Antibody-drug conjugate containing a proteolytic agent class of biologically active compounds, method for preparing the same, and use thereof

Abstract

The present disclosure relates to antibody-drug conjugates of biologically active compounds of the class of proteolytic agents represented by formula I, methods for their preparation, and use in the prevention and / or treatment of diseases associated with abnormal cell activity, including but not limited to use in the prevention and / or treatment of neoplastic diseases. JPEG2025516873000190.jpg19170

Description

Technical Field

[0001] The present disclosure belongs to the technical field of medicine and relates to antibody-drug conjugates against biologically active compounds of the protease class, biologically active compounds of the protease class, drug linker conjugates, and methods for preparing the same, and use in the prevention and / or treatment of diseases related to abnormal cell activities, including but not limited to use in the prevention and / or treatment of neoplastic diseases.

Background Art

[0002] Many diseases are associated with abnormal functions of intracellular proteins, and the main means for treating these diseases are small-molecule compounds. However, more than 80% of proteins are considered to be "undruggable" because they have no sites that can produce their druggable effects, and thus such targets cannot be drugged with conventional small molecules.

[0003] Targeted protein degradation inducers (TPD) such as proteolysis-targeting chimeric molecules (PROTAC) and molecular glue degraders (MGD) both mediate target protein degradation through the ubiquitin-proteasome pathway and can function without the need to bind closely to the sites that affect protein activity. As a result, the above-mentioned "undruggable" proteins can become new drug targets. At the same time, targeted protein degradation therapy can continuously induce rapid and efficient degradation of pathogenic proteins and reduce the generation of resistance to the target protein.

[0004] Currently, targeted protein degradation inducers have already entered the stage of clinical development. C4 Therapeutics has already presented preclinical data of the molecular glue CFT7455 targeting IKZF1 / 3 at the AACR in 2021. The company presented the clinical results of CFT7455 at the AACR meeting in April 2022, and CFT7455 showed clinical benefits of deep target degradation, but there was a dose limit for neutrophil reduction.

[0005] Currently, in the field of antibody-drug conjugates (ADCs), maleimide has been widely applied to the formation of ADC molecules through thiol coupling with antibodies via Michael addition reaction due to its high selectivity, high reactivity, and better stability. Among the 15 commercially available ADC drugs, 9 are coupled by maleimide. Thiosuccinimide bonds are unstable in the presence of thiol-containing substances (e.g., in plasma), and can be cleaved by the reverse Michael reaction or exchanged with endogenous thiols such as albumin (HAS) and glutathione (GSH), which has been reported in many literatures to result in undesirable states in pharmacodynamics, pharmacokinetics, and safety.

Summary of the Invention

[0006] The present invention relates to an antibody-drug conjugate (ADC) represented by Formula I and its use. The ADC drug combines the tumor-targeting effect of an antibody and the high activity of a biologically active compound, serving as a biological missile and having highly desirable therapeutic effects and safety advantages. The antibody induces the binding of the ADC to target cells, realizes the enrichment of tumor tissues, reduces the exposure to non-target tissues, and reduces the potential toxicity of the biologically active compound by systemic administration. The ADC that binds to tumor cells can release biologically active molecules in the tumor microenvironment and kill tumor cells. The ADC can also be internalized by tumor cells and, under the action of specific enzymes intracellularly, undergo enzymatic degradation to release small molecule drugs and treat diseases. Therefore, an ADC composed of a combination of a proteolytic agent and a tumor-targeting antibody is expected to achieve tumor enrichment, removal or reduction of toxic side effects caused by the action of the proteolytic agent on non-disease tissues, and improvement of therapeutic effects. Realizing tumor targeting of a proteolytic agent using ADC technology and reducing its toxic side effects have very high clinical value.

[0007] Therefore, in one aspect of the present disclosure, the present disclosure provides an antibody-drug conjugate represented by Formula I,

Chemical Formula

Chemical formula

Chemical formula

Chemical formula

[0008] In some embodiments, the biologically active compound of the protease class is selected from a protease-inducing chimeric molecule or a molecular glue.

[0009] In some embodiments, the biologically active compound of the protease class is selected from one or more of the following: (1) A protease-inducing chimeric molecule capable of causing the degradation of family proteins such as GSPT1, IKZF1 and / or 3, CK1α.

[0010] (2) A molecular glue capable of causing the dissociation of the binding of family proteins such as GSPT1, IKZF1 and / or 3, and CK1α.

[0011] In some embodiments, the biologically active compound of the above protease class is a molecule capable of causing the degradation of GSPT1 family proteins.

[0012] In some embodiments, q is selected from any numerical value between 0.1 and 12.0.

[0013] In some embodiments, q is selected from any numerical value between 1.0 and 10.0.

[0014] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0015] In some embodiments, q is selected from any numerical value between 2 and 8.

[0016] In some embodiments, q is selected from 2, 4, 6 or 8.

[0017] In some embodiments, L 1 is

Chemical formula

[0018] In some embodiments, L 1 is

Chemical formula

[0019] In some embodiments, L 2 is [Chemical formula] selected from, with the first position linked to L 1 and the second position linked to L 3 .

[0020] In some embodiments, L 2 is [Chemical formula] selected from, with the first position linked to L 1 and the second position linked to L 3 .

[0021] In some embodiments, Y is -CH 2 - or -OCH 2 CH 2 -.

[0022] In some embodiments, Y is -CH 2 .

[0023] In some embodiments, n is selected from any integer between 0 and 10.

[0024] In some embodiments, n is selected from 0, 1, 2, or 3.

[0025] In some embodiments, n is selected from 1, 2, or 3.

[0026] In some embodiments, X is -CR m R n - or -NR m -.

[0027] In some embodiments, X is -CH 2 -, -C(CH 3 ) 2 -, -N(CH 3 )- or -NH-.

[0028] In some embodiments, R m , R n is independently selected from hydrogen, a C1-4 alkyl group, a C2-4 alkenyl group, a C2-4 alkynyl group, a C3-6 cycloalkyl group, or a 3-6 membered heterocyclic group, or, R m and R n together with the carbon atom to which they are commonly attached form a 3-6 membered carbocyclic or 3-6 membered heterocyclic ring.

[0029] In some embodiments, R m , R n is independently selected from hydrogen or a methyl group.

[0030] In some embodiments, L 1 -L 2 is

Chemical formula

[0031] In some embodiments, L 1 -L 2 is

Chemical formula

[0032] In some embodiments, L 1 -L 2 is

Chemical formula

[0033] In some embodiments, L 3 is AA1 is selected from the amino acid residues or AAs shown in 1 dipeptides, tripeptides or tetrapeptides containing the amino acid residues shown in

[0034] In some embodiments, L 3 is AA 1 , Val-Ala, Val-AA 1 , Ala-AA 1 , Gly-AA 1 , AA 1 -Gly, AA 1 -Ala, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly, or Val-AA 1 -Ala, and is selected from amino acid residues, dipeptides, or tripeptides shown in

[0035] In some embodiments, L 3 is AA 1 , Val-AA 1 , Ala-AA 1 , Gly-AA 1 , AA 1 -Gly, AA 1 -Ala, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly or Val-AA 1 -Ala, and is selected from amino acid residues, dipeptides, or tripeptides shown in

[0036] In some embodiments, L 3 is Val-AA 1 , Val-AA 1 -Ala or Val-AA 1 -Gly, and is selected from dipeptides or tripeptides shown in

[0037] In some embodiments, L 3 is the tripeptide shown by Val-AA 1 -Gly

[0038] In some embodiments, L 3 is [Chemical formula] selected from, with the first position linked to L 2 and the second position linked to L 4 or D.

[0039] In some embodiments, L 3 is [Chemical formula] selected from, with the first position linked to L 2 and the second position linked to L 4 or D.

[0040] In some embodiments, L 3 is [Chemical formula] selected from, with the first position linked to L 2 and the second position linked to L 4 or D.

[0041] In some embodiments, one of R a , R b is hydrogen and the other is [Chemical formula] .

[0042] In some embodiments, R a and R b together with the carbon atom to which they are commonly linked form a 5- to 6-membered heterocyclic ring substituted with R 0 .

[0043] In some embodiments, R a and R b together with the carbon atom to which they are commonly linked form a 5- to 6-membered heterocyclic ring substituted with R 0forms a piperidine ring or a piperazine ring substituted with

[0044] In some embodiments, R a and R b together with the carbon atom to which they are commonly attached form a piperidine ring substituted with R 0

[0045] In some embodiments, R a and R b together with the carbon atom to which they are commonly attached,

Chemical formula

[0046] In some embodiments, r, r 1 are each independently selected from 0, 1, 2, 3, 4 or 5.

[0047] In some embodiments, r, r 1 are each independently selected from 0 or 4.

[0048] In some embodiments, r is 0 and r 1 is 4.

[0049] In some embodiments, R m1 , R n1 are each independently selected from hydrogen or a C1-6 alkyl group.

[0050] In some embodiments, R m1 , R n1 are each independently selected from a C1-6 alkyl group.

[0051] In some embodiments, R m1 , R n1 ​​is independently selected from hydrogen, a methyl group, an ethyl group, an n-propyl group, or an n-butyl group.

[0052] In some embodiments, R m1 and R n1 are independently selected from a methyl group, an ethyl group, an n-propyl group, or an n-butyl group.

[0053] In some embodiments, R m1 and R n1 together with the carbon atom to which they are commonly attached, form a 5- to 6-membered heterocyclic ring optionally substituted with R 0’ .

[0054] In some embodiments, R m1 and R n1 together with the carbon atom to which they are commonly attached, form a piperidine ring or a piperazine ring optionally substituted with R 0’ .

[0055] In some embodiments, R m1 and R n1 together with the carbon atom to which they are commonly attached,

Chemical formula

[0056] In some embodiments, R 0 , R 0’ are each independently selected from a C1-6 alkyl group, -NR m2 R n2 , or a 5- to 6-membered heterocyclic group optionally substituted with a C1-6 alkyl group.

[0057] In some embodiments, R 0is selected from a C1-6 alkyl group or a 5- to 6-membered heterocyclic group substituted with a C1-6 alkyl group, and the above 5- to 6-membered heterocyclic group is selected from a piperidinyl group and a piperazinyl group.

[0058] In some embodiments, R 0 is selected from a methyl group, an ethyl group, or a 5- to 6-membered heterocyclic group substituted with a methyl group, and the above 5- to 6-membered heterocyclic group is a piperidinyl group.

[0059] In some embodiments, R 0 is a methyl group, an ethyl group, or

Chemical formula

[0060] In some embodiments, R 0’ is a C1-6 alkyl group or -NR m2 R n2 selected from.

[0061] In some embodiments, R 0’ is a methyl group or -NR m2 R n2 selected from.

[0062] In some embodiments, R m2 , R n2 is a methyl group.

[0063] In some embodiments, AA 1 The structure of the amino acid residue shown is as follows:

Chemical formula

Chemical formula

[0064] In some embodiments, the amino acid residue shown as AA 1 is

Chemical formula

[0065] In some embodiments, the amino acid residue shown as AA 1 is

Chemical formula

[0066] In some embodiments, the amino acid residue shown as AA 1 is

Chemical formula

[0067] In some embodiments, L 4 is absent

[0068] In some embodiments, L 4 is

Chem.

[0069] In some embodiments, L 4 is

Chem.

[0070] In some embodiments,

Chem.

Table 1

[0071] with the 1-position linked to L 2 and the 2-position linked to D, and the definition of AA 1 is as described in any one form of the present disclosure.

[0072] In some embodiments,

Chem.

[0073] In some embodiments,

Chem.

Table 2

[0074] The 1-position is linked to L 2 and the 2-position is linked to D.

[0075] In some embodiments,

Chemical formula

Table 3

[0076] The 1-position is linked to L 2 and the 2-position is linked to D.

[0077] In some embodiments,

Chemical formula

Table 4

[0078] The 1-position is linked to Tb via an S atom, the 2-position is linked to D, and the definitions of Y, X, AA 1 and n are as described in any one of the forms of the present disclosure.

[0079] In some embodiments,

Chemical formula

Table 5

[0080] The 1-position is linked to Tb via an S atom, the 2-position is linked to D, and the definitions of Y, X, AA 1 and n are as described in any one of the forms of the present disclosure.

[0081] In some embodiments,

Chemical formula

Table 6

[0082] The 1-position is linked to Tb via an S atom, and the 2-position is linked to D.

[0083] In some embodiments,

Chemical formula

Table 7

[0084] The 1-position is linked to Tb via an S atom, and the 2-position is linked to D.

[0085] In some embodiments, D is a structural unit represented by Formula II, and D is linked to L 4 or L 3 via A,

Chemical formula

Chemical formula

[0086] In some embodiments, R 1 is selected from hydrogen, halogen, or a C1-4 alkyl group.

[0087] In some embodiments, R 1 is halogen.

[0088] In some embodiments, R 1 is selected from hydrogen, methyl, or chlorine.

[0089] In some embodiments, R 1 is chlorine.

[0090] In some embodiments, Z is -NH-.

[0091] In some embodiments, U 1 , U 2 are each independently selected from -CH 2 - or -C(O)-, and U 1 , U 2 are not the same.

[0092] In some embodiments, U 1 is -CH 2 -, and U 2 is -C(O)-.

[0093] In some embodiments, A is

Chemical formula

[0094] In some embodiments, A is

Chemical formula

[0095] In some embodiments, A is O, S, -NH-, -N(CH 3 ), -O(CH 2 ) m -, -S(CH 2 ) m -, -NH(CH 2 ) m -, -N(CH 3 )(CH 2 ) m -, -O(CH 2 ) m O-, -S(CH 2 ) m O-, -S(CH 2 ) m S-, -NH(CH 2 ) m O-, -N(CH 3 )(CH 2 ) m O-, -NH(CH 2 ) m S-, -N(CH 3 )(CH 2 ) m S-, -NH(CH 2 ) m NH-, -N(CH 3 )(CH2 ) m NH-, -O(CH 2 ) m C(O)NH(CH 2 ) P -, -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, -O(CH 2 ) m C(O)NH(CH 2 ) P O-, -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P O-, -S(CH 2 ) m C(O)NH(CH 2 ) P -, -S(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, -NH(CH 2 ) m C(O)NH(CH 2 ) P -, -NH(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, -O(CH 2 ) m C(O)NH(CH 2 ) P O(CH 2 ) y -, or -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P O(CH 2 ) y - selected from.

[0096] In some embodiments, A is -O(CH 2 ) m C(O)NH(CH 2 )P -, -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, -O(CH 2 ) m C(O)NH(CH 2 ) P O-, -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P O-, -S(CH 2 ) m C(O)NH(CH 2 ) P -, -S(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, -NH(CH 2 ) m C(O)NH(CH 2 ) P -, -NH(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, -O(CH 2 ) m C(O)NH(CH 2 ) P O(CH 2 ) y -, or -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P O(CH 2 ) y selected from.

[0097] In some embodiments, A is -N(CH 3 )(CH 2 ) m -, -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, or -O(CH2 ) m C(O)N(CH 3 )(CH 2 ) P O(CH 2 ) y selected from -

[0098] In some embodiments, A is -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P -, or -O(CH 2 ) m C(O)N(CH 3 )(CH 2 ) P O(CH 2 ) y selected from -

[0099] In some embodiments, A is -N(CH 3 )(CH 2 ) 3 -, -OCH 2 C(O)N(CH 3 )(CH 2 ) 3 -, or -OCH 2 C(O)N(CH 3 )(CH 2 ) 2 O(CH 2 ) 2 selected from -

[0100] In some embodiments, A is -OCH 2 C(O)N(CH 3 )(CH 2 ) 3 -, or -OCH 2 C(O)N(CH 3 )(CH 2 ) 2 O(CH 2 ) 2 selected from -

[0101] In some embodiments, R 2 , R 3 , R4 , R 5 , R 6 , R 7 , R 8 is independently selected from hydrogen, deuterium, or a C1-4 alkyl group.

[0102] In some embodiments, W is selected from O, S, or NR 9 .

[0103] In some embodiments, W is O.

[0104] In some embodiments, V is a direct bond, O, S, or NR 9 .

[0105] In some embodiments, V is selected from a direct bond or O.

[0106] In some embodiments, m, p, y are independently selected from 0, 1, 2, 3, or 4.

[0107] In some embodiments, m, p, y are independently selected from 1, 2, 3, or 4. In some embodiments, R 9 is selected from hydrogen, deuterium, or a methyl group.

[0108] In some embodiments, D is preferably a structural unit represented by II-1, II-2, or II-3:

Chemical formula

[0109] In some embodiments, D is selected from the following structures, and the 1-position is linked to L 4 or L 3linked to

Chem.

Chem.

Chem.

[0110] In some embodiments, the antibody-drug conjugate has the structure shown in Formula III-1:

Chem.

[0111] In some embodiments, the antibody-drug conjugate is selected from the group consisting of:

Table 8

[0112] q is selected from any numerical value between 1.0 and 10.0.

[0113] In some preferred embodiments, the antibody-drug conjugate is selected from the group consisting of: [Table 9]

[0114] q is selected from any numerical value between 1.0 and 10.0.

[0115] In some preferred embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0116] In some more preferred embodiments, q is selected from 2, 4, 6 or 8.

[0117] In some embodiments, the Tb is a targeting moiety, such as an antibody and its antigen-binding fragment, a ligand, a protein, a polypeptide, a non-protein reagent (such as a sugar, RNA, or DNA), an antibody mimetic, and the like.

[0118] In some embodiments, the Tb is an antibody or its antigen-binding fragment.

[0119] In some embodiments, the Tb is an antibody or its antigen-binding fragment having endocytosis activity.

[0120] In some embodiments, the Tb is an antibody or its antigen-binding fragment having the activity of binding to a tumor cell surface antigen.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has the activity of binding to a tumor cell surface antigen and has tumor cell endocytosis activity.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has antigen-binding activity and has no or weak tumor cell endocytosis activity.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that binds to a non-endocytotic antigen on the surface of tumor cells.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has no tumor cell endocytosis activity.

[0125] In some preferred embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has the activity of binding to a tumor cell surface antigen and has tumor cell endocytosis activity.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof includes Fab, Fab’, F(ab’)2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, Probody antibody, bispecific antibody, or multispecific antibody.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof is a non-human antibody, humanized antibody, chimeric antibody, or fully human antibody.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof is a Probody antibody, bispecific antibody, or multispecific antibody.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof includes Fab, Fab’, F(ab’)2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv).

[0130] In some embodiments, Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, an anti-Trop-2 antibody or antigen-binding fragment thereof, an anti-Her2 antibody or antigen-binding fragment thereof, an anti-Her3 antibody or antigen-binding fragment thereof, or an anti-EGFR antibody or antigen-binding fragment thereof.

[0131] In some embodiments of the present disclosure, Tb is an anti-B7H3 monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the anti-B7H3 antibody includes all prior art anti-B7H3 antibodies, for example, see CN112521512, WO2021027674, WO2021021543, WO2021006619, CN111662384, CN111454357, WO2020151384, WO2020140094, WO2020103100, WO2020102779, WO2020063673, WO2020047257, WO2020041626, CN110684790, CN110642948, WO2019225787, WO2019226017, US20190338030, CN110305213, WO2018209346, WO2018177393, US9150656, WO2016106004, WO2016044383, WO2016033225, WO2015181267, US20120294796, WO2011109400, CN101104639, WO2004093894, WO2002010187, WO2001018021.

[0132] In some embodiments, Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, such as 1D1-01, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab, or antigen-binding fragment thereof. In some preferred embodiments, Tb is the 2E3-02 antibody.

[0133] In some embodiments, the VH sequence of the above 1D1-01 is shown in SEQ ID NO: 1, and the VL sequence is shown in SEQ ID NO: 2. In some embodiments, the heavy chain sequence of the above 1D1-01 is shown in SEQ ID NO: 3, and the light chain sequence is shown in SEQ ID NO: 4.

[0134] In some embodiments, the VH sequence of the above 2E3-02 is shown in SEQ ID NO: 5, and the VL sequence is shown in SEQ ID NO: 6. In some embodiments, the heavy chain sequence of the above 2E3-02 is shown in SEQ ID NO: 7, and the light chain sequence is shown in SEQ ID NO: 8.

[0135] In some embodiments, the above Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown in SEQ ID NO: 2. In some preferred embodiments, the above Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having the heavy chain sequence shown in SEQ ID NO: 3 and the light chain sequence shown in SEQ ID NO: 4.

[0136] In some embodiments, the above Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having the VH sequence shown in SEQ ID NO: 5 and the VL sequence shown in SEQ ID NO: 6. In some preferred embodiments, the above Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having the heavy chain sequence shown in SEQ ID NO: 7 and the light chain sequence shown in SEQ ID NO: 8.

[0137] In some embodiments, Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof, such as datopotamab, sacituzumab or an antigen-binding fragment thereof.

[0138] In some embodiments, Tb is an anti-Her2 antibody or an antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or an antigen-binding fragment thereof. In some preferred embodiments, Tb is Trastuzumab or Pertuzumab.

[0139] In some embodiments, Tb is an anti-Her2 antibody or an antigen-binding fragment thereof.

[0140] In some embodiments, Tb is an anti-Her2 monoclonal antibody or an antigen-binding fragment thereof.

[0141] In some embodiments, Tb is a bispecific antibody having Her2-binding activity or an antigen-binding fragment thereof.

[0142] In some embodiments, Tb is a multispecific antibody having Her2-binding activity or an antigen-binding fragment thereof.

[0143] In some preferred embodiments, Tb is Trastuzumab or an antigen-binding fragment thereof.

[0144] In some preferred embodiments, Tb is Pertuzumab or an antigen-binding fragment thereof.

[0145] In some embodiments, Tb is an anti-Her3 antibody or an antigen-binding fragment thereof.

[0146] In some embodiments, Tb is an anti-EGFR antibody or an antigen-binding fragment thereof, such as, demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutumumab, Cetuximab, or an antigen-binding fragment thereof.

[0147] In some embodiments, the antibody has an antibody that binds to B7H3 antigen activity without having endocytosis activity. For example, INV721 and I7-01 of WO2021168379A1. More specifically, the antibody against Anti-B7H3 has VH of SEQ ID NO: 2 and VL of SEQ ID NO: 1 described in WO2021168379A1.

[0148] In some embodiments, the antibody has an antibody that binds to CD20 antigen activity without having endocytosis activity. So-called "type II" CD20-specific antibodies have been shown to be less likely to be internalized by CD20-positive target cells, while other so-called "type I" CD20-specific antibodies have been found to be internalized and degraded to some extent depending on the expression levels of activation and inhibition of FcγR on the target cells with which they interact. In some embodiments, the antibody that binds to CD20 antigen activity without having endocytosis activity is a "type II" CD20-specific antibody, such as obinutuzumab.

[0149] In some embodiments, the antibody is an antibody having an activity of binding to a non-endocytosis (e.g., ALCAM / CD166) antigen. In some embodiments, the antibody is an antibody comprising VH of SEQ ID NO: 73 and VL of SEQ ID NO: 74, VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76, VH of SEQ ID NO: 77 and VL of SEQ ID NO: 78 in EP3911682A1, or an antibody shown by VH of SEQ ID NO: 79 and VL of SEQ ID NO: 88.

[0150] In some embodiments, the antibody-drug conjugate is as follows:

Chemical formula

[0151] In some preferred embodiments, Tb 1 is the 2E3-02 antibody.

[0152] In some embodiments, the antibody-drug conjugate is as follows:

Chemical formula

[0153] In some embodiments, the antibody-drug conjugate is as follows:

Chemical formula

[0154] In some preferred embodiments, Tb 2 is Trastuzumab.

[0155] In some embodiments, the antibody-drug conjugate is as follows:

Chemical Structure

[0156] In some embodiments, the antibody-drug conjugate is as follows:

Chemical Structure

[0157] In some embodiments, the antibody-drug conjugate is as follows:

Chemical Structure

[0158] In some embodiments, the antibody-drug conjugate is as follows:

Chemical Structure

[0159] In some embodiments, the antibody-drug conjugate is as follows: [Chemical formula] The DAR value is 8.0.

[0160] In a second aspect of the present disclosure, the present disclosure provides a drug linker conjugate represented by Formula IV, [Chemical formula] or a stereoisomer, a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate of the above drug linker conjugate, wherein, L 1 is [Chemical formula] when, the 1-position is linked to Lg, and the 2-position is linked to L 2 and Lg is a leaving group when reacting with the antibody, and the definitions of L 2 , L 3 , L 4 and D are as described in any one form of the present disclosure.

[0161] L 1 is [Chemical formula] when, Lg-L 1 is [Chemical formula] and the definitions of L 2 , L 3 , L 4 and D are as described in any one form of the present disclosure.

[0162] In some embodiments, Lg is a halogen, a sulfone, a tertiary amine base (Me 3 N + , Et3 N + ) diazonium base, -OMs, MeSO 2 -, or CF 3 SO 3 - is selected from.

[0163] In some embodiments, Lg is F, Cl or MeSO 2 - is selected from, more preferably, Lg is F or MeSO 2 - is selected from.

[0164] In some embodiments, the above drug linker complex has the structure shown in formula V,

Chemical formula

[0165] In some embodiments, the above drug linker complex has the structure shown in formula V-1:

Chemical formula

[0166] In some embodiments, the above drug linker complex is selected from the following group:

Table 10

[0167] In a third aspect of the present disclosure, the present disclosure provides a biologically active compound of the protease class represented by Formula IIA,

Chemical formula

[0168] In some embodiments, the biologically active compound of the protease class is of the structure represented by Formula IIA-1:

Chemical formula

[0169] In some embodiments, the biologically active compound is selected from the group consisting of:

Chemical formula

Chemical formula

[0170] Preferably, the definitions of the above antibody and the fragment of the biologically active compound of the proteolytic agent class are as described for Tb and D in any one form of the present disclosure, respectively.

[0171] In a fifth aspect of the present disclosure, the present disclosure provides a method for preparing an antibody-drug conjugate represented by formula I, which includes the following: Coupling Tb with a drug linker conjugate represented by formula IV

Chemical formula

[0172] Specifically, the above method includes coupling Tb with a drug linker conjugate represented by formula IV

Chemical formula

[0173] In some embodiments, the ratio of the amount of substance of the above Tb to the drug linker conjugate is 1:(1-20) such as 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20) or 1:(18-20).

[0174] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0175] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol), or any combination thereof.

[0176] In some embodiments, the method further includes a step of purifying the composite product.

[0177] In some embodiments, the composite product is purified by a chromatography method.

[0178] In some embodiments, the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography.

[0179] In a sixth aspect of the present disclosure, the present disclosure provides a method for preparing a drug linker complex represented by Formula IV, reacting the compounds represented by General Formula IV-E-1 and General Formula IV-E-2 as shown in the following reaction,

Chemical formula

Chemical formula

Chem.

[0180]

Chem.

[0181] In some embodiments, the present disclosure provides a method for preparing a drug linker complex represented by Formula V-1, which includes reacting the compounds represented by General Formula V-A-1 and General Formula VI as follows:

Chem.

[0182] In some embodiments, the present disclosure provides a method for preparing a drug linker complex represented by Formula DL-1.

[0183] Specifically, a target compound (DL-1) is obtained by subjecting a compound INT1 represented by a general formula to an amide condensation reaction with a compound DL-1-1. By coupling an analog of INT1 with an analog of DL-1-1, drug linker complexes of various structures can be obtained.

[0184]

Chemical formula

Chemical formula

Chemical formula

[0185] In some embodiments, the above linker is selected from the group consisting of:

Table 11

[0186] The present disclosure provides the use of a biologically active compound of the above protease class, or a stereoisomer, prodrug, pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of the above compound, in the preparation of an antibody-drug conjugate (ADC).

[0187] The present disclosure provides a pharmaceutical composition comprising the above antibody-drug conjugate, or a stereoisomer of the above antibody-drug conjugate, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or a biologically active compound of the above protease class, or a stereoisomer of the above biologically active compound, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the above drug linker complex, or a stereoisomer of the above drug linker complex, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, and optionally one or more pharmaceutical adjuvants.

[0188] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value between 1.0 and 12.0.

[0189] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value between 1.0 and 10.0.

[0190] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value between 1.0 and 9.0.

[0191] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value between 1.0 and 8.5.

[0192] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value between 1.5 and 8.5.

[0193] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value between 2.0 and 8.0.

[0194] In some embodiments, among them, the drug-to-antibody ratio (DAR) is any value within 2±0.4, 4±0.4, 6±0.4, or 8±0.4.

[0195] In some embodiments, among which, the drug-to-antibody ratio (DAR) is about 2.0, 4.0, 6.0, or 8.0.

[0196] The term "drug-to-antibody ratio" or "DAR" refers to the quantity of drug, e.g., the small molecule toxin conjugated to the antibody of the ADC. The DAR of an ADC may be in the range of 1 to 16, but higher loadings (e.g., 20) are also possible depending on the number of binding sites on the antibody. The term DAR can be used when referring to the quantity of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a set of ADCs. In some embodiments, the ADCs include ADCs having a DAR distribution of 1 to 8, e.g., ADCs with drug loading species of 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8). It should be noted that degradation products can be generated such that DARs of 1, 3, 5, and 7 can also be included in the ligand-drug conjugate. Otherwise, the ADC can also have a DAR greater than 8. The ADCs are generated by interchain disulfide reduction followed by coupling. In some embodiments, the ADCs include both ADCs with a DAR of 4 or less (i.e., drug loading species of 4 or less) and ADCs with a DAR of 6 or more (i.e., drug loading species of 6 or more).

[0197] The present disclosure provides the use of the antibody-drug conjugate, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or a biologically active compound of the protease class, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the drug-linker conjugate, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the pharmaceutical composition, in the preparation of a medicament for the treatment and / or prevention of a disease related to abnormal cell activity (e.g., a cancer disease).

[0198] The present disclosure provides the above antibody-drug conjugate, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or a biologically active compound of the above protease class, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the above drug linker complex, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the above pharmaceutical composition, for the treatment and / or prevention of diseases related to abnormal cell activity (e.g., cancer diseases).

[0199] The present disclosure provides a method for preventing and / or treating a disease associated with abnormal cell activity (e.g., cancer disease), comprising administering to a subject in need thereof a prophylactically and / or therapeutically effective amount of the above antibody-drug conjugate, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or a biologically active compound of the above protease class, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the above drug linker complex, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the above pharmaceutical composition.

[0200] In some embodiments, the cancer diseases of the present disclosure are selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial cancer, solid tumor, non-Hodgkin lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer.

[0201] In some embodiments, the cancer disease is a cancer disease related to HER2, TROP2, B7H3, HER3, or EGFR.

[0202] In some embodiments, the cancer is a solid tumor or a hematological tumor.

[0203] In the present disclosure, unless otherwise specified, the scientific and technical terms used in the present disclosure have the meanings generally understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratory procedures used in the present disclosure are the commonly used procedures widely used in the art. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0204] As used in the present disclosure, the term "proteolytic agent" refers to a class of compounds that can induce ubiquitination of a protein of interest by an E3 ligase and then be degraded by a proteasome.

[0205] As used in the present disclosure, the term "proteolysis-inducing chimeric molecule (PROTAC)" generally includes three parts, namely, one E3 ubiquitin ligase ligand and one target protein ligand. The two active ligands are linked by a specially designed linker structure. The target protein recruits the E3 ubiquitin ligase onto the target protein, whereby the target protein is ubiquitinated and further degraded.

[0206] As used in the present disclosure, the term "molecular glue-like degrader (MGD)" refers to a class of small molecules that can induce a new interaction between an E3 ubiquitin ligase and a target protein, thereby resulting in the degradation of the target protein.

[0207] As used herein, examples of the term "pharmaceutically acceptable salt" are organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including, but not limited to, formates, acetates, propionates, benzoates, maleates, fumarates, succinates, tartrates, citrates, ascorbates, α-ketoglutarates, α-glycerophosphates, alkylsulfonates, or arylsulfonates. Preferably, the alkylsulfonates are methanesulfonates or ethanesulfonates, and the arylsulfonates are benzenesulfonates or p-toluenesulfonates. Suitable inorganic salts can also be formed, including, but not limited to, hydrochlorides, hydrobromides, hydroiodides, nitrates, bicarbonates and carbonates, sulfates, or phosphates.

[0208] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0209] As used herein, the term "stereoisomer" refers to isomers formed by at least one chiral center. Compounds having one or more (e.g., 1, 2, 3, or 4) chiral centers can form racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Certain individual molecules may also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention may exist as mixtures of two or more structurally different forms that are in rapid equilibrium (generally referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. It should be understood that the scope of the present disclosure covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0210] In the present disclosure, the carbon-carbon bonds of the compounds of the present invention can be depicted using a solid line (--), a solid-wedge, or a dashed-wedge. The use of a solid line to depict a bond to an asymmetric carbon atom indicates that all possible stereoisomers (e.g., a particular enantiomer, a racemic mixture, etc.) at that carbon atom are included. The use of a solid or dashed-wedge to depict a bond to an asymmetric carbon atom indicates the presence of the indicated stereoisomer. When present in a racemic mixture, solid and dashed-wedges are used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention can exhibit one or more isomerization phenomena and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0211] The compounds of the present disclosure may exist in the form of solvates (e.g., hydrates), among which the compounds of the present disclosure include polar solvents, particularly, for example, water, methanol, or ethanol, which are components of the crystal lattice of the above compounds. The amount of the polar solvent, particularly water, may exist in a stoichiometric or non-stoichiometric ratio.

[0212] The present disclosure further includes within its scope prodrugs of the compounds of the present invention. Generally, such prodrugs are functional group derivatives of the above compounds that are readily convertible in vivo to the desired therapeutically active compound. Accordingly, in these situations, the term "administering" as used in the therapeutic methods of the present disclosure should include using prodrug forms of one or more of the claimed compounds to treat various diseases or medical conditions, provided that the prodrug forms are converted in vivo to the above compounds after administration to a subject. For example, in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985, conventional methods for selecting and preparing suitable prodrug derivatives are described.

[0213] In the present disclosure, the above-mentioned pharmaceutical adjuvants refer to excipients and additives used in the preparation and formulation of pharmaceuticals, and refer to substances that are reasonably evaluated in terms of safety and are included in drug formulations in addition to the active ingredients. Pharmaceutical adjuvants act as excipients and carriers, and in addition to improving stability, they further have important functions such as solubilization, solubilization assistance, and sustained release, and are important components that can affect the quality, safety, and efficacy of pharmaceuticals. According to their origin, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their functions and uses, they can be divided into solvents, propellants, solubilizing agents, solubilization aids, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, moisturizing agents, absorbents, diluents, aggregating agents and anti-aggregating agents, filter aids, release retardants, etc. According to their administration routes, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ophthalmic administration, etc. The same pharmaceutical adjuvant can be used in pharmaceutical formulations with different administration routes and has different functions and uses.

[0214] The above-mentioned pharmaceutical composition can be prepared into various appropriate dosage forms according to the administration route. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder aerosols, sprays, etc. Among them, the above-mentioned pharmaceutical composition or appropriate dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or complex, appropriately 0.1 mg to 800 mg, preferably 0.5 mg to 500 mg, preferably 0.5 mg to 350 mg, and particularly preferably 1 mg to 250 mg.

[0215] The above pharmaceutical composition can be administered in the form of an injection, including an injection, a sterile powder for injection, and a concentrated solution for injection. Among them, carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized non-volatile oils, such as glycerides or diglycerides, can also be used as solvents or suspension media. The above pharmaceutical composition can be administered in an infusion form.

[0216] As used herein, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such effects may be prophylactic, based on the complete or partial prevention of a disease or its symptoms, and / or may be therapeutic, based on the partial or complete stabilization or cure of a disease and / or side effects caused by the disease. "Treatment" as used herein includes (a) preventing a disease or symptom from developing in a patient who is susceptible to, but not yet diagnosed with, the disease or symptom, (b) inhibiting the symptoms of a disease, i.e., preventing its progression, or (c) alleviating the symptoms of a disease, i.e., causing a deterioration of the disease or symptom, and encompasses any treatment of a patient's disease.

[0217] In the present disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) suffering from a disease (e.g., a disease described in the present disclosure) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and / or livestock animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0218] In the present disclosure, the term "effective amount" refers to the amount of an antibody-drug conjugate, drug-linker conjugate, compound, or composition that, upon administration, reduces to some extent one or more symptoms of a therapeutic condition.

[0219] In the present disclosure, the terms "antibody-drug conjugate" and "ADC" refer to a substance in which a fragment of a biologically active compound (drug molecule) is linked to an antibody or an antigen-binding fragment portion thereof. In some embodiments of the present disclosure, the fragment of the biologically active compound is linked to the target moiety via a linker. The linker can be cleaved under a specific environment (e.g., intracellular low pH environment) or a specific action (e.g., the action of lysosomal protease), thereby separating the fragment of the biologically active compound from the target moiety or the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker includes a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the fragment of the biologically active compound is directly linked to the target moiety or the antibody or its antigen-binding fragment via a covalent bond, and the covalent bond can be cleaved under a specific environment or action, thereby separating the fragment of the biologically active compound from the antibody or its antigen-binding fragment portion.

[0220] In the present disclosure, the term "drug" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0221] In the present disclosure, the statement that "the 1-position of L1 is linked to Tb via an S atom" means that those skilled in the art can understand that the 1-position of L1 is linked to the thiol group contained in Tb (e.g., an antibody) itself after the disulfide bond is opened (for example, the reduction of the disulfide bond by a reducing agent TCEP can open the disulfide bond to generate a thiol -SH), that is, the -S- between L1 and Tb is not an externally connected sulfur atom separately. For example, among them, -S- is not an externally connected sulfur atom separately, but the thiol group contained in Tb itself after the disulfide bond is opened is linked to the 1-position of L1, for example,

Chemical formula

[0222] In the present disclosure, the term "linker" refers to a fragment that links a fragment of a biologically active compound (drug molecule) to an antibody moiety.

[0223] In the present disclosure, the above "fragment of a biologically active compound" refers to a part (fragment or group) that can form a drug having biological activity (e.g., a small molecule cytotoxic drug containing a group after losing one atom or atomic group) or a derivative thereof (e.g., its precursor) after cleavage / degradation / enzyme cleavage of the linker between tumor tissues or within tumor cells in an antibody-drug conjugate (also called an antibody-drug conjugate, ADC) known in the art. To avoid ambiguity, "drug" does not only refer to "pharmaceutical products" approved by pharmaceutical regulatory agencies, but further includes any compound having potential therapeutic biological activity in clinical practice, or in development and academic research.

[0224] In the present disclosure, the term "antibody" is taken in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, as long as they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" can be used interchangeably.

[0225] In the present disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is identical except for minor natural variations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas the corresponding polyclonal antibodies contain different antibodies against different determinants (epitopes). In addition to specificity, monoclonal antibodies also have the advantage of not being contaminated by other antibodies during synthesis. Here, the modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies as a characteristic, but it should not be understood that it is necessary to be prepared by a specific method.

[0226] In some embodiments of the present disclosure, the monoclonal antibody further includes, in particular, a chimeric antibody, that is, a part of the heavy chain and / or light chain is the same as or homologous to an antibody of a certain type, class, or subclass as long as they have the desired biological activity (for example, see US 4,816,567 and Morrison et al., 1984, PNAS, 81: 6851-6855), and the remaining part is the same as or homologous to an antibody of another type, class, or subclass. The chimeric antibodies used in the present disclosure include primatized antibodies containing variable region antigen-binding sequences derived from non-human primates (such as old world monkeys, orangutans, etc.) and human constant region sequences.

[0227] The term "antibody fragment" refers to a part of an antibody, preferably the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’) 2 , Fd, Fv, dAb and complementarity-determining region fragments, diabodies, linear antibodies, and single-chain antibody molecules.

[0228] The term "bispecific antibody" is also referred to as a "bifunctional antibody complex", and refers to a complex formed by a first antibody (fragment) and a second antibody (fragment) via a complex arm. The complex has bifunctionality and bispecificity in order to retain the activities of the respective antibodies.

[0229] The term "multispecific antibody" includes, for example, a trispecific antibody having three different antigen-binding specificities, a quadraspecific antibody having four different antigen-binding specificities, and the like.

[0230] The term "complete antibody" refers to an antibody that includes an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region may be a natural sequence (for example, a human natural constant region sequence) or an amino acid sequence variant thereof. The complete antibody is preferably a complete antibody having one or more effector functions.

[0231] The term "Probody" refers to a modified antibody that can specifically bind to its target and can bind to a masking group, where the masking group is such that the dissociation constant of the antibody or antibody fragment for its target is at least 100-fold, 1000-fold, or 10,000-fold greater than the dissociation constant of the antibody or antibody fragment not bound to the masking group for its target.

[0232] In the present disclosure, the "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of the human recipient immunoglobulin have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues that have the desired specificity, affinity, and functionality (donor antibody). In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. And the humanized antibody may further contain residues that are not present in the recipient antibody or donor antibody. These modifications are for further optimizing the performance of the antibody. Humanized antibodies generally contain at least one, usually two variable regions, of which all or substantially all of the hypervariable loops correspond to non-human immunoglobulins and the FRs are completely or substantially completely of human immunoglobulin sequence. The humanized antibody can further contain at least a portion of the immunoglobulin constant region (Fc, usually human immunoglobulin Fc). See, for example, Jones et al., 1986, Nature, 321:522-525, Riechmann et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr Op Struct Bwl 2:593-596 for related details.

[0233] Complete antibodies can be divided into different "classes" according to the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, and some of these classes can also be divided into different "subclasses" (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different classes of antibodies are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.

[0234] In the present disclosure, amino acid substitutions in antibodies are, in most cases, substituted with L-amino acids, but are not limited thereto. In some embodiments, the antibody peptide chain may contain one or more D-amino acids. Peptides containing D-amino acids are more stable and less likely to degrade in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0235] The monoclonal antibodies used in the present disclosure can be produced by many methods. For example, the monoclonal antibodies used in the present disclosure can be obtained by the hybridoma method using many species (including mouse, hamster, rat, and human cells) (see, for example, Kohler et al., 1975, Nature, 256:495), or can be produced by recombinant DNA technology (see, for example, US 4,816,567), or can be isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352:624-628, and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0236] In the present disclosure, unless otherwise specified, the descriptions "each... is independently selected" and "... are each independently selected" used throughout the present disclosure are interchangeable with each other and should both be understood in a broad sense, which may refer to the fact that among specific options represented between the same or different symbols on different bases, they do not affect each other, or may represent that among specific options represented between the same or different symbols on the same base, they do not affect each other.

[0237] AA 1 The structure of the amino acid residue shown in

Chemical formula

Chemical formula

[0238] AA 1 The structure of the amino acid residue shown in

Chemical formula

[0239] In each part of this specification, the substituents of the compounds of the present disclosure are disclosed by the type or range of the groups. In particular, the present disclosure includes each independent sub-combination of these types and ranges of each member of the groups. For example, the term "C1-6 alkyl group" particularly refers to a methyl group, an ethyl group, a C3 alkyl group, a C4 alkyl group, a C5 alkyl group, and a C6 alkyl group independently disclosed.

[0240] In the present disclosure, the term "direct bond" means that the indicated substituent is absent and the two end portions of the substituent are directly connected to form a bond.

[0241] In the present disclosure, the terms "comprising", "including", "having", "containing", or "relating to", and other variations thereof in this specification are inclusive or open-ended and do not exclude other elements or method steps not mentioned.

[0242] In the present disclosure, the term "C1-6 alkyl group" refers to an alkyl group containing 1 to 6 carbon atoms in a straight chain or branched chain, and includes, for example, "C1-3 alkyl group" or "C1-4 alkyl group", a methyl group, an ethyl group, etc. Specific examples include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, and a hexyl group.

[0243] In the present disclosure, the term "C2-6 alkenyl group" refers to a linear, branched or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, and includes, for example, "C2-4 alkenyl group". Examples thereof include, but are not limited to, vinyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 1,3-butadienyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 1,3-pentadienyl group, 1,4-pentadienyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 1,4-hexadienyl group, cyclopentenyl group, 1,3-cyclopentadienyl group, cyclohexenyl group, 1,4-cyclohexadienyl group, etc.

[0244] In the present disclosure, the term "C2-6 alkynyl group" refers to a linear or branched alkynyl group containing at least one triple bond and having 2 to 6 carbon atoms, and includes, for example, "C2-4 alkynyl group". Examples thereof include, but are not limited to, ethynyl group, propynyl group, 2-butynyl group, 2-pentynyl group, 3-pentynyl group, 4-methyl-2-pentynyl group, 2-hexynyl group, 3-hexynyl group, 5-methyl-2-hexynyl group, etc.

[0245] In the present disclosure, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0246] In the present disclosure, the term "C3-6 cycloalkyl group" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms. Optionally, the carbon atoms in the cyclic structure may be substituted with an oxo group. Specific examples include, but are not limited to, cyclopropane group (i.e., cyclopropyl group), cyclobutane group (i.e., cyclobutyl group), cyclopentane group (i.e., cyclopentyl group), cyclohexyl group.

[0247] In the present disclosure, the term "C1-6 alkoxy group" refers to the alkyl group defined above that is linked to a part of the parent molecule via an oxygen atom, and includes, for example, "C1-3 alkoxy group" or "C1-4 alkoxy group". Specific examples include, but are not limited to, methoxy group, ethoxy group, propoxy group, i-propoxy group, n-propoxy group, i-propoxy group, n-butoxy group, i-butoxy group, t-butoxy group, pentyloxy group, hexyloxy group, etc.

[0248] In the present disclosure, the term "C1-6 haloalkyl group" refers to the alkyl group defined above that is substituted with one or more halogens defined above, and includes, for example, "C1-3 haloalkyl group" or "C1-4 haloalkyl group". Specific examples include, but are not limited to, chloromethyl group, fluoroethyl group, bromopropyl group, etc.

[0249] In the present disclosure, the term "4- to 10-membered heterocyclic ring" refers to a ring containing 4 to 10 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "3- to 6-membered heterocyclic ring" refers to a ring containing 3 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "3- to 7-membered heterocyclic ring" refers to a ring containing 3 to 7 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "5- to 6-membered heterocyclic ring" refers to a ring containing 5 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may be substituted with an oxo group. Specific examples include, but are not limited to, rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyranyl, pyrrolidone, etc.

[0250] In the present disclosure, the term "3- to 10-membered carbocyclic ring" refers to a ring having 3 to 10 ring atoms that are carbon atoms. Optionally, the carbon atoms in the cyclic structure may be substituted with oxo groups. For example, it may be a 3- to 6-membered carbocyclic ring, a 3- to 7-membered carbocyclic ring, a 5- to 8-membered carbocyclic ring, etc. Specific examples include, but are not limited to, cyclopentane, cyclohexane, etc.

[0251] In the present disclosure, the term "4- to 10-membered heterocyclic group" refers to a cyclic group containing 4 to 10 ring atoms, at least one of which is a heteroatom such as an oxygen atom, a nitrogen atom, or a sulfur atom. The term "4- to 6-membered heterocyclic group" refers to a cyclic group containing 4 to 6 ring atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. The term "3- to 6-membered heterocyclic group" refers to a cyclic group containing 3 to 6 ring atoms, at least one of which is a heteroatom such as an oxygen atom, a nitrogen atom, or a sulfur atom. Optionally, the ring atoms in the cyclic structure (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) may be substituted with oxo groups. The "4- to 8-membered heterocyclic group" includes, for example, "4- to 8-membered nitrogen-containing heterocyclic group", "4- to 8-membered oxygen-containing heterocyclic group", "4- to 7-membered heterocyclic group", "4- to 7-membered oxygen-containing heterocyclic group", "4- to 7-membered heterocyclic group", "4- to 6-membered heterocyclic group", "5- to 7-membered heterocyclic group", "5- to 6-membered heterocyclic group", "5- to 6-membered nitrogen-containing heterocyclic group". Specific examples include, but are not limited to, oxocyclobutane group, pyrrolidinyl group, tetrahydrofuranyl group, piperidinyl group, piperazinyl group, tetrahydropyranyl group, homopiperazinyl group, etc.

[0252] In the present specification, the term "aryl group" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity, for example, a 6- to 10-membered aryl group, a 5- to 8-membered aryl group, etc. Specific examples include, but are not limited to, phenyl group, naphthyl group, anthracenyl group, phenanthryl group, etc. The above "6- to 10-membered aryl group" refers to an aryl group containing 6 to 10 ring atoms. The above "C6-10 aryl group" refers to an aryl group containing 6 to 10 carbon atoms.

[0253] In the present disclosure, the term "heteroaryl group" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may be substituted with oxo groups. Specific examples include 5- to 10-membered heteroaryl groups such as furanyl group, thienyl group, pyrrolyl group, thiazolyl group, isothiazolyl group, thiadiazolyl group, oxazolyl group, isoxazolyl group, oxadiazolyl group, imidazolyl group, pyrazolyl group, 1,2,3-triazole group, 1,2,4-triazole group, 1,2,3-oxadiazolyl group, 1,2,4-oxadiazolyl group, 1,2,5-oxadiazolyl group, 1,3,4-oxadiazolyl group, pyridyl group, 2-pyridonyl group, 4-pyridonyl group, pyrimidinyl group, 1,4-dioxyheterocyclohexenyl group, 2H-1,2-oxazinyl group, 4H-1,2-oxazinyl group, 6H-1,2-oxazinyl group, 4H-1,3-oxazinyl group, 6H-1,3-oxazinyl group, 4H-1,4-oxazinyl group, pyridazinyl group, pyrazinyl group, 1,2,3-triazinyl group, 1,3,5-triazinyl group, 1,2,4,5-tetrazinyl group, azacycloheptatrienyl group, 1,3-diazacycloheptatrienyl group, azacyclooctatetraenyl group, etc., 5- to 6-membered heteroaryl groups, 5- to 10-membered nitrogen-containing heteroaryl groups, 6- to 10-membered oxygen-containing heteroaryl groups, 6- to 8-membered nitrogen-containing heteroaryl groups, 5- to 8-membered oxygen-containing heteroaryl groups, etc., but are not limited thereto.

[0254] In the structural formula indicated by the wavy line "~~" of the present disclosure, the bond means that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any ratio.

[0255] In the present disclosure, the term "linking unit" is a composition of an antibody-drug conjugate or a drug linker conjugate or a linker that functions to link an antibody or an antigen-binding fragment thereof bound to a target to the remaining part of the antibody-drug conjugate. The linking unit can link the Tb unit to L 2 and specific examples are [Chemical formula] (wherein the 1-position is linked to an antibody or an antigen-binding fragment thereof, and the 2-position is linked to L 2 or L 3 linked thereto), but is not limited thereto.

[0256] In the present disclosure, the term "linking unit" is a composition of an antibody-drug conjugate or a drug-linker conjugate or a linker that acts to bind the linking unit to an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues. When a linking unit is present, L 1 can be linked to L 3 . Specific examples include [Chemical formula] (wherein the 1-position is linked to the linking unit and the 2-position is linked to L 3 linked thereto), but is not limited thereto.

[0257] Without departing from the common general knowledge in the art, each of the above preferred conditions can be arbitrarily combined to obtain each preferred example of the present invention.

[0258] The reagents and raw materials used in the present disclosure are all commercially available. [Advantages of the Invention]

[0259] Through many studies, the present disclosure has obtained a GSPT1 protease (biologically active compound or payload) with a novel structure and its antibody-drug conjugate (ADC), both of which have extremely strong biological activities and can achieve the following various technical effects: (1) The GSPT1 protease inhibitor shows extremely strong growth inhibitory activity against different cells, is significantly superior to the control compounds (3 - 40 times), and has the potential for a strong bystander effect after forming an ADC. The molecule has no basic group. On the one hand, it can reduce or eliminate the lysosome capture phenomenon (that is, molecules containing basic groups are bound in lysosomes with relatively strong acidity and thus cannot function). On the other hand, it is rapidly distributed to other non-tumor tissues and does not cause "off-target" toxicity.

[0260] (2) To overcome the stability problems of conventional maleimide conjugate linkers, the ADC has high stability in the circulatory system and can reduce the shedding of biologically active compounds in non-target tissues.

[0261] (3) The ADC can release the payload simultaneously inside and outside tumor cells, increase the efficient release of biologically active compounds at the tumor site. The biologically active compounds have a higher tumor-to-blood concentration ratio and thus have a higher therapeutic index.

[0262] (4) The ADC shows extremely strong growth inhibitory activity on cells, shows a significantly superior tumor suppression effect in in vivo efficacy compared to the control compounds. During the administration period, the animals in each group have no obvious weight loss and obvious drug toxicity and have good safety.

[0263] Therefore, the GSPT1 protease inhibitor and ADC of the present invention have very high clinical application value.

Brief Description of the Drawings

[0264]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Modes for Carrying Out the Invention

[0265] The present disclosure will be further described below by way of specific embodiments, which do not limit the present disclosure. Those skilled in the art can make various modifications and improvements based on the suggestions of the present disclosure without departing from the basic idea and scope of the present disclosure. When the reagents or equipment used are not specified by the manufacturer, they may all be ordinary commercially available products.

[0266] The abbreviations in the present disclosure have the following meanings:

Table 12

[0267] Array and its specific information

Table 13

[0268] Preparation method The structures of the compounds described in the following examples were identified by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0269] For nuclear magnetic resonance ( 1 H NMR), a Bruker 400 MHz nuclear magnetic resonance apparatus was used, and the measurement solvents were deuterated methanol (CD 3 OD), deuterated chloroform (CDCl 3 ), or hexadeuterodimethyl sulfoxide (DMSO-d 6 ), and the internal standard substance was tetramethylsilane (TMS).

[0270] The abbreviations of the nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

[0271] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d 6 : Deuterated dimethyl sulfoxide. The δ value is expressed in ppm value.

[0272] For mass spectrometry (MS), the measuring instrument used was an Agilent (ESI) mass spectrometer with model number Agilent 6120B.

[0273] For ultra-high performance liquid chromatography (UPLC), the measuring instrument used was AB SCIEX with model number ExionLC.

[0274] For high-resolution mass analysis, AB SCIEX with model number X500B was used.

[0275] Example 1. Synthesis of Intermediate Example 1.1: Synthesis of (10S,13S)-10-(4-(dipropylamino)butyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosan-19-oic acid (INT1)

Chemical formula

[0276] Step 1: Compound INT1-1 (117 g, 206.2 mmol) was added to dioxane (1.5 L) and stirred for 10 minutes. Then, HCl (0.75 L, 4 N in dioxane) was further added and the reaction was allowed to proceed overnight at room temperature. Methyl tert-butyl ether (2 L) was added to the reaction mixture and stirred for 30 minutes. The mixture was then filtered, and the solid was transferred to a blower dryer and dried to obtain the hydrochloride salt of the target compound INT1-2 (94 g, yield: 91%, purity 94%). LCMS (ESI) [M+H] + = 468.3. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.24 (d, J = 7.3 Hz, 1H), 8.07 (brs, 3H), 7.90 (d, J = 7.5 Hz, 2H), 7.77 (dd, J = 7.1, 3.4 Hz, 2H), 7.47 - 7.37 (m, 3H), 7.34 (td, J = 7.4, 1.7 Hz, 2H), 4.34 - 4.17 (m, 4H), 3.96 (dd, J = 8.6, 7.4 Hz, 1H), 2.83 - 2.65 (m, 2H), 2.04 (dd, J = 13.5, 6.7 Hz, 1H), 1.82 - 1.52 (m, 4H), 1.51 - 1.30 (m, 2H), 0.96 - 0.84 (m, 6H).

[0277] Step 2: The hydrochloride salt of compound INT1-2 (55.0 g, 109.1 mmol), acetic acid (10 mL) were dissolved in methanol (500 mL). Under an ice-water bath, n-propionaldehyde (25.35 g, 436.4 mmol) was added to the reaction solution, and the reaction was carried out with stirring at room temperature for 30 minutes. Under an ice-water bath, sodium cyanoborohydride (27.43 g, 436.4 mmol) was further added to the reaction solution, and the reaction was carried out with stirring at room temperature for 1 h. Next, n-propionaldehyde (12.67 g, 218.2 mmol) and sodium cyanoborohydride (13.72 g, 218.2 mmol) were further added to the reaction solution, and the reaction continued for 0.5 h. The reaction solution was filtered, the filtrate was concentrated, purified water (300 mL) was added to the concentrated solution, and it was purified with DCM (300 mL × 2). The organic phase was washed with saturated brine and dried over anhydrous Na 2 SO 4 . After concentration, a crude product was obtained. The crude product was purified by column chromatography (the eluent was MeOH and DCM, among which MeOH:DCM (V / V) = 1:4 - 2:3), and compound INT1-3 (39 g, yield 65%, purity 98%) was obtained. LCMS (ESI) [M+H] + = 552.3. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.95 - 7.85 (m, 3H), 7.76 (t, J = 7.3 Hz, 2H), 7.51 - 7.40 (m, 3H), 7.35 (t, J = 7.4 Hz, 2H), 4.34 - 4.22 (m, 3H), 4.13 (dd, J = 12.6, 7.2 Hz, 1H), 3.91 (dd, J = 8.8, 7.1 Hz, 1H), 2.52 - 2.41 (m, 6H), 2.03 (dt, J = 13.5, 6.7 Hz, 1H), 1.79 - 1.67 (m, 1H), 1.66 - 1.56 (m, 1H), 1.42 - 1.36 (m, 6H), 1.36 - 1.21 (m, 2H), 0.90 (t, J = 6.9 Hz, 6H), 0.82 (t, J = 6.9 Hz, 6H).

[0278] Step 3: Dissolve compound INT1-3 (6.6 g, 12 mmol) in DMF (50 mL), sequentially add compound INT1-4 (3 g, 12 mmol), HATU (4 g, 11 mmol), and N,N-diisopropylethylamine (3.5 g, 30 mmol), stir at room temperature for 1 h, then add water (100 mL) to the reaction solution, purify with ethyl acetate (200 mL × 3), combine the organic phases, wash with saturated brine (200 mL × 2), dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain a crude product, and purify the crude product by column chromatography (methanol:dichloromethane = 5% - 10%) to obtain the target compound INT1-5 (9 g). LCMS (ESI) [M+H] + = 787.0, 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.87 (s, 1H), 8.70 (t, J = 6.5 Hz, 1H), 8.27 - 8.18 (m, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 7.2 Hz, 2H), 7.47 - 7.40 (m, 3H), 7.38 - 7.30 (m, 6H), 5.15 (s, 2H), 4.71 - 4.55 (m, 2H), 4.37 - 4.27 (m, 2H), 4.26 - 4.19 (m, 2H), 4.15 (s, 2H), 3.92 - 3.83 (m, 1H), 3.73 (d, J = 5.5 Hz, 2H), 3.04 - 2.89 (m, 6H), 2.01 - 1.94 (m, 1H), 1.81 - 1.63 (m, 2H), 1.61 - 1.52 (m, 6H), 1.36 - 1.27 (m, 2H), 0.91 - 0.82 (m, 12H).

[0279] Step 4: Compound INT1-5 (3 g, 3.8 mmol) was dissolved in a mixed solvent of ethanol (20 mL) and ethyl acetate (10 mL). Under the protection of nitrogen gas, Pd / C (1.5 g) was added, and the reaction mixture was purged with hydrogen gas three times. The reaction solution was stirred at room temperature overnight under a hydrogen atmosphere, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT1-6 (2.8 g), which was used directly in the next reaction without further purification. LCMS (ESI) [M+H] + = 696.4. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.67 (t, J = 6.5 Hz, 1H), 8.25 (t, J = 5.7 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 7.3 Hz, 2H), 7.49 - 7.39 (m, 3H), 7.33 (t, J = 7.4 Hz, 2H), 4.65 - 4.55 (m, 2H), 4.35 - 4.26 (m, 2H), 4.24 - 4.14 (m, 2H), 3.97 (s, 2H), 3.92 - 3.83 (m, 1H), 3.73 (d, J = 5.6 Hz, 2H), 2.94 - 2.84 (m, 6H), 1.73 - 1.64 (m, 1H), 1.63 - 1.47 (m, 8H), 1.34 - 1.26 (m, 3H), 0.89 - 0.82 (m, 12H).

[0280] Step 5: Compound INT1-6 (1 g, 1.43 mmol) was dissolved in THF (30 mL), diethylamine (1.0 g, 14.3 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the target compound INT1-7 (680 mg), which was used directly in the next reaction without further purification. LCMS (ESI) [M+H] + = 474.1.

[0281] Step 6: Compound INT1-8 (340 mg, 1.27 mmol) was dissolved in DMF (50 mL), and HATU (4 g, 11 mmol) and N,N-diisopropylethylamine (3.5 g, 30 mmol) were sequentially added. After stirring at room temperature for 30 min, compound INT1-7 (0.6 g, 1.27 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h, and the reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile:water = 5% - 50%) to obtain the target compound INT1 (200 mg). LCMS (ESI) [M+H] + = 724.8, 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.13 (s, 2H), 8.72 - 8.67 (m, 1H), 8.20 (t, J = 5.1 Hz, 1H), 8.11 (d, J = 7.3 Hz, 1H), 8.01 (d, J = 8.7 Hz, 1H), 4.62 (d, J = 6.2 Hz, 2H), 4.26 - 4.21 (m, 1H), 4.18 - 4.14 (m, 1H), 3.90 (s, 2H), 3.70 (d, J = 5.6 Hz, 2H), 3.42 (s, 3H), 3.17 (s, 2H), 2.42 - 2.32 (m, 4H), 2.05 - 1.94 (m, 3H), 1.87 - 1.78 (m, 2H), 1.75 - 1.67 (m, 2H), 1.63 - 1.53 (m, 2H), 1.48 - 1.40 (m, 9H), 0.87 - 0.84 (m, 12H).

[0282] Example 1.2: Synthesis of 1-(3-chloro-4-(3-(methylamino)propyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea (INT2)

Chemical Structure

[0283] Step 1: Compound INT2-1 (4.0 g, 23.65 mmol), triethylamine (0.33 mL, 2.36 mmol) were dissolved in Compound INT2-2 (3.18 g, 24.84 mmol), zirconium dichloride hydrate (691 mg, 2.36 mmol) was added, and the reaction solution was reacted with stirring at 60 °C for 8 h under nitrogen gas protection. The reaction was monitored by LCMS. The reaction solution was concentrated directly under reduced pressure to obtain a crude product, and the crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 40:1) to obtain the target compound INT2-3 (2.92 g). 1 H NMR (400 MHz, DMSO-d 6 ) δ 6.38 (dt, J = 18.0, 4.3 Hz, 1H), 5.32 (d, J = 18.1 Hz, 1H), 3.83 (d, J = 3.7 Hz, 2H), 2.74 (s, 3H), 1.39 (s, 9H), 1.20 (s, 12H).

[0284] Step 2: Compound INT2-4 (1.86 g, 7.88 mmol), Compound INT2-3 (2.34 g, 7.88 mmol), potassium phosphate (5.02 g, 23.64 mmol), 2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl (368 mg, 0.788 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (V:V = 5:1, 50 mL), palladium acetate (88 mg, 0.394 mmol) was added, and the reaction solution was reacted with stirring at 80 °C for 5 h under nitrogen gas protection. The reaction was monitored by LCMS. The reaction solution was concentrated directly under reduced pressure to obtain a crude product, and the crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 40:1) to obtain the target compound INT2-5 (1.0 g). 1 H NMR (400 MHz, DMSO-d 6) δ 8.29 (d, J = 2.1 Hz, 1H), 8.16 - 8.14 (m, 1H), 8.02 (d, J = 8.7 Hz, 1H), 6.79 (d, J = 16.0 Hz, 1H), 6.19 - 6.05 (m, 1H), 3.78 (d, J = 5.7 Hz, 2H), 2.84 (s, 3H), 1.39 (s, 9H).

[0285] Step 3: Compound INT2-5 (500 mg, 1.53 mmol) was dissolved in a mixed solution of tetrahydrofuran, anhydrous methanol and water (V:V:V = 2:2:1, 6 mL), ammonium chloride (821 mg, 15.30 mmol) and zinc powder (997 mg, 15.30 mmol) were sequentially added, and the reaction was carried out with stirring at 50 °C for 2 h, and the reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT2-6 (320 mg). LCMS (ESI) [M+H -56] + = 241.0. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.36 (d, J = 8.5 Hz, 1H), 6.62 - 6.58 (m, 2H), 6.51 (d, J = 8.5 Hz, 1H), 5.95 - 5.93 (m, 1H), 5.50 (s, 2H), 3.89 (d, J = 5.4 Hz, 2H), 2.77 (s, 3H), 1.40 (s, 9H).

[0286] Step 4: Compound INT2-6 (300 mg, 1.01 mmol) was dissolved in THF (10 mL), platinum dioxide (50 mg) was added to the reaction solution, and the reaction was carried out with stirring in a hydrogen atmosphere for 1 h. The reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT2-7 (200 mg), which was used directly in the next reaction. LCMS (ESI) [M+H-100] + = 199.0. 1 H NMR (400 MHz, DMSO-d 6 ) δ 6.95 (d, J = 8.2 Hz, 1H), 6.59 (d, J = 2.2 Hz, 1H), 6.45 (dd, J = 8.2, 2.2 Hz, 1H), 5.16 (s, 2H), 3.17 (t, J = 7.1 Hz, 2H), 2.77 (s, 3H), 2.48 - 2.43 (m, 2H), 1.66 (s, 2H), 1.37 (s, 9H).

[0287] Step 5: Compound INT2-7 (200 mg, 0.67 mmol) was dissolved in tetrahydrofuran (5 mL), trichloromethyl chloroformate (265 mg, 0.14 mmol) was added, and the reaction mixture was reacted with stirring at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in DMF (5 mL), and then a DMF solution of hydrochloride salt of compound INT2-8 (221 mg, 0.88 mmol) and triethylamine (670 mg, 6.70 mmol) was added. The reaction was monitored by LCMS. Water (50 mL) was added to the reaction mixture, and it was purified with ethyl acetate (25 mL × 3). The organic phases were combined, the organic phase was washed with saturated brine (30 mL × 3), dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by column chromatography (DCM:MeOH = 10:1) to obtain the target compound INT2-9 (150 mg). LCMS (ESI) [M+H-100] + = 498.2. 1 H NMR (400 MHz, DMSO-d 6) δ 10.98 (s, 1H), 8.81 (s, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.51 (s, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.20 - 7.14 (m, 2H), 6.84 (t, J = 5.7 Hz, 1H), 5.17 - 5.03 (m, 1H), 4.43 - 4.39 (m, 2H), 4.34 - 4.27 (m, 1H), 3.21 - 3.17 (m, 2H), 2.96 - 2.86 (m, 1H), 2.77 (s, 3H), 2.64 - 2.52 (m, 4H), 2.43 - 2.33 (m, 1H), 2.03 - 1.98 (m, 1H), 1.76 - 1.67 (m, 2H), 1.37 (s, 9H).

[0288] Step 6: Compound INT2-9 (120 mg, 0.20 mmol) was dissolved in DCM (6 mL), and then trifluoroacetic acid (2 mL) was added. The reaction mixture was reacted with stirring at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the trifluoroacetate salt (100 mg) of the target compound INT2. LCMS (ESI) [M+H] + =498.1.

[0289] Example 1.3: Synthesis of 2-(3-chloro-4-(3-(methylamino)propyl)phenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide (INT3) [Chemical Structure]

[0290] Step 1: Under nitrogen gas protection, compound INT3-1 (10.0 g, 31.50 mmol) was dissolved in dimethyl sulfoxide (70 mL), ethyl difluorobromoacetate (12.79 g, 63.00 mmol) and copper powder (4.0 g, 63.00 mmol) were sequentially added to the reaction solution, and the reaction solution was reacted with stirring at room temperature for 48 h, and the reaction was monitored by TLC. The reaction solution was slowly poured into a saturated aqueous ammonium chloride solution (200 mL), purified with petroleum ether (200 mL × 2), the organic phases were combined, washed with saturated brine (200 mL × 2), and dried over anhydrous Na 2 SO 4 4, filtered by suction, the filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT3-2 (7.4 g). 1 1H NMR (400 MHz, DMSO-d 6 ) δ 7.98 (d, J = 8.3 Hz, 1H), 7.83 (s, 1H), 7.51 (d, J = 8.3 Hz, 1H), 4.35 - 4.30 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H).

[0291] Step 2: Under nitrogen gas protection, compound INT3-2 (3.0 g, 9.57 mmol) was dissolved in DMF (50 mL), tert-butyl methyl(prop-2-yn-1-yl)carbamate (3.2 g, 19.14 mmol), triethylamine (4.8 g, 47.84 mmol), cuprous iodide (182 mg, 0.96 mmol) and bis(triphenylphosphine)palladium(II) dichloride (672 mg, 0.96 mmol) were sequentially added, and the reaction solution was reacted with stirring at 60 °C for 4 h. The reaction was monitored by LCMS. The reaction solution was cooled to room temperature, water (150 mL) was added to the reaction solution, and it was purified with ethyl acetate (150 mL × 3), the organic phases were combined, washed with saturated brine (200 mL × 3), and dried over anhydrous Na 2 SO 4It was dried, suction filtered, the filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT3-3 (2.0 g). LCMS (ESI) [M+H-56] + = 346.0. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.75 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 8.2 Hz, 1H), 4.38 - 4.29 (m, 4H), 2.91 (s, 3H), 1.43 (s, 9H), 1.24 (t, J = 7.1 Hz, 3H).

[0292] Step 3: Compound INT3-3 (1.2 g, 2.99 mmol) was dissolved in methanol (10 mL), under the protection of nitrogen gas, Raney nickel (300 mg) was added, and the reaction mixture was replaced with hydrogen gas three times. The reaction solution was reacted under a hydrogen atmosphere at room temperature with stirring for 6 h. The reaction was monitored by LCMS. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the target compound INT3-4 (930 mg) for direct use in the next reaction, which was used directly in the next reaction as it was. LCMS (ESI) [M+H-56] + = 350.0. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.64 - 7.54 (m, 2H), 7.52 (s, 1H), 4.32 - 4.30 (m, 2H), 3.29 - 3.16 (m, 2H), 2.78 (s, 3H), 2.74 - 2.67 (m, 2H), 1.87 - 1.70 (m, 2H), 1.41 (s, 9H), 1.27 - 1.22 (m, 3H).

[0293] Step 4: Compound INT3-4 (900 mg, 2.22 mmol) was dissolved in a mixed solution of methanol (15 mL) and water (3 mL), lithium hydroxide monohydrate (212 mg, 8.87 mmol) was further added, and the reaction was carried out with stirring at room temperature for 2 h. The reaction was monitored by LCMS. Water (20 mL) was added to the reaction solution, and the reaction solution was concentrated under reduced pressure to remove methanol. The pH was adjusted to 4 with 1N hydrochloric acid aqueous solution, and it was purified with ethyl acetate (20 mL × 2). The organic phases were combined and washed with saturated brine (20 mL × 3), and dried over anhydrous Na 2 SO 4 and filtered by suction. The filtrate was concentrated under reduced pressure to obtain the target compound INT3-5 (700 mg), which was used directly in the next reaction. LCMS (ESI) [M+H-56] + = 322.2. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.49 (s, 1H), 7.40 (s, 2H), 3.23 - 3.20 (m, 2H), 2.78 (s, 3H), 2.68 - 2.64 (m, 2H), 1.77 - 1.73 (m, 2H), 1.36 (s, 9H).

[0294] Step 5: Compound INT3-5 (670 mg, 1.77 mmol), 3-(6-aminomethylene-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione hydrochloride (500 mg, 1.61 mmol) were dissolved in dichloromethane (20 mL), triethylamine (765 mg, 7.57 mmol), ethyl acetate solution of propylphosphonic acid tricyclic anhydride (2.87 g, 4.51 mmol, mass fraction 50%) were added. The reaction solution was reacted with stirring at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was diluted by adding water (20 mL), and purified with ethyl acetate (30 mL × 3). The organic phases were combined and washed with saturated brine (20 mL × 2), and dried over anhydrous Na 2 SO 4It was dried, suction filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 1:1) to obtain the target compound INT3-6 (270 mg). LCMS (ESI) [M+Na] + = 655.6. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.99 (s, 1H), 9.68 (t, J = 5.9 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.52 (d, J = 11.0 Hz, 2H), 7.43 (s, 1H), 7.37 (d, J = 7.8 Hz, 1H), 5.13 - 5.09 (m, 1H), 4.45 - 4.27 (m, 4H), 3.22 - 3.20 (m, 3H), 2.94 - 2.91 (m, 1H), 2.78 (s, 3H), 2.69 (d, J = 7.8 Hz, 2H), 2.67 - 2.62 (m, 1H), 2.42 - 2.35 (m, 1H), 1.77 (s, 2H), 1.33 (s, 9H).

[0295] Step 6: Compound INT3-6 (205 mg, 0.32 mmol) was dissolved in HCl / 1,4-dioxane solution (5 mL). The reaction solution was reacted with stirring at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the hydrochloride salt of the target compound INT3 (165 mg). LCMS (ESI) [M+H] + = 532.9.

[0296] Example 1.4: Synthesis of 1-(3-chloro-4-(3-(methylamino)propyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)methyl)urea (INT4)

Chemical Structure

[0297] Step 1: Dissolve compound INT2-7 (200 mg, 0.67 mmol) in THF (5 mL), add trichloromethyl chloroformate (265 mg, 1.34 mmol), and react the reaction solution with stirring at room temperature for 1 h. Concentrate the reaction solution under reduced pressure to obtain a crude intermediate. Dissolve the crude intermediate in DMF (5 mL), then add a DMF (5 mL) solution of compound INT4-1 (193 mg, 0.67 mmol) and triethylamine (670 mg, 6.70 mmol), and monitor the reaction by LCMS. Add water (25 mL) to the reaction solution, purify it with EA (30 mL×3), combine the organic phases, wash the organic phases with saturated brine (30 mL×2), dry over anhydrous Na 2 SO 4 , filter, concentrate the filtrate under reduced pressure, and purify the crude product by column chromatography (dichloromethane:methanol = 10:1) to obtain the target compound INT4-2 (200 mg). LCMS (ESI) [M+H-100] + =512.3. 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.12 (s, 1H), 8.86 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.85 - 7.75 (m, 2H), 7.64 (s, 1H), 7.18 (s, 2H), 6.91 (t, J = 6.1 Hz, 1H), 5.14 (dd, J = 12.9, 5.3 Hz, 1H), 4.46 (d, J = 6.1 Hz, 2H), 3.18 (t, J = 7.1 Hz, 2H), 2.94 - 2.85 (m, 1H), 2.77 (s, 3H), 2.64 - 2.52 (m, 4H), 2.09 - 2.03 (m, 1H), 1.77 - 1.64 (m, 2H), 1.36 (s, 9H).

[0298] Step 2: Compound INT4-2 (120 mg, 0.20 mmol) was dissolved in DCM (6 mL), then trifluoroacetic acid (2 mL) was added, and the reaction mixture was stirred at room temperature for 1 h while reacting. The reaction was monitored by LCMS, and the reaction mixture was concentrated under reduced pressure to obtain the trifluoroacetate salt (100 mg) of the target compound INT4. LCMS (ESI) [M+H] + =512.3. 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.12 (s, 1H), 8.98 (s, 1H), 8.34 (s, 2H), 7.90 (d, J = 7.7 Hz, 1H), 7.85 - 7.78 (m, 2H), 7.67 (d, J = 1.7 Hz, 1H), 7.23 - 7.17 (m, 2H), 7.04 (t, J = 6.0 Hz, 1H), 5.15 (dd, J = 12.8, 5.3 Hz, 1H), 4.47 (d, J = 5.8 Hz, 2H), 2.95 - 2.85 (m, 3H), 2.70 - 2.57 (m, 4H), 2.57 - 2.54 (m, 3H), 2.10 - 2.01 (m, 1H), 1.87 - 1.79 (m, 2H).

[0299] Example 1.5: Synthesis of (5S,8S)-8-(4-(dimethylamino)butyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazapentadecan-17-oic acid (INT5)

Chemical Structure

[0300] Step 1: Compound INT1-2 (50.0 g, 107.07 mmol) was dissolved in MeOH (1000 mL), sodium acetate (17.6 g, 214.1 mmol) was added, and paraformaldehyde (57.8 g, 642.4 mmol) was added under ice bath conditions. The mixture was stirred for 30 min, and sodium cyanoborohydride (13.5 g, 214.1 mmol) was further added to the reaction system. After the addition was complete, the temperature was restored to room temperature, and the reaction solution was reacted with stirring at room temperature overnight. The reaction was monitored by LCMS, and the reaction solution was concentrated under reduced pressure. The crude product was directly purified by preparative high performance liquid chromatography (acetonitrile:H 2 O, 0.05% FA = 5% - 70% content) to obtain the target compound INT5-1 (25 g). LCMS (ESI) [M+H] + = 496.6. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.96 (s, 1H), 8.19 (d, J = 7.5 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.75 (d, J = 7.4 Hz, 2H), 7.42 - 7.40 (m, 3H), 7.37 - 7.27 (m, 2H), 4.33 - 4.25 (m, 1H), 4.24 - 4.18 (m, 2H), 3.94 - 3.85 (m, 1H), 3.01 - 2.89 (m, 2H), 2.68 (d, J = 4.7 Hz, 6H), 2.06 - 1.91 (m, 2H), 1.78 - 1.68 (m, 1H), 1.65 - 1.58 (m, 2H), 1.38 - 1.28 (m, 2H), 1.27 - 1.22 (m, 1H), 0.93 - 0.83 (m, 6H).

[0301] Step 2: Compound INT5-1 (4.7 g, 9.5 mmol) and compound INT1-4 (2.4 g, 9.5 mmol) were dissolved in DMF (50 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.2 g, 8.6 mmol) and N,N-diisopropylethylamine (3.7 g, 28.5 mmol) were added, and the reaction mixture was reacted with stirring at room temperature for 1 h. The reaction was monitored by LCMS. Water (200 mL) was added to the reaction mixture, and it was purified with EA (200 mL × 3). The organic phases were combined, and the organic phase was washed with saturated brine (150 mL × 3) and dried over anhydrous Na 2 SO 4 and filtered. After that, the filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH = 10:1) to obtain the target compound INT5-2 (1.5 g). LCMS (ESI) [M+H] + = 730.4. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.70 (t, J = 6.6 Hz, 1H), 8.25 (t, J = 6.2 Hz, 1H), 8.08 (d, J = 7.3 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.74 (d, J = 7.3 Hz, 2H), 7.43 - 7.32 (m, 10H), 5.15 (s, 2H), 4.66 - 4.60 (m, 2H), 4.33 - 4.24 (m, 2H), 4.24 - 4.19 (m, 2H), 4.16 (s, 2H), 3.92 - 3.86 (m, 1H), 3.73 (d, J = 5.9 Hz, 2H), 2.91 - 2.82 (m, 2H), 2.66 (s, 6H), 2.02 - 1.94 (m, 1H), 1.74 - 1.64 (m, 1H), 1.60 - 1.51 (m, 3H), 1.31 - 1.26 (m, 2H), 0.85 (t, J = 6.0 Hz, 6H).

[0302] Step 3: Compound INT5-2 (1.3 g, 1.7 mmol) was dissolved in a mixed solvent of EtOH (40 mL) and EA (20 mL), Pd / C (400 mg) was added, the reaction solution was purged with hydrogen gas three times, and the reaction was carried out with stirring for 16 h under a hydrogen atmosphere while monitoring the reaction by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT5 (780 mg). LCMS (ESI) [M+H] + =640.4.

[0303] Example 1.6: Synthesis of 1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-5-yl)methyl)-3-(4-(3-(methylamino)propyl)phenyl)urea (INT6)

Chemical Structure

[0304] Step 1: Compound INT6-1 (3.0 g, 17.60 mmol) was dissolved in DMF (100 mL), INT6-2 (5.0 g, 17.60 mmol), triethylamine (8.9 g, 88.20 mmol), cuprous iodide (335 mg, 1.76 mmol) and palladium dichloride triphenylphosphine (1.2 g, 1.76 mmol) were successively added to the reaction solution. The reaction was carried out with stirring at room temperature for 4 h under nitrogen gas protection while monitoring the reaction by TLC. Water (100 mL) was added to the reaction solution to quench it, and it was purified with ethyl acetate (100 mL×3). The combined organic phases were washed with saturated brine (100 mL×2) and water (100 mL×2), dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT6-3 (3.5 g). 1 H NMR (400 MHz, DMSO-d 6) δ 8.39 (d, J = 2.3Hz, 1H), 8.18 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 4.38 (s, 2H), 2.91 (s, 3H), 1.42 (s, 9H).

[0305] Step 2: Compound INT6-3 (1.0 g, 3.09 mmol) was dissolved in anhydrous MeOH (20 mL), and THF (20 mL), distilled water (10 mL), zinc powder (2.0 g, 30.86 mmol), and ammonium chloride (1.6 g, 30.86 mmol) were sequentially added to the reaction solution. The reaction solution was stirred at 50 °C for 4 h, the reaction was monitored by LCMS, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure. Then, EA was added for purification (50 mL × 3), the organic phases were combined and washed successively with saturated brine (50 mL × 2) and water (50 mL × 2), dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by a chromatography column (petroleum ether:ethyl acetate = 5:1) to obtain the target compound INT6-4 (900 mg). LCMS (ESI) [M+H-56] + = 239.0. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.16 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 2.1 Hz, 1H), 6.45 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 5.78 (s, 2H), 4.22 (s, 2H), 2.86 (s, 3H), 1.41 (s, 9H).

[0306] Step 3: Compound INT6-4 (5.0 g, 17.00 mmol) was dissolved in MeOH (50 mL), ammonium formate (5.36 g, 85.00 mmol) and palladium on carbon (360 mg, 3.40 mmol) were added sequentially, the reaction mixture was purged with hydrogen gas three times, and the reaction solution was reacted at 60 °C overnight under a hydrogen atmosphere. The reaction was monitored by LCMS, the reaction solution was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: H 2 O, containing 0.05% FA = 5% - 50%) to obtain the target compound INT6-5 (2.6 g). LCMS (ESI) [M+H-100] + = 165.1. 1 H NMR (400 MHz, CDCl 3 ) δ 6.97 (d, J = 8.2 Hz, 2H), 6.62 (d, J = 8.3 Hz, 2H), 3.18 - 3.23 (m, 2H), 2.83 (s, 3H), 2.52 - 2.44 (m, 2H), 1.83 - 1.72 (m, 2H), 1.45 (s, 9H).

[0307] Step 4: Compound INT6-5 (1.0 g, 3.78 mmol) was dissolved in THF (20 mL), triphosgene (1.50 g, 7.56 mmol) was added, and the reaction was carried out with stirring at room temperature for 30 min. The formation of the intermediate state of the reaction was detected by LCMS, the reaction solution was concentrated under reduced pressure, DMF (5 mL) was added and dissolved for use. The hydrochloride salt of compound INT2-8 (1.04 g, 3.78 mmol) and triethylamine (3.03 g, 30.04 mmol) were dissolved in DMF (5 mL), and the above-mentioned preliminary reaction solution was slowly added dropwise. After the addition was completed, the reaction was carried out with stirring at room temperature for 2 h. The reaction was monitored by LCMS, water (10 mL) was added to the reaction solution to quench the reaction, and it was purified with EA (10 mL × 3). The organic phases were combined and washed with saturated brine (10 mL × 3), anhydrous Na 2 SO 4It was dried, filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by a chromatography column (methanol:dichloromethane = 0% - 7%) to obtain the target compound INT6-6 (800 mg). LCMS (ESI) [M+H-100] + = 464.3. 1 H NMR (400 MHz, CDCl 3 ) δ 8.97 (s, 1H), 7.57 - 7.45 (m, 2H), 7.29 (s, 1H), 7.26 - 7.21 (m, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 7.7 Hz, 2H), 5.95 (s, 1H), 5.04 (dd, J = 13.1, 4.8 Hz, 1H), 4.43 - 4.30 (m, 2H), 4.24 - 4.11 (m, 2H), 3.21 (t, J = 7.0 Hz, 2H), 2.81 (s, 3H), 2.80 - 2.63 (m, 2H), 2.50 (t, J = 7.1 Hz, 2H), 2.27 - 2.05 (m, 2H), 1.79 - 1.74 (m, 2H), 1.43 (s, 9H).

[0308] Step 5: Compound INT6-6 (200 mg, 0.35 mmol) was dissolved in DCM (5 mL), trifluoroacetic acid (1 mL) was slowly added dropwise to the reaction solution, and then the reaction was carried out with stirring at room temperature for 30 min. The reaction was monitored by LCMS, and the reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of the target compound INT6 (150 mg). LCMS (ESI) [M+H] + = 464.2. 1 H NMR (400 MHz, DMSO-d 6) δ 10.90 (s, 1H), 8.54 (s, 1H), 8.25 (s, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.69 (t, J = 6.0 Hz, 1H), 5.03 (dd, J = 13.3, 4.9 Hz, 1H), 4.42-4.24 (m, 4H), 2.90-2.83 (m, 2H), 2.82-2.75 (m, 2H), 2.62-2.47 (m, 5H), 2.37-2.24 (m, 1H), 1.96-1.90 (m, 1H), 1.81-1.70 (m, 2H).

[0309] Example 1.7: Synthesis of 2,2-dimethyl-4-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butyric acid (INT7)

Chemical formula

[0310] Step 1: Compound INT7-1 (500 mg, 4.39 mmol) was dissolved in ultradry DCM (5 mL), and boron tribromide (1.15 g, 4.6 mmol) was added dropwise at 0 °C. After the addition was complete, the temperature was raised to room temperature and stirred for 24 h, then cooled to 0 °C, and methanol (5 mL) was added dropwise to the reaction solution to quench the reaction. The reaction solution was continuously stirred at room temperature for 24 h. Saturated aqueous sodium hydrogen carbonate solution (30 mL) was added to the reaction solution, and then purified with DCM (30 mL × 3). The organic phases were combined and dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure to obtain the target compound INT7-2 (830 mg). 1 H NMR (400 MHz, CDCl 3) δ 3.69 (s, 3H), 3.40 - 3.27 (m, 2H), 2.20 - 2.09 (m, 2H), 1.21 (s, 6H).

[0311] Step 2: Compound INT7 - 2 (650 mg, 3.13 mmol) was dissolved in DMF (10 mL), sodium azide (1.02 g, 15.63 mmol) was added, the temperature was raised to 80 °C, and the mixture was stirred overnight. Then, the temperature was lowered to room temperature, water (30 mL) was added to the reaction solution, and then it was purified with MTBE (30 mL × 3). The organic phases were combined and dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under pressure to obtain the target compound INT7 - 3 (470 mg). 1 H NMR (400 MHz, CDCl 3 ) δ 3.69 (s, 3H), 3.31 - 3.23 (m, 2H), 1.89 - 1.81 (m, 2H), 1.22 (s, 6H).

[0312] Step 3: Compound INT7 - 3 (365 mg, 2.13 mmol) and INT7 - 4 (320 mg, 2.13 mmol) were dissolved in tert - butanol (5 mL) and water (5 mL), sodium ascorbate (84 mg, 0.43 mmol) and anhydrous copper sulfate (34 mg, 0.21 mmol) were added, and the reaction solution was stirred at room temperature for 1 h. Water (25 mL) was added to the reaction solution, and then it was purified with ethyl acetate (30 mL × 3). The organic phases were combined and dried over anhydrous Na 2 SO 4 and filtered. The filtrate was evaporated to dryness under reduced pressure, and the crude product was purified by column chromatography (PE:EA = 3:1) to obtain the target compound INT7 - 5 (600 mg). LCMS (ESI) [M + H] + = 322.1. 1 H NMR (400 MHz, CDCl 3) δ 8.95 (s, 2H), 7.85 (s, 1H), 4.53 - 4.39 (m, 2H), 3.69 (s, 3H), 2.61 (s, 3H), 2.29 - 2.17 (m, 2H), 1.31 (s, 6H).

[0313] Step 4: Compound INT7 - 5 (500 mg, 1.60 mmol) was dissolved in THF (5 mL) and water (5 mL), lithium hydroxide (112 mg, 4.70 mmol) was added, and the reaction mixture was stirred at room temperature for 5 h. 1 N hydrochloric acid was added to the reaction mixture to adjust the pH to 3 - 4, a large amount of white solid precipitated, filtered, and the filter cake was dried to obtain the target compound INT7 - 6 (450 mg). LCMS (ESI) [M + H] + = 308.0. 1 H NMR (400 MHz, DMSO - d 6 ) δ 12.43 (s, 1H), 9.06 (s, 2H), 8.76 (s, 1H), 4.55 - 4.31 (m, 2H), 2.56 (s, 3H), 2.15 - 2.04 (m, 2H), 1.20 (s, 6H).

[0314] Step 5: Compound INT7 - 6 (1.5 g, 4.90 mmol) was dissolved in THF (10 mL) and water (10 mL), Oxane (15.0 g, 24.5 mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into water (200 mL), filtered, and the filter cake was dried to obtain the target compound INT7 (1.6 g). LCMS (ESI) [M + H] + = 340.1. 1 H NMR (400 MHz, DMSO - d 6 ) δ 9.48 (s, 2H), 8.98 (s, 1H), 4.54 - 4.48 (m, 2H), 3.44 (s, 3H), 2.16 - 2.09 (m, 2H), 1.21 (s, 6H).

[0315] Example 2. Synthesis of Biologically Active Compound (Payload) Example 2.1: Synthesis of N-(3-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)propyl)-2-hydroxy-N-methylacetamide (PL-1)

Chemical Structure

[0316] Step 1: Dissolve the trifluoroacetate of compound INT2 (20 mg, 0.04 mmol) and glycolic acid (3 mg, 0.04 mmol) in DMF (3 mL), and add triethylamine (12 mg, 0.12 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (17 mg, 0.06 mmol). React the reaction solution with stirring at room temperature for 2 h. Monitor the reaction by LCMS. Purify the reaction solution by direct preparative high-performance liquid chromatography (acetonitrile:H 2 2O, containing 0.05% FA = 5% - 50%) to obtain the target compound PL-1 (10 mg). LCMS (ESI) [M+H] + = 556.2, 1 1H NMR (400 MHz, DMSO-d 6) δ 10.97 (s, 1H), 8.95 - 8.86 (m, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.66 (s, 1H), 7.51 (s, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.22 - 7.15 (m, 2H), 6.98 - 6.90 (m, 1H), 5.18 - 5.03 (m, 1H), 4.46 - 4.36 (m, 4H), 4.33 - 4.28 (m, 1H), 4.09 - 4.01 (m, 2H), 3.24 - 3.17 (m, 1H), 2.96 - 2.86 (m, 1H), 2.84 (s, 3H), 2.64 - 2.58 (m, 1H), 2.58 - 2.54 (m, 2H), 2.44 - 2.32 (m, 1H), 2.02 - 1.97 (m, 1H), 1.80 - 1.66 (m, 2H).

[0317] Example 2.2: Synthesis of 2-(3-chloro-4-(3-(2-hydroxy-N-methylacetamido)propyl)phenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide (PL-2)

Chemical formula

[0318] Step 1: The hydrochloride salt of compound INT3 (40 mg, 0.08 mmol) and glycolic acid (6 mg, 0.08 mmol) were dissolved in DMF (2 mL), and triethylamine (24 mg, 0.24 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (33 mg, 0.12 mmol) were added. The reaction solution was reacted with stirring at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was directly purified by preparative high performance liquid chromatography (CH 3 CN:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the target compound PL-2 (15 mg). LCMS (ESI) [M+H] + = 591.2, 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.98 (s, 1H), 9.69 (t, J = 5.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.42 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 5.16 - 5.05 (m, 1H), 4.45 (d, J = 6.1 Hz, 2H), 4.41 - 4.38 (m, 1H), 4.40 - 4.30 (m, 1H), 4.07 (t, J = 4.8 Hz, 2H), 3.30 - 3.23 (m, 2H), 2.97 - 2.88 (m, 1H), 2.86 (s, 3H), 2.74 - 2.67 (m, 2H), 2.64 - 2.57 (m, 1H), 2.44 - 2.32 (m, 1H), 2.03 - 1.98 (m, 1H), 1.84 - 1.72 (m, 2H).

[0319] Example 2.3: Synthesis of N-(3-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)methyl)ureido)phenyl)propyl)-2-hydroxy-N-methylacetamide (PL-3)

Chemical Structure

[0320] Step 1: Compound INT4 (50.0 mg, 0.10 mmol) was dissolved in DMF (1 mL), and glycolic acid (7.0 mg, 0.10 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.10 mmol), and N,N-diisopropylethylamine (32 mg, 0.25 mmol) were sequentially added. The reaction solution was reacted with stirring at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was directly purified by preparative high-performance liquid chromatography (acetonitrile:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the target compound PL-3 (15 mg). LCMS (ESI) [M+H] + = 570.3, 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.11 (s, 1H), 8.86 (d, J = 5.0 Hz, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.82 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.24 - 7.15 (m, 2H), 6.92 (t, J = 6.4 Hz, 1H), 5.14 (dd, J = 13.0, 5.4 Hz, 1H), 4.51 - 4.44 (m, 2H), 4.44 - 4.32 (m, 1H), 4.08 - 3.99 (m, 2H), 3.37 - 3.35 (m, 1H), 3.25 - 3.16 (m, 1H), 2.95 - 2.86 (m, 1H), 2.84 (d, J = 5.1 Hz, 3H), 2.65 (d, J = 19.8 Hz, 1H), 2.59 - 2.53 (m, 3H), 2.11 - 2.01 (m, 1H), 1.81 - 1.65 (m, 2H).

[0321] Example 2.4: Synthesis of N-(2-chloro-4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-methyl)ureido)phenyl ethoxy)ethyl-2-hydroxy-N-methylacetamide (PL-4) [Chemical]

[0322] Step 1: Dissolve compound PL-4-1 (50.0 mg, 0.09 mmol, prepared with reference to Patent Document WO2021198965) in DMF (1 mL), and sequentially add glycolic acid (6.9 mg, 0.09 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (37 mg, 0.09 mmol), and N,N-diisopropylethylamine (31 mg, 0.22 mmol). React the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, and purify the reaction solution by direct preparative high-performance liquid chromatography (acetonitrile:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the target compound PL-4 (23 mg). LCMS (ESI) [M+H] + = 586.4, 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.97 (s, 1H), 8.77 (s, 1H), 7.68 (dd, J = 9.5, 4.8 Hz, 2H), 7.51 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.20 - 7.11 (m, 2H), 6.80 (t, J = 5.8 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.48 - 4.26 (m, 4H), 4.05 (d, J = 17.8 Hz, 2H), 3.57 - 3.53 (m, 2H), 3.52 - 3.48 (m, 2H), 3.46 - 3.41 (m, 2H), 3.36 - 3.30 (m, 1H), 2.96 - 2.84 (m, 2H), 2.83 (d, J = 4.6 Hz, 3H), 2.60 - 2.51 (m, 1H), 2.45 - 2.30 (m, 1H), 2.02 - 1.95 (m, 1H).

[0323] Example 2.5: Synthesis of N-(3-(4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-methyl)ureido)phenyl)propyl)-2-hydroxy-N-methylacetamide (PL-5)

Chemical Structure

[0324] Step 1: Dissolve the trifluoroacetate of compound INT6 (50.0 mg, 0.10 mmol) in DMF (1 mL), and sequentially add glycolic acid (8.0 mg, 0.10 mmol), 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (41 mg, 0.10 mmol), and N,N-diisopropylethylamine (35 mg, 0.25 mmol). Stir the reaction solution at room temperature for 1 h, monitor the reaction by LCMS, and purify the reaction solution directly by preparative high-performance liquid chromatography (acetonitrile:H 2 2O, containing 0.05% FA = 5% - 50%) to obtain the target compound PL-5 (18 mg). LCMS (ESI) [M+H] + = 522.4. 1 H NMR (400 MHz, DMSO-d 6) δ 10.97 (s, 1H), 8.52 (d, J = 5.7 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.35 - 7.27 (m, 2H), 7.07 (t, J = 6.8 Hz, 2H), 6.68 (t, J = 5.6 Hz, 1H), 5.10 (dd, J = 13.3, 5.0 Hz, 1H), 4.48 - 4.27 (m, 4H), 4.03 - 3.99 (m, 2H), 3.85 (s, 1H), 3.31 (t, J = 7.3 Hz, 1H), 3.20 - 3.10 (m, 1H), 2.97 - 2.86 (m, 1H), 2.83 (s, 3H), 2.61 - 2.57 (m, 1H), 2.49 - 2.42 (m, 2H), 2.41 - 2.30 (m, 1H), 2.04 - 1.93 (m, 1H), 1.82 - 1.67 (m, 2H).

[0325] Example 3. Synthesis of Toxin - Linker Example 3.1: Synthesis of N - ((16S,19S) - 1 - (2 - chloro - 4 - (3 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 5 - yl)methyl)ureido)phenyl) - 16 - (4 - (dipropylamino)butyl) - 6,20 - dimethyl - 7,12,15,18 - tetraoxo - 3,9 - dioxo - 6,11,14,17 - tetraazoundecan - 19 - yl) - 6 - (2 - (methylsulfonyl)pyrimidin - 5 - yl)hex - 5 - enamide (DL - 1) Method 1: [Chemical formula] Compound INT1 (22 mg) was dissolved in DMF (1 mL), and HATU (15 mg) and N,N-diisopropylethylamine (100 μL) were sequentially added. After stirring at room temperature for 5 min, compound PL-4-1 (16 mg) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h, and the reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: water = 5% - 50%) to obtain the formate salt (18 mg) of the target compound DL-1. LCMS (ESI) [M+H] + = 1233.6. 1 H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 9.11 (s, 2H), 8.82 (s, 1H), 8.63 - 8.57 (m, 1H), 8.16 (s, 2H), 8.03 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.70 - 7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.19 - 7.14 (m, 2H), 6.85 (s, 1H), 5.10 (dd, J = 13.3, 5.0 Hz, 1H), 4.60 - 4.55 (m, 2H), 4.48 - 4.38 (m, 4H), 4.34 - 4.28 (m, 1H), 4.24 - 4.16 (m, 2H), 4.11 (d, J = 15.5 Hz, 2H), 3.74 - 3.68 (m, 2H), 3.56 - 3.45 (m, 6H), 3.41 (s, 3H), 2.96 - 2.89 (m, 1H), 2.87 - 2.78 (m, 6H), 2.62 (s, 1H), 2.59 - 2.51 (m, 4H), 2.42 - 2.29 (m, 4H), 2.02 - 1.96 (m, 2H), 1.85 - 1.78 (m, 2H), 1.70 - 1.65 (m, 1H), 1.59 - 1.53 (m, 1H), 1.45 - 1.38 (m, 7H), 1.33 - 1.27 (m, 2H), 0.85 - 0.81 (m, 12H).

[0326] Method 2:

Chemical Structure

[0327] Step 1: Dissolve compound PL-4-1 (200 mg, 0.38 mmol) in DMF (2 mL), sequentially add compound IN1-6 (263 mg, 0.38 mmol), 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (144 mg, 0.38 mmol) and N,N-diisopropylethylamine (122 mg, 0.95 mmol). After the addition is complete, react the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, slowly add methyl tert-butyl ether (80 mL) to the reaction solution for dilution, let it stand for 10 min, then discard the supernatant, directly concentrate the residue under reduced pressure with an oil pump to remove the excess solvent, and purify the crude product by preparative TLC (dichloromethane:methanol = 8:1) to obtain the target compound DL-1-1 (280 mg). LCMS (ESI) [M+H] + = 1205.5. 1 H NMR (400 MHz, DMSO-d 6) δ 10.99 (s, 1H), 8.81 (s, 1H), 8.65 (t, J = 8.3 Hz, 1H), 8.30 - 8.18 (m, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 - 7.70 (m, 2H), 7.67 (d, J = 5.7 Hz, 2H), 7.51 (s, 1H), 7.47 - 7.39 (m, 4H), 7.32 (t, J = 7.4 Hz, 2H), 7.20 - 7.13 (m, 2H), 6.83 (t, J = 5.4 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.61 - 4.54 (m, 2H), 4.43 - 4.39 (m, 2H), 4.35 - 4.29 (m, 2H), 4.21 (d, J = 6.8 Hz, 2H), 4.11 (d, J = 15.8 Hz, 2H), 3.92 - 3.85 (m, 1H), 3.72 (t, J = 6.0 Hz, 2H), 3.57 - 3.45 (m, 6H), 2.96 - 2.87 (m, 2H), 2.86 (s, 2H), 2.83 (d, J = 6.2 Hz, 2H), 2.78 (s, 2H), 2.61 (d, J = 4.5 Hz, 1H), 2.59 - 2.56 (m, 1H), 2.42 - 2.32 (m, 2H), 2.04 - 1.94 (m, 3H), 1.74 - 1.51 (m, 4H), 1.50 - 1.37 (m, 6H), 1.32 - 1.25 (m, 3H), 0.87 - 0.81 (m, 12H).

[0328] Step 2: Dissolve compound DL-1-1 (100 mg, 0.08 mmol) in DMF (2 mL), add diethylamine (25.6 mg, 0.32 mmol) to the reaction solution, react the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, directly concentrate the reaction solution under reduced pressure with an oil pump to obtain the target compound DL-1-2 (60 mg), and use it directly in the next reaction. LCMS (ESI) [M+H] + = 983.5.

[0329] Step 3: Dissolve compound DL-1-2 (60 mg, 0.06 mmol), compound INT1-8 (18 mg, 0.06 mmol), 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (25 mg, 0.06 mmol), and N,N-diisopropylethylamine (21 mg, 0.15 mmol) in DMF (3 mL), react the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, and directly purify the reaction solution by preparative high performance liquid chromatography (acetonitrile:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the formate salt (23 mg) of the target compound DL-1. LCMS (ESI) [M+H] + = 1233.9. 1 H NMR (400 MHz, DMSO-d 6) δ 10.97 (s, 1H), 9.11 (s, 2H), 8.82 (s, 1H), 8.63-8.57 (m, 1H), 8.16 (s, 2H), 8.03 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.19-7.14 (m, 2H), 6.85 (s, 1H), 5.10 (dd, J = 13.3, 5.0 Hz, 1H), 4.60-4.55 (m, 2H), 4.48-4.38 (m, 4H), 4.34-4.28 (m, 1H), 4.24-4.16 (m, 2H), 4.11 (d, J = 15.5 Hz, 2H), 3.74-3.68 (m, 2H), 3.56-3.45 (m, 6H), 3.41 (s, 3H), 2.96-2.89 (m, 1H), 2.87-2.78 (m, 6H), 2.62 (s, 1H), 2.59-2.51 (m, 4H), 2.42-2.29 (m, 4H), 2.02-1.96 (m, 2H), 1.85-1.78 (m, 2H), 1.70-1.65 (m, 1H), 1.59-1.53 (m, 1H), 1.45-1.38 (m, 7H), 1.33-1.27 (m, 2H), 0.85-0.81 (m, 12H).

[0330] Example 3.2: Synthesis of N-(14S,17S)-1-(2-chloro-4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)-14-(4-(dipropylamino)butyl)-4,18-dimethyl-5,10,13,16-tetraoxo-7-oxa-4,9,12,15-tetraazanonadecane-17-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-hexanamide (DL-2) [Chemical formula]

[0331] Step 1: The trifluoroacetate salt of compound INT2 (100 mg, 0.20 mmol), compound INT1-6 (139 mg, 0.20 mmol) were dissolved in DMF (5 mL), and N,N-diisopropylethylamine (77 mg, 0.60 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76 mg, 0.20 mmol) were added. The reaction mixture was reacted with stirring at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was added to water (45 mL) and purified with ethyl acetate (25 mL×3). The combined organic phases were washed with saturated brine (30 mL×3) and dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by preparative thin layer chromatography (DCM:MeOH = 10:1) to obtain the target compound DL-2-1 (100 mg). LCMS (ESI) [M+H] + = 1175.3. 1 H NMR (400 MHz, DMSO-d 6) δ 10.98 (s, 1H), 8.85 - 8.73 (m, 1H), 8.68 - 8.56 (m, 1H), 8.31 - 8.18 (m, 1H), 8.07 - 7.97 (m, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.74 - 7.64 (m, 4H), 7.51 (s, 1H), 7.46 - 7.39 (m, 4H), 7.31 (t, J = 6.2 Hz, 2H), 7.20 - 7.15 (m, 2H), 6.86 - 6.77 (m, 1H), 5.17 - 5.04 (m, 1H), 4.61 - 4.54 (m, 2H), 4.47 - 4.38 (m, 4H), 4.34 - 4.27 (m, 2H), 4.23 (d, J = 6.8 Hz, 2H), 4.10 (s, 2H), 3.92 - 3.84 (m, 1H), 3.76 - 3.67 (m, 2H), 2.97 - 2.86 (m, 2H), 2.81 (d, J = 21.5 Hz, 4H), 2.63 - 2.53 (m, 6H), 2.40 - 2.33 (m, 2H), 2.05 - 1.94 (m, 4H), 1.77 - 1.64 (m, 4H), 1.60 - 1.48 (m, 2H), 1.47 - 1.34 (m, 6H), 0.87 - 0.83 (m, 6H), 0.83 - 0.77 (m, 6H).

[0332] Step 2: Compound DL-2-1 (50 mg, 0.04 mmol) was dissolved in DMF (2 mL), diethylamine (15 mg, 0.20 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h while reacting. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the target compound DL-2-2 (50 mg), which was used directly in the next reaction. LCMS (ESI) [M+H] + = 953.5.

[0333] Step 3: Compound DL-2-2 (50 mg, 0.04 mmol) and compound INT1-8 (14 mg, 0.04 mmol) were dissolved in DMF (3 mL), and N,N-diisopropylethylamine (15 mg, 0.12 mmol) and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15 mg, 0.04 mmol) were added. The reaction mixture was reacted with stirring at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was purified directly by preparative high performance liquid chromatography (CH 3 CN:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the formate salt (10 mg) of the target compound DL-2. LCMS (ESI) [M+H] + = 1203.6. 1 H NMR (400 MHz, DMSO-d 6) δ 10.97 (s, 1H), 9.11 (s, 2H), 8.80 (d, J = 5.4 Hz, 1H), 8.61 (t, J = 6.5 Hz, 1H), 8.20 - 8.15 (m, 2H), 8.05 - 8.03 (m, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.51 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.21 - 7.16 (m, 2H), 6.86 - 6.80 (m, 1H), 5.10 (dd, J = 13.4, 5.0 Hz, 1H), 4.61 - 4.56 (m, 2H), 4.47 - 4.29 (m, 4H), 4.22 - 4.14 (m, 2H), 4.10 (s, 2H), 3.72 - 3.69 (m, 2H), 3.41 (s, 3H), 2.95 - 2.88 (m, 2H), 2.86 (s, 2H), 2.79 (s, 1H), 2.63 - 2.54 (m, 12H), 2.41 - 2.31 (m, 4H), 2.02 - 1.97 (m, 2H), 1.84 - 1.78 (m, 2H), 1.70 - 1.66 (m, 2H), 1.59 - 1.54 (m, 1H), 1.47 - 1.42 (m, 6H), 1.31 - 1.26 (m, 2H), 0.86 - 0.82 (m, 12H).

[0334] Example 3.3: Synthesis of N - ((14S,17S) - 1 - (2 - chloro - 4 - (2 - (((2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 5 - yl)methyl)amino) - 1,1 - difluoro - 2 - oxoethyl)phenyl) - 14 - (4 - (dipropylamino)butyl) - 4,18 - dimethyl - 5,10,13,16 - tetraoxo - 7 - oxa - 4,9,12,15 - tetraazanonadecane - 17 - yl) - 6 - (2 - (methylsulfonyl)pyrimidin - 5 - yl)hex - 5 - enamide (DL - 3)

Chemical Structure

[0335] Step 1: Dissolve the hydrochloride salt of compound INT3 (165 mg, 0.31 mmol) in DMF (8 mL), and sequentially add compound INT1-6 (216 mg, 0.31 mmol), N,N-diisopropylethylamine (80 mg, 0.62 mmol), and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (129 mg, 0.47 mmol). React the reaction solution with stirring at room temperature for 1 h. Monitor the reaction by LCMS. Add water (45 mL) to the reaction solution, purify it with ethyl acetate (25 mL × 3), combine the organic phases, wash them with saturated brine (30 mL × 3), dry over anhydrous Na 2 SO 4 and filter. Concentrate the filtrate under reduced pressure to obtain a crude product, and purify the crude product by preparative thin-layer chromatography (DCM:MeOH = 10:1) to obtain the target compound DL-3-1 (150 mg). LCMS (ESI) [M+H] + = 1210.2. 1 H NMR (400 MHz, DMSO-d 6) δ 10.98 (s, 1H), 9.68 (t, J = 6.0 Hz, 1H), 8.70 - 8.58 (m, 1H), 8.29 - 8.19 (m, 1H), 8.02 (t, J = 9.1 Hz, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.75 - 7.70 (m, 2H), 7.67 (d, J = 7.9 Hz, 1H), 7.58 (s, 1H), 7.56 - 7.52 (m, 1H), 7.51 - 7.44 (m, 2H), 7.44 - 7.37 (m, 4H), 7.31 (t, J = 7.0 Hz, 2H), 5.33 (t, J = 4.6 Hz, 1H), 5.13 - 5.06 (m, 1H), 4.61 - 4.55 (m, 2H), 4.45 (d, J = 5.9 Hz, 2H), 4.30 (s, 1H), 4.23 - 4.20 (m, 2H), 4.11 - 4.09 (m, 1H), 3.87 (t, J = 8.2 Hz, 1H), 3.77 - 3.65 (m, 2H), 2.94 - 2.89 (m, 1H), 2.87 - 2.79 (m, 3H), 2.69 - 2.66 (m, 2H), 2.64 - 2.60 (m, 1H), 2.59 - 2.56 (m, 1H), 2.43 - 2.35 (m, 2H), 2.34 - 2.32 (m, 1H), 2.28 - 2.22 (m, 2H), 2.05 - 1.93 (m, 6H), 1.78 - 1.71 (m, 2H), 1.68 - 1.61 (m, 2H), 1.56 - 1.52 (m, 1H), 1.47 - 1.44 (m, 1H), 1.37 - 1.26 (m, 8H), 0.86 - 0.78 (m, 12H).

[0336] Step 2: Compound DL-3-1 (100 mg, 0.08 mmol) was dissolved in DMF (2 mL), and diethylamine (2 mg, 0.24 mmol) was added. The reaction solution was reacted with stirring at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain the target compound DL-3-2 (70 mg), which was used directly in the next reaction. LCMS (ESI) [M+H] + = 988.3.

[0337] Step 3: Compound DL-3-2 (70 mg, 0.06 mmol) and compound INT1-8 (15 mg, 0.06 mmol) were dissolved in DMF (2 mL), and N,N-diisopropylethylamine (23 mg, 0.18 mmol) and 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (23 mg, 0.06 mmol) were added. The reaction mixture was reacted with stirring at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was purified by preparative high performance liquid chromatography (CH 3 CN:H 2 O, 0.05% FA = 5% - 50% content) to obtain the formate salt (15 mg) of the target compound DL-3. LCMS (ESI) [M+H] + = 1238.4. 1 H NMR (400 MHz, DMSO-d 6) δ 10.98 (s, 1H), 9.67 (t, J = 6.4 Hz, 1H), 9.11 (s, 2H), 8.59 (t, J = 6.0 Hz, 1H), 8.23 (s, 1H), 8.17 (t, J = 5.8 Hz, 1H), 8.02 (t, J = 7.2 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.60 - 7.57 (m, 1H), 7.57 - 7.53 (m, 1H), 7.52 - 7.47 (m, 1H), 7.42 (d, J = 5.9 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H), 5.16 - 5.04 (m, 1H), 4.62 - 4.55 (m, 2H), 4.47 - 4.39 (m, 3H), 4.29 - 4.25 (m, 1H), 4.20 - 4.14 (m, 2H), 4.12 (d, J = 5.5 Hz, 2H), 3.75 - 3.66 (m, 2H), 3.41 (s, 3H), 2.96 - 2.89 (m, 1H), 2.87 (s, 3H), 2.72 - 2.69 (m, 1H), 2.68 - 2.66 (m, 1H), 2.64 - 2.58 (m, 1H), 2.57 - 2.52 (m, 4H), 2.42 - 2.36 (m, 2H), 2.35 - 2.31 (m, 2H), 2.31 - 2.25 (m, 6H), 2.04 - 1.96 (m, 3H), 1.84 - 1.72 (m, 4H), 1.40 - 1.28 (m, 8H), 0.85 - 0.78 (m, 12H).

[0338] Example 3.4: Synthesis of N-((13S,16S)-1-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenoxy)-13-(4-(dipropylamino)butyl)-3,17-dimethyl-4,9,12,15-tetraoxo-6-oxa-3,8,11,14-tetraazanonadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enamide (DL-4)

Chemical Structure

[0339] Step 1: Dissolve compound INT6 (150 mg, 0.32 mmol) in DMF (2 mL), and sequentially add compound INT1-6 (192 mg, 0.32 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (123 mg, 0.32 mmol), and N,N-diisopropylethylamine (104 mg, 0.80 mmol). After the addition is complete, react the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, slowly add methyl tert-butyl ether (80 mL) to the reaction solution for dilution, let it stand for 10 min, then discard the supernatant. The residue is directly concentrated under reduced pressure to remove the excess solvent, and the crude product is purified by preparative TLC (dichloromethane:methanol = 8:1) to obtain the target compound DL-4-1 (250 mg). LCMS (ESI) [M+H] + = 1141.7. 1 H NMR (400 MHz, DMSO-d 6) δ 10.98 (s, 1H), 8.65 (t, J = 6.2 Hz, 1H), 8.54 (d, J = 5.3 Hz, 1H), 8.27-8.19 (m, 1H), 8.08-8.01 (m, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.75-7.66 (m, 3H), 7.51 (s, 1H), 7.46-7.39 (m, 4H), 7.35-7.29 (m, 4H), 7.07 (t, J = 9.1 Hz, 2H), 6.74-6.67 (m, 1H), 5.10 (dd, J = 13.3, 5.0 Hz, 1H), 4.63-4.53 (m, 2H), 4.41 (d, J = 6.2 Hz, 2H), 4.35-4.20 (m, 5H), 4.11-4.05 (m, 2H), 3.88 (t, J = 8.2 Hz, 1H), 3.73 (d, J = 2.5 Hz, 2H), 3.31-3.24 (m, 4H), 3.21-3.15 (m, 2H), 2.88-2.85 (m, 1H), 2.80-2.77 (m, 3H), 2.64-2.56 (m, 2H), 2.48-2.42 (m, 2H), 2.40-2.33 (m, 1H), 2.04-1.93 (m, 3H), 1.82-1.60 (m, 4H), 1.56-1.42 (m, 7H), 1.33-1.27 (m, 2H), 0.88-0.83 (m, 12H).

[0340] Step 2: Dissolve compound DL-4-1 (100 mg, 0.08 mmol) in DMF (2 mL), add diethylamine (25.6 mg, 0.32 mmol) to the reaction solution, react the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, and concentrate the reaction solution under reduced pressure to obtain the target compound DL-4-2 (55 mg), which was directly used in the next reaction. LCMS (ESI) [M+H] + = 919.6.

[0341] Step 3: Compound DL-4-2 (55 mg, 0.06 mmol), compound INT1-8 (18 mg, 0.06 mmol), 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (25 mg, 0.06 mmol), and N,N-diisopropylethylamine (21 mg, 0.15 mmol) were added to DMF (2 mL), and the reaction solution was reacted with stirring at room temperature for 1 h. The reaction was monitored by LCMS, and the reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the formate salt (15 mg) of the target compound DL-4. LCMS (ESI) [M+H] + = 1169.7. 1 H NMR (400 MHz, DMSO-d 6) δ 10.97 (s, 1H), 9.11 (s, 2H), 8.67 - 8.57 (m, 2H), 8.23 - 8.17 (m, 2H), 8.09 - 8.03 (m, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.35 - 7.29 (m, 2H), 7.07 (t, J = 8.6 Hz, 2H), 6.85 - 6.78 (m, 1H), 5.10 (dd, J = 13.2, 5.0 Hz, 1H), 4.62 - 4.54 (m, 2H), 4.44 - 4.28 (m, 4H), 4.23 - 4.14 (m, 2H), 4.10 - 4.07 (m, 2H), 3.74 - 3.69 (m, 4H), 3.41 (s, 3H), 3.30 - 3.23 (m, 2H), 3.20 - 3.14 (m, 1H), 2.96 - 2.89 (m, 1H), 2.81 - 2.73 (m, 3H), 2.60 - 2.57 (m, 1H), 2.56 - 2.52 (m, 6H), 2.48 - 2.43 (m, 2H), 2.38 - 2.32 (m, 2H), 2.02 - 1.95 (m, 2H), 1.86 - 1.75 (m, 3H), 1.72 - 1.66 (m, 2H), 1.60 - 1.52 (m, 1H), 1.48 - 1.40 (m, 6H), 1.33 - 1.25 (m, 2H), 0.84 (t, J = 6.8 Hz, 12H).

[0342] Example 3.5: Synthesis of (2S)-N-(2-(((2-((3-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)propyl)(methyl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-2-((S)-2-(2,2-dimethyl-4-(4)-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanamide)-3-methylbutanamide)-6-(dimethylamino)hexanamide (DL-5) [Chemical]

[0343] Step 1: Dissolve compound INT2 (100 mg, 0.20 mmol) in DMF (2 mL). Next, sequentially add compound INT5 (128 mg, 0.20 mmol), 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (76 mg, 0.20 mmol), and N,N-diisopropylethylamine (26 mg, 0.50 mmol). After the addition is complete, stir the reaction solution at room temperature for 1 h, monitor the reaction by LCMS, add methyl tert-butyl ether (80 mL) to the reaction solution, let it stand for 10 min, then discard the supernatant, directly concentrate the residue under reduced pressure with an oil pump to remove the excess solvent, and directly purify the crude product by preparative TLC (DCM:MeOH = 8:1) to obtain the target compound DL-5-1 (150 mg). LCMS (ESI) [M+H] + = 1119.5. 1 H NMR (400 MHz, DMSO-d 6) δ 10.97 (s, 1H), 8.85 (t, J = 8.3 Hz, 1H), 8.67-8.61 (m, 1H), 8.38-8.21 (m, 2H), 8.19-8.10 (m, 1H), 8.07-7.98 (m, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.84 (d, J = 7.5 Hz, 1H), 7.75-7.70 (m, 1H), 7.70-7.64 (m, 2H), 7.51 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.42-7.40 (m, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.33-7.29 (m, 1H), 7.20-7.16 (m, 2H), 6.92-6.83 (m, 1H), 5.10 (dd, J = 13.6, 5.2 Hz, 1H), 4.61-4.56 (m, 2H), 4.41 (d, J = 5.8 Hz, 2H), 4.35-4.28 (m, 2H), 4.24-4.20 (m, 1H), 4.10 (s, 2H), 3.92-3.84 (m, 1H), 3.75-3.68 (m, 3H), 3.63-3.59 (m, 2H), 2.94-2.90 (m, 1H), 2.86 (s, 2H), 2.79 (s, 1H), 2.59-2.54 (m, 2H), 2.41-2.37 (m, 2H), 2.29-2.26 (m, 6H), 2.02-1.97 (m, 2H), 1.71-1.66 (m, 2H), 1.45-1.41 (m, 2H), 1.31-1.28 (m, 2H), 0.91-0.78 (m, 12H).

[0344] Step 2: Dissolve compound DL-5-1 (100 mg, 0.09 mmol) in DMF (2 mL), add diethylamine (26 mg, 0.36 mmol) to the reaction solution, react the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, and directly concentrate the reaction solution under reduced pressure with an oil pump to obtain the target compound DL-5-2 (80 mg), which was used directly in the next reaction. LCMS (ESI) [M+H] += 897.5、

[0345] Step 3: Dissolve compound DL-5-2 (80 mg, 0.09 mmol) and compound INT7 (23 mg, 0.09 mmol) in DMF (3 mL), and add N,N-diisopropylethylamine (21 mg, 0.22 mmol) and 2-(7-azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (25 mg, 0.09 mmol). React the reaction solution with stirring at room temperature for 1 h, monitor the reaction by LCMS, and purify the reaction solution by preparative high performance liquid chromatography (acetonitrile:H 2 O, containing 0.05% FA = 5% - 50%) to obtain the formate salt (13 mg) of the target compound DL-5. LCMS (ESI) [M+H] + = 1218.8. 1 H NMR (400 MHz, DMSO-d 6) δ 10.98 (s, 1H), 9.47 (s, 2H), 8.93 (s, 1H), 8.85 (d, J = 7.0 Hz, 1H), 8.64 (t, J = 6.4 Hz, 1H), 8.27 (t, J = 5.3 Hz, 1H), 8.20 (s, 1H), 8.06 (d, J = 7.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.51 (s, 1H), 7.43 (t, J = 6.7 Hz, 2H), 7.23 - 7.13 (m, 2H), 6.93 - 6.84 (m, 1H), 5.10 (dd, J = 13.2, 5.0 Hz, 1H), 4.62 - 4.52 (m, 2H), 4.44 - 4.39 (m, 4H), 4.31 (d, J = 17.5 Hz, 1H), 4.28 - 4.22 (m, 1H), 4.14 (t, J = 7.9 Hz, 1H), 4.09 (s, 2H), 3.74 - 3.69 (m, 2H), 3.44 (s, 3H), 2.96 - 2.89 (m, 1H), 2.82 (d, J = 29.0 Hz, 3H), 2.64 - 2.53 (m, 4H), 2.43 - 2.36 (m, 2H), 2.35 (s, 6H), 2.21 - 2.10 (m, 2H), 2.05 - 1.96 (m, 2H), 1.80 - 1.72 (m, 1H), 1.71 - 1.63 (m, 2H), 1.62 - 1.38 (m, 4H), 1.30 - 1.23 (m, 4H), 1.22 (d, J = 3.2 Hz, 6H), 0.87 (t, J = 7.1 Hz, 6H).

[0346] Example 4. Preparation and Identification of Antibodies 4.1.1 Preparation of B7H3 Antigen and Anti - B7H3 Antibody The nucleic acid sequence of amino acids 29 - 245 of human 2Ig B7H3 (NCBI protein number NP_001316557.1) was selected, and among them, a detectable 6×His tag was added to the C-terminus, which was named human B7H3-2IgG-his. The nucleic acid sequence of amino acids 27 - 461 of human 4Ig B7H3 (Uniprot protein number Q5ZPR3-1) was selected, and among them, a purified 6×His tag was added to the C-terminus, which was named human B7H3-4IgG-his. The nucleic acid sequence of amino acids 27 - 465 of monkey B7H3 4Ig (Uniprot protein number F7G5V3) was selected, and among them, a purified 6×His tag was added to the C-terminus, which was named monkey B7H3-4IgG-his. B7H3-C1 referred to the antibody with the number M30-H1-L4 in CN103687945B, and B7H3-C2 referred to the antibody with the number mAb-C-DUBA in CN109069633A.

[0347] The genes of the above three types of B7H3 antigens were each synthesized, and the codons were optimized to synthesize the genes of B7H3-C1 and B7H3-C2 antibodies (Shanghai Sangon Biotech Co., Ltd.), which were constructed into the PTT5 expression vector, and a large amount of plasmid was extracted. The expression vectors prepared by extraction were transiently expressed in HEK293F for 7 days respectively, and the antigen protein, B7H3-C1 antibody and B7H3-C2 antibody were prepared by Protein A treatment of the expression supernatant.

[0348] The constant region amino acid sequence of IgG1 (SEQ ID NO: 9) was added to SEQ ID NO: 1 and SEQ ID NO: 5 of the VH amino acid sequence respectively. After codon optimization and gene synthesis (Shanghai Sangon Biotech Co., Ltd.), it was then constructed into the PTT5 vector and named PTT5-01-CH and PPT5-02-CH respectively. The kappa constant region sequence (SEQ ID NO: 10) was added to SEQ ID NO: 2 and SEQ ID NO: 6 of the VL amino acid sequence. After codon optimization and gene synthesis (Shanghai Sangon Biotech Co., Ltd.), it was then constructed into the PTT5 vector and named PTT5-01-CL and PTT5-02-CL respectively. The antibody B7H3 antibody expression plasmids PTT5-01-CH / PTT5-01-CL and PTT5-02-CH / PTT5-02-CL were co-transfected into HEK293F cells for expression with the PEI max reagent, and shaken at 37 °C in 5% CO 2 for 7 days, and the supernatant was collected and purified by ProA magnetic beads. The obtained anti-B7H3 antibodies were named 1D1-01 (its heavy chain sequence is SEQ ID NO: 3 and its light chain sequence is SEQ ID NO: 4) and 2E3-02 (its heavy chain sequence is SEQ ID NO: 7 and its light chain sequence is SEQ ID NO: 8) respectively.

[0349] 4.1.2 Detection of anti-B7H3 antibody protein affinity The affinity of the B7H3 fully humanized antibody for human B7H3-4IgG-his and monkey B7H3-4IgG-his proteins was detected by ELISA. Specific experimental operations: Dilute human B7H3-4IgG-his or monkey B7H3-4IgG-his protein with carbonate buffer at pH 9.6 until the final concentration reaches 1 μg / mL, and add 100 μL / well to each well of a 96-well microplate, coat overnight at 4°C, discard the coating solution, wash once with PBST, add 100 μL of PBS (containing 2% BSA) to each well, block at 37°C for 1 hour. Next, add 100 μL of B7H3 fully humanized antibody diluted with PBS (containing 2% BSA) (starting from 10 μg, 3-fold dilution, two parallel wells) to each well, incubate at 37°C for 2 h, wash 3 times with PBST, add anti-human Fc antibody labeled with horseradish peroxidase (diluted 1:10000 with PBS), 100 μL / well, act at 37°C for 1 hour, and wash the plate 5 times with PBST. Add TMB chromogenic solution to develop color, add stop solution at 100 μL / well, 37°C, 7 min to stop the reaction, and measure the absorbance at 450 nm at 50 μL / well. The EC50 value was calculated from the raw data.

[0350] The results are shown in Figures 1A, 1B, 2A and 2B, and the detailed results are shown in Tables 1-1 and 1-2. 1D1-01 and 2E3-02 both have relatively high affinity for human B7H3-4IgG, which are 43.56 pM and 17.54 pM respectively. Among them, 2E3-02 has stronger affinity than 51.85 pM of B7H3-C1. 1D1-01 and 2E3-02 have relatively high binding ability for monkey B7H3-4IgG, which are 35.82 pM and 27.72 pM respectively.

Table 14

Table 15

[0351] 4.1.3 Detection of the cellular affinity of anti-B7H3 antibody The B7H3 protein is highly expressed in various types of solid tumor cells. The affinity between the fully humanized B7H3 antibody and tumor cells is detected by the FACS experimental method. Specific experimental operations: MCF-7 breast cancer cells (from Nanjing Kebai, product number CBP60380), A549 human non-small cell lung cancer (from ATCC, product number CRM-CCL-185), and PC3 human prostate cancer cells (from ATCC, product number CRL-1435) that have grown to the logarithmic phase are digested with pancreatin and resuspended in PBS buffer containing 2% BSA at a concentration of 5×10 5 cells per milliliter. 100 μL of the cell suspension is added to a 96-well plate. B7H3-C1, B7H3-C2, 1D1-01, and 2E3-02 are diluted with PBS buffer containing 2% BSA, with an initial concentration of 40 μg / mL, and then serially diluted three-fold. Next, 100 μL of the antibody suspension is added to the 96-well plate and mixed uniformly. Incubate at 4°C for 1 hour, wash twice with pre-cooled PBS at 300 μL / well each time, and centrifuge at 500 g for 5 minutes. Add 100 μL / well of a 1:50 fluorescent secondary antibody, APC anti-human IgG or PE anti-human IgG (purchased from Biolegend), and mix uniformly. Incubate at 4°C for 0.5 hour, wash twice with pre-cooled PBS at 300 μL / well each time, centrifuge at 500 g for 5 minutes, resuspend in 500 μL of PBS, and detect the mean fluorescence signal value using a Beckman flow cytometer. Calculate the EC50 value based on the mean fluorescence signal value, and the results are shown in Figures 3A, 3B, 4A, 4B, and 5, and the specific data are shown in Tables 2-1 and 2-2.

Table 16

Table 17

[0352] As shown in Tables 2-1 and 2-2, in B7H3-high-expressing tumor cells MCF-7, the affinities of 1D1-01 and 2E3-02 were 0.524 nM and 0.398 nM, respectively, both much higher than that of B7H3-C1. At the same time, the maximum fluorescence signal values were 12% and 23% higher than that of B7H3-C1, respectively. Among them, the maximum fluorescence signal value of 2E3-02 was 2.78 times higher than that of control antibody 2. In the expression of tumor cells A549 in B7H3, the affinities of 1D1-01 and 2E3-02 were 0.322 nM and 0.150 nM, respectively, both much higher than that of B7H3-C1. At the same time, the maximum fluorescence signal values were 36% and 77% higher than that of B7H3-C1, respectively. In B7H3-low-expressing tumor cells PC-3, the affinity of 2E3-02, 0.06 nM, was much higher than that of B7H3-C1, 0.618 nM, and also higher than that of B7H3-C1, 0.167 nM. At the same time, the maximum fluorescence signal values were 1.51 times and 4.48 times that of B7H3-C1 and B7H3-C2, respectively. Overall, anti-B7H3 antibodies 1D1-01 and 2E3-02 have better tumor cell affinities than B7H3-C1 and B7H3-C1.

[0353] 4.1.4 Detection of Endocytosis Activity of Anti-B7H3 Antibody The strength of cellular endocytosis of anti-B7H3 antibody in A549 human non-small cell lung cancer cells was detected using the FACS method. A549 cells grown to the logarithmic growth phase were digested with pancreatin, the cells were collected by centrifugation, and washed three times with pre-cooled PBS. The cells were resuspended in PBS (containing 1% BSA), and the anti-B7H3 antibody to be detected was added respectively, with a concentration of 10 μg / mL, and incubated at 4 °C for 1 hour. The cells were collected by centrifugation, washed three times with PBS, resuspended in DMEM + 10% FBS, and the resuspended cells were divided into four parts, and incubated at 37 °C for 0, 1, 2, and 4 hours respectively. After the incubation phase was completed, the cells were collected by centrifugation, washed three times with pre-cooled PBS, resuspended in 50 μL of 1% BSA (in PBS), 1 μL of the fluorescent secondary antibody APC anti-human IgG (Biolegend) was added to each well, mixed uniformly, and incubated at 4 °C for 0.5 hour. The cells were collected by centrifugation, washed three times with pre-cooled PBS, resuspended in 200 μL of PBS, and loaded for detection. Formula: Endocytosis rate (%) = [1 - (average fluorescence value of the sample detected at that time point - average fluorescence value of the negative control sample at that time point) / (average fluorescence value of the sample detected at 0 hour - average fluorescence value of the negative control sample at 0 hour)] × 100. The results of 1D1-01 and 2E3-02 are shown in Figures 6A and 6B respectively. The anti-B7H3 antibodies 1D1-01 and 2E3-02 have relatively strong endocytosis activity in A549 tumor cells, reaching about 40% at 4 hours, which is equivalent to that of antibody B7H3-C1.

[0354] Example 5. Synthesis of Antibody-Drug Conjugate (ADC) Example 5.1 Preparation of B7H3-ADC Example 5.1.1 Preparation of B7H3-ADC-001 Take 25 mL of 2E3-02 antibody (anti-B7H3, concentration 28.5 mg / mL, 20 mM acetate buffer), add 25 mL of 20 mM acetate buffer to dilute it, then add 1 mL of aqueous solution containing 0.25 M EDTA and mix uniformly. After adjusting the sample pH to 7.6 with 0.5 M disodium hydrogen phosphate aqueous solution, add a 4.5-fold equivalent amount of 20 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) solution of the antibody and mix uniformly, and react at room temperature for 90 min. Finally, add a 10-fold equivalent amount of compound DL-1 dissolved in DMSO to the antibody, mix uniformly, and then continue the reaction at room temperature for 2 h. After the reaction is completed, use a 30 KDa ultrafiltration tube to replace the sample with 10 mM histidine buffer at pH 5.5, remove low molecular weight substances, and finally concentrate the sample to obtain solution B7H3-ADC-001 containing anti-B7H3 antibody ADC. Measured by mass spectrometry, the DAR value was 8.0.

Chemical formula

[0355] Example 5.2 Preparation of HER2-ADC Example 5.2.1: Preparation of HER2-ADC-001 Take 0.8 mL of trastuzumab (20.4 mg / mL), dilute it with 0.008 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), adjust the pH to 7.81 with 0.5 M Na 2 HPO 4 solution, add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0303 mL, 0.606 μmol) solution and mix uniformly, and leave it at room temperature for 90 min. Add a solution of DL-2 (1.654 mg) in dimethyl sulfoxide (0.1323 mL) to the above solution, mix uniformly, let it stand at room temperature for 2 h, and after completion, change the solution by a centrifugal filtration tube (Merck, Amicon Ultra-15). The composite product HER2-ADC-001 of DL-2 and trastuzumab was obtained. Measured by mass spectrometry, the DAR value was 8.0.

Chemical formula

Table 18

[0356] In the table, LC represents the antibody light chain, HC represents the antibody heavy chain, DAR1 represents a complex containing one drug linker conjugated by the light chain or heavy chain, DAR2 represents a complex containing two drug linkers conjugated by the light chain or heavy chain, and DAR3 represents a complex containing three drug linkers conjugated by the light chain or heavy chain. The explanations of LC, HC, DAR1, DAR2, and DAR3 below are as above.

[0357] One trastuzumab light chain was conjugated to one toxin molecule (the ratio of LC+DAR1 was 100%), and one trastuzumab heavy chain was conjugated to three toxin molecules (the ratio of HC+DAR3 was 100%). As a result, the antibody-drug conjugation ratio (DAR value) of HER2-ADC-001 (DAR8) was calculated to be 8.0.

[0358] Method for detecting the antibody-drug conjugation ratio (DAR value): Take 50 μg of the ADC sample, dilute it to 0.5 mg / mL with ultrapure water, add 1 μL of 1 M DTT, mix uniformly, then centrifuge and take the supernatant for sampling injection.

[0359] Liquid phase parameters:

Table 19

[0360] Mass spectrometry parameters:

Table 20

[0361] Example 5.2.2: Preparation of HER2-ADC-002 In the same manner as in Example 5.2.1, DL-2 was replaced with DL-3 to obtain the conjugate product HER2-ADC-002 of DL-3 and trastuzumab. Measured by mass spectrometry, the DAR value was 8.0.

[0362]

Chem.

Table 21

[0363] Example 5.2.3: Preparation of HER2-ADC-003 In the same manner as in Example 5.2.1, DL-2 was replaced with DL-1 to obtain a conjugate product HER2-ADC-003 of DL-1 and trastuzumab. Measured by mass spectrometry, the DAR value was 8.0.

Chem.

Table 22

[0364] Example 5.2.4: Preparation of HER2-ADC-004 In the same manner as in Example 5.2.1, DL-2 was replaced with DL-5 to obtain a conjugate product HER2-ADC-004 of DL-5 and trastuzumab. Measured by mass spectrometry, the DAR value was 8.0.

[0365]

Chem.

Table 23

[0366] Example 5.2.5: Preparation of HER2-ADC-005 In the same manner as in Example 5.2.1, DL-2 was replaced with DL-4 to obtain a conjugate product HER2-ADC-005 of DL-4 and trastuzumab. Measured by mass spectrometry, the DAR value was 7.0.

[0367]

Chem.

Table 24

[0368] Example 5.2.6: Preparation of HER2-ADC-006

Chemical Structure

[0369]

Chemical Structure

Table 25

[0370] Example 6. In Vitro Cell Activity Test of Biological Active Compound (Payload) The structure of control compound 1 is as follows:

Chemical Structure

[0371]

Table 26

[0372]

Table 27

[0373] Example 7. In vitro cell activity test of antibody-drug conjugate (ADC) Example 7.1 ADC growth inhibitory activity test on in vitro cells (NCI-H358 tumor cells) The NCI-H358 tumor cells were digested by trypsin in the usual way, and the cells were collected in a tube, counted, resuspended in the corresponding detection medium (containing 2% FBS), and 5000 cells / well were added to a 96-well plate. The ADC diluted with the 2% FBS medium was added to the 96-well plate at 100 μL, starting from a concentration of 150 μg / mL and diluted 3-fold (12 concentration gradients). After culturing at 37 °C in 5% CO 2 for 7 days, 20 μL of CCK8 reagent was added to each well, reacted for 4 hours, and read with a microplate reader (the detection wavelength was 450 nm). The test results showed that the ADC molecule of the present disclosure had a tumor cell killing effect.

[0374] Example 7.2: ADC growth inhibition activity test on in vitro cells (N87 tumor cells) I. Test method: The N87 cells were resuscitated and cultured with 1640 + 10% FBS, seeded in a 96-well flat-bottom plate at a density of 5000 / well and 100 μL / well, and cultured overnight in a 37 °C incubator. The next day, the ADC molecule to be measured was diluted with 1640 + 10% FBS. The highest concentration of the ADC molecule to be measured - 002 was 200 nM, and the highest concentrations of the remaining 4 samples were 2000 nM, and they were diluted 10-fold at 4-fold. 100 μL of the diluted drug was added to the cell culture wells. After incubating at 37 °C for 4 days, 50 μL of Cell titer glo substrate was added to each well, incubated for 5 minutes, and then the fluorescence value was detected. Among them, the suppliers of the key materials are shown in Table 3.

[0375]

Table 28

[0376] II. Experimental conclusion: The test results showed that the ADC molecules of the present disclosure all had obvious growth inhibition activity in the N87 tumor cell line. The test results are shown in Table 4.

[0377]

Table 29

[0378] Example 8. In Vivo Activity Test of Antibody-Drug Conjugate (ADC) Example 8.1 Efficacy Experiment of Antibody-Drug Conjugate (ADC) against NCI-N87 Xenograft Tumor (Dose 1 mg / kg) 1. Experimental Materials Test compounds: HER2-ADC-001, HER2-ADC-003, HER2-ADC-006, with physiological saline as the negative control. Experimental cells: NCI-N87 cells, purchased from Nanjing Kebai Biotechnology Co., Ltd. Experimental animals: Balb / c nu nude mice, female, 5 - 6 weeks old, purchased from Vital River Laboratory Animal Technology Co., Ltd.

[0379] 2. Experimental Plan 2.1. Cell Treatment The NCI-N87 cells were cultured in a 15-cm diameter culture dish with 1640 medium containing 10% FBS. When the confluence reached about 80% - 90%, they were digested with trypsin-EDTA, washed twice with PBS, centrifuged, resuspended in pre-cooled PBS, counted with a cell counter, and diluted with PBS to a cell concentration of 5×10 7 / mL. 2.2. Tumor Cell Implantation The Balb / c nu mice were adapted to the laboratory environment for 2 - 5 days, and NCI-N87 cells were subcutaneously inoculated into the right flank. The inoculated cell amount was 5×10 6 / mouse, and the inoculation volume was 0.2 mL (containing 50% Matrigel). When the tumor grew to about 200 mm 3 , the experiment was conducted. 2.3. Animal Administration and Detection The tumor-bearing nude mice were grouped and administered according to the following method:

Table 30

[0380] 3. Experimental results The test results showed that all the ADCs of the present disclosure exhibited a relatively obvious tumor suppression effect. Among them, the tumor suppression effects of HER2-ADC-001 and HER2-ADC-003 were significantly superior to those of the control compound HER2-ADC-006. During the administration period, the animals in each group had no obvious weight loss and obvious drug toxicity. The specific results are shown in Table 6, Figures 7A and 7B.

[0381]

Table 31

[0382] Example 8.2 Pharmacodynamic experiment of antibody-drug conjugate (ADC) against NCI-N87 transplanted tumor (dosages 2 mg / kg and 6 mg / kg) 1. Experimental materials Test compounds: HER2-ADC-001 and HER2-ADC-003, with physiological saline as the negative control. Experimental cells: NCI-N87 cells, purchased from Nanjing Kebai Biotechnology Co., Ltd. Experimental animals: Balb / c nu nude mice, female, 5 - 6 weeks old, purchased from Vital River Laboratory Animal Technology Co., Ltd.

[0383] 2. Experimental plan 2.1. Cell treatment The NCI-N87 cells were cultured in a 15-cm diameter culture dish with RPMI 1640 medium containing 10% FBS. When the confluence reached about 80% - 90%, the cells were digested with trypsin-EDTA, washed twice with PBS, centrifuged, resuspended in pre-cooled PBS, counted using a cell counter, and diluted with PBS to a cell concentration of 5×10 7 / mL. 2.2. Transplantation of tumor cells Balb / c nu mice were allowed to adapt to the laboratory environment for 2 - 5 days, and then NCI-N87 cells were subcutaneously inoculated into the right flank. The inoculation cell amount was 5×10 6 / mouse, and the inoculation volume was 0.2 mL (containing 50% Matrigel). When the tumor grew to about 165 mm 3 , the experiment was conducted. 2.3. Animal administration and detection The tumor-bearing nude mice grouped were administered according to the following method:

Table 32

[0384] 3. Experimental results The test results showed that all the ADCs of the present disclosure exhibited a relatively obvious tumor suppression effect. Among them, the tumor suppression rate of HER2-ADC-001 at 2 mg / kg was 62.47%, and the tumor suppression rate at 6 mg / kg was 98.30%. The tumor suppression rate of HER2-ADC-003 at 2 mg / kg was 44.08%, and the tumor suppression rate at 6 mg / kg was 94.15%. During the administration period, the animals in each group had no obvious weight loss and no obvious drug toxicity. The specific results are shown in Table 8, Figures 8A and 8B.

[0385]

Table 33

[0386] Example 9 ADC Plasma Stability Test Solution Preparation: Each ADC prepared in Example 5 was diluted to a concentration of 2 mg / mL with PBS.

[0387] Sample Incubation: The bacteriostatic agent ProClin was added to human plasma so that the final concentration of ProClin was 0.1%, and the operation was performed aseptically. Each ADC prepared in Example 5 was added to the above sterile plasma at a final concentration of 200 μg / mL, respectively, and placed in a 37°C cell incubator and incubated at a vibration speed of 80 times per minute. Incubate for 0 days and 14 days, take out the ADC sample, add 100 μL of Protein A to each tube, adsorb for 2 h, elute, and obtain the ADC after incubation. The DAR value of the ADC after incubation was detected (the detection and calculation methods were the same as those in Example 5.2.1), and the plasma stability of the test sample was determined.

[0388] The test results showed that for each ADC prepared in Example 5, the DAR value was basically maintained during the plasma incubation process, and the ADC having a maleimide linker (for example, the control compound HER2-ADC-006) showed a significant decrease in the DAR value after 14 days of incubation. From this, it was shown that all the ADCs provided by the present invention have better plasma stability, and thus can reduce the shedding of biologically active compounds in non-target tissues.

[0389] Example 10 Stability Test of Antibody-Drug Conjugates in Tumor Tissue Homogenate and Muscle Tissue Homogenate Referring to Example 6.11 of Patent WO2022 / 170971A1, the lysis of each ADC prepared in Example 5 in tumor tissue / tumor microenvironment was simulated, and the stability of each ADC prepared in Example 5 in 22RV1 Balb / c nu nude mouse tumor tissue homogenate, Balb / c mouse muscle tissue homogenate, and homogenate matrix physiological saline was examined in vitro, and the payload was measured in the incubation system. The results of the tumor tissue homogenate were compared with the results of plasma stability, and the release efficiency of the payload in the tumor tissue / tumor microenvironment was evaluated.

[0390] The test results showed that each ADC prepared in Example 5 could release a large amount of payload in the tumor tissue homogenate, but there was no obvious release in the case of the normal tissue homogenate. Therefore, it was shown that the ADCs provided by the present invention can increase the efficient release of biologically active compounds at the tumor site, and thus have a higher therapeutic index.

[0391] Unless otherwise specified, the terms used in the present invention have meanings generally understood by those skilled in the art.

[0392] The embodiments described in the present invention are used only for illustrative purposes and do not limit the claims of the present invention. Those skilled in the art can make various other substitutions, changes, and improvements within the scope of the present invention. Therefore, the present invention is not limited to the above embodiments and is limited only by the claims.

Claims

1. An antibody-drug conjugate as shown in formula (I): 【Chemistry 1】 During the ceremony, Tb is an antibody or an antigen-binding fragment thereof; q is L １ -L ２ -L ３ -L ４ - the number of Tb coupled to D, D is a fragment of a biologically active compound of the proteolytic agent class; L １ is a linker unit, L ２ is the linkage unit, L ３ AA １ or AA １ The amino acid residues are selected from short peptides consisting of 2 to 10 amino acid residues, each of which is selected from the group consisting of natural amino acid residues, non-natural amino acid residues, AA １ or a stereoisomer thereof, A.A. １ The structure of the amino acid residues shown in is as follows: 【Chemistry 2】 During the ceremony, R ａ , R ｂ are each independently hydrogen or 【Chemistry 3】 and R ａ , R ｂ is not hydrogen at the same time, Or, R ａ and R ｂ together with the carbon atom to which they are commonly attached form a 4- to 10-membered heterocyclic ring, said 4- to 10-membered heterocyclic ring being bound by one or more R ０ and optionally replaced by r,r １ are each independently selected from any integer between 0 and 20; R ｍ１ , R ｎ１ are each independently selected from hydrogen, a C1-6 alkyl group, or a C3-6 cycloalkyl group; Or, R ｍ１ and R ｎ１ together with the nitrogen atom to which they are commonly attached form a 4- to 10-membered heterocyclic ring, said 4- to 10-membered heterocyclic ring being bound by one or more R ０’ and optionally replaced by R ０ , R ０’ are each independently a C1-6 alkyl group, a C3-6 cycloalkyl group, or -NR ｍ２ R ｎ２ or a 4- to 10-membered heterocyclic group optionally substituted by a C1-6 alkyl group; R ｍ２ , R ｎ２ are each independently selected from hydrogen or a C1-6 alkyl group; L ４ is absent or present, L ４ If there is L ４ teeth, 【Chemistry 4】 The first place was chosen by L ３ and position 2 is linked to D; Or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

2. One or more of the following conditions are met: (1) The q is selected from any number between 0.1 and 12.0, preferably, the q is selected from any number between 1.0 and 10.0, more preferably, the q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; (2) Said L １ teeth, 【Chemistry 5】 , wherein the 1st position is linked to Tb via an S atom, and the 2nd position is L ２ is connected to (3) Said L ２ teeth, 【Chemistry 6】 The first place was chosen by L １ It is connected to, and the second place is L ３ is connected to Preferably, the L ２ teeth, 【Chemistry 7】 The first place was chosen by L １ It is connected to, and the second place is L ３ is connected to (4) The n is selected from any integer between 0 and 10, and preferably, the n is selected from 0, 1, 2, or 3; (5) The above Y is -CH ２ -or-OCH ２ CH ２ Selected from - (6) The X is -CR ｍ R ｎ - or -NR ｍ Selected from - (7) The above R ｍ , R ｎ are each independently selected from hydrogen, a C1-4 alkyl group, a C2-4 alkenyl group, a C2-4 alkynyl group, a C3-6 cycloalkyl group, or a 3- to 6-membered heterocyclic group; Alternatively, the R ｍ and R ｎ together with the carbon atom to which they are commonly attached form a 3- to 6-membered carbocyclic or 3- to 6-membered heterocyclic ring; Preferably, the R ｍ , R ｎ are each independently selected from hydrogen or a methyl group; (8) Said L ３ AA １ or AA １ Preferably, the L is selected from a dipeptide, tripeptide, or tetrapeptide containing the amino acid residues shown in ３ AA １ , Val-AA １ , Ala-AA １ , Gly-AA １ , A.A. １ -Gly, AA １ -Ala, Val-AA １ -Gly, Ala-AA １ -Gly, Gly-AA １ -Gly, or Val-AA １ -Ala, a dipeptide, or a tripeptide, and more preferably, the L ３ is Val-AA １ , Val-AA １ -Ala or Val-AA １ -Gly, and particularly preferably selected from dipeptides or tripeptides represented by L ３ is Val-AA １ -Gly, (9) The R ａ , R ｂ Of these, one is H and the other is 【Chemistry 8】 Alternatively, the R ａ and R ｂ together with the carbon atom to which they are commonly attached, R ０ forming a 5- to 6-membered heterocyclic ring substituted with (10) The r and r １ are each independently selected from 0, 1, 2, 3, 4, or 5; Preferably, the r and r １ are each independently selected from 0 or 4; More preferably, r is 0, and r １ is 4, (11) The R ｍ１ , R ｎ１ are each independently selected from hydrogen or a C1-6 alkyl group, or ｍ１ and R ｎ１ together with the nitrogen atom to which they are commonly attached, R ０’ forming a 5- to 6-membered heterocycle optionally substituted with Preferably, the R ｍ１ , R ｎ１ are each independently selected from hydrogen, a methyl group, an ethyl group, an n-propyl group, or an n-butyl group, or ｍ１ and R ｎ１ together with the nitrogen atom to which they are commonly attached, R ０’ and more preferably, R ｍ１ , R ｎ１ are each independently selected from a methyl group, an ethyl group, an n-propyl group, or an n-butyl group; (12) The R ０ , R ０’ each independently represents a C1-6 alkyl group, -NR ｍ２ R ｎ２ or a 5- to 6-membered heterocyclic group optionally substituted with a C alkyl group; (13) The R ｍ２ , R ｎ２ is a methyl group, (14) The biologically active compounds of the protein degrader class are molecules capable of causing degradation of GSPT1 family proteins. The antibody-drug conjugate according to claim 1, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

3. One or more of the following conditions are met: (1) Said L １ -L ２ teeth, 【Chemistry 9】 , wherein the 1st position is linked to Tb via an S atom, and the 2nd position is L ３ is connected to Preferably, the L １ -L ２ teeth, 【Chemistry 10】 , wherein the 1st position is linked to Tb via an S atom, and the 2nd position is L ３ is connected to (2) Said L ３ teeth, 【Chemistry 11】 The first place was chosen by L ２ It is connected to, and the second place is L ４ or linked to D, Preferably, the L ３ teeth, 【Chemistry 12】 The first place was chosen by L ２ It is connected to, and the second place is L ４ or linked to D, (3) The above AA １ The amino acid residues shown in 【Chemistry 13】 Selected from Preferably, the AA １ The amino acid residues shown in 【Chemistry 14】 Selected from 3. The antibody-drug conjugate according to claim 1 or 2, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

4. 【Chemical 15】 is chosen from: 【Table 1】 1st place is L ２ , and the 2nd position is linked to D, and AA １ is as defined in any one of claims 1 to 3, Preferably, 【Chemistry 16】 is chosen from: 【Table 2】 1st place is L ２ and the second position is linked to D, More preferably, 【Chemistry 17】 is chosen from the following: 【Table 3】 Position 1 is linked to Tb via an S atom, position 2 is linked to D, and Y, X, AA １ and n are as defined in any one of claims 1 to 3, More preferably, 【Chemistry 18】 is chosen from: 【Table 4】 【change】 Position 1 is linked to Tb via an S atom and position 2 is linked to D; The antibody-drug conjugate according to any one of claims 1 to 3, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

5. D is a structural unit as shown in formula II, wherein the 1-position is L ４ Or L ３ is connected to 【Chemistry 19】 During the ceremony, R １ is selected from hydrogen, deuterium, halogen, a cyano group, an amino group, a nitro group, a C1-4 alkoxy group, or a C1-4 alkyl group; Z is -NH-, -CR ２ａ R ３ａ -, or -C(O)-, R ２ａ , R ３ａ are each independently selected from F or OH; U １ , U ２ each independently represents -CH ２ - or -C(O)-, and U １ , U ２ At the same time, -CH ２ -But, A is O, S, NR ９ , 【Chemistry 20】 The first place was chosen by L ４ Or L ３ and the 2-position is connected to a benzene ring, or the 1-position is connected to a benzene ring and the 2-position is L ４ Or L ３ is connected to R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ are each independently selected from hydrogen, deuterium, a C1-4 alkyl group, a C3-6 cycloalkyl group, or a 3- to 6-membered heterocyclic group; Or, R ２ , R ３ together with the carbon atoms to which they are attached form a 3- to 7-membered carbocyclic or 3- to 7-membered heterocyclic ring; R ４ , R ５ form together with the carbon and nitrogen atoms to which they are attached a 4- to 7-membered heterocyclic ring; R ５ , R ６ together with the carbon atoms to which they are attached form a 3- to 7-membered carbocyclic or 3- to 7-membered heterocyclic ring; R ７ , R ８ together with the carbon atoms to which they are attached form a 3- to 7-membered carbocyclic or 3- to 7-membered heterocyclic ring; m, p, and y are each independently selected from any integer between 0 and 8; W and V each independently represent a direct bond, O, S, or NR ９ Selected from R ９ is selected from hydrogen, deuterium, a C1-4 alkyl group, or a C3-6 cycloalkyl group; The antibody-drug conjugate according to any one of claims 1 to 4, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

6. One or more of the following conditions are met: (1) The R １ is selected from hydrogen, halogen, or a C1-4 alkyl group, and preferably, １ is a halogen, (2) the Z is -NH-, (3) Said U １ , U ２ are each independently -CH ２ - or -C(O)-, and U １ , U ２ are not the same, and preferably, １ Ha-CH ２ -, and said U ２ is —C(O)—, (4) The above A is 【Chemistry 21】 And the first place is L ４ Or L ３ The 2-position is connected to a benzene ring, and W, V, R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ The definitions of m, p and y are as defined in claim 5, and preferably, A is -O(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ -, -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, -O(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ O-, -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ O-, -S(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ -, -S(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, -NH(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ -, -NH(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, -O(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ O (CH ２ ) ｙ - or -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ O (CH ２ ) ｙ -, preferably, A is -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ - or -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ O (CH ２ ) ｙ -, preferably, A is -OCH ２ C(O)N(CH ３ ) (CH ２ ) ３ -or-OCH ２ C(O)N(CH ３ ) (CH ２ ) ２ O (CH ２ ) ２ Selected from - (5) W is O, S or NR ９ Preferably, W is O; (6) The V is a direct bond, O, S, or NR ９ Preferably, V is selected from a direct bond or O; (7) The above R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ are each independently selected from hydrogen, deuterium, or a C1-4 alkyl group; (8) m, p, and y are each independently selected from 0, 1, 2, 3, or 4; (9) The R ９ is selected from hydrogen, deuterium, or a methyl group; The antibody-drug conjugate according to claim 5, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

7. D is a structural unit shown in II-1, II-2, or II-3; 【Chemical 22】 In the formula, R １ , A, Z are as defined in claim 5 or 6, and position 1 is L ４ Or L ３ is connected to The antibody-drug conjugate according to claim 5 or 6, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

8. D is selected from the following structures, and the first position is L ４ Or L ３ is connected to 【Chemistry 23】 The antibody-drug conjugate according to any one of claims 1 to 7, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

9. The antibody-drug conjugate has the structure shown in formula III: 【Chemistry 24】 In the formula, Tb, q, L １ , L ２ , L ３ , L ４ , R １ , A, Z, U １ and U ２ is as defined in any one of claims 1 to 8, The antibody-drug conjugate according to any one of claims 1 to 8, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

10. The antibody-drug conjugate is selected from the group consisting of: 【Table 5】 In the formula, q is selected from any numerical value between 1.0 and 10.0, preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably, q is selected from 2, 4, 6, or 8. The antibody-drug conjugate according to any one of claims 1 to 9, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

11. The Tb is selected from one or more of the following: (1) The Tb is an antibody or an antigen-binding fragment thereof having endocytosis activity, (2) the Tb is an antibody or an antigen-binding fragment thereof having an activity of binding to a tumor cell surface antigen, (3) the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, an anti-Trop-2 antibody or an antigen-binding fragment thereof, an anti-Her2 antibody or an antigen-binding fragment thereof, an anti-Her3 antibody or an antigen-binding fragment thereof, or an anti-EGFR antibody or an antigen-binding fragment thereof; (4) The Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, such as 1D1-01, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab, or an antigen-binding fragment thereof; (5) The Tb is an anti-Her2 antibody or an antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, mergetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or an antigen-binding fragment thereof, preferably, the Tb is trastuzumab or pertuzumab; (6) The Tb is an anti-EGFR antibody or an antigen-binding fragment thereof, such as demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serculutamab, tomuzotuximab, zalutumumab, cetuximab, or an antigen-binding fragment thereof; (7) The Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a VH sequence shown in SEQ ID NO: 1 and a VL sequence shown in SEQ ID NO: 2, and preferably, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a heavy chain sequence shown in SEQ ID NO: 3 and a light chain sequence shown in SEQ ID NO: 4, (8) The Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a VH sequence shown in SEQ ID NO: 5 and a VL sequence shown in SEQ ID NO: 6, and preferably, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a heavy chain sequence shown in SEQ ID NO: 7 and a light chain sequence shown in SEQ ID NO:

8.

11. The antibody-drug conjugate according to any one of claims 1 to 10, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

12. A drug linker conjugate as shown in formula IV: 【Chemistry 25】 During the ceremony, L １ but, 【Chemistry 26】 If, then, position 1 is linked to Lg and position 2 is linked to L ２ is connected to Lg is a leaving group when reacting with an antibody, and L ２ , L ３ , L ４ and D are as defined in any one of claims 1 to 10, L １ but 【Chemical 27】 If Lg-L １ teeth 【Chemistry 28】 And L ２ , L ３ , L ４ and D is as defined in any one of claims 1 to 10. Or a stereoisomer of said drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

13. The Lg is a halogen, a sulfone group, a tertiary amine base (Me ３ N ＋ , Et ３ N ＋ ), diazonium base, -OMs, MeSO ２ - or CF ３ SO ３ -, preferably, Lg is selected from F, Cl or MeSO ２ -, more preferably, said Lg is selected from F or MeSO ２ Selected from: The drug linker conjugate of claim 12, or a stereoisomer of the drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

14. The drug-linker conjugate has the structure shown in Formula V: 【Chemical 29】 In the formula, Lg, L １ , L ２ , L ３ , L ４ , R １ , A, Z, U １ and U ２ is as defined in any one of claims 1 to 13, A drug linker conjugate as described in claim 12 or 13, or a stereoisomer of the drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

15. The drug linker conjugate is selected from the group consisting of: 【Table 6】 A drug linker conjugate according to any one of claims 12 to 14, or a stereoisomer of the drug linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

16. A biologically active compound according to formula IIA: 【Chemistry 30】 During the ceremony, R １ is selected from hydrogen, halogen, or a C1-4 alkyl group, and preferably, １ is a halogen, Z is -NH-, U １ , U ２ are each independently -CH ２ - or -C(O)-, and U １ , U ２ are not the same, and preferably, １ Ga-CH ２ -, and said U ２ is —C(O)—, A is 【Chemistry 31】 And the first place is L ４ Or L ３ and the 2-position is connected to a benzene ring, and preferably, A is -O(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ -, -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, -O(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ O-, -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ O-, -S(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ -, -S(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, -NH(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ -, -NH(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, -O(CH ２ ) ｍ C(O)NH(CH ２ ) Ｐ O (CH ２ ) ｙ -, or -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ O (CH ２ ) ｙ -, preferably, A is -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ -, or -O(CH ２ ) ｍ C(O)N(CH ３ ) (CH ２ ) Ｐ O (CH ２ ) ｙ -, preferably, A is -OCH ２ C(O)N(CH ３ ) (CH ２ ) ３ - or -OCH ２ C(O)N(CH ３ ) (CH ２ ) ２ O (CH ２ ) ２ Selected from - W is O, S or NR ９ Preferably, W is O; V is a direct bond, O, S, or NR ９ Preferably, V is selected from a direct bond or O; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ are each independently selected from hydrogen, deuterium, or a C1-4 alkyl group; m, p, and y are each independently selected from 0, 1, 2, 3, or 4; R ９ is selected from hydrogen, deuterium, or a methyl group; Or a stereoisomer of said biologically active compound, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof.

17. The biologically active compound is selected from the group consisting of: 【Chemistry 32】 17. The biologically active compound of claim 16, or a stereoisomer of said biologically active compound, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a solvate of a pharma- ceutically acceptable salt thereof.

18. A method for preparing an antibody-drug conjugate of formula I, comprising: 【Chemical Formula 33】 wherein Tb, L １ , L ２ , L ３ , L ４ , Lg and D are as defined in any one of claims 1 to 15.

19. Tb is a drug-linker conjugate as shown in formula IV 【Chemical 34】 to form a C-S bond; the molar ratio of Tb to the drug-linker conjugate is 1:(1-20), for example, 1:(2-20), 1:(4-20), 1:(6-20), 1:(8-20), 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20), or 1:(18-20); The coupling reaction is preferably carried out in water and / or an organic solvent, and the organic solvent is preferably one or more of N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, nitrile solvents and alcohol solvents; 20. The method according to claim 18.

20. A linker as shown in formula VA-1, 【Chemistry 35】 In the formula, Lg, L １ , L ２ , L ３ , L ４ ,W.,R. ２ , R ３ and m are as defined in any one of claims 12 to 15, and Lg ２ is selected from a hydroxy group, a halogen, and an activated hydroxy group, and is preferably a hydroxy group, a chlorine group, a bromine group, 【Chemical 36】 A linker selected from the group consisting of

21. The linker is selected from the group consisting of: 【Table 7】 The linker according to claim 20 .

22. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 11, or a stereoisomer of said antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, or a drug-linker conjugate according to any one of claims 12 to 15, or a stereoisomer of said drug-linker conjugate, a prodrug thereof, a pharma-ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, or a biologically active compound according to any one of claims 16 to 17, or a stereoisomer of said biologically active compound, a prodrug thereof, a pharma-ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, and optionally one or more pharmaceutical auxiliary materials; Pharmaceutical compositions.

23. In the antibody-drug conjugate, the drug-to-antibody ratio (DAR) is any value between 1.0 and 12.0, and preferably, the drug-to-antibody ratio (DAR) is any value within 2±0.4, 4±0.4, 6±0.4, or 8±0.

4.

23. The pharmaceutical composition of claim 22.

24. Use of the antibody-drug conjugate according to any one of claims 1 to 11, or a stereoisomer of said antibody-drug conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, or the drug-linker conjugate according to any one of claims 12 to 15, or a stereoisomer of said drug-linker conjugate, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, or the biologically active compound according to any one of claims 16 to 17, or a stereoisomer of said biologically active compound, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutical composition according to claim 22 or 23, in the preparation of a medicament for the treatment and / or prevention of a disease related to abnormal cellular activity (e.g., a cancer disease); Preferably, the cancer disease is selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer.

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