WO2025130990A1 - Tumor microenvironment-activated drug conjugate and antibody-drug conjugate - Google Patents

Abstract

The present invention relates to a tumor microenvironment-activated drug conjugate structure and an antibody-drug conjugate. Specifically, the present invention provides a drug conjugate having a chemical structure represented by formula R1-R2-D or R2'-D, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R1 is a chemical structure conjugated to a biomolecule or is absent, R2 and R2' are each independently an adjustment chemical structure for enhancing the structure-activity relationship, and D is selected from structures represented by X1-X5. The present invention also provides an antibody-drug conjugate comprising the drug conjugate.

Description

Tumor microenvironment activated drug conjugates and antibody drug conjugates Technical Field

The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a tumor microenvironment activated drug conjugate and an antibody drug conjugate.

Background Art

In order to improve the therapeutic window of antibody or protein-coupled drugs, people have been committed to improving the tumor specificity and release efficiency of coupled drugs. In recent years, driven by the development of tumor molecular biology and other basic disciplines, the research and development of tumor targeted therapeutic drugs has made great progress in many aspects.

The method of connecting effector molecules to antibodies by using natural cysteine in antibody sequences has been widely used, such as cytotoxins, immune agonists, immune antagonists, etc., further promoting the use of antibodies. Generally, antibody-drug conjugates need to enter tumor cells through endocytosis to produce drug effects, and release effector molecules with cell-killing effects through degradation in the cells, thereby achieving tumor inhibition. However, antibody-drug conjugates have been developed for many years, and currently only two types of linkers that can be activated by cathepsin B have been successful, and they must enter tumor cells through endocytosis, which has become the main influencing factor limiting the drug development of various antibodies.

The tetrapeptide Gly-Gly-Phe-Gly (GGFG) is a cathepsin B-activatable linker, and the ADC drug Enhertu uses this type of linker. Daiichi Sankyo's Enhertu is a plasma-stable ADC with a DAR of 7.7. It undergoes protease degradation in lysosomes to release DX-8951f derivatives, which is an effective topoisomerase I inhibitor derived from Exatecan. Due to the specificity of the GGFG linker, it is very impressive to achieve such a high DAR, which also shows that the structure-activity relationship between the linker and the toxin molecule is the key to developing an ADC drug. However, the GGFG linker combined with the DX-8951f derivative will cause interstitial pneumonia toxicity, including Enhertu (T-Dxd), Datopotamab deruxtecan (Dato-DXd), Raludotatug deruxtecan (R-DXd), Ifinatamab deruxtecan (I-DXd) all clinically showed interstitial pneumonia. Comprehensive analysis of 8 monotherapy trials of T-DXd: 879 patients with different tumor types, 139 patients (15.8%) have been diagnosed with ILD. 108 people had grade 1 or 2 events, and 21 people had grade 5 events. This may be because cathepsin B is expressed in lung epithelial cells, and GGFG is a cathepsin B activation linker, which will produce a certain release in the lungs. Comprehensive analysis of 23 PD-1 monotherapy trials: 3284 patients with different tumor types, 4% of patients developed ILD. In the future, if T-DXd is combined with PD-1 treatment, it may further increase the incidence of ILD.

We found that leucopenia is highly expressed in tumors, and developed a leucopenia-activated small molecule conjugate drug, leucopenia. Through clinical findings, hundreds of patients treated with new chemotherapy did not develop interstitial pneumonia, indicating that leucopenia activation is more specific to tumor tissues than cathepsin B. DX-8951f derivatives and immune agonist T785 are far more toxic than conventional chemotherapy drugs such as doxorubicin and paclitaxel, and it is more difficult to develop them into broad-spectrum small molecule conjugate drugs. Through the multiple connections and enzyme activation of leucopenia that can cut linkers and toxins, we screened for AAN-toxin combinations suitable for development, and through R1 and R2 combination linker and toxin screening, we obtained small molecule conjugate drugs that we believe can be developed with extremely high preclinical therapeutic indexes. These small molecule compounds can be made into drugs alone, or they can be conjugated to antibodies through EMC or other conjugation methods to form antibody-conjugated drugs.

Summary of the invention

The purpose of the present invention is to provide a drug conjugate and an antibody drug conjugate with strong specificity and high stability, and their use in treating and/or preventing cancer and/or inflammation. The drug conjugate and the antibody drug conjugate of the present invention are activated only in a pathological microenvironment (e.g., in a tumor microenvironment or an inflammatory site), release active molecules, overcome drug resistance, and reduce toxicity.

Specifically, the present invention provides a drug conjugate having a chemical structure represented by formula (I) or (II), and a stereoisomer thereof or a pharmaceutically acceptable salt thereof,
R ₁ -R ₂ -D (I)
R ₂ '-D (II)

Wherein, R ₁ is a chemical structure coupled to a biological molecule or does not exist; R ₂ and R ₂ ' are each independently a chemical structure for enhancing the structure-activity relationship; D contains -AAN- and a cytotoxic structure, and D is selected from X1-X5.

The present invention also provides an antibody-drug conjugate, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is represented by formula (III):
R ₃ -(R ₁ -R ₂ -D) _x Formula (III);

Wherein, R ₃ is an antibody; R ₁ , R ₂ , and D are as defined in any embodiment herein; and x is an integer selected from 1-8.

The present invention also provides a pharmaceutical composition, comprising: (i) the drug conjugate and/or antibody drug conjugate described in any embodiment herein, or a stereoisomer or a pharmaceutically acceptable salt thereof; and (iii) a pharmaceutically acceptable carrier.

The present invention also provides use of the drug conjugate and/or antibody drug conjugate described in any embodiment herein in the preparation of a drug for treating and/or preventing tumors and/or inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Enzyme cleavage spectrum of control compound Y.

Figure 2: Enzyme cleavage spectrum of compound S5.

Figure 3: Enzyme cleavage spectrum of compound S16.

Figure 4: Enzymatic cleavage spectrum of Deruxtecan under the action of Cathepsin B enzyme.

Figure 5: Enzyme cleavage spectrum of S16 in lung tissue.

Figure 6: Enzymatic cleavage spectrum of Deruxtecan in lung tissue.

Figure 7: Hydrophobic interaction chromatography (HIC) of Trastuzumab-PEG6-AAN-AM-Dxd (DAR8).

Figure 8: MS spectrum of Atezolizumab Ab-C5-AAN-PABC-T785 (DAR8).

Figure 9: HIC spectrum of Atezolizumab Ab-AAN-T785 (DAR8).

Figure 10: MS spectrum of Atezolizumab Ab-C5-AAN-T785 (DAR8).

Figure 11: Efficacy of high-dose small molecule drug conjugates based on TLR7/8 agonists (S5) alone and in combination with legubicin.

Figure 12: Efficacy of high-dose small molecule drug conjugates based on TLR7/8 agonists (S5) alone and in combination with CTLA-4 antibodies.

Figure 13: Inhibitory effect of anti-HER2 antibody coupled with Dxd drug molecule based on Affinity linker2 on gastric cancer tumors.

Figure 14: Inhibitory effect of anti-LY6G6D antibody-coupled Dxd drug molecule based on Affinity linker2 on colon cancer tumor model.

FIG. 15 : Inhibitory effect of PDL1-Dxd in the MC38 tumor model.

Figure 16: Inhibitory effect of Affinity linker4-based anti-PD-L1 antibody coupled with T785 drug on large tumors.

FIG. 17 : Tumor growth inhibition effect of 7B6-DXD in the mouse subcutaneous HT-55 transplanted tumor model.

Figure 18: Comparison of the stability of S15 coupled to albumin and after coupling and ring opening.

Figure 19: Schematic diagram of the structure of bispecific antibody-drug conjugates.

FIG. 20 : Inhibitory effect of EGFR/Cmet-T785 in the NCI-H292 tumor model.

FIG. 21 : Inhibitory effect of EGFR/Cmet-Dxd in the NCI-H292 tumor model.

Figure 22: Tumor volume of human fibrosarcoma HT-1080 nude mouse xenograft tumor model after administration of S41.

Figure 23: Tumor volume in the human fibrosarcoma HT-1080 nude mouse xenograft model after administration of S41.

Figure 24: Tumor volume in the mouse colon cancer CT-26 nude mouse transplant tumor model after administration of S41.

Figure 25: Tumor volume in the mouse colon cancer CT-26 nude mouse xenograft model after administration of S41 and/or anti-PD1 antibodies.

Figure 26: Tumor volume in the mouse colon cancer CT-26 nude mouse transplant tumor model after administration of S41.

DETAILED DESCRIPTION

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies and reference to "the antibody" includes reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for use of the terminology "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

The small molecule drug Legumain activated by the inventor previously developed contains AANL-DOX, and Legutaxel contains AAN-PABC-taxel and paclitaxel. At present, it has been patented in China and is undergoing key clinical phase II/III and clinical phase I experiments respectively. However, the toxicity of doxorubicin and paclitaxel cannot meet the more severe toxicity requirements used by ADC, so it is necessary to innovate and produce a conjugate structure with a new structure-activity relationship. Studies have found that Legumain forms a complex with integrin, which can efficiently activate a specific substrate structure on the surface of tumor cells. Therefore, the inventor used Legumain as an activating enzyme, combined with various toxins and immune agonist chemical structures, as well as auxiliary structures and connecting structures for a large number of structural screenings, and obtained a restrictive special conjugate structure through biological mechanism of action, in vitro and in vivo efficacy. These conjugate structures are highly stable in human plasma, and can efficiently activate and release compatible chemical molecules in the tumor microenvironment (outside tumor cells). Therefore, compared with traditional ADCs, this new release method can better exert the bystander effect and activate adjacent immune cells when the payload drug is an immune agonist. It has a different drug-making mechanism and scalability from traditional ADCs.

Drug conjugates

Provided herein are compounds having a chemical structure represented by formula (I), and stereoisomers thereof or pharmaceutically acceptable salts thereof:
R ₁ -R ₂ -D (I);

The compound can be used as a drug conjugate for preparing ADC.

In formula (I), _R1 is a chemical structure coupled to a biological molecule or does not exist; _R2 is an adjustment chemical structure for enhancing the structure-activity relationship; and D is -AAN-linker-drug.

Herein, R ₁ is preferably selected from:

and;

The wavy line indicates the position where _R1 and _R2 are connected.

Preferably, _R1 is selected from:

and

In some embodiments, R ₁ is absent.

In some embodiments, _R2 is selected from:

-C _1-6 alkylene-CO-*;

-C _1-6 alkylene-O-*;

-C _1-6 alkylene-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*;

-C _1-6 alkylene-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*;

-C _1-6 alkylene-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*;

-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*;

-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*;

-NH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*;

-NH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*;

-NH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*;

-NH-C ₁ - ₅₅ alkylene-CO-*;

-NH-C _1-55 alkylene-NH-*;

-NH-C _1-55 alkylene-O-*;

-CO-C _1-55 alkylene-CO-*;

-CO-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*;

-CO-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*;

-CO-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*;

-CO-C _1-6 alkylene-NH-*;

-CO-C _1-6 alkylene-NHCO-SO ₃ H substituted C _1-6 alkylene-NH-*;

in:

* indicates the position connected to D;

R ₄ is a monovalent group or a divalent group, and when R ₄ is connected to other parts of the compound, it is a divalent group, selected from: C _1-6 alkyl or C _1-6 alkylene; C _1-6 alkyl-C _3-8 cycloalkylene- or C _1-6 alkylene-C _3-8 cycloalkylene-; -CO-C _1-6 alkylene-; C _1-6 alkyl-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene- or C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; -NH-C _1-6 alkylene; C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; and -NH-C _1-6 alkylene-C _3-8 cycloalkylene;

R ₅ is a monovalent group or a divalent group, and when R ₅ is connected to other parts of the compound, it is a divalent group, selected from: C _1-6 alkyl-(O-CH ₂ CH ₂ -O) _m -; C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -; -CO-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -;

One of _R4 and _R5 is a divalent group, and the other is a monovalent group;

R ₆ is absent or is -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylenecarbonyl or -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NHCO-C _1-6 alkyleneoxy _- C _1-6 alkylenecarbonyl;

L ₂ and L ₃ are each independently a bond or a C _1-6 alkylene group;

L ₄ are each independently C _1-6 alkylene;

L ₅ is a bond, C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl or C _1-6 alkylenecarbonyl;

L ₆ is C _1-6 alkylene;

L ₇ is C _1-6 alkylene or -C _3-8 cycloalkylene-C _1-6 alkylene;

Each of n and m is independently an integer from 1-50.

Herein, alkylene has a well-known meaning in the art and refers to a divalent alkyl group, such as methylene (-CH ₂ -), ethylene (-CH ₂ CH ₂ -), etc. The number of carbon atoms in the alkylene group can be, for example, 1-55, 1-20, 1-10, 1-6 or 1-4.

Herein, cycloalkyl has the well-known meaning herein and refers to a saturated carbocyclic ring. The number of carbon atoms in the ring of cycloalkyl can generally be 3-8, such as 3-6. Exemplary cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, etc. Herein, cycloalkylene to divalent cycloalkyl, i.e., there are two ring carbon atoms for connection with the other parts of the compound.

In some embodiments, _R2 is:

in:

_R4 is a monovalent group or a divalent group, and is a divalent group when connected to other parts of the compound, selected from: _{C1-6 alkyl} or _C1-6 alkylene; and _C1-6 alkyl- _C3-8 cycloalkylene- or _C1-6 alkylene- _C3-8 cycloalkylene-;

R ₅ is a monovalent group or a divalent group, and when R ₅ is connected to other parts of the compound, it is a divalent group, selected from: C _1-6 alkyl-(O-CH ₂ CH ₂ -O) _m -; or C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -;

One of _R4 and _R5 is a divalent group, and the other is a monovalent group;

R ₆ does not exist;

L ₂ and L ₃ are each independently a bond or a C _1-6 alkylene group;

L ₄ are each independently C _1-6 alkylene;

L ₅ is a bond, C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl or C _1-6 alkylenecarbonyl;

L ₆ is C _1-6 alkylene;

L ₇ is C _1-6 alkylene or -C _3-8 cycloalkylene-C _1-6 alkylene;

Each of n and m is independently an integer from 1-50.

In some embodiments, _R2 is:

wherein L ₄ , L ₅ , L ₆ , L ₇ and n are as described in any of the above embodiments.

In formula (I), preferably, the R ₂ is selected from:

wherein o is independently an integer of 1-20, and m and n are independently an integer of 1-50.

Preferably, in R ₂ , o is independently an integer of 1 to 10. Preferably, in R ₂ , m and n are each independently an integer of 1 to 40, or are each independently an integer of 1 to 30, 2 to 40, 5 to 35, 10 to 25, 15 to 20, or 2 to 10.

Preferably, _R2 is selected from:

Here, o is independently an integer of 1-10, and n and m are independently an integer of 2-20.

In formula (I), the drug can be selected from: camptothecin, camptothecin derivatives (such as 10-hydroxycamptothecin, irinotecan and SN38 compounds), topotecan, floxuridine, doxifluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, daunorubicin, epirubicin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, paclitaxel, docetaxel, mitomycin, exotecan, exotecan derivative DXd, Sting agonists, resiquimod and Toll-like receptor agonists, etc.

In some embodiments, the drug is a TLR7/8 agonist, such as T785, resiquimod, and STING agonists, or a topoisomerase I inhibitor, such as exotecan and exotecan derivatives (DXd), and the like.

In some embodiments, the drug is selected from:

In formula (I), the drug can be directly linked to -AAN- or linked via a suitable linker. Usually, the drug is linked to the -AAN- linker via a suitable chemical bond, which can be a peptide bond (-NH-CO-) or -COO-. In some embodiments, the linker is a PABC (-NH-phenyl-CH ₂ -O-CO-) group or an AM (-NH-CH ₂ -) group.

In some embodiments, D of formula (I) is selected from the following X1 to X5:

The wavy line indicates where D and _R2 are connected.

In some embodiments, the drug conjugate represented by formula (I) is selected from:

In each formula, R ₁ is as described in any embodiment herein.

In some embodiments, the compound of formula (I) is selected from the following compounds S1 to S59:

In some embodiments, the drug conjugate described herein has a chemical structure represented by R ₂ ′-D (Formula II), wherein R ₂ ′ is a chemical structure for enhancing the structure-activity relationship, and the structure of D is represented by any one of X1-X5.

Preferably, R ₂ 'is selected from: C _1-6 alkyl-CO-*; C _1-6 alkyl-O-*; C _1-6 alkyl-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; C _1-6 alkyl-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*C _1-6 alkyl-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; C _1-6 alkyl-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; NH ₂ -C 1-6 _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; NH ₂ -C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C 1-6 alkylene-NH-*; NH ₂ -C _1-6 alkylene-(O-CH 2 CH ₂ -O) _n -C _1-6 alkylene-O-*; NH _{2 -C 1-55} alkylene-CO-*; NH ₂ -C _1-55 alkylene-NH-*; NH ₂ -C _1-55 alkylene-O-*; HOOC-C 1-55 alkylene-CO-*; HOOC- _{C 1-6} alkylene-CONH-C _1-6 alkylene-(O-CH 2 CH 2 -O) n -C _1-6 alkylene-CO-*; HOOC-C _1-6 alkylene-CONH-C 1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO- _* ; C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _n —C _1-6 alkylene-NH—*; HOOC—C _1-6 alkylene-CONH—C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _n —C _1-6 alkylene-O—*; HOOC—C _1-6 alkylene-NH—*; HOOC—C _1-6 alkylene-NHCO—SO ₃ H substituted C _1-6 alkylene-NH—*; N ₃ -C _1-6 alkylene-CO-*; N ₃ -C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _n —C _1-6 alkylene-NHCO—C _1-6 alkylene-OC _1-6 alkylene-CO-*; HOOC—C _1-6 alkylene-OC _1-6 alkylene-CO-*;

in:

* indicates the position connected to D;

R ₄ is selected from: C _1-6 alkyl; C _1-6 alkyl-C _3-8 cycloalkylene-; COOH-C _1-6 alkylene-; C _1-6 alkyl-(O-CH ₂ CH ₂ -O) m C _1-6 alkylene-; NH ₂ -C _1-6 alkylene; C _1-6 alkyl-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; and NH ₂ -C _1-6 alkylene-C _3-8 cycloalkylene;

R ₅ is selected from: C _1-6 alkyl-(O—CH ₂ CH ₂ —O) _m -; C _1-6 alkyl-CONH-C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _m -; COOH-C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _n -;

R ₆ is absent, or is -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylenecarbonyl, or -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NHCO-C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl;

L ₂ and L ₃ are each independently a bond or a C _1-6 alkylene group;

L ₄ are each independently C _1-6 alkylene;

L ₅ is a bond, C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl or C _1-6 alkylenecarbonyl;

L ₆ is C _1-6 alkylene;

L ₇ is C _1-6 alkylene or -C _3-8 cycloalkylene-C _1-6 alkylene;

Each of n and m is independently an integer of 1-50, and p is 0 or 1; when p is 0, the -NH-[CO-R ₄ ] _p group is -NH ₂ .

Preferably, R ₂ 'is selected from:



wherein o is independently an integer of 1-20, and m and n are each independently an integer of 1-50.

More preferably, R ₂ 'is selected from:

Preferably, n and m are each independently an integer of 2-20.

In some embodiments, the drug conjugate represented by formula (II) is selected from:

Antibody Drug Conjugates

Provided herein is an antibody drug conjugate represented by the following formula (III), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
R ₃ -(R ₁ -R ₂ -D) _x Formula (III);

In the formula: R ₃ is an antibody; R ₁ , R ₂ and D are as described in any of the above embodiments; x is an integer selected from 1-8.

In formula III, when R ₁ is:

hour,

The connection method of _R3 - _R1 is:

Wherein, S is the S atom of the cysteine residue in R ₃ ;

When _R1 is:

hour,

The connection method of _R3 - _R1 is:

Among them, _R3 is coupled to the alkynyl group in the _R1 group.

An azido modification can be introduced into the sugar chain of the antibody or the reconstructed sugar chain, which undergoes a click reaction with DBCO (dibenzocyclooctyne) or BCN (bicyclo[6,1,0]nonyne) to form a triazole ring, thereby achieving the connection between R ₁ and R ₃ .

The drug conjugate or antibody drug conjugate of the present invention may exist in the form of any one of geometric isomers or a mixture thereof.

Antibody

In the content disclosed in this article, "antibody" is used in the broadest sense and covers biological molecules such as monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), and antibody fragments, as long as they can exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170: 4854-4861).

Antibodies specifically bind to antigens and include at least two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH), and the heavy chain constant region includes three constant domains CH1, CH2 and CH3. Each light chain includes a light chain variable region (VL) and a light chain constant region (CL), and the light chain constant region includes one constant domain. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conservative regions called framework regions (FRs). In general, from the N-terminus to the C-terminus, the light chain and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acids are generally assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th edition; NIH Publication No. 91-3242 (1991): Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia et al., (1987) J Mol. Biol. 196: 901-917 or Chothia et al., (1989) Nature 341: 878-883.

The carboxyl terminal portion of the heavy chain can define the constant region that is primarily responsible for effector function. Typically, human light chains are divided into kappa chains and lambda chains. Human heavy chains are generally divided into μ, δ, γ, α, or ε. Typically, the isotype of an antibody can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG subclasses are well known to those skilled in the art and include, but are not limited to, IgG1, IgG2, IgG3, and IgG4.

Herein, the antibody may be a naturally occurring antibody or a non-naturally occurring antibody; may be a monoclonal antibody or a polyclonal antibody; may be a mouse antibody or a humanized antibody; or may be a chimeric antibody.

Chimeric antibodies refer to antibodies in which a portion of the heavy and/or light chains is identical or homologous to the corresponding sequence in antibodies derived from a particular species (e.g., humans) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species (e.g., mice) or belonging to another antibody class or subclass. Humanized antibodies refer to antibody forms that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins.

Herein, an antibody fragment or antigen-binding fragment refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability of a full-length antibody to specifically bind to an antigen, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibodies (scFv); nanobodies and multispecific antibodies formed from antibody fragments.

The antibody used in the present invention may be an antibody known in the art or a functional fragment thereof. For example, the antibody or its functional fragment may be selected from: anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, or anti-ICOS antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD3 antibody, anti-CD22 antibody, anti-CCR6 antibody, anti-CD38 antibody, anti-CD5 2 antibody, anti-complement C5 antibody, anti-RSV F protein antibody, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib/Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 anti-body, anti-α4 integrin antibody, anti-lgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody, anti-SSTR2 antibody, anti-LY6G6D antibody, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody.

In some embodiments, the antibody used herein is a multispecific antibody, such as a bispecific antibody. A multispecific antibody refers to an artificial antibody containing two or more specific antigen binding sites, which can bridge the target cell and the functional molecule (cell) to stimulate a directed immune response. An antibody molecule comprising at least two antigen binding domains can be referred to as a bispecific antibody molecule, wherein each antigen binding domain can bind to a different target. The VH region and the VL region contain framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDR. The structure of an exemplary bispecific antibody is shown in Figure 19. As shown in Figure 19 (left), from N-terminus to C-terminus, the VH region contains the following structure: {N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]}-linker-{[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus}, and the VL region contains the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus. As shown in Figure 19 (right), from N-terminus to C-terminus, the VH region contains the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region contains the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

In a preferred embodiment, the antibody used in the present invention is selected from the group consisting of: Utomilumab, Urelumab, ADG106, Poteligeo ^™ (Mogamulizumab), Poteligeo ^™ (Mogamulizumab), Bexxar ^™ (tositumomab), Zevalin ^™ (ibritumomab tiuxetan), Rituxan ^™ (rituximab), Arzerra ^™ (Ofatumumab), Gazyva ^™ (Obinutuzumab), Besponsa ^™ (Inotuzumab ozogamicin), Zenapax ^™ (daclizumab), Varlilumab, Theralizumab, Adcetris ^™ (Brentuximab vedotin), Myelotarg ^™ (gemtuzumab), Darzalex ^™ (Daratumumab), CDX-1140, SEA-CD40, RO7009789, JNJ-64457107, APX-005M, Chi Lob 7/4, Campath ^TM (alemtuzumab), Raptiva ^TM (efalizumab), Soliris ^TM (eculizumab), Yervoy ^TM (ipilimumab), tremelimumab, Erbitux ^TM (cetuximab), Vectibix ^TM (panitumumab), Portrazza ^TM (Necitumumab), TheraCIM ^TM (Nimotuzumab), Synagis ^TM (palivizumab), Unituxin ^TM (Dinutuximab), TRX-518, MK-4166, MK-1248, GWN-323, INCAGN0186, BMS-986156, AMG-228, ReoPro ^TM (abiciximab), Herceptin ^TM (trastuzumab), Perjeta ^TM (Pertuzumab), Kadcyla ^TM (Ado-trastuzumab) emtansine), GSK-3359609, JTX-2011, Simulect ^TM (basiliximab), Tysabri ^TM (natalizumab), BMS-986016, REGN3767, LAG525, Xolair ^TM (omalizumab), Tavolimab, PF-04518600, BMS-986178, MOXR-0916, GSK-3174998, INCAGN01949, IBI-101, Keytruda ^TM (Pembrolizumab), Opdivo ^TM (Nivolumab), Lartruvo ^TM (Olaratumab), Tencentriq ^TM (Atezolizumab), BMS-936559, Bavencio ^TM (Avelumab), Imfinzi ^TM (Duralumab), Prolia ^TM (Denosumab), Empliciti ^TM (Elotuzumab), MTIG7192A, TSR-022, MBG-453, Remicade ^TM (infliximab), Humira ^TM (adalimumab), Avastin ^TM (bevacizumab), Lucentis ^™ (ranibizumab), Cyramza ^™ (Ramucirumab), and JNJ-61610588.

In some embodiments, the bioactive macromolecular portion (A) used in the ADC described herein is a fusion protein containing an antigen binding domain of an antibody and an optional cytokine. The antigen binding domain in the fusion protein can be selected from the antigen binding domains for the following antigens: HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylyl cyclase C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Eph rin-A, LAMP1, MUC1, PDL1, HER2, NY-ESO-1, BCMA, WT1, MUC1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC-1, SSTR2, LY6G6D, MAGE-A3, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody, or an antigen-binding domain (such as an antigen-binding fragment) of any one of the antibodies mentioned herein.

In some embodiments, the fusion protein can be a bispecific antibody comprising an antigen binding domain that specifically binds to an antigen selected from the group consisting of HER2, CD19, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylylcyclase C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1, CD19, PDL1, NY-ESO-1, WT1, MUC1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, EGFR, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, Mesothelin, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC-1, MAGE-A3, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody, or an antigen binding domain selected from any one of the antibodies mentioned in the present invention. Preferably, the bispecific antibody is a single-chain bispecific antibody comprising two scFvs from the same or different antibodies.

Pharmaceutical composition

The pharmaceutical composition of the present invention comprises: (i) a drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and/or an antibody drug conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier. The carrier may be any pharmaceutically acceptable carrier or excipient, which may vary according to the dosage form and mode of administration. Pharmaceutically acceptable carriers are generally safe and non-toxic, and may contain any known substance used in the pharmaceutical industry to prepare pharmaceutical compositions, including fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, and disintegrants, etc. Suitable pharmaceutically acceptable carriers include sugars, such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; starches, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; silicon dioxide, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate; and/or polyethylene glycol, etc. When selecting a pharmaceutically acceptable carrier, the main consideration is the mode of administration of the pharmaceutical dosage form. This is well known in the art.

The pharmaceutical composition may contain a therapeutically or prophylactically effective amount of a drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and/or an antibody drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof. An "effective amount" means that the amount of a component is sufficient to produce the desired response. The specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition, such as body weight, age and sex, the duration of treatment, the co-administered treatment (if any), and the specific formulation used. Typically, an "effective amount" as described herein is a conventional amount of a biomolecule. However, in some embodiments, the therapeutically or prophylactically effective amount of the conjugate contained in the pharmaceutical composition of the present invention may be lower than the conventional amount of the biomolecule but may produce a better therapeutic or prophylactic effect because the biomolecule is protected by a protective group before reaching the pathological microenvironment to bind to its ligand or receptor.

The pharmaceutical composition of the present invention can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered by any suitable route to achieve the desired purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally or externally. The dosage of the drug can depend on the patient's age, health status and weight, concurrent treatments, and the frequency of treatment, etc. The pharmaceutical composition of the present invention can be applied to any subject in need thereof, for example, as a mammal, especially a human being.

The conjugate of the present invention can release toxins (D) through the pathway of biomolecule endocytosis into lysosomes, or can be released through the pathway of proteolytic enzyme hydrolysis in the acidic microenvironment of the tumor, especially Legumain or granzyme. For example, the biomolecule in the antibody-drug conjugate of the present invention is Trastuzumab, anti-Trop-2 antibody or anti-PD-L1 antibody, etc. In terms of toxin release, the proteolytic enzyme hydrolysis pathway of the acidic microenvironment of the tumor can release toxins more efficiently, thereby exerting better drug efficacy. Therefore, the conjugate of the present invention can effectively break through the problem of bioconjugate drug release efficiency, and the toxin is efficiently released in the pathological microenvironment.

use

Each drug conjugate or its stereoisomer or its pharmaceutically acceptable salt, and each antibody drug conjugate or its stereoisomer or its pharmaceutically acceptable salt disclosed in the present invention can be used to treat and/or prevent tumors or inflammation, or can be used as an active ingredient for preparing a drug for treating tumors or inflammation.

The diseases that can be treated by the drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof, and antibody drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof disclosed in the present invention are related to the active ingredients contained in the conjugates. The indications of these active ingredients are well known in the art.

In some embodiments, the tumors described herein may include hematological tumors and solid tumors, including but not limited to sarcomas (such as fibrosarcomas), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer, etc.

The present invention also includes a method for treating or preventing tumors or inflammation, which comprises administering to a subject in need thereof a therapeutically or prophylactically effective amount of a drug conjugate as described herein or its stereoisomer or a pharmaceutically acceptable salt thereof, and an antibody drug conjugate, or its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein. This method can be used in combination with any known radiotherapy or immunotherapy.

The present invention also provides the use of the drug conjugate described herein or its stereoisomer or its pharmaceutically acceptable salt, and/or the antibody drug conjugate, or its stereoisomer or its pharmaceutically acceptable salt in the preparation of an anti-tumor drug. Preferably, the tumor is selected from a blood tumor and a solid tumor. Preferably, the tumor includes but is not limited to sarcoma (such as fibrosarcoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer.

Advantages of the present invention include:

The inventors have prepared trastuzumab-drug conjugates, atezolizumab-drug conjugates, anti-SSTR2 antibody-drug conjugates, and anti-LY6G6D antibody-drug conjugates through continuous experimental screening. These are tumor-targeted and efficiently activated macromolecular conjugates with innovative multifunctional targeted activation properties: (1) The tumor microenvironment-targeted activated antibody-drug conjugates of the present invention have aggregation, retention and activation effects at the tumor site, and have the characteristics of targeting the tumor microenvironment and efficiently releasing the load. (2) The tumor microenvironment-targeted conjugates of the present invention can be specifically activated or broken by tumor tissue to locally generate effective drugs. (3) Exotecan, resimod or their derivatives containing the AAN sequence in the linker are directly converted into macromolecules by conjugation with trastuzumab or certolizumab, which changes the various properties of the drug, improves the efficacy, and can change the indication restrictions of the drug. (4) When tumor cells metastasize, they usually secrete a large amount of protease hydrolases to degrade the intercellular matrix, so the protease hydrolases activated conjugates have special therapeutic effects on the treatment of tumor metastasis, and the antibody-drug conjugates of the present invention have high activation efficiency.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

It should be understood that the terms "comprising" and "including" or similar expressions used in the present invention also mean "consisting of..." etc. The sum of all weight percentages or volume percentages should be equal to 100%. Unless otherwise stated, the various reagents and products used in the examples are commercial products. Unless otherwise stated, the methods mentioned in the examples are implemented according to conventional techniques. The following examples are not intended to limit the scope of the present invention.

Example 1: Preparation of drug conjugates S1-S21, S26-S59

A typical preparation method of the compound is as follows:

Synthesis route general formula (I)

1) Compound R ₁ -R ₂ -Ala-Ala-Asn-PABOH (1.0 eq) was dissolved in an appropriate amount of DMF solvent, and then compound Bis-PNP (2.0 eq) was added. DIPEA (2.0 eq) was added to the reaction solution under an ice bath, and then the temperature was slowly restored to room temperature and stirred for 10-15 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then an appropriate amount of dichloromethane was used to dissolve and dilute it. The organic phase was washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and then column chromatographed. The eluent was dichloromethane/methanol, and the mixture was spin-dried to obtain R ₁ -R ₂ -Ala-Ala-Asn-PABC-PNP.

2) Compound R ₁ -R ₂ -Ala-Ala-Asn-PABC-PNP (1.0 eq) was dissolved in an appropriate amount of DMF solvent, and then compound D-NH ₂ (1.0 eq) was added. DIPEA (2.0 eq) was added to the reaction solution under an ice bath, and then the temperature was slowly restored to room temperature and stirred for 10-15 h. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain the target product R ₁ -R ₂ -Ala-Ala-Asn-PABC-(C=O)-NHD.

The compounds shown in Table 1 can be obtained by using the corresponding raw materials according to the above method. The properties of each compound in Table 1 are shown in Table 2.

Table 1

Table 2

Synthesis route general formula (II)

Compound R ₁ -R ₂ -Ala-Ala-Asn-OH (1.0 eq) was dissolved in an appropriate amount of DMF solvent, and then compound D-NH ₂ and condensing agent HBTU were added in sequence. DIPEA was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain the target product.

The corresponding raw materials were used to obtain the compounds shown in Table 3 according to the above method. The properties of each compound in Table 3 are shown in Table 4.

Table 3

Table 4

Synthesis route general formula (III)

1) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-OAc

Dissolve Fmoc-Ala-Ala-Asn(Trt)-Gly-OH (1.0 eq) in dichloromethane/tetrahydrofuran (volume ratio of 15/5), cool to 0°C, add AcOH (1.0 eq), lead acetate (1.2 eq) and copper acetate (0.2 eq) under nitrogen protection, and stir at 40-50°C under nitrogen protection for 2-3 hours. TLC monitors the reaction to be complete. Dilute the reaction solution with dichloromethane, wash with water in turn, separate the liquids, extract the aqueous phase with dichloromethane, combine the organic phases and dry with anhydrous sodium sulfate, spin dry, and obtain Fmoc-Ala-Ala-Asn-AM-OAc by column chromatography.

2) Synthesis of compound Fmoc-Ala-Ala-Asn(Trt)-AM-Ac-OBn

Add compound Fmoc-Ala-Ala-Asn-AM-OAc (1.0 eq) and benzyl glycolate (2.0 eq) to a three-necked flask, add trifluoroacetic acid/dichloromethane (TFA/DCM volume ratio = 1/5) under nitrogen protection and ice bath, and stir at room temperature for 2-3 hours. After the reaction is completed, the reaction solution is concentrated and separated by high pressure preparative chromatography to obtain Fmoc-Ala-Ala-Asn-AM-Ac-OBn as a white solid.

3) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-Ac-OH

The compound Fmoc-Ala-Ala-Asn-AM-Ac-OBn (1.0 eq) was dissolved in an appropriate amount of MeOH, Pd/C (10 wt%, 0.1 eq) was slowly added, and stirred for 2-3 h under hydrogen protection. The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH was prepared under high pressure as a light yellow product.

4) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-DXd

The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH (1.0 eq) and isoproterenol methanesulfonate (1.0 eq) were dissolved in DMF, cooled to 0°C, and the condensation agent DEPBT (1.2 eq) and the organic base DIPEA (2.0 eq) were added in sequence under nitrogen protection, and stirred at room temperature for 2-3 hours. TLC monitored that the reaction was complete. The reaction solution was concentrated in vacuo, redissolved in DMF, filtered, and the compound Fmoc-Ala-Ala-Asn-AM-DXd was prepared by high pressure liquid phase as a light yellow product.

5) Synthesis of compound H-Ala-Ala-Asn-AM-DXd

The compound Fmoc-Ala-Ala-Asn-AM-DXd (1.0 eq) was dissolved in DMF, piperidine (2.0 eq) was added, and the mixture was stirred at room temperature for 2-3 h under nitrogen protection. TLC monitored that the reaction was complete. The reaction solution was redissolved, the solution was filtered, and then the compound H-Ala-Ala-Asn-AM-DXd was prepared by high pressure liquid phase as a light yellow product.

6) Synthesis of Compound R ₁ -R ₂ -Ala-Ala-Asn-AM-DXd

Compound R ₁ -R ₂ -OH (1.0 eq) was dissolved in an appropriate amount of DMF, H-Ala-Ala-Asn-AM-DXd (1.0 eq) was added, HBTU (1.2 eq) and DIPEA (2.0 eq) were added under nitrogen protection, and the mixture was stirred at room temperature for 1-2 h. TLC monitored that the reaction was complete. The crude product of the reaction solution was filtered, and then the compound R ₁ -R ₂ -Ala-Ala-Asn-AM-DXd was prepared by high pressure liquid chromatography as a light yellow product.

Using the corresponding raw materials, the compounds shown in Table 5 can be obtained according to the above method. The properties of each compound in Table 5 are shown in Table 6.

Table 5

Table 6

Example 2: Synthesis of Compound S22

1) Synthesis of compound Boc-Glu(PEG8)-OtBu

Compound Boc-Glu(OH)-OtBu (300 mg, 0.989 mmol) and PEG8-NH ₂ (379 mg, 0.988 mmol) were dissolved in ultra-dry DMF (5 ml), and condensation agent HBTU (450 mg, 1.19 mmol) was added. DIPEA (255 mg, 1.98 mmol) was added to the reaction solution under ice bath, and stirred at room temperature for 2-3 h. After the reaction solution was evaporated to dryness under reduced pressure, the crude product was mixed with silica gel and column chromatography (petroleum ether: ethyl acetate = 10/1 to 1/1) was performed to obtain oily compound Boc-Glu(PEG8)-OtBu (600 mg, yield: 91%).

2) Synthesis of compound H-Glu(PEG8)-OH

The compound Boc-Glu(PEG8)-OtBu (600 mg, 0.897 mmol) was dissolved in a mixed solvent (10 mL) of DCM/TFA with a volume ratio of 1/1 and stirred for 1 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was dissolved in DCM and repeatedly concentrated to remove residual TFA. The oily compound H-Glu(PEG8)-OH (450 mg, yield 98%) was obtained.

3) Synthesis of compound S22-1

Compound H-Glu(PEG8)-OH (300 mg, 0.585 mmol) and BCN-PNP (185 mg, 0.587 mmol) were dissolved in ultra-dry DMF (5 ml), and diisopropylethylamine (151 mg, 1.17 mmol) was added dropwise and stirred at room temperature for 12-16 h. After the reaction solution was evaporated to dryness under reduced pressure, it was slurried with an organic solvent of MTBE to obtain an oily liquid compound S22-1 (350 mg, yield: 87%).

4) Synthesis of compound S22

Compound S22-1 (100 mg, 0.145 mmol) and X3 (82 mg, 0.144 mmol) were dissolved in ultra-dry DMF (5 ml), and condensation agent HBTU (66 mg, 0.174 mmol) was added. Diisopropylethylamine (38 mg, 0.29 mmol) was added dropwise under ice bath and stirred at room temperature for 2-3 h. LC-MS detected that the reaction was complete, the reaction solution was filtered, and the filtrate was purified by high pressure reverse phase preparative chromatography to obtain S22 (69 mg, yield: 38%) as a white solid.

Example 3: Synthesis of Compound S23

1) Synthesis of compound Cbz-Glu(PEG8)-OtBu

Compound Cbz-Glu(OH)-OtBu (334 mg, 0.991 mmol) and PEG8-NH ₂ (379 mg, 0.988 mmol) were dissolved in ultra-dry DMF (5 ml), and condensation agent HBTU (450 mg, 1.19 mmol) was added. DIPEA (255 mg, 1.98 mmol) was added to the reaction solution in an ice bath, and stirred at room temperature for 2-3 h. After the reaction solution was evaporated to dryness under reduced pressure, the crude product was mixed with silica gel and column chromatography (petroleum ether: ethyl acetate = 10/1 to 1/1) was performed to obtain an oily compound Cbz-Glu(PEG8)-OtBu (610 mg, yield: 88%).

2) Synthesis of compound H-Glu(PEG8)-OtBu

The compound Cbz-Glu(PEG8)-OtBu (600 mg, 0.854 mmol) was dissolved in a mixed solvent (10 mL) of MeOH/THF with a volume ratio of 1/1 and stirred for 1 h, then Pd/C (60 mg, 10 wt%) was added, and the reaction solution was stirred for 2-3 h. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure to obtain an oily liquid compound H-Glu(PEG8)-OtBu (480 mg, yield 99%).

3) Synthesis of compound S23-1

Compound H-Glu(PEG8)-OtBu (400 mg, 0.703 mmol) and MI-C2-COOH (119 mg, 0.704 mmol) were dissolved in ultra-dry DMF (8 ml), and condensation agent HBTU (320 mg, 0.844 mmol) was added. DIPEA (181 mg, 1.40 mmol) was added to the reaction solution under ice bath, and stirred at room temperature for 2-3 h. After the reaction solution was evaporated to dryness under reduced pressure, the crude product was mixed with silica gel and column chromatography (dichloromethane: methanol = 100/1 to 20/1) was performed to obtain oily compound S23-1 (400 mg, yield: 79%).

4) Synthesis of compound S23-2

Compound S23-1 (400 mg, 0.556 mmol) was dissolved in a mixed solvent (10 mL) of DCM/TFA with a volume ratio of 1/1 and stirred for 2-3 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was dissolved in DCM and repeatedly concentrated to remove residual TFA to obtain oily compound S23-2 (360 mg, yield 97.5%).

5) Synthesis of compound S23

Compound S23-2 (100 mg, 0.150 mmol) and X3 (80 mg, 0.141 mmol) were dissolved in ultra-dry DMF (5 ml), and condensing agent HBTU (65 mg, 0.172 mmol) was added. Diisopropylethylamine (36 mg, 0.28 mmol) was added dropwise under ice bath and stirred at room temperature for 2-3 h. LC-MS detected that the reaction was complete, the reaction solution was filtered, and the filtrate was purified by high pressure reverse phase preparative chromatography to obtain S23 (70 mg, yield: 41%) as a white solid. LCMS: [M+H] = 1213.64.

Example 4: Synthesis of Compound S24

Compound S23-2 (100 mg, 0.151 mmol) was dissolved in an appropriate amount of DMF solvent, and then compound X2 (100 mg, 0.140 mmol) and condensing agent HBTU (63 mg, 0.166 mmol) were added in sequence. DIPEA (40 mg, 0.31 mmol) was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain white powder S24 (50 mg, yield 26%). LCMS: [M+H] = 1362.69.

Example 5: Preparation of Compound S25

1) Compound S25-1 (2 g, 4.9 mmol), HBTU (2.24 g, 5.9 mmol), DIPEA (3.2 g, 24.5 mmol) and compound S25-2 (2.24 g, 5.9 mmol) were dissolved in DMF (30 mL), and the mixture was stirred at room temperature for 3 h. The reaction was completed by TLC. Water (20 mL) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was spin-dried to obtain the target compound S25-3 (3.5 g, yield 89.7%).

2) Compound S25-3 (3.5 g, 4.4 mmol) was dissolved in 1% DBU in DCM (40 mL), and the mixture was stirred at room temperature for 1.5 h. The reaction was confirmed to be complete by TLC plate. The organic phase was spin-dried, mixed with silica gel, and separated by normal phase column to obtain the target product S25-4 (2.35 g, yield 92.1%).

3) Compound S25-4 (2.35 g, 4.06 mmol), DEPBT (1.7 g, 5.7 mmol), DIEA (1.5 g, 12 mmol) and compound S25-5 (1.77 g, 5.7 mmol) were dissolved in DMF (30 mL), and the mixture was stirred at room temperature for 3 h. The reaction was completed as determined by TLC plate. Water (20 mL) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was spin-dried. The target product S25-6 (2.5 g, yield 71.4%) was obtained by silica gel column separation.

4) Compound S25-6 (1.5 g, 1.72 mmol) was added to 15 mL of a solution of hydrogen chloride in dioxane, and stirred at room temperature for 2 hours. The reaction solution was concentrated and dried to give the product compound S25-7 (1.2 g, yield 85.7%).

5) DIPEA (379 mg, 2.94 mmol), HBTU (1.1 g, 2.94 mmol) and compound S25-8 (516 mg, 1.76 mmol, TFAsalt) were added to a 100 mL DMF solution containing compound S25-7 (1.2 g, 1.47 mmol), and the reaction was stirred at 25 °C for 0.5 hours. The reaction solution was concentrated and purified by silica gel column chromatography (DCM/MeOH=20:1) to obtain the product compound S25-9 (800 mg, yield 54%).

6) Compound S25-9 (800 mg, 0.806 mmol) and 0.1 eq of tetrakistriphenylphosphine palladium (73 mg) were placed in a dry three-necked flask, and anhydrous dichloromethane was added under nitrogen protection in an ice bath. The mixture was returned to room temperature and reacted for 1 h. The reaction solution was filtered, concentrated, and purified by silica gel column chromatography (DCM/MeOH=20:1) to obtain the product compound S25-10 (500 mg, yield 65%).

7) Compound S25-10 (120 mg, 0.126 mmol) and compound X5 (76 mg, 0.098 mmol) were dissolved in DMF (3 mL), HBTU (57 mg, 0.150 mmol) and DIPEA (49 mg, 0.38 mmol) were added thereto, and the mixture was reacted for 2 hours. The light yellow solid compound S25 (57 mg, yield 26.3%) was obtained by reverse preparation. LCMS: [M+H] ⁺ =1713.81.

Example 6: Enzyme cleavage test of S1-S59 drug conjugates

The methods for testing the stability and enzyme cleavage efficiency of drug conjugate compounds are as follows, and the test results are shown in Table 7.

Weigh the drug conjugate compound accurately, add a certain amount of DMSO solution to make the stock solution concentration 10μmol/mL, then take 10μL and add 80μL DMSO to dilute to 1μmol/mL, then add pure water at a ratio of 1:4 to dilute to a sample solution concentration of 0.2μmol/mL. After the sample is clarified, place it in a 25℃/37℃ water bath, take samples after 0 hour and 24 hours, and use HPLC to detect (Agilent1260, chromatographic column: Eclipse Plus C18, 4.6*250mm, 5μm, mobile phase A: 0.1% TFA+H ₂ O, mobile phase B: CH3CN, flow rate: 1.0ml/min, column temperature: 30℃, detection wavelength: 214nm) the content of drug conjugate relative to 0 hour, and the solution stability data of different compounds can be obtained.

An appropriate amount of sample was weighed, and DMSO was used to prepare a 1 mM solution. Human plasma was added at a volume ratio of 1:9, and the solution was incubated in a 37°C incubator. Samples were taken after 0, 2, 4, 6, and 24 h, and DMSO/MeOH (1:1) was added at a volume ratio of 1: ₃ to remove protein. The solution was centrifuged at 12000 rpm for 5 min, and the supernatant was taken. The HPLC method was as described above (Agilent 1260, chromatographic column: Eclipse Plus C18, 4.6*250 mm, 5 μm, mobile phase A: 0.1% TFA+H2O, mobile phase B: CH3CN, flow rate: 1.0 ml/min, column temperature: 30°C, detection wavelength: 214 nm).

Weigh a certain amount of drug conjugates, dissolve and dilute them tenfold to a concentration of 0.1mM/ml. Add the drug conjugates to 100μg of acidified CT-26 tumor tissue homogenate (pH 5.0) at 37°C at a concentration of 1mg/ml. The enzymes in the tumor tissue homogenate can be released and detected by HPLC to compare the activation efficiency of the tumor tissue to the linker.

Table 7

Control Compound C

As can be seen from Table 7, the stability of the compounds S1-S59 of the present invention is relatively high, wherein the aqueous solution stability for 24 hours is higher than 85%, the plasma stability for 24 hours is higher than 75%, and the enzyme cleavage efficiency for 2 hours is higher than 90%. The aqueous solution stability and plasma stability of the control compound C (compound S1 disclosed in CN104262455B) for 24 hours are lower than those of the compounds S1-S59 of the present invention, and the enzyme cleavage efficiency for 2 hours is lower than that of the compounds S1-S59 of the present invention, because the stability of the amide bond and the aminoester bond between the linker and the load in the present invention is much higher than that of the carbonate bond in the control compound C, and the ester bond is more susceptible to hydrolysis.

The above results show that the compounds S1-S59 provided by the present invention have higher stability in aqueous solution and plasma, and the corresponding compounds are not easily degraded or deteriorated into other compounds after entering the body, and the targeted therapeutic effect after entering tumor cells or tumor tissues is stable. In addition, after entering tumor cells, and being cleaved by specific enzymes within 2 hours, the compounds S1-S59 provided by the present invention release toxic drugs with a higher killing rate on tumor cells.

Example 7: Comparison of activation and release of Dxd by S5, S16, and control compound Y

Weigh a certain amount of drug conjugates (S5, S16, Deruxtecan, control compound Y, control compound Y is compound S1 in CN103011521B), dissolve and dilute them tenfold to a concentration of 0.1mmol/ml. Add the drug conjugates to 100μg of acidified CT-26 tumor tissue homogenate (pH 5.0) at a concentration of 1mg/ml at 37°C. The enzyme in the tumor tissue homogenate can be released and detected by HPLC to compare the activation efficiency of the tumor tissue to the linker.

The results are shown in Figures 1 to 4. Figure 1 shows that the control compound Y began to release Leudox at 0.5h under the action of the enzyme (wherein the release of Leudox was not detected at 5min), and the release was complete in about 2h. Figure 2 shows that compound S5 began to release T785 at 5min. Figure 3 shows that compound S16 began to release Dxd at 5min. Figure 4 shows that Deruxtecan began to release Dxd at 6h under the action of Cathepsin B enzyme.

Therefore, the activation efficiency of compounds S5 and S16 is significantly better than that of control compounds Y and Deruxtecan.

Example 8: Comparison of lung tissue stability between S16 and Deruxtecan

method:

Preparation of experimental solution:

Assay buffer: 25 mM MES, pH 5.0.

Protein precipitant: DMSO/MEOH=1:1.

Preparation of sample solution: Weigh appropriate amounts of 6PEG-lys(MI-6PEG)-AAN-AM-Dxd and EMC-GGFG-AM-Dxd, add a certain amount of DMSO solution to make the stock solution concentration 10umol/mL, then take 10uL and add 80uLDMSO to dilute to 1umol/mL, then add pure water at a ratio of 1:4 to dilute to a sample solution concentration of 0.2umol/mL.

Preparation of lung tissue homogenate:

Take an appropriate amount of liquid nitrogen quick-frozen lungs and place them in a 4 mL EP tube. Weigh it, add pH 5.0 detection buffer at a mass-to-volume ratio of 1:5, homogenize the tissue with a tissue grinder for 3 minutes, then centrifuge the lung tissue homogenate at 12000 rpm for 5 minutes, take the supernatant, and place it in a -40°C refrigerator for later use.

Stability of lung tissue homogenate:

Take the sample solution and lung tissue homogenate in a volume ratio of 1:9 and shake them thoroughly. Take another sample solution and buffer (pH5.0) in a volume ratio of 1:9 and shake them thoroughly as a blank control solution. Place it in a 37°C constant temperature water bath. Take 30uL at 0h, 2h, 4h, and 20h, add 90uL protein precipitant and shake well. Centrifuge at 12000rpm for 5min, take the supernatant into an injection bottle, and inject it by HPLC.

The percentage of DXD released after the drug enters the lung tissue homogenate is calculated using the following formula:

Drug loading% = (Compound molar amount - newly generated Dxd molar amount) / Compound molar amount * 100%

The results are shown in Figures 5 and 6 and Table 8 below.

Table 8

The results showed that S16 produced less Dxd in lung tissue than Deruxtecan and had less impact on normal lung tissue.

Example 9: Preparation of conjugate intermediates L1-L51

Some compounds were prepared using the following reaction scheme:

1) Compound Fmoc-R ₂ '-OH (1.0 eq) was dissolved in an appropriate amount of DMF solvent, and then compound D-NH ₂ and condensing agent HBTU were added in sequence. DIPEA was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain the target product.

2) Compound Fmoc-R ₂ '-D (1.0 eq) was dissolved in DMF solvent and stirred for 30 min, then piperidine (3.0 eq) was added and the reaction solution was stirred at room temperature for 2-3 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was slurried with a mixed solvent of MTBE to obtain the target product NH ₂ -R ₂ '-D.

Using the corresponding raw materials, the compounds shown in Table 9 can be obtained according to the above method. The properties of each compound in Table 9 are shown in Table 10.

Table 9

Table 10

Some compounds were prepared using the following reaction scheme:

1) Compound Cbz-R ₂ '-OH (1.0 eq) was dissolved in an appropriate amount of DMF solvent, and then compound D-NH ₂ and condensing agent HBTU were added in sequence. DIPEA was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain the target product.

2) Compound Cbz-R ₂ '-D (1.0 eq) was dissolved in methanol and stirred for 30 min, then Pd/C (0.1 eq) was added and the reaction solution was stirred at room temperature for 2-3 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was slurried with a mixed solvent of MTBE and ethyl acetate to obtain the target product NH ₂ -R ₂ '-D.

Using the corresponding raw materials, the compounds shown in Table 11 can be obtained according to the above method. The properties of each compound in Table 11 are shown in Table 12.

Table 11

Table 12

Compounds L14 and L23 were prepared by the following method:

Compound L14-1 (100 mg, 0.299 mmol) was dissolved in an appropriate amount of DMF solvent, and then compound X4 (250 mg, 0.297 mmol) and condensing agent HBTU (136 mg, 0.359 mmol) were added in sequence. DIPEA (116 mg, 0.899 mmol) was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain a light yellow powder L14 (100 mg, yield 29%). LCMS: [M+H] = 1157.46.

By replacing X4 with X5, compound L23 can be prepared under the same reaction conditions.

Compounds L13 and L22 were prepared by the following method:

Compound diglycolic anhydride (14 mg, 0.12 mmol) was dissolved in an appropriate amount of DMF solvent, and then compound X4 (100 mg, 0.119 mmol) was added in sequence. DIPEA (31 mg, 0.24 mmol) was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain a light yellow powder L14 (80 mg, yield 70%). LCMS: [M+H] = 957.34

By replacing X4 with X5, compound L22 can be prepared under the same reaction conditions.

Compounds L28 and L29 were prepared by the following method:

The compound succinic anhydride (3 mg, 0.03 mmol) was dissolved in an appropriate amount of DMF solvent, and then the compound L26 (20 mg, 0.015 mmol) was added in sequence. DIPEA (31 mg, 0.24 mmol) was added to the reaction solution under ice bath, and then slowly returned to room temperature and continued to stir for 3-5 hours. Most of the solvent was removed from the obtained reaction solution under high vacuum, and then diluted with an appropriate amount of methanol, purified by reverse phase high pressure preparative chromatography, and finally freeze-dried to obtain a light yellow powder L28 (20 mg, yield 95%). LCMS: [M+H] = 1431.65

L26 was replaced by L27 and compound L29 was prepared under the same reaction conditions.

Example 10: Preparation of Compounds X1-X5

Synthesis of compound X1:

1) Synthesis of compound X1-1

Compound Fmoc-Ala-Ala-Asn-OH (500 mg, 1.01 mmol) and resimod (314 mg, 1.00 mmol) were dissolved in ultra-dry DMF (5 ml), and condensation agent HBTU (455 mg, 1.20 mmol) and diisopropylethylamine (387 mg, 3.00 mmol) were added and stirred at room temperature for 16 h. After the reaction solution was evaporated to dryness under reduced pressure, it was slurried and filtered with a mixed solvent of ethanol and MTBE to obtain a white solid compound X1-1 (500 mg, yield: 63%).

2) Synthesis of compound X1

Compound X1-1 (500 mg, 0.631 mmol) was dissolved in DMF solvent (10 mL) and stirred for 30 min, then piperidine (160 mg, 1.88 mmol) was added, and the reaction solution was stirred at room temperature for 2-3 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was slurried with a mixed solvent of MTBE to obtain a white solid compound X1 (300 mg, yield 83%).

Synthesis of compound X2:

1) Synthesis of compound Fmoc-Ala-Ala-Asn-PABC-PNP

The compound Fmoc-Ala-Ala-Asn-PABC (800 mg, 1.33 mmol) and Bis-PNP (808 mg, 2.66 mmol) were dissolved in ultra-dry DMF (5 ml), and diisopropylethylamine (343 mg, 2.66 mmol) was added and stirred at room temperature for 16 h. After the reaction solution was evaporated to dryness under reduced pressure, it was slurried and filtered with a mixed solvent of ethanol and MTBE to obtain a white solid compound Fmoc-Ala-Ala-Asn-PABC-PNP (820 mg, yield: 80%).

2) Synthesis of Compound X2-1

The compound Fmoc-Ala-Ala-Asn-PABC-PNP (200 mg, 0.261 mmol) was dissolved in DMF solvent (20 mL) and stirred for 1 h. Then the compound T785 (80 mg, 0.257 mmol) and diisopropylethylamine (60 mg, 0.465 mmol) were added. The reaction solution was evaporated to dryness under reduced pressure and the residue was slurried with a mixed solvent of MTBE to give a white solid compound X2-1 (200 mg, yield 83%).

3) Synthesis of Compound X2

Compound X2-1 (200 mg, 0.213 mmol) was dissolved in DMF solvent (5 mL) and stirred for 1 h, then piperidine (55 mg, 0.647 mmol) was added and the reaction solution was evaporated to dryness under reduced pressure. The residue was slurried with a mixed solvent of MTBE to obtain a white solid compound X2 (110 mg, yield 72%).

Synthesis of compound X3:

1) Synthesis of compound X3-1

The compound Fmoc-Ala-Ala-Asn-OH (450 mg, 0.907 mmol) and T785 (188 mg, 0.605 mmol) were dissolved in ultra-dry DMF (5 ml), and condensation agent DEPBT (217 mg, 0.726 mmol) and diisopropylethylamine (234 mg, 1.82 mmol) were added and stirred at room temperature for 16 h. After the reaction solution was evaporated under reduced pressure, it was slurried and filtered with a mixed solvent of ethanol and MTBE to obtain a white solid compound X3-1 (300 mg, yield: 63%).

2) Synthesis of compound X3

Compound X3-1 (300 mg, 0.380 mmol) was dissolved in DMF solvent (20 mL) and stirred for 1 h, then piperidine (100 mg, 1.18 mmol) was added and the reaction solution was evaporated to dryness under reduced pressure. The residue was slurried with a mixed solvent of MTBE to obtain a white solid compound X3 (200 mg, yield 93%).

Synthesis of compound X4:

1) Synthesis of compound X4-1

Compound Fmoc-Ala-Ala-Asn-PABC-PNP (500 mg, 0.652 mmol) and isotecan mesylate (346 mg, 0.652 mmol) were dissolved in ultra-dry DMF (8 ml), and organic base diisopropylethylamine (168 mg, 1.30 mmol) was added and stirred at room temperature for 16 h. After the reaction solution was evaporated to dryness under reduced pressure, it was slurried and filtered with a mixed solvent of ethyl acetate and MTBE to obtain a yellow solid compound X4-1 (600 mg, yield: 86.6%).

2) Synthesis of compound X4

Compound X4-1 (600 mg, 0.564 mmol) was dissolved in DMF solvent (10 mL) and stirred for 1 h, then piperidine (144 mg, 1.69 mmol) was added, and the reaction solution was continued to react for 2 h, monitored by TLC, and the reaction was stopped when the reaction was complete. The residue was evaporated to dryness under reduced pressure, and the residue was slurried with a mixed solvent of MTBE and ethyl acetate in turn to obtain a light yellow solid compound X4 (450 mg, yield 95%).

Synthesis of compound X5:

1) Synthesis of compound X5-1

1) Dissolve the compound Fmoc-Ala-Ala-Asn-AM-Ac-OH (500 mg, 0.857 mmol) and isotecan methanesulfonate (455 mg, 0.857 mmol) in DMF, cool to 0°C, add the condensing agent DEPBT (307 mg, 1.03 mmol) and the organic base DIPEA (332 mg, 2.57 mmol) in sequence under nitrogen protection, and stir at room temperature for 2-3 hours. TLC monitoring shows that the reaction is complete. The reaction solution is concentrated in vacuo, redissolved in DMF, filtered, and prepared by high pressure liquid phase to obtain compound X5-1 as a light yellow product (510 mg, yield 60%).

2) Synthesis of compound X5

Compound X5-1 (500 mg, 0.50 mmol) was dissolved in DMF, piperidine (128 mg, 1.51 mmol) was added, and the mixture was stirred at room temperature for 2-3 h under nitrogen protection. TLC monitored that the reaction was complete. The reaction solution was redissolved, the solution was filtered, and then high pressure liquid phase was used to prepare compound X5 as a light yellow product (300 mg, yield 77%).

Example 11: Preparation of Antibody Drug Conjugates

The preparation method of the antibody drug conjugate is as follows: tri(2-carboxyethyl)phosphine (TCEP) (1-40eq) is added to the antibody buffer for reduction (0.5-16 hours), and the above-mentioned drug conjugate (2-40eq) is added for reaction (0.5-16 hours) to form the antibody drug conjugate. Then, the antibody drug conjugate is purified by column chromatography and filtration to obtain a pure product.

The following ADCs were prepared by the above method: trastuzumab-S15 (HER2-S15), anti-LY6G6D antibody-S15 (LY6G6D-PEG6-AAN-AM-DXD), atezolizumab-S5 (PD-L1-TLR7/8), atezolizumab-S16 (PD-L1-PEG-K (MI-PEG)-AAN-AM-DXd), anti-GCC antibody-S16, and the DAR value of each ADC was about 8. Note: The names in brackets correspond to the names in the figure.

In the above-mentioned ADCs, "HER2" refers to trastuzumab (Chemical Biotechnology (Shanghai) Co., Ltd.); "PD-L1" refers to atezolizumab (the amino acid sequences of its light chain variable region and heavy chain variable region are shown in SEQ ID NOs: 1 and 2, respectively; the light chain and heavy chain are shown in SEQ ID NOs: 3 and 4, respectively); "Anti-SSTR2" refers to anti-SSTR2 antibody; "LY6G6D" refers to anti-LY6G6D antibody (the amino acid sequences of its light chain variable region and heavy chain variable region are shown in SEQ ID NOs: 5 and 6, respectively); the amino acid sequences of the light chain variable region and heavy chain variable region of the anti-GCC antibody are shown in SEQ ID NOs: 7 and 8, respectively.

In the above ADC, the drug conjugates used are as follows:

Affinity linker2＝EMC-PEG6-AAN-AM-Dxd(S15);

Affinity linker4＝EMC-C5-AAN-T785(S5).

Affinity linker5＝PEG-K(MI-PEG)-AAN-AM-DXd(S16) Figure 7 shows the hydrophobic interaction chromatography (HIC) of trastuzumab-S15; Figure 8 shows the MS spectrum of atezolizumab-S5; Figure 9 shows the HIC spectrum of atezolizumab-S5; Figure 10 shows the MS spectrum of atezolizumab-S5.

Example 12

1. Animals: BALB/c mice, 8-week-old female (Shanghai Lingchang Biotechnology Co., Ltd.).

2. Cells: Cells from the Chinese Academy of Sciences Cell Bank were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum at 37°C, 5% _CO2 , and passaged every 3 days. Cells were used within 15 generations.

3. Tumor generation: 0.3×10 ⁶ CT26 cells were subcutaneously injected into the back of BALB/c mice. When the tumor grew to about 160 mm ³ , the mice were randomly divided into groups and treatment began.

4. Treatment course: Administer the drug through the tail vein once a week for three weeks.

The groups and outcome measures are shown in Table 13 and Figure 11 .

Table 13

The TGI% in the present invention is the tumor growth inhibition rate of the drug. TMEA-TLR7/8 is compound S5.

Example 13

1. Animals: BALB/c mice, 8-week-old female (Shanghai Lingchang Biotechnology Co., Ltd.).

2. Cells: Cells from the Chinese Academy of Sciences Cell Bank were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum at 37°C, 5% _CO2 , and passaged every 3 days. Cells were used within 15 generations.

3. Tumor generation: 0.3×10 ⁶ CT26 cells were subcutaneously injected into the back of BALB/c mice. When the tumor grew to about 160 mm ³ , the mice were randomly divided into groups and treatment began.

4. Treatment process: Tail vein administration, CTLA-4 is administered twice a week, TMEA-TRL7/8 is administered once a week, for a total of three weeks.

The groups and outcome measures are shown in Table 14 and Figure 12 .

Table 14

Embodiment 14

1. Animals: NCG mice, 8-week-old female (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.).

2. Cells: Cells from the Chinese Academy of Sciences Cell Bank were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum at 37°C, 5% _CO2 , and passaged every 7 days. Cells were used within 15 generations.

3. Tumor generation: 5×10 ⁶ NCI-N87 cells were subcutaneously injected into the back of NCG mice. When the tumor grew to about 100 mm ³ , the mice were randomly divided into groups and treatment began.

4. Treatment process: Administer the drug once through the tail vein.

The groups and outcome measures are shown in Table 15 and Figure 13 .

Table 15

Embodiment 15

1. Animals: BALB/c mice, 8-week-old female (Shanghai Lingchang Biotechnology Co., Ltd.).

2. Cells: Wuhan Huamei Biotechnology. Cells were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum and 10 μg/ml Puromecin (10 mg/ml) at 37°C and 5% _CO2 . Cells were passaged every 3 days and were used within 15 generations.

3. Tumor formation: 0.3×10 ⁶ CT26 cells were subcutaneously injected into the back of BALB/c mice. When the tumor grew to about 100 mm ³ , the mice were randomly divided into groups and treatment began.

4. Treatment course: Intraperitoneal administration, twice a week, for 3 weeks.

The groups and outcome measures are shown in Table 16 and Figure 14 .

Table 16

Example 16

6-8 week old female C57BL/6J-Pdedleml(hPDCD1)/Smoc mice were purchased from Shanghai Model Organism Center. After the mice arrived and were unpacked, they were housed in an SPF animal breeding center at a density of 5 per cage. The general condition of the animals (mental state, activity, hair color) was observed, and the body weight of the first week was recorded. MC38 (mPD-L1 knockout; hPD-L1 knockin) cells were harvested during the logarithmic growth phase and placed in sterile saline before animal inoculation. 1×10 ⁶ cells were subcutaneously inoculated in the upper groin of the lateral hind limb of the mice. The administration method was: Gr1-control PBS (twice a week, for a total of 2 times); Gr2-PDL1-PEG-K(MI-PEG)-AAN-AM-DXd (10 mg/kg, twice a week, for a total of 2 times); Gr3-Atezolizumab (10 mg/kg, twice a week, for a total of 2 times). Tumor volume and mouse body weight were measured twice a week, where tumor volume was calculated using the formula (0.5×L×W ² ), where L and W refer to the length and width of the tumor, respectively. When the tumor reached 50-150 mm ³ , the mice were randomly divided into groups according to a random number table. At the end of the experiment, the mice were killed by cervical dislocation.

The results are shown in Figure 15 and Table 17 below.

Table 17

From the results of Figure 15 and Table 17, it can be seen that on day 18, the TGI of the drug PDL1-PEG-K(MI-PEG)-AAN-AM-DXd in the MC38 tumor model was 98.6%, and PDL1-PEG-K(MI-PEG)-AAN-AM-DXd could completely inhibit the growth of the tumor between day 7 and day 18. Therefore, the drug PDL1-Dxd showed a good tumor inhibition effect in the MC38 tumor model.

Embodiment 17

1. Animals: NCG mice, 8-week-old female (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.).

2. Cells: Cells from the Chinese Academy of Sciences Cell Bank were cultured in DMEM (1x) medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged every 3 days and were used within 15 generations.

3. Tumor generation: 5×10 ⁶ MDA-MB-231 cells were subcutaneously injected into the back of NCG mice. When the tumor grew to about 1000 mm ³ , the mice were randomly divided into groups and treatment began.

4. Treatment process: Administer the drug once through the tail vein.

The groups and outcome measures are shown in Table 18 and Figure 16 .

Table 18

Example 18: In vivo characterization of GCC TMEAbody in a mouse tumor model

Human colon cancer cell HT-55 cells were subcutaneously transplanted into NDG mice to construct an in vivo model. HT-55 cells were cultured according to the culture conditions, and the cells were collected and counted during the exponential growth phase. 1×10 ⁶ human colon cancer cell HT-55 (suspended in 0.1 ml basal culture medium) was inoculated on the right side of each mouse. After inoculation, when the tumor grew to 56-86 mm ³ (7 days after tumor loading), the mice were randomly divided into groups according to their weight and tumor volume. The experiment was divided into: PBS group, 7B6-S16 (5 mg/kg) group.

Treatment started on the day of grouping. The mice were weighed and the tumor volume was measured twice a week. The volume was calculated as follows: tumor volume = 1/2 × length × width × width (mm ³ ). Tumor inhibition rate TGI (%) = [1-(Tt-T0)/(Ct-C0)] × 100%; tumor inhibition rate T/C (%) = (Tt/T0)/(Ct/C0) × 100%. T0 is the tumor volume of the experimental group at the time of grouping; Tt is the tumor volume of the experimental group during each measurement; C0 is the average tumor volume of the PBS group at the time of grouping; Ct is the average tumor volume of the PBS group at each measurement.

The results are shown in Figure 17. 7B6-S16 showed significant tumor growth inhibition in the subcutaneous HT-55 transplanted tumor model in mice. After 35 days of treatment, the inhibition rate of the 7B6-S16 (5 mg/kg) group was 106% TGI and 2% T/C. The results showed that 7B6-S16 can be activated in the tumor microenvironment and stimulate anti-tumor immune response.

Embodiment 19

1. Animals: Nude mice, 6-8 weeks old, all female (Shanghai Lingchang Biotechnology Co., Ltd.).

2. Cells: The corresponding cells were purchased from the American type culture collection (ATCC) and identified according to the instructions provided by ATCC. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37°C and 5% carbon dioxide. The cells were passaged every 3 days and used within 15 generations.

3. Tumor formation: 1.5×10 ⁶ HT1080 cells were subcutaneously injected into the back of nude mice. When the tumor grew to about 100 mm3, the mice were randomly divided into groups and treatment began. The day of treatment was regarded as the first day.

4. Treatment process

The dose (5 μmol/Kg) was administered once a week for 3 weeks.

The groups and outcome measures are shown in Table 19.

Table 19: Effects of corresponding compounds and control group on tumor inhibition

5. Results and discussion: The compounds S26, S41, S43, S49, S57 and S58 of the present invention have greatly improved therapeutic effects compared to the exotecan derivatives and can almost achieve the effect of curing tumors.

Embodiment 20

1. Experimental purpose: To understand the acute toxicity of the drug of the present invention in vivo by determining the MTD experiment of intravenous administration in mice.

2. Experimental drugs: Exotecan derivatives, compounds S26, S41, S43, S49, S57 and S58 were dissolved in DMSO and diluted to the corresponding dose with physiological saline during the experiment.

3. Animals: Primary BALB/C mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.), weighing 19-21 g, all female.

4. Methods and results: 48 BALB/C mice were tested, weighing 19-21g, all female, and were randomly divided into 7 groups according to body weight, with 6 mice in each group. The doses shown in Table 20 were intravenously injected with exotecan derivatives, compounds S26, S41, S43, S49, S57 and S58 at one time. A normal saline group control test was also performed, with a dosing volume of 0.2 ml for each mouse. The animals were observed continuously for 17 days, and the animals were observed daily to see if they had erect hair, messy and dull, lethargic, hunched over, overreaction, etc., and the body weight and death were recorded. Blood samples were collected on the 3rd, 5th and 14th days for complete blood counts, and the heart, liver, kidney, lung, spleen and pancreas were dissected on the 14th day for HE staining observation.

Table 20: Comparison of mortality results of test mice receiving different doses of compound injection and normal saline

5. Results and discussion: Compared with the ixotecan derivatives, when the animals were injected with S26, S41, S43, S49, S57 and S58 of the present invention, there was no piloerection, matted luster, lethargy, hunchback, overreaction or death, indicating that the toxicity of the small molecule conjugated drug is significantly lower than that of the unconjugated drug.

Example 21: Ring opening method after S15 coupling to albumin and antibody

Coupling process: Measure 1.8 mL of albumin solution (1.43 umol/mL) and put it into a 5 mL PEEK tube. Then add 2 mL S15 (2 umol/mL) and mix thoroughly. Place it at room temperature. After 2 hours, dialyze it overnight with 10K dialysis card 10 mM NaH2PO4 buffer (pH 5.0) to obtain a compound coupled with albumin.

Ring-opening process: After the compound is coupled to albumin, it is incubated at 25-50°C for 24-60h in a 30-80mM sodium bicarbonate buffer with a pH of 5.0-10.0. After incubation, a 3K ultrafiltration tube is used to detect the ring-opening condition by LC-MS high-resolution mass spectrometry. The molecular weight after coupling with albumin is 67702.78, and the molecular weight after ring-opening of coupling with albumin is 67721.57.

Experimental steps for plasma stability of compounds after coupling and ring opening process:

1. Take 20uL of HSA-Compound and HSA-Compound-open-ring samples (hereinafter referred to as HSA-Compound-OR) (0.5umol/mL) and add them to 380uL of human plasma, mix thoroughly, and place in a 37°C water bath;

2. Take 50uL of the mixed solution at 0h, 2h, 4h, 24h, 48h and 96h, add 150uL of protein precipitant ( _CH3OH /DMSO=1:1), centrifuge at 12000rpm for 5min, and take the supernatant for HPLC injection.

The percentage of DXD released after the drug enters the lung tissue homogenate is calculated using the following formula:

The results are shown in Figure 18.

Example 22: Preparation of dual-antibody drug conjugates

The bispecific conjugate drug can target two antigens at the same time, and its targeting method is the two forms shown in Figure 19, wherein Anti-Antigen 1 is selected from any one of EGFR, Trop2, and Cmet; Anti-Antigen 2 is selected from any one of EGFR, Trop2, and Cmet.

Among the prepared bispecific antibody drug conjugates, the heavy chain amino acid sequence of the bispecific antibody in EGFR/Cmet-T785 is shown in SEQ ID NO: 9, and the light chain amino acid sequence is shown in SEQ ID NO: 11; the heavy chain amino acid sequence of the bispecific antibody in EGFR/Cmet-Dxd is shown in SEQ ID NO: 10, and the light chain amino acid sequence is shown in SEQ ID NO: 12; the light chain amino acid sequence of the bispecific antibody in EGFR/Trop2 is shown in SEQ ID NO: 13, and the heavy chain amino acid sequence is shown in SEQ ID NO: 14.

1. EGFR/Cmet-S5:

2.0×10 ⁶ NCI-H292 cells expressing human HGF were mixed with Matrigel in a 1:1 ratio in PBS and inoculated subcutaneously in the right flank of SCID Beige mice in a total volume of 100 μL. Treatment was started when the average tumor size reached 139 mm3. The dosage of the test article and the number of animals in each study group are shown in Table 21 below. The date of tumor cell inoculation was recorded as day 0.

Table 21

Prior to starting treatment, all animals were weighed and tumor volumes were measured. Because tumor volume can affect the effectiveness of any given treatment, mice were divided into groups using a randomized block design based on tumor volume. This ensures that all groups are comparable at baseline. A randomized block design is used to divide experimental animals into groups. First, the experimental animals are divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals are randomized to treatment. Using a randomized block design to assign experimental animals ensures that each animal has the same probability of being assigned to a given treatment, thereby reducing systematic error.

During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimates of food and water consumption, weight gain/loss (body weights were measured twice weekly), eye/hair matting, and any other abnormal effects.

The endpoint of the study is whether the tumor growth can be delayed or whether the tumor-bearing mice can be cured. The tumor size was measured twice a week in two dimensions using a caliper. The volume was expressed in mm ³ using the formula: V = 0.5a × b ² , where a and b are the long and short diameters of the tumor, respectively.

The results are shown in Figure 20. The results showed that on day 18, the TGI of the drug EGFR/Cmet-T785 in the NCI-H292 tumor model was 76.9%, indicating that EGFR/Cmet-S5 can significantly slow down the growth rate of the tumor, thereby effectively prolonging the survival of the mice. Therefore, the drug EGFR/Cmet-S5 showed a good tumor inhibition effect in the NCI-H292 tumor model.

2. EGFR/Cmet-S16:

2.0×10 ⁶ NCI-H292 cells expressing human HGF were mixed with Matrigel in a 1:1 ratio in PBS and inoculated subcutaneously in the right flank of SCID Beige mice in a total volume of 100 μL. Treatment was started when the average tumor size reached 139 mm3. The dosage of the test article and the number of animals in each study group are shown in Table 22 below. The date of tumor cell inoculation was recorded as day 0.

Table 22

Prior to starting treatment, all animals were weighed and tumor volumes were measured. Because tumor volume can affect the effectiveness of any given treatment, mice were divided into groups using a randomized block design based on tumor volume. This ensures that all groups are comparable at baseline. A randomized block design is used to divide experimental animals into groups. First, the experimental animals are divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals are randomized to treatment. Using a randomized block design to assign experimental animals ensures that each animal has the same probability of being assigned to a given treatment, thereby reducing systematic error.

During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimates of food and water consumption, weight gain/loss (body weights were measured twice weekly), eye/hair matting, and any other abnormal effects.

The endpoint of the study is whether the tumor growth can be delayed or whether the tumor-bearing mice can be cured. The tumor size was measured twice a week in two dimensions using a caliper. The volume was expressed in mm ³ using the formula: V = 0.5a × b ² , where a and b are the long and short diameters of the tumor, respectively.

The results are shown in Figure 21. The results showed that on day 18, the TGI of the drug EGFR/Cmet-S16 in the NCI-H292 tumor model was 87.2%, and EGFR/Cmet-S16 could completely inhibit the growth of the tumor between day 7 and day 18. Therefore, the drug EGFR/Cmet-S16 showed a good tumor inhibition effect in the NCI-H292 tumor model.

3. EGFR/Trop2-S5:

2.0×10 ⁶ NCI-H292 (TROP2 ^high EGFR ^high ) cells were mixed with Matrigel in a 1:1 ratio in PBS and inoculated subcutaneously in the right side of SCID Beige mice in a total volume of 100 μL. Treatment was started when the average tumor size reached about 100 mm3. The dosage of the test article and the number of animals in each study group are shown in Table 23 below. The date of tumor cell inoculation was recorded as day 0.

Table 23

Prior to starting treatment, all animals were weighed and tumor volumes were measured. Because tumor volume can affect the effectiveness of any given treatment, mice were divided into groups using a randomized block design based on tumor volume. This ensures that all groups are comparable at baseline. A randomized block design is used to divide experimental animals into groups. First, the experimental animals are divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals are randomized to treatment. Using a randomized block design to assign experimental animals ensures that each animal has the same probability of being assigned to a given treatment, thereby reducing systematic error.

During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimates of food and water consumption, weight gain/loss (body weights were measured twice weekly), eye/hair matting, and any other abnormal effects.

The endpoint of the study is whether the tumor growth can be delayed or whether the tumor-bearing mice can be cured. The tumor size was measured twice a week in two dimensions using a caliper. The volume was expressed in mm ³ using the formula: V = 0.5a × b ² , where a and b are the long and short diameters of the tumor, respectively.

The results are shown in Table 24 below.

Table 24

4. EGFR/Trop2-S16:

2.0×10 ⁶ NCI-H292 (TROP2 ^high EGFR ^high ) cells were mixed with Matrigel in a 1:1 ratio in PBS and inoculated subcutaneously in the right side of SCID Beige mice in a total volume of 100 μL. Treatment was started when the average tumor size reached about 100 mm ^3. The dosage of the test article and the number of animals in each study group are shown in Table 25 below. The date of tumor cell inoculation was recorded as day 0.

Table 25

Prior to starting treatment, all animals were weighed and tumor volumes were measured. Because tumor volume can affect the effectiveness of any given treatment, mice were divided into groups using a randomized block design based on tumor volume. This ensures that all groups are comparable at baseline. A randomized block design is used to divide experimental animals into groups. First, the experimental animals are divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals are randomized to treatment. Using a randomized block design to assign experimental animals ensures that each animal has the same probability of being assigned to a given treatment, thereby reducing systematic error.

During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimates of food and water consumption, weight gain/loss (body weights were measured twice weekly), eye/hair matting, and any other abnormal effects.

The endpoint of the study is whether the tumor growth can be delayed or whether the tumor-bearing mice can be cured. The tumor size was measured twice a week in two dimensions using a caliper. The volume was expressed in mm ³ using the formula: V = 0.5a × b ² , where a and b are the long and short diameters of the tumor, respectively.

The results are shown in Table 26 below.

Table 26

Through the above experiments, it was found that small molecule drugs or protein-coupled drugs whose linkers contain the AAN sequence can release the load more efficiently, thereby improving the therapeutic effect.

Example 23: Pharmacodynamics and drug safety evaluation of S41 (QHL-1618) on human fibrosarcoma cell HT-1080 in a BALB/c nude mouse subcutaneous tumor model

In this study, 32 female Balb/c nude mice were subcutaneously inoculated with human fibrosarcoma cells HT-1080 to construct a transplanted tumor model. When the average volume of the transplanted tumor reached 100 ^mm3 , they were randomly divided into 4 groups, with 8 mice in each group, and given vehicle (Vehicle), 2.47 mg/kg or 5 μmol/kg CPT-780 (positive control, S41 payload Dxd), 7.12 mg/kg or 5 μmol/kg S41, and 14.25 mg/kg or 10 μmol/kg S41 by tail vein injection. The dosage was 10 ml/kg, and the mice were given once a week for 3 weeks.

The results are shown in Figure 22. S41 showed a significant inhibitory effect on the growth of HT-1080 tumor cells. On the 38th day of the experiment, 10 μmol/kg of S41 was an effective dose, and no significant drug toxicity was shown in mice.

Example 24: In vivo pharmacodynamic study of the test compound on human fibrosarcoma HT-1080 in a BALB/c nude mouse subcutaneous transplant tumor model

In this study, 30 female Balb/c mice were subcutaneously inoculated with human fibrosarcoma HT-1080 cells to construct a transplant tumor model. When the average volume of the transplant tumor reached 161 ^mm3 , they were randomly divided into 5 groups, with 6 mice in each group, and given lysozyme (Vehicle), 1.63 mg/kg or 3.3 μmol/kg CPT-780 (positive control, S41 payload Dxd), 1.57 mg/kg or 1.1 μmol/kg S41, 4.70 mg/kg or 3.3 μmol/kg S41, and 14.25 mg/kg or 10 μmol/kg S41 by tail vein injection. The dosage was 10 μl/g, and the mice were given once a week for 17 days.

The results are shown in Figure 23. The results showed that S41 exhibited a significant inhibitory effect on the growth of tumor cell HT-1080 transplanted tumors. On the 17th day of the experiment, tumor regression was observed in all mice (N=6) in the S41 4.70 mg/kg and 14.25 mg/kg treatment groups, of which 80% of the mice had complete tumor regression to zero in the S41 4.70 mg/kg and 14.25 mg/kg treatment groups.

Example 25: Pharmacodynamics and drug safety evaluation of S41 on mouse colon cancer cell CT-26 in a BALB/c mouse subcutaneous tumor model

This study used 30 female Balb/c mice, which were subcutaneously inoculated with mouse colon cancer cells CT-26 to construct a transplant tumor model. When the average volume of the transplanted tumor reached 127 ^mm3 , they were randomly divided into 5 groups, with 6 mice in each group, and were given lysozyme (Vehicle), 3 mg/kg or 6 μmol/kg CPT-780 (positive control, S41 payload Dxd), 3 mg/kg or 6 μmol/kg S41, and 9 mg/kg or 1 μmol/kg S41 by tail vein injection. The dosage was 10 ml/kg, and the mice were given once a week for 4 weeks.

The results are shown in Figure 24. The results showed that S41 showed a significant inhibitory effect on the growth of tumor cell CT-26 transplanted tumors. On the 27th day of the experiment, S41 (18 μmol/kg) was an effective dose. At the same time, no obvious drug toxicity was shown in mice.

Example 26: Pharmacodynamics and drug safety evaluation of S41 and anti-mPD-1 combined therapy on mouse colon cancer cell CT-26 in a BALB/c mouse subcutaneous tumor model

This study used 24 female Balb/c mice, subcutaneously inoculated with mouse colon cancer cells CT-26, to construct a transplant tumor model. When the average volume of the transplant tumor reached 127mm ³ , they were randomly divided into 4 groups, 6 mice in each group, and given lysozyme (Vehicle), 9mg/kg or 18μmol/kg S41, 5mg/kg anti-mPD-1 antibody, 9mg/kg S41 combined with 5mg/kg anti-mPD-1 antibody, respectively, by tail vein injection, and the administration volume was 10ml/kg. S41 was administered once a week, and anti-mPD-1 antibody was administered twice a week for 4 weeks.

The results are shown in Figure 25. The results showed that S41 and anti-mPD-1 exhibited a synergistic inhibitory effect on the growth of tumor cell CT-26 transplanted tumors.

Example 27: Pharmacodynamics and drug safety evaluation of S41 on mouse colon cancer cell CT-26 in a BALB/e mouse subcutaneous tumor model

In this study, 24 female Balb/c mice were subcutaneously inoculated with mouse colon cancer cells CT-26 to construct a transplant tumor model. When the average volume of the transplanted tumor reached 150-200 ^mm3 , they were randomly divided into 4 groups, with 6 mice in each group, and given lysozyme (Vehicle), 10 mg/kg T-DXD analog (positive control), 16 mg/kg S41, and 24 mg/kg S41 (HSNTD dose) by tail vein injection. The dosage was 10 ml/kg, and the mice were given once a week for 2 weeks.

The results are shown in Figure 26. The results showed that S41 showed a significant inhibitory effect on the growth of tumor cell CT-26 transplanted tumors. According to the results of the rat toxicity test, 16mg/kg S41 and 10mg/kg T-DXD had similar toxicity. At high doses, S41 was significantly more effective than T-DXD.

Example 28: Toxicity test of SD rats given intravenous injection of S41 for four weeks and repeated administration for four weeks during the recovery period

This study selected 160 rats and randomly divided them into 4 groups, 40 rats in each group, half male and half female, including 30 main test groups and 10 TK groups. They were given normal saline, 3, 6, and 12 mg/kg of S41, respectively, once a week for 4 consecutive weeks. The drug was then stopped for 4 weeks. The following examinations were performed during the experiment, including general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, coagulation), body temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weight, bone marrow smear, histopathological examination, and toxicokinetics. The results showed that the HNSTD dose was 12 mg/kg.

Example 29: Toxicity test of S41 administered intravenously to Beagle dogs for four weeks with a recovery period of four weeks and repeated administration

This study selected 40 beagle dogs and randomly divided them into 4 groups, 10 in each group, half male and half female. They were given normal saline, 1, 2.5, and 5 mg/kg of S41, respectively, once a week for 4 consecutive weeks. The drug was then discontinued for 4 weeks. The following examinations were performed during the experiment, including general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, coagulation), body temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weight, bone marrow smear, histopathological examination, and toxicokinetics. The results showed that the HNSTD dose was 2.5 mg/kg.

In summary, the antibody-drug conjugate provided by the present invention can target and aggregate around tumor cells, and can only activate the drug on the surface of tumor cells, effectively killing tumors, and the toxicity of the drug is also reduced to a certain extent, and has very good application prospects.

Claims (15)

A drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, characterized in that the drug conjugate has a chemical structure shown in formula (I) or (II),
R ₁ -R ₂ -D (I)
R ₂ '-D (II) in: R ₁ is a chemical structure coupled to a biomolecule, or R ₁ does not exist; R ₂ and R ₂ ' are each independently an adjusted chemical structure that enhances the structure-activity relationship; D is selected from the following structures represented by X1-X5:

The wavy line indicates the position where D is connected to R ₂ or R ₂ '. The drug conjugate, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that R ₁ is absent or is selected from:
Preferably, _R1 is selected from: The wavy line indicates the position where _R1 and _R2 are connected. The drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that R ₂ is selected from: -C _1-6 alkylene-CO-*; -C _1-6 alkylene-O-*; -C _1-6 alkylene-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; -C _1-6 alkylene-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-* -C _1-6 alkylene-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-* -(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; -C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; -NH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; -NH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*; -NH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*; -NH-C _1-55 alkylene-CO-*; -NH-C _1-55 alkylene-NH-*; -NH-C _1-55 alkylene-O-*; -CO-C _1-55 alkylene-CO-*; -CO-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; -CO-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*; -CO-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*; -CO-C _1-6 alkylene-NH-*; -CO-C _1-6 alkylene-NHCO-SO ₃ H substituted C _1-6 alkylene-NH-*;
in: * indicates the position connected to D; R ₄ is a monovalent group or a divalent group, and when R ₄ is connected to other parts of the compound, it is a divalent group, selected from: C _1-6 alkyl or C _1-6 alkylene; C _1-6 alkyl-C _3-8 cycloalkylene- or C _1-6 alkylene-C _3-8 cycloalkylene-; -CO-C _1-6 alkylene-; C _1-6 alkyl-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene- or C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; -NH-C _1-6 alkylene; C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; and -NH-C _1-6 alkylene-C _3-8 cycloalkylene; R ₅ is a monovalent group or a divalent group, and when R ₅ is connected to other parts of the compound, it is a divalent group, selected from: C _1-6 alkyl-(O-CH ₂ CH ₂ -O) _m -; C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -; -CO-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -; One of _R4 and _R5 is a divalent group, and the other is a monovalent group; R ₆ is absent or is -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylenecarbonyl or -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NHCO-C _1-6 alkyleneoxy _- C _1-6 alkylenecarbonyl; L ₂ and L ₃ are each independently a bond or a C _1-6 alkylene group; L ₄ are each independently C _1-6 alkylene; L ₅ is a bond, C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl or C _1-6 alkylenecarbonyl; L ₆ is C _1-6 alkylene; L ₇ is C _1-6 alkylene or -C _3-8 cycloalkylene-C _1-6 alkylene; Each n and m is independently an integer from 1 to 50; Preferably, _R2 is selected from:



Wherein, o is independently an integer of 1-20, and m and n are each independently an integer of 1-50; More preferably, _R2 is selected from:

Preferably, n and m are each independently an integer of 2-20. The drug conjugate, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the drug conjugate represented by formula (I) is selected from:











In each formula, R ₁ is as described in claim 1 or 2. The drug conjugate, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the drug conjugate of formula (I) is selected from:










The drug conjugate, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that R ₂ 'is selected from: C _1-6 alkyl-CO-*; C _1-6 alkyl-O-*; C _1-6 alkyl-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; C _1-6 alkyl-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-* C _1-6 alkyl-OCONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; C _1-6 alkyl-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; NH ₂ -C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; NH ₂ -C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*; NH ₂ -C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*; NH ₂ -C _1-55 alkylene-CO-*; NH ₂ -C _1-55 alkylene-NH-*; NH ₂ -C _1-55 alkylene-O-*; HOOC-C _1-55 alkylene-CO-*; HOOC-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-CO-*; HOOC-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NH-*; HOOC-C _1-6 alkylene-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-O-*; HOOC-C _1-6 alkylene-NH-*; HOOC-C _1-6 alkylene-NHCO-SO ₃ H substituted C _1-6 alkylene-NH-*; N ₃ -C _1-6 alkylene-CO-*; N ₃ -C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NHCO-C _1-6 alkylene-OC _1-6 alkylene-CO-*; HOOC-C _1-6 alkylene-OC _1-6 alkylene-CO-*;
in: * indicates the position connected to D; R ₄ is selected from: C _1-6 alkyl; C _1-6 alkyl-C _3-8 cycloalkylene-; COOH-C _1-6 alkylene-; C _1-6 alkyl-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; NH ₂ -C _1-6 alkylene; C _1-6 alkyl-CONH-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _m -C _1-6 alkylene-; and NH ₂ -C _1-6 alkylene-C _3-8 cycloalkylene; R ₅ is selected from: C _1-6 alkyl-(O—CH ₂ CH ₂ —O) _m -; C _1-6 alkyl-CONH-C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _m -; COOH-C _1-6 alkylene-(O—CH ₂ CH ₂ —O) _n -; R ₆ is absent, or is -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylenecarbonyl, or -NH-C _1-6 alkylene-triazolyl-C _1-6 alkylene-(O-CH ₂ CH ₂ -O) _n -C _1-6 alkylene-NHCO-C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl; L ₂ and L ₃ are each independently a bond or a C _1-6 alkylene group; L ₄ are each independently C _1-6 alkylene; L ₅ is a bond, C _1-6 alkyleneoxy-C _1-6 alkylenecarbonyl or C _1-6 alkylenecarbonyl; L ₆ is C _1-6 alkylene; L ₇ is C _1-6 alkylene or -C _3-8 cycloalkylene-C _1-6 alkylene; Each n and m is independently an integer of 1-50; p is 0 or 1; when p is 0, the -NH-[CO-R ₄ ] _p group is -NH ₂ ; Preferably, R ₂ 'is selected from:



Wherein, o is independently an integer of 1-20, and m and n are each independently an integer of 1-50; More preferably, R ₂ 'is selected from:

Preferably, n and m are each independently an integer of 2-20. The drug conjugate, its stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the drug conjugate represented by formula (II) is selected from:










An antibody-drug conjugate, a stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that the antibody-drug conjugate is as shown in formula (III),
R ₃ -(R ₁ -R ₂ -D) _x Formula (III); Wherein, R ₃ is an antibody; R ₁ , R ₂ , and D are as described in any one of claims 1 to 4; x is an integer selected from 1-8. The antibody-drug conjugate, its stereoisomer or pharmaceutically acceptable salt according to claim 8, characterized in that: _R1 is:
The connection method of _R3 - _R1 is:
wherein S is the S atom of a cysteine residue in _R3 ; or _R1 is:
The connection method of _R3 - _R1 is:
Among them, _R3 containing an azide group is coupled with the alkynyl group in the _R1 group. The antibody-drug conjugate, its stereoisomer or pharmaceutically acceptable salt according to claim 8, characterized in that the antibody is a monoclonal antibody or a multispecific antibody; Preferably, the antibody is selected from the group consisting of anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, anti-ICOS antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD3 antibody, anti-CD22 antibody, anti-CCR6 antibody, anti-CD38 antibody, anti-CD52 antibody, anti-complement C5 antibody, anti-R SV F protein antibody, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib/Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-α4 integrin antibody, anti-1gE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody, anti-SSTR2 antibody and anti-LY6G6D antibody, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody, or a multispecific antibody formed by any two or more of these antibodies. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: (i) the drug conjugate according to any one of claims 1 to 7, or its stereoisomer or pharmaceutically acceptable salt and/or the antibody drug conjugate according to any one of claims 8 to 10, or its stereoisomer or pharmaceutically acceptable salt; and (ii) a pharmaceutically acceptable carrier. A drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, characterized in that the drug conjugate is selected from:
A pharmaceutical composition, characterized in that it contains the drug conjugate according to claim 12, its stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition according to any one of claims 11 to 13, characterized in that the pharmaceutical composition further contains one or more other anticancer drugs; preferably, the other anticancer drug is legubicin. Use of the drug conjugate or its stereoisomer or pharmaceutically acceptable salt according to any one of claims 1 to 7 or the antibody drug conjugate or its stereoisomer or pharmaceutically acceptable salt according to any one of claims 8 to 11 in the preparation of a medicament for treating and/or preventing tumors and/or inflammation; preferably, the tumor is a blood tumor or a solid tumor; more preferably, the tumor is selected from the group consisting of sarcoma (such as fibrosarcoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer.