Methods for preparing drug-linker conjugates

Abstract

The present invention provides a method for preparing a drug-linker conjugate. The present invention also provides a method for preparing the intermediate or its salt used, and the use of the intermediate or its salt. The raw materials for the preparation method of the present invention are easily available, the operation is simple, the product has high purity, and is suitable for large-scale synthesis. JPEG2025502931000097.jpg81155

Description

[Technical field]

[0001] This application claims priority from Chinese patent application No. 2022100957718, filed on January 26, 2022. This application incorporates the above-mentioned Chinese patent application in its entirety. The present application is in the field of medicinal chemistry, and specifically relates to methods for preparing drug-linker conjugates or salts thereof, as well as related intermediates or salts thereof. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are conjugates of antibodies and small molecule drugs, which combine the tumor targeting effect of antibodies with the activity of bioactive molecules to become biological missiles with highly promising therapeutic effects and safety advantages. The antibody induces the ADC to bind to the target cells / tissues, and then the small molecule drug is released into the cells / tissues through the action of specific enzymes to treat the disease. Among them, the drug-linker conjugate (including the cytotoxic payload and linker) plays a very important role in the preparation of ADCs. Therefore, the development of a simple, efficient, low-cost, and large-scale synthesis-friendly drug-linker conjugate synthesis method is of great importance for the development and use of ADC drugs. Summary of the Invention

[0003] The present application provides a method for preparing the compound of formula I, which is a drug-linker conjugate, a method for preparing the intermediate or its salt used, and the use of the intermediate or its salt. The preparation method described in the present application has easy availability of raw materials, simple operation, high purity of the product, and is suitable for large-scale synthesis.

[0004] In one aspect, the present application provides a method for preparing a compound of Formula I or a salt thereof.

[0005] [ka]

[0006] The method includes the following steps.

[0007] c) reacting a compound of formula 6 or a salt thereof with a compound of formula 7 or a salt thereof to obtain a compound of formula 8 or a salt thereof.

[0008] [ka]

[0009] d) reacting a compound of formula 8 or a salt thereof to obtain a compound of formula 9 or a salt thereof.

[0010] [ka]

[0011] e) reacting a compound of formula 9 or a salt thereof with a compound of formula 10 to obtain a compound of formula I or a salt thereof.

[0012] [ka]

[0013] however, R1 and R2 are each independently C 1-4 alkyl, R3 is selected from hydroxy and halogen; PG1 is selected from amino protecting groups.

[0014] In some embodiments, the method of preparing the compound of formula I or a salt thereof further comprises the steps of:

[0015] a) reacting a compound of formula 3 or a salt thereof with a compound of formula 4 to obtain a compound of formula 5 or a salt thereof.

[0016] [ka]

[0017] b) reacting a compound of formula 5 or a salt thereof to obtain a compound of formula 6 or a salt thereof.

[0018] [ka]

[0019] wherein PG2 is selected from amino protecting groups.

[0020] In another aspect, the present application provides a method for preparing a compound of formula 7 or a salt thereof, the method comprising the steps of:

[0021] f-1) reacting a compound of formula 7-A1 with a compound of formula 7-A2 or a salt thereof to obtain a compound of formula 7-A3.

[0022] [ka]

[0023] f-2) reacting a compound of formula 7-A3 to obtain a compound of formula 7-A4 or a salt thereof.

[0024] [ka]

[0025] f-3) reacting a compound of formula 7-A4 or a salt thereof to obtain a compound of formula 7 or a salt thereof.

[0026] [ka]

[0027] however, R1 and R2 are each independently C 1-4 alkyl, PG1 and PG3 are each independently selected from amino protecting groups, and PG1 and PG3 are different.

[0028] In another aspect, the present application provides a method for preparing a compound of formula 7 or a salt thereof, the method comprising the steps of:

[0029] g-1) A step of reacting a compound of formula 7-B1 or a salt thereof to obtain a compound of formula 7-B2 or a salt thereof.

[0030] [ka]

[0031] g-2) reacting a compound of formula 7-B2 or a salt thereof to obtain a compound of formula 7-B3 or a salt thereof.

[0032] [ka]

[0033] g-3) A step of reacting a compound of formula 7-B3 or a salt thereof with a compound of formula 7-A1 to obtain a compound of formula 7 or a salt thereof.

[0034] [ka]

[0035] however, R1 and R2 are each independently C 1-4 alkyl, PG1 and PG4 are each independently selected from amino protecting groups.

[0036] In another aspect, the present application provides a method for preparing a compound of formula 3 or a salt thereof, the method comprising the steps of:

[0037] h) reacting a compound of formula 1 or a salt thereof with a compound of formula 2 to obtain a compound of formula 3 or a salt thereof.

[0038] [ka]

[0039] however, R4 and R5 are independently -OC 1-4 Alkyl, -SC 1-4 Alkyl and -NHC 1-4 alkyl, or R4 and R5 together with the carbon atom connected thereto form a carbonyl; Alternatively, R4 and R5 together with the carbon atom(s) attached thereto may form

[0040] [ka]

[0041] where X and Y are each independently O, S, NH, or NC 1-4 alkyl, and n is selected from 1, 2 and 3.

[0042] In another aspect, the present application provides a method for preparing a compound of formula 1, the method comprising the steps of:

[0043] Step i-1) A compound of formula 1-A1 is reacted with a compound of formula 1-A2 to obtain a compound of formula 1-A3.

[0044] Step i-2) The compound of formula 1-A3 is reacted to obtain the compound of formula 1.

[0045] [ka]

[0046] In another aspect, the present application provides a method for preparing a compound of formula 1 or a salt thereof, the method comprising the steps of:

[0047] Step j-1) A compound of formula 1-B1 is reacted with a compound of formula 1-B2 to obtain a compound of formula 1-B3.

[0048] Step j-2) The compound of formula 1-B3 is reacted to obtain a compound of formula 1-B4.

[0049] Step j-3) The compound of formula 1-B4 is reacted to obtain a compound of formula 1-B5 or a salt thereof.

[0050] Step j-4) The compound of formula 1-B5 or a salt thereof is reacted to obtain the compound of formula 1 or a salt thereof.

[0051] [ka]

[0052] wherein X is selected from halogens.

[0053] In some embodiments, R1 and R2 are each independently selected from methyl, ethyl, and propyl, and preferably, R1 and R2 are the same.

[0054] In some embodiments, R1 and R2 are both propyl.

[0055] In some embodiments, R3 is selected from hydroxy, fluorine, chlorine, bromine, and iodine.

[0056] In some embodiments, R3 is selected from hydroxy and chlorine.

[0057] In some embodiments, X is selected from fluorine, chlorine, bromine, and iodine.

[0058] In some embodiments, X is chlorine.

[0059] In some embodiments, PG1 is selected from an alkoxycarbonyl protecting group, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc).

[0060] In some embodiments, PG2 is selected from an alkoxycarbonyl protecting group, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc).

[0061] In some embodiments, PG3 is selected from an alkoxycarbonyl protecting group, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably tert-butoxycarbonyl (Boc).

[0062] In some embodiments, PG4 is selected from an alkoxycarbonyl protecting group, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc).

[0063] In some embodiments, R4 and R5 are each independently selected from methoxy, ethoxy, propoxy, methylthio, ethylthio, propylthio, methylamino, ethylamino, and propylamino; or R4 and R5 together with the carbon atom connected thereto form a carbonyl; Alternatively, R4 and R5 together with the carbon atom(s) attached thereto may form

[0064] [ka]

[0065] where X and Y are each independently O, S, NH, or NC 1-4 alkyl, and n is selected from 1, 2 and 3.

[0066] In some embodiments, R4 and R5 together with the carbon atom connected thereto form a carbonyl, or R4 and R5 together with the carbon atom connected thereto form a carbonyl.

[0067] [ka]

[0068] Form.

[0069] In some embodiments, in step a), the reaction is carried out under acidic conditions, the acid being selected from hydrogen chloride, hydrobromic acid, sulfuric acid, formic acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, zinc acetate, aluminum trichloride (AlCl3), ferric chloride (FeCl3) and boron trifluoride diethyl etherate (BF3·Et2O), for example hydrogen chloride or boron trifluoride diethyl etherate, preferably hydrogen chloride, p-toluenesulfonic acid or zinc acetate.

[0070] In some embodiments, in step a), the molar ratio of reacting the compound of formula 3 or a salt thereof with the acid is selected from 1:0.2 to 1:2.

[0071] In some embodiments, in step a), the reaction is carried out under basic conditions, the base being selected from sodium hydroxide, potassium tert-butoxide, potassium carbonate, triethylamine (EtN), N,N-diisopropylethylamine (DIPEA) and pyridine, preferably potassium tert-butoxide or sodium hydroxide.

[0072] In some embodiments, in step a), the molar ratio of reacting the compound of formula 3 or a salt thereof with the base is selected from 1:0.5 to 1:3.

[0073] In some embodiments, in step a), the molar ratio of reacting the compound of formula 3 or a salt thereof with the compound of formula 4 is selected from 1:1 to 1:10, preferably 1:2 to 1:6.

[0074] In some embodiments, in step a), the reaction solvent is selected from aprotic solvents, preferably ethers, halogenated alkanes, ketones, nitriles, amides, or sulfones, more preferably ethyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, dichloromethane, 1,2-dichloroethane, acetone, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethylsulfoxide, and even more preferably isopropyl ether, tetrahydrofuran, 1,4-dioxane, or N,N-dimethylformamide.

[0075] In some embodiments, in step a), the mass-to-volume ratio (g / mL) of the compound of formula 3 or a salt thereof to the solvent is selected from 1:5 to 1:50, preferably 1:8 to 1:30.

[0076] In some embodiments, in step a), the reaction temperature is selected from -10 to 110°C, preferably 0 to 60°C.

[0077] In some embodiments, in step b), the protecting group PG2 on the amino group is removed from a compound of formula 5 or a salt thereof to give a compound of formula 6 or a salt thereof.

[0078] In some embodiments, in step b), the reaction of removing the protecting group PG2 on the amino group from the compound of formula 5 or a salt thereof can be carried out using conventional reaction conditions well known to those skilled in the art, for example, removal under acidic conditions, removal under basic conditions, and removal under the catalysis of a palladium catalyst.

[0079] In some embodiments, in step b), when PG2 is 9-fluorenylmethyloxycarbonyl (Fmoc), the compound of formula 5 or a salt thereof is subjected to removal of the protecting group PG2 under basic conditions, the base being selected from piperidine, diethylamine, morpholine, diisopropylamine, DBU, triethylamine and N,N-diisopropylethylamine, preferably piperidine, diethylamine or morpholine.

[0080] In some embodiments, in step b), when PG2 is 9-fluorenylmethyloxycarbonyl (Fmoc), the molar ratio of the compound of formula 5 or a salt thereof to the base is selected from 1:0.2 to 1:40, preferably 1:1 to 1:10.

[0081] In some embodiments, in step b), when PG2 is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction solvent is selected from one or any combination of DMF, DMAc, dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, methanol and ethanol, preferably selected from one or any combination of DMF and dichloromethane.

[0082] In some embodiments, in step b), when PG2 is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass-to-volume ratio (g / mL) of the compound of formula 5 or a salt thereof to the reaction solvent is selected from 1:5 to 1:50, preferably 1:10 to 1:30.

[0083] In some embodiments, in step b), when PG2 is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction temperature is selected from 0 to 50°C, preferably 10 to 30°C.

[0084] In some embodiments, in step c), a compound of formula 6 or a salt thereof and a compound of formula 7 or a salt thereof are reacted with a condensing agent to obtain a compound of formula 8 or a salt thereof, the condensing agent being selected from DMTMM, HATU, HBTU, HOBt / EDCI and T3P, preferably DMTMM or HOBt / EDCI.

[0085] In some embodiments, in step c), the molar ratio of the compound of formula 6 or a salt thereof to the condensing agent is selected from 1:0.7 to 1:5, preferably 1:0.7 to 1:2.

[0086] In some embodiments, in step c), the molar ratio of the compound of formula 6 or a salt thereof to the compound of formula 7 or a salt thereof is selected from 1:0.7 to 1:3, preferably 1:0.7 to 1:2.

[0087] In some embodiments, in step c), the reaction solvent is selected from one or any combination of DMF, DMAc, NMP, tetrahydrofuran, 1,4-dioxane, acetonitrile, and dichloromethane, preferably selected from one or any combination of DMF, dichloromethane, and tetrahydrofuran.

[0088] In some embodiments, in step c), the mass-to-volume ratio (g / mL) of the compound of formula 6 or a salt thereof to the reaction solvent is 1:5 to 1:50.

[0089] In some embodiments, in step c), the reaction temperature is selected from -20 to 50°C, preferably -5 to 25°C.

[0090] In some embodiments, in step d), the protecting group PG1 on the amino group is removed from the compound of formula 8 or a salt thereof to obtain the compound of formula 9 or a salt thereof. This step can be carried out in a manner similar to step b).

[0091] In some embodiments, in step d), when PG1 is 9-fluorenylmethyloxycarbonyl (Fmoc), the compound of formula 8 or a salt thereof is subjected to removal of the protecting group PG1 under basic conditions, the base being selected from piperidine, diethylamine, morpholine, diisopropylamine, DBU, triethylamine and N,N-diisopropylethylamine, for example, diethylamine or N,N-diisopropylethylamine, preferably piperidine, diethylamine or morpholine.

[0092] In some embodiments, in step d), when PG1 is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction solvent is selected from one or any combination of DMF, DMAc, dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, and ethanol.

[0093] In some embodiments, in step d), when PG1 is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass-to-volume ratio (g / mL) of the compound of formula 8 or a salt thereof to the reaction solvent is selected from 1:5 to 1:50.

[0094] In some embodiments, in step d), when PG1 is 9-fluorenylmethyloxycarbonyl (Fmoc), the volume ratio of the base to the reaction solvent is selected from 1:5 to 1:50.

[0095] In some embodiments, in step d), when PG1 is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction temperature is selected from -10 to 50°C, preferably 0 to 30°C.

[0096] In some embodiments, in step e), when R3 is hydroxy, the compound of formula 9 or a salt thereof is reacted with the compound of formula 10 under the action of a condensing agent to obtain the compound of formula I. This step can be carried out in a manner similar to step c).

[0097] In some embodiments, in step e), when R3 is hydroxy, the condensing agent is selected from DMTMM, HATU, HBTU, HOBt / EDCI and T3P, preferably DMTMM or HOBt / EDCI.

[0098] In some embodiments, in step e), when R3 is hydroxy, the molar ratio of the compound of formula 9 or a salt thereof to the condensing agent is selected from 1:1 to 1:5, preferably 1:1 to 1:2.

[0099] In some embodiments, in step e), when R3 is hydroxy, the molar ratio of the compound of formula 9 or a salt thereof to the compound of formula 10 is selected from 1:0.8 to 1:2.0.

[0100] In some embodiments, in step e), when R3 is hydroxy, the reaction solvent is selected from one of DMF, tetrahydrofuran, and dichloromethane, or any combination thereof.

[0101] In some embodiments, in step e), when R3 is hydroxy, the mass-to-volume ratio (g / mL) of the compound of formula 9 or a salt thereof to the reaction solvent is selected from 1:5 to 1:50.

[0102] In some embodiments, in step e), when R3 is hydroxy, the reaction temperature is selected from -20 to 50°C, preferably 0 to 30°C.

[0103] In some embodiments, in step e), when R3 is selected from halogen (eg, chlorine, fluorine or bromine), it can be obtained by conversion of a hydroxyl group with a halogenating reagent via a halogenation reaction.

[0104] In some embodiments, in step e), when R3 is selected from halogen (e.g., chlorine, fluorine, or bromine), the compound of formula 9 or a salt thereof is reacted with the compound of formula 10 under basic conditions, the base being selected from triethylamine, N,N-diisopropylethylamine, DBU, N-methylpiperidine, and N-methylmorpholine.

[0105] In some embodiments, in step e), when R3 is chlorine, it is obtained by converting hydroxyl group by chlorination reaction with a chlorination reagent. The chlorination reagent is selected from thionyl chloride, oxalyl chloride and phosphorus oxychloride, preferably thionyl chloride. The chlorination reaction solvent is selected from tetrahydrofuran and dichloromethane, preferably dichloromethane. The chlorination temperature is selected from 0 to 50°C, preferably 0 to 30°C.

[0106] In some embodiments, in step e), when R3 is halogen (eg, chlorine, fluorine or bromine), the molar ratio of reacting the compound of formula 9 or a salt thereof with the base is selected from 1:1 to 1:5.

[0107] In some embodiments, in step e), when R3 is halogen (e.g., chlorine, fluorine or bromine), the molar ratio of the compound of formula 9 or a salt thereof to the compound of formula 10 is selected from 1:0.8 to 1:2.0, preferably 1:1 to 1:1.5.

[0108] In some embodiments, in step e), when R3 is halogen (eg, chlorine, fluorine, or bromine), the reaction solvent is selected from one or any combination of DMF, tetrahydrofuran, dichloromethane.

[0109] In some embodiments, in step e), when R3 is a halogen (e.g., chlorine, fluorine or bromine), the mass-to-volume ratio (g / mL) of the compound of formula 9 or a salt thereof to the reaction solvent is selected from 1:5 to 1:50.

[0110] In some embodiments, in step e), when R3 is halogen (eg, chlorine, fluorine or bromine), the reaction temperature is selected from -20 to 50°C, preferably 0 to 30°C.

[0111] In some embodiments, in step f-1), the reaction is carried out under basic conditions, the base being selected from sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate and potassium bicarbonate, for example sodium bicarbonate or sodium carbonate, preferably sodium bicarbonate, potassium bicarbonate or potassium carbonate.

[0112] In some embodiments, in step f-1), the solvent is selected from one or any combination of tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, and water, preferably selected from one or any combination of tetrahydrofuran, acetone, acetonitrile, and water.

[0113] In some embodiments, in step f-1), the mass-to-volume ratio (g / mL) of the compound of Formula 7-A1 or a salt thereof to the reaction solvent is selected from 1:5 to 1:20.

[0114] In some embodiments, in step f-1), the molar ratio of the compound of formula 7-A1 to the compound of formula 7-A2 or a salt thereof is selected from 1:1 to 1:3.

[0115] In some embodiments, in step f-1), the reaction temperature is selected from 0 to 50°C, preferably 0 to 30°C.

[0116] In some embodiments, in step f-2), the protecting group PG3 on the amino group is removed from the compound of formula 7-A3 to give a compound of formula 7-A4.

[0117] In some embodiments, in step f-2), when PG3 is tert-butoxycarbonyl (Boc), the compound of formula 7-A3 is subjected to removal of the protecting group PG3 under acidic conditions, the acid being selected from trifluoroacetic acid, hydrogen chloride, hydrobromic acid and sulfuric acid, preferably hydrogen chloride or trifluoroacetic acid.

[0118] In some embodiments, in step f-2), when PG3 is tert-butoxycarbonyl (Boc), the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, ethyl acetate, and methyl tert-butyl ether or any combination thereof, preferably tetrahydrofuran or 1,4-dioxane.

[0119] In some embodiments, in step f-2), when PG3 is tert-butoxycarbonyl (Boc), the mass-to-volume ratio (g / mL) of the compound of Formula 7-A3 to the reaction solvent is selected from 1:4 to 1:20, preferably 1:5 to 1:15.

[0120] In some embodiments, in step f-2), when PG3 is tert-butoxycarbonyl (Boc), the molar ratio of the compound of Formula 7-A3 to the acid is selected from 1:5 to 1:50, preferably 1:10 to 1:30.

[0121] In some embodiments, in step f-2), when PG3 is tert-butoxycarbonyl (Boc), the reaction temperature is selected from 0 to 50°C, preferably 0 to 30°C.

[0122] In some embodiments, in step f-3), the compound of formula 7-A4 or its salt is subjected to reductive amination with an aldehyde under the action of a reducing agent and an acid to obtain a compound of formula 7 or its salt. The reducing agent is selected from sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride and Pd / C-H2, preferably sodium cyanoborohydride and / or sodium triacetoxyborohydride. The acid is selected from acetic acid, formic acid, propionic acid, trifluoroacetic acid and hydrochloric acid, preferably acetic acid or propionic acid.

[0123] In some embodiments, in step f-3), the reaction solvent is selected from one or any combination of methanol, ethanol, propanol, tetrahydrofuran, 1,4-dioxane, and ethyl acetate, preferably methanol or ethanol.

[0124] In some embodiments, in step f-3), the mass-to-volume ratio (g / mL) of the compound of formula 7-A4 or a salt thereof to the solvent is selected from 1:5 to 1:20, preferably 1:6 to 1:15.

[0125] In some embodiments, in step f-3), the molar ratio of the compound of formula 7-A4 or a salt thereof to the reducing agent is selected from 1:2 to 1:10, preferably 1:3 to 1:8.

[0126] In some embodiments, in step f-3), the molar ratio of the compound of Formula 7-A4 or a salt thereof to the aldehyde is selected from 1:2 to 1:10, preferably 1:4 to 1:8.

[0127] In some embodiments, in step f-3), the temperature at which the reducing agent is added is selected from -20 to 30°C, and preferably 0°C. The reaction temperature is selected from -20 to 50°C, and preferably 0 to 30°C.

[0128] In some embodiments, in step g-1), the compound of formula 7-B1 or a salt thereof is subjected to reductive amination with an aldehyde under the action of a reducing agent and an acid to obtain a compound of formula 7-B2 or a salt thereof, which step can be carried out in the same manner as step f-3).

[0129] In some embodiments, in step g-1), the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride and Pd / C—H2, preferably sodium cyanoborohydride or sodium triacetoxyborohydride.

[0130] In some embodiments, in step g-1), the acid is selected from acetic acid, formic acid, propionic acid, trifluoroacetic acid, and hydrochloric acid, preferably acetic acid or propionic acid.

[0131] In some embodiments, in step g-1), the reaction solvent is selected from one of methanol, ethanol, propanol, tetrahydrofuran, 1,4-dioxane, and ethyl acetate or any combination thereof, preferably methanol or ethanol.

[0132] In some embodiments, in step g-1), the mass-to-volume ratio (g / mL) of the compound of Formula 7-B1 or a salt thereof to the solvent is selected from 1:5 to 1:20.

[0133] In some embodiments, in step g-1), the molar ratio of the compound of Formula 7-B1 or a salt thereof to the reducing agent is selected from 1:2 to 1:10.

[0134] In some embodiments, in step g-1), the molar ratio of the compound of Formula 7-B1 or a salt thereof to the aldehyde is selected from 1:2 to 1:10.

[0135] In some embodiments, in step g-1), the temperature at which the reducing agent is added is selected from -20 to 30°C. The reaction temperature is selected from -20 to 50°C, preferably 0 to 30°C.

[0136] In some embodiments, in step g-2), the protecting group PG4 on the amino group is removed from the compound of formula 7-B2 or a salt thereof to obtain the compound of formula 7-B3 or a salt thereof, which can be carried out in the same manner as in step b).

[0137] In some embodiments, in step g-2), when PG4 is 9-fluorenylmethyloxycarbonyl (Fmoc), the compound of formula 7-B2 or a salt thereof is subjected to removal of the protecting group PG4 under basic conditions, the base being selected from piperidine, diethylamine, morpholine, diisopropylamine, DBU, triethylamine, and N,N-diisopropylethylamine, and preferably piperidine, diethylamine, or morpholine.

[0138] In some embodiments, in step g-2), when PG4 is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction solvent is selected from one or any combination of DMF, DMAc, dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, methanol and ethanol, preferably one or any combination of DMF, tetrahydrofuran and dichloromethane.

[0139] In some embodiments, in step g-2), when PG4 is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass-to-volume ratio (g / mL) of the compound of formula 7-B2 or a salt thereof to the reaction solvent is selected from 1:2 to 1:20.

[0140] In some embodiments, in step g-2), when PG4 is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass-to-volume ratio (g / mL) of the base to the reaction solvent is selected from 1:5 to 1:50.

[0141] In some embodiments, in step g-2), when PG4 is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction temperature is selected from 0 to 50°C, preferably 0 to 30°C.

[0142] In some embodiments, in step g-3), the reaction is carried out under basic conditions, the base being selected from sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate and potassium bicarbonate, preferably sodium bicarbonate, potassium bicarbonate or potassium carbonate.

[0143] In some embodiments, in step g-3), the reaction solvent is selected from one or any combination of tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, and water, preferably one or any combination of tetrahydrofuran, acetone, and acetonitrile, more preferably a combination of acetone and water.

[0144] In some embodiments, in step g-3), the reaction solvent is selected from a combination of acetone and water, where the volume ratio of acetone to water is selected from 1:0.2 to 1:10.

[0145] In some embodiments, in step g-3), the molar ratio of the compound of formula 7-B3 or a salt thereof to the compound of formula 7-A1 is selected from 1:1 to 1:5.

[0146] In some embodiments, in step g-3), the reaction temperature is selected from 0 to 50°C.

[0147] In some embodiments, in step h), the reaction is carried out under acidic conditions, the acid being selected from one or any combination of p-toluenesulfonic acid monohydrate, p-toluenesulfonic acid pyridinium salt, trifluoroacetic acid, and acetic acid, preferably p-toluenesulfonic acid monohydrate.

[0148] In some embodiments, in step h), the molar ratio of the compound of formula 2 to the acid is selected from 1:0.2 to 1:2, preferably 1:0.2 to 1:0.4.

[0149] In some embodiments, in step h), the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, toluene, o-methylphenol, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone, or any combination thereof, preferably, one of toluene, o-methylphenol, N-methylpyrrolidone, or any combination thereof, more preferably, N-methylpyrrolidone.

[0150] In some embodiments, in step h), the mass to volume ratio (g / mL) of the compound of formula 2 to the solvent is selected from 1:1 to 1:60.

[0151] In some embodiments, in step h), the molar ratio of the compound of formula 1 or a salt thereof to the compound of formula 2 is selected from 3:1 to 1:1, preferably 2:1 to 1:1.

[0152] In some embodiments, in step h), the compound of formula 1 is added to the reaction system in batches, preferably, based on the total amount added, 25-35 mol% of the compound of formula 1 or its salt and the compound of formula 2 are reacted under acidic conditions at 100-120°C, and the remaining compound of formula 1 is added to the reaction system in 2-5 batches, more preferably, based on the total amount added, 25-35 mol% of the compound of formula 1 or its salt and the compound of formula 2 are reacted under acidic conditions at 100-120°C for 20-40 minutes, and the remaining compound of formula 1 is added to the reaction system in 2-5 batches, with an interval of 20-40 minutes each time, and reacted for 30-90 minutes after addition.

[0153] In some embodiments, in step h), the reaction temperature is selected from 70 to 130°C, preferably 80 to 120°C, and more preferably 100 to 120°C.

[0154] In some embodiments, in step i-1), the compound of formula 1-A1 and the compound of formula 1-A2 are reacted with a palladium catalyst, a copper catalyst and a base to obtain a compound of formula 1-A3, wherein the palladium catalyst is selected from Pd(PPh3)4, Pd(OAc)2, Pd(PPh3)2Cl2 and Pd2(dba)3, preferably Pd(PPh3)2Cl2, the copper catalyst is selected from CuI, CuBr and CuCl, preferably CuI, and the base is selected from triethylamine, N,N-diisopropylethylamine, DBU, potassium carbonate and cesium carbonate, preferably triethylamine or N,N-diisopropylethylamine.

[0155] In some embodiments, in step i-1), the molar ratio of the palladium catalyst reacted with the compound of Formula 1-A1 is selected from 0.01:1 to 0.2:1, preferably 0.01:1 to 0.10:1, more preferably 0.01:1 to 0.03:1.

[0156] In some embodiments, in step i-1), the molar ratio of reacting the copper catalyst with the compound of formula 1-A1 is selected from 0.01:1 to 0.2:1, preferably 0.02:1 to 0.10:1, more preferably 0.02:1 to 0.05:1.

[0157] In some embodiments, in step i-1), the molar ratio of the compound of formula 1-A1 to the compound of formula 1-A2 is selected from 1:1 to 1:5, preferably 1:1 to 1:3, and more preferably 1:1 to 1:2.

[0158] In some embodiments, in step i-1), the molar ratio of reacting the base with the compound of formula 1-A1 is selected from 10:1 to 1:1, preferably 5:1 to 2:1.

[0159] In some embodiments, in step i-1), the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, toluene, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, or any combination thereof, for example, the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, or any combination thereof, also for example, tetrahydrofuran, N-methylpyrrolidone or N,N-dimethylformamide, preferably tetrahydrofuran or 1,4-dioxane.

[0160] In some embodiments, in step i-1), the mass-to-volume ratio (g / mL) of the compound of formula 1-A1 to the solvent is selected from 1:5 to 1:20, preferably 1:10 to 1:20.

[0161] In some embodiments, in step i-1), the reaction temperature is selected from 40 to 80°C, preferably 40 to 60°C.

[0162] In some embodiments, in step i-2), the compound of formula 1-A3 is reacted with Sn and Na2S to give the compound of formula 1 or a salt thereof, and Na2S is added to the reaction system in the form of Na2S·2H2O and / or Na2S·9H2O.

[0163] In some embodiments, in step i-2), the molar ratio of Sn to the compound of formula 1-A3 is selected from 5:1 to 1:1, preferably 3:1 to 1:1.

[0164] In some embodiments, in step i-2), the molar ratio of Na2S to the compound of formula 1-A3 is selected from 0.1:1 to 2:1, preferably 0.2:1 to 1:1.

[0165] In some embodiments, in step i-2), the reaction solvent is selected from one of tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, methanol, ethanol, propanol, and water, or any combination thereof, preferably one of tetrahydrofuran, ethanol, and methanol, or any combination thereof, more preferably a combination of ethanol and water, wherein the volume ratio of ethanol to water is selected from 20:1 to 1:1, preferably 10:1 to 5:1.

[0166] In some embodiments, in step i-2), the reaction temperature is selected from 40 to 100°C, for example, 50 to 80°C, and preferably 60 to 90°C.

[0167] In some embodiments, in step j-1), a compound of formula 1-B1 is subjected to a Grignard reaction with a compound of formula 1-B2 to provide a compound of formula 1-B3.

[0168] In some embodiments, in step j-1), the molar ratio of the compound of formula 1-B1 to the compound of formula 1-B2 is selected from 1:0.8 to 1:2, preferably selected from 1:1 to 1:1.5.

[0169] In some embodiments, in step j-1), the reaction solvent is selected from one of tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl ether, and diethyl ether, or any combination thereof, preferably tetrahydrofuran or isopropyl ether.

[0170] In some embodiments, in step j-1), the reaction temperature is selected from -20 to 60°C, preferably -20 to 50°C, and more preferably 0 to 30°C.

[0171] In some embodiments, in step j-2), the compound of formula 1-B3 can undergo a nitration reaction to give a compound of formula 1-B4.

[0172] In some embodiments, in step j-2), the nitrification system of the reaction is selected from concentrated nitric acid, concentrated nitric acid / acetic acid, concentrated sulfuric acid / potassium nitrate.

[0173] In some embodiments, in step j-2), the mass-to-volume ratio (g / mL) of the compound of formula 1-B3 to the nitrification system is selected from 1:4 to 1:20, preferably 1:6 to 1:15.

[0174] In some embodiments, in step j-2), the reaction temperature is selected from -20 to 30°C, preferably -20 to 0°C.

[0175] In some embodiments, in step j-3), the nitro group of the compound of formula 1-B4 is reduced to an amino group to give a compound of formula 1-B5 or a salt thereof, which may be carried out using conventional reaction conditions well known to those skilled in the art.

[0176] In some embodiments, in step j-3), the reducing agent of the reaction is selected from hydrogen gas, TiCl3, SnCl2, sodium dithionite, zinc powder, and iron powder, preferably hydrogen gas, SnCl2, or sodium dithionite.

[0177] In some embodiments, in step j-3), the reaction is carried out under an atmosphere of hydrogen gas with the addition of a transition metal catalyst, the transition metal catalyst being selected from platinum dioxide, palladium on carbon, rhodium on carbon, and Raney nickel.

[0178] In some embodiments, in step j-3), when the reaction is carried out in a hydrogen gas-platinum dioxide system, the mass ratio of the compound of formula 1-B4 to platinum dioxide is selected from 1:0.01 to 1:0.4, preferably 1:0.01 to 1:0.1.

[0179] In some embodiments, in step j-3), when the reaction is carried out in a hydrogen gas-platinum dioxide system, the pressure of the hydrogen gas is selected from 1 to 10 atmospheres, preferably 1 to 5 atmospheres.

[0180] In some embodiments, in step j-3), when the reaction is carried out in a hydrogen gas-platinum dioxide system, the reaction temperature is selected from -10 to 100°C, preferably -10 to 80°C.

[0181] In some embodiments, in step j-3), when the reaction is carried out in a hydrogen gas-platinum dioxide system, the reaction solvent is selected from one of methanol, ethanol, ethyl acetate, tetrahydrofuran and 1,4-dioxane or any combination thereof, preferably ethyl acetate or tetrahydrofuran.

[0182] In some embodiments, in step j-4), the reaction is carried out under basic conditions, the base being selected from potassium carbonate, potassium hydroxide, sodium hydroxide, cesium carbonate and sodium carbonate, for example, the base being selected from potassium carbonate, sodium hydroxide, cesium carbonate and sodium carbonate, for example, potassium carbonate or potassium hydroxide, preferably potassium carbonate or sodium carbonate.

[0183] In some embodiments, in step j-4), the molar ratio of the compound of Formula 1-B5 to the base is selected from 1:1 to 1:5, preferably 1:1 to 1:3.

[0184] In some embodiments, in step j-4), the reaction solvent is selected from one or any combination of water, tetrahydrofuran, 1,4-dioxane, acetonitrile, hexamethylphosphonotriamide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and dimethylsulfoxide, preferably one or any combination of water, acetonitrile, tetrahydrofuran, dimethylsulfoxide, and hexamethylphosphonotriamide.

[0185] In some embodiments, in step j-4), the reaction solvent is selected from a combination of acetonitrile and water, where the volume ratio of acetonitrile to water is selected from 1:5 to 10:1, preferably 1:2 to 5:1.

[0186] In some embodiments, in step j-4), the mass-to-volume ratio (g / mL) of the compound of formula 1-B5 or a salt thereof to the reaction solvent is selected from 1:5 to 1:20, preferably 1:5 to 1:10.

[0187] In some embodiments, the reaction temperature in step j-4) is selected from 20 to 100°C, preferably 40 to 80°C.

[0188] Intermediates In another aspect, the present application provides a compound of the following formula 1-B4, a compound of formula 1-B5 or a salt thereof, a compound of formula 1 or a salt thereof, a compound of formula 7 or a salt thereof, a compound of formula 8 or a salt thereof, or a compound of formula 9 or a salt thereof.

[0189] [ka]

[0190] Where: R1 and R2 are each independently C 1-4 alkyl, X is selected from halogens; PG1 is selected from amine protecting groups; The compound of formula 7

[0191] [ka]

[0192] isn't it.

[0193] In another aspect, the present application provides use of a compound of formula 1-B4 or a salt thereof, or a compound of formula 1-B5 in preparing a compound of formula 1 or a salt thereof, or a compound of formula 3 or a salt thereof, or a compound of formula I or a salt thereof.

[0194] [ka]

[0195] Where: R1 and R2 are each independently C 1-4 alkyl, X is selected from halogens.

[0196] In another aspect, the present application provides the use of a compound of formula 1, or a salt thereof, in the preparation of a compound of formula 3, or a salt thereof, or a compound of formula I, or a salt thereof.

[0197] [ka]

[0198] Where: R1 and R2 are each independently C 1-4 is selected from alkyl.

[0199] In another aspect, the present application provides the use of a compound of formula 7 or a salt thereof, a compound of formula 8 or a salt thereof, or a compound of formula 9 or a salt thereof in the preparation of a compound of formula I or a salt thereof.

[0200] [ka]

[0201] Where: R1 and R2 are each independently C 1-4 alkyl, PG1 is selected from amine protecting groups.

[0202] In some embodiments, in the above intermediate or use thereof, R1 and R2 are each independently selected from methyl, ethyl and propyl, and preferably, R1 and R2 are the same.

[0203] In some embodiments, in the above intermediate or use thereof, R1 and R2 are both propyl.

[0204] In some embodiments, in the above intermediate or use thereof, said X is selected from fluorine, chlorine, bromine and iodine.

[0205] In some embodiments, in the above intermediate or use thereof, said X is chlorine.

[0206] In some embodiments, PG1 is selected from an alkoxycarbonyl protecting group, preferably selected from benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc).

[0207] In some embodiments of the present application, the compound of formula 1-B4 is

[0208] [ka]

[0209] is selected from.

[0210] In some embodiments of the present application, the compound of formula 1-B5 or a salt thereof is

[0211] [ka]

[0212] is selected from.

[0213] In some embodiments of the present application, the compound of formula 7 or a salt thereof is

[0214] [ka]

[0215] is selected from.

[0216] In some embodiments of the present application, the compound of formula 8 or a salt thereof is

[0217] [ka]

[0218] is selected from.

[0219] In some embodiments of the present application, the compound of formula 9 or a salt thereof is

[0220] [ka]

[0221] is selected from.

[0222] [Definitions and Terms] Unless otherwise specified, the terms and phrases used herein have the meanings given below. A particular term or phrase should not be considered uncertain or unclear without a specific definition, but should be interpreted according to the meaning generally understood by those skilled in the art. When a trade name appears herein, it is intended to refer to the corresponding trade name or its active ingredient.

[0223] In this application, singular words include the plural unless otherwise stated or implied by context. Thus, references to singular terms and "the", "said" and "said" generally include the plural of the respective terms. The terms "including" and "containing" are to be interpreted inclusively rather than exclusively.

[0224] In this application, unless otherwise stated or implied by the context, the term "salt thereof" as used refers to the salt form of a compound (e.g., a compound of formula 1). A compound in salt form contains one or more internal salt forms and / or another molecule, such as acetate, succinate, or other counterions. The counterions of a compound in salt form are usually organic or inorganic moieties that stabilize the charge of the parent compound. A salt form of a compound has one or more charged atoms in its structure. When multiple charged atoms are part of a salt form, multiple counterions and / or multiple charged counterions are present. Thus, a salt form of a compound generally has one or more charged atoms and one or more counterions that correspond to the non-salt form of the compound. In some embodiments, the non-salt form of a compound contains at least one amino or other basic moiety, such that in the presence of an acid, an acid addition salt with a basic moiety is obtained. In other embodiments, the non-salt form of a compound contains at least one carboxylic acid group or other acidic moiety, such that in the presence of a base, a carboxylate or other anionic moiety is obtained.

[0225] In the present application, exemplary counter anions and counter cations in the salt form of the compounds include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, glycolate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthoate)).

[0226] In this application, the term “C 1-4 "Alkyl" refers to a straight or branched chain alkyl containing 1 to 4 carbon atoms. Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 1-n-butyl, 2-n-butyl, isobutyl, tert-butyl, and the like.

[0227] In this application, the term "room temperature" refers to 25±5°C.

[0228] In the present application, the protecting group and its attachment or removal method can be carried out using conventional methods in the art, which may be a one-step reaction or a multi-step reaction, and can be realized by referring to, for example, but not limited to, "Greene's Protective Groups in Organic Synthesis - 4th Edition" published by Wiley Publishing Co., or "Protective Group Chemistry" published by Chemical Industry Publishing Co., Ltd.

[0229] In this application, the term "amino protecting group" refers to a group that protects an amino group in a compound, and may be selected from amino protecting groups known in the art. The medium for the protection reaction described in this application is preferably selected from aprotic solvents, preferably dichloromethane. The upper protecting group reaction for protecting an amino may be carried out using conventional reaction conditions well known to those skilled in the art. Preferably, the reaction for removing the protecting group on an amino may be carried out using conventional reaction conditions well known to those skilled in the art. Preferably, the amino protecting group used in the present invention is selected from alkoxycarbonyl-based protecting groups, which may be unsubstituted or substituted, such as benzyloxycarbonyl, tert-butoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, methoxycarbonyl or ethoxycarbonyl.

[0230] In this application, when the term "about" is used in conjunction with a numerical value or a numerical range, it modifies the numerical value or range by extending the boundaries of the numerical value upward and / or downward. For example, the term "about" is intended to modify the numerical value by ≦20% above and below the stated value, more preferably ≦10%.

[0231] In this application, when both a compound name and a structural formula are given for a compound and there is a discrepancy between the two, the compound's structure shall prevail unless the context indicates that the compound's structure is incorrect and the name is correct.

[0232] In this application, compounds may exist in specific geometric or stereoisomeric forms. This application contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as mixtures in enantiomeric or diastereomeric excess, all of which mixtures are included within the scope of this application. Other asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, and mixtures thereof, are included within the scope of the present invention.

[0233] All of the reagents and materials used in the present invention are commercially available.

[0234] Positive advancement effects of the present invention: The raw materials of the preparation method of this application are easily available and low-cost. The separation and purification of intermediates and products are simple to operate. The reaction conditions are mild, carried out under normal pressure, and the reaction temperature is easy to control. The purity of the final product is high. In addition, the preparation method described in this application can be scaled up and is suitable for industrialized production. Furthermore, the drug-linker conjugate prepared in this application can be conjugated with an antibody to obtain an antibody-drug conjugate (ADC) with good antitumor activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0235] Sequence information Sequence information relevant to this application is set forth in the table below.

[0236] [Table 1-1]

[0237] All features disclosed in this specification, or all steps of any method or process disclosed, may be combined in any manner, except for mutually exclusive features and / or steps.

[0238] Any feature disclosed in this specification, unless expressly stated otherwise, may be replaced by other equivalent or alternative features serving a similar purpose, i.e., unless expressly stated otherwise, each feature is only one example in a series of equivalent or similar features. EXAMPLES

[0239] The operating conditions used in the examples may be further adjusted according to specific requirements, and the operating conditions not specified are usually those in routine experiments.

[0240] All the reagents used in the following examples are commercially available.

[0241] The meanings of the abbreviations or English terms used in the conventional synthesis methods, examples, and intermediate synthesis examples are as shown in the table below.

[0242] [Table 1-2]

[0243] [Table 1-3]

[0244] In an exemplary embodiment of the invention, compound 1 is synthesized by the following route:

[0245] Scheme (1)

[0246] [ka]

[0247] Scheme (2)

[0248] [ka]

[0249] In an exemplary embodiment of the invention, compound 3 is synthesized by the following route:

[0250] Scheme (1)

[0251] [ka]

[0252] Scheme (2)

[0253] [ka]

[0254] In an exemplary embodiment of the invention, compound 7a is synthesized by the following route:

[0255] Scheme (1)

[0256] [ka]

[0257] In an exemplary embodiment of the invention, drug-linker conjugate Ia is synthesized by the following route:

[0258] [ka]

[0259] Example 1 Preparation of 4-(6-nitrobenzo[d][1,3]dioxolan-5-yl)butan-3-yn-1-ol (Compound 1-A3)

[0260] [ka]

[0261] Method 1: Compounds 1-A1 (100 g, 408 mmol) and 1-A2 (57.2 g, 816 mmol) were dissolved in THF (1.5 L), CuI (3.88 g, 20.4 mmol), Pd(PPh3)2Cl2 (8.60 g, 12.24 mmol), and triethylamine (102 g, 1.02 mol, 141 mL) were added, and the mixture was reacted overnight at 50 °C under nitrogen gas protection. The reaction was monitored by LCMS. The reaction solution was filtered through diatomaceous earth while still hot, and the crude product was obtained after concentrating the filtrate. The crude product was slurried with ethyl acetate (300 ml) to obtain the target product 1-A3 (65 g, yield: 67%, purity: 98.9%).

[0262] Method 2: Compound 1-A1 (100 g, 408 mmol) and 1-A2 (11.4 g, 163 mmol) were dissolved in NMP (300 mL), triethylamine (102 g, 1.02 mol, 141 mL) was added, and the mixture was protected with nitrogen gas. CuI (3.88 g, 20.4 mmol) and Pd(PPh3)2Cl2 (2.9 g, 4.1 mmol) were added in sequence, and the mixture was reacted at 50 ° C for 0.5 h. 1-A2 (22.8 g, 326 mmol) was added dropwise, and after the addition was completed, the mixture was reacted at 50 ° C for 4 h. The reaction solution was cooled to room temperature and poured into water (4.5 L) containing ammonia water (3.26 mol), and the solid was precipitated and stirred for 0.5 h. The mixture was filtered by suction, and the cake was added to water (4.5 L) and stirred for 0.5 h. After suction filtration, the cake was added to water (4.5 L) and stirred for 0.5 h. The cake was dried to give the target product 1-A3 (85.0 g, yield: 88%, purity: 96.4%).

[0263] Method 3: Compound 1-A1 (100 g, 408 mmol) and 1-A2 (34.1 g, 490 mmol) were dissolved in DMF (0.5 L), and CuI (1.55 g, 8.16 mmol) and Pd(PPh3)2Cl2 (2.87 g, 4.08 mmol) were added. Triethylamine (204 g, 2.02 mol) was added dropwise, and after the addition was completed, the reaction was carried out at 50 °C for 3 h. The reaction solution was cooled to below 30 °C, and the reaction solution was added to 1% ammonia water (1 L) and stirred for 30 min. The mixture was filtered with suction, and the cake was slurried in water (500 mL) for 20 min and filtered with suction. The cake was dried to obtain the target product 1-A3 (86 g, yield: 90%, purity: 96.6%).

[0264] Compound 1-A3:LCMS (ESI) [M+H] + = 236.0.

[0265] Compound 1-A3: 1 H NMR (400 MHz, DMSO-d6) δ 7.66 (s, 1H), 7.18 (s, 1H), 6.25 (s, 2H), 4.91 (t, J = 5.6 Hz, 1H), 3.59 (dd, J = 12.6, 6.7 Hz, 2H), 2.59 (t, J = 6.9 Hz, 2H).

[0266] Example 2 Preparation of 1-(6-aminobenzo[d][1,3]dioxolan-5-yl)-4-hydroxybutan-1-one (Compound 1)

[0267] [ka]

[0268] Method 1: Compound 1-A3 (8.42 g, 35.79 mmol) was dissolved in a solution of EtOH:H2O=9:1 (v:v, 152 mL), Sn (8.46 g, 71.58 mmol), Na2S·2H2O (0.84 g, 10.74 mmol) were added, then concentrated hydrochloric acid (30 mL, 358 mmol) was added, and the mixture was stirred at 78 °C for 1 h, and the reaction was monitored by LCMS. The reaction solution was filtered through diatomaceous earth while hot, and the filtrate was concentrated to obtain a crude product. Ethyl acetate (20 ml) was added to make a slurry, then filtered with suction, and the cake was washed twice with ethyl acetate (5 ml) to obtain the crude product of compound 1. The crude product of compound 1 (5.0 g) was added to water (50 mL), and a saturated solution of sodium bicarbonate was added dropwise until the pH value was about 8.0, and the mixture was stirred at room temperature for 1 h. The cake and filtrate were obtained by suction filtration. The filtrate was further extracted with ethyl acetate (50 mL x 2) and concentrated to obtain a solid. The solid and cake obtained by concentration were dried to obtain compound 1 (3.55 g, yield: 51%, purity: 99.7%).

[0269] Method 2: Compound 1-A3 (80 g, 340 mmol) was dissolved in a solution of EtOH:H2O=9:1 (v:v, 1600 mL), Sn (80.4 g, 680 mmol), Na2S·9H2O (24.5 g, 102 mmol) were added, followed by concentrated hydrochloric acid (142 mL, 1.7 mol) and stirred at 55 °C for 1 h. The reaction solution was filtered hot through diatomaceous earth with suction, rinsed with a small amount of ethanol, the filtrate was combined, concentrated to dryness, dissolved in ethyl acetate (800 mL), diluted slowly with aqueous sodium carbonate, and finally adjusted to pH=8. The filtrate was collected by filtration, separated, and the organic phase was concentrated until a large amount of brown solid precipitated, cooled to room temperature, filtered with suction, and dried to give compound 1 (50.1 g, yield: 66.0%, purity: 93.2%).

[0270] Compound 1:LCMS (ESI) [M-18+H] + =206.2.

[0271] Compound 1: 1H NMR (400 MHz, CD3OD) δ 7.18 (s, 1H), 6.24 (s, 1H), 5.87 (s, 2H), 3.62 (t, J = 6.7 Hz, 2H), 2.95 - 2.84 (m, 2H), 1.95 - 1.82 (m, 2H).

[0272] Example 3 Preparation of 1-(benzo[d][1,3]dioxolan-5-yl)-4-chlorobutan-1-one (compound 1-B3-a)

[0273] [ka]

[0274] Method 1: Add magnesium chips (12.60g, 518mmol), iodine particles (0.50g) and magneton to a reaction bottle. Attach a reflux condenser, add THF (200ml) under nitrogen gas protection, and start stirring. First, add 4-bromo-1,2-methylenedioxybenzene (10.00g, 50mmol, 0.12eq) and heat with hot air from a hair dryer to induce the reaction, and the color of the reaction system changed from pale yellow to colorless. Then add THF (300ml) and add 4-bromo-1,2-methylenedioxybenzene (90.00g, 450mmol, 1.08eq) in several portions while the reaction system is boiling slightly. Most of the magnesium chips are consumed, the reaction system stops generating heat and returns to room temperature naturally, and the newly prepared Grignard reagent 1-B2-a is used later.

[0275] Compound 1-B1-a (68.66 g, 415 mmol), THF (500 ml) and magnetone were added to the reaction bottle. The reaction was cooled to 0-5°C, stirring was started and the THF solution of the freshly prepared Grignard reagent 1-B2-a was slowly added using a constant pressure addition funnel, while maintaining the temperature of the reaction at 0-10°C. After the addition of the raw materials was completed, the reaction was allowed to return to room temperature and reacted overnight.

[0276] After the reaction was completed, the reaction system was quenched by adding a saturated aqueous ammonium chloride solution (40 ml), and EtOAc (1000 ml) and saturated saline (1500 ml) were added to dilute the reaction solution. 2M HCl (250 ml) was added to adjust the pH to 6-7, and the liquid was separated by shaking, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation to obtain a crude product. The crude product was dissolved in methyl tert-butyl ether (80 ml), and n-heptane (200 ml) was slowly added while stirring, and a solid was precipitated. The reaction system was cooled to -30 ° C, stirred for 15 minutes, and filtered by suction at low temperature. The solid was transferred and then dried to obtain compound 1-B3-a (87.21 g, yield: 92%, purity: 96.8%).

[0277] Method 2: Add magnesium chips (14.71g, 613mmol), iodine particles (0.58g) and magneton to a reaction bottle. Attach a reflux condenser, add isopropyl ether (200ml) under nitrogen gas protection, and start stirring. First, add 4-bromo-1,2-methylenedioxybenzene (11.66g, 58mmol, 0.14eq) and heat with hot air from a hair dryer to induce the reaction, and the color of the reaction system changed from pale yellow to colorless. Then add isopropyl ether (300ml) and add 4-bromo-1,2-methylenedioxybenzene (105.1g, 523mmol, 1.26eq) in several portions while the reaction system is boiling slightly. Most of the magnesium chips are consumed, the reaction system stops generating heat and returns to room temperature naturally, and the newly prepared Grignard reagent 1-B2-a is used later.

[0278] Compound 1-B1-a (68.66 g, 415 mmol), isopropyl ether (500 ml) and magnetone were added to the reaction bottle. The reaction was cooled to 0-5°C, stirring was started and the above freshly prepared Grignard reagent 1-B2-a in isopropyl ether solution was slowly added using a constant pressure addition funnel, while maintaining the temperature of the reaction at 0-10°C. After the addition was complete, the reaction was allowed to return to room temperature and reacted overnight.

[0279] After the reaction was completed, the reaction system was quenched by adding a saturated aqueous ammonium chloride solution (40 ml), and EtOAc (1000 ml) and saturated saline (1500 ml) were added to dilute the reaction solution. 2M HCl (250 ml) was added to adjust the pH to 6-7, the liquid was separated by shaking, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation to obtain a crude product. The crude product was dissolved in methyl tert-butyl ether (80 ml), n-heptane (200 ml) was slowly added while stirring, a solid was precipitated, the reaction system was cooled to -30 ° C, stirred for 15 minutes, suction filtered at low temperature, and the solid was transferred and dried to obtain compound 1-B3-a (86.52 g, yield: 91.3%, purity: 95.1%).

[0280] Compound 1-B3-a:LCMS (ESI) [M+H] + = 227.2.

[0281] Compound 1-B3-a: 1 H NMR (400 MHz, DMSO-d6) δ 7.61 (dd, J = 8.2, 1.8 Hz, 1H), 7.43 (d, J = 1.7 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 6.13 (s, 2H), 3.69 (t, J = 6.7 Hz, 2H), 3.09 (t, J = 7.1 Hz, 2H), 2.05 (p, J = 6.9 Hz, 2H).

[0282] Example 4 Preparation of 4-chloro-1-(6-nitrobenzo[d][1,3]dioxolan-5-yl)butanone (compound 1-B4-a)

[0283] [ka]

[0284] Method 1: Concentrated nitric acid (800 mL) was slowly added to a 2 L reaction flask and cooled to -5°C, and compound 1-B3-a (80 g, 0.353 mol) was weighed and added in batches to the reaction flask. Stirred at -5°C for 4 hours and monitored the reaction by TLC (PE:EA=5:1). The reaction solution was slowly added to 2 L of ice water, and a large amount of solid precipitated. Filtered, and the cake was slurried twice with saturated saline (2 L x 2). Filtered, and the cake was slurried twice with aqueous sodium bicarbonate (1 L x 2), and then slurried once with 2 L of water. Filtered, and the cake was dissolved in ethyl acetate (1 L), dried over anhydrous sodium sulfate, filtered, concentrated, and evaporated to dryness to obtain target compound 1-B4-a (81 g, yield: 76%, purity: 89.3%).

[0285] Method 2: Concentrated nitric acid (12 mL) was added slowly to the reaction flask, the temperature was cooled to -5°C, and compound 1-B3-a (4.0 g, 17.6 mmol) was weighed and dissolved in acetic acid (12 mL), and slowly added dropwise to the reaction flask. The mixture was stirred at -5°C for 4 hours, and the reaction was monitored by TLC (PE:EA=5:1). The reaction solution was slowly added to 100 mL of ice water, and a large amount of solid precipitated. After filtration, the cake was slurried twice with saturated saline (100 mL x 2). After filtration, the cake was slurried twice with saturated aqueous sodium bicarbonate (100 mL x 2), and then slurried once with 100 mL of water. After filtration, the cake was dissolved in ethyl acetate (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and evaporated to dryness to obtain the target compound 1-B4-a (4 g, yield: 75.7%, purity: 90.7%).

[0286] Compound 1-B4-a: 1 H-NMR (400 MHz, DMSO-d6) δ 7.72 (s, 1H), 7.27 (s, 1H), 6.30 (s, 2H), 3.70 (t, J = 6.7 Hz, 2H), 2.95 (t, J = 7.1 Hz, 2H), 2.19 - 1.93 (m, 2H).

[0287] Example 5 Preparation of 1-(6-aminobenzo[d][1,3]dioxolan-5-yl)-4-chlorobutan-1-one (compound 1-B5-a)

[0288] [ka]

[0289] Method 1: Compound 1-B4-a (250 g, purity: 89.3%, 0.92 mol) was added to ethyl acetate (3 L), platinum dioxide (12.5 g) was added, and the mixture was replaced with hydrogen gas three times. The mixture was reacted overnight at room temperature under a hydrogen gas atmosphere, and the reaction was monitored by LCMS. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the crude product of target compound 1-B5-a (200 g), which was directly used in the next conversion step.

[0290] Method 2: Compound 1-B4-a (5 g, purity: 89.3%, 0.016 mol) was added to tetrahydrofuran (50 mL), platinum dioxide (0.2 g) was added, and the mixture was replaced with hydrogen gas three times. The mixture was reacted overnight at room temperature under hydrogen gas atmosphere, and the reaction was monitored by LCMS. After the reaction was completed, the reaction system was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the crude product of target compound 1-B5-a (4 g), which was directly used for the next conversion.

[0291] Compound 1-B5-a:LCMS (ESI) [M+H] + =242.0.

[0292] Compound 1-B5-a: 1 H NMR (400 MHz, CDCl3) δ 7.15 (s, 1H), 6.27 (s, 1H), 5.93 (s, 2H), 3.66 (t, J = 6.3 Hz, 2H), 3.02 (t, J = 7.0 Hz, 2H), 2.18 (p, J = 6.7 Hz, 2H).

[0293] Example 6 Preparation of 1-(6-aminobenzo[d][1,3]dioxolan-5-yl)-4-hydroxybutan-1-one (Compound 1)

[0294] [ka]

[0295] Method 1: Compound 1-B5-a (200.0 g) was dissolved in acetonitrile (1 L), and then water (1 L) was added, followed by potassium carbonate (137.3 g, 0.994 mol). The reaction was stirred at 50° C. for 8 hours, and the reaction was monitored by LCMS. After the reaction was completed, the reaction solution was concentrated to remove acetonitrile, and ethyl acetate (6 L) was added to the residue for extraction. The organic phase was washed twice with pure water (2 L), and then washed once with saturated saline (2 L). The organic phase was dried over anhydrous Na2SO4 and then concentrated to obtain a crude product. The crude product was slurried in ethyl acetate (400 ml) for 1.5 h. After filtration, the cake was slurried in ethyl acetate (250 ml) for 2 h. After filtration, the cake was dried under reduced pressure to obtain compound 1 (112 g, calculated yield for two steps from 1-B4-a: 54%, purity: 97.7%).

[0296] Method 2: Compound 1-B5-a (4.0 g) was dissolved in acetonitrile (20 mL), and then water (20 mL) was added, followed by potassium hydroxide (1.12 g, 0.02 mol). The reaction was stirred at 50° C. for 8 hours, and the reaction was monitored by LCMS. After the reaction was completed, the reaction solution was concentrated to remove acetonitrile, and ethyl acetate (120 mL) was added to the residue for extraction. The organic phase was washed twice with pure water (120 mL), and then once with saturated saline (120 mL). The organic phase was dried over anhydrous Na2SO4 and then concentrated to obtain the crude product. The crude product was slurried in ethyl acetate (8 mL) for 1.5 h. After filtration, the cake was slurried in ethyl acetate (5 mL) for 2 h. After filtration, the cake was dried under reduced pressure to obtain compound 1 (2.2 g, calculated yield for two steps from 1-B4-a: 53%, purity: 98.0%).

[0297] Compound 1:LCMS (ESI) [M-18+H] + =206.2.

[0298] Compound 1: 1 H NMR (400 MHz, CD3OD) δ 7.18 (s, 1H), 6.24 (s, 1H), 5.87 (s, 2H), 3.62 (t, J = 6.7 Hz, 2H), 2.95 - 2.84 (m, 2H), 1.95 - 1.82 (m, 2H).

[0299] Example 7 Preparation of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4';6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Compound 3)

[0300] [ka]

[0301] Method 1: Compound 1 (41.80 g, 0.19 mol), compound 2a (110.00 g, 0.42 mol) and p-toluenesulfonic acid monohydrate (31.90 g, 0.17 mol) were added to a three-neck flask. 275 ml of NMP was weighed into the flask, protected with nitrogen gas, and heated to 110°C and stirred. Compound 1 (31.35 g, 0.14 mol) was added to the reaction system every 0.5 h for a total of three times. After the addition was completed, the mixture was stirred for 1 hour and the reaction was monitored by HPLC. After the reaction was completed, isopropyl alcohol (550 ml) was added to the reaction solution and stirred for 1.0 hour. The resulting suspension was added to methyl tert-butyl ether (11 L) and stirred for 1 hour. The mixture was filtered by suction, and the cake was slurried in water (5.5 L) for 1 hour. The cake was obtained by filtration, air-dried at 50° C. for 16 hours, and then dried under reduced pressure at 80° C. for 4 hours to obtain compound 3 (182.00 g, yield: 77%, purity: 80.2%).

[0302] Method 2: Compound 1 (41.80 g, 0.19 mol), compound 2a (110.00 g, 0.42 mol) and p-toluenesulfonic acid monohydrate (31.90 g, 0.17 mol) were added to a three-neck flask, 550 ml of NMP was weighed into the flask and protected with nitrogen gas. The mixture was heated to 110°C and stirred, and compound 1 (31.35 g, 0.14 mol) was added to the reaction system every 0.5 h for a total of three times. After the addition was completed, the mixture was stirred for 1 hour. Isopropyl alcohol (110 mL) was added to the reaction solution, and water (1.32 L) was further added dropwise, cooled to 5°C and stirred for 1.0 hour. The mixture was filtered with suction, the cake was washed with water, slurried with water (13.75 L) for 2 hours, filtered with suction, and the cake was further washed with water. The cake was added to a reaction bottle, and DMF (165 mL) and methanol (1.32 L) were added successively. The mixture was stirred at 25°C for 12 h, suction filtered, the cake was washed with methanol, and vacuum dried at 50°C for 48 h to obtain compound 3 (184.0 g, yield: 77.9%, purity: 93%).

[0303] Comparative Example 1: Compound 1 (1.23 g, 5.5 mmol), compound 2a (1.0 g, 3.8 mmol) and p-toluenesulfonic acid monohydrate (289 mg, 1.5 mmol) were added to a three-neck flask. 2.5 mL of NMP was weighed into the flask, protected with nitrogen gas, heated to 110 ° C and stirred for 2 hours. Isopropyl alcohol (1.0 mL) was added to the reaction solution, and water (20 mL) was further added dropwise, cooled to 5 ° C and stirred for 1.0 hour. Suction filtration was performed, the cake was washed with water, slurried with water (20 mL) for 2 hours, suction filtration was performed, and the cake was further washed with water. The cake was added to a reaction bottle, and DMF (1.5 mL) and methanol (12 mL) were added successively. The mixture was stirred at 25°C for 12 h, suction filtered, the cake was washed with methanol, and vacuum dried at 50°C for 48 h to obtain compound 3 (0.68 g, yield: 39.7%, purity: 91%).

[0304] Comparative Example 2: Compound 1 (0.39 g, 1.7 mmol), compound 2a (1.0 g, 3.8 mmol) and p-toluenesulfonic acid monohydrate (289 mg, 1.5 mmol) were added to a three-neck flask. 2.5 mL of NMP was weighed into the flask, protected with nitrogen gas, heated to 65 ° C and stirred. Compound 1 (0.28 g, 1.26 mmol) was added to the reaction system three times in total every 0.5 h. After the addition was completed, the mixture was stirred for 1 hour, and the reaction was monitored by HPLC to show that there were many intermediate states that were not cyclized, and there was little product.

[0305] Compound 3: LCMS (ESI) [M+H] + = 451.4.

[0306] Compound 3: 1 H NMR (400 MHz, DMSO-d6) δ 7.59 (s, 1H), 7.49 (s, 1H), 7.25 (s, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.54 - 5.36 (m, 2H), 5.19 (s, 2H), 4.68 (t, J = 5.0 Hz, 1H), 3.51 (q, J = 5.6 Hz, 2H), 3.14 (t, J = 7.6 Hz, 2H), 1.99 - 1.73 (m, 4H), 0.92 (t, J = 7.3 Hz, 3H).

[0307] Example 8 Preparation of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4';6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (Compound 3)

[0308] [ka]

[0309] Compound 1 (0.076 g, 0.34 mmol), compound 2b (0.2 g, 0.65 mmol) and p-toluenesulfonic acid monohydrate (0.05 g, 0.26 mmol) were added to a 25 mL three-neck flask, NMP (0.50 mL) was added, magneton was added, the mixture was protected with argon gas, and the mixture was stirred at 110 ° C. Compound 1 was added to the reaction system every 0.5 h, and the amount of compound 1 added was (0.057 g, 0.26 mmol), (0.057 g, 0.26 mmol), and (0.038 g, 0.17 mmol) in sequence. After the addition was completed, the mixture was stirred for 0.5 h. The mixture was sampled and detected by LCMS. After the reaction was completed, 2 ml of isopropyl alcohol was added to the reaction solution while stirring, and the mixture was stirred for 1.0 h. The resulting suspension was added to 40 ml of methyl tert-butyl ether and stirred for 0.5 h. Suction filtered, the cake was slurried with 40ml water for 0.5h, the cake was obtained and blast dried at 50°C for 12h to give crude compound 3 (0.20g, yield: 56%, purity: 80%).

[0310] Compound 3: LCMS (ESI) [M+H] + = 451.4.

[0311] Compound 3: 1 H NMR (400 MHz, DMSO-d6) δ 7.59 (s, 1H), 7.49 (s, 1H), 7.25 (s, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.54 - 5.36 (m, 2H), 5.19 (s, 2H), 4.68 (t, J = 5.0 Hz, 1H), 3.51 (q, J = 5.6 Hz, 2H), 3.14 (t, J = 7.6 Hz, 2H), 1.99 - 1.73 (m, 4H), 0.92 (t, J = 7.3 Hz, 3H).

[0312] Example 9 (9H-Fluoren-9-yl)methyl (S)-(2-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 5a)

[0313] [ka]

[0314] Method 1: DMF (1.75 L) was added to a reaction bottle at room temperature. Compound 3 (175.66 g, purity: 80.2%, 313 mmol) and compound 4a (345.93 g, 939 mmol) were added in sequence. The atmosphere was replaced with argon gas three times, and HCl / EA (2N, 94 ml, 187.8 mmol) was added dropwise at room temperature. The reaction was monitored by HPLC.

[0315] After the reaction was completed, the reaction solution was added dropwise to water (17.5 L) at room temperature. After the addition was completed, the mixture was stirred for 1 hour. The mixture was filtered under suction. The cake was added to water (8.75 L) and stirred for 1 hour. The cake was filtered under suction, and added to DCM (3.5 L) and stirred to dissolve. The resulting DCM system was washed with water (3.5 L x 2). The organic phase was separated and dried by adding anhydrous sodium sulfate (875.00 g). The mixture was filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (eluent: DCM / THF / EA / Acetone) to obtain compound 5a (198.44 g, yield: 70%, purity: 84%).

[0316] Method 2: DMF (20 mL) was added to a reaction bottle at 30° C. Compound 3 (2.0 g, purity: 80.2%, 3.56 mmol) and compound 4a (3.94 g, 10.7 mmol) were added in sequence. The mixture was purged with argon gas three times, and BF3.Et2O (1.01 g, 7.12 mmol) was added at room temperature.

[0317] After the reaction was completed, the reaction solution was added dropwise to water (200 mL) at room temperature. After the addition was completed, the mixture was stirred for 1 hour. The mixture was filtered under suction. The cake was added to water (100 mL) and stirred for 1 hour. The cake was filtered under suction, and added to DCM (40 mL) and stirred to dissolve. The resulting DCM system was washed with water (40 mL x 2). The organic phase was separated and dried by adding anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (eluent: DCM / THF / EA / Acetone) to obtain compound 5a (2.72 g, yield: 67.8%, purity: 84%).

[0318] Compound 5a: LCMS (ESI) [M+H] + = 759.5.

[0319] Compound 5a: 1 H NMR (400 MHz, DMSO-d6) δ 8.65 (t, J = 6.4 Hz, 1H), 7.84 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 7.4 Hz, 2H), 7.56 - 7.50 (m, 2H), 7.45 (s, 1H), 7.38 (t, J = 7.3 Hz, 2H), 7.29 (t, J = 7.3 Hz, 2H), 7.22 (s, 1H), 6.45 (s, 1H), 6.25 (s, 2H), 5.47 - 5.34 (m, 2H), 5.20 - 5.03 (m, 2H), 4.60 (d, J = 6.5 Hz, 2H), 4.27 (d, J = 6.8 Hz, 2H), 4.22 - 4.16 (m, 1H), 3.66 (d, J = 6.0 Hz, 2H), 3.50 (t, J = 6.0 Hz, 2H), 3.13 - 2.95 (m, 2H), 1.94 - 1.77 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0320] Example 10 Preparation of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (Compound 6a)

[0321] [ka]

[0322] Method 1: At room temperature, compound 5a (33.30 g, purity: 84%, 36.9 mmol) and DMF (333 ml) were added to a reaction bottle and stirred to dissolve, and the above solution was added dropwise to a solution of diethylamine (4.86 g, 66.42 mmol) in DMF (333 ml). After stirring at room temperature for 1 h, the above reaction solution was added dropwise to a mixed solvent of methyl tert-butyl ether (7 L) and n-heptane (7 L) previously cooled in an ice bath, and after the addition was completed, the mixture was stirred for 2 hours. The mixture was filtered by suction, and the filtered solid was dried under reduced pressure to obtain compound 6a (22.53 g, yield: 88%, purity: 77.7%).

[0323] Method 2: At room temperature, compound 5a (2.0 g, purity: 84%, 2.2 mmol) and DMF (20 ml) were added to a reaction bottle and stirred to dissolve, and the above solution was added dropwise to a solution of pyridine (0.34 g, 4.0 mmol) in DMF (20 ml). After stirring at room temperature for 1 h, the above reaction solution was added dropwise to a mixed solvent of methyl tert-butyl ether (40 mL) and n-heptane (40 mL) previously cooled in an ice bath, and after the addition was completed, the mixture was stirred for 2 hours. The mixture was filtered by suction, and the filtered solid was dried under reduced pressure to obtain compound 6a (1.3 g, yield: 82.6%, purity: 75.0%).

[0324] Compound 6a:LCMS (ESI) [M+H] + = 537.4.

[0325] The NMR data of compound 6a formate is as follows:

[0326] 1 H NMR (400 MHz, DMSO-d6) δ 8.78 (t, J= 6.6 Hz, 1H), 8.23 ​​(s, 1H), 7.60 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 6.49 (s, 1H), 6.29 (s, 2H), 5.43 (s, 2H), 5.22 (s, 2H), 4.62 (d, J = 6.5 Hz, 2H), 3.53 - 3.46 (m, 2H), 3.29 (s, 2H), 3.15 - 3.06 (m, 2H), 1.98 - 1.75 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0327] Example 11 N 2 -((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of -(tert-butoxycarbonyl)-L-lysine (Compound 7-A3-a)

[0328] [ka]

[0329] Method 1: Compound 7-A1-a (100 g, 0.229 mol) and compound 7-A2-a (56.4 g, 0.229 mol) were placed in a reaction bottle, and acetone / water = 1:1 (v:v, 1 L) was added under stirring conditions, and NaHCO3 (76.95 g, 0.916 mol) was further added. Stirred at room temperature overnight. The reaction was monitored by LCMS. The solids in the reaction solution were filtered, the filtrate was concentrated, and the concentrated solution was added with HCl (4N) to adjust the pH to about 5, and extracted with ethyl acetate (0.75 L x 2). The combined organic phase was washed with saturated brine (300 mL x 2), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to obtain the target compound 7-A3-a (117 g, yield: 90%, purity: 97%).

[0330] Method 2: Compound 7-A1-a (100 g, 0.229 mol) and compound 7-A2-a (56.4 g, 0.229 mol) were placed in a reaction bottle, and tetrahydrofuran / water = 1:1 (v:v, 1 L) was added under stirring conditions, and Na2CO3 (97.1 g, 0.916 mol) was further added. Stirred at room temperature overnight. The reaction was monitored by LCMS. The solids in the reaction solution were filtered, the filtrate was concentrated, and the concentrated solution was added with HCl (4N) to adjust the pH to about 5, and extracted with ethyl acetate (0.75 L x 2). The combined organic phase was washed with saturated brine (300 mL x 2), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to obtain the target compound 7-A3-a (118.3 g, yield: 91.0%, purity: 97%).

[0331] Compound 7-A3-a:LCMS (ESI) [M+H] + = 568.3.

[0332] Compound 7-A3-a: 1 H NMR (400 MHz, DMSO-d6) δ 7.91 (d, J = 7.5 Hz, 2H), 7.82 - 7.73 (m, 2H), 7.64 (d, J = 6.9 Hz, 1H), 7.58 (d, J = 9.1 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.40 - 7.31 (m, 2H), 6.68 (brs, 1H), 4.38 - 4.21 (m, 3H), 3.99 - 3.91 (m, 1H), 3.87 (dd, J = 8.8, 6.8 Hz, 1H), 2.92 - 2.80 (m, 2H), 2.17 - 2.01 (m, 1H), 1.70-1.67 (m, 1H), 1.64 - 1.52 (m, 1H), 1.42 - 1.16 (m, 13H), 0.93 - 0.83 (m, 6H).

[0333] Example 12 Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-lysine hydrochloride (hydrochloride of compound 7-A4-a)

[0334] [ka]

[0335] Compound 7-A3-a (117 g, 206.2 mmol) was added to dioxane (1.5 L) and stirred for 10 min, then HCl (0.75 L, 4N in dioxane) was added and reacted at room temperature overnight. The reaction was monitored by LCMS. Methyl tert-butyl ether (2 L) was added to the reaction solution and stirred for 30 min, then filtered, and the solid was transferred to an air drying oven to dry, yielding the hydrochloride salt of target compound 7-A4-a (94 g, yield: 91%, purity 94%).

[0336] LCMS (ESI) [M+H] + = 468.3.

[0337] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 7.3 Hz, 1H), 8.07 (brs, 3H), 7.90 (d, J = 7.5 Hz, 2H), 7.77 (dd, J = 7.1, 3.4 Hz, 2H), 7.47 - 7.37 (m, 3H), 7.34 (td, J = 7.4, 1.7 Hz, 2H), 4.34 - 4.17 (m, 4H), 3.96 (dd, J = 8.6, 7.4 Hz, 1H), 2.83 - 2.65 (m, 2H), 2.04 (dd, J = 13.5, 6.7Hz, 1H), 1.82- 1.52 (m, 4H), 1.51- 1.30 (m, 2H), 0.96- 0.84 (m, 6H).

[0338] Example 13 N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 ,N 6 Preparation of -dipropyl-L-lysine (compound 7a)

[0339] [ka]

[0340] Method 1: Compound 7-A4-a hydrochloride (55.0g, 109.1mmol) and acetic acid (10ml) were dissolved in methanol (500mL) solution, and n-propionaldehyde (25.35g, 436.4mmol) was added to the reaction solution under ice water bath, and the reaction solution was stirred at room temperature for 30 minutes, and sodium cyanoborohydride (27.43g, 436.4mmol) was added to the reaction solution under ice water bath, and the reaction solution was stirred at room temperature for 1h. LCMS showed that the raw material was not completely reacted. n-propionaldehyde (12.67g, 218.2mmol) and sodium cyanoborohydride (13.72g, 218.2mmol) were added, and the reaction was continued for 0.5h. The reaction solution was filtered, the filtrate was concentrated, and the concentrate was added to pure water (300 ml) and extracted with DCM (300 ml x 2). The organic phase was washed with saturated saline, dried over anhydrous Na2SO4, and then concentrated to obtain a crude product. The crude product was purified by column chromatography (eluent: MeOH and DCM) to obtain compound 7a (39 g, yield: 65%, purity: 98%).

[0341] Method 2: Compound 7-A4-a hydrochloride (55.0 g, 109.1 mmol) and acetic acid (10 ml) were dissolved in methanol (500 mL) solution, and sodium cyanoborohydride (27.43 g, 436.4 mmol) was added to the reaction solution in an ice-water bath, and the reaction solution was stirred at room temperature for 30 minutes to react. Furthermore, n-propionaldehyde (25.35 g, 436.4 mmol) was added to the reaction solution in an ice-water bath, and the reaction system was stirred at room temperature for 1 h to react. The reaction solution was filtered, the filtrate was concentrated, and the concentrate was added to water (300 ml) and extracted with DCM (300 ml x 2). The organic phase was washed with saturated saline, dried over anhydrous Na2SO4, and then concentrated to obtain a crude product. The crude product was dissolved in 110 mL of DCM, added dropwise to 1.1 L of methyl tert-butyl ether, stirred for 30 min, and filtered by suction to give compound 7a (48 g, yield: 80%, purity: 93%).

[0342] Compound 7a:LCMS (ESI) [M+H] + = 552.3.

[0343] Compound 7a: 1 H NMR (400 MHz, DMSO-d6) δ 7.95 - 7.85 (m, 3H), 7.76 (t, J = 7.3 Hz, 2H), 7.51 - 7.40 (m, 3H), 7.35 (t, J = 7.4 Hz, 2H), 4.34 - 4.22 (m, 3H), 4.13 (dd, J = 12.6, 7.2 Hz, 1H), 3.91 (dd, J = 8.8, 7.1 Hz, 1H), 2.52 - 2.41 (m, 6H), 2.03 (dt, J = 13.5, 6.7 Hz, 1H), 1.79 - 1.67 (m, 1H), 1.66 - 1.56 (m, 1H), 1.42-1.36 (m, 6H), 1.36 - 1.21 (m, 2H), 0.90 (t, J = 6.9 Hz, 6H), 0.82 (t, J = 6.9 Hz, 6H).

[0344] Example 14 Preparation of (9H-fluoren-9-yl)methyl ((11S,14S)-11-(4-(dipropylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)carbamate (compound 8a)

[0345] [ka]

[0346] Method 1: Compound 6a (19.88 g, purity: 77.7%, 28.79 mmol), compound 7a (19.06 g, 34.55 mmol) and DMF (500 ml) were added to a reaction bottle at room temperature. The mixture was stirred for 15 minutes at room temperature, protected with argon gas, cooled to 0°C, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride monohydrate (10.17 g, 34.55 mmol) was added. The mixture was protected with argon gas, cooled to 0°C, and after the addition was complete, the reaction was continued at 0°C, and the reaction was monitored by LCMS.

[0347] After the reaction was completed, the reaction solution was dropped into methyl tert-butyl ether (10 L), stirred for 1.5 hours, filtered, and the filtered solid was dissolved in 600 ml of a DCM / MeOH mixed solvent (DCM:MeOH=10:1), and the resulting solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: DCM:MeOH) to obtain compound 8a (24.00 g, yield: 74%, purity: 95.1%).

[0348] Method 2: Compound 6a (20 g, purity: 77.7%, 2.9 mmol), compound 7a (2.08 g, 3.77 mmol) and DMF (50 ml) were added to a reaction bottle at room temperature. The mixture was stirred at room temperature for 15 minutes, cooled to -5°C, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride monohydrate (1.11 g, 3.77 mmol) was added. The mixture was protected with argon gas and cooled to -5°C. After the addition was complete, the mixture was reacted at -5°C and monitored by LCMS.

[0349] After the reaction was completed, the reaction solution was dropped into methyl tert-butyl ether (1 L), stirred for 1.5 hours, filtered, and the filtered solid was dissolved in 60 ml of a DCM / MeOH mixed solvent (DCM:MeOH=10:1), and the resulting solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: DCM:MeOH) to obtain compound 8a (2.35 g, yield: 71.9%, purity: 95.0%).

[0350] Compound 8a:LCMS (ESI) [M+H] + = 1070.7.

[0351] Compound 8a: 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (t, J = 6.6 Hz, 1H), 8.28 (t, J = 5.9 Hz, 1H), 8.09 (d, J = 7.4 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.77 - 7.70 (m, 2H), 7.57 (s, 1H), 7.53 - 7.37 (m, 4H), 7.36 - 7.28 (m, 2H), 7.26 (s, 1H), 6.50 (s, 1H), 6.30 (s, 2H), 5.77 (s, 1H), 5.44 (d, J = 2.5 Hz, 2H), 5.20 (s, 2H), 4.67 - 4.53 (m, 2H), 4.36 - 4.17 (m, 4H), 3.93 - 3.85 (m, 1H), 3.84 - 3.70 (m, 2H), 3.52 (t, J = 6.1 Hz, 2H), 3.16 - 3.04 (m, 2H), 3.02 - 2.80 (m, 5H), 2.01 - 1.50 (m, 13H), 1.38 - 1.28 (m, 2H), 0.95 - 0.80 (m, 15H).

[0352] Example 15 Preparation of (S)-2-((S)-2-amino-3-methylbutylamino)-6-(dipropylamino)-N-(2-(((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)amino)-2-oxoethyl)hexanoamide (Compound 9a)

[0353] [ka]

[0354] Method 1: Under ice bath conditions, compound 8a (29.50 g, 27.56 mmol) in DMF (147.5 ml) was added to a reaction bottle under argon gas protection, stirring was started, and diethylamine (7.4 ml) was added dropwise. After the addition was completed, the ice bath was removed, and the reaction was continued and monitored by LCMS. Tetrahydrofuran (295 ml) was added to the reaction solution, and the solution was concentrated under reduced pressure. This operation was repeated three times. A DMF solution of compound 9a was obtained and used directly in the next step reaction (calculated as 100% conversion).

[0355] Method 2: Compound 8a (2.0 g, 1.87 mmol) and THF (20 mL) were added to the reaction solution, stirring was started, and N,N-diisopropylethylamine (2 mL) was added dropwise. After the addition was completed, the mixture was allowed to react at room temperature for 6 h. A THF solution of compound 9a was obtained and used directly in the next reaction (the conversion rate was calculated as 100%).

[0356] Compound 9a:LCMS (ESI) [M+H] + = 848.6.

[0357] Compound 9a: 1H NMR (400 MHz, DMSO-d6, trifluoroacetate of compound 9a) δ 9.22 (s, 1H), 8.68 (t, J = 6.5 Hz, 1H), 8.54 (d, J = 7.6 Hz, 1H), 8.36 (t, J = 5.8 Hz, 1H), 8.08 (d, J = 3.6 Hz, 3H), 7.59 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 6.50 (brs, 1H),6.29 (d, J = 1.1 Hz, 2H), 5.42 (s, 2H), 5.23 (s, 2H), 4.67 - 4.53 (m, 2H), 4.35 (q, J = 7.3 Hz, 1H), 3.83 - 3.69 (m, 2H), 3.66 - 3.61 (m, 1H), 3.15 - 3.06 (m, 2H), 3.04 - 2.94 (m, 6H), 2.10 - 1.95 (m, 1H), 1.93- 1.78 (m, 4H), 1.76 - 1.54 (m, 8H), 1.42 - 1.26 (m, 2H), 0.95 - 0.85 (m, 15H).

[0358] Example 16 Preparation of 6-(2-(methanesulfonyl)-5-pyrimidinyl)-5-hexynyl chloride (compound 10a)

[0359] [ka]

[0360] Under room temperature conditions and under argon gas protection, compound 6-[2-(methanesulfonyl)-5-pyrimidinyl]-5-hexynoic acid (7.76 g, 28.95 mmol) and DCM (77.6 ml) were added to a reaction bottle, stirring was started, and thionyl chloride (6.88 g, 57.83 mmol) was added dropwise. After the addition was completed, the reaction was continued for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure. DCM (77.6 ml) was added to the concentrated solution, and the solution was concentrated under reduced pressure. This operation was repeated twice, and then DCM (100 ml) was added to the concentrated solution to obtain a DCM solution of acyl chloride intermediate 10a, which was directly used in the next reaction (calculated with a conversion rate of 100%).

[0361] Compound 10a was quenched with methanol and the resulting methyl ester was + = 283.0.

[0362] Example 17 Preparation of N-((11S,14S)-11-(4-(dipropylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexa-5-ynamide (Compound Ia)

[0363] Method 1:

[0364] [ka]

[0365] Under room temperature conditions, under argon gas protection, a DMF solution of compound 9a (prepared in Example 15, Method 1, 23.36 g, 27.56 mmol) and DIPEA (5.34 g, 41.38 mmol) were added to a reaction bottle, and stirring was started. Under ice bath conditions, a DCM solution of compound 10a (prepared in Example 16, 8.30 g, 28.95 mmol) was added dropwise. After the dropwise addition was completed, the reaction was continued under ice bath conditions and monitored by LCMS. The reaction solution was diluted with DCM (1.4 L). The organic phase was washed successively with 2% aqueous sodium bicarbonate (1.4 L x 2) and saturated saline (1.4 L x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by preparative HPLC to obtain the formate salt of compound Ia (18 g, calculated yield based on compound 8a: 57%), with HPLC purity (method 1): 94.4% and chiral purity: 98.68%.

[0366] Method 2:

[0367] [ka]

[0368] 6-[2-(methanesulfonyl)-5-pyrimidinyl]-5-hexynoic acid (0.5 g, 1.87 mmol) was dissolved in DMF (5 mL) and added to a THF solution of 9a (prepared by Method 2 in Example 15, 1.58 g, 1.87 mmol), and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride monohydrate (0.55 g, 1.87 mmol) was added under ice bath conditions. The ice bath was removed and the reaction was carried out at room temperature for 3 hours. The reaction solution was separated and purified by preparative HPLC to obtain the formate salt of compound Ia (1.23 g, calculated yield for compound 8a: 60%).

[0369] Formate salt of compound Ia: LCMS (ESI) [M+H] + = 1098.5.

[0370] Formate salt of compound Ia: 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 2H), 8.56 (t, J = 6.5 Hz, 1H), 8.17 (s, 1H), 8.14 (t, J = 5.7 Hz, 1H), 7.97 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.58 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.45 (s, 1H), 6.28 (s, 2H), 5.48 - 5.35 (m, 2H), 5.24 (s, 2H), 4.64 - 4.54 (m, 2H), 4.23 - 4.07 (m, 2H), 3.79 - 3.66 (m, 2H), 3.50 (t, J = 5.9 Hz, 2H), 3.40 (s, 3H), 3.14 - 3.08 (t, J = 8.22 Hz, 2H), 2.59 - 2.52 (m, 2H), 2.36 - 2.27 (m, 8H), 2.00 - 1.76 (m, 7H), 1.72- 1.62 (m, 1H), 1.58- 1.48 (m, 1H), 1.40 - 1.20 (m, 8H), 0.88 - 0.77 (m, 15H).

[0371] Conditions for fractionation of HPLC クロマトグラフィー カラム: C18-divided カラム (100mm×250mm, 10μm), wavelength: 214nm, flow rate: 250ml / min. Mobile phase A: 0.1% aqueous acid solution. Moving phase B: Ayator.

[0372]

Table 1-4

[0373]

Table 1-5

[0374]

Table 1-6

[0375] The following drug-linker conjugates were synthesized by the above method:

[0376] [ka]

[0377] Example 18 Preparation of 2E3-02 antibody The heavy chain amino acid sequence (SEQ ID NO: 1) of 2E3-02 was optimized through codon optimization and gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), then constructed into PTT5 vector, and named PPT5-02-CH. The light chain amino acid sequence (SEQ ID NO: 2) of 2E3-02 was optimized through codon optimization and gene synthesis (Sangon Biotech (Shanghai) Co., Ltd.), then constructed into PTT5 vector, and named PPT5-02-CL. The anti-B7H3 antibody expression plasmids PTT5-02-CH / PTT5-02-CL were co-transfected into HEK293F cells (ATCC) using PEI max reagent and expressed for 7 days in a shaker at 37°C and 5% CO2, and the supernatant was collected and purified with ProA magnetic beads to obtain the anti-B7H3 antibody 2E3-02 (heavy chain sequence is SEQ ID NO: 1, light chain sequence is SEQ ID NO: 2).

[0378] Example 19 Preparation of ADC 25 ml of 2E3-02 antibody (anti-B7H3 antibody, concentration: 28.5 mg / ml, 20 mM acetate buffer) was taken, and 25 ml of 20 mM acetate buffer was added to dilute it. Next, 1 ml of an aqueous solution containing 0.25 M EDTA was added and mixed well. The pH of the sample was adjusted to 7.6 with a 0.5 M aqueous solution of disodium hydrogen phosphate, and then a 20 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) solution was added in an amount 4.5 times the antibody equivalent, mixed well, and reacted at room temperature for 90 minutes. Finally, Ia dissolved in DMSO in an amount 10 times the antibody equivalent was added, mixed well, and then reacted at room temperature for 2 hours. After the reaction was completed, the sample was replaced with a 10 mM histidine buffer solution at pH 5.5 using a 30 KDa ultrafiltration tube to remove low molecular weight substances, and finally the sample was concentrated to obtain a solution containing an anti-B7H3 antibody ADC composition (ADC-01). The DAR value measured by mass spectrometry was 7.98.

[0379] [ka]

[0380] Here, Ab is the B7H3 antibody 2E3-02.

[0381] The method for measuring the DAR value is as follows: Chromatography conditions: Chromatography column: PLRP-S, 2.1*50mm, 5μm; Mobile phase A: 0.1% FA / H2O, Mobile phase B: 0.1% FA / ACN Column temperature: 30°C, sample chamber temperature: 8°C, flow rate: 0.6 ml / min, injection volume: 2 μl

[0382] [Table 1-7]

[0383] Sample treatment: 50 μg of ADC-01 sample was taken, 2 μl of 1 M DTT was added, and ultrapure water was added up to 50 μl to dilute to a concentration of approximately 1.0 mg / ml, mixed well, and reduced at room temperature for 30 minutes. LC / MS model: Agilent 1290-6545XT Q-TOF. Mass spectrometry conditions: Gas temp: 320℃, Drying Gas: Nebulizer: 35 psi; Sheath Gas Temp: 350℃; Sheath Gas Flow: 11 l / min; m / z 500~3000.

[0384] The results are as follows:

[0385] [Table 1-8]

[0386] In the table, mAb represents unbound antibody, LC represents antibody light chain, HC represents antibody heavy chain, DAR1 represents a complex containing a light chain or heavy chain bound to one toxin molecule, DAR2 represents a complex containing a light chain or heavy chain bound to two toxin molecules, and DAR3 represents a complex containing a light chain or heavy chain bound to three toxin molecules, where the theoretical molecular weight of the monoclonal antibody was calculated based on the G0F glycoform. In the following, mAb, LC, HC, DAR1, DAR2, and DAR3 are as above.

[0387] The light chain of the 2E3-02 antibody bound to 0-1 toxin molecule (LC, DAR1 ratios were 1.0% and 99.0%, respectively) and the heavy chain bound to 0-3 toxin molecules (mAb, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100%, respectively) were measured, and the antibody-drug binding ratio (DAR value) of ADC-01 was calculated to be 7.98. Based on the distribution of 0-1 toxin molecule bound to the light chain and 0-3 toxin molecule bound to the heavy chain, it was inferred that n may be 1, 2, 3, 4, 5, 6, 7, or 8.

[0388] Using the same antibodies, methods and reaction conditions as above, different drug-linker conjugates were used to obtain the target products, and the DAR values ​​were measured by mass spectrometry.

[0389] [Table 1-9]

[0390] Test Example 1: ADC efficacy test against NCI-HT29 transplanted tumors 1. Experimental Materials Test compounds: ADC-01, ADC-02 Experimental cells: NCI-HT29 cells, purchased from ATCC; Experimental animals: Balb / c nu nude mice, female, 5-6 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0391] 2. Experimental Protocol 2.1. Cell treatment NCI-HT29 cells were cultured in 1640 medium containing 10% FBS in 15 cm culture dishes, and when the cells reached approximately 80-90% confluence, they were digested with trypsin-EDTA, washed twice with PBS, centrifuged, resuspended in pre-chilled PBS, counted with a cell counter, and diluted with PBS to a cell concentration of 5 × 10 7 / ml.

[0392] 2.2. Tumor cell transplantation

[0393] Balb / c nu mice were adapted to the laboratory environment for 2 to 5 days and then subcutaneously inoculated with NCI-HT29 cells in the right flank. The cell volume was 5 × 10 6 The inoculation volume was 0.2 ml (containing 50% Matrigel), and the tumor size was approximately 250 mm 3 The experiments were carried out at the time when the plants had grown to .

[0394] 2.3. Animal administration and detection

[0395] Enrolled tumor-bearing nude mice were administered according to the following regimen:

[0396] [Table 1-10]

[0397] After the end of the administration cycle, the mice were observed for 1 to 2 weeks. After the final tumor volume was measured, the tumors were isolated, accurately weighed, and photographed to record the tumor weight.

[0398] 2.4. Measurement of tumor volume and body weight: Tumor volumes and body weights were measured twice weekly, and TGI% was calculated. Tumor volume (V) calculation formula: V = 1 / 2 × L long × L short 2 .

[0399] 3. Experimental Results

[0400] [Table 1-11]

[0401] Conclusion: As shown in Table 1, all the ADCs of the present application showed inhibitory effects on tumor growth, and ADC-01 and ADC-02 in particular had strong tumor inhibitory effects. During the administration period, the animals in each group did not show significant weight loss or significant drug toxicity.

[0402] The invention is not limited to the particular embodiments described above, but extends to any novel feature or any novel combination disclosed herein, and to any novel method or process step or any novel combination disclosed.

Claims

1. c) reacting a compound of formula 6 or a salt thereof with a compound of formula 7 or a salt thereof to obtain a compound of formula 8 or a salt thereof; 【Chemistry 1】 d) reacting a compound of formula 8 or a salt thereof to obtain a compound of formula 9 or a salt thereof; 【Chemistry 2】 e) reacting a compound of formula 9 or a salt thereof with a compound of formula 10 to obtain a compound of formula I or a salt thereof; 【Chemistry 3】 Including, Where: R １ , R ２ are each independently C １－４ alkyl, R ３ is selected from hydroxyl and halogen; P.G. １ is selected from an amino protecting group, or a salt thereof.

2. (1) The R １ , R ２ are each independently selected from methyl, ethyl and propyl, preferably R １ , R ２ are the same, and preferably, １ , R ２ are all propyl; (2) The above R ３ is selected from hydroxy, fluorine, chlorine, bromine and iodine, preferably ３ is selected from hydroxy and chlorine; (3) The PG １ is selected from alkoxycarbonyl-based protecting groups, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc); (4) In step c), a compound of formula 6 or a salt thereof and a compound of formula 7 or a salt thereof are reacted with a condensing agent to obtain a compound of formula 8 or a salt thereof; Further, the condensing agent is DMTMM, HATU, HBTU, HOBt / EDCI and T ３ P, preferably DMTMM or HOBt / EDCI; Furthermore, the molar ratio of the compound of formula 6 or its salt to the condensing agent is selected from 1:0.7 to 1:5, preferably 1:0.7 to 1:2; (5) in step c), the molar ratio of the compound of formula 6 or a salt thereof to the compound of formula 7 or a salt thereof is selected from 1:0.7 to 1:3, preferably 1:0.7 to 1:2; (6) in step c), the reaction solvent is selected from one of DMF, DMAc, NMP, tetrahydrofuran, 1,4-dioxane, acetonitrile and dichloromethane or any combination thereof, preferably selected from one of DMF, dichloromethane and tetrahydrofuran or any combination thereof; (7) In step c), the mass-to-volume ratio (g / mL) of the compound of formula 6 or a salt thereof to the reaction solvent is selected from 1:5 to 1:50; (8) in step c), the reaction temperature is selected from −20 to 50° C., preferably −5 to 25° C.; (9) In step d), a protecting group PG on the amino group is obtained from the compound of formula 8 or a salt thereof. １ to obtain a compound of formula 9 or a salt thereof; Preferably, the PG １ When is 9-fluorenylmethyloxycarbonyl (Fmoc), the compound of formula 8 or a salt thereof can be reacted with the protecting group PG under basic conditions. １ Remove; Further, the base is selected from piperidine, diethylamine, morpholine, diisopropylamine, DBU, triethylamine and N,N-diisopropylethylamine, for example diethylamine or N,N-diisopropylethylamine, preferably piperidine, diethylamine or morpholine; Further, the volume ratio of the base to the reaction solvent is selected from 1:5 to 1:50; Further, the reaction solvent is selected from one or any combination of DMF, DMAc, dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, methanol, and ethanol; Further, the mass to volume ratio (g / mL) of the compound of formula 8 or salt thereof to the reaction solvent is selected from 1:5 to 1:50; Furthermore, the reaction temperature is selected from −10 to 50° C., preferably 0 to 30° C.; (10) In step e), R ３ is hydroxy, a compound of formula 9 or a salt thereof is reacted with a compound of formula 10 under the action of a condensing agent to obtain a compound of formula I; Further, the condensing agent is DMTMM, HATU, HBTU, HOBt / EDCI and T ３ P, preferably DMTMM or HOBt / EDCI; Furthermore, the molar ratio of the compound of formula 9 or its salt to the condensing agent is selected from 1:1 to 1:5, preferably 1:1 to 1:2; Further, the molar ratio of the compound of formula 9 or a salt thereof to the compound of formula 10 is selected from 1:0.8 to 1:2.0; Further, the reaction solvent is selected from one of DMF, tetrahydrofuran, and dichloromethane, or any combination thereof; Further, the mass to volume ratio (g / mL) of the compound of formula 9 or salt thereof to the reaction solvent is selected from 1:5 to 1:50; Furthermore, the reaction temperature is selected from −20 to 50° C., preferably 0 to 30° C.; (11) In step e), R ３ is selected from halogen (e.g., chlorine, fluorine or bromine), reacting a compound of formula 9 or a salt thereof with a compound of formula 10 under basic conditions to obtain a compound of formula I or a salt thereof; Further, the base is selected from triethylamine, N,N-diisopropylethylamine, DBU, N-methylpiperidine, and N-methylmorpholine; Further, the molar ratio of reacting the compound of formula 9 or a salt thereof with the base is selected from 1:1 to 1:5; Furthermore, the molar ratio of the compound of formula 9 or its salt to the compound of formula 10 is selected from 1:0.8 to 1:2.0, preferably 1:1 to 1:1.5; Further, the reaction solvent is selected from one or any combination of DMF, tetrahydrofuran, and dichloromethane; Further, the mass to volume ratio (g / mL) of the compound of formula 9 or salt thereof to the reaction solvent is selected from 1:5 to 1:50; Furthermore, the reaction temperature is selected from −20 to 50° C., preferably 0 to 30° C.; 2. A process for the preparation of a compound of formula I or a salt thereof according to claim 1, characterized in that one or more of the following conditions are met:

3. a) reacting a compound of formula 3 or a salt thereof with a compound of formula 4 to obtain a compound of formula 5 or a salt thereof; 【Chemistry 4】 b) reacting a compound of formula 5 or a salt thereof to obtain a compound of formula 6 or a salt thereof; 【Chemistry 5】 Here, P.G. ２ is selected from amine protecting groups; 2. A process for preparing a compound of formula I or a salt thereof according to claim 1, further comprising the step of:

4. (1) The PG ２ is selected from alkoxycarbonyl-based protecting groups, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc); (2) In step a), the reaction is carried out under acidic conditions, and the acid is selected from the group consisting of hydrogen chloride, hydrobromic acid, sulfuric acid, formic acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, zinc acetate, aluminum trichloride (AlCl ３ ), ferric chloride (FeCl ３ ), boron trifluoride diethyl etherate (BF ３ Et ２ O), for example hydrogen chloride or boron trifluoride diethyl etherate, preferably hydrogen chloride, p-toluenesulfonic acid or zinc acetate, and further, in step a), the molar ratio of reacting the compound of formula 3 or a salt thereof with said acid is selected from 1:0.2 to 1:2; Alternatively, in step a), the reaction is carried out under basic conditions, and further, the base is selected from the group consisting of sodium hydroxide, potassium tert-butoxide, potassium carbonate, triethylamine (Et ３ N), N,N-diisopropylethylamine (DIPEA) and pyridine, preferably potassium tert-butoxide or sodium hydroxide, and further, in step a), the molar ratio of reacting the compound of formula 3 or a salt thereof with said base is selected from 1:0.5 to 1:3; (3) in step a), the molar ratio of the compound of formula 3 or a salt thereof to the compound of formula 4 is selected from 1:1 to 1:10, preferably 1:2 to 1:6; (4) In step a), the reaction solvent is selected from aprotic solvents, preferably ethers, halogenated alkanes, ketones, nitriles, amides, or sulfones, more preferably ethyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, dichloromethane, 1,2-dichloroethane, acetone, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethylsulfoxide, and even more preferably isopropyl ether, tetrahydrofuran, 1,4-dioxane, or N,N-dimethylformamide; Furthermore, in step a), the mass to volume ratio (g / mL) of the compound of formula 3 or its salt to the solvent is selected from 1:5 to 1:50, preferably 1:8 to 1:30; (5) In step a), the reaction temperature is selected from −10 to 110° C., preferably 0 to 60° C.; (6) In step b), a protecting group PG on the amino group is reacted with the compound of formula 5 or a salt thereof. ２ to obtain a compound of formula 6 or a salt thereof, Preferably, the PG ２ When is 9-fluorenylmethyloxycarbonyl (Fmoc), the compound of formula 5 or a salt thereof can be reacted with the protecting group PG under basic conditions. ２ Remove; Further, the base is selected from piperidine, diethylamine, morpholine, diisopropylamine, DBU, triethylamine and N,N-diisopropylethylamine, preferably piperidine, diethylamine or morpholine; Furthermore, the molar ratio of the compound of formula 5 or its salt to the base is selected from 1:0.2 to 1:40, preferably 1:1 to 1:10; (7) In step b), the PG ２ is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction solvent is selected from one or any combination of DMF, DMAc, dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, methanol and ethanol, preferably selected from one or any combination of DMF and dichloromethane; (8) In step b), the PG ２ is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass to volume ratio (g / mL) of the compound of formula 5 or a salt thereof to the reaction solvent is selected from 1:5 to 1:50, preferably 1:10 to 1:30; (9) In step b), the PG ２ is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction temperature is selected from 0 to 50° C., preferably 10 to 30° C.; A process for the preparation of a compound of formula I or a salt thereof according to claim 3, characterized in that one or more of the following conditions are met:

5. h) reacting a compound of formula 1 or a salt thereof with a compound of formula 2 to obtain a compound of formula 3 or a salt thereof; 【Chemistry 6】 Where: R ４ , R ５ are each independently -OC １－４ Alkyl, -SC １－４ Alkyl and -NHC １－４ alkyl; Or, R ４ and R ５ form a carbonyl together with the carbon atom attached thereto; Or, R ４ and R ５ together with the carbon atoms connected to them 【Chemistry 7】 wherein X and Y each independently represent O, S, NH, or NC １－４ alkyl, where n is selected from 1, 2 and 3; 4. A process for preparing a compound of formula I or a salt thereof according to claim 3, further comprising the step of:

6. (1) The R ４ , R ５ are each independently selected from methoxy, ethoxy, propoxy, methylthio, ethylthio, propylthio, methylamino, ethylamino, and propylamino; or R ４ and R ５ together with the carbon atom attached thereto form a carbonyl, or R ４ and R ５ together with the carbon atoms connected to them 【Chemistry 8】 wherein X and Y are each independently O, S, NH, or NC １－４ alkyl, where n is selected from 1, 2 and 3; Furthermore, the R ４ and R ５ together with the carbon atom attached thereto form a carbonyl, or R ４ and R ５ together with the carbon atoms connected to them 【Chemistry 9】 Forming (2) in step h), the reaction is carried out under acidic conditions; Further, the acid is selected from one or any combination of p-toluenesulfonic acid monohydrate, p-toluenesulfonic acid pyridinium salt, trifluoroacetic acid, and acetic acid, preferably p-toluenesulfonic acid monohydrate; Furthermore, the molar ratio of the compound of formula 2 to the acid is selected from 1:0.2 to 1:2, preferably 1:0.2 to 1:0.4; Further, the compound of formula 1 is added to the reaction system in batches, preferably, based on the total amount added, under acidic conditions, 25-35 mol% of the compound of formula 1 or its salt and the compound of formula 2 are reacted at 100-120°C, and the remaining compound of formula 1 is added to the reaction system in 2-5 portions, more preferably, based on the total amount added, under acidic conditions, 25-35 mol% of the compound of formula 1 or its salt and the compound of formula 2 are reacted at 100-120°C for 20-40 minutes, and the remaining compound of formula 1 is added to the reaction system in 2-5 portions, with an interval of 20-40 minutes each time, and continued to react for 30-90 minutes after the completion of the addition; (3) In step h), the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, toluene, o-methylphenol, N,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidone or any combination thereof, preferably one of toluene, o-methylphenol and N-methylpyrrolidone or any combination thereof, more preferably N-methylpyrrolidone; (4) in step h), the mass to volume ratio (g / mL) of the compound of formula 2 to the reaction solvent is selected from 1:1 to 1:60; (5) in step h), the molar ratio of the compound of formula 1 or a salt thereof to the compound of formula 2 is selected from 3:1 to 1:1, preferably 2:1 to 1:1; (6) In step h), the reaction temperature is selected from 70 to 130° C., preferably 80 to 120° C., more preferably 100 to 120° C.; 6. A process for the preparation of a compound of formula I or a salt thereof according to claim 5, characterized in that one or more of the following conditions are met:

7. Method 1 includes the following steps: Step i-1) reacting a compound of formula 1-A1 with a compound of formula 1-A2 to obtain a compound of formula 1-A3; Step i-2) reacting a compound of formula 1-A3 to obtain a compound of formula 1 or a salt thereof; 【Chemistry 10】 Method 2 includes the following steps: Step j-1) reacting a compound of formula 1-B1 with a compound of formula 1-B2 to obtain a compound of formula 1-B3; Step j-2) reacting a compound of formula 1-B3 to obtain a compound of formula 1-B4; Step j-3) reacting a compound of formula 1-B4 to obtain a compound of formula 1-B5 or a salt thereof; Step j-4) reacting a compound of formula 1-B5 or a salt thereof to obtain a compound of formula 1 or a salt thereof; 【Chemistry 11】 Wherein X is selected from halogen; A process for preparing a compound of formula I or a salt thereof according to claim 5, further comprising process 1 or process 2.

8. (1) said X is selected from fluorine, chlorine, bromine and iodine, preferably chlorine; (2) In step i-1), a compound of formula 1-A1 and a compound of formula 1-A2 are reacted with a palladium catalyst, a copper catalyst and a base to obtain a compound of formula 1-A3; Furthermore, the palladium catalyst is Pd(PPh ３ ) ４ , Pd(OAc) ２ , Pd(PPh ３ ) ２ C ２ and Pd ２ (dba) ３ and preferably Pd(PPh ３ ) ２ C ２ and further wherein the molar ratio of the palladium catalyst to the compound of formula 1-A1 is selected from 0.01:1 to 0.2:1, preferably 0.01:1 to 0.10:1, more preferably 0.01:1 to 0.03:1; Further, the copper catalyst is selected from CuI, CuBr and CuCl, preferably CuI, and the molar ratio of reacting the copper catalyst with the compound of formula 1-A1 is selected from 0.01:1 to 0.2:1, preferably 0.02:1 to 0.10:1, more preferably 0.02:1 to 0.05:1; Further, the base is selected from triethylamine, N,N-diisopropylethylamine, DBU, potassium carbonate and cesium carbonate, preferably triethylamine or N,N-diisopropylethylamine, and the molar ratio of reacting the base with the compound of formula 1-A1 is selected from 10:1 to 1:1, preferably 5:1 to 2:1; (3) in step i-1), the molar ratio of the compound of formula 1-A1 to the compound of formula 1-A2 is selected from 1:1 to 1:5, preferably 1:1 to 1:3, more preferably 1:1 to 1:2; (4) In step i-1), the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, toluene, N-methylpyrrolidone, N,N-dimethylformamide, and N,N-dimethylacetamide, or any combination thereof, for example, the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, toluene, N,N-dimethylformamide, and N,N-dimethylacetamide, or any combination thereof, also for example, tetrahydrofuran, N-methylpyrrolidone, or N,N-dimethylformamide, preferably tetrahydrofuran or 1,4-dioxane; (5) In step i-1), the mass-to-volume ratio (g / mL) of the compound of formula 1-A1 to the reaction solvent is selected from 1:5 to 1:20, preferably 1:10 to 1:20; (6) In step i-1), the reaction temperature is selected from 40 to 80° C., preferably 40 to 60° C.; (7) In step i-2), the compound of formula 1-A3 is reacted with Sn and Na ２ S to obtain a compound of formula 1 or a salt thereof, ２ S is Na ２ S.2H ２ O and / or Na ２ S.9H ２ is added to the reaction system in the form of O; Furthermore, the molar ratio of Sn to the compound of formula 1-A3 is selected from 5:1 to 1:1, preferably 3:1 to 1:1; Furthermore, the Na ２ the molar ratio of S to the compound of formula 1-A3 is selected from 0.1:1 to 2:1, preferably 0.2:1 to 1:1; (8) In step i-2), the reaction solvent is selected from one of tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, methanol, ethanol, propanol and water, or any combination thereof, preferably one of tetrahydrofuran, ethanol and methanol, or any combination thereof, more preferably a combination of ethanol and water, wherein the volume ratio of ethanol to water is selected from 20:1 to 1:1, preferably 10:1 to 5:1; (9) In step i-2), the reaction temperature is selected from 40 to 100° C., for example, 50 to 80° C., preferably 60 to 90° C.; (10) in step j-1), a compound of formula 1-B1 is reacted with a compound of formula 1-B2 to obtain a compound of formula 1-B3; (11) in step j-1), the molar ratio of the compound of formula 1-B1 to the compound of formula 1-B2 is selected from 1:0.8 to 1:2, preferably selected from 1:1 to 1:1.5; (12) In step j-1), the reaction solvent is selected from one of tetrahydrofuran, 2-methyltetrahydrofuran, isopropyl ether and diethyl ether or any combination thereof, preferably tetrahydrofuran or isopropyl ether; (13) In step j-1), the reaction temperature is selected from −20 to 60° C., preferably −20 to 50° C., more preferably 0 to 30° C.; (14) In step j-2), the compound of formula 1-B3 is subjected to a nitration reaction to obtain a compound of formula 1-B4; Further, the nitrification system of the reaction is selected from concentrated nitric acid, concentrated nitric acid / acetic acid, concentrated sulfuric acid / potassium nitrate; Furthermore, the mass to volume ratio (g / mL) of the compound of formula 1-B3 to the nitrification system is selected from 1:4 to 1:20, preferably 1:6 to 1:15; (15) In step j-2), the reaction temperature is selected from -20 to 30°C, preferably -20 to 0°C; (16) in step j-3), reducing the nitro group of the compound of formula 1-B4 to an amino group under the action of a reducing agent to obtain a compound of formula 1-B5 or a salt thereof; Furthermore, the reducing agent is hydrogen gas, TiCl ３ , SnCl ２ , sodium dithionite, zinc powder and iron powder, preferably hydrogen gas, SnCl ２ or sodium dithionite; Further, the reaction needs to be carried out under hydrogen gas atmosphere, and a transition metal catalyst is added, and the transition metal catalyst is preferably selected from platinum dioxide, palladium on carbon, rhodium on carbon and Raney nickel; further, when the reaction is carried out in hydrogen gas-platinum dioxide system, the mass ratio of the compound of formula 1-B4 to platinum dioxide is selected from 1:0.01 to 1:0.4, preferably 1:0.01 to 1:0.1; when the reaction is carried out in hydrogen gas-platinum dioxide system, the pressure of hydrogen gas is selected from 1 to 10 atm, preferably 1 to 5 atm; the reaction temperature is selected from -10 to 100°C, preferably -10 to 80°C; the reaction solvent is selected from one of methanol, ethanol, ethyl acetate, tetrahydrofuran and 1,4-dioxane or any combination thereof, preferably ethyl acetate or tetrahydrofuran; (17) In step j-4), the reaction is carried out under basic conditions, and the base is selected from potassium carbonate, potassium hydroxide, sodium hydroxide, cesium carbonate, and sodium carbonate, for example, the base is selected from potassium carbonate, sodium hydroxide, cesium carbonate, and sodium carbonate, and is, for example, potassium carbonate or potassium hydroxide, preferably potassium carbonate or sodium carbonate, and the molar ratio of the compound of formula 1-B5 to the base is selected from 1:1 to 1:5, preferably 1:1 to 1:3; (18) In step j-4), the reaction solvent is selected from one or any combination of water, tetrahydrofuran, 1,4-dioxane, acetonitrile, hexamethylphosphonotriamide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone and dimethylsulfoxide, preferably one or any combination of water, acetonitrile, tetrahydrofuran, dimethylsulfoxide and hexamethylphosphonotriamide; further, in step j-4), the reaction solvent is selected from a combination of acetonitrile and water, wherein the volume ratio of acetonitrile to water is selected from 1:5 to 10:1, preferably 1:2 to 5:1; (19) In step j-4), the mass-to-volume ratio (g / mL) of the compound of formula 1-B5 or a salt thereof to the reaction solvent is selected from 1:5 to 1:20, preferably 1:5 to 1:10; (20) In step j-4), the reaction temperature is selected from 20 to 100° C., preferably 40 to 80° C.; 8. A process for the preparation of a compound of formula I or a salt thereof according to claim 7, characterized in that one or more of the following conditions are met:

9. Method 1 includes the following steps: f-1) reacting a compound of formula 7-A1 with a compound of formula 7-A2 or a salt thereof to obtain a compound of formula 7-A3; 【Chemistry 12】 f-2) reacting a compound of formula 7-A3 to obtain a compound of formula 7-A4 or a salt thereof; 【Chemistry 13】 f-3) reacting a compound of formula 7-A4 or a salt thereof to obtain a compound of formula 7 or a salt thereof; 【Chemistry 14】 Where: R １ , R ２ are each independently C １－４ alkyl; P.G. １ , P.G. ３ are each independently selected from an amine protecting group, and PG １ and P.G. ３ is different; Method 2 includes the following steps: g-1) reacting a compound of formula 7-B1 or a salt thereof to obtain a compound of formula 7-B2 or a salt thereof; 【Chemistry 15】 g-2) reacting a compound of formula 7-B2 or a salt thereof to obtain a compound of formula 7-B3 or a salt thereof; 【Chemistry 16】 g-3) reacting a compound of formula 7-B3 or a salt thereof with a compound of formula 7-A1 to obtain a compound of formula 7 or a salt thereof; 【Chemistry 17】 Where: R １ , R ２ are each independently C １－４ alkyl, P.G. １ , P.G. ４ are each independently selected from an amine protecting group; A process for preparing a compound of formula I or a salt thereof according to claim 1, further comprising process 1 or process 2.

10. (1) The R １ , R ２ is selected from methyl, ethyl and propyl, preferably R １ , R ２ are the same, and preferably, １ , R ２ are all propyl; (2) The PG １ is selected from alkoxycarbonyl-based protecting groups, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc); (3) The PG ３ is selected from alkoxycarbonyl-based protecting groups, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably tert-butoxycarbonyl (Boc); (4) The PG ４ is selected from alkoxycarbonyl-based protecting groups, preferably selected from benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc); (5) in step f-1), the reaction is carried out under basic conditions, and the base is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate and potassium bicarbonate, for example, sodium bicarbonate or sodium carbonate, preferably sodium bicarbonate, potassium bicarbonate or potassium carbonate; (6) In step f-1), the reaction solvent is selected from one of tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide and water, or any combination thereof, preferably selected from one of tetrahydrofuran, acetone, acetonitrile and water, or any combination thereof; (7) In step f-1), the mass-to-volume ratio (g / mL) of the compound of formula 7-A1 or a salt thereof to the reaction solvent is selected from 1:5 to 1:20; (8) in step f-1), the molar ratio of the compound of formula 7-A1 to the compound of formula 7-A2 or a salt thereof is selected from 1:1 to 1:3; (9) In step f-1), the reaction temperature is selected from 0 to 50° C., preferably 0 to 30° C.; (10) In step f-2), a protecting group PG on the amino group is reacted with the compound of formula 7-A3. ３ to obtain a compound of formula 7-A4; Preferably, the PG ３ is tert-butoxycarbonyl (Boc), the compound of formula 7-A3 can be prepared by converting the protecting group PG ３ Remove; Further, the acid is selected from trifluoroacetic acid, hydrogen chloride, hydrobromic acid and sulfuric acid, preferably hydrogen chloride or trifluoroacetic acid; Furthermore, the PG ３ is tert-butoxycarbonyl (Boc), the molar ratio of the compound of formula 7-A3 to said acid is selected from 1:5 to 1:50, preferably 1:10 to 1:30; (11) In step f-2), the PG ３ is tert-butoxycarbonyl (Boc), the reaction solvent is selected from one of tetrahydrofuran, 1,4-dioxane, ethyl acetate and methyl tert-butyl ether or any combination thereof, preferably tetrahydrofuran or 1,4-dioxane; (12) In step f-2), the PG ３ is tert-butoxycarbonyl (Boc), the mass to volume ratio (g / mL) of the compound of formula 7-A3 to the reaction solvent is selected from 1:4 to 1:20, preferably 1:5 to 1:15; (13) In step f-2), the PG ３ is tert-butoxycarbonyl (Boc), the reaction temperature is selected from 0 to 50° C., preferably 0 to 30° C.; (14) In step f-3), the compound of formula 7-A4 or a salt thereof undergoes reductive amination with an aldehyde in the presence of a reducing agent and an acid to obtain a compound of formula 7 or a salt thereof; Further, the reducing agent is sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride and Pd / C—H ２ preferably sodium cyanoborohydride and / or sodium triacetoxyborohydride; Further, the molar ratio of the compound of formula 7-A4 or its salt to the reducing agent is selected from 1:2 to 1:10, preferably 1:3 to 1:8, and the temperature at which the reducing agent is added is selected from -20 to 30°C, preferably 0°C; Further, the acid is selected from acetic acid, formic acid, propionic acid, trifluoroacetic acid and hydrochloric acid, preferably acetic acid or propionic acid; Furthermore, the molar ratio of the compound of formula 7-A4 or its salt to the aldehyde is selected from 1:2 to 1:10, preferably 1:4 to 1:8; (15) In step f-3), the reaction solvent is selected from one of methanol, ethanol, propanol, tetrahydrofuran, 1,4-dioxane and ethyl acetate or any combination thereof, preferably methanol or ethanol; (16) In step f-3), the mass-to-volume ratio (g / mL) of the compound of formula 7-A4 or a salt thereof to the reaction solvent is selected from 1:5 to 1:20, preferably 1:6 to 1:15; (17) In step f-3), the reaction temperature is selected from -20 to 50°C, preferably 0 to 30°C; (18) In step g-1), the compound of formula 7-B1 or a salt thereof undergoes reductive amination with an aldehyde in the presence of a reducing agent and an acid to obtain a compound of formula 7-B2 or a salt thereof; Further, the reducing agent is sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride and Pd / C—H ２ preferably sodium cyanoborohydride or sodium triacetoxyborohydride; Further, the molar ratio of the compound of formula 7-B1 or a salt thereof to said reducing agent is selected from 1:2 to 1:10; Furthermore, the temperature at which the reducing agent is added is selected from −20 to 30° C.; Further, the acid is selected from acetic acid, formic acid, propionic acid, trifluoroacetic acid and hydrochloric acid, preferably acetic acid or propionic acid; Further, the molar ratio of the compound of formula 7-B1 or a salt thereof to the aldehyde is selected from 1:2 to 1:10; (19) In step g-1), the reaction solvent is selected from one of methanol, ethanol, propanol, tetrahydrofuran, 1,4-dioxane and ethyl acetate or any combination thereof, preferably methanol or ethanol; (20) In step g-1), the mass-to-volume ratio (g / mL) of the compound of formula 7-B1 or a salt thereof to the reaction solvent is selected from 1:5 to 1:20; (21) In step g-1), the reaction temperature is selected from -20 to 50°C, preferably 0 to 30°C; (22) In step g-2), a protecting group PG on the amino group is obtained by converting a compound of formula 7-B2 or a salt thereof into ４ to obtain a compound of formula 7-B3 or a salt thereof; Preferably, the PG ４ When is 9-fluorenylmethyloxycarbonyl (Fmoc), the compound of formula 7-B2 or a salt thereof can be reacted with the protecting group PG under basic conditions. ４ Remove; Furthermore, the PG ４ is 9-fluorenylmethyloxycarbonyl (Fmoc), the base is selected from piperidine, diethylamine, morpholine, diisopropylamine, DBU, triethylamine and N,N-diisopropylethylamine, preferably piperidine, diethylamine or morpholine; Furthermore, the PG ４ is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass to volume ratio (g / mL) of said base to reaction solvent is selected from 1:5 to 1:50; Furthermore, the PG ４ is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction solvent is selected from one or any combination of DMF, DMAc, dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, methanol and ethanol, preferably one or any combination of DMF, tetrahydrofuran and dichloromethane; Furthermore, the PG ４ is 9-fluorenylmethyloxycarbonyl (Fmoc), the mass to volume ratio (g / mL) of the compound of formula 7-B2 or a salt thereof to the reaction solvent is selected from 1:2 to 1:20; Furthermore, the PG ４ is 9-fluorenylmethyloxycarbonyl (Fmoc), the reaction temperature is selected from 0 to 50° C., preferably 0 to 30° C.; (23) In step g-3), the reaction is carried out under basic conditions; Further, the base is selected from sodium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate and potassium bicarbonate, preferably sodium bicarbonate, potassium bicarbonate or potassium carbonate; (24) In step g-3), the reaction solvent is selected from one of tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide and water or any combination thereof, preferably one of tetrahydrofuran, acetone and acetonitrile or any combination thereof, more preferably a combination of acetone and water, further, the reaction solvent is selected from a combination of acetone and water, wherein the volume ratio of acetone to water is selected from 1:0.2 to 1:10; (25) In step g-3), the molar ratio of the compound of formula 7-B3 or a salt thereof to the compound of formula 7-A1 is selected from 1:1 to 1:5; (26) In step g-3), the reaction temperature is selected from 0 to 50° C.; 10. A process for the preparation of a compound of formula I or a salt thereof according to claim 9, characterized in that one or more of the following conditions are met:

11. h) reacting a compound of formula 1 or a salt thereof with a compound of formula 2 to obtain a compound of formula 3 or a salt thereof; 【Chemistry 18】 Where: R ４ , R ５ are each independently -OC １－４ Alkyl, -SC １－４ Alkyl and -NHC １－４ alkyl; Or, R ４ and R ５ form a carbonyl together with the carbon atom attached to them, Or, R ４ and R ５ together with the carbon atoms connected to them 【Chemistry 19】 wherein X and Y each independently represent O, S, NH, or NC １－４ alkyl, n is selected from 1, 2 and 3; Furthermore, R ４ and R ５ are defined independently as set forth in claim 6, Furthermore, the operations and conditions of step h) are independently as set forth in claim 6. A process for preparing a compound of formula 3 or a salt thereof.

12. Method 1 includes the following steps: Step i-1) reacting a compound of formula 1-A1 with a compound of formula 1-A2 to obtain a compound of formula 1-A3; Step i-2) reacting a compound of formula 1-A3 to obtain a compound of formula 1 or a salt thereof; 【Chemistry 20】 Method 2 includes the following steps: Step j-1) reacting a compound of formula 1-B1 with a compound of formula 1-B2 to obtain a compound of formula 1-B3; Step j-2) reacting a compound of formula 1-B3 to obtain a compound of formula 1-B4; Step j-3) reacting a compound of formula 1-B4 to obtain a compound of formula 1-B5 or a salt thereof; Step j-4) reacting a compound of formula 1-B5 or a salt thereof to obtain a compound of formula 1 or a salt thereof; 【Chemistry 21】 Wherein X is selected from halogen; Furthermore, X is independently defined as set forth in claim 8; Further, the operations and conditions of steps i-1), i-2), j-1), j-2), j-3) and j-4) are independently as set forth in claim 8; A process for preparing a compound of formula 1, which is process 1 or process 2.

13. Method 1 includes the following steps: f-1) reacting a compound of formula 7-A1 with a compound of formula 7-A2 or a salt thereof to obtain a compound of formula 7-A3; 【Chemical 22】 f-2) reacting a compound of formula 7-A3 to obtain a compound of formula 7-A4 or a salt thereof; 【Chemistry 23】 f-3) reacting a compound of formula 7-A4 or a salt thereof to obtain a compound of formula 7 or a salt thereof; 【Chemistry 24】 Where: R １ , R ２ are each independently C １－４ alkyl; P.G. １ , P.G. ３ are each independently selected from an amine protecting group, and PG １ and P.G. ３ is different; Method 2 includes the following steps: g-1) reacting a compound of formula 7-B1 or a salt thereof to obtain a compound of formula 7-B2 or a salt thereof; 【Chemistry 25】 g-2) reacting a compound of formula 7-B2 or a salt thereof to obtain a compound of formula 7-B3 or a salt thereof; 【Chemistry 26】 g-3) reacting a compound of formula 7-B3 or a salt thereof with a compound of formula 7-A1 to obtain a compound of formula 7 or a salt thereof; 【Chemical 27】 Where: R １ , R ２ are each independently C １－４ alkyl; P.G. １ , P.G. ４ are each independently selected from an amine protecting group; Furthermore, R １ , R ２ , P.G. １ , P.G. ３ and P.G. ４ are defined independently as set forth in claim 10; Further, the operations and conditions of steps f-1), f-2), f-3), g-1), g-2) and g-3) are independently as set forth in claim 10; A process for preparing a compound of formula 7, which is process 1 or process 2.

14. A compound represented by the following formula or a salt thereof. 【Chemistry 28】 (where: R １ , R ２ are each independently C １－４ alkyl; X is selected from halogen; P.G. １ is selected from amine protecting groups; The compound of formula 7 【Chemical 29】 isn't it.)

15. (1) The R １ , R ２ are each independently selected from methyl, ethyl and propyl, preferably R １ , R ２ are the same, and preferably, １ , R ２ are all propyl; (2) said X is selected from fluorine, chlorine, bromine and iodine, preferably said X is chlorine; (3) The PG １ is selected from alkoxycarbonyl-based protecting groups, preferably benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), methoxycarbonyl or ethoxycarbonyl, more preferably 9-fluorenylmethyloxycarbonyl (Fmoc); 15. A compound or a salt thereof according to claim 14, characterized in that it satisfies one or more of the conditions.

16. 16. The compound or salt thereof according to claim 15, characterized in that the compound is selected from the following: 【Chemistry 30】

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