Novel degradation agent conjugates

Abstract

The invention relates to novel degradation agent conjugates. Novel degradation agents and conjugates of novel degradation agents and binding moieties are provided. Also provided are compositions comprising the conjugates. The compounds and compositions are useful for treating a disease or condition, such as cancer, in an individual in need thereof.

Description

[0001] This application is a divisional application of the invention application with an application date of June 2, 2022, Chinese application number 202280052606.2, and invention name “New Degrader Conjugate”.

[0002] Reference to a sequence listing submitted electronically

[0003] The contents of the electronically submitted Sequence Listing as an ASCII text file (name 4547_016PC02_Seqlisting_ST25; size: 24,777 bytes; and creation date: May 31, 2022) submitted with this application are incorporated herein by reference in their entirety. Technical Field

[0004] The present disclosure provides a neoDegrader conjugate, wherein the neoDegrader is conjugated to a binding moiety. Compositions comprising the conjugate are also provided. The conjugates and compositions can be used to treat cancer in an individual in need thereof. Background Art

[0005] Protein degradation has been demonstrated as a therapeutic strategy through the effectiveness of immunomodulatory imide drugs. These compounds have the ability to bind cereblon (CRBN) and promote the production of CRL4 CRBN E3 ubiquitin ligases mediate recruitment and ubiquitination of substrate proteins. It is thought that immunomodulatory imides act as a "molecular glue" filling the binding interface as a hydrophobic patch, reprogramming the protein interaction between the ligase and the new substrate.

[0006] Although these compounds are exciting as novel treatments for cancer, to date, they have been limited to use in hematological malignancies, such as multiple myeloma and myelodysplastic syndrome (MDS). Expanding the library of compounds that can act by degrading other oncoproteins, many of which are considered "undruggable," is an active area of ​​drug development. Therefore, there is a continuing need for new compounds that can target these alternative oncoproteins and treat a variety of cancers. Summary of the invention

[0007] Treatment of cancer patients with small molecule GSPT1 degraders has been shown to drive clinical responses but is associated with serious adverse events (AEs). Cancers often express antigens on their surface that are not expressed or expressed at much lower levels on healthy cells. The present invention is based on the discovery that combining GSPT1 degrading payload molecules with antibodies that bind to cell surface antigens on cancer cells can improve the clinical efficacy and tolerability of GSPT1 degraders.

[0008] In certain aspects, the present disclosure provides a conjugate of formula (I):

[0009]

[0010] or a pharmaceutically acceptable salt thereof, wherein:

[0011] a is 1 to 10;

[0012] L is a linker selected from the following:

[0013]

[0014] in:

[0015] is the point of attachment to the nitrogen atom; and

[0016] is the connection point with Bm; and

[0017] Bm is a binding moiety capable of specifically binding to a protein (eg, a protein that is a cell surface antigen).

[0018] In certain aspects, the binding moiety is an antibody, antibody fragment or antigen binding fragment. In some aspects, a is 2 to 8.

[0019] In some aspects, L is

[0020]

[0021] In some aspects, L is

[0022]

[0023] In some aspects, Bm is an antibody or an antigen binding portion thereof. In some aspects, the protein to which the binding portion binds is a surface antigen. In some aspects, the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5 、CD51、CD52、CD54、CD56、CD62E、CD62P、CD62L、CD70、CD71、CD72、CD74、CD79a、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、Clumping factor、cKit、Claudin 3、Claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Crypto 1 Growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), IL-2Rγ (i.e. CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzyme, pr ostein, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras, Ras mutant, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1 or a combination thereof.

[0024] In some aspects, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof. In some aspects, the surface antigen comprises CD33.

[0025] In some aspects, the antibody is selected from the group consisting of rituximab, trastuzumab, gemtuzumab, CD33AB, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuximab, isatuximab, mab), Sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, balantamab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab and veltuzumab. In some aspects, the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab. In some aspects, the antibody or antigen binding portion thereof comprises: a heavy chain variable region (VH) complementarity determining region (CDR) 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a light chain variable region (VL) CDR1 (VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5; a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antibody or antigen binding portion thereof comprises: a VH comprising the amino acid sequence set forth in SEQ ID NO: 4; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8.In some aspects, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. In some aspects, the antibody is CD33AB.

[0026] In certain aspects, the present disclosure provides a compound of formula (II):

[0027]

[0028] or a pharmaceutically acceptable salt thereof.

[0029] In certain aspects, the present disclosure provides a compound of formula (III):

[0030]

[0031] or a pharmaceutically acceptable salt thereof.

[0032] In certain aspects, the present disclosure provides a compound of formula (IV):

[0033]

[0034] or a pharmaceutically acceptable salt thereof, wherein Bm is a binding portion that specifically binds to a protein (eg, a protein that is a cell surface antigen).

[0035] In certain aspects, the present disclosure provides a compound of formula (V):

[0036]

[0037] or a pharmaceutically acceptable salt thereof, wherein Bm is a binding portion that specifically binds to a protein (eg, a protein that is a cell surface antigen).

[0038] In certain aspects, the present disclosure provides a compound of formula (IV) or (V), wherein Bm is an antibody or an antigen-binding portion thereof. In certain aspects, the protein to which the binding portion specifically binds is a surface antigen. In some aspects, the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5 、CD51、CD52、CD54、CD56、CD62E、CD62P、CD62L、CD70、CD71、CD72、CD74、CD79a、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、Clumping factor、cKit、Claudin 3、Claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Crypto 1 Growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPVE6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), IL-2Rγ (i.e. CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate Enzymes, prostaglandins, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1 or a combination thereof.

[0039] In some aspects, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof.

[0040] In some aspects, the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, entuximab, isatuximab, sacetuzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, belantamab, indalatuximab, cetuximab, detuximab, anti-CD38 A2 antibody, HuAT13 / 5 antibody, CD33AB, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, panitumumab, tremelimumab, tesitumumab, catumaxomab, oregovomab, or veltuzumab.

[0041] In some aspects, the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab. In some aspects, the antibody or antigen-binding portion thereof comprises: VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5; VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; and VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antibody, or antigen-binding portion thereof, comprises: a VH comprising the amino acid sequence set forth in SEQ ID NO: 4; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. In some aspects, the antibody is CD33AB.

[0042] In some aspects, the present disclosure provides a pharmaceutical composition comprising a conjugate or compound as described above, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0043] In some aspects, the present disclosure provides a method of treating cancer or myelodysplastic syndrome (MDS) in an individual in need thereof, the method comprising administering to the individual a pharmaceutically acceptable amount of a conjugate, compound or composition as described above, or a pharmaceutically acceptable salt thereof. In some aspects, the cancer is a solid tumor. In some aspects, the cancer is a hematological cancer / blood cancer. In some aspects, the cancer is breast cancer, gastric cancer, lymphoma, acute myeloid leukemia, multiple myeloma, head and neck cancer, squamous cell carcinoma and / or hepatocellular carcinoma. In some aspects, the cancer is refractory or resistant to Mylotarg.

[0044] In some aspects, the method further comprises administering to the individual an additional agent of a pharmaceutically acceptable amount before, after, or simultaneously with the conjugate or compound or a pharmaceutically acceptable salt thereof. In some aspects, the additional agent is a cytotoxic agent or an immune response modifier. In some aspects, the immune response modifier is a checkpoint inhibitor. In some aspects, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, and / or a LAG-3 inhibitor.

[0045] In certain aspects, the present disclosure provides a method for preparing a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, the method comprising reacting a binding moiety with a compound of formula (I-1):

[0046]

[0047] or a pharmaceutically acceptable salt thereof, wherein:

[0048] L' is selected from

[0049]

[0050] in:

[0051] is the point of attachment to the nitrogen atom.

[0052] In some aspects, the method further comprises reducing the binding moiety prior to reacting with the compound of formula (I-1).

[0053] In some aspects, L' is

[0054]

[0055] In some aspects, L' is

[0056]

[0057] In some aspects, the compound of Formula (I-1) is reacted with a binding moiety comprising an antibody or an antigen binding portion thereof.

[0058] In some aspects, the antibody or its antigen-binding portion binds to a surface antigen. In some aspects, the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5 、CD51、CD52、CD54、CD56、CD62E、CD62P、CD62L、CD70、CD71、CD72、CD74、CD79a、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、Clumping factor、cKit、Claudin 3、Claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Crypto 1 Growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPVE6, hTERT, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), IL-2Rγ (i.e. CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate Enzymes, prostaglandins, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1 or a combination thereof.

[0059] In some aspects, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, Trop-2, or a combination thereof.

[0060] In some aspects, the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, entuximab, isatuximab, sacetuzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, belantamab, indalatuximab, cetuximab, detuximab, anti-CD38 A2 antibody, CD33AB, HuAT13 / 5 antibody, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, panitumumab, tremelimumab, tesitumumab, catumaxomab, oregovomab, or veltuzumab.

[0061] In some aspects, the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMY9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab. In some aspects, the antibody or antigen-binding portion thereof comprises: VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5; VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; and VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antibody or antigen-binding portion thereof comprises: a VH comprising the amino acid sequence set forth in SEQ ID NO: 4; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antibody comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A Depicted are the in vivo activities of representative novel degrader conjugates against MV411 (CD33+) tumors. The X-axis shows the number of days after dosing. The Y-axis shows the tumor volume (mm) after dosing with vehicle, 3.02 mg / kg CD33AB-Compound (Ia), 2.94 mg / kg CD33AB-Compound (Ib), 0.1 mg / kg Mylotarg, 50 mg / kg x 21 Venetoclax, or 5 mg / kg bid x 10 CC-90009. 3 ).

[0063] Figure 1BDepicted are representative novel degrader conjugates for MV411 (CD33+) tumor in vivo activity. The X-axis shows the number of days after dosing. The Y-axis shows tumor volume (mm) after dosing with vehicle, 3 mg / kg CD33AB-compound (Ic), 2.83 mg / kg CD33AB-compound (Ie), 3.02 mg / kg CD33AB-compound (Ia), 2.99 mg / kg CD33AB-compound (Ih), 2.94 mg / kg CD33AB-compound (Ib), 0.1 mg / kg Mylotarg, 50 mg / kg x 21 Venetoclax, or 5 mg / kg bid x 10 CC-90009. 3 ).

[0064] Figure 2 The in vitro activities of CD33AB-compound (Ia), CC-90009 and Mylotarg on AML (CD33+) cells and non-AML (CD33-) cells are depicted.

[0065] Figure 3 Depicted is the in vitro activity of CD33AB-Compound (Ia) conjugates against MV4-11 (CD33+) cells. The X-axis shows the concentration. The Y-axis shows the % cell viability after administration of non-CD33 binding antibody-new degrader conjugates, CD33AB, new degrader P1, Venetoclax, CC-885, CC-90009, Mylotarg, and CD33AB-Compound (Ia).

[0066] Figure 4 The in vitro activities of CD33AB-Compound (Ia) conjugate, Mylotarg, and CC-90009 on patient-derived primary relapsed / refractory AML cells are depicted.

[0067] Figure 5 Depicted are the in vitro activities of CD33AB-Compound (Ia) conjugates, CC-90009, and Mylotarg in normal erythroid, myeloid, and megakaryocyte precursor cells, as measured in a colony forming cell (CFC) assay.

[0068] Figure 6 Depicted is the in vivo activity of CD33AB-Compound (Ia) against MV4-11 (CD33+) tumors. In the left panel, the X-axis shows the days after dosing, and the Y-axis shows the tumor volume (mm) after dosing with vehicle, 5 mg / kg bid CD-90009, 0.1 mg / kg Mylotarg, 50 mg / kg qd Venetoclax, and 3 mg / kg CD33AB-Compound (Ia). 3In the right figure, the X-axis shows the days after administration, and the Y-axis shows the tumor volume (mm) after administration with vehicle, 3 mg / kg CD33 non-binding antibody new degrader conjugate, 8 mg / kg azacitidine and 50 mg / kg venetoclax, 1 mg / kg CD33AB-compound (Ia) and 3 mg / kg CD33AB-compound (Ia). 3 ).

[0069] Figure 7 The in vivo activity of CD33AB-compound (Ia) in MV4-11 and OCI-AML2 cells is depicted.

[0070] Figure 8 Western blot showing GSPT1 degradation after in vitro treatment of MV4-11 cells with CD33AB-Compound (Ia) conjugate. GSPT1 was completely degraded within 12 hours when treated with CD33AB-Compound (Ia) conjugate.

[0071] Fig. 9 Depicted are the sustained in vitro effects of CD33AB-Compound (Ia) on GSPT1 (upper panel) and the pharmacokinetics of the CD33AB-Compound (Ia) conjugate (QD x1 IV) (lower panel).

[0072] Fig. 10A and Fig. 10B Describes the effect of CD33AB-Compound (Ia) conjugate on Mylotarg-insensitive AML cells (AML-193 ( Fig. 10A ) and Kasumi-6( Fig. 10B The X-axis shows the concentration, and the Y-axis shows the survival percentage of the cell lines after treatment.

[0073] Fig.11A and Fig. 11B Depicted are the synthesis of CD33AB-compound (Ia) and the synthesis of CD33AB-compound (Ib). DETAILED DESCRIPTION

[0074] The present disclosure relates to a conjugate of formula (I):

[0075]

[0076] or a pharmaceutically acceptable salt thereof, wherein:

[0077] a is 1 to 10;

[0078] L is a linker selected from the following:

[0079] and

[0080]

[0081] in:

[0082] is the point of attachment to the nitrogen atom; and

[0083] is the connection point with Bm; and

[0084] Bm is a binding moiety capable of specifically binding to a protein. In some aspects, the binding moiety is an antibody, an antibody fragment, or an antigen binding fragment.

[0085] The present disclosure also provides the above-described compounds fused to a binding moiety, compositions comprising the compounds or conjugates, or methods of using or making the compounds or conjugates.

[0086] I. Definitions.

[0087] To make this specification more easily understood, some terms are first defined. Additional definitions are set forth throughout the detailed description.

[0088] It should be noted that the term "a / an" entity refers to one or more of that entity; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a / an", "one or more", and "at least one" can be used interchangeably herein. It should be further noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent basis for using exclusive terminology such as "only", "only" or a negative limitation in conjunction with the description of claim elements.

[0089] In addition, "and / or" when used herein should be regarded as a specific disclosure of each of the two specified features or components in the presence or absence of the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this article is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0090] It should be understood that wherever herein an aspect is described with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art related to the present disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary of many of the terms used in the present disclosure for those of skill in the art.

[0092] Units, prefixes and symbols are expressed in a form accepted by the International System of Units (SI). Numerical ranges include numbers that define ranges. In the case of a range of enumerated values, it should be understood that each intermediate integer value and each fraction thereof between the upper and lower limits of the range are also explicitly disclosed, as well as each subrange between such values. The upper and lower limits of any range may be independently included in or excluded from the range, and each range including any one, zero or two limits is also included in the present disclosure. Therefore, the ranges listed herein should be understood as abbreviations for all values ​​(including the enumerated endpoints) within the range. For example, a range of 1 to 10 should be understood to include any number, combination of numbers or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0093] Where values ​​are explicitly recited, it is understood that values ​​of approximately the same quantity or amount as the recited value are also within the scope of the present disclosure. Where combinations are disclosed, each subcombination of the elements of the combination is also explicitly disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed individually, combinations thereof are also disclosed. Where any element of the disclosure is disclosed as having a plurality of alternatives, embodiments of the disclosure are also disclosed herein where each alternative is excluded, either individually or in any combination with other alternatives; more than one element disclosed may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0094] As used herein, the term "DAR" refers to the drug-antibody ratio of the conjugate, which is the average number of new degrader-linker complexes attached to each antibody. In certain aspects, the DAR of the conjugates described herein is 1 to 10. In some aspects, the DAR of the conjugates described herein is 1 to 8. In some aspects, the DAR of the conjugates described herein is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.

[0095] As used herein, the term "antibody" also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, such targets include but are not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species. However, in one aspect, the immunoglobulin is of human, mouse, or rabbit origin.

[0096] The term "single domain antibody", also known as nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of about 12 kDa to about 15 kDa. The monomeric antibody can be based on a heavy chain variable domain or a light chain. Examples of single domain antibodies include, but are not limited to, V H H fragment and V NAR Fragment.

[0097] "Antibody fragments" comprise a portion of an intact antibody, typically its antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0098] An "intact antibody" is an antibody comprising an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

[0099] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical, except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, compared to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be prepared by a hybridoma method, or can be prepared by a recombinant DNA method. "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0100] The monoclonal antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they express the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

[0101] Various methods have been used to produce monoclonal antibodies (MAbs). Hybridoma technology refers to clonal cell lines that produce a single type of antibody, using cells from various species, including mice (rat), hamsters, rats, and humans. Another method for preparing MAbs uses genetic engineering, including recombinant DNA technology. Monoclonal antibodies made by these technologies include chimeric antibodies and humanized antibodies, among others. Chimeric antibodies combine DNA coding regions from more than one species. For example, chimeric antibodies can derive variable regions from mice and constant regions from humans. Humanized antibodies are mainly derived from humans, although they contain non-human parts. Like chimeric antibodies, humanized antibodies can contain completely human constant regions. But unlike chimeric antibodies, variable regions can be partially derived from humans. The non-human synthetic parts of humanized antibodies are often derived from CDRs in mouse antibodies. In any case, these regions are crucial for allowing antibodies to recognize and bind to specific antigens. Although they can be used for diagnosis and short-term treatment, it is impossible to administer mouse antibodies to humans for a long time without increasing the risk of harmful immunogenic reactions. This reaction, called human anti-mouse antibodies (HAMA), occurs when the human immune system recognizes the mouse antibodies as foreign and attacks them. A HAMA reaction can lead to toxic shock or even death.

[0102] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody. In addition, chimeric and humanized antibodies may have the added benefit of activating secondary human immune responses, such as antibody-dependent cellular cytotoxicity.

[0103] An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0104] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be divided into different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into "subclasses" (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0105] The term "about" is used herein to mean approximately, roughly, about, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" can modify numerical values ​​above and below the set forth value by, for example, a 10% variation up or down (increase or decrease).

[0106] The terms "administration", "administering" and grammatical variations thereof refer to the introduction of a composition, such as an EV (e.g., exosome) disclosed herein, into an individual via a pharmaceutically acceptable route. A composition, such as an EV (e.g., exosome) disclosed herein, is introduced into an individual by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular or topical. Administration includes self-administration and administration by others. Suitable routes of administration allow the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the individual.

[0107] As used herein, the term "antibody" encompasses immunoglobulins and fragments thereof produced naturally or partially or completely synthetically. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibodies" further include polypeptides that specifically bind and recognize antigens comprising framework regions from immunoglobulin genes or fragments thereof. The use of the term antibody is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, mouse antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bifunctional antibodies, and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies, as long as they express the desired biological activity or function. In some aspects of the present disclosure, the bioactive molecule is an antibody or a molecule comprising an antigen-binding fragment thereof.

[0108] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to, for example, an antibody covalently linked to one or more therapeutic agents (sometimes referred to herein as agents, drugs, or active pharmaceutical ingredients). In some aspects of the disclosure, the biologically active molecule is an antibody-drug conjugate.

[0109] As used herein, the term "approximately" as applied to one or more values ​​of interest refers to a value similar to the reference value. In certain aspects, the term "approximately" refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) of the reference value, unless otherwise specified or apparent from the context (unless such number exceeds 100% of the possible values).

[0110] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if one amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids may be conservatively replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0111] As used herein, the term "conserved" refers to a nucleotide or amino acid residue, respectively, of a polynucleotide sequence or polypeptide sequence that is a residue that occurs unchanged in the same position of two or more sequences being compared. Relatively conserved nucleotides or amino acids are conserved nucleotides or amino acids in sequences that are more related than nucleotides or amino acids that occur elsewhere in the sequence.

[0112] In some aspects, if two or more sequences are 100% identical to each other, they are called "completely conserved" or "identical". In some aspects, if two or more sequences are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other, they are called "highly conserved". In some aspects, if two or more sequences are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other, they are called "conserved". Sequence conservation can be applied to the entire length of a polynucleotide or polypeptide, or can be applied to a portion, region, or feature thereof.

[0113] As used herein, the terms "linked" and "conjugated" are used interchangeably and each refers to the covalent or non-covalent connection of two or more moieties comprising a novel degrader and a binding moiety. In some aspects, the link or conjugation may comprise a linker.

[0114] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Typically, amino acid sequence variants have at least about 70% sequence identity with at least one receptor binding domain of a native antibody or with at least one ligand binding domain of a native receptor, and typically, they will be at least about 80%, more typically at least about 90% homologous in sequence to such receptor or ligand binding domains. Amino acid sequence variants have substitutions, deletions and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are named by conventional names, one-letter and three-letter codes.

[0115] "Sequence identity" is defined as the percentage of residues that are identical in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2" written by Genentech, Inc., which was submitted to the U.S. Copyright Office, Washington, DC 20559, on December 10, 1991, along with user documentation.

[0116] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. In addition, the FcR may be a receptor that binds to IgG antibodies (gamma receptors) and includes receptors of the Fc.γ.RI, Fc.γ.RII, and Fc.γ.RIII subclasses, including allelic variants and alternative splice forms of these receptors. Fc.γ.RII receptors include Fc.γ.RIIA ("activating receptor") and Fc.γ.RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc.γ.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc.γ.RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0117] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed.

[0118] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. 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 domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain.

[0119] The term "variable" refers to the fact that certain portions of the variable domain differ greatly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, primarily in a β-sheet configuration, connected by three hypervariable regions, forming loops that connect the β-sheet structure and in some cases form a part of it. The hypervariable regions in each chain are held tightly together by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen binding site of the antibody. The constant domains are not directly involved in the binding of the antibody to the antigen, but express various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity (ADCC).

[0120] When used in this article, the term "hypervariable region" refers to the amino acid residues responsible for antigen binding in an antibody. The hypervariable region generally includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., supra) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain). "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein.

[0121] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to be easily crystallized. Pepsin treatment produces a F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0122] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six hypervariable regions together give the antibody its antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, but its affinity is lower than that of the entire binding site.

[0123] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical conjugations of antibody fragments are also known.

[0124] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0125] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0126] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable light domain (VL) and a variable heavy domain (VH) connected in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0127] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Humanization is a method of transferring mouse antigen binding information to non-immunogenic human antibody receptors, and many therapeutically useful drugs have been produced. The humanization method usually first transfers all six mouse complementary determining regions (CDRs) to the human antibody framework. These CDR-grafted antibodies usually do not retain their original affinity for antigen binding, and in fact, the affinity is usually severely weakened. In addition to CDRs, selected non-human antibody framework residues must also be incorporated to maintain appropriate CDR conformations. It has been shown that transferring key mouse framework residues to human receptors to support the structural conformation of the transplanted CDRs restores antigen binding and affinity. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable regions are replaced by residues from non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates with desired specificity, affinity, and ability. In some cases, the framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies will include substantially all of at least one, and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. Humanized antibodies also optionally include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.

[0128] "Separated" antibodies are antibodies that have been identified and separated and / or recovered from components of their natural environment. The contaminating components of their natural environment are materials that interfere with the diagnosis or therapeutic use of the antibody, and may include enzymes, hormones, and other proteins or non-protein solutes. In certain aspects, the antibody is purified to (1) an antibody greater than 95% by weight as determined by the Lowry method, or greater than 99% by weight, (2) a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a gas phase protein sequencer, or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Separated antibodies include in situ antibodies in recombinant cells, because at least one component in the antibody natural environment will not be present. However, typically, separated antibodies will be prepared by at least one purification step.

[0129] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic, and recurrent cancers.

[0130] As used herein, the term "immune response" refers to a biological response in vertebrates to foreign factors that protects the organism from these factors and the diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) and soluble macromolecules (including antibodies, cytokines and complement) produced by any of these cells or the liver, resulting in selective targeting, binding, damage, destruction and / or elimination of invading pathogens, cells or tissues infected with pathogens, cancer cells or other abnormal cells in the vertebrate body, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. The immune response includes, for example, activation or inhibition of T cells, such as effector T cells or Th cells, such as CD4 + or CD8 + T cells, or suppress Treg cells. As used herein, the terms "T cells" and "T lymphocytes" are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some aspects, T cells are CD4+T cells. In some aspects, T cells are CD8+T cells. In some aspects, T cells are NKT cells.

[0131] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0132] The term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of an agent (e.g., a novel degrader or novel degrader conjugate disclosed herein) that provides a desired biological, therapeutic, and / or preventive result. The result may be reduction, improvement, modification, alleviation, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount comprises an amount sufficient to cause tumor shrinkage and / or reduce tumor growth rate (such as inhibiting tumor growth) or prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. An effective amount of the composition can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down and prevent the infiltration of cancer cells into surrounding organs to a certain extent; (iv) inhibit (i.e., slow down to a certain extent) and prevent tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent.

[0133] In some aspects, a "therapeutically effective amount" is an amount of a novel degrader or novel degrader conjugate that has been clinically demonstrated to achieve a significant reduction in cancer, such as advanced solid tumors, or slow the progression (regression) of the cancer. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

[0134] As used herein, the term "standard of care" refers to a treatment that is accepted by medical experts as an appropriate treatment for a certain type of disease and is widely used by healthcare professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard medical care," and "standard therapy."

[0135] For example, an "anti-cancer agent" promotes regression of cancer in an individual or prevents further tumor growth. In certain aspects, a therapeutically effective amount of a drug promotes regression of cancer to the point of eliminating the cancer.

[0136] The terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and / or biological level caused by the administration of the drug.

[0137] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. Many checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4):252-64(2012). These proteins are responsible for the co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0138] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or conditions, such as the development or spread of cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or amelioration of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival compared to the expected survival if not receiving treatment. Those in need of treatment include those already suffering from a disease or condition as well as those susceptible to a disease or condition or those for whom a disease or condition is to be prevented.

[0139] II. New Degraders

[0140] The present disclosure provides a novel degradation agent of formula (P1):

[0141]

[0142] In some aspects, the novel degraders are molecules that form a ternary complex with an E3 ubiquitin ligase that is capable of targeting a protein for degradation.

[0143] III. New Degrader Conjugates

[0144] The present disclosure provides conjugates of one or more novel degraders disclosed herein with binding moieties. These conjugates can promote the degradation of CRL4 by binding cereblon (CRBN). CRBN E3 ubiquitin ligases mediate the recruitment of substrate proteins and their ubiquitination for protein degradation. These agents act as "molecular glues" that fill the binding interface as hydrophobic patches, reprogramming protein interactions between the ligase and new substrates.

[0145] In some aspects, the present disclosure provides a compound of formula (I):

[0146]

[0147]

[0148] or a pharmaceutically acceptable salt thereof, wherein:

[0149] a is 1 to 10;

[0150] L is a linker selected from the following:

[0151] and

[0152]

[0153] in:

[0154] is the point of attachment to the nitrogen atom; and

[0155] is the connection point with Bm; and

[0156] In some aspects, the novel degrader conjugates described herein have in vitro antiproliferative activity against tumor cell lines. In some aspects, the novel degrader conjugates comprising the novel degrader and the binding moiety have an in vitro antiproliferative activity that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% higher than the novel degrader alone or the binding moiety alone. In some aspects, the novel degrader conjugates comprising the novel degrader and the binding moiety have an in vitro antiproliferative activity that is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times higher than the novel degrader alone or the binding moiety alone.

[0157] In some aspects, the novel degrader conjugates described herein have in vitro antiproliferative activity against BT-474 breast cancer cell lines, such as higher in vitro antiproliferative activity against BT-474 breast cancer cell lines compared to the novel degrader alone or the binding moiety alone. In some aspects, the novel degrader conjugates described herein have in vitro antiproliferative activity against SK-BR-3 breast cancer cell lines, such as higher in vitro antiproliferative activity against SK-BR-3 breast cancer cell lines compared to the novel degrader alone or the binding moiety alone. In some aspects, the novel degrader conjugates described herein have in vitro antiproliferative activity against NCI-N87 gastric cancer cell lines, such as higher in vitro antiproliferative activity against NCI-N87 gastric cancer cell lines compared to the novel degrader alone or the binding moiety alone. In some aspects, the novel degrader conjugates described herein have in vitro antiproliferative activity against Daudi lymphoma cell lines, such as higher in vitro antiproliferative activity against Daudi lymphoma cell lines compared to the novel degrader alone or the binding moiety alone. In some aspects, the new degrader conjugates described herein have in vitro antiproliferative activity against HL-60 acute myeloid leukemia cell lines, such as higher in vitro antiproliferative activity against HL-60 acute myeloid leukemia cell lines compared to a single new degrader or a single binding moiety. In some aspects, the new degrader conjugates described herein have in vitro antiproliferative activity against Ramos non-Hodgkin lymphoma cell lines, such as higher in vitro antiproliferative activity against Ramos non-Hodgkin lymphoma cell lines compared to a single new degrader or a single binding moiety. In some aspects, the new degrader conjugates described herein have in vitro antiproliferative activity against MV411 AML cell lines, such as higher in vitro antiproliferative activity against MV411 AML cell lines compared to a single new degrader or a single binding moiety. In some aspects, the new degrader conjugates described herein are capable of maintaining their antiproliferative activity in the presence of human serum. The new degrader conjugates described herein can be used to treat cancer.

[0158] In some aspects, the antibody neo-degrader conjugate (AnDC) is a conjugate of one or more neo-degraders disclosed herein and an antibody or antigen-binding portion thereof disclosed herein.

[0159] III.A. Connectors

[0160] The novel degraders of the present disclosure are linked to the binding moiety via a linker. As used herein, the term "linker" refers to any chemical moiety capable of linking the binding moiety (Bm) to the novel degrader P1.

[0161] In certain aspects, the linker may contain a heterobifunctional group. In the present disclosure, the term "heterobifunctional group" refers to a chemical moiety that connects the linker to the binding portion, which chemical moiety is a part of the linker. A heterobifunctional group is characterized by having different reactive groups at either end of the chemical moiety. The connection to "Bm" can be accomplished by chemical or enzymatic conjugation or a combination of both. Chemical conjugation involves a controlled reaction of accessible amino acid residues on the surface of the binding portion with a reactive handle on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via non-natural amino acids incorporated by genetic engineering, wherein the non-natural amino acid residues with the desired reactive handle are installed on "Bm". In enzymatic conjugation, the enzyme mediates the coupling of the linker to an accessible amino residue on the binding portion. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using a sortase, transpeptidation using a microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzyme conjugation can also be used to install a unique reaction handle on "Bm" for subsequent chemical conjugation.

[0162] In some aspects, the heterobifunctional group is

[0163]

[0164] in

[0165] is the point of connection to the remainder of the joint; and

[0166] It is the connection point with Bm.

[0167] In certain aspects, L is a β-glucuronidase cleavable linker. In some aspects, L is a β-glucuronidase cleavable linker selected from the following:

[0168] and

[0169]

[0170] in:

[0171] is the connection point with the new degradation agent P1; and

[0172] It is the connection point with the binding part.

[0173] III.B. Binding part

[0174] The present disclosure provides conjugates of new degradation agents and binding moieties. As used herein, the term "binding moiety" refers to any molecule that recognizes and binds to a cell surface marker or receptor. In some aspects, the binding moiety binds to a protein, not limited to a polypeptide moiety. In addition to targeting the new degradation agent to a specific cell, tissue, or location, the binding moiety may also have certain therapeutic effects, such as anti-proliferation (cytostatic and / or cytotoxic) activity for a target cell or pathway. In some aspects, the binding moiety may include or may be engineered to include at least one chemically reactive group, such as a carboxylic acid, an amine, a thiol, or a chemically reactive amino acid moiety or side chain. In some aspects, the binding moiety may include a targeting moiety that binds or compounds to a cell surface molecule such as a cell surface receptor or antigen of a given target cell population. After specifically binding or compounding with a receptor, the cell allows the targeting moiety or the new degradation agent conjugate to be taken up and then internalized into the cell.

[0175] In some aspects, the group "Bm" can be a moiety that can specifically bind to a cell surface molecule. In some aspects, the group "Bm" can be a peptide or protein that binds to a cell surface receptor or antigen.

[0176] In certain aspects, the group "Bm" may be an antibody, an antibody fragment, or an antigen binding fragment. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. Target antigens typically have multiple binding sites, also referred to as epitopes, which are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen may have more than one corresponding antibody. The term "antibody" is used herein in the broadest sense, and specifically encompasses monoclonal antibodies, single domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they express the desired biological activity. Antibodies may be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or derived from other species.

[0177] The monoclonal antibodies that can be conjugated to the new degraders are homogeneous populations of antibodies directed against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) directed against antigens of interest can be prepared using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas that produce mAbs for use in the present disclosure can be cultured in vitro or in vivo.

[0178] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be prepared by any of a variety of techniques known in the art.

[0179] Antibodies may also be bispecific antibodies. Methods for preparing bispecific antibodies are known in the art. The generation of traditional full-length bispecific antibodies is based on the coexpression of two heavy chain immunoglobulin-light chain pairs, in which case the two chains have different specificities. Due to the random distribution of heavy chain immunoglobulin and light chain, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. It is usually quite troublesome to use affinity chromatography steps to purify the correct molecules, and the product yield is low.

[0180] According to a different approach, antibody variable domains with desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion can be to an immunoglobulin heavy chain constant domain, which includes the hinge, C H2 and C H3 The first heavy chain constant region (C H1 ) may contain a site necessary for light chain binding, which is present in at least one of the fusions. Nucleic acids with sequences encoding immunoglobulin heavy chain fusions and, if desired, immunoglobulin light chains are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments in aspects where unequal ratios of the three polypeptide chains used for construction provide the best yield. However, when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance, it is possible to insert the coding sequences of two or all three polypeptide chains into one expression vector.

[0181] Bispecific antibodies can have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure helps separate the desired bispecific compound from unwanted immunoglobulin chain combinations, because the presence of the immunoglobulin light chain in only half of the bispecific molecule provides a convenient means of separation. Using such technology, bispecific antibodies can be prepared for conjugation with new degraders in the treatment or prevention of diseases as defined herein.

[0182] Hybrid or bifunctional antibodies may be derived biologically, ie, by cell fusion techniques, or chemically, in particular using cross-linking agents or disulfide bond forming agents, and may comprise whole antibodies or fragments thereof.

[0183] The antibody may be an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen, or a microbial antigen, or a functionally active fragment, derivative, or analog of other antibodies that bind to a tumor cell or matrix. In this regard, "functional activity" means that the fragment, derivative, or analog is able to elicit an anti-anti-idiotypic antibody that recognizes the same antigen as the antigen recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in exemplary aspects, the antigenicity of the idiotypic antigen of the immunoglobulin molecule can be enhanced by the framework and CDR sequence of the C-terminus of the CDR sequence that specifically recognizes the antigen. In order to determine which CDR sequences bind to the antigen, a synthetic peptide containing a CDR sequence can be used for binding assays with the antigen by any binding assay method known in the art.

[0184] Other useful antibodies include antibody fragments, such as but not limited to F(ab')2 fragments, which contain variable regions, light chain constant regions, and heavy chain CH1 domains, which can be produced by digesting antibody molecules with pepsin; and Fab fragments, which can be generated by reducing the disulfide bonds of F(ab')2 fragments. Other useful antibodies are antibody heavy and light chain dimers, or any minimal fragment thereof, such as Fv or single chain antibodies (SCA), or any other molecule having the same specificity as an antibody.

[0185] In addition, recombinant antibodies comprising human and non-human parts, such as chimeric antibodies and humanized monoclonal antibodies, which can be prepared using standard recombinant DNA techniques, are available antibodies. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those with variable regions derived from mouse monoclonal and human immunoglobulin constant regions. Humanized antibodies are antibody molecules from non-human species, which have one or more complementary determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. Such chimeric antibodies and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0186] Transgenic mice that cannot express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes can be used to produce fully human antibodies. Transgenic mice are immunized with selected antigens, such as all or part of the polypeptides disclosed in the normal manner. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation, and class switching and somatic mutations occur subsequently. Therefore, using this technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). Other human antibodies can be purchased from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0187] Fully human antibodies that recognize selected epitopes can be generated using a technique called "guided selection". In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope. Human antibodies can also be produced using various techniques known in the art, including phage display libraries.

[0188] The antibody may be a fusion protein of an antibody or a functionally active fragment thereof, for example, in which the antibody is fused to an amino acid sequence of another protein (or a portion thereof, such as at least a 10, 20 or 50 amino acid portion of a protein) that is not an antibody via a covalent bond (e.g., a peptide bond) at the N-terminus or C-terminus. The antibody or its fragment may be covalently linked to the other protein at the N-terminus of the constant domain.

[0189] Antibodies include analogs and derivatives modified in any way, i.e., modified by covalent attachment of any type of molecule, as long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, derivatives and analogs of antibodies include those further modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, attachment to cell antibody units or other proteins, etc. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, analogs or derivatives may contain one or more non-natural amino acids.

[0190] The antibodies in the new degrader conjugates may include antibodies with modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors. In particular, the antibodies include antibodies with modifications in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor. Antibodies immunospecific for cancer cell antigens may be commercially available, for example, from Genentech (San Francisco, Calif.) or produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens may be obtained, for example, from the GenBank database or a database similar thereto, literature publications, or by conventional cloning and sequencing.

[0191] In certain aspects, the antibody of the novel degrader conjugate can be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. In some aspects, the antibody can be an antibody fragment, such as a Fab fragment.

[0192] Known antibodies for treating or preventing cancer can be conjugated to the new degradation agents described herein. Antibodies with immunospecificity to cancer cell antigens can be commercially available or produced by any method known to those skilled in the art, such as recombinant expression technology. Nucleotide sequences encoding antibodies with immunospecificity to cancer cell antigens can be obtained, for example, from the GenBank database or a database similar thereto, a literature publication, or by conventional cloning and sequencing. Examples of antibodies that can be used to treat cancer include, but are not limited to, humanized anti-HER2 monoclonal antibodies for treating patients with metastatic breast cancer. (trastuzumab); (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody used to treat patients with non-Hodgkin's lymphoma; OvaRex (oregovomab; AltaRex Corporation, MA), a murine antibody used to treat ovarian cancer; Panorex (edrecolomab, GlaxoWellcome, NC), a murine IgG2a antibody used to treat colorectal cancer; Cetuximab Erbitux (cetuximab, Imclone Systems Inc., NY), an anti-EGFR IgG chimeric antibody used to treat epidermal growth factor-positive cancers such as head and neck cancer; Vitaxin (etaracizumab, MedImmune, Inc., MD), a humanized antibody used to treat sarcomas; Campath I / H (alemtuzumab, Leukosite, MA), a humanized IgG1 antibody used to treat chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA), a humanized anti-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (epratuzumab, Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA), a radiolabeled mouse anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA), a humanized anti-CD2 mAb; Avastin (bevacizumab, Genentech, Inc., CA), an anti-VEGF humanized antibody used to treat lung and colorectal cancer; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA), an anti-CD22 antibody used to treat non-Hodgkin lymphoma; and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody used to treat colorectal cancer.

[0193] Other antibodies that can be used in the new degrader conjugates include, but are not limited to, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, daratumumab, STI-6129, lintuzumab, huMy9-6, belantamab, indatumomab, detuximab, anti-CD38 A2 antibody, HuAT13 / 5H3s antibody, ibritumomab tiuxetan, tositumomab, panitumumab, tremelimumab, tesitumumab, catumaxomab, and veltuzumab. In certain aspects, the antibody is selected from the group consisting of rituximab, trastuzumab, pertuzumab, huMy9-6-IgG4-S228P, lintuzumab, and gemtuzumab.

[0194] Other antibodies that can be used in the novel degrader conjugates include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), α-fetoprotein (cancer), CA 242 (colorectal cancer), placental alkaline phosphatase (cancer), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (cancer), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE-4 (cancer), anti-transferrin receptor (cancer), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (cancer), CD38 (multiple myeloma), CD40 (lymphoma), mucin (cancer), P21 (cancer), MPG (melanoma), and Neu oncogene products (cancer). Some specific, useful antibodies include, but are not limited to, BR96 mAb (Trail, PA et al. Science (1993) 261, 212-215), BR64 (Trail, PA et al. Cancer Research (1997) 57, 100-105), mAbs against the CD40 antigen such as S2C6 mAb (Francisco, JA et al. Cancer Res. (2000) 60: 3225-3231), mAbs against the CD70 antigen such as 1F6 mAb, and mAbs against the CD30 antigen such as AC10. Many other internalizing antibodies that bind to tumor-associated antigens can be used and have been reviewed.

[0195] Other antigens that the conjugates of the present invention can bind to include, but are not limited to, 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79 a、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、agglutinin、cKit、claudin 3、claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Cripto protein、CS1、CTLA-4、CXCR2、CXORF61、cyclin Bl、CYP1B1、cadherin-3、cadherin-6、DLL3、E7、EDNRB、EFNA4、EGFR、EGFRvIII、ELF2M、EMR2、ENPP3、EPCAM、EphA2、Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPVE6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), IL-2Rγ (i.e. CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, αIIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, muthsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostein, Pseudomonas aeruginosaaeruginosa), rabies, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and / or XAGE1.

[0196] Antibodies that bind to antigens associated with antigen-presenting cells, such as CD40, OX40L, Endoglin, DEC-205, 4-1BBL, CD36, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, BDCA-2, XCR-1, and CCR2, can also be conjugated to the new degraders.

[0197] The antibody of the new degrader conjugate can bind to a receptor or receptor complex expressed on activated lymphocytes. The receptor or receptor complex may include an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major tissue compatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD 19, CD22, CD28, CD79, CD90, CD 152 / CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoclast inhibitory factor, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD 103 and CD 104. Non-limiting examples of suitable lectins are C-type, S-type and I-type lectins.

[0198] In some aspects, antibodies useful in the present disclosure include, but are not limited to, 3F8, 8H9, abagovomab, abciximab abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab Alirocumab altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab ascrinvacumab, aselizumab, atidortoxumab, atlizumab (tocilizumab, ), atezolizumab atinumab, torolimumab, avelumab (Bavencio), azintuxizumab, belantamab, bapineuzumab, basiliximab Bavituximab, BCD-100, bectumomab begelomab, belantamab, belimumab bemarituzumab, benralizumab bermekimab, bersanlimab, bertilimumab, besilesomab Bevacizumab Bezlotoxumab Biciromab bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab (SILIQ TM ), brolucizumab Brontictuzumab, burosumab Cabiralizumab, caplacizumab Camidanlumab, camrelizumab, canakinumab cantuzumab, capromab, carlumab, carotuximab, catumaxomab cBR96, CC49, cedelizumab, cemiplimab Cergutuzumab, certrelimab, certolizumab, cetuximab cibisatamab, cirmtuzumab, citatuzumab, cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab Crotedumab, cusatuzumab, dacetuzumab, daclizumab Dalotuzumab, dapirolizumab, daratumumab Dectrekumab, demcizumab, denintuzumab, denosumab depatuxizumab, derlotuximab, detumomab, dezamizumab, dituximab diridavumab, domagrozumab, dostarlimab, dorlimomab, dorlixizumab, drozitumab, DS-8201, duligotuzumab, dupilumab Durvalumab dusigitumab, ecromeximab, and eculizumab Edobacumab, edrecolomab Efalizumab Efungumab Eldelumab, elezanumab, elgemtumab, elotuzumab Elsilimomab, emactuzumab, emapalumab Emibetuzumab, emicizumab Enpotamab, enavatuzumab, enfortumab enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, eptinezumab Epratuzumab, erenumab Erlizumab, ertumaxomab Etaracizumab Etilimab, etrolizumab, evinacumab, evolocumab Exbivirumab, fanolesomab faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab foralumab, foravirumab, fremanezumab fresolimumab, frovocimab, frunevetmab, fulranumab, futuximab, galcanezumab galiximab, gancotamab, ganitumab, gantenerumab, gavilimomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, golimumab Gomiliximab, guselkumab huMy9-6, huMY9-6-IgG4-S228P, ianalumab, ibalizumab IBI308, ibritumomab tiuxetan, icrucumab, idarucizumab ifabotuzumab, igovomab (INDIMACIS-125), iladatuzumab, IMAB362, imalumab, imaprelimab, imciromab imgatuzumab, inclacumab, indazumab, indusatumab, inebilizumab, infliximab intetumumab, inolimomab, inotuzumab, iomab-B, ipilimumab, iratumumab, isatuximab iscalimab, istiratumab, itolizumab, ixekizumab Keliximab, labetuzumab (CEA-CIDE TM), lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab landogrozumab, laprituximab, larcaviximab, lebrikizumab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, lilotomab, lintuzumab, lirilumab , lodelcizumab, lokivetmab, loncastuximab, lorvotuzumab, losatuxizumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, matuzumab, mavrilimumab, mepolizumab metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab, mitumomab, modotuximab, molalizumab, mogamulizumab morolimumab, mosunetuzumab, motavizumab Moxetumomab muromonab-CD3 (ORTHOCLONE ), nacolomab, namilumab, naptumomab, naratuximab, narnatumab, natalizumab Navicixizumab, navivumab, naxitamab, nebacumab, necitumumab nemolizumab, NEOD001, nerelimomab, nesvacumab, netakimab, nimotuzumab Nirsevimab, nivolumab, nofetumomab, obiltoxaximab Obinutuzumab, ocaratuzumab, ocrelizumab Odulimomab, Ofatumumab Olaratumab Oleclumab, olendalizumab, olokizumab, omalizumab Omburtamab, OMS721, onartuzumab, ontecizumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, OVAREX, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozogamicin, ozoralizumab, pagibaximab, palivizumab Pamrevlumab, panitumumab pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab Perakizumab, Pertuzumab pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, polatuzumab (Polivy), prezalumab, plozalizumab, pogalizumab, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140. Quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab Ravagalimab, ravulizumab raxibacumab, refanezumab, regavirumab, REGN-EB3, renatlimab, remtolumab, reslizumab Rilotumumab, rinucumab, risankizumab Rituximab rivabazumab, rmab, robatumumab, rodumab, romilkimab, romosozumab rontalizumab, rosmantuzumab, rovalvituzumab, rovelizumab rozanolixizumab, ruplizumab (ANTOVA), SA237, sacizumab, samalizumab, samrotamab, sarilumab Satralizumab, satumomab pendetide, secukinumab selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, SGN-CD19A, SHP647, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab mab), sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, STI-6129, sulesomab Suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tavolimab, tefibazumab telimomab, telisotuzumab, tesidolumab, tetraxetan, tetulomab, tenatumomab, teneliximab, teprotumumab Teplizumab, tezepelumab, TGN1412, tibulizumab, texitumomab Tigatuzumab, timigutuzumab, timolumab, tiragolumab, tiragotumab, tislelizumab, tisotumab, tiuxetan, tildrakizumab TNX-650, Tocilizumab (Atelimumab, ), tomozuotuximab, toralizumab, tosatoxumab, tositumomab tovetumab, tralokinumab, trastuzumab TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab ublituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab Vobarilizumab, volociximab Vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, XMAB-5574, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), zatuximab, zenocutuzumab, ziralimumab, zolbetuximab, or zolimomab.

[0199] In some aspects, binding moieties useful in the present disclosure comprise anti-CD33 antibodies or antigen binding portions thereof. CD33 is expressed in approximately 90% of acute myeloid leukemia (AML) cases and has been shown to be useful as a target for therapeutic antibodies. High CD33 expression on AML blasts was reported approximately thirty years ago. CD33 is detected on blasts of 85-90% of AML patients as well as normal bone marrow precursors and bone marrow cells. CD33 is restricted to hematopoietic cells but is absent on normal hematopoietic stem cells, making it an ideal target for AML treatment.

[0200] The anti-CD33 antibodies of the conjugates of the present disclosure are capable of specifically binding to CD33. In some aspects, the anti-CD33 antibodies described herein bind to CD33 with high affinity, for example, with 10 -6 M or lower, 10-7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M or 10 -9 M to 10 -7 M of K D Binds human CD33.

[0201] In some aspects, the anti-CD33 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH), and the light chain comprises a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1 (VH-CDR1), a VH-CDR2, and a VH-CDR3, and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 3. In some aspects, the anti-CD33 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some aspects, the anti-CD33 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some aspects, the anti-CD33 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some aspects, the anti-CD33 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some aspects, the anti-CD33 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some aspects, the CDR comprises the sequence shown in Table 1 below.

[0202] Table 1. CDR sequences and variable region sequences

[0203]

[0204] In some aspects, the anti-CD33 antibody heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some aspects, the anti-CD33 antibody light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0205] In some aspects, the anti-CD33 antibody comprises: a heavy chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:4; and a light chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0206] In some aspects, the anti-CD33 antibody heavy chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 11. In some aspects, the anti-CD33 antibody comprises a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 12.

[0207] Table 2. Anti-CD33 antibody amino acid sequences.

[0208]

[0209] In some aspects, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 9, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10. The term "CD33AB" comprises a heavy chain as set forth in SEQ ID NO: 9 and a light chain as set forth in SEQ ID NO: 10.

[0210] In some aspects, the anti-CD33 antibody comprises: a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11; and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 12.

[0211] In some aspects, anti-CD33 antibodies are disclosed in US Pat. Nos. 5,585,089, 5,693,762, each of which is specifically incorporated herein by reference.

[0212] In some aspects, the anti-CD33 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH), and the light chain comprises a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1 (VH-CDR1), a VH-CDR2, and a VH-CDR3, and the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15. In some aspects, the anti-CD33 antibody comprises a VH-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some aspects, the anti-CD33 antibody comprises a VH-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some aspects, the anti-CD33 antibody comprises a VL-CDR1 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some aspects, the anti-CD33 antibody comprises a VL-CDR2 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some aspects, the anti-CD33 antibody comprises a VL-CDR3 comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 20. In some aspects, the CDR comprises the sequence shown in Table 3 below.

[0213] Table 3. CDR sequences and variable region sequences of huMy9-6 and huMy9-6-IgG4-S228P

[0214]

[0215] In some aspects, the anti-CD33 antibody heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some aspects, the anti-CD33 antibody light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0216] In some aspects, the anti-CD33 antibody comprises: a heavy chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 16; and a light chain variable region comprising a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0217] In some aspects, the anti-CD33 antibody heavy chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21. In some aspects, the anti-CD33 antibody comprises a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 22.

[0218] Table 4. huMy9-6-IgG4-S228P antibody amino acid sequence.

[0219]

[0220] In some aspects, the anti-CD33 antibody comprises a heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21, and a light chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 22. The anti-CD33 antibody comprises a heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 22.

[0221] An antibody that "binds" a molecular target or antigen of interest is one that is capable of binding the antigen with sufficient affinity to allow the antibody to be used to target cells expressing the antigen.

[0222] In the present disclosure, the group "Bm" can be conjugated to more than one new degrader. In some aspects, "Bm" can be conjugated to 1 to 10 new degraders. In some aspects, "Bm" can be conjugated to 1 to 9 new degraders. In some aspects, "Bm" can be conjugated to 1 to 8 new degraders. In some aspects, "Bm" can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 new degraders. In some aspects, "Bm" can be conjugated to 7 or 8 new degraders. In some aspects, "Bm" is conjugated to 5 new degraders. In some aspects, "Bm" is conjugated to 6 new degraders. In some aspects, "Bm" is conjugated to 7 new degraders. In some aspects, "Bm" is conjugated to 8 new degraders. In some aspects, "Bm" is conjugated to 9 new degraders.

[0223] IV. Compositions and Methods of Use

[0224] The conjugates and / or compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some aspects, such salts are derived from inorganic or organic acids or bases.

[0225] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, and undecanoate.

[0226] Examples of suitable base addition salts include ammonium salts; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine and the like.

[0227] For example, Berge lists the following FDA-approved commercial salts: anionic acetate, besylate / benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, edetate (EDTA), camsylate / camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate (EDTA), edisylate (1,2-ethanedisulfonate), estolate / lauryl sulfate sulfate), esylate / ethanesulfonate, fumarate, gluceptate / glucoheptonate, gluconate, glutamine, glycollylarsanilate / glycollamidophenylarsonate, hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate / meth anesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthalenesulfonate (2-naphthalenesulfonate), nitrate, pamoate / embonate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), and triethyl iodide; the organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and the metal cations aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0228] Berge additionally lists the following non-FDA approved commercially available (outside the U.S.) salts: anionic adipates, alginate, aminosalicylate, anhydrous methylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), naphthalene disulfonate (1,5-naphthalene disulfonate), oxalate, jellyrate, persulfate, phenethylbarbiturate, picrate, propionate, thiocyanate, toluenesulfonate, and undecanoate salts; organic cations benethamine (N-benzylphenethylamine), clemizole (1-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and metal cations barium and bismuth.

[0229] Pharmaceutical compositions comprising the novel degrader conjugates described herein may also include suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0230] In some aspects, the pharmaceutical composition can be formulated into a suitable parenteral dosage form. The formulation can be prepared by various methods known in the art. The pharmaceutical composition can be directly administered into the bloodstream, into a muscle, or directly into an organ. Suitable means for parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, intrathecal administration, intraventricular administration, intraurethral administration, intrasternal administration, intracranial administration, intramuscular administration, and subcutaneous administration. Suitable devices for parenteral administration include needle syringes, needle-free syringes, and infusion techniques.

[0231] Parenteral compositions are usually aqueous solutions, which may contain excipients such as salts, carbohydrates and buffers. However, the compositions may also be formulated as sterile non-aqueous solutions or in dry form for use with a suitable solvent such as sterile pyrogen-free water.

[0232] The preparation of parenteral compositions under sterile conditions, for example by lyophilization, may readily be accomplished using standard techniques well known to those skilled in the art.

[0233] Compositions for parenteral administration can be formulated to release immediately and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Thus, the composition can be formulated as a solid, semisolid, or thixotropic liquid to be administered as an implanted reservoir providing modified release of an active agent.

[0234] The parenteral formulation may be mixed with other suitable pharmaceutically acceptable excipients for parenteral dosage forms such as, but not limited to, preservatives.

[0235] In another aspect, the pharmaceutical composition can be formulated into a suitable oral dosage form, such as tablets, capsules, powders, pills, suspensions, solutions, emulsions and the like. Other suitable carriers may be present, such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, extenders, anti-adherents and the like.

[0236] Oral dosage forms may also contain other suitable pharmaceutical excipients, such as sweeteners, solvents / wetting agents, colorants, flavoring agents, preservatives, viscosity / thickening agents and the like.

[0237] The novel degrader conjugates described herein can be used to treat various cancers. Certain conjugates disclosed herein may be superior in terms of efficacy expression, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interaction with other agents (e.g., drug metabolizing enzyme inhibition), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity) and / or stability (e.g., chemical stability, stability to enzymes), and may be used as medicaments.

[0238] The novel degrader conjugates of the present disclosure can be used as a medicament, such as an agent for preventing or treating a disease, such as cancer, for example, colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, stomach / gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., ovarian epithelial carcinoma, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low-malignant potential tumor) tumor), testicular tumors, prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (e.g., medullary thyroid cancer), kidney cancer (e.g., renal cell carcinoma (e.g., clear cell renal cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, uterine corpus cancer, uterine sarcoma), gestational choriocarcinoma, brain tumors (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocyte astrocytoma), cytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell tumor, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, blood cancer (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disease), cancer of unknown primary; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; agents for treating precancerous lesions (e.g., myelodysplastic syndrome); and the like.

[0239] In certain aspects, the novel degrader conjugates of the present disclosure can be used as agents for treating breast cancer, gastric cancer, ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, liver cancer, lymphoma, or blood cancer.

[0240] In addition, the novel degrader conjugates of the present disclosure can be used simultaneously with, before, or after non-drug therapies. Specifically, the conjugates can be combined with non-drug therapies such as (1) surgery, (2) chemotherapy for hypertension using angiotensin II, etc., (3) gene therapy, (4) thermal therapy, (5) cryotherapy, (6) laser ablation, and (7) radiotherapy.

[0241] For example, by using the novel degrader conjugates of the present disclosure before or after the above-mentioned surgery and the like, effects such as preventing the emergence of drug resistance, prolonging disease-free survival, inhibiting cancer metastasis or recurrence, prolonging life span, and the like can be provided.

[0242] Furthermore, it is possible to combine treatment with the novel degrader conjugates of the present disclosure with the following supportive therapies: (i) administration of antibiotics (e.g., β-lactams such as pansporin and the like, macrolides such as clarithromycin and the like) for complications of various infectious diseases, (ii) administration of high-calorie infusions, amino acid preparations, or general vitamin preparations to improve malnutrition, (iii) administration of analgesics to relieve pain, (iv) administration of agents for improving side effects such as nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, alopecia, liver disease, kidney disease, DIC, fever, and the like, and (v) administration of agents for suppressing multidrug resistance of cancer, and the like.

[0243] In some aspects, the new degraders or new degrader conjugates disclosed herein can be used in combination with standard care therapies, such as one or more therapeutic agents (e.g., anticancer agents and / or immunomodulators). Therefore, in certain aspects, the method for treating a tumor disclosed herein comprises administering the new degraders or new degrader conjugates disclosed herein in combination with one or more additional therapeutic agents. In some aspects, the new degraders or new degrader conjugates disclosed herein can be used in combination with one or more anticancer agents, for example, so that multiple components of an immune pathway can be targeted. In some aspects, the anticancer agent comprises an immune checkpoint inhibitor (i.e., blocking the signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies) or combinations thereof.

[0244] In some aspects, the novel degraders or novel degraders conjugates of the present disclosure are administered to an individual before or after the administration of an additional therapeutic agent. In other aspects, the novel degraders or novel degraders conjugates of the present disclosure are administered to an individual simultaneously with the additional therapeutic agent. In certain aspects, the novel degraders or novel degraders conjugates of the present disclosure and the additional therapeutic agent can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other aspects, the novel degraders or novel degraders conjugates of the present disclosure and the additional therapeutic agent are administered simultaneously as separate compositions.

[0245] In some aspects, the subject that can be treated with the novel degraders or novel degrader conjugates of the present disclosure is a non-human animal, such as a rat or mouse. In some aspects, the subject that can be treated is a human.

[0246] V. Methods of Preparing New Degrading Agents and Compositions

[0247] The present disclosure provides a method for preparing a novel degrader conjugate, the method comprising reacting a binding moiety with a compound of formula (I-1):

[0248]

[0249] or a pharmaceutically acceptable salt thereof, wherein:

[0250] L' is a linker precursor selected from the following:

[0251]

[0252] in:

[0253] is the point of attachment to the nitrogen atom.

[0254] As described herein, the linker precursor contains a heterobifunctional group attached to a binding moiety.

[0255] In some aspects, the binding moiety is pretreated before reacting with the compound of formula (I-1). In certain aspects, the compound of formula (I-1) is reacted with a binding moiety that comprises an antibody or an antigen-binding portion thereof. In aspects where the binding moiety is an antibody, the antibody may be pretreated to reduce interchain disulfide bonds prior to reacting with the compound of formula (I-1).

[0256] Example

[0257] General synthetic methods and intermediates

[0258] The compounds of the present disclosure can be prepared by those of ordinary skill in the art according to the knowledge of the present disclosure and the art, and / or by reference to the schemes and synthetic examples shown below. Exemplary synthetic routes are described in the following schemes and examples. It should be understood that the variables (e.g., "R" groups) appearing in the following schemes and examples should be read independently of the variables appearing elsewhere in the application. Those of ordinary skill in the art will readily understand how the schemes and examples shown below illustrate the preparation of the compounds described herein.

[0259] Abbreviations used in the schemes generally follow the conventions used in the art. Chemical abbreviations used in the specification and examples are defined as follows: "Me" represents methyl; "Bu" represents butyl; "Ph" represents phenyl; "TFA" represents trifluoroacetic acid; "DCM" represents dichloromethane; "HATU" represents N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanamine hexafluorophosphate N-oxide; "THF" represents tetrahydrofuran; "BOC" or "Boc" represents tributyloxycarbonyl; "TEA" represents triethylamine; "EtOH" represents ethanol; "DMF" stands for N,N-dimethylformamide; "PE" stands for petroleum ether; "EtOAc" stands for ethyl acetate; "DIEA" stands for diisopropylethylamine; "MeOH" stands for "methanol"; h stands for hour; min stands for minute; "Ac" stands for acetate; "EDCI" stands for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; "HOBT" stands for 1-hydroxybenzotriazole hydrate; "ACN" stands for acetonitrile; "TCEP" stands for (tris(2-carboxyethyl)phosphine); "DMA" stands for N,N-dimethylacetamide;

[0260]

[0261] Scheme 1: Preparation of new degrader P1

[0262]

[0263] Example 1: Synthesis of new degradation agent P1

[0264]

[0265] Step 1: Synthesis of compound 2

[0266] To a stirred solution of 2-chloro-4-nitrophenyl)acetic acid (Compound 1, 5.00 g, 23.19 mmol, 1.00 equiv) in THF (75.00 mL) at 0 ° C. under nitrogen atmosphere, BH3-Me2S (10 M in THF) (5.80 mL, 58.0 mmol, 2.50 equiv) was added dropwise. The resulting mixture was stirred at 70 ° C. under nitrogen atmosphere for 2 h. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EtOAc = 1: 1) to give 2- (2-chloro-4-nitrophenyl) ethanol (3 g, 64%) as a yellow solid. 1H NMR (400MHz, CDCl3) δ8.26 (d, J = 4.0 Hz, 1H), 8.10-8.05 (m, 1H), 7.50 (d, J = 8.0 Hz, 1H), 3.99-3.91 (m, 2H), 3.16-3.09 (m, 2H).

[0267]

[0268] Step 2: Synthesis of compound 3

[0269] To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (Compound 2, 5.00 g, 24.800 mmol, 1.00 equiv) and tert-butyl 2-bromoacetate (29.0 mL, 148.28 mmol, 8.00 equiv) in toluene (150.00 mL) was added Bu4NHSO4 (6.74 g, 19.84 mmol, 0.80 equiv). To the above mixture was added NaOH (5 M in H2O) (500.00 mL) dropwise at 0°C over 40 min. The resulting mixture was stirred at 25°C for another 2 h. The resulting mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (400 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to give tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (8 g, 65%) as a yellow oil. 1 H NMR(400MHz, CDCl3)δ8.23(d,J=4.0Hz,1H),8.10-8.04(m,1H),7.60(d,J=8 .0Hz,1H),4.09(s,2H),3.83-3.80(m,2H),3.17-3.14(m,2H),1.45(s,9H).

[0270]

[0271] Step 3: Synthesis of compound 4

[0272] At room temperature, TFA (16.00 mL) was added dropwise to a stirred solution of tertiary butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (compound 3, 8.00 g, 16.14 mmol, 1.00 equiv., 63.7%) in DCM (80.00 mL). The resulting mixture was stirred at room temperature for 1 h. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with water (500 mL). The mixture was extracted with EtOAc (3 x 500 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This produced [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (6.5 g, crude) as a yellow oil. LCMS (ESI): 517 (2M-H)-

[0273]

[0274] Step 4: Synthesis of compound 5

[0275] To a stirred solution of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 4, 6.30 g, 21.84 mmol, 1.00 equiv, 90%) and HATU (12.46 g, 32.76 mmol, 1.50 equiv) in DMF (65.00 mL) at room temperature, CH3NH2.HCl (1.77 g, 26.21 mmol, 1.20 equiv) and DIEA (15.20 g, 117.8 mmol, 4.00 equiv) were added dropwise. The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was diluted with water. The resulting mixture was extracted with EtOAc (2×100 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (10 g, purity: 50%, yield: 84%) as a yellow oil. LCMS (ESI): 273.28 (M+H) +

[0276]

[0277] Step 5: Synthesis of compound 6

[0278] To a stirred solution of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 5, 3.3 g, 12.10 mmol, 1.00 equiv) in THF (35.00 mL) was added dropwise BH3-THF (1 M in THF) (12.10 mL, 12.10 mmol, 1.00 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 70 ° C under nitrogen atmosphere for 2 h. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO3 (saturated aqueous solution). The resulting mixture was extracted with EtOAc (3 x 100 mL), washed with brine (50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (2.5 g, 80%) as a yellow oil. LCMS (ESI): 259.26 (M+H) +

[0279]

[0280] Step 6. Synthesis of compound 7

[0281] To a stirred solution of [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (Compound 6, 2.50 g, 9.69 mmol, 1.00 equiv) and Boc2O (2.53 g, 11.6 mmol, 1.20 equiv) in THF (40 mL) was added TEA (1.17 g, 11.6 mmol, 1.20 equiv) dropwise at 25°C. The mixture was stirred at 25°C for 2 h. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography (DCM:MeOH=5:1) to give tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (1.70 g, 50%) as a yellow oil. LCMS (ESI): 359.36 (M+H) +

[0282]

[0283] Step 7: Synthesis of compound 8

[0284] To a stirred solution of tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 7, 1.70 g, 4.74 mmol, 1.00 equiv) and NH4Cl (750 mg, 14.2 mmol, 3.00 equiv) in EtOH (85 mL) and H2O (17 mL) at 25°C was added Fe (1.3 g, 23.7 mmol, 5.00 equiv). The mixture was stirred at 80°C for 2 h. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with EtOH (3 x 50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to obtain tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (900 mg, 58%) as a yellow oil. LCMS (ESI): 329.33 (M+H) +

[0285]

[0286] Step 8: Synthesis of compound 9

[0287] To a stirred solution of tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 8, 500 mg, 1.52 mmol, 1.00 equiv) in THF (10 mL) was added diphosgene (601 mg, 3.04 mmol, 2.00 equiv) dropwise at 25° C. The mixture was stirred at 25° C. for 1 h. The resulting mixture was concentrated under vacuum and redissolved in DMF (5 mL). To a stirred mixture of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, prepared as described below, 499 mg, 1.82 mmol, 1.20 equiv) and TEA (1.56 g, 15.45 mmol, 10.00 equiv) in DMF (20 mL) at 25 °C was added the above-mentioned solution dropwise. The mixture was stirred at 25 °C for 1 h. The resulting mixture was diluted with 40 mL of ice water. The resulting mixture was extracted with EtOAc (3 x 40 mL). The combined organic layers were washed with brine (5 x 40 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give tert-butyl (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (670 mg, 70%) as a white solid. LCMS: (ESI): 628.63 (M+H) +

[0288]

[0289] Step 9: Synthesis of new degrader P1

[0290] To a stirred solution of tert-butyl N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate (compound 9, 670 mg, 1.07 mmol, 1 equiv) in DCM (10 mL) at 0°C was added TFA (2.5 mL) dropwise. The mixture was stirred at 25°C for 1 h. The resulting mixture was concentrated under vacuum. The crude product was purified by preparative HPLC under the following conditions: column, SunFire C18 OBD preparative column, 100 μm, 19×250 mm; mobile phase, water (0.05% TFA) and ACN (5% phase B, reaching 60% in 30 min); detector, UV 220 nm. The collected fractions were lyophilized to give 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (500 mg, 89%) as a white solid. LCMS (ESI): 528.53 (M+H) + . 1 H NMR (400MHz, methanol-d4) δ7.77(d,J=8.0Hz,1H),7.57-7.53(m,2H),7.49(d,J=8.0Hz,1H),7.21(d,J=4.0Hz,2H),5.19-5.1(m,1H),4.55-4.41(m,4H), 3.75-3.67(m,4H),3.21-3.15(m,2H),3.03-3.96(m,2H),2.96-2.84(m,1 H),2.83-2.73(m,2H),2.69(s,3H),2.55-2.42(m,1H),2.21-2.12(m,1H).

[0291]

[0292] Scheme 2: Synthesis of the novel degradation agent P1-β-glucuronide linker complex (Compound (Ia))

[0293] Example 2: Synthesis of Compound (Ia)

[0294]

[0295] Step 1: Synthesis of compound 12

[0296] To a stirred solution of 5-formyl-2-hydroxybenzoic acid, 10 (20 g, 120.38 mmol, 1.00 equiv) in DMF (200 mL) at 0 °C under nitrogen atmosphere was added EDCI (28 g, 144.44 mmol, 1.20 equiv), HOBT (20 g, 144.46 mmol, 1.20 equiv) and tert-butyl N-(2-aminoethyl)carbamate, 11 (23 g, 144.46 mmol, 1.20 equiv) in portions. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. LCMS indicated the reaction was complete. The reaction mixture was quenched with water and extracted with ethyl acetate (3x200 mL). The combined organics were washed with brine (200 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EA (2:3) to give tert-butyl N-[2-[(5-formyl-2-hydroxyphenyl)formamido]ethyl]carbamate, 12 (23 g, 53%) as a white solid. LCMS (ES, m / z): 209 [M+H-100] + ,309[M+H] + ,331[M+Na] + .

[0297]

[0298] Step 2: Synthesis of compound 14

[0299] To a stirred solution of tert-butyl N-[2-[(5-formyl-2-hydroxyphenyl)formamido]ethyl]carbamate, 12 (23 g, 74.59 mmol, 1.00 equiv) in ACN (600 mL) at 0°C under nitrogen atmosphere, AgO (34.57 g, 149.17 mmol, 2.00 equiv) and methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate, 13 (32.6 g, 82.05 mmol, 1.10 equiv) were added portionwise. The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / A (1:4) to obtain (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-([2-[(tert-butyloxycarbonyl)amino]ethyl]carbamoyl)-4-formylphenoxy]oxane-2-carboxylic acid methyl ester, 14 (38 g, 76%) as a green solid. LCMS (ES, m / z): 525 [M+H-100] + ,625[M+H] + ,647[M+Na]+ .

[0300]

[0301] Step 3: Synthesis of compound 15

[0302] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-formylphenoxy]oxane-2-carboxylic acid methyl ester, 14 (20 g, 32.02 mmol, 1.00 equiv) in EA (200 mL) was added Pd / C (4.0 g, 10%) in portions at room temperature. The resulting mixture was stirred at room temperature under hydrogen atmosphere overnight. LCMS indicated the reaction was complete. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (19:1) to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetoxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester, 15 (12.5 g, 58%) as a white solid. LCMS (ES, m / z): 527 [M+H-100] + ,627[M+H] + ,649[M+Na] + .

[0303]

[0304] Step 4: Synthesis of compound 16

[0305] To a stirred solution of methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate, 15 (5.0 g, 7.98 mmol, 1.00 equiv) in DMF (50 mL) at 0°C under nitrogen atmosphere was added bis(4-nitrophenyl) carbonate (2.67 g, 8.77 mmol, 1.10 equiv) and DIEA (2.0 g, 15.94 mmol, 2.00 equiv) portionwise. The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 70% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness under vacuum to give (2S, 3S, 4S, 5R, 6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-{[(4-nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxane-2-carboxylic acid methyl ester, 16 (5.3 g, 78%) as a white solid. LCMS (ES, m / z): 692 [M+H-100] + ,792[M+H] + ,814[M+Na] + .

[0306]

[0307] Step 5: Synthesis of compound 17

[0308] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-{[(4-nitrophenoxycarbonyl)oxy]methyl}phenoxy]oxane-2-carboxylic acid methyl ester, 16 (550 mg, 0.69 mmol, 1.00 equiv) and 1-(3 To a solution of 4-{2-[2-(methylamino)ethoxy]ethyl}phenyl)-3-{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}urea, P1 (367 mg, 0.69 mmol, 1.00 equiv) in DMF (6.0 mL) was added DIEA (180 mg, 1.38 mmol, 2.00 equiv) in portions. The resulting mixture was stirred at room temperature for 2 h. LCMS indicated the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 70% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]oxane-2-carboxylic acid methyl ester, 17 (670 mg, 77%) as a green solid. LCMS (ES, m / z): 1080 [M+H-100] + ,1180[M+H] + ,1202[M+Na] + .

[0309]

[0310] Step 6: Synthesis of compound 18

[0311] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-({2-[(tert-butyloxycarbonyl)amino]ethyl}carbamoyl)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]oxane-2-carboxylic acid methyl ester, 17 (660 mg, 0.56 mmol, 1.00 equiv) in THF (12 mL) was added HCl (12 mL, 6.0 N) portionwise at 45 °C under nitrogen atmosphere. The resulting mixture was stirred at 45 °C under nitrogen atmosphere for 4 h. LCMS indicated the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 40% gradient in 30 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (320 mg, 54%) as a white solid, 18. LCMS (ES, m / z): 940 [M+H] + ,962[M+Na] + .

[0312]

[0313] Step 7: Synthesis of compound (Ia)

[0314] Under nitrogen atmosphere at room temperature, stirred (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy To a solution of 1-(2-(2,5-dioxopyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-ester, 19 (36 mg, 0.12 mmol, 1.10 equiv) in DMF (1.00 mL) was added DIEA (27 mg, 0.21 mmol, 2.0 equiv) in portions. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was purified by preparative HPLC under the following conditions: Xselect CSH F-Phenyl OBD column, 19x250 mm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25 mL / min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-({2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]ethyl}carbamoyl)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid, Compound (Ia) (46 mg, 37%) as a white solid. LCMS (ES, m / z): 568 [M / 2+H] + ,1133[M+H] + ,1155[M+Na] + . 1H-NMR(300MHz,DMSO-d6):10.98(s,1H),8.87(br s,1H),8.30(t,J=6Hz,1H),7.86(t,J=3Hz,1H),7.77(s,1H),7.70-7.69(m,2H),7.51-7.42(m,3H),7.30-7.10(m,3H),6.99(s,2H),6.93(br s,1H),5.79-5.77(m,1H),5.36(d,J=4.2Hz,1H),5.13-5.02(m,4H),4. 47-4.28(m,4H),3.95(d,J=9.0Hz,1H),3.540-3.49(m,4H),3.39-3.36 (m,4H),3.24-3.22(m,2H),2.86-2.81(m,7H),2.62-2.51(m,1H),2.49 -2.41(m,3H),2.06-1.99(m,3H),1.49-1.42(m,4H),1.23-1.16(m,2H).

[0315]

[0316] Scheme 3: Synthesis of the novel degradation agent P1-β-glucuronide linker complex (Compound (Ib))

[0317]

[0318] Example 3: Synthesis of Compound (Ib)

[0319] To a stirred solution of bis(2,5-dioxopyrrolidin-1-yl glutarate) (62 mg, 0.19 mmol, 1.2 equiv) and (2S,3S,4S,5R,6S)-6-{2-[(2-aminoethyl)carbamoyl]-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid, 9 (150 mg, 0.16 mmol, 1.00 equiv) in DMF (3.00 mL) was added portionwise DIEA (41 mg, 0.32 mmol, 2.00 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS indicated that the reaction was complete. The crude product was purified by preparative HPLC under the following conditions (column: mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL / min). The solution was lyophilized to give (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidine-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-[(2-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanamido}ethyl)carbamoyl]phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid, Compound (Ib) (50 mg, 26%) as a white solid. LCMS (ES, m / z): 576 [M / 2+H] + ,1151[M+H] + ,1173[M+Na] + . 1H-NMR(300MHz,DMSO-d6):12.90(br s,1H),10.98(s,1H),8.78(s,1H),8.31(t,J=3.0Hz,1H),7.92(t,J=3.0Hz,1H),7.77(d,J=3.0Hz,1H),7 .70-7.66(m,2H),7.51-7.43(m,3H),7.25-7.09(m,3H),6.82(t,J=3.0Hz,1H),5.80-5.20(m,2H),5.12- 5.02(m,4H),4.47-4.27(m,4H),3.98(d,J=9.3Hz,1H),3.52-3.44(m,5H),3.30-3.05(m,7H),2.97-2.73 (m,9H),2.72-2.62(m,3H),2.45-2.35(m,1H),2.30-2.15(m,2H),2.05-1.90(m,1H),1.90-1.70(m,2H).

[0320]

[0321] Scheme 4: Preparation of compound (Ic)

[0322]

[0323] Example 4: Synthesis of Compound (Ic)

[0324] To a stirred solution of 1-(3-chloro-4-[2-[2-(methylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (new degradation agent P1, 200 mg, 0.38 mmol, 1.00 equiv) and lutidine (81 mg, 0.76 mmol, 2.00 equiv) in DMF (1 0 mL) were added HOBT (26 mg, 0.19 mmol, 0.50 equiv) and [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl 4-nitrophenyl carbonate (279 mg, 0.38 mmol, 1.00 equiv) in portions. The reaction mixture was stirred at 40 °C under nitrogen atmosphere for 12 hours. After cooling to room temperature, the reaction was quenched with water (30 mL). The resulting mixture was extracted with DCM (3 x 30 mL). The combined organic layers were washed with water (2 x 30 mL), brine (30 mL) and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified by reverse phase column (C18, mobile phase A: water containing 0.1% FA, B: ACN). The collected fractions were concentrated to dryness under vacuum. The crude product (60 mg) was purified by preparative HPLC under the following conditions (column: Xselect CSH OBD column 30x150 mm 5um, n; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 33B to 50B in 7 min; 220 nm; RT1: 5.27 min). The collected fractions were lyophilized to give [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidine-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-N-methylcarbamate (23.8 mg, 5%) as a white solid. LCMS (ESI): 1126.11 (M+H) + . 1H NMR (400MHz, DMSO-d6) δ10.99(s,1H),10.00(s,1H),8.88(s,1H),8.12-8.08(m,1H),7.85-7.81(m,2H),7.70-7.67(m ,2H),7.60-7.58(m,1H),7.51(s,1H),7.47-7.44(m,1H),7.28-7.25(m,2H),7.18-7.12(m,2H),7.00(s,2H),6.90(br s,1H),5.97-5.95(m,1H),5.42(s,2H),5.12-5.05(m,1H),4.98(s,2H),4.42-4.32(m ,4H),4.18-4.15(m,1H),3.56-3.40(m,4H),3.37-3.36(m,3H),3.05-2.90(m,3H),2. 89-2.85(m,5H),2.72-2.55(m,2H),2.40-2.33(m,2H),2.25-2.15(m,2H),2.00-1.87 (m,2H),1.74-1.57(m,2H),1.50-1.42(m,5H),1.22-1.10(m,3H),0.85-0.80(m,6H).

[0325]

[0326] Scheme 5A: Synthesis of the novel degrader P1-β-glucuronide linker complex

[0327] Step 1. Synthesis of compound 25

[0328] To a stirred mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propionic acid (compound 24, 5.00 g, 16.06 mmol, 1.00 equiv) in SOCl2 (25 mL) at room temperature. The resulting mixture was stirred at 80 ° C for 16 h. The desired product can be detected by LCMS (MS as MeOH derivative=326). LCMS indicated that the reaction was complete. The resulting mixture was concentrated under vacuum to give 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 25, 7.5 g, crude) as a yellow oil. The crude product was used directly in the next step without further purification. 1 H-NMR analysis showed it to be the desired product (as a MeOH derivative). 1H-NMR (300MHz, CDCl3) δ7.81-7.77(m,2H),7.63-7.59(m,2H),7.46-7.40(m,2H),7.40-7.31(m,2H),5.33(s,1H ), 4.42 (d, J = 3.0Hz, 2H), 4.24 (t, J = 6.0Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J = 3.0Hz, 2H), 2.59 (t, J = 6.0Hz, 2H).

[0329] Step 2. Synthesis of compound 28

[0330] To a stirred solution of 4-formyl-2-nitrophenol (compound 27, 4.21 g, 25.19 mmol, 1.00 equiv) and AgO (7.00 g, 30.20 mmol, 1.20 equiv) in ACN (100 mL, 190.24 mmol, 75.00 equiv) was added compound 26 (10.00 g, 25.17 mmol, 1.00 equiv) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred overnight at room temperature under N2 atmosphere. LCMS indicated that the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 ml x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (PE:EA=1:2) to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylic acid methyl ester (Compound 28, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed that it was the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300MHz, CDCl3) δ10.00 (s, 1H), 8.34 (s, 1H), 8.13-8.09 (m, 1H), 7.52 (d, J = 3.0 Hz,1H),5.47-5.29(m,4H),4.37-4.35(m,1H),3.75-3.73(m,3H),2.17-2.06(m,9H).

[0331] Step 3. Synthesis of compound 29

[0332] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylic acid methyl ester (compound 28, 6.00 g, 12.41 mmol, 1.00 equiv) in MeOH (50 mL) was added NaBH4 (0.47 g, 12.42 mmol, 1.00 equiv) in portions at RT under N2 atmosphere. The resulting mixture was stirred at room temperature under N2 atmosphere for 2 h. LCMS indicated the reaction was complete. The reaction was quenched with water at room temperature. The resultant was dried over Na2SO4. The resulting mixture was filtered and the filter cake was washed with DCM. The resulting mixture was concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylic acid methyl ester (Compound 29, 5.5 g, 91%) as a solid. LCMS (ES, m / z): 486 [M+H]+.

[0333] Step 4. Synthesis of compound 30

[0334] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylic acid methyl ester (compound 29, 5.50 g, 11.33 mmol, 1.00 equiv) in EA (60 mL) was added Pd / C (1.10 g, 10%) in portions at room temperature. The resulting mixture was stirred at room temperature under H2 atmosphere for 16 h. LCMS indicated the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM and MeOH. The filtrate was concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (compound 30, 4.0 g, 77%) as a solid. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 456 [M+H] + .

[0335] Step 5. Synthesis of compound 31

[0336] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (compound 30, 1.00 g, 2.19 mmol, 1.00 equiv) and NaHCO (0.20 g, 2.40 mmol, 1.1 equiv) in THF (10 mL) was added compound 25 (0.87 g, 2.62 mmol, 1.20 equiv) in portions at 0°C under N2 atmosphere. The resulting mixture was stirred at 0°C under N2 atmosphere for 6 h. LCMS indicated the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (EA=100%) to obtain (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propionamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (Compound 31, 1.1 g, 66%) as a pale yellow solid. LCMS (ES, m / z): 749 [M+H] + .

[0337] Step 6. Synthesis of compound 33

[0338] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetoxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (compound 31, 1.50 g, 2.00 mmol, 1.00 equiv) and bis(4-nitrophenyl carbonate) (compound 32, 0.68 g, 2.24 mmol, 1.12 equiv) in DMF (15 mL) at 0°C under N2 atmosphere was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv) in portions. The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 90% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness in vacuo to give (2S, 3S, 4S, 5R, 6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylic acid methyl ester (Compound 33, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] + .

[0339]

[0340] Scheme 5B: Synthesis of the novel degrader P1-β-glucuronide linker complex

[0341] Step 7. Synthesis of compound 34

[0342] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylic acid methyl ester (Compound 33, 1.00 g, 1.09 mmol, 1.00 equiv) and 1-(3-chloro-4-[2-[2-(methylamino)-1-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate was added under N2 atmosphere at room temperature. To a mixture of 2-[(2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl)methyl]urea (new degradation agent P1, 0.58 g, 1.09 mmol, 1.00 equiv) in DMF (10 mL) were added HOBT (1.18 g, 8.72 mmol, 8.00 equiv) and 2,4-lutidine (1.07 g, 8.72 mmol, 8.00 equiv) in batches. The resulting mixture was stirred at room temperature under N2 atmosphere for 16 h. LCMS indicated that the reaction was complete. The resulting mixture was further purified. The residue was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water containing ACN (0.1% FA), 10% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]oxane-2-carboxylic acid methyl ester (Compound 34, 800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] + .

[0343] Step 8. Synthesis of compound 35

[0344] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]oxane-2-carboxylic acid methyl ester (compound 34, 800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) was added HCl (6N, 80 mL) portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at 50°C under N2 atmosphere for 3 h. LCMS indicated the reaction was complete. The resulting mixture was concentrated under vacuum. The residue was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 80% in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound 35, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] + .

[0345] Step 9. Synthesis of compound 36

[0346] To a stirred solution of (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 0.2 mmol, 1.00 equiv) in DMF (2 mL) was added piperidine (0.4 mL) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 10 min. LCMS indicated the reaction was complete. The resulting mixture was further purified directly by preparative HPLC under the following conditions (column: XSelect CSH preparative C18 OBD column, 19x250 mm, 5um; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B in 7 min; 220 nm; RT 1: 5.78 min) to give (2S, 3S, 4S, 5R, 6S)-6-[2-(3-aminopropionamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound 36, 35 mg, 18%) as a white solid. LCMS (ES, m / z): 940 [M+H]+.

[0347] Step 10. Synthesis of compound (Ie)

[0348] To a stirred solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropionamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 36, 30 mg, 0.03 mmol, 1.00 equiv) in DMF (3 mL) was added DIEA (13 mg, 0.10 mmol, 3.00 equiv) and compound 37 (30 mg, 0.10 mmol, 3.00 equiv) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS indicated the reaction was complete. The resulting mixture was purified by preparative HPLC under the following conditions (column: Xselect CSH OBD column 30 x 150 mm 5 um, mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 21 B to 36B in 10 min; 220 nm; RT 1: 11.15 min). The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-(methyl)carbamoyl]oxy)methyl]-2-[3-[6-(2,5-dioxopyrrol-1-yl)hexanamido]propionamido]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound (Ie), 10.5 mg, 28%) as a white solid. LCMS (ES, m / z): 1133 [M+H] + . 1H-NMR(300MHz,DMSO-d6)δ10.9(s,1H),9.13(s,1H),8.16(s,1H),7.92-7.68(m,4H),7.52 (s,1H),7.44(d,J=3.0Hz,1H),7.18-6.99(m,7H),5.76(s,1H),5.20-5.10(m,2H),4.98(br s,2H),4.76-4.74(m,1H),4.42-4.33(m,4H),3.65(br s,1H),3.58-3.54(m,5H),3.35(d,J=6Hz,2H),2.90-2.83(m,7H),2.57-2.55(m, 3H),2.45-2.30(m,1H),2.02-1.98(m,4H),1.48-1.42(m,5H),1.40-1.20(m,3H).

[0349]

[0350] Scheme 6: Synthesis of the novel degrader P1-β-glucuronide linker complex (Compound (Ih))

[0351]

[0352] Example 6: Synthesis of Compound (Ih)

[0353] Step 1. Synthesis of compound 63

[0354] To a stirred mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionic acid (compound 62, 5.00 g, 16.06 mmol, 1.00 equiv) was added SOCl2 (25 mL) at room temperature. The resulting mixture was stirred at 80 ° C for 16 h. The desired product can be detected by LCMS (MS as MeOH derivative = 326). LCMS indicated that the reaction was complete. The resulting mixture was concentrated under vacuum to give 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 63, 7.5 g, crude) as a yellow oil. The crude product was used directly in the next step without further purification. 1 H NMR analysis showed it to be the desired product (as a MeOH derivative). 1H-NMR (300MHz, CDCl3) δ7.81-7.77(m,2H),7.63-7.59(m,2H),7.46-7.40(m,2H),7.40-7.31(m,2H),5.33(s,1H ), 4.42 (d, J = 3.0Hz, 2H), 4.24 (t, J = 6.0Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J = 3.0Hz, 2H), 2.59 (t, J = 6.0Hz, 2H).

[0355] Step 2. Synthesis of compound 66

[0356] To a stirred solution of 4-formyl-2-nitrophenol (compound 65, 4.21 g, 25.19 mmol, 1.00 equiv) and AgO (7.00 g, 30.20 mmol, 1.20 equiv) in ACN (100 mL, 190.24 mmol, 75.00 equiv) was added (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylic acid methyl ester (compound 64, 10.00 g, 25.17 mmol, 1.00 equiv) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature under N2 atmosphere overnight. LCMS indicated the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (PE:EA=1:2) to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylic acid methyl ester (Compound 66, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed that it was the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300MHz, CDCl3) δ10.00 (s, 1H), 8.34 (s, 1H), 8.13-8.09 (m, 1H), 7.52 (d, J = 3.0 Hz,1H),5.47-5.29(m,4H),4.37-4.35(m,1H),3.75-3.73(m,3H),2.17-2.06(m,9H).

[0357] Step 3. Synthesis of compound 67

[0358] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylic acid methyl ester (compound 66, 6.00 g, 12.41 mmol, 1.00 equiv) in MeOH (50 mL) was added NaBH4 (0.47 g, 12.42 mmol, 1.00 equiv) in portions at room temperature under N2 atmosphere. The resulting mixture was stirred at room temperature under N2 atmosphere for 2 h. LCMS indicated the reaction was complete. The reaction was quenched with water at room temperature. The resultant was dried over Na2SO4. The resulting mixture was filtered and the filter cake was washed with DCM. The resulting mixture was concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylic acid methyl ester (Compound 67, 5.5 g, 91%) as a solid. LCMS (ES, m / z): 486 [M+H] + .

[0359] Step 4. Synthesis of Compound 68

[0360] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylic acid methyl ester (compound 67, 5.50 g, 11.33 mmol, 1.00 equiv) in EA (60 mL) was added Pd / C (1.10 g, 10%) in portions at room temperature. The resulting mixture was stirred at room temperature under H2 atmosphere for 16 h. LCMS indicated the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM and MeOH. The filtrate was concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (compound 68, 4.0 g, 77%) as a solid. The crude product was used directly in the next step without further purification. LCMS (ES, m / z): 456 [M+H] + .

[0361] Step 5. Synthesis of compound 70

[0362] To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (compound 68, 1.00 g, 2.19 mmol, 1.00 equiv) and NaHCO (0.20 g, 2.40 mmol, 1.1 equiv) in THF (10 mL) was added portionwise 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 69, 0.87 g, 2.62 mmol, 1.20 equiv) at 0°C under N2 atmosphere. The resulting mixture was stirred at 0°C under N2 atmosphere for 6 h. LCMS indicated the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with DCM. The combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (EA=100%) to obtain (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propionamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (Compound 70, 1.1 g, 66%) as a pale yellow solid. LCMS (ES, m / z): 749 [M+H] + .

[0363] Step 6. Synthesis of Compound 72

[0364] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetoxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylic acid methyl ester (compound 70, 1.50 g, 2.00 mmol, 1.00 equiv) and bis(4-nitrophenyl carbonate) (compound 71, 0.68 g, 2.24 mmol, 1.12 equiv) in DMF (15 mL) at 0° C. under N2 atmosphere was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv) in portions. The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. LCMS indicated the reaction was complete. The reaction mixture was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), 10% to 90% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated to dryness in vacuo to give (2S, 3S, 4S, 5R, 6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propionamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylic acid methyl ester (Compound 72, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] + .

[0365] Step 7. Synthesis of Compound 73

[0366] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylic acid methyl ester (compound 72, 1.00 g, 1.09 mmol, 1.00 equiv) and 1-(3-chloro-4-[2-[2-(methylamino)-1-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate was added under N2 atmosphere at room temperature. To a mixture of 2-[(2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl)methyl]urea (new degradation agent P1, 0.58 g, 1.09 mmol, 1.00 equiv) in DMF (10 mL) were added HOBT (1.18 g, 8.72 mmol, 8.00 equiv) and 2,4-lutidine (1.07 g, 8.72 mmol, 8.00 equiv) in batches. The resulting mixture was stirred at room temperature under N2 atmosphere for 16 h. LCMS indicated that the reaction was complete. The resulting mixture was further purified. The residue was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water containing ACN (0.1% FA), 10% to 80% gradient in 40 min; detector, UV 254 nm. The collected fractions were concentrated under vacuum to give (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]oxane-2-carboxylic acid methyl ester (Compound 73, 800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] + .

[0367] Step 8. Synthesis of Compound 74

[0368] To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]oxane-2-carboxylic acid methyl ester (compound 73, 800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) was added HCl (6N, 80 mL) portionwise at room temperature under N2 atmosphere. The resulting mixture was stirred at 50°C under N2 atmosphere for 3 h. LCMS indicated the reaction was complete. The resulting mixture was concentrated under vacuum. The residue was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 80% in 40 min; detector, UV 254 nm. The collected fractions were lyophilized to give (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound 74, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] + .

[0369] Step 9. Synthesis of Compound 75

[0370] To a stirred solution of (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]-2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propionamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, 74 (230 mg, 0.2 mmol, 1.00 equiv) in DMF (2 mL) was added piperidine (0.4 mL) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 10 min. LCMS indicated the reaction was complete. The resulting mixture was further purified directly by preparative HPLC under the following conditions (column: XSelect CSH preparative C18 OBD column, 19x250 mm, 5um; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 20B to 40B in 7 min; 220 nm; RT1: 5.78 min) to give (2S, 3S, 4S, 5R, 6S)-6-[2-(3-aminopropionamido)-4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl](methyl)carbamoyl]oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound 75, 35 mg, 18%) as a white solid. LCMS (ES, m / z): 940 [M+H] + .

[0371] Step 10. Synthesis of compound (Ih)

[0372] To a stirred solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropionamido)-4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 75, 110 mg, 0.12 mmol, 1.00 equiv) and bis(2,5-dioxopyrrolidin-1-yl) glutarate (compound 76, 46 mg, 0.14 mmol, 1.2 equiv) in DMF (2.0 mL) was added DIEA (30 mg, 0.23 mmol, 2.0 equiv) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was purified by preparative HPLC under the following conditions (column: Kinetex EVO preparative C18, 30*150, 5um; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 21% B to 41% B, 41% in 7 min. B; wavelength: 254 nm; RT1 (min): 5.8. The collected fractions were lyophilized to obtain (2S, 3S, 4S, 5R, 6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-(3-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanamido}propionamido)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound (Ih), 48 mg, 34%) as a white solid. LCMS (ES, m / z): 1151 [M+H] + ,1173[M+Na] + . 1H-NMR(300MHz,DMSO-d6):12.80(br s,1H),10.98(s,1H),9.08(s,1H),8.79(s,1H),8.18(s,1H),7.96(s,1H),7.68-7.66(m,2H),7.51(s,1H),7.44(d,J= 8.1Hz,1H),7.25-7.00(m,4H),6.82-6.80(m,1H),5.86(s,1H),5.39-5.30(m,2H),5.14-5.07(m,1H),4.97(s,2H),4. 84(d,J=7.2Hz,1H),4.47-4.27(m,4H),3.90(d,J=9.6Hz,1H),3.56-3.48(m,4H),3.45-3.36(m,6H),2.95-2.80(m,8H ),2.75-2.65(m,3H),2.62-2.55(m,2H),2.49-2.35(m,1H),2.21-2.16(m,2H),2.01-1.95(m,1H),1.85-1.80(m,2H).

[0373] Example 7: General Procedure for Preparation and Characterization of New Degrader Conjugates

[0374] Scheme 7: Synthesis of CD33AB-compound (Ia)

[0375] Synthesis of CD33AB-compound (Ia)

[0376] 2.25 molar equivalents of TCEP were added to a solution of 7.8 mg / mL CD33AB in 50 mM EPPS, 5 mM EDTA pH 7.0, and the mixture was incubated at 37°C for 2 hours. After the partially reduced antibody was cooled to ambient temperature, 8 molar equivalents of compound (Ia) were added as a stock solution in DMA, resulting in a final concentration of 7.0 mg / mL antibody and a final concentration of 10% (v / v) in DMA. The reaction was incubated at ambient temperature for 1 hour. The resulting conjugate was purified by gel filtration using a Zeba 40K desalting column followed by dialysis using a Slide-a-Lyzer cassette (10K MWCO) into 20 mM sodium succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer. Purified AnDC was found to be 100% monomeric by SEC, with an average drug loading of 3.1 drug / antibody by reducing RPLC-MS, and <1.5% unconjugated compound (Ia) by RPLC.

[0377] Scheme 8: Synthesis of CD33AB-compound (Ib)

[0378] Synthesis of CD33AB-compound (Ib)

[0379] 3.8 molar equivalents of compound (Ib) in a stock solution in DMA were added to a solution of CD33AB in 50mM EPPS pH 8.0 buffer, resulting in a final concentration of 6.4 mg / mL of antibody and a final concentration of 10% (v / v) in DMA. The reaction was incubated at ambient temperature for 3 hours. The resulting conjugate was purified into 20mM sodium succinate, 8% sucrose, 0.01% Tween-20 pH 5.5 formulation buffer by gel filtration using a Zeba 40K desalting column and then dialyzed using a Slide-a-Lyzer cassette (10K MWCO). The purified AnDC was found to have 100% monomer by SEC, an average drug loading of 3.1 drug / antibody by complete RPLC-MS, and <1.5% unconjugated compound (Ib) by RPLC.

[0380] Concentration and monomer were determined by size exclusion chromatography using a 7.8 x 300 mM TSKGel 3000SWXL column and 5 μm particles (Tosoh Bioscience), isocratic elution with 400 mM sodium perchlorate, 50 mM sodium phosphate, 5% (v / v) isopropanol mobile phase, running at 0.5 mg / mL for 30 min. The new degrader conjugate was quantified based on an antibody standard curve, detected at 214 nm.

[0381] Drug to antibody ratio (DAR) was determined by hydrophobic interaction chromatography using a 4.6 x 35 mm TSKgel Butyl-NPR column and 2.5 μm particles. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0. Mobile phase B was 25 mM sodium phosphate pH 7.0, 25% (v / v) isopropanol. Analytes were eluted with a linear gradient of 0-100% B in 12 min at a flow rate of 0.6 mL / min. Detection was at 214 nm.

[0382] Free linker payload was determined by mixed mode chromatography using a 4.6 x 250 mm HISEP column with 2.5 μm particles (Supelco). Mobile phase A was 100 mM ammonium acetate. Mobile phase B was 100% acetonitrile. Analytes were eluted with a gradient of 25-40% B in 25 min, then 40-100% B in 2 min at a flow rate of 0.7 mL / min. The column temperature was 35° C. Free linker payload was quantified using an external standard curve with detection at 254 nm.

[0383] Additional neodegrader conjugates can be prepared using the above procedure, substituting the appropriate linker-neodegrader with a lysine- or cysteine-reactive conjugation handle.

[0384] Example 8: Treatment of Acute Myeloid Leukemia (AML) with Anti-CD33 Antibody-New Degrader Conjugates

[0385] Testing athymic nude mice (Crl:NU(NCr)-Foxn1 nu , Charles River) in CD33AB-new degrader compound. 1×10 7 MV411 human acute monocytic leukemia cells ( CRL-5991 TM ) was injected subcutaneously into the flank of the mouse (0.1 mL / mouse). Once the tumor reached 100–150 mm 3 Based on the average size of CD33, mice were administered anti-CD33 antibody-neo-degrader conjugates, non-targeted neo-degrader conjugates, and vehicle control.

[0386] The stock solutions of CD33AB-compound (Ia) and CD33AB-compound (Ib) were diluted with vehicle to obtain 0.302 mg / mL and 0.294 mg / mL dosing solutions, which provided 3.02 mg / kg and 2.94 mg / kg in a dosing volume of 10 mL / kg (0.2 mL per 20 g mouse) adjusted according to the body weight of each animal. This dosing strategy ensures that the same amount of payload is provided to each test group. Mylotarg was diluted to 0.01 mg / mL in a 0.9% sodium chloride solution, which provided 3 mg / kg in a dosing volume of 10 mL / kg (0.2 mL per 20 g mouse). Venetoclax was formulated in a solvent consisting of 60% PG, 30% PEG400, and 10% ethanol via ultrasonic treatment to obtain a 5 mg / mL dosing suspension, which delivers 50 mg / kg when administered in a volume of 10 mL / kg. CC-90009 was centrifuged to collect the bottom powder; N-methyl-2-pyrrolidone (NMP), PEG400, and saline were then added and mixed thoroughly one by one to obtain a 0.5 mg / mL dosing solution in 5% NMP, 45% PEG400, and 50% saline, which delivered 5 mg / kg when administered in a volume of 10 mL / kg.

[0387] Mice were divided into 6 treatment groups (N=9 / group) as follows: 1) vehicle; 2) CD33AB-compound (Ia) (3.02 mg / kg, iv, qd x 1); 3) CD33AB-compound (Ib) (2.94 mg / kg, iv, qd x 1); 4) Mylotarg (0.1 mg / kg, iv, qd x 1); 5) Venetoclax (50 mg / kg, po, qd x 21); 6) CC-90009 (5 mg / kg, ip, bid x 10). The test articles for groups 1-4 were administered as a single dose (qd x 1) intravenously (iv) in a volume adjusted for body weight (0.200 mL / 20 g mouse). Venetoclax was administered orally (po), while CC-90009 was administered intraperitoneally (ip) in a dosing volume of 10 mL / kg (0.2 mL per 20 g mouse) adjusted to the BW of each animal.

[0388] Tumors were measured twice weekly using calipers and were counted when each animal's tumor reached the endpoint volume (2,000 mm 3 ) or on the last day of the study (Day 45), whichever comes first, each animal was euthanized. MTV (n) was defined as the median tumor volume of the remaining number of animals (n) whose tumors did not reach the endpoint volume on the last day of the study.

[0389] like Figure 1A As shown, both novel degrader conjugates provided slower tumor growth over time compared to vehicle.

[0390] To further confirm activity and evaluate alternative conjugation and linker release methods, a panel of CD33AB-based conjugates were tested in clinical trials at clinical dosing levels and compared with Mylotarg (a clinically approved CD33-targeted ADC) and CC-90009 (a small molecule GSPT1 degrader). Figure 1B As shown, and consistent with in vitro observations, in vivo treatment of CD33-positive AML model tumors (MV4-11) with CD33AB-based conjugates, release of the novel degrader P1 resulted in tumor regression, with the most robust effects seen with conjugates containing a β-glucuronide release trigger and cysteine ​​conjugation. Comparison of two variants of the β-glu linker (compound (Ie) versus compound (Ia)) showed that the compound (Ia) conjugate expressed a longer-lasting response compared to compound (Ie).

[0391] Example 9: Treatment of human leukemia models with anti-CD33 antibody-novel degrader conjugates

[0392] To confirm activity in a range of CD33-positive and CD33-negative models, the efficacy of the CD33AB-Compound (Ia) conjugate in inducing tumor cell killing in a panel of in vitro human leukemia models, including CD33-positive AML and CD33-negative malignancies, was evaluated. The cytotoxicity of the test article (TA) was measured using a panel of CD33-positive acute myeloid leukemia cell lines and a panel of non-AML CD33-negative cells. Cells were plated into 96-well plates at a predetermined concentration and after incubation overnight at 37°C / 5% CO2, serial dilutions of each test article (TA) were added to the cells. Cells were incubated with the test article for 72 hours and incubated with The viability was detected by using the ELISA kit (Promega). The luminescence value of each cell line was standardized, and the IC50 was calculated using Prizm software. Figure 2 Although the conjugate showed good activity in several CD33+ cells, advantageously, the conjugate was inactive in CD33-negative cell models.

[0393] Example 10: Cytotoxicity of anti-CD33 antibody-novel degrader conjugates

[0394] The cytotoxicity of the conjugates was measured using MV4-11 CD33-positive acute myeloid leukemia cells. Cells were plated in assay medium in 96-well plates and serial dilutions of CD33AB-Compound I(a) conjugates, venetoclax, Mylotarg, CC-885, CC-90009, CD33AB (unconjugated antibody), or non-binding AnDC controls prepared in assay medium were added to the cells. Cells were incubated for 72 hours and then counted using a cell counting kit-8 (Dojindo) or Reagent (Promega) was used to detect cell viability. Figure 3 As shown, the results indicate that the conjugate expressed overall in vitro efficacy comparable to, and in some cases superior to, CC885 or Mylotarg in CD33-positive AML cells.

[0395] Example 11: Activity of anti-CD33 antibody-novel degrader conjugates in AML patient-derived cells

[0396] The CD33AB-compound (Ia) conjugate was evaluated in vitro in primary blast cells from several AML patients. Frozen bone marrow samples collected from adult AML patients after the last treatment were thawed and plated into 96-well plates containing serial dilutions of the conjugate, Mylotarg, or CC-90009. Each plate was incubated at 37°C / 5% CO2 for 48 hours, then the red blood cells in each well were lysed, and the remaining cells were stained with blast marker antibodies (the best blast antibody for each donor was previously determined) and annexin V. The samples were analyzed by flow cytometry. Tumor cell survival was determined by the absolute count of surviving tumor cells in each sample and normalized by the count in the untreated wells. Figure 4 As shown, the anti-CD33 antibody-neo-degrader conjugate expressed superior activity in patient-derived cells (as measured by cytotoxicity) compared to standard of care treatment (Mylotarg) or an exploratory small molecule GSPT1 degrader (CC-90009).

[0397] Example 12: Activity of anti-CD33 antibody-new degrader conjugates on proliferation of human precursor cells CFC

[0398] Since CD33 is expressed in normal bone marrow precursor cell populations and normal cell myelosuppression is an observed adverse event (AE) of Mylotarg, the activity of CD33AB-compound (Ia) conjugate, Mylotarg and CC-90009 on normal erythroid, bone marrow and megakaryocyte precursor cells was measured in a colony formation assay. Normal human bone marrow light density cells were thawed, washed and plated into 24-well plates in XVivo 15 medium supplemented with rhIL-3 (10 ng / mL), rhGMCSF (10 ng / mL) and rhSCF (50 ng / mL) on the day of the experiment. CD33AB-compound I(a) conjugate, Mylotarg and CC-90009 were added to each well and the cells were incubated for 72 hours. After incubation, for bone marrow and erythroid precursors, 400uL of each cell suspension was added to a methylcellulose-based medium supplemented with rhIL-3 (10ng / mL), rh SCF (50ng / mL), rhGM-CSF (10ng / mL), and rhEpo (3U / mL). For megakaryocyte precursors, cells were added to 35mm culture dishes containing a semisolid, collagen-based matrix supplemented with hIL-3 (10ng / mL), rhIL-6 (10ng / mL), and (50ng / mL). After 14 days of incubation, colonies of bone marrow and erythroid precursors were assessed microscopically. Colonies are divided into several categories based on size: CFU-E (colony forming unit - erythrocyte; this colony forming cell produces small colonies containing less than 200 erythrocyte blasts) and BFU-E - erythrocyte precursor cells (bursting colony forming unit - erythrocyte; this is a more primitive colony forming cell and it produces larger colonies containing more than 200 erythrocyte blasts), CFU-GM - granulocyte-monocyte precursor cells (colony forming unit - granulocyte, macrophage; this bone marrow colony forming cell is able to produce colonies with 40 or more granulocytes-monocytes and / or macrophages) and CFU-GEMM - multipotent precursor cells (colony forming unit - granulocyte, erythrocyte, macrophage, megakaryocyte; this primitive colony forming cell is able to produce colonies containing erythrocytes and 20 or more granulocytes, macrophages and megakaryocytes).

[0399] For megakaryocytes, after 14 days of incubation, cells were transferred to slides, fixed, and stained with CD41 antibody and alkaline phosphate detection system. Colonies were divided into three categories according to size: CFU-Mk (3-20), CFU-Mk (21-49), CFU-Mk (≥50). Results were normalized by the number of colonies in untreated samples, and IC50 was calculated using Prizm software. Results are shown in Figure 5 shown.

[0400] Example 13: Activity of anti-CD33 antibody-novel degrader conjugates in AML tumors compared to current standard of care

[0401] Subcutaneous tumor model MV4-11 human acute myeloid leukemia cells (1x10 in 0.1 mL) were added. 6 cells) were inoculated subcutaneously into the right flank of female athymic nude mice. 3 Starting at 400 μg / mL, mice were treated with TA by intravenous injection into the lateral tail vein, intraperitoneal injection, oral gavage, or a combination thereof. Tumor size and mouse body weight were measured twice a week. Figure 6 As shown, the CD33AB-Compound (Ia) conjugate demonstrated efficacy superior to the best care option.

[0402] Example 14: Activity of anti-CD33 antibody-novel degrader conjugates in disseminated AML models

[0403] Since AML is a disease that often disseminate throughout the patient's body, with lesions in multiple bone marrow niches and circulating in the bloodstream, the activity of the CD33AB-Compound (Ia) conjugate in an in vivo model of disseminated AML was investigated.

[0404] The disseminated model MV4-11 cells (3 × 10 6 100 cells) were injected intravenously into the lateral tail vein of female NCG mice. Treatment was started thirteen days after tumor cell injection. The mice were checked for morbidity, mortality and clinical observations every day. Body weight was measured twice a week. Imaging analysis of tumor development was performed under anesthesia once a week by injecting 5 μL / g D-luciferin 10 minutes before bioluminescence imaging.

[0405] The disseminated model OCI-AML2 human acute myeloid leukocytes (1x10 in 0.2 mL) were 7 100 cells) were injected intravenously into the lateral tail vein of female NOG mice. Treatment was started nine days after tumor cell injection. Mice were checked daily for morbidity, mortality, and clinical observations. Body weight was measured twice a week. Imaging analysis of tumor development was performed under anesthesia once a week by injecting 5 μL / g D-luciferin 10 minutes before bioluminescence imaging.

[0406] like Figure 7 As shown, robust single-dose responses were observed in both models (MV4-11 and OCI-AML2), with sustained regression of the fluorescein signal, a marker of tumor cell killing.

[0407] Example 15: Degradation of GSPT1 by CD33AB-Compound (Ia) Conjugate

[0408] The degradation of GSPT1 was monitored by Western blot, confirming the mechanism of action of the CD33AB-Compound (Ia) conjugate. Whole cell lysates were prepared from MV4-11 CD33-positive AML cells treated with CD33AB-Compound (Ia) conjugate, new degrader P1, CC-90009 or Mylotarg for 6, 12 and 18 hours, and the proteins were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. GSPT1 was detected by commercially available rabbit anti-GSPT1 antibody (Abcam), followed by anti-rabbit HRP-conjugated secondary antibody (Cell Signaling Technology), and detected by chemiluminescent substrate (ThermoFisher). The PVDF membrane was then stripped and re-probed with β-actin-HRP-conjugated antibody (Cell Signaling Technology). The results are shown in Figure 2. Figure 8 As shown. These data support that conjugation of the new degrader P1 to CD33AB enhances intracellular exposure that drives selective GSPT1 degradation. Similar dose-dependent reductions in GSPT1 levels were observed for the conjugate and small molecules, new degrader P1 and CC-90009, within just 6 hours after administration. Compared to the transient depletion observed with the new degraders P1 and CC-90009, which showed a rebound in GSPT1 levels at the 12h time point, treatment with the conjugate showed persistent depletion to 18h after administration. These data support the possibility of prolonged exposure of the active payload after administration, consistent with the possibility of longer response duration and lower dosing frequency.

[0409] Example 16: Pharmacokinetics and pharmacodynamics of anti-CD33 antibody-new degrader conjugates

[0410] The exposure and pharmacodynamic activity of the CD33AB-Compound (Ia) conjugate were evaluated relative to the doses previously observed to establish tumor regression. MV4-11 human acute myeloid leukemia cells (1x10 in 0.1 mL) were injected into the subcutaneous tumor model. 6 Cells) were inoculated subcutaneously into the right flank of female athymic nude mice. Mice were treated with 0.5 mg / kg, 1 mg / kg and 3 mg / kg CD33AB-Compound (Ia) intravenous intra-tail vein injection. Terminal cardiac puncture blood and tumors were collected from subcutaneous MV4-11 tumor model mice before dosing, 10 minutes, 30 minutes, 1 hour, 6 hours, 24 hours, 72 hours and 120 hours after dosing. Blood was processed into EDTA plasma and tumors were snap frozen in liquid nitrogen.

[0411] Pharmacokinetic Analysis: Conjugate payload levels in plasma samples were quantified by LC-MS / MS. The process involved b-glucuronidase digestion to release the new degrader P1 payload from the conjugate, followed by protein precipitation extraction to collect the free and released payload, and LC-MS / MS analysis using MRM acquisition. The method was qualified and met the acceptance criteria for non-GLP bioanalytical quantitative LC-MS / MS for small molecule analysis based on linearity, specificity, retention effect, accuracy, and precision. Relevant pharmacokinetic parameters were calculated using WinNonLin (V8.3).

[0412] Pharmacodynamic Analysis - Tumor tissues were homogenized using RIPA lysis buffer with protease and phosphatase inhibitors, and Western blotting was performed using a polyclonal rabbit anti-GSPT1 antibody (Abcam ab126090). Consistent with the sustained depletion of GSPT1 observed in vitro, a single in vivo dose of the conjugate was sufficient to maintain reduced GSPT1 levels for up to 120 h ( Fig. 9 , top). In addition, conjugation of the new degrader P1 to CD33AB resulted in a sustained half-life of P1 exceeding 64 h ( Fig. 9 , bottom), supporting the potential for sustained exposure of the payload and less frequent dosing.

[0413] Example 17: Activity of the novel anti-CD33 degrader conjugate against Mylotarg-insensitive cell lines

[0414] The in vitro cytotoxicity of the test articles (TA) was measured using a panel of CD33-positive acute myeloid leukemia cell lines (AML193 and Kasumi-6) known to be insensitive to Mylotarg. Cells were plated into 96-well plates at a predetermined concentration and incubated overnight at 37°C / 5% CO2. Serial dilutions of each test article (TA) were added to the cells. The cells were incubated with the test articles for 72 hours and the cells were incubated with The viability was detected by using ELISA kit (Promega). The luminescence value of each cell line was normalized and the IC50 was calculated using Prizm software.

[0415] like Fig. 10A and Fig. 10B As shown, the conjugate had good activity against both cell lines.

[0416] It should be understood that the [Implementation] section, rather than the [Summary of the Invention] and [Abstract of the Invention] sections, is intended to be used to interpret the claims. The [Summary of the Invention] and [Abstract of the Invention] sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventors, and therefore are not intended to limit the present disclosure and the appended claims in any way.

[0417] The present disclosure has been described above with the aid of functional building blocks that illustrate implementations of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may be defined as long as the specific functions and their relationships are appropriately performed.

[0418] The foregoing description of specific aspects will disclose the general nature of the disclosure so fully that others can easily modify and / or rewrite various applications of these specific aspects without departing from the general concepts of the disclosure by applying the knowledge of those skilled in the art without undue experimentation. Therefore, based on the teachings and guidance presented herein, such rewriting and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects. It should be understood that the words or terms herein are for descriptive and not limiting purposes, so that the terms or words of this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0419] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following terms and their equivalents.

[0420] The present disclosure relates to the following embodiments:

[0421] 1. A conjugate of formula (I):

[0422]

[0423] or a pharmaceutically acceptable salt thereof, wherein:

[0424] a is 1 to 10;

[0425] L is a linker selected from the following:

[0426] and

[0427]

[0428] in:

[0429] is the point of attachment to the nitrogen atom; and

[0430] is the connection point with Bm; and

[0431] Bm is a binding moiety capable of specifically binding to a protein serving as a cell surface antigen.

[0432] 2. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein a is 2 to 8.

[0433] 3. The conjugate of item 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L is

[0434]

[0435] 4. The conjugate of item 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L is

[0436]

[0437] 5. The conjugate of any one of items 1 to 4, or a pharmaceutically acceptable salt thereof, wherein Bm is an antibody or an antigen-binding portion thereof.

[0438] 6. The conjugate of any one of items 1 to 5, wherein the surface antigen is selected from the group consisting of 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5 、CD51、CD52、CD54、CD56、CD62E、CD62P、CD62L、CD70、CD71、CD72、CD74、CD79a、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、Clumping factor、cKit、Claudin 3、Claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Crypto 1 Growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, EphrinA4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), IL-2Rγ (i.e., CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzyme, prostein, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin and WTl, XAGE1.

[0439] 7. A conjugate according to any one of items 1 to 5, or a pharmaceutically acceptable salt thereof, wherein the surface antigen is selected from the group consisting of HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248 and TROP-2.

[0440] 8. The conjugate of any one of items 1 to 5, or a pharmaceutically acceptable salt thereof, wherein the surface antigen is CD33.

[0441] 9. The conjugate of claim 5 or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of rituximab, trastuzumab, gemtuzumab, CD33AB, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuximab, isatuximab isatuximab, Sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, balantamab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, and veltuzumab.

[0442] 10. The conjugate of claim 5 or a pharmaceutically acceptable salt thereof, wherein the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab.

[0443] 11. A conjugate of claim 5 or a pharmaceutically acceptable salt thereof, wherein the antibody or its antigen-binding portion comprises: a heavy chain variable region (VH) complementarity determining region (CDR) 1 (VH-CDR1), which comprises the amino acid sequence as set forth in SEQ ID NO: 1; VH-CDR2, which comprises the amino acid sequence as set forth in SEQ ID NO: 2; VH-CDR3, which comprises the amino acid sequence as set forth in SEQ ID NO: 3; a light chain variable region (VL) CDR1 (VL-CDR1), which comprises the amino acid sequence as set forth in SEQ ID NO: 5; VL-CDR2, which comprises the amino acid sequence as set forth in SEQ ID NO: 6; and VL-CDR3, which comprises the amino acid sequence as set forth in SEQ ID NO: 7.

[0444] 12. A conjugate as described in claim 12 or a pharmaceutically acceptable salt thereof, wherein the antibody or its antigen-binding portion comprises: VH, which comprises the amino acid sequence listed in SEQ ID NO:4; and VL, which comprises the amino acid sequence listed in SEQ ID NO:8.

[0445] 13. A conjugate of claim 11 or a pharmaceutically acceptable salt thereof, wherein the antibody comprises: a heavy chain comprising the amino acid sequence listed in SEQ ID NO:9; and a light chain comprising the amino acid sequence listed in SEQ ID NO:10.

[0446] 14. A compound of formula (II):

[0447]

[0448] or a pharmaceutically acceptable salt thereof.

[0449] 15. A compound of formula (III):

[0450]

[0451] or a pharmaceutically acceptable salt thereof.

[0452] 16. A conjugate of formula (IV):

[0453]

[0454] or a pharmaceutically acceptable salt thereof, wherein Bm is a binding portion that specifically binds to a protein that is a cell surface antigen.

[0455] 17. A conjugate of formula (V):

[0456]

[0457] or a pharmaceutically acceptable salt thereof, wherein Bm is a binding portion that specifically binds to a protein that is a cell surface antigen.

[0458] 18. The conjugate of claim 16 or 17, or a pharmaceutically acceptable salt thereof, wherein Bm is an antibody or an antigen-binding portion thereof.

[0459] 19. A conjugate as in any one of items 16 to 18, wherein the surface antigen is selected from the group consisting of: 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5 、CD51、CD52、CD54、CD56、CD62E、CD62P、CD62L、CD70、CD71、CD72、CD74、CD79a、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、Clumping factor、cKit、Claudin 3、Claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Crypto 1 Growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, EphrinA4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2 ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), IL-2Rγ (i.e. CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, muthsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine Acid, PIK3CA, PLAC1, polysialic acid, prostate enzyme, prostein, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and XAGE1.

[0460] 20. A conjugate according to any one of items 16 to 18, or a pharmaceutically acceptable salt thereof, wherein the surface antigen is selected from the group consisting of HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR, GD2, PDGFR, TEM1 / CD248 and TROP-2.

[0461] 21. A conjugate of claim 18 or a pharmaceutically acceptable salt thereof, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, entuximab, isatuximab, sacetuzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMY9-6-IgG4-S228P, belantamab, indalatuximab, cetuximab, detuximab, anti-CD38A2 antibody, HuAT13 / 5 antibody, CD33AB, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, panitumumab, tremelimumab, tesitumumab, catumaxomab, oregovozumab or veltuzumab.

[0462] 22. The conjugate of claim 21 or a pharmaceutically acceptable salt thereof, wherein the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab.

[0463] 23. A conjugate as described in claim 18 or a pharmaceutically acceptable salt thereof, wherein the antibody or its antigen-binding portion comprises: VH-CDR1, which comprises the amino acid sequence as listed in SEQ ID NO:1; VH-CDR2, which comprises the amino acid sequence as listed in SEQ ID NO:2; VH-CDR3, which comprises the amino acid sequence as listed in SEQ ID NO:3; VL-CDR1, which comprises the amino acid sequence as listed in SEQ ID NO:5; VL-CDR2, which comprises the amino acid sequence as listed in SEQ ID NO:6; and VL-CDR3, which comprises the amino acid sequence as listed in SEQ ID NO:7.

[0464] 24. A conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein the antibody or its antigen-binding portion comprises: VH comprising the amino acid sequence listed in SEQ ID NO:4; and VL comprising the amino acid sequence listed in SEQ ID NO:8.

[0465] 25. A conjugate of claim 22 or a pharmaceutically acceptable salt thereof, wherein the antibody comprises: a heavy chain comprising the amino acid sequence listed in SEQ ID NO:9; and a light chain comprising the amino acid sequence listed in SEQ ID NO:10.

[0466] 26. A pharmaceutical composition comprising a conjugate or compound as described in any one of items 1 to 25, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0467] 27. A method of treating cancer in an individual in need thereof, the method comprising administering to the individual a pharmaceutically acceptable amount of a conjugate, compound or composition of any one of items 1 to 20, or a pharmaceutically acceptable salt thereof.

[0468] 28. The method of claim 27, wherein the cancer is a solid tumor.

[0469] 29. The method of claim 27, wherein the cancer is a hematological cancer / blood cancer.

[0470] 30. The method of claim 27, wherein the cancer is breast cancer, gastric cancer, lymphoma, acute myeloid leukemia, multiple myeloma, head and neck cancer, squamous cell carcinoma and / or hepatocellular carcinoma.

[0471] 31. The method of any one of items 27 to 30, wherein the cancer is refractory or resistant to Mylotarg.

[0472] 32. A method of treating myelodysplastic syndrome (MDS) in an individual in need thereof, the method comprising administering to the individual a pharmaceutically acceptable amount of a conjugate, compound or composition of any one of items 1 to 20, or a pharmaceutically acceptable salt thereof.

[0473] 33. The method of any one of items 27 to 32, further comprising administering to the individual a pharmaceutically acceptable amount of an additional agent before, after or simultaneously with the conjugate, compound or composition of any one of items 1 to 26, or a pharmaceutically acceptable salt thereof.

[0474] 34. A method as described in claim 33, wherein the additional agent is a cytotoxic agent or an immune response regulator.

[0475] 35. The method of claim 34, wherein the immune response regulator is a checkpoint inhibitor.

[0476] 36. A method as described in claim 35, wherein the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor and / or a LAG-3 inhibitor.

[0477] 37. A method for preparing a conjugate of item 1 or a pharmaceutically acceptable salt thereof, the method comprising reacting a binding moiety with a compound of formula (I-1):

[0478]

[0479] or a pharmaceutically acceptable salt thereof, wherein:

[0480] L' is selected from

[0481] and

[0482]

[0483] in:

[0484] is the point of attachment to the nitrogen atom.

[0485] 38. A method as described in item 37, which further comprises reducing the binding part before reacting with the compound of formula (I-1).

[0486] 39. A method as in item 37 or 38, wherein L' is

[0487]

[0488] 40. A method as in item 37 or 38, wherein L' is

[0489]

[0490] 41. A method as described in any one of items 37 to 40, wherein the compound of formula (I-1) is reacted with a binding moiety, which binding moiety comprises an antibody or an antigen binding portion thereof.

[0491] 42. A method as described in any one of items 37 to 41, wherein the binding portion is capable of specifically binding to a surface antigen selected from the group consisting of: 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, C D3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a 、CD79b、CD80、CD90、CD97、CD125、CD138、CD141、CD147、CD152、CD154、CD326、CEA、CEACAM5、CFTR、agglutinin、cKit、claudin 3、claudin 18.2、CLDN6、CLEC12A、CLL-1、cll3、c-MET、Crypto1 growth factor、CS1、CTLA-4、CXCR2、CXORF61、cyclin Bl、CYP1B1、cadherin-3、cadherin-6、DLL3、E7、EDNRB、EFNA4、EGFR、EGFRvIII、ELF2M、EMR2、ENPP3、EPCAM、EphA2、Ephrin A4, Ephrin B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor (IL-2Rα (i.e. CD25), IL-2Rβ (i.e. CD122), IL-2Rγ (i.e. CD132)), IL-4 receptor (IL-4R, IL-2Rγ / IL-13Rα1), IL-13 receptor (IL-13Rα1, IL-13Rα2, IL-4R) IL-1 receptor (IL-1 lRa), IL-12 receptor (IL-12Rβ1, IL-12Rβ2), IL-23 receptor (IL-12Rβ1, IL-23R), IL-22 receptor (IL-22Rα1, IL-22Rα2, IL-10Rβ), IL-5 receptor (IL-5Rα, CSF2RB), IL-6 receptor (IL-6Rα, gp130), interferon receptor, integrin (including α4, α. v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, legumin, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA / MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, Nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzyme, prostein, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, SART3, SLAMF7, SLC44A4, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and XAGE1.

[0492] 43. A method as described in claim 42, wherein the surface antigen is selected from the group consisting of: HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248 and Trop-2.

[0493] 44. The method of claim 41, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, entuximab, isatuximab, sacetuzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, huMy9-6-IgG4-S228P, belantamab, indalatuximab, cetuximab, detuximab, anti-CD38 A2 antibody, CD33AB, HuAT13 / 5 antibody, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, panitumumab, tremelimumab, tesitumumab, catumaxomab, oregovozumab or veltuzumab.

[0494] 45. The method of claim 41, wherein the antibody is rituximab, trastuzumab, pertuzumab, huMy9-6, huMy9-6-IgG4-S228P, CD33AB, lintuzumab or gemtuzumab.

[0495] 46. ​​A method as described in claim 41, wherein the antibody or its antigen-binding portion comprises: VH-CDR1, which comprises the amino acid sequence listed in SEQ ID NO:1; VH-CDR2, which comprises the amino acid sequence listed in SEQ ID NO:2; VH-CDR3, which comprises the amino acid sequence listed in SEQ ID NO:3; VL-CDR1, which comprises the amino acid sequence listed in SEQ ID NO:5; VL-CDR2, which comprises the amino acid sequence listed in SEQ ID NO:6; and VL-CDR3, which comprises the amino acid sequence listed in SEQ ID NO:7.

[0496] 47. A method as described in claim 46, wherein the antibody or its antigen-binding portion comprises: VH, which comprises the amino acid sequence listed in SEQ ID NO:4; and VL, which comprises the amino acid sequence listed in SEQ ID NO:8.

[0497] 48. A method as described in claim 47, wherein the antibody comprises: a heavy chain comprising the amino acid sequence listed in SEQ ID NO:9; and a light chain comprising the amino acid sequence listed in SEQ ID NO:10.

Claims

1. A conjugate of formula (I): or a pharmaceutically acceptable salt thereof; in: a is 1 to 10; L is is the point of attachment to the nitrogen atom; is the connection point with Bm; and Bm is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:9, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

10.

2. The conjugate according to claim 1, wherein the conjugate is of formula (I).

3. The conjugate according to claim 1, wherein the conjugate is a pharmaceutically acceptable salt of formula (I).

4. The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein a is 2 to 8. 5 . The conjugate according to claim 4 , or a pharmaceutically acceptable salt thereof, wherein a is 3 to 5.

6. A conjugate of formula (I): or a pharmaceutically acceptable salt thereof; in: a is 1 to 10; L is is the point of attachment to the nitrogen atom; is the connection point with Bm; and Bm is an antibody or an antigen-binding portion thereof that can specifically bind to CD33.

7. The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein a is 2 to 8, and L is 8. The conjugate according to claim 7 or a pharmaceutically acceptable salt thereof, wherein a is 3 to 5.

9. The conjugate according to claim 6, 7 or 8, or a pharmaceutically acceptable salt thereof, wherein Bm is an antibody or an antigen-binding portion thereof, comprising a heavy chain variable region (VH) complementarity determining region (CDR) 1 (VH-CDR1) comprising the amino acid sequence as shown in SEQ ID NO: 1; VH-CDR2 comprising the amino acid sequence as shown in SEQ ID NO: 2; VH-CDR3 comprising the amino acid sequence as shown in SEQ ID NO: 3; a light chain variable region (VL) CDR1 (VL-CDR1) comprising the amino acid sequence as shown in SEQ ID NO: 5; VL-CDR2 comprising the amino acid sequence as shown in SEQ ID NO: 6; and VL-CDR3 comprising the amino acid sequence as shown in SEQ ID NO:

7.

10. The conjugate according to claim 6, 7 or 8, or a pharmaceutically acceptable salt thereof, wherein Bm is an antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion thereof comprises: VH comprising the amino acid sequence as set forth in SEQ ID NO:4; and VL comprising the amino acid sequence as set forth in SEQ ID NO:8.

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