Modular linker compounds for target-bound drug conjugates

Abstract

The present invention relates to modular linker compounds for target binding drug conjugates (TBDCs), and in particular to modular linker compounds for antibody-drug conjugates (ADCs). The modular linker compounds comprise a solubilizing group and a self-immolative spacer. The present invention further relates to methods of synthesizing the modular linker compounds, to target binding drug conjugates comprising the modular linker compounds of the present invention, and to pharmaceutical compositions comprising the target binding drug conjugate compounds of the present invention. Embodiments of the present invention are specifically developed for target binding drug conjugates for use in cancer therapy, and will be described hereinafter with reference to the present application. However, it will be appreciated that the present invention is not limited to this particular field of use.

Description

Technical Field

[0001] This invention relates to modular adapter compounds for target-binding drug conjugates (TBDCs), and particularly to modular adapter compounds for antibody-drug conjugates (ADCs). The invention further relates to methods for synthesizing said modular adapter compounds, to target-binding drug conjugates comprising the modular adapter compounds of the invention, and to pharmaceutical compositions comprising target-binding drug conjugate compounds of the invention. Embodiments of the invention have been specifically developed for target-binding drug conjugates for use in cancer therapy and will be described below with reference to this application. However, it will be understood that the invention is not limited to this particular field of use. Background Technology

[0002] Aside from surgical resection and radiotherapy, chemotherapy remains a primary treatment option for cancer. However, it is well established that chemotherapy, due to its limited selectivity and specificity against cancer cells, typically provides only a small therapeutic window for administering high doses of chemotherapeutic agents. This often results in severe side effects on patients due to toxicity to non-cancer cells, while still delivering only low efficacy against malignant cancer cells.

[0003] Targeted drug therapy strategies have been developed to increase the specific delivery of cytotoxic drugs directly to malignant cancer cells. Specifically, target-binding drug conjugates based on the principle of recognition and binding to known target sequences or epitopes of immunoglobulins / antibodies offer new therapeutic options that allow for the highly specific delivery of drug substances to malignant cancer cells with minimal or no effect on non-cancer cells.

[0004] Target-binding drug conjugates (TBDCs), particularly antibody-drug conjugates (ADCs), typically consist of a target-binding moiety covalently linked to a cytotoxic drug substance via a synthetic linker. While (a) the target-binding moiety ensures high specificity and (b) the cytotoxic drug substance ensures post-delivery action on cancer cells, (c) the linker compound significantly affects the therapeutic index, efficacy, and pharmacokinetics of such conjugates.

[0005] Specifically, the adapter compound must (i) ensure high plasma stability of the conjugate in circulation to prevent premature drug release and the resulting undesirable systemic off-target effects; (ii) maintain the properties of the target-binding partially ethyl cytotoxic drug substance; (iii) provide the conjugate with high water solubility to allow delivery of hydrophobic / lipophilic drug substances in the pharmaceutical composition and prevent TBDC aggregation; and (iv) ensure that the targeted cancer cells are appropriately exposed to the cytotoxic drug substance to maximize therapeutic efficacy.

[0006] The lipophilicity of many cytotoxic drug substances can adversely affect the properties of TBDCs, to the point that they cannot effectively deliver the payload to target cells. Modulating TBDC properties by immobilizing polar groups in the linker can reduce aggregation during conjugation and improve the physicochemical properties of the ADC, thereby improving the pharmacokinetics (PK) and therapeutic index of the ADC (see, for example, Nature Biotechnology, 2015, 33, 733-735). Modulating the linker to achieve changes in the polarity or charge of the final metabolite can improve activity against multidrug-resistant (MDR) cells by better retaining the payload inside the cell (see, for example, Cancer Res; 70(6) March 15, 2010, p2528). Chemical components on the connector (e.g., polyethylene glycol (PEG), sugar or sulfate groups (see, for example, WO 2014062697, US20100323973)) not only affect the conjugation efficiency and ease of production of ADCs, but are also often retained as part of metabolites, thereby affecting the activity and safety of ADCs.

[0007] Regarding the delivery of cytotoxic drugs to target sites, both inelastic and cleavable linkers have been developed. Inelastic linkers (e.g., containing thioether or maleimide-hexanoyl linkages) typically require internalization of the entire TBDC by the target cell and subsequent lysosomal enzymatic degradation to release the cytotoxic drug. Cleavable linkers can be categorized into chemically cleavable and enzymatically cleavable linkers. Chemically cleavable linkers typically depend on pH sensitivity (lysosomal / endosomal release of the cytotoxic drug) or reductosensitivity (cytoplasmic release of the cytotoxic drug), while various known enzymatically cleavable linkers depend on the enzymatic release of the cytotoxic drug, typically within the lysosomal compartment of the target cell. For example, combinations of cleavable peptide structures with self-immolative spacers between the targeted binding antibody and drug in ADCs have been described as suitable for payloads carrying primary or secondary amines. Jeffrey et al., 2005 (Design, Synthesis, and in Vitro Evaluation of Dipeptide-Based Antibody Minor Groove Binder Conjugates. J. Med. Chem. Vol. 48: 1344-1358). Nevertheless, in many cases, the released cytotoxic drug substance still contains adaptor-derived adducts. In some cases, the ingenious design of cleavable links allows for positive modification of the drug substance. In other cases, the efficacy of the drug substance is negatively affected by residual adaptor adducts.

[0008] However, the therapeutic efficacy of TBDCs depends not only on the appropriate release of the cytotoxic drug substance, but also on the effective bioconjugation of the drug substance to the target-binding moiety, and further on the number of cytotoxic drug substance molecules successfully conjugated to a single target-binding moiety. Given that TBDCs have been primarily developed as ADCs to date, this number of molecules is referred to as the drug-to-antibody ratio (DAR). Low DAR is known to reduce TBDC efficacy and require conjugation of highly toxic drug substances, while high DAR is associated with potential TBDC instability, which leads to increased systemic effects, shortened TBDC half-life, and overall alterations in molecular pharmacokinetics. However, given that random chemistry is typically applied to conjugating drug substances to antibodies, even FDA-approved ADCs are composed of mixtures of conjugates, which are heterogeneous with respect to their DAR.

[0009] According to the prior art, there is a need in the art for a widely applicable linker compound for improving target-binding drug conjugates (TBDCs) that mediates the stable conjugation of cytotoxic drug substances to target-binding moieties, while allowing the drug-antibody ratio (DAR) and / or water solubility of the TBDC to be easily adjusted according to the cytotoxicity and hydrophobicity of the drug substance to be conjugated.

[0010] The objective of this invention is to overcome or improve at least one drawback of the prior art, or to provide a useful alternative. Specifically, the objective of this invention is to provide improved linker compounds for target-binding drug conjugates (TBDCs), particularly for antibody-drug conjugates (ADCs). Summary of the Invention

[0011] As indicated above, the present invention aims to provide adapter compounds for target-binding drug conjugates (TBDCs) that can be readily adjusted with respect to their drug-antibody ratio (DAR) and / or water solubility, respectively, based on the cytotoxicity and hydrophobicity of the drug substance to be conjugated, while ensuring uniform labeling (DOL).

[0012] The linker compounds described herein offer high tunability due to their modular architecture. Specifically, the modular linker compounds of the present invention comprise at least one functional unit of a cleavable peptide sequence linked to a self-degrading spacer, with a solubilizing group and a cytotoxic drug substance physically close to said functional unit. Furthermore, the chemical spacer links this functional unit to a polymer core comprising one or more branched units, wherein the number of functional units that the modular linker compounds of the present invention can carry is customizable, and can even be further adapted to have another direct link to a second cytotoxic drug substance outside the functional unit directly described above.

[0013] In modular adapter compounds, a branch unit is connected to a terminal reactive moiety via another chemical spacer, wherein the reactive moiety is adapted for reliable conjugation of the modular adapter compound to a selected target-binding moiety. Typically, this target-binding moiety relies on immunoglobulin principles to recognize and bind to a target sequence or epitope of an antigen with high specificity.

[0014] Therefore, in a first aspect, the present invention relates to a modular connector compound of formula (I).

[0015]

[0016] Formula (I)

[0017] Or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0018] X is a self-degrading spacer. ,

[0019] in:

[0020] R 1 X is covalently linked to D at the para, meta, or ortho position of the amino group of X, and the linkage is selected from the bond. , , , ,and ;

[0021] R 2 Connect X to SE and is , , Or -O-;

[0022] D is a pharmaceutically active substance;

[0023] SE is a solubilizing group;

[0024] L is a spacer containing a peptide covalently linked to X;

[0025] n is an integer of at least 2, preferably an integer from 2 to 5, such as 2, 3, 4 or 5;

[0026] Y is the branch unit.

[0027] A is the reactive portion used to bind with the target.

[0028] In some implementations, L is

[0029] ,

[0030] in:

[0031] W is connected to Z. 3The cleavable and incleavable peptide sequences of X, selected from:

[0032] Val-Ala, Val-Cit, Met-Thr, Thr-Thr, Phe-Lys, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, Gly-Gly-Tyr-Gly, cBu-Ala, cBu-Cit, Glu-cBu-Ala, Glu-cBu-Cit, Asp-cBu-Ala, Asp-cBu-Cit, Asp-Pro-Val, Asn-Pro-Val, iGlu-cBu-Ala, iGlu-cBu-Cit, Val-Lys, Val-Arg, Val-Gln, Val-Met, Val-Thr, AcLys-Val-Cit, AcLys-Va l-Ala, Asp-Val-Ala, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Tyr-Arg, Tyr-Met, Phe-Cit, Phe-Ala, Phe-Arg, Phe-Met, Phe-Gln, Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly, Ala-Lys, Leu-Cit, Leu-Gln, Ile-Cit, Ala-Ala-Asn, Ala-Asn, Ala-Ala, Asn-Asn, Asn, β-Ala and Lys or combinations thereof; and

[0033] Z 3 It is a bond or spacer, the spacer containing C 1-20 Alkyl chains or 1 to 10 polyethylene glycol (PEG) 1-10 or C2-amide-PEG 1-10 ,

[0034] Among them (PEG) 1-10 yes And C2-amide-PEG 1-10 yes ;

[0035] Preferably, L is

[0036] or .

[0037] In a specific implementation, in the modular connector compound of formula (Ia), if the ClogP of D is less than -1,

[0038]

[0039] Formula (Ia)

[0040] Or its pharmaceutically acceptable salts or solvates, R 2 It is H and has no SE attached to the modular connector compound.

[0041] As disclosed (PEG) 1-10 and C2-amide-PEG 1-10 Specify the chain lengths including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0042] In a second aspect, the present invention relates to a method for synthesizing a modular connector compound according to formula (I) of the first aspect, the method comprising:

[0043] (i) Make a first compound comprising the following:

[0044] -Degradation portion,

[0045] - A cleavable peptide sequence W,

[0046] -Amino protecting group Pg 1 It is covalently connected to W.

[0047] -Pharmaceutical active substance D, and

[0048] - First complementary group Cg 1

[0049] Contact with a second compound comprising the following:

[0050] -Solubilizing group SE and

[0051] -Second complementary group Cg 2

[0052] Intermediate form of Formula II:

[0053] ,

[0054] Formula II

[0055] SE via R 2 Connected to X, and where R 2 Through Cg 1 With Cg 2 The reaction forms;

[0056] (ii) Remove the ammonia protecting group Pg from formula II 1 To expose primary amines;

[0057] (iii) Contacting the primary amine with a third compound comprising:

[0058] -spacer Z 3 ,

[0059] -amine reactive groups, and

[0060] -Third complementary group Cg 3

[0061] Intermediate form of Formula III:

[0062] ;

[0063] Formula III

[0064] Where L is

[0065] (iv) Contact the intermediate of Formula III with a fourth branched compound comprising:

[0066] -Fourth complementary group Cg 4 and

[0067] -Fifth complementary group Cg 5 ,

[0068] Cg 3 With Cg 4 The reaction forms Z 2 ;

[0069] (v) Contact the product of (iv) with a fifth compound comprising:

[0070] -Reactive portion A for binding with the target and

[0071] -Sixth complementary group Cg 6

[0072] Cg 5 With Cg 6 The reaction forms Z 1 To form the modular connector compound of Formula I:

[0073]

[0074] Formula (I).

[0075] In a specific embodiment of the second aspect, if the ClogP of D is less than -1, the present invention relates to a method for synthesizing a modular connector compound according to formula (Ia) of the first aspect, the method comprising:

[0076] (i) Select a first compound that includes the following:

[0077] -Degradation portion,

[0078] - A cleavable peptide sequence W,

[0079] -Amino protecting group Pg 1 It is covalently connected to W.

[0080] -Pharmaceutical active substance D

[0081] As an intermediate of formula IIa:

[0082] ,

[0083] Formula IIa

[0084] (ii) Remove the amino protecting group Pg from formula IIa 1 To expose primary amines;

[0085] (iii) Contacting the primary amine with a third compound comprising:

[0086] -spacer Z 3 ,

[0087] -amine reactive groups, and

[0088] -Third complementary group Cg 3

[0089] Intermediates of Form IIIa:

[0090] ;

[0091] Formula IIIa

[0092] Where L is

[0093] (iv) Contact the intermediate of formula IIIa with a fourth branched compound comprising:

[0094] -Fourth complementary group Cg 4 and

[0095] -Fifth complementary group Cg 5 ,

[0096] Cg 3 With Cg 4 The reaction forms Z 2 ;

[0097] (v) Contact the product of (iv) with a fifth compound comprising:

[0098] -Reactive portion A for binding with the target and

[0099] -Sixth complementary group Cg6

[0100] Cg 5 With Cg 6 The reaction forms Z 1 To form a modular connector compound of formula Ia:

[0101]

[0102] Formula (Ia).

[0103] In a third aspect, the present invention relates to a target-binding drug conjugate (TBDC) comprising at least one reaction product of a modular connector compound according to the first aspect and a corresponding reactive group of a target-binding moiety, wherein the at least one reaction product is the product of a reaction between A of the modular connector compound and the corresponding reactive group of the target-binding moiety, the reaction covalently coupling the modular connector compound to the target-binding moiety. In some embodiments, the corresponding reactive group of the target-binding moiety is a thiol group.

[0104] In some implementations, the target-binding portion of the third aspect's target-binding drug conjugate specifically recognizes and binds to the target sequences or epitopes of the following cancer-specific antigens or cancer-associated antigens: guanylate cyclase C (GUCY2C), differentiation group 33 (CD33), differentiation group 30 (CD30), differentiation group (CD5), differentiation group 19 (CD19), differentiation group 20 (CD20), differentiation group 37 (CD37), differentiation group 45 (CD45), differentiation group 47 (CD47), differentiation group 56 (CD56), and so on. Differentiation group 70 (CD70), differentiation group 79b (CD79b), differentiation group 117 (CD117 / c-Kit), differentiation group 123 (CD123 / IL3RA), differentiation group 137 (CD137 / 4-1BB), differentiation group 138 (CD138), differentiation group 269 (CD269 / B cell maturation antigen, BCMA), sealing protein 18.2, CEA (carcinoembryonic antigen), epithelial cell adhesion molecule (EpCam), DKK1 (Dickkopf-associated protein 1), phosphorus Inositol proteoglycan-3, mucin-1 (MUC-1 / CD227), mucin 5AC (MUC5AC), carbonic anhydrase 9 (CAIX), CXC motif chemokine receptor 1 (CXCR1, CD181), CXC motif chemokine receptor 2 (CXCR2 / CD182), human epidermal growth factor receptor 2 (HER2), differentiation cluster 22 (CD22), connexin cell adhesion molecule 4 (connexin 4), tumor-associated calcium signaling transducer 2 (Trop-2), delta-like protein 3 (DLL3), transmembrane glycoprotein NMB (gpNMB), prostate-specific membrane antigen (PSMA), hepatocyte growth factor receptor (c-Met, CD151), solute carrier family 39 member 6 (LIV-1), folate receptor 1 (FOLR1), mesothelin, carbonic anhydrase 6 (CA6), epidermal growth factor receptor (EGFR), or extracellular nucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mucin 17 (MUC17), and sealing protein 23 (CLDN23).

[0105] In some implementations, the target-binding portion of the third aspect of the drug conjugate is any one of the following: adecatumumab (anti-EpCam), amatuximab (anti-mesothelin), amivantamab (anti-EGFR), besilesomab (anti-CEA), blinatumomab (anti-CD19), brentuximab (anti-CD30), cantuzumab, cibisatamab (anti-CEACAM5), or cirmtuzumab. b. Anti-ROR1, cetuximab (anti-EGFR), clivatuzumab (anti-MUC1), gatipotuzumab (anti-MUC1), coltuximab (anti-CD19), daratumumab (anti-CD38), duligotuzumab (anti-ERBB3), edrecolomab (anti-EpCam), enfortumab (anti-connector protein-4), enoblituzumab (Anti-CD276), Ensituximab (anti-MUC5AC), Epratuzumab (anti-CD22), Farletuzumab (anti-FOLR1), Igovomab (anti-CA-125), Inebilizumab (anti-CD19), Iratumumab (anti-CD30), Labetuzumab (anti-CEA), Margetuximab (anti-HER2 / neu), Matuzumab (anti-EGFR), Motuzumab (M odotuximab (anti-EGFR extracellular domain III), naptumomab (anti-5T4), nalatuximab (anti-CD37), necitumumab (anti-EGFR), nimotuzumab (anti-EGFR), obinutuzumab (anti-CD20), obcaratuzumab (anti-CD20), ocrelizumab (anti-CD20), and ofatumumab (anti-CD20).Anti-CD37), Ontuxizumab (anti-TEM1), Pertuzumab (anti-HER2 / neu), Polotuzumab (anti-CD79b), Rituximab (anti-CD20), Rovalpituzumab (anti-DLL3), Sacituzumab (anti-TROP-2), Seribantumab (anti-ERBB3 / HER3), Tafasitamab (anti-CD19), Tetulomab (anti-CD37), Timigutuzumab (anti-HER2), Trastuzumab (anti-HER2), Ublituximab (anti-CD20), or Zolbetuximab (anti-sealing protein 18.2). In some embodiments, the aforementioned target-binding portion (e.g., antibody) has been recombinantly engineered to include at least one amino acid-substituted Fc domain, wherein the amino acid of the Fc domain has been replaced by the amino acid cysteine. The cysteine-engineered antibodies are particularly advantageous for use with the modular adapter compounds of formula (I) of this disclosure, as they allow for site-specific conjugation between the cysteine-engineered antibody and the modular adapter compound of formula (I), resulting in homogeneous antibody-drug conjugates with a labeling degree of about 2 (e.g., about 1.8, 1.9, 1.95 to about 2.0, 2.05, 2.1, 2.2, or about 1.98, 2.0 to about 2.1, 2.15, 2.2). Corresponding schemes for generating cysteine-substituted or cysteine-engineered antibodies have been disclosed in: WO 2016040856 A2 or Junutula et al., 2008b Nature Biotech., 26(8):925-932 (both are incorporated herein by reference). As disclosed above, the preferred cysteine ​​substitution in the Fc region of the antibody is the corresponding position in the D265C IgG1 Fc domain or in IgG2 and IgG4 antibodies (according to EU numbering system, Edelman et al., Proc Natl Acad Sci USA. May 1969; 63(1):78-85), as disclosed in WO 2016142049 A1.

[0106] Preferably, the antibody or target-binding moiety described above has been engineered to further include amino acid substitutions for L234A and L235A (as per the EU numbering system), said substitutions reducing the interaction of the Fc domain of the corresponding antibody with FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptors. In some embodiments, the antibody above may include amino acid substitutions such as N297A, P329G, G236R, or combinations of L234F, L235E, and D265C, said substitutions reducing interaction with FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptors.

[0107] In the fourth aspect, the target-binding drug conjugate of the third aspect is used as a drug, preferably for use in cancer treatment.

[0108] Therefore, this fourth aspect of the invention also covers a method of treating cancer, wherein the method includes administering the target-binding drug conjugate of the third aspect to a subject in need; and / or the use of the target-binding drug conjugate of the third aspect in the manufacture of a medicament for treating cancer.

[0109] In some implementations, cancer is typically characterized by the expression of at least one of the aforementioned cancer-specific antigens or cancer-associated antigens.

[0110] Therefore, in a fifth aspect, the present invention relates to a pharmaceutical composition comprising a target-binding pharmaceutical conjugate according to a third or fourth aspect.

[0111] In another aspect, the present invention also relates to a modular connector compound, preferably the modular connector compound of the first aspect when synthesized by the method of the second aspect.

[0112] In another aspect, the present invention also relates to the use of the modular connector compound according to the first aspect in the manufacture of a target-binding drug conjugate according to the fourth aspect. Attached Figure Description

[0113] Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0114] Figure 1. In vitro cytotoxicity of the linker-payload conjugate (IV.1) to (A) SKBR3 cells and (B) JIMT-1 cells. T-LALA-D265C represents a trastuzumab variant containing L234A, L235A, and D265C mutations (numbered according to the EU numbering system) in its Fc region. T-LALA-D265C-va-PAB-exa represents the control DAR2 eczemab ADC containing a self-degrading valine-alanine dipeptide linker lacking a solubilizing group.

[0115] Figure 2 shows the in vitro cytotoxicity of the anti-GCC ADCs containing the linker-loador conjugates (IV.4) and (IV.6) to (A) HEK cells expressing human GUCY2C (HEK293-GUCY2C-HDP-2B3 cells) or (B) HEK wild-type cells. The cysteine ​​adduct of the linker-loador (IV.6) was used as a free compound.

[0116] Figure 3 shows the in vitro cytotoxicity of the anti-GCC ADCs containing the adapter-payload conjugates (IV.5) and (IV.6) to (A) HEK cells expressing human GUCY2C (HEK293-GUCY2C-HDP-2B3 cells) or (B) HEK293 wild-type cells. SEC indicates that the ADCs were purified by size exclusion chromatography.

[0117] Figure 4 shows the in vitro cytotoxicity of the linker-loador conjugates (IV.8), (IV.17), and (IV.19) against (A) HEK cells overexpressing human GUCY2C (HEK293-GUCY2C-HDP-2B3 cells) or (B) HEK293 wild-type cells. SEC indicates ADC purification by size exclusion chromatography. “N / A” indicates that EC50 was not calculated.

[0118] Figure 5 shows the in vitro cytotoxicity of the anti-GCC ADCs containing adapter-payload conjugates (IV.9), (IV.12), and (IV.18) to (A) HEK cells overexpressing human GUCY2C (HEK293-GUCY2C-HDP-2B3 cells) or (B) HEK293 wild-type cells.

[0119] Figure 6 shows the in vitro cytotoxicity of the anti-GCC ADCs containing adapter-load conjugates (IV.9), (IV.12), and (IV.18) to (A) HEK cells overexpressing human GUCY2C (HEK293-GUCY2C-HDP-2B3 cells) or (B) HEK293 wild-type (wt) cells.

[0120] Figure 7 shows the in vitro cytotoxicity of the anti-GCC ADCs containing adapter-loador conjugates (IV.18), (IV.16), and (IV.6) to (A) HEK cells overexpressing human GUCY2C (HEK293-GUCY2C-HDP-2B3 cells) or (B) HEK293 wild-type cells. SEC indicates that the ADCs were purified by size exclusion chromatography.

[0121] Figure 8The mean tumor volume [mm] depicted from day 0 to day 94 after group allocation in mice treated with mediator control, eczema-TBDC IV.6 of the present invention, or delutec-ADC (as a single dose of 25 mg / kg on day 0, or 1x / week for 3 weeks) was shown. 3 (n = 10 animals / group, depicted mean ± SD). “hD” indicates a high DAR (DAR10) ADC. In the animals described, eczema-TBDC-IV.6, administered as a single dose or in multiple doses, was equally effective in reducing tumor burden compared to hD-based ADCs targeting the same target.

[0122] Figure 9 The mean tumor volume [mm] depicted from day 0 to day 67 after group allocation in mice treated with the mediator control or with eczema-TBDC IV.2 and IV.11 of the present invention (as a single dose of 15 mg / kg on day 1). 3 (n = 10 animals / group, depicted mean ± SD).

[0123] Figure 10 The mean tumor volume [mm] depicted from day 0 to day 61 after group allocation in mice treated with either the mediator control or with eczema-TBDC IV.6 of the present invention (as a single dose of 15 mg / kg on day 1). 3 (n = 10 animals / group, depicted mean ± SD).

[0124] Figure 11 The mean tumor volume [mm] depicted from day 0 to day 70 after group allocation in mice treated with a mediator control or with eczema-TBDC IV.2 (20 mg / kg, as a single dose or multiple doses) of the present invention. 3 (n = 9 animals / group, depicted mean ± SD).

[0125] Figure 12 The mean tumor volume [mm] depicted from day 0 to day 70 after group allocation in mice treated with a mediator control or with eczema-TBDC IV.5 (20 mg / kg, as a single dose or multiple doses) of the present invention. 3 (n = 9 animals / group, depicted mean ± SD).

[0126] Figure 13The mean tumor volume [mm] depicted from day 0 to day 74 after group allocation in mice treated with the mediator control or with eczema-TBDC IV.16 or IV.3 (15 mg / kg, as a single dose) of the present invention. 3 (n = 10 animals / group, depicted mean ± SD).

[0127] Figure 14 The mean tumor volume [mm] depicted from day 0 to day 66 after group allocation in mice treated with the medium control or with eczema-TBDC IV.9 of the present invention (as a single dose of 15 mg / kg on day 1, or 1x / week for 3 weeks, 10 or 15 mg / kg). 3 (n = 10 animals / group, depicted mean ± SD).

[0128] Figure 15 The mean tumor volume [mm] depicted from day 0 to day 66 post-group allocation in mice treated with either the mediator control or with eczema-TBDC IV.1 or IV.20 of the present invention (as a single dose of 15 mg / kg on day 1). 3 (n = 10 animals / group, depicted mean ± SD).

[0129] Figure 16 The mean tumor volume [mm] depicted from day 0 to day 66 after group allocation in mice treated with either the mediator control or with eczema-TBDC IV.8 of the present invention (as a single dose of 15 mg / kg on day 1). 3 (n = 10 animals / group, depicted mean ± SD).

[0130] Figure 17 The mean tumor volume [mm] depicted from day 0 to day 66 after group allocation in mice treated with the mediator control or with eczema-TBDC IV.18 of the present invention (as a single dose of 15 mg / kg on day 1). 3 (n = 10 animals / group, depicted mean ± SD).

[0131] Figure 18 The mean tumor volume [mm] plotted from day 0 to day 22 after group allocation in mice treated with the medium control or with eczema-TBDC IV.9, IV.8, IV.12, IV.18, or IV.20 of the present invention (1x / week for 3 weeks, each 5 mg / kg) was shown. 3[(n = 10 animals, depicted mean ± SD). "hD" indicates high DAR (DAR10). The eciletecan-TBDC of the present invention, comprising compounds (IV.9), (IV.12), (IV.18), and (IV.22), reduced tumor burden more effectively than hD-based derunotecan ADCs targeting the same target at the same total dose of administered ADC (or eciletecan-TBDC), while the eciletecan-TBDC of the present invention, comprising compound (IV.8), was equally effective. This indicates that lower doses of (IV.8), (IV.9), (IV.12), (IV.18), and (IV.22) are equally or more effective in reducing tumor burden in animals compared to higher doses of derunotecan-load.]

[0132] Figure 19 The mean tumor volume [mm] plotted from day 0 to day 22 after group allocation in mice treated with the medium control or with eczema-TBDC IV.9, IV.8, IV.12, IV.18, or IV.20 of the present invention (1x / week for 3 weeks, each at 2.5 mg / kg) was shown. 3 [(n = 10 animals, depicted mean ± SD). The results showed that lower doses (IV.8), (IV.9), (IV.12), (IV.18), and (IV.22) were equally or more effective than higher doses of derutec-load in reducing tumor burden in animals.]

[0133] Figure 20 Displays the average tumor volume [mm] from day 0 to day 100 after group assignment. 3 [Measured by calipers. Mice were treated with mediator control, IV.9, IV.8, IV.12, IV.18, or IV.22 (1x / week for 3 weeks, each at 5 mg / kg) (n = 10 animals, depicted mean ± SD).

[0134] Figure 21 Displays the average tumor volume [mm] from day 0 to day 100 after group assignment. 3 [Measured by calipers. Mice were treated with mediator control, IV.9, IV.8, IV.12, IV.18, or IV.22 (1x / week for 3 weeks, each at 2.5 mg / kg) (n = 10 animals, depicted mean ± SD).

[0135] Figure 22The overall survival rate is depicted from day 0 to day 100 after group allocation. Survival rate was monitored daily. Mice were treated with vector control, IV.9, IV.8, IV.12, IV.18, or IV.22 (1x / week for 3 weeks, each at 5 mg / kg) (n = 10 animals, depicted mean ± SD).

[0136] Figure 23 The overall survival rate is depicted from day 0 to day 100 after group assignment. Survival rate was monitored daily. Mice were treated with vector control, IV.9, IV.8, IV.12, IV.18, or IV.22 (1x / week for 3 weeks, each at 2.5 mg / kg) (n = 10 animals, depicted mean ± SD).

[0137] Figure 24 The average tumor volume [mm] depicted from day 0 to day 87 after group assignment is shown. 3 [Measured by calipers. Mice were treated with a mediator control, IV.9, IV.8, IV.12, IV.18, or IV.22 (a single intravenous dose of 10 mg / kg) (n = 10 animals, depicted mean ± SD).

[0138] Figure 25 The average tumor volume [mm] depicted from day 0 to day 87 after group assignment is shown. 3 [Measured by calipers. Mice were treated with a mediator control, IV.9, IV.8, IV.12, IV.18, or IV.22 (a single intravenous dose of 5 mg / kg) (n = 10 animals, depicted mean ± SD).

[0139] Figure 26 The overall survival rate is depicted from day 0 to day 87 after group allocation. Survival rate was monitored daily. Mice were treated with a vector control, IV.9, IV.8, IV.12, IV.18, or IV.22 (a single intravenous dose of 10 mg / kg) (n = 10 animals, depicted mean ± SD).

[0140] Figure 27 The overall survival rate is depicted from day 0 to day 87 after group allocation. Survival rate was monitored daily. Mice were treated with a vector control, IV.9, IV.8, IV.12, IV.18, or IV.22 (a single intravenous dose of 5 mg / kg) (n = 10 animals, depicted mean ± SD).

[0141] Figure 28The results showed that in a 96-h BrdU ELISA of Her2-expressing NCI-N87 cells, the anti-Her2 conjugates exhibited fully mature cytotoxicity, with EC50 values ​​between 1.749 x 10⁻¹¹ M (T-LALA-D265C-compound IV.27) and 3.279 x 10⁻¹² M (T-LALA-D265C-compound IV.25).

[0142] Figure 29 The results showed that the anti-Her2 conjugate had no cytotoxicity in a 96-h BrdU ELISA performed on target-negative MDA-MB-231 cells.

[0143] Figure 30 The results showed that in the co-culture of Trop2-expressing NCI-N87 cells and TLR7-expressing reporter cell line (HEK-hTLR7), the anti-Trop2 TLR7 conjugate exhibited strong dose-dependent TLR7 activation in both.

[0144] Figure 31 shows the conjugates generated using the linker / load (L / P) method. Derivatives with or without solubilizers were purified by preparative FPLC using a HiLoad 16 / 600 Superdex 200pg pre-packed XK16 column (equilibrated with 1x PBS (pH 7.4) at a flow rate of 1.6 mL / min). The elution curve at 280 nm shows the main peak corresponding to the monomer eluting at approximately 80 min, while aggregates and other high molecular weight species (HMWS) eluted earlier, between 60 and 70 min. The conjugate generated with L / P (left) with solubilizer shows significantly less aggregate in the elution curve compared to the conjugate generated without solubilizer (right). Detailed Implementation

[0145] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be defined only by the appended claims. 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.

[0146] Preferably, the terms used herein are defined as described in the following reference: "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by Leuenberger, HGW, Nagel, B. and Kolbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland. Unless otherwise specified, the definitions of chemical groups used herein shall have the meanings provided in the "Compendium of Chemical Terminology" ("Gold Book") (version 2.3.3, goldbook.iupac.org, ISBN: 0-9678550-9-8) published by the International Union of Pure and Applied Chemistry (IUPAC), and the contents of the aforementioned references are hereby incorporated by reference as defined therein.

[0147] Nevertheless, in order to provide a clear and consistent understanding of the specification and claims, as well as the scope of certain terms to be given, the following definitions are provided.

[0148] definition

[0149] In the context of this disclosure, the term "modular adapter compound" refers to an adapter comprising at least one molecule of a cytotoxic drug substance (also referred to simply as a drug or payload), several other structural modules and a functional module (also referred to as a spacer or functional unit or group, including at least one solubilizing group and a branching unit), and a reactive group suitable for stably attaching the modular adapter compound to a target-binding moiety (such as an antibody or TCR).

[0150] In the context of this disclosure, the term "solubilizing group" refers to a module within a modular connector compound that at least counteracts (or at most eliminates) the hydrophobic / lipophilic properties of a cytotoxic drug substance, for example, to provide a modular connector compound comprising a specific payload having high water solubility. A variety of soubilizing groups suitable for incorporation into modular connector compounds of this disclosure are well known to those skilled in the art and include, but are not limited to: (a) polar functional groups, such as hydroxyl (-OH), amine (-NH2), and carboxyl (-COOH) groups; polar sulfonate (-SO3H) groups; polymeric chains, such as hydrophilic polyethylene glycol (PEG) chains; surfactant groups, such as alkyl chains; aromatic ring structures; halogen substituents, such as fluorine or chlorine additions; ester (-COO-) or ether (-O-) groups; and cyclodextrin / cyclic oligosaccharides.

[0151] In the context of this disclosure, the term "one or more copolyols prepared from one or more carbohydrates" refers to a group of alcohols derived from carbohydrates. The terms "polyalcohol," "polyol," and "sugar alcohol" are used synonymously.

[0152] In the context of this disclosure, the term "PEG" refers to polyethylene glycol, and the term "(PEG)" refers to... 1-10 "Refers to connectors having 1 to 10 ethylene glycol portions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ethylene glycol portions)"

[0153] .

[0154] The term "C2-amide-PEG" 1-10 "Refers to a connector having 1 to 10 ethylene glycol moieties (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 ethylene glycol moieties) covalently attached to a propionamide group."

[0155] .

[0156] In the context of this disclosure, the term "self-degrading spacer" refers to a spacer that is stably linked to a cytotoxic drug substance but undergoes triggered self-degradation (i.e., decomposition), resulting in the elimination of its link to the cytotoxic drug substance and the release of the drug substance without any adducts or traces of the self-degrading spacer itself. In some embodiments, a change in pH may trigger the self-degradation of the spacer and the release of the payload. In other embodiments, the self-degrading spacer is also linked to an enzyme-cleavable peptide sequence, and enzymatic cleavage of said peptide triggers the self-degradation of the spacer and the release of the payload. An illustrative example of an enzyme-triggered self-degrading spacer is a p-aminobenzyl (PAB) spacer linked to an enzyme-cleavable Val-Ala dipeptide.

[0157] In the context of this disclosure, the term "cleavable linker compound" refers to a linker compound that is (i) cleavable by an enzyme or (ii) cleavable in a reducing environment. Furthermore, the terms "enzyme-cleavable" or "enzyme-cleavable" indicate that the peptide, structure, sequence, or spacer in question can be cleaved by an enzyme, particularly by lysosomal proteases (such as cathepsin B), thereby resulting in the release of a cytotoxic drug substance / payload.

[0158] In the context of this disclosure, the term "branching unit" refers to a structure within a modular connector compound that allows the compound to branch into a branched structure, wherein each branch is typically linked to at least one molecule of a cytotoxic drug substance via a self-degrading spacer. Thus, modular connector compounds typically contain as many drug molecules as branches. Therefore, modular connector compounds containing between 1 and 10 branches (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10), preferably between 2 and 8, 2 and 6, or 2 and 4, typically contain between 1 and 10 branches (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10), preferably between 2 and 8, 2 and 6, or 2 and 4, respectively.

[0159] In the context of this disclosure, the term "reactive portion for coupling with the target binding portion" refers to a chemically reactive group within the linker that can react with a corresponding reactive group of the target binding portion under suitable conditions, such that the resulting reaction product covalently couples the remaining linker compound to the target binding portion.

[0160] In the context of this disclosure, the term "reaction product of the modular connector compound with the corresponding reactive group of the target binding portion" refers to the portion formed due to the reaction between the reactive portion of the modular connector compound of the present invention and the corresponding reactive group of the target binding portion. For example, if the reactive portion of the modular connector compound of the present invention is a maleimide, the reaction product obtained from the reaction of the maleimide with the sulfur atom of the cysteine ​​residue of the target binding portion is a succinimide thioether or a derivative thereof. Furthermore, if the reactive portion of the modular connector compound of the present invention is a group that selectively reacts with the thiol group of the free cysteine ​​of the target binding portion, i.e., if the reactive portion of the modular connector compound is a "thiol reactive portion", the reaction product is generally selected from: thiol-substituted acetamide; thiol-substituted succinimide; thiol-substituted succinic acid; thiol-substituted heteroaryl groups, particularly thiol-substituted benzothiazoles, thiol-substituted phenyltetrazoles, and thiol-substituted phenyloxadiazoles; and disulfides, wherein one sulfur atom is derived from the cysteine ​​residue of the target binding portion.

[0161] In the context of this disclosure, the term "target-binding moiety" refers to a polypeptide molecule that specifically recognizes and binds to a target sequence or epitope of an antigen, relying on the concept of immunoglobulin (Ig) recognition known from conventional Ig forms (i.e., IgG, IgD, IgE, IgA, and / or IgM). Therefore, in certain embodiments, the target-binding moiety may at least include a complementarity-determining region (CDR) of Ig sufficient to confer target / epitope specificity, and in some cases, the target-binding moiety may include a variable heavy chain (V) of Ig. H ) and variable light chains (V L The target-binding moiety is sequenced to impart target / epitope specificity. In some embodiments, the target-binding moiety may be Ig itself, such as IgG. However, this does not mean that the target-binding moiety must still be in the form of that Ig. Rather, the target-binding moiety may also be an Ig fragment or a derivative thereof, such as a single-chain variable fragment (scFv) or an antigen-binding fragment (Fab, obtained by papain cleavage of IgG; F(ab'), obtained by pepsin cleavage of IgG; and / or F(ab')2, obtained by pepsin cleavage of IgG and subsequent β-mercaptoethanol treatment; etc.). Similarly, the immunoreactive molecule of this disclosure may be a novel antibody form, such as, but not limited to, bispecific or trispecific antibody constructs, biantibodies, camelid antibodies, domain antibodies, nanobodies, divalent homodimers having two chains composed of scFv, shark antibodies, antibodies consisting of a New World primate framework plus a non-New World primate CDR, or containing CH3+V. L +V H The dimerized construct.

[0162] In a particular implementation, the target-binding portion includes the T-cell receptor (TCR). The TCR is a disulfide-linked membrane-anchored heterodimer, typically composed of highly variable alpha (α) and beta (β) chains expressed on the surface of T cells as part of a complex with an invariant CD3 chain molecule. A crucial step in the formation of the TCR heterodimer is called "pairing." T cells expressing the paired receptor are called α:β (or αβ) T cells, but a minority of T cells express alternative receptors formed by variable gamma (γ) and delta (δ) chains, and are called γδ T cells. Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region, both belonging to the immunoglobulin superfamily (IgSF) domains, which form antiparallel β-sheets. The constant region is located close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex of antigen-presenting cells.

[0163] As used herein, if the dissociation constant KD of the target-binding portion with the antigen is 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less and even more preferably 10 If nM or lower, the target-binding moiety is considered to "specifically recognize and bind" the target sequence or epitope of the antigen.

[0164] In the context of this disclosure, the term "pharmaceutically acceptable salt or solvate" includes acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or with organic acids such as glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, inaleic acid, fumaric acid, tartaric acid, citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, lauryl sulfate, gluconic acid, glutamic acid, salicylic acid, mucoconic acid, etc. The term also includes base addition salts formed with the conjugate base of any of the inorganic acids listed above, wherein the conjugate base comprises a cation selected from: Na... + K + Mg 2+ Ca 2+ and NHgR' 4-g + ; where R' is a C1-3 alkyl group and g is a number selected from 0, 1, 2, 3 or 4; and the solvation form of the corresponding acid addition salt (i.e., the solvate).

[0165] As used herein, “treat,” “treating,” or “treatment” for a disease or disorder means to accomplish one or more of the following: (a) reducing the severity of the disease; (b) limiting or preventing the development of symptoms specific to one or more disorders being treated; (c) suppressing the worsening of symptoms specific to one or more disorders being treated; (d) limiting or preventing the recurrence of said one or more disorders in a patient who previously had one or more disorders; and (e) limiting or preventing the recurrence of symptoms in a patient who previously had symptoms of one or more disorders.

[0166] As used herein, "patient" means any mammal or bird that may benefit from treatment with the target-binding drug conjugates described herein. Preferably, "patient" is selected from laboratory animals (e.g., mice or rats), livestock (including, for example, guinea pigs, rabbits, chickens, pigs, sheep, goats, camels, cattle, horses, donkeys, cats, or dogs), or primates (including humans). Particularly preferably, "patient" is a human.

[0167] "Therapeutic effective amount" is the amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary depending on factors such as the nature of the agent, the route of administration, the size and species of the animal to be treated, and the purpose of administration. The effective amount in each individual case can be determined empirically by a person skilled in the art using methods recognized in the field.

[0168] In addition to the definition above, and unless the context clearly requires otherwise, throughout the specification and claims, the words “comprise”, “comprising”, etc., shall be interpreted as inclusive rather than exclusive or exhaustive; that is, they have the meaning of “including but not limited to”.

[0169] Furthermore, throughout this specification, references to "one embodiment," "some embodiments," or "implementation" mean that a particular feature, structure, or characteristic described herein in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment," "in some embodiments," or "in an embodiment" appearing in different places throughout this specification do not necessarily refer to the same embodiment, but may all refer to the same embodiment. Moreover, in one or more embodiments, a particular feature, structure, or characteristic may be combined in any suitable manner, as will be apparent to those skilled in the art based on this disclosure.

[0170] As used herein, unless otherwise specified, the use of ordinal adjectives such as “first,” “second,” “third,” etc., to describe common objects merely indicates reference to different instances of similar objects and is not intended to imply that the objects described in this way must have a given order in time, space, rank, or any other manner.

[0171] As used herein, the term "exemplary" is used in the sense of providing an example, not in the sense of quality. That is, an "exemplary implementation" is an implementation provided as an example, and not necessarily an implementation with exemplary quality.

[0172] As used herein, the partition coefficient (abbreviated as PC) is defined as a specific ratio of the concentrations of the solute between two solvents (liquid-phase two-phase), particularly for unionized solutes, and the logarithm of this ratio is thus clog P. When one solvent is water and the other is n-octanol, the log P value is a measure of lipophilicity or hydrophobicity. (See Comer J, Tam K (2001). "Lipophilicity Profiles: Theory and Measurement", Testa B, van deWaterbed H, Folkers G, Guy R, Comer J, Tam K (eds.). Pharmacokinetic Optimization in Drug Research: Biological, Physicochemical, and Computational Strategies. Weinheim: Wiley-VCH. pp. 275-304.)

[0173] Several references are cited throughout the text of this specification. Each of the references cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number serial submissions, etc.) is incorporated herein by reference in its entirety to the fullest extent permitted by the applicable patent law.

[0174] Modular connector compound

[0175] The inventors of this invention have developed a modular connector compound comprising modules (also referred to as groups, units, elements, etc.) that are associated with the function and efficacy of target-binding drug conjugates (TBDCs) and can be readily assembled in a combinatorial manner to provide the modular connector compound.

[0176] The modular architecture of the modular adapter compound allows for a high degree of flexibility with respect to the selected modules, enabling the modular adapter compound to be adapted to various physicochemical properties of the relevant cytotoxic drug substance and the target-binding moiety to be conjugated with the modular adapter compound. Therefore, it has been found that the modular adapter compounds of this disclosure allow for the generation of TBDCs in a relatively simple manner, which target selected cancer-associated antigens or cancer-specific antigens and contain the preferred cytotoxic drug substance for the treatment of a specific cancer type.

[0177] Furthermore, and as will become clear from the following description, the generation of TBDCs with both uniform labeling (DOL) and drug-antibody ratio (DAR) can be readily achieved through the conjugation chemistry employed in modular linker compounds.

[0178] In a first aspect, the present invention relates to a modular connector compound of formula (I).

[0179]

[0180] Formula (I)

[0181] Or its pharmaceutically acceptable salts or solvates;

[0182] in:

[0183] X is a self-degrading spacer. ,

[0184] in:

[0185] R 1 X is covalently linked to D at the para, meta, or ortho position of the amino group of X, and the bond is a kinetic link. , , , or ;and

[0186] R 2 Connect X to SE and is , , Or -O-;

[0187] D is a pharmaceutically active substance;

[0188] SE is a solubilizing group;

[0189] L is a spacer containing a peptide covalently linked to X;

[0190] n is an integer of at least 2;

[0191] Y is a branching unit; and

[0192] A is the reactive portion used to bind with the target.

[0193] Components of formula (I) The modular connector compound of the first aspect is repeated at least twice (i.e., n is an integer of at least 2), but preferably between 2 and 5 times, such as 2, 3, 4 or 5 times. This advantageous increase in the DOL of a single modular connector compound depends on the polymer core of the compound defined by the degree of branching observed in the branching unit Y.

[0194] As defined above, the self-degrading spacer of the modular linker compound of the present invention is a para- or ortho-aminobenzyl spacer stably linked to a cytotoxic drug substance D and a solubilizing group SE, and undergoes triggered self-degradation (i.e., decomposition) such that at least the cytotoxic drug substance D is released as a metabolite of the self-degradation reaction, and no chemical moiety of the linker compound remains attached to the drug. Specifically, in some embodiments, the amino group of the para-aminobenzyl spacer acts as an electron donor in the electron cascade of the 1,6-self-degradation reaction releasing the cytotoxic drug substance D. Advantageously, this traceless release mechanism ensures that the cytotoxic drug substance is not altered by its connection to the linker compound and subsequent release.

[0195] In a specific embodiment of the first aspect, if the ClogP of the payload (D) is -1 or lower, the present invention relates to a modular connector compound of formula (Ia).

[0196]

[0197] Formula (Ia)

[0198] Or its pharmaceutically acceptable salts or solvates;

[0199] in:

[0200] X is a self-degrading spacer. ,

[0201] in:

[0202] R 1 X is covalently linked to D at the para, meta, or ortho position of the amino group of X, and the bond is a kinetic link. , , , or ;and

[0203] R 2 It is H

[0204] D is a pharmaceutically active substance;

[0205] L is a spacer containing a peptide covalently linked to X;

[0206] n is an integer of at least 2;

[0207] Y is a branching unit; and

[0208] A is the reactive portion used to bind with the target.

[0209] Components of formula (Ia) The modular connector compound of the first aspect is repeated at least twice (i.e., n is an integer of at least 2), but preferably between 2 and 5 times, such as 2, 3, 4 or 5 times. This advantageous increase in the DOL of a single modular connector compound depends on the polymer core of the compound defined by the degree of branching observed in the branching unit Y.

[0210] As defined above, the self-degrading spacer of the modular linker compound of the present invention is a para- or ortho-aminobenzyl spacer stably linked to a cytotoxic drug substance D and a solubilizing group SE, and undergoes triggered self-degradation (i.e., decomposition) such that at least the cytotoxic drug substance D is released as a metabolite of the self-degradation reaction, and no chemical moiety of the linker compound remains attached to the drug. Specifically, in some embodiments, the amino group of the para-aminobenzyl spacer acts as an electron donor in the electron cascade of the 1,6-self-degradation reaction releasing the cytotoxic drug substance D. Advantageously, this traceless release mechanism ensures that the cytotoxic drug substance is not altered by its connection to the linker compound and subsequent release.

[0211] In some embodiments, the self-degradation reaction also results in the release of the solubilizing group when the solubilizing group SE is also attached to the para or ortho position of the aminobenzyl spacer. However, as will be understood, in contrast to the release of the cytotoxic drug substance D, the release of the solubilizing group is not particularly related to the functionality and efficacy of the TBDC based on the modular connector compound. In contrast, the modular connector compound contains an SE group attached at the meta position of the aminobenzyl spacer to the self-degrading spacer, and a cytotoxic drug substance attached at the adjacent para position of the aminobenzyl spacer. Without being bound by theory, the close proximity of the solubilizing group SE to the cytotoxic drug substance D may lead to better masking of the lipophilic / hydrophobic properties of the drug substance, for example, to further increase the solubility of the modular connector compound.

[0212] In a preferred embodiment, the degradation spacer X is a component containing R 1 With R 2 One of the specific combinations of X1.1 to X1.3, X2.1 to X2.3, X3.1 to X3.3, and X4.1 to X4.3, is shown directly in the table below:

[0213]

[0214] In one or more of the above implementation schemes, L is

[0215] ,

[0216] in:

[0217] W is selected from the following cleavable or non-cleavable peptide sequences:

[0218] Val-Ala, Val-Cit, Met-Thr, Thr-Thr, Phe-Lys, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, Gly-Gly-Tyr-Gly, cBu-Ala, cBu-Cit, Glu-cBu-Ala, Glu-cBu-Cit, Asp-cBu-Ala, Asp-cBu-Cit, Asp-Pro-Val, Asn-Pro-Val, iGlu-cBu-Ala, iGlu-cBu-Cit, Val-Lys, Val-Arg, Val-Gln, Val-Met, Val-Thr, AcLys-Val-Cit, AcLys-Val -Ala, Asp-Val-Ala, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Tyr-Arg, Tyr-Met, Phe-Cit, Phe-Ala, Phe-Arg, Phe-Met, Phe-Gln, Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly, Ala-Lys, Leu-Cit, Leu-Gln, Ile-Cit, Ala-Ala-Asn, Ala-Asn, Ala-Ala, Asn-Asn, Asn, β-Ala and Lys or combinations thereof; and where Z 3 It is a bond or spacer, the spacer containing C 1-20 Alkyl chains or 1 to 10 polyethylene glycol (PEG) 1-10 or C2-amide-PEG 1-10 .

[0219] Preferably, L is

[0220] or .

[0221] The cleavable peptide sequence W of L is a target of lysosomal proteases, making TBDC, which internalizes after recognizing and binding to a target sequence or target antigen and contains the modular linker compound of the present invention, a substrate for lysosomal proteases. In human lysosomes, the primary proteases are cysteine ​​proteases of the cathepsin family (including cathepsins B, C, D, H, L, S, and Z). Other relevant human lysosomal proteases involved in lysosomal protein degradation include: lentinan (also known as asparagine endopeptidase (AEP)), an asparagine endopeptidase; pepsin-insensitive protease 1, pepsin-sensitive protease A, and pepsin-insensitive protease B, all of which are aspartic proteases. Preferably, the peptide sequence W is cleavable by cathepsin B. Once the peptide sequence W of L is cleaved, an electronic cascade of self-degradation is triggered, resulting in the traceless release of the drug substance from the self-degrading spacer X.

[0222] The cleavable peptide sequence W is a sequence targeted by the lysosomal proteases discussed above and may contain non-protein amino acids (such as citrulline (Cit)) or modified amino acids (such as cysteine ​​butyl ester (cBu)), non-natural amino acid isomers (such as isofpartic-glutamic acid (iGlu) or N-acetyllysine (AcLys)). As described, once cleaved, an electronic cascade leading to the elimination of self-degrading spacers and the traceless release of drug substance D is initiated. In a preferred embodiment, the dipeptide valine-alanine (Val-Ala) constitutes W. In an alternative preferred embodiment, the dipeptide valine-citrulline (Val-Cit) constitutes W. In yet another preferred embodiment, the dipeptide phenylalanine-lysine (Phe-Lys) constitutes W. In yet another preferred embodiment, W comprises the tripeptide glutamic-valine-alanine (Glu-Val-Ala) or glutamic-valine-citrulline (Glu-Val-Cit).

[0223] In some implementation schemes, Z 3 This is the key that directly links L to branch unit Y. In an alternative implementation, Z... 3 Includes: C 1-20 Alkyl chain, preferably C 2-18 Or C 4-16 Or C 6-14 Or C 8-12 Alkyl chain; or 1 to 10 polyethylene glycol (PEG). 1-10 PEG is preferred 2-8 or PEG 4-6 Or PEG5; or C2-amide-PEG 1-10 C2-amide-PEG is preferred. 2-8 or C2-amide-PEG 2-6 Or C2-amide-PEG4.

[0224] Thus, in a preferred embodiment, L is

[0225] ,

[0226] Where W stands for Val-Ala.

[0227] In another preferred embodiment, L is

[0228] ,

[0229] Where W stands for Val-Cit.

[0230] In another preferred embodiment, L is

[0231] ,

[0232] Where W stands for Phe-Lys.

[0233] In another preferred embodiment, L is

[0234] ,

[0235] Where W stands for Glu-Val-Ala.

[0236] In another preferred embodiment, L is

[0237] ,

[0238] Where W stands for Glu-Val-Cit.

[0239] In some implementations, the Y of the modular connector compound of the first aspect is:

[0240] or .

[0241] In such implementations:

[0242] m is an integer from 1 to 6, preferably 1, 2, 3, 4, 5 or 6;

[0243] Z 1 Connect Y to A and it is

[0244] or

[0245] ,in

[0246] R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-, and

[0247] p is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, or 8 to 12.

[0248] q is an integer from 1 to 8, such as 2, 3, 4, 5, 6, 7, or 8.

[0249] r is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, 8 to 12, 2, 3, 4, 5, 6, 7 or 8;

[0250] Z 2 Connect Y to L and select from:

[0251] , , , , , , , , , , , , , , , , , , , , , , , , , ,and ;

[0252] B is:

[0253] -Cap, preferably acyl, and n' is n; or

[0254] - or ,in

[0255] i is an integer from 1 to 20.

[0256] j is an integer from 1 to 10, and

[0257] n' is n minus 1; or

[0258] - ,in

[0259] D' is a pharmaceutically active substance different from D; and

[0260] X' is a self-degrading spacer. ,in:

[0261] R 5 X' is covalently linked to D' at the para or ortho position of the amino group of X', and it is a bond. , or ,

[0262] And n' is n; and

[0263] R 4 It is OH or NH2.

[0264] In the preferred embodiment, Z 2 It is selected from the following 1,2,3-triazole, amide, or urea derivatives: , , , , , , , , , , and .

[0265] If Z 2 Selected from

[0266] , , , , , , , , , , , , , ,and ;

[0267] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0268] In some implementations, B is Z in L 3 It may further include a terminal carbonyl moiety (such as a dicarboxylic acid) for the amino linkage of L and Y.

[0269] Regardless of B

[0270] The cap, preferably an acyl group, wherein n' is n; or

[0271] or ,in

[0272] i is an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0273] j is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and

[0274] n' is n minus 1; or

[0275] unit,

[0276] B constitutes the end of the final branching unit Y in the polymeric core of the modular connector compound in these implementation schemes.

[0277] As shown above, the multi-core can contain n' = 2 to 5 (preferably 2, 3, 4 or 5) branch units Y, wherein the branch units closest to A are connected via Z. 1 Connected to A, and the final branch unit (i.e., the branch unit furthest from A) contains B. Each branch unit Y in this embodiment is connected via spacer Z. 2 Spacer L and degradation spacer X are indirectly linked to a molecule of pharmaceutical drug substance D.

[0278] Thus, if B is a cap structure, the loading degree (DOL) of the modular connector compound containing a polymer core with n' = 5 branch units Y is 5, or the loading degree is 6 if B is:

[0279] or ,in

[0280] i is an integer from 1 to 20.

[0281] j is an integer from 1 to 10, and

[0282] n' is n minus 1; or

[0283] unit.

[0284] Therefore: when n'=4, the DOL is 4 or 5; when n'=3, the DOL is 3 or 4; and when n'=2, the DOL is 2 or 3. Preferably, n' = 2, B is the terminal acyl group (such as acetyl) as the cap, and the DOL of the modular connector compound is 2.

[0285] In a particularly preferred embodiment, branch unit Y is

[0286]

[0287] in:

[0288] B is an acyl cap, preferably an acetyl group.

[0289] n'=2,

[0290] Z 2 It is 1,2,3-triazole And m=4, and

[0291] Z 1 yes ,in

[0292] R 3 It is -NH-, q=3 and r=4.

[0293] In an alternative implementation, the branching unit Y of the modular connector compound of the first aspect is:

[0294] , ,

[0295] , ,

[0296] , ,

[0297] or ;

[0298] Z 1 Connect Y to A and it is or ,in

[0299] R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-.

[0300] p is an integer from 1 to 24, preferably from 4 to 24, more preferably from 8 to 24, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0301] q is an integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0302] r is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, 8 to 12, 2, 3, 4, 5, 6, 7 or 8;

[0303] s is an integer from 0 to 4, such as 0, 1, 2, 3 or 4;

[0304] Z 2 Connect Y to L and select from:

[0305] , , , , , , , , , , , , , , , , , , , , , , , , , ,and ;

[0306] In the preferred embodiment, Z 2 Connect Y to L and it is selected from the following 1,2,3-triazole, amide, amide, or urea derivative:

[0307] , , , , , , , , , , and .

[0308] If Z 2 Selected from , , , , , , , , , , , , , ,and It can be attached or used in the following reactions: thiol-alkynyl / alkene reaction, cross metathesis and cross coupling reaction, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0309] Depending on which of the alternative branch units Y shown above is used, the DOL of the modular connector compound of the present invention can be 2, 3, 4 or 5.

[0310] Advantageously, the modular adapter compounds of the present invention allow for variability in the cytotoxic pharmaceutical substances relating to TBDCs, enabling the use of different mechanisms of action, depending on the type of cancerous target cells and the desired therapeutic outcome. Typically, the pharmaceutically active substances D and D' are sufficiently potent to kill or inhibit the growth of cancerous target cells at relatively low concentrations (e.g., equal to or below about 1 µM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM) while possessing low immunogenicity characteristics, for example, to ensure patient tolerability. Importantly, they are generally stable in systemic circulation and remain attached to the adapter compounds.

[0311] Therefore, in some embodiments of the modular adapter compound of the present invention, the pharmaceutically active substances D and D' (i.e., one or more cytotoxic drug substances incorporated into the modular adapter compound with the above-mentioned DOL and to be delivered to cancerous target cells) are independently selected from: camptothecin, preferably eczema, SN38 and topotecan; auristatin, preferably selected from methylauristatin E (MMAE) and methylauristatin F (MMAF); maytansine, preferably selected from mitrensine (DM1) and ravtansine (DM4); calicheamicin; Toll-like receptor 7 (TLR7) agonists; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPT...i ): Pyrrolobenzodiazepine (PBD); pyroxine; anthracyclines, preferably doxorubicin; amatoxins, preferably α-amaminine, β-amaminine, amatoxin and amaninamide; nostocin; phalloidin; open-ring-cyclopropylpyrroloindoline (open-ring-CPI); open-ring-cyclopropylbenzoindoline (open-ring-CBI); paclitaxel; vinblastine; HDAC inhibitors; colchicine; eribulin; methotrexate (MTX); triptolide; and their derivatives.

[0312] However, as mentioned above, many of these substances may have unfavorable low solubility in aqueous solutions (such as physiological / systemic solutions). Therefore, to avoid undesirable aggregation of TBDCs containing the modular connector compounds of the present invention, the modular connector compounds must contain a solubilizing group SE. The solubilizing group SE can be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, polysarcosine, PEG, galactoside, glucuronide, or a polyol prepared from carbohydrates.

[0313] Cyclodextrins are cyclic oligosaccharides composed of glucose units arranged in a ring (donut) shape. They have a hydrophobic interior and a hydrophilic exterior. This unique structure allows them to form inclusion complexes with hydrophobic or poorly soluble molecules, and the solubility of hydrophobic / lipophilic pharmaceutical substances in aqueous solutions generally increases when complexed with cyclodextrins. Therefore, hydrophobic pharmaceutically active substances can be encapsulated within the hydrophobic pockets of cyclodextrins, shielding them from the surrounding aqueous environment and thus enhancing their solubility. Nevertheless, cyclodextrins are chemically compatible with a wide variety of pharmaceutically active substances and are generally unreactive to molecules encapsulated by forming inclusion complexes. This allows for the simultaneous maintenance of the complex stability and integrity of pharmaceutically active substances.

[0314] In addition, cyclodextrins have been widely used in pharmaceuticals, food and cosmetics for many years, and their overall safety and biocompatibility have been established to be favorable.

[0315] Therefore, in the context of the linker compounds described herein, cyclodextrins are particularly suitable solubilizing groups (SEs). For example, directly or via spacers (Zs). 4 When attached to a PEG chain, α-cyclodextrin (containing six glucose molecules), β-cyclodextrin (containing seven glucose molecules), and γ-cyclodextrin (containing eight glucose molecules) are used, such as:

[0316] ,

[0317] ,

[0318] , , , ,

[0319] Z 4 yes

[0320] , , , , , , , , , , , , , , , , , , , , , , , , , ,and ;

[0321] Furthermore, the PEG chain contains between 2 and 19 PEGs, where t is 2 to 18, 4 to 16, 6 to 14, and preferably 8, 9, 10, 11, or 12.

[0322] Suitable variants that constitute the solubilizing group SE.

[0323] In the preferred embodiment, Z 4 It is selected from the following 1,2,3-triazoles, amides, amides, or urea derivatives:

[0324] , , , , , , , , , , and .

[0325] If Z 4 Selected from , , , , , , , , , , , , , ,and It can be attached or used in the following reactions: thiol-alkynyl / alkene reaction, cross metathesis and cross coupling reaction, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0326] exist In embodiments where the pharmaceutical drug substance D within the unit is camptothecin, it is particularly advantageous to connect the cyclodextrin reinforcing group of the type described above to the self-degrading spacer X via an 8- to 12-membered PEG chain (i.e., where t = 8 to 12, for example 8, 9, 10, 11, or 12). This PEG chain length favors a conformational arrangement of the linker compound that allows the inclusion complex to place the lipophilic drug substance into a pouch with a cyclodextrin-containing structure, thereby substantially eliminating the hydrophobic / lipophilic properties of camptothecin.

[0327] Other suitable solubilizing groups include other polar groups, such as

[0328] Polysarcosine Where u is at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14;

[0329] Polysarcosamide Where u is at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14;

[0330] PEG chain , where v is 4 to 24, such as 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, or 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0331] ,

[0332] PEG-glucamide ,

[0333] ,

[0334] ,

[0335] ,

[0336] ,

[0337] ,or ;

[0338] Where w is 2 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0339] The terminal reactive group A of the modular connector compound of the present invention ensures specific and controlled binding of the connector compound to the target binding site, enabling the formation of stable TBDC. In this regard, various reactive groups A have proven useful. For example, in some embodiments, reactive group A is a thiol reactive group, such as...

[0340] 3-Maleimide propionamide group ,

[0341] 6-Maleimide hexamethylene amide group or

[0342] 3-Amino-2-maleimide propionamide group .

[0343] Thiol reactive groups have been shown to provide a robust and versatile means of conjugating adapter compounds to TBDCs. They allow site-specific conjugation via corresponding reactive thiol groups in the target-binding moiety. Specifically, they allow the conjugation of the modular adapter compounds of the present invention to a naturally occurring thiol moiety in the target-binding moiety, or to a thiol moiety that has been genetically engineered into the target-binding moiety, as described, for example, in Junutula Nat Biotechnol. Aug. 2008; 26(8): 925-32. Preferably, the modular adapter compounds disclosed herein are conjugated via one of the thiol reactive groups shown above to a thiol moiety in the Fc region of the target-binding moiety conjugated to the adapter, as disclosed above, for example in antibodies containing D265C (according to EU number, Edelman et al., Proc. Natl. Acad. Sci. USA; 63 (1969) 78-85.).

[0344] Advantageously, thiol reactions are highly efficient and generally proceed rapidly under mild conditions. This means that the thiol reactive group can rapidly form conjugates with high completion. Furthermore, the thioether bond formed between the thiol group and the thiol reactive group is stable under physiological conditions. Although the thioether bond is less susceptible to hydrolysis or degradation compared to some other types of chemical bonds, further stability of the conjugate can be achieved by incorporating a basic amino group adjacent to the maleimide, as described by Lyon et al., Nat. Biotechnol. 2014, 32, 1059-1062.

[0345] Furthermore, thiol reactive chemistry is highly flexible, offering a variety of thiol reactive groups that can be incorporated into modular linker compounds, which are generally biocompatible (i.e., do not introduce highly toxic or immunogenic components into TBDCs) and allow for precise control of the drug-antibody ratio (DAR) in TBDCs, which is crucial for optimizing therapeutic efficacy and minimizing off-target effects as mentioned above.

[0346] In alternative embodiments, reactive group A can be a 3-(2-((1,2-dimethylhydrazyl)methyl)-1H-indol-1-yl)propionamide or a 7-aza derivative for conjugation to the formylglycine moiety in the target binding site via a hydrazyl-Pictet-Spengler (HIPS) linker.

[0347] or .

[0348] In alternative embodiments, reactive group A may be a compound selected from DBCO (aza-dibenzocyclooctyne) or BCN (bicyclo[6.1.0]non-4-yn-9-ylcarbonyl) as a strained cycloalkyne, intended for conjugation with antibodies containing azide or azide-modified compounds, for example via 4-azido-phenylalanine or azide-modified glycan side chains, such as “SiteClick”. Specifically, reactive group A may be

[0349] , or

[0350] In other alternative embodiments, reactive group A can be a glycine derivative, which is particularly useful for enzymatic coupling reactions using sorting enzyme A. An exemplary glycine derivative can be a polyglycine containing between 2 and 5 (e.g., 2, 3, 4, and 5) glycine residues:

[0351] .

[0352] Glycine is a particularly small amino acid, and its incorporation into the modular connector compound of the present invention and ultimately into the TBDC to be prepared minimizes steric hindrance while allowing flexible attachment without disrupting the structure of the connector compound or the target binding site.

[0353] In other alternative embodiments, reactive group A may be a primary amine, for example, a lysine group. Or any group containing a primary amine (e.g., amino-modified PEG). Such conjugation can be performed, for example, using transglutaminase, as described in, for example, WO 2015 / 162563 A1.

[0354] Using lysine groups allows modular linker compounds to be controlled conjugated to selected corresponding reactive sites in the target binding moiety (e.g., antibodies modified to contain site-specific maleimide or N-hydroxysuccinimide (NHS) ester adducts) via their reaction with the primary amine group (-NH2) of lysine.

[0355] The versatility of lysine residues as conjugation sites in target-binding moieties (such as antibodies) makes them valuable in ADC development, as they have long been used for the controlled (but still random) attachment of drugs or other therapeutic molecules to antibodies. However, it has been shown that specific modifications to specific residues within the target-binding moieties backbone structure, such as to present specific attachment sites for linker compounds carrying lysine, are useful in generating TBDCs with specific DOL and DAR.

[0356] In a preferred embodiment, the modular connector compound of the present invention is selected from...

[0357]

[0358] SE is selected from

[0359] - Polysarcosine Where u is at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14.

[0360] - Polysarcosinate Where u is at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14;

[0361] -PEG chain , where v is 4 to 24, such as 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, or 4, 5, 6, 7, 8, 9, 10, 11, 12 or 14;

[0362] ,

[0363] PEG-glucamide ,

[0364] ,

[0365] ,

[0366] ,

[0367]

[0368] ,or ;,

[0369] Where w is 2 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or

[0370] - Directly or via spacer Z 4 α-Cyclodextrin, β-Cyclodextrin, or γ-Cyclodextrin attached to the PEG chain, such as

[0371] , , , , , ,

[0372] Z 4 yes

[0373] , , , , , , , , , , , , , , , , , , , , , , , , , ,and ;

[0374] Furthermore, the PEG chain contains between 2 and 19 PEGs, i.e., t is 2 to 18, 4 to 16, 6 to 14, and preferably 8 to 12.

[0375] In the preferred embodiment, Z 4 It is selected from the following 1,2,3-triazoles, amides, amides, or urea derivatives:

[0376] , , , , , , , , , , and .

[0377] If Z 4 Selected from

[0378] , , , , , , , , , , , , , ,and ;

[0379] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0380] In some embodiments, the solubilizing group SE used in the above-described modular connector compounds (IIIb.1) to (IIIb.6) is selected from...

[0381] ,

[0382] ,

[0383] ,

[0384] ,

[0385] ,

[0386] ,

[0387] ,

[0388] ,

[0389] ,

[0390] ,

[0391] ,

[0392] ,

[0393] ,

[0394] , ,

[0395] ,

[0396] ,

[0397]

[0398] , , ,or A particularly preferred embodiment of the modular connector compound of the present invention is:

[0399]

[0400] The inventors have further come to understand that the modular connector compound of the first aspect can be synthesized using building blocks in a particularly advantageous high-yield method, said building blocks being reliably assembled into the modular connector compound of the present invention via robust and predictable orthogonal click chemistry reactions.

[0401] Therefore, in a second aspect, the present invention relates to a method for synthesizing a modular connector compound according to formula (I) of the first aspect, the method comprising:

[0402] (i) Make a first compound comprising the following:

[0403] -Degradation portion,

[0404] - A cleavable peptide sequence W,

[0405] -Amino protecting group Pg 1 It is covalently connected to W.

[0406] -Pharmaceutical active substance D, and

[0407] - First complementary group Cg 1

[0408] Contact with a second compound comprising the following:

[0409] -Solubilizing group SE and

[0410] -Second complementary group Cg 2

[0411] Intermediate form of Formula II:

[0412] ,

[0413] Formula II

[0414] SE via R 2 Connected to X, and where R 2 Through Cg 1 With Cg 2 The reaction forms;

[0415] (ii) Remove the ammonia protecting group Pg from formula II 1 To expose primary amines;

[0416] (iii) Contacting the primary amine with a third compound comprising:

[0417] -spacer Z 3 ,

[0418] -amine reactive groups, and

[0419] -Third complementary group Cg 3

[0420] Intermediate form of Formula III:

[0421] ;

[0422] Formula III

[0423] Where L is

[0424] (iv) Contact the intermediate of Formula III with a fourth branched compound comprising:

[0425] -Fourth complementary group Cg 4 and

[0426] -Fifth complementary group Cg 5 ,

[0427] Cg 3 With Cg 4 The reaction forms Z 2 ;

[0428] (v) Contact the product of (iv) with a fifth compound comprising:

[0429] -Reactive portion A for binding with the target and

[0430] -Sixth complementary group Cg 6

[0431] Cg 5 With Cg 6 The reaction forms Z 1 To form the modular connector compound of Formula I:

[0432]

[0433] Formula (I)

[0434] In embodiments of the method for synthesizing the modular connector compound of formula (I) of the second aspect (including embodiments of the method described subsequently in the second aspect).

[0435] Cg 1 It can be an alkyne group, and Cg2 It can be an azide group, or

[0436] Cg 1 It can be a primary or secondary amine group, and Cg 2 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group.

[0437] Furthermore, in embodiments of the method for synthesizing the modular connector compound of formula (I) of the second aspect (including embodiments of the method subsequently described in the second aspect).

[0438] Cg 3 It can be an alkyne group, and Cg 4 It can be an azide group;

[0439] Cg 3 It can be an azide group, and Cg 4 It can be an alkyne group;

[0440] Cg 3 It can be a primary or secondary amine group, and Cg 4 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; an amide; a carbamate; a carbamoyl halide; or a urea group; or

[0441] Cg 3 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; an amide; a carbamate; a carbamoyl halide; or a urea group, and Cg 4 It can be a primary amine or a secondary amine group.

[0442] In other embodiments of the method for synthesizing the modular connector compound of formula (I) of the second aspect (including embodiments of the method subsequently described in the second aspect),

[0443] Cg 5 It can be a primary or secondary amine group, and Cg 6 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group; or

[0444] Cg 5 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group, and Cg6 It can be a primary amine or a secondary amine group.

[0445] In some embodiments of the method for synthesizing the modular connector compound of formula (I) of the second aspect (including embodiments of the method subsequently described in the second aspect), the amino protecting group Pg 1 It can be benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-bethoxybenzyl (PMB), p-toluenesulfonyl (Ts) or carbamate group.

[0446] As will be described in more detail in the examples below, in some embodiments, during the steps of the method for synthesizing the modular connector compound of the second aspect, a well-known copper-catalyzed azide-alkyne cycloaddition click chemistry reaction (CuAAC reaction) is employed to efficiently link various building blocks of the modular connector compound of form (I).

[0447] In this method for synthesizing modular connector compounds of formula (I),

[0448] X is a self-degrading spacer. ,

[0449] in:

[0450] R 1 X is covalently linked to D at the para, meta, or ortho position of the amino group of X, and

[0451] Is it a key or , or ;and

[0452] R 2 Connect X to SE and is , Or -O-;

[0453] In some implementations, the cleavable or non-cleavable peptide sequence W is selected from:

[0454] Val-Ala, Val-Cit, Met-Thr, Thr-Thr, Phe-Lys, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, Gly-Gly-Tyr-Gly, cBu-Ala, cBu-Cit, Glu-cBu-Ala, Glu-cBu-Cit, Asp-cBu-Ala, Asp-cBu-Cit, Asp-Pro-Val, Asn-Pro-Val, iGlu-cBu-Ala, iGlu-cBu-Cit, Val-Lys, Val-Arg, Val-Gln, Val-Met, Val-Thr, AcLys-Val-Cit, AcLys-Va l-Ala, Asp-Val-Ala, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Tyr-Arg, Tyr-Met, Phe-Cit, Phe-Ala, Phe-Arg, Phe-Met, Phe-Gln, Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly, Ala-Lys, Leu-Cit, Leu-Gln, Ile-Cit, Ala-Ala-Asn, Ala-Asn, Ala-Ala, Asn-Asn, Asn, β-Ala and Lys or combinations thereof; and

[0455] Z 3 It is a bond or spacer, the spacer containing C 1-20 Alkyl chains or 1 to 10 polyethylene glycol (PEG) 1-10 or C2-amide-PEG 1-10 ;

[0456] Where L is preferably

[0457] or .

[0458] As described above, the branching unit Y of the modular connector compound of formula (I) synthesized in the method of the second aspect

[0459]

[0460] Formula (I)

[0461] yes:

[0462] or ;

[0463] m is an integer from 1 to 6;

[0464] Z 1 Connect Y to A and it is or ,in

[0465] R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-, and

[0466] p is an integer from 1 to 24, preferably from 4 to 24, more preferably from 8 to 24, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0467] q is an integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0468] r is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, 8 to 12, 2, 3, 4, 5, 6, 7 or 8;

[0469] Z 2 Connect Y to L and it is selected from the following 1,2,3-triazole, amide, or urea derivatives:

[0470] , , , , , , , , , , , , , , , , , , , , , , , , , ,and .

[0471] B is:

[0472] The cap, preferably an acyl group, and n' is n; or

[0473] or ,in

[0474] i is an integer from 1 to 20.

[0475] j is an integer from 1 to 10, and

[0476] n' is n minus 1; or

[0477] ,in

[0478] D' is a pharmaceutically active substance different from D; and

[0479] X' is a self-degrading spacer. ,in:

[0480] R 5 X' is covalently linked to D' at the para or ortho position of the amino group of X', and it is a bond. , or ,

[0481] And n' is n;

[0482] R 4 It is OH or NH2.

[0483] If Z 4 Selected from

[0484] , , , , , , , , , , , , , ,and ,

[0485] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0486] In B is In the implementation plan, Z in L 3It may further include a terminal carbonyl moiety (such as a dicarboxylic acid) for the amino linkage of L and Y.

[0487] Regardless of B

[0488] The cap, preferably an acyl group, wherein n' is n; or

[0489] or ,in

[0490] i is an integer from 1 to 20.

[0491] j is an integer from 1 to 10, and

[0492] n' is n minus 1; or

[0493] unit,

[0494] B constitutes the end of the final branching unit Y in the polymeric core of the modular connector compound of formula (I) synthesized in these embodiments of the second aspect.

[0495] As described above, the polymeric core in the modular connector compound of formula (I) synthesized by the method of the second aspect can contain n=2 to 5 (preferably 2, 3, 4 or 5) branching units Y, wherein the branching units closest to A are connected via Z. 1 Connected to A, and the final branch unit (i.e., the branch unit furthest from A) contains B. Each branch unit Y in this embodiment is connected via spacer Z. 2 Spacer L and degradation spacer X are indirectly linked to a molecule of pharmaceutical drug substance D.

[0496] In an alternative embodiment, the branching unit Y of the modular connector compound of formula (I) synthesized in the method of the second aspect is:

[0497] , ,

[0498] , , , , or

[0499] ;

[0500] Z 1 Connect Y to A and it is

[0501] or ,in

[0502] R3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-.

[0503] p is an integer from 1 to 24, preferably from 4 to 24, more preferably from 8 to 24, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0504] q is an integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0505] r is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, 8 to 12, 2, 3, 4, 5, 6, 7 or 8;

[0506] s is an integer from 0 to 4, such as 0, 1, 2, 3 or 4;

[0507] Z 2 Connect Y to L and it is selected from the following 1,2,3-triazole, amide, amide, or urea derivative:

[0508] , , , , , , , , , , , , , , , , , , , , , , , , , ,and .

[0509] If Z 4 Selected from

[0510] , , , , , , , , , , , , , ,and ,

[0511] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0512] Depending on which of the alternative branch units Y shown above is used, the DOL of the modular connector compound of formula (I) can be 2, 3, 4 or 5.

[0513] Therefore, in some embodiments of the method of the present invention for synthesizing modular adapter compounds, the pharmaceutically active substances D and D' (i.e., one or more cytotoxic drug substances incorporated into the modular adapter compound with the above-described DOL and to be delivered to cancerous target cells) are independently selected from: camptothecin, preferably eczema, SN38, and topotecan; auristatin, preferably selected from methylauristatin E (MMAE) and methylauristatin F (MMAF); maytansine, preferably selected from metansine (DM1) and lavtansine (DM4); chalcogenide; Toll-like receptor 7 (TLR7) agonists; nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPT... i ): Pyrrolobenzodiazepine (PBD); pyroxine; anthracyclines, preferably doxorubicin; amatoxins, preferably α-amaminine, β-amaminine, amatoxins and amatoxins; nostocin; phalloidin; open-ring-cyclopropylpyrroloindoline (open-ring-CPI); open-ring-cyclopropylbenzoindoline (open-ring-CBI); paclitaxel; vincristine; colchicine; eribulin; methotrexate (MTX); triptolide; and their derivatives.

[0514] To facilitate the above, cyclodextrin is a particularly suitable solubilizing group (SE) in the context of the connector compounds and their synthetic methods described herein. For example, directly or via spacer Z... 4 When attached to a PEG chain, α-cyclodextrin (containing six glucose molecules), β-cyclodextrin, and γ-cyclodextrin, such as:

[0515] ,

[0516] ,

[0517] ,

[0518] ,

[0519] ,

[0520] ,

[0521] Z 4 yes

[0522] , , , , , , , ; , , , , , , , , , , , , , , , , , ,and ;

[0523] Furthermore, the PEG chain contains between 2 and 19 PEGs, where t is 2 to 18, 4 to 16, 6 to 14, and preferably 8 to 12.

[0524] Suitable variants that constitute the solubilizing group SE.

[0525] If Z 4 Selected from

[0526] , , , , , , , , , , , , , ,and ,

[0527] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0528] The modular connector compound synthesized in the second aspect of the method In embodiments where the pharmaceutical drug substance D within the unit is camptothecin, it is particularly advantageous to link the cyclodextrin reinforcing group of the type described above to the self-degrading spacer X via an 8- to 12-membered PEG chain (i.e., where t = 8 to 12). This PEG chain length favors a conformational arrangement of the linker compound that allows the inclusion complex to place the lipophilic drug substance into a pouch with a cyclodextrin-containing structure, thereby substantially eliminating the hydrophobic / lipophilic properties of camptothecin.

[0529] Other suitable solubilizing groups for the modular connector compounds synthesized in the second aspect of the method include other polar groups, such as

[0530] Polysarcosine Where u is at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14.

[0531] Polysarcosamide Where u is at least 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 14.

[0532] PEG chain Where v is 4 to 24, such as 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, or 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0533] ,

[0534] PEG-glucamide ,

[0535] ,

[0536] ,

[0537] ,

[0538] or ;

[0539] Where w is 2 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0540] As described above, the terminal reactive group A of the modular connector compound of formula (I) (i.e., the modular connector compound synthesized in the method of the second aspect) ensures the specific controlled conjugation of the connector compound to the target binding site, thereby enabling the generation of stable TBDC.

[0541] In this regard, various reactive groups A have proven useful. For example, in some embodiments, reactive group A is a thiol reactive group, such as...

[0542] 3-Maleimide propionamide group ,

[0543] 6-Maleimide hexamethylene amide group or

[0544] 3-Amino-2-maleimide propionamide group .

[0545] In alternative embodiments, reactive group A can be a 3-(2-((1,2-dimethylhydrazyl)methyl)-1H-indol-1-yl)propionamide or a 7-aza derivative for conjugation to the formylglycine moiety in the target binding site via a hydrazyl-Pictet-Spengler (HIPS) linker.

[0546] or .

[0547] In an alternative embodiment, reactive group A may be a triazine compound, which allows for triazine-based conjugation of the linker compound to an amino group (primarily in lysine residues) in the target binding moiety. Specifically, reactive group A may be...

[0548] or .

[0549] In other alternative embodiments, reactive group A can be a glycine derivative, which is particularly useful, for example, for enzymatic coupling reactions using sorting enzyme A. An exemplary glycine derivative can be a polyglycine containing between 2 and 5 glycine residues:

[0550] .

[0551] In other alternative embodiments, reactive group A can be a primary amine, such as a lysine group. Or any group containing a primary amine (e.g., amino-modified PEG). Such conjugation can be performed, for example, using transglutaminase, as described in, for example, WO 2015 / 162563 A1.

[0552] In other alternative embodiments, reactive group A may be a bicyclic [6.1.0]non-4-yn-9-ylcarbonyl (BCN) group. .

[0553] BCNs can react with molecules containing highly strained olefin or alkyne bonds (such as tetraazines or other cyclic alkynes) via a bioorthogonal reaction known as the inverse electron demand diels-Alder (IEDDA) reaction. This reaction allows for the selective and efficient conjugation of modular linker compounds to the target-binding moiety, thus providing a means of generating TBDCs with improved specificity and reduced off-target effects. BCNs and their derivatives are gaining popularity in ADC development due to their bioorthogonal properties and ability to facilitate precise drug-antibody conjugation.

[0554] Therefore, in a specific embodiment of the third aspect, if the clogP of the pharmaceutically active compound is less than -1, the present invention relates to a method for synthesizing a modular connector compound according to formula (Ia) of the first aspect, the method comprising:

[0555] (i) Select compounds that include the following:

[0556] -Degradation portion,

[0557] - A cleavable peptide sequence W,

[0558] -Amino protecting group Pg 1 It is covalently connected to W.

[0559] -Pharmaceutical active substance D as an intermediate of formula IIa:

[0560] ,

[0561] Formula IIa

[0562] (ii) Removal of the ammonia protecting group Pg from formula IIa 1 To expose primary amines;

[0563] (iii) Contacting the primary amine with a third compound comprising:

[0564] -spacer Z 3 ,

[0565] -amine reactive groups, and

[0566] -Third complementary group Cg 3

[0567] Intermediates of Form IIIa:

[0568] ;

[0569] Formula IIIa

[0570] Where L is

[0571] (iv) Contact the intermediate of formula IIIa with a fourth branched compound comprising:

[0572] -Fourth complementary group Cg 4 and

[0573] -Fifth complementary group Cg 5 ,

[0574] Cg 3 With Cg 4 The reaction forms Z 2 ;

[0575] (v) Contact the product of (iv) with a fifth compound comprising:

[0576] -Reactive portion A for binding with the target and

[0577] -Sixth complementary group Cg 6

[0578] Cg 5 With Cg 6 The reaction forms Z 1 To form a modular connector compound of formula Ia:

[0579]

[0580] Formula (Ia)

[0581] In embodiments of the method for synthesizing the modular connector compound of formula (Ia) of the third aspect (including embodiments of the method subsequently described in the third aspect), (including embodiments of the method subsequently described in the third aspect).

[0582] Cg 3 It can be an alkyne group, and Cg 4 It can be an azide group;

[0583] Cg 3 It can be an azide group, and Cg 4 It can be an alkyne group;

[0584] Cg 3 It can be a primary or secondary amine group, and Cg4 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; an amide; a carbamate; a carbamoyl halide; or a urea group; or

[0585] Cg 3 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; an amide; a carbamate; a carbamoyl halide; or a urea group, and Cg 4 It can be a primary amine or a secondary amine group.

[0586] In other embodiments of the method for synthesizing the modular connector compound of formula (Ia) of the third aspect (including embodiments of the method of the second aspect described subsequently).

[0587] Cg 5 It can be a primary or secondary amine group, and Cg 6 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group; or

[0588] Cg 5 It can be a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group, and Cg 6 It can be a primary amine or a secondary amine group.

[0589] In some embodiments of the method for synthesizing the modular connector compound of formula (Ia) of the third aspect (including embodiments of the method subsequently described in the third aspect), the amino protecting group Pg 1 It can be benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-benzyloxybenzyl (PMB), p-toluenesulfonyl (Ts) or carbamate group.

[0590] As will be described in more detail in the examples below, in some embodiments, during the steps of the method for synthesizing the modular connector compound of the second aspect, a well-known copper-catalyzed azide-alkyne cycloaddition click chemistry reaction (CuAAC reaction) is employed to efficiently link various building blocks of the modular connector compound of form (Ia).

[0591] In this method for synthesizing modular connector compounds of formula (Ia),

[0592] X is a self-degrading spacer. ,

[0593] in:

[0594] R 1 X is covalently linked to D at the para, meta, or ortho position of the amino group of X.

[0595] Is it a key or , or ;and

[0596] R 2 It is H.

[0597] W is a cleavable and non-cleavable peptide sequence linking Z3 and X, and is selected from:

[0598] Val-Ala, Val-Cit, Met-Thr, Thr-Thr, Phe-Lys, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, Gly-Gly-Tyr-Gly, cBu-Ala, cBu-Cit, Glu-cBu-Ala, Glu-cBu-Cit, Asp-cBu-Ala, Asp-cBu-Cit, Asp-Pro-Val, Asn-Pro-Val, iGlu-cBu-Ala, iGlu-cBu-Cit, Val-Lys, Val-Arg, Val-Gln, Val-Met, Val-Thr, AcLys-Val-Cit, AcLys-Val -Ala, Asp-Val-Ala, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Tyr-Arg, Tyr-Met, Phe-Cit, Phe-Ala, Phe-Arg, Phe-Met, Phe-Gln, Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly, Ala-Lys, Leu-Cit, Leu-Gln, Ile-Cit, Ala-Ala-Asn, Ala-Asn, Ala-Ala, Asn-Asn, Asn, β-Ala and Lys or combinations thereof; and where Z 3 It is a bond or spacer, the spacer containing C 1-20 Alkyl chains or 1 to 10 polyethylene glycol (PEG) 1-10 or C2-amide-PEG 1-10 ;

[0599] Where L is preferably

[0600] or .

[0601] As described above, the branching unit Y of the modular connector compound of formula (I) synthesized in the method of the third aspect

[0602]

[0603] Formula (Ia)

[0604] yes:

[0605] or ;

[0606] m is an integer from 1 to 6;

[0607] Z 1 Connect Y to A and it is or ,in

[0608] R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-, and

[0609] p is an integer from 1 to 24, preferably from 4 to 24, more preferably from 8 to 24, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0610] q is an integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0611] r is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, 8 to 12, 2, 3, 4, 5, 6, 7 or 8;

[0612] Z 2 Connect Y to L and it is selected from the following 1,2,3-triazole, amide, or urea derivatives:

[0613] , , , , , , , , , , , , , , , , , , , , , , , , , ,and .

[0614] B is:

[0615] The cap, preferably an acyl group, and n' is n; or

[0616] or ,in

[0617] i is an integer from 1 to 20.

[0618] j is an integer from 1 to 10, and

[0619] n' is n minus 1; or

[0620] ,in

[0621] D' is a pharmaceutically active substance different from D; and

[0622] X' is a self-degrading spacer. ,in:

[0623] R 5 X' is covalently linked to D' at the para or ortho position of the amino group of X', and it is a bond. , or ,

[0624] And n' is n;

[0625] R 4 It is OH or NH2.

[0626] If Z 4 Selected from

[0627] , , , , , , , , , , , , , ,and ,

[0628] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0629] In B is In the implementation plan, Z in L 3 It may further include a terminal carbonyl moiety (such as a dicarboxylic acid) for the amino linkage of L and Y.

[0630] Regardless of B

[0631] The cap, preferably an acyl group, wherein n' is n; or

[0632] or ,in

[0633] i is an integer from 1 to 20.

[0634] j is an integer from 1 to 10, and

[0635] n' is n minus 1; or

[0636] unit,

[0637] B constitutes the end of the final branching unit Y in the polymeric core of the modular connector compound of formula (Ia) synthesized in these embodiments of the second aspect.

[0638] As described above, the polymeric core in the modular connector compound of formula (Ia) synthesized by the method of the third aspect can contain n=2 to 5 (preferably 2, 3, 4 or 5) branching units Y, wherein the branching units closest to A are connected via Z. 1 Connected to A, and the final branch unit (i.e., the branch unit furthest from A) contains B. Each branch unit Y in this embodiment is connected via spacer Z. 2 Spacer L and degradation spacer X are indirectly linked to a molecule of pharmaceutical drug substance D.

[0639] In an alternative embodiment, the branching unit Y of the modular connector compound of formula (Ia) synthesized in the method of the third aspect is:

[0640] , ,

[0641] , , , , or

[0642] ;

[0643] Z 1 Connect Y to A and it is

[0644] or ,in

[0645] R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-.

[0646] p is an integer from 1 to 24, preferably from 4 to 24, more preferably from 8 to 24, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24;

[0647] q is an integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0648] r is an integer from 1 to 24, such as 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 16, 6 to 14, 8 to 12, 2, 3, 4, 5, 6, 7 or 8;

[0649] s is an integer from 0 to 4, such as 0, 1, 2, 3 or 4;

[0650] Z 2 Connect Y to L and it is selected from the following 1,2,3-triazole, amide, or urea derivatives:

[0651] , , , , , , , , , , , , , , , , , , , , , , , , , ,and .

[0652] If Z 4 Selected from

[0653] , , , , , , , , , , , , , ,and ,

[0654] It can be attached or used in the following reactions: thiol-alkynyl / alkene reactions, cross metathesis and cross coupling reactions, such as Heck coupling, Kumada coupling, Negishi coupling, Hiyama coupling, Stille coupling, Suzuki coupling, Sonogashira coupling and Glaser coupling.

[0655] Depending on which of the alternative branch units Y shown above is used, the DOL of the modular connector compound of formula (I) can be 2, 3, 4 or 5.

[0656] As described above, the terminal reactive group A of the modular connector compound of formula (Ia) (i.e., the modular connector compound synthesized in the method of the third aspect) ensures the specific controlled conjugation of the connector compound to the target binding site, thereby enabling the generation of stable TBDC.

[0657] In this regard, various reactive groups A have proven useful. For example, in some embodiments, reactive group A is a thiol reactive group, such as...

[0658] 3-Maleimide propionamide group ,

[0659] 6-Maleimide hexamethylene amide group or

[0660] 3-Amino-2-maleimide propionamide group .

[0661] In alternative embodiments, reactive group A can be a 3-(2-((1,2-dimethylhydrazyl)methyl)-1H-indol-1-yl)propionamide or a 7-aza derivative for conjugation to the formylglycine moiety in the target binding site via a hydrazyl-Pictet-Spengler (HIPS) linker.

[0662] or .

[0663] In an alternative embodiment, reactive group A may be a triazine compound, which allows for triazine-based conjugation of the linker compound to an amino group (primarily in lysine residues) in the target binding moiety. Specifically, reactive group A may be...

[0664] or .

[0665] In other alternative embodiments, reactive group A can be a glycine derivative, which is particularly useful, for example, for enzymatic coupling reactions using sorting enzyme A. An exemplary glycine derivative can be a polyglycine containing between 2 and 5 glycine residues:

[0666] .

[0667] In other alternative embodiments, reactive group A can be a primary amine, such as a lysine group. Or any group containing a primary amine (e.g., amino-modified PEG). Such conjugation can be performed, for example, using transglutaminase, as described in, for example, WO 2015 / 162563 A1.

[0668] In other alternative embodiments, reactive group A may be a bicyclic [6.1.0]non-4-yn-9-ylcarbonyl (BCN) group. .

[0669] BCNs can react with molecules containing highly strained olefin or alkyne bonds (such as tetraazines or other cyclic alkynes) via a bioorthogonal reaction known as the inverse electron demand diels-Alder (IEDDA) reaction. This reaction allows for the selective and efficient conjugation of modular linker compounds to the target-binding moiety, thus providing a means of generating TBDCs with improved specificity and reduced off-target effects. BCNs and their derivatives are gaining popularity in ADC development due to their bioorthogonal properties and ability to facilitate precise drug-antibody conjugation.

[0670] A particularly preferred embodiment of the modular connector compound of the present invention, which can be obtained by the method of the third aspect, is the direct end product of the preferred embodiment of the method of the third aspect shown above, as well as in the further embodiments below.

[0671] In a fourth aspect, the present invention relates to a target-binding drug conjugate (TBDC) comprising at least one reaction product of a modular connector compound according to the first aspect and a corresponding reactive group of a target-binding moiety, wherein the at least one reaction product is the product of a reaction between A of the modular connector compound and a corresponding reactive group of the target-binding moiety, the reaction covalently coupling the modular connector compound to the target-binding moiety, preferably the corresponding reactive group of the target-binding moiety being a thiol group.

[0672] Preferably, the TBDC of the fourth aspect comprises more than one of the reaction products, such that more than one, preferably between two and ten, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 modular connector compounds are coupled to the target binding portion.

[0673] As described above, each modular connector compound in the fourth aspect contains at least two The unit, thus each modular connector compound has at least two (2) DOLs. In an embodiment of the fourth aspect, if the TBDC contains more than one of the above reaction products, the DOLs of the modular connector compound are multiplied by the number of reaction products. For example, if the TBDC contains four (4) of the reaction products of the reactive group A of the modular connector compound according to the fourth aspect with four (4) corresponding reactive groups of the target binding portion, the TBDC will contain four (4) such modular connector compounds and will thus have a DAR of 8.

[0674] In another aspect, the present invention also relates to a modular connector compound, preferably the modular connector compound of the first aspect when synthesized by the method of the second aspect.

[0675] In another aspect, the present invention also relates to the use of the modular connector compound according to the first aspect in the manufacture of a target-binding drug conjugate according to the fourth aspect.

[0676] As will generally be understood, based on the mechanism of action and potency of the pharmacologically active pharmaceutical ingredient D contained in the modular connector compound within the TBDC and the specific therapeutic outcome sought, the modular connector compound of this disclosure allows for the preparation of TBDCs with variable DAR scores in a relatively straightforward manner (i.e., in combination). This is also specifically shown in the further embodiments below.

[0677] The target-binding moiety is the portion that specifically recognizes and binds to the target sequence or epitope of an antigen. As further defined above, the target-binding moiety relies on the concept of an immunoglobulin (Ig; i.e., antibody) that recognizes the target / epitaxe known from the molecules in their conventional Ig forms (i.e., IgG, IgD, IgE, IgA, and / or IgM) for the specific recognition and binding of the target sequence or epitope of the antigen.

[0678] Therefore, the target binding portion can be:

[0679] - Antibodies, such as monoclonal antibodies;

[0680] - Functional antibody fragments or antibody derivatives, such as single-chain variable fragments (scFv), antigen-binding fragments such as Fab fragments, F(ab') fragments, F(ab')2 fragments, or bispecific or trispecific antibody constructs;

[0681] -Double antibody;

[0682] -Cameloid antibodies;

[0683] - Domain antibodies;

[0684] -Nanobodies;

[0685] - A divalent homodimer with two chains composed of scFv;

[0686] - Shark antibodies;

[0687] -An antibody composed of a New World primate framework sequence plus a non-New World primate complementarity-determining region (CDR); or

[0688] - A dimerized construct comprising a constant heavy chain 3 (CH3) domain and a variable light chain (V) domain. L ) and variable heavy chain (V H )

[0689] -TCR.

[0690] Preferably, the target-binding portion of the TBDC in the fourth aspect specifically recognizes and binds to the target sequences or epitopes of the following cancer-specific antigens or cancer-associated antigens: guanylate cyclase C (GUCY2C), differentiation cluster 33 (CD33), differentiation cluster 30 (CD30), differentiation cluster (CD5), differentiation cluster 20 (CD20), differentiation cluster 45 (CD45), differentiation cluster 117 (CD117 / c-Kit), differentiation cluster 123 (CD123 / IL3RA), differentiation cluster 137 (CD137 / 4-1BB), differentiation cluster 269 (CD269 / B cell maturation antigen, BCMA), sealing protein 18.2, CEA (carcinoembryonic antigen), epithelial cell adhesion molecule (EpCam), and DKK1 (Dickkopf-associated protein 1). Phosphatidylinositol proteoglycan-3, mucin-1 (MUC-1 / CD227), mucin 5AC (MUC5AC), carbonic anhydrase 9 (CAIX), CXC motif chemokine receptor 1 (CXCR1, CD181), CXC motif chemokine receptor 2 (CXCR2 / CD182), human epidermal growth factor receptor 2 (HER2), differentiation cluster 22 (CD22), differentiation cluster 79b (CD79b), connexin cell adhesion molecule 4 (connexin 4), tumor-associated calcium signaling transducer 2 (Trop-2), differentiation cluster 19 (CD19), delta-like protein 3 (DLL3), transmembrane glycoprotein NMB (gpNMB), prostate-specific membrane antigen (PSMA), hepatocyte growth factor receptor (c-Met, CD181), and other related proteins. 151), Solute carrier family 39 member 6 (LIV-1), Folate receptor 1 (FOLR1), Differentiation group 56 (CD56), Differentiation group 138 (CD138), Mesothelin, Carbonic anhydrase 6 (CA6), Epidermal growth factor receptor (EGFR), Differentiation group 37 (CD37), Extracellular nucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3).

[0691] Typically, the target binding site of a tumor-specific drug concentration (TBDC) is an antibody, preferably a monoclonal antibody (mAb). As is widely acknowledged in antibody-mediated cancer therapy, the high specificity of mAbs for a single target sequence or epitope of cancer-specific antigens or cancer-associated antigens significantly reduces off-target effects. Furthermore, the pharmacokinetic properties of mAbs (including half-life, distribution, and clearance rate) can be readily established, allowing for the generation of appropriate TBDCs (in this case, ADCs) to ensure optimal delivery of the active pharmaceutical ingredient to cancerous target cells. In addition, many mAbs targeting the target sequences or epitopes of cancer-specific antigens or cancer-associated antigens are commercially available and have undergone extensive clinical validation.

[0692] In a preferred embodiment, the target binding portion according to the invention is a monoclonal antibody selected from chimeric, humanized, or human monoclonal antibodies; more preferably, the target binding portion is a chimeric, humanized, or human IgG1 or IgG4 monoclonal antibody.

[0693] Therefore, in a preferred embodiment of this fourth aspect of the invention, the target binding portion of TBDC is any one of the following: adenomyumab (anti-EpCam), ametoumab (anti-mesothelin), epantuzumab (anti-EGFR), bexolumab (anti-CEA), belintoumab (anti-CD19), bentoximab (anti-CD30), cantuzumab, cybituzumab (anti-CEACAM5), cetuzumab (anti-ROR1), cetuzumab (anti-EGFR), crimetuzumab (anti-MUC1), gatuzumab. (Anti-MUC1), Coctuximab (anti-CD19), Daratuximab (anti-CD38), Dugoutuzumab (anti-ERBB3), Ezocurumab (anti-EpCam), Enrofloxacin (anti-connectin-4), Enrofloxacin (anti-CD276), Entoximab (anti-MUC5AC), Ipatuzumab (anti-CD22), Faletuzumab (anti-FOLR1), Igvovomab (anti-CA-125), Inellizumab (anti-CD19), Itolimumab (anti-CD30), Labezil (anti-) CEA, maggotuximab (anti-HER2 / neu), maggotuximab (anti-EGFR), mogotuximab (anti-EGFR extracellular domain III), napotumumab (anti-5T4), nagotuximab (anti-CD37), nesetuximab (anti-EGFR), nimotuzumab (anti-EGFR), obbituximab (anti-CD20), oxcartuximab (anti-CD20), ozenatuzumab (anti-CD20), olfamoluzumab (anti-CD20), olletuximab (anti-CD37), ontocitracinuximab (Anti-TEM1), pertuzumab (anti-HER2 / neu), polotuzumab (anti-CD79b), rituximab (anti-CD20), lovatotuzumab (anti-DLL3), goxatotuzumab (anti-TROP-2), seretuzumab (anti-ERBB3 / HER3), tancituzumab (anti-CD19), tetuximab (anti-CD37), telmituzumab (anti-HER2), trastuzumab (anti-HER2), ulrituximab (anti-CD20), or zotuximab (anti-sealing protein 18.2).

[0694] Furthermore, in a specific implementation of the TBDC of the fourth aspect, the commercially available mAb may have been genetically modified, for example as described in Junutula Nat Biotechnol. 2008 Aug;26(8):925-32. When the reactive group a of the linker compound is a thiol reactive group, specific modifications include incorporating a cysteine ​​residue into each heavy chain of the mAb, thereby introducing an additional thiol moiety into the target-binding portion for the conjugation of the modular linker compound of the first aspect. This incorporation can be readily achieved by substituting the amino acids A118C, S239C, and / or D265C (according to the EU numbering system) in each heavy chain of the mAb.

[0695] Preferably, the mAb of this specific embodiment of the TBDC of the fourth aspect contains a D265C substitution in each of its heavy chains and is conjugated to at least one modular linker compound of the first aspect via engineered cysteine ​​residues at said target-binding portion. More preferably, a modular linker compound of the first aspect is conjugated to two engineered cysteine ​​residues at position 265 in each heavy chain of the mAb. As stated above, if the DOL of the modular linker of the first aspect thus conjugated is two (2), then the DAR of the TBDC is four (4). Due to the variability of the modular linker compounds of the present invention, the DAR of the ADC of the mAb containing two engineered cysteine ​​residues at corresponding position 265 in each heavy chain is between four (4) and sixteen (16). In some cases, it may be preferred that the TBDC of the fourth aspect also optionally contains mutants L234A and L235A (numbered according to EU designations) to reduce Fcγ receptor interaction and thereby reduce the uptake of TBDC into immune cells (e.g., macrophages) (see, for example, Liu et al. Antibodies (2020), 9, 64). Therefore, in some embodiments, the TBDC of the present invention comprises an antibody (e.g., a human or humanized IgG1 or IgG4 antibody) containing mutants L234A, L235A, and D265C in each of its heavy chain Fc regions.

[0696] Of course, in the fifth aspect, the TBDC of the fourth aspect is specifically designed for use as a medicine, preferably for use in cancer treatment. Therefore, this fourth aspect of the invention also covers methods of treating cancer, wherein the methods include administering the target-binding drug conjugate of the third aspect to a subject in need; and / or the use of the target-binding drug conjugate of the third aspect in the manufacture of a medicine for treating cancer.

[0697] In some implementations, the cancer is typically characterized by the expression of at least one of the aforementioned cancer-specific antigens or cancer-associated antigens. Therefore, TBDCs containing a target-binding moiety specifically designed to recognize and bind to a corresponding target sequence or epitope of said antigen can be used to specifically target cells of said cancer and ultimately to treat the corresponding cancer.

[0698] Preferably, the target sequence or epitope is present on the surface of one or more tumor cell types or tumor-associated cells at an increased concentration and / or in a different spatial configuration compared to the surface of non-tumor cells. Preferably, the antigen or epitope is present on the surface of one or more tumor cell or tumor stromal cell types, but not on the surface of non-tumor cells.

[0699] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising TBDC according to a third or fourth aspect.

[0700] In certain embodiments, the pharmaceutical composition is used in the form of a systemically administered drug. This includes parenteral preparations, particularly injections and infusions. Injections are formulated in ampoules or as so-called ready-to-use injections (e.g., ready-to-use syringes or disposable syringes), or in puncture-resistant flasks for multiple draws. Injections can be administered subcutaneously (sc), intramuscularly (im), intravenously (iv), or intradermally (ie). Specifically, suitable injectable formulations can be manufactured as crystal suspensions, solutions, nanoparticles, or colloidal dispersions (such as, for example, hydrosols).

[0701] Injectable formulations can be further developed into concentrates, which can be dissolved or dispersed using aqueous isotonic diluents. Infusion formulations can also be prepared as isotonic solutions, fat emulsions, liposome formulations, and microemulsions. Similar to injections, infusion formulations can also be prepared as concentrates for dilution. Injectable formulations can also be used for both inpatient and outpatient treatment as permanent infusions, for example, using microinfusion pumps.

[0702] Examples of substances that may be added to parenteral drug formulations include albumin, plasma, expanders, surfactants, organic diluents, pH-affecting substances, complexes, or polymers, particularly as substances that affect the adsorption of TBDC of the present invention to proteins or polymers, or they may be added to reduce the adsorption of TBDC of the present invention to materials (such as injection instruments or packaging materials, e.g., plastics or glass).

[0703] The adjuvants and carriers added during the preparation of the pharmaceutical compositions of the present invention as parenteral solutions are preferably sterile water (sterile water); pH-affecting substances, such as, for example, organic or inorganic acids or bases and their salts; buffering substances for adjusting pH; substances for isotonicity, such as, for example, sodium chloride, sodium bicarbonate, glucose and fructose; respectively, surfactants and emulsifiers, such as, for example, fatty acid esters of polyoxyethylene sorbitan (e.g., Tween®) or, for example, fatty acid esters of polyoxyethylene (e.g., Cremophor®). Fatty oils, such as peanut oil, soybean oil, or castor oil; synthetic esters of fatty acids, such as ethyl oleate, isopropyl myristate, and neutral oils (e.g., Miglyol®); and polymerization adjuvants, such as gelatin, dextran, and polyvinylpyrrolidone; additives that increase the solubility of organic solvents, such as propylene glycol, ethanol, N,N-dimethylacetamide, and propylene glycol; or complex-forming substances, such as citrate and urea; preservatives, such as hydroxypropyl benzoate and methyl benzoate, and benzyl alcohol; antioxidants, such as sodium sulfite; and stabilizers, such as EDTA.

[0704] In a preferred embodiment, when formulating the pharmaceutical composition of the present invention into a suspension, a thickener is added to prevent the sedimentation of the TBDC of the present invention, or a surfactant and polyelectrolyte are added to ensure the resusceptibility of the precipitate, and / or a complex-forming agent (such as EDTA, for example) is added. Complexes of the active ingredient with various polymers can also be obtained. Examples of such polymers are polyethylene glycol, polystyrene, carboxymethyl cellulose, Pluronics®, or polyethylene glycol sorbitan fatty acid esters. The TBDC of the present invention can also be incorporated into liquid formulations in the form of inclusion compounds (e.g., with cyclodextrin). In certain embodiments, a dispersant may be added as other adjuvants. For the production of lyophilized products, scaffolding agents such as mannitol, dextran, sucrose, human albumin, lactose, PVP, or gelatin variants can be used.

[0705] Example

[0706] The invention is further described through the following non-limiting embodiments.

[0707] General information

[0708] Although the invention has been described and illustrated in detail in the accompanying drawings and the foregoing description, such description and illustration are to be regarded as illustrative or exemplary, and not restrictive; the invention is not limited to the disclosed embodiments.

[0709] All amino acid sequences disclosed in this paper are shown from the N-terminus to the C-terminus; all nucleic acid sequences disclosed in this paper are shown in 5'->3'.

[0710] Chemicals and solvents were obtained from commercial suppliers such as ABCR, Fisher Scientific, Merck Chemicals, Carl Roth, Sigma-Aldrich, VWR International, Biozol Diagnostika, TCI Deutschland, IRIS Biotech, AnalytiChem, Hycultec, Hölzel Diagnostika, Bachem, Thermo Fisher, Activate Scientific, AxisPharm, Biosynth, or BLD Pharmatech, and were used directly without further purification. Water used for reactions, post-processing, and purification was purified using the ELGA Purelab flex 2 pure water system. TLC was performed on silica gel 60-coated polyester sheets (Macherey-Nagel), and the spots were visualized by irradiation (λ = 254 nm) or by staining with ninhydrin, molybdate, or KMNO4 solutions.

[0711] General experimental methods

[0712] Analytical HPLC

[0713] Analytical RP-HPLC was performed at 25ºC on a VWR-Hitachi Chromaster system equipped with a diode array detector 5430, an autosampler 5260, a quaternary pump 5160, a column oven 6310, and Luna® C18 (2) 5 µm 100 Å 250 x 4.6 mm, Kinetex® EVO-C18 5 µm 100 Å 250 x 4.6 mm, and ACE C18-PFP 5 µm 100 Å 250 x 4.6 mm. Specified parameters are shown below.

[0714] Analytical UHPLC

[0715] Analytical RP-UHPLC was performed on a VWR-Hitachi Chromaster Ultra Rs system equipped with a diode array detector 6430, an autosampler 6270, a binary pump 6170, a column oven 6310, and a Kinetex® EVO-C18 1.7 µm 100 Å 100 x 2.1 mm column at a flow rate of 0.65 mL / min, using a linear gradient elution of acetonitrile in water. Specified parameters are shown below.

[0716] Method A

[0717]

[0718] Method B

[0719]

[0720] Method C

[0721]

[0722] Method D

[0723]

[0724] Method E

[0725]

[0726] Method F

[0727]

[0728] Method G

[0729]

[0730] Method H

[0731]

[0732] Method I

[0733]

[0734] Method J

[0735]

[0736] Method K

[0737]

[0738] Method L

[0739]

[0740] Method M

[0741]

[0742] Method N

[0743]

[0744] Method O

[0745]

[0746] Method P

[0747]

[0748] Method Q

[0749]

[0750] Method R

[0751]

[0752] Method S

[0753]

[0754] Method T

[0755]

[0756] Method U

[0757]

[0758] Method V

[0759]

[0760] Method W

[0761]

[0762] Method X

[0763]

[0764] Method Y

[0765]

[0766] Method Z

[0767]

[0768] Method AA

[0769]

[0770] Method AB

[0771]

[0772] Method AC

[0773]

[0774] Method AD

[0775]

[0776] Method AE

[0777]

[0778] Method AF

[0779]

[0780] Method AG

[0781]

[0782] NMR spectroscopy

[0783] Recording at 500 MHz at room temperature 1 H and spectra, with trimethylsilane (TMS) as a reference. Chemical shifts (δ) are reported in parts per million (ppm) from low field to high field. Coupling constants (J) are reported in Hertz (Hz), and abbreviations indicating multiplets are used as follows: s = singlet, d = doublet, t = triplet, dd = double doublet, m = multiplet.

[0784] Mass spectrometry (MS)

[0785] MS measurements were performed on an Advion Expression® CMS. Samples were submitted by direct injection using a syringe. For ionization, an electrospray ionization (ESI) or atmospheric pressure ionization (APCI) source was used. Analysis was performed in positive ion mode. For ESI, the capillary temperature was set to 250ºC. The source gas temperatures were set to 250ºC and 300ºC, respectively, with flow rates of 4 L / min and 5 L / min. The nebulizer flow rate was set to 0.5 L / min. The electrospray voltage was set to 3.5 kV. For APCI, the capillary temperature was set to 250ºC, the source gas temperature was set to 350ºC, and an APCI corona discharge of 5 µA was used.

[0786] Preparative RP-HPLC purification

[0787] Preparative RP-HPLC purification was performed on an Agilent 1260 Infinity II system equipped with a binary pump, multisampler, VWD, fraction collector, and column manager. Phenomenx Luna® C18 (2) 10 µm 100 Å 250 x 21.2 mm, Kinetex® EVO-C18 5 µm 100 Å 250 x 21.2 mm, and ACE C18-PFP 5 µm 100 Å 250 x 21.2 mm columns were used as columns at flow rates between 24 and 30 mL / min, eluting with a linear gradient of acetonitrile in water (+0.05% TFA). When an acid (e.g., TFA) was used as a solvent additive during purification, compounds containing basic groups (e.g., amines) were obtained as corresponding salts (e.g., TFA salts).

[0788] Rapid chromatography

[0789] Rapid silica gel chromatography purification was performed using a Teledyne ISCO CombiFlash NextGen 300+, equipped with UV and ELSD detection and RediSep® disposable normal phase silica gel columns with sieve sizes between 230 and 400.

[0790] General Procedure 1 (GP1): CuAAC click reaction for attaching solubilizer

[0791] Add a stirred solution of azide (0.9–1.5 equivalents) and alkyne (1 equivalent) in DMF containing tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amino (TBTA, 0.125–0.625 equivalents) or tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 0.125–2 equivalents), copper(II) sulfate pentahydrate (0.1–1 equivalent), and sodium ascorbate (0.25–3.0 equivalents) in a DMF:water mixture (5:1 or 10:1). Allow the resulting reaction mixture to be stirred at room temperature until complete. After completion, purify the reaction mixture directly by preparative HPLC without any pretreatment to yield the product upon lyophilization.

[0792] General Procedure 2 (GP2): CuAAC Click Reaction of Polyazide Scaffolds

[0793] Add water containing tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 0.63-1.35 equivalents / azide fraction), copper(II) sulfate pentahydrate (0.525-0.65 equivalents / azide fraction), and sodium ascorbate (1-2.15 equivalents / azide fraction) to a stirred solution of polyazides (1 equivalent) and alkynes (1.05-1.25 equivalents / azide fraction) in DMF. The resulting DMF / water ratio is 5:1 or 10:1. The resulting reaction mixture is allowed to be stirred at room temperature until complete. After completion, the reaction mixture is directly purified by preparative HPLC without any pretreatment to produce a polylinker-loaded product upon lyophilization.

[0794] General Program 3 (GP3): Attachment to intermediate 3.1 or 3.2

[0795] The Fmoc-protected linker-loaded material was treated with diethylamine (Et2N, 20–24 equivalents) in DMF at room temperature, and the reaction progress was monitored by RP-HPLC. The solvent was removed under vacuum, and the residue was co-evaporated once with DMF. The residue was then dissolved in DMF under argon atmosphere, and a solution of intermediate 3.1 or 3.2 (1.1–1.5 equivalents) in DMF was added, followed by the addition of N-ethyldiisopropylamine (DIPEA, 1.4–3 equivalents). The solution was stirred at room temperature, and the progress was monitored by RP-HPLC. Upon completion, the solvent was evaporated, and the crude product was purified by RP-HPLC.

[0796] General Procedure 4 (GP4): Maleimide Attachment

[0797] Mal-PEG(4)-NHS ester (1–4 equivalents) and DIPEA (1–4 equivalents) were added to a stirred solution of the polylinker-loaded material in dry DMF. The reaction mixture was stirred at room temperature for 30 min. After completion, the reaction mixture was directly purified by preparative HPLC without any pretreatment to yield the final compound after lyophilization.

[0798] General Procedure 5 (GP5): Attaching PEG-azide to mono-6-amino-6-deoxy-cyclodextrin

[0799] Dissolve 1 equivalent of azide-PEG-NHS ester in water or DMF, and add a solution of 1 equivalent of mono-6-amino-6-deoxy-cyclodextrin in water or DMF. If DMF is used as the solvent, add DIPEA (1.5–2 equivalents) if necessary. Allow the resulting reaction mixture to be stirred at room temperature until complete, and monitor by RP-HPLC. If necessary, add another part of mono-6-amino-6-deoxy-cyclodextrin (0.2 equivalents). After completion, freeze-dry the reaction mixture and use it as the crude product without further purification, using water as the solvent. If DMF is used as the solvent, separate the product by precipitation from MTBE. When using mono-6-amino-6-deoxy-β-cyclodextrin, commercially available mono-6-amino-6-deoxy-β-cyclodextrin is used. In the case of using mono-6-amino-6-deoxy-γ-cyclodextrin, the materials were synthesized from γ-cyclodextrin according to the following literature: Tang, W., Ng, S.-C. Nat.Protoc. 2008, 3, 691–697.

[0800] ADC generation - adapter - payload and antibody conjugation

[0801] Antibodies are conjugated to drug-carrying linker conjugates using a technique known as Thiomab. In this method, conjugation is achieved by coupling the terminal thiol reactive portion (such as a maleimide residue) of the drug-carrying linker to a free SH group of a genetically engineered cysteine ​​residue in the Fc region of the antibody, as shown in the following reaction protocol:

[0802]

[0803] The principle of this conjugation method is disclosed in Junutula et al. Nat Biotechnol. Aug 2008;26(8):925-32, the contents of which are incorporated herein by reference.

[0804] The anti-GUCY2C antibody used in this experiment contains D265C substitutions in both Fc domains to provide cysteine ​​residues with free SH groups. The corresponding technology is disclosed in WO 2016 / 142049 A1, the contents of which are incorporated herein by reference.

[0805] Example 1 - Synthesis of Modular Connector Compounds

[0806] Example 1.1 - Synthesis of the connector intermediate

[0807] Step 1: Synthesis of 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid

[0808]

[0809] At room temperature, propan-2-ynyl alcohol (2.16 mL, 36.5 mmol, 5 equivalents) and Cs₂CO₃ (5.96 g, 18.3 mmol, 2.5 equivalents) were added to a solution of methyl 2-(bromomethyl)-4-nitrobenzoate (CAS No. 133446-99-8, 2 g, 7.30 mmol, 1 equivalent) in 10 mL of acetonitrile. The mixture was stirred at 80ºC for 3 h and the progress was monitored by RP-HPLC. The reaction mixture was filtered. The filtrate was diluted with ethyl acetate (20 mL) and washed with water (10 mL) and extracted with NaHCO₃ (2 x 10 mL). For the precipitate, the combined aqueous layers were acidified to pH 2 using 2 M HCl (50 mL). After filtration, the obtained solid was absorbed into 1,4-dioxane and lyophilized overnight to produce 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid (1.44 g, 83%) as a pale red solid. 1 H NMR (500 MHz, DMSO) δ 8.36 (d, J = 2.4Hz, 1H), 8.22 (dd, J = 8.6, 2.5 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 4.94 (s,2H), 4.36 (d, J = 2.4 Hz, 2H), 3.53 (t, J = 2.4 Hz, 1H). HPLC (Method A): t R = 11.3min.

[0810] Step 2: Synthesis of methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate

[0811]

[0812] SOCl2 (3.56 mL, 49 mmol, 8 equivalents) was added to a solution of 4-nitro-2-((propan-2-yn-1-yloxy)methyl)benzoic acid (1.44 g, 6.12 mmol, 1 equivalent) in 30 mL of dry methanol at 0ºC. The mixture was stirred at 70ºC for 3.5 h and the progress was monitored by RP-HPLC. After completion, the solvent and volatiles were removed under reduced pressure. The resulting residue was absorbed in 1,4-dioxane and lyophilized overnight to produce methyl 4-nitro-2-((propan-2-yn-1-yloxy)methyl)benzoate (1.52 g, 99%) as a light brown solid. 1H NMR (500 MHz, DMSO) δ 8.37 (dd, J = 2.3,1.1 Hz, 1H), 8.28 – 8.22 (m, 1H), 8.08 (d, J = 8.5 Hz, 1H), 4.92 (d, J = 0.8Hz, 2H), 4.34 (d, J = 2.4 Hz, 2H), 3.90 (s, 3H), 3.49 (t, J = 2.4 Hz, 1H). HPLC (Method A): t R = 13.1 min.

[0813] Step 3: Synthesis of methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate

[0814]

[0815] Iron powder (2.73 g, 48.8 mmol, 8 equivalents) and NH4Cl (2.61 g, 48.8 mmol, 8 equivalents) were added to a stirred suspension of methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate (1.52 g, 6.1 mmol, 1 equivalent) in a mixture of EtOH (15 mL) and water (7.5 mL) at room temperature. The resulting mixture was allowed to be stirred at 80ºC for 4 h and the progress was monitored by RP-HPLC. After the starting material was completely consumed, the reaction was cooled to room temperature and filtered through diatomaceous earth and washed with ethyl acetate (15 mL). The filtrate was washed with saturated sodium bicarbonate solution, water, and brine (15 mL each). The aqueous layer was extracted with ethyl acetate (15 mL). The combined organic layers were dried over MgSO4. After filtration, the solvent was removed under reduced pressure and dried in a vacuum to obtain 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate (1.29 g, 96%) as a red solid. 1 H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 8.5 Hz, 1H), 6.99 (dt, J =2.2, 1.0 Hz, 1H), 6.59 (dd, J = 8.5, 2.5 Hz, 1H), 5.00 (s, 2H), 4.31 (d, J =2.4 Hz, 2H), 3.84 (s, 3H), 2.48 (t, J = 2.4 Hz, 1H). HPLC (Method A): t R = 10.5 min.

[0816] Step 4: Synthesis of (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol

[0817]

[0818] LiAlH4 (1 M in THF, 12.01 mmol, 12.01 mL, 2.5 equivalents) was slowly added to a cooled solution of dry THF (0ºC) (13 mL). A solution of 4-amino-2-((propane-2-yn-1-yloxy)methyl)benzoate (1.06 g, 4.8 mmol, 1 equivalent) in dry THF (10 mL) was added dropwise at 0ºC. After complete addition, the resulting mixture was allowed to stir overnight at room temperature. The reaction progress was monitored by RP-HPLC. The reaction mixture was poured into a vigorously stirred 1 M potassium sodium tartrate solution (120 mL) and stirred again at room temperature for 30 min. The reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with water and brine (30 mL each). The organic layers were dried over MgSO4. The solvent was removed under reduced pressure to obtain the crude product. (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (0.65 g, 71%) was purified by ISCO SiO2 rapid chromatography (0-100% ethyl acetate in hexane). 1 H NMR (500 MHz, DMSO) δ 6.99(d, J = 8.1 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.0, 2.4 Hz,1H), 4.93 (s, 2H), 4.62 – 4.53 (m, 1H), 4.47 (s, 2H), 4.35 (d, J = 5.4 Hz, 2H), 4.15 (d, J = 2.4 Hz, 2H), 3.42 (t, J = 2.4 Hz, 1H). HPLC (Method A): t R = 4.8 min. MS (ESI): Measured value m / z = 174.2 [M-H2O+H] + The calculated value is m / z = 174.1.

[0819] Step 5a: Synthesis of Fmoc-Val-Ala-OH

[0820] The synthesis of Fmoc-Val-Ala-OH is carried out as disclosed in WO 2017 / 149077.

[0821] Step 5b: Synthesis of Boc-Val-Ala-OH

[0822] The title compound is commercially available. CAS: 70396-18-8

[0823] Example 1.2 - Synthesis of the Joint-Payload Intermediate

[0824] Step 6a: Synthesis of intermediate 1.1

[0825]

[0826] N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 1.05 g, 4.1 mmol, 1.2 equivalent) was added to a stirred solution of Fmoc-Val-Ala-OH (1.43 g, 3.41 mmol, 1 equivalent) and (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (0.65 g, 3.41 mmol, 1 equivalent) in dry THF (20 mL). The resulting mixture was stirred at room temperature in darkness for 5 days. The solvent was removed under reduced pressure. The resulting crude intermediate was purified by silica gel rapid chromatography (gradient of 0-5% MeOH in DCM) to yield intermediate 1.1 (1.25 g, 63%) as a colorless solid. HPLC (Method A): t R = 12.9 min. MS (ESI): Measured value m / z = 601.2 [M+NH4] + The calculated value is m / z = 601.3.

[0827] Step 7: Synthesis of intermediate 1.2

[0828]

[0829] Bis-4-nitrophenyl carbonate (0.75 g, 2.48 mmol, 2 equivalents) and N-ethyldiisopropylamine (DIPEA, 3.1 mmol, 0.53 mL, 2.5 equivalents) were added to a stirred solution of intermediate 1.1 (0.72 g, 1.24 mmol) in dry DMF (15 mL) under an argon atmosphere. The reaction was allowed to proceed with stirring at room temperature for 4 h. After completion, the solvent was removed under reduced pressure. The crude intermediate was purified by silica gel rapid chromatography (gradient of 0-100% ethyl acetate in n-hexane) to yield intermediate 1.2 (0.75 g, 81%) as a pale yellow solid after lyophilization from 1,4-dioxane:water (4:1). HPLC (Method C): t R = 13.4 min. MS (ESI): Measured value m / z = 771.2 [M+Na] + The calculated value is m / z = 771.3.

[0830] Step 8a: Synthesis of intermediate 1.3

[0831]

[0832] A solution of freshly prepared intermediate 1.2 (0.82 g; 1.1 mmol, 1 equivalent) and 1-hydroxybenzotriazole (HOBt, 0.042 g, 0.275 mmol, 0.25 equivalent) in dry DMF (20 mL) was added to a stirred suspension of eczemacon mesylate (0.58 g, 1.1 mmol, 1 equivalent) in dry DMF (20 mL). DIPEA was added to the resulting mixture (0.28 g, 2.16 mmol, 0.37 mL, 2 equivalent) and 2,6-rutidine (141 mmol, 16.3 mL, 129 equivalent). The reaction mixture was stirred overnight at room temperature and the progress was monitored by RP-HPLC. The reaction mixture was concentrated under reduced pressure. Intermediate 1.3 (1.08 g, 95%) was purified by silica gel rapid chromatography (gradient of 0-5% MeOH in DCM) and lyophilized from 1,4-dioxane:water (4:1) to produce a gray solid. HPLC (Method D): t R = 13.0 min. MS (ESI): Measured value m / z = 1045.3 [M+H] + The calculated value is m / z = 1045.4.

[0833] Step 6b: Synthesis of intermediate 1.4

[0834]

[0835] N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 0.56 g, 2.27 mmol, 1.2 equivalents) was added to a stirred solution of Boc-Val-Ala-OH (0.57 g, 1.98 mmol, 1 equivalent) and (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (0.36 g, 1.89 mmol, 1.05 equivalents) in dry THF (9 mL). The resulting mixture was stirred at room temperature in darkness for 5 h. The solvent was removed under reduced pressure. The resulting crude intermediate was purified by ISCOSiO2 chromatography (0-50% hexane / ethyl acetate) to yield intermediate 1.4 (0.81 g, 93%) as a colorless solid. 1HNMR (500 MHz, DMSO) δ 9.89 (s, 1H), 7.98 (q, J = 7.1 Hz, 1H), 7.54 (d, J =7.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 1H), 4.95 (t, J = 5.4 Hz, 1H), 1.97 (s, 1H), 1.39 (s, 9H), 1.30 (d, J = 7.0 Hz, 3H), 0.85 (dd, J = 23.2, 6.8 Hz, 6H). HPLC (Method A): t R = 10.9 min. MS (ESI): Measured value m / z = 461.9 [M+H] + The calculated value is m / z = 462.26.

[0836] Step 7b: Synthesis of intermediate 1.5

[0837]

[0838] A solution of tert-butyldimethylchlorosilane (0.12 g, 0.81 mmol, 1.25 equivalent) in dry DMF (1 mL) was added dropwise to a stirred solution of intermediate 1.4 (0.3 g, 0.65 mmol, 1 equivalent) and imidazole (0.07 g, 0.96 mmol, 1.5 equivalent) in dry DMF (2 mL). The reaction was stirred at room temperature and monitored by TLC and RP-HPLC. After 2 h, the solvent was removed under reduced pressure. The residue was absorbed into 100 mL of ethyl acetate and washed with 0.2 N citric acid, saturated sodium bicarbonate, and brine (100 mL each). The organic layer was dried over MgSO4. After filtration, the solvent was removed under reduced pressure. The resulting crude intermediate was purified by ISCO SiO2 chromatography (0-50% hexane / ethyl acetate) to produce intermediate 1.5 (0.22 g, 58%) as a colorless solid. HPLC (Method P): t R = 16.7 min. MS (ESI): Measured value m / z = 576.0 [M+H] + The calculated value is m / z = 576.35.

[0839] Step 8b: Synthesis of intermediate 1.6

[0840]

[0841] Intermediate 1.6 was synthesized as described for intermediate 1.3, but using MMAE, and the reaction mixture was stirred for 45 h. Intermediate 1.6 (175.37 mg, 56%) was obtained as a colorless solid. UHPLC (Method R, with TFA as an additive, at 290 nm): t R = 4.9 min. MS (ESI): Measured value m / z = 1327.8 [M+H] + The calculated value is m / z = 1327.75.

[0842] Step 8c: Synthesis of intermediate 1.7

[0843]

[0844] Intermediate 1.7 was synthesized as described for intermediate 1.3. However, RP-HPLC analysis of the reaction mixture indicated that the reaction proceeded to completion after stirring for 3.5 h. Intermediate 1.7 (182.24 mg, 67%) was obtained as a pale yellow solid. UHPLC (Method R, with TFA as an additive, at 290 nm): t R = 4.1 min. MS (ESI): Measured value m / z = 967.2 [M+H] + The calculated value is m / z = 967.41.

[0845] Step 8d: Synthesis of intermediate 1.8

[0846]

[0847] A solution of 1,1,1-trimethyl-N-(trimethylsilyl)silaneamine lithium (LiHMDS, 1 M in THF, 0.57 mL, 0.57 mmol, 1.5 equivalent) was added dropwise to a cooled solution (0ºC) of intermediate 1.5 (0.21 g, 0.38 mmol, 1 equivalent) in dry THF (7.6 mL). After complete addition, the resulting mixture was allowed to stir for 10 min. Then, 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl, 0.13 mL, 0.76 mmol, 2 equivalent) was added, and the reaction mixture was allowed to stir again at 0ºC for 15 min. The ice bath was removed, and the reaction mixture was stirred from 0ºC to room temperature for 30 min. After the reaction was complete, a saturated sodium citrate solution (2.5 mL, pH = 6.4) was added. For phase separation, the resulting mixture was diluted with 20 mL of ethyl acetate. The organic layer was washed with sodium citrate buffer (pH = 4.76), saturated sodium bicarbonate, and brine (2.5 mL each). The organic layer was dried over MgSO4. After filtration, the solvent was removed under reduced pressure. The resulting crude intermediate was purified by ISCO SiO2 chromatography (0-50% hexane / tert-butyl methyl ether) to yield intermediate 1.8 (0.25 g, 94%) as a colorless solid. HPLC (Method P): t R = 16.1 min. MS (ESI): Measured value m / z = 706.1 [M+H] + The calculated value is m / z = 706.43.

[0848] Step 8c: Synthesis of intermediate 1.9

[0849]

[0850] To intermediate 1.2 (1 g, 1.34 mmol, 1 equivalent) in a stirred solution of dry DMF (35 mL), tert-butyl methyl (2-(methylamino)ethyl)carbamate (0.3 g, 1.6 mmol, 1.2 equivalent) was added. The resulting mixture was stirred at room temperature under an argon atmosphere for 1 h. The reaction mixture was concentrated under reduced pressure. It was then diluted with 75 mL ethyl acetate / THF 5:1 and washed with 10% citric acid, 5% sodium bicarbonate, and brine (50 mL each). The organic layer was dried over MgSO4. After filtration, the solvent was removed under reduced pressure. The resulting crude intermediate was purified by ISCO SiO2 chromatography (0-100% hexane / ethyl acetate). After evaporation of the volatiles, the residue was absorbed in 1,4-dioxane and lyophilized to produce intermediate 1.9 (0.95 g, 89%) as a colorless solid. HPLC (Method A): t R= 15.2 min. MS (ESI): Measured value m / z = 798.2 [M+H] + The calculated value is m / z = 798.41.

[0851] Step 9a: Synthesis of intermediate 1.10

[0852]

[0853] To an intermediate 1.8 (0.25 g, 0.36 mmol, 1 equivalent) in a stirred solution of dry THF (3.6 mL), N,N,N-tributylbutane-1-ammonium fluoride (TBAF, 1 M in THF, 0.43 mL, 0.43 mmol, 1.2 equivalent) was added. After completion (approximately 15 min), the reaction mixture was subjected to ISCO SiO2 chromatography (0-100% hexane / tert-butyl methyl ether) to produce intermediate 1.10 (0.23 g, quantified) as a colorless solid. HPLC (Method A): t R = 14.6 min. MS (ESI): Measured value m / z = 591.9 [M+H] + The calculated value is m / z = 592.34.

[0854] Step 9b: Synthesis of intermediate 1.11

[0855]

[0856] TFA (0.43 mL, 6.61 mmol, 19.75 equivalents) was added to a stirred solution of intermediate 1.9 (0.27 g, 0.33 mmol, 1 equivalent) in dry dichloromethane (3 mL) at room temperature. The resulting mixture was stirred at room temperature. After completion (approximately 2 h), the reaction mixture was added to 13 mL of ice-cold MTBE. The resulting precipitate was separated by centrifugation, resuspended in 10 mL of cold MTBE, and centrifuged again. The crude product (0.24 g) was absorbed into 5 mL of DMF and purified by preparative RP-HPLC to yield 190.4 mg (70%) of intermediate 1.11 (TFA salt) as a colorless solid. HPLC (Method A): t R = 15.2 min. MS(ESI): Measured value m / z = 798.2 [M+H] + The calculated value is m / z = 798.41.

[0857] Step 10a: Synthesis of intermediate 1.12

[0858]

[0859] In an inert atmosphere, a cooled solution (5ºC) of intermediate 1.10 (0.13 g, 0.22 mmol, 1 equivalent) in dry dichloromethane (1.3 mL) was added to a solution of methanesulfonic anhydride (0.046 g, 0.26 mmol, 1.2 equivalent) dissolved in 0.7 mL dry DCM and DIPEA (0.09 mL, 0.53 mmol, 2.4 equivalent). The reaction was monitored by TLC and RP-HPLC. After 3 h, sulfonation was complete (additional methanesulfonic anhydride may be added if necessary). A solution of LiBr (0.095 g, 1.11 mmol, 5 equivalent) in 1.7 mL dry THF was added, and the reaction mixture was allowed to be stirred at 5ºC for another 10 min. The cooling bath was removed, and the reaction was stirred at room temperature until complete. After completion (approximately 2 h), a 0.2 M sodium citrate solution (5 mL, pH = 6.4) was added. For phase separation, the resulting mixture was diluted with 50 mL MTBE. The organic layer was washed with 0.2 M sodium citrate buffer (5 mL, pH = 4.76), saturated sodium bicarbonate, and brine (50 mL each). The organic layer was dried over MgSO4. After filtration, the solvent was removed under reduced pressure. The resulting crude intermediate was purified by rapid chromatography (SiO2, 0-100% hexane / tert-butyl methyl ether). The solvent was removed, and the residue was absorbed into 1,4-dioxane and lyophilized to yield intermediate 1.12 (0.052 g, 36%) as a colorless lyophilized product. HPLC (Method P): t R = 9.3 min. MS (ESI): Measured value m / z = 653.8 [M+H] + The calculated value is m / z = 654.26.

[0860] Step 10b: Synthesis of intermediate 1.13

[0861]

[0862] The intermediate was prepared as described in Holte et al., Bioorg. & Med. Chem., Vol. 30, 24, 2020, 127640. The target product was obtained in 17% yield as a red solid. MS (ESI): m / z = 806.8 [M+H] + The calculated value is m / z = 807.22 [M+H]. + .

[0863] Step 10c: Synthesis of intermediate 1.14 (deacetylated colchicine)

[0864]

[0865] The intermediate was prepared as described in WO 2016 / 019340. The target product, a pale yellow solid, was obtained in 99% yield (via 3 steps). UHPLC (Method V): t R = 1.0 min. 1 ¹H NMR (500 MHz, DMSO): 8.46 (s, 3H), 7.20 – 7.13 (m, 2H), 7.10 (d, J = 11.2 Hz, 1H), 6.81 (s, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.83 – 3.74 (m, 1H), 3.78 (s, 3H), 3.61 (s, 3H), 2.66 (d, J = 6.8 Hz, 1H), 2.34 – 2.26 (m, 2H), 1.93 (dd, J = 11.8, 5.4 Hz, 1H). MS (APCI): Measured m / z = 357.8 [M+H] + The calculated value is m / z = 358.16.

[0866] Step 11a: Synthesis of intermediate 1.15

[0867]

[0868] SN-38 (0.036 g, 0.092 mmol, 1 equivalent) was suspended in 1.6 mL of dry DMF. Potassium tert-butoxide (KOtBu, 1 M in THF, 0.14 mL, 0.14 mmol, 1.5 equivalent) was added to 0.8 mL of dry DMF. The resulting KOtBu solution was added dropwise to a reaction flask at room temperature under an argon atmosphere. After 5 min, intermediate 1.12 (0.06 g, 0.092 mmol, 1 equivalent) was dissolved in 0.6 mL of dry DMF and added dropwise to the reaction mixture. The solvent was removed under reduced pressure. The resulting crude intermediate was purified by rapid chromatography (SiO2, 0-50% hexane / ethyl acetate) to produce intermediate 1.9 (0.04 g, 52%) as a colorless solid. HPLC (Method A (380 nm)): t R = 15.9 min. MS (ESI): Measured value m / z = 966.2 [M+H] + The calculated value is m / z = 966.47.

[0869] Step 11b: Synthesis of intermediate 1.16

[0870]

[0871] Under an argon atmosphere, 0.54 mL of dry DMF containing intermediate 1.11 (47.55 mg, 0.059 mmol, 1 equivalent) and DIPEA (13.62 µL, 0.078 mmol, 2 equivalent) was added to a stirred solution of intermediate 1.13 (31.62 mg, 0.039 mmol, 1.5 equivalent) in 0.54 mL of dry DMF and a stirred solution of DIPEA (13.62 µL, 0.078 mmol, 2 equivalent). The resulting mixture was stirred at room temperature for 1.5 h. After completion, the reaction mixture was purified directly by preparative HPLC without any pretreatment to yield 26.9 mg (50%) of intermediate 1.16 as a red solid. HPLC (Method A, without additives): t R = 15.8 min. MS (ESI): Measured value m / z = 1365.4 [M+H] + The calculated value is m / z = 1365.55.

[0872] Synthesis of SN-38-PNP

[0873]

[0874] DIPEA (129.6 µL, 0.74 mmol, 1.5 equivalent) was added to a suspension of SN-38 (200 mg, 0.51 mmol, 1 equivalent) and 4-nitrophenyl chloroformate (123.3 mg, 0.61 mmol, 1.2 equivalent) in THF (12 mL) at 0ºC under argon. The resulting solution was heated to room temperature and stirred for 1.5 h. The solution was concentrated under vacuum, and the resulting residue was purified by preparative HPLC to provide SN-38-PNP (169.4 mg, 60%) as a pale yellow solid. 29 H 23 N3O9. HPLC (Method A): t R = 10.0 min. MS (ESI): Measured value m / z = 558.0, calculated value m / z = 558.1 [M+H] + .

[0875] Synthesis of intermediate 1.17

[0876]

[0877] DIPEA (29.9 µL, 171.4 µmol, 2 equivalents) was added to a solution of intermediate 1.11 (69.6 mg, 85.7 µmol, 1 equivalent) and SN-38-PNP (47.8 mg, 85.7 µmol, 1 equivalent) in DMF (2 mL) under argon conditions. The resulting solution was stirred at room temperature for 2.25 h and purified by preparative HPLC to provide intermediate 1.17 (78 mg, 82%) as a pale yellow solid. 62 H 65 N7O 13 HPLC (Method A): t R = 14.0 min. MS (ESI): Measured value m / z = 1168.8, calculated value m / z = 1116.5 [M+H] + .

[0878] NAMPTi

[0879]

[0880] NAMPTi 1 was synthesized as described in WO 2018201087.

[0881] Synthesis of intermediate 1.18

[0882]

[0883] Intermediate 1.18 was synthesized as described for intermediate 1.3, but using NAMPTi 1 (150 mg, 0.36 mmol, 1 equivalent), intermediate 1.2 (402 mg; 0.54 mmol, 1.5 equivalent), 1-hydroxy-7-azabenzotriazole (HOAt, 600 µL of 0.6 M solution in DMF, 0.36 mmol, 1 equivalent), and 2,6-rutidine (5.5 mL, 45 mmol, 125 equivalent) in dry DMF (9.52 mL). The pH of the reaction mixture was periodically adjusted with DIPEA to maintain pH 8. The reaction mixture was stirred at room temperature for 6 days and purified by preparative HPLC to provide intermediate 1.18 (120.6 mg, 32%) as a colorless solid. 58 H 64 N 10 O8. HPLC (Method AA): t R = 12.8 min. MS (ESI): Measured value m / z = 1029.7 [M+H] + The calculated value is m / z = 1029.5.

[0884] TLR7 agonist

[0885]

[0886] Agonist 1 was synthesized as described in WO 2022187809.

[0887] Synthesis of intermediate 1.19

[0888]

[0889] Intermediate 1.19 was synthesized as described for intermediate 1.3, but using agonist 1 and stirred for 5 days. Intermediate 1.19 (184.1 mg, 33%) was obtained as a pale yellow solid. C 56 H 58 N8O7. HPLC (Method A): t R = 12.0 min. MS(ESI): Measured value m / z = 955.5 [M+4H] 4+ The calculated value is m / z = 955.4 [M+4H]. 4+ .

[0890] Synthesis of version 1.20

[0891]

[0892] Intermediate 1.20 was synthesized from intermediate 1.2 (258 mg, 0.344 mmol, 1 equivalent) and doxorubicin hydrochloride (200 mg, 1.0 equivalent) as described for intermediate 1.3. Intermediate 1.20 (395 mg, 97%) was obtained as a red solid. HPLC method A (480 nm): t R = 14.0 min. MS (ESI): Measured value m / z = 1173.3 [M+Na] + The calculated value is m / z = 1175.41.

[0893] Synthesis of 1.21

[0894]

[0895] DIPEA (28.9 µL, 169.74 µmol, 2 equivalents) was added to 1 mL of dry DMF containing a 4-nitrophenyl-carbonate derivative of triptolide (44.60 mg, 84.87 µmol, 1 equivalent) and intermediate 1.11 (103.35 mg, 127.30 µmol, 1.5 equivalents), and the mixture was stirred at room temperature under an argon atmosphere. After complete conversion (1 h 45 min), the solution was acidified with TFA (13 µL, 2 equivalents), filtered (0.2 µm nylon), and purified by preparative HPLC to give intermediate 1.21 (72.95 mg, 79%) as a colorless solid. HPLC method A (260 nm): t R = 13.4 min. MS (ESI): Measured value m / z = 1084.8 [M+Na] + The calculated value is m / z = 1084.5.

[0896] Intermediate 1.22

[0897] Step 1

[0898]

[0899] S-deoxy-β-amaminine was synthesized according to Lutz et al., Angewandte Chemie, International Edition (2020), 59(28), 11390-11393 [CAS: 2417149-39-2].

[0900] Step 2

[0901]

[0902] Synthesize the Fmoc-Val-Ala-(4-aminomethyl)-aniline linker according to WO 2022 / 155347 [CAS: 2803767-21-5]

[0903] Step 3

[0904]

[0905] 1-hydroxybenzotriazole hydrate (159 mg, 1040 µmol, 10 equivalents) and N,N'-diisopropylcarbodiimide (162 µL, 1040 µmol, 10 equivalents) were added to the product S-deoxy-β-amanitride (94 mg, 104 µmol) from step 1 dissolved in 3 mL of dry DMF, and the mixture was stirred for 1 h. Then, the product from step 2 (98 g, 156 µmol, 1.5 equivalents) and DIPEA (53 µL, 312 µmol, 3 equivalents) were added, and stirring was continued overnight. The reaction mixture was then added dropwise to 40 mL of ice-cold MTBE, and the resulting precipitate was separated by centrifugation, washed with 40 mL of fresh MTBE, and dried under vacuum.

[0906] The crude product was further purified by preparative HPLC to obtain 115 mg (79%) of the product from step 3.

[0907] Step 4:

[0908]

[0909] The product from step 3 (67.02 mg, 47.85 µmol) was dissolved in 3 mL of DMF, and 100 µL (960 µmol) of diethylamine was added. The reaction was stirred at room temperature for 1 h. The evaporation was evaporated under vacuum, and the remainder was co-evaporated with 3 mL of DMF. The crude product was then dissolved in 1 mL of methanol and added dropwise to 40 mL of ice-cold MTBE. The resulting precipitate was separated by centrifugation, washed with 40 mL of fresh MTBE, and dried under vacuum. Intermediate 1.22 (69.5 mg) was used without further purification.

[0910] Example 1.3 - Synthesis of intermediates 2.1 and 2.2

[0911]

[0912] 2-Chlorotriphenylmethyl resin (4 g) was swollen in anhydrous DCM (20 mL) for 10 min. The suspension was filtered, and a solution of Fmoc-Lys(N3)-OH (1.578 g, 4 mmol, 1 equivalent) and DIPEA (3.48 mL, 20 mmol, 5 equivalent) in anhydrous DCM (20 mL) was added and shaken at room temperature for 2 h. The suspension was filtered, the resin was washed with DMF (3 x 1 min) and capping solution (20 mL, MeOH / DIPEA / DCM 1 / 1 / 8) was added and shaken at room temperature for 10 min. The suspension was filtered, and the capping procedure was repeated once. The resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min) and dried under vacuum. The resin loading was determined as described in the following literature: J. Pept. Sci. 2017, 23, 757-762.

[0913] For the next step, the resin was swollen in DMF for 10 min. The suspension was filtered, and a 20% piperidine solution in DMF for Fmoc deprotection was added to the resin. The suspension was incubated for 20 min, filtered, and the deprotection procedure was repeated once. Then, the resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min). The resin was swollen in DMF for 10 min, filtered, and a solution of Fmoc-Lys(N3)-OH (3 equivalents), TBTU (3 equivalents), and DIPEA (6 equivalents) in DMF was added. The suspension was shaken at room temperature for 2 h. The resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min), and the Fmoc groups were removed with 20% piperidine as described above. For acetylation, the resin was swollen in DMF for 10 min, the suspension was filtered, and a solution of acetic anhydride (3 equivalents) and pyridine (3 equivalents) in DMF was added. The suspension was incubated at room temperature for 2 h, filtered, and the resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min) and dried in a vacuum.

[0914] For cleavage, the resin was treated with a solution of hexafluoroisopropanol in DCM (1:4, 20 mL) for 15 min. The solution was collected, and the resin was treated again with a solution of hexafluoroisopropanol in DCM (1:4, 20 mL). The resin was washed with a solution of hexafluoroisopropanol in DCM (1:4), and the combined filtrates were concentrated under vacuum. The resulting crude peptide was purified by RP-HPLC, yielding Ac-Lys(N3)-Lys(N3)-OH (731 mg, 50%) as a colorless lyophilized product.

[0915]

[0916] Ac-Lys(N3)-Lys(N3)-OH (731 mg, 1.98 mmol, 1 equivalent) and HOBt (910 mg, 5.94 mmol, 3 equivalent) were dissolved in DMF (8 mL). DIC (920 µL, 5.9 mmol, 3 equivalent) was added, and the solution was stirred at room temperature for 5 min. Then, a solution of Boc-amino-PEG(3)-amine (CAS No. 101187-40-0, 868 mg, 2.97 mmol, 1.5 equivalent) in DMF (4 mL) was added, followed by DIPEA (1.04 mL, 5.98 mmol, 3 equivalent). The reaction was stirred at room temperature and then performed by RP-HPLC. After 20 h, the solution was concentrated and the residue was dissolved in ethyl acetate. The organic layer was washed with 0.2 M citric acid (1 x 50 mL), sodium bicarbonate solution (3 x 50 mL), and brine. The organic layer was dried over magnesium sulfate and concentrated under vacuum to give 1.428 g of crude product. Purified by RP-HPLC, followed by two lyophilizations in the presence of HCl (1 equivalent) to provide an intermediate as an HCl salt, 2.1 g (705 mg, 66%). HPLC (Method H): t R = 11.2 min. MS(ESI): Measured value m / z = 543.3 [M+H] + The calculated value is m / z = 543.3 [M+H]. + .

[0917]

[0918] Intermediate 2.2 was synthesized as described for intermediate 2.1, but with two additional cycles of Lys(N3) coupling to yield a tetrapeptide. Purified by RP-HPLC and then lyophilized, intermediate 2.2 (139 mg, 37%) was provided as a viscous, colorless solid. HPLC (Method H): t R = 9.3 min. MS (ESI): Measured value m / z = 851.6 [M+H] + The calculated value is m / z = 851.51 [M+H]. + .

[0919]

[0920] Intermediate 2.3 was synthesized as described for intermediate 2.1, but with two additional cycles of Lys(N3) coupling to obtain a tetrapeptide, which was then N-terminated with azide-dPEG4-NHS ester [944251-24-5] instead of acetic anhydride. Purification by RP-HPLC followed by lyophilization provided intermediate 2.3 as a viscous, colorless solid. HPLC (Method A): tR = 10.1 min. MS (ESI): m / z = 1082.67 [M+H] + The calculated value is m / z = 1082.63[C43H80N21O12]. + .

[0921] Intermediate 2.4 was synthesized in two steps:

[0922]

[0923] Step 1: Under an argon atmosphere, wash 128 mg (4.06 mmol) of sodium hydride (60% suspension in mineral oil) twice with 5 mL of dry THF and resuspend it in 5 mL of THF. Add dropwise 653 mg (2901 µmol = 8 equivalents) of azide-PEG4-ol [CAS 86770-67-4, TCI, A2294] in 5 mL of THF and stir at room temperature until gas precipitation stops (15 min). Add 231 mg of solid hexa(bromomethyl)benzene (363 mmol, CAS: 3095-73-6, TCIH0901) fractionally and stir the mixture overnight. Pour the reaction mixture into 20 mL of saturated ammonium chloride solution and dilute with 20 mL of ethyl acetate. Separate the organic phase and wash with 20 mL of brine, dry (MgSO4) and evaporate to dryness. The crude product was purified on silica gel using a gradient of 0-50% acetone in hexane to give 392 mg (79%) of a pale yellow oily hexaazide. MS (ESI): m / z = 1482.67 [M+NH4] + The calculated value is m / z = 1482.81. ¹H NMR (500 MHz, D₆-DMSO) δ 4.59 (s, 16H), 3.63 (dd, J = 6.1, 3.7 Hz, 15H), 3.61 – 3.48 (m, 72H), 3.39 – 3.33 (m, 14H). ¹³C NMR (126 MHz, D₆-DMSO) δ 137.25, 69.75, 69.73, 69.70, 69.65, 69.62, 69.45, 69.12, 65.60, 49.93.

[0924] Step 2: 139 mg of the product from Step 1 (95 µmol) was dissolved in 4.5 mL of THF and 25 mg of triphenylphosphine (95 µmol) was added. After the phosphine dissolved, 0.5 mL of water was added, and the mixture was stirred overnight in an open container at room temperature, followed by evaporation to dryness. Triphenylphosphine oxide and trace amounts of starting material in the crude product were removed on silica gel using a gradient of 0 to 10% methanol + 0.1% triethylamine in DCM. The fraction containing the product was evaporated and purified by preparative HPLC to give 29.6 mg (20%) of intermediate 2.4. HPLC (Method A) t R = 11.2 min. MS (ESI): Measured value m / z = 1439.75 [M+2H] 2+ The calculated value m / z = 1439.80 [C 60 H 111 N 16 O 24 ] 2+ .

[0925] Intermediate 2.5

[0926]

[0927] 2-Chlorotriphenylmethyl resin (1 g) was swollen in anhydrous DCM (5 mL) for 10 min. The suspension was filtered, and a solution of 1,11-diamino-3,6,9-trioxaundecan (531 mg, 2.76 mmol, 2 equivalents) in anhydrous DCM (5 mL) was added and shaken at room temperature for 2 h. The suspension was filtered, the resin was washed with DCM (3 x 1 min) and capping solution (5 mL, MeOH / DIPEA / DCM 1 / 1 / 8) was added and shaken at room temperature for 10 min. The suspension was filtered, and the capping procedure was repeated once. The resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min) and dried under vacuum.

[0928] For the next step, the resin was swollen in DMF for 10 min. The suspension was filtered, and a 20% piperidine solution in DMF for Fmoc deprotection was added to the resin. The suspension was incubated for 20 min, filtered, and the deprotection procedure was repeated once. Then, the resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min). The resin was swollen in DMF for 10 min, filtered, and a solution of Fmoc-Lys(N3)-OH (3 equivalents), TBTU (3 equivalents), and DIPEA (6 equivalents) in DMF was added. The suspension was shaken at room temperature for 2 h. The resin was washed with DMF (3 x 1 min) and DCM (3 x 1 min), and the Fmoc groups were removed with 20% piperidine as described above. The coupling procedure was repeated with another Fmoc-Lys(N3)-OH (3 equivalents), followed by washing with DMF (3 x 1 min) and DCM (3 x 1 min) and drying under vacuum to obtain 1.584 g of resin loaded with intermediate 2.5. The loading was determined as described in J. Pept. Sci. 2017, 23, 757-762, yielding a loading of 0.2 mmol / g.

[0929] Test cleavage was performed using 200 mg of resin, which was treated twice with 1 mL DCM / TFA 95:5 for 15 min. The cleavage solutions were combined, evaporated, and purified by preparative HPLC to obtain 28 mg of Fmoc-protected diazide. HPLC (Method AF): t R = 17.3 min. MS (ESI): Measured value m / z = 723.4 [M+H] + The calculated value is m / z = 723.4 [C]. 35 H 51 N 10 O7] + .

[0930] Intermediate 2.6

[0931]

[0932] By repeating the procedure for intermediate 2.5 but performing two additional coupling steps with Fmoc-Lys(N3)-OH, a resin loaded with intermediate 2.6 (1.626 g) was obtained with a loading of 0.20 mmol / g.

[0933] Test cleavage with 200 mg of resin yielded 48.36 mg of Fmoc-protected tetraazide. HPLC (Method AF): t R= 19.9 min. MS (ESI): Measured value m / z = 1031.5 [M+H] + The calculated value is m / z = 1031.6 [C]. 47 H 71 N 18 O9] + .

[0934] Intermediate 2.7

[0935]

[0936] Intermediate 2.7 was synthesized in 7 steps:

[0937] Step 1:

[0938]

[0939] Sodium hydride (60% dispersion in mineral oil, 656 mg, 3 equivalents) was added dropwise to a solution of 2,2'-(triphenylmethylazinediyl)diethanol (CAS No.: 23202-47-3, 1.89 g, 5.47 mmol, 1.0 equivalent) in 15 mL of anhydrous DMF, and the mixture was stirred for 1 h until gas precipitation ceased. A solution of azide-PEG4-Tos (CAS No.: 168640-82-2, 4.50 g, 12.0 mmol, 2.2 equivalents) in 5 mL of DMF was added dropwise, and the mixture was stirred overnight at 50ºC, followed by stirring for 3 h. The reaction was then quenched with 50 mL of 0.1 M potassium phosphate buffer (pH 6), and the mixture was extracted with 75 mL of ethyl acetate. The organic phase was washed with brine, dried (MgSO4), and evaporated. The crude product was purified on silica gel using a gradient of 0 to 50% acetone in hexane to give 3.20 g (78% yield) of the step 1 product as a colorless oil. 1 H NMR (500MHz, CDCl3) δ 7.55 – 7.45 (m, 5H), 7.26 – 7.20 (m, 5H), 7.17 – 7.10 (m, 3H), 3.67 – 3.58 (m, 28H), 3.55 – 3.50 (m, 4H), 3.37 (dd, J = 5.5, 4.6 Hz, 4H), 2.54 (t, J = 7.0 Hz, 4H). 13C NMR (126 MHz, CDCl3) δ 143.74, 129.31, 127.52,125.99, 78.66, 71.20, 70.66, 70.63, 70.60, 70.52, 70.37, 70.00, 52.86, 50.64.

[0940] Step 2:

[0941]

[0942] 500 µl of formic acid was added to the product from step 1 (2.16 g, 2.88 mmol) dissolved in 10 ml of DCM / methanol 9:1 and stirred for 5 min. Then, 10 g of diatomaceous earth was added and all volatiles were evaporated under vacuum. The product was purified on silica gel using a gradient of 0 to 5% methanol in DCM containing 0.5% ammonia as a modifier to give 1.325 g (91%) of the product from step 2 as a colorless oil. MS (ESI): m / z = 508.3 [M+H] + The calculated value m / z = 508.3 [C 20 H 42 N7O8] + . 1 HNMR (500 MHz, DMSO) δ 3.63 – 3.59 (m, 4H), 3.58 – 3.47 (m, 24H), 3.44 (t, J =5.6 Hz, 4H), 3.41 – 3.36 (m, 4H), 3.25 (s, 1H), 2.66 (t, J = 5.7 Hz, 4H). 13 CNMR (126 MHz, DMSO) δ 70.08, 69.74, 69.71, 69.70, 69.68, 69.62, 69.54, 69.13, 49.94, 48.65.

[0943] Step 3:

[0944]

[0945] Triphenylphosphine (350 mg, 1 equivalent) was added to a solution of the product from step 1 (1.015 g, 1.355 mmol, 1 equivalent) in 22.5 ml THF, and the mixture was stirred at room temperature until the phosphine was completely dissolved. Water (2.5 ml) was then added, and the solution was stirred at room temperature for 3 days. Diboc (591 mg, 2.71 mmol, 2 equivalent) and DIPEA (461 µl, 2.71 mmol, 2 equivalent) were then added, and stirring was continued for 1 h. Diatomaceous earth was then added, and the evaporation was evaporated. The residue was thoroughly dried under high vacuum.

[0946] The product of step 3 was purified on silica gel using a gradient of 0% to 37% acetone in hexane to give 502 mg (45%) of a colorless oil. MS (ESI): m / z = 824.0 [M+H] + The calculated value m / z = 824.5 [C 44 H 66 N5O 10 ] + . 1 HNMR (500 MHz, CDCl3) δ 7.52 – 7.47 (m, 6H), 7.23 (dd, J = 8.3, 7.2 Hz, 6H), 7.17 – 7.10 (m, 3H), 5.03 (s, 1H), 3.69 – 3.58 (m, 26H), 3.56 – 3.49 (m, 6H), 3.39 – 3.34 (m, 2H), 3.30 (d, J = 5.5 Hz, 2H), 2.54 (t, J = 7.0 Hz, 4H), 1.44 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 155.97, 143.73, 129.31, 127.53, 126.00,79.12, 78.66, 71.20, 70.67, 70.63, 70.60, 70.55, 70.53, 70.49, 70.37, 70.21,70.19, 70.01, 52.85, 50.64, 40.32, 28.41.

[0947] Step 4:

[0948]

[0949] By applying the procedure of step 2 to the product of step 3, 342 mg (91%) of product 4, a colorless oil, was obtained. MS (ESI): m / z = 582.4 [M+H] + The calculated value m / z = 582.4 [C] 25 H 52 N5O 10 ] + . 1 H NMR(500 MHz, CDCl3) δ 5.12 (s, 1H), 3.72 – 3.58 (m, 31H), 3.54 (t, J = 5.2 Hz, 2H), 3.42 – 3.37 (m, 2H), 3.32 (q, J = 5.4 Hz, 2H), 2.83 (t, J = 5.4 Hz, 4H), 1.44 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 156.00, 79.09, 70.67, 70.63, 70.60,70.54, 70.50, 70.48, 70.32, 70.21, 70.01, 50.64, 49.14, 40.31, 28.40.

[0950] Step 5:

[0951]

[0952] Cyanuric acid (86 mg, 0.467 mmol, 1.0 equivalent) and DIPEA (1.675 ml, 9.85 mmol, 21 equivalent) were dissolved in 15 ml of acetonitrile, and the mixture was added to the product from step 2 (500 mg, 0.985 mmol, 2.1 equivalent) and stirred at room temperature. After 3 h, ethyl acetate was added, and the acetonitrile was evaporated under vacuum. The remaining aqueous phase was extracted twice with ethyl acetate (20 ml each time), and the combined organic phases were washed with brine, dried (MgSO4), and evaporated to dryness.

[0953] The crude product was purified on silica gel using a gradient of 0-50% acetone in hexane to yield 403 mg (77%) of the product from step 5 as a colorless oil. MS (ESI): m / z = 1126.6 [M+H] + The calculated value m / z = 1126.6 [C] 43 H 81 ClN 17 O 16 ] +. 1 H NMR (500 MHz, CDCl3) δ 3.78 (t, J = 5.7 Hz, 4H), 3.73 (t, J= 5.9 Hz, 4H), 3.69 – 3.56 (m, 64H), 3.44 – 3.34 (m, 8H). 13 C NMR (126 MHz, CDCl3) δ 168.80, 164.53, 70.64, 70.63, 70.61, 70.58, 70.57, 70.55, 70.54,70.51, 70.25, 70.00, 69.26, 68.88, 50.62, 48.12, 47.77.

[0954] Step 6:

[0955]

[0956] The product from step 6 (145 mg, 129 µmol, 1.0 equivalent) and the product from step 4 (151 mg, 258 µmol, 2.0 equivalent) were dissolved in 4.5 mL of acetonitrile, and DIPEA (217 µl, 1.29 µmol, 10 equivalent) was added. The mixture was heated to reflux for 3 days. After cooling, sufficient diatomaceous earth was added to absorb all the reaction mixture, and all volatiles were evaporated. The remainder was dried under high vacuum. The crude product was purified on silica gel using a gradient of 0 to 50% acetone in hexane to give 161 mg (75%) of the product from step 7 as a colorless oil. MS (ESI): m / z = 1671.9 [M+H] + The calculated value m / z = 1672.0 [C] 68 H 131 N 22 O 26 ] + .

[0957] Step 7:

[0958] The product from step 6 (57 mg, 34 µM) was dissolved in 250 µl of DCM and 57 µl of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, and then the evaporation was evaporated. The crude product was purified by preparative HPLC to yield 40 mg (70%) of intermediate 2.7 as a colorless oil. HPLC (Method A): t R = 9.3 min. MS (ESI): Measured value m / z = 1571.9 [M+H] + The calculated value m / z = 1571.9 [C] 63H 123 N 22 O 24 ] + .

[0959] Example 1.4 - Synthesis of Intermediate 3.1

[0960]

[0961] EDC x HCl (485.8 mg, 2.53 mmol, 1.3 equivalents) and DIPEA (1.09 mL, 6.24 mmol, 3.2 equivalents) were added to a solution of propargyl-PEG(5)-acid (CAS No. 1245823-51-1, 593.1 mg, 1.94 mmol, 1 equivalent) in anhydrous THF (8.9 mL). N-hydroxyphthalimide (413.4 mg, 2.53 mmol, 1.3 equivalents) in anhydrous THF (8.9 mL) was added dropwise over 10 minutes. The reaction was stirred at room temperature and monitored by TLC and RP-HPLC. After 21 h, the solution was diluted with ethyl acetate (60 mL), and the organic layer was washed with 1N HCl (30 mL), sodium bicarbonate solution (30 mL), and brine (30 mL). The organic layer was dried over magnesium sulfate and concentrated under vacuum. The crude product (724.7 mg) was purified by silica gel rapid chromatography using a gradient from n-hexane to ethyl acetate. Intermediate 3.1 (457.3 mg, 52%) was obtained as a pale yellow oil. 22 H 27 NO.9 1 H NMR (500 MHz, CDCl3): δ (ppm) =7.89 (dd, J = 5.5, 3.1 Hz, 2H), 7.80 (dd, J = 5.5, 3.1 Hz, 2H), 4.21 (d, J =2.3 Hz, 2H), 3.89 (t, J = 6.4 Hz, 2H), 3.73 – 3.63 (m, 17H), 2.97 (t, J = 6.4Hz, 2H), 2.44 (t, J = 2.4 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ (ppm) = 167.65, 161.73, 134.73, 128.82, 123.92, 79.61, 74.48, 70.69, 70.58, 70.54, 70.53, 70.49, 70.46, 70.33, 69.04, 65.76, 58.33, 32.14. HPLC (Method I): t R = 11.7 min. MS(ESI): Measured value m / z = 472.2 [M+Na] + The calculated value is m / z = 472.2 [M+Na]. + .

[0962] Example 1.5 - Synthesis of Intermediate 3.2

[0963]

[0964] HATU (11.64 g, 30.6 mmol, 1.6 equivalent) and DIPEA (10.7 mL, 61.2 mmol) were added to a solution of pentylene-4-acetylic acid (2 g, 20.4 mmol, 1.05 equivalent) in DMF (40 mL) at 5ºC, and the resulting solution was stirred at 5ºC for 10 min. Then, a solution of tert-butyl 1-amino-3,6,9,12-tetraoxapentadecano-15-oate (6.24 g, 19.4 mmol, 1 equivalent) in DMF (10 mL) was added dropwise, and the resulting solution was allowed to warm to room temperature. After 2 h, the solution was concentrated under vacuum, and the residue was absorbed into ethyl acetate (300 mL). The organic layer was washed with 0.2 M citric acid (150 mL), saturated sodium bicarbonate solution (150 mL), and brine (150 mL). The organic layer was dried over magnesium sulfate and concentrated under vacuum. 1-(pentane-4-acetylamido)-3,6,9,12-tetraoxapentadecano-15-oic acid tert-butyl ester (8.20 g, quantified) was obtained by gradient purification from hexane to acetone using silica gel rapid chromatography. 20 H 35 NO7. HPLC (Method A): t R = 10.6 min. MS(ESI): Measured value m / z = 424.4 [M+Na] + The calculated value is m / z = 424.2 [M+Na]. + .

[0965]

[0966] 1-(pentan-4-acetylamido)-3,6,9,12-tetraoxapentadecano-15-oic acid tert-butyl ester (8.20 g, maximum 19.4 mmol, 1 equivalent) was dissolved in TFA (15.6 mL) and stirred at 300 mbar for 1 h. The TFA was removed under vacuum, and the residue was co-evaporated twice with toluene. The resulting residue was dissolved in THF (anhydrous, 50 mL), and N-ethyl-N′-carbodiimide hydrochloride (4.66 g, 24.3 mmol, 1.25 equivalent) and DIPEA (11 mL, 63 mmol, 3.25 mmol) were added dropwise. Then, a solution of N-hydroxyphthalimide (3.96 g, 24.3 mmol, 1.25 equivalent) in THF (anhydrous, 50 mL) was added dropwise, and the resulting pale pink solution was stirred overnight at room temperature. Ethyl acetate (400 mL) was added, and the organic layer was washed with 2N HCl (100 mL), saturated sodium bicarbonate solution (100 mL), and brine (100 mL). The organic layer was dried over magnesium sulfate and concentrated under vacuum. Purification by rapid silica gel chromatography using a gradient from dichloromethane to acetone provided intermediate 3.2 (4.96 g, 52%) as a pale yellow oil. 24 H 30 N2O9. 1 H NMR (500 MHz, DMSO-d6): δ(ppm) = 8.01 – 7.89 (m, 4H), 7.87 (s, 1H), 3.77 (t, J = 6.0 Hz, 2H), 3.63 –3.46 (m, 12H), 3.40 (t, J = 5.9 Hz, 2H), 3.20 (q, J = 5.8 Hz, 2H), 3.01 (t, J= 6.0 Hz, 2H), 2.70 (t, J = 2.6 Hz, 1H), 2.39 – 2.31 (m, 2H), 2.31 – 2.23 (m,2H). 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 170.18, 168.21, 161.61, 135.39, 128.10, 123.85, 83.65, 71.00, 69.73, 69.68, 69.62, 69.60, 69.50, 69.01, 65.18, 38.50, 34.02, 31.55, 14.09. HPLC (Method A): t R = 10.5 min. MS (ESI): Measured value m / z = 491.4 [M+H]+ The calculated value is m / z = 491.2 [M+H]. + .

[0967] Example 1.6 - Intermediate 4.x (N3-PEG) n Synthesis of -β / γ-CD

[0968]

[0969] Intermediate 4.1 was synthesized from GP5 using azide-PEG(2)-NHS ester and 6-amino-6-deoxy-β-cyclodextrin. The crude product obtained was used without further purification.

[0970]

[0971] Intermediate 4.2 was synthesized from GP5 using azide-PEG(4)-NHS ester and 6-amino-6-deoxy-β-cyclodextrin. The crude product obtained was used without further purification.

[0972]

[0973] Intermediate 4.3 was synthesized from GP5 using azide-PEG(8)-NHS ester and 6-amino-6-deoxy-β-cyclodextrin. The crude product obtained was used without further purification.

[0974]

[0975] Intermediate 4.4 was synthesized from GP5 using azide-PEG(24)-NHS ester and 6-amino-6-deoxy-β-cyclodextrin. The crude product obtained was used without further purification.

[0976]

[0977] Intermediate 4.5 was synthesized from GP5 using azide-PEG(2)-NHS ester and 6-amino-6-deoxy-γ-cyclodextrin. The crude product obtained was used without further purification.

[0978]

[0979] Intermediate 4.6 was synthesized from GP5 using azide-PEG(4)-NHS ester and 6-amino-6-deoxy-γ-cyclodextrin. The crude product obtained was used without further purification.

[0980]

[0981] Intermediate 4.7 was synthesized from GP5 using azide-PEG(8)-NHS ester and 6-amino-6-deoxy-γ-cyclodextrin. The crude product obtained was used without further purification.

[0982]

[0983] β-Cyclodextrin (2.000 g, 1.762 mmol) was co-evaporated twice with 30 mL of dry DMSO (anhydrous), and then dissolved again in 30 mL of DMSO. Lithium hydride (21 mg, 2.643 mmol, 1.5 equivalent) was added, and the suspension was stirred under an argon atmosphere for 15 h. Subsequently, 1-azido-2-{2-[2-(2-bromoethoxy)ethoxy]ethoxy}ethane (497 mg, 1.762 mmol, 1.0 equivalent) dissolved in 2 mL of DMSO (anhydrous) was added, followed by lithium iodide (24 mg, 176 µmol, 0.1 equivalent), and the mixture was stirred at 55ºC for 7.5 h, and then placed at room temperature. After TLC (acetonitrile / water / ammonia, 6:3:1) indicated no further conversion, the mixture was concentrated under vacuum to approximately 6 mL, and the residue was added dropwise to 160 mL of ice-cold acetone. The resulting precipitate was separated by centrifugation, resuspended in 120 mL of cold acetone by sonication, and centrifuged again. After drying under vacuum, the solid was absorbed into 5 mL of water, and insoluble β-cyclodextrin was removed by filtration, followed by lyophilization of the filtrate. The crude product (2.263 g) was acidified to pH 2 with trifluoroacetic acid and purified by preparative RP-HPLC to give 532 mg (23%) of pure intermediate 4.8 as a colorless lyophilized product. MS (ESI): m / z = 1336.8 [M+H] + The calculated value m / z = 1336.5 [M+H] + .

[0984]

[0985] Intermediate 4.9 was synthesized using γ-cyclodextrin based on intermediate 4.8. The target product was obtained in 22% yield as a colorless lyophilized product. MS (ESI): m / z = 1498.9 [M+H] + The calculated value is m / z = 1498.5 [M+H]. + .

[0986] Example 1.7 - Synthesis of intermediates 5.1 and 5.2

[0987]

[0988] Intermediate 5.1 was synthesized according to WO 2019081455 and WO 2022207699 using Wang resin loaded with Fmoc-Sar, Fmoc-Sar-Sar-OH, Fmoc-Sar-OH, and azidoacetic acid for N-terminal capping. Fmoc deprotection was performed twice in DMF with 20% piperidine for 15 min. Amino acid coupling was performed in DMF using amino acids (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents). Resin cleavage was performed using pure TFA (10 mL / g resin), followed by precipitation from ice-cold methyl tert-butyl ether and purification by RP-HPLC. Intermediate 5.1 was given as a colorless solid (166.7 mg, 41%). 32 H 53 N 13 O 12 HPLC (Method H): t R = 7.4 min. MS (ESI): Measured value m / z = 834.2 [M+Na] + The calculated value is m / z = 834.4 [M+Na]. + .

[0989]

[0990] Intermediate 5.2 was synthesized according to WO 2019081455 and WO 2022207699 using NovaGel Rink-Amide resin loaded with Fmoc-Sar, Fmoc-Sar-Sar-OH, Fmoc-Sar-OH, and azidoacetic acid for N-terminal capping. Fmoc deprotection was performed twice in DMF with 20% piperidine for 15 min. Amino acid coupling was performed in DMF using amino acids (3 equivalents), HATU (2.9 equivalents), and DIPEA (6 equivalents). Resin cleavage was performed using TFA / TIS 95 / 5 (10 mL / g resin), followed by precipitation from ice-cold methyl tert-butyl ether and purification by RP-HPLC. Intermediate 5.2 was given as a colorless solid (83.6 mg, 54%). 32 H 54 N 14 O 11 HPLC (Method K): t R = 7.1 min. MS (ESI): Measured value m / z = 811.5 [M+H] + The calculated value is m / z = 811.4 [M+H]. + .

[0991] Example 1.8 - Synthesis of intermediates 6.1-6.4

[0992] Synthesis of intermediate 6.1

[0993]

[0994] TBTU (2.467 g, 7.8 mmol, 1 equivalent) and DIPEA (1.33 mL, 7.8 mmol, 1 equivalent) were added to a solution of Fmoc-Lys(Boc)-OH (3.056 g, 6.5 mmol, 1 equivalent) in DMF (30 mL), and the resulting solution was mixed for 1 min. Then, a pre-activated amino acid solution was added to a solution of 14-azido-3,6,9,12-tetraoxatetradecane-1-amine (1.680 g, 6.5 mmol, 1 equivalent) in DMF (10 mL), and the resulting solution was stirred at room temperature for 2 h. Water (5 mL) was added, and the solution was evaporated under vacuum. The residue was dissolved in ethyl acetate (100 mL), and the organic layer was washed with 0.2 M citric acid aqueous solution (50 mL), saturated sodium bicarbonate aqueous solution (50 mL), and brine (50 mL). The organic layer was dried over magnesium sulfate and evaporated under vacuum. Fmoc-Lys(Boc)-NH-PEG4-N3 (3.545 g, 76%) was provided as a colorless, viscous solid by gradient purification from MTBE to acetone using rapid silica gel chromatography. 36 H 52 N6O9. 1H NMR (500MHz, DMSO-d6): δ (ppm) = 7.88 (dt, J = 7.7, 0.9 Hz, 2H), 7.82 (t, J = 5.7 Hz,1H), 7.76 – 7.68 (m, 2H), 7.42 (td, J = 7.5, 1.0 Hz, 2H), 7.33 (tt, J = 7.4,1.2 Hz, 3H), 6.68 (s, 1H), 4.30 – 4.18 (m, 3H), 3.93 (td, J = 8.6, 5.2 Hz,1H), 3.62 – 3.56 (m, 2H), 3.55 – 3.48 (m, 12H), 3.41 (t, J = 6.0 Hz, 2H), 3.37 (dd, J = 5.6, 4.4 Hz, 2H), 3.21 (ddt, J = 19.6, 13.6, 7.6 Hz, 2H), 2.89 (q, J = 10.3, 8.3 Hz, 2H), 1.69 – 1.43 (m, 2H), 1.37 (s, 9H), 1.24 (s, 4H). 13 CNMR (126 MHz, DMSO-d6): δ (ppm) = 171.86, 155.78, 155.45, 143.79, 143.70, 140.60, 127.49, 126.92, 125.17, 119.95, 77.21, 69.71, 69.69, 69.67, 69.63, 69.60, 69.48, 69.11, 68.88, 65.50, 54.56, 49.93, 46.62, 38.47, 31.63, 29.08, 28.16, 22.69. HPLC (Method A): t R = 14.3 min. MS (ESI): Measured value m / z = 735.4 [M+Na] + The calculated value is m / z = 735.4 [M+Na]. + .

[0995]

[0996] Diethylamine (5.00 mL, 56.1 mmol, 17 equivalents) was added to a solution of Fmoc-Lys(Boc)-NH-PEG4-N3 (2.00 g, 2.8 mmol, 1 equivalent) in DMF (20 mL), and the resulting solution was stirred at 600 mbar at room temperature for 30 min. The solvent was removed under vacuum, and the deprotected intermediate H-Lys(Boc)-NH-PEG4-N3 (1.182 g, 86%) was purified by silica gel rapid chromatography using a gradient from dichloromethane to dichloromethane / MeOH / concentrated ammonia (9:1:0.1) to provide a colorless oily solution. As a next step, 1 M HCl (11.85 mL, 11.8 mmol, 5 equivalents) was added to a solution of H-Lys(Boc)-NH-PEG4-N3 (1.163 g, 2.4 mmol, 1 equivalent) in 20 mL of 1,4-dioxane, and the mixture was stirred at 600 mbar at room temperature for 10 min. The solvent was removed under vacuum, and the residue was dissolved in 1 M HCl (11.85 mL, 11.8 mmol, 5 equivalents) and stirred at 600 mbar at room temperature for 10 min. The solvent was removed under vacuum, and the mixture was further co-evaporated twice with 1,4-dioxane. Lys-NH-PEG4-N3 (1.119 g, quantified) was obtained as a highly viscous, colorless oil. 16 H 34 N6O5. 1 H NMR (500 MHz, DMSO-d6): δ (ppm) = 8.67 (t, J = 5.6 Hz, 1H),8.32 (s, 3H), 8.11 (s, 3H), 3.78 – 3.72 (m, 1H), 3.61 – 3.58 (m, 2H), 3.59 –3.47 (m, 12H), 3.45 (t, J = 5.9 Hz, 2H), 3.38 (dd, J = 5.5, 4.4 Hz, 2H), 3.28(d, J = 5.9 Hz, 1H), 3.23 (dq, J = 13.7, 5.8 Hz, 1H), 2.73 (d, J = 8.7 Hz,2H), 1.73 (q, J = 7.3 Hz, 2H), 1.59 (qd, J = 8.5, 7.8, 6.0 Hz, 2H), 1.42 –1.33 (m, 2H). 13C NMR (126 MHz, DMSO-d6): δ (ppm) = 168.43, 69.74, 69.70, 69.66, 69.62, 69.49, 69.13, 68.63, 51.78, 49.97, 38.66, 38.11, 30.18, 26.08, 20.97. HPLC (Method H): t R = 6.9 min. MS (ESI): Measured value m / z = 391.3 [M+H] + The calculated value is m / z = 391.3 [M+H]. + .

[0997]

[0998] A solution of Lys-NH-PEG4-N3 (500 mg, 1.1 mmol, 1 equivalent) in DMF (10 mL) was added with d-gluconic acid-1,5-lactone (481 mg, 2.7 mmol, 2.5 equivalent) and DIPEA (917 µL, 5.4 mmol, 5.0 equivalent), and the resulting solution was stirred at room temperature for 15 h, followed by stirring at 80ºC for 1 h. After a total reaction time of 16.5 h, d-gluconic acid-1,5-lactone (481 mg, 2.7 mmol, 2.5 equivalent) and DIPEA (917 µL, 5.4 mmol, 5.0 equivalent) were added. After a total reaction time of 21 h, the solvent was removed under vacuum, and the residue was co-evaporated with a mixture of methanol (10 mL) and DIPEA (917 µL), followed by co-evaporation with methanol (10 mL). The crude product was precipitated from ice-cold MTBE. Intermediate 6.1 (294 mg, 36%) was obtained as a colorless, glassy solid by preparative HPLC purification. 28 H 54 N6O 17 . 1H NMR (500 MHz, DMSO-d6): δ (ppm) = 7.90 (t, J = 5.7 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.53 (t, J= 5.9 Hz, 1H), 4.41 (s, 10H), 4.22 (td, J = 8.4, 5.0 Hz, 2H), 4.06 (d, J =3.5 Hz, 1H), 3.97 (d, J = 3.7 Hz, 1H), 3.93 – 3.86 (m, 3H), 3.62 – 3.56 (m,4H), 3.56 – 3.43 (m, 14H), 3.43 – 3.35 (m, 4H), 3.20 (qd, J = 6.0, 3.5 Hz, 4H), 3.05 (dp, J = 13.9, 6.9 Hz, 2H), 1.73 – 1.64 (m, 1H), 1.56 (tdd, J =13.5, 9.4, 5.3 Hz, 1H), 1.45 – 1.33 (m, 2H), 1.23 (s, 2H). 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 172.28, 172.17, 171.28, 73.50, 73.32, 72.36, 72.16, 71.46, 70.42, 70.10, 69.72, 69.64, 69.50, 69.14, 68.75, 63.29, 63.24, 51.99, 49.97, 38.52, 38.08, 31.75, 28.77, 22.46. HPLC (Method M): t R = 13.5 min. MS (ESI): Measured value m / z = 747.4 [M+H] + The calculated value is m / z = 747.4 [M+H]. + .

[0999] Synthesis of intermediate 6.2

[1000]

[1001] 14-Azide-3,6,9,12-tetraoxatetradecane-1-amine (262 mg, 1.0 mmol, 1 equivalent) was dissolved in DMF (2 mL). d-gluconic acid-1,5-lactone (214 mg, 1.2 mmol, 1.2 equivalent) was added as a solid, followed by DIPEA (348 µL, 2.0 mmol, 2 equivalent), and the resulting solution was stirred at 110ºC for 2 h. After cooling to room temperature, the solvent was evaporated under vacuum. Intermediate 6.1 (318 mg, 72%) was provided by rapid silica gel chromatography using a gradient purification from dichloromethane to dichloromethane / methanol / water (5:1:0.1). 16 H 32 N4O 10 HPLC (Method M): t R = 13.7 min. MS(ESI): Measured value m / z = 441.3 [M+H] + The calculated value is m / z = 441.2 [M+H]. + .

[1002] Synthesis of intermediate 6.3

[1003]

[1004] N-hydroxy-succinimide (2.32 g, 20.2 mmol, 2.3 equivalents) was added to a solution of ZL-Glu-OH (2.50 g, 8.9 mmol, 1 equivalent) in THF (anhydrous, 50 mL), and the solution was cooled to 0ºC. Then, N,N'-dicyclohexylcarbodiimide (3.85 g, 18.7 mmol, 2.1 equivalents) was added, and the resulting suspension was stirred at 0ºC for 2 h, followed by stirring overnight at room temperature. For post-treatment, the formed urea was removed by filtration, and the oily residue was dissolved in ethyl acetate (75 mL). The organic layer was washed with saturated sodium bicarbonate solution, brine, and water, dried over magnesium sulfate, and the solvent was removed under vacuum. After treatment with diethyl ether, ZL-Glu(OSu)-OSu (3.68 g, 87%) was obtained as an intermediate and used without further purification.

[1005] Next, d-glucamine (762.23 mg, 4.2 mmol, 2 equivalents) was added to a solution of ZL-Glu(OSu)-OSu (1.00 g, 2.1 mmol, 1 equivalent) in DMF (anhydrous, 40 mL), and the resulting suspension was stirred at 65ºC for 3 h. As the reaction progressed, the initial suspension became a solution. The solution was removed under vacuum, and ZL-Glu(NHDG)-NHDG (933.9 mg, 73%) was purified by RP-HPLC. 25 H 41 N3O 14 HPLC (Method H): t R = 8.0 min. MS (ESI): Measured value m / z = 630.3 [M+Na] + The calculated value is m / z = 630.2 [M+Na]. + .

[1006]

[1007] Pd / C (10%, 161 mg) was added to a solution of ZL-Glu(NHDG)-NHDG (924.9 mg, 1.5 mmol) in methanol (180 mL) under argon atmosphere. The atmosphere was changed from argon to hydrogen, and the resulting suspension was stirred at room temperature for 3.5 h. The catalyst was removed by filtration through diatomaceous earth, and the solvent of the resulting filtrate was removed under vacuum to provide HL-Glu(NHDG)-NHDG (728.7 mg, 1.5 mmol, quantified). Next, HL-Glu(NHDG)-NHDG (100.0 mg, 211.2 µmol, 1.5 equivalents) was dissolved in DMF (anhydrous, 2 mL) and added to a solution of azide-PEG4-NHS ester (CAS No. 944251-24-5, 54.68 mg, 140.8 µmol, 1 equivalent) under argon atmosphere. DIPEA (73.57 µL, 422.4 µmol, 3 equivalents) was added, and the resulting solution was stirred at room temperature for 1.5 h. The solvent was removed under vacuum, and intermediate 6.3 (82.74 mg, 79%) was purified by RP-HPLC. 28 H 54 N6O 17 HPLC (Method A): t R = 5.9 min. MS (ESI): Measured value m / z = 747.2 [M+H] + The calculated value is m / z = 747.4 [M+H]. + .

[1008] Synthesis of intermediate 6.4

[1009]

[1010] Intermediate 6.4 (91.9 mg, 71%) was synthesized as described for intermediate 6.3, but using azide-PEG8-NHS ester (CAS No. 1204834-00-3) instead of azide-PEG4-NHS ester. 36 H 70 N6O 21 HPLC (Method A): t R =6.7 min. MS (ESI): Measured value m / z = 923.6 [M+H] + The calculated value is m / z = 923.5 [M+H]. + .

[1011]

[1012] Example 3 - Synthesis of Compound IV.1

[1013] Synthesis of I.1

[1014]

[1015] Intermediate I.1 was synthesized from GP1 using m-PEG(4)-azide (40.4 mg, 173 µmol, 1.2 equivalents), intermediate 1.3 (150 mg, 144 µmol, 1.0 equivalents), copper(II) sulfate pentahydrate (3.6 mg, 14.4 µmol, 0.1 equivalents), tris(benzyltriazolylmethyl)amine (9.6 mg, 18 µmol, 0.125 equivalents), and sodium ascorbate (7.1 mg, 36 µmol, 0.25 equivalents) in DMF / water (1.65 mL, 10:1). Purification by RP-HPLC yielded intermediate I.1 (137 mg, 74%) as a pale yellow solid. HPLC (Method D): t R = 11.8 min. MS (ESI): Measured value m / z = 1278.5 [M+H] + The calculated value is m / z = 1278.5 [M+H]. + .

[1016] Synthesis of II.1

[1017]

[1018] Intermediate II.1 was synthesized from GP3 using 222 µL of diethylamine (2.14 mmol, 20 equivalents) in 1.37 mL of DMF for Fmoc deprotection, and intermediates I.1 (137 mg, 107 µmol, 1 equivalent), 3.1 (53.0 mg, 118 µmol, 1.1 equivalents), and diisopropylethylamine (56 µL, 321 µmol, 3 equivalents) in 1 mL of DMF for coupling reaction. Intermediate II.1 (101 mg, 70%) was purified by RP-HPLC as a pale yellow solid. HPLC (Method A): t R =11.1 min. MS (ESI): Measured value m / z = 1342.5 [M+H] + The calculated value is m / z = 1342.6 [M+H]. + .

[1019]

[1020] Example 6 - Synthesis of Compound IV.4

[1021] Synthesis of I.4

[1022]

[1023] Intermediate I.4 was synthesized from GP1 using intermediate 4.1 (22.4 µmol, 1 equivalent), intermediate 1.3 (26.16 mg, 25.0 µmol, 1.1 equivalent), copper(II) sulfate pentahydrate (1.23 mg, 4.9 µmol, 0.2 equivalent), tris(benzyltriazolylmethyl)amine (3.17 mg, 6.0 µmol, 0.26 equivalent), and sodium ascorbate (4.33 mg, 11.9 µmol, 0.5 equivalent) in DMF / water (0.28 mL, 10:1). Purification by RP-HPLC yielded intermediate I.4 (31.51 mg, 60%) as a pale yellow solid. 108 H 139 FN 10 O 48 HPLC (Method D): t R = 10.7 min. MS (ESI): Measured value m / z = 1182.5 [M+2H] 2+ The calculated value is m / z = 1182.4 [M+2H]. 2+ .

[1024] Synthesis of II.4

[1025]

[1026] Intermediate II.4 was synthesized from GP3 using intermediate I.4 (31.51 mg, 13.3 µmol, 1 equivalent), 33 µL of diethylamine (319.9 µmol, 24 equivalents) in 1.6 mL of DMF for Fmoc deprotection, and intermediate 3.1 (8.98 mg, 20.0 µmol, 1.5 equivalents), and diisopropylethylamine (6.8 µL, 40.0 µmol, 3 equivalents) in 2.1 mL of DMF for coupling reaction. Intermediate II.4 (15.51 mg, 50%) was purified by RP-HPLC as a pale yellow solid. 107 H 151 FN 10 O 52 HPLC (Method D): t R = 9.1 min. MS (ESI): Measured value m / z = 1214.5 [M+2H] 2+ The calculated value is m / z = 1214.5 [M+2H]. 2+ .

[1027]

[1028] Example 18 - Synthesis of Compound IV.16

[1029] Synthesis of I.16

[1030]

[1031] Intermediate I.16 was synthesized from GP1 using intermediate 5.1 (30 mg, 37.0 µmol, 1 equivalent), intermediate 1.3 (42.5 mg, 40.7 µmol, 1.1 equivalent), copper(II) sulfate pentahydrate (1.85 mg, 7.4 µmol, 0.2 equivalent), tris(benzyltriazolylmethyl)amine (4.91 mg, 9.25 µmol, 0.25 equivalent), and sodium ascorbate (3.66 mg, 18.5 µmol, 0.5 equivalent) in DMF / water (0.33 mL, 10:1). Purification by RP-HPLC yielded intermediate I.16 (51.7 mg, 74%) as a pale yellow solid. 91 H 110 FN 19 O 23 HPLC (Method A): t R = 11.1 min. MS (ESI): Measured m / z = 1855.1 [MH] - The calculated value is m / z = 1854.8 [MH]. - .

[1032]

[1033] Example 21 - Synthesis of Compound IV.19

[1034] Synthesis of I.19

[1035]

[1036] Intermediate I.19 was synthesized from GP1 using intermediate 6.1 (64.31 mg, 86.11 µmol, 1 equivalent), intermediate 1.3 (60.0 mg, 57.4 µmol, 1.0 equivalent), copper(II) sulfate pentahydrate (14.3 mg, 57.4 µmol, 1 equivalent), THPTA (50.08 mg, 115.2 µmol, 2 equivalent), and sodium ascorbate (34.38 mg, 173.5 µmol, 3 equivalent) in DMF / water (1.72 mL, 2:1). Intermediate I.17 (76.18 mg, 74%) was obtained as a pale yellow solid by RP-HPLC purification. 87 H 111 FN 12 O 28 HPLC (Method B): t R= 16.8 min. MS (ESI): Measured value m / z = 896.7 [M+2H] 2+ The calculated value is m / z = 896.4 [M+2H]. 2+ .

[1037]

[1038] Synthesis of Examples 25 - IV.23

[1039] Synthesis of I.23a

[1040]

[1041] Compound I.23a was synthesized according to GP1 using intermediate 4.6 (65 mg, 41.4 µmol, 1 equivalent), intermediate 4.6 (40 mg, 41.4 µmol, 1.0 equivalent), copper(II) sulfate pentahydrate (2.1 mg, 8.3 µmol, 0.2 equivalent), THPTA (4.5 mg, 10.3 µmol, 0.25 equivalent), and sodium ascorbate (4.1 mg, 20.7 µmol, 0.5 equivalent) in DMF / water (640 µL, 10:1). Intermediate I.23a (113 mg, quantitative) was purified by RP-HPLC (Method A (380 nm)): t R =10.5 min. MS (ESI): Measured value m / z = 1267.3 [M+2H] 2+ The calculated value is m / z = 1268.52.

[1042] Synthesis of I.23b

[1043]

[1044] Intermediate I.23a (113.5 mg, 41 µmol, 1 equivalent) was absorbed into TFA (3.14 mL, 41 mmol, 1000 equivalent). The resulting reaction mixture was rotated in a rotary evaporator at 400 mbar at room temperature. After 2 minutes, the solution was concentrated to dryness. The residue was dissolved in 3 mL of H₂O at room temperature and the pH was adjusted to 10 by dropwise addition of 1.2 mL of 3.2% ammonia. The resulting mixture was lyophilized overnight to produce intermediate I.23b (0.047 g, 45%), which was a pale yellow lyophilized product. HPLC (Method A (380 nm)): tR = 7.2 min. MS (ESI): Measured value m / z = 1152.5 [M+2H] 2+ The calculated value is m / z = 1153.46.

[1045] Synthesis of II.23

[1046]

[1047] Intermediate I.23b (0.047 g, 0.02 mmol, 1 equivalent) was dissolved in 0.28 mL of dry DMF. Intermediate 3.2 (0.01 g, 0.022 mmol, 1.1 equivalent) and DIPEA (0.0011 mL, 0.061 mmol, 3 equivalent) were added under an argon atmosphere. The solution was stirred at room temperature and the progress was monitored by RP-HPLC. After completion (approximately 1 h), the reaction mixture was neutralized with TFA and purified directly by preparative HPLC without any pretreatment to yield intermediate II.23 (0.041 mg, 78%) as a pale yellow lyophilized product. HPLC (Method N, at 380 nm): t R = 13.5 min. MS (ESI): Measured value m / z = 1316.2 [M+2H] 2+ The calculated value is m / z = 1317.04.

[1048]

[1049] Synthesis of Examples 26 - IV.24

[1050] Synthesis of I.24

[1051]

[1052] To a stirred solution of intermediates 4.6 (31.5 mg, 20.1 µmol, 1 equivalent) and 1.16 (28.8 mg, 21.1 µmol, 1.05 equivalent) in 286 µL of DMF, add 286 µL of DMF containing TBTA (2.66 mg, 5.02 µmol, 0.25 equivalent) and 57.2 µL of water containing copper(II) sulfate pentahydrate (1 mg, 4.02 µmol, 0.2 equivalent) and sodium ascorbate (1.98 mg, 10 µmol, 0.5 equivalent). Stir the reaction mixture at room temperature and monitor progress by RP-UHPLC. After 15 min, add the reaction mixture to 10 mL of ice-cold acetone. Separate the resulting precipitate by centrifugation, resuspend in 6 mL of cold acetone, and centrifuge again. The crude product (54.3 mg, 92%) is used without further purification. UHPLC (Method Q): t R =2.7 min. MS (ESI): Measured value m / z = 1467.0 [M+2H] 2+ The calculated value is m / z = 1468.06.

[1053]

[1054] Synthesis of III.40

[1055]

[1056] Intermediate III.40 was synthesized from GP2 using intermediates 2.4 (5.18 mg, 3.33 µmol, 1 equivalent) and II.39 (53.75 mg, 20.00 µmol, 6.0 equivalent) in DMF (1000 µL) and 100 µL of an aqueous catalyst containing 100 mM copper(II) sulfate pentahydrate, 120 mM tris(3-hydroxypropyltriazolylmethyl)amine, and 300 mM sodium ascorbate. The reaction was completed after 50 min, and the reaction mixture was filtered through a 0.2 µm membrane filter and purified by preparative HPLC to yield intermediate III.40 (36.27 mg, 73%) as a colorless lyophilized product. HPLC (Method N, 250 nm): t R =15.2 min. MS (ESI): Measured value m / z = 2480.73; [M+6H] 6+ The calculated value is m / z = 2479.74.

[1057] Synthesis of IV.40

[1058]

[1059] Compound IV.40 was synthesized from GP4 using a TFA salt of intermediate III.40 (36.27 mg, 2.42 µmol, 1 equivalent), a Mal-PEG12-NHS-ester solution (100 mM in DMF, 48.4 µL, 4.84 µmol, 2 equivalents), and a DIPEA ester solution (100 mM in DMF, 48.4 µL, 4.84 µmol, 2 equivalents) in 242 µL of DMF. After completion at 4.5 h, the reaction mixture was added dropwise to 10 mL of ice-cold MTBE, and the precipitate was collected by centrifugation (4000 × g, 4 min) and washed with 10 mL of fresh MTBE. The crude product was dissolved in 0.05% TFA / ACN 80:20, filtered through a 0.2 µm membrane filter (nylon), and purified by RP-HPLC to provide compound IV.40 (32.75 mg, 87%) as a colorless lyophilized product. HPLC (Method AE): t R = 15.4 min. MS (ESI): Measured value m / z = 2608.65 [M+6H] 6+ The calculated value m / z = 2604.80 [C 679 H 1069 N 63 O 345 ] 6+ .

[1060]

[1061] Example 50 - In vitro cytotoxicity assay using 2D and 3D cell cultures

[1062] The cytotoxicity of anti-GUCY2C ADCs containing the modular adapter compounds of the present invention was evaluated using in vitro cytotoxicity assays in 2D and 3D cell cultures with target-positive HEK293-GUCY2C and target-negative HEKwt cells.

[1063] At the start of the 2D cytotoxicity assay, seed 2 x 10⁶ cells per well in a 96-well plate. 3 Target-positive or target-negative cells were incubated at 37ºC and 5% CO2 for 24 h. The indicated ADC was then applied at a rate of 1x10⁻⁶ cells / mL. -6 M to 1.28x10 -12 Different concentrations of M were added to the adherent cells, and the plates were then incubated at 37ºC and 5% CO2 for 4 days. Cytotoxicity was assessed using a cell proliferation ELISA (Roche's BrdU colorimetric assay).

[1064] At the start of the 3D cytotoxicity assay, 2 x 10⁶ cells were seeded per well in an ultra-low adhesion (ULA) 96-well plate. 3 Target-positive or target-negative cells were incubated at 37ºC and 5% CO2. After 48 h, the cells were treated with the indicated ADC at a rate of 1 x 10⁻⁶. -6 M to 1.28x10 -12 Different concentrations of M were used to treat spheroids, followed by incubation at 37ºC and 5% CO2 for 5 days. Cytotoxicity was assessed using Promega's CellTiter-Glo® 2.0 cell viability assay.

[1065] Example 51 - Research Design of In Vivo Animal Models

[1066] Subcutaneous injection of 5 x 10 g of each drug into the flank of female NOD SCID mice. 6 Human embryonic kidney HEK293-GUCY2C-(HDP)-2B3 cells overexpressing GCC were cultured in 200 µL of RPMI medium containing 50% Matrigel without phenol red. Once the cells reached approximately 150 mm... 3The average tumor volume was used to assign animals to either the control or treatment group based on tumor size. Animals were treated with a single intravenous dose of an anti-GCC ADC conjugated to a mediator control or a corresponding modular connector compound of the present invention (indicated as (“IV.x”)) on the same day (Day 0) or the following day (Day 1), or once weekly for three weeks. Tumor volume was measured twice weekly by calipers, and body weight was determined in parallel. Clinical signs and survival were monitored daily. Animals were euthanized and autopsied upon the occurrence of one or more termination criteria or at the end of the study. The indicated dose of the corresponding ADC corresponds to the total ADC dose (mg / kg) indicated in each case.

[1067] Those skilled in the art will recognize many modifications and other embodiments of the invention described herein, which have the benefits of the teachings presented in the foregoing description and associated drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terminology is used herein, it is used in a general descriptive sense only and is not intended for limiting purposes.

[1068] Example 52 - Cytotoxicity against different cell lines

[1069] against NCI-N87 cytotoxicity

[1070]

[1071] Table 1: In a 96-h BrdU ELISA targeting NCI-N87 cells expressing Her2 and Trop2, anti-Her2 ADC and anti-Trop2 ADC showed EC50 values ​​of 1.705 x 10⁻⁶. -9 M and 3.279x10 -12 Cytotoxicity between M and M.

[1072] against BxPC-3 cytotoxicity

[1073]

[1074] Table 2: In a 96-h BrdU ELISA targeting Trop2-expressing BxPC-3 cells, anti-Trop2 showed an EC50 value of 3.667 x 10⁻⁶. -9 M and 1.558x10 -12 Cytotoxicity between M and M.

[1075] against SKBR-3 cytotoxicity

[1076]

[1077]

[1078] Table 3: In a 96-h BrdU ELISA targeting SKBR-3 cells expressing Her2 and Trop2, anti-Her2 ADC and anti-Trop2 ADC showed EC50 values ​​of 1.178 x 10⁻⁶. -10 M and 8.363 x 10 -12 Cytotoxicity between M and M.

[1079] against JIMT-1 cytotoxicity

[1080]

[1081] Table 4: In a 96-h BrdU ELISA targeting JIMT-1 cells expressing Her2 and Trop2, anti-Her2 ADC and anti-Trop2 ADC showed EC50 values ​​of 1.205 x 10⁻⁶. -9 M and 1.276 x 10 -12 Cytotoxicity between M and M.

[1082] against MDA-MB-231 (target negative) cytotoxicity

[1083]

[1084] Table 5: In a 96-h BrdU ELISA targeting target-negative MDA-MB-231 cells, anti-Her2 ADC and anti-Trop2 ADC showed no cytotoxicity.

[1085] against HEKwt (target negative) cytotoxicity

[1086]

[1087] Table 6: In a 96-h BrdU ELISA targeting target-negative HEKwt cells, anti-Her2 ADC and anti-Trop2 ADC showed no cytotoxicity.

[1088] against L540 cytotoxicity

[1089]

[1090] Table 7: In 96 h CTG treatment of L540 cells expressing CD30, the EC50 value of anti-CD30 ADC was 3.421 x 10⁻⁶. -10 M's cytotoxicity.

[1091] ClogP value

[1092] Table 8 depicts the ClogP values ​​of the effective load (D) with or without a solubilizer (SE) and the ClogP values ​​of the compounds according to the invention:

[1093]

Claims

1. A modular connector compound of formula (I) Or its pharmaceutically acceptable salts or solvates; in: X is a self-degrading spacer. , in: R 1 X is covalently linked to D at the para, meta, or ortho position of the amino group of X, and the bond is a kinetic link. , or ;and R 2 Connect X to SE and is , or Or -O-; D is a pharmaceutically active substance; SE is a solubilizing group; L is a spacer containing a peptide covalently linked to X; n is an integer of at least 2, and preferably an integer from 2 to 5; Y is the branch unit. A is the reactive portion used to bind with the target.

2. The modular connector compound according to claim 1, wherein... L is ， in: W is connected to Z. 3 A cleavable or non-cleavable peptide sequence of X, selected from: Val-Ala, Val-Cit, Met-Thr, Thr-Thr, Phe-Lys, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, Gly-Gly-Tyr-Gly, cBu-Ala, cBu-Cit, Glu-cBu-Ala, Glu-cBu-Cit, Asp-cBu-Ala, Asp-cBu-Cit, Asp-Pro-Val, Asn-Pro-Val, iGlu-cBu-Ala, iGlu-cBu-Cit, Val-Lys, Val-Arg, Val-Gln, Val-Met, Val-Thr, AcLys-Val-Cit, AcLys-Va l-Ala, Asp-Val-Ala, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Tyr-Arg, Tyr-Met, Phe-Cit, Phe-Ala, Phe-Arg, Phe-Met, Phe-Gln, Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly, Ala-Lys, Leu-Cit, Leu-Gln, Ile-Cit, Ala-Ala-Asn, Ala-Asn, Ala-Ala, Asn-Asn, Asn, β-Ala and Lys or combinations thereof; and Z 3 It is a bond or spacer, the spacer containing C 1-20 Alkyl chains or 1 to 10 polyethylene glycol (PEG) 1-10 or C2-amide-PEG 1-10 ; Preferably, L is or .

3. The modular connector compound according to claim 1 or claim 2, wherein... Y is: or ; m is an integer from 1 to 6; Z 1 Connect Y to A and it is or ,in R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-, and p is an integer from 1 to 24, preferably from 4 to 24, and more preferably from 8 to 24. q is an integer from 1 to 8. r is an integer from 1 to 24. Z 2 Connect Y to L and select from: , , , , , , , , , , , , , , , , , , , , , , , , , ,and ; B is: The cap, preferably an acyl group, and n' is n; or or ,in i is an integer from 1 to 20. j is an integer from 1 to 10, and n' is n minus 1; or ,in D' is a pharmaceutically active substance different from D; and X' is a self-degrading spacer. ,in: R 5 X' is covalently linked to D' at the para or ortho position of the amino group of X', and it is a bond. , or , And n' is n; R 4 It is OH or NH2.

4. The modular connector compound according to claim 1 or 2, wherein Y is: 、 、 、 、 ,or , or ; Z 1 Connect Y to A and it is or ,in R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-. p is an integer from 1 to 24. q is an integer from 1 to 8. r is an integer from 1 to 24. s is an integer from 0 to 4. Z 2 Connect Y to L and select from: , , , , , , , , , , , , , , , , , , , , , , , , , ,and .

5. The modular linker compound according to any one of claims 1 to 4, wherein D and D' are independently selected from: camptothecin, preferably eczema, SN38, and topotecan; aurestatin, preferably methylaurestatin E (MMAE) and methylaurestatin F (MMAF); maytansine, preferably methylamycin (DM1) and lavantanine (DM4); chalcogenine; Toll-like receptor 7 (TLR7) agonist; nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (NAMPT) i ): Pyrrolobenzodiazepine (PBD); pyroxine; anthracyclines, preferably doxorubicin; amatoxins, preferably α-amaminine, β-amaminine, amatoxins and amatoxins; nostocin; phalloidin; open-ring-cyclopropylpyrroloindoline (open-ring-CPI); open-ring-cyclopropylbenzoindoline (open-ring-CBI); paclitaxel; vincristine; colchicine; eribulin; methotrexate (MTX); triptolide; and their derivatives.

6. The modular connector compound according to any one of claims 1 to 5, wherein the SE comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, polysarcosine, PEG, galactoside, glucuronic acid, or a polyol prepared from carbohydrates, preferably the SE comprises: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 , ,or ; Z 4 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , ,and ; t is 2 to 18, preferably 8 to 12. u is at least 1, preferably 10; v ranges from 4 to 24; w ranges from 2 to 24.

7. The modular connector compound according to any one of claims 1 to 6, wherein A is 、 、 、 、 , , , , or .

8. The modular connector compound according to any one of claims 1 to 7, wherein the modular connector compound is: 。 9. A method for synthesizing a modular connector compound of formula (I) according to any one of claims 1 to 8, the method comprising: (i) Make a first compound comprising the following: -Degradation portion, - A cleavable peptide sequence W, -Amino protecting group Pg 1 It is covalently connected to W. -Pharmaceutical active substance D and - First complementary group Cg 1 Contact with a second compound comprising the following: -Solubilizing group SE and -Second complementary group Cg 2 Intermediate form of Formula II: SE via R 2 Connected to X, and where R 2 Through Cg 1 With Cg 2 The reaction forms; (ii) Remove the ammonia protecting group Pg from formula II 1 To expose primary amines; (iii) Contact the primary amine with a third compound comprising: -spacer Z 3 , -amine reactive groups, and -Third complementary group Cg 3 Intermediate form of Formula III: Where L is (iv) Contact the intermediate of Formula III with a fourth branched compound comprising: -Fourth complementary group Cg 4 and -Fifth complementary group Cg 5 , Cg 3 With Cg 4 The reaction forms Z 2 ;as well as (v) Contact the product of (iv) with a fifth compound comprising: -Reactive portion A for binding with the target and -Sixth complementary group Cg 6 Cg 5 With Cg 6 The reaction forms Z 1 To form the modular connector compound of Formula I: 。 10. The method according to claim 9, wherein: X is a self-degrading spacer. , in: R 1 X is covalently linked to D at the para or ortho position of the amino group of X, and Is it a key or , or ;and R 2 Connect X to SE and is , or .

11. The method according to claim 9 or claim 10, Where W is a peptide sequence selected from the following cleavable or non-cleavable sequences: Val-Ala, Val-Cit, Met-Thr, Thr-Thr, Phe-Lys, Glu-Val-Ala, Glu-Val-Cit, Gly-Gly-Phe-Gly, Gly-Gly-Tyr-Gly, cBu-Ala, cBu-Cit, Glu-cBu-Ala, Glu-cBu-Cit, Asp-cBu-Ala, Asp-cBu-Cit, Asp-Pro-Val, Asn-Pro-Val, iGlu-cBu-Ala, iGlu-cBu-Cit, Val-Lys, Val-Arg, Val-Gln, Val-Met, Val-Thr, AcLys-Val-Cit, AcLys-Va l-Ala, Asp-Val-Ala, iGlu-Val-Ala, Asp-Val-Cit, iGlu-Val-Cit, Tyr-Arg, Tyr-Met, Phe-Cit, Phe-Ala, Phe-Arg, Phe-Met, Phe-Gln, Phe-Arg-Arg-Gly, Phe-Arg-Arg-Leu, Phe-Arg-Arg-Leu-Gly, Phe-Leu-Arg-Arg-Gly, Ala-Lys, Leu-Cit, Leu-Gln, Ile-Cit, Ala-Ala-Asn, Ala-Asn, Ala-Ala, Asn-Asn, Asn, β-Ala and Lys or combinations thereof; and Z 3 It is a bond or spacer, the spacer containing C 1-20 Alkyl chains or 1 to 10 polyethylene glycol (PEG) 1-10 or C2-amide-PEG 1-10 ; Where L is preferably or .

12. The method according to any one of claims 9 to 11, wherein Y is: or ; m is an integer from 1 to 6; Z 1 Connect Y to A and it is or ,in R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-, and p is an integer from 1 to 24, preferably from 4 to 24, and more preferably from 8 to 24. q is an integer from 1 to 8. r is an integer from 1 to 24. Z 2 Connect Y to L and select from: , , , , , , , , , , , , , , , , , , , , , , , , , ,and ; B is: The cap, preferably an acyl group, and n' is n; or or ,in i is an integer from 1 to 20. j is an integer from 1 to 10, and n' is n minus 1; or ,in D' is a pharmaceutically active substance different from D; and X' is a self-degrading spacer. ,in: R 5 X' is covalently linked to D' at the para or ortho position of the amino group of X', and it is a bond. , or And n' is n; R 4 It is OH or NH2.

13. The method according to any one of claims 9 to 11, wherein Y is: 、 、 、 、 、 , or ; Z 1 Connect Y to A and it is or ,in R 3 It is -O-, -NH-, -C(=O)-, -C(=O)N(CH3)-, -C(=O)NH-, -NHC(=O)- or -N(CH3)C(=O)-. p is an integer from 1 to 24. q is an integer from 1 to 8. r is an integer from 1 to 24. s is an integer from 0 to 4. Z 2 Connect Y to L and select from: , , , , , , , , , , , , , , , , , , , , , , , , , ,and ; 14. The method according to any one of claims 9 to 13, wherein D and D' are independently selected from camptothecin, preferably eczema, SN38, and topotecan; auristatin, preferably selected from methylauristatin E (MMAE) and methylauristatin F (MMAF); maytansine, preferably selected from metansine (DM1) and lavtansine (DM4); chalcogenide; Toll-like receptor 7 (TLR7) agonist; nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (NAMPT) i ): Pyrrolobenzodiazepine (PBD); pyroxine; anthracyclines, preferably doxorubicin; amatoxins, preferably α-amaminine, β-amaminine, amatoxins and amatoxins; nostocin; phalloidin; open-ring-cyclopropylpyrroloindoline (open-ring-CPI); open-ring-cyclopropylbenzoindoline (open-ring-CBI); paclitaxel; vincristine; colchicine; eribulin; methotrexate (MTX); triptolide; and their derivatives.

15. The method according to any one of claims 9 to 14, wherein S comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, polysarcosine, PEG, galactoside, glucuronic acid, or a polyol prepared from carbohydrates, preferably. 、 、 、 、 , 、 、 、 、 、 、 、 、 、 , ,or ; Z 4 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , ,and ; t is 2 to 18, preferably 8 to 12; u is at least 1, preferably 10; v ranges from 4 to 24; w ranges from 2 to 24.

16. The method according to any one of claims 9 to 15, wherein A is 、 、 、 、 , , , , , , or .

17. The method according to any one of claims 9 to 16, wherein Cg 1 It is an alkyne group and Cg 2 It is an azide group, or Cg 1 It is a primary or secondary amine group and Cg 2 It is a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group.

18. The method according to any one of claims 9 to 17, wherein Cg 3 It is an alkyne group and Cg 4 It is an azide group; Cg 3 It is an azide group and Cg 4 It is an alkyne group; Cg 3 It is a primary or secondary amine group and Cg 4 It is a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; an amide; a carbamate; a carbamoyl halide; or a urea group; or Cg 3 It is a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; an amide; a carbamate; a carbamoyl halide; or a urea group, and Cg 4 It is a primary or secondary amine group.

19. The method according to any one of claims 9 to 18, wherein Cg 5 It is a primary or secondary amine group and Cg 6 It is a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group; or Cg 5 It is a carboxylic acid; a carboxylic acid ester, preferably an N-hydroxysuccinimide ester, a tetrafluorophenyl ester, a pentafluorophenyl ester, or an N-hydroxyphthalimide ester; a carboxylic anhydride; an acyl halide; or an amide group, and Cg 6 It is a primary or secondary amine group.

20. The method according to any one of claims 9 to 19, wherein the amino protecting group Pg 1 It is a benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-benzyloxybenzyl (PMB), p-toluenesulfonyl (Ts) or carbamate group.

21. A target-binding drug conjugate comprising at least one reaction product of a modular connector compound according to any one of claims 1 to 8 and a corresponding reactive group of a target-binding moiety, wherein the at least one reaction product is the product of a reaction between A of the modular connector compound and the corresponding reactive group of the target-binding moiety, and wherein the modular connector compound is covalently coupled to the target-binding moiety, preferably the corresponding reactive group of the target-binding moiety is a thiol group.

22. The target-binding drug conjugate of claim 21, wherein the target-binding drug conjugate comprises more than one of the reaction products, such that more than one, preferably two to ten, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 modular connector compounds are coupled to the target-binding portion.

23. The target-binding drug conjugate according to claim 21 or claim 22, wherein the target-binding moiety specifically recognizes and binds to the target sequence or epitope of the antigen, preferably the target-binding moiety is: - Antibodies, such as monoclonal antibodies; - Functional antibody fragments or antibody derivatives, such as single-chain variable fragments (scFv), antigen-binding fragments such as Fab fragments, F(ab') fragments, F(ab')2 fragments, or bispecific or trispecific antibody constructs; -Double antibody, -Cameloid antibodies, - Domain antibodies, -Nanobodies, - A divalent homodimer with two chains composed of scFv, -Shark antibodies, -Antibodies composed of New World primate framework sequences plus non-New World primate complementarity-determining regions (CDRs), or - A dimerized construct comprising a constant heavy chain 3 (CH3) domain and a variable light chain (V) domain. L ) and variable heavy chain (V H ) -TCR.

24. The target-binding drug conjugate according to any one of claims 21 to 23, wherein the target-binding moiety specifically recognizes and binds to the following target sequences or epitopes: guanylate cyclase C (GUCY2C), differentiation group 33 (CD33), differentiation group 30 (CD30), differentiation group (CD5), differentiation group 20 (CD20), differentiation group 45 (CD45), differentiation group 117 (CD117 / c-Kit), differentiation group 123 (CD123 / IL3RA), differentiation group 137 (CD137 / 4-1BB), differentiation group 269 (CD269 / B cell maturation antigen, BCMA), sealing protein 18.2, CEA (carcinoembryonic antigen), epithelial cell adhesion molecule (EpCAM), DKK1 (Dickkopf-associated protein). 1) Phosphatidylinositol proteoglycan-3, mucin-1 (MUC-1 / CD227), mucin 5AC (MUC5AC), carbonic anhydrase 9 (CAIX), CXC motif chemokine receptor 1 (CXCR1, CD181), CXC motif chemokine receptor 2 (CXCR2 / CD182), human epidermal growth factor receptor 2 (HER2), differentiation cluster 22 (CD22), differentiation cluster 79b (CD79b), connexin cell adhesion molecule 4 (connexin 4), tumor-associated calcium signaling transducer 2 (Trop-2), differentiation cluster 19 (CD19), delta-like protein 3 (DLL3), transmembrane glycoprotein NMB (gpNMB), prostate-specific membrane antigen (PSMA), hepatocyte growth factor receptor (c-Met, CD181), and other related proteins. 151) Solute carrier family 39 member 6 (LIV-1), folate receptor 1 (FOLR1), differentiation group 56 (CD56), differentiation group 138 (CD138), mesothelin, carbonic anhydrase 6 (CA6), epidermal growth factor receptor (EGFR), differentiation group 37 (CD37) or extracellular nucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3).

25. The target-binding drug conjugate according to any one of claims 21 to 24, wherein the target-binding moiety is any one of the following: adenomyumab (anti-EpCam), amemetuzumab (anti-mesothelin), epantuzumab (anti-EGFR), bexolumab (anti-CEA), belintoumab (anti-CD19), bentuximab (anti-CD30), cantuzumab, cybituximab (anti-CEACAM5), cetuximab (anti-ROR1), cetuximab (anti-EGFR), crimetuzumab (anti-MUC1). Gautotuzumab (anti-MUC1), Cortuximab (anti-CD19), Daratumumab (anti-CD38), Dugoutuzumab (anti-ERBB3), Ezocurumab (anti-EpCam), Enrotopuzab (anti-connectin-4), Enrotopuzab (anti-CD276), Entoximab (anti-MUC5AC), Ipatuzumab (anti-CD22), Faletuzumab (anti-FOLR1), Igvovomab (anti-CA-125), Inelizumab (anti-CD19), Itolimumab (anti-CD30), Labezil Anti-CEA, Magitocilizumab (anti-HER2 / neu), Magitocilizumab (anti-EGFR), Motuzumab (anti-EGFR extracellular domain III), Napotumab (anti-5T4), Natuzumab (anti-CD37), Necetuzumab (anti-EGFR), Nimotuzumab (anti-EGFR), Obbituzumab (anti-CD20), Ocalatuzumab (anti-CD20), Orelizumab (anti-CD20), Ofamumab (anti-CD20), Oletocilizumab (anti-CD37), Ontocilizumab Anti-TEM1, pertuzumab (anti-HER2 / neu), polotuzumab (anti-CD79b), rituximab (anti-CD20), lovatotuzumab (anti-DLL3), goxatotuzumab (anti-TROP-2), seretuzumab (anti-ERBB3 / HER3), tancituzumab (anti-CD19), tetuximab (anti-CD37), telmituzumab (anti-HER2), trastuzumab (anti-HER2), ulrituximab (anti-CD20), or zotuximab (anti-sealing protein 18.2).

26. The target-binding drug conjugate of claim 25, wherein the target-binding portion has been genetically engineered to have cysteine ​​substitutions selected from A118C, S239C or D265C (according to the EU numbering system), preferably D265C, in each of its heavy chains, wherein the target-binding portion is conjugated to at least one modular linker compound according to any one of claims 1 to 8 via one or more engineered cysteine ​​residues of the target-binding portion.

27. The target-binding drug conjugate according to any one of claims 21 to 26, used as a drug, preferably for use in cancer treatment.

28. A pharmaceutical composition comprising a target-binding drug conjugate according to any one of claims 21 to 26.

29. Use of the modular connector compound according to any one of claims 1 to 8 in the manufacture of the target-binding drug conjugate according to any one of claims 21 to 23.

30. A method of treating cancer, wherein the method comprises administering a target-binding drug conjugate according to any one of claims 21 to 23 to a subject in need.

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