 64%).

ESI-MS m/z：1141.4(M+H),¹H NMR(400MHz,DMSO)δ9.98(s,1H),8.55(s,1H),8.31–8.11(m,1H),7.88–7.72(m,2H),7.63–7.50(m,2H),7.28(s,3H),6.99(s,2H),6.51(s,1H),5.59(s,1H),5.50–5.32(m,2H),5.17(s,2H),4.98(s,2H),4.85(d,J＝17.3Hz,2H),4.43–4.33(m,1H),4.25–4.11(m,1H),4.03(s,2H),3.74–3.64(m,2H),3.20–3.03(m,3H),3.01–2.81(m,4H),2.36(s,3H),2.23–2.09(m,4H),2.01–1.91(m,1H),1.90–1.76(m,2H),1.55–1.39(m,4H),1.30(d,J＝6.7Hz,3H),1.22–1.11(m,2H),0.93–0.77(m,9H).

Route 2:

step 1: synthesis of Compound 4

Compound 5 (3.3 g,7.0 mmol) and paraformaldehyde (651.0 mg, as converted to formaldehyde: 21.7 mmol) synthesized according to step 7 of CN115215921A were added to 50mL of ultra-dry tetrahydrofuran, and to the resulting mixture was added dropwise trimethylchlorosilane (3.5 mL,28.0 mmol), and after the addition was completed, the resulting mixture was stirred at room temperature overnight, then the reaction solution was directly filtered, and the resulting filtrate was subjected to evaporation of the solvent under reduced pressure and vacuum-pulled on a high vacuum pump for 2 hours to give Compound 4 (3.9 g) as a foam (directly used in the next step).

Step 2a: synthesis of Compound 3a

The crude product of compound 4 (3.9 g) obtained according to step 8 of CN115215921A was dissolved in 25mL of ultra-dry 1, 4-dioxane, and then compound Dxd-a (3.7 g,7.0mmol, synthesized as in example 2) and pantidine (1.9 mL,10.5 mmol) were added thereto, and the resulting mixture was heated to 60℃and stirred for 2 hours. The solvent was distilled off from the resultant reaction solution under reduced pressure, and the crude product was dissolved in 100mL of methylene chloride, and then the crude solution was washed with 0.1mol/L of dilute hydrochloric acid (100 mL), water (100 mL) and saturated brine (100 mL) in this order, and the resultant organic phase was dried over anhydrous sodium sulfate overnight. The dried solution was distilled off to remove the solvent and then subjected to silica gel column chromatography eluting with methylene chloride/methanol=60:1 to 10:1 to give compound 3a (7.1 g, which was used directly in the next step).

Step 2b: synthesis of Compound 3b

Another batch of crude compound 4 (3.8 g) obtained according to step 8 of CN115215921A was dissolved in dry dichloromethane (150 mL), then compound Dxd-b (3.7 g,7.0mmol, synthesized as in example 3) was added, the reaction system was replaced three times with nitrogen and cooled to 0-5℃with an ice bath, and then panobidine (1.9 mL,10.5 mmol) was slowly added dropwise over 2 hours. After the resulting mixture was stirred at 0-5 ℃ for 4-5 hours, TLC monitored the reaction was complete. The obtained reaction solution was washed with saturated brine (three times, 50mL each) and dried over anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure to obtain a crude product (8.5 g, directly used in the next step) of compound 3 b.

Step 3a: synthesis of Compound 2a

Compound 3a (7.1 g) obtained according to the procedure of step 9a in CN115215921A was dissolved in 35mL of tetrahydrofuran, then 50mL of tetrahydrofuran and acetic acid buffer (125 mL,12.5 mmol) at pH 5.0 were added, and then 1M solution of trimethylphosphine in tetrahydrofuran (7.7 mL,7.7 mmol) was added. The resulting mixture was stirred at 0-5℃for 2 hours, and after completion of the reaction, 200mL of saturated brine was added to the reaction mixture, followed by extraction with 150mL of methylene chloride. The resulting organic phase was dried over anhydrous sodium sulfate overnight, the solvent was distilled off under reduced pressure, and the crude product was chromatographed on a silica gel column, eluting with dichloromethane/methanol 20:1-10:1 to give compound 2a (3.7 g, 97% purity, 53% yield based on Dxd-a).

MS：m/z＝990.4(M+H)。

Step 3b: synthesis of Compound 2b

Compound 3b (8.5 g) obtained according to the procedure of step 9b in CN115215921A was dissolved in 35mL of tetrahydrofuran, then 120mL of tetrahydrofuran and acetic acid buffer (125 mL,12.5 mmol) at pH 5.0 were added, and then 1M solution of trimethylphosphine in tetrahydrofuran (7.7 mL,7.7 mmol) was added. The resulting mixture was stirred at 0-5℃for 2 hours, and after completion of the reaction, 200mL of saturated brine was added to the reaction mixture, followed by extraction with 150mL of methylene chloride. The resulting organic phase was dried over anhydrous sodium sulfate overnight, the solvent was distilled off under reduced pressure, and the crude product was chromatographed on a silica gel column, eluting with dichloromethane/methanol 20:1-10:1 to give compound 2b (4.7 g, 97% purity, 56% yield based on Dxd-b).

MS：m/z＝1186.5(M+H)。

Step 4a: synthesis of Compound 1a

Compound 2a (3.7 g,3.7 mmol) was mixed with compound EMCS (commercially available, 0.8g,3.7 mmol) and dissolved in 20mL of dichloromethane and the resulting mixture was stirred overnight at 40 ℃. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography, eluting with methylene chloride/methanol=50:1 to 15:1 to give compound 1a (2.8 g, yield 64%).

MS:m/z＝1183.4(M+H)；

¹H NMR(500MHz,CDCl₃)δ9.22(s,1H),7.61(d,J＝9.2Hz,1H),7.59-7.53(m,3H),7.52(s,1H),7.44(d,J＝8.4Hz,1H),7.36-7.30(m,3H),7.19(t,J＝1.0Hz,1H),6.71(s,2H),5.13(s,1H),5.13-5.07(m,3H),5.05(d,J＝9.5Hz,1H),4.91-4.82(m,3H),4.76(dd,J＝12.5,1.1Hz,1H),4.57(dd,J＝8.8,6.4Hz,1H),4.46(dq,J＝8.4,5.7Hz,1H),4.15-4.09(m,2H),3.72-3.55(m,4H),3.25(td,J＝8.2,1.6Hz,2H),3.03(s,3H),2.95(ddd,J＝12.3,8.6,5.9Hz,1H),2.84(ddd,J＝12.5,8.4,5.9Hz,1H),2.26(s,3H),2.24-2.17(m,3H),2.17-2.06(m,6H),2.00(dddd,J＝12.3,Hz,1H),1.69(p,J＝6.2Hz,2H),1.55-1.45(m,2H),1.41-1.37(m,1H),1.37-1.32(m,4H),1.08-1.01(m,3H),0.89(dd,J＝6.5,2.1Hz,6H).

Step 4b: synthesis of Compound 1b

Compound 2b (4.7 g,4.0 mmol) was mixed with compound EMCS (commercially available, 0.8g,4.0 mmol) and dissolved in 60mL of dichloromethane, and the resulting mixture was stirred overnight at 40 ℃. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (eluent dichloromethane/methanol=50:1-15:1) to give compound 1b (3.8 g, yield 68%).

MS:m/z＝1379.6(M+H)；

¹H NMR(500MHz,CDCl₃)δ9.22(s,1H),7.66-7.58(m,5H),7.58-7.53(m,2H),7.53(d,J＝8.8Hz,1H),7.44(d,J＝8.4Hz,1H),7.41-7.30(m,9H),7.18(t,J＝1.0Hz,1H),6.71(s,1H),5.15-5.03(m,6H),4.91-4.83(m,3H),4.57(dd,J＝8.8,6.4Hz,1H),4.42(dq,J＝8.6,5.7Hz,1H),4.13(d,J＝4.0Hz,2H),3.72-3.62(m,2H),3.65-3.55(m,2H),3.25(td,J＝8.2,1.6Hz,2H),3.03(s,2H),2.95(ddd,J＝12.4,8.6,5.9Hz,1H),2.84(ddd,J＝12.4,8.6,5.9Hz,1H),2.27-2.13(m,4H),2.03-1.86(m,3H),1.69(p,J＝6.2Hz,2H),1.55-1.45(m,2H),1.41-1.32(m,5H),1.07-0.98(m,9H),0.89(dd,J＝6.5,2.1Hz,6H).

Step 5a: preparation of Compound 1 from Compound 1a

Compound 1a (2.8 g,2.4 mmol) was dissolved in a mixed solvent of methanol and dichloromethane (75 mL, volume ratio 1:1) in a 250mL three-necked flask, and after the three-necked flask reaction system was replaced with nitrogen gas three times, it was cooled to 0 ℃ with an ice bath under the protection of nitrogen gas. Slowly adding an acetyl chloride (0.1 g,1.2 mmol) solution dissolved in a mixed solvent (5 mL, volume ratio of 1:1) of methanol and dichloromethane into a reaction system, and maintaining the temperature of the reaction system at 0-5 ℃ in the dripping process; after the dripping is finished, the cooling device is removed to restore the reaction system to normal temperature, stirring is continued for 2-3 hours, and TLC monitoring is carried out to finish the reaction. The obtained reaction solution was washed with aqueous sodium hydrogencarbonate solution (pH 7-8) (twice, 50mL each) and saturated brine (once, 50 mL), dried over anhydrous sodium sulfate, filtered, and finally concentrated under reduced pressure to obtain a residue which was subjected to silica gel column chromatography (eluent dichloromethane/methanol=30:1-15:1) to obtain compound 1 (1.70 g, yield 62%).

MS:m/z＝1141.4(M+H)；

¹H NMR(500MHz,CDCl₃)δ9.22(s,1H),7.61(d,J＝9.2Hz,1H),7.59-7.53(m,2H),7.53(d,J＝8.8Hz,1H),7.44(d,J＝8.4Hz,1H),7.36-7.30(m,3H),7.24(t,J＝1.0Hz,1H),6.71(s,1H),5.23(dd,J＝12.4,1.1Hz,1H),5.15-5.10(m,3H),5.06(d,J＝9.5Hz,1H),4.91-4.83(m,3H),4.77-4.70(m,2H),4.57(dd,J＝8.8,6.4Hz,1H),4.46(dq,J＝8.4,5.7Hz,1H),4.13(d,J＝4.0Hz,2H),3.72-3.55(m,4H),3.25(td,J＝8.2,1.6Hz,2H),3.03(s,2H),2.95(ddd,J＝12.5,8.6,6.0Hz,1H),2.84(ddd,J＝12.4,8.6,5.9Hz,1H),2.27-2.13(m,4H),2.01-1.86(m,2H),1.79(dq,J＝13.7,8.0Hz,1H),1.69(p,J＝6.2Hz,2H),1.55-1.45(m,2H),1.41-1.34(m,2H),1.34(d,J＝5.7Hz,3H),0.97(t,J＝8.0Hz,3H),0.89(dd,J＝6.5,2.1Hz,6H).

Step 5b preparation of Compound 1 starting from Compound 1b

Compound 1b (3.8 g,2.8 mmol) was added to a mixed solution of methylene chloride and methanol (30 mL, volume ratio: 20:1), and then a mixed solution of t-butylammonium fluoride (1.1 g,4.2mmol, dissolved in a mixed solvent of methylene chloride and methanol at a volume ratio of 20:1, 5 mL) and acetic acid (0.3 g,4.2 mmol) was slowly added dropwise to the reaction system, and as the reaction proceeded, a white solid began to precipitate in the reaction system. The resulting mixture was stirred overnight at room temperature and the reaction was completed by TLC. The reaction solution was filtered, and the obtained cake was washed with methylene chloride (twice, used in an amount of 20mL and 10mL, respectively) and then the residual solvent was removed under reduced pressure to give compound 1 (3.0 g, purity 98%, yield 95%) as a white solid.

MS:m/z＝1141.4(M+H)；

Dxd derivatives: preparation of Compound Dxd-a

Step 1: synthesis of intermediate 17

In a 250mL three-necked flask was added irinotecan (4.4 g,10.0 mmol) and methylene chloride (66 mL), and then trimethylchlorosilane (1.6 mL,12.0 mmol) was added, the resulting milky mixture was heated to 45℃under reflux for 1 hour, TLC monitored that the irinotecan had been reacted, then the reaction solution was cooled to 0℃and then N, N-diisopropylethylamine (5.0 mL,30.0 mmol) and 4-methoxytriphenylmethane chloride (3.7 g,12.0 mmol) were added, the resulting mixture was stirred at room temperature overnight, and TLC monitored that the reaction was complete. The resulting reaction solution was washed with ph=5 sodium acetate buffer (2 times each 20 mL) and saturated brine (2 times each 20 mL), and the obtained organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give crude product of intermediate 17 (6.0 g, crude yield 85%).

MS：m/z＝708.3(M+H)；

¹H NMR(500MHz,CDCl₃)δ7.34-7.26(m,11H),7.24(t,J＝1.0Hz,1H),7.16-7.10(m,2H),6.89-6.84(m,2H),5.24(dd,J＝12.4,1.0Hz,1H),5.11(d,J＝9.5Hz,1H),5.04(d,J＝9.5Hz,1H),4.88(dt,J＝8.6,4.4Hz,1H),4.75(s,1H),4.70(dd,J＝12.3,1.0Hz,1H),3.85(d,J＝8.2Hz,1H),3.78(s,2H),2.93(dt,J＝12.5,6.3Hz,1H),2.82(dt,J＝12.5,6.4Hz,1H),2.08(td,J＝6.3,4.4Hz,2H),2.04-1.89(m,2H),0.97(t,J＝8.0Hz,3H).

Step 2: synthesis of intermediate 16a

In a 100mL three-necked flask were charged intermediate 17 (2.4 g,3.0 mmol), methylene chloride (36 mL), 4-dimethylaminopyridine (1.3 g,10.0 mmol) and triethylamine (0.7 mL,5.0 mmol), and the resulting reaction system was cooled to 0℃with an ice bath and replaced three times with nitrogen. A solution (12 mL) of acetic anhydride (0.4 g,4.0 mmol) in methylene chloride was slowly added dropwise, the temperature of the system being maintained at 0-5℃during the addition. After the completion of the dropwise addition, the ice bath was removed and the reaction mixture was allowed to warm to room temperature, and the resulting reaction mixture was stirred for 3 hours, followed by TLC monitoring. The reaction was added to a buffer (50 mL) of ph=5.0 sodium acetate/acetic acid, and the resulting mixture was extracted with dichloromethane (100 mL). The organic phase was washed with saturated brine (three times, 50mL each), dried over anhydrous sodium sulfate and stirred for 0.5 hour, and then concentrated by filtration to give solid intermediate 16a (1.7 g, yield 75%).

MS:m/z＝750.3(M+H)；

¹H NMR(500MHz,CDCl₃)δ7.33-7.26(m,10H),7.19(t,J＝1.0Hz,1H),7.16-7.10(m,2H),6.89-6.84(m,2H),5.14-5.08(m,2H),5.05(d,J＝9.3Hz,1H),4.88(dt,J＝8.6,4.4Hz,1H),4.74(dd,J＝12.5,0.9Hz,1H),3.85(d,J＝8.2Hz,1H),3.78(s,2H),2.93(dt,J＝12.5,6.3Hz,1H),2.82(dt,J＝12.5,6.4Hz,1H),2.26(s,2H),2.17(dq,J＝14.1,8.3Hz,1H),2.13-2.07(m,1H),2.10-2.04(m,2H),1.05(t,J＝8.3Hz,3H).

Step 3: synthesis of intermediate 15a

Intermediate 16a (1.7 g,2.3 mmol) and dichloromethane (40 mL) were added to a three-necked flask at room temperature, triethylsilane (0.7 g,5.8 mmol) was added after the solution was cleared, the resulting mixture was cooled to-5-5℃and replaced with argon three times, the reaction mixture was kept at an internal temperature of-5-5℃under argon, stirring was continued for 1-1.5 hours, and TLC detection was completed. To the resulting reaction solution was added dropwise methyl tert-butyl ether (80 mL), the internal temperature was maintained at 0-5℃and after the completion of the addition, the supernatant was poured, and the obtained viscous solid was dissolved with methylene chloride (40 mL) under stirring, the internal temperature was maintained at 0-10℃and solid was precipitated during the addition of methyl tert-butyl ether (80 mL) dropwise, and the solid was collected by filtration to give intermediate 15a (0.8 g, crude yield 75%).

MS：m/z＝478.2(M+H)；

¹H NMR(500MHz,CDCl₃)δ7.32(d,J＝7.9Hz,1H),7.19(t,J＝1.0Hz,1H),5.13-5.07(m,2H),5.05(d,J＝9.5Hz,1H),4.76(dd,J＝12.5,1.1Hz,1H),4.26(tdd,J＝6.8Hz,1H),2.93(ddd,J＝12.4Hz,1H),2.85(ddd,J＝12.4Hz,1H),2.45(t,J＝6.9Hz,1H),2.34(t,J＝6.9Hz,1H),2.26(s,2H),2.19-2.00(m,3H),1.90(ddt,J＝12.3Hz,1H),1.05(t,J＝8.3Hz,3H).

Step 4: dxd-a Synthesis

To a 100mL three-necked flask, intermediate 15a (0.8 g,1.0 mmol), glycolic acid (0.2 g,2.0 mmol) and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (0.7 g,2.0 mmol) were added, and methylene chloride (30 mL) was further added, and after the resulting reaction system was replaced with nitrogen three times, N-diisopropylethylamine (0.5 g,4.0 mmol) was added. The resulting mixture was stirred at room temperature for 13 hours, and TLC checked the reaction. The obtained reaction solution was concentrated and purified by column chromatography (using methylene chloride: methanol=50:1 as eluent) to give compound Dxd-a (0.7 g, yield 80%).

MS：m/z＝536.2(M+H)；

¹H NMR(500MHz,CDCl₃)δ7.62(d,J＝9.3Hz,1H),7.32(d,J＝7.9Hz,1H),7.19(t,J＝1.0Hz,1H),5.15-5.08(m,2H),5.05(d,J＝9.5Hz,1H),4.90(ddd,J＝9.2Hz,1H),4.74(dd,J＝12.4Hz,1H),3.96(d,J＝5.5Hz,2H),3.86-3.80(m,1H),2.95(ddd,J＝12.3Hz,1H),2.84(ddd,J＝12.5Hz,1H),2.26(s,2H),2.22-2.10(m,2H),2.13-2.04(m,1H),2.00(dddd,J＝12.3Hz,1H),1.05(t,J＝8.3Hz,3H).

Dxd derivatives: preparation of Compound Dxd-b

Step 1: synthesis of intermediate 16b

In a 250mL three-necked flask, intermediate 17 (3.5 g,5.0 mmol), N-dimethylformamide (70 mL) and N, N-diisopropylethylamine (1.1 g,10.0 mmol) were added, the resulting reaction system was cooled to 3℃and replaced three times with nitrogen, then tert-butyldiphenylchlorosilane (1.5 g,5.5 mmol) was added dropwise to the reaction system while keeping the internal temperature at 0-10℃during the dropwise addition, the reaction mixture was allowed to warm to room temperature after the dropwise addition was allowed to stir for 3 hours, and TLC was monitored to complete the reaction. The resulting reaction solution was poured into ice water (50 mL), followed by extraction with ethyl acetate (100 mL). The resulting organic phase was washed with saturated brine (three times, 80mL each), dried over anhydrous sodium sulfate, stirred, and concentrated by filtration to give solid intermediate 16b (4.4 g, yield 92%).

MS：m/z＝946.4(M+H)；

¹H NMR(500MHz,CDCl₃)δ7.66-7.58(m,4H),7.41-7.34(m,6H),7.37-7.26(m,10H),7.18(t,J＝1.0Hz,1H),7.16-7.10(m,2H),6.89-6.84(m,2H),5.15-5.02(m,4H),4.88(ddd,J＝8.6,5.8,3.0Hz,1H),3.85(d,J＝8.2Hz,1H),3.78(s,2H),2.94(ddd,J＝12.6Hz,1H),2.81(ddd,J＝12.4Hz,1H),2.19(dddd,J＝12.5Hz,1H),2.03-1.87(m,3H),1.07-0.98(m,9H).

Step 2: synthesis of intermediate 15b

Intermediate 16b (3.3 g,4.7 mmol), methylene chloride (60 mL) was added to a three-necked flask at room temperature, triethylsilane (0.7 g,6.1 mmol) was further added, the resulting reaction system was cooled to-5-5deg.C, replaced three times with argon, and stirring was continued at-5-5deg.C under argon for 1-1.5 hours, followed by completion of the TLC detection. To the resulting reaction solution was added dropwise methyl tert-butyl ether (120 mL), the internal temperature was maintained at 0-5℃and after the completion of the addition, the supernatant was poured, and the resulting viscous solid was dissolved with methylene chloride (60 mL) under stirring, the internal temperature was maintained at 0-10℃and solid was precipitated during the addition of methyl tert-butyl ether (120 mL), and the solid was collected by filtration to give intermediate 15b (2.5 g, crude yield 78%).

MS：m/z＝674.3(M+H)。

Step 3: synthesis of Compound Dxd-b

To a 250mL three-necked flask were added intermediate 15b (1.3 g,2.0 mmol), glycolic acid (0.2 g,3.0 mmol) and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (0.8 g,3.0 mmol). DCM (55 mL) was further added, and after the resulting reaction system was replaced three times with nitrogen, N-diisopropylethylamine (0.7 g,5.0 mmol) was added, and the resulting mixture was stirred at room temperature for 13 hours, and TLC was monitored for completion of the reaction. The resulting reaction was concentrated and purified by column chromatography (DCM: meoh=50:1 as eluent) to give compound Dxd-b (1.2 g, 79% yield).

MS：m/z＝732.3(M+H)；

¹H NMR(500MHz,CDCl₃)δ7.65-7.58(m,5H),7.41-7.30(m,7H),7.18(t,J＝1.0Hz,1H),5.15-5.09(m,2H),5.11-5.03(m,2H),4.89(ddd,J＝9.3Hz,1H),4.09(d,J＝5.5Hz,2H),3.83(t,J＝5.5Hz,1H),2.95(ddd,J＝12.4Hz,1H),2.84(ddd,J＝12.5Hz,1H),2.18(dddd,J＝12.3Hz,1H),2.03-1.86(m,3H),1.07-0.98(m,10H).

Route 3:

step1: synthesis of Compounds of formulas VIII-C

A crude solution of a compound of formula VI II-C was obtained by mixing a compound of formula IX-C (25.0 g,53.36 mmol) with paraformaldehyde (2.09 g,69.37 mmol) and dissolving in 250.0mL of anhydrous 1, 4-dioxane, slowly adding trimethylchlorosilane (14.43 g,133.40 mmol), reacting at 18-25deg.C for 15.0h, sampling the reaction solution, detecting under controlled conditions, filtering the reaction solution after the reaction is completed, and concentrating the filtrate to obtain a crude compound of formula VI II-C (28.40 g). (directly used in the next reaction)

Step 2: synthesis of Compounds of formula VI-C

The crude compound of formula VI II-C (28.40 g, calculated as theoretical yield, 53.36 mmol) obtained was dissolved in 250.0mL of anhydrous tetrahydrofuran, panobidine (12.43 g,80.05 mmol) and compound of formula VII-C-7 (14.12 g,80.05 mmol) were added, the reaction was heated to 60℃under nitrogen and reacted under these conditions for 4.0 to 8.0h. After the reaction of the raw materials is completed, concentrating under reduced pressure to remove tetrahydrofuran, then adding ethyl acetate and saturated saline for extraction, drying the obtained organic phase, concentrating under reduced pressure, and purifying the obtained crude product by silica gel column chromatography (n-heptane: ethyl acetate=10:1-1:1 (v/v)) to obtain a compound of formula VI-C-6 (17.88 g, purity 92.81%, combined yield of step 1 and step 2 is 51.0%);

ESI-MS m/z：657.4(M+H)；

1H NMR(400MHz,DMSO-d6)δ10.12(s,1H),8.49(d,J＝7.0Hz,1H),7.59(d,J＝8.6Hz,2H),7.42–7.21(m,2H),5.15–4.97(m,2H),4.85(s,2H),4.46(p,J＝7.0Hz,1H),4.12(dd,J＝29.5,10.7Hz,4H),3.68(dd,J＝8.7,6.0Hz,2H),3.49(d,J＝8.2Hz,1H),3.40(t,J＝7.3Hz,2H),2.97(d,J＝26.2Hz,3H),1.33(d,J＝7.1Hz,3H),1.29–1.18(m,2H),0.93(dd,J＝9.6,6.6Hz,6H),0.87–0.82(m,1H),0.15(s,9H).

Step 3: synthesis of Compound of formula V-C

The compound of formula VI-C-6 (5.0 g,7.62 mmol) was dissolved in 50.0mL of DMF, potassium fluoride (0.67 g,11.42 mmol) was added at room temperature to form a mixed solution, the reaction was heated to 60℃under nitrogen protection, and the reaction was carried out under these conditions for 5.0 to 8.0 hours. After the reaction of the raw materials is completed, concentrating under reduced pressure to remove the solvent, and purifying the obtained crude product by silica gel column chromatography (dichloromethane: methanol=10:1-1:1 (V/V)) to obtain a compound of formula V-C (3.78 g, yield 89.1%);

ESI-MS m/z：556.0(M-H)；

¹H NMR(400MHz,DMSO-d₆)δppm 10.57(d,J＝38.3Hz,1H),9.36(d,J＝94.5Hz,1H),7.61(d,J＝8.5Hz,2H),7.28(d,J＝7.2Hz,2H),5.01(d,J＝10.8Hz,2H),4.83(s,2H),4.38(s,1H),4.17(s,1H),3.71(s,2H),3.54(s,2H),3.48(d,J＝8.3Hz,1H),3.44(s,1H),3.14(s,2H),2.95(d,J＝22.9Hz,3H),1.32(d,J＝7.2Hz,3H),0.89(dd,J＝14.7,6.7Hz,6H).

Step 4: synthesis of Compounds of formula IV-C

The compound of formula V-C (2.79 g,5.00 mmol) was dissolved in 20.0mL anhydrous DMF and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM) (2.08 g,7.50 mmol) was added and the reaction was allowed to proceed for 1.0h at room temperature. N, N-diisopropylethylamine (1.30 mL,7.50 mmol) and III (Exatecan mesylate) (2.40 g,4.50 mmol) were then added and the reaction continued for 1.5-2.0 h. After the reaction of the starting materials is completed, the solvent is removed by decompression concentration, and the obtained crude product is purified by silica gel column chromatography (chloroform: methanol=10:1 (v/v)) to obtain a compound of formula IV-C (3.44 g, purity 91.30%, yield 70.5%);

ESI-MS m/z：974.5(M+H)。

Step 5: synthesis of Compounds of formula II-C

2.1ML of a 1.0mol/L solution of trimethylphosphine in tetrahydrofuran (2.1 mL,2.01 mmol) and 5.0mL of THF were mixed and added dropwise to a sodium acetate buffer (pH=5.0) containing 10.0mL, and the reaction was cooled to 0 to 5℃under nitrogen protection. Then 5.0mL of tetrahydrofuran in which the compound of the formula IV-C (1.30 g,1.34 mmol) was dissolved was slowly dropped into the reaction system, and the reaction was continued at 0-10℃for 0.5-1.0 h. After the reaction of the raw materials is finished, removing tetrahydrofuran solvent by vacuum distillation, extracting with dichloromethane, drying an obtained organic phase, removing the solvent by vacuum distillation, and purifying the obtained crude product by silica gel column chromatography (dichloromethane: methanol=10:1-1:1 (v/v)) to obtain a compound of the formula II-C (980 mg, purity 89.75%, yield 77.5%);

ESI-MS m/z：948.7(M+H)；

step 6: synthesis of Compound I

The compound of formula II-C (0.98 g,1.04 mmol) was dissolved in 30.0mL of anhydrous dichloromethane, 6- (maleimido) hexanoic acid succinimidyl ester (0.64 g,2.08 mmol) was added, the reaction was heated to 35℃under nitrogen protection, and the reaction was carried out under this condition for 3-6 hours. After the reaction of the raw materials is completed, the solvent is removed by reduced pressure distillation to obtain a crude product of the compound I (1.53 g, the purity is 71.81 percent, and the maximum single impurity content is 5.15 percent)

ESI-MS m/z：1141.6(M+H)；

¹H NMR(500MHz,DMSO-d₆)δppm 9.93(s,1H),8.49(s,1H),8.13(d,J＝6.9Hz,1H),7.76(d,J＝28.7Hz,2H),7.57(s,2H),7.28(s,3H),6.99(s,2H),6.48(s,1H),5.59(s,1H),5.47–5.32(m,2H),5.17(s,2H),5.04–4.79(m,4H),4.38(d,J＝7.1Hz,1H),4.21–4.14(m,1H),4.03(s,2H),3.69(d,J＝2.8Hz,2H),3.37(dt,J＝14.2,7.7Hz,4H),3.24–3.09(m,2H),2.93(d,J＝37.1Hz,3H),2.36(t,J＝1.8Hz,3H),2.22–2.07(m,4H),2.01–1.77(m,3H),1.52–1.41(m,4H),1.30(d,J＝7.1Hz,3H),1.19(d,J＝7.7Hz,2H),0.84(d,J＝12.0Hz,9H).

Route 4:

step 1: synthesis of Compounds VIII-D

The compound of the formula IX-D (25.00 g,53.36 mmol) was mixed with paraformaldehyde (2.09 g,69.37 mmol) and dissolved in 250.0mL of anhydrous 1, 4-dioxane, trimethylchlorosilane (17.39 g,160.08 mmol) was slowly added to the resultant mixture with stirring, the resultant reaction system was continuously stirred at 18℃to 25℃for 15.0 hours, the reaction was monitored by TLC, and after completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain the crude compound of the formula VIII-D (28.95 g). (directly used in the next reaction)

Step 2: synthesis of Compound VI-D-6

The crude compound of formula VIII-D obtained in step 1 (28.95 g, 53.37mmol calculated as theoretical yield) was dissolved in 250.0mL of anhydrous tetrahydrofuran and panpridine (12.43 g,80.05 mmol) and V II-D-7 (14.12 g,80.05 mmol) were added with stirring, and the resulting mixture was heated to 60℃under nitrogen and reacted under this condition for 6.0h. The reaction was monitored by TLC, after the starting material had reacted, tetrahydrofuran was removed by concentrating under reduced pressure, then ethyl acetate and saturated brine were added to the concentrate for extraction, the resulting organic phase was dried and concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by silica gel column chromatography (n-heptane: ethyl acetate=10:1 to 1:1 (v/v)) to give the compound of formula VI-D-6 (18.12 g, purity 93.3%, step one and step two combined yield 51.7%).

ESI-MS m/z：657.4(M+H)。

¹H-NMR(400MHz,DMSO-d₆)δ10.12(s,1H),8.49(d,J＝7.0Hz,1H),7.59(d,J＝8.6Hz,2H),7.42–7.21(m,2H),5.15–4.97(m,2H),4.85(s,2H),4.46(p,J＝7.0Hz,1H),4.12(dd,J＝29.5,10.7Hz,4H),3.68(dd,J＝8.7,6.0Hz,2H),3.49(d,J＝8.2Hz,1H),3.40(t,J＝7.3Hz,2H),2.97(d,J＝26.2Hz,3H),1.33(d,J＝7.1Hz,3H),1.29–1.18(m,2H),0.93(dd,J＝9.6,6.6Hz,6H),0.87–0.82(m,1H),0.15(s,9H).

Step 3: synthesis of Compound V-D-5

32.0ML of a 1M solution of trimethylphosphine in tetrahydrofuran and 15.0mL of THF were mixed and added dropwise to a solution of 60.0mL of sodium acetate buffer (pH=5.0) under nitrogen protection, the resulting reaction system was stirred and cooled to 0℃to 5℃and then 45.0mL of tetrahydrofuran in which the compound of the formula VI-D-6 (15.00 g,21.20 mmol) was dissolved was slowly added dropwise to the reaction system, and the resulting reaction system was kept at 0℃to 5℃for further stirring for 0.5 to 1.0 hour. Monitoring the reaction by TLC, after the reaction of the raw materials is finished, concentrating under reduced pressure to remove tetrahydrofuran in the reaction liquid, adding dichloromethane into the obtained concentrated reaction liquid for extraction, drying an obtained organic phase, concentrating under reduced pressure, and purifying the obtained crude product by silica gel column chromatography (dichloromethane: methanol=10:1-1:1 (V/V)) to obtain a compound of the formula V-D-5 (10.30 g, purity 96.5%, yield 76.9%);

ESI-MS m/z：631.4(M+H)。

¹H-NMR(400MHz,DMSO-d₆)δ10.10(s,1H),8.47(d,J＝7.2Hz,1H),7.56(d,J＝8.6Hz,2H),7.41–7.20(m,2H),5.16–4.99(m,2H),5.16(m,2H),4.86(s,2H),4.45(p,J＝7.4Hz,1H),4.10(dd,J＝29.3,10.5Hz,4H),3.66(dd,J＝8.5,6.0Hz,2H),3.47(d,J＝8.2Hz,1H),3.42(t,J＝7.5Hz,2H),2.97(d,J＝26.2Hz,3H),1.35(d,J＝7.1Hz,3H),1.27–1.16(m,2H),0.95(dd,J＝9.6,6.6Hz,6H),0.85–0.80(m,1H),0.13(s,9H).

Step 4: synthesis of Compound IV-D-4

The compound of formula V-D-5 (10.00 g,15.30 mmol) was dissolved in 300.0mL of anhydrous dichloromethane, succinimidyl 6- (maleimido) hexanoate (7.08 g,22.95 mmol) was added with stirring, the resulting reaction system was heated to 35℃under nitrogen protection, and stirring was continued under this condition for 3-6 h. The reaction was monitored by TLC, after the starting material had reacted, the resulting reaction solution was concentrated under reduced pressure, the crude product was washed with dichloromethane and water, the organic phase was dried and concentrated under reduced pressure, and the resulting concentrate was purified by silica gel column chromatography (dichloromethane: methanol=10:1 to 1:1 (v/v)) to give the compound of formula IV-D-4 (9.80 g, purity 97.31%, yield 75.4%).

ESI-MS m/z：824.5(M+H)。

¹H-NMR(400MHz,DMSO-d₆)δ9.95(s,1H),8.14(d,J＝6.9Hz,1H),7.79(d,J＝8.6Hz,1H),7.59(d,J＝8.2Hz,2H),7.39–7.22(m,2H),6.99(s,2H),5.07–5.01(m,2H),4.85(s,2H),4.38(p,J＝7.0Hz,1H),4.16(dd,J＝8.6,6.9Hz,2H),4.08(d,J＝12.3Hz,2H),3.68(dd,J＝8.8,6.0Hz,2H),3.44–3.35(m,4H),3.07(s,1H),2.97(d,J＝27.4Hz,3H),2.14(tt,J＝14.1,6.9Hz,2H),1.95(h,J＝6.7Hz,1H),1.54–1.42(m,4H),1.29(d,J＝7.1Hz,3H),1.18(q,J＝7.7Hz,2H),0.98–0.91(m,2H),0.84(dd,J＝15.4,6.8Hz,6H),0.06(s,9H).

Step 5: synthesis of Compounds II-D

The compound of formula IV-D-4 (8.50 g,10.04 mmol) was dissolved in DMF (45.0 mL), potassium fluoride (0.88 g,15.06 mmol) was added at room temperature and the resulting reaction system was heated to 60℃under nitrogen with stirring and reacted under this condition for 6h. The reaction was monitored by TLC, after the reaction of the starting materials was completed, the crude product obtained by concentrating under reduced pressure to remove the solvent was pretreated and then purified by silica gel column chromatography (dichloromethane: methanol=10:1 to 1:1 (v/v)) to give the compound of formula II-D (4.72 g, purity 83.06%, yield 65.0%; the amount of the pure compound of formula II-D actually contained was 3.92g, and the yield was 54.0% calculated as the amount of the pure compound of formula II-D).

The pretreatment steps are as follows: dissolving crude oily substance by using methylene dichloride with the volume (mL) being 10 times the weight (g) of the crude compound of the formula II-D (the ratio of the volume of methylene dichloride to the weight of the crude compound is 10 mL/g) at room temperature in an ultrasonic way, then adding methyl tertiary butyl ether with the volume being 50 times the weight of the crude compound of the formula II-D (the ratio of the volume of the methyl tertiary butyl ether to the weight of the crude compound is 50 mL/g) into the oily substance, stirring the mixture for 2 to 5 hours at normal temperature, pouring out supernatant after standing, and concentrating the oily substance at the lower part in vacuum to obtain the pretreated crude compound of the formula II-D.

ESI-MS m/z：722.3(M-H)；

¹H-NMR(400MHz,DMSO-d₆)δ10.57(d,J＝38.3Hz,1H),9.36(d,J＝94.5Hz,1H),7.61(d,J＝8.5Hz,2H),7.28(d,J＝7.2Hz,2H),5.01(d,J＝10.8Hz,2H),4.83(s,2H),4.38(s,1H),4.17(s,1H),3.71(s,2H),3.54(s,2H),3.48(d,J＝8.3Hz,1H),3.44(s,1H),3.14(s,2H),2.95(d,J＝22.9Hz,3H),1.32(d,J＝7.2Hz,3H),0.89(dd,J＝14.7,6.7Hz,6H).

Step 6: synthesis of Compound I

The compound of formula II-D (4.72 g, 83.06% purity, 5.42 mmol) was dissolved in 15.0mL anhydrous DMF and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM) (2.24 g,8.13 mmol) was added and the reaction was allowed to proceed at room temperature for 1.0h. N, N-diisopropylethylamine (1.04 g,8.13 mmol) and compound Exatecan of formula III (methanesulfonate) (2.89 g,5.42 mmol) were then added thereto and the reaction was continued for 1.5 to 2.0h. The reaction was monitored by TLC and after the starting material was reacted, the solvent was removed under reduced pressure to give crude compound I (1.53 g, purity 76.42%, maximum single impurity content 4.37%).

Example 2 verification of HPLC Condition 2 Master impurity detection

The compound I finished product prepared according to scheme 1 and scheme 2 was analyzed using HPLC condition 1 and HPLC condition 2, and the analysis results are shown in the following table:

TABLE 5 comparison of purity data for Compound I for different HPLC methods for different process routes

NA: no impurity consistent in relative retention time was detected (rrt=1.0 to 1.1).

As can be seen from table 5, the HPLC condition 1 shows that the impurity spectrum of the finished product has a significant difference in relative retention time between rrt=1.0 and 1.1, no impurity having a consistent relative retention time is found, no significant single impurity peak is found, and no main impurity having rrt=1.0 to 1.1 is detected under the detection condition, and the chromatographic peak of the main impurity is not separated from the chromatographic peak of the compound I.

Compound I obtained by different routes, using HPLC condition 2, after detection, found that the relative retention time in the finished product was found to be common impurity at rrt=1.04 positions.

Example 3 verification of the Presence of Master impurity in crude Compound I prepared by different routes

According to the invention, HPLC condition 2 is adopted to detect the crude products of the compound I prepared in the routes 1-4 respectively, and the results show that the crude products of the compound I prepared in the routes 1-4 all contain main control impurities with RRT=1.04, as shown in Table 6.

Route 1: the crude compound I was synthesized according to the synthetic route and procedure described in WO2022204947A 1. Detection by HPLC condition 2: purity 82.03%, main control impurity content: 3.15% (rrt=1.04).

Route 2: the crude compound I was synthesized according to the synthetic route and procedure in CN115215921 a. Detection by HPLC condition 2: purity 79.30%, main control impurity content: 4.77% (rrt=1.04).

Route 3: the crude compound I was synthesized according to the synthetic route and procedure of scheme 3. As shown in fig. 1, the detection was performed by HPLC under condition 2: purity 71.81%, main control impurity content: 5.15% (rrt=1.04).

Route 4: the crude compound I was synthesized according to the synthetic route and procedure of scheme 4. Detection by HPLC condition 2: purity 76.42%, main control impurity content: 4.37% (rrt=1.04).

The crude products of the compound I prepared in the schemes 1 to 4 all contain main control impurities (rrt=1.04), wherein the main control impurities in the crude product of the compound I prepared in the scheme 3 have the highest content, and further purification research is performed on the crude product of the compound I prepared in the scheme 3 in the subsequent examples.

TABLE 6 HPLC Condition 2 detection of crude Compound I products prepared in routes 1-4

	Route 1	Route 2	Route 3	Route 4
					Crude purity of Compound I	82.03％	79.30％	71.81％	76.42％
Main control impurity (RRT=1.04)	4.77％	4.77％	5.15％	4.37％

Example 4

This example is a high pressure normal phase preparative purification chromatography for purifying compound I prepared in scheme 3 of example 1.

Specifically, the crude compound I was purified in this example as follows:

step 1. Preparation of chromatographic silica gel by normal phase 8-120 As filler stationary phase, and adopting normal phase chromatography to prepare compound I.

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, detection wavelength of uv detector: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=2/98 (volume ratio), pressure 1.2MPa; the crude product of compound I (2.0 g) was weighed, dissolved in methylene chloride (40 ml) and loaded, prepared by running a separation, and the main peak component was collected.

And 2. Combining the fractions with the chromatographic content of more than 90.0%, and concentrating the combined fractions in vacuum or extracting a concentrated solution after concentrating the combined fractions in vacuum at 35 ℃ and the vacuum degree of 10Mbar for 0.5 hour to obtain a finished product of the compound I.

Detection by HPLC condition 2: purity 97.43%, main control impurity content: 0.53% (rrt=1.04), compound I yield 72.37%.

Example 5

This example is a high pressure normal phase preparative purification chromatograph for purification of crude compound I prepared in scheme 3 of example 1.

Specifically, the crude compound I was purified in this example as follows:

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, detection wavelength of uv detector: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=35/65 (volume ratio), pressure 1.2Mpa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Step 2: the fractions with a chromatographic content of more than 90.00% are combined, the combined fractions are concentrated in vacuo or the concentrated solution is extracted after concentration, the vacuum concentration temperature is 35 ℃, the vacuum degree is 10Mbar, and the vacuum concentration duration is 0.5 hour. Obtaining a compound I finished product.

Detection by HPLC condition 2: purity 95.92%, main control impurity content: 0.16% (rrt=1.04), compound I yield 61.03%.

Example 6

Step 1: preparation of chromatographic silica gel using normal phase8-120 As filler stationary phase, and adopting normal phase chromatography to prepare compound I.

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, detection wavelength of uv detector: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=25/75 (volume ratio), pressure 1.2MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 97.98%, main control impurity content: 0.18% (rrt=1.04), compound I yield 64.07%.

Example 7

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, detection wavelength of uv detector: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=20/80 (volume ratio), pressure 1.2MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 98.17%, main control impurity content: 0.15% (rrt=1.04), compound I yield 73.11%.

Example 8

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, detection wavelength of uv detector: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=10/90 (volume ratio), pressure 1.2MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 98.41%, main control impurity content: 0.07% (rrt=1.04), compound I yield 77.07%.

By comparing methods 4-8, it was found that the mobile phase methanol/dichloromethane ratio had a large effect on the purity and yield of compound I, as shown in table 7.

TABLE 7 Effect of mobile phase methanol/dichloromethane volume ratio on purification effect

The proportion of the mobile phase influences the removal and yield of the main control impurities and needs to be comprehensively considered.

In addition, compared with the content of the master control impurity (5.15%) in the crude product of the compound I obtained by the method in the route 3, the content of the master control impurity in the product of the compound I prepared by the method is obviously reduced, which indicates that the purification method can effectively remove the master control impurity in the crude product.

Example 9

Specifically, the crude compound I was purified in this example as follows:

step 1. Preparation of chromatographic silica gel by normal phase 10-120 As filler stationary phase, and adopting normal phase chromatography to prepare compound I.

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, ultraviolet detector detection wavelength: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=3/97 (volume ratio), pressure 1.1MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

And 2. Combining the fractions with the chromatographic content of more than 90.00%, and concentrating the combined fractions in vacuum or extracting concentrated solution after concentrating the combined fractions in vacuum at 35 ℃ and the vacuum degree of 10Mbar for 0.5 hour. Obtaining a compound I finished product.

Detection by HPLC condition 2: purity 97.50%, main control impurity content: 0.10% (rrt=1.04), compound I yield 78.39%.

Example 10

The crude compound I prepared in the synthetic route 3 in the example 1 is purified by high-pressure normal phase preparation and purification chromatography to obtain a compound I product with the purity of 98.73% and the main control impurity content of 0.11%.

Specifically, the crude compound I was purified in this example as follows:

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, ultraviolet detector detection wavelength: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=30/70 (volume ratio), pressure 1.5MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 98.73%, main control impurity content: 0.11% (rrt=1.04), compound I yield 75.31%.

Example 11

The crude compound I product prepared in the synthetic route 3 in the example 1 is purified by high-pressure normal-phase preparation and purification chromatography to obtain a compound I product with purity of 99.44% and main control impurity content of 0.03%.

Specifically, the crude compound I was purified in this example as follows:

step 1. Preparation of chromatographic silica gel by normal phase 20-120 As filler stationary phase, and adopting normal phase chromatography to prepare compound I.

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, ultraviolet detector detection wavelength: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=3/97 (volume ratio), pressure 1.25MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 99.44%, main control impurity content: 0.03% (rrt=1.04), yield of compound I was 76.98%.

Example 12

The crude compound I product prepared in the synthetic route 3 in the example 1 is purified by high-pressure normal-phase preparation and purification chromatography to obtain a compound I product with purity of 99.52% and main control impurity content of 0.11%.

Specifically, the crude compound I was purified in this example as follows:

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, ultraviolet detector detection wavelength: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=30/70 (volume ratio), pressure 1.3MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 99.52%, main control impurity content: 0.11% (rrt=1.04), compound I yield 73.27%.

Example 13

The crude compound I product prepared in the synthetic route 3 in the example 1 is purified by high-pressure normal-phase preparation and purification chromatography to obtain a compound I product with purity of 99.64% and main control impurity content of 0.03%.

Specifically, the crude compound I was purified in this example as follows:

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, ultraviolet detector detection wavelength: 254nm, mobile phase flow rate: 50ml/min, mobile phase: methanol/dichloromethane=10/90 (volume ratio), pressure 1.5MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC condition 2: purity 99.64%, main control impurity content: 0.03% (rrt=1.04), yield of compound I was 79.13%.

Example 14

The crude compound I prepared in the synthetic route 3 in the example 1 is purified by high-pressure normal phase preparation and purification chromatography to obtain the compound I with the purity of 99.50% and the main control impurity content of less than or equal to 0.03%.

Specifically, the crude compound I was purified in this example as follows:

Preparation, separation and purification conditions: instrument model: ZDAC200 x 1000, ultraviolet detector detection wavelength: 254nm, mobile phase flow rate: 1000ml/min, mobile phase: methanol/dichloromethane=10/90 (volume ratio), pressure 1.2MPa; the crude compound I (110.0 g) was weighed and loaded in methylene chloride (2200 ml) and isolated by running to collect the main peak fraction.

And 2. Combining the fractions with the chromatographic content of more than 90.00%, and concentrating the combined fractions in vacuum or extracting concentrated solution after concentrating the combined fractions in vacuum at 35 ℃ and 10Mbar under reduced pressure for 6.5 hours. Obtaining a compound I finished product.

Step 3. Compound I product 86.57g of yellow-green solid was obtained altogether, detected by HPLC condition 2: purity 99.82%, main control impurity content: n/a (as shown in fig. 2, the detection result shows that the content of the main impurity in the product is reduced below the detection limit of HPLC, no main impurity is detected in the product, the content of the main impurity is estimated to be 0-0.03% by the peak shape of the compound I), no main impurity is found (rrt=1.04), and the yield of the compound I is 79.90%.

The mass spectrum of the compound I product is shown in figure 3.

High resolution mass spectrometry: 1142.43[ M+1].

HNMR data are as follows:

¹H-NMR(400MHz,DMSO-d₆)δ9.95(d,J＝11.4Hz,1H),8.55(s,1H),8.16(s,1H),7.85(d,J＝8.4Hz,1H),7.67(d,J＝10.8Hz,1H),7.60–7.50(m,2H),7.30(s,1H),7.26(d,J＝8.2Hz,2H),6.93(s,2H),5.56(s,1H),5.38(q,J＝16.3Hz,2H),5.12(dt,J＝33.6,17.7Hz,2H),4.96(s,2H),4.86(d,J＝24.2Hz,2H),4.36(qd,J＝7.6,5.2Hz,1H),4.13(t,J＝7.7Hz,1H),4.06(s,2H),3.69(d,J＝7.7Hz,2H),3.37(t,J＝6.9Hz,4H),2.92(d,J＝35.3Hz,3H),2.33(d,J＝1.9Hz,3H),2.23–2.08(m,4H),1.97(h,J＝6.4Hz,1H),1.83(q,J＝6.9,6.4Hz,2H),1.48(h,J＝7.2,6.2Hz,4H),1.32(d,J＝7.1Hz,3H),1.18(p,J＝7.8Hz,2H),0.89–0.80(m,9H).

Example 15

This example uses the purification method of example 11 to purify the crude compound I prepared in synthetic route 1 of example 1 to obtain the final compound I. Detection by HPLC condition 2: purity 99.87%, main control impurity content: 0.02% (rrt=1.04), yield of compound I was 77.98%.

Example 16

This example uses the purification method of example 11 to purify the crude compound I prepared in synthetic route 2 of example 1 to obtain the final compound I. Detection by HPLC condition 2: purity 99.79%, main control impurity content: 0.02% (rrt=1.04), compound I yield 81.83%.

Example 17

This example uses the purification method of example 11 to purify the crude compound I prepared in synthesis route 4 of example 1 to obtain the final compound I. Detection by HPLC condition 2: purity 99.85%, main control impurity content: 0.01% (rrt=1.04), compound I yield 80.48%.

The purification conditions and purification effects of examples 4 to 14 are summarized in the following table:

TABLE 8 influence of purification conditions on purification effect for examples 4 to 11

According to the table, the method has a good purification effect on the crude product of the intermediate (compound I) of the antibody coupling drug and can well remove the main control impurity, the compound I product with the main control impurity content less than or equal to 0.20% can be obtained by purifying through optimizing the proportion of the silica gel filler and the eluent, and the compound I product with the main control impurity content less than or equal to 0.05% can be further obtained under the better condition; the above products can meet the requirements of the subsequent antibody coupling reaction.

Further, the purity of the product is desirably controlled to be more than or equal to 97.0 percent; under the better condition, the purity is controlled to be more than or equal to 98.5 percent.

Comparative example 1 general silica gel column chromatography purification of Compound I

In this example, the crude compound I prepared in the synthetic route 3 in example 1 was purified by 38-74 μm ordinary silica gel column chromatography (Qingdao ocean silica gel column) to obtain a compound I product with a purity of 90.0% or more.

Specifically, the crude compound I was purified in this example as follows:

Step 1, preparing a compound I by using conventional normal phase chromatographic silica gel as a filler stationary phase and adopting a column chromatography method.

The specific implementation method comprises the following steps: injecting the crude product (10.0 g) of the compound I obtained in the example 1 into a conventional glass chromatographic normal phase chromatographic column, wherein the specific chromatographic column parameters are shown in tables 1 and 2, eluting at 25 ℃, the eluent is preferably dichloromethane and methanol, the elution gradient is preferably dichloromethane: methanol=60:1-10:1 (volume ratio), when the product starts to be eluted, the elution gradient is 60:1, when the product is completely eluted, the elution gradient is 10:1, eluting the compound I by adjusting the eluent gradient mode, and collecting a target fraction;

And 2. Combining the fractions with the chromatographic content of more than 90.0%, and concentrating the combined fractions in vacuum or extracting a concentrated solution after concentrating the combined fractions in vacuum at 35 ℃ and the vacuum degree of 10Mbar for 2.5 hours to obtain a finished product of the compound I.

Detection by HPLC condition 2: purity 90.42%, main control impurity content: 2.70% (rrt=1.04), compound I yield 70.16%.

Comparative example 2 40-60 mu mGRACE silica gel column chromatography purification of Compound I

The method comprises the following steps:

in this example, the crude compound I prepared in the synthetic route 3 in example 1 was purified by 40 to 60 mu mGRACE silica gel column chromatography to obtain the compound I with a purity of 95.0% or more.

Specifically, the crude compound I was purified in this example as follows:

And step1, using conventional normal phase chromatographic silica gel as a filler stationary phase, and adopting a normal phase chromatographic method to prepare the compound I.

The specific implementation method comprises the following steps: injecting the crude product (10.0 g) of the compound I into a conventional glass chromatographic normal phase chromatographic column, eluting at 25 ℃ with dichloromethane and methanol as eluent, wherein the gradient is dichloromethane/methanol=50:1-40:1 (volume ratio) when the product starts to be eluted, the gradient is dichloromethane/methanol=30:1-20:1 (volume ratio) when the product is completely eluted, eluting the compound I by adjusting the gradient mode of the eluent, and collecting a target fraction;

Detection by HPLC condition 2: purity 97.49%, main control impurity content: 0.97% (rrt=1.04).

Step 3. Yellow-green solid of compound I6.4 g, purity: 97.49%, master impurity content (rrt=1.04) 0.97%, yield of compound I64.0%.

High resolution mass spectrometry: 1142.43[ M+1].

The second method is as follows:

In this example, crude compound I prepared in synthetic route 1 of example 1 was purified by 40-60 μm GRACE silica gel column chromatography. Detection by HPLC condition 2: purity 98.31%, main control impurity content: 0.33% (rrt=1.04), compound I yield 61.7%.

And a third method:

in this example, crude compound I prepared in synthetic route 2 of example 1 was purified by 40-60 μm GRACE silica gel column chromatography. Detection by HPLC condition 2: purity 97.97%, main control impurity content: 0.47% (rrt=1.04), compound I yield 68.88%.

The method four:

In this example, the crude compound I prepared in scheme 4 of example 1 was purified by 40-60. Mu. mGRACE silica gel column chromatography. Detection by HPLC condition 2: purity 97.31%, main control impurity content: 0.58% (rrt=1.04), compound I yield 65.60%.

The purification effect by the conventional column chromatography method was compared with the purification effect by the high pressure preparation method of the present invention, and the results are shown in the following table:

TABLE 9 comparison of the effectiveness of column chromatography and high pressure purification methods

The comparison shows that the method of the invention has better effect than the common column chromatography method in the aspects of purity and yield of the compound I and removal of main control impurities.

And, for the purification of the crude compound I obtained by different synthetic routes, the purification effect of the method of the invention is also better than that of the common column chromatography method, so the purification method of the invention is suitable for the purification of the compound I in a compound I/master impurity (rrt=1.04) system obtained by different synthetic routes.

Comparative example 3 high pressure forward preparation of isolated and purified Compound I

The main difference between this comparative example and the examples is that: the mobile phase system used in this comparative example was not halogenated hydrocarbon solvents or alcohol solvents.

The method comprises the following steps:

Step 1: preparation of chromatographic silica gel using normal phase 8-120 As filler stationary phase, and adopting normal phase chromatography to prepare compound I.

Preparation, separation and purification conditions: instrument model: ZDAC50 0 x 650, detection wavelength of uv detector: 254nm, mobile phase flow rate: 50ml/min, mobile phase: ethanol/dichloromethane=2/98 (volume ratio), pressure 1.2MPa; the crude compound I (2.0 g) was weighed and dissolved in methylene chloride (40 ml) to be loaded, and the mixture was separated and prepared by running, and the main peak component was collected.

Detection by HPLC: purity 85.12%, main control impurity content: 1.46% (rrt=1.04), compound I yield 62.73%.

The second method is as follows:

The first method is the same as that in this comparative example, except that the mobile phase: methanol/ethyl acetate=2/98 (volume ratio).

The obtained compound I finished product is detected by HPLC: purity 80.43%, main control impurity content: 2.46% (rrt=1.04), compound I yield 69.73%.

And a third method:

The first method is the same as that in this comparative example, except that the mobile phase: ethanol/ethyl acetate=2/98 (volume ratio).

The obtained compound I finished product is detected by HPLC: purity 79.55%, main control impurity content: 2.87% (rrt=1.04), compound I yield 61.07%.

The purification effect in example 4 was compared with the purification effect of this comparative example, and the results are shown in the following table:

TABLE 10 influence of the selection of the components of the mobile phase on the purification effect

The separation effect of the main control impurity is better than that of other elution systems under the methanol/dichloromethane elution system, because the compound I and the main control impurity have similar structures and are poor in solubility, and the elution system with smaller polarity cannot perform obvious elution and removal on the compound I and the main control impurity.

Example 15 Structure of Master impurity (RRT=1.04) in crude and finished Compound I

The HPLC condition 2 of the invention can be used for HPLC purity identification, the main control impurity with RRT=1.04 can be detected, and the relative retention time in the finished product is found to exist at the RRT=1.04 position when the compound I obtained in the route 1-4 in the example 1 is detected, and the main control impurity is judged by a Liquid Chromatography Mass Spectrometer (LCMS) identification result, the main control impurity has the structure that a hydroxymethyl group is introduced on the basis of the compound I, and ESI-MS m/z:1171, as shown in fig. 4. The master control impurity is difficult to separate from the compound I, and is finally coupled with the antibody in the subsequent ADC drug preparation step, so that the master control impurity is further present in the antibody coupling drug formed after the antibody coupling.

The invention purifies the main control impurity from the crude product of the compound I prepared in the synthetic route 3, and characterizes the compound structure of the main control impurity. The purification method of the main control impurity comprises the following steps: and separating the synthesized crude product of the compound I by the high-pressure normal-phase preparation purification chromatography, and collecting impurity peak components at the rear end of the main peak component to obtain a main control impurity crude product. The obtained crude master control impurity is further purified by the following method. The method comprises the following steps:

Step 1: preparation of chromatographic silica gel using normal phase 20-120 As filler stationary phase, and preparing impurity compound by normal phase chromatography.

Preparation, separation and purification settings: instrument model: DAC50, detection wavelength: 254nm, flow rate: 50ml/min, mobile phase: methanol/dichloromethane = 10/90; crude impurity compound (0.5 g) is weighed and dissolved in dichloromethane (10 ml) for loading, and the crude impurity compound is prepared by operation separation, and main peak components are collected.

Step 2: and combining the fractions with the chromatographic content of more than 85.00%, and carrying out vacuum concentration or concentration on the combined fractions to extract a concentrated solution, thereby obtaining a primary purified product.

Step 3: purifying the primary purified product for multiple times through the steps to finally obtain a main control impurity compound product, and detecting the main control impurity compound product through LCMS: the purity is more than or equal to 90.0%, and the product can be directly used for nuclear magnetic detection.

The nuclear magnetic resonance hydrogen spectrum of the main impurity is shown in fig. 5, and HNMR data thereof are as follows:

¹H NMR(400MHz,DMSO-d₆)δ10.07–9.87(m,1H),8.53(d,J＝14.5Hz,1H),8.18(dd,J＝32.8,6.7Hz,1H),7.78(t,J＝9.2Hz,2H),7.55(d,J＝8.8Hz,2H),7.29(d,J＝7.8Hz,3H),6.99(s,1H),6.50(s,1H),5.75(s,1H),5.59(s,1H),5.48–5.33(m,2H),5.18(s,2H),4.99(d,J＝11.4Hz,2H),4.90(s,2H),4.75(s,2H),4.36(ddt,J＝24.0,15.5,7.7Hz,1H),4.16(t,J＝7.7Hz,1H),4.10(d,J＝4.8Hz,2H),3.83(s,2H),3.68(t,J＝7.3Hz,2H),3.45–3.35(m,3H),3.25–3.07(m,2H),2.96(d,J＝24.5Hz,3H),2.38(s,3H),2.13(ddt,J＝20.9,13.9,5.8Hz,4H),1.87(dtd,J＝21.9,16.5,14.6,7.1Hz,3H),1.58(dt,J＝16.1,8.3Hz,4H),1.29(d,J＝7.0Hz,3H),0.87(d,J＝7.0Hz,9H).

Based on the nuclear magnetic data, the structure of the master impurity (ESI-MS m/z: 1171) was confirmed as follows:

the molecular formula: c ₅₇H₆₇FN₈O₁₆ S;

Molecular weight: 1170.44.

By analyzing routes 1-4, it was found that this master impurity was mainly generated in the chloromethylation step, the reaction formula is as follows:

And (3) a compound containing two methoxy groups, which is generated by the reaction of hydrolysis byproducts in chloromethylation reaction and excessive paraformaldehyde in a reaction system, is an initial source compound of master control impurities, and generates the master control impurities through subsequent reactions in a synthesis route, wherein the master control impurities are accompanied in a final product compound I.

Taking route 3 as an example, the generation route of the master impurity is as follows:

wherein, the initial source compound structure of the main control impurity is as follows:

The molecular formula: c ₂₈H₄₆N₆O₁₀ SSi;

molecular weight: 686.28.

Impurity separation and characterization were performed on the product obtained in step 2 of scheme 3, resulting in MS:703.95 (m+18) whose mass spectrum is shown in fig. 6; the nuclear magnetic pattern is shown in figure 7. The purification method of the initial source compound is the same as the purification method of the master impurity, and the initial source compound sample is obtained through repeated purification and is used for nuclear magnetic analysis.

HNMR data are as follows:

¹H NMR(600MHz,Chloroform-d)δ8.51(d,J＝29.3Hz,1H),7.55(d,J＝7.9Hz,2H),7.32(dd,J＝23.5,8.5Hz,3H),6.95(d,J＝7.5Hz,1H),5.14(s,2H),4.96(d,J＝10.2Hz,2H),4.79(d,J＝47.6Hz,2H),4.63(p,J＝7.1Hz,1H),4.33–4.21(m,2H),4.15(d,J＝34.3Hz,2H),3.90(d,J＝4.0Hz,1H),3.85–3.76(m,2H),3.33(d,J＝61.0Hz,2H),2.88(d,J＝83.4Hz,3H),2.48–2.36(m,1H),1.57–1.41(m,3H),1.12(d,J＝6.9Hz,3H),1.07–1.00(m,2H),0.96–0.80(m,4H),0.06(s,10H).

The mass spectrum and nuclear magnetism characterization results show that the chloromethylation step introduces side reaction, and the initial source compound of the main control impurity is generated.

The reaction is commonly applied to the four synthetic routes, and the impurities are monitored according to detection means such as MS (MS) and the like to be side reactions in the chloromethylation reaction step, and the side reaction products in the step finally generate main control impurities (RRT=1.04) through subsequent reaction steps.

Claims

1. A process for the purification of compound I, characterized in that it comprises the steps of: separating the mixture containing the compound I and the main control impurity under the high-pressure chromatography condition;

Wherein, the high-pressure chromatography conditions are as follows:

The chromatographic column is a dynamic axial compression column;

the stationary phase is unbound silicon hydroxyl;

2. The purification method of claim 1, wherein the purification method satisfies one or more of the following conditions:

(1) The main control impurity structure is as follows:

(2) The mass spectrum of the main control impurity is ESI-MSm/z:1171;

(3) The mass content of the master control impurity in the mixture is at most 10.0%, preferably 1.0-10.0%, and more preferably 3.0-5.5%;

(4) The purification process collects a fraction having a compound I content of more than 90.0%, preferably more than 95%, further preferably more than 98%;

(5) The inner diameter of the dynamic axial compression column is 50-200mm, preferably 50mm or 200mm;

(6) The length of the dynamic axial compression column is 650-1000mm, preferably 650mm or 1000mm;

(7) The particle size of the stationary phase is 5-50. Mu.m, preferably 8-20. Mu.m, further preferably 10-20. Mu.m, most preferably 20. Mu.m;

(8) The stationary phase is Or (b)PreferablyOr (b)Further preferred is

(9) The aperture of the stationary phase is

(10) The volume ratio of the alcohol solvent to the halogenated hydrocarbon solvent is 35:65 to 2:98, more preferably 35:65 to 3:97, for example 35:65, 30:70, 25:75, 20:80, 10:90 or 3:97, still more preferably 10:90 to 3:97, most preferably 10:90;

(11) The alcohol solvent is methanol, ethanol or isopropanol, preferably methanol;

(12) The halogenated alkane solvent is saturated or unsaturated chlorinated alkane of C _1-2, such as methylene chloride, chloroform, carbon tetrachloride, 1-dichloroethane, 1, 2-dichloroethane, 1-trichloroethane, 1, 2-trichloroethane 1, 2-tetrachloroethane, 1, 2-tetrachloroethane, pentachloroethane, 1-dichloroethylene, 1, 2-dichloroethylene, trichloroethylene or tetrachloroethylene, preferably dichloromethane, trichloromethane, 1-trichloroethane, trichloroethylene or tetrachloroethylene; further preferred is dichloromethane, chloroform; most preferably dichloromethane;

(13) The flow rate of the mobile phase is 50-1000 ml/min, preferably 50ml/min or 1000ml/min;

(14) The pressure of the dynamic axial compression column is 1.0-2.0 Mpa, preferably 1.1-1.5Mpa, more preferably 1.1Mpa, 1.2Mpa, 1.25Mpa, 1.3Mpa or 1.5Mpa;

(15) The purification method further comprises the following steps: and (3) dissolving the mixture in a chlorinated alkane solvent, separating under the high-pressure chromatographic condition, and collecting a main peak product to obtain the compound I.

3. The purification method of claim 2, wherein the purification method satisfies one or more of the following conditions:

(1) The relative retention time of the master impurity under any one of the following HPLC conditions is 1.03-1.05, preferably 1.04;

HPLC conditions 2-1: chromatographic column model: agilent AdvanceBio PEPTIDE MAP,3.5 μm, 2.1X1250 mm; mobile phase: phase a is 0.01mol/L potassium dihydrogen phosphate aqueous solution (ph=5.0), phase B is 10% methanolic acetonitrile solution; sample injection amount: 5uL; column temperature: 45 ℃; detection wavelength: 254nm, flow rate: 0.25mL/min;

HPLC conditions 2-2: chromatographic column model: agilent AdvanceBio PEPTIDE MAP,3.5 μm, 2.1X1250 mm; mobile phase: phase a is 0.01mol/L potassium dihydrogen phosphate aqueous solution (ph=5.0), phase B is 10% methanolic acetonitrile solution; sample injection amount: 5uL; column temperature: 45 ℃; detection wavelength: 254nm, flow rate: 0.25mL/min; high performance liquid chromatograph: agilent 187260;

(2) The model of the dynamic axial compression column is ZDAC50 0 x 650, and the flow rate of the mobile phase is 50-70 ml/min, preferably 50ml/min;

(3) The model of the dynamic axial compression column is ZDAC 200.200.times.1000, and the flow rate of the mobile phase is 800-1000 ml/min; preferably 1000ml/min;

(4) In the purification method, when the stationary phase is When the volume ratio of the alcohol solvent to the chlorinated alkane solvent is 35:65-10:90;

(5) In the purification method, when the stationary phase is When the volume ratio of the alcohol solvent to the chlorinated alkane solvent is 30:70-3:97;

(6) In the purification method, when the stationary phase is When the volume ratio of the alcohol solvent to the chlorinated alkane solvent is 30:70-3:97, preferably 10:90-3:97, and most preferably 10:90.

4. A purification method according to claim 3, wherein the purification method is any of the following schemes:

Scheme 1: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 35:65; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

scheme 2: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 25:75; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

scheme 3: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 20:80; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

scheme 4: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 10:90; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

scheme 5: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 3:97; preferably, the pressure is 1.1Mpa; the model of the dynamic axial compression column is ZDAC50 0x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

Scheme 6: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 30:70; preferably, the pressure is 1.5Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

Scheme 7: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 3:97; preferably, the pressure is 1.25Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

scheme 8: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 30:70; preferably, the pressure is 1.3Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

scheme 9: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 10:90; preferably, the pressure is 1.5Mpa; the model of the dynamic axial compression column is ZDAC50 0 x 650; the flow rate of the mobile phase is 50ml/min; the detection wavelength of the high-pressure chromatography is 254nm;

Scheme 10: in the purification method, the stationary phase is The volume ratio of the methanol to the dichloromethane is 10:90; preferably, the pressure is 1.2Mpa; the model of the dynamic axial compression column is ZDAC ×1000; the flow rate of the mobile phase is 1000ml/min; the detection wavelength of the high-pressure chromatography is 254nm.

5. A purification process according to claim 3, wherein the purification process satisfies one or both of the following conditions:

(1) The mass content of the compound I in the mixture is at least 65%, preferably 65.0-95.0%, and more preferably 70.0-85.0%;

(2) The main control impurity is generated in a chloromethylation reaction step in the preparation method of the compound I, and then the main control impurity is generated through subsequent reaction; the preparation method of the compound I comprises a route 1, a route 2, a route 3 or a route 4; the chloromethylation reaction step has the following reaction formula:

6. The purification process of claim 5, wherein route 1 comprises the steps of:

R ^1-1、R^1-2 and R ^1-3 are each independently C ₁～C₄ alkyl.

7. The purification process of claim 5, wherein said route 2 comprises the steps of:

R ^1-1、R^1-2 and R ^1-3 are each independently C ₁～C₄ alkyl;

R ⁴ is a hydroxyl protecting group.

8. The purification process of claim 5, wherein said route 3 comprises the steps of:

wherein R is-Si (C ₁～C₆)₃ substituted C ₁～C₆ alkyl).

9. The purification process of claim 5, wherein the route 4 comprises the steps of:

Wherein R is-Si (C ₁～C₆)₃ substituted C ₁-C₆ alkyl).

10. The separation and detection method of the compound I is characterized by comprising the following steps of: separating and detecting the mixture containing the compound I under the condition of high performance liquid chromatography;

wherein, the high performance liquid chromatography conditions are as follows:

The mobile phase is a mixed solution of 0.01mol/L potassium dihydrogen phosphate aqueous solution and 10 percent methanol acetonitrile solution;

The detection wavelength is 254nm;

The elution conditions were as follows:

the preparation method of the compound I is as claimed in any one of claims 6 to 9.