Anti-tumor compound and application thereof

Abstract

A ligand conjugate, or a tautomer, a mesomer, a raceme, an enantiomer, a diastereoisomer, or a pharmaceutically acceptable salt or hydrate thereof, comprises a structure as shown in formula I, and can be used as an antitumor drug. # imgabs0 #

Description

An antitumor compound and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an anti-tumor compound and its application. Background Art

[0002] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, leveraging the specificity of antibodies for binding to surface antigens on both normal and tumor cells and the high potency of cytotoxic substances, resulting in anti-tumor effects. The application of the camptothecin derivative exatecan in antibody-drug conjugates (ADCs) has been reported in the literature, but further development of ADCs with improved efficacy and safety is still needed. However, currently marketed ADCs still suffer from poor plasma stability and off-target toxic side effects, which compromise their efficacy and safety. Further development of new, stable linkers and / or linker-drug molecules is needed to address these issues.

[0003] Summary of the Invention

[0004] The present invention provides a ligand conjugate with high stability, good efficacy and high safety, and a ligand conjugate precursor, a linker, a linker precursor and the like.

[0005] In a first aspect of the present invention, a ligand conjugate is provided, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate comprises the structure shown in Formula I:

[0006] Wherein, L is an optionally substituted linker, which is connected to any O atom, S atom or N atom in the P structure;

[0007] Ab is a ligand, a is a number greater than 0, and a is a decimal or an integer;

[0008] P is a toxin, and the P has a structure shown in the following formula II:

[0009] Where n is 0 or 1;

[0010] X is selected from the following group: N or CR 0 ;

[0011] R 0 Selected from the group consisting of H, D, halogen, C1-C8 alkyl, C1-C8 alkoxy, OH, NH2, N3 or NO2;

[0012] R1 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C8 alkyl group, a C1-C8 alkoxy group, a C1-C8 haloalkyl group, a C1-C8 haloalkoxy group, N3, NO2, NH2, NH-OH, -NR'R", -COOR', -CONR'R", -NHR"'NR'R", wherein R', R" and R'" are each independently selected from the group consisting of hydrogen, deuterium, an alkyl group, an aryl group, an arylalkyl group, an acyl group, an alkoxycarbonyl group, and an aryloxycarbonyl group;

[0013] R 2 、R 3 、R 4 、R 5 and R 6 Each is independently selected from the following groups: hydrogen atom, deuterium atom, halogen, hydroxyl, cyano, NH2, NO2, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m tri(C1-C4alkyl)silyl, -(CH2) m (C3-C8 cycloalkyl), -(CH2) m (3-12 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m S(CH2) p R 7 、-(CH2) m S(O)(CH2) p R 7 、-(CH2) m S(O)2(CH2) p R 7 、-(CH2) m NH(CH2) p R 7 、-(CH2) m NHC(O)(CH2) p R 7 、-(CH2) m OC(O)(CH2) p R 7 、-(CH2) m C(O)(CH2) p R 7 , -CH=N(OtBu); wherein m and p are each independently 0, 1, 2, 3 or 4;

[0014] Or, R 2 and R 3Together with the carbon atom to which it is attached, it forms a substituted or unsubstituted C5-C8 carbocyclic ring or a substituted or unsubstituted 5-12 membered heterocyclic ring;

[0015] Or, R 3 and R 4 , or R 4 and R 5 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-12 membered carbon ring, unsubstituted or replaced by one or more R e substituted saturated or unsaturated 5-12 membered heterocycle; said R e is a substituted or unsubstituted substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C1-C6 alkyl group-NH-, a (C1-C6 alkyl)2N-, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, an allyl group, a benzyl group, a C6-C 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C3-C8 cycloalkyl, 3-12 membered heterocyclyl, -(CH2) m N(R 7 )2、-(CH2) m S(CH2) p R 7 、-(CH2) m S(O)(CH2) p R 7 、-(CH2) m S(O)2(CH2) p R 7 、-(CH2) m NH(CH2) p R 7 、-(CH2) m NHC(O)(CH2) p R 7 、-(CH2) m OC(O)(CH2) p R 7 、-(CH2) m C(O)(CH2) p R 7 ; wherein m and p are each independently 0, 1, 2, 3 or 4, preferably 0, 1 or 2;

[0016] R 7Selected from the following group: hydrogen atom, deuterium atom, halogen, substituted or unsubstituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 deuterated alkyl, substituted or unsubstituted C1-C8 alkoxy, hydroxyl, amino, cyano, nitro, mercapto, substituted or unsubstituted C1-C8 alkylene-OH, substituted or unsubstituted C1-C8 alkylene-NH2, SO2Me, -OC(O)(substituted or unsubstituted C1-C4 alkyl), -C(O)(substituted or unsubstituted C1-C4 alkyl), substituted or unsubstituted phenyl, substituted or unsubstituted 5-7 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl;

[0017] Unless otherwise specified, the term "substituted" refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C1-C6 alkyl-NH-, a (C1-C6 alkyl)2N-, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a halogenated C1-C6 alkyl group, a halogenated C2-C6 alkenyl group, a halogenated C2-C6 alkynyl group, a halogenated C1-C6 alkoxy group, an allyl group, a benzyl group, a C6-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a halogenated C1-C6 alkoxy group, an allyl group, a benzyl group, a C6-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a C1-C6 alkyl group, a C2 ... 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C3-C8 cycloalkyl, 3-12 membered heterocyclyl.

[0018] In another preferred embodiment, the L has a structure as shown in the following formula: -L1-L2-L3-L4-L5-;

[0019] Wherein, the L1 is optionally substituted R d is H, C1-C6 alkyl, C1-C6 deuterated alkyl, C3-C8 cycloalkyl or C3-C8 deuterated cycloalkyl;

[0020] The L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-, optionally substituted -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-, optionally substituted-X1-(CHROCHR) m2 -C(O)-, optionally substituted -(CHR) p1-C(O)-, optionally substituted -(CHR) m1 -X1-(CHR) m2 -C(O)-, optionally substituted -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-, optionally substituted -X1-(CHR) m1 -X2-(CHR) m2 -C(O)-, optionally substituted -(CH2CH2O) n3 -C(O)-;

[0021] X1 and X2 are each independently selected from the following groups: -O-, -C(O)-, -C(O)-NR-, optionally substituted C6-C 10 aryl, optionally substituted 5-9 membered heteroaryl, optionally substituted 3-8 membered heteroalicyclic group and optionally substituted C3-C6 alicyclic group;

[0022] Wherein, each of the R's is independently selected from the following groups: H, D, (CH2) n4 OH, (CH2) n4 NH2, (CH2O) n4 (CH2CH2O) n5 H, (CH2O) n4 (CH2CH2O) n5 CH3, (CH2) n4 OCH3, (CH2CH2O) n5 CH3, CH2C(O)NH(CH2O) n4 (CH2CH2O) n5 H、CH2C(O)NH(CH2O) n4 (CH2CH2O) n5 CH3;

[0023] wherein m1, m2, n3, n4 and n5 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; p1 is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0024] The L3 is a peptide residue; and the L3 may be substituted with one or more substituents selected from the following group: CH2C(O)R c ; The R c Select from the following groups: wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0025] The L4 is an optionally substituted -L 4a -(NR b ) n6 -R 12 -L 4b -, where L 4a Does not exist, or L 4a is optionally substituted Where n6 is 0 or 1; R 12 is a chemical bond, CH2, or CD2;

[0026] L 4b Does not exist, or L 4b is optionally substituted Among them, R a and R b are each independently selected from the group consisting of hydrogen, optionally substituted C1-C4 alkyl, and optionally substituted C1-C4 deuterated alkyl;

[0027] The L5 is absent or optionally substituted wherein Y is selected from the group consisting of O, S, or NH; v is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R 10 and R 11 are each independently selected from the group consisting of hydrogen, deuterium, optionally substituted C1-C4 alkyl, optionally substituted C1-C4 haloalkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C4-C8 cycloalkylalkyl, or R 10 and R 11 Together with the atoms to which it is attached, it forms an optionally substituted 3-6 membered cycloalkyl group, R 10 and R 11 Each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an optionally substituted C1-C8 alkyl group, an optionally substituted C1-C8 haloalkyl group, and an optionally substituted C1-C8 deuterated alkyl group.

[0028] In another preferred embodiment, the L1 is

[0029] In another preferred embodiment, X1 and X2 are each independently selected from the following groups: -O-, -C(O)-, -C(O)-NR-, optionally substituted phenyl, optionally substituted pyridyl, optionally substituted C3-C6 cycloalkyl, optionally substituted or optionally substituted

[0030] In another preferred embodiment, the L2 is a group selected from the group consisting of: optionally substituted -(CH2) m1 -X1-(CH2CH2O)n3 -(CH2) m2 -C(O)-; wherein X1 is -C(O)-NH-; preferably, m1 and m2 are each independently selected from 1, 2 or 3; n3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0031] In another preferred embodiment, the L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1 is optionally substituted or optionally substituted X2 is -C(O)-NR-; preferably, m1 and m2 are each independently selected from 0, 1 or 2; n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0032] In another preferred embodiment, the L2 is a group selected from the group consisting of: optionally substituted -X1-(CHROCHR) m2 -C(O)-; wherein X1 is an optionally substituted aryl group or an optionally substituted heteroaryl group; preferably, m2 is selected from 0, 1, 2 or 3.

[0033] In another preferred embodiment, the L2 is a group selected from the group consisting of optionally substituted (CHR) p1 -C(O)-; p1 is selected from 0, 1 or 2; R is selected from the following group: H, (CH2) n4 OH, (CH2O) n4 (CH2CH2O) n5 H; Preferably, n4 and n5 are each independently selected from 0, 1, 2 or 3.

[0034] In another preferred embodiment, the L2 is a group selected from the group consisting of: optionally substituted -(CH2) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1 is -C(O)-; preferably, m1 and m2 are each independently selected from 0, 1, 2 or 3; n3 is selected from 0, 1 or 2.

[0035] In another preferred embodiment, the L2 is a group selected from the group consisting of: optionally substituted -X1-(CH2) m1 -X2-(CHR) m2 -C(O)-; wherein X1 is an optionally substituted aryl or an optionally substituted heteroaryl; X2 is -C(O)-; preferably, m1 and m2 are each independently selected from 0, 1 or 2.

[0036] In another preferred embodiment, the L2 is a group selected from the following group: -(CHR) m1 -X1-(CHR) m2 -C(O)-; wherein X1 is an optionally substituted 3-8 membered heteroalicyclic group or an optionally substituted C3-C6 alicyclic group; preferably, m1 is 0, 1 or 2, and m2 is 0.

[0037] In another preferred embodiment, the L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 - C(O)-; wherein X1 is O; preferably, m1, n3 and m2 are each independently 0, 1 or 2. In another preferred embodiment, the L2 is a group selected from the following group: optionally substituted -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-; wherein X1 is optionally substituted -C(O)-NR-, X2 is O; preferably, m1, n3 and m2 are each independently 1, 2 or 3; R is as described above.

[0038] In another preferred embodiment, the L2 is an optionally substituted structure selected from the following group:

[0039] In a preferred embodiment, the selection of the above-mentioned L2 structure enables the maleimide group of the resulting ligand conjugate to form an open-ring structure, thereby inhibiting the Retro-Michael reaction, improving the stability of the ligand conjugate, reducing the shedding of the small molecule part, and thus improving the safety of the ligand conjugate drug.

[0040] In another preferred embodiment, the L3 is unsubstituted or replaced by CH2C(O)R c The peptide residues are composed of substituted amino acids selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine.

[0041] In another preferred embodiment, the L3 is unsubstituted or replaced by CH2C(O)R c The peptide residues are composed of substituted amino acids selected from the group consisting of glycine, alanine, lysine, phenylalanine, valine and citrulline.

[0042] In another preferred embodiment, the L3 is unsubstituted or replaced by CH2C(O)R cSubstituted peptide residues selected from the group consisting of: -Glycine-Phenylalanine-Glycine-(-Gly-Phe-Gly-), -Glycine-Glycine-Phenylalanine-Glycine-(-Gly-Gly-Phe-Gly-), -Valine-Citrulline-(-Val-Cit-), -Citrulline-Valine-(-Cit-Val-), -Citrulline-Alanine-(-Cit-Ala-), -Valine-Alanine-(-Val-Ala-), -Valine-Arginine-(-Val-Arg-), -Valine-Lysine-(-Val-Lys-), -Valine-Lysine(Ac)-(-Val-Lys(Ala-) c)-), -Lysine-Valine-(-Lys-Val-), -Leucine-Citrulline-(-Leu-Cit-), -Isoleucine-Citrulline-(-Ile-Cit-), -Tryptophan-Citrulline-(-Trp-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Lysine(Ac)-(-Phe-Lys(Ac)-), -Phenylalanine-Citrulline-(-Phe-Cit-), -Phenylalanine-Ala-(-Phe-Ala-), -Phenylalanine-Arginine-(-Phe-Arg-), -Ala-Lysine-(-Ala-Lys-), -Ala -Ala-Ala-), -Ala-Ala-Ala-Ala-), -Ala-Ala-Ala-Asparagine-(-Ala-Ala-Asn-), -Ala-Ala-Ala-Aspartic Acid-(Ala-Ala-Asp-), -Lysine-Ala-Ala-Ala-Asparagine-(-Lys-Ala-Ala-Asn-), -Lysine-Ala-Ala-Ala-Aspartic Acid-(-Lys-Ala-Ala-Asp-), -(D)-Valine-Leucine-Lysine-(-D-Val-Leu-Lys-), -Glycine-Glycine-Arginine- (-Gly-Gly-Arg-), -Glycine-Glycine-Asparagine-(-Gly-Gly-Asn-), -Glycine-Glycine-Phenylalanine-(-Gly-Gly-Phe-), -Valine-Lysine-Glycine-(-Val-Lys-Gly-), -Glutamic acid-Alanine-Alanine-(-Glu-Ala-Ala-), -Aspartic acid-Alanine-Alanine-(-Asp-Ala-Ala-), -Valine-Lysine-Glycine-Glycine-(-Val-Lys-Gly-Gly-), and -Lysine-Alanine-Asparagine-(-Lys-Ala-Asn-).

[0043] In another preferred embodiment, the L3 is unsubstituted or replaced by CH2C(O)R c A substituted structure selected from the group consisting of:

[0044] In another preferred embodiment, L4 is a chemical bond, or an optionally substituted group selected from the following group: Among them, the R a and R b Each is independently selected from the following group: hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C1-C4 deuterated alkyl.

[0045] In another preferred embodiment, L4 is a chemical bond, or an optionally substituted structure selected from the following group:

[0046] In another preferred embodiment, the L5 is a chemical bond, or a structure selected from the following groups: optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted and optionally substituted

[0047] In another preferred embodiment, the compound of formula II is selected from the following group:

[0048] In another preferred embodiment, the compound of formula II is selected from the following group:

[0049] In another preferred embodiment, the compound of formula II is selected from the following group:

[0050] In another preferred embodiment, the R 4 Selected from the following groups: hydrogen atom, deuterium atom, halogen, hydroxyl, cyano, NH2, NO2, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m (C3-C8 cycloalkyl), -(CH2) m(3-12 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m S(O)(CH2) p R 7 、- (CH2) m S(O)2(CH2) p R 7 、-(CH2) m NH(CH2) p R 7 ; wherein m and p are each independently 0, 1 or 2, R 7 is defined as above;

[0051] R 5 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, NH2, OH, a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C1-C8 alkoxy group;

[0052] Or, R 4 and R 5 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-6 membered carbon ring, unsubstituted or replaced by one or more R e Substituted saturated or unsaturated 5-6 membered heterocyclic ring; wherein R e The definition of is as described above.

[0053] In another preferred embodiment, R 2 and R 3 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, NH2, a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C1-C8 deuterated alkyl group, -(CH2) m (C3-C6 cycloalkyl), -(CH2) m (3-6 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m OC(O)R 7 ; wherein m is 0, 1, 2, 3 or 4, R 7 is defined as above;

[0054] Or, R 2 and R 3 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-6 membered ring, unsubstituted or replaced by one or more R e Substituted saturated or unsaturated 5-6 membered heterocyclic ring; wherein Re The definition of is as described above.

[0055] In another preferred embodiment, R 1 and R 6 are each independently a hydrogen atom.

[0056] In another preferred embodiment, the R 4 and R 5 Each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a hydroxyl group, NH2, a substituted or unsubstituted C1-C4 alkyl group;

[0057] Or, R 4 and R 5 The carbon atoms to which it is attached together form an unsubstituted or substituted carbon atom or carbon atoms. e Substituted oxa 5-6 membered heterocycle; wherein R e The definition of is as described above.

[0058] In another preferred embodiment, R 2 Selected from the group consisting of: deuterium atom, halogen, NH2, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m (C3-C6 cycloalkyl), -(CH2) m (3-6 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m OC(O)R 7 ; wherein m is 0, 1, 2, 3 or 4;

[0059] R 3 Each is selected from the following groups: hydrogen atom, deuterium atom, halogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m (C3-C6 cycloalkyl), -(CH2) m (3-6 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m OC(O)R 7 ; wherein m is 0, 1, 2, 3 or 4;

[0060] Or, R 2 and R 3 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-6 membered ring, unsubstituted or replaced by one or more R e a substituted saturated or unsaturated 5-6 membered heterocycle;

[0061] R 4 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a hydroxyl group, a cyano group, NH2, NO2, a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C1-C8 alkoxy group;

[0062] R 5 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, and a substituted or unsubstituted C1-C8 alkyl group;

[0063] Or, R 4 and R 5 The carbon atoms to which it is attached together form an unsubstituted or substituted carbon atom or carbon atoms. e a substituted group selected from the group consisting of: -OCH2O- or -O(CH2)2O-;

[0064] R 7 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a substituted or unsubstituted C1-C8 alkyl group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a mercapto group;

[0065] Among them, R e The definition of is as described above.

[0066] In another preferred embodiment, the compound of formula II is selected from the following group:

[0067] In a preferred embodiment, the ligand conjugate is selected from the following group:

[0068] In the structure shown above, "-L1-" is selected from the following structures:

[0069] In a preferred embodiment, the ligand conjugate is selected from the following group:

[0070] In the structure shown above, "-L1-" is selected from the following structures: Preferably More preferably

[0071] In another preferred embodiment, the ligand conjugate comprises a structure represented by formula (Ia):

[0072] wherein R4, R5 and L are as defined in the first aspect of the present invention, a is a number greater than 0, and a is a decimal or an integer.

[0073] In another preferred embodiment, the ligand conjugate is a structure selected from the following group:

[0074] Where a is a number greater than 0, and a is a decimal or an integer.

[0075] In another preferred embodiment, a is a non-zero integer or decimal from 0 to 8, preferably an integer or decimal between 1-8; more preferably, it is from 2 to 8, and can be an integer or a decimal; most preferably, it is from 3 to 8, and can be an integer such as 4.0, or a decimal such as 3.9.

[0076] In another preferred embodiment, the Ab is an antibody or an antigen-binding fragment thereof.

[0077] In another preferred embodiment, the antibody is selected from the following group: murine antibody, chimeric antibody, humanized antibody and fully human antibody.

[0078] In another preferred embodiment, the antibody is a monoclonal antibody.

[0079] In another preferred embodiment, the antigen-binding fragment is selected from the following group: Fab, Fab′, Fv fragment, F(ab′) 2 , F(ab) 2 , scFv, di-scFv, VHH and dAb.

[0080] In another preferred embodiment, the antibody is selected from the following group: Her-2 specific antibody (such as Trastuzumab), Trop2 specific antibody (such as Humanized RS7 or Sacituzumab in US7238785B2), PSMA specific antibody (such as PSMA specific monoclonal antibody AB-PG1-XG1-006 in WO2003034903A2), FR-α specific antibody (such as humanized LK26 antibody (Farletuzumab, MORAb-003) in US5952484), B7H3 antibody (such as antibody P7-C05-H4L3 in WO2021244590A1), or IGF-1R specific antibody.

[0081] In another preferred embodiment, the antibody is an IGF-1R specific antibody.

[0082] In another preferred embodiment, the IGF-1R comprises IGF-1R derived from primates.

[0083] In another preferred embodiment, the antibody is IgG1 or a mutation thereof.

[0084] In another preferred embodiment, the antibody comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.

[0085] In another preferred embodiment, the antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2.

[0086] In another preferred embodiment, the antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0087] In another preferred example, the antibody comprises a heavy chain variable region VH, the VH comprises the HCDR1, HCDR2 and HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1.

[0088] In another preferred embodiment, the antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 4.

[0089] In another preferred embodiment, the antibody has the full-length sequence shown as SEQ ID No. 5.

[0090] In another preferred embodiment, the antibody is a variant of any of the above antibodies, and the variant includes the CDR region.

[0091] In another preferred embodiment, the variant is a sequence formed by replacing, deleting and / or adding one or more amino acids (e.g., 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions) of the amino acid sequence of the antibody.

[0092] In another preferred embodiment, the variant is a homolog of the amino acid sequence of the antibody, and the homolog can be an amino acid sequence having at least about 85% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR.

[0093] In another preferred embodiment, the CDRs are determined by the Kabat numbering scheme.

[0094] In a second aspect of the present invention, there is provided a ligand conjugate precursor, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate precursor comprises a structure shown in Formula IA: L A -P(IA),

[0095] Where L is L 1A -L2-L3-L4-L5-; among them, L 1A for Among them, R d , L2, L3, L4, L5 and P are defined as described in the first aspect of the present invention.

[0096] In another preferred embodiment, the ligand conjugate precursor is selected from the following structures:

[0097] In the structure shown above, L 1A Selected from

[0098] In a third aspect of the present invention, there is provided a linker as shown in formula (L), which connects the drug unit to the ligand to form a ligand-drug conjugate: L1-L2-L3-L4-L5 (L),

[0099] Wherein, the definitions of L1, L2, L3, L4, and L5 are as described in the first aspect of the present invention.

[0100] In another preferred embodiment, the linker is selected from the following group:

[0101] In another preferred embodiment, the linker is connected to the ligand through the L1 segment and to P1 through the L5 segment to form a ligand-drug conjugate; and the P1 is selected from the following group: glycopeptide antibiotics, such as bleomycin or bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, belotecan or rubitecan, DXd, etc.), topoisomerase II inhibitors (such as actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, clarithromycin, etc. Tribine or nelarabine; drugs that act on structural proteins, such as microtubule inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; maytansine derivatives (such as DM1, DM4, etc.); calicheamicin derivatives; auristatin derivatives (such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin E, auristatin F, etc.); pyrrolobenzodiazepine dimers (PBD) derivatives; amanitin derivatives (such as α-Amanitin, etc.); anthracyclines; dukamycin; eribulin; melphalan; mitomycin C; chlorambucil; TLR agonists; STING agonists; glucocorticoids and other groups formed by dehydrogenation of active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0102] In a fourth aspect of the present invention, there is provided a linker precursor as shown in formula (L-1), which is used to obtain a ligand-drug conjugate formed by linking a drug unit to a ligand:

[0103] It is used to obtain a ligand-drug conjugate formed by connecting a drug unit to a ligand: L A -R g (L-1),

[0104] Among them, R g is H, OH, O (C1-C6 alkyl); wherein L A is as defined in the third aspect of the present invention.

[0105] In another preferred embodiment, the linker precursor is selected from the following group:

[0106] At the same time, the present invention also provides a synthesis scheme of another preferred embodiment of (L-1), which is described as follows:

[0107] Option 1:

[0108] Pg1 is selected from wait;

[0109] Pg2 is selected from Boc, Fmoc, Cbz, etc.;

[0110] Pg3 is selected from Boc, Fmoc, Cbz, etc.;

[0111] Option 2:

[0112] Pg1 is selected from wait;

[0113] Pg2 is selected from Boc, Fmoc, Cbz, etc.;

[0114] Pg4 is selected from Me, Et, i Pr, Allyl, t Bu, Bn, 4-Methybenzyl, 4-Methoxybenzyl, 2,4-Dimethoxybenzyl, 2,6-Dimethoxybenzyl, Trimethylsilyl, tert-Butyldimethylsilyl, Pentafluorophenyl, etc.

[0115] The fifth aspect of the present invention provides a pharmaceutical composition comprising the ligand conjugate described in the first aspect of the present invention, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, and optionally a pharmaceutically acceptable carrier.

[0116] In a sixth aspect, the present invention provides a ligand conjugate according to the first aspect of the present invention, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, and / or the pharmaceutical composition according to the fifth aspect of the present invention, in the preparation of a medicament for treating and / or preventing a disease or condition associated with target expression and / or abnormal expression of the ligand.

[0117] In another preferred embodiment, the disease or condition related to the target expression and / or abnormal expression of the ligand is a tumor / cancer, an autoimmune disease or an infectious disease; preferably, the tumor / cancer is a tumor / cancer with high expression, medium expression or low expression of the ligand target.

[0118] In another preferred embodiment, the tumor is selected from tumors associated with the expression of the following target sites: Her-2, Trop2, PSMA, FR-α, B7H3, or IGF-1R.

[0119] In another preferred embodiment, the tumor comprises a solid tumor and / or a blood tumor.

[0120] In another preferred embodiment, the tumor is a tumor associated with IGF-1R target expression.

[0121] In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, endometrial cancer, urothelial cancer, lung cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, and head and neck cancer. Astrocytoma, basal or squamous cell carcinoma, brain cancer, neuroblastoma, glioblastoma, liposarcoma, bladder cancer, colorectal cancer, colon cancer, chondrosarcoma, kidney cancer, choriocarcinoma, leukemia, multiple myeloma, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, liver cancer, glioma, hepatocellular carcinoma, leiomyoma, melanoma, non-small cell lung cancer, nervous system cancer, pancreatic cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymoma, thyroid cancer, testicular cancer, and osteosarcoma.

[0122] Another aspect of the present invention provides a method for preparing a linker precursor as shown in formula (L-1), characterized in that it comprises the steps of:

[0123] use and Reaction, get

[0124] Among them, L 1A for Preferably, L 1A for More preferably, L 1A for

[0125] L2 is as described in any of the above aspects; preferably, L2 is selected from the following group: More preferably, L2 is selected from

[0126] Preferably, Selected from amino acids or dipeptide fragments; preferably, Glycine or glycine-glycine fragments Preferably, Glycine-glycine fragment

[0127] Preferably, Selected from amino acids or dipeptide fragments; preferably, Glycine or glycine-glycine fragments Preferably, Glycine-glycine fragment

[0128] Preferably, is a dipeptide or tripeptide fragment; preferably, Glycine-phenylalanine-glycine fragment Phenylalanine-glycine fragment Preferably, Phenylalanine-glycine fragment

[0129] Preferably, is a dipeptide or tripeptide fragment; preferably, Glycine-phenylalanine-glycine fragment Phenylalanine-glycine fragment Preferably, Phenylalanine-glycine fragment

[0130] Pg1 is selected from Preferably, the Pg1 is selected from Preferably, the Pg1 is selected from Preferably, the Pg1 is selected from Preferably, the Pg1 is selected from

[0131] Preferably, the It is prepared by the following method:

[0132] (1) L 1A -L2-OPg5 with Reaction, get Preferably, the reaction is carried out in the absence of a base or in the presence of a base; preferably, the reaction is carried out in the absence of a base; preferably, the base is selected from triethylamine, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine, DMAP, DBU, NMM, NaHCO3; preferably, the base is NaHCO3;

[0133] Pg5 is selected from Preferably, the Pg5 is selected from Preferably, the Pg5 is selected from Preferably, the Pg5 is selected from Preferably, the Pg5 is selected from

[0134] (2) Use Pg1OH and Reaction, get

[0135] Preferably, the Pg1OH is selected from Preferably, the Pg1OH is selected from Preferably, the Pg1OH is selected from Preferably, the Pg1OH is selected from Preferably, the Pg1OH is selected from

[0136] Preferably, the It is prepared by the following method:

[0137] (i) By formula The reaction is

[0138] (ii) Use and Reaction, get

[0139] And the method further comprises: obtaining by the following method (iii) or (iv)

[0140] (iii) First, deprotection group Pg4 is carried out to obtain Then Deprotection of Pg2 yields

[0141] Preferably, the deprotection is carried out in the presence of a base; preferably, the base is an organic base; more preferably, the base is DBU; preferably, the deprotection is carried out in the presence of a reducing agent; preferably, the reducing agent is H2; preferably, the deprotection is carried out in the presence of a catalyst; preferably, the catalyst is palladium carbon Pd / C;

[0142] (iv) First deprotect the Pg2 to obtain Then Deprotection of Pg4 is performed to obtain

[0143] Preferably, the deprotection is carried out in the presence of a base; preferably, the base is an organic base; more preferably, the base is DBU; preferably, the deprotection is carried out in the presence of a reducing agent; preferably, the reducing agent is H2; preferably, the deprotection is carried out in the presence of a catalyst; preferably, the catalyst is palladium carbon Pd / C;

[0144] wherein Pg2 is selected from Boc, Fmoc, and Cbz;

[0145] Pg4 is selected from Me, Et, i Pr, Allyl, t Bu, Bn, 4-methylbenzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, trimethylsilyl, tert-butyldimethylsilyl, pentafluorophenyl.

[0146] In another preferred embodiment, the linker precursor is

[0147] Another aspect of the present invention provides a method for preparing the compound described in Formula 4 below:

[0148] Characterized in that the method comprises the steps of:

[0149] In an inert solvent, a compound of Formula 15h is reacted with a compound of Formula 1d to produce a compound of Formula 4:

[0150] In another preferred embodiment, the inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, HMPA, or a combination thereof; preferably N,N-dimethylformamide;

[0151] In another preferred embodiment, the reaction is carried out in the presence of a condensing agent; more preferably, the condensing agent is selected from the group consisting of HATU, HBTU, TBTU, EDCI, HOAt, HOBt, CDI, TCFH, TFFH, DCC, DIC, BOP, AOP, PyAOP, BrOP, PyClOP, PyBrOP, DMTMM, or a combination thereof; more preferably, the condensing agent is HATU;

[0152] In another preferred embodiment, the reaction is carried out in the presence of a base; more preferably, the base is selected from the group consisting of TEA, DIPEA, DBU, DMAP, pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, imidazole, N-methylimidazole, or a combination thereof; more preferably, the base is 2,4,6-trimethylpyridine.

[0153] Another aspect of the present invention provides a method for preparing the compound of formula 15h:

[0154] Characterized in that the method comprises the steps of:

[0155] (1) In an inert solvent, reacting a compound of Formula 15f with a compound of Formula 8e to obtain a compound of Formula 15g;

[0156] In another preferred embodiment, the inert solvent is selected from the group consisting of acetonitrile, water, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or a combination thereof; more preferably, acetonitrile, water, or a combination thereof; more preferably, a combination of acetonitrile and water in a volume ratio of 10:1 to 1:10; more preferably, the inert solvent is a combination of acetonitrile and water in a volume ratio of 2:1 to 4:1; more preferably, the inert solvent is a combination of acetonitrile and water in a volume ratio of 3:1;

[0157] In another preferred embodiment, the reaction can be carried out in the presence of a base; preferably, the base is selected from TEA, DIPEA, NMM, sodium bicarbonate, DBU, or a combination thereof; more preferably, the base is DIPEA;

[0158] (2) In an inert solvent, the compound of Formula 15g is subjected to a silicon-based removal reagent to remove the protecting group to obtain the compound of Formula 15h;

[0159] In another preferred embodiment, the silicon-based removal agent is selected from the group consisting of an acid, a fluorination agent (fluorine-containing agent); preferably, the silicon-based removal agent is selected from the group consisting of hydrogen fluoride, an aqueous solution of hydrogen fluoride, triethylamine trihydrofluoride, pyridine hydrofluoride, formic acid, acetic acid, trifluoroacetic acid, dichloroacetic acid, methanesulfonic acid, and trifluoromethanesulfonic acid; preferably, the silicon-based removal agent is formic acid;

[0160] In another preferred embodiment, the inert solvent is selected from the group consisting of tetrahydrofuran, N,N-dimethylformamide, acetonitrile, water, or a combination thereof; further preferably, acetonitrile, water, or a combination thereof; further preferably, the volume ratio of acetonitrile to water is 10:1 to 1:10, further preferably, the volume ratio of acetonitrile: water is 1:4 to 4:1, and further preferably, the volume ratio of acetonitrile: water is 1:1.5.

[0161] In another aspect, the present invention also provides a method for preparing the compound of formula 8e:

[0162] Characterized in that the method comprises the steps of:

[0163] (1) In an inert solvent, the Fmoc protecting group is removed from the compound of Formula 8c under alkaline conditions to obtain the compound of Formula 15b;

[0164] In another preferred embodiment, the inert solvent is selected from dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, or a combination thereof; more preferably, the inert solvent is dichloromethane;

[0165] In another preferred embodiment, the base is an organic base; more preferably, the base is dimethylamine, diethylamine, DBU, or piperidine; more preferably, the base is DBU.

[0166] (2) removing the benzyl protecting group using the compound of Formula 15b in an inert solvent to obtain the compound of Formula 8e;

[0167] In another preferred embodiment, the inert solvent is selected from tetrahydrofuran, water, or a combination thereof; more preferably, the inert solvent is water; more preferably, the inert solvent is a combination of tetrahydrofuran and water in a volume ratio of 1:5 to 5:1.

[0168] In another preferred embodiment, the deprotection is carried out in the presence of a base; preferably, the base is an organic base; more preferably, the base is DBU;

[0169] In another preferred embodiment, the deprotection is carried out in the presence of a reducing agent; more preferably, the reducing agent is hydrogen H2; more preferably, the deprotection is carried out in the presence of a catalyst; more preferably, the catalyst is palladium carbon Pd / C;

[0170] In another aspect of the present invention, the present invention also provides a method for preparing the compound described in Formula 8c below:

[0171] Characterized in that the method comprises the steps of:

[0172] In an inert solvent, reacting the compound of formula 8b with benzyl glycolate to obtain the compound of formula 8c;

[0173] In another preferred embodiment, the inert solvent is selected from dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, or a combination thereof; more preferably, the inert solvent is tetrahydrofuran.

[0174] In another preferred embodiment, the reaction is carried out in the presence of an acid or a base; more preferably, the acid is p-toluenesulfonic acid or p-toluenesulfonic acid monohydrate; more preferably, the base is lithium hydroxide, lithium hydroxide monohydrate, potassium tert-butoxide, sodium tert-butoxide, or sodium hydroxide; more preferably, the base is lithium hydroxide; more preferably, the base is lithium hydroxide monohydrate;

[0175] In another aspect of the present invention, the present invention also provides a method for preparing the compound described in Formula 8b below:

[0176] Characterized in that the method comprises the steps of:

[0177] (1) reacting the compound of Formula 15a with diglycine in an inert solvent to obtain the compound of Formula 8a;

[0178] In another preferred embodiment, the reaction is carried out in the presence of a base; more preferably, the base is sodium bicarbonate;

[0179] In another preferred embodiment, the inert solvent is selected from ethylene glycol dimethyl ether, water, or a combination thereof; more preferably, the inert solvent is a combination of ethylene glycol dimethyl ether and water; more preferably, the inert solvent is a combination of ethylene glycol dimethyl ether and water in a volume ratio of 1:5 to 5:1; more preferably, the inert solvent is a combination of ethylene glycol dimethyl ether and water in a volume ratio of 1:2 to 2:1;

[0180] (2) reacting with the compound of Formula 8a below in an inert solvent to obtain the compound of Formula 8b;

[0181] In another preferred embodiment, the inert solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide, and HMPA; more preferably, the inert solvent is N,N-dimethylformamide.

[0182] In another preferred embodiment, the reaction is carried out in the presence of lead tetraacetate;

[0183] In another preferred embodiment, the reaction is carried out in the presence of copper acetate;

[0184] In another preferred embodiment, the reaction is carried out in the presence of acetic acid;

[0185] In another preferred embodiment, the reaction is carried out in the presence of acetic acid, lead tetraacetate and copper acetate;

[0186] In another preferred embodiment, the reaction is carried out in the presence of acetic acid and lead tetraacetate;

[0187] In another aspect of the present invention, the present invention also provides a method for preparing the compound of formula 15f:

[0188] Characterized in that the method comprises the steps of:

[0189] (1) In an inert solvent, a compound represented by the following formula 15c is subjected to a condensation reaction with tetrafluorophenol to obtain a compound represented by the following formula 15d;

[0190] In another preferred embodiment, the inert solvent is selected from dichloromethane, tetrahydrofuran, and 2-methyltetrahydrofuran; more preferably, the inert solvent is dichloromethane;

[0191] In another preferred embodiment, the reaction is carried out in the presence of a condensing agent; more preferably, the condensing agent is selected from the group consisting of HATU, HBTU, TBTU, EDCI, HOAt, HOBt, CDI, TCFH, TFFH, DCC, DIC, BOP, AOP, PyAOP, BrOP, PyClOP, PyBrOP, DMTMM, or a combination thereof; more preferably, the condensing agent is DCC.

[0192] (2) reacting the compound of Formula 15d with diglycine in an inert solvent to obtain the compound of Formula 15e;

[0193] In another preferred embodiment, the inert solvent is selected from ethylene glycol dimethyl ether, water, or a combination thereof; more preferably, the inert solvent is a combination of ethylene glycol dimethyl ether and water; more preferably, the inert solvent is a combination of ethylene glycol dimethyl ether and water in a volume ratio of 1:5 to 5:1; more preferably, the inert solvent is a combination of ethylene glycol dimethyl ether and water in a volume ratio of 1:2 to 2:1;

[0194] In another preferred embodiment, the reaction is carried out in the absence of a base or in the presence of a base; more preferably, the base is selected from triethylamine, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine, DMAP, DBU, NMM, NaHCO3; more preferably, the base is NaHCO 3；

[0195] (3) In an inert solvent, a compound represented by the following formula 15e is subjected to a condensation reaction with tetrafluorophenol to obtain a compound represented by the following formula 15f;

[0196] In another preferred embodiment, the inert solvent is selected from dichloromethane, tetrahydrofuran, and 2-methyltetrahydrofuran; more preferably, the inert solvent is dichloromethane;

[0197] In another preferred embodiment, the reaction is carried out in the presence of a condensing agent; more preferably, the condensing agent is selected from the group consisting of HATU, HBTU, TBTU, EDCI, HOAt, HOBt, CDI, TCFH, TFFH, DCC, DIC, BOP, AOP, PyAOP, BrOP, PyClOP, PyBrOP, DMTMM, or a combination thereof; more preferably, the condensing agent is DCC.

[0198] In another preferred embodiment, the reaction is carried out in the presence or absence of a base; more preferably, the reaction does not require the participation of a base. ；

[0199] In another aspect of the present invention, the present invention also provides a method for preparing the compound represented by the following formula 15c:

[0200] Characterized in that the method comprises the steps of:

[0201] (1) reacting the compound of Formula 4a with a silicon-based reagent in an inert solvent to obtain a compound of Formula 4b;

[0202] In another preferred embodiment, the inert solvent is selected from the group consisting of acetonitrile, DMF, DMA, THF, and dichloromethane; more preferably, the inert solvent is acetonitrile;

[0203] In another preferred embodiment, the reaction is carried out in the presence of a base; more preferably, the base is selected from DBU, imidazole, DIPEA, TEA; more preferably, the base is DBU;

[0204] In another preferred embodiment, the silicon-based reagent is TBSCl or TBSOTf; more preferably, the silicon-based reagent is TBSCl;

[0205] (2) In an inert solvent, the compound of formula 4b is reacted with N-methoxycarbonylmaleimide in the presence of a base to obtain the compound of formula 15c;

[0206] In another preferred embodiment, the inert solvent is selected from the group consisting of water, THF, DMF, or a combination thereof; more preferably, the inert tetrahydrofuran and water are in a volume ratio of 1:1;

[0207] In another preferred embodiment, the base is selected from the group consisting of sodium bicarbonate, sodium carbonate, triethylamine, and diisopropylethylamine; more preferably, the base is sodium bicarbonate.

[0208] In another aspect of the present invention, the present invention also provides a method for preparing the compound described in Formula 1d below:

[0209] Characterized in that the method comprises the steps of:

[0210] (1) In an inert solvent, a compound of the following formula 14g and a compound of the following formula 14d undergo a condensation cyclization reaction in the presence of an additive to obtain a compound of the following formula 14k;

[0211] In another preferred embodiment, the inert solvent is preferably selected from toluene, acetic acid, chlorobenzene, or a combination thereof; more preferably, the inert solvent is a combination of toluene and acetic acid; more preferably, the inert solvent is a combination of toluene and acetic acid in a volume ratio of 1:5 to 5:1; more preferably, the inert solvent is a combination of toluene and acetic acid in a volume ratio of 1:2 to 2:1; more preferably, the inert solvent is a combination of toluene and acetic acid in a volume ratio of 1:1;

[0212] In another preferred embodiment, the reaction can be carried out in the presence of an additive; more preferably, the additive can be selected from the group consisting of p-toluenesulfonic acid, p-toluenesulfonic acid monohydrate, and p-toluenesulfonic acid pyridinium salt; more preferably, the additive is p-toluenesulfonic acid pyridinium salt.

[0213] (2) In an inert solvent, the Fmoc protecting group is removed using the compound of the following formula 14k under organic base conditions to obtain a mixture of formula 1d and formula 1d′;

[0214] In another preferred embodiment, the organic base is selected from secondary amines and tertiary amines; more preferably, the organic base is selected from piperidine, dimethylamine, and diethylamine; more preferably, the organic base is piperidine;

[0215] In another preferred embodiment, the inert solvent is selected from tetrahydrofuran and 2-methyltetrahydrofuran; more preferably, the inert solvent is tetrahydrofuran.

[0216] (3) chiral separation of the obtained mixture of 1d and 1d′ to obtain 1d;

[0217] In another aspect of the present invention, the present invention also provides a method for preparing the compound described in Formula 14g below:

[0218] Characterized in that the method comprises the steps of:

[0219] (1) In an inert solvent, deacetyl protection is performed using a compound of formula 14e to obtain a compound of formula 14f.

[0220] In another preferred embodiment, the inert solvent is selected from no solvent, water, tetrahydrofuran, or a combination thereof; more preferably, the inert solvent is water; more preferably, the inert solvent is no solvent;

[0221] In another preferred embodiment, the reaction is carried out in the presence of an acid; more preferably, the acid is hydrochloric acid; more preferably, the concentration of the hydrochloric acid is 3-9N; more preferably, the concentration of the hydrochloric acid is 6N;

[0222] (2) reacting the compound of Formula 14f with FmocCl in the presence of a base to obtain the compound of Formula 14g;

[0223] In another preferred embodiment, the base is selected from organic bases and inorganic bases; more preferably, the base is selected from triethylamine, diisopropylethylamine, DBU, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate; more preferably, the base is potassium carbonate;

[0224] In another preferred embodiment, the inert solvent is selected from acetonitrile, DMF, DMA, dichloromethane, water, tetrahydrofuran, or a combination thereof; more preferably, the inert solvent is a combination of water and tetrahydrofuran in a volume ratio of 1:5 to 5:1; more preferably, the inert solvent is a combination of water and tetrahydrofuran in a volume ratio of 3:4;

[0225] In another aspect of the present invention, the present invention also provides a method for preparing the compound described in Formula 14b below:

[0226] Characterized in that the method comprises the steps of:

[0227] In the presence of a base, in an inert solvent, the compound of Formula 14a is reacted with two different silicon-based reagents to perform silicon-based protection to obtain a compound of Formula 14b;

[0228] In another preferred embodiment, the silicon-based reagent is TESCl, TESOTf, TIPSCl, or TIPSOTf; more preferably, the silicon-based reagent is selected from TESCl and TIPSCl;

[0229] In another preferred embodiment, the inert solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide, HPMA, tetrahydrofuran, and dichloromethane; more preferably, the inert solvent is selected from N,N-dimethylformamide;

[0230] In another preferred embodiment, the base is preferably selected from TEA, DIPEA, NMM, pyridine, 2,6-lutidine, 2,4,6-trimethylpyridine, imidazole, and DMAP; more preferably, the base is imidazole.

[0231] In another aspect of the present invention, the present invention also provides a method for preparing the compound described in Formula 14c below:

[0232] Characterized in that the method comprises the steps of:

[0233] In an inert solvent, reacting the compound of formula 14b with Lawesson's reagent to obtain the compound of formula 14c;

[0234] In another preferred embodiment, the inert solvent is selected from tetrahydrofuran, dioxane, 2-methyltetrahydrofuran, and toluene; more preferably, the inert solvent is toluene;

[0235] In another preferred embodiment, the reaction can be carried out in the presence or absence of a base; the base is preferably selected from triethylamine, DIPEA, NMM, pyridine, 2,6-lutidine, 2,4,6-collidine, DMAP, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, potassium tert-butoxide, sodium tert-butoxide; more preferably, the base is selected from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate; more preferably, the base is sodium carbonate.

[0236] In another aspect of the present invention, the present invention also provides a method for preparing the compound of formula 14d:

[0237] Characterized in that the method comprises the steps of:

[0238] In an inert solvent, the compound of formula 14c is used to remove the TIPS and TES silyl protecting groups with a silyl removing agent to obtain the compound of formula 14d.

[0239] In another preferred embodiment, the silicon-based removal agent is selected from the following group: acid, fluorination agent (fluorine-containing agent); more preferably, the silicon-based removal agent is selected from formic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, dichloroacetic acid, hydrogen fluoride, hydrogen fluoride aqueous solution, triethylamine trihydrofluoride, hydrogen fluoride pyridinium salt; more preferably, the silicon-based removal agent is hydrogen fluoride aqueous solution.

[0240] In another preferred embodiment, the inert solvent is selected from tetrahydrofuran, 2-methyltetrahydrofuran, and toluene; more preferably, the inert solvent is tetrahydrofuran.

[0241] Another aspect of the present invention provides a method for preparing a molecule as shown in Formula 7 below:

[0242] Characterized in that the method comprises the steps of:

[0243] In an inert solvent, a compound described by the following formula 7i and a compound described by the following formula 1d are subjected to a condensation reaction to produce a compound of formula 7:

[0244] In another preferred embodiment, the inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, HMPA, or a combination thereof; preferably N,N-dimethylformamide.

[0245] In another preferred embodiment, the condensing agent is selected from the group consisting of HATU, HBTU, TBTU, EDCI, HOAt, HOBt, CDI, TCFH, TFFH, DCC, DIC, BOP, AOP, PyAOP, BrOP, PyClOP, PyBrOP, DMTMM, or a combination thereof, preferably EDCI.

[0246] In another preferred embodiment, the base is selected from the group consisting of TEA, DIPEA, NMM, DBU, DMAP, pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, imidazole, N-methylimidazole, or a combination thereof, preferably 2,4,6-trimethylpyridine.

[0247] Another aspect of the present invention provides a method for preparing a molecule as shown in Formula 7i below:

[0248] Characterized in that the method comprises the steps of:

[0249] In an inert solvent, a compound of Formula 14j is reacted with a compound of Formula 8e to obtain a compound of Formula 7i;

[0250] In another preferred embodiment, the inert solvent is selected from the group consisting of acetonitrile, water, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, or a combination thereof; more preferably tetrahydrofuran.

[0251] In another preferred embodiment, the reaction is carried out without or with the participation of a base; preferably, the base is selected from triethylamine, N,N-diisopropylethylamine, 2,6-lutidine, 2,4,6-trimethylpyridine, DBU, NMM; more preferably, the reaction does not require the participation of a base ；

[0252] In another aspect of the present invention, the present invention also provides a method for preparing a molecule represented by the following formula 14j:

[0253] Characterized in that the method comprises the steps of:

[0254] (1) In an inert solvent, a condensation reaction is carried out between the compound of the following formula 7g and the compound of the following formula 14h to obtain a compound of the following formula 14i;

[0255] In another preferred embodiment, the inert solvent is selected from the group consisting of acetonitrile, water, tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, ethyl acetate, or a combination thereof; more preferably, ethyl acetate.

[0256] In another preferred embodiment, the reaction is carried out in the absence of a base or in the presence of a base; preferably, the base is selected from triethylamine, N,N-diisopropylethylamine, 2,6-lutidine, 2,4,6-collidine, DBU, NMM; more preferably, the reaction does not require the participation of a base;

[0257] (2) In an inert solvent, the compound of the following formula 14i is subjected to detert-butyl protection under acidic conditions to obtain a compound of the formula 8g;

[0258] In another preferred embodiment, the inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, dioxane, ethyl acetate, toluene, or a combination thereof; more preferably, toluene.

[0259] In another preferred embodiment, the acid is selected from the group consisting of hydrochloric acid, sulfuric acid, hydrogen chloride in ethyl acetate, hydrogen chloride in tetrahydrofuran, hydrogen chloride in dioxane, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, and dichloroacetic acid; preferably, the acid is selected from trifluoroacetic acid;

[0260] (3) In an inert solvent, a compound of the following formula 8g is subjected to a condensation reaction with pentafluorophenol in the presence of a condensing agent to obtain a compound of the following formula 14j;

[0261] In another preferred embodiment, the condensing agent is selected from the group consisting of HATU, HBTU, TBTU, EDCI, HOAt, HOBt, CDI, TCFH, TFFH, DCC, DIC, BOP, AOP, PyAOP, BrOP, PyClOP, PyBrOP, DMTMM, or a combination thereof, preferably EDCI.

[0262] The inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, or a combination thereof; more preferably dichloromethane.

[0263] Another aspect of the present invention provides a compound represented by the following formula 14b:

[0264] Another aspect of the present invention provides a compound represented by the following formula 14c:

[0265] Another aspect of the present invention provides a compound represented by the following formula 14g:

[0266] Another aspect of the present invention provides a compound represented by the following formula 14i:

[0267] Another aspect of the present invention provides a compound represented by the following formula 14j:

[0268] Another aspect of the present invention provides a compound represented by the following formula 14k:

[0269] Another aspect of the present invention provides a compound represented by the following formula 15c:

[0270] Another aspect of the present invention provides a compound represented by the following formula 15d:

[0271] Another aspect of the present invention provides a compound represented by the following formula 15f:

[0272] Another aspect of the present invention provides a compound represented by the following formula 15g:

[0273] Another aspect of the present invention provides a compound represented by the following formula 7c:

[0274] Another aspect of the present invention provides a compound represented by the following formula 7e:

[0275] Another aspect of the present invention provides a compound represented by the following formula 7h:

[0276] Another aspect of the present invention provides a compound represented by the following formula 8b:

[0277] Another aspect of the present invention provides a compound represented by the following formula 8e:

[0278] Another aspect of the present invention provides a compound shown in the following formula 8g:

[0279] Another aspect of the present invention provides a compound represented by the following formula 9b:

[0280] Another aspect of the present invention provides a compound represented by the following formula 9c:

[0281] Another aspect of the present invention provides a compound represented by the following formula 9e:

[0282] Another aspect of the present invention provides a compound represented by the following formula 9f:

[0283] Another aspect of the present invention provides a compound represented by the following formula 9g:

[0284] Another aspect of the present invention provides a compound represented by the following formula 9h:

[0285] Another aspect of the present invention provides a compound represented by the following formula 9i:

[0286] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. DETAILED DESCRIPTION

[0287] Based on long-term and in-depth research, the inventors have developed a novel class of ligand-conjugated drugs, drug molecules (toxins) suitable for use in ligand-conjugated compounds, linker precursors suitable for use in ligand-conjugated compounds, and ligand-conjugated precursors suitable for use in ligand-conjugated compounds. These drugs exhibit improved stability, efficacy, and safety. Based on these findings, the inventors have completed the present invention.

[0288] the term

[0289] As used herein, the term "alkyl" includes straight or branched chain alkyl groups. For example, C1-C8 alkyl groups represent straight or branched chain alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

[0290] As used herein, the term "alkenyl" includes straight or branched alkenyl groups. For example, C2-C6 alkenyl refers to a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0291] As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. For example, C2-C6 alkynyl refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

[0292] As used herein, the term "C3-C 10 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be a bicyclic ring, such as a bridged ring or a spiro ring.

[0293] As used herein, the term "C1-C8 alkylamino" refers to an amino group substituted by a C1-C8 alkyl group, which may be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino, etc.

[0294] As used herein, the term "C1-C8 alkoxy" refers to a linear or branched alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, and the like.

[0295] As used herein, the term "3-10 membered heterocycloalkyl group having 1-3 heteroatoms selected from the group consisting of N, S, and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms, 1-3 of which are heteroatoms selected from the group consisting of N, S, and O. It may be a monocyclic or bicyclic ring, such as a bridged ring or a spirocyclic ring. Specific examples include oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, and pyrrolidinyl.

[0296] As used herein, the term "C6-C 10 The term "aryl" refers to an aromatic group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

[0297] As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S, and O" refers to a cyclic aromatic group having 5-10 atoms, 1-3 of which are heteroatoms selected from the group consisting of N, S, and O. It may be a monocyclic or condensed ring. Specific examples include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, and oxazolyl.

[0298] Unless otherwise specified, the chain groups herein (such as alkyl, haloalkyl or deuterated alkyl) have 1-10 carbon atoms, preferably 1-6 carbon atoms, or 1-4 carbon atoms; the non-aromatic cyclic groups herein (such as cycloalkyl, heterocycloalkyl, alicyclic, alicyclic heterocyclic, etc.) are 3-12 members, preferably 3-8 members, 3-6 members; the aromatic cyclic groups herein (such as aryl, heteroaryl, etc.) are 5-15 members, such as 6-10 membered aryl, 5-7 membered heteroaryl, 5-10 membered heteroaryl, etc.

[0299] Unless otherwise specified as "substituted or unsubstituted", the groups of the present invention may be substituted by substituents selected from the following groups: halogen, nitrile, nitro, hydroxyl, amino, C1-C6 alkyl-amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, halogenated C1-C6 alkoxy, allyl, benzyl, C6-C 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, etc.

[0300] As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl, and Br. "Halogenated" means substituted with an atom selected from F, Cl, Br, and I.

[0301] Unless otherwise specified, the structural formulas described herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers (or conformers)): for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformers) are all within the scope of the present invention.

[0302] As used herein, the term "tautomer" refers to structural isomers of different energies that can interconvert across a low energy barrier. For example, proton tautomers (i.e., prototropy) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via reorganization of some of the bonding electrons.

[0303] As used herein, the term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

[0304] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining specific embodiments with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0305] The solvents used in this application can be obtained commercially, and the compounds can be artificially or Software naming, commercially available compounds use supplier catalog names.

[0306] In this application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of a ligand can be to present a drug to a target cell population bound to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. In this application, a ligand can be represented by Ab. The ligand antigen forms a bond with a linker through a heteroatom on the ligand. It can be an antibody or an antigen-binding fragment thereof. The antibody can be selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody or an antigen-binding fragment thereof that targets a target selected from the group consisting of HER2, TROP2, PSMA, FR-α or B7H3, and IGF-1R.

[0307] In this application, the terms "Trop2" and "TROP2" generally refer to a single-pass transmembrane type I cell membrane protein. In this application, the term "Trop2" may also encompass homologs, variants, and isoforms of Trop 2, including spliced ​​isoforms. The term "Trop" also includes proteins having one or more sequences of Trop 2 homologs, variants, and isoforms, as well as fragments of the sequence, as long as they are variant proteins (including isoforms). Trop2 may be human Trop2. For example, Uniprot accession number P09758 provides a description of Trop2 and its sequence.

[0308] In this application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (HER2). For example, the term "HER2" refers to any natural HER2 from any human source. The term also encompasses "full-length" and unprocessed HER2 and any form of HER2 (e.g., mature protein) processed in the cell. The term also encompasses naturally occurring variants and isoforms of HER2, such as splice variants or allelic variants. For example, Uniprot accession number P04626 provides a description of HER2 and sequences.

[0309] In this application, the term "PSMA" refers to glutamate carboxypeptidase II. In this application, the term "PSMA" may also encompass homologs, variants, and isoforms of PSMA. The term "PSMA" also includes proteins having one or more sequences of PSMA homologs, variants, and isoforms from various sources (e.g., human), as well as fragments of such sequences. For example, Uniprot Accession No. Q04609 provides a description of PSMA and its sequence.

[0310] In this application, the term "FR-α" refers to folate receptor α, which is encoded by FOLR1. In this application, the term "FR-α" may also encompass homologs, variants, and isoforms of FR-α. The term "FR-α" also includes proteins having one or more sequences of FR-α homologs, variants, and isoforms from various sources (such as human), as well as fragments of such sequences. For example, Uniprot accession number P15328 provides a description of FR-α and sequences.

[0311] In this application, the term "B7H3" refers to CD276. In this application, the term "B7H3" can cover any homologs, variants and isoforms thereof, and can also include any possible expression form thereof in humans (such as 2Ig and 4Ig). The term "B7H3" also includes proteins having one or more sequences of B7H3 homologs, variants and isoforms from various different sources (such as human), as well as fragments of the sequence. For example, Uniprot accession number Q5ZPR3 provides a description of B7H3 (CD276) and the sequence.

[0312] In this application, the term "IGF-1R" refers to the insulin-like growth factor I receptor. In this application, the term "IGF-1R" may also encompass homologs, variants, and isoforms thereof. The term "IGF-1R" also includes proteins having one or more sequences of IGF-1R homologs, variants, and isoforms of various different origins (e.g., human), as well as fragments of such sequences. For example, Uniprot accession number P08069 provides a description of IGF-1R and sequences.

[0313] In this application, the term "peptide residue" generally refers to a residue comprising one or more amino acid residues linked together. For example, one or more amino acids in a polypeptide residue may be optionally substituted. For example, a polypeptide residue herein may comprise glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0314] In this application, the term "Drug unit" generally refers to a chemical moiety that is directly or indirectly conjugated to an antibody or antigen-binding fragment to form an immunoconjugate. For example, a "Drug unit" includes, but is not limited to, a compound with anti-tumor activity as described herein. For example, a Drug unit includes a topoisomerase inhibitor.

[0315] As used herein, the term "compound with anti-tumor activity" generally refers to a compound that has the ability to reduce the proliferation rate, viability, or metastatic activity of tumor cells. For example, anti-tumor activity can be demonstrated by a decrease in the growth rate of abnormal cells or a stabilization or reduction in tumor size during treatment, or by a prolonged survival period resulting from treatment compared to a control without treatment. Anti-tumor activity can be assessed using recognized in vitro or in vivo tumor models, such as xenograft models.

[0316] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0317] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0318] In the present application, the compounds of the present application include tautomers, meso-racemates, racemates, enantiomers, and / or diastereomers of the compounds.

[0319] In the present application, some atoms of the compounds of the present application may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon can exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also include, but are not limited to 15 N. 18 O. 17 O. 18 F. 32 P. 33 P. 129 I. 131 I. 123 I. 124 I. 125 I, or similar isotopes. Therefore, relative to the natural abundance of these isotopes, the compounds of the present application may be in one or more of these isotopically enriched forms. As known to those skilled in the art, such isotopically enriched compounds can be used for a variety of purposes. For example, with heavy isotopes such as deuterium ( 2 H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability.

[0320] As used herein, the term "pharmaceutical composition" generally refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Conventional pharmaceutical compositions can be prepared using techniques commonly used in the art.

[0321] In this application, the term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refers to a salt of the compound or ligand-drug conjugate of the present application, or a salt of the compound described in the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The antibody-antibody drug conjugate compound of the present application may form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate.

[0322] In the present application, the term "conjugate" generally refers to a compound prepared by one or more chemical reactions of the compounds of the present application, or connected to each other through one or more connecting structures such as a bridge, a spacer, or a connecting part.

[0323] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as antibodies or polypeptides, genes and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. For example, a pharmaceutically acceptable carrier can be distinguished from a nucleic acid vector used to contain a target gene in genetic engineering. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may also be present in these carriers.

[0324] In this application, "antibody" can generally cover monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies) and antibody fragments, as long as they show the desired biological activity. Antibodies can be mouse, human, humanized, chimeric antibodies or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens generally have a large number of binding sites, also known as epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to different epitopes has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing antigens or portions thereof that specifically bind to targets of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins described herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, or any mutation thereof), or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulins are derived from humans, mice, or rabbits. "Antibody fragments" can comprise a portion of a full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies; fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity determining regions); and any of the above epitope-binding fragments that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The antibodies that constitute the antibody-drug conjugates herein can retain their original antigen-binding ability in the wild-type state. Therefore, the antibodies in the present application can, for example, specifically bind to antigens. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth and differentiation (such as known or predicted functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known antibodies). The antigens bound by antibodies can be one or a subset of the above categories, while other subsets contain other molecules / antigens with special properties (compared to the target antigen). Antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the industry and can be prepared by antibody preparation methods and information well known in the industry.These targets can be specifically expressed on the surface of one or more cancer cells, while being little or not expressed on the surface of one or more non-cancerous cells. Generally, such tumor-associated polypeptides can be more overexpressed on the surface of cancer cells than on the surface of non-cancerous cells.

[0325] In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To create a chimeric antibody, a hybridoma that secretes a murine-specific monoclonal antibody can be established. The variable region genes can then be cloned from the murine hybridoma cells. The constant region genes of a human antibody can then be cloned as needed. The murine variable region genes and the human constant region genes can then be linked to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in either eukaryotic or prokaryotic systems.

[0326] In this application, the term "humanized antibody", also known as CDR-grafted antibody, generally refers to antibodies produced by transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.

[0327] In this application, the terms "fully human antibody", "fully human antibody" or "completely human antibody" are also called "fully human monoclonal antibody", and the variable region and constant region of the antibody can be both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

[0328] In the present application, term " CDR " generally refers to one of 6 hypervariable regions that mainly contribute to antigen combination in the variable domains of antibody.One of the most frequently used definitions of described 6 CDRs is provided by Kabat EA et al., or Chothia et al. and MacCallum et al. As used in the present application, the Kabat definition of CDR can be applied to CDR1, CDR2 and CDR3 (LCDR1, LCDR 2, LCDR 3 or L1, L2, L3) of light chain variable domain, and CDR1, CDR2 and CDR3 (HCDR 1, HCDR 2, HCDR 3 or H1, H2, H3) of heavy chain variable domain.

[0329] In the present application, the term "group capable of coupling with a thiol group" generally means that compound A has a thiol group, and compound B has a group capable of coupling with a thiol group, and compound B reacts with the thiol group of compound A through the group capable of coupling with a thiol group, thereby achieving the connection between compound A and compound B.

[0330] In the present application, the term "linker" generally refers to a chemical structure fragment or bond that is connected to one group at one end and to another group at the other end, and can also be connected to other linkers before being connected to the drug and / or ligand. The directly or indirectly connected ligand can refer to the group being directly connected to the ligand through a covalent bond, or it can be connected to the ligand through a linker. For example, the linker can be the structure shown in the linker described in the present application. For example, a chemical structure fragment or bond containing an acid-labile linker structure (such as a hydrazone), a protease-sensitive (such as a peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure can be used as a linker.

[0331] In this application, the term "linking group" generally refers to a group capable of connecting to another group. For example, a compound having a linking group can be connected to another group through a coupling reaction between the linking group and the other group. For example, a maleimide group can serve as a linking group.

[0332] In this application, the term "disease associated with the expression of a target" generally means that the occurrence and / or progression of the disease is associated with the expression level of the target. For example, the expression level of a target in cells from a disease area, such as a specific tissue or organ of a patient, is increased relative to the expression level of normal cells from the tissue or organ, i.e., highly expressed. Or, for example, the expression level of a target in cells from a disease area, such as a specific tissue or organ of a patient, is decreased relative to the expression level of normal cells from the tissue or organ, i.e., low expressed. Or, for example, cells from a disease area, such as a specific tissue or organ of a patient, express a target, i.e., are positive. Or, for example, cells from a disease area, such as a specific tissue or organ of a patient, do not express a target, i.e., are negative. For example, the characteristics of target expression can be determined by standard assays known in the art.

[0333] As used herein, the term "effective amount" generally refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The precise effective amount for a given subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation, which is within the judgment of the clinician.

[0334] Unless otherwise specified, all compounds mentioned in this application are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this application, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0335] In the present application, the term "compound of the present application" generally refers to the compound of the present application. The term also includes various crystal forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of the present application.

[0336] When a trade name is used herein, the trade name is intended to include the trade name product formulation, its corresponding generic drug, and the active pharmaceutical ingredient of the trade name product.

[0337] Pharmaceutical compositions and methods of administration

[0338] Since the compounds of the present invention have excellent tumor cell proliferation inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to prevent and / or treat (stabilize, alleviate or cure) diseases associated with tumor cell proliferation.

[0339] The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention and a pharmaceutically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 1-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet, or a unit dose of an injectable formulation.

[0340] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0341] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration, or subcutaneous administration).

[0342] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0343] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0344] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0345] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0346] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0347] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0348] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0349] When administered in combination, the pharmaceutical composition may further comprise one or more (2, 3, 4, or more) other pharmaceutically acceptable therapeutic agents. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable therapeutic agents may be used simultaneously, separately, or sequentially with the compound of the present invention to prevent and / or treat diseases associated with tumor cell proliferation.

[0350] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 1 to 500 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0351] A preparation process for toxin molecular intermediate

[0352] The present invention also provides a novel preparation process for the synthetic intermediate 14d (compound 1k in WO 2022262789 A1) of the toxin molecule 1d. The specific preparation process is described above. The synthetic route disclosed in the prior art (WO 2022262789 A1) is as follows:

[0353] Compared with the above prior art, the preparation process of the present invention uses TIPS silicon-based protecting group to replace one of the two TES silicon-based protecting groups in the prior art, and the main advantages are:

[0354] 1. The intermediates 14b and 14c of the synthetic route of the present application are more stable than the intermediates 1i and 1j of the prior art, and are easy to scale up production and store for a long time;

[0355] 2. The intermediates 14b and 14c of the synthetic route of this application can be separated and purified, and are easy to control in quality. The intermediates 1i and 1j of the prior art of the above formula are used crude in the next reaction.

[0356] 3. The yield of the synthetic route of the present application is increased from 49% to 73% compared with the yield in the above prior art (WO 2022262789 A1).

[0357] Preparation process of toxin molecule

[0358] The present invention also provides a novel preparation process for toxin molecule 1d (compound 1n-P1 in WO 2022262789 A1). The prior art synthesis route (WO 2022262789 A1) is as follows:

[0359] Compared with the above prior art, the preparation process of the present invention uses Fmoc protecting group to replace the acetyl Ac protecting group in the prior art, and the main advantages are:

[0360] 1. In the synthetic route of the present application, the acetyl protecting group of 11 in the prior art synthetic route was replaced with an Fmoc protecting group to obtain compound 14g, thereby increasing the synthetic yield of compound 1d (compound 1n-P1 in WO 2022262789 A1) from 7.9% to 15.6%;

[0361] 2. The deacetyl protecting group step of compound 1m in the prior art synthesis route (WO 2022262789 A1) has harsh reaction conditions, poor reproducibility, and incomplete removal of the protecting group during scale-up. The Fmoc protecting group removal step of method 14k in the present application has milder reaction conditions and is more suitable for scale-up production.

[0362] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0363] Abbreviation Definition

[0364] Example

[0365] Example 1.1

[0366] first step

[0367] Compound 1b (342 mg, 0.81 mmol) and 1a (500 mg, 0.85 mmol) were dissolved in a mixture of acetonitrile (3.42 mL) and water (6.84 mL). The mixture was cooled to 0°C and N,N-diisopropylethylamine (83 mg, 0.64 mmol) was added dropwise with stirring. The reaction was stirred for 3 hours. The reaction solution was directly separated and purified by preparative chromatography to obtain 1c (283 mg) in a 39% yield.

[0368] MS-ESI calculated value [M+Na] + =1020, the actual measured value is 1020.

[0369] Step 2

[0370] Compound 1c (85 mg, 0.19 mmol) was dissolved in N,N-dimethylformamide (1.70 mL), trifluoroacetic acid (22 mg, 0.19 mmol) was added, the mixture was cooled to 0°C, 1d (226 mg, 0.23 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72 mg, 0.38 mmol) and 2,4,6-trimethylpyridine (23 mg, 0.19 mmol) were added in sequence, and the reaction system was kept at 0°C to 10°C and stirred for 1 hour after the addition was complete. The reaction solution was eluted with HCl. aq The pH value of the reaction mixture was adjusted to 6-7 by adding 0.05 M sodium phosphate and then separated and purified by preparative liquid phase to obtain compound 1 (134 mg). The yield was 49%.

[0371] 1 H NMR (400MHz, DMSO-d6) δ8.66(t,J=6.4Hz,1H),8.60(d,J=8.8Hz,1H),8.28(t,J=6.0Hz,1H),8.15(t,J=5.6Hz,1H),8.09(d,J=8.0Hz,1H) ,8.04-7.95(m,2H),7.84-7.77(m,2H),7.28-7.14(m,5H),6.99(s,2H),6.69(s,1H),5.91(d,J=16.8Hz,1H),5.70-5.62(m,1H),5.56-5. 45(m,2H),5.35(d,J=18.8Hz,1H),4.68(d,J=6.8Hz,2H),4.51-4.42(m,1H),4.17-4.04(m,2H),3.80-3.55(m,12H),3.55-3.42(m,32H), 3.17-3.10(m,2H),3.06-2.98(m,1H),2.80-2.71(m,1H),2.41-2.28(m,7H),2.27-2.15(m,2H),1.94-1.82(m,2H),0.86(t,J=7.2Hz,3H).

[0372] Example 1.2

[0373] first step

[0374] 2a (14.72 g, 150.16 mmol) was dissolved in acetone (118 mL). 2b (20.00 g, 150.16 mmol) was added under ice-cooling. The mixture was stirred for 5 minutes, and the reaction was monitored for completion by TLC. The reaction solution was concentrated to obtain a crude solid product. The crude product was dissolved in acetic anhydride (28 mL), and sodium acetate (24.63 g, 300.32 mmol) was added. The reaction system was heated to 90°C and refluxed for 2 hours. The reaction solution was filtered to remove insoluble solids, and the filter cake was rinsed with toluene. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was then separated by silica gel column chromatography (EA: Hexanes = 0-100%) to obtain 2c (17.43 g) in a yield of 43%.

[0375] MS-ESI calculated value [M+Na] + =236, the actual measured value is 236.

[0376] Step 2

[0377] 2c (12.95 g, 60.74 mmol) and 2d (9.00 g, 60.74 mmol) were dissolved in toluene (180 mL). p-Toluenesulfonic acid (2.10 g, 12.15 mmol) was added, and the mixture was heated to 90°C and refluxed for 2 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain a residue, which was dissolved in ethyl acetate. The organic phase was washed sequentially with saturated sodium bicarbonate and saturated brine (200 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (EA: Hexanes = 0-100%) to obtain 2e (5.53 g), in a yield of 33%.

[0378] MS-ESI calculated value [M+H] + =270, the actual measured value is 270.

[0379] Step 3

[0380] 2e (3.87 g, 14.37 mmol) was dissolved in THF (77.4 mL), lithium hydroxide (1.37 g, 57.49 mmol) was dissolved in H2O (38.7 mL) and added to the above solution, the mixture was stirred at room temperature for 30 min. Ethyl acetate (15 mL) was added to the reaction solution, and HCl was used to aq The pH was adjusted to about 2 by adding (1N) and the aqueous phase was extracted with ethyl acetate (38 mL x 3). The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 2f (3.06 g).

[0381] MS-ESI calculated value [M+H] + =260, the actual measured value is 260.

[0382] Step 4

[0383] Dissolve N-hydroxysuccinimide (6.49 g, 56.37 mmol) in N,N-dimethylformamide (36.5 mL) and add trifluoroacetic anhydride (11.84 g, 56.37 mmol) dropwise under ice bath. Stir for 30 min and then add 2,4,6-trimethylpyridine (6.83 g, 56.37 mmol). After the addition is complete, the mixture is stirred for 40 min. The reaction solution is designated as A and set aside. Dissolve the crude product 2f (3.06 g) in N,N-dimethylformamide (36.5 mL) and add 2,4,6-trimethylpyridine (3.41 g, 28.15 mmol) dropwise under ice bath. Stir for 30 min and then add the above reaction solution A dropwise. The reaction system is naturally warmed to room temperature and stirred for 24 h. Add dichloromethane (180 mL) to the reaction solution and add HCl. aq (0.7N, 140mL) and then continued stirring for 30min, the liquids were separated, the aqueous phase was extracted with dichloromethane (70mL), the organic phases were combined, the organic phases were washed with water to pH = 5-7, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated by silica gel column chromatography (EA: Hexanes = 0-100%) to obtain 2 g (3.54 g), with a yield of 78%.

[0384] MS-ESI calculated value [M+H] + =339, the actual measured value is 339.

[0385] 1 H NMR (400MHz, CDCl3) δ6.77 (s, 2H), 4.79 (t, J = 4.8Hz, 1H), 4.49-4.40 (m, 2H) ,3.91(t,J=11.6Hz,2H),3.79-3.70(m,2H),3.45-3.34(m,1H),2.86(s,4H).

[0386] Step 5

[0387] 2h (850 mg, 3.38 mmol) and 2g (1.20 g, 3.55 mmol) were dissolved in N,N-dimethylformamide (8.5 mL). N,N-diisopropylethylamine (437 mg, 3.38 mmol) was added dropwise under ice-cooling. The mixture was stirred at 0°C–10°C for 2 h. The reaction mixture was purified by preparative liquid phase separation to afford 2i (740 mg) in a 46% yield.

[0388] MS-ESI calculated value [M+H] + 475, the actual measured value is 475.

[0389] Step 6

[0390] 2j (10.00 g, 13.59 mmol) was dissolved in THF (450 mL), and wet Pd / C (2 g, 20% w / w) was added. The mixture was stirred under a hydrogen atmosphere for 66 h. The reaction mixture was filtered, and the filter cake was rinsed with a DCM / MeOH mixture. The filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 2k (6.10 g) in a yield of 69%.

[0391] MS-ESI calculated value [M+H] + =668, the actual measured value is 668.

[0392] Step 7

[0393] Under nitrogen atmosphere, 1d (2.70 g, 5.97 mmol) and trifluoroacetic acid (680 mg, 5.97 mmol) were dissolved in N,N-dimethylformamide (54 mL) and stirred under ice bath for 10 min. Then, 2k (5.40 g, 8.36 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.30 g, 11.94 mmol) and 2,4,6-trimethylpyridine (722 mg, 5.97 mmol) were added to the reaction solution and the temperature was kept at 0℃~10℃ with stirring for 2 h. HCl was added dropwise to the reaction solution under ice bath. aq (0.05N, 54 mL), solid precipitated. The suspension was diluted with 2-methyltetrahydrofuran (100 mL), stirred until dissolved, and then separated. The aqueous phase was extracted with 2-methyltetrahydrofuran (100 mL x 2). The organic phases were combined and washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 21 (6.00 g), with a yield of 84%.

[0394] MS-ESI calculated value [M+H] + =1079, the actual measured value is 1079.

[0395] Step 8

[0396] 2l (6.00 g, 5.56 mmol) was dissolved in a mixture of DCM / MeOH (120 mL / 12 mL). Diethylamine (24 mL) was added dropwise under an ice bath. After complete addition, the mixture was allowed to warm to room temperature and stirred for 4 h. The reaction solution was concentrated directly to obtain a crude brown solid. This solid was then slurried with methyl tert-butyl ether and filtered. The filter cake was rinsed three times with methyl tert-butyl ether, and the filtrate was concentrated to obtain a crude yellow solid. Preparative liquid phase separation was performed on 1 g of the crude product to obtain 2m (150 mg). The remaining crude product was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 2m (800 mg), with a combined yield of 59%.

[0397] MS-ESI calculated value [M+H] + =857, the actual measured value is 857.

[0398] Step 9

[0399] 2i (74 mg, 0.16 mmol) was dissolved in N,N-dimethylformamide (1.30 mL), and the atmosphere was replaced with nitrogen three times. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (88 mg, 0.23 mmol) was added under ice bath, and stirred for 10 min. 2m (132 mg, 0.16 mmol) was added, and stirred until dissolved. 2,4,6-trimethylpyridine (54 mg, 0.45 mmol) was added, and the temperature was kept at 0℃~10℃ for 2 h. The reaction solution was washed with HCl. aq (0.05N) to adjust pH to 6-7, and perform preparative liquid separation and purification to obtain 2 (71 mg), yield: 34%.

[0400] MS-ESI calculated value [M+H] + =1313, the actual measured value is 1313.

[0401] Example 1.3

[0402] first step

[0403] Under a nitrogen atmosphere, 3a (5.00 g, 26.74 mmol) was dissolved in N,N-dimethylformamide (50 mL). The temperature was cooled to 0-5°C, and NaH (1.28 g, 32.09 mmol) was added. After stirring for 10 min, tert-butyl bromoacetate (6.23 g, 32.09 mmol) was added. The mixture was stirred at 0-5°C for 2 h. Water (200 mL) was added to the reaction solution, and the aqueous phase was extracted with ethyl acetate (200 mL). The organic phase was washed with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3b (4.60 g) in a 43% yield.

[0404] MS-ESI calculated value [M+H] + =302,304, the actual measured value is 302,304.

[0405] 1 H NMR (400MHz, CDCl3) δ8.60(d,J=2.0Hz,1H),7.84(dd,J=8.4,2.4Hz,1H),7.45(d,J=8.4Hz,1H),4.69(s,2H),4.10(s,2H),1.49(s,9H).

[0406] Step 2

[0407] Under a nitrogen atmosphere, 3b (5.00 g, 16.60 mmol), 3c (3.61 g, 19.93 mmol), Pd2(dba)3 (0.76 g, 0.83 mmol), BINAP (1.03 g, 1.66 mmol), and cesium carbonate (13.53 g, 41.52 mmol) were dissolved in anhydrous toluene (50 mL). The mixture was heated to an internal temperature of 80°C and stirred for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (EA: hexanes = 0-100%) to obtain 3d (4.50 g) in a yield of 67%.

[0408] MS-ESI calculated value [M+H] + =403, the actual measured value is 403.

[0409] Step 3

[0410] 3d (2.81 g, 6.98 mmol) was dissolved in THF (28 mL) and HCl. aq The mixture was stirred at room temperature for 3 h in a 1N, 28 mL mixed solvent. Water (100 mL) was added to the reaction solution, and the aqueous phase was washed with ethyl acetate (100 mL x 2). The organic phase was discarded. The aqueous phase was adjusted to pH 8-9 with aqueous ammonia and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford 3e (1.43 g) in an 86% yield.

[0411] MS-ESI calculated value [M+H] + =239, the actual measured value is 239.

[0412] Step 4

[0413] 3e (1.83 g, 7.68 mmol) and maleic anhydride (0.75 g, 7.68 mmol) were dissolved in acetonitrile (18.3 mL) and stirred at room temperature for 2 h. The reaction solution was directly dried to give 2.45 g of a white solid intermediate. This 2.45 g of white solid intermediate was added to a reaction flask, followed by acetic anhydride (5 mL) and sodium acetate (2.09 g, 15.36 mmol). The mixture was stirred at room temperature for 2 h. Water (50 mL) was added to the reaction solution, and the aqueous phase was extracted with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was then separated by silica gel column chromatography (EA: hexanes = 0-100%) to afford 3f (1.97 g) in an 80% yield.

[0414] MS-ESI calculated value [M+H] + =319, the actual measured value is 319.

[0415] 1 H NMR (400MHz, DMSO-d6) δ8.51(d,J=2.0Hz,1H),7.83(dd,J=8.4,2.4Hz,1H),7.6(d,J=8.4Hz,1H),7.24(s,2H),4.67(s,2H),4.15(s,2H),1.44(s,9H).

[0416] Step 5

[0417] Dissolve 3f (0.90 g, 2.83 mmol) in dichloromethane (9 mL) and add trifluoroacetic acid (1.8 mL). Stir and react at room temperature for 5 h. The reaction solution is directly spin-dried to dryness, dichloromethane (45 mL x 5) is evaporated, and concentrated using an oil pump until no oil is visible, yielding 3 g (0.93 g) of crude product.

[0418] MS-ESI calculated value [M+H] + =263, the actual measured value is 263.

[0419] Step 6

[0420] 3g (0.06g), 2m (0.20g, 0.23mmol) and N,N-dimethylformamide (4mL) were added to the reaction flask in sequence, stirred to dissolve and placed in an ice bath, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.13g, 0.35mmol) and 2,4,6-trimethylpyridine (0.26g, 2.15mmol) were added, and the mixture was stirred at 0-5°C for 1h. The reaction solution was treated with HCl aqThe reaction mixture was added with 0.5N to adjust the pH to 4-5 and then sent to a preparative liquid phase neutral pure water system for separation to obtain 3 (70 mg), with a yield of 27%.

[0421] MS-ESI calculated value [M+H] + =1101, the actual measured value is 1101.

[0422] 1 H NMR(400MHz, DMSO-d6)δ8.65(t,J=6.8Hz,1H),8.60(d,J=8.8Hz,1H),8.34-8. 24(m,2H),8.09(d,J=8.0Hz,1H),7.99(t,J=5.6Hz,1H),7.81(d,J=11.2Hz,1H ),7.79(s,1H),7.30-7.14(m,5H),7.04(s,2H),6.68(s,1H),5.91(d,J=16.8H z,1H),5.70-5.61(m,1H),5.56-5.45(m,2H),5.36(d,J=19.6Hz,1H),5.05(t,J =6.0Hz,1H),4.68(d,J=6.4Hz,2H),4.59(dd,J=9.2,6.0Hz,1H),4.52-4.42(m ,1H),4.17-4.05(m,2H),4.00-3.92(m,1H),3.89-3.79(m,1H),3.78-3.68(m, 5H),3.64-3.54(m,2H),3.01(dd,J=8.8,4.4Hz,1H),2.74(dd,J=13.6,9.6Hz, 1H),2.39(s,3H),2.26-2.18(m,2H),1.94-1.82(m,2H),0.86(t,J=7.6Hz,3H).

[0423] Example 1.4

[0424] first step

[0425] 4a (10.00 g, 95.20 mmol) was dissolved in acetonitrile (100 mL), and tert-butyldimethylsilyl chloride (15.06 g, 99.92 mmol) was added. The mixture was cooled to 0°C, and 1,8-diazobisspiro[5.4.0]undec-7-ene (13.73 g, 90.21 mmol) was added dropwise. After complete addition, the reaction system was returned to room temperature and stirred for 16 hours. The product gradually precipitated. The reaction solution was directly filtered, and the filter cake was collected to obtain 4b (11.50 g) in a yield of 55%.

[0426] MS-ESI calculated value [M+H]+ =220, the actual measured value is 220.

[0427] Step 2

[0428] To a reaction flask, 4b (5.55 g, 25.33 mmol), acetone (55.5 mL), and maleic anhydride (2.48 g, 25.33 mmol) were added sequentially and stirred at room temperature for 2 h. The reaction solution was directly spin-dried to dryness to obtain 8.25 g of a yellow oil. This yellow oil was dissolved in toluene (82 mL), and triethylamine (5.12 g, 50.66 mmol) was added. The mixture was heated to 120°C and refluxed for 2 h. The reaction solution was directly concentrated under reduced pressure to obtain a residue, which was then separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 4c (1.53 g) in a 33% yield.

[0429] MS-ESI calculated value [MH] - =184, the actual measured value is 184.

[0430] 1 H NMR (400MHz, DMSO-d6) δ7.00(s,2H),6.05(s,1H),4.30(dd,J=9.6,5.6Hz,1H),3.98(dd,J=10.8,5.6Hz,1H),3.84(dd,J=10.8,10.8Hz,1H).

[0431] Step 3

[0432] 4c (65 mg, 0.234 mmol), 2m (0.20 g, 0.23 mmol) and N,N-dimethylformamide (4 mL) were added to the reaction flask in sequence. After stirring to dissolve, the mixture was placed in an ice bath. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.13 g, 0.35 mmol) and 2,4,6-trimethylpyridine (85 mg, 0.70 mmol) were added. The mixture was stirred at 0-5°C for 1 h. The reaction solution was treated with HCl. aq After adjusting the pH to 4-5 with 0.5N, 4 (70 mg) was obtained by separation using a preparative liquid phase neutral pure water system. The yield was 14%.

[0433] MS-ESI calculated value [M+H] + =1024, the actual measured value is 1024.

[0434] 1H NMR (400MHz, DMSO-d6) δ8.65(t,J=6.8Hz,1H),8.60(d,J=8.8Hz,1H),8.29(q,J= 5.6Hz,2H),8.09(d,J=8.0Hz,1H),7.99(t,J=5.6Hz,1H),7.81(d,J=11.2Hz,1H), 7.80(s,1H),7.29-7.14(m,5H),7.04(s,2H),6.68(s,1H),5.91(d,J=16.8Hz,1H) ,5.71-5.60(m,1H),5.56-5.45(m,2H),5.36(d,J=19.6Hz,1H),5.05(t,J=5.6Hz, 1H),4.68(d,J=6.4Hz,2H),4.59(dd,J=9.2,6.0Hz,1H),4.52-4.43(m,1H),4.17 -4.04(m,2H),4.00-3.92(m,1H),3.98-3.80(m,1H),3.78-3.68(m,5H),3.64-3.5 4(m,2H),3.20-3.09(m,1H),3.00(dd,J=14.0,4.4Hz,1H),2.73(dd,J=13.6,9.6H z,1H),2.39(s,3H),2.26-2.17(m,2H),1.94-1.83(m,2H),0.86(t,J=7.2Hz,3H).

[0435] Example 1.5

[0436] first step

[0437] 5a (650 mg, 1.70 mmol) and 5b (699 mg, 1.70 mmol) were dissolved in anhydrous dichloromethane (6 mL). 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (771 mg, 2.03 mmol) and N,N-diisopropylethylamine (440 mg, 2.40 mmol) were added sequentially under ice-cooling. After complete addition, the reaction system was returned to room temperature and stirred for 1 h. The reaction solution was concentrated to obtain a residue, which was then separated by silica gel column chromatography (MeOH:DCM = 0-100%) to afford 5c (1.10 g) in an 83% yield.

[0438] MS-ESI calculated value [M+H] + =777, the actual measured value is 777.

[0439] Step 2

[0440] Dissolve 5c (1.10 g, 1.42 mmol) in anhydrous dichloromethane (2.6 mL). Add trifluoroacetic acid (2 mL) dropwise under ice-cooling. After complete addition, return the reaction system to room temperature and stir for 2 h. The reaction solution is concentrated to obtain a residue, which is then separated by silica gel column chromatography (MeOH:DCM = 0-100%) to afford crude product 5d (1.16 g).

[0441] MS-ESI calculated value [M+H] + =721, the actual measured value is 721.

[0442] Step 3

[0443] 5f (800 mg, 1.69 mmol) was dissolved in anhydrous acetonitrile (12 mL). 1,8-Diazabispiro[5.4.0]undec-7-ene (129 mg, 0.85 mmol) was added dropwise under ice-cooling. After complete addition, the reaction system was returned to room temperature and stirred for 1 h. 5e (750 mg, 1.69 mmol) was added to the above reaction solution. 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (771 mg, 2.03 mmol) and N,N-diisopropylethylamine (654 mg, 5.07 mmol) were added sequentially under ice-cooling. After complete addition, the reaction system was returned to room temperature and stirred for 2 h. The reaction solution was diluted with ethyl acetate (200 mL), and the organic phase was washed with saturated brine (50 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 5 g (440 mg) of the crude product, with a yield of 38%.

[0444] MS-ESI calculated value [M+Na] + =701, the measured value is 701.

[0445] Step 4

[0446] Dissolve 5g (420mg, 0.62mmol) in anhydrous acetonitrile (10mL) and add 1,8-diazobisspiro[5.4.0]undec-7-ene (94mg, 0.62mmol) dropwise under ice-cooling. After complete addition, the reaction mixture is returned to room temperature and stirred for 2h. Add 5d (446mg, 0.62mmol) to the above reaction mixture, and add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (353mg, 0.93mmol) and N,N-diisopropylethylamine (240mg, 1.86mmol) sequentially under ice-cooling. After complete addition, the reaction mixture is returned to room temperature and stirred for 1h. The reaction solution was diluted with ethyl acetate (100 mL), and the organic phase was washed with saturated brine (25 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (MeOH:DCM=0-100%) to obtain 5h (600 mg), yield: 83%.

[0447] MS-ESI calculated value [M+Na] + =1181, the actual measured value is 1181.

[0448] Step 5

[0449] 5h (600 mg, 0.52 mmol) was dissolved in a tetrahydrofuran / water (8 mL / 2 mL) mixture, and Pd / C (60 mg, 10% w / w) was added. The mixture was stirred under a hydrogen atmosphere for 5 h. The reaction mixture was filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 5i (360 mg) in a yield of 65%.

[0450] MS-ESI calculated value [M+Na] + =1091.5, the actual measured value is 1091.8.

[0451] Step 6

[0452] To a reaction flask, 1d (50 mg, 0.11 mmol), 5i (140 mg, 0.13 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After stirring to dissolve, the mixture was placed in an ice bath. 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (65 mg, 0.17 mmol) and N,N-diisopropylethylamine (44 mg, 0.34 mmol) were added. The mixture was stirred at 0-5°C for 1 h. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was then separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain crude product 5j (140 mg) in an 85% yield.

[0453] MS-ESI calculated value [M+H] + =1502.6, the measured value is 1503.

[0454] Step 7

[0455] 5j (55 mg, 0.037 mmol) was dissolved in anhydrous acetonitrile (2 mL). Diethylamine (27 mg, 0.37 mmol) was added dropwise under ice-cooling. After complete addition, the reaction system was returned to room temperature and stirred overnight. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then separated by silica gel column chromatography (MeOH:DCM = 0-100%) to afford 5k (30 mg) in a 63% yield.

[0456] MS-ESI calculated value [M+H] + =1280.6, the measured value is 1281.

[0457] Step 8

[0458] 5k (30 mg, 0.023 mmol) and 5l (20 mg, 0.12 mmol) were dissolved in anhydrous dichloromethane (1 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (35 mg, 0.092 mmol) and N,N-diisopropylethylamine (16 mg, 0.12 mmol) were added in sequence under ice bath, and the mixture was stirred at room temperature for 1 h. The reaction solution was treated with HCl. aq After adjusting the pH to 4-5 with 0.5N, 5 (15 mg) was obtained by separation using a preparative liquid phase neutral pure water system. The yield was 45%.

[0459] MS-ESI calculated value [M+H] + =1431.6, the actual measured value is 1432.

[0460] Example 1.6

[0461] first step

[0462] Dissolve 6a (4.00 g, 21.03 mmol) in anhydrous dichloromethane (64 mL), add Dess-Martin periodinane (11.15 g, 26.30 mmol), and after complete addition, stir the mixture at room temperature for 2 h. The reaction mixture was diluted with dichloromethane (100 mL), and the organic phase was washed sequentially with saturated sodium thiosulfate solution (50 mL x 1) and saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield crude 6b (5.30 g).

[0463] Step 2

[0464] Crude products 6b (500 mg) and 6c (1.20 g, 3.19 mmol) were dissolved in anhydrous methanol (25 mL). Acetic acid (7.56 g, 126.00 mmol) and sodium cyanoborohydride (418 mg, 6.65 mmol) were added. After complete addition, the mixture was stirred at room temperature for 3 h. The reaction solution was diluted with dichloromethane (150 mL) and the excess acetic acid was quenched with saturated sodium bicarbonate (50 mL). The layers were separated, and the organic phase was washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield crude product 6d (1.38 g).

[0465] MS-ESI calculated value [M+H] + =556, the actual measured value is 556.

[0466] Step 3

[0467] The crude product 6d (1.38 g) was dissolved in anhydrous tetrahydrofuran (30 mL), and N,N-diisopropylethylamine (482 mg, 3.73 mmol) and 9-fluorenylmethyl chloroformate (1.29 g, 4.97 mmol) were added. After complete addition, the mixture was stirred at room temperature for 3 h. The reaction solution was diluted with dichloromethane (150 mL), and the organic phase was washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the residue, which was then separated by silica gel column chromatography (MeOH:DCM = 0-100%) to afford 6e (1.00 g) in a 65% yield.

[0468] MS-ESI calculated value [M+H] + =778, the actual measured value is 778.

[0469] Step 4

[0470] 6e (1.00 g, 1.28 mmol) was dissolved in anhydrous dichloromethane (4 mL), and trifluoroacetic acid (1.46 g, 12.85 mmol) was added. The mixture was stirred at room temperature for 1 h, and then trifluoroacetic acid (1.46 g, 12.85 mmol) was added. The reaction solution was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 6f (500 mg) in a yield of 54%.

[0471] MS-ESI calculated value [M+H] + =722.4, the measured value is 722.9.

[0472] Step 5

[0473] 2j (500 mg, 0.68 mmol) was dissolved in anhydrous acetonitrile (5 mL). 1,8-Diazabispiro[5.4.0]undec-7-ene (134 mg, 0.88 mmol) was added dropwise under ice-cooling. After complete addition, the reaction system was returned to room temperature and stirred for 2 h. 6f (490 mg, 0.68 mmol) was added to the above reaction solution. 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (388 mg, 1.02 mmol) and N,N-diisopropylethylamine (220 mg, 1.70 mmol) were added sequentially under ice-cooling. After complete addition, the reaction system was returned to room temperature and stirred for 1 h. The reaction solution was diluted with dichloromethane (100 mL), and the organic phase was washed with saturated brine (25 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (MeOH:DCM=0-100%) to obtain 6 g (470 mg), with a yield of 57%.

[0474] MS-ESI calculated value [M+Na] + =1239.6, the actual measured value is 1239.9.

[0475] 1 H NMR(400MHz,DMSO-d6)δ7.84(d,J=7.6Hz,2H),7.82-7.74(m,1H),7.63(d,J=7.6Hz,2H),7.61-7 .52(m,2H),7.50-7.44(m,2H),7.44-7.24(m,15H),4.90-4.81(m,2H),4.73-4.62(m,1H),4.59(m ,J=5.6Hz,2H),4.30(s,2H),4.29-4.25(m,1H),4.15-4.02(m,1H),4.00-3.89(m,2H),3.86(d,J =5.2Hz,2H),3.80-3.45(m,34H),3.43(s,3H),3.41-3.38(m,1H),3.11(dd,J=14.0,10.0Hz,1H).

[0476] Step 6

[0477] Dissolve 6g (470mg, 0.39mmol) in a mixture of tetrahydrofuran and water (4mL / 1mL). Add wet Pd / C (62mg, 13% w / w). Stir the mixture under a hydrogen atmosphere for 2h. Filter the reaction mixture to remove insoluble matter, and concentrate the filtrate under reduced pressure to obtain the crude product 6h (360mg).

[0478] MS-ESI calculated value [M+Na] +=1149.5, the actual measured value is 1149.9.

[0479] Referring to Reference Example 5, Compound 5i and Compound 1d were reacted in three steps to synthesize Compound 5, and Compound 6h and Compound 1d were reacted in three steps to synthesize Compound 6.

[0480] MS-ESI calculated value [M+H] + =1489.6, the actual measured value is 1490.

[0481] Example 1.7

[0482] Synthesis Route 1

[0483] first step

[0484] Under a nitrogen atmosphere, 7a (10.60 g, 30.00 mmol) was dissolved in a mixture of tetrahydrofuran / toluene (200 mL / 50 mL). Lead tetraacetate (17.30 g, 39.00 mmol) and pyridine (3.08 g, 39.00 mmol) were added. The mixture was heated to 75°C and stirred for 4 h. The reaction mixture was filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure to remove most of the solvent. The residue was dissolved in ethyl acetate (400 mL). The organic phase was washed sequentially with water (100 mL x 2), saturated sodium bicarbonate solution (100 mL), dilute hydrochloric acid (0.5 N, 100 mL x 2), and saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (EA: hexanes = 0-100%) to obtain 7b (9.70 g) in an 88% yield.

[0485] MS-ESI calculated value [M+Na] + =391, the actual measured value is 391.

[0486] Step 2

[0487] Under a nitrogen atmosphere, 7b (8.00 g, 21.74 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL). p-Toluenesulfonic acid (374 mg, 2.18 mmol) and tert-butyl glycolate (8.60 g, 65.14 mmol) were added sequentially under an ice bath. After complete addition, the mixture was returned to room temperature and stirred for 1 h. The reaction mixture was diluted with ethyl acetate (200 mL). The organic phase was washed sequentially with water (50 mL x 2), saturated sodium bicarbonate (50 mL), and saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was isolated by silica gel column chromatography (EA: hexanes = 0-100%) to afford 7c (5.90 g) in a 62% yield.

[0488] MS-ESI calculated value [M+Na] + =463, the actual measured value is 463.

[0489] Step 3

[0490] Under nitrogen, 7c (4.90 g, 11.14 mmol) was dissolved in anhydrous acetonitrile (40 mL). 1,8-Diazabispiro[5.4.0]undec-7-ene (847 mg, 5.57 mmol) was added under ice-cooling. After complete addition, the mixture was returned to room temperature and stirred for 3 h. 7d (6.13 g, 11.14 mmol) was added to the reaction mixture. 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.34 mg, 16.68 mmol) and N,N-diisopropylethylamine (2.88 g, 22.32 mmol) were added sequentially under ice-cooling. After complete addition, the reaction was returned to room temperature and stirred for 1 h. The reaction solution was diluted with ethyl acetate (200 mL). The organic phase was washed with saturated brine (25 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain compound 7e (5.8 g). Yield: 74%.

[0491] MS-ESI calculated value [M+Na] + =724, the actual measured value is 724.

[0492] Step 4

[0493] Under a nitrogen atmosphere, 7e (2.80 g, 3.99 mmol) was dissolved in anhydrous acetonitrile (30 mL). 1,8-Diazabispiro[5.4.0]undec-7-ene (606 mg, 3.99 mmol) was added under ice-cooling. After complete addition, the mixture was returned to room temperature and stirred for 5 h. The reaction solution was cooled to 0°C, and 1-hydroxybenzotriazole (1.08 g, 7.98 mmol) was added portionwise. A jelly-like solid precipitated from the reaction system. Stirring was continued under ice-cooling for 1 h before filtration. The upper filter cake was slurried with isopropyl ether / n-hexane (10 mL / 40 mL) and filtered to obtain 7f (2.0 g).

[0494] MS-ESI calculated value [M+H] + =480, the actual measured value is 480.

[0495] Step 5

[0496] 7g (0.99g, 3.20mmol) and 7f (2.40g, 3.20mmol) were dissolved in a mixture of acetonitrile and water (15mL / 15mL). 2,4,6-Trimethylpyridine (387mg, 3.20mmol) was added under ice-cooling. After complete addition, the mixture was allowed to return to room temperature and stirred for 2h. The reaction solution was diluted with ethyl acetate (200mL). The organic phase was washed sequentially with water (30mL x 2), dilute hydrochloric acid (0.5N, 50mL x 2), and saturated brine (50mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was isolated by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 7h (1.25g) in a yield of 58%.

[0497] MS-ESI calculated value [M+Na] + =697, the actual measured value is 697.

[0498] Step 6

[0499] Under a nitrogen atmosphere, zinc bromide (8.00 g, 35.60 mmol) was weighed into a reaction flask, followed by nitromethane (15 mL). The mixture was stirred at room temperature for 10 min, resulting in an emulsion. The flask was placed in an ice bath, and a solution of 7h (1.20 g, 1.78 mmol) in nitromethane (20 mL) was added dropwise. After complete addition, the mixture was stirred and maintained in an ice bath for 4 h. The reaction solution was diluted with water (50 mL), and the pH of the aqueous phase was adjusted to 5-6 with aqueous ammonia (2% w / w). The phases were separated. The aqueous phase was washed with dichloromethane (50 mL x 3) and lyophilized. The lyophilized residue was isolated by silica gel column chromatography (MeOH:DCM = 0-100%) to afford 7i (450 mg) in a yield of 41%.

[0500] MS-ESI calculated value [M+Na] + =641, the actual measured value is 641.

[0501] 1 H NMR (400MHz, DMSO-d6) δ7.28-7.14(m,5H),6.97(s,2H),4.66-4.54(m,2H),4.43(dd,J=9.2,4.4Hz,1H),3.81( s,2H),3.77-3.52(m,12H),3.07(dd,J=14.0,4.8Hz,1H),2.81(dd,J=13.6,9.6Hz,1H),2.33(t,J=6.0Hz,2H).

[0502] Step 7

[0503] Compound 1d (50 mg, 0.11 mmol), 7i (74 mg, 0.12 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (31 mg, 0.16 mmol), and N,N-dimethylformamide (2 mL) were added sequentially to a 10 mL single-necked flask. 2,4,6-trimethylpyridine (60 mg, 0.49 mmol) was added dropwise to the mixture under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction solution was added dropwise to methyl tert-butyl ether (60 mL). A solid precipitated and was collected by filtration. The solid was dissolved in a dichloromethane / methanol mixture (24 mL / 4 mL) and concentrated to obtain a crude product. The crude product was purified by liquid preparative liquid separation to obtain compound 7 (16 mg) in a yield of 17%.

[0504] MS-ESI calculated value [M+H] + =1052, measured value: 1052.

[0505] 1 H NMR (400MHz, DMSO-d6) δ8.65(t,J=6.8Hz,1H),8.61(d,J=9.2Hz,1H),8.28(t,J=4. 4Hz,1H),8.14-8.06(m,2H),8.06(t,J=5.6Hz,1H),7.82(d,J=11.2Hz,1H),7.79(s ,1H),7.28-7.13(m,5H),6.99(s,2H),5.92(d,J=16.8Hz,1H),5.70-5,60(m,1H),5 .53(d,J=8.0Hz,1H),5.48(d,J=4.8Hz,1H),5.37(d,J=19.6Hz,1H),4.68(d,J=6.4 Hz,1H),4.51-4.41(m,1H),4.17-4.04(m,2H),3.72-3.68(m,2H),3.68-3.63(m,2H ),3.62-3.58(m,1H),3.58-3.54(m,3H),3.54-3.39(m,3H),3.48-3.43(m,2H),3.1 9-3.08(m,1H),3.06-2.97(m,1H),2.80-2.70(m,1H),2.70-2.65(m,1H),2.39(s,3 H),2.35-2.28(m,3H),2.27-2.18(m,2H),1.94-1.82(m,2H),0.85(t,J=7.2Hz,3H).

[0506] Synthesis Route 2

[0507] first step

[0508] Under a nitrogen atmosphere, 8a (4.50 g, 8.98 mmol) was dissolved in anhydrous N,N-dimethylformamide (25 mL). Acetic acid (1.19 g, 19.76 mmol), lead tetraacetate (7.96 g, 17.96 mmol), and copper acetate (1.10 g, 8.98 mmol) were added. The mixture was heated to 50°C and stirred for 0.5 h. The reaction mixture was quenched with water (50 mL), and the aqueous phase was extracted with ethyl acetate (500 mL). The organic phase was separated and washed sequentially with saturated sodium bicarbonate solution (100 mL x 1), water (100 mL), and saturated brine (50 mL x 4), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain crude product 8b (4.70 g).

[0509] MS-ESI calculated value [M+Na] + =538, the actual measured value is 538.

[0510] Step 2

[0511] Under a nitrogen atmosphere, 8b (4.70 g, 9.13 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL). p-Toluenesulfonic acid (235 mg, 1.37 mmol) and benzyl glycolate (5.30 g, 31.96 mmol) were added sequentially under an ice bath. After complete addition, the mixture was allowed to return to room temperature and stirred for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL). The organic phase was washed sequentially with water (50 mL x 1), saturated sodium bicarbonate solution (50 mL x 1), and saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was isolated by silica gel column chromatography (EA: hexanes = 0-100%) to afford 8c (4.50 g) in a yield of 79%.

[0512] MS-ESI calculated value [M+Na] + =644, the actual measured value is 644.

[0513] Step 3

[0514] Dissolve 8c (4.50 g, 7.25 mmol) in a mixture of tetrahydrofuran and water (50 mL / 15 mL). Add wet Pd / C (380 mg, 8% w / w). Stir and react overnight under a hydrogen atmosphere. The reaction mixture is filtered to remove insoluble matter, and the filtrate is concentrated under reduced pressure to yield crude product 8d (2.65 g).

[0515] MS-ESI calculated value [M+Na] + =554, the actual measured value is 554.

[0516] Step 4

[0517] Under a nitrogen atmosphere, 8d (2.65 g, 4.99 mmol) was dissolved in anhydrous acetonitrile (30 mL). 1,8-Diazabispiro[5.4.0]undec-7-ene (1.52 g, 9.98 mmol) was added under ice-cooling. After complete addition, the mixture was returned to room temperature and stirred for 5 h. The reaction solution was cooled to 0°C, and 1-hydroxybenzotriazole (1.35 g, 9.98 mmol) was added portionwise. A solid precipitated from the reaction system. Stirring was continued under ice-cooling for 2 h, and the mixture was filtered. The filter cake was collected and drained to obtain the crude product 8e (1.85 g).

[0518] MS-ESI calculated value [M+H] + =310, the actual measured value is 310.

[0519] Step 5

[0520] Dissolve 7g (3.10g, 10.00mmol) and 8f (1.45g, 11.00mmol) in a mixture of acetonitrile and water (25mL / 8mL). Add triethylamine (1.51g, 15.00mmol) under ice-cooling. After complete addition, return the mixture to room temperature and stir for 2h. The reaction mixture was vortexed to remove most of the acetonitrile, and the residue was diluted with dichloromethane / water (100mL / 50mL). The layers were separated, and the aqueous phase was washed with dichloromethane (50mL x 2) and lyophilized. The lyophilized residue was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to afford 8g (4.25g) of the crude product.

[0521] MS-ESI calculated value [M+H] + =328, the actual measured value is 328.

[0522] Step 6

[0523] 8g (1.03g) and N-hydroxysuccinimide (552mg, 4.80mmol) were dissolved in anhydrous dichloromethane (10mL). Dicyclohexylcarbodiimide (0.99g, 4.80mmol) was added under ice-cooling. After complete addition, the mixture was allowed to return to room temperature and stirred for 1.5h. The reaction mixture was filtered to remove insoluble solids, and the mother liquor was set aside. 8e (0.52g) and 2,4,6-trimethylpyridine (203mg, 1.68mmol) were added to the mother liquor under ice-cooling. The mixture was allowed to return to room temperature and stirred for 4h. The reaction mixture was diluted with water (50mL), and the aqueous phase was washed with dichloromethane (50mL x 2) and lyophilized. The lyophilized residue was separated by silica gel column chromatography (MeOH:DCM = 0-100%) to afford 7i (630mg).

[0524] MS-ESI calculated value [M+Na] + =641, the actual measured value is 641.

[0525] Step 7

[0526] Compounds 1d (110 mg, 0.24 mmol) and 7i (195 mg, 0.32 mmol) were added sequentially to a single-necked flask (10 mL), followed by anhydrous N,N-dimethylformamide (3 mL). The mixture was cooled to 0°C, and trifluoroacetic acid (29 mg, 0.25 mmol), 2,4,6-trimethylpyridine (133 mg, 1.10 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (68 mg, 0.35 mmol) were added dropwise. After complete addition, the mixture was returned to room temperature and stirred for 3 hours. The reaction solution was directly purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to afford compound 7 (86 mg) in a 34% yield.

[0527] MS-ESI calculated value [M+H] + =1052, the actual measured value is 1052.

[0528] Synthesis Route 3

[0529] first step

[0530] Under a nitrogen atmosphere, 9a (5.10 g, 11.48 mmol) was dissolved in anhydrous dichloromethane (100 mL). 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.54 g, 17.22 mmol), N,N-diisopropylethylamine (4.44 g, 34.44 mmol), and glycine tert-butyl ester (1.80 g, 13.78 mmol) were added sequentially. After complete addition, the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane (100 mL). The organic phase was washed sequentially with dilute hydrochloric acid (2N, 50 mL x 1), water (50 mL x 1), and saturated brine (50 mL x 2), dried, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 9b (6.80 g) as a white solid.

[0531] MS-ESI calculated value [M+H] + =558, the actual measured value is 558.

[0532] Step 2

[0533] Under nitrogen, the crude product 9b (6.30 g) from the previous step was dissolved in anhydrous acetonitrile (100 mL). 1,8-Diazacyclo[5,4,0]undecene-7 (3.44 g, 22.62 mmol) was added under ice-cooling. The mixture was allowed to return to room temperature and stirred for 5 h. The reaction flask was placed in an ice-cooling bath again, and Fmoc-glycine (4.03 g, 13.57 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.16 g, 13.57 mmol), and N,N-diisopropylethylamine (2.19 g, 16.97 mmol) were added sequentially. After the addition was complete, the reaction was stirred at room temperature for 1 h. The reaction solution was diluted with ethyl acetate (200 mL). The organic phase was washed sequentially with dilute hydrochloric acid (2N, 50 mL x 1), water (50 mL x 2), and saturated brine (50 mL x 2), then dried and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain crude product 9c (8.5 g).

[0534] MS-ESI calculated value [M+H] + =615, the actual measured value is 615.

[0535] Step 3

[0536] Under nitrogen, 9c (8.50 g, 13.83 mmol) was dissolved in anhydrous dichloromethane (20 mL). The mixture was cooled to 0°C in an ice-water bath, and trifluoroacetic acid (20 mL, 0.261 mol) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 4 hours. The mixture was concentrated under reduced pressure, and ethyl acetate (100 mL) and water (100 mL) were added to the residue. The pH of the solution was adjusted to between 4 and 5 with saturated sodium bicarbonate (solid precipitated and formed an emulsion with the aqueous phase). The layers were separated, and the aqueous emulsion was collected. The aqueous phase was washed with ethyl acetate (50 mL x 2) and the pH was adjusted to 1-2 with 1N aqueous hydrochloric acid. Dichloromethane (200 mL) was added, and the layers were separated. The aqueous phase was extracted with dichloromethane (150 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 9d (4.10 g) as a white solid.

[0537] MS-ESI calculated value [M+H] + =559, the actual measured value is 559.

[0538] Step 4

[0539] Under a nitrogen atmosphere, 9d (4.10 g, 7.34 mmol), acetic acid (1.78 g, 29.35 mmol), lead tetraacetate (13.00 g, 17.96 mmol), and copper acetate (1.80 g, 14.68 mmol) were added sequentially to a reaction flask. The mixture was heated to 50°C and stirred for 0.5 h. The reaction mixture was quenched by the addition of water (50 mL). The aqueous phase was extracted with dichloromethane (500 mL). The layers were separated, and the organic phase was washed sequentially with saturated sodium bicarbonate (100 mL x 1), water (100 mL), and saturated brine (50 mL x 4), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to yield the crude product 9e (3.84 g).

[0540] MS-ESI calculated value [M+Na] + =595, the actual measured value is 595.

[0541] Step 5

[0542] Under a nitrogen atmosphere, 9e (3.80 g, 6.60 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL). p-Toluenesulfonic acid (171 mg, 1.00 mmol) and benzyl glycolate (3.86 g, 25.20 mmol) were added sequentially under an ice bath. After complete addition, the mixture was allowed to return to room temperature and stirred for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL), and the organic phase was washed sequentially with water (50 mL x 1), saturated sodium bicarbonate solution (50 mL x 1), and saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was isolated by silica gel column chromatography (methanol:dichloromethane = 0-100%) to afford 9f (2.14 g) in a 48% yield.

[0543] MS-ESI calculated value [M+Na] + =701, the measured value is 701.

[0544] Step 6

[0545] 9f (2.10 g, 3.09 mmol) was dissolved in a mixture of tetrahydrofuran and water (50 mL / 15 mL). Wet Pd / C (168 mg, 8% w / w) was added and the mixture was stirred under a hydrogen atmosphere for 5 hours. The reaction mixture was filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure to yield 9 g (2.40 g) of the crude product.

[0546] MS-ESI calculated value [M+Na] + =611, the actual measured value is 611.

[0547] Step 7

[0548] Under a nitrogen atmosphere, 9g (2.18g, 3.70mmol) was dissolved in anhydrous acetonitrile (30mL). 1,8-Diazabisspiro[5.4.0]undec-7-ene (1.13g, 7.41mmol) was added under an ice bath. After complete addition, the mixture was returned to room temperature and stirred for 5h. The reaction solution was cooled to 0°C and 1-hydroxybenzotriazole (1.35g, 9.98mmol) was added portionwise. A solid precipitated from the reaction system. Acetonitrile (30mL) was added and the mixture was stirred at room temperature for 2h. The mixture was filtered and the filter cake was washed with acetonitrile (20mL x 2). The filter cake was collected and dried to obtain the crude product 9h (1.83g) as a white powder.

[0549] MS-ESI calculated value [M+H] + =367, the actual measured value is 367.

[0550] Step 8

[0551] 9i (500 mg, 1.43 mmol, 9i:2,4,6-trimethylpyridine = 1:0.655), 2,4,6-trimethylpyridine (494 mg, 4.08 mmol), and N-hydroxysuccinimide (206 mg, 1.79 mmol) were dissolved in anhydrous dichloromethane (25 mL). Dicyclohexylcarbodiimide (443 mg, 2.15 mmol) was added under ice-cooling. After the addition was complete, the mixture was stirred at 0°C for 3 h. 9h (600 mg, 1.60 mmol) was added to the reaction mixture under ice-cooling. The mixture was allowed to return to room temperature and stirred overnight. The reaction mixture was diluted with water (100 mL), and the aqueous phase was washed with dichloromethane (100 mL x 3). The aqueous phase was lyophilized, and the lyophilized residue was separated by silica gel column chromatography (MeOH:DCM=0-50%) to obtain 7i (550 mg) in a yield of 62%.

[0552] MS-ESI calculated value [M+Na] + =641, the actual measured value is 641.

[0553] Step 9

[0554] Compounds 1d (110 mg, 0.24 mmol) and 7i (195 mg, 0.32 mmol) were added sequentially to a single-necked flask (10 mL), followed by anhydrous N,N-dimethylformamide (3 mL). The mixture was cooled to 0°C, and trifluoroacetic acid (29 mg, 0.25 mmol), 2,4,6-trimethylpyridine (133 mg, 1.10 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (68 mg, 0.35 mmol) were added dropwise. After complete addition, the mixture was returned to room temperature and stirred for 3 hours. The reaction solution was directly purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to afford compound 7 (90 mg) in a 35% yield.

[0555] MS-ESI calculated value [M+H] + =1052, the actual measured value is 1052.

[0556] Example 1.8

[0557] first step

[0558] Under nitrogen protection, 10a (10 g, 22.91 mmol) and N6-Boc-L-lysine tert-butyl ester hydrochloride (9.32 g, 27.50 mmol) were dissolved in a tetrahydrofuran / water mixed solvent (V 四氢呋喃 :V 水 =4:1, 100 mL), the mixture was cooled to 0°C and stirred for 10 minutes, after which sodium bicarbonate (3.85 g, 45.83 mmol) was added. The mixture was stirred at 0°C for 2 hours. Water (100 mL) and ethyl acetate (100 mL) were added to the reaction solution, and the layers were separated. The aqueous phase was extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to obtain compound 10b (12.50 g) in a yield of 87%.

[0559] MS-ESI calculated value [M+Na] + 646, the actual measured value is 646.

[0560] Step 2

[0561] 10b (12.50 g, 20.04 mmol) was dissolved in a 4M solution of hydrogen chloride in dioxane (63 mL). The mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then purified by slurrying with methyl tert-butyl ether (150 mL) to obtain crude product 10c (11.40 g).

[0562] MS-ESI calculated value [M+H] + =468, the actual measured value is 468.

[0563] Step 3

[0564] Compound 10c (11.40 g) was dissolved in anhydrous dichloromethane (190 mL), and n-propionaldehyde (7.01 g, 120.70 mmol) was added. The mixture was stirred at room temperature for 15 minutes, followed by the addition of sodium triacetoxyborohydride (21.33 g, 100.64 mmol) in portions. After the addition was complete, the mixture was stirred at room temperature for 1 hour. The reaction was quenched with saturated aqueous ammonium chloride (10 mL), stirred at room temperature for 1 hour, and then water (200 mL) and ethyl acetate (100 mL) were added. The mixture was separated, and the aqueous phase was extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 10d (9.40 g) in a two-step yield of 85%.

[0565] MS-ESI calculated value [M+H] + =552, the actual measured value is 552.

[0566] Step 4

[0567] Under nitrogen, 10e (2.5 g, 5.27 mmol) was dissolved in anhydrous dichloromethane (35 mL), and 1,8-diazobisspiro[5.4.0]undec-7-ene (1.20 g, 7.88 mmol) was added. The mixture was cooled to 0°C and stirred for 10 minutes before returning to room temperature and stirring for 1 hour. The mixture was cooled to 0°C again, and 10d (3.49 g, 6.33 mmol), N,N-diisopropylethylamine (1.02 g, 7.89 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.01 g, 7.92 mmol) were added. After the addition was complete, the mixture was maintained at 0°C for 1 hour. Water (50 mL) and ethyl acetate (50 mL) were added to the reaction solution, and the layers were separated. The aqueous phase was extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to give compound 10f (2.00 g) in a yield of 48%.

[0568] MS-ESI calculated value [M+H] + =786, the actual measured value is 786.

[0569] Step 5

[0570] 10f (2.00 g, 2.54 mmol) was dissolved in a tetrahydrofuran / water mixed solvent (V 四氢呋喃 :V 水 =3:1, 40 mL), 10% wet palladium on carbon (200 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 3 hours. The reaction system was filtered to remove insoluble matter, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain a residual aqueous phase. The aqueous phase was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 10 g (930 mg), with a yield of 53%.

[0571] MS-ESI calculated value [M+H] + =696, the actual measured value is 696.

[0572] Step 6

[0573] Under a nitrogen atmosphere, 10 g (652 mg, 0.94 mmol) and 1d (330 mg, 0.73 mmol) were dissolved in anhydrous dichloromethane (5 mL). The mixture was cooled to 0°C, and N,N-diisopropylethylamine (189 mg, 1.46 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (330 mg, 0.87 mmol) were added. The mixture was stirred at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 10h (730 mg) in a yield of 88%.

[0574] MS-ESI calculated value [M+H] + =1129, the actual measured value is 1129.

[0575] Step 7

[0576] Under nitrogen, 10h (720 mg, 0.64 mmol) was dissolved in anhydrous tetrahydrofuran (7 mL), cooled to 0°C, and piperidine (272 mg, 3.19 mmol) was added. The mixture was stirred at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 10i (350 mg) in a yield of 61%.

[0577] MS-ESI calculated value [M+H] + =907, the actual measured value is 907.

[0578] 1H NMR (400MHz, DMSO-d6) δ7.78(s,1H),7.72(d,J=10.8Hz,1H),5.91(d,J=16.4Hz,1H),5.70-5.60(m,1H),5.49(d,J =16.4Hz,1H),4.74-4.62(m,2H),4.24-4.15(m,1H),4.13(d,J=6.0Hz,2H),3.81-3.61(m,2H),3.34-3.20(m,1H), 3.18(s,2H),3.16-3.07(m,1H),3.00(d,J=4.8Hz,1H),2.93-2.81(m,2H),2.37(s,3H),2.31-2.21(m,5H),1.99-1 .83(m,3H),1.67-1.46(m,4H),1.39-1.22(m,8H),0.91-0.82(m,6H),0.82(t,J=7.2Hz,6H),0.76(d,J=6.8Hz,3H).

[0579] Step 8

[0580] 10i (150 mg, 0.17 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), followed by the addition of 2,4,6-trimethylpyridine (60 mg, 0.50 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (94 mg, 0.25 mmol). The mixture was stirred at 0°C for 5 minutes before the addition of 4c (57 mg, 0.20 mmol). After complete addition, the mixture was stirred at 0°C for 1 hour. Water (5 mL) and ethyl acetate (10 mL) were added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to afford compound 10 (125 mg) in a 67% yield.

[0581] MS-ESI calculated value [M+H] + =1074, the actual measured value is 1074.

[0582] Example 1.9

[0583] first step

[0584] Under nitrogen protection, 11a (5.00 g, 12.18 mmol) and p-aminobenzyl alcohol (2.25 g, 18.27 mmol) were dissolved in anhydrous dichloromethane (100 mL). The mixture was cooled to 0°C and then N,N-diisopropylethylamine (3.94 g, 30.49 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.56 g, 14.62 mmol) were added. After the addition was complete, the mixture was stirred at 0°C for 1 hour. The reaction solution was added with water (200 mL) and a dichloromethane / methanol mixed solvent (V 二氯甲烷 :V 甲醇 =10:1, 300mL), separate the liquid, and the aqueous phase is washed with dichloromethane / methanol mixed solvent (V 二氯甲烷 :V 甲醇 =10:1, 300 mL x 2) and the organic phases were combined, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 11b (6.68 g) in a yield of 100%.

[0585] MS-ESI calculated value [M-OH] + =498, the actual measured value is 498.

[0586] Step 2

[0587] Under nitrogen protection, 11b (3.23 g, 6.26 mmol) and bis(4-nitrophenyl) carbonate (2.86 g, 9.40 mmol) were dissolved in anhydrous dichloromethane / N,N-dimethylformamide mixed solvent (V 二氯甲烷 :V N,N-二甲基甲酰胺 =2:1, 48 mL) was added with N,N-diisopropylethylamine (2.43 g, 18.80 mmol). After complete addition, the mixture was stirred at room temperature for 12 hours. The reaction solution was quenched with water (100 mL), and ethyl acetate (100 mL) was added. The layers were separated, and the aqueous phase was extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by slurrying with methyl tert-butyl ether (10 mL) to obtain compound 11c (2.00 g) in a yield of 47%.

[0588] MS-ESI calculated value [M+Na] + =703, the actual measured value is 703.

[0589] Step 3

[0590] Under nitrogen, 11c (1.27 g, 1.87 mmol) and 1d (430 mg, 0.95 mmol) were dissolved in anhydrous N,N-dimethylformamide (12 mL). The mixture was cooled to 0°C and three drops of triethylamine were added. The mixture was stirred at 0°C for 15 minutes before the addition of 1-hydroxybenzotriazole (299 mg, 2.21 mmol) and pyridine (2.15 g, 27.18 mmol). After complete addition, the mixture was stirred at 0°C for 15 minutes before returning to room temperature and stirring for 2 hours. Water (20 mL) and ethyl acetate (30 mL) were added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to afford compound 11d (550 mg) in a yield of 58%.

[0591] MS-ESI calculated value [M+H] + =993, the actual measured value is 993.

[0592] Step 4

[0593] 11d (500 mg, 0.50 mmol) was dissolved in 1,4-dioxane (5 mL), and piperidine (5 mL) was added. The mixture was stirred at room temperature for 2 hours. Petroleum ether (5 mL) was added dropwise to the reaction solution, causing a large amount of solid to precipitate. The solid was filtered, and the filter cake was dried to obtain 11e (386 mg) in a 94% yield.

[0594] MS-ESI calculated value [M+H] + =771, the actual measured value is 771.

[0595] 1H NMR (400MHz, DMSO-d6) δ7.76 (s, 1H), 7.66 (d, J = 10.4Hz, 1H), 7.60 (d, J = 8.0Hz, 2H), 7.43 (d, J = 8.4Hz, 2H), 5. 91(d,J=16.4Hz,1H),5.49(d,J=16.4Hz,1H),5.29(q,J=4.8Hz,1H),5.20-5.02(m,2H),4.50-4.39(m,1H),3.3 0-3.14(m,1H),3.14-3.02(m,1H),2.99(d,J=5.2Hz,1H),2.31(s,3H),2.30-2.21(m,1H),2.13-1.98(m,1H), 1.98-1.80(m,3H),1.30(d,J=7.2Hz,3H),0.88(d,J=6.8Hz,3H),0.84(t,J=7.2Hz,3H),0.78(d,J=6.8Hz,3H).

[0596] Step 5

[0597] 11e (150 mg, 0.20 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), followed by the addition of 2,4,6-trimethylpyridine (71 mg, 0.59 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (111 mg, 0.29 mmol). The mixture was stirred at 0°C for 5 minutes before the addition of 4c (67 mg, 0.23 mmol). After complete addition, the mixture was stirred at 0°C for 1 hour. Water (5 mL) and ethyl acetate (10 mL) were added to the reaction solution, and the layers were separated. The aqueous phase was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to afford compound 11 (38 mg) in a yield of 21%.

[0598] MS-ESI calculated value [M+H] + =938, the actual measured value is 938.

[0599] 1H NMR (400MHz, DMSO-d6) δ8.56–8.00(m,3H),7.78(s,1H),7.72(d,J=10.8Hz,1H),7.59(d,J=8.0Hz,2H),7.42(d,J=8.0Hz,2H),7.02( s,2H),5.92(d,J=16.4Hz,1H),5.65-5.42(m,2H),5.42-5.26(m,2H),5.23-4.97(m,2H),4.68-4.51(m,1H),4.41-4.27(m,1H),4.23 -4.11(m,1H),4.04-3.94(m,1H),3.92-3.77(m,1H),3.31-3.18(m,1H),3.16-2.92(m,1H),2.35(s,3H),2.30-2.20(m,1H),2.15-2. 02(m,1H),2.03-1.91(m,1H),1.92-1.82(m,2H),1.31-1.26m,3H),0.86(t,J=6.8Hz,3H),0.83-0.78(m,3H),0.77(d,J=6.8Hz,3H).

[0600] Example 1.10

[0601] first step

[0602] 12a (4.00 g, 6.65 mmol) and bis(4-nitrophenyl) carbonate (10.08 g, 33.14 mmol) were dissolved in anhydrous N,N-dimethylformamide (80 mL). N,N-diisopropylethylamine (2.40 g, 18.57 mmol) was added and the mixture was stirred at room temperature for 2 hours. Ethyl acetate (120 mL) and petroleum ether (240 mL) were added dropwise to the reaction solution. After the addition was complete, the mixture was stirred at room temperature for 10 minutes and filtered. The filter cake was collected and dried to obtain compound 12b (3.30 g) in a yield of 65%.

[0603] MS-ESI calculated value [M+H] + =767, the actual measured value is 767.

[0604] Step 2

[0605] Under nitrogen, 12b (675 mg, 0.88 mmol) and 1d (270 mg, 0.60 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). The mixture was cooled to 0°C, and three drops of triethylamine were added. After stirring for 15 minutes, 1-hydroxybenzotriazole (189 mg, 1.40 mmol) and pyridine (1.36 g, 17.19 mmol) were added. The mixture was stirred at 0°C for 15 minutes, then returned to room temperature and stirred for 2 hours. Water (20 mL) and ethyl acetate (30 mL) were added to the reaction solution, and the layers were separated. The aqueous phase was extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 12c (350 mg) in a yield of 54%.

[0606] MS-ESI calculated value [M+Na] + =1101, the actual measured value is 1101.

[0607] Step 3

[0608] 12c (570 mg, 0.53 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and piperidine (450 mg, 5.28 mmol) was added. The mixture was stirred at room temperature for 3 hours. Methyl tert-butyl ether (15 mL) was added to the reaction solution to precipitate a solid. The mixture was stirred for 30 minutes, filtered, and the filter cake was dried to obtain compound 12d (400 mg) in an 88% yield.

[0609] MS-ESI calculated value [M+H] + =857, the actual measured value is 857.

[0610] Step 4

[0611] 12d (210 mg, 0.25 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of 2,4,6-trimethylpyridine (89 mg, 0.73 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (140 mg, 0.37 mmol). The mixture was stirred at 0°C for 5 minutes before the addition of 4c (71 mg, 0.25 mmol). After complete addition, the mixture was stirred at 0°C for 1 hour. Water (5 mL) and ethyl acetate (10 mL) were added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to afford compound 12 (30 mg) in a 12% yield.

[0612] MS-ESI calculated value [M+H]+ =1024, the actual measured value is 1024.

[0613] 1 H NMR(500MHz,DMSO-d6)δ7.79(s,1H),7.75(d,J=10.5Hz,1H),7.59(d,J=7.5Hz ,1H),7.42(d,J=8.5Hz,1H),7.02(s,2H),5.92(d,J=17.0Hz,1H),5.57(d,J=2 0.0Hz,1H),5.51(d,J=16.5Hz,1H),5.37(d,J=19.5Hz,1H),5.33-5.28(m,1H) ,5.16(d,J=12.5Hz,1H),5.08(d,J=12.5Hz,1H),4.60-4.54(m,1H),4.37-4.3 0(m,1H),4.20-4.14(m,1H),4.00-3.93(m,1H),3.88-3.81(m,1H),3.30-3.21 (m,1H),3.15-3.06(m,1H),3.05-2.89(m,2H),2.36(s,3H),2.31-2.23(m,1H) ,2.14-2.03(m,1H),2.02-1.93(m,1H),1.92-1.82(m,2H),1.74-1.64(m,1H), 164-1.53(m,1H),1.48-1.31(m,2H),0.89-0.80(m,6H),0.76(d,J=6.5Hz,3H).

[0614] Example 1.11

[0615] first step

[0616] Compound 12b (427 mg, 0.56 mmol) and compound 13a (270 mg, 0.38 mmol) were dissolved in anhydrous N,N-dimethylformamide (15 mL), and 1-hydroxybenzotriazole (51 mg, 0.38 mmol) and pyridine (744 mg, 9.41 mmol) were added. The mixture was stirred at room temperature for 12 hours. Water (20 mL) and ethyl acetate (30 mL) were added to the reaction system, and the mixture was separated. The aqueous phase was extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 13b (220 mg) in a yield of 43%.

[0617] MS-ESI calculated value [(1 / 2M)+H] + =673, the actual measured value is 673.

[0618] Step 2

[0619] 13b (200 mg, 0.15 mmol) was dissolved in anhydrous tetrahydrofuran / anhydrous N,N-dimethylformamide mixed solvent (V 四氢呋喃 :V N,N-二甲基甲酰胺 =2:1, 5.4 mL) was added with piperidine (66 mg, 0.78 mmol), and the mixture was stirred at room temperature for 1 hour. The reaction solution was directly purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 13c (110 mg) in a yield of 66%.

[0620] MS-ESI calculated value [M+H] + =1123.7, the measured value is 1123.4.

[0621] Step 3

[0622] 13c (110 mg, 0.10 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), followed by the addition of 2,4,6-trimethylpyridine (25 mg, 0.21 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (59 mg, 0.16 mmol). The mixture was stirred at 0°C for 5 minutes before the addition of 4c (55 mg, 0.19 mmol). After complete addition, the mixture was stirred at 0°C for another hour. Water (5 mL) and ethyl acetate (10 mL) were added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to afford compound 13 (54 mg) in a 43% yield.

[0623] MS-ESI calculated value [M+H] + =1290.7, the actual measured value is 1290.4.

[0624] 1H NMR(500MHz,DMSO-d6)δ8.05(d,J=8.5Hz,1H),7.58-7.52(m,2H),7.36-7.21(m,5H),7.21-7.10(m,1H),7.02(s,2H),5.14-4.91(m,2H),4.60-4.5 2(m,1H),4.51-4.44(m,1H),4.44-4.36(m,1H),4.36-4.30(m,1H),4.28- 4.20(m,1H),4.20-4.14(m,1H),4.05-3.90(m,3H),3.85(dd,J=11.0,9.0 Hz,1H),3.59-3.51(m,1H),3.25–3.20(m,3H),3.21–3.15(m,3H),3.10(s ,1H),3.07-2.90(m,3H),2.88-2.80(m,2H),2.43-2.34(m,1H),2.30-2.2 0(m,1H),2.16-1.88(m,4H),1.85-1.63(m,4H),1.62-1.22(m,6H),1.06- 0.96(m,6H),0.89-0.86(m,2H),0.85-0.80(m,10H),0.80-0.70(m,12H).

[0625] Example 1.12

[0626] first step

[0627] Under nitrogen, imidazole (161.60 g, 2373.68 mmol) was dissolved in anhydrous N,N-dimethylformamide (1.25 L). The mixture was cooled to 0±5°C and triisopropylsilyl chloride (458.20 g, 2376.56 mmol) was added dropwise. The mixture was stirred at 0±5°C for 30 minutes. A solution of 14a (250.00 g, 949.67 mmol) in N,N-dimethylformamide (1250 mL) was added dropwise to the reaction system. After the addition was complete, the mixture was stirred at 0±5°C for 18 hours. Imidazole (129.30 g, 1899.24 mmol) was added and stirred for 10 minutes. Triethylsilyl chloride (286.50 g, 1900.86 mmol) was added dropwise. After the addition was complete, the mixture was stirred at 0±5°C for 18 hours. Methanol (250 mL) was added dropwise to the reaction system, and the mixture was stirred at 0°C for 1 hour. After returning to room temperature, the reaction solution was diluted with water (5000 mL), and the aqueous phase was extracted with methyl tert-butyl ether (5000 mL x 2). The organic phases were combined. The organic phase was washed with a sodium chloride solution (2500 mL x 2, 5%), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (n-heptane:ethyl acetate = 0-100%) to obtain compound 14b (479.10 g) with a purity of 99.95%, an assay of 93.5%, and an equivalent yield of 88%.

[0628] MS-ESI calculated value [M+H] + =534, the actual measured value is 534.

[0629] 1 H NMR (400MHz, CDCl3) δ6.70(s,1H),6.14(d,J=16.4Hz,1H),5.69-5.66(m,1H),5.59(d,J=16.4Hz,1H),5.16(d,J=16.0Hz,1H),4 .53(d,J=2.4Hz,2H),1.87-1.75(m,2H),1.37-1.23(m,3H),1.13(d,J=7.6Hz,18H),0.95-0.87(m,12H),0.67(q,J=7.6Hz,6H).

[0630] Step 2

[0631] Under nitrogen, 14b (450.0 g, content: 93.5%, 788.13 mmol) was dissolved in toluene (4.5 L), sodium carbonate (25.10 g, 236.81 mmol) and Lawesson's reagent (382.50 g, 945.68 mmol) were added, and the mixture was heated to 110° C. and stirred for 3 hours. The reaction system was cooled to 50-60° C., and n-heptane (4500 mL) was added dropwise to the reaction system. After stirring for 1 hour, the mixture was filtered, and the filter cake was rinsed with n-heptane (900 mL). The organic phases were combined and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (n-heptane:ethyl acetate = 0-100%) to obtain compound 14c (517.0 g), which was used directly in the next step.

[0632] MS-ESI calculated value [M+H] + =550, the actual measured value is 550.

[0633] 1 H NMR (400MHz, CDCl3) δ7.20(s,1H),6.14(d,J=16.4Hz,1H),5.87(t,J=2.0Hz,1H),5.26(d,J=16.8Hz,1H),4.82(d ,J=2.0Hz,1H),1.90-1.75(m,2H),1.41-1.27(m,3H),1.18-1.11(m,18H),0.97-0.85(m,12H),0.77-0.66(m,6H).

[0634] Step 3

[0635] Under nitrogen, the crude product 14c (517.0 g) from the previous step was dissolved in anhydrous tetrahydrofuran (4.5 L). Hydrofluoric acid (295.50 g, 48 wt.%, 7092.00 mmol) was added at room temperature. After complete addition, the reaction was stirred at room temperature for 36 hours. The reaction solution was diluted with water (4500 mL), the aqueous phase was extracted with ethyl acetate (4500 mL x 2), and the organic phases were combined. The organic phase was washed with saturated brine (2250 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate = 0-100%) to obtain the crude product. Methyl tert-butyl ether (2250 mL) was added to the crude product and beaten for 1 hour. The product was filtered to obtain compound 14d (194.60 g), with a content of 94% and a two-step yield of 83%.

[0636] MS-ESI calculated value [M+H] + =280, the actual measured value is 280.

[0637] 1H NMR (400MHz, CDCl3) δ6.63 (s, 1H), 5.82 (d, J = 17.2Hz, 1H), 5.41 (d, J = 17.2Hz, 1H), 4.45-4.30(m,2H),2.99-2.92(m,2H),1.80(q,J=7.2Hz,2H),0.78(t,J=7.2Hz,3H).

[0638] Step 4

[0639] Under nitrogen protection, water (8.0 L) and HCl (aq) 14e (800.00 g, 3196.55 mmol) was added to a 50 L reactor, followed by the addition of 14e (800.00 g, 3196.55 mmol). The mixture was heated to 100±5°C and stirred for 4 hours. The reaction system was cooled to 30°C, and aqueous ammonia (3500 mL, 25%-28%) was slowly added dropwise to adjust the pH to 4-5 (the reaction temperature was kept below 40°C during the addition). After the addition was complete, the mixture was stirred for 30 minutes, filtered, and the filter cake was rinsed with water (2500 mL x 1). The filter cake was slurried in ethanol (8 L) at room temperature for 1 hour, filtered, and rinsed with ethanol (1000 mL x 1). The filter cake was collected and air-dried at 50±5°C to yield 14f (709.00 g), yield: 91%.

[0640] MS-ESI calculated value [M+H] + =209, the actual measured value is 209.

[0641] Step 5

[0642] Tetrahydrofuran (10200 mL), water (10200 mL), and 14f (680.00 g, 2779.03 mmol) were added to a 50 L reactor. The mixture was cooled to 0-10°C, potassium carbonate (653.00 g, 4724.69 mmol) was added, and a solution of 9-fluorenylmethyl chloroformate (733.31 g, 2834.60 mmol) in tetrahydrofuran (3400 mL) was slowly added dropwise (the reaction mixture temperature was maintained at 0-10°C during the addition). The mixture was stirred at 0-10°C for 1 hour. The reaction mixture was extracted with 2-methyltetrahydrofuran (10200 mL x 2), and the organic phase was washed sequentially with water (10200 mL x 1) and saturated brine (10000 mL x 1). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. Methyl tert-butyl ether (6800 mL) was added to the residue, and the mixture was stirred at room temperature for 4 hours. The mixture was filtered, and the filter cake was rinsed with methyl tert-butyl ether (680 mL x 1). The filter cake was collected and dried under air at 50±5°C to obtain 14 g (1090.00 g), with a yield of 91%.

[0643] MS-ESI calculated value [M+H] + =431, the actual measured value is 431.

[0644] 1 H NMR (400MHz, DMSO-d6) δ7.90 (d, J = 7.2Hz, 2H), 7.80-7.73 (m, 2H), 7.53-7.28 (m, 6H), 6.39 (d, J = 12. 8Hz,1H),4.40-4.15(m,4H),3.03-2.75(m,2H),2.20-2.07(m,1H),2.04-1.87(m,1H),1.98(s,3H).

[0645] Step 6

[0646] Under nitrogen, 7 g (150.00 g, 483.47 mmol) was added to a three-necked flask (3 L), followed by ethyl acetate (1200 mL). A solution of 14h (111.20 g, 90% content, 531.69 mmol) in ethyl acetate (300 mL) was added dropwise. After complete addition, the mixture was stirred at room temperature for 2-6 hours. Saturated brine (450 mL) was added to the reaction system, stirred at room temperature for 10 minutes, and the liquids were separated, retaining the organic phase. The organic phase was washed with saturated brine (450 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to obtain compound 14i (134.00 g) in a yield of 73%.

[0647] MS-ESI calculated value [M+H] + =384, the actual measured value is 384.

[0648] 1 H NMR (400MHz, DMSO-d6) δ8.13-8.05(m,2H),7.01(s,2H),3.71(d,J=6.0Hz,2H),3.70(d, J=6.0Hz,2H),3.61-3.52(m,4H),3.50-3.44(m,2H),2.33(t,J=6.4Hz,2H),1.40(s,9H).

[0649] Step 7

[0650] Under nitrogen, 14i (184.30 g, 480.70 mmol) was added to a three-necked flask (3 L), followed by toluene (1.84 L) and trifluoroacetic acid (823.27 g, 7220.21 mmol). After the addition was complete, the mixture was stirred at room temperature for 4.5 hours. The reaction solution was concentrated under reduced pressure to obtain a residue, toluene (600 mL) was added to the residue, and the mixture was further concentrated under reduced pressure for three consecutive times until a solid precipitated. Ethyl acetate (1.7 L) was added to the solid, and the mixture was beaten for 2 hours and filtered to obtain compound 8g (150.20 g) in a yield of 96%.

[0651] MS-ESI calculated value [MH] - =326, the actual measured value is 326.

[0652] 1 H NMR (400MHz, DMSO-d6) δ8.12-8.05 (m, 2H), 7.01 (s, 2H), 3.75 (d, J = 6.0Hz, 2H), 3. 70(d,J=5.6Hz,2H),3.60-3.52(m,4H),3.50-3.44(m,2H),2.33(t,J=6.4Hz,2H).

[0653] Step 8

[0654] Under nitrogen protection, 8g (20.00g, 61.11mmol) was dissolved in dichloromethane (200mL), cooled to 0±5°C, and pentafluorophenol (16.90g, 91.82mmol) was added. The mixture was stirred for 10 minutes, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.60g, 91.82mmol) was added. The mixture was kept at 0±5°C and stirred for 4-16 hours. The reaction solution was diluted with water (200 mL), stirred for 10 minutes, and the layers were separated. The organic phase was washed with saturated brine (100 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography (acetone:dichloromethane = 0-100%) to give a crude product. Methyl tert-butyl ether (150 mL) was added to the crude product, and the mixture was stirred at room temperature for 1 hour. The mixture was filtered and the filter cake was dried at 35°C to give compound 14j (23.00 g). Yield: 76%.

[0655] MS-ESI calculated value [M+H] + =494, the actual measured value is 494.

[0656] 1H NMR (400MHz, CDCl3) δ7.12(d,J=5.6Hz,1H),6.96(t,J=5.6Hz,1H),6.70(s,1H),4.42(d,J=6.0Hz,2H),4.03 (d,J=6.0Hz,1H),3.72(t,J=5.2Hz,2H),3.69(t,J=5.6Hz,2H),3.59(t,J=5.2Hz,2H),2.49(t,J=5.6Hz,2H).

[0657] Step 9

[0658] Under nitrogen, 8e (7.50 g, 24.25 mmol) was dissolved in tetrahydrofuran (400 mL) and stirred at room temperature for 1 hour. 14j (10.00 g, 20.27 mmol) was then added. After complete addition, the mixture was stirred at room temperature for 16 hours. Ethyl acetate (400 mL) was added to the reaction solution, stirred for 1 hour, filtered, and the filter cake dried. The filter cake was dissolved in a dichloromethane / methanol (v:v = 20:1, 100 mL) mixture, and the mixture was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to give a crude product (14.51 g). The crude product was dissolved in methanol (30 mL), and ethyl acetate (450 mL) was added dropwise to the methanol solution. The mixture was stirred at room temperature for 24 hours, filtered, and the filter cake dried to give compound 7i (10.20 g) in a yield of 81%.

[0659] MS-ESI calculated value [M+Na] + =641, the actual measured value is 641.

[0660] Step 10

[0661] Under nitrogen, toluene (3200 mL), acetic acid (3200 mL), 14d (320.00 g, 1145.68 mmol), 14g (592.00 g, 1375.21 mmol), and pyridinium p-toluenesulfonate (144.00 g, 573.02 mmol) were added sequentially to a 10 L reaction flask. The mixture was heated to 110°C and stirred for 24 hours. The reaction mixture was cooled to 30±10°C and transferred to an 80 L extraction vessel. 2-Methyltetrahydrofuran (6400 mL) and water (6400 mL) were added sequentially. The mixture was stirred for 10-20 minutes, and the mixture was separated, retaining the upper organic phase. The organic phase was washed sequentially with water (4800 mL x 3) and saturated aqueous sodium chloride solution (4800 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure (50±5°C) until almost no liquid dripped out. The residue was steamed twice with methyl tert-butyl ether (640 mL x 2). The crude product 1 was added with methyl tert-butyl ether (3200 mL) and slurried for 2 to 2.5 hours. The product was filtered, and the filter cake was rinsed twice with methyl tert-butyl ether (640 mL x 2). The filter cake was collected and dried to obtain the crude product 2. The crude product 2 was dissolved in N,N-dimethylformamide (6400 mL), and methyl tert-butyl ether (25600 mL) was added dropwise. After the addition was completed, the mixture was stirred and crystallized at 20-30°C for 16-18 hours, filtered, and the filter cake was slurried with methyl tert-butyl ether (3200 mL) for 2-2.5 hours and then filtered. The filter cake was rinsed twice with methyl tert-butyl ether (640 mL×2), and the filter cake was collected and air-dried (50±5°C) to obtain 14k (490 g). The yield was 63%.

[0662] MS-ESI calculated value [M+H] + =674, the actual measured value is 674.

[0663] 1 H NMR(400MHz, DMSO-d6)δ8.16-8.06(m,1H),7.85(d,J=7.2Hz,2H),7.82-7.74(m,2H),7.74-7.6 4(m,2H),7.42-7.33(m,2H),7.33-7.23(m,2H),6.76-6.64(m,2H),5.97-5.81(m,1H),5.58-5. 40(m,2H),5.38-5.20(m,2H),4.68-4.52(m,1H),4.34(s,2H),3.28-3.14(m,1H),3.12-2.96(m ,1H),2.32(s,3H),2.28-2.18(m,1H),2.18-2.04(m,1H),2.00-1.82(m,2H),0.94-0.82(m,3H).

[0664] Step 11

[0665] Under nitrogen atmosphere, tetrahydrofuran (19200 mL) and 14k (480 g, 712.42 mmol) were added to the reactor in sequence, the mixed system was cooled to 0-5 ° C, and piperidine (546 g, 6412.21 mmol) was slowly added dropwise to the reaction flask (temperature was controlled at 0-5 ° C during the addition). After the addition was completed, the reaction system was kept at 0-5 ° C with stirring for 22-24 hours. To the reactor was slowly added aqueous hydrochloric acid solution (1440 mL, 6N) (temperature controlled at 0-10 ° C during addition), the mixed system was continued to stir for 10-15 minutes, and concentrated under reduced pressure to obtain a residue. The residue was slurried with water (9600 mL) for 2-2.5 hours, filtered, and the filter cake was rinsed with acetonitrile (480 mL x 2). The filter cake was collected and dried for preparative separation and purification (preparative instrument: Hanbang industrial-grade preparative liquid chromatograph, specification model: DAC150, preparative column packing: Welch Ultimate XB-C18, 150 * 250 mm, 10 μm). The preparative solution was adjusted to pH 7-8 using 5% ammonia water, stirred for 30-35 minutes and then filtered. The filter cake was rinsed with water (1000 mL x 1), the filter cake was collected, and the filter cake was air-dried (60 ° C) for 24 ± 2 hours to obtain 1d (97 g), with a yield of 30%.

[0666] MS-ESI calculated value [M+H] + =452, the actual measured value is 452.

[0667] Step 12

[0668] Under nitrogen protection, N,N-dimethylformamide (1200 mL) was added to the reaction flask, 1d (60.00 g, 132.89 mmol) and trifluoroacetic acid (15.20 g, 133.31 mmol) were added to the reaction flask, the mixed system was cooled to 0-10 ° C, stirred for ten minutes, and then 7i (115.10 g, 186.07 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (51.00 g, 266.04 mmol) and 2,4,6-trimethylpyridine (16.10 g, 132.86 mmol) were added, and the reaction system was stirred at 0-10 ° C for 1 hour. Aqueous hydrochloric acid (1200 mL, 0.05 N) was added dropwise to the reaction flask, followed by the addition of water (1800 mL), stirring for 10 to 20 minutes, filtering, washing the filter cake with water (300 mL x 2), collecting the filter cake, dissolving the filter cake in dichloromethane / methanol (V / V = 30 / 1, 1800 mL), drying the solution over anhydrous sodium sulfate, and filtering the filtrate. Purification by silica gel column chromatography (methanol: dichloromethane = 0-100%) afforded the crude product. The crude product was slurried with methyl tert-butyl ether (600 mL) for 1 to 1.5 hours, filtered, and the filter cake was rinsed with methyl tert-butyl ether (300 mL x 2). The filter cake was collected and dried on a rotary evaporator for 5 ± 1 hours to afford compound 7 (90.0 g), yield: 64%.

[0669] MS-ESI calculated value [M+H] + =1052, the actual measured value is 1052.

[0670] Example 1.13

[0671] first step

[0672] Under nitrogen, 15a (438.00 g, 904.0 mmol) was dissolved in ethylene glycol dimethyl ether (4380 mL). Water (2630 mL) was added and the reaction temperature was lowered to 0-5°C. Glycine (238.87 g, 1808.0 mmol) and sodium bicarbonate (151.87 g, 1808.0 mmol) were added sequentially. After the addition was complete, the reaction temperature was raised to 20-25°C and stirred for 1 hour. The reaction solution was cooled to -5-0°C, and dilute hydrochloric acid (0.5 M, 4380 mL) was added and stirred for 10 minutes. The aqueous phase was extracted with 2-methyltetrahydrofuran (8760 mL x 1) and 2-methyltetrahydrofuran (4380 mL x 1) in sequence. The organic phases were combined. The organic phase was washed with saturated brine (4380 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was spin-dried to obtain the crude product, which was slurried with acetone (8760 mL) for 16 hours and then filtered. The filter cake was dried to obtain 8a (375.00 g) in a yield of 83%.

[0673] MS-ESI calculated value [M+H] + =502, the actual measured value is 502.

[0674] Step 2

[0675] 8a (375.00 g, 747.69 mmol) was dissolved in N,N-dimethylformamide (4000 mL). Lead tetraacetate (730.11 g, 1646.70 mmol), copper acetate (135.95 g, 748.50 mmol) and acetic acid (98.80 g, 1646.70 mol) were added to the reaction flask in sequence. The mixture was heated to 45-50 °C and stirred for 0.5 h. The reaction solution was added to 2-methyltetrahydrofuran (7500 mL) and water (7500 mL), the mixture was separated, and the aqueous phase was extracted with 2-methyltetrahydrofuran (3750 mL x 1). The organic phases were combined and washed sequentially with saturated aqueous sodium bicarbonate solution (3750 mL x 1), water (3750 mL x 1), and saturated brine (3750 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain a crude product, which was slurried with methyl tert-butyl ether (3750 mL) for 16 hours and then filtered. The filter cake was air-dried for 16 hours to obtain 8b (345.00 g) in a yield of 89%.

[0676] MS-ESI calculated value [M-CH3COO] + =456, the actual measured value is 456.

[0677] 1 H NMR (400MHz, DMSO-d6) δ8.67(t,J=6.8Hz,1H),8.34(t,J=5.6Hz,1H),7.87(d,J=7.6Hz,1H),7.71-7.58(m,3H),7.50-7.10(m,9H),5.18-5.04( m,2H),4.40-4.00(m,4H),3.78(d,J=5.6Hz,2H),3.83-3.69(m,2H),3.05(dd,J=13.6,4.0Hz,1H),2.80(dd,J=13.2,10.8Hz,1H),1.98(s,3H).

[0678] Step 3

[0679] Under nitrogen, 8b (345.00 g, 669.17 mmol) was dissolved in tetrahydrofuran (3450 mL) and the reaction system was cooled to 0-5°C. Benzyl glycolate (244.47 g, 1472.17 mmol) and p-toluenesulfonic acid (17.20 g, 100.37 mmol) were added to the reaction system in sequence. After the addition was complete, the reaction system was heated to 20-25°C and stirred for 1 hour. Ethyl acetate (3450 mL) and water (3450 mL) were added to the reaction system, and the mixture was separated. The aqueous phase was extracted with ethyl acetate (3450 mL x 1). The organic phases were combined and washed sequentially with water (3450 mL x 1) and brine (3450 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to give 8c (178.00 g) in a yield of 42%.

[0680] MS-ESI calculated value [M+Na] + =644, the actual measured value is 644.

[0681] 1 H NMR (400MHz, DMSO-d6) δ8.63(t,J=6.8Hz,1H),8.35(t,J=5.6Hz,1H),7.87(d,J=7.6Hz,1H),7.71-7.60(m,3H),7.45-7.13(m,14H),5.13(s,2 H),4.67-4.61(m,2H),4.32-4.24(m,1H),4.22-4.09(m,5H),3.83-3.69(m,2H),3.06(dd,J=13.6,4.0Hz,1H),2.80(dd,J=13.6,10.4Hz,1H).

[0682] Step 4

[0683] Under nitrogen, 8c (102.00 g, 164.07 mmol) was dissolved in dichloromethane (1020 mL). The reaction system was cooled to -5-0°C, and 1,8-diazobisspiro[5.4.0]undec-7-ene (24.97 g, 164.07 mmol) was added. After complete addition, the mixture was stirred at 0-5°C for 1 hour. The reaction solution was directly purified by silica gel column chromatography (dichloromethane:isopropanol = 0-100%) to obtain 15b (43.50 g) in a 66% yield.

[0684] MS-ESI calculated value [M+H] + =400, the actual measured value is 400.

[0685] 1H NMR (400MHz, DMSO-d6) δ8.64(t,J=6.4Hz,1H),8.26-8.14(m,1H),7.45-7.15(m,10H),5.15(s,2H),4.64(d,J=6.8Hz,2H) ,4.15(s,2H),3.81-3.65(m,2H),3.44(dd,J=8.4,4.4Hz,1H),2.98(dd,J=13.6,4.8Hz,1H),2.59(dd,J=13.6,8.8Hz,1H).

[0686] Step 5

[0687] Under nitrogen, 15b (43.00 g, 107.65 mmol) was dissolved in a mixture of tetrahydrofuran (430 mL) and water (430 mL). The reaction system was cooled to 0-10°C, and wet Pd / C (6.50 g) was added. The atmosphere was replaced with hydrogen three times, and the reaction was stirred at 0-10°C for 24 hours. The reaction mixture was filtered, and the filter cake was washed sequentially with tetrahydrofuran (43 mL) and water (43 mL). The filtrate was concentrated under reduced pressure to remove tetrahydrofuran. The residual aqueous phase was washed with 2-methyltetrahydrofuran (430 mL x 2) and lyophilized to afford 8e (31.50 g) in a 95% yield.

[0688] MS-ESI calculated value [MH] - =308, the actual measured value is 308.

[0689] 1 H NMR(400MHz,DMSO-d6)δ9.07(s,1H),8.67(s,1H),7.33-7.26(m,2H),7.25-7.18(m,3H),4.68-4.56(m,2H), 3.92-3.71(m,4H),3.52(dd,J=16.4,4.0Hz,1H),3.01(dd,J=13.6,6.4Hz,1H),2.79(dd,J=13.2,7.6Hz,1H).

[0690] Step 6

[0691] 4a (1000.00 g, 9515.65 mmol) and acetonitrile (10.00 L) were added sequentially to a 20 L reaction flask. Tert-butyldimethylsilyl chloride (1505.92 g, 9991.42 mmol) was added to the reaction flask. The reaction system was cooled to 0-10°C, and 1,8-diazobisspiro[5.4.0]undec-7-ene (1376.18 g, 9039.84 mmol) was slowly added. After the addition was complete, the reaction system was returned to room temperature and stirred for 40 hours. The reaction mixture was filtered directly, and the resulting filter cake was dissolved in methanol (18.00 L) and filtered. Acetonitrile (54.00 L) was added to the filtrate, and the mixture was stirred for 2 hours to precipitate a solid. The filter cake was collected by filtration and dried to obtain 4b (1481.60 g) in a yield of 71%.

[0692] MS-ESI calculated value [MH] - =218, the actual measured value is 218.

[0693] Step 7

[0694] 4b (500.00 g, 2279.36 mmol) and tetrahydrofuran (3.75 L) were added to a reaction flask. The reaction system was cooled to 0-5°C, and N-methoxycarbonylmaleimide (395.00 g, 85% content, 2164.59 mmol) was added. After complete addition, the mixture was returned to room temperature and stirred for 16 hours. The reaction solution was cooled again to 0-5°C, and 10% aqueous sodium bicarbonate solution (3.75 L) was added (the temperature was controlled to <20°C during the addition process). After the addition was complete, the reaction system was heated to 30°C and stirred for 88 hours. The reaction solution was cooled to 0-5°C, and 5% aqueous citric acid solution was added dropwise to adjust the pH to 5-6. Ethyl acetate (3.75 L) was added, stirred, and the mixture separated. The aqueous phase was extracted with ethyl acetate (1.50 L). The organic phases were combined and washed sequentially with water (3.75 L x 1) and saturated brine (3.75 L x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to give compound 15c (350.00 g), with a content of 88% and a yield of 45%.

[0695] MS-ESI calculated value [M+H] + =300, the actual measured value is 300.

[0696] 1 H NMR (400MHz, DMSO-d6) δ13.35(br s,1H),7.14(s,2H),4.69-4.81(m,1H),4.13-3.97(m,2H),0.77(m,9H),0.00(s,3H),-0.06(s,3H).

[0697] Step 8

[0698] Dichloromethane (1260 mL) and 15c (210.00 g, 701.40 mmol) were added to a reaction flask, and the reaction system was cooled to 0-5°C. A solution of N,N'-dicyclohexylcarbodiimide (159.30 g, 772.56 mmol) in dichloromethane (420 mL) and a solution of tetrafluorophenol (122.46 g, 737.40 mmol) in dichloromethane (420 mL) were added sequentially, and the reaction system was stirred at 0-5°C for 1 hour. The reaction solution was filtered, the filter cake was washed with dichloromethane (1050 mL), and the filtrate was dried to obtain a residue. Methyl tert-butyl ether (2100 mL) was added to the residue, and the mixture was stirred for 10 minutes and then filtered. The filter cake was rinsed with methyl tert-butyl ether (630 mL), and the filtrate was concentrated to obtain a residue. n-hexane (2100 mL) was added to the residue and the mixture was beaten for 30 minutes and then filtered. The filter cake was washed with n-hexane (630 mL), filtered, and dried to obtain 15d (246.78 g). Yield: 79%.

[0699] MS-ESI calculated value [M+H] + =448, the actual measured value is 448.

[0700] 1 H NMR (400MHz, CD3OD) δ7.48-7.36(m,1H),6.98(s,2H),5.30(dd,J=9.2,6.0Hz,1H),4.34-4.23(m,2H),0.84(s,9H),0.07(s,3H),0.02(s,3H).

[0701] Step 9

[0702] Water (1470 mL) and diglycine (79.58 g, 602.91 mmol) were added to the reaction flask, stirring was started, and ethylene glycol dimethyl ether (2450 mL) was added. The reaction system was cooled to 5-10 ° C. 15d (245.00 g, 547.53 mmol) and sodium bicarbonate (92.09 g, 1096.18 mmol) were added to the reaction system, and the mixed system was stirred for 40 hours. The reaction solution was washed with methyl tert-butyl ether (2450 mL x 2), 2-methyltetrahydrofuran (7350 mL) was added to the aqueous phase, and a pre-cooled citric acid aqueous solution (3920 mL, 2.5%) was added to adjust the pH to 5-6 (the temperature was controlled at around 0°C during the dropwise addition). The liquids were separated, and the aqueous phase was extracted with 2-methyltetrahydrofuran (2450 mL x 1). The organic phases were combined and washed with water (7350 mL x 4) and a saturated sodium chloride aqueous solution (3680 mL x 1). 1) washing, the organic phase was dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure to obtain a residue, the residue was slurried with ethyl acetate (2450 mL) for 10 minutes and then filtered, the filter cake was washed with ethyl acetate (735 mL), and the obtained filtrate was concentrated under reduced pressure again to obtain a residue, the residue was dissolved in ethyl acetate (1230 mL), and n-hexane (6125 mL) was added dropwise to precipitate a solid, stirred for 16 hours and then filtered, the filter cake was rinsed with n-hexane (735 mL) and dried to obtain 15e (115.40 g), yield: 51%.

[0703] MS-ESI calculated value [M+H] + =414, the actual measured value is 414.

[0704] 1 H NMR (400MHz, DMSO-d6) δ8.41(t,J=5.6Hz,1H),8.05(t,J=5.6Hz,1H),7.11(s,2H),4.73(dd,J=10.4,5.2Hz,1H),4.11(dd,J=10. 4,5.6Hz,1H),4.00(t,J=10.4Hz,1H),3.83-3.73(m,3H),3.57(dd,J=16.8,5.6Hz,1H),0.76(s,9H),-0.01(s,3H),-0.07(s,3H).

[0705] Step 10

[0706] Dichloromethane (1150 mL) and 15e (115.00 g, 278.11 mmol) were added to the reaction flask, the temperature of the reaction system was lowered to -40 to -30 ° C, and a solution of N, N'-dicyclohexylcarbodiimide (86.10 g, 417.29 mmol) in dichloromethane (155 mL) and a solution of tetrafluorophenol (46.24 g, 278.44 mmol) in dichloromethane (155 mL) were added in sequence, and the reaction was stirred at -40 to -30 ° C for 5 hours. The reaction solution was quenched with water (2300 mL), and the reaction solution was filtered to remove insoluble matter. The filtrate was stirred and separated, and the aqueous phase was extracted with dichloromethane (1150 mL). The organic phases were combined, washed with saturated brine (1730 mL x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a residue. The residue was slurried with ethyl acetate (1150 mL) for 30 minutes and then filtered. The filter cake was rinsed with ethyl acetate (345 mL), the filtrate was collected and concentrated, and the residue was slurried with (methyl tert-butyl ether: n-hexane = 2:13, 1730 mL) for 3 hours and then filtered. The filter cake was rinsed with n-hexane (345 mL), and the filter cake was collected and dried to give 15f (146.80 g) with a yield of 94%.

[0707] MS-ESI calculated value [M+H] + =562, the actual measured value is 562.

[0708] 1 H NMR (400MHz, CDCl3) δ7.70-7.61(m,1H),7.14-7.06(m,1H),7.06-6.96(m,1H),6.75(s,2H),4.72(dd,J=8.4,6.4Hz,1H),4.5 0-4.35(m,2H),4.35-4.24(m,1H),4.16-3.94(m,2H),3.87(dd,J=10.4,6.4Hz,1H),0.87(s,9H),0.085(s,3H),0.078(s,3H).

[0709] Step 11

[0710] Acetonitrile (2628 mL), water (876 mL) and 15f (146.00 g, 260.00 mmol) were added to the reaction flask, the reaction system was cooled to -5-0 ° C, 8e (96.60 g, 312.30 mmol) and N, N-diisopropylethylamine (33.70 g, 260.65 mmol) were added, and the reaction system was stirred at -5-0 ° C for 18 hours. Methyl tert-butyl ether (2920 mL) and water (1460 mL) were added to the reaction solution, stirred and separated, the organic phase was extracted with water (1460 mL x 1), the aqueous phases were combined, the aqueous phase was washed with methyl tert-butyl ether (2190 mL x 3), the aqueous phase was collected, and a phosphoric acid system buffer solution (2190 mL, sample ratio: 6.51 g sodium dihydrogen phosphate and 0.30 g sodium hydrogen phosphate dissolved in 100.0 mL water) was added, stirred for 10 to 20 minutes, and the mixed solution was extracted with 2-methyltetrahydrofuran (2190 mL x 3), the organic phases were combined, the organic phases were washed with saturated brine (2190 mL x 2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain the residue, which was separated by HPLC preparation (preparative instrument: Hanbang industrial-grade preparative liquid chromatograph, specification model: DAC150, preparative column packing: Yuexu Xtimate C18), to the prepared solution was added a mixed solvent (ethyl acetate: 2-methyltetrahydrofuran = 1:1) in an amount of about half the volume of the prepared solution, and the mixture was stirred and separated. The aqueous phase was extracted with ethyl acetate in an amount of about half the volume of the prepared solution, and the two organic phases were combined. The organic phase was washed three times with deionized water in an amount of about half the volume of the prepared solution, and the mixture was concentrated under reduced pressure. The residue was lyophilized to obtain 15 g (83.54 g), with a yield of 46%.

[0711] MS-ESI calculated value [M+Na] + =727, the actual measured value is 727.

[0712] Step 12

[0713] Acetonitrile (664 mL), H2O (996 mL), and 15g (83.00 g, 117.8 mmol) were added to a reaction flask in sequence. The reaction system was cooled to 5-10°C, and formic acid (65.11 g, 1414.5 mmol) was added. The reaction system was stirred at 5-10°C for 40 hours. Methyl tert-butyl ether (830 mL) was added to the reaction solution, and the layers were separated. The organic phase was extracted with water (420 mL x 2). The aqueous phases were combined and lyophilized to obtain a crude product. Dichloromethane (1660 mL) was added to the crude product and the product was concentrated under reduced pressure. This operation was repeated five times (to remove residual formic acid). After dissolution in water (830 mL), the aqueous solution was lyophilized again to obtain 15h (65.30 g) with a yield of 94%.

[0714] MS-ESI calculated value [M+H] +=591, the actual measured value is 591.

[0715] 1 H NMR (400MHz, DMSO-d6) δ8.54(t,J=6.4Hz,1H),8.37-8.28(m,1H),8.12(d,J=7. 6Hz,1H),8.02(t,J=6.4Hz,1H),7.29-7.15(m,5H),7.05(s,2H),4.65-4.57(m,3 H),4.55-4.45(m,1H),4.02-3.94(m,3H),3.92-3.82(m,1H),3.80-3.68(m,4H), 3.65-3.57(m,2H),3.04(dd,J=13.6,4.4Hz,1H),2.78(dd,J=14.0,10.0Hz,1H).

[0716] Step 13

[0717] Under nitrogen protection, a solution of 1d (120.00 g, 265.77 mmol) in N,N-dimethylformamide (2400 mL) was added to the reactor, and the mixed system was cooled to -10 to -5 ° C. 15h (157.20 g, 266.19 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (151.20 g, 397.64 mmol) were added to the reactor. 2,4,6-Trimethylpyridine (64.80 g, 534.74 mmol) was slowly added dropwise to the reactor (the temperature was maintained at -10 to -5 ° C during the addition). After the addition was completed, the reaction system was stirred at -10 to -5 ° C for 4 hours. Slowly add 2% citric acid aqueous solution (7200mL) to the reactor, continue stirring for 10-15 minutes after the addition is complete, filter, wash the filter cake with water (1200mL x 3), dry, and prepare the crude compound by reverse phase chromatography (preparative instrument: Hanbang industrial-grade preparative liquid chromatograph, specification model: DAC150, preparative column packing: Kromasil100-10-C18 (W)), and prepare the solution in batches (each 25.0±5.0kg is considered as a batch). Each batch of preparation solution is transferred to a 50L reactor, stirred, acetonitrile is slowly added to clarify the solution, and then ethyl acetate (10000mL) is added, stirred for 5-10 minutes, allowed to stand for 5-10 minutes, and separated. The organic phase was washed with water (10000mL x 3), the combined aqueous phases were back-extracted with ethyl acetate (10000mL), the two organic phases were combined and washed with water (10000mL x 2), the organic phase was concentrated on a rotary evaporator until no liquid was dripped out, the material in the rotary evaporation bottle was transferred to a 10L Buchner funnel for filtration, the filter cake was filtered until no liquid was dripped out, and distilled water (1200mL x 2) was used to rinse the rotary evaporation bottle, rinse the filter cake, and filter until no liquid was dripped out of the filter cake. The above multiple batches of wet products were combined, the wet products were added to water (2400mL), stirred at 0-10°C for 2-3 hours, and filtered. The filter cake was rinsed with water (480mL), and the filter cake was collected and dried in a vacuum oven (30-35°C) for 40 hours to obtain compound 4 (152.00g), with a yield of 56%.

[0718] MS-ESI calculated value [M+H] + =1024, the actual measured value is 1024.

[0719] 1H NMR (400MHz, DMSO-d6) δ8.70-8.62(m,1H),8.56(d,J=8.8Hz,1H),8.35-8.24(m,2H ),8.09(d,J=8.0Hz,1H),8.04-7.95(m,1H),7.76(s,1H),7.69(d,J=8.8Hz,1H),7.2 8-7.13(m,5H),7.05(s,2H),6.69(s,1H),5.90(d,J=16.4Hz,1H),5.72-5.62(m,1H) ,5.48(d,J=16.8Hz,1H),5.42(d,J=20.0Hz,1H),5.17(d,J=19.6Hz,1H),5.06(t,J= 5.6Hz,1H),4.68(d,J=7.2Hz,2H),4.60(dd,J=9.6,6.0Hz,1H),4.52-4.43(m,1H),4 .18-4.04(m,2H),4.01-3.92(m,1H),3.90-3.81(m,1H),3.79-3.68(m,4H),3.65-3. 55(m,2H),3.28-3.07(m,2H),3.01(dd,J=13.6,4.0Hz,1H),2.73(dd,J=13.6,9.6Hz ,1H),2.35(s,3H),2.28-2.12(m,2H),1.88(q,J=7.2Hz,2H),0.85(t,J=7.2Hz,3H).

[0720] Example 1.14

[0721] first step

[0722] Dissolve 8a (200.00 g, 399.12 mmol) in N,N-dimethylformamide (600 mL) and set aside. Add N,N-dimethylformamide (400 mL), acetic acid (119.80 g, 1996.67 mmol), and lead tetraacetate (260.00 g, 598.66 mmol) to a reaction flask in that order. Slowly add the N,N-dimethylformamide solution of 8a dropwise to the reaction flask. After complete addition, heat the mixture to 40°C and stir for 4 hours. Dichloromethane (4000 mL) and water (4000 mL) were added to the reaction solution and stirred for 30 minutes. The insoluble matter was removed by filtration, and the filtrate was separated. The aqueous phase was extracted with dichloromethane (1000 mL x 1). The organic phases were combined, washed with water (2000 mL x 3), and concentrated under reduced pressure to obtain a crude product. The crude product was slurried with methyl tert-butyl ether (1000 mL) for 1 hour and then filtered. The filter cake was collected and dried to obtain 8b (179.63 g) with a yield of 87%.

[0723] MS-ESI calculated value [M-CH3COO] + =456, the actual measured value is 456.

[0724] Step 2

[0725] Under nitrogen, 8b (300.00 g, 582.29 mmol) and tetrahydrofuran (1200 mL) were added to a reaction flask. The reaction system was cooled to 0-5°C, and benzyl glycolate (106.39 g, 640.50 mmol) was added (the residual benzyl glycolate was rinsed with 300 mL of tetrahydrofuran and then added to the reaction flask). A solution of lithium hydroxide (16.70 g, 698.74 mmol) in water (300 mL) was then added dropwise. After the addition was complete, the reaction system was stirred at 0-5°C for 2 hours. The reaction system was diluted with water (3000 mL) and dichloromethane (3000 mL). After stirring, the mixture was separated, and the organic phase was washed with brine (4000 mL x 1). The organic phase was concentrated under reduced pressure to approximately 450 g. The resulting crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to afford 8c (269.20 g) in a yield of 74%.

[0726] MS-ESI calculated value [M+Na] + =644, the actual measured value is 644.

[0727] Step 3

[0728] Under nitrogen, 8c (264.00 g, 424.98 mmol) was dissolved in dichloromethane (26440 mL). The reaction system was cooled to -5-0°C, and 1,8-diazobisspiro[5.4.0]undec-7-ene (64.60 g, 424.72 mmol) was added. After complete addition, the mixture was stirred at 0-5°C for 2 hours. The reaction solution was directly purified by silica gel column chromatography (dichloromethane:isopropanol = 0-100%) to obtain 15b (111.00 g) in a 66% yield.

[0729] MS-ESI calculated value [M+H] + =400, the actual measured value is 400.

[0730] Step 4

[0731] 15b (108.00 g, 270.60 mmol) and water (2160 mL) were added to a reaction flask, followed by wet Pd / C (10.80 g). The atmosphere was replaced with hydrogen three times, and the reaction was stirred under a hydrogen atmosphere for 3 hours. The reaction mixture was filtered to remove insoluble matter, and the aqueous phase was directly lyophilized to afford 8e (82.16 g) in a 98% yield.

[0732] MS-ESI calculated value [MH] - =308, the actual measured value is 308.

[0733] Example 2

[0734] Method for preparing antibody-drug conjugates

[0735] The antibody was prepared using the anti-IGF-1R antibody sequence, which has the following CDR regions (Kabat definition):

[0736] CDR1: SFVMA (SEQ ID No. 1)

[0737] CDR2:AISGSGSRARYADSVKG(SEQ ID No.2)

[0738] CDR3:NPRRATPDLTQYAY(SEQ ID No.3)

[0739] Heavy chain variable region:

[0740] The full-length sequence is as follows:

[0741] The antibody was dialyzed into 50mM PB buffer to obtain an antibody intermediate. Take an appropriate amount of the antibody intermediate and add 10mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and 10mM diethylenetriaminepentaacetic acid (DTPA) stock solution in sequence. Then, add 50mM PB buffer to make the final antibody concentration in the reaction system 20mg / mL, the TCEP to antibody molar ratio of 4, and the final DTPA concentration of 1mM. After thorough mixing, place in a constant temperature mixer, reduce the reaction temperature at 25±2°C, rotate at 400rpm, and reduce for 2 hours. After the reduction is completed, add an appropriate amount of 5mM linker-drug stock solution to each reaction system in an ice-water bath, with a linker-drug to antibody molar ratio of 4.5. Add DMSO to a final DMSO volume of 20% in the coupling reaction. After thorough mixing, place in a constant temperature mixer, and react at 25±2°C, rotate at 400rpm, and react for 1 hour. After the coupling was completed, the ADC sample was dialyzed into a dialysate (10 mM His / His-HCl, pH 6.0±0.2) using an ultrafiltration centrifuge tube to obtain the ADC stock solution, which was aliquoted and stored at -80°C.

[0742] High-performance size exclusion liquid chromatography (SEC-HPLC) and hydrophobic HPLC-HPLC (HIC-HPLC) were used to test the molecular size variant purity and average drug-to-antibody ratio (DAR) of the ADCs. The main parameters are shown in Tables 1 and 2. The purity and DAR of the prepared ADCs are shown in Table 3. The test results demonstrate that the prepared ADCs all have high purity and a uniform distribution of DARs.

[0743] Table 1. Main parameters of high performance exclusion liquid chromatography (SEC-HPLC)

[0744] Table 2. Main parameters of high performance liquid phase hydrophobic chromatography (HIC-HPLC)

[0745] Table 3. Purity, average drug-antibody conjugate ratio, and DAR4 percentage of antibody-drug conjugates

[0746] Example 3

[0747] ADC plasma stability study

[0748] The linker-payload of the present application was obtained by referring to the method of Example 1 of the present application, and the antibody-drug conjugate (ADC) of the present application was obtained by referring to the method of Example 2 of the present application.

[0749] An appropriate amount of the ADC stock solution to be tested was added to 8 mL of anticoagulated human plasma to achieve an ADC concentration of 200 μg / mL. The sample was incubated at 37°C for 0, 24, 72, or 168 hours. Samples were then collected and purified by protein A chromatography. The purified ADC sample was concentrated by ultrafiltration and centrifugation. After centrifugation, 5 μL of a 5 M tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and an appropriate amount of ultrapure water were added to a total sample volume of approximately 200 μL. The sample was placed in a 56°C dry-bed thermostat for 40 minutes, removed, and ultrafiltration and centrifugation were repeated (12,000 rpm for 15 minutes). After centrifugation, 100 μL of ultrapure water was added and mixed, and then RP-MS analysis was performed. The mass spectrometer was set in positive mode with mobile phases A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. The column temperature was 40°C, and the flow rate was 0.4 mL / min.

[0750] According to the molecular weight, the mass of the conjugated small molecule drugs with different numbers was analyzed, and the average drug-to-antibody ratio (DAR) was calculated based on the peak area percentage and the number of conjugated drugs. The plasma stability of the ADC was studied by the change of DAR values ​​after different incubation times. The test results are shown in Table 4. The average coupling ratio value is calculated as follows:

[0751] The experimental results show that the ADCs prepared in this application all exhibit good plasma stability.

[0752] Table 4. Changes in DAR values ​​of antibody drug conjugates after incubation in human plasma for different times

[0753] Example 4

[0754] In vitro proliferation inhibition assay of human tumor cells

[0755] Refer to the method of Example 1 of the present application to obtain the linker-payload of the present application, and refer to the method of Example 2 of the present application to obtain the ADC of the present application.

[0756] Human tumor cells in the logarithmic growth phase were collected, digested, resuspended in fresh complete culture medium and adjusted to the appropriate concentration, added to a 96-well cell culture plate, and the cell culture plate was placed in a 37°C, 5% CO2 incubator for overnight culture. The next day, different concentrations of the ADC sample to be tested (the highest final concentration was 1 μM, 1:4 or 1:8 gradient dilution) or buffer control were added to the corresponding wells of the cell culture plate. After continuing to culture in a carbon dioxide incubator for 168 hours, the test plate was equilibrated to room temperature and the luminescence reading was detected using the CellTiter Glo assay kit (Promega, G7558) and a multi-function microplate reader. The cell inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1–(RLU ADC –RLU 空白 ) / (RLU 缓冲液 –RLU 空白 ))×100%. Graphpad Prism software was used to draw the efficacy inhibition rate curve and fit the EC 50 EC values ​​of each ADC in different human tumor cells 50 The values ​​are shown in Table 5.

[0757] The ADC prepared in this application exhibits excellent tumor cell proliferation inhibition activity in vitro.

[0758] Table 5. Inhibitory activity of ADC on proliferation of human tumor cells in vitro

[0759] Example 5

[0760] In vivo antitumor efficacy assay

[0761] Refer to the method of Example 1 of the present application to obtain the linker-payload of the present application, and refer to the method of Example 2 of the present application to obtain the ADC of the present application.

[0762] Human tumor cells in the logarithmic growth phase were harvested, counted, and adjusted to the appropriate concentration. They were then resuspended on ice in a 1:1 mixture of serum-free medium and Matrigel (Corning 356234) and inoculated subcutaneously into BALB / c nude mice, 200 μL per mouse. After the tumors grew to a measurable size, the long and short diameters of each tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = (a × b 2 ) / 2, where a represents the long diameter of the tumor and b represents the short diameter of the tumor.

[0763] When the average tumor volume reaches 100-200 mm 3The animals were randomly divided into groups according to the tumor volume. According to the predetermined dosing schedule, the nude mice bearing tumors were injected with solvent control (normal saline) or different doses of ADC (3 mg / kg or 10 mg / kg) through the tail vein. The long and short diameters of the tumors were measured twice a week, and the animal body weight was recorded. The tumor volume of each group was counted, and the tumor growth inhibition rate (TGI) was calculated according to the following formula: TGI = 100% × [1-(TV tT -TV 0T ) / (TV tC -TV 0C )]. Among them (TV tT represents the tumor volume of the drug-treated group on the day of measurement, TV 0T TV represents the tumor volume of the drug-treated group at the time of grouping; tC represents the tumor volume of the solvent control group on the day of measurement, TV 0C Represents the tumor volume of the vehicle control group at the time of grouping.

[0764] The ADC prepared in this application exhibits excellent in vivo anti-tumor activity.

[0765] Example 6

[0766] Pharmacokinetics in rats

[0767] Healthy adult Sprague Dawley rats, aged 6-8 weeks, were administered a tail vein bolus injection of ADC (20 mg / kg) for approximately 1 minute ± 10 seconds at a volume of 5 mL / kg. Blood was collected at 0.083, 1, 2, 8, 24, 48, 72, 96, 120, 144, and 168 hours after administration. Serum was separated by centrifugation within 30-120 minutes. Total antibody (Tab) and ADC concentrations in the blood samples were determined by conventional ELISA.

[0768] The detection method of total antibody is briefly described as follows: Trop2-His is coated overnight at 4°C at a concentration of 0.75ug / mL, and blocked with 5% skim milk powder at 37°C for 2h. The standard curve and quality control points are added and incubated for 2h. The detection range of the standard curve is 64ng / mL-0.5ng / mL, starting at 64ng / mL, and 2-fold serial dilution. The quality control concentration points are set to 60ng / mL, 6ng / mL, and 0.6ng / mL. The recovery rate of the quality control points needs to be between 80% and 120%. Subsequently, anti-human K light chain peroxidase antibody produced in goats (manufacturer: Sigma, product number: A7164-1ML) is added at a dilution of 1:8000. The secondary antibody is incubated for 1h, washed 8 times with PBST, and TMB is added for color development. The plate is terminated with 0.1M sulfuric acid. The OD450 plate is read on an enzyme-linked microplate reader. The blood sample concentration at different time points is calculated using the enzyme-linked microplate reader analysis software SoftMaxPro.

[0769] The ADC detection method is briefly described as follows: Trop2-His was coated overnight at 4°C at a concentration of 0.75 μg / mL, and the plate was blocked with 5% skim milk powder at 37°C for 2 hours. The standard curve and quality control points were added and incubated for 2 hours. The detection range of the standard curve was 64 ng / mL-0.5 ng / mL, starting at 64 ng / mL, and the dilution was 2-fold. The quality control concentration points were set at 60 ng / mL, 6 ng / mL, and 0.6 ng / mL. The recovery rate of the quality control points needed to be between 80% and 120%. Subsequently, anti-human K light chain peroxidase antibody produced in goats (manufacturer: Sigma, product number: A7164-1ML) was added at a dilution of 1:8000. The plate was incubated for 1 hour, washed 8 times with PBST, and TMB was added for color development. The plate was terminated with 0.1 M sulfuric acid. The plate was read at OD450 on an enzyme-linked microplate reader. The blood sample concentration at different time points was calculated using the enzyme-linked microplate reader analysis software SoftMaxPro.

[0770] ELISA was used to draw a curve of drug concentration changes in the blood. The ADC concentration basically coincided with the total antibody concentration, with extremely low shedding and very stable in the blood.

[0771] Example 7

[0772] Cell proliferation inhibition assay

[0773] KPL-4 tumor cells in the logarithmic growth phase were taken and resuspended in fresh RPMI1640 culture medium. The cells were counted and the cell suspension was adjusted to 2×10 4 Cells / mL. The cell suspension was inoculated into a 96-well cell culture plate at 100 μL / well and cultured overnight in a carbon dioxide incubator (37°C, 5% CO2). The next day, one of the 96-well plates seeded with cells was removed and equilibrated to room temperature. 100 μL of CellTiter-Glo reagent (Promega, USA), which had been pre-equilibrated to room temperature and mixed thoroughly, was added to each well of the test plate. After incubation in the dark for 30 minutes, the luminescence value was read on a microplate reader (recorded as the G0 value). Another parallel plate was taken and different concentrations of the test compound or DMSO (final concentration 0.5%) were added to the corresponding wells of the test plate. After incubation in a carbon dioxide incubator for 72 hours, the test plate was equilibrated to room temperature and the cell activity was detected using the CellTiter-Glo reagent, which was recorded as the G3 value.

[0774] The cell proliferation rate was calculated according to the following formula: Cell proliferation rate (%) = (average value of G3 in the test compound wells - average value of G0) / (average value of G3 in the DMSO control wells - average value of G0) * 100. Graphpad Prism software was used to fit the inhibition curve and calculate the GI 50 value.

[0775] The compound prepared in the present application exhibits excellent in vivo anti-tumor activity.

[0776] Example 8

[0777] Bystander killing activity experiment

[0778] BxPC3 (human pancreatic cancer cells, ATCC, CRL-1687) and MiaPaCa2 (human pancreatic cancer cells, biocytogen, B-HCL-014) cells were cultured in RPMI1640 + 10% FBS and DMEM / high glucose + 10% FBS, respectively. The cells were trypsinized, neutralized with fresh culture medium, centrifuged at 1000 rpm for 3 minutes, the supernatant discarded, and the cells resuspended in RPMI1640 + 10% FBS. After cell counting, the BxPC3 cell density was adjusted to 6 × 10 4 / mL, and adjust the MiaPaCa2-luc cell density to 1.5*10 4 Add 500 μL of BxPC3 cells and 500 μL of MiaPaCa2-luc cells to each well of a 12-well plate (plate 1). Add 500 μL of MiaPaCa2-luc cells and 500 μL of RPMI1640 culture medium (10% FBS) to each well of a 12-well plate (plate 2). Incubate at 37°C in a 5% CO2 atmosphere for 24 hours.

[0779] ADC samples were prepared into an intermediate solution (0.2 μM) at a 40x concentration. 25 μL of each sample was added to the corresponding wells of a 12-well plate. A solvent control group was set up. Culture was carried out at 37°C with 5% carbon dioxide for 6 days. The cells in the 12-well plate were trypsinized, neutralized with fresh culture medium, centrifuged at 1000 rpm for 3 minutes, the supernatant discarded, and resuspended in 1 mL of FACS buffer (PBS + 2.5% FBS). 20 μL of cells were added to 20 μL of trypan blue and counted. The cells in plate 1 were centrifuged at 1000 rpm for 3 minutes, the supernatant discarded, and resuspended in 100 μL of FACS buffer. 2 μL of monoclonal antibody was added and incubated on ice for 30 minutes. Centrifuged at 2000 rpm for 1 minute at 4°C, the supernatant discarded, and the cells were resuspended in 150 μL of FACS buffer. Detection was performed using a BD FACSVerse. Data were analyzed using Flowjo 7.6.

[0780] The results showed that the ADC disclosed in the present invention had a clear bystander killing effect. The ADC did not kill the target-negative MiaPaCa2 cells. However, after the target-expressing BxPC3 cells were mixed with the negative cells MiaPaCa2, the ADC also had a killing effect on the target-negative cells.

[0781] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A ligand conjugate, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate comprises the structure shown in Formula I: in, L is an optionally substituted linker that is connected to any O atom, S atom or N atom in the P structure; Ab is a ligand, a is a number greater than 0, and a is a decimal or an integer; preferably, a is 1-16 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16); more preferably, a is 2-8; P is a group formed by dehydrogenation of the structure described in the following formula (II): Where n is 0 or 1; X is selected from the following group: N or CR 0 ; R 0 Selected from the group consisting of H, D, halogen, C1-C8 alkyl, C1-C8 alkoxy, OH, NH2, N3 or NO2; R 1 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C8 alkyl group, a C1-C8 alkoxy group, a C1-C8 haloalkyl group, a C1-C8 haloalkoxy group, N3, NO2, NH2, NH-OH, -NR'R", -COOR', -CONR'R", -NHR"'NR'R", wherein R', R" and R'" are each independently selected from the group consisting of hydrogen, deuterium, an alkyl group, an aryl group, an arylalkyl group, an acyl group, an alkoxycarbonyl group, and an aryloxycarbonyl group; R 2 、R 3 、R 4 、R 5 and R 6 Each is independently selected from the following groups: hydrogen atom, deuterium atom, halogen, hydroxyl, cyano, NH2, NO2, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m tri(C1-C4alkyl)silyl, -(CH2) m (C3-C8 cycloalkyl), -(CH2) m (3-12 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m S(CH2) p R 7 、-(CH2) m S(O)(CH2) p R 7 、-(CH2) m S(O)2(CH2) p R 7 、-(CH2) m NH(CH2) p R 7 、-(CH2) m NHC(O)(CH2) p R 7 、-(CH2) m OC(O)(CH2) p R 7 、-(CH2) m C(O)(CH2) p R 7 , -CH=N(OtBu); wherein m and p are each independently 0, 1, 2, 3 or 4; Or, R 2 and R 3 Together with the carbon atom to which it is attached, it forms a substituted or unsubstituted C5-C8 carbocyclic ring or a substituted or unsubstituted 5-12 membered heterocyclic ring; Or, R 3 and R 4 , or R 4 and R 5 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-12 membered carbon ring, unsubstituted or replaced by one or more R e substituted saturated or unsaturated 5-12 membered heterocycle; said R e is a substituted or unsubstituted substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C1-C6 alkyl group-NH-, a (C1-C6 alkyl)2N-, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, an allyl group, a benzyl group, a C6-C 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C3-C8 cycloalkyl, 3-12 membered heterocyclyl, -(CH2) m N(R 7 )2、-(CH2) m S(CH2) p R 7 、-(CH2) m S(O)(CH2) p R 7 、-(CH2) m S(O)2(CH2) p R 7 、-(CH2) m NH(CH2) p R 7 、-(CH2) m NHC(O)(CH2) p R 7 、-(CH2) m OC(O)(CH2) p R 7 、-(CH2) m C(O)(CH2) p R 7 ; wherein m and p are each independently 0, 1, 2, 3 or 4, preferably 0, 1 or 2; R 7 Selected from the following group: hydrogen atom, deuterium atom, halogen, substituted or unsubstituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 deuterated alkyl, substituted or unsubstituted C1-C8 alkoxy, hydroxyl, amino, cyano, nitro, mercapto, substituted or unsubstituted C1-C8 alkylene-OH, substituted or unsubstituted C1-C8 alkylene-NH2, SO2Me, -OC(O)(substituted or unsubstituted C1-C4 alkyl), -C(O)(substituted or unsubstituted C1-C4 alkyl), substituted or unsubstituted phenyl, substituted or unsubstituted 5-7 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl; Unless otherwise specified, the term "substituted" refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C1-C6 alkyl-NH-, a (C1-C6 alkyl)2N-, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a halogenated C1-C6 alkyl group, a halogenated C2-C6 alkenyl group, a halogenated C2-C6 alkynyl group, a halogenated C1-C6 alkoxy group, an allyl group, a benzyl group, a C6-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a halogenated C1-C6 alkoxy group, an allyl group, a benzyl group, a C6-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a C1-C6 alkyl group, a C2 ... 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl- Carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C3-C8 cycloalkyl, 3-12 membered heterocyclyl.

2. The ligand conjugate according to claim 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, characterized in that: The L has a structure as shown in the following formula: L1-L2-L3-L4-L5; Wherein, the L1 is optionally substituted R d is H, C1-C6 alkyl, C1-C6 deuterated alkyl, C3-C8 cycloalkyl or C3-C8 deuterated cycloalkyl; The L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-, optionally substituted -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-, optionally substituted-X1-(CHROCHR) m2 -C(O)-, optionally substituted -(CHR) p1 -C(O)-, optionally substituted -(CHR) m1 -X1-(CHR) m2 -C(O)-, optionally substituted -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-, optionally substituted -X1-(CHR) m1 -X2-(CHR) m2 -C(O)-, optionally substituted -(CH2CH2O) n3 -C(O)-; X1 and X2 are each independently selected from the following groups: -O-, -C(O)-, -C(O)-NR-, optionally substituted C6-C 10 aryl, optionally substituted 5-9 membered heteroaryl, optionally substituted 3-8 membered heteroalicyclic group and optionally substituted C3-C6 alicyclic group; Wherein, each of the R's is independently selected from the following groups: H, D, (CH2) n4 OH, (CH2) n4 NH2, (CH2O) n4 (CH2CH2O) n5 H, (CH2O) n4 (CH2CH2O) n5 CH3, (CH2) n4 OCH3, (CH2CH2O) n5 CH3, CH2C(O)NH(CH2O) n4 (CH2CH2O) n5 H、CH2C(O)NH(CH2O) n4 (CH2CH2O) n5 CH3; wherein m1, m2, n3, n4 and n5 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; p1 is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8; The L3 is a peptide residue; and the L3 may be substituted with one or more substituents selected from the group consisting of: CH2C(O)R c ; The R c Select from the following groups: wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; The L4 is an optionally substituted -L 4a -(NR b ) n6 -R 12 -L 4b -, where L 4a Does not exist, or L 4a is optionally substituted Where n6 is 0 or 1; R 12 is a chemical bond, CH2, or CD2; L 4b Does not exist, or L 4b is optionally substituted Among them, R a and R b are each independently selected from the group consisting of hydrogen, optionally substituted C1-C4 alkyl, and optionally substituted C1-C4 deuterated alkyl; The L5 is absent or optionally substituted wherein Y is selected from the group consisting of O, S, or NH; v is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R 10 and R 11 are each independently selected from the group consisting of hydrogen, deuterium, optionally substituted C1-C4 alkyl, optionally substituted C1-C4 haloalkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C4-C8 cycloalkylalkyl, or R 10 and R 11 Together with the atoms to which it is attached, it forms an optionally substituted 3-6 membered cycloalkyl group, R 10 and R 11 Each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an optionally substituted C1-C8 alkyl group, an optionally substituted C1-C8 haloalkyl group, and an optionally substituted C1-C8 deuterated alkyl group.

3. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, characterized in that: The L1 is 4. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: X1 and X2 are each independently selected from the following group: -O-, -C(O)-, -C(O)-NR-, optionally substituted phenyl, optionally substituted pyridyl, optionally substituted C3-C6 cycloalkyl, optionally substituted or optionally substituted 5. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -(CH2) m1 -X1-(CH2CH2O) n3 -(CH2) m2 -C(O)-; wherein X1 is -C(O)-NH-; preferably, m1 and m2 are each independently selected from 1, 2 or 3; n3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

6. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-X2-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1 is optionally substituted or optionally substituted X2 is -C(O)-NR-; preferably, m1 and m2 are each independently selected from 0, 1 or 2; n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

7. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -X1-(CHROCHR) m2 -C(O)-; wherein X1 is an optionally substituted aryl group or an optionally substituted heteroaryl group; preferably, m2 is selected from 0, 1, 2 or 3.

8. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted (CHR) p1 -C(O)-; p1 is selected from 0, 1 or 2; R is selected from the following group: H, (CH2) n4 OH, (CH2O) n4 (CH2CH2O) n5 H; Preferably, n4 and n5 are each independently selected from 0, 1, 2 or 3.

9. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -(CH2) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1 is -C(O)-; preferably, m1 and m2 are each independently selected from 0, 1, 2 or 3; n3 is selected from 0, 1 or 2.

10. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -X1-(CH2) m1 -X2-(CHR) m2 -C(O)-; wherein X1 is an optionally substituted aryl or an optionally substituted heteroaryl; X2 is -C(O)-; preferably, m1 and m2 are each independently selected from 0, 1 or 2.

11. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the following group: -(CHR) m1 -X1-(CHR) m2 -C(O)-; wherein X1 is an optionally substituted 3-8 membered heteroalicyclic group or an optionally substituted C3-C6 alicyclic group; preferably, m1 is 0, 1 or 2, and m2 is 0.

12. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-(CH2CH2O) n3 -(CHR) m2 -C(O)-; wherein X1 is O; preferably, m1, n3 and m2 are each independently 0, 1 or 2.

13. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L2 is a group selected from the group consisting of: optionally substituted -(CHR) m1 -X1-(CHR) n3 -X2-(CHR) m2 -C(O)-; wherein X1 is optionally substituted -C(O)-NR-, X2 is O; preferably, m1, n3 and m2 are each independently 1, 2 or 3; R is as described in claim 2.

14. The ligand conjugate according to claim 2 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or hydrate thereof, wherein L2 is an optionally substituted structure selected from the group consisting of:

15. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L3 is unsubstituted or replaced by CH2C(O)R c The peptide residues are composed of substituted amino acids selected from the group consisting of phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine.

16. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L3 is unsubstituted or replaced by CH2C(O)R c The peptide residues are composed of substituted amino acids selected from the group consisting of glycine, alanine, lysine, phenylalanine, valine and citrulline.

17. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L3 is unsubstituted or replaced by CH2C(O)R c Substituted peptide residues selected from the group consisting of: -Glycine-Phenylalanine-Glycine-(-Gly-Phe-Gly-), -Glycine-Glycine-Phenylalanine-Glycine-(-Gly-Gly-Phe-Gly-), -Valine-Citrulline-(-Val-Cit-), -Citrulline-Valine-(-Cit-Val-), -Citrulline-Alanine-(-Cit-Ala-), -Valine-Alanine-(-Val-Ala-), -Valine-Arginine-(-Val-Arg-), -Valine-Lysine-(-Val-Lys-), -Valine-Lysine(Ac)-(-Val-Lys(Ala-) c)-), -Lysine-Valine-(-Lys-Val-), -Leucine-Citrulline-(-Leu-Cit-), -Isoleucine-Citrulline-(-Ile-Cit-), -Tryptophan-Citrulline-(-Trp-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Lysine(Ac)-(-Phe-Lys(Ac)-), -Phenylalanine-Citrulline-(-Phe-Cit-), -Phenylalanine-Ala-(-Phe-Ala-), -Phenylalanine-Arginine-(-Phe-Arg-), -Ala-Lysine-(-Ala-Lys-), -Ala -Ala-Ala-), -Ala-Ala-Ala-Ala-), -Ala-Ala-Ala-Asparagine-(-Ala-Ala-Asn-), -Ala-Ala-Ala-Aspartic Acid-(Ala-Ala-Asp-), -Lysine-Ala-Ala-Ala-Asparagine-(-Lys-Ala-Ala-Asn-), -Lysine-Ala-Ala-Ala-Aspartic Acid-(-Lys-Ala-Ala-Asp-), -(D)-Valine-Leucine-Lysine-(-D-Val-Leu-Lys-), -Glycine-Glycine-Arginine- (-Gly-Gly-Arg-), -Glycine-Glycine-Asparagine-(-Gly-Gly-Asn-), -Glycine-Glycine-Phenylalanine-(-Gly-Gly-Phe-), -Valine-Lysine-Glycine-(-Val-Lys-Gly-), -Glutamic acid-Alanine-Alanine-(-Glu-Ala-Ala-), -Aspartic acid-Alanine-Alanine-(-Asp-Ala-Ala-), -Valine-Lysine-Glycine-Glycine-(-Val-Lys-Gly-Gly-), and -Lysine-Alanine-Asparagine-(-Lys-Ala-Asn-).

18. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L3 is unsubstituted or replaced by CH2C(O)R c A substituted structure selected from the group consisting of:

19. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L4 is a chemical bond, or an optionally substituted group selected from the following group: Among them, the R a and R b Each is independently selected from the following group: hydrogen, optionally substituted C1-C4 alkyl, optionally substituted C1-C4 deuterated alkyl.

20. The ligand conjugate according to claim 19, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L4 is a chemical bond, or an optionally substituted structure selected from the following group:

21. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The L5 is a chemical bond, or a structure selected from the following groups: optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted Optionally substituted and optionally substituted 22. The ligand conjugate according to claim 2, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The compound of formula II is selected from the following group:

23. The ligand conjugate according to claim 22, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The R 4 Selected from the following groups: hydrogen atom, deuterium atom, halogen, hydroxyl, cyano, NH2, NO2, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m (C3-C8 cycloalkyl), -(CH2) m (3-12 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m S(O)(CH2) p R 7 、-(CH2) m S(O)2(CH2) p R 7 、-(CH2) m NH(CH2) p R 7 ; wherein m and p are each independently 0, 1 or 2, R 7 is defined as above; R 5 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, NH2, OH, a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C1-C8 alkoxy group; Or, R 4 and R 5 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-6 membered carbon ring, unsubstituted or replaced by one or more R e Substituted saturated or unsaturated 5-6 membered heterocyclic ring; wherein R e The definition of is as described above.

24. The ligand conjugate according to claim 22, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: R 2 and R 3 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, NH2, a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C1-C8 deuterated alkyl group, -(CH2) m (C3-C6 cycloalkyl), -(CH2) m (3-6 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m OC(O)R 7 ; wherein m is 0, 1, 2, 3 or 4, R 7 is defined as above; Or, R 2 and R 3 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e Replaced by full and or unsaturated 5-6 membered ring, unsubstituted or replaced by one or more R e Substituted saturated or unsaturated 5-6 membered heterocyclic ring; wherein R e The definition of is as described above.

25. The ligand conjugate according to claim 22, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The R 4 and R 5 Each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a hydroxyl group, NH2, a substituted or unsubstituted C1-C4 alkyl group; Or, R 4 and R 5 The carbon atoms to which it is attached together form an unsubstituted or substituted carbon atom or carbon atoms. e Substituted oxa 5-6 membered heterocycle; wherein R e The definition of is as described above.

26. The ligand conjugate according to claim 22, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: R 2 Selected from the group consisting of: deuterium atom, halogen, NH2, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m (C3-C6 cycloalkyl), -(CH2) m (3-6 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m OC(O)R 7 ; wherein m is 0, 1, 2, 3 or 4; R 3 Each is selected from the following groups: hydrogen atom, deuterium atom, halogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 deuterated alkyl, -(CH2) m (C3-C6 cycloalkyl), -(CH2) m (3-6 membered heterocyclic group), -(CH2) m N(R 7 )2、-(CH2) m OC(O)R 7 ; wherein m is 0, 1, 2, 3 or 4; Or, R 2 and R 3 The carbon atoms connected thereto together form a structure selected from the group consisting of: unsubstituted or substituted with one or more R e substituted saturated or unsaturated 5-6 membered ring, unsubstituted or replaced by one or more R e a substituted saturated or unsaturated 5-6 membered heterocycle; R 4 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a hydroxyl group, a cyano group, NH2, NO2, a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C1-C8 alkoxy group; R 5 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, and a substituted or unsubstituted C1-C8 alkyl group; Or, R 4 and R 5 The carbon atoms to which it is attached together form an unsubstituted or substituted carbon atom or carbon atoms. e a substituted group selected from the group consisting of: -OCH2O- or -O(CH2)2O-; R 7 Selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a substituted or unsubstituted C1-C8 alkyl group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a mercapto group; Among them, R e The definition of is as described above.

27. The ligand conjugate according to claim 22, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The compound of formula II is selected from the following group:

28. The ligand conjugate according to claim 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: Wherein the Ab is an antibody or an antigen-binding fragment thereof.

29. The ligand conjugate according to claim 28, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

30. The ligand conjugate according to claim 28, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The antibody is a monoclonal antibody, a bispecific antibody, or a polypeptide.

31. The ligand conjugate according to claim 28, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The antigen binding fragment is selected from the group consisting of Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

32. The ligand conjugate according to claim 1, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, wherein: The above-mentioned ligand conjugate is selected from the following group:

33. The ligand conjugate according to claim 1 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate comprises the structure shown in formula (Ia): in, R4, R5 and L are as defined in claim 1, a is a number greater than 0, and a is a decimal or an integer.

34. The ligand conjugate of claim 33 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate is a structure selected from the group consisting of: Where a is a number greater than 0, and a is a decimal or an integer.

35. The ligand conjugate according to any one of claims 1 to 34, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or hydrate, characterized in that The antibody is an IGF-1R specific antibody.

36. The ligand conjugate according to claim 35, wherein The IGF-1R includes an IGF-1R derived from a primate.

37. The ligand conjugate according to claim 36, wherein The antibody comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

38. The ligand conjugate according to claim 36, wherein The antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

39. The ligand conjugate according to claim 36, wherein The antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

1.

40. The ligand conjugate according to claim 36, wherein The antibody comprises a heavy chain variable region VH, wherein the VH comprises the HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

1.

41. The ligand conjugate according to claim 36, wherein The antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

4.

42. The ligand conjugate according to claim 36, wherein The antibody has a full-length sequence as shown in SEQ ID No.

5.

43. A pharmaceutical composition comprising the ligand conjugate of any one of claims 1 to 42, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and optionally a pharmaceutically acceptable carrier.

44. Use of the ligand conjugate described in any one of claims 1-42, or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, prodrug or solvate, and / or the pharmaceutical composition described in claim 43 in the preparation of a medicament for treating and / or preventing a disease or condition associated with target expression and / or abnormal expression of the ligand.

45. The use according to claim 44, wherein the disease or condition associated with target expression and / or abnormal expression of the ligand is a tumor, cancer, autoimmune disease or infectious disease; preferably, the tumor / cancer is a tumor / cancer with high expression, medium expression or low expression of the ligand's target.

46. ​​A ligand conjugate precursor, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate precursor comprises the structure shown in Formula IA: L A -P(IA), in, L A For L 1A -L2-L3-L4-L5; among them, L 1A for R d , L2, L3, L4, L5 and P are defined as described in any one of claims 1-34.

47. The ligand conjugate precursor of claim 46, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt or hydrate thereof, wherein the ligand conjugate precursor is selected from the group consisting of:

48. A linker as represented by formula (L), which connects a drug unit to a ligand to form a ligand-drug conjugate: L1-L2-L3-L4-L5 (L), Among them, among them, The definitions of L1, L2, L3, L4, and L5 are as described in any one of claims 1-34.

49. The connector according to claim 48, wherein The linker is selected from the following group:

50. The connector according to claims 48-49, characterized in that The linker is connected to the ligand through the L1 segment and to the P1 through the L5 segment to form a ligand-drug conjugate; and the P1 is selected from the following group: glycopeptide antibiotics, such as bleomycin or bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, isitecan, topotecan, belotecan or rubitecan, DXd, etc.), topoisomerase II inhibitors (such as actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or nabendazim Labine; drugs that act on structural proteins, such as microtubule inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; maytansine derivatives (such as DM1, DM4, etc.); calicheamicin derivatives; auristatin derivatives (such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin E, auristatin F, etc.); pyrrolobenzodiazepine dimers (PBD) derivatives; amanitin derivatives (such as α-Amanitin, etc.); anthracyclines; dukamycin; eribulin; melphalan; mitomycin C; chlorambucil; TLR agonists; STING agonists; glucocorticoids and other groups formed by dehydrogenation of active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

51. A linker precursor as shown in formula (L-1), which is used to obtain a ligand-drug conjugate formed by connecting a drug unit to a ligand: L A -R g (L-1), in, R g is H, OH, O (C1-C6 alkyl); wherein L A The definition as set forth in claim 46.

52. The linker precursor according to claim 51, wherein The linker precursor is selected from the following group:

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