Antibody-drug conjugate, production method thereof, and use

Abstract

A bioactive complex, a method for producing the same, and use thereof, specifically, an antibody-drug conjugate represented by formula (XV), a method for producing the same, and use thereof in the prevention and / or treatment of tumor diseases, and use thereof in the prevention and / or treatment of diseases associated with abnormal cell activity, including but not limited to, are disclosed. JPEG2025523090000399.jpg1164 Formula XV

Description

Technical Field

[0001] The present disclosure belongs to the field of medical technology, and relates to the use thereof in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to various antibodies, antibody-drug conjugates and methods for producing the same, and their use in the prevention and / or treatment of tumor diseases.

Background Art

[0002] Chemotherapy using cytotoxicity was once the standard treatment for cancer, but highly lethal cytotoxic molecules can kill normal cells and may cause serious toxic side effects. Targeted anti-tumor drugs currently have become a hot topic in the field of cancer research because they have both target characteristics and anti-tumor activity. However, due to the problem of target selectivity of targeted drugs, relatively large toxic side effects are often caused, thereby limiting the therapeutic effect of targeted drugs. Biological macromolecular drugs such as antibodies or antibody fragments are highly targeted, but have limited therapeutic effects on solid tumors or no therapeutic effect at all. An ADC is a conjugate of an antibody and a small molecule drug, which combines the target effect of the antibody and the activity of a bioactive molecule, and has become a very promising biological missile with the advantages of high efficacy and safety. The antibody guides the ADC to bind to target cells, which are then taken up by the cells, and the small molecule drug is released into the cells through enzymatic degradation by the action of specific enzymes to treat the disease.

[0003] ADC drugs have developed rapidly in recent years, and 14 kinds of ADCs are on the market. Among many targeted ADCs, antibody / ADC drugs targeting DLL3 are all in the clinical research stage. The development of differentiated, higher-quality and safer monoclonal antibodies or ADC drugs for many targets can provide a wider range of better drug options for tumor patients and also have broad market prospects.

Summary of the Invention

[0004] To improve the therapeutic effect of antibody-drug conjugates (ADCs), reduce the toxic side effects of drugs, and expand the therapeutic range, the present disclosure provides an antibody-drug conjugate represented by formula (XV) or a pharmaceutically acceptable salt or solvate thereof, which comprises a bioactive molecule (drug molecule), a linker, and a targeting moiety, and the targeting moiety is linked to the linker via a reactive group (e.g., sulfhydryl) to form an antibody-drug conjugate. The present disclosure further develops a variable light chain domain (VL) and / or variable heavy chain domain (VH) containing anti-DLL3, and the variable light chain domain and / or variable heavy chain domain antibody is derived from a humanized antibody.

[0005] Accordingly, in a first aspect of the present disclosure, the present disclosure provides an antibody-drug conjugate represented by formula XV or a stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate of the antibody-drug conjugate:

Chemical formula

Chemical formula

[0006] Furthermore, regarding "the 1-position of L1 is linked to Tb via an S atom", for those skilled in the art, after opening the disulfide bond at the 1-position of L1 (for example, opening the disulfide bond by reducing the disulfide bond with the reducing agent TCEP to generate sulfhydryl -SH), it is linked to the sulfhydryl contained in Tb (for example, an antibody) itself. That is, it can be understood that the -S- between L1 and Tb is not an additional external sulfur atom. For example,

Chemical formula

Chemical formula

[0007] The extension unit is a component of an antibody-drug conjugate or a drug-linker conjugate or a linker, and its function is to link the remaining part of the antibody-drug conjugate that binds to the target or the remaining part of the linker. The extension unit can link the Tb unit to L2 (if present) or L3. Specific examples include, but are not limited to, the following (here, the 1-position is linked to the antibody part that binds to the target, and the 2-position is linked to L2 or L3):

Chemical formula

[0008] In some embodiments, L1 is selected from the following formulas:

Chemical formula

Chemical formula

Chemical formula

[0009] The linking unit is a component of an antibody-drug conjugate or a drug-linker conjugate or a linker, and its function is to link the extension unit to an amino acid residue or a fragment or short peptide consisting of 1 to 10 amino acid residues. When the linking unit is present, L1 can be linked to L3. Specific examples include, but are not limited to, the following (where the 1-position is linked to the extension unit and the 2-position is linked to L3):

Chemical formula

[0010] In some embodiments, L2 is absent or present. When L2 is present, L2 is selected from the following formulas:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0011] In some embodiments, L1 is [Chemical Formula] .

[0012] In some embodiments, L1 is [Chemical Formula] selected from, each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aryl, a 5-10 membered heteroaryl, and an amide (preferably selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond), Rx and Ry are independently selected from H and C1-4 alkyl, each m is independently selected from 0, 1, 2, 3, 4, 5, and 6, y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 15 (e.g., 6 - 15), each y3 is independently selected from 1, 2, and 3, each y4 is independently selected from 0 and 1, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L2 or L3.

[0013] In some embodiments, L1 is [Chemical Formula] selected from, m is selected from 2, 3, 4, y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6 - 10), each y3 is independently selected from 1 or 2, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L2 or L3.

[0014] In some embodiments, L1 is [Chemical Formula] selected from.

[0015] In some embodiments, L1 is [Chemical formula] selected from, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L2 or L3.

[0016] In some embodiments, L2 is absent or present. When L2 is present, L2 is [Chemical formula] selected from, y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6 - 10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, the 1-position is linked to L1, and the 2-position is linked to L3.

[0017] In some embodiments, L2 is absent or present. When L2 is present, L2 is [Chemical formula] selected from, the 1-position is linked to L1, and the 2-position is linked to L3.

[0018] In some embodiments, L2 is absent.

[0019] In some embodiments, L2 is [Chemical formula] selected from.

[0020] In some embodiments, L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues, and the amino acid residue is selected from a natural amino acid residue, a non-natural amino acid residue, or an amino acid residue represented by AA1 or a stereoisomer thereof.

[0021] In some embodiments, L3 is an amino acid residue Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA 1 or a short peptide consisting of amino acid residues selected from 2 to 10 Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AA 1 selected from short peptides consisting of amino acid residues selected from.

[0022] In some embodiments, L3 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AA 1 , Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA 1 , Ala-AA 1 , Gly-AA 1 , AA 1 -Gly, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Asp, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Gly-Phe-Gly, Gly-Gly-Phe-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys and Lys-Ala-Asn selected from.

[0023] In some embodiments, L3 is AA 1 , AA 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 -Ala-Asn and Gly-Gly-Phe-Gly selected from.

[0024] In some embodiments, L3 is AA 1 and Val-AA 1-Gly.

[0025] In some embodiments, L3 is Val-AA 1 -Gly.

[0026] In some embodiments, L3 is

Chemical formula

Chemical formula

[0027] In some embodiments, L3 is

Chemical formula

[0028] In some embodiments, L3 is

Chemical formula

[0029] In some embodiments, AA 1 The structure of the amino acid residue represented by is as shown below:

Chemical formula

Chemical formula

[0030] In some embodiments, R a , R b , one of which is H and the other is

Chemical Formula

[0031] In some embodiments, R a and R b , one of which is H, and the other one is selected from

Chemical formula

[0032] In some embodiments, R a and R b , together with the carbon atom to which they are attached, form a 5- to 6-membered heterocyclic ring substituted by R 0 .

[0033] In some embodiments, R a and R b , together with the carbon atom to which they are attached, form a piperidine ring or a piperazine ring substituted by R 0 .

[0034] In some embodiments, R a and R b , together with the carbon atom to which they are attached, form a piperidine ring substituted by R 0 .

[0035] In some embodiments, R a and R b , together with the carbon atom to which they are attached,

Chemical formula

[0036] In some embodiments, R a and R b , together with the carbon atom to which they are attached,

Chemical formula

[0037] In some embodiments, r, r 1 are each independently selected from 0, 1, 2, 3, 4 and 5.

[0038] In some embodiments, r, r 1 are each independently selected from 0 and 4.

[0039] In some embodiments, r, r 1 wherein one of them is 0 and the other one is 4.

[0040] In some embodiments, R m1 , R n1 are each independently selected from H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 and -COOCH2CH2CH2CH3.

[0041] In some embodiments, R m1 , R n1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and tert-butoxycarbonyl.

[0042] In some embodiments, R m1 , R n1 are each independently selected from H and C1-6 alkyl.

[0043] In some embodiments, R m1 , R n1 are each independently selected from H, methyl, ethyl and n-propyl.

[0044] In some embodiments, r, r 1 where r is 4 and r 1 is 0, R m1 , Rn1 is independently selected from H, C1-6 alkyl (e.g., H, methyl), r is 0, and when r 1 is 4, R m1 , R n1 is independently selected from C1-6 alkyl (e.g., methyl, ethyl, n-propyl), and preferably is selected from C2-6 alkyl (e.g., ethyl, n-propyl).

[0045] In some embodiments, R m1 and R n1 together with the nitrogen atom to which they are attached, optionally form a 5- to 6-membered heterocyclic ring substituted by R 0’ .

[0046] In some embodiments, R m1 and R n1 together with the nitrogen atom to which they are attached, optionally form a piperidine ring or a piperazine ring substituted by R 0’ .

[0047] In some embodiments, R m1 and R n1 together with the nitrogen atom to which they are attached,

Chemical formula

[0048] In some embodiments, R z is methyl.

[0049] In some embodiments, R 0 , R 0’ are independently selected from C1-6 alkyl, -NR m2 R n2 and a 5- to 6-membered heterocyclic group optionally substituted by C1-6 alkyl.

[0050] In some embodiments, R 0 is selected from C1-6 alkyl and a 5-6 membered heterocyclic group substituted by C1-6 alkyl, and the 5-6 membered heterocyclic group is selected from piperidinyl and piperazinyl.

[0051] In some embodiments, R 0 is selected from methyl, ethyl and a 5-6 membered heterocyclic group substituted by methyl, and the 5-6 membered heterocyclic group is piperidinyl.

[0052] In some embodiments, R 0 is selected from methyl and a 5-6 membered heterocyclic group substituted by methyl, and the 5-6 membered heterocyclic group is piperidinyl.

[0053] In some embodiments, R 0 is methyl, ethyl and

Chemical formula

[0054] In some embodiments, R 0 is methyl and

Chemical formula

[0055] In some embodiments, R 0’ is selected from C1-6 alkyl and -NR m2 R n2 selected from.

[0056] In some embodiments, R 0’ is selected from methyl and -NR m2 R n2 selected from.

[0057] In some embodiments, R m2 , R n2 is methyl.

[0058] In some embodiments, AA 1 The amino acid residue represented by

Chem.

[0059] In some embodiments, AA 1 The amino acid residue represented by

Chem.

[0060] In some embodiments, AA 1 The amino acid residue represented by

Chem.

[0061] In some embodiments, L4 is absent or, if present, L4 is

Chem.

[0062] In some embodiments, L4 is absent.

[0063] In some embodiments, L4 is

Chem.

[0064] In some embodiments, L4 is

Chem.

[0065] In some embodiments,

Chemical formula

Table 1

[0066] In some embodiments, q is selected from any numerical value between 0.1 and 16.0, and in a preferred embodiment, q is selected from any integer between 0.1 and 16.0.

[0067] In some embodiments, q is selected from any numerical value between 0.1 and 8.0, and in a preferred embodiment, q is selected from any integer between 0.1 and 8.0.

[0068] In some embodiments, q is selected from any numerical value between 2 and 8.

[0069] In some embodiments, q is selected from any numerical value between 3 and 8.

[0070] In some embodiments, q is selected from any numerical value between 4 and 8.

[0071] In some embodiments, q is selected from any numerical value between 6 and 8.

[0072] In some embodiments, q is selected from any integer between 2 and 8.

[0073] In some embodiments, q is selected from any integer between 3 and 8.

[0074] In some embodiments, q is selected from any integer between 4 and 8.

[0075] In some embodiments, q is selected from any integer between 6 and 8.

[0076] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0077] In some embodiments, q is selected from 2, 4, 6, and 8.

[0078] In the present disclosure, the bioactive molecule fragment is known in the art, and after the linker is cleaved / decomposed / enzyme-cleaved in tumor tissue or tumor cells, a bioactive drug (e.g., a small molecule cytotoxic drug containing a drug group that has lost an atom or atomic group) or a derivative thereof (e.g., its precursor) can be formed. It refers to a part (fragment or group) of an antibody-drug conjugate (or an antibody-drug conjugate, also referred to as an ADC). To avoid ambiguity, "medicine" refers not only to "pharmaceuticals" approved by medical regulatory authorities, but also includes any molecule with potential therapeutic bioactivity in clinical, or research and development and academic research.

[0079] In some embodiments, D is a molecular fragment having anti-tumor bioactivity.

[0080] In some embodiments, D is a molecular fragment having anti-tumor bioactivity, where the bioactive molecule is selected from a cytotoxic agent or a derivative thereof, e.g., a DNA topoisomerase inhibitor (e.g., a camptothecin-based bioactive molecule, e.g., camptothecin, DXD, camptothecin having a modified substituent, or DXD having a modified substituent) or a tubulin inhibitor (e.g., an MMAF-based tubulin inhibitor, an MMAE-based tubulin inhibitor).

[0081] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I: [Chemical formula] (wherein, R1 and R2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or R1 and R2 together with the carbon atom to which they are attached form a 5- to 7-membered carbocyclic ring or a 5- to 7-membered heterocyclic ring, and the heterocyclic ring contains one or more O, S, N, carbonyl, sulfinyl or sulfonyl or any combination thereof, R3 is selected from H, halogen, -OH, -NH2, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or R3 and X together with the carbon atom to which they are attached form a 5- to 7-membered carbocyclic ring or a 5- to 7-membered heterocyclic ring, and the heterocyclic ring contains one or more O, S, N, carbonyl, sulfinyl or sulfonyl or any combination thereof, or R3 and R2 together with the carbon atom to which they are attached form a 5- to 7-membered carbocyclic ring or a 5- to 7-membered heterocyclic ring, and the heterocyclic ring contains one or more O, S, N, carbonyl, sulfinyl or sulfonyl or any combination thereof, W is absent or present, and when W is present, W is -O-, -S-, -NR4-, [Chemical formula] selected from, the 1-position is linked to X, and the 2-position is linked to L4 or L3, X is a direct bond, optionally substituted -O-(CH2) n3 -, -N(R4)-(CH2) n3 -, -S-(CH2) n3 -, carbonyl-(CH2) n3 , -SO2-(CH2) n3 -, [Chemical formula] -(CH2) n1- selected from C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic group, the 1-position is linked to the parent ring, the 2-position is linked to W or L4, and the substituent is selected from one or more of C1-4 alkyl, C3-6 cycloalkyl, or a plurality of C1-4 alkyl together with the carbon atoms to which they are linked form a C3-6 cycloalkyl, Each M is independently a direct bond and -CR 5a R 5b - selected from, R4, R5, R 5a 、R 5b 、R6, R7 are each independently selected from H, optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy and optionally substituted C3-6 cycloalkyl, n, n’, n1, n2, n3 are each independently selected from any integer between 0 and 6, L4 is absent or, when present, L4 is

Chemical formula

[0082] Tb, L1, L2, L3 and q have the meanings provided by any of the above and the specific embodiments described herein.

[0083] In some embodiments, R1, R2 are each independently selected from H, halogen and C1-4 alkyl.

[0084] In some embodiments, R1 and R2 together with the carbon atom to which they are linked form a 5-6 membered heterocyclic ring, and the heterocyclic ring contains one, two or three of O, S or N or any combination thereof.

[0085] In some embodiments, R1 is selected from H and halogen, and R2 is selected from H and C1-4 alkyl.

[0086] In some embodiments, R1 and R2, together with the carbon atom to which they are attached

Chemical formula

[0087] In some embodiments, R1 is H or F, and R2 is H or methyl.

[0088] In some embodiments, R1 is F, R2 is methyl, or R1 and R2, together with the carbon atom to which they are attached

Chemical formula

[0089] In some embodiments, R1 is F and R2 is methyl.

[0090] In some embodiments, R1 and R2, together with the carbon atom to which they are attached

Chemical formula

[0091] In some embodiments, R3 is selected from H and C1-4 alkyl.

[0092] In some embodiments, R3 and X, together with the carbon atom to which they are attached, form a 5- to 6-membered carbon ring.

[0093] In some embodiments, R3 is H, or R3 and X, together with the carbon atom to which they are attached

Chemical formula

[0094] In some embodiments, R3 is H.

[0095] In some embodiments, W is absent or, if present, W is selected from -O-, -S-, -NR4-,

Chemical formula

[0096] In some embodiments, W is absent or, if present, W is selected from -O-, -S-, -NR4-,

Chemical formula

[0097] In some embodiments, W is -O-, -NR4- and

Chemical formula

[0098] In some embodiments, W is selected from -O- and -NR4-, wherein the 1-position is linked to X and the 2-position is linked to L4 or L3.

[0099] In some embodiments, X is optionally substituted -(CH2) n1 -,

Chemical formula

[0100] In some embodiments, X is optionally substituted

Chemical formula

[0101] In some embodiments, X is

Chemical formula

[0102] In some embodiments, X is

Chemical formula

[0103] In some embodiments, when W is absent, X is

Chemical formula

Chemical formula

[0104] In some embodiments, W is -O-, -NR4-, and

Chemical formula

Chemical formula

[0105] In some embodiments, R4 and R5 are each independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl.

[0106] In some embodiments, each R4 is independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl, and R5 is H.

[0107] In some embodiments, each R4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl, and cyclopropyl, and R5 is H.

[0108] In some embodiments, R 5a , R 5b are each independently selected from H and C1-4 alkyl.

[0109] In some embodiments, R 5a , R 5b are each independently selected from H and methyl.

[0110] In some embodiments, each R7 is independently selected from H and C1-4 alkyl.

[0111] In some embodiments, R7 is H.

[0112] In some embodiments, n is selected from 1, 2, and 3.

[0113] In some embodiments, n is 1.

[0114] In some embodiments, n1 is selected from 1, 2, 3, and 4.

[0115] In some embodiments, n2 is 1.

[0116] In some embodiments, n3 is 0.

[0117] In some embodiments, L3 is

Chemical formula

Chemical formula

[0118] In some embodiments, L3 is

Chemical formula

[0119] In some embodiments, L3 is

Chemical formula

[0120] In some embodiments, L4 is absent or present, and when L4 is present, L4 is [Chemical formula] wherein the 1-position is connected to L3 and the 2-position is connected to W or X.

[0121] In some embodiments, L4 does not exist.

[0122] In some embodiments, L4 [Chemical formula] is selected from, the 1-position is connected to L3, and the 2-position is connected to W or X.

[0123] In some embodiments, L4 [Chemical formula] is selected from, the 1-position is connected to L3, and the 2-position is connected to W or X.

[0124] As described above, W either does not exist or exists. Thus, when W does not exist, the 1-position of L4 is connected to L3 and the 2-position is connected to X; when W exists, the 1-position of L4 is connected to L3 and the 2-position is connected to W. With reference to the above content, the following connection relationships of L4 can be understood.

[0125] In some embodiments, [Chemical formula] the structure of is selected from the following structural fragments: [Table 2-1] [Table 2-2] [Table 2-3] (In the formula, the 1-position is linked to Tb, and the 2-position is linked to W).

[0126] In some embodiments, D is

Chemical formula

Chemical formula

[0127] In some embodiments,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0128] In some embodiments, W is absent or present, and when W is present, W is -O-, -S-, -NR4-,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0129] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-1: [Chemical formula] (wherein Tb, L1, L2, L3, L4, X, R1, R2, R3, R4, and q have the meanings provided by any of the embodiments described above and specifically described herein).

[0130] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-1A or I-1B: [Chemical formula] (wherein Tb, L2, L3, L4, X, R1, R2, R3, R4, and q have the meanings provided by any of the embodiments described above and specifically described herein).

[0131] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-2: [Chemical formula] (wherein Tb, L1, L2, L3, L4, X, R1, R2, R3, and q have the meanings provided by any of the embodiments specifically described above and in this specification).

[0132] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-2A or I-2B:

Chemical formula

[0133] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-3:

Chemical formula

[0134] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-3A or I-3B:

Chemical formula

[0135] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-A:

Chemical formula

[0136] In some embodiments, the antibody-drug conjugate has a structure represented by Formula I-B: [Chemical formula] (wherein Tb, X, R1, R2, R3, R a , R b and q have the meanings provided by any of the embodiments described above and specifically described herein).

[0137] In some embodiments, the antibody-drug conjugate is selected from the following: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12]

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

[0138] In some embodiments, Tb is an anti-DLL3 antibody or an antigen-binding fragment thereof.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof and the monoclonal antibody or antigen-binding fragment thereof include Fab, Fab’, F(ab’)2, Fd, Fv (e.g., scFv), dAb, complementarity-determining region fragment, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, Probody, monoclonal antibody, bispecific antibody or multispecific antibody.

[0140] In some embodiments, Tb is an anti-DLL3 antibody or antigen-binding fragment thereof having endocytosis activity, having no endocytosis activity, or having weak endocytosis activity.

[0141] In some embodiments, Tb is an anti-DLL3 antibody or antigen-binding fragment thereof having endocytosis activity.

[0142] In some embodiments, Tb is an anti-DLL3 antibody or an antigen-binding fragment thereof that does not have endocytosis activity or has weak endocytosis activity.

[0143] In some embodiments, Tb is an anti-DLL3 antibody or an antigen-binding fragment thereof.

[0144] In some embodiments, the anti-DLL3 antibody is a non-human antibody, a humanized antibody, a chimeric antibody, or a fully human antibody.

[0145] In some embodiments, the anti-DLL3 antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0146] In some embodiments, the anti-DLL3 antibody or an antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment thereof.

[0147] In some embodiments, the anti-DLL3 antibody or an antigen-binding fragment thereof is selected from rovalpituzumab, or an antigen-binding fragment thereof, tarlatamab or an antigen-binding fragment thereof, antibody 10F2F3, antibody 87F7F10, antibody 55C11E4, antibody 59B10D3, antibody 6F11C10, antibody 55C11E4-Hz1, antibody 55C11E4-Hz2, antibody 59B10D3-Hz, antibody 10F2F3-Hz, antibody 87F7F10-Hz, antibody 6F11C10-Hz1, and antibody 6F11C10-Hz2.

[0148] In some preferred embodiments, the anti-DLL3 antibody is selected from mouse antibodies 10F2F3, 87F7F10, 55C11E4, 59B10D3, and 6F11C10 or humanized antibodies thereof.

[0149] In some preferred embodiments, the anti-DLL3 antibody is selected from antibody 55C11E4-Hz1, antibody 55C11E4-Hz2, antibody 59B10D3-Hz, antibody 10F2F3-Hz, antibody 87F7F10-Hz, antibody 6F11C10-Hz1, and antibody 6F11C10-Hz2.

[0150] The Tb antibody or its antigen-binding fragment can be produced by various methods known in the art, such as genetic engineering and recombinant techniques. For example, DNA molecules encoding the heavy chain gene and light chain gene of the antibody of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and transfected into a host cell. Next, the transfected host cell is cultured under specific conditions to express the antibody of the present disclosure.

[0151] In some preferred embodiments, in the antibody-drug conjugate described above, Tb is the anti-DLL3 antibody or its antigen-binding fragment described in the second aspect.

[0152] In the second aspect, the present disclosure provides an anti-DLL3 antibody or its antigen-binding fragment.

[0153] In some embodiments, the present disclosure provides an anti-DLL3 antibody or its antigen-binding fragment, and the antibody or its antigen-binding fragment comprises the following complementarity-determining regions (CDRs).

[0154] HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1, 2, 5, 6, 7, 10, 11, 14, 15, 18, 19 or 20, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the light chain variable region (VL) shown in SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, 17, 21 or 22.

[0155] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR1, LCDR1, LCDR2, LCDR3, and comprises an antibody and antigen-binding fragment thereof that binds to the same DLL3 epitope as any of the antibodies described in the above-described embodiments. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR1, LCDR1, LCDR2, LCDR3, and comprises an antibody and antigen-binding fragment thereof that competes with any of the antibodies described in the above-described embodiments with respect to binding to DLL3.

[0156] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof is an antibody and antigen-binding fragment thereof that binds to the same DLL3 epitope as 55C11E4-hz2, 55C11E4-hz1, 6F11C10-Hz1 or 6F11C10-Hz2.

[0157] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof is an antibody and antigen-binding fragment thereof that competes with 55C11E4-hz2, 55C11E4-hz1, 6F11C10-Hz1 or 6F11C10-Hz2 with respect to binding to DLL3.

[0158] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 1 or 2, and / or comprises LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 3 or 4.

[0159] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 5, 6, or 7, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 8 or 9.

[0160] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 10 or 11, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 12 or 13.

[0161] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 14 or 15, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 16 or 17.

[0162] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 18, 19, or 20, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 21 or 22.

[0163] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 1, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 3.

[0164] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 2, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 4.

[0165] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 5, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 8.

[0166] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 6, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 9.

[0167] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 7, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 9.

[0168] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 10, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 12.

[0169] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 11, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 13.

[0170] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in VH shown in SEQ ID NO: 14, and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in VL shown in SEQ ID NO: 16.

[0171] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises an HCDR1 or variant thereof, an HCDR2 or variant thereof, and an HCDR3 or variant thereof contained in the VH shown in SEQ ID NO: 15, and / or an LCDR1 or variant thereof, an LCDR2 or variant thereof, and an LCDR3 or variant thereof contained in the VL shown in SEQ ID NO: 17.

[0172] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises an HCDR1 or variant thereof, an HCDR2 or variant thereof, and an HCDR3 or variant thereof contained in the VH shown in SEQ ID NO: 18, and / or an LCDR1 or variant thereof, an LCDR2 or variant thereof, and an LCDR3 or variant thereof contained in the VL shown in SEQ ID NO: 21.

[0173] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises an HCDR1 or variant thereof, an HCDR2 or variant thereof, and an HCDR3 or variant thereof contained in the VH shown in SEQ ID NO: 19, and / or an LCDR1 or variant thereof, an LCDR2 or variant thereof, and an LCDR3 or variant thereof contained in the VL shown in SEQ ID NO: 22.

[0174] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises an HCDR1 or variant thereof, an HCDR2 or variant thereof, and an HCDR3 or variant thereof contained in the VH shown in SEQ ID NO: 20, and / or an LCDR1 or variant thereof, an LCDR2 or variant thereof, and an LCDR3 or variant thereof contained in the VL shown in SEQ ID NO: 22.

[0175] In certain preferred embodiments, the variant of the sequence is a CDR having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to its source CDR.

[0176] In certain preferred embodiments, said substitution is a conservative substitution.

[0177] Preferably, said CDRs are defined according to the AbM, Chothia, Kabat or IMGT numbering system.

[0178] In one aspect, the present disclosure provides an anti-DLL3 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising a heavy-chain variable region (VH) and / or a light-chain variable region (VL).

[0179] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy-chain variable region (VH) and / or light-chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system.

[0180] (a) A heavy-chain variable region (VH) comprising the following three CDRs: an HCDR1 having a sequence that is SEQ ID NO: 23 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR2 having a sequence that is SEQ ID NO: 24 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR3 having a sequence that is SEQ ID NO: 25 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and / or, A light chain variable region (VL) containing the following three CDRs: LCDR1 of a sequence that is SEQ ID NO: 26 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of a sequence that is SEQ ID NO: 27 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR3 of a sequence that is SEQ ID NO: 28 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (b) A heavy chain variable region (VH) containing the following three CDRs: HCDR1 of a sequence that is SEQ ID NO: 34 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 of a sequence that is SEQ ID NO: 35 or 36 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR3 of a sequence that is SEQ ID NO: 37 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or A light chain variable region (VL) containing the following three CDRs: LCDR1 of a sequence that is SEQ ID NO: 38 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 of a sequence that is SEQ ID NO: 39 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR3 of a sequence that is SEQ ID NO: 40 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (c) A heavy chain variable region (VH) comprising the following three CDRs: an HCDR1 having a sequence that is SEQ ID NO: 46 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR2 having a sequence that is SEQ ID NO: 47 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR3 having a sequence that is SEQ ID NO: 48 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and / or, A light chain variable region (VL) comprising the following three CDRs: an LCDR1 having a sequence that is SEQ ID NO: 49 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an LCDR2 having a sequence that is SEQ ID NO: 50 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an LCDR3 having a sequence that is SEQ ID NO: 51 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, (d) A heavy chain variable region (VH) comprising the following three CDRs: an HCDR1 having a sequence that is SEQ ID NO: 57 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR2 having a sequence that is SEQ ID NO: 58 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR3 having a sequence that is SEQ ID NO: 59 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and / or, The light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence that is SEQ ID NO: 60 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 having a sequence that is SEQ ID NO: 61 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR3 having a sequence that is SEQ ID NO: 62 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto. (e) The heavy chain variable region (VH) comprising the following three CDRs: HCDR1 having a sequence that is SEQ ID NO: 68 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR2 having a sequence that is SEQ ID NO: 69 or 70 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; HCDR3 having a sequence that is SEQ ID NO: 71 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and / or The light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence that is SEQ ID NO: 72 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR2 having a sequence that is SEQ ID NO: 73 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; LCDR3 having a sequence that is SEQ ID NO: 74 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto.

[0181] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system. (a) The VH comprises an HCDR1 having the sequence of SEQ ID NO: 23, an HCDR2 having the sequence of SEQ ID NO: 24, and an HCDR3 having the sequence of SEQ ID NO: 25, and / or, The VL comprises an LCDR1 having the sequence of SEQ ID NO: 26, an LCDR2 having the sequence of SEQ ID NO: 27, and an LCDR3 having the sequence of SEQ ID NO: 28, (b) The VH comprises an HCDR1 having the sequence of SEQ ID NO: 34, an HCDR2 having the sequence of SEQ ID NO: 35 or 36, and an HCDR3 having the sequence of SEQ ID NO: 37, and / or, The VL comprises an LCDR1 having the sequence of SEQ ID NO: 38, an LCDR2 having the sequence of SEQ ID NO: 39, and an LCDR3 having the sequence of SEQ ID NO: 40, (c) The VH comprises an HCDR1 having the sequence of SEQ ID NO: 46, an HCDR2 having the sequence of SEQ ID NO: 47, and an HCDR3 having the sequence of SEQ ID NO: 48, and / or, The VL comprises an LCDR1 having the sequence of SEQ ID NO: 49, an LCDR2 having the sequence of SEQ ID NO: 50, and an LCDR3 having the sequence of SEQ ID NO: 51, (d) The VH comprises an HCDR1 having the sequence of SEQ ID NO: 57, an HCDR2 having the sequence of SEQ ID NO: 58, and an HCDR3 having the sequence of SEQ ID NO: 59, and / or, The VL comprises an LCDR1 having the sequence of SEQ ID NO: 60, an LCDR2 having the sequence of SEQ ID NO: 61, and an LCDR3 having the sequence of SEQ ID NO: 62, Or, (e) The VH comprises an HCDR1 having the sequence of SEQ ID NO: 68, an HCDR2 having the sequence of SEQ ID NO: 69 or 70, and an HCDR3 having the sequence of SEQ ID NO: 71, and / or, The VL comprises an LCDR1 having the sequence of SEQ ID NO: 72, an LCDR2 having the sequence of SEQ ID NO: 73, and an LCDR3 having the sequence of SEQ ID NO: 74.

[0182] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system. (a) A heavy chain variable region (VH) comprising the following three CDRs: an HCDR1 having a sequence that is SEQ ID NO: 29 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; an HCDR2 having a sequence that is SEQ ID NO: 30 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; an HCDR3 having a sequence that is SEQ ID NO: 31 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; and / or, A light chain variable region (VL) comprising the following three CDRs: an LCDR1 having a sequence that is SEQ ID NO: 32 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; an LCDR2 having a sequence that is FAS or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; an LCDR3 having a sequence that is SEQ ID NO: 28 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto; (b) A heavy chain variable region (VH) comprising the following three CDRs: HCDR1 of a sequence that is SEQ ID NO: 41 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; HCDR2 of a sequence that is SEQ ID NO: 42 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; HCDR3 of a sequence that is SEQ ID NO: 43 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; and / or, A light chain variable region (VL) comprising the following three CDRs: LCDR1 of a sequence that is SEQ ID NO: 44 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; LCDR2 of a sequence that is YAS or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; LCDR3 of a sequence that is SEQ ID NO: 40 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto, (c) A heavy chain variable region (VH) comprising the following three CDRs: HCDR1 of a sequence that is SEQ ID NO: 52 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; HCDR2 of a sequence that is SEQ ID NO: 53 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; HCDR3 of a sequence that is SEQ ID NO: 54 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; and / or, The light chain variable region (VL) containing the following three CDRs: LCDR1 of a sequence that is SEQ ID NO: 55 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; LCDR2 of a sequence that is YTS or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; LCDR3 of a sequence that is SEQ ID NO: 51 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto. (d) The heavy chain variable region (VH) containing the following three CDRs: HCDR1 of a sequence that is SEQ ID NO: 63 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; HCDR2 of a sequence that is SEQ ID NO: 64 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; HCDR3 of a sequence that is SEQ ID NO: 65 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto, and / or The light chain variable region (VL) containing the following three CDRs: LCDR1 of a sequence that is SEQ ID NO: 66 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; LCDR2 of a sequence that is WAS or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto; LCDR3 of a sequence that is SEQ ID NO: 62 or has one or more amino acid substitutions, deletions, or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids) compared thereto. (e) A heavy chain variable region (VH) comprising the following three CDRs: an HCDR1 having a sequence that is SEQ ID NO: 75 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR2 having a sequence that is SEQ ID NO: 76 or 77 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an HCDR3 having a sequence that is SEQ ID NO: 78 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, and / or A light chain variable region (VL) comprising the following three CDRs: an LCDR1 having a sequence that is SEQ ID NO: 79 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an LCDR2 having a sequence that is LAS or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, an LCDR3 having a sequence that is SEQ ID NO: 74 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

[0183] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system. (a) The VH comprises an HCDR1 having a sequence that is SEQ ID NO: 29, an HCDR2 having a sequence that is SEQ ID NO: 30, and an HCDR3 having a sequence that is SEQ ID NO: 31, and / or The VL comprises an LCDR1 having a sequence that is SEQ ID NO: 32, an LCDR2 having a sequence that is FAS, and an LCDR3 having a sequence that is SEQ ID NO: 28, (b) The VH comprises an HCDR1 having a sequence that is SEQ ID NO: 41, an HCDR2 having a sequence that is SEQ ID NO: 42, and an HCDR3 having a sequence that is SEQ ID NO: 43, and / or The VL includes an LCDR1 with a sequence of SEQ ID NO: 44, an LCDR2 with a sequence of YAS, and an LCDR3 with a sequence of SEQ ID NO: 40, (c) The VH includes an HCDR1 with a sequence of SEQ ID NO: 52, an HCDR2 with a sequence of SEQ ID NO: 53, and an HCDR3 with a sequence of SEQ ID NO: 54, and / or The VL includes an LCDR1 with a sequence of SEQ ID NO: 55, an LCDR2 with a sequence of YTS, and an LCDR3 with a sequence of SEQ ID NO: 51, (d) The VH includes an HCDR1 with a sequence of SEQ ID NO: 63, an HCDR2 with a sequence of SEQ ID NO: 64, and an HCDR3 with a sequence of SEQ ID NO: 65, and / or The VL includes an LCDR1 with a sequence of SEQ ID NO: 66, an LCDR2 with a sequence of WAS, and an LCDR3 with a sequence of SEQ ID NO: 62, Or (e) The VH includes an HCDR1 with a sequence of SEQ ID NO: 75, an HCDR2 with a sequence of SEQ ID NO: 76 or 77, and an HCDR3 with a sequence of SEQ ID NO: 78, and / or The VL includes an LCDR1 with a sequence of SEQ ID NO: 79, an LCDR2 with a sequence of LAS, and an LCDR3 with a sequence of SEQ ID NO: 74.

[0184] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure includes the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein at least one CDR within the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation as compared to the CDR defined by the aforementioned Kabat numbering system or IMGT numbering system, and the mutation is a substitution, deletion, or addition of one or more amino acids or any combination thereof (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids or any combination thereof), and still retains binding activity to DLL3.

[0185] Preferably, the substitutions described in the present disclosure are conservative substitutions.

[0186] In certain embodiments, the antibody or antigen-binding fragment thereof binds to human DLL3, monkey DLL3, and / or rat DLL3.

[0187] In certain embodiments, the VH of the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a framework region (FR) derived from the variable heavy chain region (VH) of a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from the variable light chain region (VL) of a human immunoglobulin. Thus, in certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure is of fully human origin. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure is a humanized antibody.

[0188] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following. (a) A heavy chain framework region of a human immunoglobulin or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived, and / or (b) A light chain framework region of a human immunoglobulin or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived.

[0189] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% humanized.

[0190] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following. (a) A heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the following. (i) The sequence set forth in SEQ ID NO: 1, 2, 5, 6, 7, 10, 11, 14, 15, 18, 19 or 20, (ii) One or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), or (iii) A sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity compared to the sequence set forth in SEQ ID NO: 1, 2, 5, 6, 7, 10, 11, 14, 15, 18, 19 or 20, and / or (b) A light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the following. (iv) The sequence set forth in SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, 17, 21 or 22, (v) An array having one or more amino acid substitutions, deletions, additions, or any combination thereof (e.g., substitution, deletion, addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or any combination thereof) compared to the array shown in SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, 17, 21, or 22, or (vi) An array having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity compared to the array shown in SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, 17, 21, or 22.

[0191] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a VH shown in SEQ ID NO: 1 or 2 and / or a VL shown in SEQ ID NO: 3 or 4.

[0192] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a VH shown in SEQ ID NO: 5, 6, or 7 and / or a VL shown in SEQ ID NO: 8 or 9.

[0193] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a VH shown in SEQ ID NO: 10 or 11 and / or a VL shown in SEQ ID NO: 12 or 13.

[0194] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a VH shown in SEQ ID NO: 14 or 15 and / or a VL shown in SEQ ID NO: 16 or 17.

[0195] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a VH shown in SEQ ID NO: 18, 19, or 20 and / or a VL shown in SEQ ID NO: 21 or 22.

[0196] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure has a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VH of any one of the five groups of implementation states described above, and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VL of said group, or In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure has a VH having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof) compared to the VH of any one of the five groups of implementation states described above, and / or a VL having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof) compared to the VL of said group, preferably, said substitution is a conservative substitution.

[0197] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 3, (b) VH of the sequence shown in SEQ ID NO: 2 and VL of the sequence shown in SEQ ID NO: 4, (c) VH of the sequence shown in SEQ ID NO: 5 and VL of the sequence shown in SEQ ID NO: 8, (d) VH of the sequence shown in SEQ ID NO: 6 and VL of the sequence shown in SEQ ID NO: 9, (e) The VH of the sequence shown in SEQ ID NO: 7 and the VL of the sequence shown in SEQ ID NO: 9, (f) The VH of the sequence shown in SEQ ID NO: 10 and the VL of the sequence shown in SEQ ID NO: 12, (g) The VH of the sequence shown in SEQ ID NO: 11 and the VL of the sequence shown in SEQ ID NO: 13, (h) The VH of the sequence shown in SEQ ID NO: 14 and the VL of the sequence shown in SEQ ID NO: 16, (i) The VH of the sequence shown in SEQ ID NO: 15 and the VL of the sequence shown in SEQ ID NO: 17, (j) The VH of the sequence shown in SEQ ID NO: 18 and the VL of the sequence shown in SEQ ID NO: 21, (k) The VH of the sequence shown in SEQ ID NO: 19 and the VL of the sequence shown in SEQ ID NO: 22, (l) The VH of the sequence shown in SEQ ID NO: 20 and the VL of the sequence shown in SEQ ID NO: 22, (m) Independently compared to the VH and VL described in any one of the groups (a) to (l), having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity for the heavy chain variable region (VH) and the light chain variable region (VL), or, (n) Independently having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof) when compared to the VH and VL described in any one of the groups (a) to (l) respectively. Preferably, said substitution is a conservative substitution.

[0198] In certain embodiments, the anti-DLL3 antibodies of the present disclosure are chimeric antibodies, humanized antibodies or fully human antibodies. In certain embodiments, the anti-DLL3 antibodies of the present disclosure or antigen-binding fragments thereof are selected from Fab, Fab’, (Fab’)2, Fv fragments such as scFv or Fv linked by disulfide bonds (dsFv), diabodies and multispecific antibodies. In certain embodiments, the anti-DLL3 antibodies of the present disclosure are scFv.

[0199] In certain embodiments, the heavy chain of the anti-DLL3 antibody of the present disclosure or an antigen-binding fragment thereof comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having, compared to the wild-type sequence from which it is derived, a conservative substitution of up to 50 amino acids (e.g., a conservative substitution of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids, e.g., a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids). In certain embodiments, the light chain of the anti-DLL3 antibody of the present disclosure or an antigen-binding fragment thereof comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof, said variant having, compared to the wild-type sequence from which it is derived, a conservative substitution of up to 50 amino acids (e.g., a conservative substitution of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids, e.g., a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).

[0200] In some embodiments, the constant region is modified (e.g., to change one or more of the following properties: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function) to modify the properties of the anti-DLL3 antibody molecule and is, for example, mutated. The functional change can be generated by replacing at least one amino acid residue within the constant region of the antibody with another residue, for example, by changing the affinity of the antibody for an effector ligand (such as FcR or complement C1q) to change (e.g., decrease) the effector function. The Fc region of the antibody mediates several important effector functions such as ADCC, antibody-dependent cell phagocytosis (ADCP), and CDC.

[0201] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure has a heavy chain constant region (CH), which is selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, specifically, for example, selected from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, and more specifically, selected from the heavy chain constant region of IgG1 (e.g., human IgG1). In some embodiments, the human IgG1 heavy chain constant region is as shown in SEQ ID NO: 82. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure has a light chain constant region selected from, for example, a κ or λ light chain constant region, preferably a κ light chain constant region (e.g., human κ light chain constant region). In some embodiments, the light chain constant region has the sequence shown in SEQ ID NO: 83.

[0202] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a CH shown in SEQ ID NO: 82 or a variant thereof, wherein the variant has a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 20, up to 15, up to 10, or up to 5 amino acids, e.g., a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to SEQ ID NO: 82, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 70.

[0203] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof. In some embodiments, the light chain constant region comprises a κ light chain constant region. In some embodiments, the light chain constant region comprises a light chain constant region (CL) shown in SEQ ID NO: 83 or a variant thereof, wherein the variant has a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 20, up to 15, up to 10, or up to 5 amino acids, e.g., a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to SEQ ID NO: 83, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 80.

[0204] In some embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 82 and a light chain constant region (CL) shown in SEQ ID NO: 83.

[0205] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following. (a) A heavy chain consisting of or comprising an amino acid sequence selected from the following: (i) A sequence comprising VH shown in SEQ ID NO: 6 or 7 and CH shown in SEQ ID NO: 82, (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to the sequence shown in (i), or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in (i), and (b) A light chain comprising an amino acid sequence selected from the following: (iv) A sequence comprising VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 83, (v) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to the sequence shown in (iv), or A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity as compared to the sequences shown in (vi)(iv).

[0206] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0207] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises the following. (a) Heavy chain comprising an amino acid sequence selected from the following: (i) A sequence comprising VH shown in SEQ ID NO: 19 or 20 and CH shown in SEQ ID NO: 82, (ii) A sequence having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) as compared to the sequence shown in (i), or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity as compared to the sequence shown in (i), and (b) Light chain comprising an amino acid sequence selected from the following: (iv) A sequence comprising VL shown in SEQ ID NO: 22 and CL shown in SEQ ID NO: 83, (v) an array having one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids, or any combination thereof, e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the sequence shown in (iv), or (vi) an array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence shown in (iv).

[0208] In certain embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0209] In certain embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0210] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain, said heavy chain comprises the following sequence or is composed of the following sequence: (i) the sequence shown in SEQ ID NO: 33, (ii) an array having one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g., a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids, or any combination thereof, e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the sequence shown in (i), or, (iii) The sequence shown in (i), a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and The light chain comprises the following sequence or is composed of the following sequence: (iv) The sequence shown in SEQ ID NO: 56, (v) Compared with the sequence shown in (iv), a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof), or (vi) The sequence shown in (iv), a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0211] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain, The heavy chain comprises the following sequence or is composed of the following sequence: (i) The sequence shown in SEQ ID NO: 45, (ii) An array having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof, e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the array shown in (i), or (iii) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the array shown in (i), and The light chain comprises or consists of the following sequence: (iv) The sequence shown in SEQ ID NO: 56, (v) An array having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof, e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the array shown in (iv), or (vi) An array having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the array shown in (iv), Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0212] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain, The heavy chain comprises, or consists of, the following sequences: (i) The sequence shown in SEQ ID NO: 67, (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to the sequence shown in (i), or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (i), and The light chain comprises, or consists of, the following sequences: (iv) The sequence shown in SEQ ID NO: 81, (v) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to the sequence shown in (iv), or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (iv), Preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0213] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the following sequence: (i) the sequence shown in SEQ ID NO: 80, (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the sequence shown in (i), or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence shown in (i), and wherein the light chain comprises or consists of the following sequence: (iv) the sequence shown in SEQ ID NO: 81, (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the sequence shown in (iv), or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the sequence shown in (iv), Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0214] In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain selected from the group consisting of: (a) a heavy chain comprising VH shown in SEQ ID NO: 6 or 7 and CH shown in SEQ ID NO: 82, and a light chain comprising VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 83; (b) a heavy chain comprising VH shown in SEQ ID NO: 19 or 20 and CH shown in SEQ ID NO: 82, and a light chain comprising VL shown in SEQ ID NO: 22 and CL shown in SEQ ID NO: 83.

[0215] In certain embodiments, the antibody provided by the present disclosure is a multispecific antibody that binds to DLL3 and one or more other antigens. In certain preferred embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a quadrispecific antibody. In some embodiments, the multispecific antibody of the present disclosure comprises the anti-DLL3 antibody or antigen-binding fragment thereof and another antibody or fragment thereof or antibody mimetic.

[0216] The antibody derivative of the present disclosure The anti-DLL3 antibodies or antigen-binding fragments thereof of the present disclosure can be conjugated, for example, to another molecule (e.g., another polypeptide or protein) to be derivatized. Generally, derivatization (e.g., labeling) of an antibody or its antigen-binding fragment does not adversely affect its binding to DLL3. Accordingly, the anti-DLL3 antibodies or antigen-binding fragments thereof of the present disclosure are also intended to include such derivatized forms. For example, the anti-DLL3 antibodies or antigen-binding fragments thereof of the present disclosure can be conjugated (by chemical coupling, gene fusion, non-covalent binding or other means) to one or more other molecular moieties such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or polyhistidine tag) capable of mediating the binding of the antibody or its antigen-binding fragment to another molecule.

[0217] One type of derivatized antibody (e.g., a bispecific antibody) is produced by cross-linking two or more antibodies (belonging to the same type or different types). Methods for obtaining bispecific antibodies are well known in the art and include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods) or genetic engineering methods.

[0218] Another type of derivatized antibody is a labeled antibody. For example, the antibodies or antigen-binding fragments thereof of the present disclosure can be linked to a detectable label. The detectable labels described in the present disclosure can be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and examples thereof include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32P), calorimetric markers such as fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., Dynabeads®), gold colloids or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin that binds to avidin (e.g., streptavidin) modified with the above markers, but are not limited thereto. Patents teaching the use of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241 (all incorporated herein by reference). The detectable labels described above can be detected by methods well known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted light. Enzyme markers are generally detected by providing a substrate for the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and calorimetric markers are detected simply by visualizing the colored marker. In certain embodiments, such labels can be adapted for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, the detectable labels described above can be attached to the antibodies or antigen-binding fragments of the present disclosure via linkers of various lengths to reduce potential steric hindrance.

[0219] Furthermore, the antibodies or antigen-binding fragments thereof of the present invention can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups can be used to improve the biological properties of the antibodies, such as prolonging the serum half-life.

[0220] Production of Antibodies The antibodies of the present disclosure can be produced by various methods well known in the art, such as genetic engineering and recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present disclosure can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and transfected into a host cell. The transfected host cell is cultured under specific conditions to express the antibodies of the present invention.

[0221] The antigen-binding fragments of the present disclosure can be obtained by hydrolysis of a complete antibody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). In addition, these antigen-binding fragments can also be generated directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab’ fragments can be obtained directly from host cells, and Fab’ fragments can be chemically linked to form F(ab’)2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Furthermore, Fv, Fab or F(ab’)2 fragments can also be isolated directly from the recombinant host cell culture medium. Those skilled in the art are well aware of other techniques for producing such antigen-binding fragments.

[0222] In a third aspect, the present disclosure provides an isolated nucleic acid molecule comprising an antibody or an antigen-binding fragment thereof according to the second aspect of the present disclosure, or a nucleotide sequence encoding the heavy-chain variable region and / or light-chain variable region thereof, or one or more CDRs thereof, or a multispecific antibody according to the present disclosure. In certain embodiments, the nucleotide sequence is replaceable according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0223] In certain embodiments, the isolated nucleic acid molecule described in the present disclosure comprises: (i) a first nucleic acid and a second nucleic acid encoding the heavy-chain variable region and the light-chain variable region, respectively, of an antibody or an antigen-binding fragment thereof according to the second aspect of the present disclosure, or (ii) a first nucleic acid encoding the heavy-chain variable region and the heavy-chain constant region, respectively, of an antibody or an antigen-binding fragment thereof according to the second aspect of the present disclosure, and a second nucleic acid encoding the light-chain variable region and the light-chain constant region, respectively, or (iii) a first nucleic acid and a second nucleic acid encoding the heavy chain and the light chain, respectively, of an antibody or an antigen-binding fragment thereof according to the second aspect of the present disclosure. In certain embodiments, the first nucleic acid and the second nucleic acid comprise nucleic acids having a degenerate sequence or a substantially identical sequence to the first nucleic acid and the second nucleic acid of any of (i)-(iii) above. In certain embodiments, the degenerate sequence or substantially identical sequence refers to a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity compared to the nucleic acid molecule described in (i)-(iii), or a sequence having one or more nucleotide substitutions, or a sequence having a difference of 3, 6, 15, 30 or 45 or fewer nucleotides.

[0224] In a fourth aspect, a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule according to the third aspect of the present disclosure is provided. In certain embodiments, the vector of the present invention is a cloning vector or an expression vector. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector can express the antibody or an antigen-binding fragment thereof according to the present disclosure in a subject (e.g., a mammal such as a human).

[0225] In a fifth aspect, there is provided a host cell comprising the isolated nucleic acid molecule of the third aspect of the present disclosure or the vector of the fourth aspect of the present disclosure. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, SP2 / 0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present disclosure is a mammalian cell such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0226] In a sixth aspect, there is provided a method for producing the antibody or antigen-binding fragment thereof of the second aspect of the present disclosure, or the multispecific antibody of the present disclosure, the method comprising culturing the host cell of the present disclosure under conditions that allow the expression of the antibody or antigen-binding fragment thereof, or the multispecific antibody, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0227] Antibody-drug conjugate In a seventh aspect, there is provided an anti-DLL3 antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the antibody is the above-mentioned antibody or antigen-binding fragment thereof that binds to DLL3.

[0228] In some embodiments, the antibody-drug conjugate is selected from the following: [Chemical formula] [Chemical formula] [Chemical formula] (wherein Tb2 is an anti-DLL3 antibody or an antigen-binding fragment thereof, for example, rovalpituzumab, tarlatamab, 55C11E4-Hz1, 55C11E4-Hz2, 59B10D3-Hz, 10F2F3-Hz, 87F7F10-Hz, C2, 6F11C10-Hz1 and 6F11C10-Hz2 antibodies). q is selected from any numerical value between 0.1 and 16.0, preferably any numerical value between 2 and 8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0229] Manufacture of Antibody-Drug Conjugates In the eighth aspect of the present disclosure, the present disclosure provides a method for manufacturing the aforementioned antibody-drug conjugate, and this method includes the following. Perform a coupling reaction on Tb and the drug linker complex represented by Formula III under appropriate solvents and conditions:

Chemical formula

[0230] In some embodiments, Lg is selected from F, Cl, MeSO2-.

[0231] In some embodiments, Lg is selected from F, MeSO2-.

[0232] In some embodiments, the method includes coupling Tb with a drug linker complex represented by Formula III under appropriate solvents and conditions to form a C-S bond.

Chemical formula

[0233] In some embodiments, the molar ratio of the Tb to the drug linker complex is 1:(1 to 20), for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10 to 20), 1:(12 to 20), 1:(14 to 20), 1:(16 to 20), or 1:(18 to 20).

[0234] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0235] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol), or any combination thereof.

[0236] In some embodiments, the method further includes purifying the coupling product.

[0237] In some embodiments, the coupling product is purified by a chromatography method.

[0238] In some embodiments, the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography.

[0239] In a ninth aspect of the present disclosure, the present disclosure provides a group of antibody-drug conjugates comprising the aforementioned antibody-drug conjugate of the present disclosure, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or its pharmaceutically acceptable solvates, wherein the antibody-drug conjugate has one, two, or more q values.

[0240] In some embodiments, when the antibody-drug conjugate within the group has one q value, the q value is equal to the DAR value.

[0241] In some embodiments, when the antibody-drug conjugate within the group has two or more q values, the proportion of the antibody-drug conjugate having a specific q value among all the antibody-drug conjugates within the group is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%.

[0242] In some embodiments, the drug-to-antibody ratio (DAR) of the antibody-drug conjugate within the group is selected from integers or decimals from 1 to 10.

[0243] In some embodiments, the drug-to-antibody ratio (DAR) of the group is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5, and 7.5 to 8.5. In some embodiments, the drug-to-antibody ratio (DAR) of the group is selected from approximately 2.0, 4.0, 6.0, and 8.0. In some embodiments, the drug-to-antibody ratio (DAR) in the antibody-drug conjugate of the group is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.

[0244] In some embodiments, the group includes ADCs having a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 payload species). It should be noted that degradation products may be generated and the group may include DARs of 1, 3, 5, and 7. Further, the group can also have a DAR exceeding 8. The antibody-drug conjugate is generated by coupling after being reduced by an interchain disulfide. In some embodiments, the antibody-drug conjugate includes both the antibody-drug conjugate with a DAR of 4 or less (i.e., the payload species is 4 or less) and the antibody-drug conjugate with a DAR of 6 or more (i.e., the payload species is 6 or more).

[0245] In a tenth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect or the seventh aspect above, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, the anti-DLL3 antibody of the second aspect or its antigen-binding fragment, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, or the group of antibody-drug conjugates of the ninth aspect, and optionally one or more pharmaceutical excipients.

[0246] In certain embodiments, the pharmaceutical composition comprises the antibody-drug conjugate of the first aspect or the seventh aspect above, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, the anti-DLL3 antibody of the second aspect or its antigen-binding fragment, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, or the group of antibody-drug conjugates of the ninth aspect, and all of their dosages are therapeutically effective amounts.

[0247] In certain embodiments, the pharmaceutical composition comprises the antibody-drug conjugate of the first aspect or the seventh aspect above, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and a pharmaceutically acceptable carrier and / or excipient.

[0248] In certain embodiments, the pharmaceutical composition of the present disclosure comprises a group of antibody-drug conjugates of the ninth aspect, and a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition of the present disclosure comprises the above group of antibody-drug conjugates or comprises the above group of antibody-drug conjugates and a buffer. In certain further preferred embodiments, the pharmaceutical composition of the present disclosure further comprises an excipient and / or a surfactant.

[0249] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the anti-DLL3 antibody of the present disclosure or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition of the present disclosure comprises an anti-DLL3 antibody or an antigen-binding fragment thereof and a buffer. In certain further preferred embodiments, the pharmaceutical composition of the present disclosure further comprises an excipient and / or a surfactant.

[0250] In some embodiments, the buffer is a histidine buffer. In certain preferred embodiments, the buffer is a 20 mM histidine buffer with a pH of 6.0.

[0251] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises the above isolated nucleic acid molecule or vector.

[0252] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the multispecific antibody of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0253] In some embodiments, the drug-to-antibody ratio (DAR) in the pharmaceutical composition is selected from integers or decimals from 1 to 10.

[0254] In some embodiments, the drug-to-antibody ratio (DAR) in the pharmaceutical composition is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5, and 7.5 to 8.5. In some embodiments, the drug-to-antibody ratio (DAR) in the pharmaceutical composition is selected from about 2.0, 4.0, 6.0, and 8.0, In some embodiments, the drug-to-antibody ratio (DAR) in the antibody-drug conjugate in the pharmaceutical composition is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.

[0255] In a 11th aspect, there is provided the use of an antibody of the present disclosure or an antigen-binding fragment thereof in the manufacture of a kit for detecting the presence or level of DLL3 in a sample. In another aspect, the present disclosure provides a diagnostic kit or a therapeutic kit comprising one or more of the antibody or an antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, antibody-drug conjugate, group of antibody-drug conjugates, or pharmaceutical composition described in the present disclosure. Optionally, the diagnostic kit or the therapeutic kit further comprises instructions for use.

[0256] In a 12th aspect of the present disclosure, there is provided the use of the antibody-drug conjugate composition or the pharmaceutical composition in the manufacture of a medicament for treating and / or preventing a disease associated with abnormal cell activity (e.g., a cancer disease). The antibody-drug conjugate composition or the pharmaceutical composition may be in a therapeutically effective amount.

[0257] In some examples, there is provided the use of an anti-DLL3 antibody or an antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure in the manufacture of a medicament, wherein the medicament is used to modulate (inhibit or block) the activity of DLL3.

[0258] In some examples, there is provided the use of an anti-DLL3 antibody or an antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure in the manufacture of a medicament, wherein the medicament is used for the treatment or prevention of a disease associated with the activity of DLL3.

[0259] In some embodiments, provided is the use of the anti-DLL3 antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, antibody-drug conjugate, or multispecific antibody of the present disclosure in the manufacture of a medicament, wherein the medicament is used for the treatment or prevention of tumors related to the activity of DLL3.

[0260] In some embodiments, provided is the use of the antibody-drug conjugate of the first aspect or the seventh aspect above of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, the anti-DLL3 antibody or antigen-binding fragment thereof of the second aspect, the nucleic acid according to the third aspect, the vector according to the fourth aspect, the host cell according to the fifth aspect, the group of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the manufacture of a medicament, wherein the medicament is used for the treatment or prevention of tumors.

[0261] In some embodiments, provided is the use of the antibody-drug conjugate of the first aspect or the seventh aspect above of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, the group of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the manufacture of a medicament, wherein the medicament is used for the treatment or prevention of tumors related to the target of Tb.

[0262] In some embodiments, provided is the use of the antibody-drug conjugate of the first aspect or the seventh aspect above of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, the group of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the manufacture of a medicament, wherein the medicament is used for the treatment or prevention of tumors related to DLL3.

[0263] In some preferred embodiments, provided is the use of the antibody-drug conjugate of the first aspect or the seventh aspect of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate in the manufacture of a medicament, the anti-DLL3 antibody or its antigen-binding fragment of the second aspect, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the group of antibody-drug conjugates of the ninth aspect, and the pharmaceutical composition of the tenth aspect in the treatment or prevention of tumors related to DLL3.

[0264] In the thirteenth aspect of the present disclosure, the present disclosure provides a method of using the antibody-drug conjugate of the first aspect or the seventh aspect of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, the anti-DLL3 antibody or its antigen-binding fragment of the second aspect, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the group of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the treatment and / or prevention of diseases (such as tumors) related to abnormal cell activity.

[0265] In the above twelfth and thirteenth aspects, the tumor is selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer or lung adenocarcinoma), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colorectal adenocarcinoma), rectal cancer, large bowel cancer, liver cancer, kidney cancer, urothelial cancer, epidermal cancer, non-Hodgkin lymphoma, central nervous system tumors (such as glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0266] In some embodiments, the tumor is a DLL3-related tumor.

[0267] In some implementation states, the tumor is a tumor related to DLL3.

[0268] Unless otherwise specified, the scientific and technical terms used in this specification have the meanings generally understood by those skilled in the art. In addition, the experimental steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used in this specification are all ordinary steps widely used in the art. At the same time, to better understand this specification, the definitions and explanations of related terms are provided below.

[0269] Examples of the term "pharmaceutically acceptable salt" as used in this specification are organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkylsulfonate or arylsulfonate. Preferably, the alkylsulfonate is methylsulfonate or ethylsulfonate, and the arylsulfonate is benzenesulfonate or p-toluenesulfonate. Appropriate inorganic salts may also be formed, including but not limited to hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate and carbonate, sulfate or phosphate.

[0270] The term "pharmaceutically acceptable carrier and / or excipient" as used in this specification refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is known in the art (see, for example, Remington’s Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption retardants, and preservatives.

[0271] Pharmaceutically acceptable salts can be obtained by standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0272] As used herein, the pharmaceutical adjuvant refers to excipients and additives used in the manufacture and formulation of pharmaceuticals, and refers to substances contained in pharmaceutical preparations whose safety has been reasonably evaluated in addition to the active ingredient. Pharmaceutical adjuvants act as shaping agents, carriers, improve stability, and also have important functions such as solubilization, co-solubilization, sustained release, and release control. They are important components that can affect the quality, safety, and efficacy of drugs. Depending on their origin, they may be divided into natural products, semi-synthetic products, and fully synthetic products. Depending on their effects and uses, they may be classified into solvents, propellants, solubilizing agents, co-solubilizing aids, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, moisturizing agents, absorbents, diluents, aggregating and disaggregating agents, filter aids, release blocking agents, etc. Depending on the administration route, they may be classified into oral administration, injection, mucosal administration, transdermal or topical administration, nasal or oral inhalation administration, and ophthalmic administration, etc. The same pharmaceutical adjuvant can be used in pharmaceutical preparations with different administration routes and has different effects and uses.

[0273] The pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route. For example, tablets, capsules, granules, oral liquids, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye drops, pills, implants, aerosols, powder aerosols, sprays, etc. Here, the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or complex, preferably contains 0.1 mg to 800 mg, preferably contains 0.5 to 500 mg, preferably contains 0.5 to 350 mg, and particularly preferably contains 1 to 250 mg.

[0274] The pharmaceutical composition can be administered as an injection such as an injection, a sterile powder for injection, a concentrated solution for injection, etc. Here, usable carriers and solvents include water, Ringer's solution and isotonic sodium chloride solution. Further, a sterile non-volatile oil such as monoglyceride or diglyceride can also be used as a solvent or a suspension medium.

[0275] As used herein, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such an effect may be prophylactic in terms of complete or partial prevention of a disease or its symptoms, and / or may be therapeutic in terms of partial or complete stabilization or cure of a disease and / or side effects caused by the disease. "Treatment" as used herein includes any treatment of a patient's disease, including (a) prevention of a disease or symptoms in a patient who is susceptible to but not yet diagnosed with the disease or symptoms, (b) inhibition of the symptoms of a disease, i.e., prevention of its onset, or (c) alleviation of the symptoms of a disease, i.e., regression of the disease or symptoms.

[0276] In the present disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., a disease described herein) or normal individuals. In the present disclosure, the term "non-human animal" includes all vertebrates, including non-mammals (e.g., birds, amphibians, reptiles) and mammals (e.g., non-human primates, pets and / or livestock (e.g., sheep, dogs, cats, dairy cows, pigs, etc.)).

[0277] In the present disclosure, the term "effective dose" refers to the amount of a compound that, upon administration, alleviates to some extent one or more symptoms of the condition of the treatment subject.

[0278] The term "bioactive substance", "bioactive molecule" or "drug molecule" in the present disclosure refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. In some embodiments of the present disclosure, the bioactive substance, bioactive molecule or drug molecule in the complex is a molecule having anti-tumor bioactivity. For example, At 211 、I 131, I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes such as Lu; for example, metal complexes such as metal platinum complexes, metal gold complexes, oxaliplatin; for example, glycopeptide antibiotics such as bleomycin, pingyangmycin; for example, DNA topoisomerase inhibitors such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, velotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, adriamycin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide; for example, drugs that inhibit DNA synthesis such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine; for example, drugs that act on structural proteins such as tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel; for example, tumor signal transduction pathway inhibitors such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors, and further, proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cell cycle protein inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, Pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit the growth of tumor cells and promote apoptosis and necrosis of tumor cells; enzymes such as nucleases and fragments thereof; antibiotics; small molecule payloads or enzyme-active payloads, fragments and / or variants thereof derived from bacteria, fungi, plants or animals; growth inhibitors, pharmaceutical modules. The term "payload" refers to a substance that may have an adverse effect on the growth or proliferation of cells.

[0279] In the present disclosure, the term "linker" refers to a fragment that links a bioactive molecule (drug molecule) to a target moiety.

[0280] In the present disclosure, the term "target moiety" refers to a moiety in a complex that can specifically bind to a target (or a part of the target) on the cell surface. Due to the interaction between the target moiety and the target, the complex is delivered to a specific cell population.

[0281] In the present disclosure, antibodies or antigen-binding fragments thereof include derivatized antibodies or antigen-binding fragments thereof, such as antibodies having sulfhydryl, in which the antibody can have a group or ability to react with a drug-linker complex by the derivatization. The sulfhydryl -SH may be induced by opening a disulfide bond (for example, reduction by a reducing agent TCEP).

[0282] As used herein, the terms "cancer" and "tumor" are used interchangeably.

[0283] As used herein, the term "gene" includes not only DNA, but also its mRNA, its cDNA and its cRNA.

[0284] As used herein, the term "polynucleotide" is used in the same sense as nucleic acid and further includes DNA, RNA, probes, oligonucleotides and primers.

[0285] As used herein, the terms "polypeptide" and "protein" are used interchangeably.

[0286] As used herein, the term "cell" includes cells of an individual in an animal and cultured cells.

[0287] The DLL3 contained in the anti-DLL3 antibody described in the present disclosure may be a conventional DLL3 in the art, and also represents a DLL3 variant.

[0288] The term "DLL3" refers to a highly tumor-selective cell surface target that is mainly expressed in nerves or neuroendocrine tumors, including small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioblastoma multiforme, metastatic castration-resistant prostate cancer, and pulmonary neuroendocrine tumors. In particular, in the case of SCLC, more than 80% of SCLC shows positive DLL3 expression, but it is rarely present in normal lung cancer tissues and tumor-adjacent tissues. DLL3 is a type I transmembrane protein composed of 618 amino acids. Among them, positions 27 to 492 are the extracellular domain, which is the target region for ADC drug-target binding. The extracellular domain is composed of an N-terminal DSL domain followed by six tandem EGF-like domains.

[0289] KD refers to the dissociation constant obtained from the ratio of Kd (the dissociation rate of the interaction between a specific binding molecule and a target protein) and Ka (the binding rate of the interaction between a specific binding molecule and a target protein) (or Kd / Ka, expressed in molar concentration (M)). The KD value can be measured using methods well established in the art. A preferred method for measuring the KD of a binding molecule is to use a biosensor system, such as a surface plasmon resonance system like the Biacore TM (GE Healthcare Life Sciences) system.

[0290] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences. The percentage of sequence identity between two sequences is a function of the number of positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), where the number of gaps and the length of each gap need to be introduced to achieve an optimal comparison of the two sequences. The comparison of sequences and the determination of the percentage of identity can be performed using methods generally known in the art, and mathematical algorithms can be used to perform such sequence comparison and determination of the percentage of identity. For example, the algorithm of Meyers and Miller, 1988 Comput. Appl. Biosci. 4:11-17 (incorporated into the ALIGN program (version 2.0)) can be used to determine the percentage of identity between amino acid sequences and / or nucleotide sequences. Further, the GAP program (using default parameters) of the GCG software package available online from Accelrys can be used to determine the percentage of identity between amino acid sequences or nucleotide sequences. In one embodiment, the lengths of the two sequences are the same.

[0291] The term "epitope" refers to a part of an antigen polypeptide or protein that has antigenic or immunogenic activity in vivo in an animal, preferably a mammal. The epitopes of the antibodies or antigen-binding fragments thereof of the present disclosure can be determined using existing techniques such as synthetic peptide methods, immunoinformatics prediction, determination of polypeptide activity, epitope peptide scanning, phage display technology, X-ray diffraction and nuclear magnetic resonance analysis, and antibody homology modeling and protein docking prediction methods. The phrase "antibodies that bind to the same epitope" as used herein refers to different antibodies that bind to a common epitope. When a second antibody binds to a part of the peptide or a part of the tertiary structure to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope.

[0292] In the present disclosure, the term "antibody" is used in the broadest interpretation and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, as long as they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" may be used interchangeably. As used herein, an "antibody molecule" or "antibody" refers to an immunoglobulin molecule and the immunologically active portion of the immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that binds immunologically specifically to an antigen. Thus, the term antibody, in its broadest interpretation, includes not only intact antibody molecules but also the antibody fragments and variants (including derivatives) of the antibody and antibody fragments. When an "antibody molecule" or "antibody" is used in the same context as an antigen-binding fragment, the "antibody molecule" or "antibody" refers to an intact antibody molecule or full-length antibody. As used herein, the term antibody molecule includes, but is not limited to, for example, single-chain Fv (scFv), Fab fragment, Fab' fragment, F(ab')2, disulfide-linked Fv (sdFv), Fv, and intact antibodies or full-length antibodies. The term "single-chain Fv" or "scFv" refers to a polypeptide containing the VL domain of an antibody linked to the VH domain of the antibody. For example, an antibody that binds immunologically specifically to DLL3 may cross-react with other antigens. Preferably, an antibody that binds immunologically specifically to DLL3 does not cross-react with other antigens. Immunologically specific binding can be identified, for example, by immunoassay or other methods well known to those skilled in the art. An "intact" antibody or "full-length" antibody refers to a protein containing two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds, and the protein includes (1) for the heavy chain, a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region containing three domains CH1, CH2, and CH3, and (2) for the light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region containing one domain CL.The antibodies of the present disclosure include, but are not limited to, monoclonal antibodies, multispecific antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab’) fragments, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of the antibodies of the present disclosure), and epitope-binding fragments of any of the above antibodies. The immunoglobulin molecules of the present disclosure may be of any type of immunoglobulin (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass. Preferably, the antibodies of the present disclosure have a VH domain, a VH CDR (often represented herein as HCDR), a VL domain, or a VL CDR (often represented herein as LCDR) having any of the amino acid sequences or fragments or variants thereof described in the sequence and its specific information table.

[0293] In the present disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is identical except for a few natural variations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies contain different antibodies against different determinants (epitopes). In addition to specificity, an advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" as used herein is not to be construed as meaning that the antibody has the characteristic of being derived from a substantially homogeneous group of antibodies and requires a special method of production.

[0294] In some embodiments of the present disclosure, monoclonal antibodies further include chimeric antibodies, i.e., antibodies in which part of the heavy chain and / or light chain is identical or homologous to one type, class or subclass of antibody, and the remaining part is of another type of antibody, as long as they are identical or homologous to another class or subclass and have the desired biological activity (see, e.g., US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies that contain variable region antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, chimpanzees, etc.) and human constant region sequences.

[0295] The term "antigen-binding fragment" refers to a part of an antibody, preferably the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fd, Fv, dAb and complementarity-determining region fragments, diabodies, linear antibodies and single-chain antibody molecules. As used herein, the term "antigen-binding fragment" refers to a partial fragment of an antibody having antigen-binding activity, where the fragment has all or part of the function of the antibody and includes, but is not limited to, single-chain Fv (scFv), Fab, Fab’, F(ab’)2, disulfide-bonded Fv (sdFv), Fv, di-scFv, etc. The term also includes Fab’ which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab’)2 under reducing conditions. Here, the term is not limited to these molecules as long as the fragment has binding affinity for the antigen. Further, these functional fragments include not only fragments obtained by treating the full-length molecule of the antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using a genetically modified antibody gene.

[0296] As used herein, the term "Fab’" refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab’)2 under the above-mentioned reducing conditions. Here, the Fab’ of the present disclosure also includes Fab’ produced using a genetically modified antibody gene.

[0297] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, where said VL and VH are linked either by a linker or directly (e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art are composed of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers applicable to the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may exist between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by the linkage of two scFvs.

[0298] As used herein, the antibody or antigen-binding fragment thereof includes variants, amino acid substitutions, deletions or additions that still have the activity of binding to the antigen.

[0299] The term "bispecific antibody" is also referred to as "bifunctional antibody complex", and refers to a complex formed by a first antibody (fragment) and a second antibody (fragment) via a binding arm, and the complex retains the activity of each antibody and has bifunctional and bispecific properties.

[0300] The term "multispecific antibody" includes, for example, a trispecific antibody having three different antigen-binding specificities and a tetravalent antibody having four different antigen-binding specificities.

[0301] The term "complete antibody" or "full-length antibody" refers to an antibody that includes an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. The complete antibody is preferably a complete antibody having one or more effector functions.

[0302] The term "Probody" is a modified antibody that includes an antibody or antibody fragment that specifically binds to its target and can bind to a masking group, where the dissociation constant of the binding ability of the antibody or antibody fragment to its target is at least 100-fold, 1000-fold, or 10,000-fold greater than the dissociation constant of the binding ability of the antibody or antibody fragment not bound to the masking group to its target.

[0303] In the present disclosure, a "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains minimal non-human immunoglobulin sequences. Most humanized antibodies are human receptor immunoglobulins, and the residues of their hypervariable regions can be replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibodies) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Additionally, the humanized antibody may further contain residues that are not present in the recipient antibody or the donor antibody. These modifications are intended to further optimize the performance of the antibody. A humanized antibody generally contains at least one variable region, usually two variable regions, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and the FRs are the complete or substantially complete sequences of the human immunoglobulin. The humanized antibody may further contain at least a portion of the immunoglobulin constant region (Fc, typically the human immunoglobulin Fc). For details, see, for example, Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Biol 2:593-596.

[0304] Complete antibodies can be classified into different "classes" based on the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, and some of these may be further classified into different "subclasses" (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different classes of antibodies are designated α, β, ε, γ, and μ, respectively. The subunit structures and tertiary structures of the immunoglobulins of different classes are known in the art.

[0305] In this specification, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably determined using the Kabat, Chothia, or AbM or IMGT numbering systems.

[0306] As used herein, the term "framework residue region" or "FR residue" refers to the amino acid residues within the antibody variable region other than the CDR residues defined above.

[0307] As used herein, the term "germline antibody gene" refers to a gene encoding an immunoglobulin that is expressed in non-lymphoid cells and has not undergone the maturation process leading to the rearrangement and maturation of the genes that result in the expression of a particular immunoglobulin. The advantages provided by various embodiments of the present disclosure are derived from the recognition that the amino acid sequence encoded by the germline antibody gene retains more of the characteristic and important amino acid sequence structures of an individual of an animal species than the amino acid sequence encoded by the mature antibody gene. Therefore, when therapeutically applied to that species, the likelihood of being recognized as foreign by that species is reduced.

[0308] In this specification, conservative amino acid substitutions are preferred as amino acid substitutions. Conservative amino acid substitutions refer to substitutions that occur within a set of amino acids related to the side chains of amino acids. Preferred sets of amino acids are the acidic set (aspartic acid and glutamic acid), the basic set (lysine, arginine, and histidine), the nonpolar set (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), and the uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine). More preferred sets of amino acids are the aliphatic hydroxyl group (serine and threonine), the amide-containing set (asparagine and glutamine), the aliphatic set (alanine, valine, leucine, and isoleucine), and the aromatic set (phenylalanine, tryptophan, and tyrosine). Such amino acid substitutions are preferably carried out within a set that does not impair the properties of the substance having the original amino acid sequence.

[0309] It is also known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced by cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of an antibody produced by cultured mammalian cells are deleted and the proline residue newly arranged at the carboxyl terminus is amidated (Analytical Biochemistry, 360:75-83(2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cell cytotoxicity, etc.) of the antibody.

[0310] The 20 conventional amino acids included in this specification are described according to conventional usage. For example, Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)) can be referred to and is incorporated herein by reference. In this specification, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Further, in this disclosure, amino acids are usually represented using one-letter and three-letter abbreviations known in the art. For example, alanine is represented by A or Ala, arginine is represented by R or Arg, glycine is represented by G or Gly, and glutamine is represented by Q or Gln.

[0311] As used herein, the term "prevent" refers to a method implemented to prevent or delay the onset of a disease or disorder or symptom (e.g., tumor and infection) in a subject. The term "treat" as used herein refers to a method implemented to obtain a beneficial or desirable clinical outcome. For the purposes of this disclosure, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of the disease, stabilization of the disease state (i.e., prevention of further deterioration), delay or deceleration of the progression of the disease, improvement or alleviation of the disease state, and alleviation of symptoms (partial or complete, whether detectable or not). Further, "treat" may also refer to extending the survival period compared to the expected survival period (if not treated).

[0312] As used herein, the term "subject" refers to a mammalian primate, such as a mammalian non-human primate or a human. In certain embodiments, the subject (e.g., a human) is suffering from or at risk of suffering from a tumor or an infection.

[0313] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (e.g., tumor and infection) refers to an amount sufficient to prevent, inhibit, or delay the occurrence of the disease (e.g., tumor and infection), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. Measurement of such effective amounts is well within the ability of one of ordinary skill in the art. For example, the therapeutically effective amount depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the method of drug administration, and other treatments administered simultaneously.

[0314] As used herein, the term "effector function" refers to a biological activity resulting from the Fc region of an antibody (natural sequence Fc region or amino acid sequence variant Fc region), which varies depending on the antibody isotype.

[0315] The term "pharmaceutically acceptable" refers to a molecule, molecular fragment, or composition that does not cause an adverse reaction, allergic reaction, or other harmful reaction when appropriately administered to an animal or human. Specific examples of substances that may function as pharmaceutically acceptable carriers or components thereof include saccharides (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginates, and the like.

[0316] The term "group of antibody-drug conjugates" refers to a mixture composed of a set or group of the antibody-drug conjugates of the present disclosure, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers, or its pharmaceutically acceptable solvates, where the q of the antibody-drug conjugates may be the same or different. Further, it may also be referred to as an "antibody-drug conjugate mixture".

[0317] The in vivo therapeutic effect of an antibody and an antibody-drug conjugate against cancer can be determined by using experimental animals, for example, by administering the antibody to nude mice transplanted with a tumor cell line expressing DLL3 and measuring the changes in cancer cells.

[0318] In the present disclosure, the amino acid substitutions in the antibody are mainly substituted by L-amino acids, but are not limited thereto. In some embodiments, one or more D-amino acids may be included in the antibody peptide chain. Peptides containing D-amino acids are more stable than peptides containing only L-amino acids and are less susceptible to degradation in the oral cavity, intestinal tract, or plasma.

[0319] The monoclonal antibodies used in the present invention can be produced by several methods. For example, the monoclonal antibodies used in the present disclosure can be obtained by the hybridoma method using many species (including mouse, hamster, rat, and human cells) (see, for example, Kohler et al., 1975, Nature, 256:495), or can be produced by recombinant DNA technology (see, for example, US 4,816,567), or can be isolated from a phage antibody library (see, for example, Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0320] In this specification, unless otherwise specified, the expressions "each... independently selected" and "... each independently selected" used throughout this specification may be used interchangeably and should be understood in a broad sense, which may mean that specific options represented by the same or different symbols in different groups do not affect each other, and may also mean that specific options represented by the same or different symbols in the same group do not affect each other.

[0321] In this specification, the term "direct bond" indicates that the groups on both sides are directly linked. For example, in the compound represented by Formula II

Chem.

Chem.

[0322] In this specification, the term "absent" means that, for example, in the compound represented by Formula II

Chem.

Chem.

[0323] In the compound represented by Formula II

Chem.

Chem.

Chem.

[0324] In this specification, for the drug linker complex represented by Formula III

Chem.

Chem.

Chem.

Chem.

[0325] In this specification, the compound represented by formula II

Chem.

Chem.

Chem.

[0326] In this specification, when the definition of X is, for example, "X is optionally substituted

Chem.

Chem.

[0327] In this specification, when the definition of X is, for example, "X is optionally substituted [Chemistry] selected from, wherein the substituent is selected from two C1-4 alkyls (e.g., methyl), and together with the carbon atoms to which they are attached form a C3-6 cycloalkyl (e.g., cyclopropyl), X is, for example, [Chemistry] may be. Other similar definitions of X can be understood with reference to the foregoing.

[0328] AA 1 the structure of the amino acid residue represented by [Chemistry] in, when r is 0, those skilled in the art will understand that the structure of the amino acid residue represented by AA 1 changes to [Chemistry] can be understood.

[0329] AA 1 the structure of the amino acid residue represented by [Chemistry] in, R a and R b form a 4- to 10-membered heterocyclic ring together with the carbon atom to which they are attached, the 4- to 10-membered heterocyclic ring is optionally substituted by one or more R 0 , where the term "the 4- to 10-membered heterocyclic ring is optionally substituted by one or more R 0 " means that the 4- to 10-membered heterocyclic ring may not be substituted, or may be substituted by one or more R 0 , and in the plurality of R 0 , each R 0 may have the same or different definitions. Other similar definitions can be understood with reference to the foregoing.

[0330] In this specification, for example, when L3 is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, Lys-Ala-Asn, "the distal amino of the lysine (Lys) is optionally substituted with one, two or three substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O" means that the distal amino of Lys in each option of the above L3 is optionally substituted with one, two or three substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O. Here, "the distal amino of Lys" refers to the exposed amino group -NH2 in the lysine residue

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0331] In various parts of this specification, substituents of the compounds of the present disclosure are disclosed according to the type or range of the groups. In particular, the present disclosure includes each independent secondary combination of each member of these types and ranges of groups. For example, the term "C1-6 alkyl" specifically refers to methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl independently disclosed.

[0332] In this specification, the term "C1-6 alkyl" represents an alkyl containing 1 to 6 carbon atoms in a straight or branched chain, for example, "C1-3 alkyl" or "C1-4 alkyl", including methyl, ethyl, etc., and specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl.

[0333] In this specification, the term "C" 1-4 alkyl" represents an alkyl containing 1 to 4 carbon atoms in a straight or branched chain, for example, "C" 1-3 alkyl", including methyl, ethyl, etc., and specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

[0334] In this specification, the term "C" 2-6 alkenyl" refers to a straight, branched, or cyclic alkenyl containing at least one double bond and 2 to 6 carbon atoms, for example, "C" 2-4Includes "alkenyl", etc. Examples include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, etc., but are not limited thereto.

[0335] In this specification, the term "C" 2-6 "alkynyl" refers to a straight-chain or branched-chain alkynyl containing at least one triple bond and 2 to 6 carbon atoms. For example, "C" 2-4 "alkynyl", etc. Examples include ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc., but are not limited thereto.

[0336] In this specification, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0337] In this specification, the term "3- to 6-membered cycloalkyl" or "C3-6 cycloalkyl" refers to a saturated cyclic alkyl containing 3 to 6 carbon atoms, including cyclopropane (cyclopropyl), cyclobutane (cyclobutyl), cyclopentane (cyclopentyl), and cyclohexyl.

[0338] In this specification, the term "3- to 7-membered carbocycloalkyl" or "C" 3-7 "cycloalkyl" refers to a saturated cyclic alkyl containing 3 to 7 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0339] As used herein, the term "C1-6 alkoxy" refers to alkyl as defined above, which is bonded to the parent molecular moiety via an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0340] As used herein, the term "C1-4 alkoxy" refers to alkyl as defined above, which is bonded to the parent molecular moiety via an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0341] As used herein, the term "4-10 membered heterocyclyl" refers to a cyclic group containing 4 to 10 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. The term "4-6 membered heterocyclyl" refers to a cyclic group containing 4 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure may be oxidized. "4-8 membered heterocyclyl" includes, for example, "4-8 membered nitrogen-containing heterocyclyl", "4-8 membered oxygen-containing heterocyclyl", "4-7 membered heterocyclyl", "4-7 membered oxygen-containing heterocyclyl", "4-7 membered heterocyclyl", "4-6 membered heterocyclyl", "5-7 membered heterocyclyl", "5-6 membered heterocyclyl", "5-6 membered nitrogen-containing heterocyclyl", and includes, but is not limited to, oxetanyl, pyrrolidinyl, tetrahydrofuryl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, and the like.

[0342] As used herein, the term "4- to 10-membered heterocyclic ring" refers to a ring containing 4 to 10 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. The term "5- to 6-membered heterocyclic ring" refers to a ring containing 5 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom, and includes rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, etc., but is not limited thereto.

[0343] As used herein, the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon group such as 6- to 10-membered aryl, 5- to 8-membered aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl, etc. The "6- to 10-membered aryl" refers to an aryl containing 6 to 10 ring atoms. The "C6-10 aryl" refers to an aryl containing 6 to 10 carbon atoms.

[0344] As used herein, the term "heteroaryl" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure may be oxidized. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure may be oxidized. Specific examples include, but are not limited to, 5- to 10-membered heteroaryl, 5- to 6-membered heteroaryl, 5- to 10-membered nitrogen-containing heteroaryl, 6- to 10-membered oxygen-containing heteroaryl, 6- to 8-membered nitrogen-containing heteroaryl, 5- to 8-membered oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxacyclohexadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, etc., but are not limited thereto.

[0345] As used herein, the bond in the structural formula represented by the wavy line "~~" is intended to indicate that the structure represents a cis isomer or a trans isomer, or a mixture of cis isomers and trans isomers in any ratio.

[0346] The term "drug-to-antibody ratio" or "DAR" refers to the ratio of drug to antibody in a group (or mixture) or composition or ADC molecule such as a small molecule payload bound to an antibody of an ADC. The DAR of an ADC ranges from 1 to 16, but higher loadings (e.g., 20) are possible depending on the number of conjugation sites on the antibody. The term DAR can be used when referring to the amount of drug loaded on a single antibody or when referring to the average or mean DAR of a group of ADCs. It should be understood that the latter is usually referred to as the average DAR. During measurement of the DAR value by mass spectrometry, the antibody is reduced to its separated heavy and light chains, and DAR1 represents a complex containing a light or heavy chain conjugated to one payload molecule, DAR2 represents a complex containing a light or heavy chain conjugated to two payload molecules, and DAR3 represents a complex containing a light or heavy chain conjugated to three payload molecules.

[0347] Advantageous effects of the invention Through extensive research on multiple targeted antibody-drug conjugates (ADCs), the present disclosure combines enrichment of the tumor microenvironment, linker-specific in vivo enzyme cleavage properties, and coupling methods with target sites, and a number of in vivo and in vitro pharmacodynamic screening verifications to obtain a new class of antibody bioactive molecule conjugates. Using the conjugate obtained according to the above method, various surprising technical effects as described below can be achieved.

[0348] The conjugate obtained according to the above method has improved solubility and excellent chemical stability. For example, it can avoid the reversible Michael addition reaction caused by the conventional maleimide bond in ADCs, so that a high drug-antibody ratio (DAR) can be achieved. In some embodiments, the DAR value of the conjugate can reach 6 to 8.

[0349] It has a very high coupling efficiency. In some embodiments, the coupling efficiency reaches or exceeds 90%.

[0350] Through extensive research, a linker has been discovered that has high plasma stability and is cleavable in the tumor microenvironment (both inside and outside tumor cells), enabling an effective anti-tumor effect even in tumors with low or no antigen expression.

[0351] The conjugate (ADC) obtained according to the above method can improve the exposure of the entire ADC molecule in a relatively acidic tumor environment by adjusting the physicochemical properties of the linker and the entire ADC molecule. Therefore, the ADC has better targeting to tumor tissues, that is, its ability to concentrate in the tumor microenvironment is improved, the ratio of the concentration of the bioactive molecule in the tumor to that in the blood increases, and the mechanism-related toxicity of the ADC molecule (the toxicity generated after the ADC binds to the cell surface antigen of non-tumor tissues and is endocytosed, also called "on-target toxicity") is reduced, thereby achieving a higher therapeutic index.

[0352] The conjugate obtained according to the above method shows high stability in the body circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target" toxicity caused by the shedding of the payload in non-target tissues.

[0353] The bioactive molecule of the conjugate has higher anti-tumor cell activity, thereby having an excellent by-stander effect, and the ADC can more effectively kill tumor cells with high antigen expression and tumor cells with low or no antigen expression in the tumor tissue.

[0354] The payload linker of the present disclosure can utilize the extracellular cleavage ability of the linker in the tumor microenvironment to form an antibody-drug conjugate with an antibody that does not have the ability of cell endocytosis, and such an antibody-drug conjugate still has high anti-tumor activity.

[0355] The payload linker of the present disclosure utilizes its extracellular cleavage ability and enrichment ability in the tumor microenvironment to form antibody-drug conjugates with antibodies lacking cell endocytosis ability and antibodies lacking the ability to bind to extracellular antigens of tumor cells, and such antibody-drug conjugates still have high antitumor activity.

[0356] The anti-DLL3 antibody provided by the present disclosure has a highly humanized or fully human antibody, and thus can be safely administered to human subjects without inducing an immunogenic reaction.

[0357] In summary, the ADC of the present disclosure has important clinical value.

Brief Description of the Drawings

[0358]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0359] The present disclosure will be further described through the following description of specific embodiments, which does not limit the present invention. Those skilled in the art can make various changes or improvements based on the teachings of the present disclosure without departing from the basic concepts and scope of the present disclosure. When the manufacturer of the reagents or equipment used is not specified, all are common products that can be purchased commercially.

[0360] The abbreviations in the present disclosure have the following meanings.

Table 4-1

Table 4-2

Table 4-3

[0361] Array and its specific information:

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

[0362] The present disclosure will be described with reference to the following examples, which are intended to illustrate rather than limit the present disclosure.

[0363] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present disclosure are generally performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. It is well known to those skilled in the art that the examples are used to illustrate the present disclosure and are not intended to limit the scope of protection claimed in the present disclosure.

[0364] Manufacturing protocol The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0365] Nuclear magnetic resonance ( 1The equipment for detecting 1H NMR used a Bruker 400 MHz nuclear magnetic resonance apparatus. The test solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterodimethyl sulfoxide (DMSO-d6), and the internal standard substance was tetramethylsilane (TMS).

[0366] The abbreviations of the nuclear magnetic resonance (NMR) spectra used in the examples are as shown below. s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: hexadeuterodimethyl sulfoxide. The δ value is expressed in ppm value.

[0367] The apparatus for detecting mass spectrometry (MS) used an Agilent (ESI) mass spectrometer, and the model was Agilent 6120B.

[0368] I. Synthesis of bioactive molecules and intermediates used in the synthesis of "drug-linker compounds" A. Synthesis of bioactive molecules Example A1.1: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.1)

Chemical formula

[0369] Step 1: Dissolve 4-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (4.6 g) in anhydrous tetrahydrofuran (50 ml). Under the protection of nitrogen gas, cool the above solution to -78 °C with dry ice-acetone. Next, dropwise add n-butyllithium (12 ml, 2 M), stir the reaction solution at -78 °C for 20 minutes, then add methyl 2-amino-5-fluorobenzoate (1.83 g). After the addition is complete, naturally raise the temperature of the reaction system to room temperature and continue stirring for 5 hours to react. After quenching the reaction system with methanol (3 ml), add ethyl acetate (200 ml), wash the solution with water (100 ml × 3), dry the organic phase, remove the organic solvent, and separate by silica gel column chromatography to obtain the target product (2-amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone. ESI-MS (m / z): 304 [M+H] + 。

[0370] Step 2: Dissolve (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2.63 g), (2-amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone (3.03 g), and p-toluenesulfonic acid (1.74 g) in dichloromethane (50 ml). Next, remove the solvent, heat the mixture to 120 °C under the protection of nitrogen gas, and react for 4 hours. Dissolve the mixture in ethyl acetate (300 ml), wash the organic phase with water (100 ml × 2), dry it, remove the organic solvent, and separate the residue by silica gel column chromatography to obtain the target product (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.1). ESI-MS (m / z): 447 [M+H] + 。

[0371] Example A1.2: Synthesis of (S)-7-ethyl-7-hydroxy-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.2)

Chem.

[0372] Example A1.3: Synthesis of (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,33]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3)

Chem.

[0373] Example A1.4: Synthesis of (S)-14-(4-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.4)

Chemical Structure

[0374] ESI-MS (m / z): 484 [M+H] + ; 1 H NMR (400 MHz, DMSO) δ 7.56 (s, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.27 (s, 1H), 7.14 (s, 1H), 6.89 (d, J = 7.8 Hz, 2H), 6.26 (s, 2H), 5.40 (s, 2H), 5.05 (s, 2H), 1.96 - 1.78 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0375] Example A1.5: Synthesis of (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.5)

Chemical Structure

[0376] Step 1: Synthesis of (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

Chemical Structure

[0377] LCMS (ESI) [M+H] + : 468.9; 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.26 (s, 2H), 3.81 (d, J = 5.9 Hz, 2H), 3.22 (s, 2H), 1.98 (d, J = 6.7 Hz, 4H), 0.88 (t, J = 7.2 Hz, 3H).

[0378] Step 2: Synthesis of (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

Chemical formula

[0379] LCMS (ESI) [M+H] + : 476; 1 H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.25 (s, 2H), 3.53 - 3.49 (m, 2H), 3.16 - 3.12 (m, 2H), 1.93 - 1.80 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H).

[0380] Step 3: Synthesis of (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

Chemical Structure

[0381] LCMS (ESI) [M+H] + : 449.9; 1 H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 3H), 7.53 (s, 1H), 7.25 (s, 1H), 6.50 (s, 1H), 6.30 (s, 2H), 5.43 (s, 2H), 5.24 (s, 2H), 3.15 (d, J = 6.4 Hz, 2H), 3.03 (t, J = 6.9 Hz, 2H), 1.96 - 1.81 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0382] Example A1.6: Synthesis of (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6)

Chemical Structure

Chemical formula

[0383] Step 2: Synthesis of (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6)

Chemical formula

[0384] LCMS (ESI) [M+H] + : 522.0; 1 H NMR (400 MHz, DMSO) δ 7.92 (s, 1H), 7.53 (m, 1H), 7.25 (s, 1H), 6.50 (s, 1H), 6.31 (s, 2H), 5.43 (s, 2H), 5.34 (m, 2H), 4.65 (m, 1H), 4.07 (m, 2H), 3.52 (m, 2H), 3.41 (m, 2H), 2.00 (m, 2H), 1.88 (m, 2H), 1.16 - 1.04 (m, 3H), 0.89 - 0.83 (m, 3H).

[0385] Example A1.7: Synthesis of (S)-N-methyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.7) [Chemical formula]

[0386] Step 1: Preparation of compound benzylmethyl(3-(6-nitrobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)carbamate [Chemical formula] Compound 1-(6-nitrobenzo[d][1,3]dioxin-5-yl)ethan-1-one (A1.7-A, 500 mg), methylamine hydrochloride (1.6 g), and paraformaldehyde (714 mg) were dissolved in ethanol (8 mL) and reacted in a sealed container at 100 °C for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (80 mL), and the organic phase was extracted with water (50 mL × 3). The obtained aqueous phase was adjusted to pH = 9 with sodium bicarbonate, and then benzyl chloroformate (513 mg, 3.0 mmol) was added and reacted at room temperature for 16 hours. The reaction solution was extracted with ethyl acetate (30 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, suction filtered, concentrated to obtain a crude product, and the crude product was separated and purified by C18 (acetonitrile / 0.05% aqueous formic acid solution = 5 - 95%) to obtain the target compound (A1.7-B, 180 mg, yield: 23%).

[0387] LCMS (ESI) [M+H] + : 387.1. 1 H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.35 (m, 5H), 7.32 (m, 1H), 6.17 (s, 2H), 5.13 (s, 2H), 3.71 (m, 2H), 3.03 (m, 3H), 2.99 - 2.86 (m, 2H).

[0388] Step 2: Preparation of benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(methyl)carbamate (A1.7-C)

Chem.

[0389] LCMS (ESI) [M+H] + : 357.0.

[0390] Step 3: Preparation of benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(methyl)carbamate (A1.7-D)

Chem.

[0391] LCMS (ESI) [M+H] + = 584.0。

[0392] Step 4: Preparation of (S)-7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.7-E)

Chemical Structure

[0393] LCMS (ESI) [M+H] + : 450.0

[0394] Step 5: Preparation of compound (S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(methyl)amino)-2-oxoethyl acetate (A1.7-F)

Chem.

[0395] LCMS (ESI) [M5+H] + = 550.1

[0396] Step 6: Preparation of compound (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-methylacetamide (A1.7)

Chem.

[0397] LCMS (ESI) [M+H] + : 508.2

[0398] Example A1.8: Synthesis of (S)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.8)

Chemical Structure

[0399] Step 1: Preparation of compound benzyl isopropyl (3-(6-nitrobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)carbamate (A1.8-B)

Chemical Structure

[0400] LCMS (ESI) [M+H] + : 414.9; 1 H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 1H), 7.34 (br s, 6H), 6.31 (s, 2H), 5.08 (s, 2H), 4.14 - 4.10 (m, 1H), 3.48 (d, J = 7.9 Hz, 2H), 3.03 (s, 2H), 1.13 - 1.11 (m, 6H).

[0401] Step 2: Preparation of compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(isopropyl)carbamate (A1.8-C)

Chemical Structure

[0402] LCMS (ESI) [M+H] + : 395.2。

[0403] Step 3: Preparation of (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.8-D)

Chemical formula

[0404] LCMS (ESI) [M+H] + : 478.0; 1 H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 6.33 (s, 2H), 5.44 (s, 2H), 5.35 (s, 2H), 3.41 (br s, 2H), 3.23 (br s, 3H), 1.94 - 1.78 (m, 2H), 1.25 (d, J = 6.4 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0405] Step 4: Preparation of compound (S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)amino)-2-oxoethyl acetate (A1.8-E)

Chemical Structure

[0406] LCMS (ESI) [M+H] + : 578.0.

[0407] Step 5: Preparation of compound (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-isopropylacetamide (A1.8) [Chemical formula] Concentrated hydrochloric acid (0.5 mL) was added to an ethanol solution (3 mL) of compound (S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)amino)-2-oxoethyl acetate (A1.8-E, 50 mg), and the mixture was reacted at 70 °C for 1 hour. The reaction solution was concentrated to obtain a crude product. Further purification by preparative high performance liquid chromatography (acetonitrile / 0.05% aqueous formic acid) gave the target compound (A1.8, 3 mg).

[0408] LCMS (ESI) [M+H] + : 464.0; 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.53 (s, 1H), 7.26 (s, 1H), 6.31 (s, 2H), 5.43 (s, 2H), 5.36 (s, 2H), 4.22 (s, 2H), 3.99 - 3.94 (m, 1H), 3.44 (dd, J = 16.7, 7.8 Hz, 2H), 3.33 - 3.21 (m, 2H), 1.95 - 1.79 (m, 2H), 1.19 (dd, J = 16.4, 5.8 Hz, 6H), 0.88 (t, J = 7.2 Hz, 3H).

[0409] Example A1.9: Synthesis of (S)-7-Ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.9) [Chemical formula] Compound (S)-7-Ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (100 mg, 0.213 mmol) was dissolved in a 10% sulfuric acid (5 mL) solution, and the reaction system was reacted at 110 °C for 48 hours. A saturated sodium hydrogen carbonate (30 mL) solution was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL × 5), dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain a crude product. Purification by preparative high-performance liquid chromatography (acetonitrile / 0.05% aqueous formic acid solution) gave (S)-7-Ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.9, 1.78 mg).

[0410] LCMS (ESI) [M+H] + : 451.0; 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.28 (s, 2H), 5.47 - 5.37 (m, 2H), 5.32 - 5.19 (m, 2H), 3.51 - 3.46 (m, 2H), 3.17 - 3.13 (m, 2H), 1.92 - 1.76 (m, 4H), 0.90 - 0.84 (m, 3H).

[0411] Example A1.10: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.10) [Chemical formula]

[0412] Step 1: At 0 °C, 1 mol / L boron trichloride (96 mL) and 4-chlorobutyronitrile (9.9 g) were added dropwise to a solution of compound A1.10-A (10 g) in 1,2-dichloroethane (200 mL). The mixture was stirred at 80 °C for 2 hours to allow the reaction to proceed. The reaction solution was cooled to room temperature, 2 mol / L hydrochloric acid (90 mL) was added, and the mixture was refluxed at 80 °C and stirred for 0.5 hour. The reaction solution was cooled to room temperature, diluted by adding a small amount of water, and extracted with dichloromethane (200 mL × 3). The organic phase was dried over anhydrous sodium sulfate, suction filtered, concentrated to obtain a crude product, and the crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A1.10-B (4 g).

[0413] LCMS (ESI) [M+H] + : 230.0.

[0414] Step 2: To Compound A1.10-B (50 mg), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (35 mg) and p-toluenesulfonic acid monohydrate (41.4 mg) were added, and the mixture was dissolved in dichloromethane (30 mL). After uniformly mixing until the solution became clear, it was concentrated under reduced pressure and evacuated to vacuum with an oil pump. The reaction system was reacted at 120 °C under vacuum for 3 hours. LCMS indicated the completion of the reaction. The reaction solution was cooled to room temperature, water (20 mL) was added, and extraction was performed with dichloromethane (20 mL × 3). The organic phases were combined, dried successively over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 20:1) to obtain the target compound A1.10-C (80 mg), which was a white solid.

[0415] LCMS (ESI) [M+H] + : 457.0.

[0416] Step 3: Compound A1.10-C (75 mg) was dissolved in hexamethylphosphoramide, pure water (0.8 mL) was added, and the reaction solution was stirred at 100 °C for 72 hours. LCMS detected the completion of the reaction. Purification by preparative chromatography (0.01% aqueous TFA solution, MeCN) gave the target compound (10 mg).

[0417] LCMS (ESI) [M+H] + : 439.2; 11H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.50 (s, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 4.67 (t, J = 4.9 Hz, 1H), 3.55 - 3.47 (m, 2H), 3.28 - 3.20 (m, 2H), 2.51 (s, 3H), 1.93 - 1.81 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0418] Example A1.11: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-aminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.11)

Chemical formula

[0419] Step 1: Sodium azide (432 mg) was added to a solution of A1.10-C (395 mg) in N,N-dimethylformamide (10 mL). The reaction mixture was reacted at 80 °C for 16 hours, then cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (80 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered by suction, and concentrated to obtain the target product A1.11-A (290 mg).

[0420] LCMS (ESI) [M+H] + : 464.0.

[0421] Step 2: Compound A1.11-A (93 mg) was dissolved in tetrahydrofuran (5 mL), triphenylphosphine (78 mg) was added, and the mixture was reacted at room temperature for 4 hours. Next, hydrochloric acid (4 M, 1 mL) was added to the reaction solution, the temperature of the reaction solution was raised to 55 °C, and the mixture was reacted for 16 hours. LCMS detected the completion of the reaction. The reaction solution was directly concentrated, and the crude product was purified by a reverse-phase column (mobile phase A: 0.05% aqueous formic acid solution, B: acetonitrile) to obtain the target product A1.11 (28 mg, yield: 36%), which was a white solid.

[0422] LCMS (ESI) [M+H] + : 438.4; 1 H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 10.7 Hz, 1H), 7.63 (s, 1H), 5.49 (ABq, J = 78.9, 16.3 Hz, 2H), 5.31 (br s, 2H), 3.35 - 3.32 (m, 2H), 3.22 - 3.10 (m, 2H), 2.55 (s, 3H), 2.13 - 2.11 (m, 2H), 1.96 - 1.94 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).

[0423] Example A1.12: Synthesis of (S,E)-14-(3-amino-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12)

Chemical Structure

[0424] Step 1: Compound A1.12-A (200 mg, 0.39 mmol), Compound A1.12-B (112 mg, 0.39 mmol), cesium fluoride (152 mg, 0.975 mmol), and tetrakis(triphenylphosphine)palladium (45 mg, 0.039 mmol) were added to a 1,4-dioxane solution (8 mL). The reaction was carried out under microwave irradiation at 120 °C for 0.5 h in an atmosphere of nitrogen gas. LCMS indicated the completion of the reaction. A mixed solution of dichloromethane (20 mL) and methanol (10 mL) was added to the reaction solution for dilution, and then filtered. The filtrate was concentrated, and the crude product was purified by preparative TLC (dichloromethane:methanol = 30:1) to obtain the target compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-7-yl acetate (A1.12-C, 60 mg, yield: 26%), which was a brown solid. LCMS (ESI) [M+H] + = 590.3; 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.49 (s, 1H), 7.35 (s, 1H), 7.09 (d, J = 16.7 Hz, 1H), 6.93 (s, 1H), 6.45 (d, J = 16.6 Hz, 1H), 6.30 (s, 2H), 5.47 (s, 2H), 5.34 - 5.24 (m, 2H), 3.94 (s, 2H), 2.21 (br s, 3H), 2.03 - 1.96 (m, 2H), 1.45 (s, 9H), 0.91 (t, J = 6.7 Hz, 3H).

[0425] Step 2: A solution of compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-7-yl acetate (A1.12-C, 50 mg, 0.085 mmol) in methanol (15 mL) was added with sodium methoxide (9.2 mg, 0.17 mmol), and reacted with stirring at 50 °C for 2 hours. LCMS indicated the completion of the reaction. The reaction solution was concentrated to obtain the target compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12-D, 50 mg) as a crude product, which was a brown solid. LCMS (ESI) [M+H] + = 548;

[0426] Step 3: Trifluoroacetic acid (1 mL) was added to a dichloromethane solution (2 mL) of compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12-D, 50 mg, 0.091 mmol), and reacted with stirring at room temperature for 30 minutes. LCMS indicated the completion of the reaction. The reaction solution was concentrated, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile / 0.05% aqueous formic acid solution) to obtain the target compound (S,E)-14-(3-amino-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12) (8.1 mg, yield: 21%) as a brown solid.

[0427] LCMS (ESI) [M+H] + = 448.3; 1 H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 2H), 7.72 (s, 1H), 7.54 (s, 1H), 7.40 (d, J = 16.5 Hz, 1H), 7.27 (s, 1H), 6.52 - 6.46 (m, 2H), 6.31 (s, 2H), 5.42 (s, 2H), 5.27 (s, 2H), 3.86 (br s, 2H), 1.90 - 1.83 (m, 2H), 0.88 (t, J = 7.1 Hz, 3H).

[0428] Example A1.13: Synthesis of (S,E)-14-(3-hydroxy-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.13)

Chemical Structure

[0429] Step 1: Compound A1.9 (200 mg, 0.444 mmol) was dissolved in dimethyl sulfoxide (2 mL), IBX (311 mg, 1.11 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Next, IBX (186 mg, 0.666 mmol) was added to the reaction solution, and then pyrrolidine (6.3 mg, 0.089 mmol) and acetonitrile (3 mL) were added to the reaction solution. The reaction solution was stirred at room temperature overnight. LCMS detected the completion of the reaction. The mixture was extracted with ethyl acetate (20 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, and spin-dried to obtain a crude product. The crude product was purified by silica gel column (DCM:MeOH = 50:1 to 10:1) to obtain the target compound (S,E)-14-(3-oxo-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.13-A, 100 mg, purity: 50.0%, yield: 25.0%), which was a yellow solid. LCMS (ESI) [M+H] + = 447.1。

[0430] Step 2: Compound A1.13-A (40 mg, 0.09 mmol) was dissolved in tetrahydrofuran (1 mL), and then sodium cyanoborohydride (28 mg, 0.448 mmol) was added to the reaction solution. The mixture was stirred at room temperature overnight. LCMS indicated the completion of the reaction. The reaction solution was concentrated to obtain a crude product, and the crude product was purified by prep-HPLC (HCl in water / MeCN) to obtain the target compound (3.56 mg, purity: 93.6%, yield: 9.0%), which was a pale yellow solid.

[0431] LCMS (ESI) [M+H] + = 449.2; 1H NMR (400 MHz, DMSO-d6) δ 7.61 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 7.20 (d, J = 16.4 Hz, 1H), 6.69 - 6.59 (m, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.26 (s, 2H), 5.16 (br s, 1H), 4.34 (br s, 2H), 1.93 - 1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0432] Example A1.14: Synthesis of (S,E)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-hydroxy-1-propen-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.14)

Chemical formula

[0433] Step 1: To a 25 mL single-necked flask, add the compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.10, 100 mg, 0.228 mmol), DMSO (0.5 mL) and acetonitrile (0.75 mL). Then, sequentially add IBX (160 mg, 0.571 mmol) and (R)-diphenyl(pyrrolidin-2-yl)methanol (12 mg, 0.047 mmol). Stir continuously at room temperature for 16 h to carry out the reaction. Next, add a mixed solvent of methanol and dichloromethane (200 mL) (DCM:MeOH = 10:1) to the reaction solution, extract with saturated brine (300 mL), combine the organic phases, wash with water, dry, filter, evaporate the filtrate to dryness under reduced pressure to remove the solvent, obtain the crude product, and purify the crude product by flash chromatography (DCM:MeOH = 10:1) to obtain the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-oxopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolo[1,2-b]quinoline-3,14(4H)-dione (A1.14-A, 40 mg, yield: 40%).

[0434] LCMS (ESI) [M+H] + = 435.1.

[0435] Step 2: In a 50 mL three-necked flask, compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-oxopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolo[1,2-b]quinoline-3,14(4H)-dione (A1.14-A, 40 mg, 0.228 mmol) and anhydrous THF (5 mL) were added. The mixture was cooled to -78 °C under N2 protection, and a THF solution of lithium tri-sec-butylborohydride (0.11 mL, 1 N) was slowly added dropwise. Stirring was continued for 1 hour while maintaining the temperature at -78 °C. After completion of the reaction, saturated aqueous ammonium chloride solution was added to quench the reaction system, the temperature was raised to room temperature, diluted with saturated brine (50 mL), extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with water, dried, filtered, and evaporated to dryness to remove the solvent, obtaining (S,E)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxy-1-propen-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.14, 28 mg), which was an off-white solid.

[0436] LCMS (ESI) [M+H] + = 437.1.

[0437] Example A1.15: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-(n-propylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.15a) and (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-propylacetamide (A1.15b)

Chemical Structure

[0438] Step 1: Compound A1.15-A (5.0 g, 23.9 mmol), propylamine hydrochloride (22.8 g, 239 mmol), and paraformaldehyde (7.18 g, 239 mmol) were dissolved in ethanol (100 mL) and reacted in a sealed container at 110 °C for 12 hours. LCMS detected the completion of the reaction. The reaction solution was cooled to room temperature, concentrated under reduced pressure, dichloromethane (100 mL) was added to the reaction solid, washed with water (80 mL × 3), and the aqueous phase was adjusted to pH = 9 with sodium bicarbonate. Benzyloxycarbonyl chloride (3.7 g, 21.8 mmol) was added to the aqueous solution and reacted at room temperature for 2 hours. LCMS detected the completion of the reaction. Extracted with dichloromethane (100 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, suction filtered, concentrated to obtain a crude product, and the crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A1.15-B (1.5 g, yield: 20.1%), which was a yellow oily liquid.

[0439] LCMS (ESI) [M+H] + = 415.1; 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.35 (m, 6H), 6.18 (s, 2H), 5.17 - 5.12 (m, 2H), 3.69 - 3.67 (m, 2H), 3.32 - 3.30 (m, 2H), 3.09 - 2.90 (m, 2H), 1.59 (m, 2H), 0.90 (m, 3H).

[0440] Step 2: Compound A1.15-B (1.5 g, 3.63 mmol) was dissolved in a mixed solution of saturated ammonium chloride (20 mL) and ethanol (20 mL). Next, iron powder (1.02 g, 18.1 mmol) was added to the reaction solution, and the reaction system was stirred at 80 °C for 1 hour. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by a reverse-phase C18 column (acetonitrile / 0.05% aqueous FA solution: 5% - 55%) to obtain the target compound A1.15-C (600 mg, yield: 43.0%), which was a yellow solid. LCMS (ESI) [M+H] + = 385.2, t R = 1.329 min.

[0441] Step 3: At room temperature, compound A1.15-C (350 mg, 0.91 mmol), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (A1.15-D, 218 mg, 0.83 mmol), and p-toluenesulfonic acid (170 mg, 0.87 mmol) were dissolved in a dichloromethane (5 mL) solution. After the solution was uniformly mixed until it became clear, it was concentrated under reduced pressure and evacuated to vacuum with an oil pump. The reaction system was reacted at 120 °C under vacuum for 2 hours. LCMS indicated the completion of the reaction. The reaction solution was cooled to room temperature, water (50 mL) was added, and it was extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried sequentially over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1.15-E (390 mg, yield: 76%), which was a brown solid.

[0442] LCMS (ESI) [M+H] + = 612.0.

[0443] Step 4: At room temperature, compound A1.15-E (390 mg, 0.638 mmol) was dissolved in dichloromethane (5 mL) solution. At 0 °C, under nitrogen gas protection, iodotrimethylsilane (510 mg, 2.55 mmol) was added dropwise, and the reaction system was stirred at room temperature for 2 hours. Diethyl ether (2 mL) and concentrated hydrochloric acid (4 mL) were added to the reaction solution, and it was stirred for 30 minutes. The reaction system was adjusted to pH = 9 with saturated sodium bicarbonate, extracted with dichloromethane (50 mL × 5), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. Purification by preparative high-performance liquid chromatography (acetonitrile / 0.05% aqueous formic acid solution) gave compound A1.15a (200 mg, yield: 66.1%), which was an off-white solid.

[0444] LCMS (ESI) [M+H] + = 478.2; 1H NMR (400 MHz, DMSO-d6) δ 8.30 (s, 1H, HCO2H), 7.67 (s, 1H), 7.51 (s, 1H), 7.23 (s, 1H), 6.62 - 6.41 (m, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.28 (s, 2H), 3.27 - 3.25 (m, 2H), 2.91 - 2.79 (m, 2H), 2.58 - 2.56 (m, 2H), 1.91 - 1.79 (m, 2H), 1.44 - 1.42 (m, 2H), 0.90 - 0.84 (m, 6H).

[0445] Step 5: At room temperature, compound A1.15a (120 mg, 0.251 mmol) was dissolved in dichloromethane (5 mL). At 0 °C, compound acetoxyacetyl chloride (167 mg, 1.25 mmol) and triethylamine (133 mg, 1.25 mmol) were added dropwise, and the reaction system was stirred at 0 °C for 30 minutes for reaction. LCMS detected the completion of the reaction. The reaction solution was concentrated under reduced pressure to obtain a crude product, compound A1.15-F (120 mg, yield: 82.8%), which was a yellow solid. LCMS (ESI) [M+H] + = 578.3.

[0446] Step 6: At room temperature, compound A1.15-F (120 mg, 0.207 mmol) was dissolved in ethanol (4 mL). Next, concentrated hydrochloric acid (2 mL) was added to the reaction solution, and the reaction system was stirred at 70 °C for 1 hour to react. LCMS detected the completion of the reaction. Water (20 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL × 5). The organic phases were combined, dried sequentially over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. Purification by preparative high-performance liquid chromatography (acetonitrile / 0.05% aqueous formic acid solution) gave compound A1.15b (20 mg, yield: 18.1%), which was an off-white solid.

[0447] LCMS (ESI) [M+H] + = 536.2; 1H NMR (400 MHz, DMSO-d6) δ 7.93 (s, 0.7H), 7.69 (s, 0.3H), 7.51 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.35 (s, 1.5H), 5.29 (s, 0.5H), 4.65 - 4.62 (m, 1H), 4.13 - 3.97 (m, 2H), 3.51 - 3.49 (m, 2H), 3.33 - 3.30 (m, 2H), 3.24 - 3.20 (m, 2H), 1.86 - 1.84 (m, 2H), 1.60 - 1.52 (m, 2H), 0.89 - 0.86 (m, 6H).

[0448] Example A1.16: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-isopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.16)

Chemical Structure

[0449] LCMS (ESI) [M+H] + = 480.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 8.0 Hz, 1H), 8.27 (s, 1H, HCO2H) 7.92 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.53 (s, 1H), 5.45 (s, 2H), 5.30 (s, 2H), 3.30 - 3.22 (m, 3H), 3.18 - 3.10 (m, 2H), 2.54 (s, 3H), 2.06 - 1.94 (m, 2H), 1.93 - 1.80 (m, 2H), 1.26 - 1.20 (m, 6H), 0.88 (t, J = 7.3 Hz, 3H).

[0450] Synthesis of Example A1.17: (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(3-cyclopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.17) [Chemical formula] To a DMF solution (10 mL) of A1.10C (200 mg, 0.439 mmol) and cyclopropylamine (52 mg, 0.881 mmol), diisopropylethylamine (165 mg, 1.28 mmol) and sodium iodide (96 mg, 0.640 mmol) were added, and the reaction system was reacted at 50 °C in a sealed tube for 2 hours. The completion of the reaction was monitored by LCMS. Ethyl acetate was added to the reaction solution, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by TLC (dichloromethane:methanol = 10:1), and then continuously purified by preparative high performance liquid chromatography (acetonitrile / 0.05% aqueous formic acid solution) to obtain the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-cyclopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.17, 5.12 mg).

[0451] LCMS (ESI) [M+H] + = 478.2; 11H NMR (400 MHz, DMSO-d6) δ 8.22 (br s, 2H, one is HCO2H), 7.86 (br s, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.43 (s, 2H), 5.28 (s, 2H), 3.24 - 3.19 (m, 2H), 2.75 - 2.68 (m, 2H), 2.49 (s, 3H), 2.16 - 2.09 (m, 1H), 1.95 - 1.76 (m, 4H), 0.91 - 0.83(t, J = 7.3 Hz, 3H), 0.43 - 0.33 (m, 2H), 0.31 - 0.21 (m, 2H).

[0452] Example A1.18: Synthesis of (S)-4-Ethyl-8-fluoro-4-hydroxy-11-(4-aminophenyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.18) [Chemical formula]

[0453] Step 1: Under nitrogen gas protection, 3-Fluoro-4-methylaniline (A1.18A, 2.0 g, 16.3 mmol) was dissolved in 1,2-dichloroethane (40 mL). At 0 °C, boron trichloride (19.2 mL, 19.2 mmol) was added dropwise thereto, and then p-nitrobenzonitrile (2.8 g, 19.2 mmol) was slowly added. After the addition was completed, the reaction solution was heated to 80 °C and stirred overnight. The reaction solution was cooled to room temperature, hydrochloric acid (40 mL, 2 M) was added thereto, then the temperature was raised to 80 °C and stirring was continued for 30 minutes. LCMS detected the completion of the reaction. Water (80 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, spin-dried, and the crude product was purified by silica gel column chromatography (PE:EA = 10:1) to obtain the target compound (A1.18B, 2.0 g, yield: 46.5%).

[0454] LCMS (ESI) [M+H] + = 275.1; 1 H NMR (400 MHz, DMSO) δ 8.34 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.39 (s, 2H), 7.11 (d, J = 8.8 Hz, 1H), 6.63 (d, J = 12.4 Hz, 1H), 2.00 (s, 3H).

[0455] Step 2: Compound A1.18B (620 mg, 2.28 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (600 mg, 2.28 mmol), p-toluenesulfonic acid (560 mg, 2.96 mmol) were added to a reaction flask, dissolved uniformly with dichloromethane, spin-dried with dichloromethane, evacuated to vacuum, heated to 120 °C under vacuum, and maintained at this temperature for 6 hours. After LCMS detected the completion of the reaction, methanol (10 mL) was added to the reaction system, and further water (80 mL) was added to precipitate a large amount of precipitate. After filtering the precipitate and drying, the target compound (A1.18C, 800 mg, yield: 80.0%) was obtained, which was a yellow solid.

[0456] LCMS (ESI) [M+H] + = 502.2; 1 H NMR (400 MHz, DMSO) δ 8.52 (d, J = 8.2 Hz, 2H), 8.13 - 7.89 (m, 3H), 7.64 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 13.4 Hz, 1H), 6.54 (s, 1H), 5.41 (s, 2H), 5.06 (m, 2H), 2.40 (s, 3H), 1.94 - 1.84 (m, 2H), 0.87 (m, 3H).

[0457] Step 3: Raney nickel (609 mg, 10.5 mmol) was added to a mixed solution of compound A1.18C (1.0 g, 2.1 mmol) in methanol (25 mL) and tetrahydrofuran (50 mL). The reaction mixture was stirred at room temperature for 5 hours under a hydrogen gas atmosphere. LCMS detected the completion of the reaction. The reaction mixture was filtered, and the filtrate was spin-dried to obtain the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(4-aminophenyl)-9-methyl-1,12-dihydro-14H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.18, 650 mg, yield: 67%), which was a yellow solid.

[0458] LCMS (ESI) [M+H] + = 472.3; 1 H NMR (400 MHz, DMSO) δ 7.93 - 7.81 (m, 2H), 7.40 - 7.25 (m, 3H), 6.81 (d, J = 8.2 Hz, 2H), 6.51 (s, 1H), 5.62 (s, 2H), 5.41 (s, 2H), 5.10 (s, 2H), 2.41 (s, 3H), 1.93 - 1.80 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0459] B. Synthesis of Intermediates Containing Bioactive Molecular Fragments Example B1.1: Synthesis of (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)butoxy)methyl)acetamide (B1.1)

Chemical Structure

[0460] ESI-MS (m / z): 539 [M+H] + 。

[0461] Example B1.2: Synthesis of (S)-2-((2-aminoacetamido)methoxy)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.2)

Chemical Structure

[0462] Step 1: Compound B1.2-A (274 mg), diisopropylethylamine (334 mg), and HBTU (369 mg) were sequentially added to N,N-dimethylformamide (10 mL), and then compound B1.2-B (300 mg) was added. The reaction mixture was stirred at room temperature for 2 hours. Ethyl acetate (50 mL) was added to the reaction mixture, and it was washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B1.2-C (300 mg).

[0463] LCMS (ESI) [M+H] + : 830.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 7.91 - 7.84 (m, 2H), 7.80 - 7.67 (m, 2H), 7.65 - 7.54 (m, 2H), 7.53 - 7.46 (m, 1H), 7.45 - 7.37 (m, 2H), 7.36 - 7.27 (m, 2H), 7.25 - 7.23 (m, 1H), 6.57 - 6.44 (m, 1H), 6.29 (s, 2H), 5.50 - 5.19 (m, 4H), 4.71 - 4.57 (m, 2H), 4.32 - 3.97 (m, 7H), 3.80 - 3.53 (m, 4H), 3.20 - 3.14 (m, 2H), 1.92 - 1.80 (m, 2H), 1.28 - 1.22 (m, 3H), 0.87 - 0.82 (m, 3H).

[0464] Step 2: Piperidine (1 mL) was added to a solution of compound B1.2-C (300 mg) in N,N-dimethylformamide (4 mL), and the reaction mixture was stirred at room temperature for 20 minutes. After removing the low-boiling components from the reaction mixture, the target product was obtained and used directly in the next step of the synthesis.

[0465] LCMS (ESI) [M+H] + = 608.0。

[0466] Example B1.3: Synthesis of (S)-2-amino-N-((2-(((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.3)

Chemical Structure

[0467] ESI-MS (m / z): 554 [M+H] + .

[0468] Example B1.4: Synthesis of (S)-2-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (B1.4)

Chemical Structure

[0469] ESI-MS (m / z): 467 [M+H] + 。

[0470] Example B1.5: Synthesis of (S)-2-amino-N-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)acetamide (B1.4)

Chem.

[0471] ESI-MS (m / z): 479.3 [M+H] + 。

[0472] Example B1.6: Synthesis of (S)-2-amino-N-((4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)-1H-pyrazol-1-yl)methyl)acetamide (B1.6)

Chemical Structure

[0473] ESI-MS (m / z): 545 [M+H] + 。

[0474] Example B1.7: Synthesis of (S)-2-amino-N-((2-(((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.7) [Chemical Structure] Using the same method and reaction conditions as in Example B1.2, and using compound (B1.5-A) instead of compound (B1.2-B), a mixture containing the target product (S)-2-amino-N-((2-(((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.7) was obtained, and the above mixture was directly used in the following synthesis reaction.

[0475] ESI-MS (m / z): 566 [M+H] + .

[0476] Example B1.8: Synthesis of (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.8) [Chemical Structure] Using the same method and reaction conditions as in Example B1.4 and using compound (A1.3) instead of compound (B1.3-A), the target product (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.8) was obtained.

[0477] ESI-MS (m / z): 541 [M+H] + 。

[0478] Example B1.9: Synthesis of (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9)

Chemical formula

[0479] Compound A1.4 (60 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL), and (tert-butoxycarbonyl)glycine (26 mg, 0.15 mmol), HATU (56 mg, 0.15 mmol), and N,N-diisopropylethylamine (48 mg, 0.37 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour, and TLC detected the completion of the reaction. Next, TFA (1.0 mL) was directly added to the above reaction solution. Stirring was continued at room temperature for 1 hour, LCMS detected the completion of the reaction, and the reaction solution was concentrated to remove trifluoroacetic acid to obtain a crude product. The crude product was purified by preparative chromatography (0.01% aqueous trifluoroacetic acid, acetonitrile) to obtain the target product (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9) (26.3 mg, yield: 38%), which was a yellow solid.

[0480] ESI-MS (m / z): 541 [M+H] + ; 1 H NMR (400 MHz, DMSO) δ 10.71 (s, 1H), 8.16 (s, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.67-7.58 (m, 3H), 7.29 (s, 1H), 7.04 (s, 1H), 6.50 (s, 1H), 6.28 (s, 2H), 5.40 (s, 2H), 5.05 (s, 2H), 3.87 (s, 2H), 1.93-1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0481] Example B1.10: Synthesis of (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)propyl)acetamide (B1.10)

Chem.

[0482] LCMS (ESI) [M+H] + : 607.

[0483] Step 2: Trifluoroacetic acid (2 mL) was added to a solution of compound B1.10-A (100 mg) in dichloromethane (4 mL), and the mixture was stirred at room temperature for 1 hour to react. The reaction solution was concentrated, and N,N-dimethylformamide solution (3 mL) was added to the crude product. The crude product was further purified by a C18 column (acetonitrile / 0.05% aqueous formic acid solution: 5% - 60%) to obtain the target compound (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)propyl)acetamide (B1.10, 80 mg).

[0484] LCMS (ESI) [M+H] + : 507; 11H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.51 (s, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.23 (s, 2H), 3.31 (s, 2H), 3.30 - 3.27 (m, 2H), 3.13 - 3.07 (m, 2H), 2.04 - 1.76 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0485] Example B1.11: Synthesis of (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.11)

Chemical Structure

[0486] Step 1: 1-(9H-Fluoren-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazacyclo-11-carboxylic acid (242 mg), diisopropylethylamine (330 mg), and N,N,N’,N’-tetramethyluronium hexafluorophosphate (359 mg) were dissolved in an N,N-dimethylformamide (10 mL) solution. Next, (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (300 mg) was added, and the reaction system was reacted at room temperature for 2 hours. Ethyl acetate (50 mL) was added to the reaction solution, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, suction filtered, the filtrate was concentrated under reduced pressure to a solid, and the crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B1.11B (220 mg).

[0487] LCMS (ESI) [M+H] + : 844.0.

[0488] Step 2: Preparation of (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide Piperidine (1 mL) was added to a solution of compound B1.11B (220 mg, 0.261 mmol) in N,N-dimethylformamide (4 mL), and the reaction system was stirred at room temperature for 20 minutes. LCMS detected the completion of the reaction. The reaction solution was concentrated under reduced pressure to obtain the target compound (220 mg), which was a brown solid. The above product was not purified and was directly used in the synthesis of the next step.

[0489] LCMS (ESI) [M+H] + : 622.1.

[0490] Example B1.12: Synthesis of (S)-2-Amino-N-((4-(4-Ethyl-8-Fluoro-4-Hydroxy-9-Methyl-3,14-Dioxo-3,4,12,14-Tetrahydro-1H-Pyrano[3’,4’:6,7]Indolizino[1,2-b]Quinolin-11-Yl)Propoxy)Methyl)Acetamide (B1.12)

Chemical Structure

[0491] Step 1: Dissolve compound A1.10 (160 mg, 0.365 mmol) in N,N-dimethylformamide (3 mL), add (2-((((9H-Fluoren-9-yl)Methoxy)Carbonyl)Amino)Acetamide)Methyl Acetate (B1.1-A, 672 mg, 1.83 mmol), and then add ethyl acetate solution of hydrochloric acid (0.073 ml, 3M) to the reaction solution. Stir the reaction solution at room temperature overnight. LCMS detected the completion of the reaction. The reaction solution was directly purified by reverse-phase chromatography (acetonitrile / 0.05% aqueous solution of FA: 5% - 50%) to obtain the target compound (80 mg, yield: 29.0%), which was a white solid.

[0492] LCMS (ESI) [M+H] + = 747.4; 11H NMR (400 MHz, DMSO-d6) δ 8.74 (t, J = 6.4 Hz, 1H), 8.28 - 8.17 (m, 1H), 7.99 - 7.88 (m, 3H), 7.73 (d, J = 7.3 Hz, 2H), 7.63 (t, J = 5.7 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.2 Hz, 3H), 6.58 (s, 1H), 5.48 (s, 2H), 5.29 (s, 2H), 4.66 (d, J = 6.3 Hz, 2H), 4.32 (d, J = 6.9 Hz, 2H), 4.26 (d, J = 6.1 Hz, 1H), 3.71 (d, J = 5.8 Hz, 2H), 3.57 (t, J = 5.7 Hz, 2H), 3.29 - 3.20 (m, 2H), 2.55 (s, 3H), 2.00 - 1.86 (m, 4H), 0.93 (t, J = 7.2 Hz, 3H).

[0493] Step 2: B1.12-A (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, and the compound was stirred for 20 minutes to dissolve it. The low-boiling components were removed under reduced pressure, and the residue was used directly for the synthesis of the next step. ESI-MS (m / z): 525.2 [M+H] + 。

[0494] A small amount of the crude product was purified by reverse-phase chromatography (acetonitrile / 0.05% aqueous FA solution: 5% - 50%) to obtain the target compound.

[0495] ESI-MS (m / z): 525.1 [M+H] + ; 11H NMR (400 MHz, DMSO) δ 9.13 (t, J = 6.6 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.02 (brs, 2H), 7.89 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.28 (s, 2H), 4.66 (d, J = 6.5 Hz, 2H), 3.64 (s, 2H), 3.53 (t, J = 6.1 Hz, 2H), 3.25 - 3.18 (m, 2H), 2.52 (s, 3H), 1.98 - 1.84 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0496] Example B1.13: Synthesis of (S)-2-Amino-N-(3-(4-Ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)propyl)acetamide (B1.13)

Chemical Structure

[0497] Step 1: At room temperature, triethylamine (67 mg) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl) glycine (182 mg) were added to a solution of compound A1.11 (200 mg) in N,N-dimethylformamide (5 mL), and the mixture was reacted at room temperature for 1 hour. The reaction solution was diluted by adding water (30 mL), extracted with ethyl acetate (30 mL × 2), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was further purified by a C18 column (acetonitrile / 0.05% aqueous formic acid solution: 5% - 60%) to obtain the target compound (B1.13-A, 104 mg).

[0498] LCMS (ESI) [M+H] + : 595.

[0499] Step 2: To a dichloromethane solution (4 mL) of compound B1.13-A (100 mg) was added trifluoroacetic acid (2 mL), and the mixture was stirred at room temperature for 1 hour to cause a reaction. The reaction solution was concentrated, and N,N-dimethylformamide solution (3 mL) was added to the crude product, and further purified by a C18 column (acetonitrile / 0.05% aqueous formic acid solution: 5% - 60%) to obtain the target compound (S)-2-amino-N-(3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)propyl)acetamide (B1.13, 88 mg).

[0500] LCMS (ESI) [M+H] + : 495.

[0501] Example B1.14: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B1.14)

Chemical Structure

[0502] Step 1: Compound (B1.1-A) (368 mg), compound (A1.9) (440 mg) and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 ml) for 20 hours, then washed with aqueous sodium bicarbonate solution and aqueous hydrochloric acid solution respectively, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound compound B1.14A (240 mg).

[0503] LCMS (ESI) [M+H] + : 759.5.

[0504] Step 2: Dissolve B1.14 - A (240 mg) in DMF (5 ml), add piperidine (1 ml), stir the compound for 20 minutes to dissolve it, remove the low - boiling components under reduced pressure, and use the residue directly for the synthesis of the next step. A small amount of the crude product was purified by reverse - phase chromatography (acetonitrile / 0.05% aqueous FA solution: 5% - 50%) to obtain the target compound.

[0505] ESI - MS (m / z): 537.4 [M + H] + ; 1 H NMR (400 MHz, DMSO - d6) δ 9.13 (t, 1H), 8.04 (br, 2H), 7.58 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 6.29 (s, 2H), 5.43 (S, 2H), 5.21 (s, 2H), 4.65 (d, 2H), 3.63 (m, 2H), 3.53 (m, 2H), 3.11 (m, 2H), 1.87 (m, 4H), 0.88 (t, 3H).

[0506] Example B1.15: (S,E) - 2 - amino - N - (3 - (7 - ethyl - 7 - hydroxy - 8,11 - dioxo - 7,8,11,13 - tetrahydro - 10H - [1,3] dioxolo[4,5 - g] pyrano[3’,4’:6,7] indolizino[1,2 - b] quinolin - 14 - yl) allyl) acetamide

Chemical Structure

[0507] Step 1: At room temperature, triethylamine (135 mg, 1.341 mmol) and N-hydroxy-2,5-dioxopyrrolidine tert-butoxycarbonyl glycinate (183 mg, 0.671 mmol) were added to a solution of compound A1.12 (200 mg, 0.447 mmol) in N,N-dimethylformamide (5 mL), and the mixture was reacted at room temperature for 1 hour. LCMS indicated the completion of the reaction. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the target compound B1.15A (130 mg, yield: 48%), which was a brown solid.

[0508] LCMS (ESI) [M+H] + =605.6.

[0509] Step 2: At room temperature, compound B1.15-A (130 mg, 0.215 mmol) was dissolved in a 1,4-dioxane solution of hydrochloric acid (5 mL), and the mixture was reacted at room temperature for 30 minutes. LCMS indicated the completion of the reaction. The reaction solution was purified by reverse-phase chromatography to obtain the target compound (60 mg, yield: 52%), which was a brown solid.

[0510] LCMS (ESI) [M+H] + =505.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.15 (s, 2H), 7.70 (s, 1H), 7.54 (s, 1H), 7.26 (s, 1H), 7.21 (d, J = 16.2 Hz, 1H), 6.51 (d, J = 16.2 Hz, 1H), 6.31 (s, 2H), 5.42 (s, 2H), 5.28 (s, 2H), 4.19 (s, 2H), 1.86 - 1.82 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0511] Example B1.16: (S,E)-2-Amino-N-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)oxy)methyl)acetamide [Chemical formula]

[0512] Step 1: Under nitrogen gas protection, raw material A1.13 (150 mg, 0.335 mmol) was dissolved in toluene (3 mL), (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)methyl acetate (B1.1-A, 308 mg, 0.84 mmol) was added, and then zinc acetate (123 mg, 0.67 mmol) was added to the reaction solution. The reaction solution was stirred at 100 °C overnight. LCMS detected the completion of the reaction. The reaction solution was directly purified by reverse-phase chromatography (acetonitrile / 0.05% aqueous formic acid solution: 5% - 50%) to obtain the target compound (9H-fluoren-9-yl)methyl (S,E)-(2-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)allyl)oxy)methyl)amino)-2-oxoethyl)carbamate (B1.16-A, 80 mg, yield: 31%), which was a white solid.

[0513] LCMS (ESI) [M+H] + = 757.4.

[0514] Step 2: Compound B1.16-A (80 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (2 mL). Next, piperidine (0.05 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. LCMS indicated the completion of the reaction. The reaction solution was concentrated to obtain a crude product, and the crude product was separated and purified by a reverse-phase C18 column (acetonitrile / 0.05% aqueous ammonia solution: 5% - 50%) to obtain the target compound (35 mg, yield: 62%), which was a brown solid.

[0515] LCMS (ESI) [M+H]+ = 535.2, tR = 1.070 min.

[0516] Example B1.17: (S,E)-2-Amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide

Chemical Structure

[0517] Step 1: Compound A1.14 (80 mg, 0.183 mmol) and compound (2 - ((((9H - fluoren - 9 - yl)methoxy)carbonyl)amino)acetamide)methyl acetate (B1.1 - A, 135 mg, 0.367 mmol) were dissolved in toluene (4 mL), zinc acetate (190 mg, 1.03 mmol) was added, and the reaction mixture was stirred at 100 °C for 48 h. It was cooled to room temperature, water (10 mL) was added thereto, and the mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated to obtain a crude product, and the crude product was purified by reverse - phase chromatography (0.05% aqueous FA / acetonitrile: 5% - 100%) to obtain the target compound (S,E)-2-(Fmocamino)-N-(((3-(4 - ethyl - 8 - fluoro - 4 - hydroxy - 9 - methyl - 3,14 - oxo - 3,4,12,14 - tetrahydro - 1H - pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 11 - yl)allyl)oxy)methyl)acetamide (B1.17 - A, 30 mg), which was a white solid.

[0518] LCMS (ESI) [M+H] + = 745.2; 1 H NMR (400 MHz, DMSO) δ 8.82 (m 1H), 8.21 (m, 1H), 7.89 (m, 2H), 7.84 (d, J = 7.8 Hz, 2H), 7.68 (m, 1H), 7.61 (m, 1H), 7.42 (m, 1H), 7.37 (m, 1H), 7.33 - 7.28 (m, 4H), 6.72 - 6.55 (m, 1H), 6.53 (s, 1H), 5.42 (s, 2H), 5.28 (s, 2H), 4.75 (m, 2H), 4.34 (m, 2H), 4.28 - 4.24 (m, 2H), 4.20 (m, 1H), 3.70 (d, J = 6.1 Hz, 2H), 2.47 (s, 3H), 1.90 - 1.82 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0519] Step 2: Compound 4 (25 mg, 0.034 mmol) was dissolved in N,N-dimethylformamide (2 mL), and then diethylamine (0.2 mL) was added. After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. After LCMS detected the completion of the reaction, the solvent of the reaction mixture was directly evaporated to dryness to obtain the crude product target compound (S,E)-2-amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide, which was a yellow viscous product.

[0520] LCMS (ESI) [M+H]+ = 523.2; LCMS (ESI) [M+H] + = 523.1, t R = 0.446 min, 1.311 min. 1 1H NMR (400 MHz, DMSO) δ 9.26 (t, J = 6.5 Hz, 1H), 8.23 (d, J = 8.2 Hz, 1H), 8.06 (s, 2H), 7.91 (m, 1H), 7.40 (d, J = 16.3 Hz, 1H), 7.34 (s, 1H), 6.70 - 6.60 (m, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.35 (s, 2H), 4.80 (d, J = 6.6 Hz, 2H), 4.38 (d, J = 3.9 Hz, 2H), 3.68 (d, 2H), 2.52 (s, 3H), 1.90 - 1.84 (m, 2H), 0.88 (m, 3H).

[0521] C. Synthesis of Molecular Fragment Intermediates Containing a Coupling Linker Example C1.1: N 6 -(tert-Butoxycarbonyl)-N 2-((1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azapentatriacontan-31-yl)-L-valyl)-L-lysine (Compound C1.1) [Chemical formula]

[0522] Step 1: tert-Butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoate NaH (0.6 g, 60%) was added to a solution of 26-azido-3,6,9,12,15,18,21,24-octaoxahexacos-1-ol (4.39 g) in DMF (40 ml). The above mixture was stirred for 30 minutes, and tert-butyl bromoacetate (2.4 g) was added. The mixture was stirred under dry conditions for 20 hours. Next, ethyl acetate (200 ml) and water (200 mL, added slowly at first) were added to the mixture, the organic phase was washed with water (100 ml × 3), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography to obtain the target product tert-butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoate.

[0523] ESI-MS (m / z): 554 [M + H] + .

[0524] Step 2: tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoate Palladium carbon catalyst (Pd / C, 10%, 100 mg) was added to a solution of tert-butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoate (1.16 g) in ethyl acetate (20 ml). The above solution was stirred in an atmosphere of hydrogen gas for 5 hours, then the palladium carbon was filtered off, and the solvent was removed under reduced pressure to obtain the target product.

[0525] ESI-MS (m / z): 528 [M + H] + 。

[0526] Step 3: 1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azapentatriacontan-31-oic acid tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosanate (527 mg) and 2-methylthio-pyrimidine-5-carboxylic acid (170 mg) were added to dry DMF (10 ml). Next, DIPEA (0.2 ml) and HBTU (420 mg) were sequentially added to the above solution while cooling in an ice bath. The above mixture was stirred at room temperature for 20 hours. Next, it was diluted with ethyl acetate (100 ml), washed with water (100 ml × 4), the organic phase was dried over anhydrous sodium sulfate, and then the organic solvent was removed under reduced pressure. The residue was dissolved in DCM (10 ml), then TFA (10 ml) was added, the mixture was stirred at room temperature for 1 hour, then the low-boiling components were removed under reduced pressure, and the residue was separated by preparative HPLC to obtain 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azapentatriacontan-31-oic acid.

[0527] ESI-MS (m / z): 624 [M + H] + 。

[0528] Step 4: N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azapentatriacontan-31-yl)-L-valyl)-L-lysine 1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-oic acid (623 mg) was dissolved in DMF (10 ml), and DIPEA (300 μl) and HBTU (420 mg) were added thereto under 0 °C. The mixture was stirred for 30 minutes, and then the valine-(Boc)lysine dipeptide compound (345 mg) was added. The reaction system was stirred for 20 hours, and then ethyl acetate (100 ml) was added. The mixture was washed with dilute hydrochloric acid (20 ml × 3), and the organic phase was dried. Then, the organic solvent was removed under reduced pressure, and the crude product was separated by preparative HPLC to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-yl)-L-valyl)-L-lysine (Compound C1.1) was obtained.

[0529] ESI-MS (m / z): 951 [M + H] + 。

[0530] Example C1.2: N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoyl)-L-valyl)-L-lysine (Compound C1.2)

Chemical formula

[0531] Step 1: tert-Butyl 29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanate tert-Butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosanate (1.7 g) and 2-methylthio-5-ethynylpyrimidine (450 mg) were dissolved in DMSO-water (20 mL, 4:1), and copper(I) bromide (50 mg) was added to the mixture. The reaction solution was stirred at room temperature for 2 hours. Then, ethyl acetate (100 ml) was added, and the mixture was washed with water (100 ml × 3), dried, and the organic solvent was removed under reduced pressure. The crude product was separated by silica gel column chromatography to obtain the target product tert-butyl 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanate.

[0532] MS (m / z): 704 [M + H] + 。

[0533] Step 2: 29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanic acid tert-Butyl 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanate (1 g) was dissolved in dichloromethane (10 ml), and then TFA (5 ml) was added. The mixture was left at room temperature for 1 hour, and then the low-boiling components were removed under reduced pressure to obtain the target product 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanic acid.

[0534] MS (m / z): 648 [M + H] + 。

[0535] Step 3: N 6 -(tert-Butoxycarbonyl)-N 2-((29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoyl)-L-valyl)-L-lysine Dissolve 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanic acid (647 mg) in DMF (10 ml), and add DIPEA (300 μl) and HBTU (420 mg) under 0 °C. Stir the mixture for 30 minutes, and then add valine-lysine dipeptide compound (345 mg). Stir the reaction system for 20 hours, then add ethyl acetate (100 ml), wash with dilute hydrochloric acid (20 ml × 3), dry the organic phase, remove the organic solvent under reduced pressure, and separate the crude product by preparative HPLC to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoyl)-L-valyl)-L-lysine (Compound C1.2) was obtained.

[0536] ESI-MS (m / z): 975 [M + H] + 。

[0537] Example C1.3: (1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azatriacontan-31-yl)-glycyl-glycyl-L-phenylalanine (Compound C1.3)

Chem.

[0538] ESI-MS (m / z): 885 [M+H] + .

[0539] Example C1.4: (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-azahexatriacont-35-ynoyl)glycylglycyl-L-phenylalanine (compound C1.4) [ka] Using the same method and reaction conditions as in Step 4 of Example C1.1, and using the raw materials shown in the reaction scheme, the target product (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-azahexatriacont-35-ynoyl)glycylglycyl-L-phenylalanine (compound C1.4) was obtained.

[0540] ESI-MS (m / z): 951 [M+H] + .

[0541] Example C1.5:N 6 -(tert-butoxycarbonyl)-N 2 -((36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-azahexatriacont-35-ynoyl)-L-valyl)-L-lysine (C1.5) [ka] Using the same method and reaction conditions as in Step 4 of Example C1.1 and using the compound shown in the reaction formula as a raw material, the target product N 6 -(tert-Butoxycarbonyl)-N 2 -((36-(2-(Methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-azatetracont-35-enoyl)-L-valyl)-L-lysine (Compound C1.5) was obtained.

[0542] ESI-MS (m / z): 1017 [M+H] + 。

[0543] Example C1.6: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylthio)pyrimidin-5-yl)-hex-5-enoyl)-L-valyl)-L-lysine (Compound C1.6)

Chemical formula

[0544] ESI-MS (m / z): 564.3 [M+H] + 。

[0545] 1 H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.68 (s, 2H), 8.12 (d, J = 7.3 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 6.76 (s, 1H), 4.27 - 4.19 (m, 1H), 4.11 (d, J = 4.9 Hz, 1H), 2.88 (d, J = 7.9 Hz, 3H), 2.73 (s, 1H), 2.59 (s, 1H), 2.52 (s, 4H), 2.40 - 2.24 (m, 3H), 2.03 - 1.88 (m, 2H), 1.77 (d, J = 3.2 Hz, 2H), 1.68 (d, J = 7.0 Hz, 1H), 1.60 - 1.49 (m, 1H), 1.36 (s, 15H), 0.85 (dd, J = 14.8, 6.7 Hz, 7H).

[0546] Example C1.7: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)-hex-5-ynoyl)-L-valyl)-L-lysine (Compound C1.7)

Chemical formula

[0547] LCMS (ESI) [M+H] + : 596; 1 H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 9.13 (s, 2H), 8.14 (d, J = 7.0 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 6.77 (s, 1H), 4.24 (t, J = 7.7 Hz, 1H), 4.12 (br s, 1H), 3.41 (s, 3H), 2.89 (d, J = 6.0 Hz, 2H), 2.43 - 2.29 (m, 2H), 2.03 - 1.93 (m, 2H), 1.82 (br s, 2H), 1.63 - 1.60 (m, 4H), 1.37 (s, 12H), 0.88 - 0.83 (m, 6H).

[0548] Example C1.8: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylthio)pyrimidine-5-carboxamido)hexanoyl)-L-valyl)-L-lysine (Compound C1.8)

Chemical formula

[0549] ESI-MS (m / z): 611 [M+H] + .

[0550] Example C1.9:N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (Compound C1.9)

Chemical formula

[0551] Step 2:N 6 -(tert-Butoxycarbonyl)-N 2-((6-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine Compound 6-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (370 mg) and N-hydroxysuccinimide (152 mg) were dissolved in dichloromethane (5 mL) with stirring, and then dicycloethylcarbodiimide (273 mg) was added. Next, the reaction solution was stirred at room temperature for 1 hour. 10 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in N,N-dimethylformamide (10 mL) with stirring, and compound N 2 -L-valyl-N 6 -(Boc)-L-lysine was added, and the reaction solution was reacted at room temperature for 16 hours. Citric acid was slowly added to the reaction solution to adjust the pH to about 5, water was added, and the mixture was further extracted with ethyl acetate (3 × 10 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated to remove ethyl acetate, and the target product N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (Compound C1.9, 400 mg) was obtained.

[0552] ESI-MS (m / z): 635.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 9.06 (s, 2H), 9.06 (s, 1H), 8.70 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 6.76 (t, J = 5.6 Hz, 1H), 4.41 (t, J = 7.0 Hz, 2H), 4.19 (dd, J = 8.9, 7.0 Hz, 1H), 4.14 - 4.05 (m, 1H), 2.92 - 2.85 (m, 2H), 2.56 (s, 3H), 2.24 - 2.10 (m, 2H), 1.97 - 1.81 (m, 3H), 1.71 - 1.64 (m, 1H), 1.61 - 1.50 (m, 3H), 1.36 (s, 9H), 1.28 - 1.22 (m, 6H), 0.88 - 0.76 (m, 6H).

[0553] Example C1.10: (6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (Compound C1.10)

Chemical Structure

[0554] Using the same method and reaction conditions as in Example C1.6 and using different reaction raw materials, the target products in the following table were obtained.

Table 6

[0555] Example C1.13: (29-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxanonacosanoyl)glycylglycyl-L-phenylalanine (Compound C1.13)

Chemical formula

[0556] ESI-MS (m / z): 909 [M+H] + .

[0557] Example C1.14: N 6 -(tert-Butoxycarbonyl)-N 2 -((39-(2-(Methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazanonatriacont-38-inoyl)-L-valyl)-L-lysine (C1.14)

Chemical formula

[0558] Step 1: Dissolve Compound C1.14-A (336 mg) in dichloromethane (5 mL), add N-hydroxysuccinimide (98 mg, 0.85 mmol) and dicyclohexylcarbodiimide (175 mg, 0.85 mmol), stir and react at room temperature for 1 hour, and then add Compound N 2 -L-valyl-N 6-(Boc)-L-lysine (230 mg) was added to the reaction solution, and the mixture was stirred overnight at room temperature to react. The solution was concentrated to remove the solvent to obtain a crude product, which was separated and purified by a reverse-phase C18 column (acetonitrile / 0.01% aqueous FA solution: 5% - 50%) to obtain the target compound (C1.14B, 290 mg) as a colorless oil.

[0559] LCMS (ESI) [M+H] + = 882.3.

[0560] Step 2: Compound C1.14-B (300 mg) was dissolved in anhydrous methanol (5 mL), Pd / C (10%, 60 mg) was added, the mixture was purged with hydrogen gas three times, stirred overnight at room temperature to react, filtered by suction, and concentrated to obtain the target compound C1.14-C (293 mg), which was a colorless oil.

[0561] LCMS (ESI) [M+H] + : 856.1.

[0562] Step 3: 6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoic acid (84 mg) was dissolved in N,N-dimethylformamide (3 mL), and then N,N-diisopropylethylamine (61 mg) and HATU (108 mg) were sequentially added. The reaction solution was stirred at room temperature for 20 minutes, then compound C1.14-C (196 mg) was added, and the mixture was stirred at 40 °C for 3 hours to react. The crude product was separated and purified by a reverse-phase C18 column (acetonitrile / 0.01% aqueous FA solution: 5% - 65%) to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((39-(2-(Methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazahentriacont-38-inoyl)-L-valyl)-L-lysine (C1.14) (170 mg).

[0563] LCMS (ESI) [M+H] + : 1074.0; 1 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 2H), 8.23 (t, J = 5.7 Hz, 1H), 8.13 (d, J = 8.9 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.43 (d, J = 6.5 Hz, 1H), 6.69 (s, 1H), 4.09 (dd, J = 8.8, 6.5 Hz, 1H), 4.02 (s, 2H), 3.98 (d, J = 2.9 Hz, 2H), 3.74 - 3.70 (m, 1H), 3.50 (s, 30H), 3.45 (d, J = 6.2 Hz, 2H), 3.39 (d, J = 5.8 Hz, 2H), 3.26 (d, J = 6.1 Hz, 2H), 3.20 (q, J = 5.9 Hz, 2H), 2.81 (t, J = 6.6 Hz, 2H), 2.52 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 2.12 - 2.06 (m, 1H), 1.79 - 1.74 (m, 2H), 1.67 - 1.59 (m, 1H), 1.50 (d, J = 5.3 Hz, 1H), 1.36 (s, 9H), 1.24 (s, 2H), 1.20 - 1.09 (m, 2H), 0.85 (t, J = 6.3 Hz, 6H).

[0564] Example C1.15:N 6 -(tert-Butoxycarbonyl)-N 2 -((32-(4-(2-(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanoyl)-L-valyl)-L-lysine (C1.15)

Chemical formula

[0565] LCMS (ESI) [M+H] + = 1032.3.

[0566] Example C1.16: N 6 ,N 6 -dimethyl-N 2 -((6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical Structure

[0567] Step 1: A solution of hydrogen chloride-dioxane (100 mL) was added to compound C1.16-A (10.0 g), and the mixture was reacted at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the target compound C1.16-B (8.0 g) as a white solid.

[0568] LCMS (ESI) [M+H] + : 380.1.

[0569] 11H NMR (400 MHz, DMSO) δ 8.18 (d, J = 7.4 Hz, 1H), 7.40 - 7.30 (m, 5H), 7.26 (d, J = 8.8 Hz, 1H), 5.08 - 4.99 (m, 2H), 4.23 - 4.11 (m, 1H), 3.94 - 3.88 (m, 1H), 2.78 - 2.73 (m, 2H), 2.03 - 1.94 (m, 1H), 1.77 - 1.51 (m, 4H), 1.44 - 1.31 (m, 2H), 0.87 (dd, J = 17.3, 6.6 Hz, 6H).

[0570] Step 2: Compound C1.16-B (3.0 g) and sodium acetate (1.90 g) were dissolved in a methanol (100 mL) solution, reacted at room temperature for 10 minutes, paraformaldehyde (2.8 g) was added to the reaction solution, stirred at room temperature for 30 minutes to react, and then sodium cyanoborohydride (1.0 g) was added to the reaction solution, and the reaction was stirred at room temperature for 16 hours. After filtering the reaction solution, the filtrate was separated and purified by a reverse-phase C18 column (acetonitrile: 0.05% aqueous formic acid solution: 5% - 55%) to obtain the target compound C1.16-C (1.70 g).

[0571] LCMS (ESI) [M+H] + : 408.1; 1 1H NMR (400 MHz, DMSO) δ 7.86 (d, J = 7.3 Hz, 1H), 7.40 - 7.26 (m, 6H), 5.03 (s, 2H), 4.08 - 4.06 (m, 1H), 3.90 - 3.85 (m, 1H), 2.50 - 2.45 (m, 2H), 2.35 (s, 6H), 2.05 - 1.93 (m, 1H), 1.73 - 1.55 (m, 2H), 1.51 - 1.40 (m, 2H), 1.33 - 1.22 (m, 2H), 0.85 (dd, J = 16.5, 6.8 Hz, 6H).

[0572] Step 3: At room temperature, compound C1.16-C (1.6 g) was dissolved in methanol (80 mL). Next, Pd / C (10%, 0.16 g) was added to the reaction solution, and the mixture was stirred and reacted with hydrogen gas at room temperature for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.16-D (680 mg).

[0573] LCMS (ESI) [M+H] + : 274.2.

[0574] Step 4: 6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoic acid (944 mg) was dissolved in N,N-dimethylformamide (30 mL). Next, N,N-diisopropylethylamine (1.3 g) and 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU, 1.8 g) were sequentially added. After the reaction solution was stirred at room temperature for 20 minutes, compound C1.16-D (1.1 g) was added, and the mixture was stirred and reacted at 40 °C for 3 hours. The crude product was separated and purified by a reverse-phase C18 column (acetonitrile / 0.01% aqueous formic acid solution: 5% - 65%) to obtain the target compound N as a white solid 6 ,N 6 -dimethyl-N 2 -((6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine C1.16 (1.2 g).

[0575] LCMS (ESI) [M+H] + : 492.1; 11H NMR (400 MHz, DMSO) δ 8.68 (s, 2H), 8.00 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 4.20 (t, 1H), 4.08 (dd, J = 12.7, 7.7 Hz, 1H), 2.47 - 2.40 (m, 4H), 2.37 - 2.31 (m, 2H), 2.30 (s, 6H), 2.01 - 1.92 (m, 1H), 1.82 - 1.73 (m, 2H), 1.71 - 1.56 (m, 2H), 1.49 - 1.37 (m, 2H), 1.33 - 1.23 (m, 2H), 0.88 - 0.82 (m, 6H).

[0576] Example C1.17: N 6 , N 6 -Dimethyl-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine C1.17

Chemical Structure

[0577] LCMS (ESI) [M+H] + : 524.4.

[0578] 1 1H NMR (400 MHz, ) δ 9.13 (s, 2H), 7.95 (t, J = 8.8 Hz, 2H), 4.21 (dd, J = 8.8, 6.9 Hz, 1H), 4.08 - 4.03 (m, 1H), 3.41 (s, 3H), 2.55 (t, J = 7.0 Hz, 2H), 2.42 - 2.32 (m, 4H), 2.27 (s, 6H), 1.98 (dd, J = 13.6, 6.8 Hz, 1H), 1.86 - 1.77 (m, 2H), 1.74 - 1.55 (m, 2H), 1.47 - 1.37 (m, 2H), 1.31 - 1.23 (m, 2H), 0.85 (dd, J = 12.8, 6.8 Hz, 6H).

[0579] Example C1.18:N 2 -(tert-Butoxycarbonyl)-N 6 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine [Chemical formula]

[0580] Step 1: Compound C1.18-B (3 g) was dissolved in dichloromethane (30 mL), DIPEA (4 mL) was added, and then compound C1.18-A (3.48 g) was added. The mixture was stirred at room temperature for 20 hours to react. Ethyl acetate (200 mL) was added to the reaction solution, and the mixture was washed successively with hydrochloric acid (0.1 M, 30 mL × 3) and water (30 ml × 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the target mixture C1.18-C (4.7 g).

[0581] Step 2: At room temperature, compound C1.18-C (4.7 g) was dissolved in methanol (80 mL), and then Pd / C (10%, 0.6 g) was added to the reaction solution. The mixture was stirred and reacted with hydrogen gas at room temperature for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.18-D (3.4 g). LCMS (ESI) [M+H] + : 346.2

[0582] Step 3: 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (2.68 g) was dissolved in N,N-dimethylformamide (50 mL), and then triethylamine (3 mL) and HBTU (3.8 g) were added. The mixture was stirred at room temperature for 10 minutes, and then ethyl acetate (200 mL) was added. The mixture was washed with saturated sodium bicarbonate (20 mL×2), hydrochloric acid (0.1 M, 50 mL×2), and water (50 mL×2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product intermediate. The above crude product was dissolved in DMF (30 mL), DIPEA (1.6 mL) was added, and then C1.18-D (3.4 g) was added. The reaction solution was stirred at room temperature for 3 hours, then ethyl acetate (200 mL) was added, washed with hydrochloric acid (0.1 M, 50 mL×2) and water (50 mL×2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain the target compound N 2 -(tert-Butoxycarbonyl)-N 6 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (C1.18, white solid, 3.8 g) was obtained.

[0583] LCMS (ESI) [M+H] + : 596.4

[0584] Example C1.19: N 6 ,N 6 -Dimethyl-N 2 -((6-(4-(2-(Methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine [Chemistry]

[0585] Step 1: Dissolve 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (1.0 g, 3.3 mmol) in a mixed solution of tetrahydrofuran and water (40 mL, 3:1), and add potassium peroxymonosulfate (10.0 g, 16.3 mmol). Stir and react at room temperature for 3 hours, and LCMS detected the completion of the reaction. Filter the reaction solution, wash the cake with DMSO, and combine the filtrates. Purify by reverse-phase chromatography (C18, acetonitrile: 0.1% formic acid = 5% - 55%) to obtain 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (750 mg, yield: 67.9%), which was a white solid.

[0586] LCMS (ESI) [M+H] + = 340.1; 1 H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 2H), 8.95 (s, 1H), 4.49 (t, J = 7.0 Hz, 2H), 3.45 (s, 3H), 2.22 (t, J = 7.3 Hz, 2H), 1.95 - 1.84 (m, 2H), 1.61 - 1.50 (m, 2H), 1.36 - 1.26 (m, 2H).

[0587] Step 2: Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (300 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (6 mL), HATU (337 mg, 0.88 mmol) and DIPEA (286 mg, 2.21 mmol) were added, and the mixture was stirred at room temperature for 30 minutes to react. Next, dipeptide C1.16-D (243 mg, 0.88 mmol) was added, and the mixture was stirred at room temperature for 2 hours to react. LCMS detected the completion of the reaction, and the reaction solution was separated and purified by a C18 column (acetonitrile / 0.01% aqueous FA solution: 5% - 50%) to obtain the target compound N 6 ,N 6 -dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine (300 mg, yield: 57%) was obtained as a white solid.

[0588] LCMS (ESI) [M+H] + = 595.5, t R = 2.024 min.

[0589] 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 2H), 9.01 (s, 1H), 7.85 (br s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 4.48 (t, J = 6.8 Hz, 2H), 4.17 - 4.15 (m, 1H), 4.02 (br s, 1H), 3.44 (s, 3H), 2.42 (br s, 2H), 2.30 (s, 6H), 2.18 - 2.14 (m, 2H), 1.99 - 1.96 (m, 1H), 1.88 (dd, J = 14.6, 7.1 Hz, 2H), 1.67 (br s, 1H), 1.59 - 1.53 (m, 2H), 1.43 (br s, 2H), 1.28 - 1.26 (m, 5H), 0.83 - 0.89 (m, 6H).

[0590] Example C1.20:N 6 ,N 6 -diethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical formula

[0591] Step 1: Dissolve compound C1.16-B (5.0 g, 12.05 mmol) in dichloromethane (100 mL). Add acetaldehyde (3.2 g, 72.3 mmol) to the reaction solution and stir at room temperature for 10 minutes to react. Then add sodium triacetoxyborohydride (12.8 g, 60.25 mmol) to the reaction solution and stir the reaction system at room temperature for 1 hour. LCMS indicated the completion of the reaction. Add saturated ammonium chloride aqueous solution to the reaction solution, stir for 1 hour, spin dry, filter, and then separate and purify the filtrate by a reverse-phase C18 column (acetonitrile: 0.05% formic acid aqueous solution: 5% - 55%) to obtain the target compound C1.20-A (4.57 g, yield: 82.0%), which was a white solid.

[0592] LCMS (ESI) [M+H] + = 436.4; 11H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 7.0 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.38 - 7.26 (m, 5H), 5.08 - 4.99 (m, 2H), 4.00 (dd, J = 12.6, 6.5 Hz, 1H), 3.86 (dd, J = 8.6, 6.8 Hz, 1H), 2.74 (dd, J = 14.0, 6.9 Hz, 4H), 2.64 - 2.54 (m, 2H), 2.05 - 1.94 (m, 1H), 1.72 - 1.52 (m, 2H), 1.52 - 1.38 (m, 2H), 1.38 - 1.18 (m, 2H), 1.04 (t, J = 7.1 Hz, 6H), 0.87 - 0.81 (m, 6H).

[0593] Step 2: At room temperature, compound C1.20-A (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL). Next, Pd / C (0.16 g) was added to the reaction solution, and the mixture was stirred at room temperature for 12 hours under hydrogen gas to allow the reaction to proceed. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.20-B (900 mg, yield: 82%), which was an off-white solid.

[0594] 1 1H NMR (400 MHz, DMSO-d6) δ 8.04 (br s, 1H), 4.02 - 3.99 (m, 1H), 3.10 (d, J = 4.5 Hz, 1H), 2.65 (q, J = 7.1 Hz, 4H), 2.55 - 2.51 (m, 2H), 2.06 - 1.93 (m, 1H), 1.73 - 1.54 (m, 2H), 1.47 - 1.38 (m, 2H), 1.30 - 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H), 0.89 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).

[0595] Step 3: 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL), and HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added sequentially. After stirring at room temperature for 20 minutes, compound C1.20-B (301 mg, 1 mmol) was added, and stirring was continued at room temperature for 30 minutes. After LCMS detected the completion of the reaction, the reaction solution was directly purified by a reverse-phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain the target compound (280 mg, yield: 51%), which was a white solid.

[0596] LCMS (ESI) [M+H] + = 552.3; 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 2H), 7.95 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 4.18 (dd, J = 8.8, 6.8 Hz, 1H), 4.02 (dd, J = 12.8, 7.2 Hz, 1H), 3.41 (s, 3H), 2.74 - 2.69 (m, 4H), 2.62 - 2.52 (m, 4H), 2.44 - 2.29 (m, 2H), 2.04 - 1.94 (m, 1H), 1.86 - 1.77 (m, 2H), 1.72 - 1.54 (m, 2H), 1.51 - 1.39 (m, 2H), 1.33 - 1.23 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H), 0.87 - 0.82 (m, 6H).

[0597] Example C1.21: N 6 ,N 6 -Diethyl-N 2 -((6-(4-(2-(Methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine

Chemical Structure

[0598] LCMS (ESI) [M+H] + = 623.4; 1 H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 9.04 (s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 7.1 Hz, 1H), 4.47 (t, J = 7.0 Hz, 2H), 4.14 (dd, J = 8.8, 6.6 Hz, 1H), 4.06 - 3.95 (m, 1H), 3.44 (s, 3H), 2.67 - 2.64 (m, 4H), 2.55 (t, J = 7.4 Hz, 2H), 2.21 - 2.10 (m, 2H), 2.02 - 1.94 (m, 1H), 1.92 - 1.84 (m, 2H), 1.72 - 1.63 (m, 1H), 1.72 - 1.63 (m, 3H), 1.59 - 1.54 (m, 2H), 1.32 - 1.21 (m, 4H), 1.01 (t, J = 7.1 Hz, 6H), 0.81 (dd, J = 9.6, 6.8 Hz, 6H).

[0599] Example C1.22:N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chemical formula

[0600] Step 1: Dissolve compound C1.16-B (5.0 g, 12 mmol) in dichloromethane (100 mL), add n-propionaldehyde (4.2 g, 72.3 mmol) to the reaction solution, stir and react at room temperature for 10 minutes. Then add sodium triacetoxyborohydride (12.8 g, 60.25 mmol) to the reaction solution, and stir and react the reaction system at room temperature for 1 hour. LCMS indicated the completion of the reaction. Add saturated ammonium chloride aqueous solution to the reaction solution, stir for 1 hour, spin-dry, filter, and then separate and purify the filtrate by a reverse-phase C18 column (acetonitrile: 0.05% formic acid aqueous solution: 5% - 55%) to obtain the target compound C1.22-A (4.57 g, yield: 82.0%), which was a white solid.

[0601] LCMS (ESI) [M+H] + = 464.0; 1 H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 7.3 Hz, 1H), 7.38 - 7.28 (m, 5H), 5.04 (d, J = 1.7 Hz, 2H), 4.12 - 4.02 (m, 1H), 3.94 - 3.82 (m, 1H), 2.65 - 2.52 (m, 6H), 2.06 - 1.94 (m, 1H), 1.76 - 1.64 (m, 1H), 1.64 - 1.53 (m, 1H), 1.52 - 1.40 (m, 6H), 1.34 - 1.18 (m, 2H), 0.92 - 0.80 (m, 12H). -

[0602] Step 2: At room temperature, compound C1.22-A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL). Next, Pd / C (0.16 g) was added to the reaction solution, and the mixture was stirred and reacted with hydrogen gas at room temperature for 12 hours. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.22-B (1.2 g, yield: 85.5%), which was a white solid.

[0603] Step 3: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL). Next, HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added, and the reaction system was stirred for 30 minutes. Then, compound C1.22-B (122 mg, 0.371 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. After LCMS detected the completion of the reaction, the reaction solution was directly purified by a reverse-phase C18 column (acetonitrile and 0.05% formic acid aqueous solution reaction system) to obtain the target compound N 6 ,N 6 -dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (50 mg, yield: 28%), which was a pale yellow solid.

[0604] LCMS (ESI) [M+H] + = 580.0; 11H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.98 - 7.93 (m, 2H), 4.24 - 4.16 (m, 1H), 4.10 (d, J = 5.2 Hz, 1H), 3.41 (s, 3H), 2.79 - 2.64 (m, 6H), 2.55 (t, J = 7.1 Hz, 2H), 2.45 - 2.26 (m, 2H), 2.06 - 1.91 (m, 1H), 1.89 - 1.78 (m, 2H), 1.76 - 1.66 (m, 1H), 1.64 - 1.57 (m, 1H), 1.57 - 1.42 (m, 6H), 1.37 - 1.24 (m, 2H), 0.93 - 0.78 (m, 12H).

[0605] Example C1.23:N 6 ,N 6 -Dipropyl-N 2 -((6-(4-(2-(Methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valyl)-L-lysine

Chemical Structure

[0606] LCMS (ESI) [M+H] + = 651.5; 1 H NMR (400 MHz, DMSO) δ 9.49 (s, 2H), 9.00 (s, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 4.47 (t, J = 6.9 Hz, 2H), 4.21 - 4.11 (m, 1H), 4.09 - 4.01 (m, 1H), 3.44 (s, 3H), 2.45 - 2.35 (m, 6H), 2.23 - 2.09 (m, 2H), 1.97 - 1.86 (m, 3H), 1.72 - 1.64 (m, 1H), 1.61 - 1.50 (m, 3H), 1.43 - 1.34 (m, 6H), 1.32 - 1.22 (m, 4H), 0.86 - 0.77 (m, 12H).

[0607] Example C1.24: tert-Butyl (S)-(6-((4-(Hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamido)-6-oxohexyl)carbamate

Chemical formula

[0608] Step 1: Compound C1.24-A (3.70 g, 15.7 mmol), diisopropylethylamine (11.0 mL, 62.8 mmol), and HBTU (8.90 g, 23.6 mmol) were dissolved in N,N-dimethylformamide (30 mL), and the reaction system was reacted at room temperature for 30 minutes. Next, lysine protected with Boc (3.86 g, 15.7 mmol) was added, and the reaction system was reacted at room temperature for 2 hours. LCMS detected the completion of the reaction. An aqueous citric acid solution (30 mL) was added to the reaction solution to adjust the pH to 5, and the aqueous solution was extracted with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain the target compound C1.24-B (7.0 g, crude product), which was a yellow oil. LCMS (ESI) [M+H] + = 465.1, t R = 1.751 min.

[0609] Step 2: Compound C1.24-B (7.00 g, 5.60 mmol), diisopropylethylamine (10.7 mL, 60.4 mmol), and HBTU (8.60 g, 22.7 mmol) were dissolved in N,N-dimethylformamide (100 mL). Next, p-aminobenzyl alcohol (3.71 g, 30.2 mmol) was added, and the reaction system was reacted at room temperature for 2 hours. LCMS detected the completion of the reaction. Ethyl acetate (300 mL) was added to the reaction solution, washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, suction filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamido)-6-oxohexyl)carbamate (6.00 g, yield: 69.9%), which was a yellow oil.

[0610] LCMS (ESI) [M+H] + = 570.1; 11H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 8.46 (s, 2H), 7.51 - 7.47 (m, 2H), 7.31 - 7.23 (m, 3H), 7.02 - 6.95 (m, 1H), 4.83 (br s, 1H), 4.63 - 4.60 (m, 2H), 3.19 - 2.96 (m, 4H), 2.57 (s, 3H), 2.52 - 2.48 (m, 2H), 2.47 - 2.41 (m, 2H), 1.99 - 1.92 (m, 2H), 1.87 - 1.77 (m, 2H), 1.76 - 1.63 (m, 2H), 1.43 (s, 9H).

[0611] Example C1.25: tert-Butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)-6-oxohexyl)carbamate [Chemical Structure]

[0612] Step 1: Compound C1.25-A (1.60 g, 5.21 mmol), diisopropylethylamine (2.68 mL, 20.8 mmol), and HBTU (2.97 g, 7.82 mmol) were dissolved in N,N-dimethylformamide (30 mL), and the reaction system was reacted at room temperature for 30 minutes. Next, Boc-protected lysine (1.28 g, 5.21 mmol) was added, and the reaction system was reacted at room temperature for 2 hours. LCMS detected the completion of the reaction. Aqueous citric acid solution (30 mL) was added to the reaction solution to adjust the pH to 5, and the aqueous solution was extracted with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain the target compound C1.25-B (3.0 g, crude product), which was a yellow oil.

[0613] LCMS (ESI) [M+H] + = 536.0; Step 2: Compound C1.25-B (3.00 g, 5.60 mmol), diisopropylethylamine (2.89 g, 22.4 mmol), and HBTU (3.19 g, 8.40 mmol) were dissolved in N,N-dimethylformamide (30 mL). Next, p-aminobenzyl alcohol (1.38 g, 11.2 mmol) was added, and the reaction system was reacted at room temperature for 2 hours. LCMS detected the completion of the reaction. Ethyl acetate (200 mL) was added to the reaction solution, washed with saturated brine (80 mL × 3), dried over anhydrous sodium sulfate, suction filtered, the filtrate was concentrated under reduced pressure to a solid, and the crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound tert-butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)-6-oxohexyl)carbamate (2.90 g), giving a yellow oil.

[0614] LCMS (ESI) [M+H] + = 641.1; 1 H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.95 (s, 2H), 7.94 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 6.83 - 6.77 (m, 1H), 4.93 - 4.80 (m, 1H), 4.64 - 4.58 (m, 2H), 4.40 - 4.29 (m, 2H), 3.12 - 2.97 (m, 4H), 2.60 (s, 3H), 2.31 - 2.23 (m, 2H), 1.96 - 1.83 (m, 4H), 1.76 - 1.61 (m, 4H), 1.41 (s, 9H), 1.37 - 1.29 (m, 4H).

[0615] Example C1.26: N 6 ,N 6 -dibutyl-N 2-((6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine

Chem.

[0616] Step 1: Compound C1.16-B (10.0 g) was dissolved in a dichloromethane (200 mL) solution. n-Butyraldehyde (10.4 g) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes to react. Then, sodium triacetoxyborohydride (25.6 g) was added to the reaction solution in one batch, and the reaction system was stirred at room temperature for 1 hour to react. LCMS indicated the completion of the reaction. An aqueous saturated ammonium chloride solution was added to the reaction solution, and the mixture was stirred for 1 hour, spin-dried, filtered. After that, the filtrate was separated and purified by a reverse-phase C18 column (acetonitrile: 0.05% formic acid aqueous solution: 5% - 55%) to obtain the target compound C1.26-A (4.9 g), which was a white solid.

[0617] LCMS (ESI) [M+H] + = 492.7; 1 H NMR (400 MHz, CDCl3) δ 7.35 - 7.30 (m, 5H), 5.14 - 5.08 (m, 2H), 4.33 - 4.30 (m, 1H), 4.14 - 4.12 (m, 1H), 2.94 - 2.80 (m, 6H), 2.13 - 2.11 (m, 1H), 1.85 - 1.82 (m, 2H), 1.58 - 1.55 (m, 4H), 1.40 - 1.22 (m, 8H), 0.98 - 0.87 (m, 12H).

[0618] Step 2: At room temperature, compound C1.26-A (5.0 g) was dissolved in a methanol (100 mL) solution. Next, Pd / C (10%, 1 g) was added to the reaction solution, and the mixture was stirred with hydrogen gas for 12 hours to react. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.26-B (3.68 g), which was a white solid.

[0619] LCMS (ESI) [M+H] + = 358.3。

[0620] Step 3: Dissolve 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1.0 g) in N,N-dimethylformamide (15 mL), then sequentially add N,N-diisopropylethylamine (1.2 g) and HATU (1.4 g), stir the reaction system for 30 minutes, then add compound C1.26-B (1.3 g), stir the reaction solution at room temperature for 1 hour. After LCMS detected the completion of the reaction, purify the reaction solution directly by preparative chromatography (aqueous solution of 0.01% trifluoroacetic acid, acetonitrile) to obtain the target compound N 6 ,N 6 -dibutyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (C1.26, 500 mg) was obtained as a yellow solid.

[0621] LCMS (ESI) [M+H] + = 608.7; 1H NMR (400 MHz, DMSO) δ 9.37 (s, 1H), 9.13 (s, 2H), 8.19 (d, J = 7.3 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 4.25 - 4.20 (m, 1H), 4.20 - 4.13 (m, 1H), 3.42 (s, 3H), 3.06 - 2.99 (m, 6H), 2.57 - 2.53 (m, 2H), 2.41 - 2.30 (m, 2H), 1.99 - 1.95 (m, 2H), 1.87 - 1.79 (m, 2H), 1.78 - 1.71 (m, 1H), 1.62 - 1.56 (m, 4H), 1.34 - 1.32 (m, 8H), 0.94 - 0.85 (m, 12H).

[0622] Example C1.27: (S)-1-Ethyl-4-(3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexanamido)butanamido)piperidine-4-carboxylic acid

Chemical formula

[0623] Step 1: Compound C1.27-A (5.0 g, 20.49 mmol) was dissolved in DMF (100 mL), and 2,5-dioxopyrrolidin-1-yl ((benzyloxy)carbonyl)-L-valinate (7.13 g, 20.49 mmol) was added to the reaction solution, and the mixture was stirred at 100 °C for 48 hours to react. LCMS indicated the completion of the reaction. The reaction solution was separated and purified by a reverse-phase C18 column (acetonitrile: 0.05% aqueous formic acid solution: 5% - 55%) to obtain the target compound C1.27-B (1.3 g, yield: 13.3%), which was a yellow solid.

[0624] LCMS (ESI) [M-boc] + = 378.2; 1 H NMR (400 MHz, DMSO) δ 7.38 - 7.33 (m, 5H), 7.32 - 7.27 (m, 1H), 7.20 (d, J = 9.0 Hz, 1H), 5.14 - 5.02 (m, 2H), 3.97 - 3.87 (m, 1H), 3.70 - 3.59 (m, 2H), 3.08 - 2.95 (m, 2H), 2.04 - 1.97 (m, 1H), 1.95 - 1.87 (m, 2H), 1.76 - 1.69 (m, 2H), 1.40 (s, 9H), 0.91 - 0.84 (m, 6H).

[0625] Step 2: At room temperature, compound C1.27-B (1.3 g, 2.71 mmol) was dissolved in an ethyl acetate (10 mL) solution. Next, dioxane-HCl (3 N, 10 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour to allow the reaction to proceed. LCMS indicated the completion of the reaction. The reaction solution was concentrated under reduced pressure to obtain the target compound C1.27-C (1.06 g, yield: 94.4%), which was a yellow solid.

[0626] LCMS (ESI) [M+H] + = 378.4.

[0627] Step 3: Compound C1.27-C (1.06 g, 2.81 mmol) was dissolved in dichloromethane (10 mL). Acetaldehyde (0.75 g, 16.87 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes to allow the reaction to proceed. Further, sodium triacetoxyborohydride (3 g, 14.05 mmol) was added to the reaction system in one portion, and the reaction system was stirred at room temperature for 1 hour to allow the reaction to proceed. LCMS indicated the completion of the reaction. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was stirred for 1 hour and evaporated under reduced pressure to remove dichloromethane. The remaining aqueous phase was separated by reverse-phase chromatography (acetonitrile: 0.05% formic acid aqueous solution: 5% - 55%) to obtain the target compound C1.27-D (1.13 g, yield: 99.23%), which was a white solid.

[0628] LCMS (ESI) [M+H] + = 406.1; 1H NMR (400 MHz, MeOD) δ 7.42 - 7.25 (m, 5H), 5.22 - 5.06 (m, 2H), 3.91 (d, J = 7.0 Hz, 1H), 3.52 - 3.36 (m, 2H), 3.25 - 2.93 (m, 4H), 2.66 - 2.54 (m, 1H), 2.47 - 2.36 (m, 1H), 2.27 - 2.14 (m, 2H), 2.13 - 2.06 (m, 1H), 1.35 - 1.29 (m, 4H), 0.98 - 0.90 (m, 6H).

[0629] Step 4: At room temperature, compound C1.27-D (1.13 g, 2.78 mmol) was dissolved in a methanol (20 mL) solution. Next, Pd / C (0.5 g) was added to the reaction solution, and the mixture was stirred and reacted with hydrogen gas for 12 hours. LCMS indicated the completion of the reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.27-E (0.58 g, yield: 76.18%), which was a white solid. LCMS (ESI) [M+H] + = 272.4.

[0630] Step 5: Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1 g, 3.73 mmol) was dissolved in N,N-dimethylformamide (5 mL). Next, N,N-diisopropylethylamine (1.2 g, 9.32 mmol) and HATU (1.42 g, 3.73 mmol) were sequentially added, and the reaction solution was stirred for 30 minutes. Then, compound C1.27-E (1.01 g, 3.73 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. After LCMS detected the completion of the reaction, the reaction solution was directly purified by preparative chromatography (aqueous solution of 0.01% trifluoroacetic acid, acetonitrile) to obtain the target compound (S)-1-ethyl-4-(3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)piperidine-4-carboxylic acid (C1.27, 500 mg, yield: 28.7%), which was a yellow solid.

[0631] LCMS (ESI) [M+H] + = 522.5; 11H NMR (400 MHz, DMSO) δ 9.12 (s, 2H), 8.08 (s, 1H), 7.88 (d, J = 9.0 Hz, 1H), 4.28 - 4.25 (m, 1H), 3.41 (s, 3H), 2.89 - 2.78 (m, 2H), 2.57 - 2.52 (m, 4H), 2.45 - 2.30 (m, 4H), 2.07 - 2.01 (m, 2H), 1.99 - 1.96 (m, 1H), 1.95 - 1.88 (m, 2H), 1.85 - 1.79 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.88 - 0.83 (m, 6H).

[0632] II. Synthesis of Drug-Linker Compounds Example 2.1: N-((S)-1-(((S)-6-Amino-1-((2-((((S)-7-Ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-001) [Chemical Structure]

[0633] Step 1: Compound C1.6 (50 mg, 0.10 mol), compound B1.5 (71 mg, 0.12 mol), and N,N - diisopropylethylamine (39 mg, 0.30 mol) were dissolved in DMF (5 mL). 1 - Hydroxybenzotriazole (16 mg, 0.12 mmol) and 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (23 mg, 0.12 mmol) were added sequentially, and the above reaction solution was stirred at room temperature for 16 hours. 50 mL of ethyl acetate was added for dilution. The organic phase was washed three times with water (25 ml×3), dried over anhydrous sodium sulfate, concentrated to remove ethyl acetate to obtain a crude product. The crude product was separated and purified by flash silica gel column chromatography (DCM:MeOH = 100:0~90:10) to obtain the target compound 2.1A (62 mg, yellow solid, yield: 58%).

[0634] LCMS (ESI) [M+H] + : 1024.8。

[0635] Step 2: Compound 2.1A (40 mg, 0.04 mmol) was dissolved in a mixed solution of tetrahydrofuran and water (volume ratio: 1 / 1, 4 mL). Potassium peroxymonosulfate (246 mg, 0.4 mmol) was added, and the reaction solution was stirred at room temperature for 5 hours. 30 mL of ethyl acetate was added for dilution. The organic phase was washed three times with water (25 ml×3), dried over anhydrous sodium sulfate, concentrated to remove ethyl acetate to obtain the target compound 2.1B (30 mg, yellow solid, yield: 74%).

[0636] LCMS (ESI) [M+H] + = 1055.9。

[0637] Step 3: Compound 2.1B (30 mg, 0.03 mmol) was dissolved in a mixed solution of trifluoroacetic acid and dichloromethane (volume ratio 1:3, 4 mL), and the reaction solution was stirred at room temperature for 1 hour. Next, it was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by preparative chromatography (0.01% TFA in water, MeCN) to obtain the target compound N-((S)-1-(((S)-6-amino-1-((2-((((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-14-yl)methyl)amino)-2-oxoethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (9 mg, white solid, yield: 33%).

[0638] LCMS (ESI) [M+H] + : 955.8; 1 H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 2H), 8.54 (s, 1H), 8.15 (m, 2H), 7.84 (m, 1H), 7.80 (s, 1H), 7.64 (br s, 3H), 7.53 (s, 1H), 7.26 (s, 1H), 6.51 (s, 1H), 6.30 (d, J = 2.9 Hz, 2H), 5.59 - 5.46 (m, 2H), 5.43 (s, 2H), 4.87 - 4.78 (m, 1H), 4.70 - 4.60 (m, 1H), 4.17 - 4.03 (m, 2H), 3.82 - 3.58 (m, 2H), 3.41 (s, 3H), 2.80 - 2.70 (m, 2H), 2.40 - 2.22 (m, 2H), 1.93 - 1.68 (m, 6H), 1.65 - 1.57 (m, 1H), 1.56 - 1.45 (m, 3H), 1.36 - 1.22 (m, 2H), 0.87 (t, J...

Claims

Claim 1 An antibody-drug conjugate represented by formula XV, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof: 【Chemical 1】 Formula XV (In the formula, Tb is an anti-DLL3 antibody or an antigen-binding fragment thereof, q is the drug-to-antibody ratio, D is a bioactive molecule fragment, L １ is an expansion unit, L ２ does not exist or is a connecting unit, L ３ is Val-AA １ -Gly, Val-AA １ , Ala-AA １ , Gly-AA １ , AA １ -Gly, Ala-AA １ -Gly, Gly-AA １ -Gly, AA １ -Ala-Asn, AA １ selected from, and the structure of the amino acid residue represented by said AA １ is as shown below, 【Chemical Formula 2】 AA １ Among them, R ａ , R ｂ are each independently H, 【Chemical Formula 3】 selected from and R ａ R ｂ are not H at the same time, Alternatively, R ａ and R ｂ together with the carbon atoms to which they are attached form a 4- to 10-membered heterocyclic ring, and said 4- to 10-membered heterocyclic ring is optionally substituted by one or more R ０ groups r, r １ are each independently selected from any integer from 0 to 20, R ｍ１ and R ｎ１ are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and -COOR ｘ１ and R ｘ１ is selected from C1-6 alkyl, Alternatively, R ｍ１ and R ｎ１ together with a nitrogen atom connected thereto form a 4- to 10-membered heterocyclic ring, and the 4- to 10-membered heterocyclic ring is optionally substituted by one or more R ０’ s, R ｚ is selected from C1-6 alkyl, R ０ and R ０’ are each independently selected from C1-6 alkyl, C3-6 cycloalkyl, -NR ｍ２ R ｎ２ and a 4- to 10-membered heterocyclic group optionally substituted by C1-6 alkyl, R ｍ２ and R ｎ２ are each independently selected from H and C1-6 alkyl, L ４ either does not exist or, if it exists, L ４ when L exists, L ４ is 【Chemical 4】 selected from, the first position is L ３ linked to, the second position is linked to D). Claim 2 (1) Tb, the antibody or an antigen-binding fragment thereof includes Fab, Fab’, F(ab’)2, Fd, Fv, dAb, complementarity-determining region fragment, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, monoclonal antibody, bispecific antibody or multispecific antibody; (2) q is selected from any value between 0.1 and 16.0; (3) D has an anti-tumor bioactive molecule fragment; The antibody-drug conjugate according to claim 1, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized by satisfying one or more of the above conditions. Claim 3 (1) q is selected from any value between 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; (2) In the bioactive molecule fragment, the bioactive molecule is a DNA topoisomerase inhibitor or a tubulin inhibitor; (3) L １ is 【Chemical Formula 5】 selected from, each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, C6-10 aryl, 5-10 membered heteroaryl and amide, Rx, Ry are each independently selected from H and C1-4 alkyl, each m is independently selected from 0, 1, 2, 3, 4, 5 and 6, y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 15, each y3 is independently selected from 1, 2 and 3, each y4 is independently selected from 0 and 1, the 1-position is linked to Tb via an S atom, and the 2-position is L ２ or L ３ under the condition of being linked to (4) L ２ does not exist, or exists, and when L ２ exists, L ２ is 【Chemical Formula 6】 selected from, y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 10, each y3 is independently selected from 1 and 2, each y4 is independently selected from 0 and 1, and the first position is L １ connected to, and the second position is L ３ under the condition of being connected to (5) AA １ at the amino acid residue of, R ａ , R ｂ wherein any one of them is H and the other one is [Chemical Formula 7] Selected from the above conditions; Alternatively, AA １ at the amino acid residue of, R ａ and R ｂ together with the carbon atom to which they are attached together form a 5- to 6-membered heterocyclic ring substituted by R ０ under the condition of (6) AA １ at the amino acid residue of, r, r １ are each independently selected from the conditions 0, 1, 2, 3, 4, and 5, (7) AA １ at the amino acid residue of, R ｍ１ , R ｎ１ are each independently H, methyl, ethyl, n-propyl, n-butyl, -COOCH ３ , -COOCH ２ CH ３ , -COOCH ２ CH ２ CH ３ , -COOCH(CH ３ ) ２ , -COOC(CH ３ ) ３ and -COOCH ２ CH ２ CH ２ CH ３ selected from, or, R ｍ１ and R ｎ１ together with the nitrogen atom to which they are linked, form a 5- to 6-membered heterocyclic ring substituted by R ０’ , (8) AA １ In the amino acid residue of, R ｚ is a condition that it is methyl, (9) AA １ In the amino acid residue of, R ０ , R ０’ are each independently C1-6 alkyl, -NR ｍ２ R ｎ２ and a 5- to 6-membered heterocyclic group optionally substituted by C1-6 alkyl, The antibody-drug conjugate according to claim 2, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized by satisfying one or more of the above conditions. Claim 4 (1) q is selected from any value between 0.1, 1, 2, 3, 4, 5, 6, 7, 8; (2) When the bioactive molecule in the bioactive molecule fragment is a DNA topoisomerase inhibitor, the DNA topoisomerase inhibitor is a camptothecin-based bioactive molecule; (3) When the bioactive molecule in the bioactive molecule fragment is a tubulin inhibitor, the tubulin inhibitor is an MMAF-based tubulin inhibitor or an MMAE-based tubulin inhibitor; (4) L １ wherein each Z is independently selected from the group consisting of a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond (5) L １ wherein y1 is 4, 5 or 6, (6) L １ wherein each y2 is independently selected under the condition of any integer between 6 and 10 (7) L ２ does not exist, or exists, and if L ２ exists, then L ２ is 【Chemical 8】 selected from, with the first being L １ linked to, with the second being L ３ linked to, the condition that (8) AA １ In the amino acid residue represented by, R ａ , R ｂ wherein any one of them is H and the other one is, 【Chemical Formula 9】 is selected from, or R ａ and R ｂ together with the carbon atoms linked together thereto, R ０ forms a 5- to 6-membered heterocyclic ring substituted by R ０ The 5- to 6-membered heterocyclic ring substituted by R ０ is a piperidine ring or a piperazine ring substituted by R (9) AA １ In the amino acid residues of, r, r １ are each independently selected from the conditions of 0 and 4, (10) AA １ In the amino acid residue of, R ｍ１ , R ｎ１ are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl, and tert-butoxycarbonyl, or R ｍ１ and R ｎ１ together with the nitrogen atom to which they are attached form a piperidine ring or a piperazine ring substituted by R ０’ under the condition that, (11) AA １ In the amino acid residue of, R ０ is selected from a C1-6 alkyl and a 5-6 membered heterocyclic group substituted by a C1-6 alkyl, and the 5-6 membered heterocyclic group is selected from piperidinyl and piperazinyl, (12) AA １ In the amino acid residue of, R ０’ is C1-6 alkyl and -NR ｍ２ R ｎ２ selected from the conditions, (13) AA １ In the amino acid residue of, R ｍ２ R ｎ２ is a condition where R is methyl, (14) L ４ either does not exist or, if L ４ exists, then L ４ is 【Chemical Formula 10】 where the first position is linked to L ３ under the condition that the second position is linked to D The antibody-drug conjugate according to claim 3, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized by satisfying one or more of the above conditions. Claim 5 (1) The condition that q is selected from any numerical value between 2, 3, 4, 5, 6, 7, and 8, (2) L １ is 【Chemical Formula 11】 selected from, m is selected from 2, 3 and 4, y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 10, each y3 is independently selected from 1 or 2, the 1-position is linked to Tb via an S atom, and the 2-position is L ２ or L ３ under the condition of being linked to (3) L ３ is AA １ , AA １ -Gly, Val-AA １ -Gly, AA １ -Ala-Asn selected conditions, (4) AA １ In the amino acid residue represented by, R ａ and R ｂ together with the carbon atom to which they are attached, form a 5- to 6-membered heterocyclic ring substituted by R ０ , and the 5- to 6-membered heterocyclic ring substituted by R ０ is a piperidine ring substituted by R ０ under the condition that (5) AA １ In the amino acid residues of, r, r １ where one of them is 0 and the other one is 4, (6) AA １ at the amino acid residue of, R ｍ１ , R ｎ１ are each independently selected from H and C1-6 alkyl, or R ｍ１ and R ｎ１ together with the nitrogen atom to which they are attached form a piperidine ring substituted by R ０’ under the condition that, (7) AA １ In the amino acid residue of, R ０ is selected from methyl, ethyl and a 5- to 6-membered heterocyclic group substituted by methyl, and the 5- to 6-membered heterocyclic group is piperidinyl. Preferably, R ０ is methyl, ethyl and 【Chemical 12】 selected from, (8) AA １ In the amino acid residue of, R ０’ is methyl and -NR ｍ２ R ｎ２ a condition selected from, (9) When the bioactive molecule in the bioactive molecule fragment is a DNA topoisomerase inhibitor and the DNA topoisomerase inhibitor is a camptothecin-based bioactive molecule, the condition that the camptothecin-based bioactive molecule is camptothecin, DXD, camptothecin having a modified substituent, or DXD having a modified substituent, An antibody-drug conjugate according to any one of claims 1 to 4, or a stereoisomer of the antibody-drug conjugate, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, characterized by satisfying one or more of the above.

6. (1) The condition that q is selected from any numerical value between 3, 4, 5, 6, 7, and 8, preferably, q is selected from any numerical value between 4, 5, 6, 7, and 8, preferably, q is selected from any numerical value between 6, 7, and 8, (2) L １ is 【Chemical 13】 selected from, the 1-position is linked to Tb via an S atom, and the 2-position is linked to L ２ or L ３ under the condition of being linked to (3) L ２ does not exist or 【Chemical 14】 being, (4) L ３ is AA １ , Val-AA １ -Gly, preferably, L ３ is Val-AA １ -Gly selected conditions, (5) AA １ In the amino acid residue represented by, R ａ and R ｂ together with the carbon atom to which they are attached, 【Chemical Formula 15】 is formed, and the carbon atom of No. 1 is R ａ and R ｂ are carbon atoms connected together to them, condition (6) r, r １ wherein r is 4 and r １ is 0, R ｍ１ , R ｎ１ are each independently selected from H and C1-6 alkyl, r is 0 and r １ is 4, R ｍ１ , R ｎ１ are each independently selected from C1-6 alkyl, or R ｍ１ and R ｎ１ together with the nitrogen atom to which they are attached, 【Chemical 16】 is formed, and the carbon atom No. 1 is R ａ and R ｂ are conditions where they are carbon atoms linked together An antibody-drug conjugate according to any one of claims 1 to 5, or a stereoisomer of the antibody-drug conjugate, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, characterized by satisfying one or more of the above.

7. AA １ The amino acid residue represented by 【Chemical 17】 selected from, Most preferably, AA １ The amino acid residue represented by 【Chemical Formula 18】 An antibody-drug conjugate according to any one of claims 1 to 6, or a stereoisomer of the antibody-drug conjugate, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, characterized by being selected from.

8. L ３ is 【Chemical Formula 19】 selected from, X － is a halide ion, carboxylate ion, sulfate ion, bisulfite ion, and OH － selected from, the 1-position is L １ or L ２ linked to, the 2-position is L ４ or linked to D, Most preferably, L ３ is 【Chemical 20】 selected from, the first position being L １ or L ２ linked to, the second position being L ４ or D, the antibody-drug conjugate according to any one of claims 1 to 7, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof.

9. 【Fig. 21】 The structure is selected from the following structural fragments: 【Table 1】 Preferably, 【Chemical 22】 The structure is selected from the following: 【Table 2-1】 【Table 2-2】 【Table 2-3】 In the formula, the 1-position is linked to Tb and the 2-position is linked to D. An antibody-drug conjugate according to any one of claims 1 to 8, or a stereoisomer of the antibody-drug conjugate, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate.

10. The antibody-drug conjugate has a structure represented by Formula I: 【Chemical 23】 Formula I In the formula, Tb, L1, L2, L3, L4, and q are as defined in any one of claims 1 to 9, R １ 、R ２ each independently represents H, halogen, -OH, optionally substituted C1-6 alkyl or optionally substituted C1-6 alkoxy, or R １ and R ２ form a 5- to 7-membered carbocyclic ring or a 5- to 7-membered heterocyclic ring together with the carbon atoms to which they are attached, and the heterocyclic ring contains one or more of O, S, N, carbonyl, sulfinyl or sulfonyl or any combination thereof, R ３ is selected from H, halogen, -OH, -NH ２ , optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or R ３ and X, together with the carbon atoms linked thereto, form a 5- to 7-membered carbocyclic ring or a 5- to 7-membered heterocyclic ring, said heterocyclic ring containing one or more of O, S, N, carbonyl, sulfinyl or sulfonyl or any combination thereof, or R ３ and R ２ together with the carbon atoms linked thereto form a 5- to 7-membered carbocyclic ring or a 5- to 7-membered heterocyclic ring, and the heterocyclic ring contains one or more of O, S, N, carbonyl, sulfinyl or sulfonyl or any combination thereof W is absent or present, and when W is present, W is -O-, -S-, -NR ４ -, 【Chemical 24】 selected from, the first position is linked to X, and the second position is linked to L ４ or L ３ is linked to X is a direct bond, -O-(CH optionally substituted ２ ) ｎ３ -, -NR ４ -(CH ２ ) ｎ３ -, -S-(CH ２ ) ｎ３ -, carbonyl-(CH ２ ) ｎ３ -, -SO ２ -(CH ２ ) ｎ３ -, -(CH ２ ) ｎ１ -, 【Chemical 25】 Selected from C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic group, the 1-position is linked to the parent ring, and the 2-position is linked to W or L ４ linked thereto, the substituent is selected from one or more C1-4 alkyl, C3-6 cycloalkyl, or forms C3-6 cycloalkyl together with a plurality of C1-4 alkyl and the carbon atoms linked together thereto, Each M is independently selected from a direct bond and -CR ５ａ R ５ｂ - and is selected from R ４ 、 R ５ 、 R ５ａ 、 R ５ｂ 、 R ６ 、 R ７ are each independently selected from H, optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy, and optionally substituted C3-6 cycloalkyl, n, n', n1, n2, n3 are each independently selected from any integer between 0 and 6, The 1-position is linked to the camptothecin mother nucleus, and the 2-position is linked to W or L ４ An antibody-drug conjugate according to any one of claims 1 to 9, or a stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate of said antibody-drug conjugate, characterized in that it is linked thereto.

11. (1) R １ , R ２ are each independently selected from H, halogen, C1-4 alkyl, or R １ and R ２ together with the carbon atoms to which they are attached form a 5- or 6-membered heterocycle, provided that the heterocycle contains one, two or three of O, S or N or any combination thereof (2) R ３ is selected from H, C1-4 alkyl, or ３ R and X together with the carbon atom to which they are attached form a 5- to 6-membered carbon ring (3) W does not exist, or exists. When W exists, W is -O-, -S-, -NR ４ -, 【Chemical 26】 selected from, the first position is linked to X, and the second position is linked to L ４ or L ３ under the condition of being linked to (4) X is optionally substituted - (CH ２ ) ｎ１ -, ​ Selected from C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic groups, the 1-position is linked to the parent ring, and the 2-position is linked to W or L ４ wherein the substituent is selected from one or two of C1-4 alkyl, or forms a C3-6 cycloalkyl together with two C1-4 alkyl and the carbon atoms linked together thereto (5) R ４ , R ５ are each independently selected from the group consisting of H, C1-4 alkyl, and C3-6 cycloalkyl, (6) R ５ａ , R ５ｂ are each independently selected from the group consisting of H and C1-4 alkyl, (7) Each R ７ is independently selected from the conditions of H and C1-4 alkyl, (8) The condition that n is selected from 1, 2, and 3, (9) The condition that n1 is selected from 1, 2, 3, and 4, (10) The condition that n2 is 1, (10) The condition that n3 is 0, An antibody-drug conjugate according to claim 10, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of said antibody-drug conjugate, characterized by satisfying one or more of the following:

12. (1) R １ is selected from H and halogen, and R ２ is selected from H and C1-4 alkyl, or R １ and R ２ together with the carbon atom to which they are attached 【Chemical 28】 forming a condition where the dotted line represents the position where the heterocyclic ring is condensed with the benzene ring, (2) R ３ is H, or R ３ and X, together with the carbon atom to which they are attached 【Chemical formula 29】 forming a condition where the dotted line represents the position where the carbocyclic ring is condensed with the benzene ring and the pyridine ring, (3) W does not exist or exists. When W exists, W is -O-, -S-, -NR ４ -, 【Chemical 30】 selected from, the first position is linked to X, and the second position is linked to L ４ or L ３ linked to (4) Each R ４ is independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl, provided that R ５ is H (5) R ５ａ , R ５ｂ are each independently a condition selected from H and methyl, (6) R ７ is the condition that it is H, (7)The condition that n is 1, An antibody-drug conjugate according to claim 10 or 11, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of said antibody-drug conjugate, characterized by satisfying one or more of the following:

13. (1) R １ is H or F, and R ２ is H or methyl, or R １ and R ２ together with the carbon atoms to which they are attached 【Chemical 31】 forming a condition where the dotted line represents the position where the heterocyclic ring is condensed with the benzene ring, (2) W is -O-, -NR ４ -, and 【Chemical 32】 selected from, the first position is linked to X, and the second position is linked to L ４ or L ３ under the condition of being linked to (3) Each R ４ is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl and cyclopropyl, provided that R ５ is H (4)X is optionally substituted 【Chemical 33】 selected from, the 1-position is linked to the parent ring, and the 2-position is linked to W or L ４ linked to, the substituent is selected from one or two C1-4 alkyls, or forms a C3-6 cycloalkyl together with two C1-4 alkyls and the carbon atoms linked together thereto, the C1-4 alkyl is methyl, and the C3-6 cycloalkyl is cyclopropyl under the condition that An antibody-drug conjugate according to any one of claims 10 to 12, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of said antibody-drug conjugate, characterized by satisfying one or more of the following:

14. (1) W is selected from -O- and -NR ４ -, and (2)X is 【Chemical 34】 selected from, the 1-position is linked to the parent ring, and the 2-position is linked to W or L ４ An antibody-drug conjugate according to any one of claims 10 to 13, or a stereoisomer of said antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, characterized in that it is linked to ４ .

15. When W is absent, X is 【Chemical 35】 selected from, the 1-position is linked to the parent ring, and the 2-position is linked to L ４ linked to, when W is present, X is 、 【Chemical Formula 36】 selected from, with the 1-position linked to the parent ring and the 2-position linked to W, an antibody-drug conjugate according to any one of claims 10 to 14, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of said antibody-drug conjugate.

16. 【Fig. 37】 The structure of is as follows: 【Chemical 38】 【Chemical 39】 【Chemical 40】 【Chemical 41】 【Chemical 42】 【Chemical Formula 43】 【Chemical Formula 44】 (wherein the 1-position is linked to L ４ and when L ４ does not exist, the 1-position is linked to L ３ ), the antibody-drug conjugate according to any one of claims 10 to 15, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

17. The antibody-drug conjugate has a structure represented by formula I-1, formula I-2, or formula I-3, an antibody-drug conjugate according to any one of claims 10 to 16, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of said antibody-drug conjugate: 【Chemical 45】 (wherein Tb, L １ , L ２ , L ３ , L ４ , X, R １ , R ２ , R ３ , R ４ and q are as defined in any one of claims 1 to 16), 【Chemical 46】 (wherein, Tb, L １ , L ２ , L ３ , L ４ , X, R １ , R ２ , R ３ and q are as defined in any one of claims 1 to 16). 【Chemical 47】 (wherein, Tb, L １ , L ２ , L ３ , L ４ , X, R １ , R ２ , R ３ , R ４ , R ５ , n and q are as defined in any one of claims 1 to 16).

18. The antibody-drug conjugate has a structure represented by formula I-1A, formula I-1B, formula I-2A, formula I-2B, formula I-3A, or formula I-3B, an antibody-drug conjugate according to any one of claims 10 to 17, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of said antibody-drug conjugate: 【Chemical 48】 (wherein, Tb, L ２ , L ３ , L ４ , X, R １ , R ２ , R ３ , R ４ and q are as defined in any one of claims 1 to 16), 【Chemical 49】 (wherein, Tb, L ２ , L ３ , L ４ , X, R １ , R ２ , R ３ and q are as defined in any one of claims 1 to 16), 【Chemical Formula 50】 (wherein, Tb, L ２ , L ３ , L ４ , X, R １ , R ２ , R ３ , R ４ and q are as defined in any one of claims 1 to 16).

19. The antibody-drug conjugate has a structure represented by Formula I-A or Formula I-B, and is the antibody-drug conjugate according to any one of claims 10 to 18, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof: 【Chemical 51】 (wherein, Tb, X, R １ , R ２ , R ３ , R ａ , R ｂ and q are as defined in any one of claims 1 to 16). 【Chemical 52】 (wherein, Tb, X, R １ , R ２ , R ３ , R ａ , R ｂ and q are as defined in any one of claims 1 to 16).

20. The antibody-drug conjugate is selected from the following, and is the antibody-drug conjugate according to any one of claims 1 to 19, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof: 【Table 3-1】 【Table 3-2】 【Table 3-3】 【Table 3-4】 【Table 3-5】 【Table 3-6】 【Table 3-7】 【Table 3-8】 【Table 3-9】 【Table 3-10】 【Table 3-11】 【Table 3-12】 【Table 3-13】 【Table 3-14】 【Table 3-15】 【Table 3-16】 【Table 3-17】 【Table 3-18】 【Table 3-19】 。

21. Tb is an anti-DLL3 antibody or an antigen-binding fragment thereof. Preferably, the anti-DLL3 antibody is rovalpituzumab, tarlatamab, 55C11E4-Hz1, 55C11E4-Hz2, 59B10D3-Hz, 10F2F3-Hz, 87F7F10-Hz, C2, 6F11C10-Hz1, and 6F11C10-Hz2 antibody or an antigen-binding fragment thereof. The antibody-drug conjugate is the antibody-drug conjugate according to any one of claims 1 to 20, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

22. The following complementarity-determining regions (CDRs): (a) HCDR1, HCDR2, and HCDR3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO: 1, 2, 5, 6, 7, 10, 11, 14, 15, 18, 19, or 20; and / or (b) LCDR1, LCDR2, and LCDR3 contained in the light-chain variable region VL shown in SEQ ID NO: 3, 4, 8, 9, 12, 13, 16, 17, 21, or 22 An anti-DLL3 antibody or an antigen-binding fragment thereof containing the same.

23. The antibody or an antigen-binding fragment thereof contains the following, and is the anti-DLL3 antibody or an antigen-binding fragment thereof according to claim 22: ((1) VH and / or VL, where it is defined by the Kabat numbering system, (a) The VH contains HCDR1 with a sequence of SEQ ID NO: 23, HCDR2 with a sequence of SEQ ID NO: 24, and HCDR3 with a sequence of SEQ ID NO: 25, and / or The VL contains LCDR1 with a sequence of SEQ ID NO: 26, LCDR2 with a sequence of SEQ ID NO: 27, and LCDR3 with a sequence of SEQ ID NO: 28 (b) The VH includes an HCDR1 with a sequence of SEQ ID NO: 34, an HCDR2 with a sequence of SEQ ID NO: 35 or 36, and an HCDR3 with a sequence of SEQ ID NO: 37, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 38, an LCDR2 with a sequence of SEQ ID NO: 39, and an LCDR3 with a sequence of SEQ ID NO: 40, (c) The VH includes an HCDR1 with a sequence of SEQ ID NO: 46, an HCDR2 with a sequence of SEQ ID NO: 47, and an HCDR3 with a sequence of SEQ ID NO: 48, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 49, an LCDR2 with a sequence of SEQ ID NO: 50, and an LCDR3 with a sequence of SEQ ID NO: 51, (d) The VH includes an HCDR1 with a sequence of SEQ ID NO: 57, an HCDR2 with a sequence of SEQ ID NO: 58, and an HCDR3 with a sequence of SEQ ID NO: 59, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 60, an LCDR2 with a sequence of SEQ ID NO: 61, and an LCDR3 with a sequence of SEQ ID NO: 62, or, (e) The VH includes an HCDR1 with a sequence of SEQ ID NO: 68, an HCDR2 with a sequence of SEQ ID NO: 69 or 70, and an HCDR3 with a sequence of SEQ ID NO: 71, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 72, an LCDR2 with a sequence of SEQ ID NO: 73, and an LCDR3 with a sequence of SEQ ID NO: 74, (2) VH and / or VL, where defined by the IMGT numbering system, (a) The VH includes an HCDR1 with a sequence of SEQ ID NO: 29, an HCDR2 with a sequence of SEQ ID NO: 30, and an HCDR3 with a sequence of SEQ ID NO: 31, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 32, an LCDR2 with a sequence of FAS, and an LCDR3 with a sequence of SEQ ID NO: 28, (b) The VH includes an HCDR1 with a sequence of SEQ ID NO: 41, an HCDR2 with a sequence of SEQ ID NO: 42, and an HCDR3 with a sequence of SEQ ID NO: 43, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 44, an LCDR2 with a sequence of YAS, and an LCDR3 with a sequence of SEQ ID NO: 40, (c) The VH includes an HCDR1 with a sequence of SEQ ID NO: 52, an HCDR2 with a sequence of SEQ ID NO: 53, and an HCDR3 with a sequence of SEQ ID NO: 54, and / or, The VL includes an LCDR1 with a sequence of SEQ ID NO: 55, an LCDR2 with a sequence of YTS, and an LCDR3 with a sequence of SEQ ID NO: 51, (d) The VH comprises an HCDR1 with the sequence of SEQ ID NO: 63, an HCDR2 with the sequence of SEQ ID NO: 64, and an HCDR3 with the sequence of SEQ ID NO: 65, and / or the VL comprises an LCDR1 with the sequence of SEQ ID NO: 66, an LCDR2 with the sequence of WAS, and an LCDR3 with the sequence of SEQ ID NO: 62, or (e) The VH comprises an HCDR1 with the sequence of SEQ ID NO: 75, an HCDR2 with the sequence of SEQ ID NO: 76 or 77, and an HCDR3 with the sequence of SEQ ID NO: 78, and / or the VL comprises an LCDR1 with the sequence of SEQ ID NO: 79, an LCDR2 with the sequence of LAS, and an LCDR3 with the sequence of SEQ ID NO: 74, preferably, the antibody or its antigen-binding fragment binds to human DLL3, monkey DLL3, and / or rat DLL3). **Claim 24** The antibody or its antigen-binding fragment according to claim 22 or 23, comprising the following: ((a) a VH shown in SEQ ID NO: 1 or 2, and / or a VL shown in SEQ ID NO: 3 or 4, (b) a VH shown in SEQ ID NO: 5, 6 or 7, and / or a VL shown in SEQ ID NO: 8 or 9, (c) a VH shown in SEQ ID NO: 10 or 11, and / or a VL shown in SEQ ID NO: 12 or 13, (d) a VH shown in SEQ ID NO: 14 or 15, and / or a VL shown in SEQ ID NO: 16 or 17, (e) a VH shown in SEQ ID NO: 18, 19 or 20, and / or a VL shown in SEQ ID NO: 21 or 22, (f) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one VH among (a) to (e), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one VL among (a), or (g)A VH having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to any one of the VHs in (a)-(e), and / or a VL having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to any one of the VLs in (a), preferably, the substitution is a conservative substitution). **Claim 25** The antibody or antigen-binding fragment thereof according to any one of claims 22-24, wherein the antibody or antigen-binding fragment thereof comprises: ((a) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 3, (b) VH of the sequence shown in SEQ ID NO: 2 and VL of the sequence shown in SEQ ID NO: 4, (c) VH of the sequence shown in SEQ ID NO: 5 and VL of the sequence shown in SEQ ID NO: 8, (d) VH of the sequence shown in SEQ ID NO: 6 and VL of the sequence shown in SEQ ID NO: 9, (e) VH of the sequence shown in SEQ ID NO: 7 and VL of the sequence shown in SEQ ID NO: 9, (f) VH of the sequence shown in SEQ ID NO: 10 and VL of the sequence shown in SEQ ID NO: 12, (g) VH of the sequence shown in SEQ ID NO: 11 and VL of the sequence shown in SEQ ID NO: 13, (h) VH of the sequence shown in SEQ ID NO: 14 and VL of the sequence shown in SEQ ID NO: 16, (i) VH of the sequence shown in SEQ ID NO: 15 and VL of the sequence shown in SEQ ID NO: 17, (j) VH of the sequence shown in SEQ ID NO: 18 and VL of the sequence shown in SEQ ID NO: 21, (k) VH of the sequence shown in SEQ ID NO: 19 and VL of the sequence shown in SEQ ID NO: 22, (l) VH of the sequence shown in SEQ ID NO: 20 and VL of the sequence shown in SEQ ID NO: 22, (m) Compared with the VH and VL described in any one of the groups (a) to (l), the VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and / or the VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, or (n) Compared with the VH and VL described in any one of the groups (a) to (l), the VH has one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof), and / or the VL has one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof), preferably the substitution is a conservative substitution).

26. The antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody or a fully human antibody, Optionally, the antibody or its antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv fragment (e.g., scFv or Fv linked by disulfide bond (dsFv), diabody and multispecific antibody), For example, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetravalent antibody. The anti-DLL3 antibody or its antigen-binding fragment according to any one of claims 22 to 25.

27. The antibody or its antigen-binding fragment is (a) The heavy chain constant region CH of human immunoglobulin or its variant, and / or (b) The light chain constant region CL of human immunoglobulin or its variant, Further comprising the anti-DLL3 antibody or its antigen-binding fragment according to any one of claims 22 to 26: (Here, the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the wild-type sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the wild-type sequence from which it is derived, preferably the substitution is a conservative substitution, preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and / or the light chain constant region is a κ or λ light chain constant region, more preferably, the antibody or its antigen-binding fragment comprises a human IgG1 heavy chain constant region, and / or the antibody or its antigen-binding fragment comprises a human κ light chain constant region).

28. The heavy chain constant region comprises a CH shown in SEQ ID NO: 82 or a variant thereof, the variant having up to 20 conservative amino acid substitutions (e.g., up to 20, up to 15, up to 10 or up to 5 conservative amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions) compared to SEQ ID NO: 82, or having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to SEQ ID NO: 70, and / or The light chain constant region includes CL shown in SEQ ID NO: 83 or a variant thereof, and the variant has conservative substitutions of up to 20 amino acids (for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids, for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) compared to SEQ ID NO: 83, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:

80. Preferably, the anti-DLL3 antibody or an antigen-binding fragment thereof according to claim 27 comprises a heavy chain constant region CH shown in SEQ ID NO: 82 and a light chain constant region CL shown in SEQ ID NO:

83.

29. The antibody is the anti-DLL3 antibody or an antigen-binding fragment thereof according to any one of claims 22 to 28, selected from the following group: ((a) a group comprising a heavy chain of VH shown in SEQ ID NO: 6 or 7 and CH shown in SEQ ID NO: 82, and a light chain of VL shown in SEQ ID NO: 9 and CL shown in SEQ ID NO: 83, preferably comprising a heavy chain shown in SEQ ID NO: 33 and a light chain shown in SEQ ID NO: 56, preferably comprising a heavy chain shown in SEQ ID NO: 45 and a light chain shown in SEQ ID NO: 56, (b) a group comprising a heavy chain of VH shown in SEQ ID NO: 19 or 20 and CH shown in SEQ ID NO: 82, and a light chain of VL shown in SEQ ID NO: 22 and CL shown in SEQ ID NO: 83, preferably comprising a heavy chain shown in SEQ ID NO: 67 and a light chain shown in SEQ ID NO: 81, preferably comprising a heavy chain shown in SEQ ID NO: 80 and a light chain shown in SEQ ID NO: 81).

30. Tb is selected from the anti-DLL3 antibody or an antigen-binding fragment thereof according to any one of claims 22 to 29, and is characterized in that it is the antibody-drug conjugate according to any one of claims 1 to 21, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

31. The antibody-drug conjugate is selected from the following, and is the antibody-drug conjugate according to any one of claims 1 to 21 and 30, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof: 【Chemical 53】 (wherein, Tb ２ is rovalpituzumab, tarlatamab, 55C11E4-Hz1, 55C11E4-Hz2, 59B10D3-Hz, 10F2F3-Hz, 87F7F10-Hz, C2, 6F11C10-Hz1 and 6F11C10-Hz2 antibodies, q is selected from any numerical value between 0.1 and 16.0, preferably any numerical value between 2 and 8, and more preferably, q is 2, 4, 6 or 8).

32. Tb and a drug linker complex represented by formula III 【Chemical Formula 54】 A method for producing an antibody-drug conjugate according to any one of claims 1 to 21, 30, and 31, comprising the step of performing a coupling reaction on Tb and a drug linker complex represented by formula III under appropriate solvents and conditions: (wherein, L １ 、 L ２ 、 L ３ 、 L ４ 、 Tb is as defined in any one of claims 1 to 21, R １ 、R ２ 、R ３ 、X, W are as defined in claims 1 to 21, Lg is a leaving group, and Lg is selected from halogen, sulfonyl, tertiary amine groups (Me3N+, Et3N+), diazonium groups, -OMs, MeSO ２ -, CF ３ SO ３ -).

33. The method includes the step of subjecting Tb and a drug linker complex represented by formula III 【Chemical Formula 55】 to a coupling reaction under appropriate solvents and conditions to form a C-S bond, preferably, the molar ratio of the amount of substance of Tb to the drug linker complex is 1:(1 to 20), preferably, the coupling reaction is carried out in water and / or an organic solvent. Preferably, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol), or any combination thereof. The nitriles may be acetonitrile, and the alcohols may be methanol or ethanol. Preferably, the method further includes the step of purifying the coupling product. Preferably, the coupling product is purified by chromatography. Preferably, the chromatography is one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography. The method of claim 32.

34. An isolated nucleic acid molecule encoding an anti-DLL3 antibody according to any one of claims 22 to 29 or an antigen-binding fragment thereof.

35. A vector comprising the isolated nucleic acid molecule according to claim 34, preferably a cloning vector or an expression vector.

36. A host cell comprising the isolated nucleic acid molecule according to claim 34 or the vector according to claim 35.

37. Culturing the host cell according to claim 36 under conditions that enable the expression of the antibody or its antigen-binding fragment, and recovering the antibody or its antigen-binding fragment from the cultured host cell culture, a method for producing an anti-DLL3 antibody or its antigen-binding fragment according to any one of claims 22 to 29.

38. An antibody-drug conjugate, wherein the antibody therein is an anti-DLL3 antibody or its antigen-binding fragment according to any one of claims 22 to 29, linked to the conjugate moiety via a linker, and the conjugate moiety is a detectable label, radioisotope, fluorescent substance, luminescent substance, coloring substance, enzyme, polyethylene glycol, nuclide, nucleic acid, small molecule payload, polypeptide having binding activity, protein, receptor, ligand, and other active substances that inhibit the growth of tumor cells and promote apoptosis or necrosis of tumor cells, an antibody-drug conjugate.

39. An antibody-drug conjugate according to any one of claims 1 to 21, 30, 31, 38, the antibody-drug conjugate having one, two or a plurality of q values, a group of antibody-drug conjugates.

40. A stereoisomer of the antibody-drug conjugate according to any one of claims 1 to 21, 30, 31, 38, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or an anti-DLL3 antibody or its antigen-binding fragment according to any one of claims 22 to 29, or the nucleic acid molecule according to claim 35, or the vector according to claim 35, or the host cell according to claim 36, or the group of antibody-drug conjugates according to claim 39, preferably further comprising a pharmaceutically acceptable carrier and / or excipient, a pharmaceutical composition.

41. The drug-antibody ratio (DAR) is selected from integers or decimals from 1 to 10, preferably, the drug-antibody ratio (DAR) of the group is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5 and 7.5 to 8.5, preferably, the drug-antibody ratio (DAR) of the group is selected from about 2.0, 4.0, 6.0 and 8.

0. Preferably, the drug-antibody ratio (DAR) of the group is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9, and the group according to claim 39 or the pharmaceutical composition according to claim 40 is characterized in that.

42. In the manufacture of a medicament for treating and / or preventing a disease associated with abnormal cell activity, a stereoisomer of the antibody-drug conjugate according to any one of claims 1 to 21, 30, 31, 38, a prodrug thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, or an anti-DLL3 antibody or an antigen-binding fragment thereof according to any one of claims 22 to 29, or a nucleic acid molecule according to claim 34, or a vector according to claim 35, or a host cell according to claim 36, or a group of antibody-drug conjugates according to claim 39, or the use of the pharmaceutical composition according to claim 40, Optionally, the disease associated with the abnormal cell activity may be a cancer disease, Preferably, the cancer disease is selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial cancer, epidermal cancer, non-Hodgkin lymphoma, central nervous system tumor, prostate cancer or thyroid cancer. The esophageal cancer is, for example, esophageal adenocarcinoma and esophageal squamous cell carcinoma. The lung cancer is, for example, small cell lung cancer, non-small cell lung cancer or lung adenocarcinoma. The central nervous system tumor is, for example, glioma, glioblastoma multiforme, glioma or sarcoma. The colon cancer is, for example, human colon adenocarcinoma. Preferably, the cancer disease is selected from colon cancer, colorectal cancer, colon adenocarcinoma, small cell lung cancer, ovarian cancer. More preferably, the cancer disease is a cancer disease associated with DLL3.

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