WO2023098831A1 - 靶向蛋白酶降解(ted)平台 - Google Patents

靶向蛋白酶降解(ted)平台 Download PDF

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WO2023098831A1
WO2023098831A1 PCT/CN2022/136002 CN2022136002W WO2023098831A1 WO 2023098831 A1 WO2023098831 A1 WO 2023098831A1 CN 2022136002 W CN2022136002 W CN 2022136002W WO 2023098831 A1 WO2023098831 A1 WO 2023098831A1
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substituted
group
unsubstituted
alkyl
compound
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French (fr)
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曹小冬
王晓磊
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嘉兴优博生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to biomedicine, and in particular relates to a targeted protease degradation (TED) platform.
  • TED targeted protease degradation
  • the targeted protein degrader can induce the proteasome system to degrade the pathogenic target protein by simultaneously binding the E3 enzyme and the target protein in the cell, so as to achieve the purpose of treating the disease, and from the perspective of the mechanism of action, this protein degrader It can play a role under the action of catalytic amount, and it is expected to overcome the drug resistance brought by traditional small molecule drugs, but this kind of TED protein degradation agent will face poor membrane permeability, easy to be metabolized in the internal circulation, and enter normal tissues. toxicity and so on.
  • ADCs Antibody-drug conjugates
  • ADCs take advantage of the targeting effect of antibodies, using antibodies as carriers to deliver supertoxin drugs to disease sites, and have achieved great success.
  • the bottleneck encountered in the development of ADC drugs is that the therapeutic window is not wide enough.
  • the super toxins will fall off before reaching the target due to the heterogeneity of the coupling, causing serious toxic and side effects.
  • the purpose of the present invention is to provide a compound capable of degrading target protein more efficiently and reusably so as to treat related diseases.
  • the inventors inspired by ADC drugs, the inventors combined the tissue specificity of the extracellular targeting ligand with the catalytic amount of the protein degrading agent to efficiently degrade the disease-causing protein, that is, coupling the protein degrading agent to the extracellular
  • the receptor ligands such as monoclonal antibodies, peptides, small molecule ligands, etc.
  • the specific recognition function of these ligands for disease tissues can be used to successfully deliver protein degradation agents to disease tissues.
  • R E3 is an E3 ligase ligand part
  • L1 is a linker connecting RE3 and R T , and L1 is shown in formula II;
  • the subscript o is an integer of 2 to 50 (preferably, the subscript o is an integer of 2 to 20, more preferably, the subscript o is an integer of 2 to 10; most preferably, the subscript o is 2, 3, 4, 5, 6 or 7);
  • R' does not exist or is H, an active group or a leaving group; preferably, each R' is independently selected from the following group: H, -OH, -SH, -COOH, -COO-C 1 -6 alkyl, protecting group (such as amino protecting group);
  • (d2)R" is -W 3 -L P1 -W P1 -(R P ) q1 ;
  • W 3 is selected from: none, or a divalent group consisting of 1, 2 or 3 bivalent fragments each independently selected from the following group: W', and W U ; wherein, W U is a divalent group that can be spontaneously cleaved Valence fragment;
  • L P1 is -(M') t1 -W Y -(M') t2 -; wherein, W Y is a bivalent linking moiety that is free or cleavable on the cell surface or in the cytoplasm; the subscripts t1 and t2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • W P1 is None, -SS-, or Among them, * represents the part connected with L P1 ; preferably, W P1 is -SS-
  • R P is -W 4 -L P4 -R P1 , -W 4 -L P4 -R P1 -R TED or -W 4 -L P4 -R P1 -R"'; where W 4 is none or -(W ") s1 -W P2 -(W") s2 -; L P4 is -(M') t5 -; R TED is -R T -L1-R E3 or -R E3 -L1-R T ;
  • the subscripts s1 and s2 are each independently 0, 1, 2, 3 or 4; t5 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably, t5 is 0, 1, 2 or 3); W P2 is none, NH, -C(R b )(COOH)-, -C(R b )(COR"')-, -C(R b )(NR a )-(such as -CH(-NH 2 )-), -N(R"')- or -C(R b )(NH(R"'))-;
  • L P2 is -(M') t3 -;
  • L P3 is -(M') t4 -; wherein the subscripts t3 and t4 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably, t3 and t4 are each independently 0, 1, 2 or 3);
  • Each M' is independently selected from the group consisting of -C(R b ) 2 -, -O-, -S-, -N(R a )-, -C(O)-, -SO 2 -, -SO-, -PO 3 -, substituted or unsubstituted C1-10 alkylene, -(CH 2 CH 2 O) 1-10 -(ie -(CH 2 CH 2 O) 1 -, -(CH 2 CH 2 O) 2 -, -(CH 2 CH 2 O) 3 -, -(CH 2 CH 2 O) 4 -, -(CH 2 CH 2 O) 5 -, -(CH 2 CH 2 O) 6 -, -(CH 2 CH 2 O) 7 -, -(CH 2 CH 2 O) 8 -, -(CH 2 CH 2 O) 9 -, -(CH 2 CH 2 O) 10 -), amino acid residue, substituted or unsubstituted C3-8 cyclo
  • W' and W" are each independently a divalent group selected from the group consisting of none, -C(R b ) 2 -, -O-, -S-, -N(R a )-, -C(O )-, -SO 2 -, -SO-, -PO 3 -, amino acid residue, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted 4 to 10 membered heterocycloalkyl, substituted or unsubstituted Substituted C6-10 aryl, substituted or unsubstituted 5 to 10 membered heteroaryl;
  • R P1 and R P2 are each independently the same or different polypeptide elements or target molecules T; preferably, R P1 and R P2 are each independently different polypeptide elements or target molecules T;
  • Each R a is independently selected from the group consisting of H, OH, SH, substituted or unsubstituted C 1-6 alkyl, amino protecting group, 4 to 10 membered heterocycloalkyl containing N(R c ) ring atoms ;
  • Each R b is independently selected from the group consisting of H, halogen, OH, SH, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2- 6 alkynyl, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted C 1-6 alkyl acyl (-C (O) -C 1-6 alkyl), carboxyl, -COO-C 1-6 alkyl, -OC(O)-C 1-6 alkyl; or, 2 R b located on the same carbon and the carbon connected to them together form a substituted or unsubstituted C 3-8 cycloalkyl , substituted or unsubstituted 4 to 10 membered heterocycloalkyl;
  • Each R c is independently selected from the following group: H, OH, SH, substituted or unsubstituted C 1-6 alkyl, amino protecting group;
  • R a is each independently H or C 1-6 alkyl (such as methyl).
  • each R b is independently H or C 1-6 alkyl (such as methyl).
  • each R c is independently H or C 1-6 alkyl (such as methyl).
  • the 4- to 10-membered heterocycloalkyl group when the heterocycloalkyl group (such as a 4- to 10-membered heterocycloalkyl group) is a divalent group, the 4- to 10-membered heterocycloalkyl group includes: Wherein, k1 and k2 are each independently 0, 1, 2 or 3; preferably, the 4- to 10-membered heterocycloalkyl group is selected from the following group:
  • the cycloalkyl (such as C 3-8 cycloalkyl) is a divalent group
  • the cycloalkyl (such as C 3-8 cycloalkyl) includes: Wherein, k1 and k2 are each independently 1, 2 or 3; more preferably, the C 3-8 cycloalkyl is selected from the following group:
  • the heteroaryl group (such as a 5- to 10-membered heteroaryl group) is a divalent group
  • the 5- to 10-membered heteroaryl is selected from the group consisting of:
  • W' and W" are each independently a divalent group selected from the following group: -C(R b ) 2 -, -O-, -N(R a )-, -C( O)-, amino acid residues.
  • At least 1 or 2 M's are W Z .
  • each M' is independently selected from the following group: none, -C(R b ) 2 -, -O-, -S-, -N(R a )-, -C(O)-, -SO 2 -, -(CH 2 CH 2 O) 1-10 -, amino acid residues.
  • the spontaneously cleavable bivalent fragment refers to a cleavable bivalent linking part or a spontaneously cleavable bivalent fragment after -S-S cleavage.
  • the cleavable bivalent linking moiety on the cell surface or in the cytoplasm is a bivalent linking moiety consisting of two or more structural fragments selected from the following group:
  • spontaneously cleavable bivalent fragment is selected from the following group:
  • W 3 is -W'-W U -W'-.
  • W 3 is none, -C(O)- or -OC(O)-. In another preferred example, W 3 is W U .
  • W 3 is selected from the following group:
  • R P is -W 4 -L P4 -R P1 , -W 4 -L P4 -R P1 -R TED or -W 4 -L P4 -R P1 -R"'.
  • R" is -W 3 -L P1 -W P1 -R P1 .
  • W P1 when W P1 is none or When , W Y is a divalent linking moiety that can be cleaved on the cell surface or in the cytoplasm.
  • the cleavable divalent linking moiety on the cell surface or in the cytoplasm refers to a divalent linking moiety that can be cleaved in the acidic environment of the cell surface or cytoplasm or specifically cleaved by GSH enzyme.
  • W P1 is And W Y is a divalent linking moiety that is cleavable on the cell surface or in the cytoplasm, or, W P1 is -SS- and W Y is none.
  • the cleavable divalent linking moiety on the cell surface or in the cytoplasm is selected from the following group:
  • W P2 is none, -C(R b )(NR a )- (such as -CH(-NH 2 )-) or -CH(NH(R"'))-.
  • W 4 is none, -CH(COOH)-CH 2 -, -C(O)-CH(NH 2 )-CH 2 -, -C(O)-CH(NH(R"'))-CH 2 -, -NH-CH(COOH)-CH 2 -, -NH-C(O)-CH(NH 2 )-CH 2 -, or -NH-C(O)-CH(NH (R"'))- CH2- .
  • the hydrophilic divalent linking moiety refers to a divalent linking moiety in which one or more groups selected from the following group exist on the main chain or side chain: -(CH 2 CH 2 O) -, -SO3H , -PO3H2 , -COOH.
  • hydrophilic bivalent linking moiety or W Z is selected from the following group:
  • W 5 is none, -C(O)- or -OC(O)-.
  • At least one M' is W Z .
  • At least one of R P1 and R P2 is capable of binding to an extracellular receptor.
  • R P1 and R P2 are capable of binding to extracellular receptors.
  • RP1 and/or RP2 are ligands of the extracellular receptors.
  • the extracellular receptor is a receptor to which the target molecule T can bind or target (such as folate receptor, Hsp90, SSTR, PSMA, CAIX, etc.).
  • one of R P1 and R P2 is a polypeptide element and the other is a target molecule T.
  • both R P1 and R P2 are the same or different polypeptide elements.
  • both R P1 and R P2 are the same or different target molecules T.
  • R P1 and R P2 are each independently the same or different mAbs.
  • the target molecule is target molecule A or target molecule T.
  • the target molecule A or T includes: a small molecule, a nanocarrier, or a combination thereof.
  • the target molecules A and T are each independently a target molecule selected from the following group or a target molecule targeting a target (such as a respective enzyme or receptor) selected from the following group: folic acid, HSP90, TINFRm, TNFR2, NADPH oxidase (oxidase), BclIBax, C5a receptor (receptor), HMG-CoA reductase (reductase), PDE I-V, squalene cyclase inhibitors (Squalene cyclase inhibitors), CXCR1, CXCR2, Nitric oxide (NO) synthase (Nitric oxide (NO) synthase), cyclooxygenase (cyclo-oxygenase) 1-2, 5HT receptor (5HT receptors), dopamine receptors (dopamine receptors), G-protein ( G-proteins), Gq, Histamine receptors, Lipoxygenases, Tryptase serine proteas
  • the polypeptide element includes: antibody, protein, or a combination thereof.
  • the antibodies include: nanobodies, minibodies, or combinations thereof.
  • the polypeptide element is an antibody; preferably, the antibody includes a nanobody (nanobody), a small molecule antibody (minibody), an antibody fragment (such as scFv, Fab), a double antibody (Dibody), a monoclonal antibody Cloned antibodies (mAbs) and more.
  • the targets of the polypeptide include but are not limited to: EGFR, FGFR, SSTR1-14, GnRH, TRPV1-6, RGD, iRGD and so on.
  • the antibody can bind to an antigen or receptor selected from the group below (for example, with one (ie, monofunctional antibody) or two (ie, bifunctional antibody) or more selected from the group below Multiple (i.e. multifunctional antibody) antigen and/or receptor binding): DLL3, EDAR, CLL1, BMPR1B, E16, STEAP1, 0772P, MPF, 5T4, NaPi2b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4 , CRIPTO, CD21, CD22, CD79b, CD19, CD37, CD38, CD138, FcRH2, B7-H4, HER2, NCA, MDP, IL20R ⁇ , Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB
  • the target molecule of the present invention can also be combined with receptors that can be targeted by specific small molecules, such as folic acid, HSP90, glucose transporter-1 (glucose transporter 1) (GLUT1), aminopeptide Enzyme (aminopeptidase N) (APN), low-density lipoprotein receptor-related protein 1 (LRP1), prostate-specific membrane antigen (PSMA), integrin ⁇ v ⁇ 3, bombesin receptor, somatostatin receptor (SSTR), tumor hypoxic microenvironment, and carbonic anhydrase IX (CAIX) receptors.
  • specific small molecules such as folic acid, HSP90, glucose transporter-1 (glucose transporter 1) (GLUT1), aminopeptide Enzyme (aminopeptidase N) (APN), low-density lipoprotein receptor-related protein 1 (LRP1), prostate-specific membrane antigen (PSMA), integrin ⁇ v ⁇ 3, bombesin receptor, somatostatin receptor (SSTR
  • R P1 is a monovalent group or a divalent group derived from a compound selected from the following
  • R P2 is a monovalent group derived from a compound selected from the following;
  • the said derived from refers to, for example, under the condition of not affecting the binding to the target protein, that is, at a position other than the binding part of the target protein, optionally introducing an active group (such as -NH 2 , -NH-, -COOH, or -SH, etc.), or utilize existing active groups (such as -NH 2 , -NH-, -COOH, or -SH, etc.) in the compound to make the active group in the compound
  • a functional group or atom in such as H in -NH2 or -NH-, or -OH in -COOH, or H in -SH
  • R P1 is a monovalent group selected from Table E1 (when R P is -W 4 -L P4 -R P1 ) or a divalent group selected from Table E2 (when R P is - W 4 -L P4 -R P1 -R TED or -W 4 -L P4 -R P1 -R"'); and R P2 is a monovalent group selected from Table E1:
  • R T is a monovalent group selected from Table B1. In another preferred embodiment, R T is a divalent group derived from a monovalent group selected from Table B1. In another preferred example, in Formula Ia, R T is a divalent group derived from a monovalent group selected from Table B1. In another preferred embodiment, -R T -RL is selected from Table B2.
  • R E3 is selected from any of the following monovalent groups or divalent groups derived from any of the following monovalent groups (preferably, the derivation refers to The H in -NH- in the monovalent group is removed to form a divalent group):
  • R E3 is as shown in or derived from formula A1 or A2:
  • -R E3 -RL is any of the following divalent groups
  • the E3 ligase ligand part is selected from the groups shown in Table C1.
  • R E3 is or is derived from formula A1.2 or formula A2.2.
  • -O-O- does not exist in L1.
  • s 1 or 2.
  • each W is independently selected from the following group: none (bond), -N(R a )-, -C(O)-, -C ⁇ C-.
  • each W is independently selected from the following group: none (bond), -NH-, -C(O)-, -C ⁇ C-.
  • W 1 and W 2 are each independently -N(R a )-C(O)-, -C(O)-N(R a )- or -C ⁇ C-. In another preferred example, W 1 and W 2 are each independently -NHC(O)-, -C(O)NH- or -C ⁇ C-. In another preferred example, one of W 1 and W 2 is N(R a )-C(O)- or -C(O)-N(R a )-, and the other is -C ⁇ C-. In another preferred example, one of W 1 and W 2 is -NHC(O)- or -C(O)NH-, and the other is -C ⁇ C-.
  • M is each independently a divalent group selected from the group consisting of -C(R b ) 2 -, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 4 to 10-membered heterocycloalkyl, substituted or unsubstituted C 6-10 aryl, substituted or unsubstituted 5- to 10-membered heteroaryl.
  • M is each independently a divalent group selected from the group consisting of -C(R b ) 2 -, substituted or unsubstituted C 3-8 cycloalkyl, substituted or unsubstituted 4 to 10-membered heterocycloalkyl.
  • M is each independently a divalent group selected from the group consisting of -C(R b ) 2 -, substituted or unsubstituted C 4-7 cycloalkyl, substituted or unsubstituted 4 to 6 membered heterocycloalkyl.
  • M is each independently a divalent group selected from the group consisting of -C(R b ) 2 -, substituted or unsubstituted 4- to 6-membered nitrogen-containing heterocycloalkyl.
  • M is independently selected from the following group: -CH 2 -, -CH(C 1-4 alkyl)-, In another preferred example, each M is independently selected from the following group: C(R b ) 2 , Preferably, wherein, R b are each independently H or C 1-6 alkyl (such as methyl).
  • the subscript o is an integer of 2-10; more preferably, it is 2, 3, 4, 5, 6 or 7, most preferably, it is 3, 4 or 5.
  • only nitrogen heteroatoms exist on the nitrogen-containing heterocycloalkyl ring in L2.
  • L1 is -W 1 -Cr 1 -Cr 2 -(CH 2 ) o1 -W 2 -. In another preferred example, L1 is -C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ C-.
  • the conjugate is R L -RT -C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 or R T - C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 -R L .
  • the conjugate is R L -RT -C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 or R T - C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 -R L ; wherein, R T is shown in formula P5 in Table B1 or derived from formula P5 in Table B1, And R E3 is as shown in or derived from formula A1 or formula A2.
  • the conjugate is selected from Table A1. In another preferred example, the conjugate is selected from Tables D1 and D2. In another preferred example, the conjugate is selected from Table D3.
  • an ACTED compound or a pharmaceutically acceptable salt thereof wherein the ACTED compound is represented by formula IVa or IVb;
  • R R T , L1 and R E3 are as defined in the first aspect.
  • the ACTED compound is selected from Tables D1 and D2.
  • the ACTED compound is selected from Table D3.
  • a TED compound or a pharmaceutically acceptable salt thereof is provided, wherein the TED compound is represented by formula Va or Vb;
  • R', R T , L1 and R E3 are as defined in the first aspect.
  • the TED compound at R' can further be directly or indirectly coupled with a ligand of an extracellular receptor.
  • R' does not exist or is H.
  • the TED compound when R' does not exist or is H, the TED compound is not UB-PA01, UB-PA02 and UB-PA03.
  • the conjugate is R'-R T -C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 or R T - C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 -R'.
  • the conjugate is R'-R T -C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 or R T - C(O)-N(R a )-Cr 1 -Cr 2 -(CH 2 ) o1 -C ⁇ CR E3 -R'; wherein, R T is as shown in formula P5 in Table B1 or derived from formula P5 in Table B1, And R E3 is as shown in or derived from formula A1 or formula A2.
  • the TED compound is selected from Table A1.
  • a pharmaceutical composition wherein the pharmaceutical composition contains (i) the conjugate as described in the first aspect or the ACTED compound as described in the second aspect or the The TED compound described in the third aspect; and (ii) a pharmaceutically acceptable carrier.
  • the conjugate as described in the first aspect or the ACTED compound as described in the second aspect or the TED compound as described in the third aspect in preparation for treatment or prevention with a target protein Use in medicines for overdose-related disorders.
  • a method for reducing the content of a target protein in a cell wherein the cell is mixed with the conjugate as described in the first aspect or the ACTED compound as described in the second aspect or the ACTED compound as described in the first aspect
  • the TED compounds of the three aspects are contacted to reduce the content of the target protein in the cell.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • the TED conjugate of the present invention has the structure shown in formula I.
  • the TED conjugate of the present invention is very suitable for further linking with polypeptide elements (especially antibodies, protein ligands) and/or other targeting molecules, or further linking with polypeptide elements and/or other targeting molecules.
  • polypeptide elements and/or other targeting molecules in the conjugates of the molecules etc.
  • the conjugates of the present invention have excellent specificity (such as the specificity of targeting tumor cells), can significantly improve drug selectivity, implement more precise degradation of disease-causing proteins, reduce systemic toxicity that may be caused by non-specific degradation, and may overcome difficulties encountered in drug absorption and metabolism. Eliminate opportunities for drug resistance.
  • the inventors have completed the present invention on this basis.
  • the terms “compound of the present invention” and “conjugate of the present invention” are used interchangeably and refer to the compound of formula I or the conjugate described in the first aspect of the present invention.
  • alkyl by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical having the indicated number of carbon atoms (i.e., C 1-6 means 1-6 carbon ).
  • the alkyl group has 1 to 4 carbons, that is, a C 1-4 alkyl group.
  • alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl et al.
  • alkenyl refers to an unsaturated alkyl group having one or more double bonds.
  • the alkenyl group has 2 to 4 carbons, that is, a C 2-4 alkenyl group.
  • alkynyl refers to an unsaturated alkyl group having one or more triple bonds.
  • the alkynyl group has 2 to 4 carbons, that is, a C2-4 alkynyl group.
  • unsaturated alkyl groups include, but are not limited to: vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3 -(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers.
  • cycloalkyl refers to a hydrocarbon ring having the specified number of ring atoms (eg, C 3-6 cycloalkyl) and being fully saturated or having no more than one double bond between ring tips.
  • cycloalkyl refers to a hydrocarbon ring having the specified number of ring atoms (eg, C 3-8 cycloalkyl) and being fully saturated or having no more than one double bond between ring tips.
  • the term also includes bicyclic and polycyclic hydrocarbon rings, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.
  • heterocycloalkyl refers to a cycloalkyl group containing one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized.
  • Heterocycloalkyl groups can be monocyclic, bicyclic or polycyclic ring systems.
  • Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, Piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, etc.
  • a heterocycloalkyl group can be attached to the remainder of the molecule via a ring carbon or a heteroatom.
  • cycloalkylalkyl and heterocycloalkylalkyl it is meant that the cycloalkyl or heterocycloalkyl is attached to the rest of the molecule through an alkyl or alkylene linker.
  • cyclobutylmethyl- is a cyclobutyl ring attached to a methylene linker on the rest of the molecule.
  • alkylene by itself or as part of another substituent refers to a divalent radical derived from an alkane, eg -CH2CH2CH2CH2- .
  • Alkyl (or alkylene) groups generally have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • Lower alkyl or “lower alkylene” are shorter chain alkyl or alkylene groups, usually having 4 or fewer carbon atoms.
  • alkenylene or “alkynylene” refers to an unsaturated form of "alkylene” having double or triple bonds, respectively.
  • heteroalkyl by itself or in combination with other terms refers to a stable linear or branched chain or cyclic hydrocarbon group or combination thereof, consisting of the indicated number of carbon atoms and 1 to 3 carbon atoms selected from the group consisting of O , N, Si and S heteroatoms, and wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatoms are optionally quaternized.
  • the heteroatoms O, N and S can be located at any internal position of the heteroalkyl.
  • the heteroatom Si can be located anywhere on the heteroalkyl, including the point where the alkyl is attached to the rest of the molecule.
  • heteroalkenyl and “heteroalkynyl” by themselves or in combination with another term refer to an alkenyl or alkynyl group, respectively, containing the indicated number of carbons and 1 to 3 optional Heteroatoms from O, N, Si and S, and wherein the nitrogen and sulfur atoms are optionally oxidized, the nitrogen heteroatoms may be optionally quaternized.
  • the heteroatoms O, N and S can be located at any internal position of the heteroalkyl.
  • heteroatoms may also occupy either or both of the chain termini (eg, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.).
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense to refer to those attached to the rest of the molecule via an oxygen, amino or sulfur atom respectively alkyl.
  • dialkylamino groups the alkyl moieties can be the same or different, and can also combine with the nitrogen atom connected to each alkyl group to form a 3-7 membered ring. Therefore, the group represented by -NR a R b includes piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl (azetidinyl) and the like.
  • halo or halogen by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl” are meant to include monohaloalkyl or polyhaloalkyl.
  • C 1-4 haloalkyl is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl and the like.
  • aryl denotes a polyunsaturated (usually aromatic) hydrocarbon group which may be a single ring or multiple rings (up to three rings) fused together or linked covalently.
  • heteroaryl refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized .
  • a heteroaryl can be attached to the rest of the molecule through a heteroatom.
  • Non-limiting examples of aryl include phenyl, naphthyl, and biphenyl
  • non-limiting examples of heteroaryl include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, Quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl (benzotriazinyl), purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benziso Oxazolyl, isobenzofuryl (isobenzofuryl), isoindolyl, indolizyl, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzene Thiazolyl, benzofuryl, benzothienyl
  • aryl when used in combination with other terms (eg, aryloxy, arylthio, aralkyl), it includes aryl and heteroaryl rings as defined above.
  • aralkyl is meant to include those groups in which the aryl group is attached to an alkyl group which is attached to the rest of the molecule (eg, benzyl, phenethyl, pyridylmethyl, etc.).
  • alkyl e.g. "alkyl,” “aryl,” and “heteroaryl”
  • aryl e.g., aryl
  • heteroaryl e.g., aryl and heteroaryl
  • aryl and heteroaryl will refer to substituted or unsubstituted forms as provided below
  • alkyl and related aliphatic groups will refer to unsubstituted forms unless substituted is specified .
  • R', R" and R"' each independently represent hydrogen, unsubstituted C 1-8 alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted by 1-3 halogens, unsubstituted C 1-8 alkyl , C 1-8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl-C 1-4 alkyl.
  • R' and R" are attached to the same nitrogen atom, they can be combined with The nitrogen atoms combine to form 3-, 4-, 5-, 6- or 7-membered rings.
  • -NR'R is meant to include 1-pyrrolidinyl and 4-morpholinyl.
  • Two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be substituted with substituents of the formula -TC(O)-( CH2 ) q -U-, wherein T and U are independently - NH-, -O-, -CH 2 - or a single bond, and q is an integer of 0 to 2.
  • two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be represented by the formula -A-(CH 2 ) r -B-, where A and B are independently -CH 2 -, - O-, -NH-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer of 1 to 3.
  • One single bond in the new ring thus formed may optionally be replaced by a double bond.
  • two substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be replaced by substituents of the formula -( CH2 ) s -X-( CH2 ) t- , where s and t are independently is an integer of 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
  • the substituent R' in -NR'- and -S(O) 2 NR'- is selected from hydrogen or unsubstituted C 1-6 alkyl.
  • the cycloalkyl or heterocycloalkyl when the cycloalkyl or heterocycloalkyl is a divalent group, the cycloalkyl or heterocycloalkyl can lose two hydrogens located on the same ring atom (ring carbon atom) so as to be separated from the chain
  • the other chain atoms in the chain can be connected (forming a structure similar to a spiro ring), or two hydrogens on different ring atoms can be lost to connect with other chain atoms in the chain (such as -cyclopentylene-).
  • heteroatom is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).
  • a bond from a substituent (typically an R group) to the center of an aromatic ring will be understood to mean a bond providing attachment at any available vertex of the aromatic ring.
  • the description also includes on-ring linkages fused to aromatic rings.
  • a bond drawn to the center of the benzene moiety of an indole would represent a bond to any available vertex of the six- or five-membered ring portion of the indole.
  • amino acid residue refers to a group formed by removing one H from the N-terminal -NH2 of an amino acid, and removing -OH from the -COOH at the C-terminal.
  • amino acids include natural amino acids or unnatural amino acids, including D-form and/or L-form amino acids.
  • amino acids include, but are not limited to, Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H ), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y ), Val(V).
  • the amino acid is an amino acid selected from the group consisting of L-glycine (L-Gly), L-alanine (L-Ala), ⁇ -alanine ( ⁇ -Ala), L-glutamine amino acid (L-Glu), L-aspartic acid (L-Asp), L-histidine (L-His), L-arginine (L-Arg), L-lysine (L- Lys), L-valine (L-Val), L-serine (L-Ser), L-threonine (L-Thr); in addition, when there are 2 or more amino groups and/or 2 or above carboxyl group, the term also includes groups formed by the removal of one H from -NH 2 and the removal of -OH from -COOH that are not on the same carbon atom, such as the -NH 2 and non- ⁇ positions of glutamic acid -COOH is a divalent group -C(O)-(CH 2 ) 2 -C(COOH)-NH-
  • the term derived from refers to, for example, optionally introducing an active group (such as - NH 2 , -NH-, -COOH, or -SH, etc.), or use existing active groups in the compound (such as -NH 2 , -NH-, -COOH, or -SH, etc.), and make the compound in the
  • the functional group or atom in the active group such as H in -NH2 or -NH-, or -OH in -COOH, or H in -SH is removed to form a Location.
  • salts of the active compounds prepared with relatively nontoxic acids or bases are intended to include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular substituents on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like.
  • Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, Choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N- Ethylpiperidine, Glucamine, Glucosamine, Histidine, Hypamine, Isopropylamine, Lysine, Mglucosamine, Morpholine, Piperazine, Piperidine, Polyamine Resin , procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.
  • arginine betaine
  • caffeine Choline
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, Hydroiodic acid, or phosphorous acid, etc.; and salts derived from relatively nontoxic organic acids, such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc.
  • salts of amino acids such as arginine salts and the like
  • salts of organic acids such as glucuronic acid or galactunoric acid and the like.
  • Certain specific compounds of the present invention contain both basic and acidic functional groups, thereby enabling conversion of the compounds into base or acid addition salts.
  • the neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise, those salts are equivalent to the parent form of the compound for the purposes of the present invention of.
  • the present invention provides compounds in prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
  • prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an ex vivo environment.
  • prodrugs can be slowly converted to compounds of the invention when placed in a transdermal patch reservoir containing a suitable enzyme or chemical reagent.
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms.
  • the solvated forms are generally equivalent to the unsolvated forms and are intended to be within the scope of this invention.
  • Certain compounds of the present invention may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers body) should be included within the scope of the present invention.
  • compounds provided herein have defined stereochemistry (designated as R or S, or indicated with dashed lines or wedge bonds)
  • those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80% , 90%, 95%, 98%, 99% and up to 100% free of other isomers).
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the isotopic atoms that constitute such compounds.
  • An unnatural proportion of an isotope can be defined as the amount from the naturally found amount of the atom in question to 100% of that atom.
  • compounds may incorporate radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C ).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C ).
  • isotopic variants may provide additional uses beyond those described herein.
  • isotopic variants of the compounds of the invention may have additional uses, including but not limited to, as diagnostic and/or imaging reagents, or as cytotoxic/radiotoxic therapeutic agents. Additionally, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic profiles, thereby contributing to increased safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.
  • polypeptide element includes peptides (such as short peptides of 3-20 aa) or proteins. Furthermore, the term also includes intact proteins or fragments thereof. Preferred polypeptide elements include antibodies (such as whole antibodies, single chain antibodies, nanobodies, antibody fragments), especially against tumor cell markers (such as tumor markers located on the surface of tumor cells, such as receptors on the cell surface) or against inflammatory Antibodies to sexual factors such as inflammatory factors associated with autoimmune diseases.
  • antibody or "immunoglobulin” is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • VL variable region
  • Specific amino acid residues form the interface between the variable domains of the light and heavy chains.
  • single domain antibody and “nanobody” have the same meaning, and refer to cloning the variable region of the heavy chain of an antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is a fully functional the smallest antigen-binding fragment.
  • the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions in a roughly ⁇ -sheet configuration connected by three CDRs that form connecting loops, which in some cases may form a partially folded structure.
  • the CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be assigned to one of two distinct classes, termed kappa and lambda, based on the amino acid sequence of their constant regions. Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which are separated into four framework regions (FRs), four
  • FRs framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • polypeptide elements may include not only complete antibodies, but also fragments of antibodies with immunological activity (such as Fab or (Fab') 2 fragments; antibody heavy chains; or antibody light chains) or antibodies formed with other sequences fusion protein. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • a targeting ligand is a small molecule capable of binding a target protein of interest.
  • target molecules include but are not limited to: folic acid, Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, targeting Compounds of the cytoplasmic signaling protein FKBP12, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds and compounds targeting the aryl hydrocarbon receptor (AHR).
  • the targeting ligand is capable of binding kinases, BET bromodomain-containing proteins, cytoplasmic signaling proteins (e.g., FKBP12), nucleoproteins, histone deacetylases, lysine methyltransferases, Proteins that regulate angiogenesis, proteins that regulate immune response, aryl hydrocarbon receptor (AHR), estrogen receptor, androgen receptor, glucocorticoid receptor, or transcription factors (eg, SMARCA4, SMARCA2, TRIM24).
  • cytoplasmic signaling proteins e.g., FKBP12
  • nucleoproteins e.g., histone deacetylases
  • lysine methyltransferases e.g., Proteins that regulate angiogenesis, proteins that regulate immune response, aryl hydrocarbon receptor (AHR), estrogen receptor, androgen receptor, glucocorticoid receptor, or transcription factors (eg, SMARCA4, SMARCA2, TRIM24).
  • AHR
  • kinases to which a targeting ligand can bind include, but are not limited to: tyrosine kinases (e.g., AATK, ABL, ABL2, ALK, AXL, BLK, BMX, BTK, CSF1R, CSK, DDR1, DDR2 , EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR , FLT1, FLT3, FLT4, FRK, FYN, GSG2, HCK, HRAS, HSP90, IGF1R, ILK, INSR, INSRR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR, KIT, K RA S, KSP, KSR1, LCK , LM
  • kinase 2 protein kinase A, protein kinase B, protein kinase C, Raf kinase, CaM kinase, AKT1, AKT2, AKT3, ALK1, ALK2, ALK3, ALK4, Auro ra A, Auro ra B, Auro ra C , CHK1, CHK2, CLK1, CLK2, CLK3, DAPK1, DAPK2, DAPK3, DMPK, ERK1, ERK2, ERK5, GCK, GSK3, HIPK, KHS1, LKB1, LOK, MAPKAPK2, MAPKAPK, MEK, MNK1, MSSK1, MST1, MST2 , MST4, NDR, NEK2, NEK3, NEK6, NEK7, NEK9, NEK11, PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, PIM1, PIM2, PLK1, RIP2, RIP5, RSK1, RSK2,
  • the target protein is bound through the RT (target molecule part) in the conjugate.
  • the target molecule may be target molecule A, target molecule T, or a combination thereof.
  • the target molecule can be any inhibitor of the target protein.
  • the target molecule can be a highly effective inhibitor of the target protein, or an inhibitor with relatively poor activity.
  • the target molecule of the present invention may be a small molecule inhibitor known in the art against any target protein in the art.
  • the target molecule used herein has a group (such as -O-, -NR a - (where R a is H, or C 1-6 alkane) that can be attached to a linker (such as L1)
  • a linker such as L1
  • substituents such as -CO-, -COO-, etc.
  • the linker molecule of the present invention such as L1 in the present invention
  • the target protein can be various target proteins known in the art, representative examples include (but not limited to): MDM2, AKT, BCR-ABL, Tau, BET (BRD2, BRD3, BRD4), ERR ⁇ , FKBP12 , RIPK2, E RB B3, androgen receptor, MetAP2, TACC3, FRS2 ⁇ , PI3K, DHFR, GST, Halo Tag, C RA BPI, C RA BPII, R R, aryl hydrocarbon receptor, estrogen receptor.
  • MDM2 MDM2, AKT, BCR-ABL, Tau, BET (BRD2, BRD3, BRD4), ERR ⁇ , FKBP12 , RIPK2, E RB B3, androgen receptor, MetAP2, TACC3, FRS2 ⁇ , PI3K, DHFR, GST, Halo Tag, C RA BPI, C RA BPII, R R, aryl hydrocarbon receptor, estrogen receptor.
  • MDM2 its inhibitors can be found in WO2017176957,
  • R T is any monovalent group shown in Table B1, or is a divalent group derived from any monovalent group shown in Table B1 (preferably, The derivation refers to the removal of H in the group -NH- to form a divalent group)
  • R Pa is selected from the group consisting of optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl;
  • R Pb is selected from the following group: H, -OH, substituted or unsubstituted C 1-6 alkyl (such as C 1-6 hydroxyalkyl), -COR Pb1 ; wherein, RPb1 is selected from the following group: H, - N(R a ) 2 , -NH-N(R a ) 2 , -OH, -OC 1-6 alkyl, -OC 1-4 alkylene-N(R a ) 2 ; preferably, R Pb for CO-NH 2
  • X P1 is N or CH.
  • R T is the formula P5 or a divalent group derived from the formula P5.
  • formula P5 is shown as any of the following
  • formula P1 is as shown in any of the following
  • -R T -RL is as shown in any one of Table B2
  • the E3 ligase ligand moiety (R E3 ) is used to bind the E3 ligase.
  • the E3 ligase ligand part A1 is selected from the group A1 in WO2017/176957A1 (preferably, A-10, A-11, A-15 in WO2017/176957A1 , A-28, A-48, A-69, A-85, A-93, A-98, A-99 or the corresponding part of A-101).
  • the E3 ligase ligand part is selected from:
  • the dotted line indicates the position connected with other parts (that is, the position connected with R T -L1);
  • a representative E3 ligase ligand moiety has or is derived from (e.g., where -NH- is depleted of H to form a site of attachment to RL ) a structure as shown in Formula A1 or A2:
  • a representative E3 ligase ligand moiety has or is derived from (e.g., where -NH- is depleted of H to form a site of attachment to RL ) the structure shown in Formula B:
  • R z2 is H or C1-C6 alkyl (such as Me), and R z1 is H or C1-C6 alkyl (such as Me or Et).
  • the E3 ligase ligand used herein has a group (such as -O-, -NR a - (wherein, R a is H, or C1-C6 alkyl, etc.) that can be connected to the linker base), -CO-, -COO-, etc.), to dock with the linker molecules of the present invention (such as L1, etc. in the present invention) to form ethers, amines, amides, etc. at a univalent price.
  • a group such as -O-, -NR a - (wherein, R a is H, or C1-C6 alkyl, etc.) that can be connected to the linker base
  • -CO-, -COO-, etc. to dock with the linker molecules of the present invention (such as L1, etc. in the present invention) to form ethers, amines, amides, etc. at a univalent price.
  • R E3 (E3 ligase ligand moiety) is a monovalent group shown in any one of Table C1 or a divalent group derived from any one of the monovalent groups shown in Table C Group (preferably, the derivatization means that -NH- removes H so as to form a linking position with RL ):
  • R E3 is or is derived from formula A1.2 or formula A2.2.
  • R E -RL is any of the following divalent groups
  • a linker that connects the E3 ligase ligand to the target molecule (L1 as described herein)
  • the linker (or linker molecule) of the present invention is used to link target molecules and E3 ligase ligands.
  • the target molecules or E3 ligase ligands are linked via functional groups at both ends (such as -OH, -SH, -NH 2 , -SOOH or -COOH).
  • the linker (head) L1 of the present invention is used to connect the target molecule (part) R T and the E3 ligase ligand (part) R E3 .
  • the linker of the present invention may further contain various other functional groups, such as -OH, -NHR, -SH and other functional groups.
  • linker L1 of the present invention can be represented by the following general formula II:
  • W 1 , L2 and W 2 are as described in the first aspect of the present invention.
  • W 1 and W 2 are each independently a divalent group formed by the following monovalent group losing one hydrogen atom: -OH, -NH 2 , -SH, -COOH , -SO 2 H and so on.
  • linkers can be connected to target molecules through linking groups as shown below:
  • W1 and W2 each independently include a rigid part (such as a four-membered, five-membered, or six-membered alicyclic (saturated carbocyclic) part, or a five-membered or six-membered aromatic heterocyclic part, etc.)
  • a rigid part such as a four-membered, five-membered, or six-membered alicyclic (saturated carbocyclic) part, or a five-membered or six-membered aromatic heterocyclic part, etc.
  • R in the above formulas is as defined above; n is 1 or 2 or 3.
  • W and W are each independently selected from the group consisting of:
  • the term "compound of the present invention” refers to a compound or a conjugate represented by formula Ia or Ib, formula IVa or IVb, formula Va or Vb.
  • the term also includes the various crystalline forms, or pharmaceutically acceptable salts, of compounds of Formula Ia or Ib, Formula IVa or IVb, Formula Va or Vb.
  • the present invention provides a class of compounds as shown in formula IVa or IVb which are suitable for further linking with polypeptide elements (such as antibodies, protein ligands, etc.) or target molecules T or connected with polypeptide elements or target molecules T.
  • Conjugates i.e. TED or TED molecules as referred to herein;
  • R' is H or a leaving group or active group (for example, a group suitable for coupling reactions);
  • R E3 is the E3 ligase ligand part, RT is the target molecule part,
  • L1 is the linker R Connectors for T and R E3 sections.
  • R', R E3 , R T and L1 are as defined above.
  • the present invention provides a class of ligands (eg, , polypeptide element or target molecule T) conjugates or molecules shown in formula IVa or IVb connected (i.e. ACTED or ACTED molecules referred to herein)
  • ligands eg, polypeptide element or target molecule T
  • conjugates or molecules shown in formula IVa or IVb connected i.e. ACTED or ACTED molecules referred to herein
  • R" is a linker with a specific structure coupled with a ligand targeting the target tissue (such as -W 3 -L P1 -W P1 -(R P ) q1 );
  • R E3 is the ligand part of E3 ligase ,
  • RT is a part of the target molecule, and
  • L1 is a linker connecting RT and RE3 .
  • the definitions of R", RE3 , RT and L1 are as described above.
  • the present invention provides a targeted protease degradation (TED) platform based on the conjugate of the present invention, which utilizes the ubiquitin-proteasome system, the "cleaner" in the cell.
  • TED targeted protease degradation
  • the cell's own protein destruction mechanism can be used to remove specific oncogenic disease proteins from the cell, so it is an alternative method for targeted therapy.
  • the TED technology of the present invention is a bifunctional hybrid compound, one side is used to bind the target protein, and the other side is used to bind an E3 ligase, so that the target protein can be combined with the E3 ligase, and the The target protein is ubiquitinated and thereby degraded by the proteome.
  • TED technology only provides binding activity, does not need to directly inhibit the functional activity of the target protein, and can be reused. Therefore, it has excellent application prospects.
  • the optimized TED molecule of the present invention has excellent target protein degradation ability, thereby inhibiting the growth of focal cells.
  • the TED of the present invention has a linker with a specific structure (for example, there is a divalent linker (such as -SS-, or -AN-, -AAN-, -VA-, -GGFG-, -AAFG, -VCit-, -VL- and other peptide chains) and hydrophilic bivalent linking parts (such as PEG chains, side chains containing acidic functional groups such as -SO 3 H, -PO 3 H 2 , - COOH, etc.)) are coupled with ligands ( RP1 and R P2 as defined herein, such as monoclonal antibodies, double antibodies, polypeptides, folic acid, etc.) that target tumor tissues to form ACTED molecules (or coupled things).
  • ligands RP1 and R P2 as defined herein, such as monoclonal antibodies, double antibodies, polypeptides, foli
  • the linker may have a spontaneously cleavable bivalent segment that cleaves to release the TED molecule upon cleavage of the cleavable bivalent linker moiety, eg, present on the cell surface or in the cytoplasm.
  • these bivalent fragments When these bivalent fragments are connected with the peptide chain (that is, the cleavable bivalent link), they can remain stable in the circulation process in vivo. Once the peptide chain is cleaved by enzymes in the cytoplasm or broken by acidic environments, the exposed The valence fragment becomes chemically unstable and spontaneously cleaves quickly in the acidic environment of tumor tissue, releasing TED.
  • these bivalent fragments provide moieties with active linking ends to TED molecules without active linking groups (such as A02).
  • ACTED with the above structure enters the blood circulation, and the ligand part coupled with the linker of a specific structure binds to the antigen or receptor on the surface of the tumor cell, so that it can be rapidly enriched in the tumor tissue.
  • the ACTED of the present invention can have the following effects: for example, 1.
  • the present invention also provides a prodrug (Pro-drug) conjugate based on targeting the tumor microenvironment and hypoxic state.”
  • these tumor tissue-targeting ligands can be monovalent or divalent groups derived from the following compounds:
  • the advantages of the ACTED of the present invention can be divided into two aspects: 1. Enrich more TED into tumor tissue, and help TED enter tumor cells, degrade target proteins to kill tumor cells, and improve the utilization rate of TED; And 2. ACTED rarely binds to normal cells, so less TED enters normal tissues during circulation, reducing toxic side effects.
  • Some exemplary linkers for coupling with tumor-targeting ligands are as follows, wherein only representative linking fragments are exemplarily listed, and it should be understood that between each fragment (such as between W Z and Linker m) There may also be common linking groups such as -NHCO-, -NH-, -CO-, methylene, residues of common amino acids, etc.
  • linker can be covalently bonded to -SH on the cysteine in Ligand:
  • the linker can be covalently bonded to -NH 2 on the lysine in Ligand:
  • W and X are each independently -(M') t1 - or -(M') t3 -, preferably, 1 or 2 M's are hydrophilic bivalent linking moieties, more preferably one or at least one W X is a hydrophilic divalent linking moiety; examples of the hydrophilic divalent linking moiety include, but are not limited to
  • Linker can be any of the following fragments or any combination of them;
  • Ligand 1 polypeptide, FA (folate), or HSP90 binding agent, or Ligand 1 is R P1 ;
  • Ligand 2 polypeptide, FA (folate), HSP90 binding agent or Ligand 2 is defined as R P2 ;
  • R P1 , R P2 , R 20 and R 21 are as defined above (preferably, R 20 and R 21 are each independently H or methyl);
  • X is H or NH 2 .
  • the dotted line is connected with L P1 .
  • the conjugate of the present invention may also be referred to as is ACTED or an ACTED molecule or an ACTED compound.
  • TED refers to a monovalent group represented by -R T -L1-R E3 or -R E3 -L1-R T ; R P and L4 are as defined above.
  • said ACTED compound is selected from Table D1
  • said ACTED compound is selected from Table D2
  • R P , R P1 , R P2 , t1, t3 and M' are as defined in the first aspect.
  • ACTED examples of the present invention include, but are not limited to, compounds or conjugates selected from the group consisting of:
  • the conjugate TED of the present invention has high activity on tumor cells, has cell selectivity, and has good safety.
  • the conjugate TED of the present invention can exert the effect of inhibiting cell proliferation in a catalytic amount.
  • the cells can circulate and play the role of degrading the target protein, so as to reduce the dosage and prolong the cycle of administration, so as to achieve a safe and effective anti-tumor effect.
  • the linker (L1) part has an active site that can be linked with drug delivery carriers (such as antibodies, polypeptides, and other small molecule ligands).
  • drug delivery carriers such as antibodies, polypeptides, and other small molecule ligands.
  • A is the structure shown in A1 or A2.
  • compound P1.1 (20mg, 1eq.), Linker-Ligand A (1eq.), HATU (2eq.) and DIEA (3eq.) were dissolved in DMF (2mL) and reacted at room temperature for 18 hours .
  • the reaction solution was poured into 5 mL of water and extracted with ethyl acetate (5 mL*3).
  • A is the structure shown in A1 or A2.
  • A is the structure shown in A1 or A2.
  • the compound NH2-Linker-(1eq.) was dissolved in pyridine, then bis(p-nitrophenyl)carbonate (1eq) was added, and reacted at room temperature for 2 hours. Then M (1eq) and DIPEA were added to obtain a yellow reaction solution, followed by reaction at room temperature for 1 hour. The reaction solution was concentrated and purified by a silica gel column to obtain a white solid.
  • M17-a (2 g, 13.2 mmol) in t-BuOH (30 mL) was added M17-b (2.4 g, 13.2 mmol) and 3.1 ml DIPEA, and the mixture was stirred at 90° C. for 18 hours. The mixture was concentrated in vacuo to give a solid. Diethyl ether was added and sonicated for 10 minutes, followed by filtration to afford M17-c (1.8 g, 46% yield) as a white solid.
  • LCMS[M+H] + 298.1
  • UB-PA03a 500 mg, 1.35 mmol
  • 3-butyn-1-ol 94 mg, 1.35 mmol
  • Pd(PPh3)2Cl2 94 mg, 0.135 mmol
  • cuprous iodide 51 mg, 0.27 mmol
  • Compound UB-PA03b (215 mg, yield 54%) was obtained as a white solid.
  • LCMS [M+H] + 313.5
  • Step 5 UB-PA03
  • Step 1 UB-A01
  • Step 1 UB-A02
  • Step 1 UB-A03
  • UB-A04d 200 mg, 0.6 mmol
  • UB-A04e 111 mg, 1.2 mmol
  • AcOH 0.2 mL
  • NaBH3CN 148mg, 2.37mmol
  • Step 8 UB-A04
  • Step 1 UB-A05
  • LCMS: (M+H) + 557.3
  • Step 7 UB-A10
  • Step 1 UB-A06
  • Step 1 UB-A07
  • Step 1 UB-A08
  • Step 1 UB-L01a
  • Step 1 UB-I01(-PP1) and UB-I02(-PP2)
  • UB-A02 (500mg, 0.58mmol) was dissolved in DMF (15mL), and NaH (112mg, 2.9mmol) was added dropwise to the solution under nitrogen protection at zero degrees Celsius, and reacted for half an hour at zero degrees Celsius.
  • 4-nitrophenyl (2-(pyridin-2-yldithio)ethyl)carbonate (()) (2g, 5.8mmol) was dissolved in DMF (5ml), added dropwise to the above reaction solution, room temperature Response for two days.
  • reaction solution was quenched with 1M HCl (1 mL), and purified using a C-18 reverse-phase chromatography column MeCN/H2O/0.5% TFA and high pressure preparation (0.1% FA (folic acid) and 10 mmol/L NH 4 HCO 3 ) to obtain the product as a white solid -PP1 (34 mg, yield 5.3%) and -PP2 (25 mg, yield 4%).
  • LCMS [M+H] 1113.1
  • Step 2 UB-B01
  • UB-I01 35 mg, 0.03 mmol was dissolved in DMF (2 ml) and added dropwise to Oct-C (41 mg, 0.036 mmol) and TEAA (1 ml) at room temperature for half an hour at room temperature.
  • the reaction solution was prepared and purified by C-18 reverse-phase chromatographic column and medium pressure MeCN/H2O/50mmol/l TEAA to obtain white solid UB-B01 (1.9 mg, yield 3%).
  • LCMS[M/2+H] 1062.92
  • Step 3 UB-B02
  • UB-I02 25mg, 0.022mmol was dissolved in DMF (2ml) and added dropwise to Oct-C (29mg, 0.026mmol) and TEAA (1ml) at room temperature for half an hour at room temperature.
  • the reaction solution was prepared and purified by C-18 reverse-phase chromatographic column and medium pressure MeCN/H2O/50mmol/l TEAA to obtain white solid UB-B02 (1.6mg, yield 3.4%).
  • LCMS[M/2+H] 1063.45
  • Step 1 UB-A09
  • Step 1 UB-B03
  • UB-I02 (30mg, 0.027mmol) was dissolved in DMF (2ml) and added dropwise to PS-FA (56mg, 0.054mmol) and TEAA (1ml) at room temperature for half an hour at room temperature.
  • the reaction solution was purified by C-18 reverse-phase chromatography column and medium-pressure preparative chromatography MeCN/H2O/50mmol/l TEAA to obtain a yellow solid product UB-B03 (29.4mg, yield 53%).
  • LCMS [M/2+H] 1024.83.
  • PS-FA Peptide Spacer-Folic Acid
  • Step 1 UB-B04
  • UB-I01 (30mg, 0.027mmol) was dissolved in DMF (2ml) and added dropwise to PS-FA (56mg, 0.054mmol) and TEAA (1ml) at room temperature for half an hour at room temperature.
  • the reaction solution was purified by C-18 reverse-phase chromatography column and medium-pressure preparative chromatography MeCN/H2O/50mmol/l TEAA to obtain the yellow solid product UB-B04 (22.7mg, yield 41%).
  • LCMS[M/2+H] 1025.41
  • Step 1 UB-B05
  • UB-I02 (30mg, 0.027mmol) was dissolved in DMF (3ml) and added dropwise to Oct-C-PS-FA (127mg, 0.054mmol) and TEAA (1.5ml) at room temperature for half an hour at room temperature.
  • the reaction solution was purified by C-18 reverse-phase chromatography column and medium-pressure preparative chromatography MeCN/H2O/50mmol/l TEAA to obtain a yellow solid product UB-B05 (0.9mg, yield 1%).
  • LCMS[M/3+H] 1121.9
  • Oct-C-PS-FA Oletide-Cysteine-Peptide Spacer-Folic Acid
  • Step 1 UB-B06
  • UB-I01 (30mg, 0.027mmol) was dissolved in DMF (3ml) and added dropwise to Oct-C-PS-FA (127mg, 0.054mmol) and TEAA (1.5ml) at room temperature for half an hour at room temperature.
  • the reaction solution was purified by C-18 reverse-phase chromatography column and medium-pressure preparative chromatography MeCN/H2O/50mmol/l TEAA to obtain a yellow solid product UB-B06 (8.3mg, yield 9%).
  • LCMS[M/3+H] 1122.2
  • Step 1 UB-I03
  • Step 1 UB-B07a
  • Step 2 UB-B07
  • Step 1 UB-I07a
  • Step 1 UB-I09a
  • Step 1 UB-I10a
  • Step 2 UB-I10
  • Reagents RPMI-1640 medium, McCoy's5A medium, IMDM medium, MEM medium, L-15 medium, fetal bovine serum, blue-chain double antibody, trypsin, etc., 2-mercaptoethanol, NEAA, acetone Etc.
  • the cells are routinely cultured, and the cells are passed at least 2 generations before plating.
  • Cells in the logarithmic growth phase were collected, prepared into a single cell suspension and counted, the cell concentration was adjusted to the required concentration, and 100 ⁇ l was added to each well to inoculate into a 96-well cell culture plate. 100 ⁇ L of the complete medium of the test compound was added to each well, and two replicate wells were set up for each concentration, and the 5-fold gradient was diluted downward, and the culture was continued for 72 hours. All cells were subjected to EC50 determinations against the samples tested. The experimental results are shown in Test Example 4.
  • the fluorescence intensity of each well was detected by the Alarm blue method, and the IC 50 was calculated.
  • IC50 is calculated by the following formula:
  • Min, Max and Slope represent the minimum value, maximum value and slope, respectively.
  • the cells were collected by centrifugation. After washing with PBS, RIPA buffer was added to lyse the cells; SDS-PAGE gel (4%-12%). After running the gel, transfer to PVDF membrane and block with 5% skimmed milk powder at room temperature for 1 hour. Place the membrane in primary antibody diluted with 5% nonfat dry milk and shake slowly overnight at 4°C. After the primary antibody incubation, wash the membrane 3 times with a TBST shaker; add the secondary antibody corresponding to the primary antibody diluted with 5% skimmed milk powder, and shake slowly at room temperature for 1 hour. After the incubation with the secondary antibody, the membrane was washed again 3 times with a TBST shaker.
  • the conjugate (or TED molecule) of the present invention exhibits concentration-dependent degradation activity on the target protein.
  • Compounds, enzymes, substrates and ATP were diluted to desired concentrations with 1x reaction buffer. Add 1 ⁇ L of different concentrations of compounds, 2 ⁇ L of enzyme, and 2 ⁇ L of substrate/ATP mixed solution into a 384-well plate, and incubate at room temperature for 1 hour. Then add 5 ⁇ L of ADP1-Glo TM reagent to each well and incubate at room temperature for 40 minutes. Finally, 10 ⁇ L of detection reagent was added, and Envision was used to detect chemiluminescent signals after incubation at room temperature for 30 minutes.
  • the TED molecule synthesized in the present invention exhibits strong cell proliferation inhibitory activity in various tumor cell lines, and has the prospect of becoming an antitumor drug.

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Abstract

本文涉及靶向蛋白酶降解(TED)平台，具体地，本发明公开了一种式Ia或式Ib所示的靶标分子-连接体-E3连接酶配体的偶联物，R L-R T-L1-R E3(Ia)R T-L1-R E3-R L(Ib)。

Description

靶向蛋白酶降解(TED)平台 技术领域
本发明属于生物医药，具体地，涉及一种靶向蛋白酶降解(TED)平台。
背景技术
现代分子生物学从3个基本层次上调控蛋白的表达水平：首先，在DNA水平，通过基因敲除，从而使目标蛋白的DNA失活；其次，在mRNA水平，通过小分子RNA，与目标蛋白的mRNA结合，从而抑制mRNA的翻译及表达；再次，在蛋白水平，通过对翻译后靶蛋白的修饰，例如甲基化、磷酸化、糖基化等，从而调整靶蛋白的量及活性。
就药物研发的总体发展来看，小分子和大分子两种药物形式都有各自的优势与不足。如小分子药物的发展一直面临如何维持体内药物浓度以及耐药性等关键挑战。有些靶点部位的形状不利于小分子的药物设计而成为“不可成药”的靶点。针对这些靶点目前还未找到有效的调控方式。泛素-蛋白酶体系统正常生理功能负责清理细胞中变性、变异或者有害的蛋白。虽然靶向蛋白降解剂可通过同时结合细胞中的E3酶和靶蛋白，来诱导蛋白酶体系统对致病靶蛋白的降解，以达到治疗疾病的目的，并且从作用机制讲，这种蛋白降解剂能够在催化量作用下发挥作用，且有望克服传统小分子药物带来的耐药性，但是这类TED蛋白降解剂又会面临透膜性差，体内循环易被代谢，以及进入正常组织带来的毒性等不足。
抗体药物偶联体(ADC)利用抗体的靶向作用，将抗体作为载体，将超级毒素药物送达疾病部位，取得了巨大成功。ADC类药物开发遇到的瓶颈是治疗窗口不够宽，除了抗体本身引起的毒副作用外，超级毒素会因偶联的非均一性而在到达靶位前脱落，引起严重毒副作用。
综上所述，本领域迫切需要开发能够更高效、具有特异性识别功能、且可重复利用的降解靶蛋白从而治疗相关疾病的化合物。
发明内容
本发明的目的在于提供一种能够更高效、且可重复利用的降解靶蛋白从而治疗相关疾病的化合物。具体地，受ADC类药物的启发，本发明人将胞外靶向配体的组织特异性和蛋白降解剂的催化量高效降解致病蛋白的特点相结合，即将蛋白降解剂偶联到胞外受体的配体上，如单抗，多肽，小分子配体等，利用这些配体的对疾病组织的特异性识别功能，将蛋白降解剂成功递送到疾病组织。
在本发明的第一方面，提供了一种如式Ia或式Ib所示的偶联物或其药学上可接受的盐，
R L-R T-L1-R E3  (Ia)
R T-L1-R E3-R L  (Ib)
其中，
(a)所述R E3为E3连接酶配体部分；
(b)所述R T为靶标分子部分；
(c)所述L1为连接R E3和R T部分的连接头，且L1如式II所示；
-W 1-L2-W 2-  (II)
其中，
W 1和W 2各自独立地为-(W) s-；其中，s＝0、1、2、3或4(较佳地，s＝0、1或2，更佳地， s＝1或2)，W各自独立地选自下组：无(键)、-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、-C(R b)＝C(R b)-、-C≡C-、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、取代或未取代的5至10元杂芳基(较佳地，W各自独立地选自下组：-N(R a)-、-C(O)-、-C≡C-；更佳地，W各自独立地选自下组：-NH-、-C(O)-、-C≡C-)；
L2如式III所示，
-(M) o-  (III)
其中，下标o为2～50的整数(较佳地，下标o为2～20的整数，更佳地，下标o为2～10的整数；最佳地，下标o为2、3、4、5、6或7)；M各自独立地为选自下组的二价基团：-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、-C(R b)＝C(R b)-、-C≡C-、取代或未取代的C 3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C 6-10芳基、取代或未取代的5至10元杂芳基、氨基酸残基；
(d)R L为R'或R"；
(d1)R'不存在或为H、活性基团或离去基团；较佳地，R'各自独立地选自下组：H、-OH、-SH、-COOH、-COO-C 1-6烷基、保护基团(如氨基保护基团)；
(d2)R"为-W 3-L P1-W P1-(R P) q1；其中，
W 3选自：无，或由1、2或3个各自独立地选自下组的二价片段组成的二价基团：W'、和W U；其中，W U为可自发裂解的二价片段；
L P1为-(M') t1-W Y-(M') t2-；其中，W Y为无或在细胞表面或细胞质内可裂解的二价连接部分；下标t1和t2各自独立地为0、1、2、3、4、5、6、7、8、9或10；
W P1为无、-S-S-或
Figure PCTCN2022136002-appb-000001
其中，*代表与L P1连接的部分；较佳地，W P1为-S-S-
Figure PCTCN2022136002-appb-000002
下标q1＞0(较佳地，q1＝1)；
R P为-W 4-L P4-R P1、-W 4-L P4-R P1-R TED或-W 4-L P4-R P1-R"'；其中，W 4为无或-(W") s1-W P2-(W") s2-；L P4为-(M') t5-；R TED为-R T-L1-R E3或-R E3-L1-R T；
其中，下标s1和s2各自独立地为0、1、2、3或4；t5为0、1、2、3、4、5、6、7、8、9或10(较佳地，t5为0、1、2或3)；W P2为无、NH、-C(R b)(COOH)-、-C(R b)(COR"')-、-C(R b)(NR a)-(如-CH(-NH 2)-)、-N(R"')-或-C(R b)(NH(R"'))-；
R"'为-W 5-L P2-W 6-L P3-R P2；
其中，L P2为-(M') t3-；L P3为-(M') t4-；其中下标t3和t4各自独立地为0、1、2、3、4、5、6、7、8、9或10(较佳地，t3和t4各自独立地为0、1、2或3)；
W 5为-(W') s4-；其中，下标s4＝0、1或2；W 6为
Figure PCTCN2022136002-appb-000003
或-(W") s6-；其中，下标s6＝0、1、2、3或4；
M'各自独立选自下组：-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、取代或未取代的C1-10亚烷基、-(CH 2CH 2O) 1-10-(即-(CH 2CH 2O) 1-、-(CH 2CH 2O) 2-、-(CH 2CH 2O) 3-、 -(CH 2CH 2O) 4-、-(CH 2CH 2O) 5-、-(CH 2CH 2O) 6-、-(CH 2CH 2O) 7-、-(CH 2CH 2O) 8-、-(CH 2CH 2O) 9-、-(CH 2CH 2O) 10-)、氨基酸残基、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、和取代或未取代的5至10元杂芳基；并且任选地1或2个M'为W Z；并且W Z为亲水性二价连接部分；
W'和W"各自独立地为选自下组的二价基团：无、-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、氨基酸残基、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、取代或未取代的5至10元杂芳基；
R P1和R P2各自独立地为相同或不同的多肽元件或者靶标分子T；优选地，R P1和R P2各自独立地为不同的多肽元件或者靶标分子T；
R a各自独立地选自下组：H、OH、SH、取代或未取代的C 1-6烷基、氨基保护基团、含N(R c)环原子的4至10元杂环烷基；
R b各自独立地选自下组：H、卤素、OH、SH、取代或未取代的C 1-6烷基、取代或未取代的C 2-6烯基、取代或未取代的C 2-6炔基、取代或未取代的C 1-6烷氧基、取代或未取代的C 1-6烷基酰基(-C(O)-C 1-6烷基)、羧基、-COO-C 1-6烷基、-OC(O)-C 1-6烷基；或者，位于相同碳上的2个R b以及与它们相连的碳共同构成取代或未取代的C 3-8环烷基、取代或未取代的4至10元杂环烷基；
R c各自独立地选自下组：H、OH、SH、取代或未取代的C 1-6烷基、氨基保护基团；
除非特别说明，所述的取代是指基团中一个或多个(如1、2、或3个)氢被选自下组的取代基所取代：卤素(较佳地，F、Cl、Br或I)、氰基(CN)、氧代(＝O)、硫代(＝S)、羟基(-OH)、C 1-6烷基、C 1-6卤代烷基、C 2-6烯基、C 2-6炔基、C 1-6烷氧基、C 1-6烷基酰基(C 1-6烷基-C(O)-)、-COO-C 1-6烷基、-OC(O)-C 1-6烷基、NH 2、NH(C 1-6烷基)、N(C 1-6烷基) 2。
在另一优选例中，R a各自独立地为H或C 1-6烷基(如甲基)。
在另一优选例中，R b各自独立地为H或C 1-6烷基(如甲基)。
在另一优选例中，R c各自独立地为H或C 1-6烷基(如甲基)。
在另一优选例中，当所述杂环烷基(如4至10元杂环烷基)为二价基团时，所述的4至10元杂环烷基包括：
Figure PCTCN2022136002-appb-000004
其中，k1和k2各自独立地为0、1、2或3；较佳地，所述的4至10元杂环烷基选自下组：
Figure PCTCN2022136002-appb-000005
在另一优选例中，当所述环烷基(如C 3-8环烷基)为二价基团时，所述环烷基(如C 3-8环烷基)包括：
Figure PCTCN2022136002-appb-000006
其中，k1和k2各自独立地为1、2或3；更佳地，所述C 3-8环烷基选自下组：
Figure PCTCN2022136002-appb-000007
Figure PCTCN2022136002-appb-000008
在另一优选例中，当所述杂芳基(如5至10元杂芳基)为二价基团时，所述杂芳基(如5至10元杂芳基)为
Figure PCTCN2022136002-appb-000009
其中，V 1、V 2和V 4各自独立地选自：-O-、-S-、-N＝、-NH-、-CH＝、-CH 2-；V 3选自下组：-N＝、-CH＝；较佳地，所述5至10元杂芳基选自下组：
Figure PCTCN2022136002-appb-000010
Figure PCTCN2022136002-appb-000011
在另一优选例中，W'和W"各自独立地为选自下组的二价基团：-C(R b) 2-、-O-、-N(R a)-、-C(O)-、氨基酸残基。
在另一优选例中，至少1或2个M'为W Z。
在另一优选例中，M'各自独立选自下组：无、-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-(CH 2CH 2O) 1-10-、氨基酸残基。
在另一优选例中，所述可自发裂解的二价片段是指可裂解的二价连接部分或-S-S裂解后，可自发裂解二价片段。
在另一优选例中，所述细胞表面或细胞质内可裂解的二价连接部分由两个或以上选自下组的结构片段组成的二价连接部分：
Figure PCTCN2022136002-appb-000012
在另一优选例中，所述可自发裂解的二价片段选自下组：
Figure PCTCN2022136002-appb-000013
在另一优选例中，W 3为-W'-W U-W'-。
在另一优选例中，W 3为无、-C(O)-或-OC(O)-。在另一优选例中，W 3为W U。
在另一优选例中，W 3为选自下组：
Figure PCTCN2022136002-appb-000014
在另一优选例中，R P为-W 4-L P4-R P1、-W 4-L P4-R P1-R TED或-W 4-L P4-R P1-R"'。
在另一优选例中，R"为-W 3-L P1-W P1-R P1。
在另一优选例中，当W P1为无或
Figure PCTCN2022136002-appb-000015
时，W Y为在细胞表面或细胞质内可裂解的二价连接部分。
在另一优选例中，细胞表面或细胞质内可裂解的二价连接部分是指能够在细胞表面或细胞质的酸性环境裂解或者被GSH酶特异性切割的二价连接部分。
在另一优选例中，W P1为
Figure PCTCN2022136002-appb-000016
且W Y为在细胞表面或细胞质内可裂解的二价连接部分，或者，W P1为-S-S-且W Y为无。
在另一优选例中，所述细胞表面或细胞质内可裂解的二价连接部分选自下组：
Figure PCTCN2022136002-appb-000017
在另一优选例中，t1+t2≤15；较佳地，t1+t2≤10；更佳地，t1+t2＝2、3、4、5、6、7或8。
在另一优选例中，W P2为无、-C(R b)(NR a)-(如-CH(-NH 2)-)或-CH(NH(R"'))-。
在另一优选例中，W 4为无、-CH(COOH)-CH 2-、-C(O)-CH(NH 2)-CH 2-、-C(O)-CH(NH(R"'))-CH 2-、-NH-CH(COOH)-CH 2-、-NH-C(O)-CH(NH 2)-CH 2-、或-NH-C(O)-CH(NH(R"'))-CH 2-。
在另一优选例中，所述亲水性二价连接部分是指主链上或侧链存在一个或多个选自下组的基团的二价连接部分：-(CH 2CH 2O)-、-SO 3H、-PO 3H 2、-COOH。
在另一优选例中，所述亲水性二价连接部分或W Z选自下组：
Figure PCTCN2022136002-appb-000018
Figure PCTCN2022136002-appb-000019
其中，n5为0-30的整数(较佳地，n5＝0、1、2、3、4、5、6、7、8、9或10)。
在另一优选例中，W 5为无、-C(O)-或-OC(O)-。
在另一优选例中，至少一个M'为W Z。
在另一优选例中，R P1和R P2中的至少一个能够与细胞外受体结合。
在另一优选例中，R P1和R P2能够与细胞外受体结合。在另一优选例中，R P1和/或R P2为所述细胞外受体的配体。
在另一优选例中，所述细胞外受体为靶标分子T可结合或靶向的受体(如叶酸受体、Hsp90、SSTR、PSMA、CAIX等)。
在另一优选例中，R P1和R P2中的一个为多肽元件且另一个为靶标分子T。
在另一优选例中，R P1和R P2均为相同或不同的多肽元件。
在另一优选例中，R P1和R P2均为相同或不同的靶标分子T。
在另一优选例中，R P1和R P2各自独立地为相同或不同的mAb。
在另一优选例中，所述的靶标分子为靶标分子A或靶标分子T。
在另一优选例中，所述靶标分子A或T包括：小分子、纳米载体，或其组合。
在另一优选例中，所述靶标分子A和T各自独立地为选自下组的靶标分子或者靶向选自下组的靶标(如各自酶或受体)的靶标分子：叶酸、HSP90、TINFRm、TNFR2、NADPH氧化酶(oxidase)、BclIBax、C5a受体(receptor)，HMG-CoA还原酶(reductase)、PDE I-V、角鲨烯环化酶抑制剂(Squalene cyclase inhibitors)、CXCR1、CXCR2、一氧化氮(NO)合成酶(Nitric oxide(NO)synthase)、环加氧酶(cyclo-oxygenase)1-2、5HT受体(5HT receptors)、多巴胺受体(dopamine receptors)、G-蛋白(G-proteins)、Gq、组胺受体(Histamine receptors)、脂肪氧合酶(Lipoxygenases)、类胰蛋白酶丝氨酸蛋白酶(Tryptase serine protease)、胸苷酸合成酶(Thymidylate synthase)、嘌呤核苷酸磷酸化酶(Purine nucleotide phosphorylase)、GAPDH锥虫(GAPDH trypanosomal)、糖原磷酸化酶(Glycogen phosphorylase)、碳酸酐酶(Carbonic anhydrase)、趋化因子受体(Chemokine receptors)、JAW STAT、RXR及其类似物、HIV 1蛋白酶(HIV 1 protease)、HIV 1整合酶(HIV 1 integrase)、流感(Influenza)、乙型肝炎逆转录酶(hepatitis B reverse transcriptase)、神经氨酸酶(neuraminidase)、钠通道(Sodium channel)、MDR、蛋白质P1-糖蛋白(protein P1-glycoprotein)、酪氨酸激酶(Tyrosine kinases)、CD23、CD124、TK p56 lck、CD4、CD5、IL-1受体(IL-1 receptor)、IL-2受体(IL-2 receptor)、TNF-aR,ICAM1,Ca+通道(Ca+channels)、VCAM、VLA-4整合素(VLA-4 integrin)、VLA-4整合素(VLA-4 integrin)、选择素(Selectins)、CD40/40L、新霉素和受体(Newokinins and receptors)、肌苷一磷酸脱氢酶(Inosine monophosphate dehydrogenase)、p38 MAP激酶(p38 MAP kinase)、白细胞介素-1转化酶(Interleukin-1 converting enzyme)、胱天蛋白酶(Caspase)、HCV NS3蛋白酶(HCV NS3 protease)、HCV-NS3 RNA解旋酶 (HCV-NS3 RNA helicase)、甘氨酰胺核糖核苷酸甲酰转移酶(Glycinamide ribonucleotide formyl transferase)、鼻病毒3C蛋白酶(rhinovirus 3C protease)、HSV-I、CMV、ADP聚合酶(ADP1-polymerae)、CDK、VEGF、催产素受体(oxytoxin receptor)、msomal转移蛋白抑制剂(msomal transfer protein inhibitor)、胆汁酸转移蛋白抑制剂(Bile acid transfer protein inhibitor)、5-a还原酶(5-a reductase)、血管紧张素11(Angiotensin 11),甘氨酸受体(Glycine receptors)、去甲肾上腺素再摄取受体(noradrenaline reuptake receptor)、内皮素受体(Endothelin receptors)、神经肽Y和受体(Neuropeptide Y and receptors)、雌激素受体(Estrogen receptors)、AMP、AMP脱氨酶(AMP deaminase)、ACC、EGFR、法呢基转移酶(Farnesyltransferase)。
在另一优选例中，所述的多肽元件包括：抗体、蛋白，或其组合。
在另一优选例中，所述抗体包括：纳米抗体、小分子抗体(minibody)，或其组合。
在另一优选例中，所述多肽元件为抗体；优选地，所述抗体包括纳米抗体(nanobody)、小分子抗体(minibody)、抗体片段(如scFv，Fab)、双抗(Dibody)、单克隆抗体(mAb)等等。在另一优选例中，所述多肽(靶向多肽)的靶标包括但不局限于：EGFR，FGFR，SSTR1-14，GnRH，TRPV1-6，RGD，iRGD等等。
在另一优选例中，所述抗体可与选自下组的抗原或受体结合(例如，与选自下组的一种(即单功能抗体)或两种(即双功能抗体)或更多种(即多功能抗体)抗原和/或受体结合)：DLL3、EDAR、CLL1、BMPR1B、E16、STEAP1、0772P、MPF、5T4，NaPi2b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD22、CD79b、CD19、CD37、CD38、CD138、FcRH2、B7-H4、HER2、NCA、MDP、IL20Rα、短小蛋白聚(Brevican)、EphB2R、ASLG659、PSCA、GEDA、BAFF-R、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2、TENB2、PMEL17、TMEFF1、GDNF-Ra1、Ly6E、TMEM46、Ly6G6D、LGR5、RET、LY6K、GPR19、GPR54、ASPHD1、酪氨酸酶(Tyrosinase)、TMEM118、GPR172A、MUC1、CD70、CD71、MUC16、methothelin、FOLR1、TroP1-2、gpNMB、EGFR、ENPP3、PSMA、CA6、GPC-3、PTK7、CD44、CD56、TIM-1、钙粘素-6(Cadherin-6)、ASG-15ME、ASG-22ME、CanAg、AXL、CEACAM5、EphA4、cMet、FGFR2、FGFR3、CD123、Her3、LAMP1、LRRC15、TDGF1、CD66、CD25、BCMA、GCC、Noch3、cMet、EGFR和CD33，或者诸如CD70、Trop2、PD-L1、CD47、CLDN-18.2的受体。在另一优选例中，本发明的靶标分子还可与可以被特异性小分子靶向的受体结合，如叶酸，HSP90，葡萄糖转运蛋白-1(glucose transporter 1)(G LUT1)，氨肽酶(aminopeptidase N)(APN)，低密度脂蛋白受体相关蛋白1(low-density lipoprotein receptor-related protein 1)(LRP1)，前列腺特异性肽(prostate-specific membrane antigen)(PSMA)，整合素αvβ3,铃蟾素(bombesin receptor)、生长抑素受体(somatostatin receptor)(SSTR)，肿瘤乏氧微环境，以及碳酸酐酶IX(CAIX)等受体。
在另一优选例中，R P1为衍生自选自以下的化合物的一价基团或二价基团，R P2为衍生自选自以下的化合物的一价基团；
Figure PCTCN2022136002-appb-000020
其中，所述的衍生自是指，例如在不影响与靶蛋白结合的情况下即在跟靶蛋白结合部分之外 的位置上，任选地通过化学手段引入含有活性基团(如-NH 2、-NH-、-COOH、或-SH等)，或者利用化合物中已存在的活性基团(如-NH 2、-NH-、-COOH、或-SH等)，使化合物中的活性基团中的官能团或原子(如-NH 2或-NH-中的H、或者-COOH中的-OH、或-SH中的H)脱去从而形成可与偶联物中剩余部分连接的位置。
在另一优选例中，R P1为选自表E1的一价基团(R P为-W 4-L P4-R P1时)或者选自表E2的二价基团(当R P为-W 4-L P4-R P1-R TED或-W 4-L P4-R P1-R"'时)；以及R P2为选自表E1的一价基团：
表E1
Figure PCTCN2022136002-appb-000021
表E2
Figure PCTCN2022136002-appb-000022
在另一优选例中，R T为选自表B1的一价基团。在另一优选例中，R T为衍生自选自表B1的一价基团的二价基团。在另一优选例中，式Ia中，R T为衍生自选自表B1的一价基团的二价基团。在另一优选例中，-R T-R L选自表B2。
在另一优选例中，R E3选自以下任一所示的一价基团或者衍生自以下任一所示的一价基团的二 价基团(较佳地，所述的衍生是指一价基团中的-NH-中H脱去从而形成二价基团)：
Figure PCTCN2022136002-appb-000023
各式中，虚线表示与L1连接的位置；
其中，R x各自独立地选自下组：无、NH、NH-CO、O、S、SO、SO 2、SO 2(NH 2)NH、C 1-4亚烷基、C 2-5亚烯基、C 2-5亚炔基；R y为C＝O、C＝S或CH 2。
在一个具体实施方案中，R E3如式A1或A2所示或衍生自式A1或A2：
Figure PCTCN2022136002-appb-000024
式A中，R X选自：无、C1-C6烷基、C2-C6烯基、C2-C6炔基、O、NH、S、CO或SO n(n为1或2)等；R Y为CH 2、C＝S、CO。
在另一优选例中，-R E3-R L为以下任一所示的二价基团
Figure PCTCN2022136002-appb-000025
在另一优选例中，所述E3连接酶配体部分选自表C1中所示的基团。在另一优选例中，R E3为或衍生自式A1.2或式A2.2。
在另一优选例中，L1中不存在-O-O-。
在另一优选例中，s＝1或2。
在另一优选例中，W各自独立地选自下组：无(键)、-N(R a)-、-C(O)-、-C≡C-。
在另一优选例中，W各自独立地选自下组：无(键)、-NH-、-C(O)-、-C≡C-。
在另一优选例中，W 1和W 2各自独立地为-N(R a)-C(O)-、-C(O)-N(R a)-或-C≡C-。在另一优选例中，W 1和W 2各自独立地为-NHC(O)-、-C(O)NH-或-C≡C-。在另一优选例中，W 1和W 2之一为N(R a)-C(O)-或-C(O)-N(R a)-，另一个为-C≡C-。在另一优选例中，W 1和W 2之一为-NHC(O)-或-C(O)NH-，另一个为-C≡C-。
在另一优选例中，M各自独立地为选自下组的二价基团：-C(R b) 2-、取代或未取代的C 3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C 6-10芳基、取代或未取代的5至10元杂芳基。在另一优选例中，M各自独立地为选自下组的二价基团：-C(R b) 2-、取代或未取代的C 3-8环烷基、取代或未取代的4至10元杂环烷基。在另一优选例中，M各自独立地为选自下组的二价 基团：-C(R b) 2-、取代或未取代的C 4-7环烷基、取代或未取代的的4至6元杂环烷基。在另一优选例中，M各自独立地为选自下组的二价基团：-C(R b) 2-、取代或未取代的的4至6元含氮杂环烷基。在另一优选例中，M各自独立地选自下组：-CH 2-、-CH(C 1-4烷基)-、
Figure PCTCN2022136002-appb-000026
Figure PCTCN2022136002-appb-000027
在另一优选例中，M各自独立地选自下组：C(R b) 2、
Figure PCTCN2022136002-appb-000028
较佳地，其中，R b各自独立地为H或C 1-6烷基(如甲基)。
在另一优选例中，下标o为2～10的整数；更佳地，为2、3、4、5、6或7，最佳地，为3、4或5。
在另一优选例中，L2为-Cr 1-Cr 2-(CH 2) o1-；其中，下标o1＝1、2或3，Cr 1为未取代或被C 1-4烷基所取代的C 4-7环烷基或4至6元杂环基；Cr 2为未取代或被C 1-4烷基所取代的C 4-7环烷基或4至6元杂环基。在另一优选例中，L2为-Cr 1-Cr 2-(CH 2) o1-；其中，下标o1＝1、2或3，Cr 1为未取代或被C 1-4烷基所取代的4至6元杂环基；Cr 2为未取代或被C 1-4烷基所取代的4至6元杂环基。在另一优选例中，L2为-Cr 1-Cr 2-(CH 2) o1-；其中，下标o1＝1、2或3，Cr 1为未取代或被C 1-4烷基所取代的4至6元含氮杂环基；Cr 2为未取代或被C 1-4烷基所取代的4至6元含氮杂环基。在另一优选例中，L2中所述含氮杂环烷基环上仅存在氮杂原子。
在另一优选例中，L1为-W 1-Cr 1-Cr 2-(CH 2) o1-W 2-。在另一优选例中，L1为-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-。
在另一优选例中，所述偶联物为R L-R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3或R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3-R L。在另一优选例中，所述偶联物为R L-R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3或R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3-R L；其中，R T如表B1中式P5所示或者衍生自表B1中式P5，且R E3为如式A1或式A2所示或衍生自如式A1或式A2。
在另一优选例中，所述偶联物选自表A1。在另一优选例中，所述偶联物选自表D1和D2。在另一优选例中，所述偶联物选自表D3。
在本发明的第二方面，提供了一种ACTED化合物或其药学上可接受的盐，其中，所述的ACTED化合物如式IVa或IVb所示；
R"-R T-L1-R E3  (IVa)
R T-L1-R E3-R"  (IVb)
其中，R"、R T、L1和R E3如第一方面中定义。
在另一优选例中，所述ACTED化合物选自表D1和D2。
在另一优选例中，所述ACTED化合物选自表D3。
在本发明的第三方面，提供了一种TED化合物或其药学上可接受的盐，其中，所述的TED化合物如式Va或Vb所示；
R'-R T-L1-R E3  (Va)
R T-L1-R E3-R'  (Vb)
其中，R'、R T、L1和R E3如第一方面中定义。
在另一优选例中，所述TED化合物在R'处能够进一步直接或间接地与胞外受体的配体偶联。
在另一优选例中，R'不存在或为H。
在另一优选例中，当R'不存在或为H时，所述TED化合物不为UB-PA01、UB-PA02和UB-PA03。
在另一优选例中，所述偶联物为R'-R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3或R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3-R'。在另一优选例中，所述偶联物为R'-R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3或R T-C(O)-N(R a)-Cr 1-Cr 2-(CH 2) o1-C≡C-R E3-R'；其中，R T如表B1中式P5所示或者衍生自表B1中式P5，且R E3为如式A1或式A2所示或衍生自如式A1或式A2。
在另一优选例中，所述TED化合物为选自表A1。
在本发明的第四方面，提供了一种药物组合物，其中，所述的药物组合物含有(i)如第一方面所述的偶联物或如第二方面所述的ACTED化合物或如第三方面所述的TED化合物；以及(ii)药学上可接受的载体。
在本发明的第五方面，提供了如第一方面所述的偶联物或如第二方面所述的ACTED化合物或如第三方面所述的TED化合物在制备用于治疗或预防与靶标蛋白过量相关的疾病的药物中的的用途。
在本发明的第六方面，提供了一种如如第一方面所述的偶联物或如第二方面所述的ACTED化合物或如第三方面所述的TED化合物在治疗或预防与靶标蛋白过量相关的疾病中用途。
在本发明的第七方面，提供了一种减少细胞中靶标蛋白含量的方法，其中，将细胞与如如第一方面所述的偶联物或如第二方面所述的ACTED化合物或如第三方面所述的TED化合物相接触，从而减少细胞中靶标蛋白的含量。
在另一优选例中，所述的方法是体外方法。
在另一优选例中，所述的方法是非诊断性和非治疗性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
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无
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种结构新颖的TED偶联物，本发明的TED偶联物具有式I所示的结构。此外，本发明的TED偶联物非常适合进一步与多肽元件(尤其是抗体、蛋白配体)和/或其他具有靶向性的分子连接，或者进一步与多肽元件和/或其他具有靶向性的分子等进一步连接后或进一步连接了多肽元件和/或其他具有靶向性的分子的偶联物中的多肽元件和/或其他具有靶向性的分子，使得本发明偶联物具有优异的特异性(例如靶向肿瘤细胞的特异性)，能够显著提高药物选择性，对致病蛋白实施更精准降解，减少非特异性降解可能引起的周身毒性，并有可能克服药物吸收代谢遇到的困难，铲除产生耐药性的机会。在此基础上发明人完成了本发明。
术语
如本文所用，术语“本发明化合物”、“本发明偶联物”可互换使用，指本发明第一方面中所述的 式I化合物或偶联物。
如本文所用，除非另有定义，术语“烷基”本身或作为另一取代基的一部分是指具有指定碳原子数的直链或支链烃基(即，C 1-6表示1-6个碳)。较佳地，烷基具体1～4个碳即C 1-4烷基。烷基的例子包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、正戊基、正己基、正庚基、正辛基等。术语“烯基”指具有一个或多个双键的不饱和烷基。较佳地，烯基具体2～4个碳即C 2-4烯基。类似地，术语“炔基”指具有一个或多个三键的不饱和烷基。较佳地，炔基具体2～4个碳即C 2-4炔基。此类不饱和烷基的例子包括但不限于：乙烯基、2-丙烯基、巴豆基、2-异戊烯基、2-(丁二烯基)、2,4-戊二烯基、3-(1,4-戊二烯基)、乙炔基、1-和3-丙炔基、3-丁炔基和更高级的同系物和异构体。术语“环烷基”是指具有指定环原子数(例如，C 3-6环烷基)并且完全饱和的或在环顶之间具有不超过一个双键的烃环。
如本文所用，术语“环烷基”是指具有指定环原子数(例如，C 3-8环烷基)并且完全饱和的或在环顶之间具有不超过一个双键的烃环。该术语也包括双环和多环烃环，例如双环[2.2.1]庚烷、双环[2.2.2]辛烷等。术语“杂环烷基”是指含有一至五个选自N、O和S的杂原子的环烷基，其中氮和硫原子任选被氧化，且氮原子任选被季铵化。杂环烷基可以是单环、双环或多环体系。杂环烷基的非限制性例子包括吡咯烷、咪唑烷、吡唑烷、丁内酰胺、戊内酰胺、咪唑烷酮、乙内酰脲、二氧戊环、苯邻二甲酰亚胺、哌啶、1,4-二噁烷、吗啉、硫代吗啉、硫代吗啉-S-氧化物、硫代吗啉-S,S-氧化物、哌嗪、吡喃、吡啶酮、3-吡咯啉、噻喃、吡喃酮、四氢呋喃、四氢噻吩、奎宁环等。杂环烷基可以经环碳或杂原子连接于分子的其余部分。对于诸如环烷基烷基和杂环烷基烷基的术语，是指环烷基或杂环烷基通过烷基或亚烷基连接体连接到分子的其余部分。例如，环丁基甲基-是连接到分子其余部分的亚甲基连接基上的环丁基环。
术语“亚烷基”本身或作为另一取代基的一部分是指衍生自烷烃的二价基团，例如-CH 2CH 2CH 2CH 2-。烷基(或亚烷基)通常具有1-24个碳原子，其中本发明优选具有10个或更少碳原子的那些基团。“低级烷基”或“低级亚烷基”是较短链烷基或亚烷基，通常具有4个或更少的碳原子。类似地，“亚烯基”或“亚炔基”分别指具有双键或三键的不饱和形式的“亚烷基”。
除非另有说明，术语“杂烷基”本身或与其它术语组合是指的稳定的直链或支链或环状烃基或其组合，由指定数目的碳原子和和1至3个选自O，N，Si和S的杂原子组成，且其中氮和硫原子可选地被氧化，氮杂原子可任选地被季铵化。杂原子O，N和S可以位于杂烷基的任何内部位置。杂原子Si可以位于杂烷基的任何位置，包括烷基连接到分子其余部分的位置。实施例包括-CH 2-CH 2-O-CH 3,-CH 2-CH 2-NH-CH 3,-CH 2-CH 2-N(CH 3)-CH 3,-CH 2-S-CH 2-CH 3,-CH 2-CH 2,-S(O)-CH 3,-CH 2-CH 2-S(O) 2-CH 3,-CH＝CH-O-CH 3,-Si(CH 3) 3,-CH 2-CH＝N-OCH 3,和-CH＝CH-N(CH 3)-CH 3。最多两个杂原子可以是连续的，例如-CH 2-NH-OCH 3和-CH 2-O-Si(CH 3) 3。类似地，除非另有说明，术语“杂烯基”和“杂炔基”其本身或与另一个术语的组合分别指烯基或炔基，其分别含有指定数目的碳和1至3个选自O，N，Si和S的杂原子，且其中氮和硫原子可选地被氧化，氮杂原子可任选地被季铵化。杂原子O，N和S可以位于杂烷基的任何内部位置。
术语“杂亚烷基”本身或作为另一取代基的一部分是指由杂烷基衍生的饱和或不饱和或多不饱和的二价基团，例如-CH 2-CH 2-S-CH 2CH 2-和-CH 2-S-CH 2-CH 2-NH-CH 2-,-O-CH 2-CH＝CH-,-CH 2-CH＝C(H)CH 2-O-CH 2-和-S-CH 2-C≡C-。对于杂亚烷基，杂原子也可以占据链末端中的任一个或两个(例如，亚烷基氧基，亚烷基二氧基，亚烷基氨基，亚烷基二氨基等)。
术语"烷氧基"、"烷氨基"和"烷硫基"(或硫代烷氧基)以其常规意义使用，指代分别经氧原子、氨基或硫原子连接于分子的其余部分的那些烷基。此外，对于二烷基氨基，烷基部分可以相同或不同，也可和与各烷基相连的氮原子组合形成3-7元环。因此，-NR aR b所示基团表示包括哌啶基、吡咯烷基、吗啉基、氮杂环丁烷基(azetidinyl)等。
除非另有表述，术语“卤代”或“卤素”本身或作为另一取代基的一部分是指氟、氯、溴、或碘原子。此外，诸如“卤代烷基”等术语表示包括单卤代烷基或多卤代烷基。例如，术语“C 1-4卤代烷基”表示包括三氟甲基、2,2,2-三氟乙基、4-氯丁基、3-溴丙基等。
除非另有表述，术语“芳基”表示多不饱和的(通常芳香性)的烃基，其可以是单环或稠合在一起或共价连接的多环(最多三环)。术语"杂芳基"是指含有1至5个选自N、O、和S的杂原子的芳基(或环)，其中氮和硫原子任选被氧化，氮原子任选被季铵化。杂芳基可通过杂原子连接于分子的其余部分。芳基的非限制性例子包括苯基、萘基和联苯基，而杂芳基的非限制性例子包括吡啶基、哒嗪基、吡嗪基、嘧啶基、三嗪基、喹啉基、喹喔啉基、喹唑啉基、噌啉基、酞嗪基、苯并三嗪基(benzotriazinyl)、嘌呤基、苯并咪唑基、苯并吡唑基、苯并三唑基、苯并异噁唑基、异苯并呋喃基(isobenzofuryl)、异吲哚基、中氮茚基、苯并三嗪基、噻吩并吡啶基、噻吩并嘧啶基、吡唑并嘧啶基、咪唑并吡啶、苯并噻唑基、苯并呋喃基、苯并噻吩基、吲哚基、喹啉基、异喹啉基、异噻唑基、吡唑基、吲唑基、蝶啶基、咪唑基、三唑基、四唑基、噁唑基、异噁唑基、噻二唑基、吡咯基、噻唑基、呋喃基、噻吩基等等。以上芳基和杂芳基环系统各自的取代基选自下述可接受的取代基的组。
为简洁起见，当术语“芳基”与其它术语(例如芳氧基，芳硫基，芳烷基)组合使用时，包括如上所定义的芳基和杂芳基环。因此，术语“芳烷基”是指包括其中芳基连接到与分子的其余部分连接的烷基的那些基团(例如苄基，苯乙基，吡啶基甲基等)。
在一些实施例中，上述术语(如“烷基”，“芳基”和“杂芳基”)将包括指定基团的取代和未取代形式。下面提供了每种类型基团的优选取代基。为简洁起见，术语芳基和杂芳基将指代如下文所提供的取代或未取代的形式，而术语“烷基”和相关的脂肪族基团是指未取代的形式，除非指明被取代。
烷基(包括通常称为亚烷基，烯基，炔基和环烷基的那些基团)的取代基可以是选自下组的各种基团：-卤素、-OR'、-NR'R"、-SR'、-SiR'R"R"‘、-OC(O)R'、-C(O)R'、-CO 2R'、-CONR'R"、-OC(O)NR'R"、-NR"C(O)R'、-NR'-C(O)NR"R"‘、-NR"C(O) 2R'、-NH-C(NH 2)＝NH、-NR'C(NH 2)＝NH、-NH-C(NH 2)＝NR'、-S(O)R'、-S(O) 2R'、-S(O) 2NR'R"、-NR'S(O) 2R"、-CN和-NO 2，数量从零到(2M'+1)，其中M'是这种基团中的碳原子总数。R'、R"和R"‘各自独立地表示氢，未取代的C 1-8烷基，未取代的杂烷基，未取代的芳基，被1-3个卤素取代的芳基，未取代的C 1-8烷基，C 1-8烷氧基或C 1-8硫代烷氧基，或未取代的芳基-C 1-4烷基。当R'和R"连接到相同的氮原子时，它们可以与氮原子结合形成3-，4-，5-，6-或7-元环。例如，-NR'R"是指包括1-吡咯烷基和4-吗啉基。术语“酰基”，单独或作为另一基团的一部分使用，是指其中在最接近该基团的连接点的碳上两个取代基的被取代基＝O取代(例如-C(O)CH 3,-C(O)CH 2CH 2OR'等)。
类似地，芳基和杂芳基的取代基是多种的，并且通常选自：-卤素、-OR'、-OC(O)R'、-NR'R"、-SR'、-R'、-CN、-NO 2、-CO 2R'、-CONR'R"、-C(O)R'、-OC(O)NR'R"、-NR"C(O)R'、-NR"C(O) 2R'、-NR'-C(O)NR"R"'、-NH-C(NH 2)＝NH、-NR'C(NH 2)＝NH、-NH-C(NH 2)＝NR'、-S(O)R'、-S(O) 2R'、-S(O) 2NR'R"、-NR'S(O) 2R"、-N 3、全氟(C 1-C 4)烷氧基和全氟(C 1-C 4)烷基，数量从零到芳香环体系上的开放化合价的总数；其中R'、R"和R"'独立地选自氢，C 1-8烷基，C 3-6环烷基，C 2-8烯基，C 2-8炔基，未取代的芳基和杂芳基，(未取代的芳基)-C 1-4烷基和未取代的芳氧基-C 1-4烷基。其 它合适的取代基包括通过1-4个碳原子的亚烷基链连接到环原子上的每一个上述芳基取代基。
芳基或杂芳基环的相邻原子上的两个取代基可任选地被式-T-C(O)-(CH 2) q-U-的取代基取代，其中T和U独立地为-NH-，-O-，-CH 2-或单键，且q是0至2的整数。或者，芳基或杂芳基环的相邻原子上的两个取代基可任选地被式-A-(CH 2) r-B-，其中A和B独立地是-CH 2-、-O-、-NH-、-S-、-S(O)-、-S(O) 2-、-S(O) 2NR'-或单键，且r是1至3的整数。由此形成的新环中的一个单键可以任选地被双键取代。或者，芳基或杂芳基环的相邻原子上的两个取代基可任选地被式-(CH 2) s-X-(CH 2) t-的取代基替代，其中s和t独立地为0至3的整数，并且X是-O-、-NR'-、-S-、-S(O)-、-S(O) 2-、或-S(O) 2NR'-。-NR'-和-S(O) 2NR'-中的取代基R'选自氢或未取代的C 1-6烷基。
在本发明中，环烷基或杂环烷基为二价基时，所述的环烷基或杂环烷基可失去位于同一环原子(环碳原子上)的两个氢从而与链上的其他链原子连接(形成类似于螺环结构)，或者可失去位于不同环原子上的两个氢从而与链上的其他链原子连接(如-亚环戊基-)。
如本文所用，术语“杂原子”意在包括氧(O)、氮(N)、硫(S)和硅(Si)。
对于本文提供的化合物，从取代基(通常为R基团)到芳香环(例如苯，吡啶等)的中心的键将被理解为是指在芳香环的任何可用顶点提供连接的键。在一些实施例中，该描述也包括稠合在芳环上的环上的连接。例如，绘制到吲哚苯部分的中心的键将表示与吲哚的六元或五元环部分的任何可用顶点连接的键。
如本文所用，术语“氨基酸残基”是指氨基酸的N端-NH 2脱去一个H，C端的-COOH脱去-OH所形成的基团。除非另有定义，在本文中，氨基酸包括天然氨基酸或非天然氨基酸，包括D型和/或L型氨基酸。氨基酸的例子包括但不限于Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。优选地，在本文中，氨基酸为选自下组的氨基酸：L-甘氨酸(L-Gly)，L-丙氨酸(L-Ala)，β-丙氨酸(β-Ala)，L-谷氨酸(L-Glu)，L-天冬氨酸(L-Asp)，L-组氨酸(L-His)，L-精氨酸(L-Arg)，L-赖氨酸(L-Lys)，L-缬氨酸(L-Val)，L-丝氨酸(L-Ser)，L-苏氨酸(L-Thr)；此外，当氨基酸存在2个或以上的氨基和/或者2个或以上的羧基时，该术语还包括不在同一个碳原子上的-NH 2脱去一个H和-COOH脱去-OH所形成的基团，例如由谷氨酸的-NH 2和非α位-COOH分别脱去一个H后形成的二价基团-C(O)-(CH 2) 2-C(COOH)-NH-。
除非另有说明，在本文中，所述衍生自是指，例如在不影响原化合物本身活性(如与靶蛋白结合能力)的情况下，任选地通过化学手段引入含有活性基团(如-NH 2、-NH-、-COOH、或-SH等)，或者利用化合物中已存在的活性基团(如-NH 2、-NH-、-COOH、或-SH等)，并使化合物中的活性基团中的官能团或原子(如-NH 2或-NH-中的H、或者-COOH中的-OH、或-SH中的H)脱去从而形成可与偶联物中剩余部分连接的位置。
术语"药学上可接受的盐"意在包括活性化合物与相对无毒的酸或碱制备的盐，其取决于本文所述化合物上具体的取代基。当本发明化合物含有相对酸性的官能团时，可通过将中性形式的此类化合物与充足量的所需碱(无溶剂的或在合适的惰性溶剂中的)接触来获得碱加成盐。衍生自药学上可接受的无机碱的盐的例子包括铝、铵、钙、铜、铁，亚铁、锂、镁、锰，亚锰、钾、钠、锌等。衍生自药学上可接受的有机碱的盐包括伯胺、仲胺和叔胺的盐，包括取代的胺、环状胺、自然产生的胺等等，例如精氨酸、甜菜碱、咖啡因、胆碱、N,N’-二苄基乙二胺、二乙胺、2-二乙基氨基乙醇、2-二甲基氨基乙醇、乙醇胺、乙二胺、N-乙基吗啉、N-乙基哌啶、葡糖胺(glucamine)、葡萄糖胺(glucosamine)、组氨酸、海巴明、异丙胺、赖氨酸、甲葡糖胺、吗啉、哌嗪、哌啶、聚胺树脂、普 鲁卡因、嘌呤、可可碱、三乙胺、三甲胺、三丙胺、氨基丁三醇等等。当本发明化合物含有相对碱性的官能团时，可通过将中性形式的此类化合物与充足量的所需酸(无溶剂的或在合适的惰性溶剂中的)接触来获得酸加成盐。药学上可接受的酸加成盐的例子包括衍生自无机酸的那些，例如盐酸、氢溴酸、硝酸、碳酸、单氢碳酸、磷酸、单氢磷酸、二氢磷酸、硫酸、单氢硫酸、氢碘酸、或亚磷酸等等；以及衍生自相对无毒的有机酸的盐，例如乙酸、丙酸、异丁酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、扁桃酸、苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸，酒石酸、甲磺酸等等。还包括氨基酸的盐，例如精氨酸盐等等，和有机酸的盐，例如葡萄糖醛酸(glucuronic acid)或半乳糖醛酸(galactunoric acid)等。本发明的某些具体化合物同时含有碱性和酸性官能团，从而能将化合物转换成碱加成盐或酸加成盐。
通过将盐与碱或酸接触并以常规方式分离母体化合物，可以再生该化合物的中性形式。化合物的母体形式与各种盐形式在某些物理性能(例如在极性溶剂中的溶解度)上不同，但除此之外，就本发明的目的而言，那些盐与母体形式化合物是等价的。
除盐形式外，本发明提供前药形式的化合物。本文所述的化合物的前药是在生理条件下很容易经历化学变化以提供本发明化合物的那些化合物。另外，前药可以在离体环境中通过化学或生物化学方法转变为本发明化合物。例如，当置于含合适的酶或化学试剂的经皮贴片贮器中时，前药可缓慢转变为本发明的化合物。
本发明的某些化合物可以非溶剂化形式以及溶剂化形式存在，包括水化形式。溶剂化形式通常与非溶剂化形式等价，应包括在本发明范围内。本发明的某些化合物可以多晶型或无定形形式存在。通常，就本发明所考虑的应用而言，所有物理形式是等价的，应包括在本发明范围内。
本发明的某些化合物拥有不对称碳原子(光学中心)或双键；消旋体、非对映体、几何异构体、区域异构体和单独的异构体(例如，分离的对映体)均应包括在本发明范围内。当本文提供的化合物具有确定的立体化学(表示为R或S，或具有虚线或楔形键指明)时，被本领域技术人员将理解那些化合物为基本上不含其他异构体(例如至少80％，90％，95％，98％，99％和至多100％不含其他异构体)。
本发明化合物还可在构成此类化合物的一个或多个同位素原子处含有非天然比例的原子同位素。某同位素的非天然比例可以定义为从所讨论原子的天然发现的量到100％该原子的量。例如，化合物可以掺入放射性同位素，例如氚( 3H)、碘-125( 125I)或碳-14( 14C)，或非放射性同位素，例如氘( 2H)或碳-13( 13C)。除了本申请所述的那些用途，此类同位素变体可提供额外的用途。例如，本发明化合物的同位素变体可以有额外的用途，包括但不限于作为诊断的和/或成像试剂，或作为细胞毒性/放射毒性治疗剂。另外，本发明化合物的同位素变体可具有改变的药代动力学和药效学特征，从而有助于增加治疗期间的安全性、耐受性或疗效。无论是否有放射性，本发明化合物的所有同位素变体均应包括在本发明范围内。
多肽元件
如本文所用，术语“多肽元件”包括肽段(如3-20aa的短肽)或蛋白。此外，该术语还包括完整的蛋白或其片段。优选的多肽元件包括抗体(如完整抗体、单链抗体、纳米抗体、抗体片段)，尤其是针对肿瘤细胞标志物(如位于肿瘤细胞表面的肿瘤标志物，如细胞表面的受体)或针对炎性因子(如与自身免疫疾病相关的炎性因子)的抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连， 而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA,IgD,IgE,IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1,IgG2,IgG3,IgG4,IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明中，多肽元件不仅可包括完整的抗体，还包括具有免疫活性的抗体的片段(如如Fab或(Fab’) 2片段；抗体重链；或抗体轻链)或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
靶向配体
靶向配体(或靶蛋白部分或靶蛋白配体或配体)是能够结合目标靶蛋白的小分子。
本申请的一些实施方案涉及靶标分子，代表性的靶标分子其包括但不限于：叶酸、Hsp90抑制剂、激酶抑制剂、MDM2抑制剂、靶向含人BET溴结构域的蛋白的化合物、靶向胞质信号蛋白FKBP12的化合物、HDAC抑制剂、人赖氨酸甲基转移酶抑制剂、血管生成抑制剂、免疫抑制化合物和靶向芳基烃受体(AHR)的化合物。
在某些实施方案中，靶向配体是能够结合激酶、BET含溴结构域的蛋白、胞质信号蛋白(例如FKBP12)、核蛋白、组蛋白脱乙酰酶、赖氨酸甲基转移酶、调节血管生成的蛋白、调节免疫应答的蛋白、芳烃受体(AHR)、雌激素受体、雄激素受体、糖皮质激素受体或转录因子(例如，SMARCA4、SMARCA2、TRIM24)。
在某些实施方案中，靶向配体能够结合的激酶包括但不限于：酪氨酸激酶(例如，AATK、ABL、ABL2、ALK、AXL、BLK、BMX、BTK、CSF1R、CSK、DDR1、DDR2、EGFR、EPHA1、EPHA2、EPHA3、EPHA4、EPHA5、EPHA6、EPHA7、EPHA8、EPHA10、EPHB1、EPHB2、EPHB3、EPHB4、EPHB6、ERBB2、ERBB3、ERBB4、FER、FES、FGFR1、FGFR2、FGFR3、FGFR4、FGR、FLT1、FLT3、FLT4、FRK、FYN、GSG2、HCK、HRAS、HSP90、IGF1R、ILK、INSR、INSRR、IRAK4、ITK、JAK1、JAK2、JAK3、KDR、KIT、K RAS、KSP、KSR1、LCK、LMTK2、LMTK3、LTK、LYN、MATK、MERTK、MET、MLTK、MST1R、MUSK、NPR1、N RAS、NTRK1、NTRK2、NTRK3、PDGF RA、PDGF RB、PLK4、PTK2、PTK2B、PTK6、PTK7、RET、ROR1、ROR2、ROS1、RYK、SGK493、SRC、SRMS、STYK1、SYK、TEC、TEK、TEX14、TIE1、TNK1、TNK2、TNNI3K、TXK、TYK2、TYRO3、YES1或ZAP70)、丝氨酸/苏氨酸激酶(例如酪蛋白激酶2、蛋白激酶A、蛋白激酶B、蛋白激酶C、Raf激酶、CaM激酶、AKT1、AKT2、AKT3、ALK1、ALK2、ALK3、ALK4、Auro raA、Auro raB、Auro raC、CHK1、CHK2、CLK1、CLK2、CLK3、DAPK1、DAPK2、DAPK3、DMPK、ERK1、ERK2、ERK5、GCK、GSK3、HIPK、KHS1、LKB1、LOK、MAPKAPK2、MAPKAPK、MEK、MNK1、MSSK1、MST1、MST2、MST4、NDR、NEK2、NEK3、NEK6、NEK7、NEK9、NEK11、PAK1、PAK2、PAK3、PAK4、PAK5、PAK6、PIM1、PIM2、PLK1、RIP2、RIP5、RSK1、RSK2、SGK2、SGK3、SIK1、STK33、TAO1、TAO2、TGF-β、TLK2、TSSK1、TSSK2、ΜLK1或ΜLK2)、周期素依赖性蛋白激酶(例如Cdk1-Cdk11)和富含亮氨酸的重复激酶(例如LRRK2)。
靶标分子
在本发明的式I所示的偶联物中，通过偶联物中的R T(靶标分子部分)来结合靶标蛋白。
在本发明中，靶标分子可以是靶标分子A、靶标分子T、或其组合。
在本发明中，所述靶标分子可以是所述靶标蛋白的任意一种抑制剂。所述靶标分子可以是所述靶标蛋白的高效抑制剂，也可以活性比较差的抑制剂。具体地，本发明的靶标分子可以是针对本领域任一种靶标蛋白的本领域已知的小分子抑制剂。
在某些实施方案中，本文所用的靶标分子具有可与连接头(如L1)进行连接的基团(如-O-，-NR a-(其中，R a为H、或C 1-6烷基等取代基，-CO-、-COO-等等)，以一价与本发明的连接体分子(如本发明中L1)对接成醚、胺、酰胺等等，从而形成靶标分子部分。
所述靶标蛋白可以是本领域已知的各种靶标蛋白，代表性的例子包括(但并不限于)：MDM2、AKT、BCR-ABL、Tau、BET(BRD2,BRD3,BRD4)、ERRα、FKBP12、RIPK2、E RBB3、雄激素受体、MetAP2、TACC3、FRS2α、PI3K、DHFR、GST、Halo Tag、C RABPI,C RABPII、 RAR、芳烃受体、雌激素受体。不同的靶标蛋白和一些相应的抑制剂可市售获得或用常规方法制备。例如，对于MDM2，其抑制剂可参见WO2017176957、WO2017176958A1等文献。
在另一个具体实施方案中，R T为表B1中任一所示的一价基团，或者为衍生自表B1中任一所示的一价基团的二价基团(较佳地，所述的衍生是指基团中-NH-中H脱去从而形成二价基团)
表B1
Figure PCTCN2022136002-appb-000029
各式中，
R Pa选自下组：任选取代的C 1-6烷基、任选取代的C 2-6烯基、任选取代的C 2-6炔基；
R Pb选自下组：H、-OH、取代或未取代的C 1-6烷基(如C 1-6羟烷基)、-COR Pb1；其中，R Pb1选自下组：H、-N(R a) 2、-NH-N(R a) 2、-OH、-OC 1-6烷基、-O-C 1-4亚烷基-N(R a) 2；较佳地，R Pb为CO-NH 2
X P1为N或CH。
在另一个优选方案中，R T为式P5或者为衍生自式P5的二价基团。
在另一个优选方案中，式P5如下任一所示
Figure PCTCN2022136002-appb-000030
在另一个优选方案中，式P1如下任一所示
Figure PCTCN2022136002-appb-000031
在另一个优选方案中，-R T-R L如表B2中任一所示
表B2
Figure PCTCN2022136002-appb-000032
E3连接酶配体
在本发明中，E3连接酶配体部分(R E3)用于结合E3连接酶。
在一些优选的实施方案中，所述E3连接酶配体部分A1选自：WO2017/176957A1中的A 1基 团(较佳地，WO2017/176957A1中的A-10、A-11、A-15、A-28、A-48、A-69、A-85、A-93、A-98、A-99或A-101的相应部分)。
在另一优选例中，所述E3连接酶配体部分选自：
Figure PCTCN2022136002-appb-000033
各式中，虚线表示与其他部分连接的位置(即与R T-L1连接的位置)；
其中，Rx各自独立地选自下组：无、NH、NH-CO、O、S、SO、SO 2、SO 2(NH 2)NH、C1～C4亚烷基、C2～C5亚烯基、C2～C5亚炔基；R y为C＝O,C＝S或CH 2。
在一个具体实施方案中，代表性的E3连接酶配体部分具有或衍生自(例如，其中-NH-脱去H从而形成与R L连接位置)如式A1或A2所示的结构：
Figure PCTCN2022136002-appb-000034
式A中，R X选自：无、C1-C6烷基、C2-C6烯基、C2-C6炔基、O、NH、S、CO或SO n(n为1或2)等；R Y为CH 2、C＝S、CO；而且，所述E3连接酶配体(式I中的R E3)可通过其中的R X基团与本发明的L1进行连接，如-R x-L1-R T(如-O-L1-R T)；
或者，代表性的E3连接酶配体部分具有或衍生自(例如，其中-NH-脱去H从而形成与R L连接位置)如式B所示的结构：
Figure PCTCN2022136002-appb-000035
式B中，R z2为H或C1-C6烷基(如Me)，R z1为H或C1-C6烷基(如Me或Et)。
在某些实施方案中，本文所用E3连接酶配体具有可与连接头进行连接的基团(如-O-，-NR a-(其中，R a为H、或C1-C6烷基等取代基)，-CO-、-COO-等等)，以一价与本发明的连接体分子(如本发明中L1等)对接成醚、胺、酰胺等等。
在另一个具体实施方案中，R E3(E3连接酶配体部分)为表C1中任一所示的一价基团或衍生自表C中任一所示的一价基团的二价基团(较佳地，所述衍生是指-NH-脱去H从而形成与R L连接位置)：
表C1
Figure PCTCN2022136002-appb-000036
在另一优选例中，R E3为或衍生自式A1.2或式A2.2。
在另一优选例中，R E-R L为以下任一所示的二价基团
Figure PCTCN2022136002-appb-000037
连接E3连接酶配体和靶标分子的连接头(如本文中所述的L1)
本发明的连接头(或连接体分子)用于连接靶标分子和E3连接酶配体。例如，通过两端的官能团(例如-OH、-SH、-NH 2、-SOOH或-COOH)与靶标分子或E3连接酶配体连接。
连接体(头)和偶联方法
本发明的连接体(头)L1用于连接靶标分子(部分)R T和E3连接酶配体(部分)R E3。
优选地，所述靶标分子(部分)或E3连接酶配体(部分)可以通过-O-、-S-、-NH-、-NR-、-(C＝O)-、-(C＝O)O-、-SO 2-等基团与连接体连接。
在本发明的连接体上，还可进一步含有其它各种官能团，例如-OH、-NHR、-SH等官能团。
典型地，本发明的连接体L1，可以以下通式II表示：
-W 1-L2-W 2-  式II
式中，W 1、L2、W 2的定义如本发明第一方面中所述。
在另一优选例中，W 1和W 2各自独立地为以下一价基团失去1个氢原子形成的二价所形成的二价基团：-OH、-NH 2、-SH、-COOH、-SO 2H等等。例如，连接体与靶标分子的连接方式可以通过如下所示的连接基团进行连接：
Figure PCTCN2022136002-appb-000038
或者，W 1和W 2各自独立地包括具有刚性的部分(如含四元、五元、或六元脂肪环(饱和碳环)部分、或者五元或、六元芳香性杂环部分等)的二价连接基团，示例性的实例如下以及实施例中所示：
Figure PCTCN2022136002-appb-000039
其中，上述各式中的R如上定义；n为1或2或3。
在一个具体实施方案中，W 1和W 2各自独立地选自下组：
无、-N(R a)-、-C(R b) 2-、-N(R a)-C(R b) 2-、-C(O)-、-C(O)-N(R a)-、-C(R b) 2-C≡C-、-C≡C-、-C(O)-C≡C-、-CH(OH)-C≡C-、-O-、-S-、-SO 2-、-SO-、-PO 3-、-C(R b)＝C(R b)-、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、取代或未取代的5至10元杂芳基。
活性成分
如本文所用，术语“本发明化合物”指式Ia或Ib、式IVa或IVb、式Va或Vb所示的化合物或偶联物。该术语还包括及式Ia或Ib、式IVa或IVb、式Va或Vb化合物的各种晶型形式、或药学上可接受的盐。
在一个方面，本发明提供了一类具有适于进一步与多肽元件(例如，抗体、蛋白配体等)或靶标分子T连接或者连接有多肽元件或靶标分子T的如式IVa或IVb所示的偶联物(即本文所称的TED或TED分子)；
R'-R T-L1-R E3  (Va)
R T-L1-R E3-R'  (Vb)
其中，R'为H或离去基团或活性基团(例如，适于进行偶联反应的基团)；R E3为E3连接酶配体部分，R T为靶标分子部分，L1为连接R T和R E3部分的连接头。优选地，R'、R E3、R T和L1的定义如前所述。
在另一个方面，本发明提供了一类通过具有特定结构的连接头(如-W 3-L P1-W P1-)进一步连接有具有靶向特定目标组织(如肿瘤组织)的配体(例如，多肽元件或靶标分子T)连接的如式IVa或IVb所示的偶联物或分子(即本文所称的ACTED或ACTED分子)
R"-R T-L1-R E3  (IVa)
R T-L1-R E3-R"  (IVb)
其中，R”为偶联有靶向目标组织的配体的具有特定结构的连接头(如-W 3-L P1-W P1-(R P) q1)；R E3为E3连接酶配体部分，R T为靶标分子部分，L1为连接R T和R E3部分的连接头。优选地，R”、R E3、R T和L1的定义如前所述。
靶向蛋白酶降解(Targeted Enzyme Degradation,TED)平台
本发明提供基于本发明偶联物的靶向蛋白酶降解(TED)平台，该平台利用了细胞内的“清洁工”—泛素-蛋白酶体系统。
典型地，基于本发明TED技术，可利用细胞自身的蛋白质破坏机制来从细胞中去除特定致癌病蛋白，因此是一种靶向治疗的替代方法。
与传统蛋白抑制剂作用原理不同，本发明的TED技术是一个双功能杂合化合物，一边用来结合目标蛋白，另一边用来结合一个E3连接酶，使得目标蛋白可以与E3连接酶结合，把目标蛋白泛素化，从而被蛋白组降解。理论上TED技术只是提供结合活性，不需直接抑制目标蛋白的功能活性，又可以重复利用，因此，具有优异的应用前景。
特别地，本发明的经过优化的TED分子，具备优越的靶蛋白降解能力，从而抑制病灶细胞生长。此外，本发明的TED通过具有特定结构的连接头(例如，链上存在细胞表面或细胞质内可裂 解的二价连接部分(如-S-S-，或-AN-、-AAN-、-VA-、-GGFG-、-AAFG、-VCit-、-VL-等肽链)和亲水性二价连接部分(如PEG链、含酸性官能团的侧链如-SO 3H、-PO 3H 2、-COOH等))跟具有靶向肿瘤组织的配体(如本文所定义的R P1和R P2，如单抗，双抗，多肽，叶酸等)偶联形成本文所述的ACTED分子(或偶联物)。
此外，所述的连接头还可具有在例如存在细胞表面或细胞质内可裂解的二价连接部分裂解后，可自发裂解而释放出TED分子的可自发裂解的二价片段。这些二价片段跟肽链(即可裂解的二价连接部分)连接在一起的时候，在体内循环过程可保持稳定，一旦肽链被细胞质内酶裂解或者酸性环境促发断裂后，裸露的二价片段变得化学上不稳定，在肿瘤组织酸性环境下很快自发裂解，释放出TED。因此，这些二价片段为没有活性链接基团的TED分子(如A02)提供了带有活性链接端的部分。具有上述结构的ACTED进入血液循环，其通过特定结构的连接头所偶联的配体部分跟肿瘤细胞表面的抗原或受体结合，从而能够快速富集到肿瘤组织。在与肿瘤细胞结合后，本发明的ACTED可发生以下作用：例如，1.经过受体介导的内吞作用进入细胞，在细胞质中，被酸性环境，或者GSH(谷胱甘肽)，或者特异性酶切割，释放出活性分子TED，TED再与细胞内的靶蛋白和E3酶结合，通过泛素介导的蛋白酶体，降解靶蛋白，进而杀死肿瘤细胞；2.ACTED在细胞表面即被微环境的酸性环境，或者GSH，或特异性酶切割，释放出TED，TED再扩散进入细胞内发挥降解靶蛋白并杀伤肿瘤细胞的作用。可见，本发明还提供了一种基于靶向肿瘤微环境及低氧状态的前药(Pro-drug)偶联物”。
在一些实施例中，这些靶向靶向肿瘤组织的配体可以是衍生自如下化合物的一价或二价基团：
Figure PCTCN2022136002-appb-000040
因此，本发明的ACTED的优点可以分为两个方面：1.将更多的TED富集到肿瘤组织，并帮助TED进入肿瘤细胞，降解靶蛋白从而杀伤肿瘤细胞，提高了TED的利用率；和2.ACTED极少结合正常细胞，故循环过程中更少的TED进入正常组织，减少了毒副作用。
一些通过具有特定结构的连接头连接的配体(即-W 3-L P1-W P1-(R P) q1，其中下标q1＝1)的示例性结构如下任一所示：
Figure PCTCN2022136002-appb-000041
Figure PCTCN2022136002-appb-000042
各式中，L4为-(M') q-，其中M'如第一方面中定义；q为0～10的整数，n5为0-10的整数(较佳地，n5＝0、1、2、3、4、5、6、7、8、9或10)；R 20和R 21各自独立地为H或C1-6烷基(较佳地，R 20和R 21各自独立地选自下组：-H，-Me,-Et,-nPr,iPro,cPro)。
一些示例性的用于跟具有靶向肿瘤组织的配体偶联的连接头如下，其中仅示例性地列举了代表性的连接片段，应当理解，其中各片段之间(如W Z和Linker之间)还可能存在诸如-NHCO-、-NH-、-CO-、亚甲基、常见氨基酸的残基等常见的连接基团。
(1)例如，连接头可通过与Ligand里的半胱氨酸上-SH的共价结合：
Figure PCTCN2022136002-appb-000043
(2)例如，连接头可通过与Ligand里的赖氨酸上的-NH 2的共价结合：
Figure PCTCN2022136002-appb-000044
(3)双配体偶联TED的示例性结构如下
Figure PCTCN2022136002-appb-000045
各式中，
W X各自独立地为-(M') t1-或-(M') t3-，较佳地，1或2个M'为亲水性二价连接部分，更佳地一个或至少一个W X为亲水性二价连接部分；所述亲水性二价连接部分的示例包括但不限于
Figure PCTCN2022136002-appb-000046
Figure PCTCN2022136002-appb-000047
，或者这些片段的任意组合；
各式中，Linker可以是下述任一片段或它们的任意组合；
Figure PCTCN2022136002-appb-000048
Ligand 1＝多肽、FA(叶酸)、或HSP90结合剂，或者Ligand 1为R P1；Ligand 2＝多肽、FA(叶酸)、HSP90结合剂或Ligand 2为R P2定义相同；R P1、R P2、R 20和R 21如前定义(优选地，R 20和R 21各自独立地为H、甲基)；X为H或NH 2。
一些具有可自发降解的二价片段的
Figure PCTCN2022136002-appb-000049
其中，虚线与L P1连接。
ACTED
在本发明中，当靶标分子为抗体，或多肽，或环肽，或者叶酸受体配体，或者HSP90配体，或其他细胞外靶蛋白配体时，也可将本发明的偶联物简称为ACTED或ACTED分子或ACTED化合物。
一些ACTED化合物列举如下：
Figure PCTCN2022136002-appb-000050
其中，TED是指如-R T-L1-R E3或-R E3-L1-R T所示的的一价基团；R P、L4如前定义。在一个具体实施方案中，所述ACTED化合物选自表D1
表D1
Figure PCTCN2022136002-appb-000051
在一个具体实施方案中，所述ACTED化合物选自表D2
表D2
Figure PCTCN2022136002-appb-000052
Figure PCTCN2022136002-appb-000053
Figure PCTCN2022136002-appb-000054
各式中，R P、R P1、R P2、t1、t3和M'如第一方面中定义。
在一个具体实施例中，本发明的ACTED实例包括但不限于选自下组的化合物或偶联物：
本发明的主要优点包括：
(a)本发明的偶联物TED，对肿瘤细胞上活性高，且具有细胞选择性，安全性好。
(b)本发明的偶联物TED，可以催化量发挥抑制细胞增殖的效果。细胞内能够循环发挥降解靶蛋白的作用，实现减少给药剂量，延长给药周期，达到安全有效的抗肿瘤效果。
(c)本发明的偶联物TED,连接头(L1)部分带有可与药物递送载体(如抗体，多肽，其他小分子配体)链接的活性位点。
下面结合具体实施方案，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，实施例中所用的起始原料或化合物均可市售获得或者通过本领域技术人员已 知方法制备得到。
制备实施例
除非特别说明，实施例中所用的起始原料或化合物均可市售获得或者通过本领域技术人员已知方法制备得到。
通用方法
通用方法1:化合物P1.1-Linker-Ligand A的合成方法
Figure PCTCN2022136002-appb-000055
式中，A为A1或A2所示的结构。在N 2条件保护下，化合物P1.1(20mg,1eq.),Linker-Ligand A(1eq.),HATU(2eq.)and DIEA(3eq.)溶解于DMF(2mL)中，室温反应18小时。将反应液倒入5mL水中，用乙酸乙酯萃取(5mL*3)。合并有机相后用饱和食盐水洗(10mL*3)，无水Na 2SO 4干燥，减压浓缩，得到粗品进行薄层色谱硅胶板(DCM/MeOH＝10/1)分离制备得到目标物。
在上述目标物溶于DCM(3mL),加入0.5mL(HCl/二氧六环4M)，室温反应1小时。反应液浓缩后用乙醚洗(5mL*3)，过滤得到白色固体目标产物P1.1-Linker-Ligand A
通用方法2:化合物P1.1–Linker g-Ligand A的合成方法
Figure PCTCN2022136002-appb-000056
式中，A为A1或A2所示的结构。
化合物(R)-8-环戊基-7-乙基-2-((4-乙炔基-2-甲氧基苯基)氨基)-5-甲基-7,8-二氢蝶呤-6(5H)-酮(1eq)、N3-linker-Ligand A(1eq.)、TBTA(1eq.),[Cu(CH 3CN) 4]PF 6(Cat.)溶解于叔丁醇(5mL)和水，该混合物室温反应16小时至4天。反应液减压浓缩后用硅胶柱纯化(MeOH/DCM＝10％)得到白色固体化合物。
在上述目标物溶于DCM(3mL),加入0.5mL(HCl/二氧六环4M)，室温反应1小时。反应液浓缩后用乙醚洗(5mL*3)，过滤得到白色固体目标产物P1-Linker-Ligand A
通用方法3:化合物M–Linker–Ligand A的合成方法
Figure PCTCN2022136002-appb-000057
式中，A为A1或A2所示的结构。
化合物NH2-Linker-(1eq.)溶于吡啶，然后加入二(对硝基苯)碳酸酯(1eq)，室温反应2小时。然后加入M(1eq)和DIPEA后得到黄色反应液，接着室温反应1小时。反应液浓缩后用硅胶柱纯化得到白色固体。
在上述目标物溶于DCM(3mL),加入0.5mL(HCl/二氧六环4M)，室温反应1小时。反应液浓缩后用乙醚洗(5mL*3)，过滤得到白色固体目标产物M–Linker-Ligand A
制备实施例
化合物M12的合成方法：
Figure PCTCN2022136002-appb-000058
步骤1:M12b
将化合物2-氨基苯甲酰胺(6.2g,45.8mmol)置于100毫升三颈烧瓶中，加入异丙醇(100毫升)，加入2，4，5-三氯嘧啶(7g,38mmol)、二异丙基乙胺(8mL,45.8mmol)，80℃搅拌过夜。反应完成后，冷却至室温，然后加入100毫升水和乙酸乙酯。有机相用盐水洗涤，用无水硫酸镁干燥，得到黄色固体M12b(9g，产率83％).LCMS[M+H] +＝284.1. 1H NMR(400MHz,DMSO)δ12.50(s,1H),8.60(d,J＝0.6Hz,1H),8.60–8.28(m,1H),8.24(s,1H),7.89(dd,J＝8.0,1.4Hz,2H),7.72–7.57(m,1H),7.56–7.20(m,1H),7.22(td,J＝7.9,1.1Hz,1H).
步骤2:M12c
将M12b(1g,4mmol)和叔丁基4-(4-氨基苯基)哌啶-1-羧酸酯(1.03g,4mmol)溶于无水DMF(10mL)，加入Pd(OAc) 2(120mg,1mmol)和xanphos(310mg,1mmol)，130℃下搅拌过夜。反应完成后加水，用乙酸乙酯(10毫升*3)萃取。有机层用Na2SO4干燥，浓缩得到粗产物。通过硅胶色谱纯化(DCM/MeOH＝20/1)，得到产物M12c(929mg,产率51％).LCMS[M+H] +＝524.1
步骤3:M12
将化合物M12c(925mg,1.78mmol)以及盐酸二氧六环溶液(10mL,4N)加入到四氢呋喃(10mL)中,室温反应2小时，反应完成后减压旋蒸浓缩，得到化合物M12(747mg,收率100％).LCMS[M+H] +＝424.1
化合物M17的合成方法
Figure PCTCN2022136002-appb-000059
步骤1:M17-c
向在t-BuOH(30mL)溶液的M17-a(2g，13.2mmol)中加入M17-b(2.4g，13.2mmol)和3.1ml DIPEA，然后将混合物在90℃下搅拌18小时。将混合物在真空中浓缩，得到固体。加入乙醚 超声10分钟，然后过滤，得到M17-c(1.8g，46％收率)，为白色固体。LCMS[M+H] +＝298.1
步骤2:M17-e
向在n-BuOH(7mL)溶液的M17-c(300mg，1.0mmol)中加入M17-d(279mg，1.0mmol)，将HCl(0.5mL)加入混合物中，然后搅拌混合物在N 2保护下的微波150℃反应1h。将反应混合物加入到乙醚中，过滤，得到M17-e(400mg，90％产率)，为黄色固体。LCMS[M+H] +＝439.1
步骤3:M17-f
向在DCM/MeOH(20ml)溶液的M17-e(480mg,1.6mmol)中加入TEA(325mg,3.2mmol)和Boc 2O(702mg,3.2mmol)，混合物在常温下搅拌2h，浓缩反应混合物，得到粗产物然后通过硅胶色谱柱(DCM/MeOH)中得到M17-f(300mg，51％收率)，为白色固体。LCMS[M+H] +＝539.4
步骤4:M17-g
向在THF/MeOH/H 2O(100mL)溶液的M17-f(300mg，0.56mmol)中加入NaOH(111mg，2.78mmol)，然后将混合物在40℃下搅拌16小时。浓缩反应混合物，用1M HCl调节pH为5，用乙酸乙酯(200mL*1)萃取，浓缩有机相，得到M17-g(250mg)，为灰色固体。LCMS[M+H] +＝525.5
步骤5:M17-i
向在DMF(2mL)溶液的M17-g(50mg，0.095mmol)中加入HATU(109mg，0.286mmol)和DIEA(37mg，0.286mmol)，然后在常温中搅拌混合物2小时。然后将M17-h(5.8mg，0.095mmol)加入混合物中，将混合物在常温搅拌持续1小时。浓缩反应混合物，得到粗产物，通过制备TLC纯化，得到M17-i(40mg，74％收率)，为白色固体。LCMS[M+H] +＝568.6
步骤6:M17
向在DCM(5ml)溶液中的M17-i(30mg，0.056mmol)中加入4M HCl(1ml)，在常温下搅拌1h。将反应混合物加入到乙醚中，过滤，得到M17(45mg)，为白色固体。LCMS[M+H] +＝468.4
化合物M18的合成方法
Figure PCTCN2022136002-appb-000060
步骤1:M18
向在DCM(3ml)溶液中的M18-a(30mg，0.056mmol)中加入4M HCl(1ml)，在常温下搅拌1h。将反应混合物加入到乙醚中，过滤，得到M18(25mg，90％收率)，为白色固体LCMS[M+H] +＝425.4
化合物M19的合成方法
Figure PCTCN2022136002-appb-000061
步骤1:M19-c
向在DMF(10mL)溶液中的M19-a(200mg，0.381mmol)中加入HATU(434mg，1.14mmol)和DIEA(147mg，1.14mmol)，然后将混合物在常温下搅拌2小时。然后将M19-b(132mg，1.91mmol)加入混合物中，将混合物在常温搅拌持续1小时。浓缩反应混合物通过硅胶色谱纯化，得到M19-c(30mg，15％收率)，为黄色固体。LCMS[M+H] +＝540.6
步骤2:M19
向在DCM(5ml)溶液中的M19-c(30mg，0.056mmol)中加入4M HCl(1ml)，在常温下搅拌1h。将反应混合物加入到乙醚中，过滤，得到M19(25mg，90％收率)，为白色固体。LCMS[M+H] +＝440.5
化合物M23的合成方法
Figure PCTCN2022136002-appb-000062
步骤1:M23-c
化合物M23-a(70mg,0.25mmol),M23-b(68mg,0.25mmol)溶于正丁醇(2mL)后加入催化量4M HCl二氧六环溶液，微波至150℃ 1小时。反应液浓缩后通过柱层析分离(MeOH/DCM＝1/10)得到棕色固体目标产物M23-c(80mg,收率62.3％)。LCMS[M+H] +＝521.2
步骤2:M23-d
M23-c(40mg,0.07mmol)、HATU(44mg,0.11mmol)、DIEA(29mg,0.23mmol)加入DMF(1mL)在常温中搅拌混合物2小时。然后将BOC-肼(15mg,0.1mol)加入，反应体系在常温搅拌持续1小时。浓缩反应混合物，得到粗产物，制备板纯化得到白色固体M23-d(40mg,收率82％收率)。LCMS[M+H] +＝635.2
步骤3:M23
K 2CO 3(100mg)加入到M23-d(30mg,0.05mmol)的MeOH(2mL)溶液中，在常温下搅拌反应16h。将反应混合物过滤，浓缩得到黄色M23(15mg,收率58.9％)。LCMS[M+H] +＝539.2
化合物M24的合成方法
Figure PCTCN2022136002-appb-000063
步骤1:M24-c
化合物M24-a(50mg,0.18mmol),M24-b(66.25mg,0.18mmol)溶于正丁醇(12mL)后加入DIPEA(0.1mL)，并通过微波合成仪加热至150℃ 2小时。反应液浓缩后加入乙醚超声十分钟，过滤得到白色固体M24-c(100mg,收率91.2％)。 1H NMR(400MHz,DMSO-d6)δ9.63(s,1H),8.39(s,1H),7.70(dd,J＝8.0,1.2Hz,1H),7.43(dd,J＝7.6,1.6Hz,1H),7.35(td,J＝7.7,1.7Hz,1H),7.24(td,J＝7.5,1.3Hz,1H),5.67(t,J＝5.2Hz,1H),4.55(d,J＝5.1Hz,2H).LCMS[M+H] +＝298.1
步骤2:M24-e
化合物M24-c(100mg,0.37mmol),M24-d(102.9mg,0.37mmol)溶于正丁醇(5mL)后加入4M HCl二氧六环溶液(0.1mL)，并通过微波合成仪加热至150℃ 1小时。反应液浓缩后得到黄色固体M24-c M24-e(70mg,收率37％。LCMS[M+H] +＝511.2
步骤3:M24-f
将化合物M24-e(39mg,0.08mol)以及盐酸二氧六环溶液(1mL,4N)加入到二氯甲烷(5mL)中,室温反应1小时，反应完成后减压旋蒸浓缩，得到化合物M24(20mg,收率27％)。LCMS[M+H] +＝411.2
化合物M25的合成方法
Figure PCTCN2022136002-appb-000064
步骤1:M25-c
化合物M24-a M25-a(1500mg,8.29mmol),M25-b(903mg,8.29mmol)溶于正丁醇(30mL)后加入DIPEA(3mL)，反应体系加热至90℃过夜。反应液浓缩后通过硅胶色谱纯化(DCM/MeOH＝10/1)，得到黄色固体M25-c(1.1g,31.2％收率)。
1H NMR(400MHz,DMSO-d6)δ9.93(s,1H),8.84(s,1H),8.35(s,1H),7.59(dd,J＝7.9,1.7Hz,1H),7.08(dd,J＝7.8,1.7Hz,1H),6.94(dd,J＝8.1,1.4Hz,1H),6.86(d,J＝1.5Hz,1H).LCMS[M+H] +＝255.2
步骤2:M25
将化合物M25-c(100mg,0.37mmol)，M25-d(103mg,0.37mmol)以及4N盐酸二氧六环溶液(0.1mL)加入到正丁醇(4mL)中,微波150℃反应1小时，反应完成后减压旋蒸浓缩，得到黄色固体M25(80mg,收率51.6％)LCMS[M+H] +＝397.4
化合物M26的合成方法
Figure PCTCN2022136002-appb-000065
步骤1:M26-c
化合物M26-a(50mg,0.096mmol),M26-b(31mg,0.19mmol)溶于正丁醇(2mL)后加入催化量4M HCl二氧六环溶液，微波至150℃ 1小时。反应液浓缩得到黄色固体M26-c(40mg,收率62.8％)。LCMS[M+1] +＝663.4
步骤2:M26
化合物M26-c(40mg,0.06mmol)溶于甲醇(2mL)后加入K 2CO 3(42mg)，反应体系于室温搅拌过夜反应。反应液浓缩后通过柱层析分离(DCM:MeOH＝10:1)得到黄色固体M26-c(25mg,收率73％)。LCMS[M+1] +＝568.3
化合物UBI-01d的合成方法
Figure PCTCN2022136002-appb-000066
步骤1:UBI-01b(V2127-011)
将UBI-01a(20.7g，100mmol)和三乙胺(17.1g，150mmol)溶于DCM(60mL)中，缓慢加入甲基磺酰氯(12.5g，110mmol)。在室温下反应1小时后，用二氯甲烷(50mL×3)萃取，用盐水(50mL)洗涤合并的有机层，有机相用无水Na2SO4干燥， 减压浓缩，用快速色谱法(DCM:MeOH＝10:1)纯化得到UBI-01b(26.5g，产率93％)，无色油状物。LCMS:[M+1] +＝286.1. 1H NMR(400MHz,CDC13):7.38-7.31(m,5H),5.25-5.20(m,1H),5.11(s,2H),4.38-4.34(m,2H),4.19-4.16(m,2H),3.06(s,3H).
步骤2:UBI-01c(V2127-013)
将UBI-01b(285mg，1.00mmol)、叔丁基哌啶-4-基氨基甲酸酯(200mg，1.00mmol)、碳酸钾(414mg，3.00mmol)溶于DMF(9mL)中，在微波120℃下反应1小时。所得粗品用快速色谱法(石油醚/乙酸乙酯＝80％～100％，20min，二氯甲烷/甲醇＝0％～10％20min)纯化，得到无色油状物UBI-01c(110mg，收率28％)。LCMS:[M+1] +＝390.2. 1H NMR(400MHz,d 6-DMSO)δ8.10–7.15(m,5H),6.78(d,J＝7.5Hz,1H),5.02(s,2H),3.91–3.79(m,2H),3.54(d,J＝13.7Hz,2H),3.21(s,1H),2.96(dd,J＝7.4,6.9Hz,1H),2.70–2.65(m,2H),1.82(dd,J＝3.9,2.5Hz,2H),1.76–1.63(m,2H),1.41(s,9H).
步骤3:UBI-01d
将UBI-01c(110mg，27.3mmol)、10％钯炭(581mg，5.46mmol)加入到甲醇(20ml)中，在氢气环境中室温下反应12h。反应液用硅藻土过滤，滤液浓缩得到UBI-01d(72mg，收率100％)白色固体。LCMS:[M+1] +＝256.2. 1H NMR(400MHz,d 6-DMSO)δ7.57–7.21(m,1H),3.36–3.23(m,4H),3.25–3.16(m,1H),3.03–2.93(m,1H),2.67–2.55(m,2H),1.90–1.61(m,4H),1.41(s,9H),1.37–1.26(m,2H).
化合物UB-PA03的合成方法
Figure PCTCN2022136002-appb-000067
步骤1:UB-PA03b
将UB-PA03a(500mg，1.35mmol)、3-丁炔-1-醇(94mg，1.35mmol)、Pd(PPh3)2Cl2(94mg，0.135mmol)和碘化亚铜(51mg，0.27mmol)加入到DMF(2mL)中，在80℃ N2保护下反应16小时。用反相色谱柱(MeOH/H2O＝5％-95％45min)纯化，60％收集。得到化合物UB-PA03b(215mg，产率54％)白色固体。LCMS[M+H] +＝313.5
步骤2:UB-PA03c
将化合物UB-PA03b(312mg，1.00mmol)和三乙胺(171mg，1.50mmol)加入到二氯甲烷(60mL)中，缓慢滴加甲基磺酰氯(125mg，1.10mmol)。在室温下反应1小时后，用DCM(50mL×3)萃取反应液，用盐水(50mL)洗涤合并的有机层，用无水Na2SO4干燥，减压浓缩，用色谱柱层析(DCM:MeOH＝10:1)纯化得到化合物UB-PA03c(171mg，产率99％)白色固体。LCMS[M+H] +＝391.5
步骤3:UB-PA03e
将化合物UB-PA03c(78mg，0.20mmol)、N,N-二异丙基乙胺(50mg，0.39mmol)和化 合物UB-PA03d(51mg，0.20mmol)加入到乙腈(30mL)中，80℃下反应18h，浓缩得到粗品，然后用硅胶层析(PE/EtOAc＝70％～100％20min，MeOH/DCM＝0％～10％40min)纯化，得到化合物UB-PA03e(51mg，产率46％)。LCMS[M+H] +＝550.5
步骤4:UB-PA03f
将UB-PA03e(51mg,0.09mmol),、盐酸/二氧六环(10mL，4N)加入到四氢呋喃(10mL)中,室温反应2小时，反应完成后减压浓缩，得到化合物UB-PA03f(41mg,收率100％).LCMS[M+H] +＝450.5
步骤5:UB-PA03
方法类似于通用方法3。 1H NMR(400MHz,d6-DMSO)δ11.85(s,1H),10.96(s,1H),9.41(d,J＝14.8Hz,1H),8.79(d,J＝7.6Hz,1H),8.40–8.20(m,1H),7.85–7.65(m,3H),7.65–7.51(m,3H),7.48(t,J＝8.5Hz,2H),7.32–7.07(m,4H),6.17(d,J＝7.6Hz,1H),5.17–4.99(m,1H),4.44(d,J＝17.6Hz,1H),4.32(d,J＝17.6Hz,1H),4.10(d,J＝13.7Hz,2H),3.61–3.47(m,1H),3.38(d,J＝4.51Hz,1H),3.25(s,2H),2.96–2.59(m,6H),2.70–2.56(m,7H),2.54–2.17(m,3H),2.17–1.78(m,6H),1.79–1.43(m,5H),0.85(t,J＝6.8Hz,2H).LCMS[M+H] +＝898.9
化合物UB-A01的合成方法
Figure PCTCN2022136002-appb-000068
步骤1:UB-A01
以类似于通用方法3的方法合成。 1H NMR(400MHz,DMSO)δ11.60(s,1H),11.01(s,1H),9.24(s,1H),8.76(d,J＝4.8Hz,2H),8.25(s,1H),8.17(s,1H),7.74(d,J＝6.9Hz,1H),7.69(d,J＝7.9Hz,1H),7.63(s,1H),7.48(dd,J＝14.2,7.8Hz,4H),7.13(t,J＝7.3Hz,1H),6.92(d,J＝9.1Hz,2H),6.31(d,J＝7.3Hz,1H),5.11(dd,J＝13.3,5.0Hz,1H),4.45(d,J＝17.7Hz,1H),4.32(d,J＝17.5Hz,1H),3.01(s,6H),2.81(d,J＝4.5Hz,7H),2.62(s,6H),2.35(d,J＝17.9Hz,3H),2.00(d,J＝7.6Hz,3H),1.75(dd,J＝25.9,12.9Hz,4H),1.52–1.31(m,4H).LCMS[M+H] +＝913.9
化合物UB-A02的合成方法
Figure PCTCN2022136002-appb-000069
步骤1:UB-A02
以类似于通用方法3的方法合成。 1H NMR(400MHz,DMSO)δ11.86(s,1H),11.02(s,1H),9.25(s,1H),8.80(s,1H),8.31(s,1H),8.15(d,J＝9.9Hz,1H),7.81(d,J＝8.2Hz,1H),7.75(s,1H),7.70(d,J＝7.9Hz,1H),7.64(s,1H),7.49(dd,J＝19.5,10.7Hz,4H),7.22(s,1H),7.11(t,J＝7.5Hz,1H),6.93(d,J＝9.0Hz,2H),6.35(s,1H),5.11(dd,J＝13.4,4.9Hz,1H),4.45(d,J＝17.8Hz,1H),4.32(d,J＝17.5Hz,1H),3.02(s,6H),2.96–2.84(m,4H),2.75–2.60(m,6H),2.37(dd,J＝22.1,9.9 Hz,3H),2.06–1.96(m,3H),1.80(d,J＝56.2Hz,4H),1.45(s,4H).LCMS[M+H] +＝899.9
化合物UB-A03的合成方法
Figure PCTCN2022136002-appb-000070
步骤1:UB-A03
以类似于通用方法3的方法合成。 1H NMR(400MHz,DMSO)δ11.51(s,1H),11.00(s,1H),9.23(s,1H),8.75(t,J＝5.4Hz,2H),8.16(s,1H),7.80(dd,J＝7.9,1.3Hz,1H),7.74–7.65(m,2H),7.56–7.44(m,4H),7.13(t,J＝7.1Hz,1H),6.92(d,J＝9.1Hz,2H),6.37(s,1H),5.11(dd,J＝13.2,5.1Hz,1H),4.77(t,J＝5.7Hz,1H),4.46(d,J＝17.6Hz,1H),4.33(d,J＝17.6Hz,1H),3.82(s,2H),3.53(d,J＝5.8Hz,2H),3.44(s,5H),3.36(d,J＝5.9Hz,2H),3.02(s,6H),2.97–2.87(m,2H),2.66(dt,J＝34.5,25.6Hz,5H),2.46–2.31(m,3H),1.98(dd,J＝25.0,19.9Hz,3H),1.75(s,2H),1.47(s,2H).LCMS[M+H] +＝944.3
化合物UB-A04的合成方法
Figure PCTCN2022136002-appb-000071
步骤1:UB-A04c
化合物UB-A04a(3.0g,13.4mmol)溶于ACN(50mL)加入UB-A04b(4.6g,33.63mmol),K 2CO 3(7.6g,13.4mmol)80℃反应过夜。将反应液浓缩粗品用柱层析分离(PE/EtOAc＝10～50％)得到无色油状产物UB-A04c(500mg,收率8.5％)。
1H NMR(400MHz,氯仿-d)δ7.64–7.55(m,2H),7.33–7.28(m,2H),3.95–3.74(m,2H),3.68(s,3H),3.31(s,1H),2.99(s,4H),2.74(d,J＝25.5Hz,4H),2.42(s,3H),2.00(s,1H),1.24(s,1H),0.82(s,9H).LCMS[M+H] +＝453.6
步骤2:UB-A04d
化合物UB-A04c(500mg,1.1mmol),Mg(130mg,5.5mmol)加入MeOH(5mL)于80℃反应过 夜。将反应液浓缩粗品用柱层析分离(DCM/MeOH＝5-30％)得到黄色油状产物UB-A04d(200mg,收率60.4％)。LCMS[M+H] +＝303.3
步骤3:UB-A04f
将UB-A04d(200mg,0.6mmol),UB-A04e(111mg,1.2mmol),AcOH(0.2mL)加入到甲醇(20mL)，反应体系搅拌一小时。然后将NaBH3CN(148mg,2.37mmol)加入，该反应体系在30℃搅拌16小时。反应完全后用水淬火(20mL)，乙酸乙酯萃取(30mL×3)。合并有机层，硫酸钠干燥,过滤，反应液浓缩后通过硅胶柱层析分离(二氯甲烷/甲醇＝0～10％)，得到白色固体UB-A04f(180mg,55.4％收率)(400mg,收率67.2％)。LCMS[M+H]+＝458.4
步骤4:UB-A04g
UB-A04f(175mg,0.4mol),溶于HCl二氧六环(2mL)和二氯甲烷(10mL)溶液中。室温反应4小时。反应液浓缩得到目标化合物B-A04g(100mg,收率69％收率)LCMS[M+H] +＝358.4
步骤5:UB-A04h
化合物UB-A04g(100mg,0.3mmol),4-bromobut-1-yne(190mg,1.5mmol),K 2CO 3(400mg)加入ACN(10mL)于80℃反应过夜。将反应液浓缩粗品用柱层析分离(DCM/MeOH＝5-10％)得到无色油状产物UB-A04h(100mg,70％收率)。LCMS[M+H] +＝260.2
步骤6:UB-A04i
化合物UB-A04h(100mg,0.25mmol)溶于乙醇(2mL)加入2M NaOH(2mL).室温反应18小时.反应液浓缩加入水(3mL),然后用乙醚萃取(10mL*3)，除去有机杂质.水相用1M HCl中和至pH～6冻干得到白色固体产物UB-A04i(50mg,52.4％收率)。LCMS[M+H] +＝282.2
步骤7:UB-A04j
把混合物UB-A04i(50mg,0.18mmol),A3I(65.8mg,0.17mmol),Pd(PPh 3) 2Cl 2(6mg,),CuI(3mg),TEA(36mg)溶于干燥的DMF(5mL)N 2保护下80℃反应2小时反应液浓缩粗品用柱层析分离(DCM/MeOH＝0-10％)得到黄色油状产物UB-A04j(35mg,37.6％收率)。LCMS[M+H] +＝524.6
步骤8:UB-A04
以类似于通法1的方法合成。LCMS[M+H] +＝929.8.
1H NMR(400MHz,DMSO-d6)δ11.51(s,1H),11.00(s,1H),9.23(s,1H),8.75(t,J＝5.5Hz,2H),8.16(s,1H),7.79(dd,J＝8.0,1.6Hz,1H),7.71(d,J＝7.8Hz,1H),7.66(s,1H),7.54(d,J＝7.9Hz,1H),7.52–7.42(m,3H),7.17–7.09(m,1H),6.92(d,J＝9.1Hz,2H),6.34(s,1H),5.11(dd,J＝13.3,5.1Hz,1H),4.77(t,J＝5.7Hz,1H),4.49–4.29(m,2H),3.53(q,J＝6.0Hz,3H),3.44(s,5H),3.02(d,J＝5.2Hz,5H),2.96–2.87(m,2H),2.62(s,1H),2.39(dd,J＝13.1,4.5Hz,3H),2.01(dd,J＝9.8,4.5Hz,2H),1.75(s,4H),1.40(d,J＝41.7Hz,2H),1.27–1.19(m,3H).
化合物UB-A05的合成方法
Figure PCTCN2022136002-appb-000072
步骤1:UB-A05
方法类似于通用方法3。 1H NMR(400MHz,DMSO)δ11.02(s,1H),9.25(s,1H),8.74(s,1H), 8.33(s,2H),7.77–7.56(m,3H),7.56–7.30(m,4H),7.11(d,J＝7.4Hz,1H),6.91(d,J＝8.7Hz,2H),6.31(s,1H),5.11(dd,J＝13.2,5.2Hz,1H),4.45(d,J＝17.6Hz,1H),4.32(d,J＝17.6Hz,1H),3.01(s,8H),2.94–2.77(m,6H),2.68–2.58(m,6H),2.42–2.31(m,3H),2.04–1.95(m,3H),1.79–1.66(m,4H),1.40(d,J＝11.2Hz,2H).LCMS[M+H] +＝916.1
化合物UB-A10的合成方法
Figure PCTCN2022136002-appb-000073
步骤1:UB-A10b
化合物UB-A10a(1.0g,5.00mmol),二叔丁基二碳酸酯(1.6g,7.4mmol)以及DMAP(82mg)依次加入到二氯甲烷(20mL)中，室温反应2小时。反应完成后倒入10mL水，二氯甲烷提取(5mL*3)。合并有机相后用饱和食盐水洗，无水Na 2SO 4干燥，减压旋蒸浓缩，得到无色油状化合物UB-A10b(1.1g,收率76％)。LCMS:(M+H) +＝273.3
步骤2:UB-A10c
将UB-A10b(1.1g,3.64mmol)、10％Pd/C(50mg)加入到甲醇(80mL)中,在氢气环境下室温反应16小时。过滤后浓缩滤液得到粗品，用冷的乙醚洗涤(10mL*3)，干燥后得到化合物UB-A10c(996mg,收率98％)。LCMS:(M+H) +＝243.3
步骤3:UB-A10d
将UB-A10c(993mg,2.3mmol)、DIEA(500mg,3.9mmol)和2,4,5-三氯嘧啶(662mg,3.66mmol)溶解在乙腈(80mL)中，在80℃下反应18小时。然后通过快速柱层析(石油醚/乙酸乙酯＝70％～100％20min，然后MeOH/DCM＝0％～10％40min)纯化，得到化合物UB-A10d(748mg,收率49％)。LCMS[M+H] +＝420.3。
步骤4:UB-A10e
将UB-A10d(748mg,1.78mmol)、浓盐酸(0.01mL)和UB-A10d-1(486mg,1.78mmol)溶在n-BuOH(30mL)中，在150℃下反应5小时。然后通过快速柱层析(石油醚/乙酸乙酯＝70％～100％20min，然后MeOH/DCM＝0％～10％40min)纯化，得到化合物UB-A10e(545mg,收率55％)。LCMS:(M+H) +＝557.3
步骤5:UB-A10f
化合物UB-A10e(200mg,0.36mmol)、二叔丁基二碳酸酯(160mg,0.74mmol)以及DMAP(82mg)依次加入到四氢呋喃(20mL)中，室温反应2小时。反应完成后倒入10mL水，二氯甲烷提取(5mL*3)。合并有机相后用饱和食盐水洗，无水Na 2SO 4干燥，减压、旋蒸浓缩，得到无色油状化合物UB-A10f(184mg,收率78％)。LCMS[M+H] +＝657.1
步骤6:UB-A10g
将化合物UB-181251f(184mg,0.28mmol)，氢氧化钠(710mg,18mmol)依次加入到甲醇(50mL)中，30℃反应16小时。反应完成后浓缩，水相用盐酸(1M)酸化至pH＝5。然后用二氯甲烷萃取(10ml*3)，合并有机层经无水Na2SO4干燥，浓缩得到白色化合物UB-A10g(127mg,收率81％)。LCMS[M+H] +＝561.3
步骤7:UB-A10
方法类似于通用方法3。 1H NMR(400MHz,DMSO)δ11.85(s,1H),11.05(s,1H),9.41(d,J＝14.8Hz,1H),8.79(d,J＝7.6Hz,1H),8.40–8.20(m,2H),7.85–7.65(m,3H),7.65–7.51(m,3H),7.48(t,J＝8.5Hz,2H),7.32–7.07(m,4H),6.17(d,J＝7.6Hz,1H),5.17–4.99(m,1H),4.44(d,J＝17.6Hz,1H),4.32(d,J＝17.6Hz,1H),4.10(d,J＝13.7Hz,2H),3.61–3.47(m,1H),3.25(s,1H),3.25(s,1H),2.96–2.70(m,6H),2.70–2.56(m,7H),2.17–1.78(m,6H),1.79–1.43(m,9H),1.43–1.15(m,5H),0.81(t,J＝6.8Hz,3H).LCMS[M+H] +＝936.1
化合物UB-A06的合成方法
Figure PCTCN2022136002-appb-000074
步骤1:UB-A06
方法类似于通用方法3。 1H NMR(400MHz,DMSO-d6)δ11.00(s,2H),9.32(s,1H),8.94(s,1H),8.23(s,1H),8.04(dd,J＝8.1,1.7Hz,1H),7.69(d,J＝7.9Hz,1H),7.66–7.56(m,2H),7.50(dd,J＝15.0,8.1Hz,3H),7.27–7.14(m,2H),6.94(d,J＝9.0Hz,2H),6.65(s,1H),6.33(s,1H),5.32(t,J＝4.7Hz,1H),5.11(dd,J＝13.3,5.2Hz,1H),4.50–4.28(m,2H),3.91(s,3H),3.44(s,5H),3.03(s,4H),2.96–2.86(m,2H),2.60(d,J＝16.7Hz,1H),2.42–2.37(m,1H),1.99(p,J＝7.2Hz,5H),1.73(s,4H),1.45(s,4H)LCMS[M/2+H] +＝915.2.
化合物UB-A07的合成方法
Figure PCTCN2022136002-appb-000075
步骤1:UB-A07
方法类似于通用方法3。 1H NMR(400MHz,DMSO-d6)δ11.00(s,2H),9.32(s,1H),8.94(s,1H),8.23(s,1H),8.04(dd,J＝8.1,1.7Hz,1H),7.69(d,J＝7.9Hz,1H),7.66–7.56(m,2H),7.50(dd,J＝ 15.0,8.1Hz,3H),7.27–7.14(m,2H),6.94(d,J＝9.0Hz,2H),6.65(s,1H),6.33(s,1H),5.32(t,J＝4.7Hz,1H),5.11(dd,J＝13.3,5.2Hz,1H),4.50–4.28(m,2H),3.91(s,3H),3.44(s,5H),3.03(s,4H),2.96–2.86(m,2H),2.60(d,J＝16.7Hz,1H),2.42–2.37(m,1H),1.99(p,J＝7.2Hz,5H),1.73(s,4H),1.45(s,4H)。LCMS[M+H] +＝887.2
化合物UB-A08的合成方法
Figure PCTCN2022136002-appb-000076
步骤1:UB-A08
方法类似于通用方法3。LCMS[M+H] +＝872.1
化合物UB-L01d的合成方法
Figure PCTCN2022136002-appb-000077
步骤1:UB-L01a
将奥曲肽(200mg,0.19mmol)和DIEA(48mg,0.37mmol)溶于DMF(5mL)并冷却至-40℃。然后加入BocOSu(40mg,0.19mmol)并在氮气保护下室温搅拌2小时。反应液浓缩后通过反相柱层析得到白色固体目标产物UB-L01a(200mg,收率91％)。LCMS[M+H] +＝1120.0
步骤2:UB-L01c
化合物UB-L01a(200mg,0.18mmol)溶于DMF(5mL)后加入UB-L01b(100mg,0.18mmol)以及DIEA(35mg,0.27mmol)并在氮气保护下室温搅拌过夜。反应液通过反相柱层析得到白色固体目标产物UB-L01c(130mg,收率47％)。LCMS[M+H] +＝1565.5
步骤3:UB-L01d
化合物UB-L01c(930mg,0.10mmol)溶于TFA(3mL)后加入催化量的iPr 3SiH并室温搅拌10分钟。反应液在低温下浓缩后用异丙醚打浆，固体滤出烘干后得到白色固体目标产物UB-L01d(880mg,收率100％)。LCMS[M+H] +＝1123.2.
化合物UB-I01,UB-B01,的合成方法
Figure PCTCN2022136002-appb-000078
式中，Py是指
Figure PCTCN2022136002-appb-000079
Oct是指
Figure PCTCN2022136002-appb-000080
步骤1:UB-I01(-PP1)和UB-I02(-PP2)
UB-A02(500mg,0.58mmol)溶于DMF(15mL)，在零摄氏度氮气保护下NaH(112mg,2.9mmol)滴加到溶液，零摄氏度反应半个小。4-硝基苯基(2-(吡啶-2-基二硫基)乙基)碳酸酯(())(2g,5.8mmol)溶于DMF(5ml)，滴加到上述反应液中，室温反应两天。反应液用1M HCl(1mL)淬灭,使用C-18反相色谱柱MeCN/H2O/0.5％TFA和高压制备纯化(0.1％FA(叶酸)和10mmol/L NH 4HCO 3)得到产品白色固体-PP1(34mg,收率5.3％)和-PP2(25mg,收率4％)。LCMS[M+H]＝1113.1
UB-I01: 1H NMR(400MHz,DMSO)δ12.32(s,1H),11.00(s,1H),8.44(d,J＝11.3Hz,2H),8.33(d,J＝8.1Hz,2H),8.17(s,1H),7.84–7.77(m,2H),7.69(ddd,J＝21.8,11.3,6.4Hz,4H),7.51(d,J＝8.0Hz,1H),7.26(t,J＝7.9Hz,1H),7.23–7.18(m,1H),7.14(d,J＝8.8Hz,2H),7.07(t,J＝7.4Hz,1H),7.00(d,J＝9.0Hz,2H),6.33(d,J＝7.2Hz,1H),5.11(dd,J＝13.3,5.1Hz,1H),4.45(d,J＝17.6Hz,1H),4.34(dd,J＝16.4,11.7Hz,3H),3.52(d,J＝5.4Hz,3H),3.44(s,4H),3.13(s,4H),3.09(t,J＝6.1Hz,2H),3.03–2.86(m,5H),2.71(dd,J＝13.0,6.9Hz,4H),2.60(d,J＝16.4Hz,1H),2.53(s,1H),2.45–2.32(m,1H),2.01(d,J＝5.4Hz,1H),1.83(t,J＝10.8Hz,2H),1.72(d,J＝10.3Hz,2H),1.42(d,J＝9.6Hz,2H).
UB-I02: 1H NMR(400MHz,DMSO)δ11.00(s,1H),10.34(s,1H),9.17(s,1H),8.51–8.44(m,2H),8.21–8.16(m,2H),7.83–7.76(m,3H),7.68(t,J＝6.3Hz,1H),7.62(s,1H),7.56(t,J＝7.9Hz,1H),7.48(dd,J＝12.4,8.8Hz,3H),7.27–7.15(m,2H),6.89(d,J＝9.1Hz,2H),6.29(d,J＝7.2Hz,1H),5.11(dd,J＝13.3,5.1Hz,1H),4.44(d,J＝17.6Hz,1H),4.32(dd,J＝11.8,5.5Hz,3H),3.44(d,J＝9.3Hz,7H),3.11(t,J＝6.2Hz,2H),3.00(s,4H),2.85(s,3H),2.69–2.61(m,5H),2.58(s,1H),2.47(d,J＝7.0Hz,2H),2.41–2.32(m,1H),2.05–1.96(m,1H),1.78(t,J＝11.2Hz,2H),1.71(d,J＝10.5Hz,2H),1.41(d,J＝9.6Hz,2H).
步骤2:UB-B01
在室温下UB-I01(35mg,0.03mmol)溶于DMF(2ml)滴加到Oct-C(41mg,0.036mmol)和TEAA(1ml)中，室温反应半个小时。反应液使用C-18反相色谱柱和中压制备纯化 MeCN/H2O/50mmol/l TEAA得到白色固体UB-B01(1.9mg,收率3％)。LCMS[M/2+H]＝1062.92
步骤3:UB-B02
在室温下UB-I02(25mg,0.022mmol)溶于DMF(2ml)滴加到Oct-C(29mg,0.026mmol)和TEAA(1ml)中，室温反应半个小时。反应液使用C-18反相色谱柱和中压制备纯化MeCN/H2O/50mmol/l TEAA得到白色固体UB-B02(1.6mg,收率3.4％)。LCMS[M/2+H]＝1063.45
化合物UB-A09的合成
Figure PCTCN2022136002-appb-000081
步骤1:UB-A09
通用方法4。LCMS[M+H] +＝943.2
化合物UB-B03的合成
Figure PCTCN2022136002-appb-000082
步骤1:UB-B03
UB-I02(30mg,0.027mmol)溶于DMF(2ml)室温滴加到PS-FA(56mg,0.054mmol)和TEAA(1ml)里，室温反应半小时。反应液使用C-18反相色谱柱纯化和中压制备色谱MeCN/H2O/50mmol/l TEAA得到黄色固体产品UB-B03(29.4mg,收率53％)。LCMS[M/2+H]＝1024.83。
PS-FA＝Peptide Spacer-Folic Acid
Figure PCTCN2022136002-appb-000083
化合物UB-B04的合成
Figure PCTCN2022136002-appb-000084
步骤1:UB-B04
UB-I01(30mg,0.027mmol)溶于DMF(2ml)室温滴加到PS-FA(56mg,0.054mmol)和TEAA(1ml)里，室温反应半小时。反应液使用C-18反相色谱柱纯化和中压制备色谱MeCN/H2O/50mmol/l TEAA得到黄色固体产品UB-B04(22.7mg,收率41％)。LCMS[M/2+H]＝1025.41
化合物UB-B05的合成
Figure PCTCN2022136002-appb-000085
步骤1:UB-B05
UB-I02(30mg,0.027mmol)溶于DMF(3ml)室温滴加到Oct-C-PS-FA(127mg,0.054mmol)和TEAA(1.5ml)里，室温反应半小时。反应液使用C-18反相色谱柱纯化和中压制备色谱MeCN/H2O/50mmol/l TEAA得到黄色固体产品UB-B05(0.9mg,收率1％)。LCMS[M/3+H]＝1121.9
Oct-C-PS-FA(Octreotide-Cysteine-Peptide Spacer-Folic Acid),其结构如下：
Figure PCTCN2022136002-appb-000086
化合物UB-B06的合成
Figure PCTCN2022136002-appb-000087
步骤1:UB-B06
UB-I01(30mg,0.027mmol)溶于DMF(3ml)室温滴加到Oct-C-PS-FA(127mg,0.054mmol)和TEAA(1.5ml)里，室温反应半小时。反应液使用C-18反相色谱柱纯化和中压制备色谱MeCN/H2O/50mmol/l TEAA得到黄色固体产品UB-B06(8.3mg,收率9％)。LCMS[M/3+H]＝1122.2
化合物UB-I03&UB-I04&UB-I05&UB-I06的合成
Figure PCTCN2022136002-appb-000088
步骤1:UB-I03
化合物UB-PA01(100mg,0.10mmol)溶于DMF(5mL)后加入LiHMDS(0.31mL)并在-50℃条件下反应15分钟,然后将UB-I07a(112mg,0.31mmol)在-20℃条件下滴加进反应液并反应5分钟，然后室温反应2小时。反应液通过饱和氯化铵水溶液淬灭后用MeOH/DCM＝1/10溶液萃取，有机相浓缩后通过柱层析分离(甲醇/二氯甲烷＝1/10)得到棕色固体目标产物UB-I03(75mg,收率60％)。LCMS[M/2+H] +＝544.3； 1H NMR(400MHz,DMSO-d 6)δ11.85(s,1H),9.22(s,1H),8.80(s,1H),8.29(s,1H),8.16(s,1H),7.81(dd,J＝7.9,1.6Hz,1H),7.77–7.68(m,2H),7.63(s,1H),7.56–7.37(m,4H),7.15(d,J＝8.1Hz,1H),7.14–7.03(m,1H),6.96–6.77(m,2H),6.10(s,1H),5.68(d,J＝9.6Hz,1H),5.60(dd,J＝9.6,4.8Hz,1H),5.36–5.20(m,1H),4.49–4.36(m,1H),4.26(t,J＝18.8Hz,1H),4.11(q,J＝5.3Hz,2H),3.80(dd,J＝14.3,7.0Hz,2H),3.49(t,J＝5.0Hz,4H),3.38(s,3H),3.17(d,J＝5.2Hz,6H),3.03(t,J＝5.0Hz,4H),2.81(d,J＝17.4Hz,1H),2.69(d,J＝8.1Hz,2H),2.35(d,J＝13.3Hz,1H),2.13–1.91(m,3H),1.85(d,J＝12.5Hz,4H),1.50(s,4H),1.43(s,9H),1.36(d,J＝1.7Hz,9H),0.83(dd,J＝7.3,2.8Hz,6H).
步骤2:UB-I04
化合物UB-I03(80mg,0.07mmol)溶于甲醇/二氯甲烷(1/1mL)后加入HCl/二氧六环(1mL)，并室温反应15分钟。反应液低温浓缩后通过二氯甲烷/乙醚＝1/10打浆得到黄色固体目标产物UB-I04(50mg,收率75％)。LCMS[M+H] +＝987.7； 1H NMR(400MHz,DMSO-d 6)δ11.90(s,1H),9.32(s,1H),9.08(s,2H),8.80(s,1H),8.45(s,2H),8.31(s,1H),8.18(s,1H),7.83(dd,J＝8.0,1.6Hz,1H),7.74(dd,J＝7.9,3.3Hz,2H),7.60(dd,J＝7.8,1.4Hz,1H),7.55–7.43(m,2H),7.12(t,J＝7.8Hz,1H),6.99(s,2H),6.09(d,J＝4.4Hz,1H),5.82(dd,J＝9.7,3.9Hz,1H),5.72(t,J＝9.7Hz,1H),5.29(dt,J＝13.4,4.4Hz,1H),4.50(d,J＝17.5Hz,1H),4.32(dd,J＝17.5,13.8Hz,1H),3.92(s,1H),3.74–3.66(m,1H),3.52(s,4H),3.08(s,6H),2.97(t,J＝7.5Hz,2H),2.92–2.78(m,1H),2.13(ddt,J＝11.9,7.0,3.5Hz,2H),2.00(q,J＝7.3Hz,1H),1.93–1.70(m,5H),1.52(s,2H),0.93(dd,J＝6.8,4.7Hz,6H)。
步骤3:UB-I05
化合物UB-I04(247mg,0.25mmol)溶于DMF(3mL)后加入(叔丁氧基羰基)-L-缬氨酰-L-丙氨酸(72mg,0.25mmol),HATU(143mg,0.38mmol)以及DIEA(65mg,0.50mmol)，并室温反应过夜。反应液浓缩后通过柱层析分离(甲醇/二氯甲烷＝1/20)得到棕色固体目标产物UB-I05(150mg,收率48％)。LCMS[M/2+H] +＝629.4。 1H NMR(400MHz,DMSO-d 6)δ11.85(s,1H),9.23(s,1H),8.80(s,1H),8.29(s,1H),8.16(s,1H),8.00(s,1H),7.88–7.63(m,5H),7.61–7.42(m,4H),7.37–7.17(m,2H),7.11(t,J＝7.8Hz,1H),6.92(d,J＝9.1Hz,2H),6.73(dd,J＝38.3,8.9Hz,1H),6.19(d,J＝6.7Hz,1H),5.78–5.65(m,1H),5.64–5.50(m,1H),5.28(td,J＝11.5,11.0,5.0Hz,1H),4.53–4.37(m,2H),4.34–4.23(m,1H),4.21–4.05(m,1H),3.79(t,J＝7.9Hz,1H),3.54(s,1H),3.44(t,J＝5.1Hz,4H),3.11(d,J＝17.4Hz,1H),3.02(t,J＝5.1Hz,4H),2.91–2.78(m,3H),2.75(s,1H),2.63(d,J＝6.2Hz,2H),2.36(dd,J＝24.9,8.1Hz,2H),2.01(s,3H),1.72–1.57(m,4H),1.49(s,4H),1.36(d,J＝2.1Hz,9H),1.28–1.09(m,3H),0.84(qd,J＝7.3,6.6,3.4Hz,12H).
步骤4:UB-I06
化合物UB-I05(150mg,0.12mmol)溶于甲醇/二氯甲烷(3/3mL)后加入HCl/二氧六环(3mL)并室温反应15分钟。反应液低温浓缩后通过二氯甲烷/乙醚＝1/10打浆得到黄色固体目标产物UB-I06(100mg,收率72％)。LCMS[M+H] +＝1157.9。 1H NMR(400MHz,DMSO-d 6)δ12.20(s,1H),10.03(s,1H),9.36(s,2H),8.67(d,J＝8.1Hz,2H),8.45–8.28(m,3H),8.19(s,3H),7.92–7.80(m,2H),7.77–7.48(m,8H),7.45–7.04(m,4H),6.26(s,1H),5.85–5.41(m,2H),5.41–5.17(m,1H),4.65–4.38(m,3H),4.37–4.09(m,5H),3.65(dd,J＝12.2,5.3Hz,3H),3.54–3.28(m,5H),3.28–2.95(m,8H),2.84(d,J＝16.8Hz,1H),2.48–2.31(m,1H),2.06(tt,J＝12.8,6.7Hz,4H),1.87(d,J＝11.1Hz,5H),1.52(s,2H),1.41–1.12(m,4H),1.05–0.68(m,12H).
化合物UB-B07的合成
Figure PCTCN2022136002-appb-000089
步骤1:UB-B07a
化合物UB-I06(100mg,0.086mmol)溶于DMF(5ml)后加入MPOSu(23mg,0.086mmol)以及DIEA(33mg,0.259mmol)并室温反应1小时。反应液浓缩后通过柱层析分离(二氯甲烷(0.5％冰醋酸)/甲醇)得到白色固体目标产物UB-B07a(100mg,收率88％)。LCMS[M/2+H] +＝654.9
步骤2:UB-B07
化合物UB-181228(30mg,0.03mmol)溶于TEAA(0.5mL)后加入UB-B07a(17mg,0.01mmol)的DMF(1mL)溶液，并室温反应2小时。反应液通过反相柱层析得到粗产物，该粗产物通过制备得到白色固体目标产物UB-B07(5.4mg,收率64％)。LCMS[M/2+H] +＝1216.3
UB-I07的合成
Figure PCTCN2022136002-appb-000090
步骤1:UB-I07a
向在丙酮(50ml)溶液中的(S)-2-[(叔丁氧基羰基)氨基]-3-甲基丁酸氯甲酯(3g,11.29mmol)中加入碘化钠(8.5g,56.45mmol)，然后将反应回流3h。将溶液过滤并通过硅胶PE/EA色谱纯化，得到UB-I07a(2.7g，67％收率)，为透明油。LCMS[M+1] +＝358.2
步骤2:UB-I07b
在-20℃和N 2保护下，将LiHMDS(0.7ml，0.698mmol)滴加到在DMF(6mL)溶液中的PA02(200mg，0.233mmol)中，将溶液在-20℃搅拌0.5小时。添加在DMF(4mL)中的UB-I07a(332mg,0.931mmol)并将溶液在-20℃搅拌2小时。溶液用1M HCl淬灭，通过DCM/MeOH＝10/1纯化，得到UB-I07b(118mg，46％产率)，为无色固体。LCMS[M/2+H] +＝545.4
步骤3:UB-I07
向UB-I07b(150mg,0.138mmol)中加入5ml TFA/DCM＝1/3，并将溶液在室温下搅拌1小时。浓缩反应混合物以除去大部分TFA。将混合物通过硅胶DCM/MeOH色谱纯化，得到呈黄色固体状的UB-I07(100mg，74％产率)。LCMS[M/2+H] +＝495.4。 1H NMR(400MHz,DMSO) δ9.54(s,1H),9.15(s,2H),8.58(s,3H),7.98–7.84(m,3H),7.74(d,J＝7.6Hz,1H),7.68(d,J＝7.0Hz,1H),7.60(d,J＝6.5Hz,2H),7.54(t,J＝7.6Hz,1H),5.82(d,J＝9.7Hz,1H),5.72(dd,J＝9.5,8.3Hz,1H),5.39–5.29(m,1H),4.55–4.43(m,2H),4.34(dd,J＝17.7,12.2Hz,1H),4.19(dd,J＝17.3,8.6Hz,1H),3.94(s,4H),3.90(s,1H),3.84(t,J＝5.2Hz,2H),3.70(t,J＝6.5Hz,2H),3.22(s,3H),3.12(dd,J＝18.2,5.1Hz,4H),2.87(s,1H),2.80(t,J＝6.7Hz,2H),2.54(s,1H),2.24–2.08(m,2H),2.03–1.78(m,12H),1.50(dd,J＝18.0,10.6Hz,6H),0.94(dt,J＝12.2,4.6Hz,6H),0.76(t,J＝7.4Hz,3H).
UB-I09的合成
Figure PCTCN2022136002-appb-000091
步骤1:UB-I09a
向在DMF(5ml)溶液中的(叔丁氧基羰基)-L-缬氨酰基-L-脯氨酸(30mg,0.1mmol)中加入HATU(73mg,0.19mmol)和DIEA(37mg,0.29mmol)，然后反应在室温下搅拌30分钟。然后将UB-I07(95mg,0.1mmol)加入混合物中并将反应混合物在室温下搅拌2小时。将混合物浓缩并通过硅胶dcm/MeOH色谱纯化，得到呈黄色固体状的UB-I09a(75mg，61％产率)。LCMS[M/2+1] +＝643.4。
步骤2:UB-I09
将2ml 4M HCl加入到在DCM(3ml)中的UB-I09a(75mg,0.058mmol)中，然后在室温下搅拌溶液1小时。浓缩反应混合物以除去DCM和HCl。然后将粗产物通过TLC纯化，得到呈黄色固体状的UB-I09(21.3mg，30％产率)。LCMS[M/2+H] +＝593.4。 1H NMR(400MHz,DMSO)δ8.49(d,J＝8.5Hz,1H),8.31(d,J＝9.1Hz,1H),7.93–7.88(m,2H),7.80(d,J＝7.6Hz,1H),7.73(d,J＝7.7Hz,1H),7.66(s,1H),7.63–7.58(m,2H),7.56(d,J＝1.7Hz,1H),5.80(dd,J＝9.6,4.5Hz,1H),5.69(dd,J＝15.6,9.6Hz,1H),5.40(td,J＝13.0,5.1Hz,1H),4.57(t,J＝13.4Hz,2H),4.49–4.26(m,4H),4.02(s,3H),3.98(s,1H),3.89(s,1H),3.75(dd,J＝18.0,11.4Hz,5H),3.52(d,J＝9.3Hz,1H),3.31(s,3H),3.27–3.00(m,5H),2.86(dd,J＝20.4,13.5Hz,3H),2.54–2.51(m,1H),2.21–2.04(m,5H),2.03–1.76(m,16H),1.76–1.53(m,6H),1.04(t,J＝9.0Hz,3H),0.98(d,J＝6.9Hz,3H),0.93–0.79(m,9H).
化合物UB-I08的合成
Figure PCTCN2022136002-appb-000092
步骤1:UB-I08b
化合物UB-I07(50mg,0.05mmol)溶于DMF(3mL)后加入UB-I08a(16mg,0.05mmol)以及DIEA(13mg,0.10mmol)，并室温反应过夜。反应液通过反相柱层析分离得到白色固体目标产物UB-I08b(40mg,收率67％)。LCMS[M/2+H] +＝594.9
步骤2:UB-I08
化合物UB-I08b(40mg,0.03mmol)溶于DCM(3mL)后加入HCl/二氧六环(1mL)，并室温反应30分钟。反应液浓缩后通过乙醚打浆得到白色固体目标产物UB-I08(20mg,收率55％)。LCMS[M+H] +＝1088.4。 1H NMR(400MHz,DMSO-d 6)δ10.43(s,1H),9.07(s,2H),8.84(dd,J＝8.5,1.9Hz,1H),8.28(s,1H),8.18(s,3H),7.94(d,J＝5.7Hz,2H),7.85(s,1H),7.75(d,J＝7.5Hz,1H),7.68(d,J＝7.6Hz,1H),7.63–7.48(m,3H),5.83–5.54(m,2H),5.35(ddd,J＝13.0,7.7,5.1Hz,1H),4.62–4.40(m,2H),4.41–4.09(m,3H),3.95(s,4H),3.80(dt,J＝17.2,5.4Hz,3H),3.75–3.66(m,2H),3.23(s,3H),3.20–3.07(m,4H),2.86(d,J＝4.1Hz,1H),2.81(q,J＝6.9,5.3Hz,2H),2.60(s,1H),2.19–1.67(m,16H),1.64–1.39(m,6H),1.24(d,J＝3.2Hz,2H),1.10(t,J＝7.0Hz,1H),1.05–0.83(m,13H),0.76(t,J＝7.4Hz,3H).
UB-I10的合成
Figure PCTCN2022136002-appb-000093
步骤1:UB-I10a
向在DMF(5ml)溶液中的UB-I09(50mg,0.041mmol)中加入叔丁氧羰基-L-缬氨酸N-羟基琥珀酰亚胺酯(66.24mg,0.211mmol)和DIEA(27mg,0.211mmol)，然后将混合物在室温下搅拌4小时。将溶液浓缩并通过DCM/MeOH＝10/1纯化，得UB-I10a(42mg，72.4％产率)，为无色固体。LCMS[M/2+H] +＝693.0
步骤2:UB-I10
将1ml 4M HCl添加到在DCM(3ml)中的UB-I10a(42mg,0.03mmol)中，然后在室温下搅拌溶液1小时。浓缩反应混合物以除去DCM和HCl。然后将粗产物通过硅胶色谱法(DCM/MeOH)纯化，得到呈黄色固体状的UB-I10(17.9mg，46％产率)。LCMS[M/2+H] +＝643.0. 1H NMR(400 MHz,DMSO)δ8.41(d,J＝8.3Hz,1H),8.35–8.24(m,1H),8.15(d,J＝9.1Hz,1H),7.91(s,1H),7.84(s,1H),7.74(d,J＝7.5Hz,1H),7.65(d,J＝7.2Hz,1H),7.59(s,1H),7.52(dd,J＝18.4,10.1Hz,3H),5.73(d,J＝5.4Hz,1H),5.63(dd,J＝14.9,9.6Hz,1H),5.34(s,1H),4.48(d,J＝17.5Hz,2H),4.35(s,2H),4.23(d,J＝5.9Hz,2H),3.94(s,3H),3.86(s,1H),3.67(dd,J＝21.7,15.2Hz,5H),3.25(s,3H),2.91–2.67(m,6H),2.33(s,1H),2.00(s,6H),1.73(dd,J＝83.9,31.2Hz,20H),1.51(s,2H),0.93(d,J＝6.6Hz,3H),0.87(d,J＝6.8Hz,6H),0.85–0.80(m,9H),0.77(d,J＝7.4Hz,3H).
本申请示例性的TED、ACTED、中间体如下表所示：
表A1 示例性的TED化合物
Figure PCTCN2022136002-appb-000094
Figure PCTCN2022136002-appb-000095
表A2
Figure PCTCN2022136002-appb-000096
表F2
Figure PCTCN2022136002-appb-000097
Figure PCTCN2022136002-appb-000098
Figure PCTCN2022136002-appb-000099
表D3 示例性的ACTED偶联物
Figure PCTCN2022136002-appb-000100
Figure PCTCN2022136002-appb-000101
测试例
测试例1 细胞增殖实验：
试剂：RPMI-1640培养基、McCoy’s5A培养基、IMDM培养基、MEM培养基、L-15培养基、胎牛血清、青-链双抗、胰蛋白酶等、2-巯基乙醇、NEAA、丙酮酸酯等。
本实验使用的部分细胞株，如下表1：
表1.细胞株列表
Figure PCTCN2022136002-appb-000102
细胞进行常规培养，细胞铺板前至少传2代以上。收集处于对数生长期细胞，制备成单细胞悬液并计数，调整细胞浓度至所需浓度，以每孔加入100μl接种到96孔细胞培养板内。每孔加入100μL受试化合物的完全培养基，每个浓度皆设2个复孔，5倍梯度往下稀释，继续培养72h。所有细胞都进行相对应受试样品的EC 50测定。实验结果如测试例4所示。
使用Alarm blue法检测各孔荧光强度，并计算IC 50。
IC 50由以下公式计算：
Y＝Max+(Min-Max)/[1+(X/IC 50)×Slope]
其中Min、Max和Slope分别表示最小值、最大值和斜率。
测试例2 蛋白质印迹
细胞用化合物处理一定时间后，离心收集细胞，PBS清洗后，加入RIPA缓冲液裂解细胞；细胞裂解液加入加样缓冲液(Loading buffer)后取适量体积缓慢加到胶板对应的孔中，跑SDS-PAGE胶(4％-12％)。跑胶结束后转到PVDF膜上，用5％脱脂奶粉室温封闭1小时。将膜放到用5％脱脂 奶粉稀释的一抗中，4℃慢摇过夜。一抗孵育结束后，用TBST摇床洗膜3次；加入与一抗对应的用5％脱脂奶粉稀释的二抗，室温慢摇1小时。二抗孵育结束后，再次用TBST摇床洗膜3次。将PVDF膜平放到暗盒中，用ECL显影液均匀浸润条带，置于ChemDoc XRS+凝胶成像仪中拍照。使用ImageJ软件定量分析蛋白条带强度，结果如图1和图2所示。
可见，本发明的偶联物(或TED分子)对靶蛋白表现出浓度相关的降解活性。
测试例3 体外激酶活性实验
用1x反应缓冲液(reaction buffer)将化合物、酶、底物和ATP稀释到所需浓度。384孔板中加入1μL不同浓度化合物、2μL酶、2μL底物/ATP混合溶液，室温孵育1小时。然后每孔加入5μL ADP1-Glo TM试剂，室温孵育40分钟。最后加入10μL检测试剂，室温孵育30分钟后使用Envision检测化学发光信号。
可见，本发明中合成制备的TED分子，在多种肿瘤细胞系中表现出很强的细胞增殖抑制活性，具有成为抗肿瘤药物的前景。
测试例4
按照前述测试例的方法对表A和D中部分化合物(或偶联物)进行了活性测试，测试结果汇总如表2所示：
表2
Figure PCTCN2022136002-appb-000103
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种如式Ia或式Ib所示的偶联物或其药学上可接受的盐，
    R L-R T-L1-R E3  (Ia)
    R T-L1-R E3-R L  (Ib)
    其中，
    (a)所述R E3为E3连接酶配体部分；
    (b)所述R T为靶标分子部分；
    (c)所述L1为连接R E3和R T部分的连接头，且L1如式II所示；
    -W 1-L2-W 2-  (II)
    其中，
    W 1和W 2各自独立地为-(W) s-；其中，s＝0、1、2、3或4(较佳地，s＝0、1或2，更佳地，s＝1或2)，W各自独立地选自下组：无(键)、-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、-C(R b)＝C(R b)-、-C≡C-、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、取代或未取代的5至10元杂芳基(较佳地，W各自独立地选自下组：-N(R a)-、-C(O)-、-C≡C-；更佳地，W各自独立地选自下组：-NH-、-C(O)-、-C≡C-)；
    L2如式III所示，
    -(M) o-  (III)
    其中，下标o为2～50的整数(较佳地，下标o为2～20的整数，更佳地，下标o为2～10的整数；最佳地，下标o为2、3、4、5、6或7)；M各自独立地为选自下组的二价基团：-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、-C(R b)＝C(R b)-、-C≡C-、取代或未取代的C 3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C 6-10芳基、取代或未取代的5至10元杂芳基、氨基酸残基；
    (d)R L为R'或R"；
    (d1)R'不存在或为H、活性基团或离去基团；较佳地，R'各自独立地选自下组：H、-OH、-SH、-COOH、-COO-C 1-6烷基、保护基团(如氨基保护基团)；
    (d2)R"为-W 3-L P1-W P1-(R P) q1；其中，
    W 3选自：无，或由1、2或3个各自独立地选自下组的二价片段组成的二价基团：W'、和W U；其中，W U为可自发裂解的二价片段；
    L P1为-(M') t1-W Y-(M') t2-；其中，W Y为无或在细胞表面或细胞质内可裂解的二价连接部分；下标t1和t2各自独立地为0、1、2、3、4、5、6、7、8、9或10；
    W P1为无、-S-S-或
    Figure PCTCN2022136002-appb-100001
    其中，*代表与L P1连接的部分；较佳地，W P1为-S-S-或
    Figure PCTCN2022136002-appb-100002
    下标q1＞0(较佳地，q1＝1)；
    R P为-W 4-L P4-R P1、-W 4-L P4-R P1-R TED或-W 4-L P4-R P1-R"'；其中，W 4为无或-(W") s1-W P2-(W") s2-；L P4为-(M') t5-；R TED为-R T-L1-R E3或-R E3-L1-R T；
    其中，下标s1和s2各自独立地为0、1、2、3或4；t5为0、1、2、3、4、5、6、7、8、9或10(较佳地，t5为0、1、2或3)；W P2为无、NH、-C(R b)(COOH)-、-C(R b)(COR"')-、-C(R b)(NR a)-(如-CH(-NH 2)-)、-N(R"')-或-C(R b)(NH(R"'))-；
    R"'为-W 5-L P2-W 6-L P3-R P2；
    其中，L P2为-(M') t3-；L P3为-(M') t4-；其中下标t3和t4各自独立地为0、1、2、3、4、5、6、7、8、9或10(较佳地，t3和t4各自独立地为0、1、2或3)；
    W 5为-(W') s4-；其中，下标s4＝0、1或2；W 6为
    Figure PCTCN2022136002-appb-100003
    或-(W") s6-；其中，下标s6＝0、1、2、3或4；
    M'各自独立选自下组：-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、取代或未取代的C1-10亚烷基、-(CH 2CH 2O) 1-10-(即-(CH 2CH 2O) 1-、-(CH 2CH 2O) 2-、-(CH 2CH 2O) 3-、-(CH 2CH 2O) 4-、-(CH 2CH 2O) 5-、-(CH 2CH 2O) 6-、-(CH 2CH 2O) 7-、-(CH 2CH 2O) 8-、-(CH 2CH 2O) 9-、-(CH 2CH 2O) 10-)、氨基酸残基、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、和取代或未取代的5至10元杂芳基；并且任选地1或2个M'为W Z；并且W Z为亲水性二价连接部分；
    W'和W"各自独立地为选自下组的二价基团：无、-C(R b) 2-、-O-、-S-、-N(R a)-、-C(O)-、-SO 2-、-SO-、-PO 3-、氨基酸残基、取代或未取代的C3-8环烷基、取代或未取代的4至10元杂环烷基、取代或未取代的C6-10芳基、取代或未取代的5至10元杂芳基；
    R P1和R P2各自独立地为相同或不同的多肽元件或者靶标分子T；优选地，R P1和R P2各自独立地为不同的多肽元件或者靶标分子T；
    R a各自独立地选自下组：H、OH、SH、取代或未取代的C 1-6烷基、氨基保护基团、含N(R c)环原子的4至10元杂环烷基；
    R b各自独立地选自下组：H、卤素、OH、SH、取代或未取代的C 1-6烷基、取代或未取代的C 2-6烯基、取代或未取代的C 2-6炔基、取代或未取代的C 1-6烷氧基、取代或未取代的C 1-6烷基酰基(-C(O)-C 1-6烷基)、羧基、-COO-C 1-6烷基、-OC(O)-C 1-6烷基；或者，位于相同碳上的2个R b以及与它们相连的碳共同构成取代或未取代的C 3-8环烷基、取代或未取代的4至10元杂环烷基；
    R c各自独立地选自下组：H、OH、SH、取代或未取代的C 1-6烷基、氨基保护基团；
    除非特别说明，所述的取代是指基团中一个或多个(如1、2、或3个)氢被选自下组的取代基所取代：卤素(较佳地，F、Cl、Br或I)、氰基(CN)、氧代(＝O)、硫代(＝S)、羟基(-OH)、C 1-6烷基、C 1-6卤代烷基、C 2-6烯基、C 2-6炔基、C 1-6烷氧基、C 1-6烷基酰基(C 1-6烷基-C(O)-)、-COO-C 1-6烷基、-OC(O)-C 1-6烷基、NH 2、NH(C 1-6烷基)、N(C 1-6烷基) 2。
  2. 如权利要求1所述的偶联物，其特征在于，R L为R"。
  3. 如权利要求1或2所述的偶联物，其特征在于，
    所述细胞表面或细胞质内可裂解的二价连接部分选自下组：
    Figure PCTCN2022136002-appb-100004
    和/或
    所述亲水性二价连接部分或W Z选自下组：
    Figure PCTCN2022136002-appb-100005
    其中，n5为0-30的整数；
    和/或
    R P1为选自表E1的一价基团或者选自表E2的二价基团；
    R P2为选自表E1的一价基团。
  4. 如权利要求1或2所述的偶联物，其特征在于，
    R T为选自表B1的一价基团，或者为衍生自选自表B1的一价基团的二价基团；
    表B1
    Figure PCTCN2022136002-appb-100006
    Figure PCTCN2022136002-appb-100007
    各式中，
    R Pa选自下组：任选取代的C 1-6烷基、任选取代的C 2-6烯基、任选取代的C 2-6炔基；
    R Pb选自下组：H、-OH、取代或未取代的C 1-6烷基(如C 1-6羟烷基)、-COR Pb1；其中，R Pb1选自下组：H、-N(R a) 2、-NH-N(R a) 2、-OH、-OC 1-6烷基、-O-C 1-4亚烷基-N(R a) 2；较佳地，R Pb为CO-NH 2
    X P1为N或CH；和/或
    R E3如式A1或A2所示或衍生自式A1或A2：
    Figure PCTCN2022136002-appb-100008
    式A中，R X选自：无、C1-C6烷基、C2-C6烯基、C2-C6炔基、O、NH、S、CO或SO n(n为1或2)等；R Y为CH 2、C＝S、CO；和/或
    L2为-Cr 1-Cr 2-(CH 2) o1-；其中，下标o1＝1、2或3，Cr 1为未取代或被C 1-4烷基所取代的C 4-7环烷基或4至6元杂环基；Cr 2为未取代或被C 1-4烷基所取代的C 4-7环烷基或4至6元杂环基；和/或
    W 1和W 2各自独立地为-N(R a)-C(O)-、-C(O)-N(R a)-或-C≡C-。
  5. 如权利要求1或2所述的偶联物，其特征在于，
    -R E3-R L为以下任一所示的二价基团
    Figure PCTCN2022136002-appb-100009
    或者，
    -R T-R L如表B2中任一所示
    表B2
    Figure PCTCN2022136002-appb-100010
  6. 一种ACTED化合物或其药学上可接受的盐，其特征在于，所述的ACTED化合物如式IVa或IVb所示；
    R"-R T-L1-R E3  (IVa)
    R T-L1-R E3-R"  (IVb)
    其中，R"、R T、L1和R E3如权利要求1中定义。
  7. 如权利要求6所述的ACTED化合物，其特征在于，所述ACTED化合物选自表D3。
  8. 一种TED化合物或其药学上可接受的盐，其中，所述的TED化合物如式Va或Vb所示；
    R'-R T-L1-R E3  (Va)
    R T-L1-R E3-R'  (Vb)
    其中，R'、R T、L1和R E3如权利要求1中定义。
  9. 如权利要求8所述的TED化合物，其特征在于，所述TED化合物选自表A1。
  10. 一种药物组合物，其中，所述的药物组合物含有
    (i)如权利要求1或2所述的偶联物或如权利要求6所述的ACTED化合物或如权利要求8所述的TED化合物；以及
    (ii)药学上可接受的载体。
  11. 一种如权利要求1所述的偶联物或如权利要求6所述的ACTED化合物或如权利要求8所述的TED化合物在制备用于治疗或预防与靶标蛋白过量相关的疾病的药物中的的用途。
  12. 一种如权利要求1所述的偶联物或如权利要求6所述的ACTED化合物或如权利要求8所述的TED化合物在治疗或预防与靶标蛋白过量相关的疾病中用途。
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