Coupling Linker

Abstract

Provided herein is a conjugated linker, as well as a linker-payload conjugate and ADC comprising the linker.

Description

Technical Field

[0001] The present invention relates to the field of drug conjugates, and in particular to antibody drug conjugates. Background Art

[0002] Antibody-drug conjugates (ADCs) are macromolecular drugs that use the specific binding of antibodies to antigens (located on the surface of cancer cells) to bring cytotoxic substances (drugs) to cells and kill them. They can be considered as macromolecular prodrugs that are both site-directed and site-specific in release. ADCs are actually a three-component system that includes a potent drug connected to an antibody (usually a monoclonal antibody mAb) via a degradable or non-degradable (cleavable / non-cleavable) linker.

[0003] Generally, ADC is internalized after binding to the antigen on the cell, and then the drug is released from the antibody inside the cell and exerts its effect. Compared with small molecules, the number of ADC molecules reaching the target cell is relatively small, and the internalization rate is also low. Therefore, it is hoped that the speed at which the linker is cleaved to release the drug can be increased.

[0004] Linkers are crucial to ADCs, for example, they have a significant impact on the stability and drug release mechanism of ADCs. The linker in the ADC usually includes a short peptide (e.g., 2 to 4 amino acids), which will be cleaved in the lysosome after the ADC is internalized, thereby releasing the drug to exert a cell-killing effect. In the already slow ADC mechanism of action, the speed of enzymatic cleavage (one or more enzymes) of the linker determines the speed of the drug's onset. Most ADCs currently use cleavable linkers because they can quickly release the drug in the target cell. For example, brentuximab vedotin trastuzumab deruxtecan loncastuximab tesirine-lpyl The peptide fragments used are VC, GGFG and VA. These peptides are mainly cleaved by tissue proteases (mainly cathepsin B) in lysosomes. The cleavage rate varies depending on many factors, including the characteristics of the relevant enzymes, regional specific enzyme-substrate interactions, etc. For example, The linker of VC contains the tetrapeptide GGFG, and the amide bond of the last glycine is a cleavable bond, which is cleaved by lysosomal proteases (such as cathepsin). The cleavage rate of GGFG is not as fast as that of VC.

[0005] One of the ideal features of ADCs is that the linker can be rapidly cleaved at a suitable location, such as in the lysosome. We need better linkers and linker-payloads to produce ADC products with better performance and to be able to produce ADCs in a more operational and high-yield process. Summary of the invention

[0006] The inventors have designed a series of linkers suitable for producing linker-payloads and ADCs with improved performance, such as higher shearability (e.g., as manifested by higher shear rates), improved DAR distribution, improved homogeneity, improved stability and / or improved therapeutic-related efficacy. Based on the linkers and linker-payloads containing linkers, ADCs can be manufactured through more operable and higher-yield processes, such as making the ADC products purer, wherein the residual linker-payload content that is not bound to the antibody is lower and / or the residual linker-payload is easier to remove.

[0007] In a first aspect, the present invention provides a compound of formula I:

[0008]

[0009] its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters;

[0010] wherein "*" indicates a chiral center, which is S- or R- or racemic; and hydrogen atoms attached to chiral carbon atoms and hydrogen atoms attached to carbon atoms having R2 substituents are omitted from the formula;

[0011] L1 is -(CH2) a -, wherein a is an integer from 0 to 10, or -(CH2CH2O) b -, wherein b is an integer from 1 to 36;

[0012] L2 is -(CH2) c -, wherein c is an integer from 1 to 10, or -(CH2CH2O) d -, wherein d is an integer from 1 to 36;

[0013] L3 does not exist or is -(CH2) e -, wherein e is an integer from 1 to 10, or -(CH2CH2O) f -, wherein f is an integer from 1 to 36;

[0014] R1 is -CF3, -NR a R b 、-NR a (C=O)R b or -O(CH2) g CH3, wherein g is an integer from 0 to 3, Ra Is H or -C 1-6 Alkyl, R b Is H or -C 1-6 alkyl;

[0015] R2 is -H, -C 1-6 Alkyl or -O(CH2) h CH3, wherein h is an integer from 0 to 3;

[0016] X is halogen, -OR3 or -NR4R5;

[0017] R3 is -H, -C 1-6 Alkyl or halogen;

[0018] R4 and R5 are independently -H or -C 1-6 alkyl;

[0019] n = 0 or 1; and

[0020] m=0 or 1.

[0021] In another aspect, the present invention provides a coupled compound of formula II:

[0022]

[0023]

[0024] Its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; wherein, "*", L1, L2, L3, R1, R2, n and m are as defined in Formula I; and "DRUG" is the drug moiety covalently coupled to the linker.

[0025] In another aspect, the present invention provides an antibody-drug conjugate of Formula III:

[0026]

[0027] Its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; wherein "*", L1, L2, L3, R1, R2, n and m and "DRUG" are as defined in Formula II; and p is 1-8, for example 1, 2, 3, 4, 5, 6, 7 and 8; Ab refers to an antibody.

[0028] In another aspect, the present invention provides a method of producing a linker-load compound, comprising coupling a drug to a linker compound of the present invention. In some embodiments, the drug is exatecan.

[0029] In another aspect, the present invention provides a method for producing an antibody-drug-conjugate, comprising:

[0030] (a) coupling a drug with a linker compound of the present invention to obtain a linker-load compound; wherein preferably

[0031] Preferably, the drug is exatecan; and

[0032] (b) coupling the antibody to the linker-load compound obtained in step (a). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : One of the representative embodiments of the present invention, the synthesis scheme of linker-load compounds (also labeled as "Deruxtecan analogs" in the accompanying drawings) 1-7.

[0034] Figure 2 : One of the representative embodiments of the present invention, a synthesis scheme of the linker-load compound 3-1.

[0035] Figure 3 : One of the representative embodiments of the present invention, a synthesis scheme of the linker-load compound 1-1.

[0036] Figure 4 : One of the representative embodiments of the present invention, a synthesis scheme of linker-load compound 1-2.

[0037] Figure 5 : One of the representative embodiments of the present invention, a synthesis scheme of linker-loaded compounds 1-3.

[0038] Figure 6 : One of the representative embodiments of the present invention, a synthesis scheme of linker-loaded compounds 1-4.

[0039] Figure 7 : One of the representative embodiments of the present invention, a synthesis scheme of linker-loaded compounds 1-8.

[0040] Figure 8 : One of the representative embodiments of the present invention, the synthesis scheme of the linker-load compound 3-4.

[0041] Fig. 9 : (a) Schematic diagram of the modification site (circled) and cleavage site (dashed line) of the linker-load compound of the present invention compared with the original linker-load compound; (b) Comparison of the cleavage percentage (%) of the linker-load compound of the present invention with that of the original linker-load compound over time.

[0042] Fig.10: (a) Schematic diagram of the modification sites (circled) and cleavage sites (dashed lines) of the ADC containing the linker-payload portion of the present invention compared with the ADC containing the original linker-payload portion; (b) Comparison of the shearing percentage (%) of the linker-payload portion of the present invention in the ADC with the original linker-payload portion in the ADC over time.

[0043] Fig.11 : Affinity of ADCs containing different linker-payload moieties binding to Her2 antigen on different cell lines.

[0044] Fig.12 : Cytotoxicity of ADCs containing different linker-payload moieties against different cell lines. DETAILED DESCRIPTION

[0045] Through the following detailed description, other purposes, features and advantages of the present invention will be apparent. However, it should be understood that the detailed description and specific examples are only exemplary descriptions while showing the preferred embodiments of the present invention, because those skilled in the art can understand various changes and adjustments within the concept and scope of the present invention from these detailed descriptions.

[0046] Terms and Definitions

[0047] Herein, the singular form introduced by "a", "an" and "the / said" includes the plural meaning unless otherwise specified. Moreover, the terms "one", "one or more" and "at least one" can be used interchangeably in this article.

[0048] Herein, unless otherwise specified, whether or not there is “about” before a numerical value or range, it includes a reasonable approximate range that can be understood by technicians in the relevant field, such as ±10%, ±5%, ±3%, ±2%, ±1% or ±0.5% of the specified value.

[0049] Herein, the term "substantially free" refers to not only the absence (i.e., "none", "zero", etc.) of a certain situation or the existence of a certain substance, but also the presence or amount of the substance that is not significant or below the detection limit and thus cannot be detected. This is well known to those skilled in the art.

[0050] One or more features of one embodiment herein may be combined with one or more features of another embodiment without departing from the spirit and concept of the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The public documents and patent documents cited in the text are incorporated herein by reference and are applicable to all purposes. The cited documents can all be regarded as indicating the skill level of those skilled in the art, but this cannot be understood as an admission that they are prior to the present invention because the present invention was invented first.

[0052] Overview

[0053] The cleavage rate of polypeptide linkers varies depending on a variety of factors, including the properties of the relevant enzymes, regional specific enzyme-substrate interactions, and the like. We found that changing the structure on both sides of the tetrapeptide GGFG changes the enzyme binding of the linker, which is manifested as an increase in the kcat / Km value of cathepsin B. Based on this, we designed a series of linkers. The use of these linkers can obtain linker-load compounds and ADCs with better performance. As shown in the examples below, the linker-load compounds of the present invention comprising the linker portion designed by the present invention, whether as an independent compound or as a component of an ADC, show a higher cathepsin B cleavage rate than the reference containing a similar existing linker portion. We found that the linker designed by the present invention is more easily cleaved by enzymes, which helps to improve the efficacy of ADCs. We also found that ADCs comprising the linker-load portion of the present invention can show higher stability (e.g., storage stability, such as freeze-thaw stability) and / or comparable or even better therapeutically relevant efficacy (e.g., binding affinity and / or cytotoxicity) compared to existing similar ADCs.

[0054] At the same time, we have also found that the manufacture of ADCs using the linker-load compounds of the present invention comprising the linker moieties designed by the present invention can provide a purer product, such as a high drug-antibody ratio (DAR). compared to. The DAR is 7.5. The higher the DAR, the more linker-payload is required for the coupling reaction. Therefore, it is very troublesome to remove the residual linker-payload. In the present invention, for example, compared with deludec in Enhertu, the linker-payload of the present invention is easier and more thorough to remove, so a purer ADC product can be produced. The reason may be that the linker-payload of the present invention has a higher hydrophilicity. At the same time, when a large excess of linker-payload is used in the coupling to achieve a high DAR, non-specific binding of the linker-payload to the antibody heavy chain will occur, thereby producing 4-drug coupled heavy chains, but the ideal number is not more than 3. In the present invention, the percentage of 4-drug coupled heavy chains can be reduced to Therefore, the linker and linker-load of the present invention can provide ADC products with higher homogeneity.

[0055] We have also found that ADCs prepared using the linker-payloads of the invention are easier to purify. Compared with the products produced by the linker-payload, the free linker-payload remaining in the ADC after coupling is significantly reduced after UFDF (ultrafiltration & depth filtration) purification. Therefore, the linker and linker-payload of the present invention can produce ADC with higher purity and can produce ADC with a more operational and productive process.

[0056] Specific implementation example

[0057] 1. Linker

[0058] In the present disclosure, the term "linker", as can be understood from the context, can refer to a separate linker compound of the present invention or a linker moiety incorporated into and thus a part of a linker-load conjugate or antibody-drug conjugate of the present invention. It is understood that a linker moiety refers to a moiety derived from the corresponding linker compound (when incorporated into a conjugate by conjugation).

[0059] In one aspect, the present invention provides a linker compound having the structure of Formula I:

[0060]

[0061] its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters;

[0062] wherein "*" indicates a chiral center, which is S- or R- or racemic; and hydrogen atoms attached to chiral carbon atoms and hydrogen atoms attached to carbon atoms having R2 substituents are omitted from the formula;

[0063] L1 is -(CH2) a -, wherein a is an integer from 1 to 10, preferably from 1 to 8, more preferably from 2 to 6 or 4 to 5, or -(CH2CH2O) b -, wherein b is an integer from 1 to 36, preferably from 2 to 30, more preferably from 3 to 25 or from 4 to 20;

[0064] L2 is -(CH2) c -, wherein c is an integer from 1 to 10, preferably from 1 to 8 or from 1 to 6, more preferably from 1 to 2, or -(CH2CH2O) d -, wherein d is an integer from 1 to 36, preferably from 2 to 30, more preferably from 3 to 25 or from 4 to 20;

[0065] L3 does not exist or is -(CH2) e-, wherein e is an integer from 1 to 10, preferably from 1 to 8 or from 1 to 6, more preferably from 1 to 2, or -(CH2CH2O) f -, wherein f is an integer from 1 to 36, preferably from 1 to 20, more preferably from 1 to 2, 3 to 25 or 4 to 20;

[0066] R1 is -CF3, -NR a R b 、-NR a (C=O)R b or -O(CH2) g CH3, wherein g is an integer from 0 to 3, preferably from 1 to 2, R a Is H or -C 1-6 Alkyl, preferably -H or -CH3; R b Is H or -C 1-6 Alkyl, preferably -H or -CH3; preferably, R1 is -CF3, -N(CH3)2, -NH(C=O)CH3 or -O(CH2)2CH3;

[0067] R2 is -H, -C 1-6 Alkyl or -O(CH2) h CH3, wherein h is an integer from 0 to 3, preferably, R2 is -H or -CH3;

[0068] X is halogen, -OR3 or -NR4R5, preferably -OR3;

[0069] R3 is -H, -C 1-6 Alkyl or halogen, preferably -H, -CH3, tert-butyl or Cl;

[0070] R4 and R5 are independently -H or -C 1-6 alkyl;

[0071] n = 0 or 1; and

[0072] m = 0 or 1;

[0073] Specific examples of linker compounds include compounds L-1-1, L-1-2, L-1-3, L-1-4, L-1-7, L-1-8, L-3-1 and L-3-4, or pharmaceutically acceptable salts or esters thereof as shown below:

[0074]

[0075]

[0076] Wherein "*" indicates a chiral center, which is racemic.

[0077] The designed linkers provide unique features and chemical properties. For example, in L-1-1, L-1-2, L-1-3, L-1-4, L-1-7 and L-1-8, a CF3 group is introduced into the α position of the amine residue, thereby generating a strong dipole moiety on the N side of the GGFG peptide. This group is likely to reduce the Km of the interaction of cathepsin B with GGFG, thereby accelerating the enzyme catalytic rate of peptide hydrolysis. Obviously, the introduction of tetramine or acetamide in similar positions of linkers (e.g., such as L-3-1 and L-3-4) also leads to an increase in catalytic rate.

[0078] We further explored the N-side modification of the tetrapeptide GGFG. We found that the introduction of additional methyl / methylene groups (e.g., such as L-1-2, L-1-3, L-1-8) or glycol groups (e.g., L-1-7 and L-1-8) near the linker bond of the drug moiety (e.g., exotecan or Dxd) did not slow down the cathepsin B cleavage rate.

[0079] 2. Connector-Payload

[0080] 2.1. Linker-Payload Conjugates of the Invention

[0081] In the present disclosure, the term "linker-payload" (hereinafter also referred to as "LP"), as can be understood from the context, can refer to a linker-payload compound of the present invention alone or a linker-payload moiety incorporated into and thus part of an antibody-drug conjugate according to the present invention. A linker-payload moiety can share the same numerical code with its corresponding linker-payload compound from which it is derived by conjugation.

[0082] In the present disclosure, the term "linker-load compound" refers to a conjugated compound formed by covalently coupling a linker moiety to a drug moiety, wherein the drug moiety is also referred to as a "load." The linker-load compound can be further coupled to an antibody, thereby providing an ADC comprising a linker-load moiety of the present invention.

[0083] Therefore, in one aspect, the present invention provides a linker-load coupling compound having a structure of Formula II:

[0084]

[0085] Its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; wherein, "*", L1, L2, L3, R1, R2, n and m are as defined in Formula I; and "DRUG" is a drug moiety covalently coupled to a linker moiety.

[0086] In some embodiments, the linker-load conjugate can be a compound having any of the following formulae, or a pharmaceutically acceptable salt or ester thereof:

[0087]

[0088]

[0089] Wherein "*" indicates a chiral center, which is racemic.

[0090] The drug that can be used in the present invention is not particularly limited, as long as it has or can be modified to have a functional group coupled to the linker compound at the opposite end to the maleimide moiety. In some embodiments, the functional group for coupling can be -NHR, where R is an alkyl group or H.

[0091] As used herein, the term "drug (DRUG)", as can be understood from the context, can refer to a drug that forms a drug moiety covalently coupled to a linker-load compound or a linker moiety in an ADC, or a drug that is released from a linker-load or ADC by enzymatic cleavage.

[0092] Drugs that can be used in the present invention include cytotoxic drugs, especially drugs for cancer treatment. Such drugs include, but are not limited to, DNA damaging agents, DNA binding agents, nucleic acid synthesis inhibitors, transcription inhibitors, antimetabolites, enzyme inhibitors such as thymidylate synthase inhibitors and topoisomerase inhibitors, microtubule protein inhibitors, and toxins such as toxins of bacterial, fungal, plant or animal origin. Specific examples include, for example, paclitaxel, methotrexate, methotrexate, dichloromethotrene, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including paclitaxel, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, and 1,2-tetracycline. spermidine), camptothecin, calicheamicin, esperamicin, enediynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterlins, maytansinoids (including DM1, DM2, DM3, DM4), auristatins including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) and monomethyl auristatin D (MMAD), camptothecin, irinotecan, topotecan, exatecan, etoposide and its derivatives. In some embodiments, the drug is a topoisomerase inhibitor, such as camptothecin, irinotecan, topotecan, exatecan, etoposide and its derivatives, such as hydroxycamptothecin and Dxd. In some embodiments, the drug is Dxd. In some other embodiments, the drug is exotecan. In some embodiments, when released from the linker-payload or ADC, the drug is Dxd. In some other embodiments, when coupled to the linker to form a drug moiety, the drug is exotecan.

[0093] Accordingly, the linker-load conjugate may be a compound having a structure of Formula IIa, an enantiomer, diastereomer, racemate, solvate, hydrate or a pharmaceutically acceptable salt or ester thereof:

[0094]

[0095] wherein, "*", L1, L2, L3, R1, R2, n and m are as defined in Formula II.

[0096] In some specific embodiments, the linker-load conjugate can be a compound selected from the group consisting of: 1-1, 1-2, 1-3, 1-4, 1-7, 1-8, 3-1 or 3-4, or a pharmaceutically acceptable salt or ester thereof:

[0097]

[0098]

[0099]

[0100] Wherein "*" indicates a chiral center, which is racemic.

[0101] 2.2. Synthesis of linker-payload conjugates

[0102] As one aspect of the present invention, a method for producing a linker-loaded conjugated compound is provided, which comprises conjugating a drug to the linker compound of the present invention. In some embodiments, the drug is exotecan.

[0103] The coupling of the drug to the linker compound can be carried out by a coupling reaction known in the art (such as an esterification reaction or an amidation reaction) or an ester exchange reaction, depending on the type of the one or more functional groups at the end of the linker compound and the type of the one or more functional groups on the drug.

[0104] 3. Antibody-drug conjugates

[0105] 3.1. Antibody-drug conjugates of the present invention

[0106] In one aspect, the present invention provides an antibody-drug conjugate comprising an antibody coupled to one or more drug molecules via a linker moiety of the present invention, which can be represented by Formula III:

[0107]

[0108] Its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; wherein, "*", L1, L2, L3, R1, R2, n and m and "DRUG" are as defined in Formula II; and, p is 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7 and 8; "Ab" refers to an antibody.

[0109] In some specific examples, the antibody-drug conjugate can be a compound selected from the following group of compounds:

[0110]

[0111]

[0112]

[0113] Wherein "*" indicates a chiral center, which is racemic,

[0114] wherein p is 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 and 8; in some embodiments, p is 2, 4 or 6; and in some embodiments, p is 4.

[0115] There is no restriction on the antibodies available for the present invention. It can be antibodies of various specificities, configurations and sources. In some embodiments, the antibody specifically binds to tumor antigens (TA), such as tumor-specific antigens (TSA) and tumor-associated antigens (TAA). Examples of tumor antigens include, but are not limited to, CD20, CD38, CD123, ROR1, ROR2, BCMA, PSMA, SSTR2, SSTR5, CD19, FLT3, CD33, PSCA, ADAM 17, CEA, Her2, EGFR, EGFR-vIII, CD30, FOLR1, GD-2, CA-IX, Trop2, CD70, CD38, mesothelin, EphA2, CD22, CD79b, GPNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON and ERBB2. Examples of TA-specific antibodies include, but are not limited to, Trastuzumab, Rituximab, Cetuximab, Bevacizumab, Panitumumab, Alemtuzumab, Matuzumab, Gemtuzumab, Polatuzumab, Inotuzumab, etc. In some embodiments, the antibody (Ab) is Trastuzumab.

[0116] For the ADC of the present invention, the term "antibody" includes antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments and scFv fragments. In addition, the term "antibody" extends to include functional equivalents, such as ligands and binding proteins that specifically recognize and bind to target molecules (such as antigens, such as tumor antigens), receptors or other surface molecules on target cells (such as disease-related cells, such as cancer cells or tumor cells), as long as these equivalent molecules have or can be modified to have functional groups that can react with the maleamide groups of the linker to covalently bind thereto. In some embodiments, the functional groups are thiol groups, such as those released by reduction of interchain disulfide bonds, and the antibody can thus be coupled to the linker portion via a sulfur-maleamide linkage.

[0117] 3.2. Preparation of Antibody-Drug Conjugates

[0118] In one aspect, the invention provides a method of producing an antibody-drug conjugate comprising conjugating an antibody to a linker-load compound of the invention.

[0119] In some embodiments, the method may include:

[0120] (a) coupling a drug to a linker compound of the present invention to obtain a linker-loaded compound; preferably, the drug is exotecan; and

[0121] (b) coupling the antibody to the linker-load compound obtained in step (a).

[0122] Step (a) can be carried out by a coupling reaction known in the art (such as an esterification reaction or an amidation reaction) or an ester exchange reaction, depending on the type of the one or more functional groups at the end of the linker compound and the type of the one or more functional groups on the drug.

[0123] Step (b) can be carried out by reacting the maleimide moiety with free thiol groups in the antibody by Michael addition reaction. For example, one or more free thiol groups can be derived from one or more cysteine ​​residues, such as those released by reduction of interchain disulfide bonds, so that the antibody can be coupled to the linker moiety via a thio-maleimide linkage.

[0124] 4. Application

[0125] The antibody-drug conjugate (ADC) of the present invention can be formulated into a pharmaceutical composition using a pharmaceutically acceptable formulation. In some embodiments, the composition can include a therapeutically effective amount of the antibody-drug conjugate. In some embodiments, the composition can include an effective amount of the antibody-drug conjugate that can achieve the desired dose.

[0126] The antibody-drug conjugates of the present invention can be used to treat a disease, disorder or condition in a subject in need thereof, wherein the treatment comprises administering to the subject a therapeutically effective amount of the antibody-drug conjugate. The present application also provides antibody-drug conjugates of the present invention for treating a disease, disorder or condition in a subject in need thereof. The diseases treated include, but are not limited to, cancers, including solid tumors and blood cancers. Examples of the cancers include, but are not limited to, breast cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer (e.g., NSCLC), head and neck cancer, colorectal cancer, B cell lymphoma (e.g., non-Hawkins lymphoma (NHL)), and leukemia.

[0127] As used herein, the term "subject" refers to a human or non-human animal subject. A non-human animal can be a mammal, such as a primate. Examples of non-human mammalian subjects include, but are not limited to, captive animals, livestock animals, and zoo animals, competitive animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, and bears. Preferably, the subject is a human. A "subject in need" refers to a subject for whom diagnosis, prognosis, alleviation, prevention, and / or treatment of a disease, disorder, or condition is desired.

[0128] Example

[0129] The following examples are for illustration only and are not intended to limit the scope of the present invention.

[0130] Example 1: Synthesis of linker-payload (a) Compound 1-7 (in Figure 1 Synthesis of delutec analogs 1-7)

[0131] The synthesis process is schematically described in Figure 1 middle.

[0132] Step 1: Methyl 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetate (a-1)

[0133] 2-((tert-butyldimethylsilyl)oxy)ethanol (6.51 g, 42.5 mmol) was added to anhydrous tert-butyl alcohol (50.0 mL). Potassium tert-butylate (3.18 g, 28.3 mmol) was added under stirring and stirred at 0° C. for 1 hour, and then a solution of 2-bromoacetic acid methyl ester (5.00 g, 28.3 mmol) in tert-butyl alcohol (50.00 mL) was added dropwise. The resulting solution was warmed to 0-25° C. and stirred for 2 hours. TLC (petroleum ether / ethyl acetate=10:1, R f=0.2) shows that the reaction is complete. At 0 ° C, water (100mL) is added to quench the reaction mixture and extracted with dichloromethane 50.0mL (50.0mL*3). The combined organic layer is washed with brine (20.0mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue is purified by column chromatography (silica gel, petroleum ether / ethyl acetate=50 / 1 to 2 / 1) to obtain colorless oily 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetic acid methyl ester (3.00g, 42.5% yield). 1 H NMR (400MHz, CDCl3): δppm 4.19 (s, 2H), 3.79-3.84 (m, 2H), 3.76 (s, 2H), 3.62-3.67 (m, 2H), 0.90 (s, 9H), 0.07 (s, 6H).

[0134] Step 2: 2-(2-Hydroxyethoxy)benzyl acetate (a-2)

[0135] To a mixture of methyl 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetate, a-1 (800 mg, 4.03 mmol) in tetrahydrofuran (10.0 mL) and water (10.0 mL) was added lithium hydroxide monohydrate (168 mg, 4.03 mmol). The mixture was stirred at 25° C. for 2 hours. TLC (petroleum ether / ethyl acetate=5:1, R f =0.2) showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove water and tetrahydrofuran to obtain a residue. The residue was dissolved in N,N-dimethylformamide (5.00 mL), potassium carbonate (556 mg, 4.03 mmol) and benzyl bromide (1.38 g, 8.05 mmol) were added, and the mixture was stirred at 25 ° C for 16 hours. TLC (petroleum ether / ethyl acetate = 10:1, R f =0.5) showed that the intermediate was completely consumed, and the reaction was chaotic according to TLC. The reaction mixture was concentrated under reduced pressure to remove N, N-dimethylformamide, and purified by column chromatography (SiO2, petroleum ether / ethyl acetate=100 / 1 to 50 / 1) to obtain the intermediate (1.30 g) as a light yellow liquid. The intermediate was de-TBS in 1M HCl (5.00 mL). TLC (petroleum ether / ethyl acetate=2:1, R f =0.5) indicated that the intermediate was completely consumed, but TLC showed that the reaction was chaotic. The reaction mixture was purified by column chromatography (silica gel, petroleum ether / ethyl acetate=50 / 1 to 2 / 1) to give 2-(2-hydroxyethoxy)acetic acid benzyl ester (600 mg, yield 88.6%) as a light yellow liquid. 1H NMR (400MHz, CDCl3): δppm 7.28-7.41 (m, 5H), 5.20 (s, 2H), 4.22 (s, 2H), 3.81 (t, J = 5.1Hz, 3H), 3.64-3.68 (m, 2H), 0.90 (s, 9H), 0.07 (s, 6H).

[0136] Step 3: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid benzyl ester (a-4)

[0137] To a mixture of 2-(2-hydroxyethoxy)benzyl acetate a-2 (400 mg, 1.09 mmol) and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate a-3 (342 mg, 1.63 mmol) in tetrahydrofuran (4.00 mL) was added 4-methylbenzenesulfonic acid monohydrate (10.4 mg, 54.2 umol), and the mixture was stirred at 25 ° C for 1 hour. TLC (petroleum ether / ethyl acetate = 1:1, R f =0.2) showed that the reaction was complete. The mixture was diluted with sodium bicarbonate (10.0 mL) and extracted with ethyl acetate (30.0 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel) and eluted with (petroleum ether / ethyl acetate=50 / 1 to 1 / 1) to obtain 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester (0.40 g, yield 71.0%) as a white solid. 1 H NMR (400MHz, CDCl3): δppm 7.77(d,J=7.5Hz,2H),7.61(d,J=7.5Hz,2H),7.30-7.44(m,9H),5.13-5.22(m,2H),4.81(d,J=6.8Hz,2H), 4.44(d,J=6.8Hz,2H), 4.15-4.18(m,2H), 3.92(d,J=5.5Hz,2H), 3.73-3.76(m,2H), 3.68(d,J=2.5Hz,2H).

[0138] Step 4: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid (a-5)

[0139] To a mixture of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid benzyl ester a-4 (0.40 g, 771 umol) in ethanol (20.0 mL) and ethyl acetate (20.0 mL) was added dry Pd / C (0.80 g), and stirred at 25° C. for 3 hours in a hydrogen atmosphere (15 psi). TLC (DCM / MeOH=10:1, R f =0.3) showed that the reaction was complete. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid (0.25 g, yield 75.6%) as a colorless oil. The crude product was used directly in the next step without purification. 1 H NMR (400MHz, CDCl3): δppm 8.64-8.74(m,1H),7.83-7.95(m,2H),7.73(d,J=7.4Hz,1H),7.55-7.62(m,1H),7.26-7.49 (m,4H),4.56(d,J=6.5Hz,2H),4.26-4.35(m,2H),3.64(d,J=4.4Hz,2H),3.49-3.60(m,4H).

[0140] Step 5: (a-6, i.e., L-1-7)

[0141] To a mixture containing CTC-resin (0.50 g, 14.1 mmol) and 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid, a-5 (0.20 g, 520 umol), N,N-diisopropylethylamine (0.25 g, 1.95 mmol), dichloromethane (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. Then, the resin was washed 3 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 5 times with N,N-dimethylformamide. Then Fmoc-Phe-OH (0.58 g, 1.31 mmol) was added and mixed for 30 seconds, followed by the addition of a solution of O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) (0.54 g, 1.43 mmol) and N,N-diisopropylethylamine (0.25 g, 1.95 mmol) in N,N-dimethylformamide and nitrogen was bubbled for 30 minutes. The resin was washed 3 times with N,N-dimethylformamide. The above steps were repeated at 25°C for the next coupling: amino acid Fmoc-Gly-OH (0.53 g, 1.50 mmol) and 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol). The reaction was monitored by the ninhydrin test. The coupling reaction was monitored by ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and vacuum drying. 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to the flask containing the peptide resin and stirred for 2 minutes twice. The HFIP mixture was removed in vacuo to obtain a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to obtain white solid compound a-6 (100 mg, crude product, purity 90.0%). LCMS (ESI, m / z): 713.68 [M+H] + :715 (Xbrige C18, 3.5um, 2.1*30mm column, wavelength: UV 220nm&254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_2 minutes). HPLC (Gemini-NX C18 5um 110A 150*4.6mm column, wavelength: UV220nm&254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_20 minutes).

[0142] Step 6: (Compound 1-7)

[0143] Compound a-6 (59.7 mg, 79.2 umol,) and 1-hydroxybenzotriazole (HOBt) (2.80 mg, 20.6 umol,) were dissolved in N, N-dimethylformamide (1.00 mL). Ixitecan mesylate (10.0 mg, 18.8 umol) and N, N-diisopropylcarbodiimide (DIC) (9.50 mg, 75.24 umol) were added to the mixture, and then the mixture was stirred at 40 ° C for 2 hours. TLC (DCM / MeOH=10 / 1, Rf=0.4) showed that the reaction was complete. Traces of N, N-dimethylformamide were removed in vacuo to obtain a residue. The residue was purified by flash chromatography (10-50% water / acetonitrile eluent, C-18 column chromatography). The resulting product was then freeze-dried to give compound 1-7 (8.00 mg, yield 37.4%, purity 95.7%) as a light yellow solid. LCMS (ESI, m / z): 1131.1 [M+H] + :1132.6 (Xbrige C18, 3.5um, 2.1*30mm column, wavelength: UV 220nm&254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_2 minutes). HPLC (Gemini-NXC18 5um 110A 150*4.6mm column, wavelength: UV 220nm&254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (35-65_20+3 minutes).

[0144] (b) Compound 3-1 (in Figure 2 Synthesis of delutec analog 3-1)

[0145] The synthesis process is schematically described in Figure 2 middle.

[0146] Step 1: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid benzyl ester (b-3)

[0147] To a mixture of (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (500 mg, 1.36 mmol) and benzyl 3-hydroxypropionate (366 mg, 2.04 mmol) in tetrahydrofuran (5.00 mL) was added 4-methylbenzenesulfonic acid monohydrate (12.9 mg, 67.8 umol), and the mixture was stirred at 25° C. for 3 hours. LC-MS showed that the starting material was completely consumed, and a major peak with the expected mass was detected. The mixture was diluted with saturated sodium bicarbonate (50.0 mL) and extracted with ethyl acetate (50.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel) and eluted with (petroleum ether / ethyl acetate = 50 / 1 to 1 / 1) to give 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid benzyl ester, b-3 (400 mg, yield 60.3%) as a white solid. 1 H NMR (400MHz, CDCl3): δppm 7.90 (d, J = 7.4Hz, 2H), 7.73 (d, J = 7.0Hz, 2H), 7.24-7.64 (m, 9H), 5.05-5.14 (m, 2H), 4.48-4.62 (m, 4H), 4.16-4.34 (m, 3H), 3.54-3.72 (m, 4H).

[0148] Step 2: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid (a-3)

[0149] To a mixture of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid benzyl ester (400 mg, 818 umol) in ethanol (10.0 mL) and ethyl acetate (10.0 mL) was added dry Pd / C (0.05 g), and the reaction mixture was stirred at 25° C. in a hydrogen atmosphere (15 psi) for 5 hours. TLC (DCM / MeOH=10:1, R f =0.2) showed that the starting material was consumed and a new spot was formed. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid, a-3 (300 mg, yield 91.9%) as a white solid. 1H NMR (400MHz, CDCl3): δppm 7.90(d,J=7.4Hz,2H),7.73(d,J=7.0Hz,2H),7.24-7.64(m,4H),5.05-5.14(m,2H),4.48-4.62(m,2H),4.16-4.34(m,3H),3.54-3.72(m,4H).

[0150] Step 3: (S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanoic acid (b-6)

[0151] A solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (5.00 g, 10.67 mmol) in N,N-dimethylformamide (10.0 mL) and triethylamine (2.50 mL) was stirred at 25° C. for 16 hours. TLC (DCM:MeOH=10:1, R f =0.2) shows that the reaction is complete. The reaction mixture is added dropwise to a solution of isopropyl ether (100 mL) in stirring, the resulting solid is filtered, and the solid is washed with isopropyl ether (50 mL). Trace amounts of isopropyl ether are removed in vacuo to give (S)-2-amino-6-((tert-butyloxycarbonyl)amino)hexanoic acid, b-6 (2.50 g, 95.1% yield) as a white solid. The crude product can be used in the next step without further purification. 1 H NMR (400MHz, CDCl3): δppm3.67(t,J=6.1Hz,1H), 3.03(t,J=6.7Hz,2H), 1.73-1.90(m,2H), 1.23-1.56(m,12H).

[0152] Step 4: (S)-6-((tert-Butyloxycarbonyl)amino)-2-(dimethylamino)hexanoic acid (b-7)

[0153] To a solution of (S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanoic acid (2.00 g, 8.12 mmol) in trifluoroethanol (14.0 mL) at 25°C was added formaldehyde (1.32 g, 16.2 mmol, 37% purity) dropwise, stirred at 25°C for 30 minutes, then sodium borohydride (614 mg, 16.2 mmol) was added dropwise in an ice bath over a period of 30 minutes, then maintained at 25°C for an additional 30 minutes. LC-MS showed complete consumption of the starting material, and a major peak with the expected mass was detected. Traces of trifluoroethanol (14.0 mL) were removed in vacuo to give a residue, which was then diluted with 1% acetic acid (20.0 mL). The mixture was purified by flash chromatography (eluent: 10-50% H2O (0.1% TFA / CH3CN, C-18 column chromatography). The resulting product was then freeze-dried to give (S)-6-((tert-butoxycarbonyl)amino)-2-(dimethylamino)hexanoic acid, b-7 (1.20 g, 53.8% yield) as a white solid. 1 HNMR (400MHz, CDCl3): δppm 3.16(t,J=7.0Hz,1H),3.02(t,J=6.8Hz,2H),2.52(s,6H),1.66-1.73(m, 2H), 1.46 (ddd, J=11.0, 7.0, 4.0Hz, 2H), 1.38 (s, 9H), 1.22-1.31 (m, 2H).

[0154] Step 5: (S)-2-(Dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (b-9)

[0155] A solution of (S)-6-((tert-butyloxycarbonyl)amino)-2-(dimethylamino)hexanoic acid b-7 (0.60 g, 2.19 mmol) in trifluoroacetic acid (0.60 mL) and dichloromethane (6.00 mL) was stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and a major peak with the expected mass was detected. The reaction mixture was concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid to give the de-Boc product as a colorless oil. The colorless oil was dissolved in saturated sodium bicarbonate (6.00 mL) at 0°C, and then 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid methyl ester (407 mg, 2.62 mmol) was added to the reaction mixture and stirred at 0°C for 1 hour and then kept at 25°C for another 3 hours. LC-MS showed that the intermediate was completely consumed and a major peak with the expected mass was detected. The reaction mixture was acidified by adding 1M HCl (5.00 mL) and purified by flash chromatography (eluent: 10-50% H2O (0.1% TFA) / CH3CN, C-18 column chromatography). The resulting product was then freeze-dried to give (S)-2-(dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid, b-9 (500 mg, 89.9% yield) as a white solid. 1H NMR (400MHz, CDCl3): δppm 6.80(s,2H),3.79(dd,J=8.5,4.4Hz,1H),3.49(t,J=6.8Hz,2H),2.88(d, J=13.4Hz,6H),1.89-2.00(m,2H),1.56-1.64(m,2H),1.30-1.40(m,2H).

[0156] Step 6: (3S, 12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl)-2-methyl-4,7,10,13,16-pentaoxo-19-oxa-2,5,8,11,14,17-hexaazadocosa-22-oic acid (b-10, ie, L-3-1)

[0157] To a mixture containing CTC-resin (0.50 g, 14.1 mmol) and 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, a-3 (0.20 g, 520 umol), N,N-diisopropylethylamine (DIEA) (0.25 g, 1.95 mmol), dichloromethane (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. Then, the resin was washed 3 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 5 times with N,N-dimethylformamide. Then Fmoc-Phe-OH (0.58 g, 1.31 mmol) was added and mixed for 30 seconds, followed by the addition of a solution of O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) (0.54 g, 1.43 mmol) and N,N-diisopropylethylamine (DIEA) (0.25 g, 1.95 mmol) in N,N-dimethylformamide and nitrogen was bubbled for 30 minutes. The resin was washed three times with N,N-dimethylformamide. The above steps were repeated for the next coupling of the amino acid Fmoc-Gly-Gly-OH (0.53 g, 1.50 mmol) and the special amino acid (S)-2-(dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid, b-9 (0.20 g, 1.31 mmol) at 25°C. The reaction was detected by the ninhydrin test. The coupling reaction was monitored by the ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and vacuum drying, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to the flask containing the peptide resin and stirred for 2 minutes 2 times. The HFIP mixture was removed in vacuo to obtain a residue. The residue was purified by flash chromatography (C-18 column chromatography, 10-50% H2O / CH3CN eluent). The resulting product was then freeze-dried to give (3S,12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl)-2-methyl-4,7,10,13,16-pentaoxo-19-oxa-2,5,8,11,14,17-hexaazadocosa-22-oic acid b-10 as a white solid (30.0 mg, yield 4.39%).

[0158] Step 7: Compound 3-1

[0159] To a solution of (3S,12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl)-2-methyl-4,7,10,13,16-pentaoxo-19-oxa-2,5,8,11,14,17-hexaazadocosapentaenoic acid, b-10 (18.6 mg, 28.2 umol), and 1-hydroxybenzotriazole (HOBt) (2.80 mg, 20.6 umol,) in N,N-dimethylformamide (1.00 mL) were added exitecan mesylate (10.0 mg, 18.8 umol) and N,N-diisopropylcarbodiimide (DIC) (9.50 mg, 75.24 umol), and the mixture was stirred at 25°C for 3 hours. TLC (DCM:MeOH=10:1, R f =0.3) showed that the reaction was complete. Trace amounts of N,N-dimethylformamide were removed in vacuo to give a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to give compound 3-1 (7.20 mg, 35.4% yield) as a light yellow solid. LCMS (ESI, m / z): 1077.1 [M+H] + :1077.6 (Xbrige C18, 3.5um, 2.1*30mm column, wavelength: UV 220nm&254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_2 minutes). HPLC (Gemini-NX C18 5um 110A150*4.6mm column, wavelength: UV 220nm&254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (25-55_20+3 minutes).

[0160] (c) Synthesis of other compounds

[0161] Compound 1-1 (in Figure 3 Synthesis of delutec analog 1-1)

[0162] Step 1: 10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxa-5,8,11,14,17-pentaazatricosane-1-oic acid (c-1, ie, L-1-1)

[0163] Peptide Synthesis:

[0164] Peptides were synthesized using standard Fmoc chemistry.

[0165] 1) DCM was added to a vessel containing CTC resin (0.25 mmol, 0.44 g, Sub = 0.57 mmol / g) and 5-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14-oic acid (0.10 g, 0.25 mmol, 1.0 equiv) while nitrogen was bubbling.

[0166] 2) Add DIEA (6.0 eq.) dropwise and mix for 2 hours.

[0167] 3) Add MeOH (0.8 mL) and mix for 30 minutes.

[0168] 4) Drain and wash 5 times with DMF.

[0169] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0170] 6) Drain and wash 3 times with DMF.

[0171] 7) Add the Fmoc amino acid solution and mix for 30 seconds, then add the activation buffer and bubble N2 for about 1 hour.

[0172] 8) Repeat steps 5 to 7 for the next amino acid coupling.

[0173] Note:

[0174]

[0175] Fmoc deprotection was performed using 20% ​​piperidine in DMF for 30 min. The coupling reaction was monitored by ninhydrin test and the resin was washed 5 times with DMF.

[0176] Peptide shearing and purification:

[0177] 1) Add shearing buffer (20% HFIP / DCM) to the peptide resin and stir for 3 minutes 3 times.

[0178] 2) DCM was concentrated under reduced pressure.

[0179] 3) Dry the peptide under high vacuum for 2 hours.

[0180] 4) The crude peptide was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) to give compound c-1 (26.0 mg, 95.0% purity, 7.36% yield).

[0181] Step 2: (Drutecan analogue 1-1)

[0182] To a solution of compound c-1 (21.0 mg, 31.3 umol, 1 eq.) and exotecan mesylate (16.6 mg, 31.3 umol) in DMF (0.5 mL) was added HOAt (12.8 mg, 93.9 umol, 3 eq.), DIC (15.8 mg, 125.2 umol, 4 eq.) and DIEA (12.14 mg, 93.9 umol, 3 eq.), and the reaction mixture was stirred at 25° C. for 16 hours. LCMS showed that compound c-1 was consumed, and a main peak with the expected mass was detected. The reaction mixture was directly purified by preparative HPLC (natural conditions, pure water), and two peaks with the expected mass were detected in the preparative HPLC. They were separated and lyophilized to obtain the delutec analog 1-1 as a white solid (peak 1: 1.4 mg, 85.97% purity, peak 2: 7.1 mg, 90.32% purity, 22.3% yield).

[0183] Compound 1-2 (in Figure 4 Synthesis of delutec analogs 1-2)

[0184] Step 1: (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (d-3)

[0185] To a mixture of (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (5 g, 13.57 mmol) and (S)-2-hydroxypropionic acid benzyl ester (d-2, 4.89 g, 27.15 mmol) in tetrahydrofuran (5.00 mL) was added 4-methylbenzenesulfonic acid hydrate (129.09 mg, 678.64 umol), and the mixture was stirred at 25 ° C for 5 hours. LC-MS showed that the starting material was completely consumed, and a major peak with the desired mass was detected. The residue was purified by preparative HPLC (TFA conditions). The yellow oily compound (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester, d-3 (4.10 g, 8.38 mmol, 61.77% yield) was obtained.

[0186] Step 2: (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (d-4)

[0187] To a solution of (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester, d-3 (2.00 g, 4.09 umol) in tetrahydrofuran (20.0 mL) was added dry Pd / C (200 mg), and the reaction mixture was stirred at 25° C. in a hydrogen atmosphere (15 psi) for 4 hours. TLC (DCM / MeOH=10:1, R f =0.3) showed that the starting material was consumed and a new spot was formed. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid, d-4 (1.28 g, 3.21 mmol, 78.4% yield) as a white solid.

[0188] Step 3: (2S,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxa-5,8,11,14,17-pentaazatricosane-1-oic acid (d-5, ie L-1-2)

[0189] To a mixture containing CTC-resin (0.50 g, 0.50 mmol) and (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid, d-4 (0.50 g, 500 umol), N,N-diisopropylethylamine (DIEA) (2.00 mmol), dichloromethane (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. Then, the resin was washed 5 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 3 times with N,N-dimethylformamide. Then Fmoc-Phe-OH (1.50 mmol) was added and mixed for 30 seconds, followed by the addition of a solution of O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) (1.50 mmol) and N,N-diisopropylethylamine (DIEA) (3.00 mmol) in N,N-dimethylformamide, and nitrogen was bubbled for 30 minutes. The resin was washed 3 times with N,N-dimethylformamide. The above steps were repeated for the next coupling of the amino acid Fmoc-Gly-OH (1.50 mmol) and the special amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol) at 25°C. The reaction was detected by the ninhydrin test. The coupling reaction was monitored by the ninhydrin color reaction. After washing with 10.0 mL of methanol and vacuum drying. Add 20.0 mL of lysis buffer (20% HFIP / 80% DCM) to the flask containing the peptide resin and stir for 2 minutes twice. Remove the HFIP mixture in vacuo to obtain a residue. Purify the residue by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product is then freeze-dried to obtain a white solid (2S, 10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxa-5,8,11,14,17-pentaazatricosane-1-acid, d-5, (55.0 mg, yield 9.98%).

[0190] Step 4: (Drutecan analogs 1-2)

[0191] To (2S, 10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxo-5,8,11,14,17-pentaazatricosane-1-oic acid, d-5 (20.0 mg, 29.2 umol), and 1-hydroxy-7-azabenzotriazole (HOAt) (7.95 mg, 58 .4umol) in N, N-dimethylformamide (0.50mL) was added to a solution of isotecan mesylate (15.5mg, 29.1umol), N, N-diisopropylcarbodiimide (DIC) (22.1mg, 175umol, 27.1uL) and N, N-diisopropylethylamine (DIEA) (7.55mg, 58.4umol, 10.1uL), and the mixture was stirred at 25°C for 3 hours. LC-MS showed that the raw material was completely consumed and a main peak with the expected mass was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by preparative HPLC (TFA conditions). The resulting product was then freeze-dried to obtain a light yellow solid derutecan analog 1-2 (18.8mg, 17.0umol, yield 58.4%, purity 96.3%).

[0192] Compound 1-3 (in Figure 5 Synthesis of delutec analogs 1-3)

[0193] Step 1: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid benzyl ester (e-3)

[0194] To the mixture of compound a-3 (500mg, 1.36mmol) and compound e-2 (366mg, 2.04mmol) in THF (5.00mL), add 4-methylbenzenesulfonic acid monohydrate (12.9mg, 67.8umol), then the mixture was stirred at 25 ℃ for 3 hours. LC-MS shows that compound a-3 is completely consumed, and a main peak with desired quality is detected. The mixture is diluted with NaHCO (50.0mL) and extracted with EtOAc (50.0mL). The organic layer combined is dried with Na SO, filtered and concentrated under reduced pressure to obtain residue. The residue is purified by silica gel column chromatography (100-200 mesh silica gel), eluted with (petroleum ether / ethyl acetate=50 / 1 to 1 / 1), and compound e-3 (400mg, 60.3% yield) as a white solid is obtained. 1H NMR (400MHz, CDCl3): δppm 7.90 (d, J = 7.4Hz, 2H), 7.73 (d, J = 7.0Hz, 2H), 7.24-7.64 (m, 9H), 5.05-5.14 (m, 2H), 4.48-4.62 (m, 4H), 4.16-4.34 (m, 3H), 3.54-3.72 (m, 4H).

[0195] Step 2: 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid (e-4)

[0196] To a mixture of compound e-3 (400 mg, 818 umol) in ethanol (10.0 mL) and ethyl acetate (10.0 mL) was added dry Pd / C (0.05 g), and the reaction mixture was stirred at 25° C. in a hydrogen atmosphere (15 psi) for 5 hours. TLC (DCM / MeOH=10:1, R f =0.2) showed that compound e-3 was consumed and a new spot was formed. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain compound e-4 (300 mg, yield 91.9%) as a white solid. 1 HNMR (400MHz, CDCl3): δppm 7.90(d,J=7.4Hz,2H),7.73(d,J=7.0Hz,2H),7.24-7.64(m,4H),5.05-5.14(m,2H),4.48-4.62(m,2H),4.16-4.34(m,3H),3.54-3.72(m,4H).

[0197] Step 3: (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentaazatetracosanoic acid (e-5, i.e., L-1-3)

[0198] To a mixture containing CTC-resin (0.50 g, 14.1 mmol) and compound e-4 (0.20 g, 520 umol), DIEA (0.25 g, 1.95 mmol) and DCM (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then MeOH (5.00 mL) was added and mixed for 30 minutes. Then, the resin was washed 3 times with DMF (10.0 mL). The resin was treated with 20% piperidine in DMF for 30 minutes for Fmoc deprotection. The resin was washed 5 times with DMF. Then Fmoc-Phe-OH (0.58 g, 1.31 mmol) was added and mixed for 30 seconds, then a DMF solution of HBTU (0.54 g, 1.43 mmol) and DIEA (0.25 g, 1.95 mmol) was added, and nitrogen was bubbled for 30 minutes. The resin was washed 3 times with DMF. The above steps were repeated for the coupling of the next amino acid Fmoc-Gly-OH (0.53 g, 1.50 mmol) and the special amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol) at 25°C. The reaction was detected by the ninhydrin test. The coupling reaction was monitored by the ninhydrin color reaction. After washing with 10.0 mL MeOH and vacuum drying. 20.0 mL of cleavage buffer (20% HFIP / 80% DCM) was added to the flask containing the peptide resin and stirred for 2 minutes twice. The HFIP mixture was removed in vacuo to obtain a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The obtained product was then freeze-dried to obtain compound e-5 (30.0 mg, yield 4.39%) as a white solid.

[0199] Step 4: (Drutecan analogs 1-3)

[0200] HATU (9.30 mg, 24.4 umol) was added to a solution of compound e-5 (27.2 mg, 37.6 umol) in DMF (1.00 mL). The reaction mixture was stirred at 25 ° C for 20 minutes. Exotecan mesylate (10.0 mg, 18.8 umol) and DIEA (4.86 mg, 37.6 umol) were added to the reaction mixture and kept at 40 ° C for 2 hours. TLC (DCM: MeOH = 10: 1, R f =0.4) indicated that the reaction was complete. Trace amounts of DMF were removed in vacuo to give a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to give delutecan analog 1-3 (7.20 mg, 34.7% yield) as a light yellow solid.

[0201] Compound 1-4 (in Figure 6 Synthesis of delutec analogs 1-4)

[0202] Step 1: (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid methyl ester (f-3)

[0203] To a mixture of (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (5 g, 13.5 mmol) and (S)-3-hydroxy-2-methylpropanoic acid methyl ester (3.21 g, 27.1 mmol) in tetrahydrofuran (50.0 mL) was added 4-methylbenzenesulfonic acid hydrate (129 mg, 678 umol), and the mixture was stirred at 25 ° C for 1 hour. LC-MS showed that the starting material was completely consumed and a main peak with the expected mass was detected. The residue was purified by preparative HPLC (TFA conditions). A yellow oily compound (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid methyl ester, f-3 (4.79 g, 11.2 mmol, 82.7% yield) was obtained.

[0204] Step 2: (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid (f-4)

[0205] To a solution of (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid methyl ester, f-3 (4.79 g, 11.2 mmol) in tetrahydrofuran (20.0 mL) and water (20.0 mL) was added lithium hydroxide monohydrate (942 mg, 22.4 mmol) and the mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and a major peak with the expected mass was detected. Acetic acid was added for acidification, followed by ammonium for alkalization, followed by (2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-yl methyl carbonate (8.21 g, 24.3 mmol) and sodium bicarbonate (2.04 g, 24.3 mmol). The mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting material was completely consumed and a main peak with the expected mass was detected. TLC (dichloromethane / methanol = 10:1, R f=0.3) showed that the starting material was exhausted and a new spot was formed. The reaction mixture was concentrated under reduced pressure to remove water and tetrahydrofuran to obtain a residue. The crude product was purified by column chromatography (SiO2, dichloromethane / methanol=100 / 1 to 20 / 1) to obtain a light yellow oily substance (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, f-4 (1.25 g, 3.03 mmol, 12.45% yield) as a light yellow liquid.

[0206] Step 3: (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentaazatetracosanoic acid-1-oic acid (f-5, i.e., L-1-4)

[0207] To a mixture containing CTC-resin (1.5 g, 1.50 mmol) and (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oic acid, f-4 (0.60 g, 1.50 mmol), N,N-diisopropylethylamine (DIEA) (6.00 mmol), dichloromethane (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. The resin was then washed 5 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 3 times with N,N-dimethylformamide. Then Fmoc-Phe-OH (4.50 mmol) was added and mixed for 30 seconds, followed by the addition of a solution of O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) (4.50 mmol) and N,N-diisopropylethylamine (DIEA) (9.00 mmol) in N,N-dimethylformamide and nitrogen was bubbled for 30 minutes. The resin was washed 3 times with N,N-dimethylformamide. The above steps were repeated for the next coupling of the amino acid Fmoc-Gly-OH (0.53 g, 4.50 mmol) and the special amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol) at 25°C. The reaction was monitored by the ninhydrin test. The coupling reaction was monitored by ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and vacuum drying, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to the flask containing the peptide resin and stirred for 2 minutes twice. The HFIP mixture was removed in vacuo to obtain a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to obtain a white solid (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentaazatetracosane-1-acid, f-5, (167 mg, 9.80% yield).

[0208] Step 4: (Drutecan analogs 1-4)

[0209] To (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentaazatetracosanoic acid, f-5 (20.0 mg, 28.6 umol) and 1-hydroxy-7-azabenzotriazole (HOAt) (7.79 mg, 57.2 umol) in N, N-dimethylformamide (0.50mL) was added to a mixture of isotecan mesylate (15.2mg, 28.6umol), N, N-diisopropylcarbodiimide (DIC) (21.6mg, 171umol, 26.5uL) and N, N-diisopropylethylamine (DIEA) (7.40mg, 57.2umol, 9.97uL), and the mixture was stirred at 25°C for 3 hours. LC-MS showed that the raw material was completely consumed and a main peak with the expected mass was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by preparative HPLC (TFA conditions). The resulting product was then freeze-dried to obtain a light yellow solid derutecan analog 1-4 (6mg, 5.20umol, yield 18.1%, purity 96.2%).

[0210] Compound 1-8 (in Figure 7 Synthesis of delutec analogs 1-8)

[0211] Step 1: Benzyl 2-(2-(Benzyloxy)ethoxy)propionate (g-2)

[0212] At 0°C, NaH (3.33 g, 83.2 mmol, 60% purity) was added to the stirred DMF (100 mL) in N2 in batches. (2S)-2-hydroxypropionic acid benzyl ester (d-2, 10.0 g, 55.5 mmol) was added dropwise to the above solution at 0°C and stirred at 25°C for 0.5 hours. Then, 2-bromoethoxytoluene (14.3 g, 66.6 mmol) was added to the above solution at 0°C. The reaction was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 5:1, R f=0.2) shows that the reaction is complete.By adding 0 ℃ water (1000mL), reaction mixture is quenched, and extracted with dichloromethane (200mL x3).The organic layer merged is washed with salt water (200mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain residue.Residue passes through column chromatography (silica gel, petroleum ether / ethyl acetate=50 / 1 to 5 / 1) purifying, obtains 2-(2-(benzyloxy) ethoxy) benzyl propionate, g-2 (790mg, 4.53% productive rate) of colorless oil. δppm 7.29-7.43(m,10H),5.10-5.30(m,2H),4.58(d,J=2.01Hz,2H),4.14(q,J= 6.86Hz, 1H), 3.75-3.87 (m, 1H), 3.60-3.73 (m, 3H), 1.47 (d, J = 7.03Hz, 3H).

[0213] Step 2: 2-(2-Hydroxyethoxy)propionic acid (g-3)

[0214] A 100 mL round bottom flask was purged with Ar 3 times, and dry Pd / C (160 mg) was carefully added. Methanol (~10.0 mL) was then added to completely permeate the Pd / C, and then a solution of (2S)-2-(2-(benzyloxy)ethoxy)benzyl propionate, g-2 (790 mg, 2.51 mmol) in MeOH (20.0 mL) was slowly added in Ar. The resulting mixture was degassed and purged with H2 3 times, and then the mixture was stirred at 25 ° C under H2 atmosphere for 4 hours. TLC (petroleum ether / ethyl acetate=5:1, R f =0.2) showed that the reaction was complete. The reactant was filtered and concentrated under reduced pressure to obtain the product 2-(2-hydroxyethoxy)propionic acid, g-3 (320 mg, crude product) as a light yellow liquid, which was used directly in the next step without purification. 1 H NMR (400MHz, DMSO): δppm 4.02 (q, J = 6.78Hz, 1H), 3.66-3.70 (m, 2H), 3.60-3.66 (m, 1H), 3.49-3.56 (m, 1H), 1.39 (d, J = 7.03Hz, 3H).

[0215] Step 3: Benzyl 2-(2-hydroxyethoxy)propionate (g-4)

[0216] (2S)-2-(2-hydroxyethoxy)propionic acid, g-3 (320 mg, 2.39 mmol) was dissolved in MeOH (4 mL) and H2O (0.8 mL) and Cs2CO3 (389 mg, 1.19 mmol) was added to the solution. The mixture was stirred at 25°C for 0.5 hours and concentrated. Then bromotoluene (449 mg, 2.62 mmol) and DMF (2 ml) were added. The mixture was stirred at 25°C for 16 hours. TLC (petroleum ether / ethyl acetate = 3:1, R f =0.4) shows that the reaction is complete. Water (50.0mL) dilutes the mixture, and extracts with DCM (30mL x 3). The organic layer merged is dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue is purified by silica gel column chromatography (100-200 mesh silica gel), eluted with (petroleum ether / ethyl acetate=20 / 1 to 5 / 1), and 2-(2-hydroxyethoxy)benzyl propionate, g-4 (340mg, 64% yield) is obtained as a white solid. 1 H NMR (400MHz, CDCl3): δppm 7.37 (s, 5H), 5.15-5.26 (m, 2H), 4.04-4.15 (m, 1H), 3.71-3.76 (m, 2H), 3.60-3.68 (m, 2H), 1.46 (d, J = 7.03Hz, 3H).

[0217] Step 4: 1-(9H-fluoren-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid benzyl ester (g-6)

[0218] To a solution of [[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]methyl acetate (a-3) (614 mg, 1.67 mmol) in THF (8 mL) was added TsOH.H2O (14.4 mg, 75.8 umol) and 2-(2-hydroxyethoxy)benzyl propionate, g-4 (340 mg, 1.52 mmol). The mixture was stirred at 25 ° C for 6 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with saturated NaHCO3 (50.0 mL) and extracted with DCM (30 mL x 3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. TLC (petroleum ether / ethyl acetate=1:1, R f=0.4) showed that the reaction was complete. The residue was purified by silica gel column chromatography (100-200 mesh silica gel) and eluted with (petroleum ether / ethyl acetate=20 / 1 to 5 / 1) to obtain 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester, g-6 (416 mg, 781 umol, yield 51.5%) as a white solid. 1 HNMR (400MHz, CDCl3): δppm 7.77(d,J=7.53Hz,2H),7.61(br d,J=7.28Hz,2H),7.39-7.43(m,2H),7.28-7.38(m,7H),7.21(br s,1H),5.53(br s,1H),5.19(s,2H),4.93-5.03(m,1H),4.67(br s,1H),4.44(d,J=7.03Hz,2H),4.22-4.27(m,1H),4.05(q,J=7.03Hz,1H),3.92(br s,2H),3.76(br s,1H),3.71(br d,J=9.03Hz,2H),3.55(br d, J = 8.53Hz, 1H), 1.44 (d, J = 6.78Hz, 3H).

[0219] Step 5: 1-(9H-fluoren-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid (g-7)

[0220] A 100 mL round-bottom flask was purged with Ar 3 times, and dry Pd / C (60 mg) was carefully added. Then THF (5 mL) was added to completely permeate Pd / C, and then 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester, g-6 (416 mg, 781 umol) in THF (10 mL) was slowly added under Ar. The resulting mixture was degassed and purged with H2 3 times, and then stirred at 25 ° C for 2 hours in H2 (15 psi). TLC (petroleum ether / ethyl acetate=1:1, R f =0.4) showed that the reaction was complete. The reactant was filtered and concentrated under reduced pressure to obtain a white gum product 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid, g-7 (320 mg, 92.6% yield), and the crude product was used for the next step without purification. 1H NMR (400MHz, CDCl3): δppm 7.77(d,J=7.53Hz,2H),7.60(br d,J=7.28Hz,2H),7.38-7.44(m,2H),7.29-7.35(m,2H),5.48-5.65(m,1H),4.95(br s,1H),4.70(dd,J=10.54,6.02Hz,1H),4.42-4.52(m,2H),4.23(t,J=6.78Hz,1H),4.02(q,J=6.86Hz,1H),3.94(br s,1H),3.83-3.92(m,1H),3.75-3.82(m,2H),3.73(br d,J=6.78Hz,2H),3.58-3.67(m,1H),1.42-1.48(m,3H).

[0221] Step 6: (13S)-13-benzyl-26-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl-9,12,15,18-tetraoxo-21-(trifluoromethyl)-3,6-dioxa-8,11,14,17,20-pentaazahexacosanoic acid (g-8, i.e. L-1-8)

[0222] Peptide Synthesis:

[0223] Peptides were synthesized using standard Fmoc chemistry.

[0224] 1) DCM was added to a vessel containing CTC resin (1.5 mmol, 1.5 g, Sub = 1.01 mmol / g) and 1-(9H-fluoren-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-oic acid (0.6 g, 1.5 mmol 1.0 equiv) while N2 was bubbled.

[0225] 2) Add DIEA (4.0 eq.) dropwise and mix for 2 hours.

[0226] 3) Add MeOH (0.5 mL) and mix for 30 minutes.

[0227] 4) Drain and wash 5 times with DMF.

[0228] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0229] 6) Drain and wash 3 times with DMF.

[0230] 7) Add the Fmoc amino acid solution and mix for 30 seconds, then add the activation buffer and bubble N2 for about 0.5 hours.

[0231] 8) Repeat steps 5 to 7 for the next amino acid coupling.

[0232] Note:

[0233]

[0234] Fmoc deprotection was performed using 20% ​​piperidine in DMF for 30 min. The coupling reaction was monitored by ninhydrin test and the resin was washed 5 times with DMF.

[0235] Peptide shearing and purification:

[0236] 1) The resin was washed 3 times with MeOH and dried by vacuum.

[0237] 2) Add shearing buffer (20% HFIP / DCM) to the peptide resin and stir for 0.5 hours three times.

[0238] 3) DCM was concentrated under reduced pressure.

[0239] 4) The peptide was dried under high vacuum for 2 hours to obtain compound g-8 (100 mg, 90%).

[0240] Step 7: (Drutecan analogs 1-8)

[0241] To a solution of compound g-8 (20.0 mg, 27.5 umol) in DMF (200 uL) were added DIC (19.8 mg, 157 umol), HOAT (7.12 mg, 52.3 umol), (10S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexyl[14.7.1.02,14.04,13.06,11.020,24]tetracosane-1,6(11),12,14,16(24),17,19-heptane-5,9-dione (13.9 mg, 26.1 umol, MsOH) and DIEA (6.76 mg, 52.3 umol). The mixture was stirred at 25°C for 8 hours. LC-MS showed that compound g-8 was completely consumed and a main peak with the expected m / z was detected. Trace amounts of N,N-dimethylformamide were removed in vacuo to obtain a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to obtain compound derutec analog 1-8 (12 mg, 10.5 umol) as a light yellow solid. LCMS (ESI, m / z): 1145.4 [M+H] +: 1146.6 (Xbrige C18, 3.5um, 2.1*30mm column, wavelength: UV220 nm & 254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_2 minutes). HPLC (Gemini-NX C18 5um110A 150*4.6mm column, wavelength: UV 220nm & 254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (40-70_20+3 minutes).

[0242] Compound 3-4 (in Figure 8 Synthesis of delutec analogs 3-4)

[0243] Step 1: (S)-2-Acetylamino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (h-3)

[0244] To a solution of (2S)-2-acetylamino-6-amino-hexanoic acid (2.00 g, 10.6 mmol) in saturated NaHCO3 (50 mL) was added 2,5-dioxopyrrole-1-carboxylic acid methyl ester (1.65 g, 10.6 mmol). The mixture was stirred at 25°C for 4 hours. LC-MS showed that compound h-1 was completely consumed and a major peak with the desired m / z was detected. The reaction mixture was quenched by adding 0°C H2SO4 to pH = 3-4 and then extracted with EA (100 mL x 3). The combined organic layers were washed with brine (50 mL x 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, DCM / MeOH=100 / 1 to 20 / 1) to give (S)-2-acetylamino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid, h-3 (460 mg, 1.71 mmol). 1 H NMR (400MHz, CDCl3): δppm 6.71 (s, 2H), 6.30 (br d,J=7.28Hz,1H),4.48-4.59(m,1H),3.54(t,J=6.78Hz,2H),2.08(s,3H),1 .90-2.00(m,1H),1.75-1.84(m,1H),1.56-1.70(m,2H),1.31-1.45(m,2H).

[0245] Step 2: (19S)-10-benzyl-19-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl)-6,9,12,15,18,21-hexaoxo-3-oxa-5,8,11,14,17,20-hexaazadocosa-1-oic acid (h-4, i.e., L-3-4)

[0246] Peptide Synthesis:

[0247] Peptides were synthesized using standard Fmoc chemistry.

[0248] 1) To a vessel containing CTC resin (0.5 mmol, 0.5 g, Sub = 1.01 mmol / g) and 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid (0.2 g, 0.5 mmol, 1.0 equiv) was added DCM while N2 was bubbled.

[0249] 2) Add DIEA (4.0 eq.) dropwise and mix for 2 hours.

[0250] 3) Add MeOH (0.5 mL) and mix for 30 minutes.

[0251] 4) Drain and wash 5 times with DMF.

[0252] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0253] 6) Drain and wash 3 times with DMF.

[0254] 7) Add the Fmoc amino acid solution and mix for 30 seconds, then add the activation buffer and bubble N2 for about 0.5 hours.

[0255] 8) Repeat steps 5 to 7 for the next amino acid coupling.

[0256] Note:

[0257]

[0258] Fmoc deprotection was performed using 20% ​​piperidine in DMF for 30 min. The coupling reaction was monitored by ninhydrin test and the resin was washed 5 times with DMF.

[0259] Peptide shearing and purification:

[0260] 1) The resin was washed 3 times with MeOH and dried by vacuum.

[0261] 2) Add shearing buffer (20% HFIP / DCM) to the peptide resin and stir for 0.5 hours three times.

[0262] 3) DCM was concentrated under reduced pressure.

[0263] 4) The peptide was dried under high vacuum for 2 hours to obtain compound h-4 (100 mg, 95%).

[0264] Step 3: (Drutecan analogs 3-4)

[0265] To a solution of compound h-4 (20.0 mg, 29.7 umol) in DMF (500 μL) was added DIC (15.6 mg, 124 umol), exotecan mesylate (13.2 mg, 24.7 ummol) and HOBt (3.68 mg, 27.2 umol). The mixture was stirred at 25 ° C for 16 hours. TLC (DCM / MeOH=10 / 1, Rf=0.4) showed that the reaction was complete. Trace amounts of N,N-dimethylformamide were removed in vacuo to obtain a residue. The residue was purified by flash chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to give compound derutecan analog 3-4 (7 mg, 6.42 umol) as a light yellow solid. LCMS (ESI, m / z): 1090.4 [M+H] + : 1091.4 (Xbrige C18, 3.5um, 2.1*30mm column, wavelength: UV220 nm & 254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_2min). HPLC (Gemini-NX C18 5um 110A 150*4.6mm column, wavelength: UV 220nm & 254nm; column temperature: 30°C) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (25-55_20+3min).

[0266] (d) Synthesis of “original linker-payload”

[0267] In the context of the present disclosure, the term "native linker-payload" (hereinafter also referred to as "native-LP" or "nat-LP") refers to a compound having the following structure:

[0268]

[0269] Original-LP has the same structure as Delutec in Enhertu, both containing a maleimide-GGFG linker. In this example, Original-LP was purchased from MedChemExpress under the name "Delutec".

[0270] Example 2: Synthesis of Antibody-Drug Conjugates

[0271] Antibody-drug conjugates were synthesized according to the following general procedure: first, the antibody in pH 7 PBS solution was reduced with 2 to 20 equivalents of TCEP for 0.5 to 18 hours; an excess of linker-load (15 to 18 molar excess) was introduced with or without removal of residual TCEP by column or membrane; the coupling reaction was completed within half an hour to several hours at a temperature range of 4°C to room temperature (RT), followed by HPLC purification to provide the final ADC product.

[0272]

[0273] Note: 1: Expressed as Ab, followed by the numeric code for each linker-payload part

[0274] 2: Molar ratio

[0275] 3: Also referred to below as "Her-Dxd" or "Her-nat-LP"

[0276] Example 3: Shearing of Linker-Load

[0277] method

[0278] Reaction buffer (40mM H3PO4 / H3BO3 / HAc, 1mM EDTA, pH 4.5), 135mM cysteine, DMA (N,N'-dimethylacetamide, 2%, v / v), sample (0.015μmol linker-load compound) and cathepsin B (final 16U / μmol linker-load compound) were added to a 1.5mL EP tube in sequence. The final concentration of cysteine ​​in the 300μL lysis system thus formed was 10mM. The EP tube was placed in a 37°C water bath.

[0279] After about 15 minutes (min), 2 hours (hr), 5 hours and 16 hours of cleavage, the mixture was thoroughly vortexed and then sampled (25 μL). The cathepsin B inhibitor E64 (2.5 equivalents compared to cathepsin B) was added to the sampled mixture to inactivate cathepsin B, and then analyzed by RP-HPLC and RP-MS. The shear percentage was calculated from the ratio of the peak area of ​​the cleaved linker-payload to the sum of the non-cleaved plus cleaved linker-payload areas (as measured by RP-HPLC and RP-MS).

[0280] result

[0281] Table 1 summarizes the shearing conditions (expressed as a percentage) for about 16 hours (overnight), showing the comparison of cathepsin B digestion efficiency between linkers and payloads. The results show that the shearing of the linkers-payloads of the present invention tested is faster than that of the original linker-payload, at least equivalent (compound 3-1).

[0282] Table 1

[0283]

[0284] exist Fig. 9 In the figure, panel (a) shows the cleavage sites and modifications of the linker portion in compound 1-1 compared to the original linker-payload; panel (b) shows the cleavage percentage, calculated based on the peak area of ​​the released drug detected by reverse phase HPLC chromatography (mixed mode). Consistent with the results in Table 1, the linker-payload of the present invention is cleaved faster.

[0285] Example 4: Cleavage of the Linker-Payload Moiety in ADC

[0286] method

[0287] The reaction conditions were the same as those described for the cleavage of the linker-load compound itself in Example 3. Specifically, reaction buffer (40 mM H3PO4 / H3BO3 / HAc, 1 mM EDTA, pH 4.5), 135 mM cysteine, DMA (2%, v / v), sample (0.015 μmol ADC) and cathepsin B (16 U / μmol ADC) were added sequentially to a 1.5 mL EP tube. The final concentration of cysteine ​​in the 300 μL cleavage system thus formed was 10 mM. The EP tube was placed in a 37°C water bath.

[0288] After 10 min, 5 hr, 24 hr and 48 hr of lysis, the mixture was vortexed thoroughly and then 25 μL was sampled. The cathepsin inhibitor E64 (2.5 equivalents compared to cathepsin B) was added to the sampled mixture to inactivate protease B, and then reversed phase LC-MS analysis was performed. The cleavage rate was calculated based on the reduction in DAR measured by mass spectrometry MS.

[0289] result

[0290] The digestion percentages over time are summarized in Table 2, showing a comparison of the digestion efficiency of different linker-payload moieties in the ADC by cathepsin B. Clearly, ADCs containing linker-payloads 1-2, 1-3, 3-1, and 3-4 released drug faster in the presence of cathepsin B. Of note, another cleavage experiment using a mixed-mode column to quantify the shear-released drug showed that the ADC containing linker-payload moiety 1-1 released drug slightly faster than the ADC containing the original linker-payload.

[0291] Table 2

[0292]

[0293] exist Fig.10In the figure, sub-figure (a) shows the cleavage sites and modifications of the linker-payload portion in the ADC compared to the original linker-payload in the ADC, and sub-figure (b) shows the time-dependent cleavage percentage (%) of the linker-payload in the ADC, which is calculated based on the peak area of ​​drug release (same as above). After 48 hours, the cleavage percentage of ADC-1-1 (i.e., ADC containing linker-payload 1-1) was 18.5%, while that of ADC-original LP was 15.5%.

[0294] Example 5: Freeze-thaw stability

[0295] method

[0296] Take out the ADC sample (~10 mg / mL, dissolved in 20 mM histidine, 150 mM NaCl, pH 6.0) in the Eppendorf tube frozen at -80℃ and thaw at room temperature for 30 minutes. Then, freeze the ADC sample at -80℃ for 2 days and then thaw at room temperature for 30 minutes; repeat the freeze / thaw process once more. Then, take 20 μl of each sample for SEC and MS to determine DAR and drug distribution. Determine the percentage (molar ratio) of various substances (L0, L1, L2, H0, H1, H2, H4, etc., where "L" refers to the light chain, "H" refers to the heavy chain, and the number refers to the number of drug molecules connected to the chain) in each sample based on the peak area of ​​the substance.

[0297] result

[0298] After two rounds of freeze-thaw (F / T) cycles, the changes in unwanted impurity formation such as L2 (ADC with two drug molecules attached to the light chain) and H4 (ADC with four drug molecules attached to the heavy chain) and the changes in DAR are summarized in Table 3. The results show that after two rounds of freeze-thaw cycles, L2 in the ADC containing the linker-load of the present invention remained zero (0), while L2 in Her-Dxd (i.e., Dexi trastuzumab) increased to 0.76%. The H4% in Her-Dxd was also the highest. The DAR of ADC containing linker-load 1-8 decreased the least after two rounds of freeze-thaw cycles. In general, the unwanted H4 content in the 8 ADC analogs after freeze-thaw cycles was lower than that of Her-Dxd and all maintained a stable DAR.

[0299] Table 3

[0300]

[0301] Note: “Her” = “Herceptin”, also known as “trastuzumab”; “Fresh” means newly synthesized without freeze-thaw cycles.

[0302] Ring ADC.

[0303] Example 6: Detection of H4 in ADC

[0304] method

[0305] ADC was prepared in 20 mM histidine buffer (containing 150 mM NaCl, pH 6.0). The resulting ADC samples were subjected to LC-MS analysis to determine the distribution of the drug on the light chain and heavy chain. Ideally, only five substances, namely L0, H0, L1, H1 and H2, were detected. HPLC was used to detect the aggregation percentage.

[0306] result

[0307] Table 4 shows the comparison of non-specific impurity H4% between ADCs. The H4% (molar ratio) of the Her-Dxd control prepared with the original linker-load was 3% after synthesis, and the impurity content in Her-1-8 was significantly reduced to 0.8%. As shown in the results, the ADC products produced with the linker-load of the present invention have reduced impurities.

[0308] Table 4

[0309] ADC Name <![CDATA[C ADC (mg / ml)]]> MS-DAR H4％ Aggregation (%) Her-Dxd(control) NA 7.75 3.0 NA Her-1-2 5.28 7.49 1.9 1.98％ Her-1-4 4.86 7.84 1.4 1.94％ Her-1-8 5.51 7.78 0.8 1.66％ Her-3-4 5.67 7.82 2.5 1.13％

[0310] Example 7: Removal of Uncoupled Linker-Payload

[0311] After coupling as described in Example 2, the ADC was exchanged into 20 mM histidine buffer (containing 150 mM NaCl, pH 6.0) using a rotary desalting column (40 kDa). Table 5 shows a comparison of free linker-load (i.e., uncoupled linker-load) clearance between ADCs made with different linker-loads. Free linker-load was cleared using UFDF. The residual free linker-load content in the Her-Dxd (i.e., Dexi trastuzumab) coupling product was close to 5%, but the content was below 2% in the product produced with the linker-load of the present invention.

[0312] As shown in the results, the linker-payload of the present invention improves the operability of purification, for example, it is easier and more thorough to remove the residual free linker-payload in the ADC conjugated product. Without being limited to any particular theory, it is believed that the introduction of polar groups in the linker portion improves the water solubility of the linker-payload and promotes its removal, for example, by UFDF. This is of great significance for the ADC manufacturing process.

[0313] Table 5

[0314]

[0315] Example 8: Affinity detection

[0316] method

[0317] The in vitro binding of ADC to human HER2 was tested using FACS (fluorescence activated cell flow cytometry). 5 Tumor cells (cells / well) were incubated with serially diluted ADC at 4°C for 1-2 hours. Her-Dxd prepared as described in Example 2 was used as a reference ADC and positive control. The buffer used to dissolve the ADC (20mM histidine buffer, containing 150mM NaCl, pH6.0) was used as a negative control. After incubation, the cells were washed with FACS staining buffer, and then the secondary antibody Alexa647-conjugated goat anti-human IgG Fc (Jackson) diluted in FACS staining buffer was added. The culture plate was incubated at 4°C in the dark for 20-60 minutes. Fluorescence intensity was measured using a flow cytometer (BD FACS Canto II), and data analysis was performed using FlowJo. EC was calculated using GraphPad Prism 50 value.

[0318] result

[0319] The results are as follows Fig.11 According to N87 cell detection, the EC of the ADC tested in the present invention is 50 The EC of the ADC tested in the present invention is about 1 nM, while that of Her-Dxd is 0.9 nM. 50 According to the MDA-MB-231 cell test, the EC of the ADCs tested in the present invention (except Her3-1) is 50 The binding affinity of the ADC of the present invention with the linker of the present invention and the linker-load of the present invention is at least equivalent to that of trastuzumab, or even better.

[0320] Example 9: Cytotoxicity Assay

[0321] method

[0322] The ability to inhibit tumor cell growth was determined by in vitro cytotoxicity assays. Tumor cell lines NCI-N87, HCC1954, MDA-MB-231 and JIMT-1 (purchased from ATCC) were routinely cultured in RPMI1640 medium or DMEM medium. The day before the test, cells were seeded in culture medium on a 96-well plate at an appropriate cell density. The next day, ADCs serially diluted with culture medium were added to each well. Her-Dxd prepared as described in Example 2 was used as a reference ADC and a positive control. The buffer used to dissolve the ADC was used as a negative control. The culture plates were kept warm in an incubator at 37°C and 5% CO2. After 4-6 days, cell viability was determined using CellTiter-Glo (Promega). IC was calculated using GraphPad Prism 50 value.

[0323] result

[0324] The results are as follows Fig.12 The ADC and Her-Dxd tested in the present invention were not found to be cytotoxic to JIMT-1 cells and MDA-MB-231 cells (IC 50 >1 nM), in N87 cells and HCC1954 cells, the cytotoxicity of all the ADCs tested in the present invention was comparable to that of the original ADCs, IC 50 The results show that the ADC of the present invention having the linker of the present invention and the linker-payload of the present invention has a cytotoxicity at least equivalent to that of trastuzumab.

Claims

1. A compound of formula I: its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; in, "*" indicates a chiral center, which is S- or R- or racemic; and hydrogen atoms attached to chiral carbon atoms and hydrogen atoms attached to carbon atoms having R2 substituents are omitted from the formula; L1 is -(CH2) a -, where a is 4 L2 is -(CH2) c -, where c is 1; L3 does not exist; R1 is -NH(C=O)CH3; R2 is -H; X is halogen or -OH; n＝1； and m＝1。 2. The compound of claim 1, wherein the compound is selected from L-3-4, or a pharmaceutically acceptable salt or ester thereof:

3. The coupling compound of formula II: its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; in, "*" indicates a chiral center, which is S- or R- or racemic; and hydrogen atoms attached to chiral carbon atoms and hydrogen atoms attached to carbon atoms having R2 substituents are omitted from the formula; L1 is -(CH2) a -, where a is 4; L2 is -(CH2) c -, where c is 1; L3 does not exist; R1 is -NH(C=O)CH3; R2 is -H; n＝1； m = 1; and "DRUG" is the Drug moiety covalently coupled to the Linker moiety.

4. The coupling compound as claimed in claim 3, which has the structure of formula IIa: Its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; wherein, "*" indicates a chiral center, which is S- or R- or racemic; and hydrogen atoms attached to chiral carbon atoms and hydrogen atoms attached to carbon atoms having R2 substituents are omitted from the formula; L1 is -(CH2) a -, where a is 4; L2 is -(CH2) c -, where c is 1; L3 does not exist; R1 is -NH(C=O)CH3; R2 is -H; n＝1； m＝1。 5. The coupling compound as claimed in claim 3, having a structure selected from the group consisting of:

6. Antibody-drug conjugate of formula III: its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; in, "*" indicates a chiral center which is S- or R- or racemic and the hydrogen atoms attached to the chiral carbon atom and the hydrogen atoms attached to the carbon atom having the R2 substituent are omitted from the formula; L1 is -(CH2) a -, where a is 4; L2 is -(CH2) c -, where c is 1; L3 does not exist; R1 is -NH(C=O)CH3; R2 is -H; n＝1； m＝1； "DRUG" is the drug moiety covalently coupled to the linker moiety; p is 2, 4, or 6, and Ab is antibody.

7. The antibody-drug conjugate of claim 6, wherein "DRUG" is exotecan; p is 4; and / or Ab is trastuzumab.

8. The antibody-drug conjugate of claim 6, having a structure selected from the group consisting of:

9. The antibody-drug conjugate according to claim 8, wherein p is 2, 4 or 6; and / or Ab is trastuzumab.

10. A method for producing a linker-loaded compound comprising coupling a drug to the compound of claim 1.

11. The method of claim 10, wherein: The drug is exotecan.

12. A method for producing an antibody-drug-conjugate, comprising: (a) coupling a drug with the compound of claim 1 to obtain a linker-loaded compound; and (b) coupling the antibody to the linker-load compound obtained in step (a).

13. The method of claim 12, wherein: The drug is exotecan.

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