Hydrophilic linker, intermediate of linker, linker-drug conjugate, antibody-drug conjugate, and use thereof

Abstract

A hydrophilic linker, an intermediate of the hydrophilic linker, a linker-drug conjugate prepared from the linker and a preparation method therefor, and an antibody-drug conjugate and use thereof.

Description

Hydrophilic linkers, intermediates of linkers, linker-drug conjugates, antibody-drug conjugates and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to structural improvements of linkers, and linker-drug conjugates and antibody-drug conjugates prepared therefrom and uses thereof. BACKGROUND

[0002] The main anti-tumor mechanism of ADC drugs is to target the delivery of cytotoxic payloads to tumor cells. That is, after entering the body, the antibody part of the ADC selectively binds to the target antigen on the surface of tumor cells, enters the tumor cells through endocytosis, and finally the ADC is broken down in the lysosome of 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 concentration of drugs 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 payloads in cells are related.

[0004] Therefore, under the premise that other factors remain unchanged, in theory, the concentration of drugs delivered to 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, which 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 a hydrophilic linker intermediate, 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 amino, carboxyl or its protected form.

[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 units,

[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 unit being connected to a nitrogen atom in the azacrown ether, the azacrown ether group being connected to L2 or the amino acid units 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 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] 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 = 1-20, an integer;

[0020] W1, W2, W3, W4, W5, W6, W7, W8, W9 are the same or different and are each independently selected from oxygen, sulfur or R1 is selected from X or n 18 = 0-100, an integer; K1 is selected from hydrogen or C1-20 alkyl;

[0021] 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.

[0022] 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.

[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 a sugar unit, which is optionally substituted with a monosaccharide group, a disaccharide group, or a polysaccharide group.

[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 sugar unit structure is:

[0025] wherein,

[0026] 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.

[0027] 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.

[0028] 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.

[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 = 0-10, 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.

[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 = 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.

[0031] 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:

[0032] wherein n 22 = an integer from 1 to 100.

[0033] 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.

[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 30.

[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 20.

[0036] 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:

[0037] wherein n 23 = an integer from 1 to 100, and K2is selected from hydrogen or C1-20alkyl.

[0038] 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.

[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 30, and K2is selected from hydrogen or C1-10alkyl.

[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 = 1-20, and K2 is selected from hydrogen or C1-8 alkyl.

[0041] 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:

[0042] wherein n 24 = 1-20.

[0043] 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.

[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 = 1-10.

[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 = 1-8.

[0046] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein,

[0047] 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

[0048] n7 = 1-12, n8 = 1-12, n9 = 1-12, n 18 = 0-50; and K1 is selected from hydrogen or C1-12 alkyl.

[0049] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein,

[0050] 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 = an integer from 1-10,

[0051] said n 18 = an integer from 0-30; said K1 is selected from hydrogen or C1-10 alkyl.

[0052] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein,

[0053] 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 = an integer from 1-8,

[0054] said n 18 = an integer from 0-20; said K1 is selected from hydrogen or C1-8 alkyl.

[0055] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3 = an integer from 0-50, n4 = an integer from 0-50, n5 = an integer from 0-50 or n6 = an integer from 0-50.

[0056] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3 = an integer from 0-30, n4 = an integer from 0-30, n5 = an integer from 0-30 or n6 = an integer from 0-30.

[0057] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3 = an integer from 0-20, n4 = an integer from 0-20, n5 = an integer from 0-20 or n6 = an integer from 0-20.

[0058] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from the group consisting of 9-18 membered ring azacrown ether groups, the hydrophilic unit is selected from the group consisting of sugar units, and the sugar units are selected from the group consisting of:

[0059] wherein n 19 is an integer from 1-3, R2 is hydrogen or methyl, n 20an integer selected from 1-3, R3is independently selected from H or a C5-C6monosaccharide, n 21 selected from 3 or 4,

[0060] or wherein n 20 an integer selected from 1-3, R3is independently selected from H or a C5-C6monosaccharide, n 21 selected from 3 or 4.

[0061] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, one of R3is selected from a C5-C6monosaccharide and the rest are H.

[0062] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from a 9, 12, 15, 18-membered ring of the azacrown ether.

[0063] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from: wherein at least one X is a saccharide unit and the other X is independently selected from H or a saccharide unit.

[0064] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the saccharide unit is selected from:

[0065] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, R3is

[0066] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from:

[0067] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, L2is present and is selected from n3is an integer selected from 8-12.

[0068] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3= 11.

[0069] The hydrophilic linker of the second aspect described herein comprises one or two of the side chains.

[0070] In some embodiments of the linker intermediate of the first aspect herein or the hydrophilic linker of the second aspect herein, -L2-E is selected from the structures shown in the following table:

[0071] In some embodiments of the hydrophilic linker of the second aspect herein, further comprising a first linker subunit for attachment to a targeting unit, and a second linker subunit for attachment to a drug, the first linker subunit and the second linker subunit being attached to the amino acid unit.

[0072] In some embodiments of the hydrophilic linker of the second aspect herein, the targeting unit is selected from an antibody or an antigen-binding fragment thereof.

[0073] In some embodiments of the hydrophilic linker of the second aspect herein, the amino acid unit comprises one or more amino acid subunits selected from:

[0074] or one or more combinations of stereoisomers thereof,

[0075] * represents a point of attachment to L2, or to E when L2 is absent;

[0076] # represents a point of attachment to the first linker subunit or the amino acid subunit,

[0077] ## represents a point of attachment to the second linker subunit or the amino acid subunit,

[0078] 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, n53 = 0-20 and n 54 = 0-20 is an integer.

[0079] In the hydrophilic linker of the second aspect herein, 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 = 0-12 is an integer.

[0080] In the hydrophilic linker of the second aspect herein, 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 = 0-10 is an integer.

[0081] In some embodiments of the hydrophilic linker of the second aspect herein, n 37 = 0-8, n 38 = 0-8, n 39 = 0-8, n 40 = 0-8, n 41 = 0-8, n42 =0-8, n 43 =0-8, n 44 =0-8, n 45 =0-8, n 46 =0-8, n

[0082] In some embodiments of the hydrophilic linker of the second aspect herein, 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 =1, n

[0083] The third aspect herein relates to a linker-drug conjugate formed by the combination of the aforementioned linker with a drug, having the structure as shown in Formula II,

[0084] wherein,

[0085] n2 = an integer from 1 to 8, A1 is selected from an amino acid subunit, and M is a linker for connecting with a targeting unit,

[0086] L1 is absent or selected from one or more combinations of ; wherein 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 =0-20, the left wave line of L1 is used to represent the site of connection with M, and the right wave line is used to represent the site of connection with A1,

[0087] A1 is selected from or a stereoisomer thereof,

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

[0089] # represents the connection site with L1,

[0090] ## represents the connection site with A2,

[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, n 54 = 0-20, n

[0092] A2is absent or selected from:

[0093] A2is absent or selected from:

[0094] D is a fragment formed by the loss of one or more atoms or groups from a molecule having anti-tumor biological activity.

[0095] In some embodiments of the linker-drug conjugate of the third aspect herein, M is selected from or a stereoisomer thereof.

[0096] In some embodiments of the linker-drug conjugate of the third aspect herein, M-L2- is

[0097] In some embodiments of the linker-drug conjugate of the third aspect herein, A2is

[0098] In some embodiments of the linker-drug conjugate of the third aspect herein, n 30 = 1-12, n 31 = 0-50, n 32 = 1-12, n 33 = 0-50, n 34 = 0-50, n 35 = 0-50, n 36 = 0-12, n

[0099] In some embodiments of the linker-drug conjugate of the third aspect herein, 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 = 0-10.

[0100] In some embodiments of the linker-drug conjugate of the third aspect herein, n 30 = 1-8, n 31 = 0-20, n 32 = 1-8, and n 33 = 0-20.

[0101] In some embodiments of the linker-drug conjugate of the third aspect herein, n2 = an integer from 1-8.

[0102] In some embodiments of the linker-drug conjugate of the third aspect herein, n2 = an integer from 1-6.

[0103] In some embodiments of the linker-drug conjugate of the third aspect herein, n2 = an integer from 1-4.

[0104] In some embodiments of the linker-drug conjugate of the third aspect herein, n2 is 1 or 2.

[0105] In some embodiments of the linker-drug conjugate of the third aspect herein, the biologically active molecule is selected from the group consisting of a tubulin inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalator, a DNA-reactive agent, a DNA alkylating agent, an immunomodulator, a nucleic acid, a BCL-XL inhibitor, a kinase inhibitor, a PROTAC, a molecular glue, and a radioisotope.

[0106] In some embodiments of the linker-drug conjugate of the third aspect herein, the linker is covalently bound to a nitrogen, oxygen, or sulfur in the drug.

[0107] In some embodiments of the linker-drug conjugate of the third aspect herein, the linker is selected from any of the structures of LP1-47:

[0108] The fourth aspect of the present application relates to an antibody-drug conjugate, which is obtained by conjugating the aforementioned linker-drug with an antibody, and has a structure as shown in the general formula III, wherein,

[0109] Ab is an antibody or an antigen binding fragment, and n1 is any number between 1 and 20.

[0110] The antibody-drug conjugate according to the fourth aspect of the present application is selected from any one of ADC-1 to ADC-67:

[0111] The fourth aspect herein relates to some embodiments of the antibody-drug conjugate, wherein the antibody is a monoclonal antibody selected from the group consisting of: Ablximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Cirmtuzumab, 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, Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab, Pinatuzumab,Polatuzumab, 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 an antigen-binding fragment or immunologically active portion thereof.

[0112] The fifth aspect of the present disclosure relates to a method for preparing the intermediate of the preceding aspect, wherein the sugar unit is comprising the steps of:

[0113] CbzHN-L2-OTs is synthesized with an azacrown ether via a bimolecular nucleophilic substitution reaction to form CbzHN-L2-azacrown ether;

[0114] CbzHN-L2-azacrown ether is reacted with to form CbzHN-L2-E via a reductive amination reaction;

[0115] CbzHN-L2-E is optionally reacted via a de-Cbz reaction to form H2N-L2-E.

[0116] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, E is selected from

[0117] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, E is selected from

[0118] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, E is selected from

[0119] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the -L2-E segment is selected from the structures shown in H1-H12.

[0120] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the -L 2- E segment is selected from H1, H2, H5, H6, H7, H8, H10, H11.

[0121] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, -L 2- E segment is selected from H1.

[0122] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the sugar unit is selected from:

[0123] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the sugar unit is selected from:

[0124] The main advantage of the present application is that:

[0125] 1. The antibody drug conjugate prepared by using the hydrophilic linker of the present application can improve the hydrophilic property. Especially suitable for different hydrophobic loads to produce high DAR value of antibody drug conjugate to reduce or avoid the problem of aggregation.

[0126] 2. Based on the improvement of the hydrophilic property mentioned above, the prepared antibody drug conjugate is expected to further improve the in vitro activity and in vivo efficacy. In some specific embodiments, the antibody drug conjugate prepared by using the linker of the present application has strong in vitro cytotoxicity (<1 nM), in addition, also has 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.

[0127] 3. The antibody drug conjugate based on the novel hydrophilic linker provided in the present application has a shorter retention time on HIC HPLC. BRIEF DESCRIPTION OF DRAWINGS

[0128] Figure 1 shows the in vivo anti-tumor effect in a xenograft JIMT-1 breast cancer mouse model using the ADC drug, comparative example and blank group in the present application;

[0129] Figure 2 shows the mouse weight change in a xenograft JIMT-1 breast cancer mouse model using the ADC drug, comparative example and blank group in the present application. DETAILED DESCRIPTION

[0130] 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. If there is a conflict between the definitions and the description, the definition will control.

[0131] The first aspect of the present application provides an intermediate of a hydrophilic linker, characterized by comprising a structure represented by general formula V or a salt or ester thereof,

[0132] R4-L2-E (V), wherein,

[0133] R4 is a functional group capable of binding to an amino acid unit in a linker, L2 is absent or a PEG unit, and E is selected from an azacrown ether group substituted with one or more hydrophilic units, the azacrown ether group comprising two or more nitrogen atoms and optionally comprising an oxygen atom, the hydrophilic groups being attached to the nitrogen atoms in the azacrown ether group, the azacrown ether group being connected to L2 or R4 via another nitrogen atom depending on the presence of L2.

[0134] As used herein, the term "azacrown ether group" refers to a group in which some or all of the oxygen atoms in a crown ether ring structure are replaced by nitrogen atoms.

[0135] In some embodiments of the hydrophilic linker of the first aspect described herein, R4 is selected from an amino group, a carboxyl group, or a protected form thereof. A protected form refers to a modification of an amino group or a carboxyl group by chemical modification to a derivative that is relatively stable and less likely to undergo unintended reactions, thereby avoiding its destruction or participation in unwanted side reactions in subsequent reactions. When the target reaction is completed, the protective group is removed (i.e., "deprotection") by a specific condition to restore the activity of the original group. Common protective groups for modifying amino groups can be benzyloxy carbonyl, tert-butyloxy carbonyl; common protective groups for modifying carboxyl groups can be methyl ester, ethyl ester, tert-butyl ester.

[0136] The second aspect described herein provides a hydrophilic linker comprising an amino acid unit, characterized in that it further comprises one or more side chains of the structure of Formula I,

[0137] wherein,

[0138] the wavy line in Formula I indicates a connection site to the amino acid unit,

[0139] L2 is absent or a PEG unit, and E is selected from an azacrown ether group substituted with a hydrophilic unit, the azacrown ether group comprising two or more nitrogen atoms and optionally comprising an oxygen atom, the hydrophilic groups being attached to the nitrogen atoms in the azacrown ether group, the azacrown ether group being connected to L2 or the amino acid unit via another nitrogen atom depending on the presence of L2.

[0140] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein,

[0141] when L2 is present, is selected from one or more combinations of

[0142] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from: ​

[0143] 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, n 17 = an integer from 1 to 20, n

[0144] W1, W2, W3, W4, W5, W6, W7, W8, and W9 are the same or different and are each independently selected from oxygen, sulfur, or R1is selected from X or n 18 = an integer from 0 to 100; and K1is selected from hydrogen or C1-20alkyl;

[0145] At least one X is selected from a hydrophilic unit, and the others 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.

[0146] 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 saccharide unit, a polysarcosine unit, a polyethylene glycol unit, or an alkyl sulfonic acid unit.

[0147] 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 saccharide unit, which is optionally substituted with a monosaccharide group, a disaccharide group, or a polysaccharide group.

[0148] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the saccharide unit has the structure:

[0149] wherein,

[0150] n 19 = an integer from 0 to 20, n 20 = an integer from 1 to 20, n 21 = an integer from 1 to 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.

[0151] 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, disaccharide group or 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 pinobiose 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.

[0152] 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.

[0153] 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, n 21 = 1-12, R2is selected from hydrogen or C1-10alkyl, and Y3is selected from hydrogen, hydroxyl or C1-10alkoxy.

[0154] 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 = 0-8, n 20 = 1-8, n 21 = 1-10, R2is selected from hydrogen or C1-8alkyl, and Y3is selected from hydrogen, hydroxyl or C1-8alkoxy.

[0155] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, the poly-sarcosine unit structure is:

[0156] wherein n 22 = 1-100.

[0157] 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 = 1-50.

[0158] 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.

[0159] 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.

[0160] 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:

[0161] wherein n 23 = an integer from 1 to 100, and K2is selected from hydrogen or a Ci-20alkyl group.

[0162] 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 a Ci-12alkyl group.

[0163] 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 a Ci-10alkyl group.

[0164] 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 20, and K2is selected from hydrogen or a Ci-8alkyl group.

[0165] 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:

[0166] wherein n 24 = an integer from 1 to 20.

[0167] 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 = an integer from 1 to 12.

[0168] 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 = an integer from 1 to 10.

[0169] 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= an integer from 1 to 8.

[0170] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein,

[0171] 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 = an integer from 1 to 12,

[0172] The n 18 = an integer from 0 to 50; the K1 is selected from hydrogen or C1-12 alkyl.

[0173] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein,

[0174] 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 = an integer from 1 to 10,

[0175] The n 18 = an integer from 0 to 30; the K1 is selected from hydrogen or C1-10 alkyl.

[0176] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein,

[0177] 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 = an integer from 1 to 8,

[0178] The n18 = an integer from 0 to 20; said K1 is selected from hydrogen or Ci-8alkyl.

[0179] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3 = an integer from 0 to 50, n4 = an integer from 0 to 50, n5 = an integer from 0 to 50, or n6 = an integer from 0 to 50.

[0180] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3 = an integer from 0 to 30, n4 = an integer from 0 to 30, n5 = an integer from 0 to 30, or n6 = an integer from 0 to 30.

[0181] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, n3 = an integer from 0 to 20, n4 = an integer from 0 to 20, n5 = an integer from 0 to 20, or n6 = an integer from 0 to 20.

[0182] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from the group consisting of 9-18 membered ring aza crown ether groups, the hydrophilic unit is selected from the group consisting of sugar units, and the sugar units are selected from the group consisting of:

[0183] wherein n 19 is an integer from 1 to 3, R2 is hydrogen or methyl, and n 20 is an integer from 1 to 3, R3 is independently selected from H or C5-C6 monosaccharides, and n 21 is 3 or 4,

[0184] or wherein n 20 is an integer from 1 to 3, R3 is independently selected from H or C5-C6 monosaccharides, and n 21 is 3 or 4.

[0185] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, one of R3 is selected from C5-C6 monosaccharides and the rest are H.

[0186] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from the group consisting of 9, 12, 15, 18 membered ring aza crowns.

[0187] In some embodiments of the linker intermediate of the first aspect described herein or the hydrophilic linker of the second aspect described herein, E is selected from the group consisting of:

[0188] wherein at least one X is a sugar unit and the other X is independently selected from H or a sugar unit.

[0189] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein, the sugar unit is selected from:

[0190] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein, R3is

[0191] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein, E is selected from:

[0192] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein, L2is present and is selected from n3 is selected from an integer from 8 to 12.

[0193] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein, n3 = 11.

[0194] In some embodiments of the hydrophilic linker of the second aspect described herein, the linker comprises one or two of the side chains.

[0195] In some embodiments of the linker intermediate of the first aspect or the hydrophilic linker of the second aspect described herein, -L2-E is selected from the structures shown in the following table:

[0196] In some embodiments of the hydrophilic linker of the second aspect described herein, the linker further comprises a first linker subunit for attachment to a targeting unit and a second linker subunit for attachment to a drug, the first linker subunit and the second linker subunit being attached to the amino acid unit.

[0197] The first linker subunit for attachment to a targeting unit and the second linker subunit for attachment to a drug referred to herein are selected by the person skilled in the art depending on the targeting unit and the drug to be attached.

[0198] In some embodiments of the hydrophilic linker of the second aspect described herein, the targeting unit is selected from an antibody or an antigen binding fragment thereof.

[0199] In some embodiments of the hydrophilic linker of the second aspect herein, the amino acid unit comprises one or more amino acid subunits selected from:

[0200] or a combination of one or more of its stereoisomers,

[0201] * represents a site of attachment to L2, or to E when L2 is absent;

[0202] # represents a site of attachment to the first linker subunit or amino acid subunit,

[0203] ## represents a site of attachment to the second linker subunit or amino acid subunit,

[0204] 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 = 0-20, n

[0205] In some embodiments of the hydrophilic linker of the second aspect herein, 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 = an integer from 0-12.

[0206] In some embodiments of the hydrophilic linker of the second aspect herein, 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 = an integer from 0-10.

[0207] In some embodiments of the hydrophilic linker of the second aspect herein, 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 = an integer from 0-8.

[0208] In some embodiments of the hydrophilic linker of the second aspect herein, 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 from 1.

[0209] A third aspect of the present disclosure relates to a linker-drug conjugate formed by conjugating a linker as described above to a drug, having the structure of Formula II,

[0210] wherein,

[0211] n2 = an integer from 1 to 8, A1 is selected from the amino acid subunits described above, and M is a linker for attachment to the targeting unit,

[0212] L1 is absent or selected from in one or more combinations; wherein 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 = 0-20, the left-hand wavy line indicates the site of attachment to M, and the right-hand wavy line indicates the site of attachment to A1,

[0213] A1 is selected from or a stereoisomer thereof,

[0214] * represents the site of attachment to L2, or to E when L2 is absent;

[0215] # represents the site of attachment to L1,

[0216] ## represents the site of attachment to A2,

[0217] 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= an integer from 0 to 20,

[0218] A2 is absent or selected from:

[0219] A2 left wavy line is used to indicate the site of attachment to A1 and the right wavy line is used to indicate the site of attachment to D.

[0220] Said D is a fragment formed by the loss of one or more atoms or groups from a molecule having anti-tumor biological activity.

[0221] The anti-tumor bioactive molecules include, but are not limited to, tubulin inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, intercalators, DNA-reactive agents, DNA-alkylating agents, immunomodulators, nucleic acids, BCL-XL inhibitors, kinase inhibitors, anti-angiogenic agents, PROTACs, molecular glues, and radioisotopes, etc. The tubulin inhibitors such as monomethyl auristatin (e.g., monomethyl auristatin E or monomethyl auristatin F, etc.), maytansinoid (e.g., maytansinol, DM1, DM2, DM3, or DM4, etc.), or eribulin; RNA polymerase inhibitors such as a-amanitin and its derivatives, etc.; topoisomerase inhibitors such as etoposide, teniposide, amsacrine, PNU-159682, SN-38, exetecan, DXD and their derivatives, etc.; intercalators such as pyrrolobenzodiazepine (PBD) and its derivatives, etc.; DNA-reactive agents such as calicheamicin, tiancimycins, other enediyne and their derivatives, etc.; DNA-alkylating agents such as trabectedin, rubitecan, etc. didemnin derivatives, etc.; immunomodulators such as STING, TLR7, TLR8, and / or TLR9 agonists, PD-1 inhibitors and their derivatives, etc.; anti-angiogenic agents including linomide, bevacizumab, angiostatin, and razoxane, etc.; PROTAC protein degradation targeting chimeras such as described in US2018228907, US2016058872, US2017327469, etc.; molecular glues such as cyclosporin A, tacrolimus, rapamycin, thalidomide, lenalidomide, pomalidomide, indisulam and their derivatives, etc.; radioisotopes including iodine (131I), yttrium (90Y), technetium (99Tc), copper (67Cu), gallium (66Ga), rhenium (188Re), rhenium (186Re), indium (67In), indium (111In), indium (114In), indium (115In), strontium (89Sr), strontium (153Sr), lutetium (177Lu), actinium (225Ac), and lead (212Pb), etc. The anti-tumor bioactive molecules can be covalently bound to the linker through known binding sites and binding modes by losing one or more atoms or groups.

[0222] In some embodiments of the linker-drug conjugate of the third aspect herein, M is selected from or stereoisomers thereof.

[0223] In some embodiments of the linker-drug conjugate of the third aspect herein, the M-L2- is

[0224] In some embodiments of the linker-drug conjugate of the third aspect herein, the A2is

[0225] In some embodiments of the linker-drug conjugate of the third aspect herein, 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 = 0-12.

[0226] In some embodiments of the linker-drug conjugate of the third aspect herein, 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 = 0-10.

[0227] In some embodiments of the linker-drug conjugate of the third aspect herein, n 30 = 1-8, n 31 = 0-20, n 32 = 1-8, and n 33 = 0-20.

[0228] In some embodiments of the linker-drug conjugate of the third aspect herein, n2= an integer from 1-8.

[0229] In some embodiments of the linker-drug conjugate of the third aspect herein, n2= an integer from 1-6.

[0230] In some embodiments of the linker-drug conjugate of the third aspect herein, n2= an integer from 1-4.

[0231] In some embodiments of the linker-drug conjugate of the third aspect herein, n2is 1 or 2.

[0232] In some embodiments of the linker-drug conjugate of the third aspect herein, the biologically active molecule is selected from the group consisting of tubulin inhibitors, RNA polymerase inhibitors, topoisomerase inhibitors, intercalators, DNA-reactive agents, DNA alkylating agents, immunomodulators, nucleic acids, BCL-XL inhibitors, kinase inhibitors, PROTACs, molecular glues, and radioisotopes.

[0233] In some embodiments of the linker-drug conjugate of the third aspect herein, the linker is covalently bound to a nitrogen, oxygen, or sulfur in the drug.

[0234] In some embodiments of the linker-drug conjugate of the third aspect herein, the linker is selected from any of the structures of LPl-47:

[0235] The fourth aspect herein relates to antibody-drug conjugates, which are obtained by conjugating the aforementioned linker-drugs to antibodies, and have the structure shown in general formula III,

[0236] wherein,

[0237] Ab is an antibody or antigen-binding fragment, and n1 is any number between 1 and 20.

[0238] The antibody-drug conjugates of the fourth aspect herein are selected from any of ADC-1-67:

[0239] The fourth aspect herein relates to some embodiments of the antibody-drug conjugate, wherein the antibody is a monoclonal antibody selected from the group consisting of: Ablximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Cirmtuzumab, 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, Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Patritumab, Pembrolizumab, Pertuzumab, Pinatuzumab,Polatuzumab, 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 an antigen-binding fragment or immunologically active portion thereof.

[0240] The fifth aspect herein relates to a method for preparing the aforementioned intermediate, wherein the sugar unit is characterized in comprising the steps of:

[0241] CbzHN-L2-OTs is synthesized with CbzHN-L2-azacrown ether via a bimolecular nucleophilic substitution reaction on the azacrown ether;

[0242] CbzHN-L2-azacrown ether is reacted with CbzHN-L2-E is prepared via a reductive amination reaction,

[0243] CbzHN-L2-E can be optionally synthesized into H2N-L2-E via a de-Cbz reaction.

[0244] (n 20 -1 means one less than the selected n 20

[0245] In the intermediate of the first aspect herein or the hydrophilic linker of the second aspect, E is selected from

[0246] In the intermediate of the first aspect herein or the hydrophilic linker of the second aspect, E is selected from

[0247] In the intermediate of the first aspect herein or the hydrophilic linker of the second aspect, E is selected from

[0248] In the intermediate of the first aspect herein or the hydrophilic linker of the second aspect, the segment of -L2-E is selected from the structures shown in H1-H12.

[0249] ​In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the -L2-E segment is selected from H1, H2, H5, H6, H7, H8, H10, H11.

[0250] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the -L2-E segment is selected from H1.

[0251] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the sugar unit is selected from:

[0252] In the intermediate of the first aspect or the hydrophilic linker of the second aspect, the sugar unit is selected from:

[0253] The antibody drug conjugate prepared by using the hydrophilic linker of the present application can improve the hydrophilic property. It is especially suitable for conjugating with different hydrophobic payloads to generate high DAR value antibody drug conjugate to reduce or avoid the problem of aggregation.

[0254] Based on the aforementioned improvement of hydrophilic property, the prepared antibody drug conjugate is expected to further improve the in vitro activity and in vivo efficacy. In some specific embodiments, the antibody drug conjugate prepared by using the linker of the present application all have strong in vitro cytotoxicity (<1 nM), and in addition, also have 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. A novel intermediate is successfully prepared in the present application, and the hydrophilic linker can be prepared by using the intermediate.

[0255] The antibody drug conjugate prepared by using the novel hydrophilic linker provided in the present application has a shorter retention time on HIC HPLC.

[0256] The present application is further illustrated by the following examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the instructions of the commercial product.

[0257] Synthetic route of linker intermediate

[0258] H2N-L2-E can be synthesized according to the following synthesis method:

[0259] Synthetic route 1:

[0260] Step 1

[0261] The compound of Formula I-1 is obtained by a double nucleophilic substitution reaction (SN2) between the compound of Formula I-intermediate 1 and the compound of Formula I-raw material 1.

[0262] Step 2

[0263] The compound of Formula I-intermediate 3 is obtained by a reductive amination reaction between the compound of Formula I-intermediate 2 and a sugar compound.

[0264] Step 3

[0265] The compound of Formula I-1 is obtained by a de-Cbz reaction of the compound of Formula I-intermediate 3.

[0266] Synthetic Route 2:

[0267] Step 1

[0268] The compound of Formula I-intermediate 4 is obtained by a reductive amination reaction between the compound of Formula I-intermediate 2 and a phosphocholine compound containing an aldehyde group.

[0269] Step 2

[0270] The compound of Formula I-2 is obtained by a de-Cbz reaction of the compound of Formula I-intermediate 4.

[0271] Synthetic Route 3 is as follows:

[0272] Step 1

[0273] The compound of Formula I-intermediate 5 is obtained by a double nucleophilic substitution reaction (SN2) between the compound of Formula I-intermediate 2 and a polyethylene glycol compound containing a -OTs or halogen substituent.

[0274] Step 2

[0275] The compound of Formula I-3 is obtained by a de-Cbz reaction of the compound of Formula I-intermediate 5.

[0276] Synthetic Route 4 is as follows:

[0277] Step 1

[0278] The compound of Formula I-intermediate 6 is obtained by a double nucleophilic substitution reaction (SN2) between the compound of Formula I-intermediate 2 and an alkyl sulfonic acid compound containing a -OTs or halogen substituent.

[0279] Step 2

[0280] The compound of Formula I-4 is obtained by a de-Cbz reaction of the compound of Formula I-intermediate 6.

[0281] Synthesis route 5 is as follows:

[0282] Step 1

[0283] The compound of general formula I-7 is obtained by condensation reaction of the compound of general formula I-intermediate 2 and a polyarginine (poly-N-methyl glycine) compound or its activated ester containing a carboxyl group.

[0284] Step 2

[0285] The compound of general formula I-5 is obtained by de-Cbz reaction of the compound of general formula I-intermediate 7.

[0286] Synthesis route 6

[0287] Step 1

[0288] The compound of general formula I-intermediate 8 is obtained by condensation reaction of the compound of general formula I-intermediate 2 and a phosphorylcholine compound or its activated ester containing a carboxyl group.

[0289] Step 2

[0290] The compound of general formula I-6 is obtained by de-Cbz reaction of the compound of general formula I-intermediate 8.

[0291] Synthesis method of HO2C-L2-E:

[0292] Synthesis route 1:

[0293] Step 1

[0294] The compound of general formula I-intermediate 10 is obtained by bimolecular nucleophilic substitution (SN2) reaction of the compound of general formula I-intermediate 9 and the compound of general formula I-raw material 1.

[0295] Step 2

[0296] The compound of general formula I-intermediate 11 is obtained by reductive amination reaction of the compound of general formula I-intermediate 10 and a sugar compound.

[0297] Step 3

[0298] The compound of general formula I-7 is obtained by debenzyl reaction of the compound of general formula I-intermediate 11.

[0299] Synthesis route 2:

[0300] Step 1

[0301] The compound of Formula I-8 is obtained by debenzylating the compound of Formula I-12.

[0302] Step 2

[0303] The compound of Formula I-8 is obtained by debenzylating the compound of Formula I-12.

[0304] Synthetic Route 3:

[0305] Step 1

[0306] The compound of Formula I-13 is obtained by bimolecular nucleophilic substitution (SN2) reaction of the compound of Formula I-10 with a polyethylene glycol compound containing -OTs or halogen substituent.

[0307] Step 2

[0308] The compound of Formula I-9 is obtained by debenzylating the compound of Formula I-13.

[0309] Synthetic Route 4:

[0310] Step 1

[0311] The compound of Formula I-14 is obtained by bimolecular nucleophilic substitution (SN2) reaction of the compound of Formula I-10 with an alkyl sulfonic acid compound containing -OTs or halogen substituent.

[0312] Step 2

[0313] The compound of Formula I-10 is obtained by debenzylating the compound of Formula I-14.

[0314] Synthetic Route 5:

[0315] Step 1

[0316] The compound of Formula I-15 is obtained by condensation reaction of the compound of Formula I-10 with a poly-muscle acid (poly-N-methyl glycine) compound or its activated ester containing carboxyl group.

[0317] Step 2

[0318] The compound of Formula I-11 is obtained by debenzylating the compound of Formula I-15.

[0319] Synthetic Route 6

[0320] Step 1

[0321] The compound of Formula I-12 is obtained by debenzylating the compound of Formula I-16.

[0322] Step 2

[0323] The compound of Formula I-12 is obtained by debenzylating the compound of Formula I-16.

[0324] It is noted that the aza crown ether portion of the compounds of Formula I-1 to Formula I-12 is not limited to the examples described herein, but can also be other crown ether forms described herein.

[0325] The intermediates for the linkers described herein are prepared using the methods described above, including but not limited to the structures shown in any of the preceding numbered H1-H44, where the wavy line represents an amino, carboxyl, or protected form as described in the preceding scheme.

[0326] Example: Preparation of Linker Intermediates LP11-10, LP11-11

[0327] Step 1: Synthesis of LP11-7

[0328] 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) was added Et3N (835 mg, 8.25 mmol) and stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The solvent was removed by concentration, and then extracted with dichloromethane after adding appropriate amount of water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the target product (5.8 g, crude) as a yellow oil.

[0329] LC-MS (ESI) m / z: 680.7 [M+H] +

[0330] Step 2: Synthesis of LP11-8

[0331] 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 reaction mixture was washed with water, extracted with dichloromethane, and the organic layer was concentrated. The target product (4.9 g, yield 71.3%) was obtained as a yellow oil by purification with silica gel flash column chromatography (DCM / MeOH = 100%-30:1).

[0332] LC-MS (ESI) m / z: 834.7 [M+H] +

[0333] 1 H NMR (400 MHz, Chloroform-d) δ 7.81 - 7.75 (m, 2H), 7.38 - 7.27 (m, 7H), 5.51 (s, 1H), 5.08 (s, 2H), 3.70 - 3.65 (m, 2H), 3.64 - 3.58 (m, 38H), 3.57 (s, 6H), 3.38 (q, J = 5.3 Hz, 2H), 2.44 (s, 3H).

[0334] Step 3: Synthesis of LP11-9

[0335] LP11-8 (2.3 g, 2.76 mmol), 1,4,7-triazonane (2.85 g, 22 mmol) and K2CO3 (1.52 g, 11 mmol) were dissolved in acetonitrile (40 mL) and stirred at room temperature overnight. LCMS showed the reaction was complete. The resulting filtrate was filtered and concentrated. Purification by reverse phase column chromatography (0.1% FA in water / CH3CN) gave the target product (1.6 g, yield 36.3%) as a colorless oil.

[0336] LC-MS (ESI) m / z: 791.8 [M+H] +

[0337] 1 H NMR (400 MHz, Chloroform-d) δ 7.37 - 7.27 (m, 5H), 5.55 (s, 1H), 5.08 (s, 2H), 3.64 - 3.57 (m, 39H), 3.57 - 3.50 (m, 4H), 3.37 (q, J = 5.3 Hz, 2H), 2.81 - 2.50 (m, 18H).

[0338] Step 4: Synthesis of LP11-10

[0339] 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. LCMS showed the reaction was complete. The solvent was removed by concentration and purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (850 mg, yield 84.3%) as a colorless oil.

[0340] LC-MS (ESI) m / z: 986.0 [M+H] +

[0341] 1 H NMR (400 MHz, Chloroform-d) δ 7.41 - 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 = 5.1 Hz, 5H), 3.41 (q, J = 5.4 Hz, 4H), 3.08 (s, 17H).

[0342] Step 5: Synthesis of LP11-11

[0343] LP11-10 (1 g, 0.89 mmol) was dissolved in TFA (5 mL) and stirred at room temperature overnight. LCMS showed the reaction was complete. The solution was diluted with 25 mL water and purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (735 mg, yield 83.5%) as a colorless oil.

[0344] LC-MS (ESI) m / z: 986.0 [M+H] +

[0345] Linker-drug conjugate examples

[0346] The linkers and drugs herein can be prepared into linker-drug conjugates by known chemical methods. They include, but are not limited to, the structures shown in any of the preceding Examples LP1-LP67. The synthesis can refer to the specific preparation methods of LP11, LP13 or LP23.

[0347] Example: Synthesis of compound LP11

[0348] The synthetic route is shown below:

[0349] Step 1: Synthesis of LP11-2

[0350] 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 stirred at room temperature for 3 hours under H2 protection. LCMS showed the reaction was complete. The filter cake was filtered and washed with DMF (10 mL). The resulting filtrate was concentrated and purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (137 mg, yield 31.2%) as a white solid.

[0351] LC-MS (ESI) m / z: 668.2 [M+Na] +

[0352] Step 2 and 3: Synthesis of LP11-5

[0353] 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), then EDCI (96 mg, 0.5 mmol) was added. Stirring at room temperature for 1-2 hours. LCMS showed the reaction was completed. Without further purification, 0.4 mL diethylamine was added and stirred at 0 °C for 30 minutes. LCMS showed the reaction was completed. The solution was purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (48.35 mg, yield 42.2%) as a white solid.

[0354] LC-MS (ESI) m / z: 1176.1 [M+H] +

[0355] Step 4: Synthesis of LP11-13

[0356] To the solution of LP11-11 (700 mg, 0.71 mmol), LP11-12 (371 mg, 0.71 mmol) in DMF (5 mL) was added DIEA (183 mg, 1.42 mmol) and stirred at room temperature for 1 hour. LCMS showed the reaction was completed. The solution was purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (135 mg, yield 13.6%) as a yellow oil.

[0357] LC-MS (ESI) m / z: 697.2 [M / 2+H] +

[0358] Step 5: Synthesis of LP11-14.

[0359] 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 the reaction was completed. The solution was purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (117 mg, yield 87.7%) as a yellow oil.

[0360] LC-MS (ESI) m / z: 669.2 [M / 2+H] +

[0361] Steps 6 and 7: Synthesis of LP11-16

[0362] LP11-14 (20 mg, 0.015 mmol), HATU (10.26 mg, 0.027 mmol), 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 diethylamine was added and stirred at 0 °C for 30 min. LCMS showed the reaction was complete. The solution was purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (12 mg, yield 35.2%) as a white solid.

[0363] LC-MS (ESI) m / z: 1136.5 [M / 2+H] +

[0364] Step 8: Synthesis of LP11

[0365] LP11-14 (20 mg, 0.015 mmol), HATU (10.26 mg, 0.027 mmol), 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 diethylamine was added and stirred at 0 °C for 30 min. LCMS showed the reaction was complete. The solution was purified by reverse phase column chromatography (0.1% FA in water / CH3CN) to give the target product (12 mg, yield 35.2%) as a white solid.

[0366] LC-MS (ESI) m / z: 1136.5 [M / 2+H] +

[0367] 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 7.5 Hz, 2H), 8.67 (s, 1H), 8.34 (d, J = 6.3 Hz, 1H), 8.26 (s, 1H), 8.14 (d, J = 8.8 Hz, 2H), 8.04 (t, J = 5.8 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.87 (d, J = 5.7 Hz, 1H), 7.75 (s, 1H), 7.27 - 7.14 (m, 6H), 6.99 (s, 2H), 6.43 (d, J = 7.0 Hz, 2H), 6.14 (s, 2H), 5.04 (d, J = 11.5 Hz, 1H), 4.67 (d, J = 7.0 Hz, 3H), 4.49 (s, 3H), 4.19 (t, J = 6.4 Hz, 2H), 4.11 (d, J = 1.9 Hz, 1H), 4.06 (d, J = 10.9 Hz, 1H), 4.02 (s, 2H), 3.83 (s, 3H), 3.70 (d, J = 5.6 Hz, 6H), 3.63 (s, 5H), 3.61 (d, J = 2.8 Hz, 8H), 3.56 (s, 6H), 3.53 - 3.49 (m, 74H), 3.47 - 3.42 (m, 14H), 3.24 - 3.16 (m, 6H), 3.03 (d, J = 11.1 Hz, 5H), 2.89 - 2.73 (m, 15H), 2.36 (d, J = 16.9 Hz, 1H), 2.29 (s, 3H), 2.21 (s, 4H), 2.15 (d, J = 14.8 Hz, 1H), 2.10 (t, J = 7.6 Hz, 5H), 2.05 (s, 4H), 1.97 (s, 3H), 1.47 (d, J = 9.8 Hz, 8H).

[0368] Example: Synthesis of compound LP13

[0369] Steps 1 and 2: Synthesis of LP13-2

[0370] LP11-14 (26.5 mg, 0.0198 mmol), HATU (14 mg, 0.0357 mmol), DIEA (8 mg, 0.059 mmol) were dissolved in DMF (2 mL) at 0 °C and stirred for 30 min. LP11-16 (45 mg, 0.0198 mmol) was added and stirred for 1 h. LCMS showed the reaction was completed. Diethylamine (0.4 mL) was added directly without further purification. The reaction solution was purified by preparative high performance liquid chromatography to give the target product (20 mg, yield 50.9%) as a colorless oil.

[0371] LC-MS (ESI) m / z: 1123.6 [M / 3+H]+

[0372] Step 2: Synthesis of LP13

[0373] Dissolve 6-maleimidohexanoic acid (1.4 mg, 0.0065 mmol), HATU (3.7 mg, 0.0098 mmol) and DIEA (1.7 mg, 0.013 mmol) in DMF (3 mL) and stir at 0 °C for 30 min. Then, add LP13-2 (20 mg, 0.0059 mmol) and stir at 0 °C for 2 h. LCMS shows the reaction is complete. Purify by preparative high performance liquid chromatography (0.1% FA in water / CH3CN) to give the target product (2.58 mg, 12.2% yield) as a white solid.

[0374] LC-MS (ESI) m / z: 1188.1 [M / 3+H] +

[0375] Example: Synthesis of compound LP23

[0376] Step 1: Synthesis of LP23-3

[0377] To a solution of LP23-1 (150.2 mg, 0.283 mmol, 1.0 eq.) and DIPEA (0.1 mL, 0.574 mmol, 2.0 eq.) in DMF (3.0 mL) was added LP23-2 (221 mg, 0.288 mmol, 1.02 eq.). The mixture was stirred at room temperature for 5 h, and the crude product solution was used for the next step without further purification.

[0378] LC-MS (ESI) m / z: 1063.3 [M+H] +

[0379] Step 2: Synthesis of LP23-4

[0380] To the above crude product solution (crude, 0.283 mmol, 1.0 eq.) was added diethylamine (0.29 mL, 2.8 mmol, 10.0 eq.). The reaction solution was stirred at room temperature for 1 h, and then purified by preparative liquid chromatography (0.1% NH4HCO3 in water / CH3CN) to give the desired product (57.5 mg, yield for two steps: 24.2%) as a white solid.

[0381] LC-MS (ESI) m / z: 841.3 [M+H] +

[0382] Step 3: Synthesis of LP23-5

[0383] To a solution of LP23-4 (57.53 mg, 0.068 mmol, 1.0 eq.) in DMF (1.9 mL) was added 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.). The resulting reaction was stirred at room temperature for 1 h, the crude product solution was used for the next step without further purification.

[0384] LC-MS (ESI) m / z: 1079.7 [M / 2+H] +

[0385] Step 4: Synthesis of LP23-6

[0386] To a solution of the crude product of LP23-5 (crude, 0.068 mmol, 1.0 eq.) was added diethylamine (0.07 mL, 0.68 mmol, 10.0 eq.). The reaction was stirred at room temperature for 1 h, then purified by preparative liquid chromatography (0.1% NH4HCO3 in water / CH3CN) to give the desired product (22.1 mg, yield over two steps: 16.8%) as a white solid.

[0387] LC-MS (ESI) m / z: 646.5 [M / 3+H] +

[0388] Step 5: Synthesis of LP23

[0389] A mixture solution of 6-maleimidopropanedioic 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 h, 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.

[0390] LC-MS (ESI) m / z: 1065.8 [M / 2+H] +

[0391] 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.85 (dd, J1 = J2 = 6.0 Hz, 1H), 7.78 (d, J = 6.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 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).

[0392] Preparation and characterization of antibody drug conjugates

[0393] The following are examples of methods for preparing and analyzing 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 antibodies are not limited to the antibodies shown herein and can be any other antibody. The preparation methods described below can be used to prepare antibody drug conjugates including, but not limited to, the structures shown in ADCs 1-67 above.

[0394] General conjugation procedure for preparing antibody drug conjugates based on hydrophilic stable linkers

[0395] The monoclonal antibody (1.0 eq.) was placed in a 0.5 mL centrifuge tube, 25 mM Na2B4O7, 25 mM NaCl, 1 mM DTPA buffer was added, the antibody concentration was diluted to 5-10 mg / mL, after shaking, 2 mg / mL TCEP (2 eq. to 10 eq.) aqueous solution was added to the mixture for antibody reduction. After shaking, the reaction was placed on a heating and refrigeration type constant temperature shaker for 2-3 hours at 20-25°C. Then 10 mg / mL DMSO dissolved toxin linker solution (4 eq. to 30 eq.) was added, 10-20% DMSO was added according to the total volume of the final reaction solution, and after shaking, the reaction was placed on a heating and refrigeration type constant temperature shaker for 0.5-4 hours at 20-25°C.

[0396] The activated carbon coated with 300 mg / mL dextran (manufacturer: Sigma) was used to remove the residual toxin linker in the reaction solution which was not coupled with the antibody. A 10% volume of activated carbon solution was added to the reaction solution, and after shaking, it was shaken at 20-25°C for 1-2 hours. The supernatant was taken to detect the content of residual toxin linker. If the content of free toxin linker was greater than 1%, the above process should be repeated multiple times until the detection result reached the standard (usually 3 times of treatment could meet the requirements). After the treatment was completed, the mixture was centrifuged, and the supernatant was collected with a syringe and a hydrophilic membrane filter to filter out the activated carbon. The buffer of the desired antibody drug conjugate was replaced with the appropriate storage buffer by 3-6 times of ultrafiltration, and stored at -80°C.

[0397] General characterization method of antibody drug conjugate

[0398] (a) HIC-HPLC analysis of DAR value of ADC

[0399] High performance liquid chromatograph: e2695 high performance liquid chromatograph system or 1260 Agilent liquid chromatograph.

[0400] Chromatographic column: MabPac TM HIC-Butyl 5 μm 4.6 x 100 mm (manufacturer: Thermo).

[0401] Mobile phase: Mobile phase A (MPA): 1.5 M (NH4)2SO4+ 50 mM potassium phosphate salt (pH 7.0); Mobile phase B (MPB): 50 mM sodium phosphate salt (pH 7.0) / isopropanol (75:25 V / 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%.

[0402] Detection condition: set the flow rate of mobile phase to 1 ml / min, detection wavelength to 280 nm, column temperature to 30 °C.

[0403] Experimental procedure: take 50 μg of the sample after conjugation (volume according to sample concentration), inject into the high performance liquid chromatograph, elute using the above elution program, and record the chromatogram.

[0404] DAR value calculation formula: DAR =∑ (relative peak area x number of loaded drugs) / 100

[0405] (b) C18-HPLC analysis of Free Linker Payload (mol / mol%)

[0406] High performance liquid chromatograph: e2695 high performance liquid chromatograph system.

[0407] Chromatographic column: C18 3.5 μm 4.6 x 150 mm (manufacturer: Waters).

[0408] Mobile phase: Mobile phase A (MPA): 0.1% TFA-H2O; Mobile phase B (MPB): 0.1% TFA-ACN; elution was performed according to the following elution 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%

[0409] Detection condition: set the flow rate of mobile phase to 0.5 ml / min, detection wavelength to 254 nm, column temperature to 30 °C.

[0410] Reagent I preparation example: Take 30 mL of anhydrous methanol and 50 mL of acetonitrile, weigh 10 g of sodium chloride, mix in a container, stir at room temperature for more than 1 hour, and then stand for 1 hour. Take the supernatant, filter with a 0.22 μm organic membrane, and store at room temperature for 3 months.

[0411] Reagent II preparation example: Take 100 mL of reagent I, 15 mL of DMSO, and 85 mL of ADC sample storage buffer, respectively, mix uniformly in a container, and store at room temperature for 2 months.

[0412] Experimental steps:

[0413] Sample solution preparation:

[0414] Free toxin reference: Take the toxin corresponding to the test sample as the reference, and prepare it into a final concentration of 1.0 mg / ml with the above reagent II.

[0415] Take 10 μl of the above concentration of 1.0 mg / ml toxin reference, add to 90 μl of reagent II, and prepare into a concentration of 100 μg / ml; then dilute the sample with reagent II according to the following table to prepare the required concentration of sample. Note: The sample prepared above is injected into HPLC in the order of concentration from small to large.

[0416] Sample testing: Take 85 μg of the coupled sample, mix the sample with 3 μl of DMSO for 5 min, then add 60 μl of reagent I to the sample system, mix uniformly for 5-10 min, centrifuge at 2000 rpm for 2 min, take 20 μL of the supernatant, inject into the high performance liquid chromatograph, elute with the above elution program, and record the chromatogram.

[0417] According to the calculated standard binary linear regression curve equation, the content of free toxin in ADC is calculated,

[0418] Free Drug (mol / mol%) = residual small molecule molar concentration / antibody molar concentration x 100.

[0419] (c) LC-MS / MS method for determining free linker-payload content

[0420] Internal reference compounds: 50 ng / ml tolbutamide, labetalol, etofenprox and buspirone

[0421] Extraction agent: ethyl acetate

[0422] Standard gradient dilution: dilute the standard to 20000, 16000, 10000, 4000, 1000, 200, 40, 20 ng / ml with 50% methanol aqueous solution.

[0423] Sample pretreatment before machine: take 20 μL sample, then add 20 μL blank buffer and 240 μL extractant. After vortex mixing, stand until stratification, then take 160 μL supernatant into a 96-well plate and blow dry, add 200 μL 50% acetonitrile water, mix well, and finally enter LC-MS / MS analysis.

[0424] Standard pretreatment before machine: take 2 μL working solution and add it to 38 μL blank buffer, then add 240 μL extractant. After vortex mixing, stand until stratification, take 160 μL supernatant into a 96-well plate and blow dry, add 200 μL 50% acetonitrile water, mix well.

[0425] Mass spectrometry conditions:

[0426] Data processing: according to the concentration of the standard and the corresponding mass spectrometry response value, a standard curve is drawn, and through linear fitting curve (formula) and the mass spectrometry response value of the detected sample, the residual amount of free linker-payload is calculated.

[0427] (d) SEC-HPLC analysis of ADC aggregates

[0428] High performance liquid chromatograph: 1260 Agilent liquid chromatograph.

[0429] Chromatographic column: Waters Xbridge BEH200 SEC (7.8 x 300 mm, 3.5 μm)

[0430] Mobile phase: 50 mM PB + 200 mM Arg (pH 6.80) + 10% IPA, eluted according to the following elution program, 0-30 min mobile phase A volume 100%-100%;

[0431] Detection conditions: set the flow rate of mobile phase to 0.5 ml / min, the detection wavelength to 280 nm, and the column temperature to 26°C.

[0432] Experimental procedure: take 20 μg of the coupled sample (volume according to sample concentration), inject it into the high performance liquid chromatograph, elute it using the above elution program, and record the chromatogram.

[0433] Calculation formula: monomer purity (%) = Amonomer / Atotal x 100%; polymer purity (%) = Apolymer / Atotal x 100%

[0434] (e) ADC product was analyzed by LC / TOF (Agilent, 6230 LC / TOF) system

[0435] The liquid chromatographic column was: PLRP-S, 1000A column (Agilent 8 μm, 2.1 x 150 mm)

[0436] The mobile phase A was 0.1% formic acid

[0437] The mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 0.4 mL / min.

[0438] The mass spectrometry was analyzed in positive ion mode, and the parameters were set as follows: the scanning range was 500-5000 m / z,

[0439] Gas temperature: 320 °C

[0440] Vcap: 4500 V

[0441] Fragmentor: 200 V

[0442] Skimmer: 65 V.

[0443] Preparation of Antibody Drug Conjugate Comparative Example 1

[0444] n1= 7.96

[0445] According to the general conjugation process for preparing antibody drug conjugates, TCEP (10 mg / mL, 12 eq.) aqueous solution was added to the buffer solution of trastuzumab (5 mg, 21.07 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0, and reduced at 22 °C for 2 hours; then RLP1 (10 mg / mL, 10 eq., purchased from MCE) DMSO solution was added, and the conjugation reaction was carried out at 22 °C for 1 hour. After purification by dextran-coated activated carbon for 2 times, the buffer solution of the desired antibody drug conjugate was obtained by 3 times of ultrafiltration to obtain Comparative Example 1 (CA DC (mg / mL): 6.74, V (mL): 0.6, yield 80.9%).

[0446] RLP1:

[0447] The following characterization results were obtained by the general characterization method of antibody drug conjugates:

[0448] HIC-DAR: 7.96, SEC purity: 97.7%, Free Linker Payload (mol / mol%): 0.04221.

[0449] Preparation of antibody drug conjugate comparative example-2

[0450] n1= 7.8

[0451] According to the general conjugation process for preparing antibody drug conjugates, an aqueous solution of TCEP (10 mM, 15 eq.) was added to the buffer solution of trastuzumab (30.0 mg, 20.18 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0, and reduced at 22 °C for 2 hours; then a DMSO solution of RLP2 (10 mg / mL, 10 eq.) was added, and the conjugation reaction was carried out at 22 °C for 1 hour. After purification by dextran-coated activated charcoal treatment twice, the buffer solution of the desired antibody drug conjugate was obtained by ultrafiltration three times to obtain comparative example-2 (CA DC (mg / mL): 5.1, V (mL): 0.06, yield 61.2%).

[0452] RLP2:

[0453] The following characterization results were obtained by the general characterization method of antibody drug conjugates:

[0454] HIC-DAR: 7.8, SEC purity: 97.39%, Free Linker Payload (mol / mol%): 0.01122%.

[0455] Preparation of antibody drug conjugate ADC-11

[0456] n1= 7.4

[0457] According to the general conjugation process for preparing antibody drug conjugates, an aqueous solution of TCEP (10 mM, 10 eq.) was added to the buffer solution of trastuzumab (1.0 mg, 20.18 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0, and reduced at 22 °C for 2 hours; then a DMSO solution of LP11 (10 mg / mL, 10 eq.) was added, and the conjugation reaction was carried out at 22 °C for 1 hour. After purification by dextran-coated activated charcoal treatment twice, the buffer solution of the desired antibody drug conjugate was obtained by ultrafiltration three times to obtain ADC-11 (CA DC (mg / mL): 5.1, V (mL): 0.06, yield 61.2%).

[0458] The following characterization results were obtained by the general characterization method of antibody drug conjugates:

[0459] HIC-DAR: 7.9, SEC purity: 95.4%, Free Linker Payload (mol / mol%): not detected.

[0460] Example: Preparation of antibody drug conjugate ADC-13

[0461] n1= 7.9

[0462] According to the general conjugation procedure for preparing antibody drug conjugates, to trastuzumab (1.5 mg, 20.18 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer was added TCEP (10 mM, 10 eq.) in water and reduced at 22 °C for 2 h; then LP13 (10 mg / mL, 20 eq.) in DMSO was added and the conjugation reaction was carried out at 22 °C for 1 h. After purification by dextran-coated charcoal treatment twice, the buffer of the desired antibody drug conjugate was exchanged by ultrafiltration three times to give ADC-13 (CA DC (mg / mL): 6.8, V (mL): 0.06, yield 40.8%).

[0463] The following characterization results were obtained by the general characterization method for antibody drug conjugates:

[0464] HIC-DAR: 7.9, SEC purity: 95.4%, Free Linker Payload (mol / mol%): not detected.

[0465] Example: Preparation of antibody drug conjugate ADC-23

[0466] n1= 7.7

[0467] According to the general conjugation procedure for preparing antibody drug conjugates, to trastuzumab (2.0 mg, 20.18 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer was added TCEP (10 mM, 15 eq.) in water and reduced at 22 °C for 2 h; then LP23 (10 mM, 16 eq.) in DMSO was added and the conjugation reaction was carried out at 22 °C for 1 h. After purification by dextran-coated charcoal treatment three times, the buffer of the desired antibody drug conjugate was exchanged by ultrafiltration three times to give ADC-23 (CA ADC (mg / mL): 6.8, V (mL): 0.06, yield 40.8%).

[0468] The following characterization results were obtained by the general characterization method for antibody drug conjugates:

[0469] HIC-DAR: 7.7, SEC purity: 100%, Free Linker Payload (mol / mol%): not detected.

[0470] HIC retention time of antibody-drug conjugates

[0471] The HIC retention time of antibody-drug conjugates ADC-11 and ADC-13 with hydrophilic linker is earlier than that of antibody-drug conjugate comparative example-2 with PEG chain linker. And the HIC retention time of LP13 is further earlier than that of ADC-11 with one side chain and comparative example 2 with one side chain, because LP13 contains two hydrophilic side chains. The hydrophilicity of high DAR value antibody-drug conjugates can be improved to avoid aggregation. In addition, unlike the antibody-drug conjugates based on MC-VC-PAB-Exatecan, which are prone to aggregation at high DAR values such as DAR8 (Mol Cancer Ther (2024) 23(2): 199-211), ADC-23 based on LP23 containing hydrophilic side chains has no aggregation at DAR7.7.

[0472] And further, when different aza crown ether groups are selected, the greater the proportion of NH and O in the aza crown ether, the more improvement in the hydrophilicity of the ADC can be expected.

[0473] Biological evaluation

[0474] 1) Tumor cell proliferation inhibition experiment of antibody-drug conjugates based on hydrophilic linker

[0475] NCI-N87 and JIMT-1 cells with a certain cell concentration (1500 cells per well, in 80 μl) were added to a 96-well plate. The cells were incubated in a constant temperature incubator (37°C, 5% CO2) overnight. Prepare antibody-drug conjugate solutions based on hydrophilic linker with a highest concentration of 400 nM, and dilute with PBS at a gradient of 1:4. After incubating the diluted solution (20 μl per well) with the cells (37°C, 5% CO2) for five days, add CellTitre-Glo reagent (40 μl per well), and measure the fluorescence value using i3X microplate reader.

[0476] In vitro cytotoxicity of antibody-drug conjugates (+++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[0477] All ADCs showed certain anti-tumor activity, for example, in HER2 high expression cell NCI-N87 cells, the in vitro cell killing activity of ADC-11, ADC-13 and ADC-23 was equivalent to that of the reference ADC pair Comparative Example-1 and the reference ADC pair Comparative Example-2, all less than 1 nM. In HER2 low expression cell JIMT-1 cells, the in vitro cell killing activity of ADC-23 was stronger than that of the reference ADC pair Comparative Example-1. It was confirmed through the experiment that the antibody-drug conjugate with in vitro cell killing activity could be prepared by using the linker herein.

[0478] 2) JIMT-1 tumor-bearing mouse efficacy evaluation experiment of antibody-drug conjugate based on hydrophilic linker

[0479] Purpose of the experiment

[0480] The conjugated drug of the present application was evaluated using Nu / Nu nude mice as test animals.

[0481] Efficacy of ADC-23 and reference ADC pair Comparative Example-1 on JIMT-1 transplanted tumor nude mice after tail vein injection.

[0482] Test drugs and materials

[0483] 1. Test drugs

[0484] ADC-23: 3 mg / kg

[0485] Reference ADC pair Comparative Example-1: 3 mg / kg

[0486] Blank control (Blank): PBS

[0487] Preparation method:

[0488] 2. All were diluted with PBS for preparation.

[0489] 3. Test animals

[0490] Nu / Nu nude mice were purchased from Beijing Weitong Lihua.

[0491] Test method

[0492] JIMT-1 cells were inoculated subcutaneously in the right flank of the mice, and the tumor grew for 7 days. The animals were randomly divided into groups, 5 animals per group (4 animals in the PBS group), a total of three groups.

[0493] Drug administration was performed by tail vein injection, a total of 1 time. Tumor volume and mouse body weight were measured twice a week, and the data were recorded.

[0494] Data statistics using Excel Microsoft 365 statistical software: average value is calculated as avg; SD value is calculated as STDEV; SEM value is calculated as STDEV / SQRT; P value of difference between groups is calculated as TTEST.

[0495] Tail vein injection administration 1 time, observation to the end of the test. The test results are shown in Figures 1 and 2, and the ADC-23 molecule can significantly (P = 0.011) reduce the tumor volume, and has a better tumor inhibition effect than the reference ADC comparative example-1. At the same time, there is no significant difference in the body weight of mice in the ADC-23 group and the reference ADC comparative example-1 group.

[0496] The main advantages of the present application are:

[0497] 1. The antibody drug conjugate prepared by using the hydrophilic linker in the present application can improve the hydrophilic property. It is especially suitable for producing high DAR value antibody drug conjugates with hydrophobic load to reduce or avoid the problem of aggregation.

[0498] 2. Based on the improvement of the aforementioned hydrophilic property, the prepared antibody drug conjugate is expected to further improve the in vitro activity and in vivo efficacy. In some specific embodiments, the antibody drug conjugates prepared by using the linker in the present application all have strong in vitro cytotoxicity (<1 nM), in addition, they also have 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.

[0499] 3. The antibody drug conjugate based on the novel hydrophilic linker provided in the present application has a shorter retention time on HIC HPLC.

[0500] The examples in the present application are only used to illustrate the present application and do not constitute a limitation on the scope of the claims. Other substantially equivalent alternatives that can be conceived by those skilled in the art are within the scope of protection of the present application.

Claims

1. An intermediate of a hydrophilic linker, characterized in that comprises a structure represented by Formula V or a salt or ester thereof, R4-L2-E (V), wherein, R4 is a functional group capable of binding to an amino acid unit in a linker, L2 is absent or is a PEG unit, and E is selected from an azacrown ether group substituted with one or more hydrophilic units, the azacrown ether comprising two or more nitrogen atoms and optionally comprising an oxygen atom, the hydrophilic units being attached to the nitrogen atoms in the azacrown ether, the azacrown ether group being attached to L2 or R4 via another nitrogen atom depending on the presence of L2.

2. The intermediate of claim 1, wherein R4 is selected from an amino group, a carboxyl group, or a protected form thereof.

3. A hydrophilic linker characterized in that comprise one or more side chains of the general structure I, or salts or esters thereof, wherein, the wavy line in Formula I represents a binding site to an amino acid unit, L2 is absent or is a PEG unit, and 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 units being attached to the nitrogen atoms in the azacrown ether, the azacrown ether group being attached to L2 or the amino acid unit via another nitrogen atom depending on the presence of L2.

4. The linker intermediate of any one of claims 1 to 2 or the hydrophilic linker of claim 3, wherein: when present, L2is selected from one or more of the combinations of n3 = 0-50, n4 = 0-50, n5 = 0-50, or n6 = 0-50.

5. The intermediate of any one of claims 1-2 or the hydrophilic linker of claim 3, wherein E is selected from the group consisting of: 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. W1, W2, W3, W4, W5, W6, W7, W8, W9are the same or different and are each independently selected from oxygen, sulfur or said R1 is selected from X or said n 18 = an integer from 0 to 100; said 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 independently selected from hydrogen or a hydrophilic unit, and when the azacrown ether unit comprises a plurality of Xs, the Xs can be the same or different.

6. The intermediate or hydrophilic linker of claim 5, wherein the hydrophilic unit is selected from a sugar unit, a polysarcosine unit, a polyethylene glycol unit, or an alkyl sulfonic acid unit.

7. The intermediate or hydrophilic linker of claim 6, wherein the hydrophilic unit is a sugar unit, and the sugar unit is optionally substituted with a monosaccharide group, a disaccharide group, or a polysaccharide group.

8. The intermediate or hydrophilic linker of claim 6 or 7, wherein The sugar unit structure is: wherein, n 19 = 0-20, n 20 = 1-20, n 21 = 1-20, an integer, said R2 is selected from hydrogen or C 1-20 alkyl; said Y1 is independently selected from -NH- or oxygen; said R3 is independently selected from hydrogen, acetyl, monosaccharidyl, disaccharidyl or polysaccharidyl; Y2 is selected from said Y3is selected from hydrogen, hydroxyl or C 1-20 alkoxy.

9. The intermediate or hydrophilic linker of claim 8, wherein 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 a-cyclodextrin group, a β-cyclodextrin group, or a γ-cyclodextrin group.

10. The intermediate or hydrophilic linker of claim 8, wherein n 19 = 0-12, n 20 = 1-12, n 21 = 1-15, R2is selected from hydrogen or C 1-12 alkyl, Y3is selected from hydrogen, hydroxy or C 1-12 alkoxy.

11. The intermediate or hydrophilic linker of claim 10, wherein n 19 = 0-10, n 20 = 1-10, n 21 = 1-12, R2is selected from hydrogen or C 1-10 alkyl, Y3is selected from hydrogen, hydroxyl or C 1-10 alkoxy.

12. The intermediate or hydrophilic linker of claim 11, wherein n 19 = 0-8, n 20 = 1-8, n 21 = 1-10, R2is selected from hydrogen or C 1-8 alkyl, Y3is selected from hydrogen, hydroxy or C 1-8 alkoxy.

13. The intermediate or hydrophilic linker of claim 6, wherein The poly sarcosine unit structure is: wherein n 22 is an integer from 1 to 100.

14. The intermediate or hydrophilic linker of claim 13, wherein n 22 = an integer from 1 to 50.

15. The intermediate or hydrophilic linker of claim 14, wherein n 22 = an integer from 1 to 30.

16. The intermediate or hydrophilic linker of claim 15, wherein n 22 = an integer from 1 to 20.

17. The intermediate or hydrophilic linker of claim 6, wherein The polyethylene glycol unit structure is: wherein n 23 = an integer from 1 to 100, K2 is selected from hydrogen or C 1-20 alkyl.

18. The intermediate or hydrophilic linker of claim 17, wherein n 23 = 1-50, said K2is selected from hydrogen or C 1-12 alkyl.

19. The intermediate or hydrophilic linker of claim 18, wherein n 23 = 1-30, said K2is selected from hydrogen or C 1-10 alkyl.

20. The intermediate or hydrophilic linker of claim 19, wherein n 23 = 1-20, said K2is selected from hydrogen or C 1-8 alkyl.

21. The hydrophilic linker of claim 6, wherein The alkylsulfonic acid unit structure is: wherein n 24 is an integer of 1-20.

22. The intermediate or hydrophilic linker of claim 21, wherein n 24 = an integer from 1 to 12.

23. The intermediate or hydrophilic linker of claim 22, wherein n 24 = an integer from 1 to 10.

24. The intermediate or hydrophilic linker of claim 23, wherein n 24 = an integer from 1 to 8.

25. The intermediate or the hydrophilic linker of claim 5, wherein: n7 = an integer from 1 to 12, n8 = an integer from 1 to 12, n9 = an integer from 1 to 12, n 10 = an integer from 1 to 12, n 11 = an integer from 1 to 12, n 12 = an integer from 1 to 12, n 13 = an integer from 1 to 12, n 14 = an integer from 1 to 12, n 15 = an integer from 1 to 12, n 16 = an integer from 1 to 12, or n 17 = an integer from 1 to 12, said n 18 = an integer from 0 to 50; said K1is selected from hydrogen or C 1-12 alkyl.

26. The intermediate or the hydrophilic linker of claim 25, wherein: n7 = an integer from 1 to 10, n8 = an integer from 1 to 10, n9 = an integer from 1 to 10, n 10 = an integer from 1 to 10, n 11 = an integer from 1 to 10, n 12 = an integer from 1 to 10, n 13 = an integer from 1 to 10, n 14 = an integer from 1 to 10, n 15 = an integer from 1 to 10, n 16 = an integer from 1 to 10, or n 17 = an integer from 1 to 10, said n 18 = an integer from 0 to 30; said K1is selected from hydrogen or C 1-10 alkyl.

27. The intermediate or the hydrophilic linker of claim 26, wherein: n7 = an integer from 1 to 8, n8 = an integer from 1 to 8, n9 = an integer from 1 to 8, n 10 = an integer from 1 to 8, n 11 = an integer from 1 to 8, n 12 = an integer from 1 to 8, n 13 = an integer from 1 to 8, n 14 = an integer from 1 to 8, n 15 = an integer from 1 to 8, n 16 = an integer from 1 to 8, or n 17 = an integer from 1 to 8, said n 18 = an integer from 0 to 20; said K1is selected from hydrogen or C 1-8 alkyl.

28. The intermediate or hydrophilic linker of claim 4, wherein n3 = 0-30, n4 = 0-30, n5 = 0-30, or n6 = 0-30.

29. The intermediate or hydrophilic linker of claim 28, wherein n3 = 0-20, n4 = 0-20, n5 = 0-20, or n6 = 0-20.

30. The intermediate of any one of claims 1-2 or the hydrophilic linker of claim 3, wherein E is selected from the azacrown ether group of a 9-18 membered ring, and the hydrophilic unit is selected from a sugar unit, and the sugar unit is selected from: wherein n 19 is an integer selected from 1 to 3, R2is hydrogen or methyl, n 20 is an integer selected from 1 to 3, R3is independently selected from H or a C5to C6monosaccharide, n 21 is selected from 3 or 4, or wherein n 20 is an integer selected from 1 to 3, R3is independently selected from H or a C5-C6monosaccharide, n 21 is selected from 3 or 4.

31. The intermediate or hydrophilic linker of claim 30, wherein E is selected from the azacrown ether of a 9-, 12-, 15-, or 18-membered ring.

32. The intermediate or hydrophilic linker of claim 31, wherein E is selected from the group consisting of: wherein at least one X is a sugar unit and the other X is independently selected from H or a sugar unit.

33. The intermediate or hydrophilic linker of claim 32, wherein E is selected from the group consisting of:

34. The intermediate or hydrophilic linker of claim 30, wherein said sugar units are selected from the group consisting of:

35. The intermediate or hydrophilic linker of claim 34, wherein R3 is 36. The intermediate or hydrophilic linker of claim 34, wherein said sugar units are selected from the group consisting of:

37. The intermediate or hydrophilic linker of claim 36, wherein said sugar units are selected from the group consisting of:

38. The intermediate or hydrophilic linker of claim 32, wherein E is selected from the group consisting of:

39. The intermediate or hydrophilic linker of claim 38, wherein E is selected from the group consisting of:

40. The intermediate or hydrophilic linker of claim 39, wherein E is selected from the group consisting of 41. The intermediate or hydrophilic linker of claim 30, wherein L2 is present and is selected from n3 is selected from an integer from 8 to 12.

42. The intermediate or hydrophilic linker of claim 41, wherein n3 = 11.

43. The hydrophilic linker of claim 30, wherein comprises one or two of the side chains.

44. The intermediate of claims 1-2 or the hydrophilic linker of claim 3, wherein The -L2-E segment is selected from the structures shown in the following table:

45. The hydrophilic linker of claim 44, wherein The L2-E segment is selected from the structure shown in H1-H12.

46. The intermediate or hydrophilic linker of claim 45, wherein The L2-E segment is selected from the structure shown in H1, H2, H5, H6, H7, H8, H10, H11.

47. The intermediate or hydrophilic linker of claim 46, wherein The L2-E segment is selected from the structure shown in H1.

48. The linker of claim 3, wherein Also included is a first linker subunit for connecting with the targeting unit, and a second linker subunit for connecting with the drug, the first linker subunit and the second linker subunit are connected with the amino acid unit.

49. The linker of claim 48, wherein The targeting unit is selected from an antibody or an antigen-binding fragment thereof.

50. The linker of claim 48, wherein The amino acid units comprise one or more amino acid subunits selected from: or one or more combinations of stereoisomers thereof, * represents a connection site with L2, or when L2 is absent, represents a connection site with E; # represents a connection site with the first linker subunit or the amino acid subunit, ## represents a connection site with the second linker subunit or the 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 = 0-20.

51. A linker-drug conjugate characterized in that The linker of any one of claims 3-50 is formed in combination with a drug, having a structure according to Formula II, wherein, n2 is an integer from 1 to 8, A1 is selected from the amino acid subunit, and M is a linker for connecting with the targeting unit, L1is absent or selected from the group consisting of in combination with one or more of the following: wherein 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 = an integer from 0-20, A1is selected from or a stereoisomer thereof, * represents a connection site with L2, or when L2 is absent, represents a connection site with E; # represents a connection site with L1, ## represents a 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, n 54 = 0-20, n A2is absent or selected from: The D is a fragment formed by losing one or more atoms or groups from a biologically active molecule having anti-tumor activity.

52. The linker drug conjugate of claim 51, wherein M is selected from or stereoisomers thereof.

53. The linker drug conjugate of claim 52, wherein M-L2- is 54. The linker drug conjugate of claim 51, wherein A2 is 55. The linker drug conjugate of claim 51, wherein 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 = an integer from 0 to 12.

56. The linker drug conjugate of claim 55, wherein 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 = 0-10.

57. The linker drug conjugate of claim 56, wherein n 30 = 1-8, n 31 = 0-20, n 32 = 1-8 and n 33 = 0-20.

58. The linker drug conjugate of claim 51, wherein n2 is an integer from 1 to 6.

59. The linker drug conjugate of claim 58, wherein n2 is an integer from 1 to 4.

60. The linker drug conjugate of claim 59, wherein The n2 is 1 or 2.

61. The linker drug conjugate of claim 51, wherein The biologically active molecule is selected from a tubulin inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalator, a DNA-reactive agent, a DNA alkylating agent, an immunomodulator, a nucleic acid, a BCL-XL inhibitor, a kinase inhibitor, a PROTAC, a molecular glue, and a radioisotope.

62. The linker drug conjugate of claim 51, wherein The linker is covalently bound to a nitrogen, oxygen, or sulfur in the drug.

63. The linker drug conjugate of claim 51, wherein Any structure selected from the group consisting of LP1-67:

64. The linker of claim 50 or the linker drug conjugate of claim 51, wherein The n37 is an integer from 0 to 12, n38 is an integer from 0 to 12, n39 is an integer from 0 to 12, n40 is an integer from 0 to 12, n41 is an integer from 0 to 12, n42 is an integer from 0 to 12, n43 is an integer from 0 to 12, n44 is an integer from 0 to 12, n45 is an integer from 0 to 12, n46 is an integer from 0 to 12, n47 is an integer from 0 to 12, n48 is an integer from 0 to 12, n49 is an integer from 0 to 12, n50 is an integer from 0 to 12, n51 is an integer from 0 to 12, n52 is an integer from 0 to 12, n53 is an integer from 0 to 12, and n54 is an integer from 0 to 12.

65. The linker or linker-drug conjugate of claim 64, wherein The n37 is an integer from 0 to 10, n38 is an integer from 0 to 10, n39 is an integer from 0 to 10, n40 is an integer from 0 to 10, n41 is an integer from 0 to 10, n42 is an integer from 0 to 10, n43 is an integer from 0 to 10, n44 is an integer from 0 to 10, n45 is an integer from 0 to 10, n46 is an integer from 0 to 10, n47 is an integer from 0 to 10, n48 is an integer from 0 to 10, n49 is an integer from 0 to 10, n50 is an integer from 0 to 10, n51 is an integer from 0 to 10, n52 is an integer from 0 to 10, n53 is an integer from 0 to 10, and n54 is an integer from 0 to 10.

66. The hydrophilic linker or linker drug conjugate of claim 65, wherein The n37 is an integer from 0 to 8, n38 is an integer from 0 to 8, n39 is an integer from 0 to 8, n40 is an integer from 0 to 8, n41 is an integer from 0 to 8, n42 is an integer from 0 to 8, n43 is an integer from 0 to 8, n44 is an integer from 0 to 8, n45 is an integer from 0 to 8, n46 is an integer from 0 to 8.

67. The hydrophilic linker or linker drug conjugate of claim 66, wherein 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 by obtained from the conjugate-drug and antibody of any one of claims 51 to 67, having the structure of Formula III, wherein, Ab is an antibody or antigen binding fragment, n1 = any number between 1 and 20.

69. The antibody-drug conjugate of claim 68, wherein ADC-1 to ADC-67:

70. The antibody-drug conjugate of claim 60, wherein The antibody is a monoclonal antibody selected from the group consisting of abciximab, alemtuzumab, anatumomab mafenatox, atezolizumab, avelumab, basiliximab, bevacizumab, blinatumomab, brentuximab vedotin, catumaxomab, cetuximab, cirmtuzumab, conatumumab, daratumumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, dinutuximab, disitamab emtansine, durvalumab, elotuzumab, gavdorimab, gemtuzumab ozogamicin, ibritumomab tiuxetan, indatuxizumab ravtuxizumab, inotuzumab ozogamicin, ipilimumab, labetuzumab, labetuzumab, labetuzumab, lifastuzumab, lilotomab, mirvetuximab, milatuzumab, naptumomab estafenatox, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, patitumab, pembrolizumab, pertuzumab, pinatuzumab vedotin, polatuzumab, ramucirumab, rovalpituzumab, sacituzumab, secolizumab, sipuleucel-t, sofituzumab, vadastuximab, vesnatinib, trastuzumab, tositumomab, anti-b7-h3 antibody, anti-cd4 antibody, anti-cd5 antibody, anti-cd13 antibody, and anti-cd30 antibody, or antigen binding fragments or immunologically active portions thereof.

71. A method of making an intermediate of any of claims 30-42, 44-47, wherein, The sugar units are characterized by comprising the steps of: CbzHN-L2-OTs and azacrown ether are reacted by a bimolecular nucleophilic substitution reaction to synthesize CbzHN-L2-azacrown ether; CbzHN-L2-azacrown ether with CbzHN-L2-E is prepared by a reductive amination reaction. CbzHN-L2-E can be alternatively synthesized to H2N-L2-E by a de-Cbz reaction.

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