TW202402736A - Linker for conjugation - Google Patents

Abstract

The invention provides a coupling linker, and a linker-load conjugate and an ADC (Analog to Digital Converter) comprising the linker.

Description

coupling linker

The present invention relates to the field of drug conjugates, specifically to antibody drug conjugates.

Antibody drug conjugates (ADCs) are large molecule 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 both site-directed and site-specific release macromolecular prodrugs. An ADC is actually a three-component system consisting of a potent drug linked to an antibody (usually a monoclonal antibody mAb) via a degradable or nondegradable (cleavable/non-cleavable) linker.

Typically, ADC is endocytosed after binding to an antigen on a cell, where the drug is then released from the antibody and takes effect. Compared with small molecules, the number of ADC molecules reaching target cells is relatively small, and the internalization rate is also low. It is therefore desirable to increase the rate at which the linker is cleaved to release the drug.

The linker is crucial to ADC, for example, it has a great impact on the stability and drug release mechanism of ADC. Enzymatically cleavable linkers, such as Trastuzumab deruxtecan and the linker in ^Enhertu® , usually include a short peptide (for example, 2 to 4 amino acids), which will be cleaved in lysosomes after endocytosis by ADC. , thereby releasing the drug to exert cell killing effect. In the already slow ADC mechanism of action, the speed of enzymatic cleavage (one or more enzymes) of the linker determines how quickly the drug takes effect. Most ADCs currently use cleavable linkers because they can rapidly release drugs in target cells. For example, brentuximab vedotin (Adcetris ^® ), trastuzumab deruxtecan (Enhertu ^® ), and loncastuximab tesirine-lpyl (Zynlonta ^® ) all use peptide cut fragments, which are VC, GGFG and VA respectively. These peptides are mainly cleaved by cathepsins (mainly cathepsin B) in lysosomes. Shearing rates vary depending on a variety of factors, including the properties of the enzyme involved, regiospecific enzyme-substrate interactions, and more. For example, the linker of ^Enhertu® contains the tetrapeptide GGFG, and the amide bond of the last glycine is a cleavable bond that is cleaved by lysosomal proteases (such as cathepsin). The cutting speed of GGFG is not as fast as that of VC.

One of the desirable features of ADCs is the rapid cleavage of the linker at a suitable location, such as the lysosome. We need better connectors and connector-loading to produce better-performing ADC products, and to produce ADCs with more operable and higher-yield processes.

The inventors have designed a series of connectors suitable for producing connectors with better performance - load and ADC, such as higher shearability (e.g., manifested as higher shear speeds), improved DAR distribution, improved homogeneity, Improved stability and/or treatment-related efficacy. The linker-based and linker-containing linker-payloads enable the manufacture of ADCs through a more operable and higher-yield process, such as making the ADC product purer with residual linker-payloads that are not bound to the antibody. Lower levels and/or residual linker-loading are easier to remove.

In a first aspect, the invention provides compounds of formula I: I its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; where "*" represents the chiral center, which is S- or R- or racemic; and the hydrogen atom attached to the chiral carbon atom and the hydrogen atom attached to the carbon atom having the _R substituent are omitted from the formula; L ₁ is -(CH ₂ ) _a -, where a is an integer from 0 to 10, or -(CH ₂ CH ₂ O) _b -, where b is an integer from 1 to 36; L ₂ is -(CH ₂ ) _c -, where c is an integer from 1 to 10, or -( CH ₂ CH ₂ O) _d -, where d is an integer from 1 to 36; L ₃ is absent, or -(CH ₂ ) _e -, where e is an integer from 1 to 10, or -(CH ₂ CH ₂ O ) _f -, where f is an integer from 1 to 36; R ₁ is -CF ₃ , -NR _a R _b , -NR _a (C=O)R _b or -O(CH ₂ ) _g CH ₃ , where g is An integer from 0 to 3, R _a is H or -C _1-6 alkyl, R _b is H or -C _1-6 alkyl; R ₂ is -H, -C _1-6 alkyl or -O(CH ₂ ) _h CH ₃ , where h is an integer from 0 to 3; X is halogen, -OR ₃ or -NR ₄ R ₅ ; R ₃ is -H, -C _1-6 alkyl or halogen; R ₄ and R ₅ is independently -H or -C _1-6 alkyl; n=0 or 1; and m=0 or 1.

In another aspect, the invention provides coupling compounds of formula II: II its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; among them, "*", L ₁ , L ₂ , L ₃ , R ₁ , R ₂ , n and m are as defined in Formula I; and, “DRUG” is the drug moiety covalently coupled to the linker.

In another aspect, the invention provides an antibody-drug conjugate of formula III: III its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; among them, "*", L ₁ , L ₂ , L ₃ , _R1 , _R2 , n and m and "DRUG" are as defined in Formula II; and, p is 1-8, such as 1, 2, 3, 4, 5, 6, 7 and 8; Ab refers to an antibody.

In another aspect, the invention provides a method of producing a linker-loaded compound comprising coupling a drug to a linker compound of the invention. In some embodiments, the drug is exatecan.

In another aspect, the invention provides a method for producing an antibody-drug-conjugate, which includes: (a) coupling a drug with a linker compound of the invention to obtain a linker-loading compound; wherein, preferably , the drug is exatecan; and (b) coupling the antibody to the linker-loading compound obtained in step (a).

Other objects, features and advantages of the present invention will be apparent from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are exemplary only, since those skilled in the art will be able to appreciate various changes and adaptations within the spirit and scope of the invention from the detailed description.

Terms and Definitions

In this document, the singular forms introduced by "a", "an" and "the/said" include the plural unless otherwise stated. Furthermore, the terms "a (species)", "one (species) or more (species)" and "at least one (species)" may be used interchangeably herein.

In this article, unless otherwise stated, regardless of whether there is "about" before the numerical value or range, it covers a reasonable approximate range that can be understood by those skilled in the relevant field, such as ±10%, ±5%, ±3%, ± 2%, ±1% or ±0.5% range.

In this article, the term "basically absent" means not only the absence of a certain situation or the existence of a certain substance (i.e. "none", "zero", etc.) but also the presence of no significant significance or below the inspection limit. An unfathomable existence or quantity of existence. This is well known to those skilled in the art.

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 invention.

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 to which this invention belongs. The publications and patent documents cited herein are incorporated by reference and are suitable for all purposes. The cited documents may be regarded as indicating the level of skill of those skilled in the art, but this should not be construed as an admission that they precede the present invention, since the present invention precedes the present invention.

Overview

The cleavage rate of a peptide linker varies depending on a variety of factors, including the properties of the enzyme involved, regiospecific enzyme-substrate interactions, and more. We found that changing the structure on both sides of the tetrapeptide GGFG will change the enzyme binding property of the linker, which is manifested as an increase in the kcat/Km value of cathepsin B (cathepsin B). Based on this we designed a series of connectors. Using these linkers can lead to better performing linker-loaded compounds and ADCs. As shown in the following examples, the linker-loaded compounds of the present invention containing the linker moiety designed by the present invention, whether as a stand-alone compound or as a component of an ADC, perform better than references containing similar existing linker moieties. Has a higher cathepsin B shearing speed. We found that the linker designed in the present invention is easier to be cleaved by enzymes, which helps to improve the efficacy of ADC. We have also found that ADCs containing the linker-payload moiety of the present invention can exhibit higher stability (e.g., storage stability, such as freeze-thaw stability) and/or comparable or even better therapeutics compared to existing similar ADCs. Relevant potency (e.g. binding affinity and/or cytotoxicity).

At the same time, we also found that making ADCs with the linker-loading compound of the invention containing the linker moiety designed by the invention can provide a purer product, for example compared to ^Enhertu® with a high drug-to-antibody ratio (DAR). ^Enhertu® has a DAR of 7.5. The higher the DAR, the more linker-loading is required for the coupling reaction. Therefore, removing residual linker-loaders can be very cumbersome. In the present invention, for example, compared to drotecan in Enhertu, the linker-payload of the present invention can be cleaned more easily and completely, and therefore a purer ADC product can be produced. The reason for this may be the higher hydrophilicity of the linker-payload of the present invention. At the same time, when a large excess of linker-load is used to achieve high DAR in coupling, non-specific binding of the linker-load to the antibody heavy chain will occur, resulting in a 4-drug coupled heavy chain, but the ideal number is not more than 3. In the present invention, the percentage of 4-drug coupling heavy chain can be reduced to 1/3 of the ^Enhertu® process. Therefore, the connectors and connector-loads of the present invention can provide more homogeneous ADC products.

We have also found that ADCs prepared using the linker-loaders of the present invention are easier to purify. In some cases, after UFDF (Ultrafiltration & Depth Filtration) purification, the residual free linker-loading in the ADC after coupling is significantly reduced compared to products produced with the linker-loading used in, for example, ^Enhertu® . Therefore, the connectors and connector-loads of the present invention can produce ADCs with higher purity, and can produce ADCs with more operable and productive processes.

Examples of specific implementations

1. Connector

In the present disclosure, the term "linker", as will be understood from the context, may refer to a linker compound of the invention alone or incorporated into a linker-loader conjugate or antibody-drug conjugate of the invention. and thus the connecting subparts that are part of it. It will be understood that a linker moiety refers to a moiety derived from the corresponding linker compound when incorporated into a conjugate by coupling.

In one aspect, the invention provides linker compounds having the structure of Formula I: I its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; where "*" represents the chiral center, which is S- or R- or racemic; and the hydrogen atom attached to the chiral carbon atom and the hydrogen atom attached to the carbon atom having the _R substituent are omitted from the formula; L ₁ is -(CH ₂ ) _a -, where a is an integer of 1 to 10, preferably 1 to 8, more preferably 2 to 6 or 4 to 5, or -(CH ₂ CH ₂ O) _b -, wherein b is 1 to 36, preferably 2 to 30, more preferably 3 to 25 or an integer from 4 to 20; L ₂ is -(CH ₂ ) _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 -(CH ₂ CH ₂ O ) _d -, where d is an integer from 1 to 36, preferably from 2 to 30, more preferably from 3 to 25 or 4 to 20; L ₃ is absent, or -(CH ₂ ) _e -, where e is from 1 to 10, Preferably it is an integer of 1 to 8 or 1 to 6, more preferably 1 to 2, or -(CH ₂ CH ₂ O) _f -, where f is 1 to 36, preferably 1 to 20, more preferably 1 to 2, 3 to 25 or an integer from 4 to 20; R ₁ is -CF ₃ , -NR _a R _b , -NR _a (C=O)R _b or -O(CH ₂ ) _g CH ₃ , where g is an integer from 0 to 3, Preferably 1 to 2, R _a is H or -C _1-6 alkyl, preferably -H or -CH ₃ ; R _b is H or -C _1-6 alkyl, preferably -H or -CH ₃ ; preferably , R ₁ is -CF ₃ , -N(CH ₃ ) ₂ , -NH(C=O)CH ₃ or -O(CH ₂ ) ₂ CH ₃ ; R ₂ is -H, -C _1-6 alkyl or -O(CH ₂ ) _h CH ₃ , where h is an integer from 0 to 3, preferably, R ₂ is -H or -CH ₃ ; X is halogen, -OR ₃ or -NR ₄ R ₅ , preferably -OR ₃ ; R ₃ is -H, -C _1-6 alkyl or halogen, preferably -H, -CH ₃ , tert-butyl or Cl; R ₄ and R ₅ are independently -H or -C _1-6 alkyl ; n=0 or 1; and m=0 or 1; Specific examples of the linker compound 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: L-1-1 L-1-2 L-1-3 L-1-4 L-1-7 L-1-8 L-3-1 L-3-4 where "*" represents the chiral center, which is racemic.

The designed linkers offer unique features and chemistries. For example, in L-1-1, L-1-2, L-1-3, L-1-4, L-1-7 and L-1-8, a CF ₃ group is introduced into the amine residue α position, thereby creating a strong dipole moiety on the N side of the GGFG peptide. This group is likely to reduce the Km of the interaction between cathepsin B and GGFG, thus accelerating the enzymatic rate of peptide hydrolysis. Apparently, the introduction of tetraamine or acetylamine at similar positions of the linker (e.g., as L-3-1 and L-3-4) also leads to an increase in the catalytic rate.

We further explored the N-side modification of the tetrapeptide GGFG. We found that introducing additional methyl/methylene groups (e.g., L-1-2, L-1-3, L-1-8) near the linkage of the drug moiety (e.g., ixotecan or Dxd) ) or glycol groups (e.g., L-1-7 and L-1-8) do not slow down the cathepsin B cleavage rate.

2. Connector - load

2.1. Linker-loaded conjugate of the present invention

In the present disclosure, the term "linker-loader" (hereinafter also referred to as "LP"), as will be understood from the context, may refer to the linker-loader compound of the invention alone or to the incorporation and therefore as a linker-loader compound according to the present invention. The linker-payload moiety is part of the inventive antibody-drug conjugate. A linker-payload moiety may share the same numerical code as the corresponding linker-payload compound from which it is derived by coupling.

In the present disclosure, the term "linker-load compound" refers to a coupling compound formed by the covalent coupling of a linker moiety and a drug moiety, where the drug moiety is also referred to as the "load". The linker-payload compound can be further coupled to an antibody, thereby providing an ADC comprising a linker-payload moiety of the invention.

Therefore, in one aspect, the present invention provides a linker-load coupling compound having the structure of Formula II: II its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; among them, "*", L ₁ , L ₂ , L ₃ , _R1 , _R2 , n and m are as defined in Formula I; and, "DRUG" is the drug moiety covalently coupled to the linker moiety.

In some embodiments, the linker-loader conjugate can be a compound having any of the following formulas, or a pharmaceutically acceptable salt or ester thereof: ,LP-1-1 , LP-1-2 ,LP-1-3 ,LP-1-4 ,LP-1-7 , LP-1-8 , and LP-3-1 . LP-3-4 where "*" represents the chiral center, which is racemic.

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 used for coupling can be -NHR, where R is alkyl or H.

As used herein, the term "drug (DRUG)", as will be understood from the context, may refer to a drug that forms a drug moiety covalently coupled to a linker-loaded compound or linker moiety in an ADC, or is derived from a drug by enzymatic cleavage Linker - payload or drug released from the ADC.

Drugs useful in the present invention include cytotoxic drugs, especially drugs used in 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, tubulin Inhibitors and toxins such as those of bacterial, fungal, plant or animal origin. Specific examples include, for example, paclitaxel, methotrexate, methotrexate, methotrexate, 5-fluorouracil, 6-mercaptopurine, arabinoside cytosine, melphalan, cyclomethacrylate 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 include Paclitaxel, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ethylenediamine Alkynes, duocarmycin A (duocarmycin A), duocarmycin SA (duocarmycin SA), kachibactins, camptothecins, hemiasterlins, maytansinoids, including DM1 , DM2, DM3, DM4), auristatins include 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, ixotecan, etoposide and derivatives thereof, such as hydroxycamptothecin (hodroxycamptothecin) and Dxd. In some embodiments, the drug is Dxd. In some other embodiments, the drug is ixotecan. In some embodiments, the drug is Dxd when released from the linker-payload or ADC. In some other embodiments, when coupled to a linker to form a drug moiety, the drug is ixotecan.

Accordingly, the linker-load conjugate may be a compound having the structure of Formula IIa, its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable Salt or ester: Iia wherein "*", L ₁ , L ₂ , L ₃ , R ₁ , R ₂ , n and m are as defined in Formula II.

In some specific embodiments, the linker-loader conjugate can be a compound selected from the group shown below: 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: Compound 1-1 Compound 1-2 Compound 1-3 Compound 1-4 Compound 1-7 Compound 1-8 Compound 3-1 . Compound 3-4 where "*" represents the chiral center, which is racemic.

2.2. Synthesis of linker-loader conjugates

As one aspect of the invention, provided herein is a method of producing a linker-loader coupling compound, which includes coupling a drug to the linker compound of the invention. In some embodiments, the drug is ixotecan.

The coupling of the drug and the linker compound can be carried out through a coupling reaction known in the art (such as esterification reaction or amidation reaction) or transesterification reaction, depending on one or more terminals of the linker compound. Types of functional groups and types of one or more functional groups on the drug.

3. antibody - drug conjugates

3.1. Antibody-drug conjugates of the present invention

In one aspect, the invention provides an antibody-drug conjugate comprising coupling an antibody to one or more drug molecules via a linker moiety of the invention, which may be represented by Formula III: III its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; among them, "*", L ₁ , L ₂ , L ₃ , R ₁ , R ₂ , n and m and "DRUG" are as defined in Formula II; and, p is 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 and 8; "Ab" means antibody.

In some specific examples, the antibody-drug conjugate can be a compound selected from the following group of compounds: , conjugate 1-1 Conjugate 1-2 Conjugate 1-3 Conjugates 1-4 Conjugates 1-7 Conjugates 1-8 , and conjugate 3-1 . Conjugate 3-4 where "*" represents a chiral center, which is racemic, where 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.

There are essentially no limitations on the antibodies useful in the present invention. Antibodies can be of various specificities, configurations and origins. In some embodiments, the antibody specifically binds a tumor antigen (TA), such as a tumor-specific antigen (TSA) and a tumor-associated antigen (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 (Panitumumab), Alemtuzumab, Matuzumab, Gemtuzumab, Polatuzumab, Inotuzumab, etc. In some embodiments, the antibody (Ab) is trastuzumab.

For the purposes of the ADCs of the invention, the term "antibody" includes antibody fragments such as Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments and scFv fragments. Furthermore, the term “antibody” extends to include functional equivalents, such as ligands and binding proteins that specifically recognize and bind to target molecules (e.g., antigens, e.g., tumor antigens), receptors, or target cells (e.g., disease-related cells, e.g., cancer cells) or other surface molecules on tumor cells), as long as these equivalent molecules have or can be modified to have functional groups capable of reacting with the maleamide group of the linker to covalently bind thereto. In some embodiments, the functional group is a thiol group, such as those released by reduction of an interchain disulfide bond, whereby the antibody can be coupled to the linker moiety via a sulfur-maleamide linkage.

3.2. Preparation of antibody-drug conjugates

In one aspect, the invention provides a method of producing an antibody-drug conjugate comprising coupling an antibody to a linker-loading compound of the invention.

In some embodiments, the method may include: (a) coupling a drug to a linker compound of the invention to obtain a linker-load compound; preferably, the drug is ixotecan; and (b) Conjugate the antibody to the linker-loading compound obtained in step (a).

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

Step (b) can be performed by reacting the maleimide moiety with free thiol groups in the antibody via a Michael addition reaction. For example, the one or more free thiol groups may originate from one or more cysteine residues, such as those liberated by reduction of interchain disulfide bonds, such that Antibodies can be coupled to the linker moiety via a thio-maleamide linkage.

4. Application

The antibody-drug conjugate (ADC) of the present invention can be formulated into a pharmaceutical composition with a pharmaceutically acceptable preparation. In some embodiments, the composition may comprise a therapeutically effective amount of an antibody-drug conjugate. In some embodiments, the composition may comprise an effective amount of the antibody-drug conjugate to achieve the desired dosage.

The antibody-drug conjugates of the invention may be used to treat a disease, disorder or condition in an object in need thereof, said treatment comprising administering to the object a therapeutically effective amount of the antibody-drug conjugate. The application also provides antibody drug-conjugates of the invention for use in treating a disease, disorder or condition in an article in need thereof. Diseases treated include, but are not limited to, cancer, including solid tumors and hematological cancers. Examples of such cancers include, but are not limited to, breast cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer (eg, NSCLC), head and neck cancer, colorectal cancer, B-cell lymphoma (eg, non-Hawkins lymphoma (NHL)), and leukemia .

As used herein, the term "object" refers to a human or non-human animal object. The non-human animal may be a mammal, such as a primate. Examples of non-human mammalian objects include, but are not limited to, captive animals, livestock and zoo animals, competitive animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, and bears. Preferably, the subject is a human being. "Item of Need" means a subject in need of diagnosis, prognosis, mitigation, prevention and/or treatment of a disease, disorder or condition.

Example

The following examples are for illustration only and do not limit the scope of the invention.

Example 1 : Connector - load synthesis

(a ) Compound 1-7 (In Chapter 1 Also known as drotecan analogues 1-7 in the figure )Synthesis

The synthesis process is schematically depicted in Figure 1.

Step 1 :2-(2-(( tert-butyldimethylsilyl) oxygen group) ethoxy) Methyl acetate (a-1 )

2-((tert-Butyldimethylsilyl)oxy)ethanol (6.51 g, 42.5 mmol) was added to anhydrous tert-butanol (50.0 mL). Add potassium tert-butoxide (3.18g, 28.3mmol) with stirring and stir at 0°C for 1 hour, then add methyl 2-bromoacetate (5.00g, 28.3mmol) in tert-butanol (50.00 mL) solution. 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) showed that the reaction was complete. The reaction mixture was quenched by adding water (100 mL) at 0°C and extracted with 50.0 mL of dichloromethane (50.0 mL*3). The combined organic layers were washed with brine (20.0 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, petroleum ether/ethyl acetate = 50/1 to 2/1) to obtain 2-(2-((tert-butyldimethylsilyl)oxy) as a colorless oil )ethoxy)methyl acetate (3.00g, 42.5% yield). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 4.19 (s, 2 H), 3.79 - 3.84 (m, 2 H), 3.76 (s, 2 H), 3.62 - 3.67 (m, 2 H), 0.90 (s, 9 H), 0.07 (s, 6 H).

Step 2 :2-(2- Hydroxyethoxy) Benzyl acetate (a-2 )

To methyl 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetate, a-1 (800 mg, 4.03 mmol) was dissolved in tetrahydrofuran (10.0 mL) and water (10.0 mL). Lithium hydroxide monohydrate (168 mg, 4.03 mmol) was added to the mixture. 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 incubated at 25 °C. Stir 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 (SiO ₂ , petroleum ether/ethyl acetate = 100/1 to 50/1) to obtain light yellow liquid Intermediate (1.30g). The intermediate was de-TBS in 1M HCl (5.00 mL). TLC (petroleum ether/ethyl acetate = 2:1, R _f =0.5) showed 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 obtain light yellow liquid 2-(2-hydroxyethoxy)benzyl acetate (600 mg, yield 88.6%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.28 - 7.41 (m, 5 H), 5.20 (s, 2 H), 4.22 (s, 2 H), 3.81 (t, J = 5.1 Hz, 3 H ), 3.64 - 3.68 (m, 2 H), 0.90 (s, 9 H), 0.07 (s, 6 H).

Step 3 ：1-(9H- Fluorene-9- base)-3,6- Dioxo-2,9,12- Trioxa-4,7- Diazatetradecane-14- Acid benzyl ester (a-4 )

To 2-(2-hydroxyethoxy)acetic acid benzyl ester a-2 (400 mg, 1.09 mmol) and acetic acid (2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino) To a mixture of acetyl)methyl ester 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 added Stir 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 layers were 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-fluoren-9-yl) as a white solid )-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester (0.40 g, yield 71.0%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.77 (d, J = 7.5 Hz, 2 H), 7.61 (d, J = 7.5 Hz, 2 H), 7.30 - 7.44 (m, 9 H), 5.13 - 5.22 (m, 2 H), 4.81 (d, J = 6.8 Hz, 2 H), 4.44 (d, J = 6.8 Hz, 2 H), 4.15 - 4.18 (m, 2 H), 3.92 (d, J = 5.5 Hz, 2 H), 3.73 - 3.76 (m, 2 H), 3.68 (d, J = 2.5 Hz, 2 H).

Step 4 ：1-(9H- Fluorene-9- base)-3,6- Dioxo-2,9,12- Trioxa-4,7- Diazatetradecane-14- Acid (a-5 )

To 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester a-4 (0.40 g, 771 umol) to a mixture of 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 h 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 celite, and the filtrate was concentrated under reduced pressure to obtain 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxa as a colorless oil -4,7-diazatetradecan-14-acid (0.25 g, yield 75.6%). The crude product was used directly in the next step without purification. ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 8.64 - 8.74 (m, 1 H), 7.83 - 7.95 (m, 2 H), 7.73 (d, J = 7.4 Hz, 1 H), 7.55 - 7.62 ( m, 1 H), 7.26 - 7.49 (m, 4 H), 4.56 (d, J = 6.5 Hz, 2 H), 4.26 - 4.35 (m, 2 H), 3.64 (d, J = 4.4 Hz, 2 H ), 3.49 - 3.60 (m, 4 H).

Step 5 :(a-6) , that is, L-1-7)

To a solution containing CTC-resin (0.50 g, 14.1 mmol) and 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diaza To the mixture of tetradecane-14-acid, a-5 (0.20 g, 520 umol), add N,N-diisopropylethylamine (0.25 g, 1.95 mmol) and dichloromethane (10.0 mL) for swelling. . Mix the resin for 2 hours, then add methanol (5.00 mL) and mix for 30 minutes. Then, the resin was washed three times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piridine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 5 times with N,N-dimethylformamide. Then add Fmoc-Phe-OH (0.58g, 1.31 mmol) and mix for 30 seconds, then add 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, bubbled with nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. Repeat the above steps at 25°C for the next couplings: amino acids 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-trifluoroheptan-2-yl)amino)acetic acid (0.20 g, 1.31 mmol). The reaction was detected by the ninhydrin test. The coupling reaction was monitored by ninhydrin color reaction. After washing with 10.0 mL methanol and vacuum drying. Add 20.0 mL of lysis buffer (20% HFIP/80% DCM) to the flask containing the peptide resin and stir twice for 2 minutes. The HFIP mixture was removed in vacuo to give 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 a-6 (100 mg, crude product, purity 90.0%) as a white solid. LCMS (ESI, m/z): 713.68 [M+H] ⁺ : 715 (Xbrige C18, 3.5 um, 2.1 * 30 mm column, Wavelength: UV 220 nm & 254 nm; Column temperature: 30 ℃) in water 0.1% TFA: 0.1% TFA in acetonitrile elutes (10-80_2 minutes). HPLC (Gemini-NX C18 5um 110A 150 * 4.6mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30 ℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80_20 minutes ).

Step 6 :(Compounds 1-7 )

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

(b) compound 3-1 (In Chapter 2 Also known as drotecan analogues in the picture 3-1 )Synthesis

The synthesis process is schematically depicted in Figure 2.

Step 1 ：1-(9H- Fluorene-9- base)-3,6- Dioxo-2,9- Dioxa-4,7- Diazadodecane-12- Benzyl acid ester (b-3 )

To (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)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 h. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. 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 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-fluoren-9-yl) as a white solid )-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester, b-3 (400 mg, yield 60.3%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.90 (d, J =7.4 Hz, 2 H), 7.73 (d, J =7.0 Hz, 2 H), 7.24 - 7.64 (m, 9 H), 5.05 - 5.14 (m, 2 H), 4.48 - 4.62 (m, 4 H), 4.16 - 4.34 (m, 3 H), 3.54 - 3.72 (m, 4 H).

Step 2 ：1-(9H- Fluorene-9- base)-3,6- Dioxo-2,9- Dioxa-4,7- Diazadodecane-12- Acid (a-3 )

To 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester (400 mg, 818 To a mixture of 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 under a hydrogen atmosphere (15 psi) for 5 h. TLC (DCM/MeOH=10:1, R _f =0.2) showed that the starting material was consumed and a new spot 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 as a white solid, 7-diazadodecane-12-acid, a-3 (300 mg, yield 91.9%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.90 (d, J =7.4 Hz, 2 H), 7.73 (d, J =7.0 Hz, 2 H), 7.24 - 7.64 (m, 4 H), 5.05 - 5.14 (m, 2 H), 4.48 - 4.62 (m, 2 H), 4.16 - 4.34 (m, 3 H), 3.54 - 3.72 (m, 4 H).

Step 3 :(S)-2- Amino-6-(( tert-butoxycarbonyl) amine group) Caproic acid (b-6 )

(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 h. TLC (DCM:MeOH=10:1, R _f =0.2) showed that the reaction was complete. The reaction mixture was added dropwise to a stirring solution of isopropyl ether (100 mL), the resulting solid was filtered, and the solid was washed with isopropyl ether (50 mL). Traces of isopropyl ether were removed in vacuo to give (S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanoic acid, b-6 (2.50 g, 95.1% yield) as a white solid ). The crude product was used in the next step without further purification. ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 3.67 (t, J = 6.1 Hz, 1 H), 3.03 (t, J = 6.7 Hz, 2 H), 1.73 - 1.90 (m, 2 H), 1.23 - 1.56 (m, 12 H).

Step 4 :(S)-6-(( tert-butoxycarbonyl) Amino)-2-( dimethylamino) Caproic acid (b-7 )

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, formaldehyde was added dropwise (1.32 g, 16.2 mmol, purity 37%), stir at 25°C for 30 min, then add sodium borohydride (614 mg, 16.2 mmol) dropwise in an ice bath over a period of 30 min, then at 25°C. Keep at C for another 30 minutes. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. Traces of trifluoroethanol (14.0 mL) were removed in vacuo to give a residue, which was diluted with 1% acetic acid (20.0 mL). The mixture was purified by flash chromatography (eluent: 10~50% H ₂ O (0.1% TFA/CH ₃ CN, C-18 column chromatography). The resulting product was then freeze-dried to obtain (S)-6 as a white solid -((tert-butoxycarbonyl)amino)-2-(dimethylamino)hexanoic acid, b-7 (1.20 g, yield 53.8%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 3.16 (t, J = 7.0 Hz, 1 H), 3.02 (t, J = 6.8 Hz, 2 H), 2.52 (s, 6 H), 1.66 - 1.73 (m, 2 H), 1.46 (ddd, J = 11.0 , 7.0, 4.0 Hz, 2 H), 1.38 (s, 9 H), 1.22 - 1.31 (m, 2 H).

steps 5 : (S)-2-( dimethylamino )-6-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base ) Caproic acid ( b-9 )

(S)-6-((tert-Butoxycarbonyl)amino)-2-(dimethylamino)hexanoic acid b-7 (0.60 g, 2.19 mmol) was dissolved in trifluoroacetic acid (0.60 mL) and dichloromethane (6.00 mL) was stirred at 25 °C for 2 h. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. The reaction mixture was concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid to obtain the de-Boc product as a colorless oil. The colorless oil was dissolved in saturated sodium bicarbonate (6.00 mL) at 0 °C, and 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid was added to the reaction mixture. methyl ester (407 mg, 2.62 mmol) and stirred at 0 °C for 1 h and then at 25 °C for an additional 3 h. LC-MS showed complete consumption of the intermediate and detected a main peak with the expected quality. The reaction mixture was acidified by adding 1 M HCl (5.00 mL) and purified by flash chromatography (eluent: 10~50% H ₂ O (0.1% TFA)/CH ₃ CN, C-18 column chromatography). The resulting product was then freeze-dried to obtain (S)-2-(dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) as a white solid Caproic acid, b-9 (500 mg, 89.9% yield). 1H NMR (400 MHz, CDCl3): δ ppm 6.80 (s, 2 H), 3.79 (dd, J = 8.5, 4.4 Hz, 1 H), 3.49 (t, J = 6.8 Hz, 2 H), 2.88 ( d , J = 13.4 Hz, 6 H), 1.89 - 2.00 (m, 2 H), 1.56 - 1.64 (m, 2 H), 1.30 - 1.40 (m, 2 H).

steps 6 : (3S , 12S)-12- Benzyl -3-(4-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base ) Butyl )-2- methyl -4,7,10,13,16- Pentaoxo -19- Oxa -2,5,8,11,14,17- hexaazadocane -twenty two- acid( b-10 ,Right now L-3-1 )

To a solution containing CTC-resin (0.50 g, 14.1 mmol) and 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane To the mixture of alkane-12-acid, a-3 (0.20 g, 520 umol), add N,N-diisopropylethylamine (DIEA) (0.25 g, 1.95 mmol) and dichloromethane (10.0 mL). swelling. Mix the resin for 2 hours, then add methanol (5.00 mL) and mix for 30 minutes. Then, the resin was washed three times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piridine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 5 times with N,N-dimethylformamide. Then add Fmoc-Phe-OH (0.58g, 1.31 mmol) and mix for 30 seconds, then add 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, bubbled with nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. For the next amino acid Fmoc-Gly-Gly-OH (0.53 g, 1.50 mmol) and the specific amino acid (S)-2-(dimethylamino)-6-(2,5-dioxo-2 Coupling of ,5-dihydro-1H-pyrrol-1-yl)hexanoic acid, b-9 (0.20 g, 1.31 mmol) at 25°C was repeated. The reaction was detected by the ninhydrin test. The coupling reaction was monitored by ninhydrin color reaction. After washing with 10.0 mL methanol and drying under vacuum, add 20.0 mL lysis buffer (20% HFIP/80% DCM) to the flask containing the peptide resin and stir twice for 2 minutes. The HFIP mixture was removed in vacuo to give 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 obtain (3S,12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) as a white solid )butyl)-2-methyl-4,7,10,13,16-pentaoxo-19-oxa-2,5,8,11,14,17-hexaazadocane-22 -Acid b-10 , (30.0 mg, yield 4.39%).

Step 7 : Compound 3-1

To (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-hexaazadocanoic acid, b-10 (18.6 mg, To a solution of 28.2 umol), and 1-hydroxybenzotriazole (HOBt) (2.80 mg, 20.6 umol,) in N,N-dimethylformamide (1.00 mL) was added ixotecan mesylate salt (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 h. TLC (DCM:MeOH=10:1, R _f =0.3) showed that the reaction was complete. Traces 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 obtained product was then freeze-dried to obtain compound 3-1 as a light yellow solid (7.20 mg, yield 35.4%). LCMS (ESI, m/z): 1077.1 [M+H] ⁺ : 1077.6 (Xbrige C18, 3.5 um, 2.1 * 30 mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30 ℃) in water 0.1% TFA: 0.1% TFA in acetonitrile elutes (10-80_2 minutes). HPLC (Gemini-NX C18 5um 110A 150 * 4.6mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30 ℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (25-55_20+ 3 minutes).

(c) Synthesis of other compounds

compound 1-1 (In Chapter 3 Also known as drotecan analogues in the picture 1-1 )Synthesis

Step 1 :10- Benzyl-23-(2,5- Dioxo-2,5- Dihydro-1H- Pyrrole-1- base)-6,9,12,15- Tetraoxo-18- (Trifluoromethyl)-3- Oxa-5,8,11,14,17- Pentaazatricosane-1- Acid (c-1 , that is, L-1-1 )

Peptide synthesis:

Peptides were synthesized using standard Fmoc chemistry.

1) Add DCM to the solution 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-triazatetradecan-14-acid (0.10 g, 0.25 mmol, 1.0 equiv) in a container while nitrogen was bubbled.

2) Add DIEA (6.0 equivalent) dropwise and mix for 2 hours.

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

4) Drain and wash 5 times with DMF.

5) Add 20% pyridine/DMF and react for 30 minutes.

6) Drain and wash 3 times with DMF.

7) Add Fmoc amino acid solution and mix for 30 seconds, then add activation buffer and bubble _N2 for about 1 hour.

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

Note: # Material Coupling agent 1 5-Benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2.12-dioxa-4,7,10-triazatetradecane-14-acid ( 1.0 equivalent) DIEA (6.0 equivalent) 2 Fmoc-Phe-OH (3.0 equiv) HBTU (2.85 equivalents) and DIEA (6.0 equivalents) 3 Fmoc-Gly-OH (3.0 equiv) twice HBTU (2.85 equivalents) and DIEA (6.0 equivalents) 4 1-(7,7,7-Trifluoro-6-hydroxyheptyl)-1H-pyrrole-2,5-dione (2.0 equivalent) HOAT (1.00 equivalent) and DIC (1.0 equivalent)

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

Peptide shearing and purification:

1) Add shear buffer (20% HFIP/DCM) to the peptide resin and stir three times for 3 minutes.

2) DCM is concentrated under reduced pressure.

3) Dry the peptide in high vacuum for 2 hours.

4) The crude peptide was purified by preparative HPLC (A: 0.075% TFA in H ₂ O, B: ACN) to obtain compound c-1 (26.0 mg, 95.0% purity, 7.36% yield).

steps 2 :(Drutican analogues 1-1 )

To a solution of compound c-1 (21.0 mg, 31.3 umol, 1 equiv) and ixotecan mesylate (16.6 mg, 31.3 umol) in DMF (0.5 mL) was added HOAt (12.8 mg, 93.9 umol, 3 equiv), DIC (15.8 mg, 125.2 umol, 4 equiv) and DIEA (12.14 mg, 93.9 umol, 3 equiv), and the reaction mixture was stirred at 25°C for 16 h. LCMS showed that compound c-1 was consumed and a main peak with the expected quality was detected. The reaction mixture was directly purified by preparative HPLC (natural conditions, pure water). Two peaks with the desired quality were detected in the preparative HPLC, separated and lyophilized to obtain a white solid of drutecan analogue 1-1. (Peak 1: 1.4 mg, 85.97% purity, Peak 2: 7.1 mg, 90.32% purity, 22.3% yield).

compound 1-2 (In Chapter 4 Also known as drotecan analogues in the picture 1-2 )Synthesis

steps 1 : (S)-1-(9H- Fluorene -9- base )-10- methyl -3,6- Dioxo -2,9- Dioxa -4,7- diazaundecane -11- acid benzyl ester ( d-3 )

To (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)methyl acetate (5 g, 13.57 mmol) and (S)-2-hydroxypropionic acid benzyl To a mixture of 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 h. . LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. The residue was purified by preparative HPLC (TFA conditions). Obtained yellow oily compound (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadeca Monoalkyl-11-acid benzyl ester, d-3 (4.10 g, 8.38 mmol, 61.77% yield).

steps 2 : (S)-1-(9H- Fluorene -9- base )-10- methyl -3,6- Dioxo -2,9- Dioxa -4,7- diazaundecane -11- acid ( d-4 )

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

steps 3 : (2S,10S)-10- Benzyl -23-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base )-2- methyl -6,9,12,15- Tetraoxo -18- (trifluoromethyl) -3- Oxa -5,8,11,14,17- Pentaazatricosane -1- acid( d-5 ,Right now L-1-2 )

To a solution containing CTC-resin (0.50 g, 0.50 mmol) and (S)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa- To the mixture of 4,7-diazaundecane-11-acid, d-4 (0.50 g, 500 umol), add N,N-diisopropylethylamine (DIEA) (2.00 mmol), dichloro Methane (10.0 mL) for swelling. Mix the resin for 2 hours, then add methanol (5.00 mL) and mix for 30 minutes. Then, the resin was washed 5 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piridine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed three times with N,N-dimethylformamide. Then add Fmoc-Phe-OH (1.50 mmol) and mix for 30 seconds, then add O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) (1.50 mmol) and N,N-diisopropylethylamine (DIEA) (3.00 mmol) in N,N-dimethylformamide, bubbled with nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. For the next amino acid Fmoc-Gly-OH (1.50 mmol) and the special amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) Repeat the above procedure for coupling of -1,1,1-trifluoroheptan-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 ninhydrin color reaction. After washing with 10.0 mL methanol and drying under vacuum. Add 20.0 mL of lysis buffer (20% HFIP/80% DCM) to the flask containing the peptide resin and stir twice for 2 minutes. The HFIP mixture was 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 obtain (2S,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2 as a white solid -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%).

steps 4 :(Drutican analogues 1-2 )

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-acid, d-5 (20.0 mg, 29.2 umol), and 1-hydroxy-7-azabenzotriazole (HOAt) (7.95 mg, 58.4 umol) in N,N-dimethylformamide (0.50 mL) was added with ixotecan Methanesulfonate (15.5 mg, 29.1 umol), N,N-diisopropylcarbodiimide (DIC) (22.1 mg, 175 umol, 27.1 uL) and N,N-diisopropylethylamine (DIEA) (7.55 mg, 58.4 umol, 10.1 uL) and the mixture was stirred at 25°C for 3 hours. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. The reaction mixture was filtered to remove undissolved residue. The residue was purified by preparative HPLC (TFA conditions). The obtained product was then freeze-dried to obtain a light yellow solid of drutecan analogue 1-2 (18.8 mg, 17.0umol, yield 58.4%, purity 96.3%).

compound 1-3 (In Chapter 5 Also known as drotecan analogues in the picture 1-3 )Synthesis

steps 1 : 1-(9H- Fluorene -9- base )-3,6- Dioxo -2,9- Dioxa -4,7- Diazadodecane -12- Benzyl acid ester ( e-3 )

To a mixture of compound a-3 (500 mg, 1.36 mmol) and compound e-2 (366 mg, 2.04 mmol) in THF (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 compound a-3 was completely consumed and a main peak with the expected quality was detected. The mixture was diluted with _NaHCO3 (50.0 mL) and extracted with EtOAc (50.0 mL). The combined organic layers were dried over _Na2SO4 , filtered and concentrated _under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel), eluting with (petroleum ether/ethyl acetate = 50/1 to 1/1) to obtain compound e-3 as a white solid (400 mg, yield 60.3%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.90 (d, J=7.4 Hz, 2 H), 7.73 (d, J=7.0 Hz, 2 H), 7.24 - 7.64 (m, 9 H), 5.05 - 5.14 (m, 2 H), 4.48 - 4.62 (m, 4 H), 4.16 - 4.34 (m, 3 H), 3.54 - 3.72 (m, 4 H).

steps 2 : 1-(9H- Fluorene -9- base )-3,6- Dioxo -2,9- Dioxa -4,7- Diazadodecane -12- acid( e-4 )

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 incubated at 25 °C. Stir under hydrogen atmosphere (15 psi) for 5 hours. TLC (DCM/MeOH=10:1, R _f =0.2) showed that compound e-3 was depleted and a new spot 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 H NMR (400 MHz, CDCl ₃ ): δ ppm 7.90 (d, J=7.4 Hz, 2 H), 7.73 (d, J=7.0 Hz, 2 H), 7.24 - 7.64 (m, 4 H), 5.05 - 5.14 (m, 2 H), 4.48 - 4.62 (m, 2 H), 4.16 - 4.34 (m, 3 H), 3.54 - 3.72 (m, 4 H).

steps 3 : (11S)-11- Benzyl -24-(2,5- dioxo -2,5- dihydrogen -1H- Pyrrole -1- base )-7,10,13,16- Tetraoxo -19-( trifluoromethyl )-4- Oxa -6,9,12,15,18- Pentaazatetrasane -1- acid( e-5 ,Right now L-1-3 )

To the 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. Mix the resin for 2 hours, then add MeOH (5.00 mL) and mix for 30 minutes. Then, the resin was washed three times with DMF (10.0 mL). The resin was treated with 20% piridine 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 s, then a solution of HBTU (0.54 g, 1.43 mmol) and DIEA (0.25 g, 1.95 mmol) in DMF was added and nitrogen was bubbled for 30 min. The resin was washed 3 times with DMF. For the next amino acid Fmoc-Gly-OH (0.53 g, 1.50 mmol) and the specific amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1) The above procedure was repeated for the coupling of -1,1,1-trifluoroheptan-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 ninhydrin color reaction. After washing with 10.0 mL MeOH and drying under vacuum. Add 20.0 mL of lysis buffer (20% HFIP/80% DCM) to the flask containing the peptide resin and stir twice for 2 minutes. The HFIP mixture was removed in vacuo to give 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 as a white solid (30.0 mg, yield 4.39%).

steps 4 :(Drutican analogues 1-3 )

To a solution of compound e-5 (27.2 mg, 37.6 umol) in DMF (1.00 mL) was added HATU (9.30 mg, 24.4 umol). The reaction mixture was stirred at 25°C for 20 minutes. Ixotecan mesylate (10.0 mg, 18.8 umol) and DIEA (4.86 mg, 37.6 umol) were added to the reaction mixture and maintained at 40 °C for 2 h. TLC (DCM:MeOH=10:1, R _f =0.4) showed that the reaction was complete. Traces 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 resultant product was then freeze-dried to obtain drotecan analog 1-3 as a pale yellow solid (7.20 mg, 34.7% yield).

compound 1-4 (In Chapter 6 Also known as drotecan analogues in the picture 1-4 )Synthesis

steps 1 : (S)-1-(9H- Fluorene -9- base )-11- methyl -3,6- dioxo -2,9- Dioxa -4,7- Diazadodecane -12- acid methyl ester ( f-3 )

To (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)methyl acetate (5 g, 13.5 mmol) and (S)-3-hydroxy-2- To a mixture of methyl methacrylate (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 hours. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. The residue was purified by preparative HPLC (TFA conditions). Obtained yellow oily compound (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadeca Methyl dioxan-12-acid, f-3 (4.79 g, 11.2 mmol, 82.7% yield).

steps 2 : (S)-1-(9H- Fluorene -9- base )-11- methyl -3,6- Dioxo -2,9- Dioxa -4,7- Diazadodecane -12- acid (f-4)

To (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-diox-2,9-dioxa-4,7-diazadodecane-12- To a solution of 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 added Stir for 2 hours at 25°C. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. Add acetic acid to acidify, then add ammonium to alkalize, then add (2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-yl methyl carbonate (8.21g, 24.3mmol) and sodium bicarbonate ( 2.04g, 24.3 mmol). The mixture was stirred at 25°C for 2 hours. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. TLC (dichloromethane/methanol = 10:1, R _f =0.3) showed consumption of starting material and formation of a new spot. 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 (SiO ₂ , dichloromethane/methanol = 100/1 to 20/1) to obtain a light yellow oil (S)-1-(9H-fluoren-9-yl) as a light yellow liquid )-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, f-4 (1.25 g, 3.03 mmol, 12.45% yield).

steps 3 : (11S)-11- Benzyl -24-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base )-2- methyl -7,10,13,16- Tetraoxo -19-( trifluoromethyl )-4- Oxa -6,9,12,15,18- Pentaazatetrasane -1- acid( f-5 ,Right now L-1-4 )

To a solution containing CTC-resin (1.5 g, 1.50 mmol) and (S)-1-(9H-fluoren-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa- To the mixture of 4,7-diazadodecane-12-acid, f-4 (0.60 g, 1.50 mmol), add N,N-diisopropylethylamine (DIEA) (6.00 mmol), dichloro Methane (10.0 mL) for swelling. Mix the resin for 2 hours, then add methanol (5.00 mL) and mix for 30 minutes. Then, the resin was washed 5 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piridine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed three times with N,N-dimethylformamide. Then add Fmoc-Phe-OH (4.50 mmol) and mix for 30 seconds, then add O-benzotriazole-N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU) (4.50 mmol) and N,N-diisopropylethylamine (DIEA) (9.00 mmol) in N,N-dimethylformamide, bubbled with nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. For the next amino acid Fmoc-Gly-OH (0.53 g, 4.50 mmol) and the specific amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1) The above procedure was repeated for the coupling of -1,1,1-trifluoroheptan-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 ninhydrin color reaction. After washing with 10.0 mL methanol and drying under vacuum, add 20.0 mL lysis buffer (20% HFIP/80% DCM) to the flask containing the peptide resin and stir twice for 2 minutes. The HFIP mixture was 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 obtain (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-methyl as a white solid Base-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentaazatetracosane-1-acid, f-5 , (167 mg, yield 9.80%).

steps 4 :(Drutican analogues 1-4 )

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-pentaazatetracosane-1-acid, f-5 (20.0 mg, 28.6 umol) To a mixture of 1-hydroxy-7-azabenzotriazole (HOAt) (7.79 mg, 57.2 umol) in N,N-dimethylformamide (0.50 mL) was added ixotecan mesylate Salt (15.2 mg, 28.6 umol), N,N-diisopropylcarbodiimide (DIC) (21.6 mg, 171 umol, 26.5 uL) and N,N-diisopropylethylamine (DIEA) (7.40 mg , 57.2 umol, 9.97 uL), and the mixture was stirred at 25°C for 3 hours. LC-MS showed complete consumption of starting material and detected a main peak with the expected quality. The reaction mixture was filtered to remove undissolved residue. The residue was purified by preparative HPLC (TFA conditions). The obtained product was then freeze-dried to obtain a pale yellow solid of drotecan analog 1-4 (6 mg, 5.20 umol, yield 18.1%, purity 96.2%).

compound 1-8 (In Chapter 7 Also known as drotecan analogues in the picture 1-8 )Synthesis

steps 1 : 2-(2-( Benzyloxy ) Ethoxy ) Benzyl propionate ( g-2 )

To stirring DMF (100 mL) was added NaH (3.33 g, 83.2 mmol, 60% purity) portionwise in _N at 0 °C. (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) showed that the reaction was complete. The reaction mixture was quenched by adding 0 °C water (1000 mL) and extracted with dichloromethane (200 mL x 3). The combined organic layers were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, petroleum ether/ethyl acetate = 50/1 to 5/1) to obtain colorless oily benzyl 2-(2-(benzyloxy)ethoxy)propionate. g-2 (790 mg, 4.53% yield). δ ppm 7.29 - 7.43 (m, 10 H), 5.10 - 5.30 (m, 2 H), 4.58 (d, J =2.01 Hz, 2 H), 4.14 (q, J =6.86 Hz, 1 H), 3.75 - 3.87 (m, 1 H), 3.60 - 3.73 (m, 3 H), 1.47 (d, J =7.03 Hz, 3 H).

steps 2 : 2-(2- Hydroxyethoxy ) Propionic acid ( g-3 )

The 100 mL round bottom flask was purged 3 times with Ar and dry Pd/C (160 mg) was carefully added. Methanol (~10.0 mL) was then added to completely penetrate the Pd/C, followed by the slow addition of (2S)-2-(2-(benzyloxy)ethoxy)benzylpropionate, g-2 (790 mg) in Ar , 2.51 mmol) in MeOH (20.0 mL). The resulting mixture was degassed and purged three times with _H2 , then the mixture was stirred under _H2 atmosphere at 25 °C for 4 h. TLC (petroleum ether/ethyl acetate=5:1, R _f =0.2) showed that the reaction was complete. The reaction 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. The crude product was used directly in the next step without purification. ¹ H NMR (400 MHz, DMSO): δ ppm 4.02 (q, J =6.78 Hz, 1 H), 3.66 - 3.70 (m, 2 H), 3.60 - 3.66 (m, 1 H), 3.49 - 3.56 (m , 1 H), 1.39 (d, J =7.03 Hz, 3 H).

Step 3 :2-(2- Hydroxyethoxy) Benzyl propionate (g-4 )

Dissolve (2S)-2-(2-hydroxyethoxy)propionic acid, g-3 (320 mg, 2.39 mmol) in MeOH (4 mL) and H ₂ O (0.8 mL) and add Cs to this solution ₂ CO ₃ (389 mg, 1.19 mmol). The mixture was stirred at 25°C for 0.5 hours and concentrated. Then toluene bromide (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) showed that the reaction was complete. Dilute the mixture with water (50.0 mL) and extract with DCM (30 mL x 3). The combined organic layers were 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 = 20/1 to 5/1) to obtain 2-(2-hydroxyethoxy) as a white solid. Benzyl propionate, g-4 (340 mg, yield 64%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.37 (s, 5 H), 5.15 - 5.26 (m, 2 H), 4.04 - 4.15 (m, 1 H), 3.71 - 3.76 (m, 2 H) , 3.60 - 3.68 (m, 2 H), 1.46 (d, J =7.03 Hz, 3 H).

Step 4 ：1-(9H- Fluorene-9- base)-13- Methyl-3,6- Dioxo-2,9,12- Trioxa-4,7- Diazatetradecane-14- Benzyl acid ester (g-6 )

To [[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]methyl acetate ( a-3 ) (614 mg, 1.67 mmol) in THF (8 mL) TsOH.H ₂ O (14.4 mg, 75.8 umol) and benzyl 2-(2-hydroxyethoxy)propionate, g-4 (340 mg, 1.52 mmol) were added to the solution. 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 layers were washed with brine (30 mL), dried _{over Na2SO4} , filtered and concentrated under reduced pressure to give 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-fluoren-9-yl) as a white solid )-13-Methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecan-14-acid benzyl ester, g-6 (416 mg, 781 umol, yield 51.5%). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.77 (d, J =7.53 Hz, 2 H), 7.61 (br d, J =7.28 Hz, 2 H), 7.39 - 7.43 (m, 2 H), 7.28 - 7.38 (m, 7 H), 7.21 (br s, 1 H), 5.53 (br s, 1 H), 5.19 (s, 2 H), 4.93 - 5.03 (m, 1 H), 4.67 (br s , 1 H), 4.44 (d, J =7.03 Hz, 2 H), 4.22 - 4.27 (m, 1 H), 4.05 (q, J =7.03 Hz, 1 H), 3.92 (br s, 2 H), 3.76 (br s, 1 H), 3.71 (br d, J =9.03 Hz, 2 H), 3.55 (br d, J =8.53 Hz, 1 H), 1.44 (d, J =6.78 Hz, 3 H).

Step 5 ：1-(9H- Fluorene-9- base)-13- Methyl-3,6- Dioxo-2,9,12- Trioxa-4,7- Diazatetradecane-14- Acid (g-7 )

Purge the 100 mL round-bottom flask with Ar three times and carefully add dry Pd/C (60 mg). THF (5 mL) was then added to completely penetrate the Pd/C, followed by the slow addition of 1-(9H-fluoren-9-yl)-13-methyl-3,6-dioxo-2,9,12 under Ar - A solution of trioxa-4,7-diazatetradecan-14-acid benzyl ester, g-6 (416 mg, 781 umol) in THF (10 mL). The resulting mixture was degassed and purged 3 times with _H2 , then stirred in _H2 (15 psi) at 25 °C for 2 h. TLC (petroleum ether/ethyl acetate = 1:1, R _f = 0.4) showed that the reaction was complete. The reaction was filtered and concentrated under reduced pressure to obtain the product 1-(9H-fluoren-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa as a white gum. -4,7-diazatetradecan-14-acid, g-7 (320 mg, 92.6% yield), crude product was used in the next step without purification. ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 7.77 (d, J =7.53 Hz, 2 H), 7.60 (br d, J =7.28 Hz, 2 H), 7.38 - 7.44 (m, 2 H), 7.29 - 7.35 (m, 2 H), 5.48 - 5.65 (m, 1 H), 4.95 (br s, 1 H), 4.70 (dd, J =10.54, 6.02 Hz, 1 H), 4.42 - 4.52 (m, 2 H), 4.23 (t, J =6.78 Hz, 1 H), 4.02 (q, J =6.86 Hz, 1 H), 3.94 (br s, 1 H), 3.83 - 3.92 (m, 1 H), 3.75 - 3.82 (m, 2 H), 3.73 (br d, J =6.78 Hz, 2 H), 3.58 - 3.67 (m, 1 H), 1.42 - 1.48 (m, 3 H).

steps 6 : (13S)-13- Benzyl -26-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base )-2- methyl -9,12,15,18- Tetraoxo -twenty one-( trifluoromethyl )-3,6- Dioxa -8,11,14,17,20- Pentaazahexadecane -1- acid( g-8 ,Right now L-1-8 )

Peptide synthesis:

Peptides were synthesized using standard Fmoc chemistry.

1) To the solution 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, To a container of 12-trioxa-4,7-diazatetradecan-14-acid (0.6 g, 1.5 mmol 1.0 equiv), add DCM while bubbling N ₂ .

2) Add DIEA (4.0 equivalent) dropwise and mix for 2 hours.

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

4) Drain and wash 5 times with DMF.

5) Add 20% pyridine/DMF and react for 30 minutes.

6) Drain and wash 3 times with DMF.

7) Add Fmoc amino acid solution and mix for 30 seconds, then add activation buffer and bubble _N2 for about 0.5 hours.

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

Note: # Material Coupling agent 1 1-(9H-fluoren-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid ( 1.0 equivalent) DIEA (4.0 equivalent) Fmoc-Phe-OH (3.0 equiv) HBTU (3.0 equivalent) and DIEA (6.0 equivalent) 2 Fmoc-Gly-OH (3.0 equiv) HBTU (3.0 equivalent) and DIEA (6.0 equivalent) 3 Fmoc-Gly-OH (3.0 equiv) HBTU (3.0 equivalent) and DIEA (6.0 equivalent) 1-(7,7,7-Trifluoro-6-hydroxyheptyl)-1H-pyrrole-2,5-dione (2.0 equivalent) HOAT (2.0 equivalent) and DIC (2.0 equivalent)

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

Peptide shearing and purification:

1) Wash the resin 3 times with MeOH and dry by vacuum.

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

3) DCM is concentrated under reduced pressure.

4) Dry the peptide in high vacuum for 2 hours to obtain compound g-8 (100 mg, 90%).

Step 7 :(Drutican analogues 1-8 )

To a solution of compound g-8 (20.0 mg, 27.5 umol) in DMF (200 uL) was 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-diazacyclo[14.7.1.02,14.04,13.06,11.020,24]tetradecane- 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. Traces 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 obtain compound delutecan 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, 3.5 um, 2.1 * 30 mm column, wavelength: UV220 nm&254 nm; column temperature: 30 ℃) 0.1% TFA in water : Elution with 0.1% TFA in acetonitrile (10-80_2 minutes). HPLC (Gemini-NX C18 5um 110A 150 * 4.6mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30 ℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (40-70_20+ 3 minutes).

Compound 3-4 (In Chapter 8 Also known as drotecan analogues 3-4 in the figure )Synthesis

steps 1 : (S)-2- acetamide -6-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base ) Caproic acid ( h-3 )

To a solution of (2S)-2-acetylamido-6-amino-hexanoic acid (2.00 g, 10.6 mmol) in saturated NaHCO ₃ (50 mL) was added 2,5-dioxopyrrole-1-carboxylic acid Acid methyl ester (1.65g, 10.6mmol). The mixture was stirred at 25°C for 4 hours. LC-MS showed that compound h-1 was completely consumed and a main peak with the expected m/z was detected. The reaction mixture was quenched by adding _H2SO4 at 0°C to _pH =3-4, 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 obtain (S)-2-acetylamide-6-(2,5-dioxo-2,5- Dihydro-1H-pyrrol-1-yl)hexanoic acid, h-3 (460 mg, 1.71 mmol). ¹ H NMR (400 MHz, CDCl ₃ ): δ ppm 6.71 (s, 2 H), 6.30 (br d, J =7.28 Hz, 1 H), 4.48 - 4.59 (m, 1 H), 3.54 (t, J =6.78 Hz, 2 H), 2.08 (s, 3 H), 1.90 - 2.00 (m, 1 H), 1.75 - 1.84 (m, 1 H), 1.56 - 1.70 (m, 2 H), 1.31 - 1.45 ( m, 2 H).

steps 2 : (19S)-10- Benzyl -19-(4-(2,5- Dioxo -2,5- dihydrogen -1H- Pyrrole -1- base ) Butyl )-6,9,12,15,18,21- Hexaoxo -3- Oxa -5,8,11,14,17,20- hexaazadocane -1- acid( h-4 ,Right now L-3-4 )

Peptide synthesis:

Peptides were synthesized using standard Fmoc chemistry.

1) To the solution 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-acid (0.2 g, 0.5 mmol, 1.0 equiv) was added to a container with DCM while _N was bubbled.

2) Add DIEA (4.0 equivalent) dropwise and mix for 2 hours.

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

4) Drain and wash 5 times with DMF.

5) Add 20% pyridine/DMF and react for 30 minutes.

6) Drain and wash 3 times with DMF.

7) Add Fmoc amino acid solution and mix for 30 seconds, then add activation buffer and bubble _N2 for about 0.5 hours.

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

Note: # Material Coupling agent 1 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid (1.0 equiv) DIEA (4.0 equivalent) Fmoc-Phe-OH (3.0 equiv) HBTU (3.0 equivalent) and DIEA (6.0 equivalent) 2 Fmoc-Gly-OH (3.0 equiv) HBTU (3.0 equivalent) and DIEA (6.0 equivalent) 3 Fmoc-Gly-OH (3.0 equiv) HBTU (3.0 equivalent) and DIEA (6.0 equivalent) 2-Acetamide-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (2.0 equiv) HOAT (2.0 equivalent) and DIC (2.0 equivalent)

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

Peptide shearing and purification:

1) Wash the resin 3 times with MeOH and dry by vacuum.

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

3) DCM is concentrated under reduced pressure.

4) Dry the peptide in high vacuum for 2 hours to obtain compound h-4 (100 mg, 95%).

Step 3 :(Drutican analogues 3-4 )

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), ixotecan 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 the reaction was complete. Traces 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 obtained product was then freeze-dried to obtain compound delutecan analogue 3-4 (7 mg, 6.42 umol) as a light yellow solid. LCMS (ESI, m/z): 1090.4 [M+H] ⁺ : 1091.4 (Xbrige C18, 3.5 um, 2.1 * 30 mm column, wavelength: UV220 nm&254 nm; column temperature: 30 ℃) using 0.1% TFA in water :Elution with 0.1% TFA in acetonitrile (10-80_2 min). HPLC (Gemini-NX C18 5um 110A 150 * 4.6mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30 ℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (25-55_20+ 3 minutes).

(d ) "Original connector - "Load" synthesis

In the context of this disclosure, the term "nat-linker-load" (hereinafter also referred to as "nat-LP" or "nat-LP") refers to compounds having the following structure:

Original-LP has the same structure as drotecan in Enhertu, both containing maleimide-GGFG linkers. In this example, the original-LP was purchased from MedChemExpress under the name "Drutican" .

Example 2 :antibody- Synthesis of drug conjugates

Antibody-drug conjugates are synthesized according to the following general procedure: first reduce the antibody in a pH7 PBS solution with 2 to 20 equivalents of TCEP for 0.5 to 18 hours; with or without removal of residual TCEP by column or membrane, An excess of linker-loading is introduced (15~18 molar excess); the coupling reaction is completed in half an hour to a few hours at a temperature ranging from 4°C to room temperature (RT), followed by HPLC purification to provide the final ADC products. ADC restore coupling TCEP equivalent Temperature(℃) time(hour) Residual TCEP LP/Ab ² Temperature(℃) time(hour) Herceptin -1-1 ¹ 15.0 twenty two 3 remove 15 4 2 Herceptin -1-2 7.5 twenty two 3 Do not remove 18 4 2 Herceptin -1-3 15.0 twenty two 3 remove 15 4 2 Herceptin -1-4 7.5 twenty two 3 Do not remove 18 4 2 Herceptin -1-7 15.0 twenty two 3 remove 15 4 2 Herceptin -1-8 7.5 twenty two 3 Do not remove 18 4 2 Herceptin -3-1 15.0 twenty two 3 remove 15 4 2 Herceptin -3-4 7.5 twenty two 3 Do not remove 18 4 2 Herceptin - original - LP ³ 15.0 twenty two 3 remove 15 4 2 Note: 1: Expressed as Ab, followed by the digital code of each linker-payload part 2: Molar ratio 3: Hereinafter also referred to as "Her-Dxd" or "Her-nat-LP"

Example 3 : Connector - load shear

method

Reaction buffer (40 mM H ₃ PO ₄ /H ₃ BO ₃ /HAc, 1 mM EDTA, pH 4.5), 135 mM cysteine, DMA (N,N'-dimethylacetamide, 2%, v/v), sample (0.015 µmol linker-loading compound) and cathepsin B (final 16 U/µmol linker-loading compound) were added sequentially to a 1.5 mL EP tube. The resulting final cysteine concentration in the 300 µL lysis system is 10 mM. Place the EP tube in a 37°C water bath.

After approximately 15 minutes (min), 2 hours (hr), 5 hours, and 16 hours of lysis, the mixture was vortexed thoroughly and a sample (25 µL) was taken. The cathepsin B inhibitor E64 (2.5 equivalents compared to cathepsin B) was added to the collected mixture sample to inactivate the protease B, and then analyzed by RP-HPLC and RP-MS. Percent shear was calculated from the ratio of the cleaved linker-loaded peak area to the sum of the non-cleaved plus cleaved linker-loaded areas (as measured by RP-HPLC and RP-MS).

result

Table 1 summarizes the cleavage profile (expressed as percentage) over approximately 16 hours (overnight), showing a linker-load comparison of cathepsin B digestion efficiency. The results show that the tested connector-loads of the present invention all shear faster than the original connector-loads, at least equivalent to (compound 3-1). Table 1 Connector-load Digestive efficiency 15 minutes 2 hours 5 hours 16 hours Original connector-load 0 NA 17.99% 76.16% 1-2 7.05% 100% 100% NA 1-3 4.75% 77.21% 97.52% 100% 1-4 0 53.6% 83.92% 100% 1-7 30.02% 81% 100% NA 1-8 0 52.49% 81.97% 100% 3-1 0 NA 16.18% 61.92% 3-4 0 100% 100% NA

In Figure 9, panel (a) shows the shear sites and modifications of the linker moiety in compound 1-1 compared to the original linker-loading; panel (b) shows the percent shear, based on The peak area of the released drug detected by reversed-phase HPLC chromatography (mixed mode) was calculated. Consistent with the results in Table 1, the inventive linker-load sheared faster.

Example 4 :ADC Central connector- Shearing of the loaded part

method

The reaction conditions were the same as described in Example 3 for the cleavage of the linker-load compound itself. Specifically, reaction buffer (40 mM H ₃ PO ₄ /H ₃ BO ₃ /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 resulting final cysteine concentration in the 300 µL lysis system is 10 mM. Place the EP tube in a 37°C water bath.

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

result

The digestion percentages over time are summarized in Table 2, showing the comparison of cathepsin B digestion efficiencies for different linker-loaded moieties in ADC. Apparently, ADCs containing linker-loaded 1-2, 1-3, 3-1 and 3-4 released the drug faster in the presence of cathepsin B. It is worth noting that in another cleavage experiment, a mixed-mode column was used to quantify the drug released by shear, and the results showed that the ADC drug containing the linker-loaded part 1-1 released the drug slightly faster than the ADC containing the original linker-loaded part. . Table 2 ADC Digestive efficiency 10 minutes 5 hours 24 hours 48 hours Herceptin - original -LP 0.24% 8.42% 38.98% 46.52% Herceptin -1-1 0.41% 3.92% 27.71% 35.31% Herceptin -1-2 0.17% 24.26% 84.59% 87.56% Herceptin -1-3 0.00% 13.95% 64.99% 74.36% Herceptin -1-4 0.57% 8.42% 24.30% 29.02% Herceptin -1-7 0.00% 12.75% 58.02% 59.20% Herceptin -1-8 0.24% 8.95% 42.94% 50.65% Herceptin -3-1 1.41% 17.75% 71.66% 79.11% Herceptin -3-4 0.57% 23.99% 71.22% 82.05%

In Figure 10, panel (a) shows the shear bit points and modifications of the linker-load portion in the ADC compared to the original linker-load in the ADC, panel (b) shows the linker in the ADC -Percent shear (%) of load over time, calculated based on drug release peak area (ibid.). After 48 hours, the cleavage percentage for ADC-1-1 (i.e. ADC containing linker-loading 1-1) was 18.5%, and for ADC-original LP it was 15.5%.

Example 5 : Freeze-thaw stability

method

Take out the ADC sample (~10mg/mL, dissolved in 20mM histidine, 150mM NaCl, pH6.0) frozen at -80°C in the Eppendorf tube, and thaw at room temperature for 30 minutes. The ADC samples were then frozen at -80°C for 2 days and then thawed at room temperature for 30 minutes; the freeze/thaw process was repeated again. Then, 20 µl of each sample was taken for SEC and MS to determine DAR and drug distribution. Determination of various substances (L0, L1, L2, H0, H1, H2, H4, etc.) in each sample based on the peak area of the substance, where "L" refers to the light chain, "H" refers to the heavy chain, and the number refers to the drug molecules connected to the chain. number) percentage (molar ratio).

result

After two freeze-thaw (F/T) cycles, changes in unwanted impurity products 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 DAR The changes are summarized in Table 3. The results showed that after two rounds of freeze-thaw cycles, L2 in the ADC containing the linker-loaded product of the present invention was still zero (0), while in Her-Dxd (i.e., Dexi trastuzumab), L2 increased to 0.76%. H4% in Her-Dxd is also the highest. The ADC containing linker-loading 1-8 showed the smallest DAR decrease after two freeze-thaw cycles. Overall, the unwanted H4 content in the eight ADC analogs after freeze-thaw cycles was lower than Her-Dxd and all maintained stable DAR. table 3 ADC L2% H4% MS-DAR fresh Two-wheel F/T fresh Two-wheel F/T fresh Two-wheel F/T Her-Dxd NA 0.76% NA 7.28% 8 7.27 Her-1-1 NA 0.00% NA 7.58% 8 7.63 Her-1-2 0.00% 0.00% 1.90% 3.61% 7.49 7.65 Her-1-3 NA 0.00% NA 7.00% 7.96 7.15 Her-1-4 0.00% 0.00% 1.40% 3.28% 7.84 7.59 Her-1-7 NA 0.00% NA 2.38% 8 7.12 Her-1-8 0.00% 0.00% 1.61% 3.62% 7.78 7.75 Her-3-1 NA 0.00% NA 1.62% 7.98 7.45 Her-3-4 0.00% 0.00% 2.50% 2.33% 7.82 7.69 Note: "Her" = "Herceptin", which is also "Trastuzumab";"Fresh" refers to newly synthesized ADC that has not been subjected to freeze-thaw cycles.

Example 6 :ADC Medium H4 detection

method

Prepare ADC in 20 mM histidine buffer (containing 150mM NaCl, pH 6.0). The obtained ADC sample was subjected to LC-MS analysis to determine the distribution of the drug on the light chain and heavy chain. Ideally, only 5 substances, namely L0, H0, L1, H1 and H2, should be detected. Aggregation percentage was detected by HPLC.

result

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-loaded product after synthesis was 3%. The content of this impurity in Her-1-8 was significantly reduced, down to 0.8%. As shown in the results, ADC products produced using the linker-load of the present invention have reduced impurities. Table 4 ADC name C _ADC (mg/ml) MS-DAR H4% Aggregate ( % ) 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%

Example 7 : Uncoupled linker - Clearance of payload

After coupling as described in Example 2, the ADC was exchanged into 20mM histidine buffer (containing 150mM NaCl, pH 6.0) using a rotating desalting column (40 kDa). Table 5 shows a comparison of free linker-loading (i.e., uncoupled linker-loading) clearance between ADCs made with different linker-loadings. Use UFDF to remove free linker-loads. The residual free linker-load content in the Her-Dxd (i.e., Desi trastuzumab) coupling product is close to 5%, but in the products produced with the linker-load of the present invention, the content is less than 2%.

As shown in the results, the linker-loading agent of the present invention improves the operability of purification, such as easier and more complete removal of residual free linker-loading agents in ADC coupling products. Without being bound to any particular theory, it is believed that the introduction of polar groups on the linker moiety increases the water solubility of the linker-payload, facilitating its clearance, for example by UFDF. This has important implications for the ADC manufacturing process. table 5 Name C _ADC (mg/ml) MS-DAR Aggregate ( % ) Free LP (%mol/mol) Endotoxins (EU/mg) Her-Dxd 2.76 8.00 0.00 <5.0 <0.14 Her-1-1 2.69 8.00 3.87 <2.0 <0.15 Her-1-3 3.14 7.96 0.01 <2.0 <0.13 Her-3-1 2.80 7.98 0.00 <2.0 0.20 Her-1-7 3.00 8.00 0.01 <2.0 <0.13

Example 8 : Affinity detection

method

The in vitro binding ability of ADC to human HER2 was tested using FACS (fluorescence live cell flow cytometry) technology. On the day of detection, tumor cells expressing HER-2 (1×10 ⁵ cells/well) were co-cultured with serially diluted ADC at 4°C for 1-2 hours. Her-Dxd prepared as described in Example 2 was used as the reference ADC and positive control. The buffer used to dissolve ADC (20 mM histidine buffer, containing 150mM NaCl, pH 6.0) was used as a negative control. After culture, 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 ₅₀ values were calculated using GraphPad Prism.

result

The results are shown in Figure 11. According to N87 cell testing, the EC ₅₀ of the ADC tested in the present invention is about 1 nM, while Her-Dxd is 0.9 nM. According to JIMT-1 cell detection, the EC ₅₀ of the ADC tested in the present invention is 0.4~0.7 nM, while Her-Dxd is 0.4 nM. According to MDA-MB-231 cell detection, the EC ₅₀ of the ADC tested in the present invention (except Her3-1) is 0.5~0.7 nM, while Her-Dxd is 0.6 nM. The results show that the binding affinity of the ADC of the present invention having the linker of the present invention and the linker-loader of the present invention is at least equivalent to that of Desitrastuzumab, and may even be better.

Example 9 : Cytotoxicity assay

method

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 into the culture medium on a 96-well plate at an appropriate cell density. The next day, ADC serially diluted in culture medium was added to each well. Her-Dxd prepared as described in Example 2 was used as the reference ADC and positive control. The buffer used to dissolve ADC was used as a negative control. Culture plates were incubated in an incubator at 37°C and 5% _CO2 . After 4-6 days, cell viability was determined using CellTiter-Glo (Promega). _IC50 values were calculated using GraphPad Prism.

result

The results are shown in Figures 12A and 12B. Neither the ADCs tested in the present invention nor Her-Dxd were found to be cytotoxic to JIMT-1 cells and MDA-MB-231 cells (IC ₅₀ >1 nM). In N87 cells and HCC1954 cells, all ADCs tested in the present invention were The cytotoxicity is comparable to the original ADC, with an IC ₅₀ of approximately 0.1 nM. The results show that the cytotoxicity of the ADC of the invention having the linker of the invention and having the linker-loader of the invention is at least equivalent to that of Trastuzumab.

without

When the following detailed description is read in conjunction with the accompanying drawings, the various features of the present disclosure may not be drawn to scale. In fact, the dimensions of the various features may be arbitrarily increased or reduced for clarity of discussion. In order to make the above and other objects, features, advantages and embodiments of the present disclosure more obvious and understandable, the accompanying drawings are described as follows: Figure 1: One of the representative embodiments of the present invention, the synthesis scheme of linker-loading compounds (also marked as "Deruxtecan analog" in the drawing) 1-7. Figure 2: One of the representative embodiments of the present invention, the synthesis scheme of the linker-loading compound 3-1. Figure 3: One of the representative embodiments of the present invention, the synthesis scheme of the linker-loading compound 1-1. Figure 4: One of the representative embodiments of the present invention, the synthesis scheme of the linker-loading compound 1-2. Figure 5: One of the representative embodiments of the present invention, the synthesis scheme of linker-loading compounds 1-3. Figure 6: One of the representative embodiments of the present invention, the synthesis scheme of linker-loading compounds 1-4. Figure 7: One of the representative embodiments of the present invention, the synthesis scheme of linker-loading compounds 1-8. Figure 8: One of the representative embodiments of the present invention, the synthesis scheme of linker-loading compound 3-4. Figure 9: (a) Schematic diagram of modification sites (circled) and cleavage sites (dashed lines) of the linker-loaded compound of the present invention compared with the original linker-loaded compound; (b) Linker of the present invention - Comparison of load compound shear percentage (%) over time with original linker-load compound. Figure 10: a) Schematic diagram of modification sites (circled) and cleavage sites (dashed lines) of ADC containing the linker-payload part of the present invention compared with ADC containing the original linker-loading part; (b) Comparison of shear percentage (%) over time of the connector-loaded part of the invention in ADC and the original connector-loaded part in ADC. Figure 11: Binding affinity of ADCs containing different linker-payload moieties to Her2 antigen on different cell lines. Figures 12A and 12B: Cytotoxicity of ADCs containing different linker-payload moieties on different cell lines.

Claims (18)

A compound of formula I: I its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; where "*" represents the chiral center, which is S- or R- or racemic; and the hydrogen atom attached to the chiral carbon atom and the hydrogen atom attached to the carbon atom having the _R substituent are omitted from the formula; L ₁ is -(CH ₂ ) _a -, where a is an integer from 0 to 10, or -(CH ₂ CH ₂ O) _b -, where b is an integer from 1 to 36; L ₂ is -(CH ₂ ) _c -, where c is an integer from 1 to 10, or -( CH ₂ CH ₂ O) _d -, where d is an integer from 1 to 36; L ₃ is absent, or -(CH ₂ ) _e -, where e is an integer from 1 to 10, or -(CH ₂ CH ₂ O ) _f -, where f is an integer from 1 to 36; R ₁ is -CF ₃ , -NR _a R _b , -NR _a (C=O)R _b or -O(CH ₂ ) _g CH ₃ , where g is An integer from 0 to 3, R _a is H or -C _1-6 alkyl, R _b is H or -C _1-6 alkyl; R ₂ is -H, -C _1-6 alkyl or -O(CH ₂ ) _h CH ₃ , where h is an integer from 0 to 3; X is halogen, -OR ₃ or -NR ₄ R ₅ ; R ₃ is -H, -C _1-6 alkyl or halogen; R ₄ and R ₅ is independently -H or -C _1-6 alkyl; n=0 or 1; and m=0 or 1. The compound of claim 1, wherein L ₁ is -(CH ₂ ) _a -, where a is an integer from 2 to 6; L ₂ is -(CH ₂ ) _c -, where c is an integer from 1 to 6 ; L ₃ is absent, or -(CH ₂ ) _e -, where e is an integer from 1 to 6, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 20; R ₁ is - CF ₃ , -N(CH ₃ ) ₂ , -NH(C=O)CH ₃ or -O(CH ₂ ) ₂ CH ₃ ; R ₂ is -H or -C _1-6 alkyl; and/or R ₃ It is -H, -CH ₃ , tert-butyl or Cl. The compound of claim 1, wherein L ₁ is -(CH ₂ ) _a -, a is an integer from 4 to 5; L ₂ is -(CH ₂ ) _c -, wherein c is an integer from 1 to 2; L ₃ is not present, or -(CH ₂ ) _e -, where e is an integer from 1 to 2, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 2; R ₁ is -CF ₃ , -N(CH ₃ ) ₂ or -NH(C=O)CH ₃ ; R ₂ is -H or -CH ₃ ; and/or R ₃ is -H, -CH ₃ , tert-butyl or Cl. The compound according to claim 1, wherein the compound is selected from the group shown below: 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 their pharmaceutically acceptable salts or esters: L-1-1 L-1-2 L-1-3 L-1-4 L-1-7 L-1-8 L-3-1 L-3-4 where "*" represents the chiral center, which is racemic. A coupling compound of formula II: II its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; where “*” represents the chiral center, which is S- or R- or racemic; and the hydrogen atom attached to the chiral carbon atom and the hydrogen atom attached to the carbon atom having the _R substituent are omitted from the formula; L ₁ is -(CH ₂ ) _a -, where a is an integer from 0 to 10, or -(CH ₂ CH ₂ O) _b -, where b is an integer from 1 to 36; L ₂ is -(CH ₂ ) _c -, where c is an integer from 1 to 10, or -( CH ₂ CH ₂ O) _d -, where d is an integer from 1 to 36; L ₃ is absent, or -(CH ₂ ) _e -, where e is an integer from 1 to 10, or -(CH ₂ CH ₂ O ) _f -, where f is an integer from 1 to 36; R ₁ is -CF ₃ , -NR _a R _b , -NR _a (C=O)R _b or -O(CH ₂ ) _g CH ₃ , where g is An integer from 0 to 3, R _a is H or -C _1-6 alkyl, R _b is H or -C _1-6 alkyl; R ₂ is -H, -C _1-6 alkyl or -O(CH ₂ ) _{hCH3 ,} where h is an integer from 0 to 3; n=0 or 1; m=0 or 1; and "DRUG" is the drug moiety covalently coupled to the linker moiety. The coupling compound as claimed in claim 5, wherein L ₁ is -(CH ₂ ) _a -, wherein a is an integer from 2 to 6; L ₂ is -(CH ₂ ) _c -, wherein c is an integer from 1 to 6 integer; L ₃ is not present, or -(CH ₂ ) _e -, where e is an integer from 1 to 6, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 20; R ₁ is -CF ₃ , -N(CH ₃ ) ₂ , -NH(C=O)CH ₃ or -O(CH ₂ ) ₂ CH ₃ ; and/or R ₂ is -H or -C _1-6 alkyl. The coupling compound as claimed in claim 5, wherein L ₁ is -(CH ₂ ) _a -, wherein a is an integer from 4 to 5; L ₂ is -(CH ₂ ) _c -, wherein c is an integer from 1 to 2 integer; L ₃ is not present, or -(CH ₂ ) _e -, where e is an integer from 1 to 2, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 2; R ₁ is -CF ₃ , -N(CH ₃ ) ₂ or -NH(C=O)CH ₃ ; and/or R ₂ is -H or -CH ₃ . The coupling compound as described in claim 5, which has the structure of formula IIa: IIa its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; among them, "*", L ₁ , L ₂ , L ₃ , R ₁ , R ₂ , n and m are as defined in claim 5. The coupling compound as described in claim 5, which has a structure selected from the following group: Compound 1-1 Compound 1-2 Compound 1-3 Compound 1-4 Compound 1-7 Compound 1-8 Compound 3-1, and . Compound 3-4 where "*" represents the chiral center, which is racemic. An antibody-drug conjugate of formula III: III Its enantiomers, diastereomers, racemates, solvates, hydrates or pharmaceutically acceptable salts or esters; where “*” represents the chiral center, which is S- or R- or racemic and the hydrogen atom attached to the chiral carbon atom and the hydrogen atom attached to the carbon atom having the _R substituent are omitted from the formula; L ₁ is -(CH ₂ ) _a -, where a is 0 to 10, or -(CH ₂ CH ₂ O) _b -, where b is an integer from 1 to 36; L ₂ is -(CH ₂ ) _c -, where c is an integer from 1 to 10, or -(CH ₂ CH ₂ O) _d -, where d is an integer from 1 to 36; L ₃ is not present, or -(CH ₂ ) _e -, where e is an integer from 1 to 10, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 36; R ₁ is -CF ₃ , -NR _a R _b , -NR _a (C=O)R _b or -O(CH ₂ ) _g CH ₃ , where g is 0 to an integer of 3, R _a is H or -C _1-6 alkyl, R _b is H or -C _1-6 alkyl; R ₂ is -H, -C _1-6 alkyl or -O(CH ₂ ) _h CH ₃ , where h is an integer from 0 to 3; n = 0 or 1; m = 0 or 1; “DRUG” is the drug moiety covalently coupled to the linker moiety; p is 1 to 8, and Ab It's an antibody. The antibody-drug conjugate of claim 10, wherein L ₁ is -(CH ₂ ) _a -, where a is an integer from 2 to 6; L ₂ is -(CH ₂ ) _c -, where c is 1 an integer from 1 to 6; L ₃ is absent, or -(CH ₂ ) _e -, where e is an integer from 1 to 6, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 20; R ₁ is -CF ₃ , -N(CH ₃ ) ₂ , -NH(C=O)CH ₃ or -O(CH ₂ ) ₂ CH ₃ ; R ₂ is -H or -C _1-6 alkyl; and /or p is 2, 4 or 6. The antibody-drug conjugate of claim 10, wherein L ₁ is -(CH ₂ ) _a -, where a is an integer from 4 to 5; L ₂ is -(CH ₂ ) _c -, where c is 1 to an integer from 2; L ₃ does not exist, or -(CH ₂ ) _e -, where e is an integer from 1 to 2, or -(CH ₂ CH ₂ O) _f -, where f is an integer from 1 to 2; R ₁ is -CF ₃ , -N(CH ₃ ) ₂ or -NH(C=O)CH ₃ ; R ₂ is -H or -CH ₃ ; "DRUG" is ixotecan; p is 4; and/ Or Ab is trastuzumab. The antibody-drug conjugate of claim 10, which has a structure selected from the following group: Conjugate 1-1 Conjugate 1-2 Conjugate 1-3 Conjugates 1-4 Conjugates 1-7 Conjugates 1-8 Conjugate 3-1, and . Conjugate 3-4 where "*" represents the chiral center, which is racemic. The antibody-drug conjugate of claim 13, wherein p is 2, 4 or 6; and/or Ab is trastuzumab. A method of producing a linker-loaded compound, which comprises coupling a drug to the compound described in claim 1. The method of claim 15, wherein the drug is ixotecan. A method of producing an antibody-drug-conjugate, comprising: (a) coupling a drug to a compound described in claim 1 to obtain a linker-loading compound; and (b) Conjugate the antibody to the linker-loading compound obtained in step (a). The method of claim 17, wherein the drug is ixotecan.