Coupling connector

Abstract

This invention provides a coupling connector, as well as a connector-load coupling and an ADC comprising said connector.

Description

Technical Field

[0001] This invention relates to the field of drug conjugates, and more specifically to antibody-drug conjugates. Background Technology

[0002] Antibody-drug conjugates (ADCs) are large molecule drugs that utilize the specific binding of antibodies to antigens (located on the surface of cancer cells) to deliver cytotoxic substances (drugs) to the cells and kill them. They can be considered as large molecule prodrugs that are both site-directed and site-specific releases. ADCs are actually a three-component system, consisting of a potent drug linked to an antibody (usually a monoclonal antibody mAb) via a degradable or non-degradable (cleavable / non-cleavable) linker.

[0003] Typically, ADCs are internalized after binding to antigens on cells, and then the drug is released from the antibody within the cell to exert its effect. Compared to small molecules, the number of ADC molecules reaching target cells is relatively small, and the internalization rate is also low. Therefore, it is desirable to increase the rate at which the linker is cleaved to release the drug.

[0004] Linkers are crucial for ADCs, significantly impacting their stability and drug release mechanisms. Linkers can be cleaved by enzymes, such as Trastuzumab deruxtecan. The linker in an ADC typically consists of a short peptide (e.g., 2-4 amino acids). This peptide, after being internalized by the ADC, is cleaved in the lysosome, releasing the drug to exert its cytotoxic effect. In the already slow mechanism of action of ADCs, the rate of enzymatic cleavage (by one or more enzymes) of the linker determines the drug's onset of action. Currently, most ADCs use cleavable linkers because they can rapidly release the drug into the target cells. For example, brentuximab vedotin... trastuzumab deruxtecan loncastuximab tesirine-lpyl The peptide cleavage fragments used are VC, GGFG, and VA. These peptides are primarily cleaved by cathepsins (mainly cathepsin B) in lysosomes. The cleavage rate varies due to various factors, including the characteristics of the relevant enzymes, region-specific enzyme-substrate interactions, etc. For example, The linker contains the tetrapeptide GGFG, with the amide bond of the last glycine residue being a cleavable bond, which is cleaved by lysosomal proteases (such as cathepsins). The cleavage rate of GGFG is slower than that of VC.

[0005] One of the ideal characteristics of an ADC is that the linker can be rapidly sheared in a suitable location, such as a lysosome. We need better linkers and linker-loads to produce ADCs with superior performance and to manufacture them using more operable and higher-yield processes. Summary of the Invention

[0006] The inventors have designed a series of linkers suitable for producing linker-loads and ADCs with superior performance, such as higher shearability (e.g., manifested as higher shear rates), improved DAR distribution, increased homogeneity, stability, and / or enhanced therapeutic efficacy. Based on these linkers and linker-loads containing the linkers, ADCs can be manufactured using more operable and higher-yield processes, for example, resulting in purer ADC products with lower levels of residual unbound linker-loads and / or easier removal of residual linker-loads.

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

[0008]

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

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

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

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

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

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

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

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

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

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

[0019] n = 0 or 1; and

[0020] m = 0 or 1.

[0021] In another respect, the present invention provides coupling compounds of formula II:

[0022]

[0023]

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

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

[0026]

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

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

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

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

[0031] The chosen site and the medication are exatecan; and

[0032] (b) The antibody is coupled with the linker-loaded compound obtained in step (a). Attached Figure Description

[0033] Figure 1 One of the representative embodiments of the present invention is the synthesis scheme of linker-loaded compounds (also labeled as "Deruxtecan analog" in the figures) 1-7.

[0034] Figure 2 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compound 3-1.

[0035] Figure 3 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compound 1-1.

[0036] Figure 4 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compounds 1-2.

[0037] Figure 5 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compounds 1-3.

[0038] Figure 6 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compounds 1-4.

[0039] Figure 7 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compounds 1-8.

[0040] Figure 8 One of the representative embodiments of the present invention is the synthesis scheme of linker-loader compounds 3-4.

[0041] Figure 9 (a) A schematic diagram of the 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) A comparison of the cleavage percentage (%) of the linker-loaded compound of the present invention with that of the original linker-loaded compound.

[0042] Figure 10(a) A schematic diagram of the modified sites (circled) and shearing sites (dashed lines) of the ADC containing the connector-load portion of the present invention compared with the ADC containing the original connector-load portion; (b) A comparison of the percentage (%) of time-transferred shearing of the connector-load portion of the present invention in the ADC with that of the original connector-load portion in the ADC.

[0043] Figure 11 The affinity of ADCs containing different linker-loaded parts for binding to Her2 antigen on different cell lines.

[0044] Figure 12 Cytotoxicity of ADCs containing different linker-loaded portions to different cell lines. Detailed Implementation

[0045] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments are merely illustrative while showing preferred embodiments of the invention, as those skilled in the art will appreciate various changes and modifications within the concept and scope of the invention from these detailed descriptions.

[0046] Terms and Definitions

[0047] In this document, the singular forms introduced by “a,” “an,” and “the,” etc., include the plural meaning, unless otherwise stated. Furthermore, the terms “a,” “a or more,” and “at least one” are used interchangeably herein.

[0048] In this document, unless otherwise stated, whether the numerical value or range is preceded by "approximately", it encompasses a reasonable approximation range that would be understood by one of those skilled in the art, such as ±10%, ±5%, ±3%, ±2%, ±1%, or ±0.5% of the specified value.

[0049] In this document, the term "substantially absent" refers not only to the absence of a substance (i.e., "nothing," "zero," etc.) but also to its insignificant presence or its presence or quantity below the detection limit and therefore undetectable. This is well known to those skilled in the art.

[0050] One or more features in one embodiment of this invention may be combined with one or more features in another embodiment without departing from the concept and idea of ​​the invention.

[0051] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publicly available and patent documents cited herein are incorporated by reference and are suitable for various purposes. The cited documents are to be regarded as indicating the skill level of someone skilled in the art, but this should not be construed as an admission that they preceded this invention, which was invented prior to it.

[0052] Overview

[0053] The cleavage rate of peptide linkers varies due to various factors, including the characteristics of the associated enzyme, region-specific enzyme-substrate interactions, and so on. We found that altering the structure flanking the tetrapeptide GGFG changes the enzyme binding of the linker, manifested as an increase in the kcat / Km value of cathepsin B. Based on this, we designed a series of linkers. Using these linkers, we were able to obtain linker-loaded compounds and ADCs with superior performance. As shown in the examples below, the linker-loaded compounds of this invention, incorporating the linker moiety designed in this invention, exhibited a higher cathepsin B cleavage rate compared to reference compounds containing similar existing linker moieties, whether used as standalone compounds or as components of ADCs. We found that the linker designed in this invention is more easily cleaved, which helps to improve the potency of the ADC. We also found that ADCs containing the linker-loaded moiety of this invention exhibit higher stability (e.g., storage stability, such as freeze-thaw stability) and / or comparable or even better therapeutic efficacy (e.g., binding affinity and / or cytotoxicity) compared to similar existing ADCs.

[0054] Furthermore, we have found that fabricating ADCs using the linker-loaded compounds of this invention, which include the linker portion designed in this invention, yields purer products, for example, compared to high drug-to-antibody ratios (DARs). compared to. The DAR is 7.5. A higher DAR requires more linker-loads for the coupling reaction. Therefore, removing residual linker-loads becomes very difficult. In this invention, for example, compared to delutecan in Enhertu, the linker-loads of this invention are easier and more thorough to remove, thus resulting in a purer ADC product. This may be because the linker-loads of this invention have higher hydrophilicity. Meanwhile, when using a large excess of linker-loads to achieve a high DAR during coupling, non-specific binding of the linker-loads to the antibody heavy chain occurs, resulting in a 4-drug coupled heavy chain, but ideally the number should not exceed 3. In this invention, the percentage of the 4-drug coupled heavy chain can be reduced to... One-third of the process. Therefore, the connector and connector-load of the present invention can provide ADC products with higher homogeneity.

[0055] We also found that ADCs prepared using the linker-loader of this invention are easier to purify. In some cases, with, for example... Compared to products produced using the linker-loaded method, UFDF (ultrafiltration & depth filtration) purification significantly reduces the amount of residual free linker-loads in the coupled ADC. Therefore, the linker and linker-loaded method of this invention can produce ADCs with higher purity and enable the production of ADCs using a more operable and high-capacity process.

[0056] Detailed implementation examples

[0057] 1. Connector

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

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

[0060]

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

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

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

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

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

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

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

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

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

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

[0071] n = 0 or 1; and

[0072] m = 0 or 1;

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

[0074]

[0075]

[0076] The asterisk (*) indicates a chiral center, which is racemic.

[0077] The designed linkers provide unique characteristics and chemical properties. For example, in L-1-1, L-1-2, L-1-3, L-1-4, L-1-7, and L-1-8, the introduction of a CF3 group at the α-position of the amine residue creates a strong dipole moiety on the N-side of the GGFG peptide. This group likely reduces the Km of the interaction between cathepsin B and GGFG, thereby accelerating the enzymatic catalytic rate of peptide hydrolysis. Clearly, the introduction of tetraamine or acetamide at similar positions in linkers (e.g., L-3-1 and L-3-4) also leads to an increased catalytic rate.

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

[0079] 2. Connector-Load

[0080] 2.1. The connector-load coupling of the present invention

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

[0082] In this disclosure, the term "linker-loader compound" refers to a conjugate compound formed by covalently linking a linker portion to a drug portion, wherein the drug portion is also referred to as the "loader". This linker-loader compound can be further conjugated to an antibody to provide an ADC comprising the linker-loader portion of the present invention.

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

[0084]

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

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

[0087]

[0088]

[0089] The asterisk (*) indicates a chiral center, which is racemic.

[0090] There are no particular limitations on the pharmaceutical products that can be used in this invention, as long as they have or can be modified to have functional groups coupled to the linker compound at the opposite end to the maleimide moiety. In some embodiments, the functional group used for coupling may be -NHR, where R is an alkyl group or H.

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

[0092] Drugs that can be used in this invention include cytotoxic drugs, particularly those used for cancer treatment. Such drugs include, but are not limited to, DNA damaging agents, DNA binding agents, nucleic acid synthesis inhibitors, transcription inhibitors, antimetabolites, enzyme inhibitors such as thymidylate synthase inhibitors and topoisomerase inhibitors, tubulin inhibitors, and toxins such as bacterial, fungal, plant, or animal-derived toxins. Specific examples include, for instance, paclitaxel, methotrexate, methopterin, dichloromethhotrezine, 5-fluorouracil, 6-mercaptopurine, arabinoside cytosine, melphalan, leurosine, leurosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vinblastine, taxanes including paclitaxel, taxotere retinoic acid, butyric acid, N8-acetylspermidine. Spermidine, camptothecin, chalcogenide, esperacin, enediyne derivatives, duocarmycin A, duocarmycin SA, chalcogenide, camptothecin, hemiasterlins, maytansinoids (including DM1, DM2, DM3, DM4), olprestatins (including monomethylolprestatin E (MMAE), monomethylolprestatin F (MMAF), and monomethylolprestatin D (MMAD)), camptothecin, irinotecan, topotecan, exatecan, etoposide, and their derivatives. In some embodiments, the drug is a topoisomerase inhibitor, such as camptothecin, irinotecan, topotecan, exatecan, etoposide, and their derivatives, such as hodroxycamptothecin and Dxd. In some embodiments, the drug is Dxd. In some other embodiments, the drug is eczemab. In some embodiments, the drug is Dxd when released from the linker-load or ADC. In some other embodiments, the drug is eczemab when coupled to the linker to form the drug portion.

[0093] Accordingly, the linker-load coupling can be a compound having the structure of formula IIa, its enantiomer, diastereomer, racemate, solvate, hydrate, or pharmaceutically acceptable salt or ester:

[0094]

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

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

[0097]

[0098]

[0099]

[0100] The asterisk (*) indicates a chiral center, which is racemic.

[0101] 2.2. Synthesis of Connector-Load Couplings

[0102] As one aspect of the invention, this document provides a method for producing a linker-load coupling compound, comprising coupling a drug substance to a linker compound of the invention. In some embodiments, the drug substance is eczema.

[0103] The coupling of a drug with a linker compound can be carried out by coupling reactions known in the art (such as esterification or amidation) or transesterification reactions, 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.

[0104] 3. Antibody-drug conjugates

[0105] 3.1. The antibody-drug conjugate of the present invention

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

[0107]

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

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

[0110]

[0111]

[0112]

[0113] The asterisk (*) indicates a chiral center, which is racemic.

[0114] 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.

[0115] There are essentially no limitations on the antibodies that can be used in this invention. Antibodies of various specificities, conformations, and origins can be used. In some embodiments, the antibody specifically binds to tumor antigens (TAs), such as tumor-specific antigens (TSA) and tumor-associated antigens (TAA). Examples of tumor antigens include, but are not limited to: CD20, CD38, CD123, ROR1, ROR2, BCMA, PSMA, SSTR2, SSTR5, CD19, FLT3, CD33, PSCA, ADAM 17, CEA, Her2, EGFR, EGFR-vIII, CD30, FORR1, GD-2, CA-IX, Trop2, CD70, CD38, mesothelin, EphA2, CD22, CD79b, GPNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON, and ERBB2. Examples of TA-specific antibodies include, but are not limited to: trastuzumab, rituximab, cetuximab, bevacizumab, panitumumab, alemtuzumab, matuzumab, gemtuzumab, polotuzumab, and inotuzumab. In some embodiments, the antibody (Ab) is trastuzumab.

[0116] In the context of the ADC of this 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, such as tumor antigens), and other surface molecules on receptors or target cells (e.g., disease-associated cells, such as cancer cells or tumor cells), provided that these equivalent molecules possess, or can be modified to possess, a functional group capable of reacting with and covalently binding to the maleamide group of a linker. In some embodiments, the functional group is a thiol group, such as those released by the reduction of interchain disulfide bonds, thereby allowing the antibody to be coupled to the linker moiety via a thio-maleamide linker bond.

[0117] 3.2. Preparation of antibody-drug conjugates

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

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

[0120] (a) Coupling a drug with the linker compound of the present invention to obtain a linker-loaded compound; preferably, the drug...

[0121] The substance is ipsilate; and

[0122] (b) The antibody is coupled with the linker-loaded compound obtained in step (a).

[0123] Step (a) can be carried out by coupling reactions (such as esterification or amidation) or transesterification reactions known in the art, 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.

[0124] Step (b) can be performed by a Michael addition reaction, in which the maleimide moiety reacts with a free thiol group in the antibody. For example, one or more free thiol groups may be derived from one or more cysteine ​​residues, such as those released by the reduction of interchain disulfide bonds, allowing the antibody to be coupled to the linker moiety via a thio-maleamide linker bond.

[0125] 4. Application

[0126] The antibody-drug conjugates (ADCs) of this invention can be formulated into pharmaceutical compositions using pharmaceutically acceptable formulations. In some embodiments, the composition may contain a therapeutically effective amount of the antibody-drug conjugate. In some embodiments, the composition may contain an effective amount of the antibody-drug conjugate to achieve the desired dosage.

[0127] The antibody-drug conjugates of the present invention can be used to treat diseases, disorders, or conditions in subjects in need, the treatment comprising administering a therapeutically effective amount of the antibody-drug conjugate to the subject. This application also provides antibody-drug conjugates of the present invention for treating diseases, disorders, or conditions in subjects in need. Diseases treated include, but are not limited to, cancers, including solid tumors and hematologic malignancies. Examples of said cancers include, but are not limited to, breast cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer (e.g., NSCLC), head and neck cancer, colorectal cancer, B-cell lymphoma (e.g., non-Hodgkin's lymphoma (NHL)), and leukemia.

[0128] In this document, the term "object" refers to a human or non-human animal object. Non-human animals can be mammals, such as primates. Examples of non-human mammal objects include, but are not limited to, domesticated animals, livestock and zoo animals, sports animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, and bears. Preferably, the object is a human being. "Object in need" refers to an object requiring diagnosis, prognosis, mitigation, prevention, and / or treatment for a disease, disorder, or condition.

[0129] Example

[0130] The following examples are for illustrative purposes only and do not constitute a limitation on the scope of the present invention.

[0131] Example 1: Synthesis of Connector-Load (a) Compounds 1-7 (in) Figure 1 Synthesis of (also known as derutin analogues 1-7)

[0132] The synthesis process is schematically described in Figure 1 middle.

[0133] Step 1: 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)methyl acetate (a-1)

[0134] 2-((tert-butyldimethylsilyl)oxy)ethanol (6.51 g, 42.5 mmol) was added to anhydrous tert-butanol (50.0 mL). Potassium tert-butoxide (3.18 g, 28.3 mmol) was added with stirring, and the mixture was stirred at 0 °C for 1 hour. Then, a solution of methyl 2-bromoacetate (5.00 g, 28.3 mmol) in tert-butanol (50.00 mL) was added dropwise. The resulting solution was heated to 0–25 °C and stirred for 2 hours. TLC (petroleum ether / ethyl acetate = 10:1, R0) was performed.f =0.2) indicates the reaction is 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 the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 50 / 1 to 2 / 1) to give a colorless oil of methyl 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetate (3.00 g, 42.5% yield). 1 H NMR (400MHz, CDCl3): δppm 4.19 (s, 2H), 3.79-3.84 (m, 2H), 3.76 (s, 2H), 3.62-3.67 (m, 2H), 0.90 (s, 9H), 0.07 (s, 6H).

[0135] Step 2: 2-(2-hydroxyethoxy)benzyl acetate (a-2)

[0136] Lithium hydroxide monohydrate (168 mg, 4.03 mmol) was added to a mixture of methyl 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)acetate, α-1 (800 mg, 4.03 mmol) in tetrahydrofuran (10.0 mL) and water (10.0 mL). The mixture was stirred at 25 °C for 2 hours. TLC (petroleum ether / ethyl acetate = 5:1, R f =0.2) indicates the reaction is complete. The reaction mixture was concentrated under reduced pressure to remove water and tetrahydrofuran, giving a residue. The residue was dissolved in N,N-dimethylformamide (5.00 mL), potassium carbonate (556 mg, 4.03 mmol) and benzyl bromide (1.38 g, 8.05 mmol) were added, and the mixture was stirred at 25 °C for 16 hours. TLC (petroleum ether / ethyl acetate = 10:1, R f =0.5) indicates that the intermediate was completely consumed, and TLC showed disordered reaction. The reaction mixture was concentrated under reduced pressure to remove N,N-dimethylformamide and purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 50 / 1) to give an intermediate (1.30 g) as a pale yellow liquid. The intermediate was deTBSedated in 1M HCl (5.00 mL). TLC (petroleum ether / ethyl acetate = 2:1, R f =0.5) indicates that the intermediate was completely consumed, but TLC showed a disordered reaction. The reaction mixture was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 50 / 1 to 2 / 1) to give a pale yellow liquid of benzyl 2-(2-hydroxyethoxy)acetate (600 mg, yield 88.6%). 1H NMR (400MHz, CDCl3): δppm 7.28-7.41 (m, 5H), 5.20 (s, 2H), 4.22 (s, 2H), 3.81 (t, J = 5.1Hz, 3H), 3.64-3.68 (m, 2H), 0.90 (s, 9H), 0.07 (s, 6H).

[0137] Step 3: 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester (a-4)

[0138] To a mixture of 2-(2-hydroxyethoxy)benzyl acetate a-2 (400 mg, 1.09 mmol) and methyl acetate (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamoxy)methyl ester a-3 (342 mg, 1.63 mmol) in tetrahydrofuran (4.00 mL), 4-methylbenzenesulfonic acid monohydrate (10.4 mg, 54.2 μmol) was added, and the mixture was stirred at 25 °C for 1 hour. TLC (petroleum ether / ethyl acetate = 1:1, R f =0.2) indicates the reaction is 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 give the residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel) by elution with (petroleum ether / ethyl acetate = 50 / 1 to 1 / 1) to give a white solid of 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester (0.40 g, yield 71.0%). 1 H NMR (400MHz, CDCl3): δppm 7.77(d,J=7.5Hz,2H),7.61(d,J=7.5Hz,2H),7.30-7.44(m,9H),5.13-5.22(m,2H),4.81(d,J=6.8Hz,2H), 4.44(d,J=6.8Hz,2H), 4.15-4.18(m,2H), 3.92(d,J=5.5Hz,2H), 3.73-3.76(m,2H), 3.68(d,J=2.5Hz,2H).

[0139] Step 4: 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid (a-5)

[0140] 0.40 g (771 μmol) of benzyl 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid ester a-4 (ethanol) in 20.0 mL of ethanol and 20.0 mL of ethyl acetate was added to a mixture of dried Pd / C (0.80 g) and stirred at 25 °C for 3 hours under a hydrogen atmosphere (15 psi). TLC (DCM / MeOH = 10:1, R f =0.3) indicates the reaction is complete. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a colorless oily 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid (0.25 g, yield 75.6%). The crude product was used directly in the next step without purification. 1 H NMR (400MHz, CDCl3): δppm 8.64-8.74(m,1H),7.83-7.95(m,2H),7.73(d,J=7.4Hz,1H),7.55-7.62(m,1H),7.26-7.49 (m,4H),4.56(d,J=6.5Hz,2H),4.26-4.35(m,2H),3.64(d,J=4.4Hz,2H),3.49-3.60(m,4H).

[0141] Step 5: (a-6, i.e., L-1-7)

[0142] To a mixture containing CTC-resin (0.50 g, 14.1 mmol) and 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid, a-5 (0.20 g, 520 μmol), N,N-diisopropylethylamine (0.25 g, 1.95 mmol) and dichloromethane (10.0 mL) were added to induce swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. The resin was then washed three times with N,N-dimethylformamide (10.0 mL). The resin was then treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed five times with N,N-dimethylformamide. Then, Fmoc-Phe-OH (0.58 g, 1.31 mmol) was added and mixed for 30 seconds, followed by the addition of a solution of O-benzotriazole-N,N,N-tetramethyluronium hexafluorophosphate (HBTU) (0.54 g, 1.43 mmol) and N,N-diisopropylethylamine (0.25 g, 1.95 mmol) in N,N-dimethylformamide, and the mixture was bubbled under nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. The above steps were repeated at 25 °C for the next coupling step: amino acid Fmoc-Gly-OH (0.53 g, 1.50 mmol) and 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol). The reaction was detected by ninhydrin assay. The coupling reaction was monitored by a ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and drying under vacuum, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to a flask containing the peptide resin, and the mixture was stirred twice for 2 minutes each time. The HFIP mixture was removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H₂O / CH₃CN eluent, C-18 column chromatography). The obtained product was then lyophilized to give a white solid compound a-6 (100 mg, crude, 90.0% purity). LCMS (ESI, m / z): 713.68 [M+H] + HPLC (Gemini-NX C18 5um 110A 150*4.6mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) was performed using a 715 column (Xbrige C18, 3.5um, 2.1*30mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) and eluted with a 0.1% TFA solution in water and 0.1% TFA solution in acetonitrile (10-80-20 minutes).

[0143] Step 6: (Compounds 1-7)

[0144] Compound a-6 (59.7 mg, 79.2 μmol) and 1-hydroxybenzotriazole (HOBt) (2.80 mg, 20.6 μmol) were dissolved in N,N-dimethylformamide (1.00 mL). Ecinotecan mesylate (10.0 mg, 18.8 μmol) and N,N-diisopropylcarbodiimide (DIC) (9.50 mg, 75.24 μmol) were added to the mixture, and the mixture was stirred at 40 °C for 2 hours. TLC (DCM / MeOH = 10 / 1, Rf = 0.4) showed that the reaction was complete. Trace amounts of N,N-dimethylformamide were removed under vacuum, yielding the residue. The residue was purified by rapid chromatography (10-50% water / acetonitrile eluent, C-18 column chromatography). The resulting product was then lyophilized to give compounds 1-7 (8.00 mg, yield 37.4%, purity 95.7%) as a pale yellow solid. LCMS(ESI,m / z):1131.1[M+H] + 1132.6 (Gemini-NXC18, 3.5µm, 2.1*30mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80 ± 2 min). HPLC (Gemini-NXC18 5µm 110A 150*4.6mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (35-65 ± 20 ± 3 min).

[0145] (b) Compound 3-1 (in) Figure 2 Synthesis of (also known as derutin analogue 3-1)

[0146] The synthesis process is schematically described in Figure 2 middle.

[0147] Step 1: 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester (b-3)

[0148] To a mixture of methyl acetate (500 mg, 1.36 mmol) and benzyl 3-hydroxypropionate (366 mg, 2.04 mmol) in tetrahydrofuran (5.00 mL), 4-methylbenzenesulfonic acid monohydrate (12.9 mg, 67.8 μmol) was added, and the mixture was stirred at 25 °C for 3 h. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The mixture was diluted with saturated sodium bicarbonate (50.0 mL) and extracted with ethyl acetate (50.0 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (100-200 mesh silica gel), eluted with petroleum ether / ethyl acetate = 50 / 1 to 1 / 1, to give a white solid of 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester, b-3 (400 mg, yield 60.3%). 1 H NMR (400MHz, CDCl3): δppm 7.90(d,J=7.4Hz,2H),7.73(d,J=7.0Hz,2H),7.24-7.64(m,9H),5.05-5.14(m,2H),4.48-4.62(m,4H),4.16-4.34(m,3H),3.54-3.72(m,4H).

[0149] Step 2: 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid (a-3)

[0150] To a mixture of 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester (400 mg, 818 μmol) in ethanol (10.0 mL) and ethyl acetate (10.0 mL), dry Pd / C (0.05 g) was added, and the reaction mixture was then stirred at 25 °C under a hydrogen atmosphere (15 psi) for 5 hours. TLC (DCM / MeOH = 10:1, R f =0.2) indicates that the raw material was consumed and a new point was formed. The reaction mixture was filtered through diatomaceous earth and the filtrate was concentrated under reduced pressure to give 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, a-3 (300 mg, yield 91.9%) as a white solid. 1H NMR (400MHz, CDCl3): δppm 7.90 (d, J = 7.4Hz, 2H), 7.73 (d, J = 7.0Hz, 2H), 7.24-7.64 (m, 4H), 5.05-5.14 (m, 2H), 4.48-4.62 (m, 2H), 4.16-4.34 (m, 3H), 3.54-3.72 (m, 4H).

[0151] Step 3: (S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanoic acid (b-6)

[0152] A solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (5.00 g, 10.67 mmol) in N,N-dimethylformamide (10.0 mL) and triethylamine (2.50 mL) was stirred at 25 °C for 16 hours. TLC (DCM:MeOH = 10:1, R f =0.2) indicates the reaction is complete. The reaction mixture was added dropwise to a solution of isopropyl ether (100 mL) under stirring. The resulting solid was filtered and washed with isopropyl ether (50 mL). Trace amounts of isopropyl ether were removed under vacuum 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 ready for use in the next step without further purification. 1 H NMR (400MHz, CDCl3): δppm3.67 (t, J=6.1Hz, 1H), 3.03 (t, J=6.7Hz, 2H), 1.73-1.90 (m, 2H), 1.23-1.56 (m, 12H).

[0153] Step 4: (S)-6-((tert-Butoxycarbonyl)amino)-2-(dimethylamino)hexanoic acid (b-7)

[0154] Formaldehyde (1.32 g, 16.2 mmol, 37% purity) was added dropwise 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. The mixture was stirred at 25 °C for 30 min, and then sodium borohydride (614 mg, 16.2 mmol) was added dropwise over a 30-minute period in an ice bath. The mixture was then kept at 25 °C for another 30 min. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. Trace amounts of trifluoroethanol (14.0 mL) were removed under vacuum, yielding a residue, which was then diluted with 1% acetic acid (20.0 mL). The mixture was purified by rapid chromatography (eluent: 10–50% H₂O (0.1% TFA / CH₃CN, C-18 column chromatography)). The resulting product was then freeze-dried to give (S)-6-((tert-butoxycarbonyl)amino)-2-(dimethylamino)hexanoic acid, b-7 (1.20 g, yield 53.8%) as a white solid. 1 HNMR (400MHz, CDCl3): δppm 3.16(t,J=7.0Hz,1H),3.02(t,J=6.8Hz,2H),2.52(s,6H),1.66-1.73(m, 2H), 1.46 (ddd, J=11.0, 7.0, 4.0Hz, 2H), 1.38 (s, 9H), 1.22-1.31 (m, 2H).

[0155] Step 5: (S)-2-(dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid (b-9)

[0156] A solution of (S)-6-((tert-butoxycarbonyl)amino)-2-(dimethylamino)hexanoic acid b-7 (0.60 g, 2.19 mmol) in trifluoroacetic acid (0.60 mL) and dichloromethane (6.00 mL) was stirred at 25 °C for 2 h. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid, giving a colorless oily de-Boc product. The colorless oily product was dissolved in saturated sodium bicarbonate (6.00 mL) at 0 °C, and then methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid (407 mg, 2.62 mmol) was added to the reaction mixture, and the mixture was stirred at 0 °C for 1 h, and then maintained at 25 °C for another 3 h. LC-MS showed that the intermediate was completely consumed and a main peak with the desired mass was detected. The reaction mixture was acidified with 1M HCl (5.00 mL) and purified by rapid chromatography (elution buffer: 10–50% H₂O (0.1% TFA) / CH₃CN, C-18 column chromatography). The resulting product was then lyophilized to give (S)-2-(dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid, b-9 (500 mg, 89.9% yield), a white solid. 1H NMR (400MHz, CDCl3): δppm 6.80(s,2H),3.79(dd,J=8.5,4.4Hz,1H),3.49(t,J=6.8Hz,2H),2.88(d, J=13.4Hz,6H),1.89-2.00(m,2H),1.56-1.64(m,2H),1.30-1.40(m,2H).

[0157] Step 6: (3S, 12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)-2-methyl-4,7,10,13,16-pentoxo-19-oxa-2,5,8,11,14,17-hexaazadocosane-22-acid (b-10, i.e., L-3-1)

[0158] To a mixture containing CTC-resin (0.50 g, 14.1 mmol) and 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, a-3 (0.20 g, 520 μmol), N,N-diisopropylethylamine (DIEA) (0.25 g, 1.95 mmol) and dichloromethane (10.0 mL) were added to induce swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. The resin was then washed three times with N,N-dimethylformamide (10.0 mL). The resin was then treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed five times with N,N-dimethylformamide. Then, Fmoc-Phe-OH (0.58 g, 1.31 mmol) was added and mixed for 30 seconds, followed by the addition of an N,N-dimethylformamide solution of O-benzotriazole-N,N,N-tetramethyluronium hexafluorophosphate (HBTU) (0.54 g, 1.43 mmol) and N,N-diisopropylethylamine (DIEA) (0.25 g, 1.95 mmol), and the mixture was bubbled under nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. The above steps were repeated for the coupling of the following amino acids, Fmoc-Gly-Gly-OH (0.53 g, 1.50 mmol) and the specific amino acid (S)-2-(dimethylamino)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid, b-9 (0.20 g, 1.31 mmol), at 25 °C. The reaction was detected by the ninhydrin assay. The coupling reaction was monitored by the ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and drying under vacuum, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to the flask containing the peptide resin, and the mixture was stirred twice for 2 minutes each time. The HFIP mixture was removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (C-18 column chromatography, 10-50% H2O / CH3CN eluent). The resulting product was then freeze-dried to give (3S,12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)-2-methyl-4,7,10,13,16-pentoxo-19-oxa-2,5,8,11,14,17-hexaazadocosane-22-acid b-10, a white solid (30.0 mg, yield 4.39%).

[0159] Step 7: Compound 3-1

[0160] To a solution of (3S,12S)-12-benzyl-3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)-2-methyl-4,7,10,13,16-pentoxo-19-oxa-2,5,8,11,14,17-hexaazadodecane-22-acid, b-10 (18.6 mg, 28.2 μmol), and 1-hydroxybenzotriazole (HOBt) (2.80 mg, 20.6 μmol) in N,N-dimethylformamide (1.00 mL), eczemab methanesulfonate (10.0 mg, 18.8 μmol) and N,N-diisopropylcarbodiimide (DIC) (9.50 mg, 75.24 μmol) were added, and the mixture was stirred at 25 °C for 3 hours. TLC(DCM:MeOH=10:1, R) f =0.3) indicates the reaction is complete. Trace amounts of N,N-dimethylformamide were removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H₂O / CH₃CN eluent, C-18 column chromatography). The resulting product was then lyophilized to give compound 3-1 (7.20 mg, 35.4% yield) as a pale yellow solid. LCMS (ESI, m / z): 1077.1 [M+H] + 1077.6 (Gemini-NX C18, 3.5µm, 2.1*30mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80 ± 2 min). HPLC (Gemini-NX C18 5µm 110A150*4.6mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (25-55 ± 20 ± 3 min).

[0161] (c) Synthesis of other compounds

[0162] Compound 1-1 (in) Figure 3 Synthesis of (also known as) derutin analogue 1-1

[0163] Step 1: 10-Benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxa-5,8,11,14,17-pentazatridecane-1-acid (c-1, i.e., L-1-1)

[0164] Peptide synthesis:

[0165] Peptides were synthesized using standard Fmoc chemical synthesis.

[0166] 1) Add DCM to a container containing CTC resin (0.25 mmol, 0.44 g, Sub = 0.57 mmol / g) and 5-benzyl-1-(9H-fluorene-9-yl)-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14-acid (0.10 g, 0.25 mmol, 1.0 equivalent) while bubbling with nitrogen.

[0167] 2) Add DIEA (6.0 equivalents) dropwise and mix for 2 hours.

[0168] 3) Add MeOH (0.8 mL) and mix for 30 minutes.

[0169] 4) Drain and wash 5 times with DMF.

[0170] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0171] 6) Drain and wash 3 times with DMF.

[0172] 7) Add Fmoc amino acid solution and mix for 30 seconds, then add activation buffer and bubble with N2 for about 1 hour.

[0173] 8) Repeat steps 5 to 7 for the next amino acid coupling.

[0174] Note:

[0175]

[0176] Fmoc was deprotected for 30 minutes using 20% ​​piperidine in DMF. The coupling reaction was monitored by ninhydrin test, and the resin was washed 5 times with DMF.

[0177] Peptide cleavage and purification:

[0178] 1) Add shear buffer (20% HFIP / DCM) to the peptide resin and stir for 3 minutes three times.

[0179] 2) DCM is concentrated under reduced pressure.

[0180] 3) Dry the peptides in a high vacuum for 2 hours.

[0181] 4) The crude peptide was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) to obtain compound c-1 (26.0 mg, 95.0% purity, 7.36% yield).

[0182] Step 2: (druticon analogue 1-1)

[0183] To a solution of compound c-1 (21.0 mg, 31.3 μmol, 1 equivalent) and eczema mesylate (16.6 mg, 31.3 μmol) in DMF (0.5 mL), HOAt (12.8 mg, 93.9 μmol, 3 equivalents), DIC (15.8 mg, 125.2 μmol, 4 equivalents), and DIEA (12.14 mg, 93.9 μmol, 3 equivalents) were added, and the reaction mixture was stirred at 25 °C for 16 hours. LCMS showed that compound c-1 was consumed, and a main peak with the desired mass was detected. The reaction mixture was directly purified by preparative HPLC (natural conditions, pure water). Two peaks with the desired mass were detected in the preparative HPLC. The peaks were separated and lyophilized to give a white solid of derutec analog 1-1 (peak 1: 1.4 mg, 85.97% purity; peak 2: 7.1 mg, 90.32% purity, 22.3% yield).

[0184] Compounds 1-2 (in) Figure 4 Synthesis of (also known as) derutin analogues 1-2

[0185] Step 1: (S)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid benzyl ester (d-3)

[0186] To a mixture of methyl acetate (5 g, 13.57 mmol) and benzyl (S)-2-hydroxypropionate (d-2, 4.89 g, 27.15 mmol) in tetrahydrofuran (5.00 mL), 4-methylbenzenesulfonic acid hydrate (129.09 mg, 678.64 μmol) was added, and the mixture was stirred at 25 °C for 5 h. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (TFA conditions). The yellow oily compound (S)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid benzyl ester, d-3 (4.10 g, 8.38 mmol, 61.77% yield) was obtained.

[0187] Step 2: (S)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid (d-4)

[0188] To a solution of (S)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid benzyl ester, d-3 (2.00 g, 4.09 μmol) in tetrahydrofuran (20.0 mL), dry Pd / C (200 mg) was added, and the reaction mixture was stirred at 25 °C under a hydrogen atmosphere (15 psi) for 4 hours. TLC (DCM / MeOH = 10:1, R f =0.3) indicates that the starting material was exhausted and a new spot was formed. The reaction mixture was filtered through diatomaceous earth and the filtrate was concentrated under reduced pressure to give (S)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid, d-4 (1.28 g, 3.21 mmol, 78.4% yield), a white solid.

[0189] Step 3: (2S,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxa-5,8,11,14,17-pentazatridecane-1-acid (d-5, i.e., L-1-2)

[0190] To a mixture containing CTC-resin (0.50 g, 0.50 mmol) and (S)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid, d-4 (0.50 g, 500 μmol), N,N-diisopropylethylamine (DIEA) (2.00 mmol) and dichloromethane (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. The resin was then washed 5 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 3 times with N,N-dimethylformamide. Then, Fmoc-Phe-OH (1.50 mmol) was added and mixed for 30 seconds, followed by the addition of a solution of O-benzotriazole-N,N,N-tetramethyluronium hexafluorophosphate (HBTU) (1.50 mmol) and N,N-diisopropylethylamine (DIEA) (3.00 mmol) in N,N-dimethylformamide, and the mixture was bubbled under nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. The above steps were repeated for the coupling of the amino acid Fmoc-Gly-OH (1.50 mmol) and the specific amino acid 2-((7-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol) at 25 °C. The reaction was detected by the ninhydrin test. The coupling reaction was monitored by the ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and drying under vacuum, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to a flask containing the peptide resin, and the mixture was stirred twice for 2 minutes. The HFIP mixture was removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then lyophilized to give a white solid (2S,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxa-5,8,11,14,17-pentazatridecane-1-acid, d-5, (55.0 mg, yield 9.98%).

[0191] Step 4: (druticon analogues 1-2)

[0192] To (2S,10S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-6,9,12,15-tetraoxo-18-(trifluoromethyl)-3-oxo-5,8,11,14,17-pentazatridecane-1-acid, d-5 (20.0 mg, 29.2 μmol), and 1-hydroxy-7-azabenzotriazole (HOAt) (7.95 mg, 58 μmol). To a solution of N,N-dimethylformamide (0.50 mL), eczemacon mesylate (15.5 mg, 29.1 μL), N,N-diisopropylcarbodiimide (DIC) (22.1 mg, 175 μL, 27.1 μL), and N,N-diisopropylethylamine (DIEA) (7.55 mg, 58.4 μL, 10.1 μL) were added, and the mixture was stirred at 25 °C for 3 hours. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reaction mixture was filtered to remove undissolved residues. The residues were purified by preparative HPLC (TFA conditions). The resulting product was then lyophilized to give a pale yellow solid of delutec analogues 1-2 (18.8 mg, 17.0 μL, yield 58.4%, purity 96.3%).

[0193] Compounds 1-3 (in) Figure 5 Synthesis of (also known as) derutin analogues 1-3

[0194] Step 1: 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid benzyl ester (e-3)

[0195] 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), 4-methylbenzenesulfonic acid monohydrate (12.9 mg, 67.8 μmol) was added, 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 desired mass 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 the 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 give compound e-3 (400 mg, 60.3% yield) as a white solid. 1H NMR (400MHz, CDCl3): δppm 7.90(d,J=7.4Hz,2H),7.73(d,J=7.0Hz,2H),7.24-7.64(m,9H),5.05-5.14(m,2H),4.48-4.62(m,4H),4.16-4.34(m,3H),3.54-3.72(m,4H).

[0196] Step 2: 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid (e-4)

[0197] Dry Pd / C (0.05 g) was added to a mixture of compound e-3 (400 mg, 818 μmol) in ethanol (10.0 mL) and ethyl acetate (10.0 mL), and the reaction mixture was then stirred at 25 °C under a hydrogen atmosphere (15 psi) for 5 hours. TLC (DCM / MeOH = 10:1, R f =0.2) indicates that compound e-3 was depleted and formed a new spot. The reaction mixture was filtered through diatomaceous earth and the filtrate was concentrated under reduced pressure to give compound e-4 as a white solid (300 mg, yield 91.9%). 1 HNMR (400MHz, CDCl3): δppm 7.90 (d, J = 7.4Hz, 2H), 7.73 (d, J = 7.0Hz, 2H), 7.24-7.64 (m, 4H), 5.05-5.14 (m, 2H), 4.48-4.62 (m, 2H), 4.16-4.34 (m, 3H), 3.54-3.72 (m, 4H).

[0198] Step 3: (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentazatetracosane-1-acid (e-5, i.e., L-1-3)

[0199] To a mixture containing CTC-resin (0.50 g, 14.1 mmol) and compound e-4 (0.20 g, 520 μmol), DIEA (0.25 g, 1.95 mmol) and DCM (10.0 mL) were added to induce swelling. The resin was mixed for 2 hours, then MeOH (5.00 mL) was added and mixed for 30 minutes. The resin was then washed three times with DMF (10.0 mL). The resin was treated with 20% piperidine in DMF for 30 minutes for Fmoc deprotection. The resin was washed five times with DMF. Then Fmoc-Phe-OH (0.58 g, 1.31 mmol) was added and mixed for 30 seconds, followed by the addition of a DMF solution of HBTU (0.54 g, 1.43 mmol) and DIEA (0.25 g, 1.95 mmol), and bubbled under nitrogen for 30 minutes. The resin was washed three times with DMF. The above steps were repeated for the coupling of the following 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-pyrrolo-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol) at 25 °C. The reaction was detected by ninhydrin assay. The coupling reaction was monitored by ninhydrin colorimetric reaction. After washing with 10.0 mL MeOH and drying under vacuum, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to a flask containing the peptide resin, and the mixture was stirred twice for 2 minutes. The HFIP mixture was removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then freeze-dried to give compound e-5 as a white solid (30.0 mg, yield 4.39%).

[0200] Step 4: (drutidine analogues 1-3)

[0201] HATU (9.30 mg, 24.4 μmol) was added to a solution of compound e-5 (27.2 mg, 37.6 μmol) in DMF (1.00 mL). The reaction mixture was stirred at 25 °C for 20 minutes. Ecinotecan mesylate (10.0 mg, 18.8 μmol) and DIEA (4.86 mg, 37.6 μmol) were added to the reaction mixture, and the mixture was kept at 40 °C for 2 hours. TLC (DCM:MeOH = 10:1, R f =0.4) indicates the reaction is complete. Trace amounts of DMF were removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The resulting product was then lyophilized to give a pale yellow solid of delutec analogue 1-3 (7.20 mg, 34.7% yield).

[0202] Compounds 1-4 (in) Figure 6 Synthesis of (also known as derutin analogues 1-4)

[0203] Step 1: (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-olate (f-3)

[0204] To a mixture of methyl acetate (5 g, 13.5 mmol) and methyl (S)-3-hydroxy-2-methylpropionate (3.21 g, 27.1 mmol) in tetrahydrofuran (50.0 mL), 4-methylbenzenesulfonic acid hydrate (129 mg, 678 μmol) was added, and the mixture was stirred at 25 °C for 1 h. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (TFA conditions). A yellow oily compound, (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oate, f-3 (4.79 g, 11.2 mmol, 82.7% yield), was given.

[0205] Step 2: (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid (f-4)

[0206] To a solution of (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-oate, f-3 (4.79 g, 11.2 mmol) in tetrahydrofuran (20.0 mL) and water (20.0 mL), lithium hydroxide monohydrate (942 mg, 22.4 mmol) was added, and the mixture was stirred at 25 °C for 2 hours. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. Acetic acid was added for acidification, followed by ammonium alkalization, and then (2,5-dioxopyrrolidone-1-yl)9H-fluorene-9-ylmethyl carbonate (8.21 g, 24.3 mmol) and sodium bicarbonate (2.04 g, 24.3 mmol). The mixture was stirred at 25 °C for 2 hours. LC-MS showed that the feedstock was completely consumed and detected a main peak with the desired quality. TLC (dichloromethane / methanol = 10:1, Rm) f=0.3) indicates that the starting material was exhausted and a new spot was formed. The reaction mixture was concentrated under reduced pressure to remove water and tetrahydrofuran, giving the residue. The crude product was purified by column chromatography (SiO2, dichloromethane / methanol = 100 / 1 to 20 / 1) to give a pale yellow liquid followed by a pale yellow oil (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, f-4 (1.25 g, 3.03 mmol, 12.45% yield).

[0207] Step 3: (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentazatetracosane-1-acid (f-5, i.e., L-1-4)

[0208] To a mixture containing CTC-resin (1.5 g, 1.50 mmol) and (S)-1-(9H-fluorene-9-yl)-11-methyl-3,6-dioxo-2,9-dioxa-4,7-diazadodecane-12-acid, f-4 (0.60 g, 1.50 mmol), N,N-diisopropylethylamine (DIEA) (6.00 mmol) and dichloromethane (10.0 mL) were added for swelling. The resin was mixed for 2 hours, then methanol (5.00 mL) was added and mixed for 30 minutes. The resin was then washed 5 times with N,N-dimethylformamide (10.0 mL). The resin was treated with 20% piperidine in N,N-dimethylformamide for 30 minutes for Fmoc deprotection. The resin was washed 3 times with N,N-dimethylformamide. Then, Fmoc-Phe-OH (4.50 mmol) was added and mixed for 30 seconds, followed by the addition of an N,N-dimethylformamide solution of O-benzotriazole-N,N,N-tetramethyl-uronium hexafluorophosphate (HBTU) (4.50 mmol) and N,N-diisopropylethylamine (DIEA) (9.00 mmol), and the mixture was bubbled under nitrogen for 30 minutes. The resin was washed three times with N,N-dimethylformamide. The above steps were repeated for the coupling of the 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-pyrrolo-1-yl)-1,1,1-trifluoroheptane-2-yl)amino)acetic acid (0.20 g, 1.31 mmol) at 25 °C. The reaction was detected by ninhydrin assay. The coupling reaction was monitored by a ninhydrin colorimetric reaction. After washing with 10.0 mL of methanol and drying under vacuum, 20.0 mL of lysis buffer (20% HFIP / 80% DCM) was added to a flask containing the peptide resin, and the mixture was stirred twice for 2 minutes. The HFIP mixture was removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H2O / CH3CN eluent, C-18 column chromatography). The product was then lyophilized to give a white solid (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentazatetracosane-1-acid, f-5, (167 mg, yield 9.80%).

[0209] Step 4: (Drutecan analogues 1-4)

[0210] To (11S)-11-benzyl-24-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-7,10,13,16-tetraoxo-19-(trifluoromethyl)-4-oxa-6,9,12,15,18-pentazatetracosane-1-acid, f-5 (20.0 mg, 28.6 μmol) and 1-hydroxy-7-azabenzotriazole (HOAt) (7.79 mg, 57.2 μmol). To a mixture of N,N-dimethylformamide (0.50 mL), eczemacon mesylate (15.2 mg, 28.6 μL), N,N-diisopropylcarbodiimide (DIC) (21.6 mg, 171 μL, 26.5 μL), and N,N-diisopropylethylamine (DIEA) (7.40 mg, 57.2 μL, 9.97 μL) were added, and the mixture was stirred at 25 °C for 3 hours. LC-MS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reaction mixture was filtered to remove undissolved residues. The residues were purified by preparative HPLC (TFA conditions). The resulting product was then lyophilized to give a pale yellow solid of delutec analogue 1-4 (6 mg, 5.20 μL, yield 18.1%, purity 96.2%).

[0211] Compounds 1-8 (in) Figure 7 Synthesis of (also known as derutin analogues 1-8)

[0212] Step 1: Benzyl 2-(2-(benzyloxy)ethoxy)propionate (g-2)

[0213] NaH (3.33 g, 83.2 mmol, 60% purity) was added dropwise to 100 mL of DMF under stirring in N2 at 0 °C. Benzyl (2S)-2-hydroxypropionate (d-2, 10.0 g, 55.5 mmol) was added dropwise to the above solution at 0 °C, and the mixture was stirred at 25 °C for 0.5 h. 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 h. TLC (petroleum ether / ethyl acetate = 5:1, R0) was performed. f=0.2) indicates the reaction is complete. The reaction mixture was quenched by adding 1000 mL of water at 0 °C 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 the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 50 / 1 to 5 / 1) to give benzyl 2-(2-(benzyloxy)ethoxy)propionate, g-2 (790 mg, 4.53% yield), a colorless oil. δppm 7.29-7.43(m,10H),5.10-5.30(m,2H),4.58(d,J=2.01Hz,2H),4.14(q,J= 6.86Hz, 1H), 3.75-3.87 (m, 1H), 3.60-3.73 (m, 3H), 1.47 (d, J = 7.03Hz, 3H).

[0214] Step 2: 2-(2-hydroxyethoxy)propionic acid (g-3)

[0215] A 100 mL round-bottom flask was purged three times with Ar, and dry Pd / C (160 mg) was carefully added. Methanol (~10.0 mL) was then added to completely permeate the Pd / C, followed by the slow addition of a solution of (2S)-2-(2-(benzyloxy)ethoxy)propionate, g-2 (790 mg, 2.51 mmol), in MeOH (20.0 mL) to Ar. The resulting mixture was degassed and purged three times with H2, and then stirred at 25 °C under H2 atmosphere for 4 hours. TLC (petroleum ether / ethyl acetate = 5:1, R f =0.2) indicates that the reaction is complete. The reactants were filtered and concentrated under reduced pressure to give the pale yellow liquid product 2-(2-hydroxyethoxy)propionic acid, g-3 (320 mg, crude product). The crude product was used directly in the next step without further purification. 1 H NMR (400MHz, DMSO): δppm 4.02 (q, J = 6.78Hz, 1H), 3.66-3.70 (m, 2H), 3.60-3.66 (m, 1H), 3.49-3.56 (m, 1H), 1.39 (d, J = 7.03Hz, 3H).

[0216] Step 3: Benzyl 2-(2-hydroxyethoxy)propionate (g-4)

[0217] (2S)-2-(2-hydroxyethoxy)propionic acid, g-3 (320 mg, 2.39 mmol), was dissolved in MeOH (4 mL) and H2O (0.8 mL), and Cs2CO3 (389 mg, 1.19 mmol) was added to this solution. The mixture was stirred at 25 °C for 0.5 h and concentrated. Then bromotoluene (449 mg, 2.62 mmol) and DMF (2 mL) were added. The mixture was stirred at 25 °C for 16 h. TLC (petroleum ether / ethyl acetate = 3:1, R f =0.4) indicates the reaction is complete. Dilute the mixture with water (50.0 mL) and extract with DCM (30 mL x 3). Dry the combined organic layers with anhydrous sodium sulfate, filter and concentrate under reduced pressure to give the residue. Purify the residue by silica gel column chromatography (100-200 mesh silica gel), eluting with (petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give benzyl 2-(2-hydroxyethoxy)propionate, g-4 (340 mg, yield 64%), as a white solid. 1 H NMR (400MHz, CDCl3): δppm 7.37 (s, 5H), 5.15-5.26 (m, 2H), 4.04-4.15 (m, 1H), 3.71-3.76 (m, 2H), 3.60-3.68 (m, 2H), 1.46 (d, J = 7.03Hz, 3H).

[0218] Step 4: 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester (g-6)

[0219] To a solution of methyl acetate [[2-(9H-fluorene-9-ylmethoxycarbonylamino)acetyl]amino]methyl ester (a-3) (614 mg, 1.67 mmol) in THF (8 mL), TsOH·H₂O (14.4 mg, 75.8 μmol) and benzyl 2-(2-hydroxyethoxy)propionate, g-4 (340 mg, 1.52 mmol) were added. 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 the residue. TLC (petroleum ether / ethyl acetate = 1:1, R f=0.4) indicates that the reaction was complete. The residue was purified by silica gel column chromatography (100-200 mesh silica gel), eluted with (petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give a white solid of 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester, g-6 (416 mg, 781 μmol, yield 51.5%). 1 HNMR (400MHz, CDCl3): δppm 7.77(d,J=7.53Hz,2H),7.61(br d,J=7.28Hz,2H),7.39-7.43(m,2H),7.28-7.38(m,7H),7.21(br s,1H),5.53(br s,1H),5.19(s,2H),4.93-5.03(m,1H),4.67(br s,1H),4.44(d,J=7.03Hz,2H),4.22-4.27(m,1H),4.05(q,J=7.03Hz,1H),3.92(br s,2H),3.76(br s,1H),3.71(br d,J=9.03Hz,2H),3.55(br d, J = 8.53Hz, 1H), 1.44 (d, J = 6.78Hz, 3H).

[0220] Step 5: 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid (g-7)

[0221] A 100 mL round-bottom flask was purged three times with Ar, and dry Pd / C (60 mg) was carefully added. Then, THF (5 mL) was added to completely permeate the Pd / C, followed by the slow addition of a solution of 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid benzyl ester, g-6 (416 mg, 781 μmol) in THF (10 mL) under Ar. The resulting mixture was degassed and purged three times with H2, then stirred for 2 hours at 25 °C in H2 (15 psi). TLC (petroleum ether / ethyl acetate = 1:1, R f =0.4) indicates that the reaction is complete. The reactants were filtered and concentrated under reduced pressure to give a white, gelatinous product 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid, g-7 (320 mg, 92.6% yield). The crude product was used for the next step without purification. 1H NMR (400MHz, CDCl3): δppm 7.77(d,J=7.53Hz,2H),7.60(br d,J=7.28Hz,2H),7.38-7.44(m,2H),7.29-7.35(m,2H),5.48-5.65(m,1H),4.95(br s,1H),4.70(dd,J=10.54,6.02Hz,1H),4.42-4.52(m,2H),4.23(t,J=6.78Hz,1H),4.02(q,J=6.86Hz,1H),3.94(br s,1H),3.83-3.92(m,1H),3.75-3.82(m,2H),3.73(br d,J=6.78Hz,2H),3.58-3.67(m,1H),1.42-1.48(m,3H).

[0222] Step 6: (13S)-13-benzyl-26-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-methyl-9,12,15,18-tetraoxo-21-(trifluoromethyl)-3,6-dioxa-8,11,14,17,20-pentazahexacosane-1-acid (g-8, i.e., L-1-8)

[0223] Peptide synthesis:

[0224] Peptides were synthesized using standard Fmoc chemical synthesis.

[0225] 1) Add DCM to a container containing CTC resin (1.5 mmol, 1.5 g, Sub = 1.01 mmol / g) and 1-(9H-fluorene-9-yl)-13-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatetradecane-14-acid (0.6 g, 1.5 mmol, 1.0 equivalent) while bubbling N2.

[0226] 2) Add DIEA (4.0 equivalents) dropwise and mix for 2 hours.

[0227] 3) Add MeOH (0.5 mL) and mix for 30 minutes.

[0228] 4) Drain and wash 5 times with DMF.

[0229] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0230] 6) Drain and wash 3 times with DMF.

[0231] 7) Add Fmoc amino acid solution and mix for 30 seconds, then add activation buffer and bubble with N2 for about 0.5 hours.

[0232] 8) Repeat steps 5 to 7 for the next amino acid coupling.

[0233] Note:

[0234]

[0235] Fmoc was deprotected for 30 minutes using 20% ​​piperidine in DMF. The coupling reaction was monitored by ninhydrin test, and the resin was washed 5 times with DMF.

[0236] Peptide cleavage and purification:

[0237] 1) Wash the resin three times with MeOH and dry it under vacuum.

[0238] 2) Add shear buffer (20% HFIP / DCM) to the peptide resin and stir for 0.5 hours three times.

[0239] 3) DCM is concentrated under reduced pressure.

[0240] 4) The peptide was dried in a high vacuum for 2 hours to obtain compound g-8 (100 mg, 90%).

[0241] Step 7: (Drutecan analogues 1-8)

[0242] To a solution of compound g-8 (20.0 mg, 27.5 μmol) in DMF (200 μL), DIC (19.8 mg, 157 μmol), HOAT (7.12 mg, 52.3 μmol), (10S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexane[14.7.1.02,14.04,13.06,11.020,24]teicosode-1,6(11),12,14,16(24),17,19-heptane-5,9-dione (13.9 mg, 26.1 μmol, MsOH) and DIEA (6.76 mg, 52.3 μmol) were added. 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 desired m / z was detected. Trace amounts of N,N-dimethylformamide were removed under vacuum to obtain the residue. The residue was purified by rapid chromatography (10-50% H₂O / CH₃CN eluent, C-18 column chromatography). The resulting product was then lyophilized to give a pale yellow solid of the derutec analogue 1-8 (12 mg, 10.5 μmol). LC-MS (ESI, m / z): 1145.4 [M+H] +1146.6 (Gemini-NX C18, 3.5µm, 2.1*30mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80 ± 2 min). HPLC (Gemini-NX C18 5µm 110A 150*4.6mm column, wavelength: UV 220nm & 254nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (40-70 ± 20 ± 3 min).

[0243] Compounds 3-4 (in) Figure 8 Synthesis of (also known as derutin analogues 3-4)

[0244] Step 1: (S)-2-acetamido-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid (h-3)

[0245] Methyl 2,5-dioxopyrrole-1-carboxylic acid (1.65 g, 10.6 mmol) was added to a solution of (2S)-2-acetamido-6-amino-hexanoic acid (2.00 g, 10.6 mmol) in saturated NaHCO3 (50 mL). The mixture was stirred at 25 °C for 4 h. LC-MS showed that compound h-1 was completely consumed and a main peak with the desired m / z was detected. The reaction mixture was quenched by adding H2SO4 at 0 °C to pH 3-4, and then extracted with EA (100 mL x 3). The combined organic layers were washed with brine (50 mL x 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, DCM / MeOH = 100 / 1 to 20 / 1) to give (S)-2-acetamido-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid, h-3 (460 mg, 1.71 mmol). 1 H NMR (400MHz, CDCl3): δppm 6.71 (s, 2H), 6.30 (br d,J=7.28Hz,1H),4.48-4.59(m,1H),3.54(t,J=6.78Hz,2H),2.08(s,3H),1 .90-2.00(m,1H),1.75-1.84(m,1H),1.56-1.70(m,2H),1.31-1.45(m,2H).

[0246] Step 2: (19S)-10-benzyl-19-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)-6,9,12,15,18,21-hexaoxo-3-oxa-5,8,11,14,17,20-hexaazadocosane-1-acid (h-4, i.e., L-3-4)

[0247] Peptide synthesis:

[0248] Peptides were synthesized using standard Fmoc chemical synthesis.

[0249] 1) Add DCM to a container containing CTC resin (0.5 mmol, 0.5 g, Sub = 1.01 mmol / g) and 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid (0.2 g, 0.5 mmol, 1.0 equivalent) while bubbling N2.

[0250] 2) Add DIEA (4.0 equivalents) dropwise and mix for 2 hours.

[0251] 3) Add MeOH (0.5 mL) and mix for 30 minutes.

[0252] 4) Drain and wash 5 times with DMF.

[0253] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0254] 6) Drain and wash 3 times with DMF.

[0255] 7) Add Fmoc amino acid solution and mix for 30 seconds, then add activation buffer and bubble with N2 for about 0.5 hours.

[0256] 8) Repeat steps 5 to 7 for the next amino acid coupling.

[0257] Note:

[0258]

[0259] Fmoc was deprotected for 30 minutes using 20% ​​piperidine in DMF. The coupling reaction was monitored by ninhydrin test, and the resin was washed 5 times with DMF.

[0260] Peptide cleavage and purification:

[0261] 1) Wash the resin three times with MeOH and dry it under vacuum.

[0262] 2) Add shear buffer (20% HFIP / DCM) to the peptide resin and stir for 0.5 hours three times.

[0263] 3) DCM is concentrated under reduced pressure.

[0264] 4) The peptide was dried in a high vacuum for 2 hours to obtain compound h-4 (100 mg, 95%).

[0265] Step 3: (druticon analogues 3-4)

[0266] To a solution of compound h-4 (20.0 mg, 29.7 μol) in DMF (500 μL), DIC (15.6 mg, 124 μol), eczetidine mesylate (13.2 mg, 24.7 μmmol), and HOBt (3.68 mg, 27.2 μol) were added. The mixture was stirred at 25 °C for 16 hours. TLC (DCM / MeOH = 10 / 1, Rf = 0.4) showed that the reaction was complete. Trace amounts of N,N-dimethylformamide were removed under vacuum to give the residue. The residue was purified by rapid chromatography (10-50% H₂O / CH₃CN eluent, C-18 column chromatography). The product was then lyophilized to give a pale yellow solid of the delutec analogue 3-4 (7 mg, 6.42 μol). LCMS (ESI, m / z): 1090.4 [M+H] + 1091.4 (Gemini-NX C18, 3.5µm, 2.1*30mm column, wavelength: UV 220 nm & 254 nm; column temperature: 30℃) eluted with 0.1% TFA in water: 0.1% TFA in acetonitrile (10-80 ± 2 min). HPLC (Gemini-NX C18 5µm 110A150*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 min).

[0267] (d) Synthesis of "existing connector-load"

[0268] In the context of this disclosure, the term "native linker-load" (hereinafter also referred to as "native-LP" or "nat-LP") refers to a compound having the following structure:

[0269]

[0270] The original-LP has the same structure as the derutecan in Enhertu, both containing a maleimide-GGFG linker. In this embodiment, the original-LP was purchased from MedChemExpress under the name "drutecan".

[0271] Example 2: Synthesis of antibody-drug conjugates

[0272] Antibody-drug conjugates are synthesized according to the following general procedure: First, the antibody in pH 7 PBS solution is reduced with 2–20 equivalents of TCEP for 0.5 to 18 hours; with or without removing residual TCEP by column or membrane, an excess of linker-loading (15–18 moles excess) is introduced; the conjugation reaction is completed within half an hour to several hours in the temperature range of 4°C to room temperature (RT), followed by HPLC purification to provide the final ADC product.

[0273]

[0274] Note: 1: Indicates Ab, followed by the numerical code for each connector-load section.

[0275] 2: Molar ratio

[0276] 3: Also referred to below as "Her-Dxd" or "Her-nat-LP"

[0277] Example 3: Shearing of Connector-Load

[0278] method

[0279] Reaction buffer (40 mM H3PO4 / H3BO3 / HAc, 1 mM EDTA, pH 4.5), 135 mM cysteine, DMA (N,N'-dimethylacetamide, 2%, v / v), sample (0.015 μmol linker-loaded compound), and cathepsin B (final 16 U / μmol linker-loaded compound) were sequentially added to 1.5 mL EP tubes. The resulting 300 μL lysis system had a final cysteine ​​concentration of 10 mM. The EP tubes were placed in a 37°C water bath.

[0280] After lysis for approximately 15 minutes, 2 hours, 5 hours, and 16 hours, the mixture was thoroughly vortexed and then sampled (25 μL). The collected mixture samples were inactivated by adding cathepsin B inhibitor E64 (2.5 equivalents to cathepsin B), followed by RP-HPLC and RP-MS analysis. The shear percentage was calculated as the ratio of the peak area of ​​the cleaved linker-loaded structure to the sum of the areas of the uncleaved and cleaved linker-loaded structures (as measured by RP-HPLC and RP-MS).

[0281] result

[0282] Table 1 summarizes the shearing results (expressed as a percentage) over approximately 16 hours (overnight), showing a comparison of cathepsin B digestion efficiency between linkers and loads. The results show that the shearing of the tested linker-loads of the present invention is faster than, or at least comparable to, that of the original linker-loads (compounds 3-1).

[0283] Table 1

[0284]

[0285] exist Figure 9 In the figure, subplot (a) shows the cleavage sites and modifications of the linker portion in compound 1-1 compared to the original linker-loading; subplot (b) shows the cleavage percentage, calculated based on the peak area of ​​the released drug detected by reversed-phase HPLC (mixed mode). Consistent with the results in Table 1, the linker-loading of the present invention is cleaved more rapidly.

[0286] Example 4: Shearing of the connector-load section in an ADC

[0287] method

[0288] The reaction conditions were the same as those described in Example 3 regarding the lysis of the linker-loaded compound itself. Specifically, reaction buffer (40 mM H3PO4 / H3BO3 / HAc, 1 mM EDTA, pH 4.5), 135 mM cysteine, DMA (2%, v / v), sample (0.015 μmol ADC), and cathepsin B (16 U / μmol ADC) were sequentially added to a 1.5 mL EP tube. The resulting 300 μL lysis system had a final cysteine ​​concentration of 10 mM. The EP tube was placed in a 37°C water bath.

[0289] After lysis for 10 min, 5 hr, 24 hr, and 48 hr, the mixture was thoroughly vortexed, and then 25 μL samples were taken. The collected mixture samples were inactivated with cathepsin inhibitor E64 (2.5 equivalents to cathepsin B), followed by reversed-phase LC-MS analysis. The lysis rate was calculated based on the decrease in DAR measured by mass spectrometry (MS).

[0290] result

[0291] Table 2 summarizes the percentage digestion over time, showing a comparison of cathepsin B digestion efficiency for different linker-load portions of the ADC. Clearly, ADCs containing linker-loads 1-2, 1-3, 3-1, and 3-4 release the drug more rapidly in the presence of cathepsin B. Notably, in another lysis assay using a mixed-mode column to quantify the released drug, the results showed that the ADC containing linker-load portion 1-1 released the drug slightly faster than the ADC containing the original linker-load.

[0292] Table 2

[0293]

[0294] exist Figure 10In the figure, subplot (a) shows the cleavage sites and modifications of the connector-load portion in the ADC compared to the original connector-load in the ADC, and subplot (b) shows the time-dependent cleavage percentage (%) of the connector-load in the ADC, calculated based on the drug release peak area (ibid.). After 48 hours, the cleavage percentage of ADC-1-1 (i.e., the ADC containing connector-load 1-1) was 18.5%, while that of the original ADC-LP was 15.5%.

[0295] Example 5: Freeze-thaw stability

[0296] method

[0297] Remove the ADC sample (~10 mg / mL, dissolved in 20 mM histidine, 150 mM NaCl, pH 6.0) from the -80℃ frozen Eppendorf tube and thaw at room temperature for 30 minutes. Then freeze the ADC sample at -80℃ for 2 days, followed by thawing at room temperature for 30 minutes; repeat this freeze / thaw process once more. Then, take 20 μl of each sample for SEC and MS to determine DAR and drug distribution. Measure the percentage (molar ratio) of each substance (L0, L1, L2, H0, H1, H2, H4, etc., where "L" refers to the light chain, "H" refers to the heavy chain, and the numbers indicate the number of drug molecules attached to the chain) in each sample based on the peak area.

[0298] result

[0299] Table 3 summarizes the changes in unwanted impurity formations, such as L2 (ADCs with two drug molecules linked to the light chain) and H4 (ADCs with four drug molecules linked to the heavy chain), and DAR after two freeze-thaw cycles. The results show that after two freeze-thaw cycles, L2 remained zero (0) in the ADCs containing the linker-loadings of this invention, while L2 increased to 0.76% in Her-Dxd (i.e., trastuzumab). H4% was also the highest in Her-Dxd. ADCs containing linker-loadings 1-8 showed the smallest decrease in DAR after two freeze-thaw cycles. Overall, the unwanted H4 content in all eight ADC analogs was lower than in Her-Dxd after freeze-thaw cycles, and all maintained stable DAR.

[0300] Table 3

[0301]

[0302] Note: "Her" = "Herceptin", also known as "trastuzumab"; "fresh" refers to newly synthesized ADC that has not undergone freeze-thaw cycles.

[0303] Example 6: Detection of H4 in ADC

[0304] method

[0305] The ADC was prepared in 20 mM histidine buffer (containing 150 mM NaCl, pH 6.0). The resulting ADC sample was analyzed by LC-MS to determine the distribution of the drug on the light and heavy chains. Ideally, only five substances, L0, H0, L1, H1, and H2, should be detected. The aggregation percentage was determined by HPLC.

[0306] result

[0307] Table 4 shows a comparison of the nonspecific impurity H4% among ADCs. The H4% (molar ratio) of the trastuzumab Her-Dxd control prepared using the original linker-loaded formulation was 3% after synthesis, while the content of this impurity in Her-1-8 was significantly reduced to 0.8%. As the results show, the ADC products produced using the linker-loaded formulation of this invention have reduced impurities.

[0308] Table 4

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

[0310] Example 7: Removal of load from uncoupled connectors

[0311] After coupling as described in Example 2, the ADC was replaced with 20 mM histidine buffer (containing 150 mM NaCl, pH 6.0) using a rotating desalting column (40 kDa). Table 5 shows a comparison of the removal of free linker-loads (i.e., uncoupled linker-loads) between ADCs prepared with different linker-loads. Free linker-loads were removed using UFDF. The residual free linker-load content in the Her-Dxd (i.e., trastuzumab) conjugate product was close to 5%, but this content was less than 2% in the product produced using the linker-loads of this invention.

[0312] As the results show, the linker-loading of this invention improves the operability of purification, for example, making it easier and more thorough to remove residual free linker-loading from ADC coupling products. Not limited to any particular theory, it is believed that introducing polar groups into the linker moiety increases the water solubility of the linker-loading, promoting its removal, for example, by UFDF. This has significant implications for ADC manufacturing processes.

[0313] Table 5

[0314]

[0315] Example 8: Affinity Detection

[0316] method

[0317] The in vitro binding affinity of ADC to human HER2 was detected using FACS (fluorescence live cell flow cytometry). On the day of detection, HER-2 expression (1×10⁻⁶) was measured. 5 Tumor cells (cells / well) were co-incubated with serially diluted ADC at 4°C for 1-2 hours. Her-Dxd, prepared as described in Example 2, served as a reference ADC and positive control. A buffer (20 mM histidine buffer containing 150 mM NaCl, pH 6.0) used to dissolve the ADC served as a negative control. After incubation, cells were washed with FACS staining buffer, and then secondary antibody Alexa647-conjugated goat anti-human IgG Fc (Jackson) diluted with FACS staining buffer was added. The culture plates were 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. EC50 was calculated using GraphPad Prism. 50 value.

[0318] result

[0319] The results are as follows Figure 11 As shown. According to N87 cell assay, the ECG of the ADC used in this invention... 50 The concentration is approximately 1 nM, while Her-Dxd is 0.9 nM. According to JIMT-1 cell assays, the EC50 of the ADC used in this invention... 50 The concentration was 0.4–0.7 nM, while Her-Dxd was 0.4 nM. According to MDA-MB-231 cell assays, the EC50 of the ADCs used in this invention (excluding Her3-1) was [data missing]. 50 The binding affinity was 0.5–0.7 nM for Her-Dxd and 0.6 nM for Her-Dxd. The results showed that the binding affinity of the ADC of this invention, having the linker of this invention and the linker-loader of this invention, was at least comparable to, and even better than, that of trastuzumab.

[0320] Example 9: Cytotoxicity Detection

[0321] method

[0322] The ability to inhibit tumor cell growth was determined by in vitro cytotoxicity assay. Tumor cell lines NCI-N87, HCC1954, MDA-MB-231, and JIMT-1 (purchased from ATCC) were cultured routinely in RPMI 1640 or DMEM medium. The day before the assay, cells were seeded at an appropriate cell density into 96-well plates. The following day, serially diluted ADCs were added to each well. Her-Dxd, prepared as described in Example 2, was used as a reference ADC and positive control. Buffer used to dissolve the ADC was used as a negative control. The culture plates were incubated at 37°C with 5% CO2. Cell viability was measured using CellTiter-Glo (Promega) after 4–6 days. IC50 was calculated using GraphPad Prism. 50 value.

[0323] result

[0324] The results are as follows Figure 12 As shown. Neither the ADC nor Her-Dxd tested in this invention showed detectable cytotoxicity (IC50) against JIMT-1 cells or MDA-MB-231 cells. 50 >1nM), in N87 cells and HCC1954 cells, the cytotoxicity of all the ADCs tested in this invention was comparable to that of the original ADCs, IC50. 50 Approximately 0.1 nM. Results showed that the cytotoxicity of the ADC of the present invention, having the linker of the present invention and having the linker-loader of the present invention, was at least comparable to that of trastuzumab.

Claims

1. A compound selected from the group consisting of the following: ###0001### L-3-1 or a pharmaceutically acceptable salt or ester thereof.

2. A conjugate compound having a structure selected from the group consisting of the following: ###0002### or a pharmaceutically acceptable salt or ester thereof.

3. An antibody-drug conjugate having a structure selected from the group consisting of the following: ###0003### or a pharmaceutically acceptable salt or ester thereof. wherein p is 1 to 8, and Ab is an antibody; or a pharmaceutically acceptable salt or ester thereof. p is 2, 4 or 6; and / or Ab is trastuzumab.

4. The antibody-drug conjugate of claim 3, wherein, 5. A method of producing a linker-payload compound comprising conjugating a drug to a compound of claim 1. The drug is exatecan.

6. The method of claim 5, wherein, 7. A method of producing an antibody-drug-conjugate comprising: (a) conjugating a drug to a compound of claim 1 to obtain a linker-payload compound; and (b) conjugating an antibody to the linker-payload compound obtained in step (a). The drug is exatecan.

8. The method of claim 7, wherein, ​

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