CN114106088A

CN114106088A - Bromomethylpyrazine-based drug conjugates and ADCs

Info

Publication number: CN114106088A
Application number: CN202110466799.3A
Authority: CN
Inventors: 郭茂君; 李辉; 李海泓; 郑兴旺; 贾忠全; 王威威; 张扬; 许喆
Original assignee: Levena Biopharma Co ltd
Current assignee: Levena Biopharma Co ltd
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2022-03-01

Abstract

The invention provides bromomethylpyrazine-based drug conjugates and ADCs. The structural formula of the drug conjugate is shown as the formula I: r₁‑L₁‑L₂‑R₂-D, wherein R₁、L₁、L₂、R₂And D is as described herein. The drug conjugate can be connected with an antibody to prepare the antibody drug conjugate.

Description

Bromomethylpyrazine-based drug conjugates and ADCs

Technical Field

The invention relates to bromomethylpyrazine-based drug conjugates and ADCs.

Background

An antibody-drug conjugate (ADC) is prepared by linking a bioactive small molecule drug to a monoclonal antibody through a chemical linkage, and the monoclonal antibody is used as a carrier to target and transport the small molecule drug to a target cell. In the target cells, the drug is released, thereby achieving the therapeutic objective.

ADC drug structures are complex and there are large differences between different ADC drug designs. Even different drugs at the same target point have obvious toxicity differences due to differences in recognition sites, ligation sites, linkers and small molecules ligated.

Disclosure of Invention

In a first aspect, the present invention provides a compound having the structure shown in formula I below:

R₁-L₁-L₂-R₂-D (I)

in the formula (I), the compound is shown in the specification,

R₁selected from any of the following structural formulas:

wherein R is₆Is bromomethyl, i.e. CH₂Br-, o is 1 or 2, the wavy line represents₁The connection position of (a);

L₁is-L_a-L_b-, wherein L_aIs C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene or- (CH)₂)_p-CONH-[CH₂CH₂-O]_q-(CH₂)_p-；L_bIs absent, or is-CO-, -NH-, -COO-or-NH-SO₂Or is:

wherein R 'and R' are each independently- (CH)₂)_p-O-CO-or- [ CH₂CH₂-O]_q-(CH₂)_p-O-CO-, with the dotted line representing L_bAnd L_aPosition of connection, wavy line L_bAnd L₂The location of the connection;

L₂is a peptide that can be recognized and hydrolyzed by enzymes in the pathological environment;

R₂is an optionally substituted alkanediamine group, or is p-aminobenzyloxy or a derivative thereof;

d represents a drug molecule covalently linked to the remainder of the compound of formula I through the oxygen in its own hydroxyl group or the nitrogen in an amine group;

each p is independently an integer from 1 to 4; and

each q is independently an integer from 1 to 8.

In a second aspect, the present invention provides an antibody drug conjugate having a structure represented by formula III below:

Ab-[X-R₁’-L₁-L₂-R₂-D]_m (III)

in the formula:

ab is an antibody or antigen-binding fragment thereof, bound by X and R₁' covalently linked;

x is a free amino group of a thiol group or a lysine group in a cysteine residue of the antibody or the antigen-binding fragment thereof and R₁' group obtained after carrying out coupling reaction;

L₁、L₂、R₂and D is as described herein for any embodiment of formula I;

R₁' is a divalent group obtained by removing 1 or 2 Br from a group of the formula:

in the formula, R₆Is bromomethyl, i.e. CH₂Br-, o is 1 or 2, the wavy line represents₁The connection position of (a);

m is a number between 1 and 8.

In a third aspect, the invention provides a pharmaceutical composition comprising an antibody drug conjugate according to any one of the embodiments herein and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides the use of a compound according to any one of the embodiments herein in the preparation of an antibody drug conjugate, and the use of an antibody drug conjugate according to any one of the embodiments herein in the preparation of a medicament for the treatment or prevention of cancer.

The detailed description and preferred versions of each aspect of the present disclosure are described in detail in various portions of this application.

Drawings

FIG. 1: nuclear magnetic hydrogen spectrum of compound 1.

FIG. 2: nuclear magnetic hydrogen spectrum of compound 2.

FIG. 3: nuclear magnetic hydrogen spectrum of compound 3.

FIG. 4: nuclear magnetic hydrogen spectrum of compound 4.

FIG. 5: nuclear magnetic hydrogen spectrum of compound 7.

FIG. 6: HIC map of LN 401-20-1.

FIG. 7: SEC profile of LN 401-20-1.

FIG. 8: HIC map of LN 401-20-2.

FIG. 9: SEC profile of LN 401-20-2.

FIG. 10: HIC profile of LN 401-89-5.

FIG. 11: SEC profile of LN 401-89-5.

FIG. 12: HIC profile of LN 401-89-6.

FIG. 13: SEC profile of LN 401-89-6.

FIG. 14: HIC profile of LN 401-88-5.

FIG. 15: SEC profile of LN 401-88-5.

FIG. 16: HIC map of LN 401-31-1.

FIG. 17: SEC profile of LN 401-31-1.

FIG. 18: HIC profile of LN 401-31-2.

FIG. 19: SEC profile of LN 401-31-2.

FIG. 20: HIC profile of LN 401-90-5.

FIG. 21: SEC profile of LN 401-90-5.

FIG. 22: HIC profile of LN 401-90-6.

FIG. 23: SEC profile of LN 401-90-6.

FIG. 24: HIC profile of LN 401-90-4.

FIG. 25: SEC profile of LN 401-90-4.

FIG. 26: dose response curves of anti-CD 25 ADC after 5 days of treatment of SU-DHL-1, Karpas 299T, HDLM-2 and L-540 and CD25 negative (-) expressing CD25 (+).

FIG. 27 is a schematic view showing: dose response curves of anti-HER 2 ADC on human tumor cells expressing (+) SKBr-3 and HCC1954 and HER2 negative (-) MDA-MB-468 after 5 days of treatment.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

The invention aims to provide a drug conjugate based on bromomethylpyrazine and an antibody drug conjugate obtained by covalently linking the bromomethylpyrazine and an antibody. Compared with the coupling method of the bis-bromomethylbenzopyrazine of WO2013/173391 and CN104379168B, the coupling method of the invention has different reactivity, the hydrophilicity of the pyrazine part is better than that of the benzopyrazine, and the corresponding synthesis and coupling are simpler and easier to control.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety unless otherwise indicated.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter claimed. In this application, the use of the singular also includes the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the use of "or", "or" means "and/or" unless stated otherwise. Furthermore, the term "comprising" as well as other forms, such as "includes," "including," and "containing," are not limiting and can be open, semi-closed, and closed. In other words, the term also includes the meaning of "consisting essentially of …," or "consisting of ….

Definitions for the terms of the standardization sector can be found in the literature references including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED." Vols.A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods within the skill of the art are employed, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy, and pharmacological methods. Unless a specific definition is set forth, the terms used herein in the pertinent description of analytical chemistry, organic synthetic chemistry, and pharmaceutical chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. For example, the reaction and purification can be carried out using the instructions of the kit from the manufacturer, or according to the methods known in the art or the instructions of the present invention. The techniques and methods described above can generally be practiced according to conventional methods well known in the art, as described in various general and more specific documents referred to and discussed in this specification. In the present specification, groups and substituents thereof may be selected by one skilled in the art to provide stable moieties and compounds.

When a substituent is described by a general formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the formula is written from right to left. For example, -CH₂O-is equivalent to-OCH₂-。

Certain chemical groups defined herein are preceded by a shorthand notation to indicate the total number of carbon atoms present in the group. For example, C1-C6 alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms. The total number of carbon atoms in the shorthand notation excludes carbons that may be present in a substituent of the group.

In addition to the foregoing, the following terms, when used in the specification and claims of this application, have the meanings indicated below, unless otherwise specifically indicated.

In the present application, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

"hydroxy" means an-OH group.

"hydroxyalkyl" refers to an alkyl group as defined below substituted with a hydroxyl group (-OH).

"carbonyl" refers to a-C (═ O) -group.

"nitro" means-NO₂。

"cyano" means-CN.

"amino" means-NH₂。

"carboxyl" means-COOH.

"carbonitrideamino" refers to-CH₂-NH-。

In the present application, "alkyl" as a group or as part of another group refers to a fully saturated straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, having, for example, from 1 to 10 (preferably from 1 to 8, more preferably from 1 to 6) carbon atoms, and being attached to the rest of the molecule by a single bond. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl, decyl, and the like. In some embodiments, the alkyl group is a C1-C4 alkyl group.

In the present application, "alkenyl" as a group or as part of another group refers to a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, containing at least one double bond, having, for example, 2 to 10 (preferably 2 to 8, more preferably 2 to 6, more preferably 2 to 4) carbon atoms, and connected to the rest of the molecule by a single bond. Alkenyl groups include, but are not limited to, ethenyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1, 4-dienyl, and the like.

In the present application, "alkynyl" as a group or as part of another group refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having, for example, from 2 to 10 (preferably from 2 to 8, more preferably from 2 to 6, more preferably from 2 to 4) carbon atoms, and being attached to the rest of the molecule by a single bond. Alkynyl groups include, but are not limited to, ethynyl, propynyl, and the like.

As used herein, "alkylene", "alkenylene" and "alkynylene" refer to monovalent alkyl, alkenyl or alkynyl groups that are divalent radicals formed by the loss of 1 hydrogen, such as-CH₂-、-CH₂CH₂-、-CH₂CH＝CH-、-CH₂C≡CCH₂-and the like.

In this application, "cycloalkyl" as a group or as part of another group refers to a stable nonaromatic monocyclic hydrocarbon radical consisting only of carbon and hydrogen atoms. The number of ring carbon atoms of the cycloalkyl group is usually 3 to 8. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

In this application, "heterocyclyl" as a group or part of another group refers to a stable 5-to 10-membered non-aromatic cyclic group consisting of 2 to 14 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically indicated in the specification, a heterocyclic group may be a monocyclic or bicyclic ring system. The heterocyclyl group may be partially or fully saturated. The heterocyclic group may be attached to the rest of the molecule via a carbon atom or a heteroatom and by a single bond. Examples of heterocyclyl groups include, but are not limited to: azetidinyl, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxolanyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, quinolizinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, dihydroindolyl, octahydroindolyl, octahydroisoindolyl, pyrazolidinyl, and the like.

In this application, "aryl" as a group or as part of another group means a conjugated hydrocarbon ring system group having 6 to 14 carbon atoms (preferably having 6 to 10 carbon atoms, e.g., 6, 7, 8, 9, or 10 carbon atoms). The aryl group may be a monocyclic or bicyclic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

In the present application, "aralkyl" refers to an alkyl group as defined above substituted with an aryl group as defined above.

In this application, "heteroaryl" as a group or part of another group means a 5-to 16-membered, preferably 5-to 10-membered conjugated ring system group having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur in the ring. Unless otherwise specifically indicated in the specification, a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or higher ring system. Examples of heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindolyl, purinyl, quinolyl, isoquinolyl, diazonaphthyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, pyrrolopyridyl, benzopyrolyl, benzothienyl, oxazolyl, cinnolinyl, quinazolinyl, thiophenyl, indolizinyl, orthophenanthrolinyl, isoxazolyl, phenoxazinyl, phenothiazinyl, and the like.

In the present application, the groups may be optionally substituted. "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted aryl" means that the aryl group is substituted or unsubstituted, and the description includes both substituted and unsubstituted aryl groups. The "optional" substituents described in the claims and the description section of this invention include, but are not limited to, one or more of alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloepoxy, haloalkenyl, haloalkynyl, cyano, cycloalkyl-O-, nitro, amino, optionally substituted amino, alkoxy, cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocyclyl. In preferred embodiments, substituents include, but are not limited to, one or more of C1-C6 alkyl, haloalkyl, cycloalkyl, amino, C1-C6 alkyl substituted amino, halogen, cyano, C3-C8 cycloalkyl-O-, aryl, and heteroaryl. In the present application, when substituted, the number of substituents may be 1 to 5, such as 1 to 3, depending on the structure of the group being substituted. For example, phenyl may be substituted with 1 to 3 substituents selected from C1-C6 alkoxy, C3-C8 cycloalkyl-O-, halo and amino.

It will also be appreciated by those skilled in the art that in the processes described below, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable hydroxy protecting groups include trialkylsilyl or diarylalkylsilyl groups (e.g.tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butyloxycarbonyl, benzyloxycarbonyl and the like. Suitable thiol protecting groups include-C (O) -R "(where R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl and the like. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.

Protecting groups may be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting Groups is described in detail in Greene, T.W. and P.G.M.Wuts, Protective Groups in organic Synthesis, (1999),4th Ed., Wiley. In some embodiments, the protecting group herein is an amino protecting group, such as Boc (tert-butyloxycarbonyl). The protecting group may also be a polymeric resin.

I. Drug conjugates

Provided herein is a drug modified or conjugated with bromomethylpyrazine derivatives. Specifically, the drug conjugate of the present invention has a structure represented by the following formula I:

R₁-L₁-L₂-R₂-D (I)

in the formula (I), the compound is shown in the specification,

R₁a group which is reactive with a thiol group in a cysteine residue or a free amino group of lysine in a polypeptide to thereby attach the drug conjugate of formula I to the polypeptide;

each p is independently an integer from 1 to 4; and

each q is independently an integer from 1 to 8.

Preferably, in formula I, R₁Selected from any of the following structural formulas:

in the formula, R₆Is bromomethyl, i.e. CH₂Br-, o is 1 or 2, the wavy line represents₁The connection position of (2).

Further preferably, R₁Selected from the following groups:

wherein the wavy line represents the relationship L₁The location of the connection.

Preferably, in formula I, L_aIs C1-C6 alkylene or- (CH)₂)_p-CONH-[CH₂CH₂-O]_q-(CH₂)_p-. In a specific embodiment, L_aIs- (CH)₂)₃-CONH-[(CH₂)₂-O]₄-(CH₂)₂-。

Preferably, in formula I, L₁Through L_bAnd L₂Covalent attachment; preferably, L_bAnd L₂Form an amide group therebetween. Preferably, L_bis-CO-or

In some preferred embodimentsIn the embodiment, L₁Is: - (CH)₂)₃-CONH-[(CH₂)₂-O]₄-(CH₂)₂-CO-、*-(CH₂)₃-CO-、*-(CH₂)₅-CO-or- (CH)₂)₃-CO-N(-CH₂CH₂-OCO-)₂Indicates with R₁The connection position of (2).

In formula I, preferably, L₂The peptide is dipeptide, tripeptide, tetrapeptide or pentapeptide. In one or more embodiments, the amino acid residues in the peptide are selected from the group consisting of: one or more of valine, ornithine, lysine, serine, glycine, and citrulline. In one or more embodiments, the peptide is Val-Cit, Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Gly-Gly-Val-Lys, Gly-Gly, or Gly-Gly-Val-Lys. In a preferred embodiment, the peptide is Val-Cit, Gly-Gly or Val-Lys.

Preferably, one or more amino acid residues in the peptide are modified with phosphorylcholine groups. Exemplary phosphorylcholine group-modified amino acids are shown in formula II below:

R₈-L₃-N(R₉)-R₁₀ (II)

in the formula, R₈Is a phosphorylcholine group; n represents a nitrogen atom on an amino acid molecule; r₉Is H, C1-C4 alkyl or-L₃-a phosphorylcholine group; each L₃Independently is a linking group; r₁₀Represents the remainder of the amino acid molecule other than the N atom shown, said remainder being optionally protected by a protecting group.

The phosphorylcholine group may be represented by the formula:

wherein the wavy line represents the phosphorylcholine group and L₃The location of the connection.

Each L₃Independently is represented by-L_c-L_d-, wherein L_cSelected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; l is_dIs absent, or is carbonyl, ester (-COO-), -O-SO₂-or-NH-SO₂-; preferably, L_dAbsent or as a carbonyl group; preferably, L₃Through L_d(ii) is covalently linked to the N; preferably, L_dAnd the N shown forms an amide group or a carbonitrideamino group (-CH)₂-NH-); preferably, each L₃Independently is a C1-C6 alkylene group or a C1-C6 alkylenecarbonyl group.

R₁₀The amino acid molecule is selected from: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, pyrrolysine and ornithine; preferably, the amino acid molecule is selected from: lysine, ornithine, valine, tyrosine, glutamine, alanine, arginine, asparagine, leucine, isoleucine, threonine, serine, phenylalanine, valine, and glycine; more preferably, the amino acid molecule is selected from: valine, ornithine, lysine, serine, glycine and citrulline. More preferably, the amino acid molecule is selected from: ornithine, lysine, serine and glycine.

The "N" in formula II is not a ring nitrogen atom on an aryl, heteroaryl or heterocyclyl group in the amino acid molecule. The "N" may be the nitrogen atom in the amino acid molecule used to form the peptide bond, or may be the nitrogen atom on the free amino group present in the amino acid molecule.

In some preferred embodiments, the peptide is Val-Lys, wherein the nitrogen of the free amino group on the side chain of Lys is modified with a phosphorylcholine group. Further preferably, in said phosphorylcholine-modified Lys, L₃is-CH₂-CO-，R₉Is hydrogen, L₃Forming an amide group with the nitrogen of the free amino group on the Lys side chain.

For more details on phosphorylcholine-modified amino acids and peptides containing phosphorylcholine-modified amino acids that can be recognized and hydrolyzed by enzymes in the pathological environment, reference may be made to CN 202110303888.6, the entire content of which is incorporated herein by reference. Preferably, the phosphorylcholine-modified amino acid may be a compound represented by any one of LN001 to LN010 and LN012 to LN025 disclosed in CN 202110303888.6; in the peptide which is recognized and hydrolyzed by an enzyme in a pathological environment, the amino acid residue may be selected from: one or more of valine, ornithine, lysine, serine, glycine, and citrulline; the phosphorylcholine group-modified amino acid residue in the peptide is selected from: one or more of ornithine, lysine, serine and glycine; preferably, the peptide is selected from one or more of Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Gly-Gly-Val-Lys and Gly-Gly-Val-Lys, preferably one or more of ornithine, lysine, serine and glycine in the peptide is modified by a phosphorylcholine group.

Preferably, in formula I, the enzyme is a proteolytic enzyme, a protease, or a peptidase. In one or more embodiments, the enzyme is selected from the group consisting of: one or more of a cysteine protease, an aspartic protease, a glutamic protease, a threonine protease, a gelatinase, a metalloprotease, and an asparagine peptide cleaving enzyme. Particularly preferably, the enzyme is asparagine endopeptidase (Legumain).

Preferably, R of formula I₂Wherein the p-aminobenzyloxy derivative has the following structure:

in the formula (I), the compound is shown in the specification,

R₃is H, halogen, C1-C4 alkyl, C3-C8 cycloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C8 cycloalkenyl, C6-14 aryl or C6-14 aryl C1-4 alkyl; preferably H or C1-C4 alkyl, more preferably H;

R₄is H or C1-C4 alkyl; preferably H, methyl or ethyl;

R₅is absent fromOr is-NR₆-(CH₂)_s-NR₇-CO-, wherein s is 2 or 3, R₆And R₇Each independently is H or C1-C4 alkyl, which indicates the position of attachment to D;

the dotted line represents R₂And L₂The position of the connection, the wavy line, R₂The position of the connection with D.

R₂The optionally substituted alkanediamine group may be represented by-NR₆-(CH₂)_s-NR₇-, where s is 2 or 3, R₆And R₇Each independently is H or C1-C4 alkyl.

In general, L₂And R₂Are connected through amide groups.

In formula I, preferably, the drug is selected from: MMAE, Duo-5, DXD, maytansine, PNU-159682(CAS No.:202350-68-3), irinotecan, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, fluorouracil, doxifluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, daunorubicin, epirubicin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin. Preferably the drug is MMAE, maytansine, PNU-159682, camptothecin or 7-ethyl-10-hydroxycamptothecin. It will be appreciated that when the drug is covalently linked to the remainder of formula I through the nitrogen of its amine group or the oxygen of its hydroxyl group, the attachment site on the drug does not affect the biological function and activity of the drug itself. For example, the remainder of formula I may be covalently linked via an amino or hydroxyl group at a position remote from the active center, according to the known active center of these drugs.

Preferably, it is capable of reacting with R₁The polypeptides that are covalently linked are polypeptides having the desired biological activity, including but not limited to enzymes and antibodies, particularly the various antibodies described herein. The antibody is preferably a monoclonal antibody.

Preferably, the compound of formula I is a compound 1-10 herein.

II.ADC

The drug conjugates of the present invention may be used to prepare Antibody Drug Conjugates (ADCs). Specifically, the present invention provides an antibody drug conjugate represented by the following formula III:

Ab-[X-R₁’-L₁-L₂-R₂-D]_m (III)

in the formula:

L₁、L₂、R₂and D is as described in any one of the embodiments above;

R₁' is a divalent or trivalent radical obtained by removing 1 or 2 Br from a group of the formula:

m represents Drug-to-Antibody Ratio (DAR) and is a value between 1 and 8.

In a preferred embodiment, R₁' is:

wherein the dotted line represents R₁' position of attachment to Ab, wavy line means R₁' and L₁The location of the connection;

L₁through L_aAnd R₁' covalent attachment, via L_bAnd L₂Covalently linked, wherein L_aIs C1-C6 alkylene or- (CH)₂)_p-CONH-[CH₂CH₂-O]_q-(CH₂)_p-，L_bis-CO-or

Wherein R 'and R' are each independently- (CH)₂)_p-O-CO-or- [ CH₂CH₂-O]_q-(CH₂)_p-O-CO-, with the dotted line representing L_bAnd L_aPosition of connection, wavy line L_bAnd L₂The position of the connection, p is an integer of 1 to 4, and q is an integer of 1 to 8;

L₂is Val-Cit, Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Gly-Gly-Val-Lys, Gly-Gly or Gly-Gly-Val-Lys; preferably Val-Cit; wherein L is_bAnd L₂Are connected through amido; preferably, L₂One or two amino acid residues in (a) are phosphorylcholine-modified amino acid residues described herein;

R₂has the following structure:

in the formula, R₃Is H or C1-C4 alkyl; r₄Is H or C1-C4 alkyl; r₅Is absent; the dotted line represents R₂And L₂The position of the connection, the wavy line, R₂The position of the linkage to D; wherein L is₂And R₂Through an amide group;

d is selected from the following medicines: MMAE, Duo-5, DXD, maytansine, PNU-159682(CAS No.:202350-68-3), irinotecan, camptothecin, 7-ethyl-10-hydroxycamptothecin (SN38), topotecan, fluorouracil, doxifluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, daunorubicin, epirubicin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin; preferably MMAE, maytansine, PNU-159682, camptothecin or 7-ethyl-10-hydroxycamptothecin.

Herein, "antibody" has a meaning well known in the art and includes any form of antibody having the desired biological activity, such as monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, camelized single domain antibodies, and the like.

By "monoclonal antibody" is meant an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.

"full-length antibody" refers to an immunoglobulin molecule that, when naturally occurring, comprises at least four peptide chains: the two heavy (H) chains and the two light (L) chains are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

An "antigen-binding fragment" of an antibody includes a fragment or derivative of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity. The term "binding compound" refers to both antibodies and binding fragments thereof.

"Single chain Fv" or "scFv" antibodies refer to antibody fragments that comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Fv polypeptides also typically comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The 2 VH regions of the bivalent domain antibody may target the same or different antigens. A "bivalent antibody" comprises 2 antigen binding sites. In some cases, 2 binding sites have the same antigen specificity. However, bivalent antibodies may be bispecific. "diabodies" refer to small antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is short enough not to allow pairing between two domains of the same strand, this domain is forced to pair with the complementary domain of the other strand and two antigen binding sites are created.

Preferably, in formula III, the antibody binds to R through the free amino group of the thiol group of cysteine or lysine contained in the antibody₁' connected to each other. Thus, X may be an-S-group or an-NH-group.

Preferably, in formula III, the antibody can be any of a variety of antibodies or antigen-binding fragments thereof known in the art to have the desired biological activity. For example, the antibody or functional fragment thereof may be selected from: anti-Her 2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD 20 antibody, anti-CD 33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNF α antibody, anti-CD 28 antibody, anti-4-1 BB antibody, anti-OX 40 antibody, anti-GITR antibody, anti-CD 27 antibody, anti-b-CD 40 antibody, or anti-ICOS antibody, anti-CD 25 antibody, anti-CD 30 antibody, anti-CD 3 antibody, anti-CD 22 antibody, anti-CCR 4 antibody, anti-CD 38 antibody, anti-CD 52 antibody, anti-complement C5 antibody, anti-RSV F protein, anti-GD 2 antibody, anti-CD GITR antibody, anti-receptor lib/ICla II glycoprotein, anti-ICOS antibody, anti-IL 2R antibody, anti-LAG 3 antibody, anti-Integrin α 4 antibody, anti-lgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF 7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR 2 antibody, anti-VISTA antibody, anti-C-Met antibody, anti-BCMA antibody, anti-Claudin 18 antibody, anti-Nectin-4 antibody, anti-CD 79b antibody, and anti-Trop 2 antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-CD 25 antibody or anti-binding fragment thereof, or an anti-Her 2 antibody or antigen-binding fragment thereof.

The number of moieties conjugated to the antibody in formula III (DAR, i.e.the number of m) may be a number between 1 and 8, which number is generally related to the number of disulfide bonds or lysine free amino groups in the antibody, as well as to the groups involved in the conjugation reaction and the conditions of the conjugation reaction. In some embodiments, m has a value between 3 and 6. The Drug Antibody Ratio (DAR) of the ADC can be determined by one skilled in the art using methods known in the art, such as LC-MS and UV/VIS.

In a preferred embodiment, the compound of formula III is an ADC represented by LN401-20-1, LN401-20-2, LN401-89-5, LN401-89-6, LN401-88-5, LN401-31-1, LN401-31-2, LN401-90-5, LN401-90-6, and LN401-90-4 of the present invention.

Preparation method

The compounds of formulae I, II and III herein can be prepared by methods described in the examples herein. The corresponding preparation process is described below by way of example.

A compound of formula I

In the present invention, L having an ester group can be introduced by alkylation with oxygen (O) starting from appropriately substituted methylpyrazinyl alcohol or dimethylpyrazinol₁Then bromine is introduced into the methyl group of methylpyrazine, and R with carboxyl is generated after degreasing₁L₁Intermediate, and further L₂R₂D or an analogue thereof to give a compound of formula I.

Preparation of ADC

The ADC of the present invention can be obtained by reducing an antibody, mixing the reduced antibody with the compound of formula I of the present invention, standing at room temperature for a sufficient time, and then separating and purifying. Methods for reducing antibodies are well known in the art. The purpose of the reduction includes reduction of disulfide bonds in the antibody to sulfhydryl groups. Suitable reducing agents include TCEP and the like. The reducing agent and the buffer system can be selected according to different antibody types.

Pharmaceutical compositions, uses and methods of treatment and prevention of disease

The present application provides a pharmaceutical composition comprising an effective amount of an ADC according to any of the embodiments of the present application and a pharmaceutically acceptable carrier. The present application also provides a method of treating or preventing a disease comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of an ADC of the present application or a pharmaceutical composition thereof. Also provided is the use of a compound of formula I as described herein for the preparation of an ADC, and the use of an ADC as described in any of the embodiments herein for the preparation of a medicament for the treatment or prevention of a disease.

Herein, "preventing" and "prevention" includes reducing the likelihood of occurrence or worsening of a disease or disorder in a subject; the term also includes: prevention of a disease or condition occurs in a mammal, particularly when such mammal is susceptible to the disease or condition, but has not yet been diagnosed as having the disease or condition. "treatment" and other similar synonyms include the following meanings: (i) inhibiting the disease or disorder, i.e., arresting its development; (ii) alleviating the disease or condition, i.e., causing regression of the state of the disease or condition; or (iii) alleviating the symptoms caused by the disease or disorder.

Herein, "administering" refers to a method capable of delivering a compound or composition to a desired site for biological action. Methods of administration well known in the art may be used in the present invention. These methods include, but are not limited to, oral routes, via the duodenal route, parenteral injection (including intrapulmonary, intranasal, intrathecal, intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Administration techniques useful for The compounds and methods described herein are well known to those skilled in The art, for example, in Goodman and Gilman, The pharmaceutical Basis of Therapeutics, current ed.; pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.

Herein, an effective amount includes both a therapeutically effective amount and a prophylactically effective amount, and refers to an amount of an ADC of the present application that, when administered to a subject, either alone or in combination with other therapeutic agents, is effective to prevent or ameliorate one or more symptoms of a disease or condition, or the development of the disease or condition. A therapeutically effective amount also refers to an amount of ADC sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition. The specific effective amount will depend upon a variety of factors, such as the particular disease to be treated, the physical condition of the patient, such as weight, age and sex, the duration of the treatment, the treatment being co-administered (if any), and the specific formulation employed.

A pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical formulation or composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersing agents, suspending agents, stabilizing agents, isotonicity agents, solvents, or emulsifiers. The pharmaceutical compositions of the present application can be formulated using pharmaceutically acceptable carriers well known in the art.

The pharmaceutical compositions of the present application may be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, and the like, and may be administered by any suitable route to achieve the intended purpose. For example, it may be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally or externally. The dosage of the drug may depend on the age, health and weight of the patient, concurrent treatment, and frequency of treatment, etc. The pharmaceutical compositions of the present application can be administered to any subject in need thereof, e.g., a mammal, particularly a human.

Depending on the antibody or antigen-binding fragment thereof in the ADC and the drug, the pharmaceutical composition of the present application may be used to treat or prevent the corresponding disease. For example, when the drug is an anticancer drug, the pharmaceutical composition of the present application can be used to treat cancers treatable by the anticancer drug, including but not limited to bladder, brain, breast, cervix, colon-rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis, and blood, among others. Specifically, the cancer includes bladder cancer, kidney cancer, urinary tract cancer, chorioepithelial cancer, glioblastoma multiforme, melanoma, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and leukemia. Preferably, the ADCs of the present application are used in the treatment or prevention of solid and hematological tumors, such as lung, chorioepithelial, kidney, urinary, colon, rectal, prostate, glioblastoma multiforme, ovarian, pancreatic, breast, melanoma, liver, bladder, gastric or esophageal cancer.

In some embodiments, the drug molecule in the ADC described herein is MMAE, maytansine, PNU-159682, camptothecin, or SN38, and the disease is an indication of MMAE, maytansine, PNU-159682, camptothecin, or SN38, including gastric cancer, esophageal cancer, cardiac cancer, colon cancer, rectal cancer, primary liver cancer, acute and chronic myelogenous leukemias, chorioepithelial cancer, lung cancer, and bladder cancer.

The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to be limiting of the scope of the invention. Test methods without specific conditions noted in the following examples are generally performed according to conventional conditions or according to conditions recommended by the manufacturer. All percentages, ratios, or parts are by weight unless otherwise specified. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only. The materials and reagents used in the examples are commercially available, for example, anti-CD 25 antibodies are available from Zhang Jiang Biotechnology, Inc., Shanghai.

Example 1: synthesis of Compound 1

Synthesis reaction formula of Compound 1

Step 1: synthesis of Compounds 1-3

Compound 1-1(4.0g, 36.33mmol, 1.0eq) was dissolved in 100mL DMF, NaI (5.4g,36.33mmol, 1.0eq) was added, compound 1-2(9.7g, 43.59mmol, 1.2eq) was added, and K was added portionwise with stirring₂CO₃(7.5g,54.55mol,1.5eq), after the addition was complete, the temperature was raised to 60-65 ℃ for 2 hours, the reaction was monitored by HPLC and assisted by LC-MS, indicating that Compound 1-1 was essentially absent.

The reaction mixture was cooled to room temperature, 400mL of EA was added to the reaction mixture, and the organic phase was washed with 150mL of a saturated ammonium chloride solution and 100mL of water 3 in this order. Reuse of the organic layer with anhydrous Na₂SO₄After drying, concentration gave about 15g of brown oil, which was purified by reverse phase preparation at medium pressure to give 4.0g of compound 1-3 as brown oil in 44% yield. (additional N-alkylated by-product 1-3-BP 3.0 g).

¹H NMR(400MHz,CDCl₃)δ:8.13(s,1H),7.92(s,1H),4.33(dd,J＝8Hz,4Hz,2H)2.47(s,3H),2.41(t,J＝8Hz,2H),2.06-2.11(m,2H),1.46(s,9H)。

MS(+)::253.46。

The preparation method comprises the following steps:

the instrument comprises the following steps: medium pressure chromatography on Biotage Isolera One;

preparing a column: trityl SW220 topical C18, 20-45 μm, 120A;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

Time (minutes)	Gradient (B%)
		0-10	20％
10-35	20％-43％
		35-60	43％
60-75	43％-95％
		75-100	95％
100-115	95％-20％

Step 2: synthesis of Compounds 1-4

Dissolve Compound 1-3(3.0g,11.85mmol,1.0eq) in CCl₄Adding NBS (2.1g,11.85mmol and 1.0eq) into 50mL, adding AIBN (1.9g,11.85mmol and 1.0eq) under the protection of nitrogen, replacing nitrogen for 3 times after the addition is finished, heating to 80 ℃ for reaction, after the reaction is carried out for 2 hours, detecting the reaction by TLC, LC-MS and HPLC, and treating the rest of raw materials with target products.

Cooling the reaction solution to about 30 ℃ of room temperature, concentrating the reaction solution in vacuum to obtain an oily crude product, adding 30mL of acetonitrile and 15mL of water, stirring for a while until the acetonitrile and the water are dissolved, preparing the mixture in a medium-pressure reverse phase manner, and freeze-drying to obtain 2.1g of a compound 1-4 dark green oily product with the yield of 54%.

¹H NMR(400MHz,CDCl₃)δ:8.20(s,1H),8.17(s,1H)，4.56(s,2H),4.38(dd,J＝8Hz,4Hz,2H),2.42(t,J＝8Hz,2H),2.10(t,8Hz,2H),1.47(s,9H)。

MS(+)::331.88。

The preparation method comprises the following steps:

preparing a column: trityl SW220 topical C18, 20-45 μm, 120A;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

Time (minutes)	Gradient (B%)
		0-15	20％
15-55	20％-43％
		55-90	43％
90-95	43％-95％
		95-110	95％
110-115	95％-20％

And step 3: synthesis of Compounds 1-5

Dissolving the compound 1-4(1.8g, 5.43mmol, 1.0eq) in 10mL of DCM, slowly adding 2mL of trifluoroacetic acid dropwise at the temperature of 10-15 ℃ under ice bath, reacting for 3 hours at the temperature of 20-25 ℃ in water bath, detecting by LC-MS and HPLC to obtain 1.4% of the compound 1-4 after the reaction is finished, concentrating the reaction solution in vacuum, carrying the residue with 20mL of DCM 3, adding 100mL of dichloromethane into the residue, washing with 15mL of water 4, drying the organic phase with anhydrous sodium sulfate, and concentrating to obtain 1.5g of gray solid with the yield of 100%.

¹H NMR(400MHz,CDCl₃)δ:8.21(s,1H),8.19(s,1H)，4.57(s,2H),4.42(dd,J＝8Hz,4Hz,2H),2.58(dd,J＝8Hz,4Hz,2H),2.17(dd,J＝8Hz,4Hz,2H).

MS(+)::276.07。

And 4, step 4: synthesis of Compound 1

Compounds 1-5(50.0mg,0.1817mmol,1.0eq) and compounds 1-6(200.0mg,0.1817mmol,1.0eq) were added to a single vial, dissolved in 8mL THF, clarified, and DIC (35.0mg,0.2727mmol,1.5eq) added. After the addition is finished, reacting for 1 hour at the room temperature of 25-30 ℃, detecting the reaction by LC-MS and HPLC, generating a target product, and processing and reacting the residual raw materials. The reaction solution was concentrated in vacuo to give an oil, which was then added with 10mL acetonitrile and 10mL water, and the mixture was prepared under high pressure and lyophilized to give 90mg of Compound 1 as a white solid with a yield of 20%. MS (+): 1381.34. Process for preparation of Compound 1¹H NMR is shown in FIG. 1.

The preparation method comprises the following steps:

the instrument comprises the following steps: waters2487 high pressure chromatography;

preparing a column: gemini 5 μm NX-C18, 150 x 50;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

Time (minutes)	Gradient (B%)
		0-30	30％-90％
30-31	95％
		31-34	95

Example 2: synthesis of Compound 2

Step 1: synthesis of Compound 2-2

Compound 2-1(4.0g, 36.33mmol, 1.0eq) was dissolved in 100mL of DMF, NaI (5.4g,36.33mmol, 1.0eq) and compound 1-2(9.7g, 43.59mmol, 1.2eq) were added, and K was added portionwise with stirring₂CO₃(7.5g,54.55mol,1.5eq), after the addition was complete, the temperature was raised to 60-65 ℃ for 4 hours, the reaction was monitored by HPLC, and LC-MS assisted indicated that Compound 2-1 remained, but the reaction did not proceed any more, and was stoppedAnd (5) reaction treatment.

Cooling the reaction solution to room temperature, adding 200mL of EA into the reaction solution, pouring water into about 300mL of ice water, separating the solution, extracting the aqueous phase with 100mL of EA by 3, combining the organic phases, and washing the organic phase with 100mL of water by 2. Reuse of the organic layer with anhydrous Na₂SO₄After drying, the mixture was concentrated to give about 12g of a brown oily substance, and the brown oily substance compound 2-2(1.3g, yield 13% was purified by reverse phase at medium pressure to give 3.2g of N-alkylated by-product 2-3-BP.

¹H NMR(400MHz,CDCl₃)δ:8.01(d,4HZ,1H),7.97(s,1H)，4.39(dd,J＝8Hz,4Hz,2H),2.53(s,3H),2.44(t,J＝8Hz,2H),2.12(t,J＝8Hz,2H),1.47(s,9H).

MS(+)::253.40。

The preparation method comprises the following steps:

preparing a column: trityl SW120 topical C18, 20-45 μm, 120A;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 30 ml/min.

Time (minutes)	Gradient (B%)
		0-10	30％
10-15	30％-45％
		15-25	45％
25-30	45-55
		30-45	55％-65％
45-75	65％
		75-80	65％-85％
80-85	85％

Step 2: synthesis of Compounds 2-3

Compound 2-2(1.0g,3.95mmol,1.0eq) was dissolved in CCl₄Adding NBS (0.7g,3.95mmol and 1.0eq) into 15mL of the mixture, adding AIBN (0.7g,3.95mmol and 1.0eq) under the protection of nitrogen, replacing the nitrogen for 3 times again after the addition is finished, heating to 80 ℃ for reaction, after the reaction is carried out for 4 hours, detecting the reaction by LC-MS and HPLC, wherein a target product exists, the raw material remains, the reaction does not proceed any more, and treating the reaction.

Cooling the reaction liquid to about 30 ℃ at room temperature, concentrating the reaction liquid in vacuum to obtain a crude product, adding 15mL of acetonitrile and 5mL of water, stirring for a moment until the acetonitrile and the water are dissolved, carrying out medium-pressure reverse phase preparation, and freeze-drying to obtain 0.7g of a compound 2-3 which is purple oily matter with the yield of 54%.

¹H NMR(400MHz,CDCl₃)δ:8.11(s,1H),8.09(s,1H)，4.61(s,2H),4.47(dd,J＝8Hz,4Hz,2H),2.48(dd,J＝4Hz,8Hz,2H),2.14(dd,J＝8Hz,4Hz,2H)，1.47(s,9H)。

MS(+)::276.07。

The preparation method comprises the following steps:

preparing a column: trityl SW120 topical C18, 20-45 μm, 120A;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 30 ml/min.

Time (minutes)	Gradient (B%)
		0-10	30％
10-12	30％-45％
		12-22	45％
22-24	45-55
		24-40	55％
40-44	55％-65％
		44-74	65％
74-76	65％-85％
		76-86	85％

And step 3: synthesis of Compounds 2-4

Dissolving the compound 2-3(0.7g, 2.11mmol, 1.0eq) in 20mL of DCM, slowly adding 4mL of trifluoroacetic acid dropwise at the temperature of 10-15 ℃ under ice bath, after the addition is finished, reacting for 5 hours in water bath at 20-25 ℃, detecting by LC-MS and HPLC, and finishing the reaction, wherein about 1.5% of raw materials are remained.

The reaction was concentrated in vacuo, the residue was taken up in 15mL _ 3 of DCM, 50mL _ dichloromethane was added to the residue, washed with 10mL _ 3 of water, the organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 2-4 as a violet solid 0.5g, 86% yield.

¹H NMR(400MHz,CDCl₃)δ:8.125(d,J＝4Hz,1H),8.08(s,1H)，4.60(s,2H),4.51(d,J＝4Hz,2H),2.48(d,J＝8Hz,2H),2.22(d,J＝8Hz,2H)。

MS(+):276.07。

And 4, step 4: synthesis of Compound 2

Adding the compounds 2-4(50.0mg,0.1817mmol,1.0eq) and the compounds 1-6(200.0mg,0.1817mmol,1.0eq) into a single-mouth bottle, adding into 8mL THF, dissolving and clarifying, adding DIC (35.0mg,0.2727mmol,1.5eq), reacting at room temperature of 25-30 ℃ for 2 hours, detecting by LC-MS and HPLC, generating the target product, and processing and reacting if the raw material is left.

And (3) concentrating the reaction solution in vacuum to obtain an oily substance, adding 10mL of acetonitrile and 10mL of water, performing medium-pressure reverse phase preparation, and performing freeze-drying to obtain 50mg of a white solid of the compound 2, wherein the yield is 27%. Process for preparation of Compound 2¹The H NMR is shown in FIG. 2.

MS(+):1381.34。

preparing a column: trityl SW40 spheical C18, 20-45 μm, 120A;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 25 ml/min.

Example 3: synthesis of Compound 3

Step 1: synthesis of Compound 3-3

Mixing compound 3-1(4.0g, 32.22mmol, 1.0eq), compound 3-2(5.2g, 32.22mmol, 1.0eq), PPh₃(10.1g, 38.66mmol, 1.2eq) was dissolved in 200mL THF, the temperature was reduced to about 5 ℃ in an ice bath, DIAD (7.8g,38.66mmol, 1.2eq) was added dropwise, after completion of the addition, the mixture was allowed to react at 0-10 ℃ for 2 hours, and HPLC detection showed that the reaction of compound 3-1 was complete, the reaction mixture was concentrated to prepare sand, and EA/PE was 30/100 column min to give compound 3-3 as a yellow oil (7.5g, 88% yield).

¹H NMR(400MHZ,CDCl₃)δ:7.93(s,1H),4.32(dd,J＝8HZ,4HZ,2H),2.45(s,3H),2.43(s,3H),2.40-2.42(m,2H),2.06-2.11(m,2H)，1.47(s,9H)。

MS(+)::267.05。

Step 2: synthesis of Compounds 3-4

Dissolving the compound 3-3(2.0g,7.51mmol,1.0eq) in CCl 450 mL, adding NBS (2.8g,15.77mmol,2.1eq), adding AIBN (0.6g,7.51mmol,1.0eq) under the protection of nitrogen, after the addition is finished, replacing the nitrogen again for 3 times, heating to 75 ℃ for reaction, after the reaction is carried out for 2 hours, detecting the reaction by LC-MS and HPLC, if the target product exists, eliminating the raw material compound 3-3, and processing the reaction.

Cooling the reaction liquid to about 30 ℃ at room temperature, filtering the reaction liquid, concentrating the filtrate in vacuum to obtain a crude product, and making sand from the crude product, wherein EA/PE is 3%. Compound 3-4 was obtained as a yellow oil (1.1g, yield 34.6%).

¹H NMR(400MHZ,CDCl₃)δ:8.12(s,1H),4.70(s,2H),4.59(s,2H),4.41(dd,J＝4Hz,8Hz,2H),2.43(t,8Hz,2H),2.10(dd,J＝8Hz,4Hz,2H)，1.48(s,9H)。

MS(+):369.20。

And step 3: synthesis of Compounds 3-5

Dissolving the compound 3-4(1.0g, 2.36mmol, 1.0eq) in 20mL of DCM, slowly adding 4mL of trifluoroacetic acid dropwise at the temperature of 10-15 ℃ under ice bath, after the addition is finished, reacting for 5 hours in water bath at 20-25 ℃, detecting by LC-MS and HPLC, and finishing the reaction, wherein about 1.5% of raw materials are remained.

The reaction was concentrated in vacuo, the residue was taken up in DCM 15mL _ 3, 100mL _ dichloromethane was added to the residue, washed with water 10mL _ 3, and the organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 3-5 as a violet solid (0.8g, 93% yield).

¹H NMR(400MHZ,CDCl₃)δ:8.13(s,1H),4.70(s,2H),4.59(s,2H),4.45(dd,J＝4Hz,8Hz,2H),2.60(t,J＝8Hz,2H),2.17(dd,J＝8Hz,4Hz,2H)，1.48(s,9H)。

MS(+):369.20。

And step 3: synthesis of Compound 3

Compounds 3-5(100.0mg,0.27mmol,1.0eq) and compounds 1-6(300.0mg,0.27mmol,1.0eq) were added to a single vial, dissolved in 20mL THF, clarified, and added PyAOP (283mg,0.54mmol,2.0eq). After the addition is finished, reacting for 2 hours at the room temperature of 20-25 ℃, detecting the reaction by LC-MS and HPLC, generating a target product, and processing and reacting the residual raw materials.

The reaction solution was concentrated in vacuo to give an oil, which was added with acetonitrile 10mL and water 10mL, prepared under high pressure in reverse phase, and lyophilized to give compound 3 as a white solid 90mg with a yield of 23%. Process for preparation of Compound 3¹H NMR is shown in FIG. 3.

MS(+):1473.46。

The preparation method comprises the following steps:

preparing a column: gemini 5 μm NX-C18, 150 x 50;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

Time (minutes)	Gradient (B%)
		0-30	10％-90％
31-32	90％-95％
		32-34	95％-10％

Example 4: synthesis of Compound 4

Step 1: synthesis of Compound 4-2

Adding compound 4-1(4.8g, 38.68mmol, 1.0eq), compound 1-2(10.4g, 46.41mmol, 1.2eq) and NaI (5.8g, 38.68mmol, 1.0eq) into 200mL DMF, adding K2CO3(8.0g, 58.00mmol, 1.5eq) in batches, heating to 65 ℃ for 5 hours after the addition is finished, LC-MS, HPLC detection, LND1070-F-1 completely reacting, cooling to room temperature, pouring into 300mL ice water, and EA 200mL x 3 extracting. The organic phases are combined and washed with H₂O100 × 2 washes the organic phase, dries the organic phase and then column-casts sand, PE-PE: EA ═ 20:1, collects the product and concentrates to give compound 4-2 as a colorless oil (3.9g, 37% yield).

¹H NMR(400MHZ,CDCl₃)δ:7.76(s,1H),4.33(dd,J＝8Hz,4Hz,2H),2.46(s,2H)，2.44(s,2H),2.10(dd,J＝8Hz,4Hz,2H),1.47(s,9H)。

MS(+):267.27。

Step 2: synthesis of Compound 4-3

Compound 4-2(3.0g,11.27mmol,1.0eq) was dissolved in CCl₄Adding NBS (4.0g,22.54mmol and 2.0eq) into 120mL, adding AIBN (3.7g,55.54mmol and 2.0eq) under the protection of nitrogen, replacing nitrogen for 3 times again after the addition is finished, heating to 85 ℃ for reaction, after 2 hours of reaction, detecting by LC-MS and HPLC that a large amount of raw materials remain, supplementing NBS (3.0g,16.90mmol and 1.5eq), continuing the reaction at 85 ℃, after 2 hours of reaction, detecting by LC-MS and HPLC that a main target product is generated, and processing the reaction.

Cooling the reaction liquid to about 20 ℃ at room temperature, filtering the reaction liquid, concentrating the filtrate in vacuum to obtain a crude product, adding acetonitrile 10ml of water 5ml of the crude product, and preparing under high pressure. Freeze-drying gave compound 4-3 as a dark green oil (1.0g, 22% yield).

¹H NMR(400MHZ,CDCl₃)δ:8.16(s,1H),4.58(s,2H),4.55(s,2H)，4.47(dd,J＝8Hz,4Hz,2H)，2.47(dd,J＝8Hz,4Hz,2H)，2.14(t,J＝8Hz,2H)，1.47(s,9H)。

MS(+):424.72。

The preparation method comprises the following steps:

the instrument comprises the following steps: waters 2767 high pressure chromatography;

preparing a column: gemini 5 μm NX-C18, 150 x 50;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

And step 3: synthesis of Compound 4-4

Dissolving the compound 4-3(1.0g, 2.36mmol, 1.0eq) in 10mL of DCM, slowly adding 3mL of trifluoroacetic acid dropwise at the temperature of 10-15 ℃ under ice bath, after the addition is finished, reacting for 4 hours in water bath at 20-25 ℃, detecting by LC-MS and HPLC, and finishing the reaction, wherein about 1% of raw materials are remained.

The reaction was concentrated in vacuo, the residue was taken up in DCM 15mL _ 3, 100mL _ dichloromethane was added to the residue, washed with water 10mL _ 3, and the organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 4-4 as a violet solid (750mg, 87% yield).

¹H NMR(400MHZ,CDCl₃)δ:8.17(s,1H),4.57(s,2H),4.55(s,2H)，4.49(dd,J＝8Hz,4Hz,2H)，2.65(t,J＝8Hz,2H)，2.18-2.24(m,2H)。

MS(+):369.13。

And 4, step 4: synthesis of Compound 4

Adding the compounds 4-4(100.0mg,0.27mmol,1.0eq) and the compounds 1-6(300.0mg,0.27mmol,1.0eq) into a single-mouth bottle, adding 20mL THF, dissolving and clarifying, adding PyAOP (283mg,0.54mmol,2.0eq), reacting at room temperature of 20-25 ℃ for 2 hours, detecting by LC-MS and HPLC, generating the target product, remaining the raw material, and processing for reaction.

The reaction was concentrated in vacuo to give an oil, which was added to acetonitrile 10mL, water 10mL, prepared under reverse pressure and lyophilized to give compound 4 as a white solid (90mg, 23% yield). The nuclear magnetic hydrogen spectrum of compound 4 is shown in FIG. 4. MS (+): 1473.46.

The preparation method comprises the following steps:

the instrument comprises the following steps: waters high pressure chromatography;

preparing a column: gemini 5 μm NX-C18, 150 x 50;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

Example 5: synthesis of Compound 5

Synthesis of Compound 5-2:

compound 5-1(200mg, 0.41mmol) and HATU (233mg, 0.61mmol) were dissolved in DMF (2ml), DIPEA (105mg, 0.82mmol) was added and the reaction stirred at room temperature for 30min, Fmoc-Val-Lys-PAB-OH (373mg, 0.41mmol) was added and the reaction stirred at room temperature for 30min, LCMS indicated complete reaction of starting material. The reaction solution was purified by reverse phase under medium pressure with acetonitrile/water as a mobile phase, and lyophilized to obtain compound 5-2(256mg, 51% yield). MS: 795.4.

synthesis of Compounds 5-3:

compound 5-2(250mg) was dissolved in 5ml of DMF, and DNPC (408mg) and DIPEA (9mg) were added. The reaction was carried out at room temperature for 18 hours. The reaction solution was purified by medium pressure reverse phase, mobile phase acetonitrile/water, collected and lyophilized to give compound 5-3(180mg, yield 67%), MS: 960.4.

synthesis of Compounds 5-4:

dissolving the compound 5-3(170mg) in 2ml of DMF, sequentially adding MMAE (140mg), HOBT (16mg) and DIPEA (50mg), and adjusting the alkalinity to a pH value of 9-10. The reaction was carried out at room temperature for 16 hours. The reaction solution was purified by medium pressure reverse phase, mobile phase acetonitrile/water, collected and lyophilized to give compound 5-4(130mg, 47% yield), MS: 1538.8.

synthesis of Compounds 5-5:

compound 5-4(130mg) was dissolved in 2ml of DMF, and DEA (0.4ml) was added. The reaction was carried out at room temperature for 2 hours. The reaction solution was purified by medium pressure reverse phase, mobile phase acetonitrile/water, collected and lyophilized to give compound 5-5(80mg, yield 57%), MS: 1316.8.

synthesis of Compound 5:

compound 5-5(50mg, 0.038mmol) and compound 1-5(16mg, 0.057 mmol) were added to THF (3ml), and DIC (15mg, 0.114mmol) was added. The reaction was carried out at room temperature for 5 hours. High pressure preparation (% acetonitrile) gave the product compound 5(23mg), MS: 1572.8.

example 6: synthesis of Compound 6

The method comprises the following steps:

synthesis of Compound 6-1:

Fmoc-ADE (10g, 30.6mmol) and DNPC (18.6g, 61.2mmol) were dissolved in 100ml DMF, DIPEA (392mg, 3.04mmol) was added, the reaction was stirred at room temperature for 16 hours, TLC showed complete reaction of the starting materials, 500ml of 5% citric acid aqueous solution was added to the reaction solution, extraction was performed three times with 500ml of ethyl acetate, organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, purified over silica gel column, mobile phase ethyl acetate: petroleum ether 30:70 gave 6-1(16.7g, 84% yield).

Step two

Synthesis of Compound 6-2:

compound 6-1(143mg, 0.216mmol), HOBt (58mg, 0.433mmol) were dissolved in DMF (7ml), DIEA (112mg, 0.865mmol) was added, stirring was carried out at room temperature for 30min, and compound 5-5(570mg, 0.433mmol) was added. The reaction was carried out at room temperature for 24 hours, and no starting material remained as determined by LCMS.

Step three:

synthesis of Compounds 6-3:

3ml of DEA was added to the above reaction system, and stirred at room temperature for 1H. Medium pressure reverse phase to obtain 125mg of product, MS: 2790.6.

Step four:

synthesis of Compound 6:

compound 6-2(50mg, 0.018mmol), Compound 1-5(8mg, 0.027mmol) was added to 4ml of THF, DIC (12mg, 0.09 mmol) was added and the reaction was allowed to proceed at ambient temperature for 14 hours under high pressure to afford 15mg of Compound 6 as a product, MS: 3046.6.

Example 7: synthesis of Compound 7

The method comprises the following steps:

synthesis of Compound 7-2: mixing compound 7-1(3g, 25mmol), compound 1-2(5g,30mmol), K₂CO₃(5.3g,37.5mmol) was dissolved in DMF (50 ml). The temperature is raised to 65 ℃ for reaction for three hours. The TLC plate detects that a small amount of the starting material remains and the reaction is stopped. Filtering the reaction solution, adding 500ml of EA and 500ml of saturated NaCl solution, washing the organic phase twice, adding anhydrous sodium sulfate into the organic phase, drying, and evaporating to dryness to obtain a crude product of 6.14 g; normal phase preparation, 10g of silica gel sand, 80g of silica gel column, 4% EA, PE to 1.69g of Compound 7-2.

¹H NMR(400MHZ,DMSO-d6)δ:7.91(d,J＝8Hz,1H),7.78(d,J＝8Hz,2H),7.66(t,J＝8Hz,1H)，7.58(t,J＝8Hz,1H),4.46(t,J＝8Hz,2H)，2.56(s,3H)，2.44(t,J＝8Hz,2H)，2.01-2.08(m,2H),1.39(s,9H)。MS(+):303.43。

Step two:

synthesis of Compounds 7-3: compound 7-2(1.69g, 5.6mmol), NBS (997mg, 5.6mmol) and AIBN (920mg,5.6mmol) were dissolved in CCl₄(20ml), the temperature was raised to 80 ℃ to react for 1 hour. The TLC plate detects that a small amount of the starting material remains and the reaction is stopped. The reaction solution was directly evaporated to dryness to give 3.61g of crude product. Normal phase preparation, 5g of silica gel sand, 40g of silica gel column, 3% EA: PE to 500mg of compound 7-3 as product, MS: 381.29.

¹H NMR(400MHZ,CDCl₃)δ:8.00(d,J＝8Hz,1H),7.84(t,J＝8Hz,2H),7.67-7.71(m,1H)，7.57-7.61(m,1H)，4.73(s,2H)，4.62(dd,J＝8Hz,4Hz2H),，2.53(t,J＝8Hz,2H)，2.21(t,J＝8Hz,2H)1.48(s,9H)。MS(+):382.23。

step three:

synthesis of Compounds 7-4: compound 7-3(500mg) was dissolved in 5ml DCM, and TFA (1ml) was added under ice bath. The reaction was carried out at room temperature for 16 hours. TLC plate check no raw material. Adding 50ml DCM into the reaction solution, washing with water for three times, drying the organic phase with anhydrous sodium sulfate, and directly evaporating to dryness to obtain 400mg of a compound 7-4 as a product.

¹H NMR(400MHZ,CDCl₃)δ:8.01(d,J＝8Hz,1H),7.84(t,J＝8Hz,2H),7.69(t,J＝8Hz,1H)，7.58-7.61(m,1H)，4.72(s,2H)，4.66(dd,J＝8Hz,4Hz 2H),，2.71(t,J＝8Hz,2H)，2.25-2.32(m,2H)。MS(+):326.25。

Step four:

synthesis of compound 11: compounds 1 to 6(50mg, 0.0445mmol) and 7 to 4(21.7mg, 0.0667mmol) were dissolved in THF (3ml), and DIC (20mg,0.155mmol) was added. The reaction was carried out at room temperature for four hours. High pressure preparation (% acetonitrile) gave the product compound 12(20 mg). The nuclear magnetic hydrogen spectrum of compound 7 is shown in FIG. 5.

MS(+)::1430.22。

The preparation method comprises the following steps:

preparing a column: gemini 5 μm NX-C18, 150 x 50;

mobile phase A: water; mobile phase B: acetonitrile;

flow rate: 50 ml/min.

Example 8: synthesis of Compound 8

The method comprises the following steps:

synthesis of Compounds 8-4:

compound 8-2(PCT Patent Application No. PCT/CN2020/088565) (25.3g, 50.0mmol), compound 8-1(36.1g, 50.0mmol)) was dissolved in 240ml of DMF, DIPEA (645mg,5mmol) and HOBt (7.26g, 53.7mmol) were added via an isopiestic dropping funnel, the reaction was stirred at room temperature for 18 hours, and HPLC showed completion of the reaction of the starting material to give compound 8-3. Diethylamine (40ml) was added to the reaction solution, and stirring was continued at room temperature for 1 hour, whereupon completion of the reaction was detected by HPLC. The reaction solution was purified by medium pressure reverse phase with mobile phase acetonitrile/water, and lyophilized to obtain compound 8-4(36.5g, 81% yield). MS: 911.4.

synthesis of Compounds 8-6:

Fmoc-Peg4-COOH (200mg, 0.41mmol) and HATU (233mg, 0.61mmol) were dissolved in DMF (2ml), DIPEA (105mg, 0.82mmol) was added, the reaction was stirred at room temperature for 30min, Compound 8-4(373mg, 0.41mmol) was added, the reaction was stirred at room temperature for 30min, and LCMS indicated that the starting material was completely reacted to give Compound 8-5. Diethylamine (0.5ml) was added to the reaction solution, and stirring was continued at room temperature for 1 hour, whereupon completion of the reaction was detected by HPLC. The reaction solution was purified by reverse phase under medium pressure, mobile phase acetonitrile/water, collected and lyophilized to give 8-6(256mg, 51% yield). MS: 1158.6.

synthesis of compound 8:

compounds 8-6(60mg, 0.052mmol), compounds 1-5(57mg, 0.207mg) were dissolved in THF (3ml), and DIC (20mg,0.155mmol) was added. The reaction was carried out at room temperature for four hours. Reverse phase high pressure preparation, mobile phase acetonitrile/water. Product 8(17mg), MS: 1414.5.

example 9: synthesis of Compound 9

Compound LND1018(100mg, 0.127mmol), compound 1-5(52.65mg, 0.191mmol) was dissolved in THF (3ml) and DIC (64mg,0.508mmol) was added. Reacting for 24 hours at normal temperature. High pressure preparation (% acetonitrile) gave compound 9(10mg), MS: 1042.61.

example 10: synthesis of Compound 10

Synthesis of Compound 10-2:

compound 10-1(300mg, 0.46mmol), compound 8-1(333mg, 0.46mmol)) was dissolved in 5ml of DMF, DIPEA (129mg,1.0mmol) was added, HOBt (62g, 0.46mmol) was added, the reaction was stirred at room temperature for 18 hours, and HPLC analysis revealed that the starting material had reacted completely. The reaction solution was purified by medium pressure reverse phase, mobile phase acetonitrile/water, collected and lyophilized to give 10-2(260mg, yield 35%), MS: 1276.6.

synthesis of Compound 10-3:

compound 10-2(260mg, 0.2mmol) was dissolved in 2ml of DMF, and 0.5ml of DEA was added thereto to react at room temperature for 1 hour, and the starting material was found to react completely by HPLC. The reaction solution was purified by medium pressure reverse phase, mobile phase acetonitrile/water, collected and lyophilized to give compound 10-3(150mg, yield 45%), MS: 1054.5.

synthesis of Compound 10-4:

compound 10-3(100mg, 0.0947mmol), compound 1-5(39mg, 0.142mmol) were dissolved in THF (3ml), and DIC (48mg,0.3788mmol) was added. The reaction was carried out at room temperature for 1 hour. Reverse phase high pressure preparation gave product compound 10(17mg), MS: 1312.7.

example 11: conjugation with CD25 antibody

The CD25 antibody (0.75 mL,14.85mg/mL, 11mg in total) was added with 5.0eq TCEP (10mM), 37. mu.L, stirred at 37 ℃ for 2 hours, and 7.0eq of the corresponding compound (5mM), 44. mu.L, were added. Preserving the heat for half an hour at 37 ℃, and performing replacement and purification by a 30KDa ultrafiltration tube to finally obtain the corresponding ADC:

LN401-20-1 (CD25+ Compound 1);

LN401-20-2 (CD25+ Compound 2);

LN401-89-5 (CD25+ Compound 3);

LN401-89-6 (CD25+ Compound 4);

LN401-88-5 (CD25+ Compound 7).

HIC and SEC tests were performed according to the following table.

HIC：

SEC：

Chromatographic column	TSKgel G3000SW_XL 5μm7.8mm300mm
		Mobile phase	200mM PBS+10％IPA pH7.0
Sample volume	10μL(50μg)
		Detection wavelength	280nm
Column temperature
		25℃
Flow rate of flow			0.8ml/min
	Elution method	Equal degree
Analysis time			30min

HIC and SEC profiles of LN401-20-1 are shown in FIGS. 6 and 7, respectively;

HIC and SEC profiles of LN401-20-2 are shown in FIGS. 8 and 9, respectively.

HIC and SEC profiles of LN401-89-5 are shown in FIGS. 10 and 11, respectively.

HIC and SEC profiles of LN401-89-6 are shown in FIGS. 12 and 13, respectively.

HIC and SEC profiles of LN401-88-5 are shown in FIGS. 14 and 15, respectively.

LN401-20-1 has the structure shown below, where a is CD25 antibody and the average DAR is 4.5:

LN401-20-2 has the structure shown below, where a is CD25 antibody and the average DAR is 5.2:

LN401-89-5 has the structure shown below, where a is CD25 antibody and the average DAR is 3.6:

LN401-89-6 has the structure shown below, where a is CD25 antibody and the average DAR is 4.3:

LN401-88-5 has the structure shown below, where a is CD25 antibody and the average DAR is 5.2:

example 12: and Her2 antibody conjugation

Take Her2 antibody 0.5mL, 11.33mg/mL, 5.67mg total, add 5.0eq TCEP (10mM) 19. mu.L, stir at 37 ℃ for 2 hours, add 7.0eq of the corresponding compound (5mM), 27. mu.L. Preserving the heat for half an hour at 37 ℃, and performing replacement and purification by a 30KDa ultrafiltration tube to finally obtain the corresponding ADC:

LN401-31-1(Her2+ Compound 1);

LN401-31-2(Her2+ Compound 2);

LN401-90-5(Her2+ Compound 3);

LN401-90-6(Her2+ Compound 4);

LN401-90-4(Her2+ Compound 7).

HIC and SEC were performed in the same manner as in example 12.

HIC and SEC profiles of LN401-31-1 are shown in FIGS. 16 and 17, respectively;

HIC and SEC profiles of LN401-31-2 are shown in FIGS. 18 and 19, respectively;

HIC and SEC profiles of LN401-90-5 are shown in FIGS. 20 and 21, respectively;

HIC and SEC profiles of LN401-90-6 are shown in FIGS. 22 and 23, respectively;

HIC and SEC profiles of LN401-90-4 are shown in FIGS. 24 and 25, respectively.

LN401-31-1 has the structure shown below, where B is Her2 antibody and the average DAR is 3.6:

LN401-31-2 has the structure shown below, where B is Her2 antibody and the average DAR is 4.7:

LN401-90-5 has the structure shown below, where B is Her2 antibody and the average DAR is 3.2:

LN401-90-6 has the structure shown in the following figure, where B is Her2 antibody and the average DAR is 4.0:

LN401-90-4 has the structure shown below, where B is Her2 antibody and the average DAR is 4.1:

example 13: ADC antitumor cell activity assay

Reagent

Solutions of 10mM toxin small molecules were prepared using 100% DMSO. Samples of Antibody Drug Conjugates (ADCs) were all provided in PBS.

Cell culture

The human SU-DHL-1, Daudi, SKBr-3, HCC1954 and MDA-MB-468 cancer cell lines used in the activity tests were purchased from Type Culture Collection (ATCC; Manassas, VA), U.S.A., L-540 and HDLM-2 were purchased from Liebnizz institute DSMZ (Mannheim, Germany), while Karpas 299T was purchased from Sigma (St. Louis, MO). All cell lines were cultured in RPMI-1640 medium (Gibco) supplemented with 10-20% heat-inactivated fetal bovine serum (FBS, Corning; Corning, NY, USA) based on the recommendations of ATCC or DSMZ, except that MDA-MB-468 was routinely cultured in DMEM/F:12 medium (Gibco; Waltham, MA). 5% CO at 37 deg.C₂Culturing in a humidified environment.

Cell viability assay

Adherent tumor cells were harvested by non-enzymatic separation from cell dissociation reagents (Corning), and suspension cell lines were harvested by centrifugation at 300 × g for 5 min. They were seeded into 384-well clear-bottom white wall plates (875 cells per well for adherent cells and 2500 cells/well for suspension cells in 12.5 μ L of whole medium) and left to adhere at 37 ℃ for 2-4 hours. Cells were then treated with 12.5 μ L of test sample at a final concentration of 2X in duplicate, then serially diluted in complete medium, which was incubated at 37 ℃ for 120 hours. According to the use scheme of the instrument manufacturer, use

Cell viability assay (Promega; Madison, Wis., USA) determined inhibition of cancer cell growth. Luminescence was measured using a Tecan Spark multimode microplate reader (Tecan Group Ltd.; Switzerland Mandoff).

Data analysis

Data were normalized using Microsoft Excel (redmond, washington) with control treated with no test sample and analyzed using GraphPad Prism software (version 8; rajohn, ca, usa). The half maximal effective concentration for inhibition was derived from the dose response curve using non-linear regression analysis fitted to a 4-parameter logistic equation (EC 50).

Results

The inhibitory effect of LN401-20-1 and LN401-20-2 on each cell is shown in Table 1 below and FIG. 26. As shown in FIG. 26, LN401-20-1 and LN401-20-2 both dose-dependently inhibited cell viability of SU-DHL-1, Karpas 299T, L-540, and HDLM-2 cells expressing CD25, as well as CD25 negative Daudi cells.

The inhibitory effects of LN401-31-1 and LN401-31-2 on the following tables of human tumors are shown in Table 2 and FIG. 27.

Table 1: EC50 values of anti-CD 25 ADC in human tumor cells

Table 2: EC50 values of anti-HER 2 ADC in human tumor cells

Claims

1. A compound having the structure shown in formula I:

R₁-L₁-L₂-R₂-D (I)

in the formula (I), the compound is shown in the specification,

R₁selected from any of the following structural formulas:

L₁is-L_a-L_b-, wherein L_aIs C1-C6 alkylene, C2-C6 methyleneAlkenyl, C2-C6 alkynylene or- (CH)₂)_p-CONH-[CH₂CH₂-O]_q-(CH₂)_p-；L_bIs absent, or is-CO-, -NH-, -COO-or-NH-SO₂Or is:

each p is independently an integer from 1 to 4; and

each q is independently an integer from 1 to 8.

2. The compound of claim 1, wherein R is₁Selected from the following groups:

3. The compound of claim 1 or 2,

L_ais C1-C6 alkylene or- (CH)₂)_p-CONH-[CH₂CH₂-O]_q-(CH₂)_p-; preferably, L_aIs- (CH)₂)₃-CONH-[(CH₂)₂-O]₄-(CH₂)₂-；

L_bis-CO-or

Wherein L is₁Through L_bWith amide groups and L₂Covalent attachment;

preferably, L₁Is- (CH)₂)₃-CONH-[(CH₂)₂-O]₄-(CH₂)₂-CO-、-(CH₂)₃-CO-、-(CH₂)₅-CO-or- (CH)₂)₃-CO-N(-CH₂CH₂-OCO-)₂。

4. A compound according to any one of claims 1 to 3 wherein L is₂The peptide is dipeptide, tripeptide, tetrapeptide or pentapeptide; preferably, the amino acid residues in the peptide are selected from: one or more of valine, ornithine, lysine, serine, glycine, and citrulline;

preferably, the peptide is Val-Cit, Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Gly-Gly-Val-Lys, Gly-Gly or Gly-Gly-Val-Lys;

preferably, the enzyme is a proteolytic enzyme, protease or peptidase; more preferably, the enzyme is selected from: one or more of a cysteine protease, an aspartic protease, a glutamic protease, a threonine protease, a gelatinase, a metalloprotease, and an asparagine peptide cleaving enzyme.

5. The compound of claim 4, wherein one or more amino acid residues in the peptide are modified with a phosphorylcholine group; preferably, the phosphorylcholine group-modified amino acid is represented by formula II below:

R₈-L₃-N(R₉)-R₁₀ (II)

in the formula, R₈Is a phosphorylcholine group; n represents a nitrogen atom on an amino acid molecule; r₉Is H, C1-C4 alkyl or-L₃-a phosphorylcholine group; each L₃Independently is a linking group; r₁₀Represents the remainder of the amino acid molecule other than the indicated N atom, said remainder being optionally protected by a protecting group;

wherein the phosphorylcholine group is represented by the following formula:

wherein the wavy line represents the phosphorylcholine group and L₃The location of the connection;

preferably, each L₃Independently is represented by-L_c-L_d-, wherein L_cSelected from C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene; l is_dIs absent, or is carbonyl, ester (-COO-), -O-SO₂-or-NH-SO₂-; preferably, L_dAbsent or as a carbonyl group; preferably, L₃Through L_d(ii) is covalently linked to the N; preferably, L_dAnd the N shown forms an amide group or a carbonitrideamino group (-CH)₂-NH-); preferably, each L₃Independently is a C1-C6 alkylene group or a C1-C6 alkylenecarbonyl group.

6. The compound of any one of claims 1-5, wherein R is₂Wherein the p-aminobenzyloxy derivative has the following structure:

in the formula (I), the compound is shown in the specification,

R₄is H or C1-C4 alkyl; preferably H, methyl or ethyl;

R₅is absent, or is-NR₆-(CH₂)_s-NR₇-CO-, wherein s is 2 or 3, R₆And R₇Each independently is H or C1-C4 alkyl, which indicates the position of attachment to D;

the dotted line represents R₂And L₂The position of the connection, the wavy line, R₂The position of the linkage to D;

the optionally substituted alkanediamine group is represented by-NR₆-(CH₂)_s-NR₇-, where s is 2 or 3, R₆And R₇Each independently is H or C1-C4 alkyl;

preferably, L₂And R₂Are connected through amide groups.

7. The compound of any one of claims 1 to 6, wherein the drug is selected from the group consisting of: MMAE, Duo-5, DXD, maytansine, PNU-159682, irinotecan, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, fluorouracil, doxifluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, daunorubicin, epirubicin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin; preferred drugs are MMAE, maytansine, PNU-159682, camptothecin or 7-ethyl-10-hydroxycamptothecin.

8. The compound of claim 1, wherein the compound is from 1 to 10:

9. an antibody drug conjugate having the structure shown in formula III below:

Ab-[X-R₁’-L₁-L₂-R₂-D]_m (III)

in the formula:

L₁、L₂、R₂and D is as described in any one of claims 1-7;

m is a number between 1 and 8.

10. The antibody drug conjugate of claim 9, wherein R is₁' is:

therein, deficiency of bloodLine represents R₁' position of attachment to Ab, wavy line means R₁' and L₁The location of the connection;

L₂is Val-Cit, Val-Orn, Val-Lys, Ser-Val-Lys, Gly-Val-Lys, Ser-Val-Cit, Gly-Gly-Gly-Val-Lys, Gly-Gly or Gly-Gly-Val-Lys; preferably Val-Cit; wherein L is_bAnd L₂Are connected through amido; preferably, L₂One or two of the amino acid residues in (a) are the phosphorylcholine-modified amino acid residue of claim 5;

R₂has the following structure:

d is selected from the following medicines: MMAE, Duo-5, DXD, maytansine, PNU-159682, irinotecan, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, fluorouracil, doxifluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, daunorubicin, epirubicin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, doxorubicin, and mitomycin; preferably MMAE, maytansine, PNU-159682, camptothecin or 7-ethyl-10-hydroxycamptothecin.

11. The antibody drug conjugate of claim 9, wherein the antibody drug conjugate is selected from the group consisting of:

an ADC of the formula wherein a is a CD25 antibody and m has a value between 1 and 8:

an ADC of the formula wherein B is Her2 antibody and m has a value between 1 and 8:

12. the antibody drug conjugate of claim 11, wherein the antibody drug conjugate is selected from the group consisting of:

LN401-20-1 of the formula wherein a is a CD25 antibody and m is 4.5:

LN401-20-2 of formula wherein a is a CD25 antibody, and m is 5.2:

LN401-89-5 of the formula wherein a is a CD25 antibody and m is 3.6:

LN401-89-6 of formula wherein a is a CD25 antibody and m is 4.3:

LN401-88-5 of the formula wherein a is a CD25 antibody and m is 5.2:

LN401-31-1 of formula wherein B is Her2 antibody and m is 3.6:

LN401-31-2 of formula wherein B is Her2 antibody and m is 4.7:

LN401-90-5 of formula wherein B is Her2 antibody and m is 3.2:

LN401-90-6 of formula wherein B is Her2 antibody and m is 4.0:

LN401-90-4 of formula wherein B is Her2 antibody and m is 4.1:

13. a pharmaceutical composition comprising the antibody drug conjugate of any one of claims 9-12 and a pharmaceutically acceptable carrier.

14. Use of a compound according to any one of claims 1 to 8 for the manufacture of an antibody drug conjugate, and use of an antibody drug conjugate according to any one of claims 9 to 12 for the manufacture of a medicament for the treatment or prevention of cancer.

15. The use of claim 14, wherein the cancer is selected from the group consisting of: bladder cancer, kidney cancer, urinary tract cancer, chorioepithelial cancer, glioblastoma multiforme, melanoma, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and leukemia;

preferably, the drug molecule in the antibody drug conjugate is MMAE, maytansine, PNU-159682, camptothecin or 7-ethyl-10-hydroxycamptothecin, and the disease is an indication for MMAE, maytansine, PNU-159682, camptothecin or 7-ethyl-10-hydroxycamptothecin, including gastric cancer, esophageal cancer, cardiac cancer, colon cancer, rectal cancer, primary liver cancer, acute and chronic myelogenous leukemias, chorioepithelial cancer, lung cancer and bladder cancer.