Hydrophilic linker, linker intermediate, linker-drug conjugate, antibody drug conjugate and application thereof

Abstract

The invention relates to a hydrophilic linker, an intermediate of the hydrophilic linker, a linker-drug conjugate prepared from the linker, a preparation method of the linker-drug conjugate, an antibody drug conjugate and application of the antibody drug conjugate. The antibody drug conjugate prepared from the hydrophilic linker can improve the hydrophilic performance. The antibody drug conjugate is especially suitable for generating a high DAR value through hydrophobic loading, so that the problem of aggregation is reduced or avoided. Based on the improvement of the hydrophilic performance, the prepared antibody drug conjugate is expected to further improve the in-vitro activity and the in-vivo drug effect.

Description

TECHNICAL FIELD

[0001] This document relates to structural improvements of linkers, and linker-drug conjugates and antibody-drug conjugates prepared therefrom, and their uses. BACKGROUND

[0002] The main anti-tumor mechanism of ADC drugs is to target the delivery of cytotoxic payloads to tumor cells. That is, after ADC enters the body, its antibody part and tumor cell surface target antigen selectively bind, enter the tumor cell through endocytosis, and finally the ADC is broken down in the lysosome in the cell to release the payload efficiently, thereby exerting an anti-tumor effect.

[0003] Since the anti-tumor effect of ADC drugs depends on the concentration of payloads that can ultimately be delivered to tumor cells, and the drug concentration in tumor cells, as can be seen from the mechanism of action of ADC, the expression level of target antigens on the surface of tumor cells that can be specifically recognized by the antibody, the DAR value (Drug-antibody ratio) of ADC, and the stability of the linker in the circulatory system and the efficiency of releasing the payload in the cell are related.

[0004] Therefore, under the premise that other factors remain unchanged, in theory, the drug concentration delivered into tumor cells can be increased by simply increasing the DAR value of ADC. However, in practice, since most payloads are inherently lipophilic, a high DAR value will usually make the ADC more hydrophobic, resulting in poor physicochemical properties and PK, and it will be cleared more quickly in the body, and may not necessarily bring better in vivo efficacy (Clin Cancer Res. 2004, 10, 7063-70), but rather an increase in toxicity due to the easier aggregation of ADC in the body (Signal Transduction and Targeted Therapy 2020, 5, 132).

[0005] It can be seen that the physicochemical properties of Linker play an important role in the design of ADC, and its chemical properties can significantly affect the physicochemical properties and PK of ADC, and thus affect its efficacy and safety. In order to improve the selectivity and anti-tumor activity of ADC, the Linker used should meet the following characteristics (Pharmaceuticals 2021, 14, 442): (1) high stability in the circulatory system to avoid premature release of Payload; (2) high water solubility, which helps to stabilize ADC with high DAR value and avoid aggregation; (3) efficient release of effective payload. The success of Daiichi Sankyo's DS-8201a is largely due to the screening of a linker that meets the above characteristics (Chem Pharm Bull. 2019, 67, 173-185). Most of the ADCs currently on the market, including DS-8201a, still face serious safety problems, and it is imperative to further improve their safety, and by modifying the linker, ADCs with better physicochemical properties and PK can further improve the safety and effectiveness of ADCs.

[0006] In summary, an ideal ADC should remain intact in the circulation without aggregation after injection, and only release the payload inside or near the targeted tumor cells. Therefore, there is still a high clinical demand to improve the DAR value (drug loading) of ADC (or other more extensive drug conjugates) while maintaining good physicochemical properties, PK and efficacy. SUMMARY

[0007] The main purpose of this paper is to provide a hydrophilic linker structure to improve the hydrophilic performance of antibody conjugate drugs using this structure.

[0008] The first aspect of this paper provides an intermediate of a hydrophilic linker comprising a structure represented by general formula V or a salt or ester thereof,

[0009] R4-L2-E (V), wherein,

[0010] R4 is a functional group capable of binding to an amino acid unit in the linker, L2 is absent or a PEG unit, and E is selected from an azacrown ether group substituted with one or more hydrophilic units, an azacrown ether containing two or more nitrogen atoms and optionally containing an oxygen atom, the hydrophilic group being connected to the nitrogen atom in the azacrown ether, the azacrown ether group being connected to L2 or R4 through other nitrogen atoms according to the presence or absence of L2.

[0011] In some embodiments of the hydrophilic linker of the first aspect of this paper, R4 is selected from an amino group, a carboxyl group or a protected form thereof.

[0012] The second aspect of the present disclosure provides a hydrophilic linker comprising amino acid units, further comprising one or more side chains of a structural segment of Formula I, or a salt or ester thereof,

[0013] wherein,

[0014] the wavy line in Formula I represents a connection site to the amino acid unit,

[0015] L2 is absent or a PEG unit, E is selected from an azacrown ether group substituted with a hydrophilic unit, the azacrown ether comprising two or more nitrogen atoms and optionally comprising an oxygen atom, the hydrophilic group being connected to a nitrogen atom in the azacrown ether, the azacrown ether group being connected to L2 or the amino acid unit via another nitrogen atom depending on the presence of L2.

[0016] In some embodiments of the linker intermediate of the first aspect of the present disclosure or the hydrophilic linker of the second aspect of the present disclosure,

[0017] when L2 is present, L2 is selected from one or more combinations thereof.

[0018] In some embodiments of the linker intermediate of the first aspect of the present disclosure or the hydrophilic linker of the second aspect of the present disclosure, E is selected from:

[0019]

[0020] n7 = an integer from 1 to 20, n8 = an integer from 1 to 20, n9 = an integer from 1 to 20, n 10 = an integer from 1 to 20, n 11 = an integer from 1 to 20, n 12 = an integer from 1 to 20, n 13 = an integer from 1 to 20, n 14 = an integer from 1 to 20, n 15 = an integer from 1 to 20, n 16 = an integer from 1 to 20, or n 17 = an integer from 1 to 20;

[0021] W1, W2, W3, W4, W5, W6, W7, W8, and W9 are the same or different and are each independently selected from oxygen, sulfur, or R1 is selected from X or n 18 = an integer from 0 to 100; K1 is selected from hydrogen or C1-20 alkyl;

[0022] at least one X is selected from a hydrophilic unit, and the other Xs are each independently selected from hydrogen or a hydrophilic unit, when the azacrown ether unit contains multiple Xs, each X can be the same or different.

[0023] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the hydrophilic unit is selected from a sugar unit, a polysarcosine unit, a polyethylene glycol unit, or an alkyl sulfonic acid unit.

[0024] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the hydrophilic unit is a sugar unit, which is optionally substituted with a monosaccharide group, a disaccharide group, or a polysaccharide group.

[0025] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the sugar unit structure is:

[0026] wherein,

[0027] n 19 = 0-20, n 20 = 1-20, n 21 = 1-20, R2is selected from hydrogen or C1-20alkyl, Y1is independently selected from -NH- or oxygen, R3is independently selected from hydrogen, acetyl, a monosaccharide group, a disaccharide group, or a polysaccharide group, and Y2is selected from Y3is selected from hydrogen, hydroxyl, or C1-20alkoxy.

[0028] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the monosaccharide group, the disaccharide group, or the polysaccharide group is selected from a glyceraldehyde group, an erythrose group, a threose group, an arabinose group, a ribose group, a xylose group, a lyxose group; a glucose group, a mannose group, a fructose group, a galactose group, a galacturonic acid group, a meglumine group, a gluconic acid group, a glucuronic acid group, a mannuronic acid group, a maltose group, a maltobionic acid group, a lactose group, a lactobionic acid group, a sucrose group, a sophorose group, a cellobiose group, a xylobiose group, a melibiose group, a mannobiose group, a gentiobiose group, a laminaribiose group, a cellobiose group, an alpha-cyclodextrin group, a beta-cyclodextrin group, or a gamma-cyclodextrin group.

[0029] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 19 = 0-12, n 20 = 1-12, n 21 = 1-15, R2is selected from hydrogen or C1-12alkyl, and Y3is selected from hydrogen, hydroxyl, or C1-12alkoxy.

[0030] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 19 = 0-10, n 20 = 1-10, n21 = an integer from 1 to 12, R2is selected from hydrogen or C1-10alkyl, and Y3is selected from hydrogen, hydroxyl, or C1-10alkoxy.

[0031] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 19 = an integer from 0 to 8, n 20 = an integer from 1 to 8, n 21 = an integer from 1 to 10, R2is selected from hydrogen or C1-8alkyl, and Y3is selected from hydrogen, hydroxyl, or C1-8alkoxy.

[0032] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the polyomithine unit structure is:

[0033] wherein n 22 = an integer from 1 to 100.

[0034] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 22 = an integer from 1 to 50.

[0035] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 22 = an integer from 1 to 30.

[0036] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 22 = an integer from 1 to 20.

[0037] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the polyethylene glycol unit structure is:

[0038] wherein n 23 = an integer from 1 to 100, and K2is selected from hydrogen or C1-20alkyl.

[0039] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 23 = an integer from 1 to 50, and K2is selected from hydrogen or C1-12alkyl.

[0040] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 23 = an integer from 1 to 30, and K2is selected from hydrogen or C1-10alkyl.

[0041] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 23 = 1-20, and K2 is selected from hydrogen or C1-8 alkyl.

[0042] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the alkyl sulfonic acid unit structure is:

[0043] wherein n 24 = 1-20.

[0044] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 24 = 1-12.

[0045] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 24 = 1-10.

[0046] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n 24 = 1-8.

[0047] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein,

[0048] n7 = 1-12, n8 = 1-12, n9 = 1-12, n 10 = 1-12, n 11 = 1-12, n 12 = 1-12, n 13 = 1-12, n 14 = 1-12, n 15 = 1-12, n 16 = 1-12, or n 17 = 1-12, n

[0049] n7 = 1-12, n8 = 1-12, n9 = 1-12, n 18 = 0-50; and K1 is selected from hydrogen or C1-12 alkyl.

[0050] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein,

[0051] n7 = 1-10, n8 = 1-10, n9 = 1-10, n 10 = 1-10, n 11 = 1-10, n 12 = 1-10, n13 =1-10, n 14 =1-10, n 15 =1-10, n 16 =1-10 or n 17 =Integers from 1 to 10

[0052] The n 18 = Integers from 0 to 30; K1 is selected from hydrogen or C1-10 alkyl.

[0053] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document,

[0054] n7=1-8, n8=1-8, n9=1-8, n 10 =1-8, n 11 =1-8, n 12 =1-8, n 13 =1-8, n 14 =1-8, n 15 =1-8, n 16 =1-8 or n 17 =Integers from 1 to 8

[0055] The n 18 = Integers from 0 to 20; K1 is selected from hydrogen or C1-8 alkyl.

[0056] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, n3 = 0-50, n4 = 0-50, n5 = 0-50 or n6 = 0-50.

[0057] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, n3 = 0-30, n4 = 0-30, n5 = 0-30 or n6 = 0-30.

[0058] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, n3 = 0-20, n4 = 0-20, n5 = 0-20 or n6 = 0-20.

[0059] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, E is selected from the azirconium crown ether group of a 9- to 18-membered ring, and the hydrophilic unit is selected from a sugar unit, wherein the sugar unit is selected from:

[0060] Where, n 19 Selected from integers 1 to 3, R2 is hydrogen or methyl, n 20R3 is selected from integers from 1 to 3, and R3 is independently selected from H or C5 to C6 monosaccharides. 21 Choose from 3 or 4.

[0061] or Where, n 20 R3 is selected from integers from 1 to 3, and R3 is independently selected from H or C5 to C6 monosaccharides. 21 Choose from 3 or 4.

[0062] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, R3 contains one monosaccharide selected from C5 to C6, and the remainder is H.

[0063] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document, E is selected from the azircon ethers of 9, 12, 15, and 18-membered rings.

[0064] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, E is selected from:

[0065] In this configuration, at least one X is a sugar unit, and the other Xs are independently selected from H or sugar units.

[0066] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the sugar unit is selected from:

[0067]

[0068] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, R3 is...

[0069]

[0070] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, E is selected from:

[0071]

[0072]

[0073] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the L2 is present and selected from... n3 is an integer selected from 8 to 12.

[0074] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n3 = 11.

[0075] The hydrophilic linker of the second aspect of this article comprises one or two of the described side chains.

[0076] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers of the second aspect of this document, -L2-E is selected from the structures shown in the table below:

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] In some embodiments of the hydrophilic linker of the second aspect of this document, a first linker subunit for linking to a target unit and a second linker subunit for linking to a drug are also included, wherein the first and second linker subunits are linked to the amino acid unit.

[0085] In some embodiments of the hydrophilic linker of the second aspect of this article, the targeting unit is selected from antibodies or their antigen-binding fragments.

[0086] In some embodiments of the hydrophilic linker of the second aspect of this document, the amino acid unit comprises one or more amino acid subunits, the amino acid subunits being selected from:

[0087] Or one or more combinations of its stereoisomers.

[0088] * Represents the connection site with L2, or, when L2 is absent, the connection site with E;

[0089] # represents the connection site with the first linker subunit or amino acid subunit.

[0090] ## represents the connection site with the second linker subunit or amino acid subunit.

[0091] n 37 =0-20, n38 =0-20, n 39 =0-20, n 40 =0-20, n 41 =0-20, n 42 =0-20, n 43 =0-20, n 44 =0-20, n 45 =0-20, n 46 =0-20, n 47 =0-20, n 48 =0-20, n 49 =0-20, n 50 =0-20, n 51 =0-20, n 52 =0-20, n 53 =0-20 and n 54 = Integers between 0 and 20.

[0092] In the hydrophilic linker of the second aspect of this paper, n 37 =0-12, n 38 =0-12, n 39 =0-12, n 40 =0-12, n 41 =0-12, n 42 =0-12, n 43 =0-12, n 44 =0-12, n 45 =0-12, n 46 =0-12, n 47 =0-12, n 48 =0-12, n 49 =0-12, n 50 =0-12, n 51 =0-12, n 52 =0-12, n 53 =0-12 and n 54 = Integers between 0 and 12.

[0093] In the hydrophilic linker of the second aspect of this paper, n 37 =0-10, n 38 =0-10, n 39 =0-10, n 40 =0-10, n 41 =0-10, n 42 =0-10, n 43 =0-10, n 44 =0-10, n 45 =0-10, n 46 =0-10, n 47=0-10, n 48 =0-10, n 49 =0-10, n 50 =0-10, n 51 =0-10, n 52 =0-10, n 53 =0-10 and n 54 = Integers between 0 and 10.

[0094] In some embodiments of the hydrophilic linker of the second aspect of this article, n 37 =0-8, n 38 =0-8, n 39 =0-8, n 40 =0-8, n 41 =0-8, n 42 =0-8, n 43 =0-8, n 44 =0-8, n 45 =0-8, n 46 = Integers from 0 to 8.

[0095] In some embodiments of the hydrophilic linker of the second aspect of this article, n 37 =1, n 38 =1, n 39 =1, n 40 =1, n 41 =1, n 42 =1, n 43 =1, n 44 =1, n 45 =1, n 46 An integer equal to 1.

[0096] The third aspect of this paper relates to linker-drug conjugates, which are formed by combining the aforementioned linker with a drug, and have the structure shown in Formula II.

[0097] in,

[0098] n2 is an integer from 1 to 8, A1 is selected from amino acid subunits, and M is a connector used to link with the target unit.

[0099] L1 does not exist or is selected from One or more combinations of n; where n 30 =1-20, n 31 =0-100, n 32 =1-20, n 33 =0-100, n 34 =0-100, n 35 =0-100 or n 36=Integers between 0 and 20. The wavy line to the left of L1 indicates the site connected to M, and the wavy line to the right indicates the site connected to A1.

[0100] A1 is selected from Or its stereoisomers,

[0101] * Represents the connection site with L2, or, when L2 is absent, the connection site with E;

[0102] # represents the connection site with L1.

[0103] ## represents the connection site with A2.

[0104] n 37 =0-20, n 38 =0-20, n 39 =0-20, n 40 =0-20, n 41 =0-20, n 42 =0-20, n 43 =0-20, n 44 =0-20, n 45 =0-20, n 46 =0-20, n 47 =0-20, n 48 =0-20, n 49 =0-20, n 50 =0-20, n 51 =0-20, n 52 =0-20, n 53 =0-20 and n 54 =Integers between 0 and 20

[0105] A2 does not exist or is selected from:

[0106] The wavy line to the left of A2 indicates the site connected to A1, and the wavy line to the right indicates the site connected to D.

[0107] The term D refers to a fragment formed by the loss of one or more atoms or groups from a molecule with antitumor biological activity.

[0108] In some embodiments of the linker-drug conjugates of the third aspect of this article, M is selected from... Or its stereoisomers.

[0109] In some embodiments of the linker-drug conjugates of the third aspect of this article, the M-L2- is

[0110] In some embodiments of the linker-drug conjugate of the third aspect of this article, A2 is...

[0111] In the third aspect of this paper, in the linker-drug conjugate, n 30 =1-12, n 31 =0-50, n 32 =1-12, n 33 =0-50, n 34 =0-50, n 35 =0-50 or n 36 = Integers between 0 and 12.

[0112] In some embodiments of the linker-drug conjugates of the third aspect of this article, n 30 =1-10, n 31 =0-30, n 32 =1-10, n 33 =0-30, n 34 =0-30, n 35 =0-30 and n 36 = Integers between 0 and 10.

[0113] In some embodiments of the linker-drug conjugates of the third aspect of this article, n 30 =1-8, n 31 =0-20, n 32 =1-8 and n 33 = Integers between 0 and 20.

[0114] In some embodiments of the linker-drug conjugates of the third aspect of this article, n2 is an integer from 1 to 8.

[0115] In some embodiments of the linker-drug conjugates of the third aspect of this article, n2 is an integer from 1 to 6.

[0116] In some embodiments of the linker-drug conjugates of the third aspect of this article, n2 is an integer from 1 to 4.

[0117] In some embodiments of the linker-drug conjugate of the third aspect of this article, n2 is 1 or 2.

[0118] In some embodiments of the linker-drug conjugates of the third aspect of this article, the bioactive molecule is selected from microtubule inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, intercalators, DNA-reactants, DNA alkylating agents, immunomodulators, nucleic acids, BCL-XL inhibitors, kinase inhibitors, PROTACs, molecular glues, and radioisotopes.

[0119] In some embodiments of the linker-drug conjugate of the third aspect of this article, the linker is covalently bound to nitrogen, oxygen, or sulfur in the drug.

[0120] In some embodiments of the linker-drug conjugates of the third aspect of this article, any structure selected from LP1 to 47 is used:

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] The fourth aspect of this article relates to antibody-drug conjugates, obtained by conjugating the aforementioned linker-drug with an antibody, with the structure shown in general formula III. in,

[0135] Ab is an antibody or antigen-binding fragment, and n1 is any value between 1 and 20.

[0136] The antibody-drug conjugates involved in the fourth aspect of this article are selected from any one of ADC-1 to ADC-67:

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150] The fourth aspect of this document relates to some examples of antibody-drug conjugates, wherein the antibodies are monoclonal antibodies selected from: abciximab, alemtuzumab, anetumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatomumab, brentuximab, catutoxomab, cetuximab, and cimetuzumab. umab), Coltuximab, Daclizumab, Daratumumab, Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab, Disitamab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab ), Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Lifastuzumab, Lorvotuzumab, Milatuzumab, Mirvetuximab, Naratu (ximab), Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratuzumab, Omalizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab, PinatuzumabPolotuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab, Sirtratumab, Sofituzumab, Vadastuximab, Vorsetuzumab, Trastuzumab, Tisotumab, anti-B7-H3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody, and anti-CD30 antibody, or their antigen-binding fragments or immunologically active portions.

[0151] The fifth aspect of this article relates to a method for preparing the aforementioned intermediate, wherein the sugar unit is... Including the following steps:

[0152] CbzHN-L2-azacrown ethers were synthesized from CbzHN-L2-azacrown ethers via a bimolecular nucleophilic substitution reaction.

[0153] CbzHN-L2-azacrown ether and CbzHN-L2-E was prepared by reductive amination.

[0154] CbzHN-L2-E can be optionally synthesized via a Cbz removal reaction to form H2N-L2-E.

[0155] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, E is selected from...

[0156]

[0157] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, E is selected from...

[0158]

[0159] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, E is selected from:

[0160] In the intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect, the -L2-E segments are selected from the structures shown in H1 to H12.

[0161] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, -L 2- The E-segment is selected from H1, H2, H5, H6, H7, H8, H10, and H11.

[0162] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, -L 2- The E segment is selected from H1.

[0163] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, the sugar units are selected from:

[0164]

[0165] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, the sugar units are selected from:

[0166]

[0167] The main advantage of this article is:

[0168] 1. Antibody-drug conjugates prepared using the hydrophilic linkers described in this paper can improve hydrophilic properties. They are particularly suitable for use with different hydrophobic loads to generate antibody-drug conjugates with high DAR values, thereby reducing or avoiding aggregation problems.

[0169] 2. Based on the aforementioned improvements in hydrophilic properties, the prepared antibody-drug conjugates are expected to further enhance in vitro activity and in vivo efficacy. In some specific embodiments, the antibody-drug conjugates prepared using the linkers described herein exhibit strong in vitro cytotoxicity (<1 nM) and also demonstrate good in vivo efficacy. For example, in the JIMT-1 tumor-bearing mouse efficacy evaluation experiment, the antibody-drug conjugate ADC-23 based on the hydrophilic linker showed better efficacy than the reference ADC comparative example-1.

[0170] 3. The antibody-drug conjugates based on novel hydrophilic linkers provided in this paper have shorter retention times on HIC HPLC. Attached Figure Description

[0171] Figure 1 The in vivo antitumor effect in xenografted JIMT-1 breast cancer mouse models using the ADC drug described in this article, comparative examples, and blank control groups is shown in the figure.

[0172] Figure 2 The graph shows the changes in body weight in mice in the xenograft JIMT-1 breast cancer mouse model using the ADC drug described in this paper, the comparative group, and the blank control group. Detailed Implementation

[0173] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the descriptions and definitions herein shall prevail.

[0174] The first aspect of this document provides an intermediate for a hydrophilic linker, characterized in that it comprises a structure of general formula V or a salt or ester thereof.

[0175] R4-L2-E(V), where,

[0176] R4 is a functional group that can bind to an amino acid unit in the linker. L2 is absent or is a PEG unit. E is selected from an azacrown ether group substituted with one or more hydrophilic units. The azacrown ether contains two or more nitrogen atoms and optionally contains oxygen atoms. The hydrophilic group is connected to a nitrogen atom in the azacrown ether. Depending on the presence or absence of L2, the azacrown ether group is connected to L2 or R4 through other nitrogen atoms.

[0177] The term "azacrown ether" as used in this article refers to a class of groups in which some or all of the oxygen atoms in the crown ether ring structure are replaced by nitrogen atoms.

[0178] In some embodiments of the hydrophilic linker of the first aspect of this article, R4 is selected from amino, carboxyl, or their protected forms. A protected form refers to a derivative of the amino or carboxyl group that has been chemically modified to be relatively stable and less prone to unintended reactions, thus preventing its destruction or participation in unwanted side reactions in subsequent reactions. After the target reaction is completed, the protecting group is removed under specific conditions (i.e., "deprotection") to restore the activity of the original group. Common protecting groups used to modify amino groups include benzyloxycarbonyl and tert-butyloxycarbonyl; common protecting groups used to modify carboxyl groups include methyl ester, ethyl ester, and tert-butyl ester.

[0179] The second aspect of this document provides a hydrophilic linker comprising amino acid units, characterized in that it further comprises side chains of one or more general formula I structural segments or their salts or esters.

[0180] in,

[0181] The wavy lines in Formula I represent the connection sites with amino acid units.

[0182] L2 is absent or is a PEG unit, and E is selected from a azeotropic crown ether group substituted with a hydrophilic unit. The azeotropic crown ether contains two or more nitrogen atoms and optionally contains oxygen atoms. The hydrophilic group is connected to a nitrogen atom in the azeotropic crown ether. Depending on the presence or absence of L2, the azeotropic crown ether group is connected to L2 or an amino acid unit through other nitrogen atoms.

[0183] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document,

[0184] When L2 exists, it is selected from One or more combinations thereof.

[0185] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, E is selected from:

[0186]

[0187] n7=1-20, n8=1-20, n9=1-20, n 10 =1-20, n 11 =1-20, n 12 =1-20, n 13 =1-20, n 14 =1-20, n 15 =1-20, n 16 =1-20 or n 17 =Integers from 1 to 20;

[0188] W1, W2, W3, W4, W5, W6, W7, W8, and W9 may be the same or different, and are each independently selected from oxygen, sulfur, or... R1 is selected from X or The n 18 = Integers from 0 to 100; K1 is selected from hydrogen or C1-20 alkyl;

[0189] At least one X is selected from a hydrophilic unit, and the other Xs are each independently selected from hydrogen or a hydrophilic unit. When the azacrown ether unit contains multiple Xs, the Xs may be the same or different.

[0190] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, the hydrophilic unit is selected from sugar units, polysarcosine units, polyethylene glycol units, or alkyl sulfonic acid units.

[0191] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, the hydrophilic unit is a sugar unit, which may optionally be replaced by a monosaccharide, disaccharide, or polysaccharide.

[0192] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the glycounit structure is as follows:

[0193] in,

[0194] n 19 =0-20, n 20 =1-20, n 21= an integer from 1 to 20, wherein R2 is selected from hydrogen or C1-20 alkyl; wherein Y1 is independently selected from -NH- or oxygen; wherein R3 is independently selected from hydrogen, acetyl, monosaccharide, disaccharide, or polysaccharide; and Y2 is selected from... The Y3 is selected from hydrogen, hydroxyl, or C1-20 alkoxy.

[0195] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers of the second aspect of this document, the monosaccharide, disaccharide, or polysaccharide is selected from glyceraldehyde, erythrosyl, threoyl, arabinose, ribosyl, xylose, lythose; glucosyl, mannose, fructose, galactosyl, galacturonic acid, glucuronic acid, glucuronic acid, mannuronic acid, maltose, maltodextrin, lactose, lactobionic acid, sucrose, sophorose, chitobiose, xylobiose, pinobiose, mesobiobiose, mannobiose, gentiobiose, laminabiose, cellobiose, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

[0196] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 19 =0-12, n 20 =1-12, n 21 = Integers from 1 to 15, R2 is selected from hydrogen or C1-12 alkyl, and Y3 is selected from hydrogen, hydroxyl or C1-12 alkoxy.

[0197] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 19 =0-10, n 20 =1-10, n 21 = Integers from 1 to 12, R2 is selected from hydrogen or C1-10 alkyl, and Y3 is selected from hydrogen, hydroxyl or C1-10 alkoxy.

[0198] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 19 =0-8, n 20 =1-8, n 21 = an integer from 1 to 10, wherein R2 is selected from hydrogen or C1-8 alkyl, and Y3 is selected from hydrogen, hydroxyl or C1-8 alkoxy.

[0199] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the polysarcosine unit structure is as follows:

[0200] Where, n 22 = Integers from 1 to 100.

[0201] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 22 = Integers from 1 to 50.

[0202] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 22 = Integers from 1 to 30.

[0203] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 22 = Integers from 1 to 20.

[0204] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the polyethylene glycol unit structure is as follows:

[0205] Where, n 23 = Integers from 1 to 100, K2 is selected from hydrogen or C1-20 alkyl.

[0206] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 23 =1-50, wherein K2 is selected from hydrogen or C1-12 alkyl.

[0207] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 23 =1-30, wherein K2 is selected from hydrogen or C1-10 alkyl.

[0208] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 23 =1-20, wherein K2 is selected from hydrogen or C1-8 alkyl.

[0209] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the alkyl sulfonic acid unit structure is as follows:

[0210] Where, n 24 = Integers from 1 to 20.

[0211] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 24 = Integers from 1 to 12.

[0212] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n24 = Integers from 1 to 10.

[0213] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n 24 = Integers from 1 to 8.

[0214] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document,

[0215] n7=1-12, n8=1-12, n9=1-12, n 10 =1-12, n 11 =1-12, n 12 =1-12, n 13 =1-12, n 14 =1-12, n 15 =1-12, n 16 =1-12 or n 17 =Integers from 1 to 12,

[0216] The n 18 = Integers from 0 to 50; K1 is selected from hydrogen or C1-12 alkyl.

[0217] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document,

[0218] n7=1-10, n8=1-10, n9=1-10, n 10 =1-10, n 11 =1-10, n 12 =1-10, n 13 =1-10, n 14 =1-10, n 15 =1-10, n 16 =1-10 or n 17 =Integers from 1 to 10

[0219] The n 18 = Integers from 0 to 30; K1 is selected from hydrogen or C1-10 alkyl.

[0220] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document,

[0221] n7=1-8, n8=1-8, n9=1-8, n 10 =1-8, n 11 =1-8, n 12 =1-8, n 13 =1-8, n 14 =1-8, n15 =1-8, n 16 =1-8 or n 17 =Integers from 1 to 8

[0222] The n 18 = Integers from 0 to 20; K1 is selected from hydrogen or C1-8 alkyl.

[0223] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, n3 = 0-50, n4 = 0-50, n5 = 0-50 or n6 = 0-50.

[0224] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, n3 = 0-30, n4 = 0-30, n5 = 0-30 or n6 = 0-30.

[0225] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, n3 = 0-20, n4 = 0-20, n5 = 0-20 or n6 = 0-20.

[0226] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, E is selected from the azirconium crown ether group of a 9- to 18-membered ring, and the hydrophilic unit is selected from a sugar unit, wherein the sugar unit is selected from:

[0227] Where, n 19 Selected from integers 1 to 3, R2 is hydrogen or methyl, n 20 R3 is selected from integers from 1 to 3, and R3 is independently selected from H or C5 to C6 monosaccharides. 21 Choose from 3 or 4.

[0228] or Where, n 20 R3 is selected from integers from 1 to 3, and R3 is independently selected from H or C5 to C6 monosaccharides. 21 Choose from 3 or 4.

[0229] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker of the second aspect of this document, R3 contains one monosaccharide selected from C5 to C6, and the remainder is H.

[0230] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect of this document, E is selected from the azircon ethers of 9, 12, 15, and 18-membered rings.

[0231] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, E is selected from:

[0232] In this configuration, at least one X is a sugar unit, and the other Xs are independently selected from H or sugar units.

[0233] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the sugar unit is selected from:

[0234]

[0235] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, R3 is...

[0236] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, E is selected from:

[0237]

[0238]

[0239] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, the L2 is present and selected from... n3 is an integer selected from 8 to 12.

[0240] In some embodiments of the linker intermediate described in the first aspect of this document or the hydrophilic linker described in the second aspect of this document, n3 = 11.

[0241] The hydrophilic linker of the second aspect of this article comprises one or two of the described side chains.

[0242] In some embodiments of the linker intermediates described in the first aspect of this document or the hydrophilic linkers of the second aspect of this document, -L2-E is selected from the structures shown in the table below:

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249] In some embodiments of the hydrophilic linker of the second aspect of this document, a first linker subunit for linking to a target unit and a second linker subunit for linking to a drug are also included, wherein the first and second linker subunits are linked to the amino acid unit.

[0250] The first connector subunit for connecting the target unit and the second connector subunit for connecting to the drug, as referred to herein, can be selected by those skilled in the art based on the target unit and the drug to be connected.

[0251] In some embodiments of the hydrophilic linker of the second aspect of this article, the targeting unit is selected from antibodies or their antigen-binding fragments.

[0252] In some embodiments of the hydrophilic linker of the second aspect of this document, the amino acid unit comprises one or more amino acid subunits, the amino acid subunits being selected from:

[0253] Or one or more combinations of its stereoisomers.

[0254] * Represents the connection site with L2, or, when L2 is absent, the connection site with E;

[0255] # represents the connection site with the first linker subunit or amino acid subunit.

[0256] ## represents the connection site with the second linker subunit or amino acid subunit.

[0257] n 37 =0-20, n 38 =0-20, n 39 =0-20, n 40 =0-20, n 41 =0-20, n 42 =0-20, n 43 =0-20, n 44 =0-20, n 45 =0-20, n 46 =0-20, n 47 =0-20, n 48 =0-20, n 49 =0-20, n 50 =0-20, n 51 =0-20, n 52 =0-20, n 53 =0-20 and n 54 = Integers between 0 and 20.

[0258] In some embodiments of the hydrophilic linker of the second aspect of this article, n 37 =0-12, n 38 =0-12, n 39 =0-12, n 40 =0-12, n 41 =0-12, n 42 =0-12, n 43 =0-12, n 44 =0-12, n 45 =0-12, n 46 =0-12, n 47 =0-12, n 48 =0-12, n 49 =0-12, n 50 =0-12, n 51 =0-12, n 52 =0-12, n 53 =0-12 and n 54 = Integers between 0 and 12.

[0259] In some embodiments of the hydrophilic linker of the second aspect of this article, n 37 =0-10, n 38 =0-10, n 39 =0-10, n 40 =0-10, n 41 =0-10, n 42 =0-10, n 43 =0-10, n 44 =0-10, n 45 =0-10, n 46 =0-10, n 47 =0-10, n 48 =0-10, n 49 =0-10, n 50 =0-10, n 51 =0-10, n 52 =0-10, n 53 =0-10 and n 54 = Integers between 0 and 10.

[0260] In some embodiments of the hydrophilic linker of the second aspect of this article, n 37 =0-8, n 38 =0-8, n 39 =0-8, n 40 =0-8, n 41 =0-8, n 42 =0-8, n 43 =0-8, n44 =0-8, n 45 =0-8, n 46 = Integers from 0 to 8.

[0261] In some embodiments of the hydrophilic linker of the second aspect of this article, n 37 =1, n 38 =1, n 39 =1, n 40 =1, n 41 =1, n 42 =1, n 43 =1, n 44 =1, n 45 =1, n 46 An integer equal to 1.

[0262] The third aspect of this paper relates to linker-drug conjugates, which are formed by combining the aforementioned linker with a drug, and have the structure shown in Formula II.

[0263] in,

[0264] n2 is an integer from 1 to 8, A1 is selected from the amino acid subunits, and M is a connector for connecting to the target unit.

[0265] L1 does not exist or is selected from One or more combinations of n; where n 30 =1-20, n 31 =0-100, n 32 =1-20, n 33 =0-100, n 34 =0-100, n 35 =0-100 or n 36 =Integers between 0 and 20. The wavy line to the left of L1 indicates the site connected to M, and the wavy line to the right indicates the site connected to A1.

[0266] A1 is selected from Or its stereoisomers,

[0267] * Represents the connection site with L2, or, when L2 is absent, the connection site with E;

[0268] # represents the connection site with L1.

[0269] ## represents the connection site with A2.

[0270] n 37 =0-20, n 38 =0-20, n 39 =0-20, n40 =0-20, n 41 =0-20, n 42 =0-20, n 43 =0-20, n 44 =0-20, n 45 =0-20, n 46 =0-20, n 47 =0-20, n 48 =0-20, n 49 =0-20, n 50 =0-20, n 51 =0-20, n 52 =0-20, n 53 =0-20 and n 54 =Integers between 0 and 20

[0271] A2 does not exist or is selected from:

[0272] The wavy line to the left of A2 indicates the site connected to A1, and the wavy line to the right indicates the site connected to D.

[0273] The term D refers to a fragment formed by the loss of one or more atoms or groups from a molecule with antitumor biological activity.

[0274] The antitumor bioactive molecules include, but are not limited to, microtubule inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, intercalating agents, DNA-reacting agents, DNA alkylating agents, immunomodulators, nucleic acids, BCL-XL inhibitors, kinase inhibitors, anti-angiogenic agents, PROTAC, molecular glues, and radioisotopes. Microtubule inhibitors include, for example, monomethyl auristatin. Auristatin (e.g., methylauritstatin E or methylauritstatin F, etc.), maytansinoid (e.g., maytansinol, DM1, DM2, DM3 or DM4, etc.) or eribulin; RNA polymerase inhibitors such as α-amanitin and its derivatives; topoisomerase inhibitors such as etoposide, teniposide, acridine, PNU-159682, SN-38, eczetidine, DXD and their derivatives; intercalators such as pyrrolobenzodiazepine (PBD) and its derivatives. etc.; DNA-reactive agents such as chazim, tiancimycins, other enediynes and their derivatives, etc.; DNA alkylating agents such as trabectedin, rubitidine and other senna derivatives, etc.; immunomodulators such as STING, TLR7, TLR8 and / or TLR9 agonists, PD-1 inhibitors and their derivatives, etc.; anti-angiogenic agents including linolamine, bevacizumab, angiostatin and raszosen, etc.; PROTAC protein degradation targeting chimeras such as those described in US2018228907, US2016058872, US2017327469, etc.; molecular gels such as cyclosporin A. A) Tacrolimus, rapamycin, thalidomide, lenalidomide, pomalidomide, indisulam and their derivatives; radioactive isotopes include iodine (131I), yttrium (90Y), technetium (99Tc), copper (67Cu), gallium (66Ga), rhenium (188Re), rhenium (186Re), indium (67In), indium (111In), indium (114... I Indium (115In), strontium (89Sr), strontium (153Sr), lutetium (177Lu), actinium (225Ac), and lead (212Pb), etc. The aforementioned antitumor bioactive molecules can covalently bind to linkers by losing one or more atoms or groups through known binding sites and binding modes.

[0275] In some embodiments of the linker-drug conjugates of the third aspect of this article, M is selected from... Or its stereoisomers.

[0276] In some embodiments of the linker-drug conjugates of the third aspect of this article, the M-L2- is

[0277] In some embodiments of the linker-drug conjugate of the third aspect of this article, A2 is...

[0278] In some embodiments of the linker-drug conjugates of the third aspect of this article, n 30 =1-12, n 31 =0-50, n 32 =1-12, n 33 =0-50, n 34 =0-50, n 35 =0-50 or n 36 = Integers between 0 and 12.

[0279] In some embodiments of the linker-drug conjugates of the third aspect of this article, n 30 =1-10, n 31 =0-30, n 32 =1-10, n 33 =0-30, n 34 =0-30, n 35 =0-30 and n 36 = Integers between 0 and 10.

[0280] In some embodiments of the linker-drug conjugates of the third aspect of this article, n 30 =1-8, n 31 =0-20, n 32 =1-8 and n 33 = Integers between 0 and 20.

[0281] In some embodiments of the linker-drug conjugates of the third aspect of this article, n2 is an integer from 1 to 8.

[0282] In some embodiments of the linker-drug conjugates of the third aspect of this article, n2 is an integer from 1 to 6.

[0283] In some embodiments of the linker-drug conjugates of the third aspect of this article, n2 is an integer from 1 to 4.

[0284] In some embodiments of the linker-drug conjugate of the third aspect of this article, n2 is 1 or 2.

[0285] In some embodiments of the linker-drug conjugates of the third aspect of this article, the bioactive molecule is selected from microtubule inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, intercalators, DNA-reactants, DNA alkylating agents, immunomodulators, nucleic acids, BCL-XL inhibitors, kinase inhibitors, PROTACs, molecular glues, and radioisotopes.

[0286] In some embodiments of the linker-drug conjugates of the third aspect of this invention, the linker is covalently bonded to nitrogen, oxygen, or sulfur in the drug. In some embodiments of the linker-drug conjugates of the third aspect of this invention, any structure selected from LP1 to 47 is used:

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] The fourth aspect of this article relates to antibody-drug conjugates, obtained by conjugating the aforementioned linker-drug with an antibody, with the structure shown in general formula III. in,

[0300] Ab is an antibody or antigen-binding fragment, and n1 is any value between 1 and 20.

[0301] The antibody-drug conjugates involved in the fourth aspect of this article are selected from any one of ADC-1 to 67:

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315] The fourth aspect of this document relates to some examples of antibody-drug conjugates, wherein the antibodies are monoclonal antibodies selected from: abciximab, alemtuzumab, anetumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatomumab, brentuximab, catutoxomab, cetuximab, and cimetuzumab. umab), Coltuximab, Daclizumab, Daratumumab, Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab, Disitamab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab ), Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Lifastuzumab, Lorvotuzumab, Milatuzumab, Mirvetuximab, Naratu (ximab), Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratuzumab, Omalizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab, PinatuzumabPolotuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab, Sirtratumab, Sofituzumab, Vadastuximab, Vorsetuzumab, Trastuzumab, Tisotumab, anti-B7-H3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody, and anti-CD30 antibody, or their antigen-binding fragments or immunologically active portions.

[0316] The fifth aspect of this article relates to a method for preparing the aforementioned intermediate, wherein the sugar unit is... Its features include the following steps:

[0317] CbzHN-L2-azacrown ethers were synthesized from CbzHN-L2-azacrown ethers via a bimolecular nucleophilic substitution reaction.

[0318] CbzHN-L2-azacrown ether and CbzHN-L2-E was prepared by reductive amination.

[0319] CbzHN-L2-E can be optionally synthesized via a Cbz removal reaction to form H2N-L2-E.

[0320] The (n) here 20 )-1 refers to the difference between the selected n and n. 20 There is one less than the number.

[0321] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, E is selected from...

[0322]

[0323] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, E is selected from...

[0324]

[0325] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, E is selected from:

[0326] In the intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect, the -L2-E segments are selected from the structures shown in H1 to H12.

[0327] In the intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect, the -L2-E segments are selected from H1, H2, H5, H6, H7, H8, H10, and H11.

[0328] In the intermediates described in the first aspect of this document or the hydrophilic linkers described in the second aspect, the -L2-E segment is selected from H1.

[0329] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, the sugar units are selected from:

[0330]

[0331] In the intermediates described in the first aspect or the hydrophilic linkers described in the second aspect of this document, the sugar units are selected from:

[0332]

[0333] Antibody-drug conjugates prepared using the hydrophilic linkers described in this paper can improve hydrophilic properties. They are particularly suitable for use with different hydrophobic loadings to generate antibody-drug conjugates with high DAR values, thereby reducing or avoiding aggregation problems.

[0334] Based on the aforementioned improvements in hydrophilicity, the prepared antibody-drug conjugates are expected to further enhance in vitro activity and in vivo efficacy. In some specific embodiments, the antibody-drug conjugates prepared using the linker described herein all exhibited strong in vitro cytotoxicity (<1 nM) and good in vivo efficacy. For example, in the JIMT-1 tumor-bearing mouse efficacy evaluation experiment, the antibody-drug conjugate ADC-23 based on the hydrophilic linker showed better efficacy than the reference ADC Comparative Example-1. This paper successfully prepared a novel intermediate, which can be used to prepare hydrophilic linkers.

[0335] The antibody-drug conjugates prepared using the novel hydrophilic linker presented in this article exhibit shorter retention times on HIC HPLC.

[0336] The invention is further illustrated below by way of examples, but this does not limit the scope of the embodiments described herein. Experimental methods not specifically described in the following examples are performed according to conventional methods and conditions, or as selected in the product manual.

[0337] Synthetic route for linker intermediates

[0338] H2N-L2-E can be synthesized according to the following method:

[0339] Synthesis Route 1:

[0340]

[0341] Step 1

[0342] Compound I-intermediate 2 is obtained by a bimolecular nucleophilic substitution reaction (SN2) between compound I-intermediate 1 and compound I-starter 1.

[0343] Step 2

[0344] Compound I-intermediate 3 is obtained by reductive amination of compound I-intermediate 2 and a carbohydrate compound.

[0345] Step 3

[0346] Compound of general formula I-1 is obtained by deCbz reaction of compound of general formula I-intermediate 3.

[0347] Synthesis Route 2:

[0348]

[0349] Step 1

[0350] Compound I-intermediate 4 is obtained by reductive amination of compound I-intermediate 2 with a phosphorylcholine compound containing an aldehyde group.

[0351] Step 2

[0352] Compound of general formula I-2 is obtained by deCbz reaction of compound of general formula I-intermediate 4.

[0353] Synthesis route 3 is as follows:

[0354]

[0355] Step 1

[0356] Compound I-intermediate 5 is obtained by a bimolecular nucleophilic substitution (SN2) reaction between compound I-intermediate 2 and a polyethylene glycol compound containing -OTs or halogen substituents.

[0357] Step 2

[0358] Compound of general formula I-3 is obtained by deCbz reaction of compound of general formula I-intermediate 5.

[0359] Synthesis route 4 is as follows:

[0360]

[0361] Step 1

[0362] Compound I-intermediate 6 is obtained by a bimolecular nucleophilic substitution (SN2) reaction between compound I-intermediate 2 and an alkyl sulfonic acid compound containing -OTs or halogen substituents.

[0363] Step 2

[0364] Compound of general formula I-4 is obtained by deCbz reaction of compound of general formula I-intermediate 6.

[0365] Synthesis route 5 is as follows:

[0366]

[0367] Step 1

[0368] Compound I-intermediate 7 is obtained by condensation reaction of compound I-intermediate 2 with a carboxyl-containing polysarcosine (poly-N-methylglycine) compound or its activated ester.

[0369] Step 2

[0370] Compound of general formula I-5 is obtained by deCbz reaction of compound of general formula I-intermediate 7.

[0371] Synthesis Route 6

[0372]

[0373] Step 1

[0374] Compound I-intermediate 8 is obtained by condensation reaction of compound I-intermediate 2 with a carboxyl-containing phosphorylcholine compound or its activated ester.

[0375] Step 2

[0376] Compound of general formula I-6 is obtained by deCbz reaction of compound of general formula I-intermediate 8.

[0377] Synthesis method of HO2C-L2-E:

[0378] Synthesis Route 1:

[0379]

[0380] Step 1

[0381] Compound I-intermediate 10 is obtained by a bimolecular nucleophilic substitution reaction (SN2) between compound I-intermediate 9 and compound I-starter 1.

[0382] Step 2

[0383] Compound I-intermediate 11 is obtained by reductive amination of compound I-intermediate 10 and a carbohydrate compound.

[0384] Step 3

[0385] Compound of general formula I-7 is obtained by debenzylation of compound of general formula I-intermediate 11.

[0386] Synthesis Route 2:

[0387]

[0388] Step 1

[0389] Compound I-intermediate 12 is obtained by reductive amination of compound I-intermediate 10 with a phosphorylcholine compound containing an aldehyde group.

[0390] Step 2

[0391] Compound of general formula I-8 is obtained by debenzylation of compound of general formula I-intermediate 12.

[0392] Synthesis Route 3:

[0393]

[0394] Step 1

[0395] Compound I-intermediate 13 is obtained by a bimolecular nucleophilic substitution (SN2) reaction between compound I-intermediate 10 and a polyethylene glycol compound containing -OTs or halogen substituents.

[0396] Step 2

[0397] Compound of general formula I-9 is obtained by debenzylation of compound of general formula I-intermediate 13.

[0398] Synthesis Route 4:

[0399]

[0400] Step 1

[0401] Compound I-intermediate 14 is obtained by a bimolecular nucleophilic substitution (SN2) reaction between compound I-intermediate 10 and an alkyl sulfonic acid compound containing -OTs or halogen substituents.

[0402] Step 2

[0403] Compound of general formula I-10 is obtained by debenzylation of compound of general formula I-intermediate 14.

[0404] Synthesis Route 5:

[0405]

[0406] Step 1

[0407] Compound I-Intermediate 15 is obtained by condensation reaction of compound I-Intermediate 10 with a carboxyl-containing polysarcosine (poly-N-methylglycine) compound or its activated ester.

[0408] Step 2

[0409] Compound of general formula I-11 is obtained by debenzylation of compound of general formula I-intermediate 15.

[0410] Synthesis Route 6

[0411]

[0412] Step 1

[0413] Compound I-intermediate 16 is obtained by condensation reaction of compound I-intermediate 10 with a carboxyl-containing phosphorylcholine compound or its activated ester.

[0414] Step 2

[0415] Compound of general formula I-12 is obtained by debenzylation of compound of general formula I-intermediate 16.

[0416] It should be noted that the azacrown ether portion of the compounds in general formulas I-1 to I-12 is not limited to the examples described herein, but may also be other crown ether forms described herein.

[0417] The intermediates for preparing the linkers described above include, but are not limited to, any of the structures numbered H1 to H44 above, where the wavy lines represent amino, carboxyl, or protected forms in the aforementioned pathway.

[0418] Example: Preparation of linker intermediates LP11-10 and LP11-11

[0419]

[0420] Step 1: Synthesis of LP11-7

[0421] Et3N (835 mg, 8.25 mmol) was added to a solution of LP11-6 (4.5 g, 8.25 mmol) and CbzOSu (2.67 g, 10.73 mmol) in anhydrous tetrahydrofuran (40 mL). The mixture was stirred at room temperature for 2 hours, and LC-MS showed that the reaction was complete. The solvent was removed by concentration, and after adding an appropriate amount of water, the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the target product (5.8 g, crude product) as a yellow oil.

[0422] LC-MS (ESI) m / z: 680.7 [M+H] +

[0423] Step 2: Synthesis of LP11-8

[0424] LP11-7 (5.6 g, 8.24 mmol), TsCl (2.35 g, 12.36 mmol), Et3N (2.5 g, 24.72 mmol), and DMAP (503 mg, 4.12 mmol) were dissolved in dichloromethane (35 mL) and stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The mixture was washed with water, extracted with dichloromethane, and the organic layer was concentrated. Purification was performed by rapid silica gel column chromatography (DCM / MeOH = 100% - 30:1) to obtain the target product (4.9 g, yield 71.3%) as a yellow oil.

[0425] LC-MS (ESI) m / z: 834.7 [M+H] +

[0426] 1 H NMR(400MHz,Chloroform-d)δ7.81–7.75(m,2H),7.38–7.27(m,7H),5.51(s,1H),5.08(s,2 H),3.70–3.65(m,2H),3.64–3.58(m,38H),3.57(s,6H),3.38(q,J=5.3Hz,2H),2.44(s,3H).

[0427] Step 3: Synthesis of LP11-9

[0428] LP11-8 (2.3 g, 2.76 mmol), 1,4,7-triazacyclononane (2.85 g, 22 mmol), and K2CO3 (1.52 g, 11 mmol) were dissolved in acetonitrile (40 mL) and stirred overnight at room temperature. LCMS showed that the reaction was complete. The mixture was filtered, and the filtrate was concentrated. The filtrate was purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (1.6 g, yield 36.3%) as a colorless oil.

[0429] LC-MS (ESI) m / z: 791.8 [M+H] +

[0430] 1 H NMR(400MHz,Chloroform-d)δ7.37–7.27(m,5H),5.55(s,1H),5.08(s,2H),3.6 4–3.57(m,39H),3.57–3.50(m,4H),3.37(q,J=5.3Hz,2H),2.81–2.50(m,18H).

[0431] Step 4: Synthesis of LP11-10

[0432] LP11-9 (703 mg, 0.9 mmol), glucose (3.24 g, 18 mmol), and acetic acid (1.08 g, 18 mmol) were dissolved in anhydrous methanol (80 mL) and stirred at 45 °C for 1 hour. Sodium cyanoborohydride (1.3 g, 20.7 mmol) was added to the solution and stirred at 45 °C for 16 hours. LC-MS showed that the reaction was complete. The solvent was removed by concentration, and the product was purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (850 mg, yield 84.3%) as a colorless oil.

[0433] LC-MS (ESI) m / z: 1120.2 [M+H] +

[0434] 1 H NMR(400MHz,Chloroform-d)δ7.41–7.31(m,5H),5.12(s,2H),4.04(s,3H),3.76(s, 7H),3.73–3.61(m,44H),3.59(t,J=5.1Hz,5H),3.41(q,J=5.4Hz,4H),3.08(s,17H).

[0435] Step 5: Synthesis of LP11-11

[0436] LP11-10 (1 g, 0.89 mmol) was dissolved in TFA (5 mL) and stirred overnight at room temperature. LCMS showed that the reaction was complete. The solution was diluted with 25 mL of water and purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (735 mg, yield 83.5%) as a colorless oil.

[0437] LC-MS (ESI) m / z: 986.0 [M+H] +

[0438] Linker-drug conjugate examples

[0439] The linkers described herein and drugs can be prepared into linker-drug conjugates using known chemical methods. These include, but are not limited to, any of the structures numbered LP1–LP67 above. Synthesis can be performed using the specific preparation methods for LP11, LP13, or LP23.

[0440] Example: Synthesis of compound LP11

[0441] The synthesis route is shown below:

[0442]

[0443] Step 1: Synthesis of LP11-2

[0444] LP11-1 (500 mg, 0.68 mmol) was dissolved in EtOH / EA (3 / 2, v / v, 5 mL). 50 mg of 10% wet Pd / C was added, and the mixture was stirred at room temperature for 3 hours under H2 protection. LC-MS showed the reaction was complete. The mixture was filtered, and the filter cake was washed with DMF (10 mL). The filtrate was concentrated and purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (137 mg, yield 31.2%) as a white solid.

[0445] LC-MS (ESI) m / z: 668.2 [M+Na] +

[0446] Steps 2 and 3: Synthesis of LP11-5

[0447] LP11-2 (97 mg, 0.15 mmol), LP11-3 (75 mg, 0.1 mmol), HOAt (68 mg, 0.5 mmol), and DMAP (6.1 mg, 0.05 mmol) were dissolved in DMF (4 mL), and then EDCI (96 mg, 0.5 mmol) was added. The mixture was stirred at room temperature for 1–2 hours. LCMS showed that the reaction was complete. Without further purification, 0.4 mL of diethylamine was added and the mixture was stirred at 0 °C for 30 minutes. LCMS showed that the reaction was complete. The solution was purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (48.35 mg, yield 42.2%) as a white solid.

[0448] LC-MS (ESI) m / z: 1176.1 [M+H] +

[0449] Step 4: Synthesis of LP11-13

[0450] DIEA (183 mg, 1.42 mmol) was added to a DMF (5 mL) solution of LP11-11 (700 mg, 0.71 mmol) and LP11-12 (371 mg, 0.71 mmol) and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The solution was purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (135 mg, yield 13.6%) as a yellow oil.

[0451] LC-MS (ESI) m / z: 697.2 [M / 2+H] +

[0452] Step 5: Synthesis of LP11-14.

[0453] LP11-13 (135 mg, 0.097 mmol) was dissolved in 6 M HCl (2 mL) and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The solution was purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (117 mg, yield 87.7%) as a yellow oil.

[0454] LC-MS (ESI) m / z: 669.2 [M / 2+H] +

[0455] Steps 6 and 7: Synthesis of LP11-16

[0456] LP11-14 (20 mg, 0.015 mmol), HATU (10.26 mg, 0.027 mmol), and DIEA (3.88 mg, 0.03 mmol) were dissolved in DMF (2 mL) and stirred at 0 °C for 15 min. Then, LP11-5 (17.63 mg, 0.015 mmol) was added and stirred at 0 °C for 2 h. LCMS showed the reaction was complete. Without further purification, 0.2 mL of diethylamine was added and stirred at 0 °C for 30 min. LCMS showed the reaction was complete. The solution was purified by reversed-phase column chromatography (0.1% FA in water / CH3CN) to give the target product (12 mg, yield 35.2%) as a white solid.

[0457] LC-MS (ESI) m / z: 1136.5 [M / 2+H] +

[0458] Step 8: Synthesize LP11

[0459] 6-maleiminohexanoic acid (2.45 mg, 0.0116 mmol), HATU (6.58 mg, 0.0133 mmol), and DIEA (2.7 mg, 0.021 mmol) were dissolved in DMF (1 mL) and stirred at 0 °C for 30 min. Then, LP11-16 (24 mg, 0.0105 mmol) was added, and the mixture was stirred at 0 °C for 2 h. LCMS showed that the reaction was complete. The target product (19.95 mg, 76.6% yield) was purified by preparative HPLC (0.1% FA in water / CH3CN) as a white solid (76.6%).

[0460] LC-MS (ESI) m / z: 1233.2 [M / 2+H] +

[0461] 1 H NMR (400MHz, DMSO-d6) δ8.73(d,J=7.5Hz,2H),8.67(s,1H),8.34(d,J=6.3Hz,1H),8.26(s,1 H),8.14(d,J=8.8Hz,2H),8.04(t,J=5.8Hz,1H),8.00(d,J=7.5Hz,1H),7.87(d,J=5.7Hz,1H) ,7.75(s,1H),7.27–7.14(m,6H),6.99(s,2H),6.43(d,J=7.0Hz,2H),6.14(s,2H),5.04(d,J= 11.5Hz,1H),4.67(d,J=7.0Hz,3H),4.49(s,3H),4.19(t,J=6.4Hz,2H),4.11(d,J=1.9Hz,1H) ,4.06(d,J=10.9Hz,1H),4.02(s,2H),3.83(s,3H),3.70(d,J=5.6Hz,6H),3.63(s,5H),3.61( d,J=2.8Hz,8H),3.56(s,6H),3.53–3.49(m,74H),3.47–3.42(m,14H),3.24–3.16(m,6H),3.0 3(d,J=11.1Hz,5H),2.89–2.73(m,15H),2.36(d,J=16.9Hz,1H),2.29(s,3H),2.21(s,4H),2. 15(d,J=14.8Hz,1H),2.10(t,J=7.6Hz,5H),2.05(s,4H),1.97(s,3H),1.47(d,J=9.8Hz,8H).

[0462] Example: Synthesis of compound LP13

[0463]

[0464] Steps 1 and 2: Synthesis of LP13-2

[0465] LP11-14 (26.5 mg, 0.0198 mmol), HATU (14 mg, 0.0357 mmol), and DIEA (8 mg, 0.059 mmol) were dissolved in DMF (2 mL) at 0 °C and stirred for 30 minutes. LP11-16 (45 mg, 0.0198 mmol) was added and stirred for 1 hour. LCMS showed the reaction was complete. Diethylamine (0.4 mL) was added directly without further purification. The reaction solution was purified by preparative high-performance liquid chromatography to obtain the target product (20 mg, yield 50.9%) as a colorless oil.

[0466] LC-MS (ESI) m / z: 1123.6 [M / 3+H] +

[0467] Step 2: Synthesize LP13

[0468] 6-maleimide hexanoic acid (1.4 mg, 0.0065 mmol), HATU (3.7 mg, 0.0098 mmol), and DIEA (1.7 mg, 0.013 mmol) were dissolved in DMF (3 mL) and stirred at 0 °C for 30 min. Then, LP13-2 (20 mg, 0.0059 mmol) was added and stirred at 0 °C for 2 h. LCMS showed that the reaction was complete. The target product (2.58 mg, yield 12.2%) was purified by preparative high performance liquid chromatography (0.1% FAinwater / CH3CN) to obtain a white solid.

[0469] LC-MS (ESI) m / z: 1188.1 [M / 3+H] +

[0470] Example: Synthesis of compound LP23

[0471]

[0472] Step 1: Synthesis of LP23-3

[0473] Add LP23-2 (221 mg, 0.283 mmol, 1.0 eq.) to a DMF (3.0 mL) solution of LP23-1 (150.2 mg, 0.283 mmol, 1.0 eq.) and DIPEA (0.1 mL, 0.574 mmol, 2.0 eq.). Stir the mixture at room temperature for 5 hours. Use the crude product solution for the next step without further purification.

[0474] LC-MS (ESI) m / z: 1063.3 [M+H] +

[0475] Step 2: Synthesis of LP23-4

[0476] Diethylamine (0.29 mL, 2.8 mmol, 10.0 eq.) was added to the above crude product solution (crude product, 0.283 mmol, 1.0 eq.). The reaction solution was stirred at room temperature for 1 hour, and then purified by preparative liquid chromatography (0.1% NH4HCO3 inwater / CH3CN) to give the desired product (57.5 mg, two-step yield: 24.2%) as a white solid.

[0477] LC-MS (ESI) m / z: 841.3 [M+H] +

[0478] Step 3: Synthesis of LP23-5

[0479] To a DMF (1.9 mL) solution of LP23-4 (57.53 mg, 0.068 mmol, 1.0 eq.), LP11-14 (91.4 mg, 0.068 mmol, 1.0 eq.), DIEA (0.072 mL, 0.413 mmol, 6.0 eq.), and HATU (41.6 mg, 0.109 mmol, 1.6 eq.) were added. The resulting reaction solution was stirred at room temperature for 1 hour. The crude product solution was used for the next step without further purification.

[0480] LC-MS (ESI) m / z: 1079.7 [M / 2+H] +

[0481] Step 4: Synthesis of LP23-6

[0482] Diethylamine (0.07 mL, 0.68 mmol, 10.0 eq.) was added to the crude product solution of LP23-5 (crude product, 0.068 mmol, 1.0 eq.). The reaction solution was stirred at room temperature for 1 hour, and then purified by preparative liquid chromatography (0.1% NH4HCO3 in water / CH3CN) to give the desired product (22.1 mg, two-step yield: 16.8%) as a white solid.

[0483] LC-MS (ESI) m / z: 646.5 [M / 3+H] +

[0484] Step 5: Synthesis of LP23

[0485] A mixture of 6-maleimide malonic acid (2.8 mg, 0.0133 mmol, 1.2 eq.), LP23-6 (22.1 mg, 0.0114 mmol, 1.0 eq.), HATU (7.3 mg, 0.019 mmol, 1.7 eq.), and DIEA (0.008 mL, 0.046 mmol, 4.0 eq.) in DMF (0.3 mL) was stirred at room temperature for 1 hour, and then purified by preparative liquid chromatography (0.1% TFA in water / CH3CN) to give the desired product (5.4 mg, yield: 22.2%) as a white solid.

[0486] LC-MS (ESI) m / z: 1065.8 [M / 2+H] +

[0487] 1 H NMR (400MHz, DMSO-d6) δ10.02(s,1H),8.15(d,J=8.0Hz,1H),8.05(d,J=8.0Hz,1H) ,8.00(d,J=8.0Hz,1H),7.85(dd,J1=J2=6.0Hz,1H),7.78(d,J=6.0Hz,1H),7.67(d ,J=8.0Hz,1H),7.60(d,J=8.0Hz,2H),7.36(d,J=8.0Hz,2H),7.32(s,1H),6.98(s, 2H),6.52(br.,1H),5.99(br.,1H),5.45(s,3H),5.29(s,4H),5.08(s,3H),4.41–4. 35(m,2H),4.27–4.18(m,3H),3.93-3.80(m,4H),3.62–3.58(m,8H),3.53-3.49(m, 54H),3.17–3.19(m,5H),2.96-2.89(m,5H),2.69–2.66(m,1H),2.38(s,3H),2.34– 2.32(m,1H),2.25-2.07(m,7H),2.01-1.95(m,1H),1.92-1.82(m,3H),1.74-1.65( m,2H),1.62-1.55(m,1H),1.52–1.32(m,7H),1.24-1.17(m,3H),0.90-0.81(m,9H).

[0488] Preparation and characterization of antibody drug conjugates

[0489] The following are examples of methods for the preparation and analytical characterization of antibody-drug conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments described herein. For example, the antibody is not limited to the antibody shown herein, but can be any other antibody. The preparation methods described below can be used to prepare structures including, but not limited to, those shown in ADC1–67 above.

[0490] A universal conjugation process for preparing antibody-drug conjugates based on hydrophilic stable linkers

[0491] Place 1.0 eq. of monoclonal antibody in a 0.5 mL centrifuge tube, add 25 mM Na2B4O7, 25 mM NaCl, and 1 mM DTPA buffer to dilute the antibody concentration to 5-10 mg / mL. After vortexing and mixing, add 2 mg / mL TCEP (2 eq. to 10 eq.) aqueous solution to the mixture for antibody reduction. After vortexing and mixing, place the reaction mixture on a thermostatic mixer at 20-25°C for 2-3 hours. Then add 4 eq. to 30 eq. of toxin linker solution dissolved in 10 mg / mL DMSO, and supplement with DMSO at 10-20% of the final reaction volume. After vortexing and mixing, place the mixture on a thermostatic mixer at 20-25°C for 0.5-4 hours for coupling.

[0492] Use 300 mg / mL dextran-coated activated charcoal (manufacturer: Sigma) to remove residual toxin linkers not conjugated with the antibody from the reaction solution. Add 10% (v / v) of activated charcoal solution to the reaction solution, mix well, and shake at 20-25°C for 1-2 hours. Take the supernatant to test the content of residual toxin linkers. If the content of free toxin linkers is greater than 1%, the above process should be repeated multiple times until the test results meet the standard (usually 3 treatments are sufficient). After treatment, centrifuge the mixture, collect the supernatant using a syringe and a hydrophilic membrane filter, and filter out the activated charcoal. Replace the buffer solution for the desired antibody-drug conjugate with a suitable storage buffer through 3-6 ultrafiltrations and store at -80°C.

[0493] General characterization methods for antibody-drug conjugates

[0494] (a) DAR value of ADC analyzed by HIC-HPLC

[0495] High performance liquid chromatograph: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0496] Column: MabPac TM HIC-Butyl 5μm 4.6×100mm (Manufacturer: Thermo).

[0497] Mobile phase: Mobile phase A (MPA): 1.5M (NH4)2SO4 + 50mM potassium phosphate (pH 7.0); Mobile phase B (MPB): 50mM sodium phosphate (pH 7.0) / isopropanol (75:25V / V); Elution was performed according to the following elution program (5%-95%), wherein: 0-2 min, mobile phase A volume was 100%-95%, mobile phase B volume was 0%-5%; 2-22 min, mobile phase A volume was 95%-5%, mobile phase B volume was 5%-95%; 22-24 min, mobile phase A volume was 5%-0%, mobile phase B volume was 95%-100%; 24-26 min, mobile phase A volume was 0%-100%, mobile phase B volume was 100%-0%; 26-30 min, mobile phase A volume was 100%-100%, mobile phase B volume was 0%-0%.

[0498] Detection conditions: The mobile phase flow rate was set to 1 ml / min, the detection wavelength to 280 nm, and the column temperature to 30 ℃.

[0499] Experimental procedure: Take 50 μg of the coupled sample (volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0500] DAR value calculation formula: DAR = Σ(relative peak area × number of loaded drugs) / 100

[0501] (b) C18-HPLC analysis of Free Linker Payload (mol / mol%)

[0502] High performance liquid chromatograph: e2695 high performance liquid chromatography system.

[0503] Chromatographic column: C18 3.5μm 4.6×150mm (Manufacturer: Waters).

[0504] Mobile phases: Mobile phase A (MPA): 0.1% TFA-H2O; Mobile phase B (MPB): 0.1% TFA-ACN; Elution was performed according to the following program (5%-95%), wherein: 0-30 min, mobile phase A volume was 90%-20%, mobile phase B volume was 10%-80%; 30-31 min, mobile phase A volume was 20%-90%, mobile phase B volume was 80%-10%; 31-35 min, mobile phase A volume was 90%-90%, mobile phase B volume was 10%-10%.

[0505] Detection conditions: The mobile phase flow rate was set to 0.5 ml / min, the detection wavelength to 254 nm, and the column temperature to 30 °C.

[0506] Example of reagent I preparation: Measure 30 mL of anhydrous methanol and 50 mL of acetonitrile, weigh 10 g of sodium chloride, mix them in a container, stir at room temperature for at least 1 hour, and then let stand for 1 hour. Take the supernatant, filter it through a 0.22 μm organic membrane, store at room temperature, and it has a shelf life of 3 months.

[0507] Example of Reagent II preparation: Measure 100 mL of Reagent I, 15 mL of DMSO, and 85 mL of ADC sample storage buffer, mix them thoroughly in a container, store at room temperature, and the shelf life is 2 months.

[0508] Experimental steps:

[0509] Sample solution preparation:

[0510] Free toxin reference: Take the toxin corresponding to the test sample as the reference and prepare it with the above reagent II to a final concentration of 1.0 mg / ml.

[0511] Take 10 μl of the above-mentioned toxin reference standard with a concentration of 1.0 mg / ml and add it to 90 μl of reagent II to prepare a concentration of 100 μg / ml; then dilute the sample sequentially with reagent II according to the table below to prepare the sample of the required concentration.

[0512] Sample number Sample concentration to be taken Volume of sample to be taken Volume of reagent II to be taken Concentration required to prepare sample 1 100 μg / ml 40 μl 360 μl 10 μg / ml 2 10 μg / ml 180 μl 180 μl 5.0 μg / ml 3 5.0 μg / ml 120 μl 180 μl 2.0 μg / ml 4 2.0 μg / ml 180 μl 180 μl 1.0 μg / ml 5 1.0 μg / ml 180 μl 180 μl 0.5 μg / ml 6 0.5 μg / ml 120 μl 180 μl 0.2 μg / ml 7 0.2 μg / ml 180 μl 180 μl 0.1 μg / ml

[0513] Note: Inject the samples into the HPLC in ascending order of concentration to prepare the required concentration.

[0514] Sample testing: Take 85 μg of the coupled sample, mix it with 3 μl of DMSO for 5 min, then add 60 μl of reagent I to the sample system, mix well for 5-10 min, centrifuge at 2000 rpm for 2 min, take 20 μL of the supernatant, inject it into the high performance liquid chromatograph, elute using the above elution program, and record the chromatogram.

[0515] The content of free toxins in the ADC was calculated based on the obtained standard bivariate linear regression curve equation.

[0516] Free Drug (mol / mol%) = Residual small molecule molar concentration / Antibody molar concentration × 100.

[0517] (c) Determination of free linker-payload content by LC-MS / MS method

[0518] Internal control compounds: 50 ng / ml tolbutamide, labetalol, etofesamide, and buspirone

[0519] Extractant: Ethyl acetate

[0520] Gradual dilution of standards: Dilute standards with 50% methanol aqueous solution to 20000, 16000, 10000, 4000, 1000, 200, 40, and 20 ng / ml.

[0521] Sample pretreatment: Take 20 μL of sample, then add 20 μL of blank buffer and 240 μL of extraction solvent. Vortex mix, let stand until layering, then take 160 μL of supernatant into a 96-well plate and dry it. Add 200 μL of 50% acetonitrile water, mix well, and finally perform LC-MS / MS analysis.

[0522] Pretreatment of standards before use: Add 2 μL of working solution to 38 μL of blank buffer, then add 240 μL of extraction solvent. Vortex to mix and allow to stand until separation occurs. Take 160 μL of supernatant and place it in a 96-well plate. Dry the plate and add 200 μL of 50% acetonitrile solution. Mix well.

[0523] Mass spectrometry conditions:

[0524]

[0525] Data processing: A standard curve is generated based on the concentration of the standard and the corresponding mass spectrometry response value. The residual amount of free linker-payload is calculated by linearly fitting the curve (formula) and the mass spectrometry response value of the sample.

[0526] (d) SEC-HPLC analysis of ADC aggregates

[0527] High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

[0528] Column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0529] Mobile phase: 50mM PB + 200mM Arg (pH 6.80) + 10% IPA. Elute according to the following procedure, with the volume of mobile phase A being 100%-100% for 0-30 min.

[0530] Detection conditions: The mobile phase flow rate was set to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 26 ℃.

[0531] Experimental procedure: Take 20 μg of the coupled sample (volume depends on sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0532] Calculation formulas: Monomer purity (%) = (A monomer / A total) × 100%; Polymer purity (%) = (A polymer / A total) × 100%

[0533] (e) ADC products were analyzed using an LC / TOF (Agilent, 6230LC / TOF) liquid chromatography-mass spectrometry system.

[0534] The liquid chromatography column was a PLRP-S, 1000A column (Agilent 8μm, 2.1×150mm).

[0535] Mobile phase A is 0.1% formic acid.

[0536] Mobile phase B is 0.1% formic acid acetonitrile. The flow rate is 0.4 mL / min.

[0537] For mass spectrometry analysis of positive ions, the parameters are set as follows: scan range 500-5000 m / z.

[0538] Gas temperature: 320℃

[0539] VCap: 4500V

[0540] Fragmenter: 200V

[0541] Sampling device (Skimmer): 65V.

[0542] Comparative Example: Preparation of Antibody-Drug Conjugate Comparative Example-1

[0543]

[0544] Following the general conjugation procedure for preparing antibody-drug conjugates, TCEP (10 mg / mL, 12 eq.) aqueous solution was added to 50 mM PBS pH 7.0 buffer containing trastuzumab (5 mg, 21.07 mg / mL, 1.0 eq.), and the mixture was reduced at 22°C for 2 hours. Then, RLP1 (10 mg / mL, 10 eq., purchased from MCE) in DMSO solution was added, and the conjugation reaction was carried out at 22°C for 1 hour. After two purifications using dextran-coated activated carbon, the buffer solution containing the desired antibody-drug conjugate was subjected to three ultrafiltrations to obtain Comparative Example-1 (CA). DC (mg / mL): 6.74, V(mL): 0.6, yield 80.9%).

[0545] RLP1:

[0546]

[0547] The following characterization results were obtained using a general characterization method for antibody-drug conjugates:

[0548] HIC-DAR: 7.96, SEC purity: 97.7%, Free Linker Payload (mol / mol%): 0.04221.

[0549] Comparative Example: Preparation of Antibody-Drug Conjugate Comparative Example-2

[0550]

[0551] Following the general conjugation process for preparing antibody-drug conjugates, TCEP (10 mM, 15 eq.) aqueous solution was added to trastuzumab (30.0 mg, 20.18 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer, and the mixture was reduced at 22°C for 2 hours. Then, RLP2 (10 mg / mL, 10 eq., synthesis method can be found in WO2024186264A1) in DMSO solution was added, and the conjugation reaction was carried out at 22°C for 1 hour. After two purifications via dextran-coated activated carbon treatment, the buffer solution for the desired antibody-drug conjugate was subjected to three ultrafiltrations to obtain comparative example-2 (CA). DC (mg / mL): 8.56, V(mL): 3, yield 85.6%).

[0552] RLP2:

[0553]

[0554] The following characterization results were obtained using a general characterization method for antibody-drug conjugates:

[0555] HIC-DAR: 7.8, SEC purity: 97.39%, Free Linker Payload (mol / mol%): 0.01122%.

[0556] Example: Preparation of antibody-drug conjugate ADC-11

[0557]

[0558] Following the general conjugation procedure for preparing antibody-drug conjugates, TCEP (10 mM, 10 eq.) aqueous solution was added to 50 mM PBS pH 7.0 buffer containing trastuzumab (1.0 mg, 20.18 mg / mL, 1.0 eq.), and the mixture was reduced at 22°C for 2 hours. Then, DMSO solution containing LP11 (10 mg / mL, 10 eq.) was added, and the conjugation reaction was carried out at 22°C for 1 hour. After two purifications via dextran-coated activated carbon treatment, the buffer for the desired antibody-drug conjugate was subjected to three ultrafiltrations to obtain ADC-11 (CA). DC (mg / mL): 5.1, V(mL): 0.06, yield 61.2%).

[0559] The following characterization results were obtained using a general characterization method for antibody-drug conjugates:

[0560] HIC-DAR: 7.4, SEC purity: 96.76%, Free Linker Payload (mol / mol%): notdetected.

[0561] Example: Preparation of antibody-drug conjugate ADC-13

[0562]

[0563] Following the general conjugation procedure for preparing antibody-drug conjugates, TCEP (10 mM, 10 eq.) aqueous solution was added to 50 mM PBS pH 7.0 buffer containing trastuzumab (1.5 mg, 20.18 mg / mL, 1.0 eq.), and the mixture was reduced at 22°C for 2 hours. Then, DMSO solution containing LP13 (10 mg / mL, 20 eq.) was added, and the conjugation reaction was carried out at 22°C for 1 hour. After two purifications via dextran-coated activated carbon treatment, the buffer for the desired antibody-drug conjugate was subjected to three ultrafiltrations to obtain ADC-13 (CA). DC (mg / mL): 5.79, V(mL): 0.1, yield 38.6%).

[0564] The following characterization results were obtained using a general characterization method for antibody-drug conjugates:

[0565] HIC-DAR: 7.9, SEC purity: 95.4%, Free Linker Payload (mol / mol%): notdetected.

[0566] Example: Preparation of antibody-drug conjugate ADC-23

[0567]

[0568] Following the general conjugation procedure for preparing antibody-drug conjugates, TCEP (10 mM, 15 eq.) aqueous solution was added to trastuzumab (2.0 mg, 20.18 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer, and the mixture was reduced at 22°C for 2 hours. Then, LP23 (10 mM, 16 eq.) in DMSO solution was added, and the conjugation reaction was carried out at 22°C for 1 hour. After purification three times by dextran-coated activated carbon treatment, the buffer for the desired antibody-drug conjugate was subjected to three ultrafiltrations to obtain ADC-23 (C ADC (mg / mL): 6.8, V(mL): 0.06, yield 40.8%).

[0569] The following characterization results were obtained using a general characterization method for antibody-drug conjugates:

[0570] HIC-DAR: 7.7, SEC purity: 100%, Free Linker Payload (mol / mol%): notdetected.

[0571] HIC retention time of antibody-drug conjugates

[0572] ADC number Antibody Linker-drug conjugate number HIC retention time (min) Comparative Example-2 Trastuzumab RLP2 20.1 ADC-11 Trastuzumab LP11 16.7 ADC-13 Trastuzumab LP13 14.7 ADC-23 Trastuzumab LP23 11.4

[0573] Compared to Comparative Example 2, which contains a PEG chain linker, the HIC retention times of antibody-drug conjugates ADC-11 and ADC-13, which contain hydrophilic linkers, were both shortened. Furthermore, LP13, containing two hydrophilic side chains, exhibited an even shorter HIC retention time compared to ADC-11 and Comparative Example 2, which contain only one side chain. This improves the hydrophilicity of antibody-drug conjugates with high DAR values, thus preventing aggregation. In addition, unlike MC-VC-PAB-Exatecan-based antibody-drug conjugates which are prone to aggregation at high DAR values, such as DAR 8 (Mol CancerTher (2024) 23(2):199–211), ADC-23, based on LP23 containing hydrophilic side chains, did not aggregate at a DAR of 7.7.

[0574] Furthermore, when different azeotropic crown ether groups are used, the greater the proportion of NH and O in the azeotropic crown ether, the more likely it is to achieve the same or greater improvement in the hydrophilicity of the ADC.

[0575] Biological evaluation

[0576] 1) Tumor cell proliferation inhibition experiment based on antibody-drug conjugates with hydrophilic linkers

[0577] A specific concentration of NCI-N87 and JIMT-1 cells (1500 cells per well, 80 μl) was added to a 96-well plate. Cells were cultured overnight in a constant temperature incubator (37°C, 5% CO2). Antibody-drug conjugate solutions with a maximum concentration of 400 nM based on hydrophilic linkers were prepared and serially diluted 1:4 with PBS. After co-incubating the diluted solutions (20 μl per well) with the cells (37°C, 5% CO2) for five days, CellTitre-Glo reagent (40 μl per well) was added, and fluorescence values ​​were measured using an i3X microplate reader.

[0578] In vitro cytotoxicity of antibody-drug conjugates (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[0579]

[0580] All ADCs exhibited certain antitumor activity. For example, in HER2-overexpressing NCI-N87 cells, the in vitro cytotoxic activities of ADC-11, ADC-13, and ADC-23 were comparable to those of the control ADCs Comparative Example-1 and Comparative Example-2, all less than 1 nM. In HER2-underexpressing JIMT-1 cells, the in vitro cytotoxic activity of ADC-23 was stronger than that of the control ADC Comparative Example-1. This experiment confirms that antibody-drug conjugates with in vitro cytotoxic activity can be prepared using the linker described in this paper.

[0581] 2) Evaluation of the efficacy of JIMT-1 tumor-bearing mice based on antibody-drug conjugates using hydrophilic linkers

[0582] Experimental Objective

[0583] The conjugate drug of this application was evaluated using Nu / Nu nude mice as test animals.

[0584] The therapeutic effects of ADC-23 and the control ADC, and the control ADC, on JIMT-1 xenograft nude mice after tail vein injection.

[0585] Test drugs and materials

[0586] 1. Test drug

[0587] ADC-23: 3 mg / kg

[0588] Reference ADC Comparative Example-1: 3 mg / kg

[0589] Blank control: PBS

[0590] Preparation method:

[0591] 2. All were diluted with PBS.

[0592] 3. Experimental animals

[0593] Nu / Nu nude mice, purchased from Beijing Vital River.

[0594] Test methods

[0595] JIMT-1 cells were subcutaneously injected into the right rib area of ​​mice. After 7 days of tumor growth, the animals were randomly divided into three groups of 5 (4 in the PBS group).

[0596] The drug was administered via tail vein injection once. Tumor volume and mouse body weight were measured twice a week, and the data were recorded.

[0597] Data were statistically analyzed using Microsoft 365 Excel software: mean values ​​were calculated as averages; SD values ​​were calculated as standard deviations (STDEV); SEM values ​​were calculated as STDEV / SQRT; and p-values ​​for intergroup differences were calculated as TTEST.

[0598] The drug was administered once via tail vein injection, and observation continued until the end of the experiment. The results are as follows: Figure 1 and Figure 2 As shown, ADC-23 significantly reduced tumor volume (P = 0.011), exhibiting superior tumor inhibition compared to the control ADC Comparative Example-1. Meanwhile, there was no significant difference in body weight between the ADC-23 group and the control ADC Comparative Example-1 group.

[0599] The main advantage of this article is:

[0600] 1. Antibody-drug conjugates prepared using the hydrophilic linkers described in this paper can improve hydrophilic properties. They are particularly suitable for use with hydrophobic loads to generate antibody-drug conjugates with high DAR values, thereby reducing or avoiding aggregation problems.

[0601] 2. Based on the aforementioned improvements in hydrophilicity, the prepared antibody-drug conjugates are expected to further enhance in vitro activity and in vivo efficacy. In some specific embodiments, the antibody-drug conjugates prepared using the linkers described herein all exhibit strong in vitro cytotoxicity (<1 nM) and good in vivo efficacy. For example, in the JIMT-1 tumor-bearing mouse efficacy evaluation experiment, the antibody-drug conjugate ADC-23 based on the hydrophilic linker showed better efficacy than the reference ADC Comparative Example-1.

[0602] 3. The antibody-drug conjugates based on novel hydrophilic linkers provided in this paper have shorter retention times on HIC HPLC.

[0603] The embodiments described herein are for illustrative purposes only and do not constitute a limitation on the scope of the claims. Other substantially equivalent alternatives that may be conceived by those skilled in the art are within the scope of protection of this document.

Claims

1. An intermediate for a hydrophilic linker, characterized in that... It includes structures represented by general formula V or their salts or esters. R4-L2-E(V), where, R4 is a functional group that can bind to an amino acid unit in the linker. L2 is absent or is a PEG unit. E is selected from an azacrown ether group substituted with one or more hydrophilic units. The azacrown ether contains two or more nitrogen atoms and optionally contains oxygen atoms. The hydrophilic group is connected to a nitrogen atom in the azacrown ether. Depending on the presence or absence of L2, the azacrown ether group is connected to L2 or R4 through other nitrogen atoms.

2. The intermediate as described in claim 1, characterized in that... The R4 is selected from amino, carboxyl, or their protected forms.

3. A hydrophilic linker, characterized in that... It includes amino acid units, and also contains side chains of one or more general formula I structural segments, or their salts or esters. in, The wavy lines in Formula I represent the connection sites with amino acid units. L2 is absent or is a PEG unit, and E is selected from a azeotropic crown ether group substituted with a hydrophilic unit. The azeotropic crown ether contains two or more nitrogen atoms and optionally contains oxygen atoms. The hydrophilic group is connected to a nitrogen atom in the azeotropic crown ether. Depending on the presence or absence of L2, the azeotropic crown ether group is connected to L2 or an amino acid unit through other nitrogen atoms.

4. The linker intermediate as described in any one of claims 1 to 2 or the hydrophilic linker as described in claim 3, characterized in that: When L2 exists, it is selected from One or more combinations of n3 = 0-50, n4 = 0-50, n5 = 0-50 or n6 = 0-50.

5. The intermediate as described in any one of claims 1 to 2 or the hydrophilic linker as described in claim 3, characterized in that... The E mentioned is selected from: n7=1-20, n8=1-20, n9=1-20, n 10 =1-20, n 11 =1-20, n 12 =1-20, n 13 =1-20, n 14 =1-20, n 15 =1-20, n 16 =1-20 or n 17 =Integers from 1 to 20; W1, W2, W3, W4, W5, W6, W7, W8, and W9 may be the same or different, and are each independently selected from oxygen, sulfur, or... R1 is selected from X or The n 18 = Integers from 0 to 100; K1 is selected from hydrogen or C 1-20 alkyl; At least one X is selected from a hydrophilic unit, and the other Xs are each independently selected from hydrogen or a hydrophilic unit. When the azacrown ether unit contains multiple Xs, the Xs can be the same or different.

6. The intermediate or hydrophilic linker as described in claim 5, characterized in that... The hydrophilic unit is selected from sugar units, polysarcosine units, polyethylene glycol units, or alkyl sulfonic acid units.

7. The intermediate or hydrophilic linker as described in claim 6, characterized in that... The hydrophilic unit is a sugar unit, which may be optionally replaced by a monosaccharide, disaccharide, or polysaccharide.

8. The intermediate or hydrophilic linker as described in claim 6 or 7, characterized in that... The structure of the sugar unit is as follows: in, n 19 =0-20, n 20 =1-20, n 21 = an integer from 1 to 20, wherein R2 is selected from hydrogen or C 1-20 Alkyl group; Y1 is independently selected from -NH- or oxygen; R3 is independently selected from hydrogen, acetyl, monosaccharide, disaccharide, or polysaccharide; Y2 is selected from... The Y3 is selected from hydrogen, hydroxyl, or C. 1-20 Alkyl group.

9. The intermediate or hydrophilic linker as described in claim 8, characterized in that... The monosaccharide, disaccharide, or polysaccharide is selected from glyceraldehyde, erythrosyl, threoyl, arabinose, ribosyl, xylose, lysolosyl; glucosyl, mannose, fructose, galactosyl, galacturonic acid, glucuronic acid, gluconic acid, mannuronic acid, maltose, maltodextrin, lactose, lactobionic acid, sucrose, sophorose, chitobiose, xylobiose, pinobiose, mesobiobiose, mannobiose, gentiobiose, laminabiose, cellobiose, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

10. The intermediate or hydrophilic linker as described in claim 8, characterized in that... n 19 =0-12, n 20 =1-12, n 21 =Integers from 1 to 15, R2 is selected from hydrogen or C 1-12 Alkyl group, Y3 is selected from hydrogen, hydroxyl group or C 1-12 Alkyl group.

11. The intermediate or hydrophilic linker as described in claim 10, characterized in that... n 19 =0-10, n 20 =1-10, n 21 =Integers from 1 to 12, R2 is selected from hydrogen or C 1-10 Alkyl group, Y3 is selected from hydrogen, hydroxyl group or C 1-10 Alkyl group.

12. The intermediate or hydrophilic linker as described in claim 11, characterized in that... n 19 =0-8, n 20 =1-8, n 21 = an integer from 1 to 10, wherein R2 is selected from hydrogen or C 1-8 Alkyl group, wherein Y3 is selected from hydrogen, hydroxyl or C 1-8 Alkyl group.

13. The intermediate or hydrophilic linker as described in claim 6, characterized in that... The polysarcosine unit structure is as follows: Where, n 22 = Integers from 1 to 100.

14. The intermediate or hydrophilic linker as described in claim 13, characterized in that... n 22 = Integers from 1 to 50.

15. The intermediate or hydrophilic linker as described in claim 14, characterized in that... n 22 = Integers from 1 to 30.

16. The intermediate or hydrophilic linker as described in claim 15, characterized in that... n 22 = Integers from 1 to 20.

17. The intermediate or hydrophilic linker as described in claim 6, characterized in that... The polyethylene glycol unit structure is as follows: Where, n 23 =Integers from 1 to 100, K2 is selected from hydrogen or C 1-20 alkyl.

18. The intermediate or hydrophilic linker as described in claim 17, characterized in that... n 23 =1-50, wherein K2 is selected from hydrogen or C 1-12 alkyl.

19. The intermediate or hydrophilic linker as described in claim 18, characterized in that... n 23 =1-30, wherein K2 is selected from hydrogen or C 1-10 alkyl.

20. The intermediate or hydrophilic linker as described in claim 19, characterized in that... n 23 =1-20, wherein K2 is selected from hydrogen or C 1-8 alkyl.

21. The hydrophilic linker as described in claim 6, characterized in that... The alkyl sulfonic acid unit structure is as follows: Where, n 24 = Integers from 1 to 20.

22. The intermediate or hydrophilic linker as described in claim 21, characterized in that... n 24 = Integers from 1 to 12.

23. The intermediate or hydrophilic linker as described in claim 22, characterized in that... n 24 = Integers from 1 to 10.

24. The intermediate or hydrophilic linker as described in claim 23, characterized in that... n 24 =Integers from 1 to 8.

25. The intermediate or hydrophilic linker as described in claim 5, characterized in that: n7=1-12, n8=1-12, n9=1-12, n 10 =1-12, n 11 =1-12, n 12 =1-12, n 13 =1-12, n 14 =1-12, n 15 =1-12, n 16 =1-12 or n 17 =Integers from 1 to 12, The n 18 = Integers from 0 to 50; K1 is selected from hydrogen or C 1-12 alkyl.

26. The intermediate or hydrophilic linker as described in claim 25, characterized in that: n7=1-10, n8=1-10, n9=1-10, n 10 =1-10, n 11 =1-10, n 12 =1-10, n 13 =1-10, n 14 =1-10, n 15 =1-10, n 16 =1-10 or n 17 =Integers from 1 to 10 The n 18 = Integers from 0 to 30; K1 is selected from hydrogen or C 1-10 alkyl.

27. The intermediate or hydrophilic linker as described in claim 26, characterized in that: n7=1-8, n8=1-8, n9=1-8, n 10 =1-8, n 11 =1-8, n 12 =1-8, n 13 =1-8, n 14 =1-8, n 15 =1-8, n 16 =1-8 or n 17 =Integers from 1 to 8 The n 18 = Integers between 0 and 20; K1 is selected from hydrogen or C 1-8 alkyl.

28. The intermediate or hydrophilic linker as described in claim 4, characterized in that... n3 = 0-30, n4 = 0-30, n5 = 0-30 or n6 = 0-30.

29. The intermediate or hydrophilic linker as described in claim 28, characterized in that... n3 = 0-20, n4 = 0-20, n5 = 0-20 or n6 = 0-20.

30. The intermediate as described in any one of claims 1 to 2 or the hydrophilic linker as described in claim 3, characterized in that... E is selected from the azirocrown ether group of a 9- to 18-membered ring, and the hydrophilic unit is selected from a sugar unit, wherein the sugar unit is selected from: Where, n 19 Selected from integers 1 to 3, R2 is hydrogen or methyl, n 20 R3 is selected from integers from 1 to 3, and R3 is independently selected from H or C5 to C6 monosaccharides. 21 Choose from 3 or 4. or Where, n 20 R3 is selected from integers from 1 to 3, and R3 is independently selected from H or C5 to C6 monosaccharides. 21 Choose from 3 or 4.

31. The intermediate or hydrophilic linker as described in claim 30, characterized in that... The E is selected from the 9, 12, 15, and 18-membered rings of the azacrown ether.

32. The intermediate or hydrophilic linker as described in claim 31, characterized in that... The E mentioned is selected from: In this configuration, at least one X is a sugar unit, and the other Xs are independently selected from H or sugar units.

33. The intermediate or hydrophilic linker as described in claim 32, characterized in that... The E mentioned is selected from:

34. The intermediate or hydrophilic linker as described in claim 30, characterized in that... The sugar units are selected from:

35. The intermediate or hydrophilic linker as described in claim 34, characterized in that... R3 is 36. The intermediate or hydrophilic linker as described in claim 34, characterized in that... The sugar units are selected from:

37. The intermediate or hydrophilic linker as described in claim 36, characterized in that... The sugar units are selected from:

38. The intermediate or hydrophilic linker as described in claim 32, characterized in that... The E mentioned is selected from:

39. The intermediate or hydrophilic linker as described in claim 38, characterized in that... The E mentioned is selected from:

40. The intermediate or hydrophilic linker as described in claim 39, characterized in that... The E mentioned is selected from 41. The intermediate or hydrophilic linker as described in claim 30, characterized in that... The L2 mentioned exists and is selected from n3 is an integer selected from 8 to 12.

42. The intermediate or hydrophilic linker as described in claim 41, characterized in that... The n3 = 11.

43. The hydrophilic linker as described in claim 30, characterized in that... It contains one or two of the aforementioned side chains.

44. The intermediate as described in claims 1-2 or the hydrophilic linker as described in claim 3, characterized in that... -L2-E segments are selected from the structures shown in the table below:

45. The hydrophilic linker of the intermediate as described in claim 44, characterized in that... -L2-E segments are selected from the structures shown in H1 to H12.

46. ​​The intermediate or hydrophilic linker as described in claim 45, characterized in that... The -L2-E chain segment is selected from the structures shown in H1, H2, H5, H6, H7, H8, H10, and H11.

47. The intermediate or hydrophilic linker as described in claim 46, characterized in that... The -L2-E segment is selected from the structure shown in H1.

48. The connector as described in claim 3, characterized in that... It also includes a first linker subunit for connecting to a target unit and a second linker subunit for connecting to a drug, wherein the first linker subunit and the second linker subunit are connected to the amino acid unit.

49. The connector as claimed in claim 48, characterized in that... The targeting unit is selected from antibodies or their antigen-binding fragments.

50. The connector as claimed in claim 48, characterized in that... The amino acid unit comprises one or more amino acid subunits, wherein the amino acid subunits are selected from: Or one or more combinations of its stereoisomers. * Represents the connection site with L2, or, when L2 is absent, the connection site with E; # represents the connection site with the first linker subunit or amino acid subunit. ## represents the connection site with the second linker subunit or amino acid subunit. n 37 =0-20, n 38 =0-20, n 39 =0-20, n 40 =0-20, n 41 =0-20, n 42 =0-20, n 43 =0-20, n 44 =0-20, n 45 =0-20, n 46 =0-20, n 47 =0-20, n 48 =0-20, n 49 =0-20, n 50 =0-20, n 51 =0-20, n 52 =0-20, n 53 =0-20 and n 54 = Integers between 0 and 20.

51. A linker-drug conjugate, characterized in that... The structure is formed by combining the linker as described in any one of claims 3 to 50 with a drug, as shown in Formula II. in, n2 is an integer from 1 to 8, A1 is selected from the amino acid subunits, and M is a connector for connecting to the target unit. L1 does not exist or is selected from One or more combinations of n; where n 30 =1-20, n 31 =0-100, n 32 =1-20, n 33 =0-100, n 34 =0-100, n 35 =0-100 or n 36 =Integers between 0 and 20 A1 is selected from Or its stereoisomers, * Represents the connection site with L2, or, when L2 is absent, the connection site with E; # represents the connection site with L1. ## represents the connection site with A2. n 37 =0-20, n 38 =0-20, n 39 =0-20, n 40 =0-20, n 41 =0-20, n 42 =0-20, n 43 =0-20, n 44 =0-20, n 45 =0-20, n 46 =0-20, n 47 =0-20, n 48 =0-20, n 49 =0-20, n 50 =0-20, n 51 =0-20, n 52 =0-20, n 53 =0-20 and n 54 =Integers between 0 and 20 A2 does not exist or is selected from: The term D refers to a fragment formed by the loss of one or more atoms or groups from a molecule with antitumor biological activity.

52. The linker drug conjugate as described in claim 51, characterized in that... M is selected from Or its stereoisomers.

53. The linker drug conjugate as described in claim 52, characterized in that... The M-L2- is 54. The linker drug conjugate as described in claim 51, characterized in that... The A2 mentioned above is 55. The linker drug conjugate as described in claim 51, characterized in that... n 30 =1-12, n 31 =0-50, n 32 =1-12, n 33 =0-50, n 34 =0-50, n 35 =0-50 or n 36 =Integers from 0 to 12.

56. The linker drug conjugate as described in claim 55, characterized in that... n 30 =1-10, n 31 =0-30, n 32 =1-10, n 33 =0-30, n 34 =0-30, n 35 =0-30 and n 36 = Integers between 0 and 10.

57. The linker drug conjugate as described in claim 56, characterized in that... n 30 =1-8, n 31 =0-20, n 32 =1-8 and n 33 = Integers between 0 and 20.

58. The linker drug conjugate as described in claim 51, characterized in that... n² = integers from 1 to 6.

59. The linker drug conjugate as described in claim 58, characterized in that... n² = integers from 1 to 4.

60. The linker drug conjugate as described in claim 59, characterized in that... The n2 mentioned is 1 or 2.

61. The linker drug conjugate as described in claim 51, characterized in that... The bioactive molecules are selected from microtubule inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, inserters, DNA-reactants, DNA alkylating agents, immunomodulators, nucleic acids, BCL-XL inhibitors, kinase inhibitors, PROTAC, molecular glues, and radioisotopes.

62. The linker drug conjugate as described in claim 51, characterized in that... The linker is covalently bound to nitrogen, oxygen, or sulfur in the drug.

63. The linker drug conjugate as described in claim 51, characterized in that... Any structure selected from LP1 to 67:

64. The linker as described in claim 50 or the linker-drug conjugate as described in claim 51, characterized in that... The integers n37 = 0-12, n38 = 0-12, n39 = 0-12, n40 = 0-12, n41 = 0-12, n42 = 0-12, n43 = 0-12, n44 = 0-12, n45 = 0-12, n46 = 0-12, n47 = 0-12, n48 = 0-12, n49 = 0-12, n50 = 0-12, n51 = 0-12, n52 = 0-12, n53 = 0-12, and n54 = 0-12.

65. The linker or linker drug conjugate as described in claim 64, characterized in that... The integers n37 = 0-10, n38 = 0-10, n39 = 0-10, n40 = 0-10, n41 = 0-10, n42 = 0-10, n43 = 0-10, n44 = 0-10, n45 = 0-10, n46 = 0-10, n47 = 0-10, n48 = 0-10, n49 = 0-10, n50 = 0-10, n51 = 0-10, n52 = 0-10, n53 = 0-10, and n54 = 0-10.

66. The hydrophilic linker or linker drug conjugate as described in claim 65, characterized in that... Integers n37 = 0-8, n38 = 0-8, n39 = 0-8, n40 = 0-8, n41 = 0-8, n42 = 0-8, n43 = 0-8, n44 = 0-8, n45 = 0-8, n46 = 0-8.

67. The hydrophilic linker or linker drug conjugate as described in claim 66, characterized in that... Integers n37=1, n38=1, n39=1, n40=1, n41=1, n42=1, n43=1, n44=1, n45=1, n46=1.

68. An antibody-drug conjugate, characterized in that... The drug is obtained by conjugating the linker-drug with the antibody as described in any one of claims 51 to 67, and the structure is shown in general formula III. in, Ab is an antibody or antigen-binding fragment, and n1 is any value between 1 and 20.

69. The antibody-drug conjugate as described in claim 68, characterized in that... Choose any one of ADC-1 to ADC-67:

70. The antibody-drug conjugate as described in claim 60, characterized in that... The antibody is a monoclonal antibody selected from: abciximab, alenmab, annatuzumab, atezolizumab, avelumab, baribizumab, bevacizumab, bonatumab, bentoximab, caputoxumab, cetuximab, cetuximab, cotoximab, dalizumab, daratumumab, dinetuzumab, dinotoximab, depertuzumab, dinotoximab, dinetuzumab, dinetuzumab, dinetuzumab, dinetuzumab, dinetuzumab, dinetuzumab, dinetuzumab, dinetuzumab, dinetuzumab, enrotoximab, gerbutuzumab, gebutuzumab, teimozumab, indatuzumab, indutuzumab, olibutuzumab, ipilimumab, labetuzumab, latoximab, latoximab, lifatoximab, lovotoximab. Monoclonal antibodies, including mirtuzumab, mitoxicumab, naltuzumab, netoxicumab, nitoxicumab, nivolumab, oxantuzumab, oxantuzumab, oxantuzumab, oxantuzumab, pallizumab, panitumumab, pertratuzumab, pembrolizumab, pertuzumab, pineutuzumab, polotuzumab, ramucirumab, lovatoxicumab, saxituzumab, storutuzumab, storutuzumab, sofostoxicumab, varadatoxicumab, vortexutuzumab, trastuzumab, tesutoxicumab, anti-B7-H3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody, and anti-CD30 antibody, or their antigen-binding fragments or immunologically active portions.

71. A method for preparing the intermediate according to any one of claims 30-42, 44-47, wherein, Sugar unit is Its features include the following steps: CbzHN-L2-Azacrown ethers were synthesized from CbzHN-L2-Azacrown ethers via a bimolecular nucleophilic substitution reaction. CbzHN-L2-azacrown ether and CbzHN-L2-E was prepared by reductive amination. CbzHN-L2-E can be optionally synthesized via a Cbz removal reaction to form H2N-L2-E.

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