Neodegrader conjugates

Abstract

To provide neoDegraders, and neoDegraders conjugated to binding moieties.SOLUTION: The compounds and compositions are useful for treating a disease or condition, e.g., cancer, in a subject in need thereof. According to one embodiment, the present disclosure provides a conjugate represented by formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the binding moiety is selected from an antibody, an antibody fragment, or an antibody-binding fragment. In some embodiments, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof in which a is an integer ranging from 2 to 8.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure provides neoDegrader conjugates, in which the neoDegrader is conjugated to a binding moiety. Compositions comprising the conjugates are also provided. The conjugates and compositions are useful for treating cancer in a subject in need thereof. [Background technology]

[0002] Protein degradation has been validated as a therapeutic strategy by the effectiveness of immunomodulatory imide drugs. These compounds bind to cereblon (CRBN) and inhibit CRL4. CRBN They have the ability to promote the recruitment and ubiquitination of substrate proteins mediated by E3 ubiquitin ligases. Immunomodulatory imides are thought to act as "molecular glue" filling the binding interface as a hydrophobic patch that reprograms protein interactions between the ligase and the new substrate.

[0003] Despite the excitement surrounding these compounds as novel cancer treatments, their use has thus far been limited to hematological malignancies such as multiple myeloma and myelodysplastic syndromes (MDS). Expanding the library of compounds that can function by degrading other oncoproteins, many of which have been considered "undruggable," is an active area of ​​drug development. Thus, there is a continuing need for new compounds that can target these alternative oncoproteins and treat a variety of cancers. Summary of the Invention [Means for solving the problem]

[0004] In certain aspects, the present disclosure provides a conjugate of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 10, A is phenyl or C4-C 10 is a cycloalkyl ring, U is selected from NH and CF; R 1 is independently selected from hydrogen and halo; X is -NR 2 -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from: Q and Q' are each independently O, S, or N(R 2 ) V and v is 1 or 2, Each R 2 are independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6, m is an integer from 2 to 6, where the left side of each group is bonded to L and the right side is bonded to A, However, X is NH or -Q-(CH2) n -When R 1 is a halo, L is a cleavable or non-cleavable linker; Bm is a binding moiety capable of specifically binding to a protein.

[0005] In some embodiments, the binding moiety is an antibody, an antibody fragment, or an antibody-binding fragment.

[0006] In some embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein a is an integer from 2 to 8.

[0007] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker. In some embodiments, L is selected from the group consisting of: [ka] During the ceremony, p is an integer from 1 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0008] In some embodiments, L is [ka] is.

[0009] In some embodiments, p is 5.

[0010] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker. In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, L is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue in the L- or D-configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0011] In some embodiments, Z 1 , Z 2 , Z 3 , and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 At least two of the residues are amino acid residues.

[0012] In some embodiments, Z 1 is absent or is glycine, and Z 2 is absent or is selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4 is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0013] In certain embodiments, L is [ka] is.

[0014] In some embodiments, q is 5.

[0015] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a bioreducible linker. In some embodiments, L is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, R, R', R", and R'" are each independently selected from hydrogen, C-C alkoxyC-C alkyl, (C-C) NC-C alkyl, and C-C alkyl, or two geminal R groups together with the carbon atoms to which they are attached are can form a cyclobutyl or cyclopropyl ring, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0016] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is an acid-cleavable linker. In some embodiments, L is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0017] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a click-to-release linker. In some embodiments, L is selected from: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0018] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a pyrophosphatase-cleavable linker. [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0019] In certain embodiments, the present disclosure provides a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a beta-glucosidase cleavable linker. In some embodiments, L is selected from: [ka] During the ceremony, q is an integer from 2 to 10, ---- is absent or is a bond, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0020] In certain embodiments, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein Bm is an antibody or an antigen-binding portion thereof. In some embodiments, the protein to which the binding moiety binds is a surface antigen.

[0021] In some embodiments, 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-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD17 9a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, D25, CD27L, CD28, CD3, CD30, CD31, CD 300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD 56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, C D138, 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 B1, 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, gp1OO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24、HMWMAA、HPV E6 hTERT was about to see photos of ICAM ICOS-L IFN-α IFN- γ、IGF-I merges with IGLL1 and IL-2 with IL-4 and IL-13Ra2 with IL-1 IRa、CH-1、CH-12、CH-23、CH-13、CH-22、CH- 4|IL-5、IL-6、and the α4α. v β3、α v β5、α v β6、α1β4、α4β1、α4β7、α5β1、α6β4、α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, 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-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein, Pseudomonas aeruginosa, rabies, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, respiratory syncytial virus, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, 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, XAGE1, or a combination thereof.

[0022] In certain embodiments, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof.

[0023] In some embodiments, Bm is an antibody, and the antibody is selected from the group consisting of rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, balantumab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, and veltuzumab. In some embodiments, the antibody is rituximab, trastuzumab, pertuzumab, OR000213 (huMy9-6 IgG4 S228P), lintuzumab, or gemtuzumab.

[0024] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R 1 But it's a halo, X is -N(R 2 ) v (CH2) m O(CH2) n - in which v is 1, m and n are 2; R 2 is methyl.

[0025] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R1 But it's a halo, X is -N(R 2 ) v (CH2) m O(CH2) n - in which v is 2, m and n are 2; Each R 2 is methyl.

[0026] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R 1 But it's a halo, X is -O(CH2) n - in which n is 2.

[0027] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R 1 But it's a halo, X is -S(CH2) n - in which n is 2.

[0028] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R 1 is hydrogen, X is --NR 2 - in which R 2 is methyl.

[0029] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R 1 But it's a halo, X is --NR 2 - in which R 2 is hydrogen.

[0030] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is phenyl; U is NH, R 1 is hydrogen, X is -C(CH3)=

[0031] In certain aspects, the present disclosure provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is C4-C 10 is a cycloalkyl ring, U is NH, R 1 is hydrogen, X is -N(R 2 )(CH2) m O(CH2) n - in which n is 1, m is 2, R 2 is methyl.

[0032] In certain aspects, the present disclosure provides a compound of formula (II) [ka] or a pharmaceutically acceptable salt thereof, wherein: A is phenyl or C4-C 10 is a cycloalkyl ring, R 1 is independently selected from hydrogen and halo; U is selected from NH and CF; R2 But -C(O)R 3 , -N(R 4 )2, -(CH2) n OH, -(CH2) n SH, -(CH2) n N(R 4 )2, -(CH2) n Q'(CH2) m OH, -(CH2) n Q'(CH2) m SH, and -(CH2) n Q'(CH2) m N(R 4 )2, wherein: R 3 is hydrogen or C1-C6 alkyl; Each R 4 are independently hydrogen or C1-C6 alkyl; Q' is O, S, or NR 4 and n is 1 to 6; m is 2 to 5; However, R 2 NH2, -(CH2) n NH2, or -(CH2) n If OH, then R 1 is a halo.

[0033] In certain aspects, the present disclosure provides a compound of formula (III) [ka] or a pharmaceutically acceptable salt thereof.

[0034] In certain aspects, the present disclosure provides a compound of formula (IV) [ka] or a pharmaceutically acceptable salt thereof.

[0035] In some embodiments, the present disclosure provides a conjugate of formula (V) [ka] or a pharmaceutically acceptable salt thereof, wherein Bm is a binding moiety that specifically binds to a protein. In some embodiments, Bm is an antibody or an antigen-binding portion thereof. In some embodiments, the protein to which the binding moiety specifically binds is a surface antigen.

[0036] In some embodiments, 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-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD17 9a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, D25, CD27L, CD28, CD3, CD30, CD31, CD 300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD 56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, C D138, 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 B1, 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, gp1OO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24、HMWMAA、HPV E6 hTERT was about to see photos of ICAM ICOS-L IFN-α IFN- γ、IGF-I merges with IGLL1 and IL-2 with IL-4 and IL-13Ra2 with IL-1 IRa、CH-1、CH-12、CH-23、CH-13、CH-22、CH- 4|IL-5、IL-6、and the α4α. v β3、α v β5、α v β6、α1β4、α4β1、α4β7、α5β1、α6β4、α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, 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-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein, Pseudomonas aeruginosa, rabies, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, respiratory syncytial virus, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, 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, XAGE1, or a combination thereof.

[0037] In some embodiments, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof.

[0038] In some embodiments, Bm is an antibody, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, balantumab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, or veltuzumab. In some embodiments, the antibody is rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, or gemtuzumab.

[0039] In certain aspects, the present disclosure provides a pharmaceutical composition comprising a conjugate or compound according to any one of the preceding aspects, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0040] In certain aspects, the present disclosure provides methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a conjugate, compound, or composition of any of the preceding aspects, or a pharmaceutically acceptable salt thereof. 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.

[0041] In some embodiments, the method further comprises administering to the subject a pharmaceutically acceptable amount of an additional agent before, after, or simultaneously with the conjugate or compound of any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof. In some embodiments, the additional agent is a cytotoxic agent or an immune response modifier. In some embodiments, the immune response modifier is a checkpoint inhibitor. In some embodiments, 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.

[0042] In certain aspects, the present disclosure provides a method for preparing a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, the process comprising: [ka] or a pharmaceutically acceptable salt thereof, wherein a is an integer from 1 to 10, A is phenyl or C4-C 10 is a cycloalkyl ring, R 1 is independently selected from hydrogen and halo; U is selected from NH and CF; X is -N(R 2 ) V -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from: v is 1 or 2, Q and Q' are each independently O, S, or NR 2 and Each R 2 are independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6, m is an integer from 2 to 6, where the left side of each group is bonded to L and the right side is bonded to A, However, X is NH or -Q-(CH2) n -When R 1 is a halo, L' is a precursor of a cleavable or non-cleavable linker that is conjugated to a binding moiety.

[0043] In some embodiments, the method further comprises reducing the binding moiety prior to reacting it with the compound of formula (I-1).

[0044] In some embodiments, a is an integer from 2 to 8.

[0045] In some embodiments, L' is a precursor of a non-cleavable linker. In some embodiments, L' is selected from the group consisting of: [ka] During the ceremony, p is an integer from 1 to 10, [ka] is the point of attachment to X.

[0046] In some embodiments, L' is [ka] is.

[0047] In some embodiments, p is 5.

[0048] In certain embodiments, L' is a precursor of a cleavable linker. In some embodiments, the precursor of the cleavable linker is cleavable by a protease. In some embodiments, L' is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, Z1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue in the L- or D-configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues, [ka] is the point of attachment to X.

[0049] In some embodiments, Z 1 , Z 2 , Z 3 , and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 At least two of the residues are amino acid residues.

[0050] In certain embodiments, Z 1 is absent or is glycine, and Z 2 is absent or is selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0051] In some embodiments, L' is [ka] is.

[0052] In some embodiments, q is 5.

[0053] In certain embodiments, L' is a precursor of a bioreducible linker. In some embodiments, L' is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, R, R', R", and R'" are each independently selected from hydrogen, C-C alkoxyC-C alkyl, (C-C) NC-C alkyl, and C-C alkyl, or two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring; [ka] is the point of attachment to X.

[0054] In certain embodiments, L' is a precursor of an acid cleavable linker. In some embodiments, L' is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X.

[0055] In certain embodiments, L' is a precursor of a click-to-release linker. In some embodiments, L' is selected from: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X.

[0056] In certain embodiments, L' is a precursor of a pyrophosphate cleavable linker. [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X.

[0057] In some embodiments, L' is a precursor of a beta-glucoronidase cleavable linker. In certain embodiments, L' is selected from: [ka] During the ceremony, q is an integer from 2 to 10, ---- is absent or is a bond, [ka] is the point of attachment to X.

[0058] In some embodiments, the compound of formula (I-1) is reacted with a binding moiety comprising an antibody or antigen-binding portion thereof. In some embodiments, the antibody or antigen-binding portion thereof binds to a surface antigen.

[0059] In certain embodiments, 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-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD17 9a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, D25, CD27L, CD28, CD3, CD30, CD31, CD 300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD 56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, C D138, 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 B1, 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, gp1OO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24、HMWMAA、HPV E6 hTERT was about to see photos of ICAM ICOS-L IFN-α IFN- γ、IGF-I merges with IGLL1 and IL-2 with IL-4 and IL-13Ra2 with IL-1 IRa、CH-1、CH-12、CH-23、CH-13、CH-22、CH- 4|IL-5、IL-6、and the α4α. v β3、α v β5、α v β6、α1β4、α4β1、α4β7、α5β1、α6β4、α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, 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-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein, Pseudomonas aeruginosa, rabies, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, respiratory syncytial virus, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, 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, XAGE1, or a combination thereof.

[0060] In some embodiments, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1 / CD248, TROP-2, or a combination thereof.

[0061] In some embodiments, Bm is an antibody, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, balantumab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13 / 5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, or veltuzumab. In certain aspects, the antibody is rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, or gemtuzumab.

[0062] In certain aspects, the present disclosure provides a method for preparing a conjugate of formula (I) from a compound of formula (I-1), wherein: A is phenyl; U is NH, R 1 But it's a halo, X is -N(R 2 ) v (CH2) m O(CH2) n - in which v is 1, m and n are 2; R 2 is methyl.

[0063] In some embodiments, A is phenyl; U is NH, R 1 But it's a halo, X is -N(R 2 ) v(CH2) m O(CH2) n - in which v is 2, m and n are 2; Each R 2 is methyl.

[0064] In certain embodiments, A is phenyl; U is NH, R 1 But it's a halo, X is -O(CH2) n - in which n is 2.

[0065] In certain embodiments, A is phenyl; U is NH, R 1 But it's a halo, X is -S(CH2) n - in which n is 2.

[0066] In some embodiments, A is phenyl; U is NH, R 1 is hydrogen, X is --NR 2 - in which R 2 is methyl.

[0067] In some embodiments, A is phenyl; U is NH, R 1 But it's a halo, X is --NR 2 - in which R 2 is hydrogen.

[0068] In certain embodiments, A is phenyl; U is NH, R 1 is hydrogen, X is -C(CH3)=

[0069] In some embodiments, A is C4-C 10 is a cycloalkyl ring, U is NH, R 1 is hydrogen, X is -N(R 2 )(CH2) m O(CH2) n - in which n is 1, m is 2, R 2 is methyl.

[0070] In some embodiments, the compound of formula (I-1) is [ka] is. [Brief explanation of the drawings]

[0071] [Figure 1] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the BT-474 cell line. The X-axis represents the antibody concentration log (M). The Y-axis represents the % survival of BT-474 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (triangles, solid line), trastuzumab alone (triangles, dotted line), Kadcyla (diamonds), neoDegrader P1 alone (crosses), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (circles). L is the linker. [Figure 2]1 illustrates the in vitro activity of representative neoDegrader conjugates against the BT-474 cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of BT-474 cells when treated with Pertuzumab-L-P1 (e.g., Pertuzumab-Compound (Ia)) (triangles, solid line), Pertuzumab alone (triangles, dotted line), Kadcyla (diamonds), neoDegrader P1 alone (crosses), and Rituximab-L-P1 (e.g., Rituximab-Compound (Ia)) (circles). [Figure 3] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the BT-474 cancer cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of BT-474 cells when treated with trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)) (triangles, solid line), trastuzumab (triangles, dotted line), Kadcyla (diamonds), neoDegrader P4 alone (crosses), and rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circles). [Figure 4] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the BT-474 cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of BT-474 cells when treated with pertuzumab-L-P4 (e.g., pertuzumab-compound (Ic)) (triangles, solid line), pertuzumab (triangles, dotted line), Kadcyla (diamonds), neoDegrader P4 alone (crosses), and rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circles). [Figure 5]1 illustrates the in vitro activity of representative neoDegrader conjugates at varying drug:antibody ratios (DAR) against the BT-474 cell line. The X-axis shows antibody concentration log (M). The Y-axis shows the DAR of trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) at 1.6 (upward triangles, solid line), trastuzumab-L-P3 (e.g., trastuzumab-compound (Ib)) at 1.5 (downward triangles, solid line), trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)) at 1.6 (circles, solid line), trastuzumab-L-P1 (e.g., trastuzumab-compound (Id)) at 1.6 (circles, solid line). Figure 1 shows the % survival of BT-474 cells upon treatment with trastuzumab-L-P1 (e.g., trastuzumab-compound (Id)) DAR1.6 (squares, solid line), trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) DAR8 (triangles, dotted line), trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)) DAR8 (filled circles, dotted line), Enhertu® (diamonds, dotted line), and trastuzumab (circles, dotted line). [Figure 6] 1 illustrates the in vitro activity of representative neoDegraders against the BT-474 cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of BT-474 cells when treated with Pertuzumab-L-P1 (e.g., Pertuzumab-Compound (Ia)) DAR8 (upward triangles, solid line), Pertuzumab-L-P4 (e.g., Pertuzumab-Compound (Ic)) DAR8 (downward triangles, solid line), and Enhertz® (diamonds, dotted line). [Figure 7] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the SK-BR-3 cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of SK-BR-3 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (triangles, solid line), trastuzumab (triangles, dotted line), Kadcyla (diamonds), neoDegrader P1 alone (circles), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (crosses). [Figure 8]1 illustrates the in vitro activity of representative neoDegrader conjugates against the SK-BR-3 cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of SK-BR-3 cells when treated with Pertuzumab-L-P1 (e.g., Pertuzumab-Compound (Ia)) (triangles, solid line), Pertuzumab (triangles, dotted line), Kadcyla (diamonds), neoDegrader P1 alone (circles), and Rituximab-L-P1 (e.g., Rituximab-Compound (Ia)) (crosses). [Figure 9] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the HL-60 cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of HL-60 cells when treated with OR000213-L-P1 (e.g., OR000213-Compound (Ia)) (triangles), Mylotarg® (diamonds), and trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (circles). [Figure 10] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the HL-60 cell line. The X-axis shows antibody concentration log (M). The Y-axis shows the DAR8 (upward-pointing filled triangle, solid line) of huMy9-6(IgG1)-L-P1 (e.g., huMy9-6(IgG1)-Compound (Ia)), huMy9-6(IgG1)-L-P1 (e.g., huMy9-6(IgG1)-Compound (Id)), and lintuzumab IgG1-L-P1 (e.g., lintuzumab IgG1-Compound (Ia)). Figure 1 shows the % survival of HL-60 cells upon treatment with rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circles, dotted line), lintuzumab IgG1-L-P1 (e.g., lintuzumab IgG1-compound (Id)) DAR8 (downward-pointing open triangles, dotted line), OR000213-L-P1 (e.g., OR000213-compound (Ia)) DAR8 (squares), and rituximab-L-P4 (e.g., rituximab-compound (Ic)) (circles, dotted line). [Figure 11]1 illustrates the in vitro activity of conjugates of compound (Ia) at varying drug:antibody ratios (DAR) against the HL60 cell line. The X-axis represents the antibody concentration log (M). The Y-axis shows the % survival of HL-60 cells when treated with huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1-Compound (Ia)) DAR of 1.9 (upward triangles, solid line), huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1-Compound (Ia)) DAR of 3.9 (downward triangles, solid line), huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1-Compound (Ia)) DAR of 5.5 (diamonds, solid line), huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1-Compound (Ia)) DAR of 8 (squares, solid line), and rituximab-L-P4 (e.g., rituximab-Compound (Ic)) (circles, dotted line). [Figure 12] 1 illustrates the in vitro activity of conjugates of compound (Ia) at varying drug:antibody ratios (DAR) against the HL60 cell line. The X-axis represents the antibody concentration log (M). The Y-axis shows the % survival of HL-60 cells when treated with OR000213-L-P1 (e.g., OR000213-Compound (Ia)) DAR1.2 (upward triangles, solid line), OR000213-L-P1 (e.g., OR000213-Compound (Ia)) DAR1.8 (downward triangles, solid line), OR000213-L-P1 (e.g., OR000213-Compound (Ia)) DAR2.3 (diamonds, solid line), OR000213-L-P1 (e.g., OR000213-Compound (Ia)) DAR8 (squares, solid line), and Rituximab-L-P4 (e.g., Rituximab-Compound (Ic)) (triangles, dotted line). [Figure 13]1 illustrates the in vitro activity of representative neoDegrader conjugates against the Ramos cell line. The X-axis shows the antibody concentration log (M), and the Y-axis shows % Ramos cell viability when treated with rituximab-L-P4 (e.g., rituximab-compound (Ic)) (upward-pointing triangle, solid line), rituximab-L-P1 (e.g., rituximab-compound (Ia)) (downward-pointing triangle, solid line), rituximab (triangle, dotted line), neoDegrader P1 alone (cross, dotted line), neoDegrader P4 alone (star, dotted line), and trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (circle, dotted line). [Figure 14] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the Daudi cell line. The X-axis shows the antibody concentration log (M), and the Y-axis shows the % survival of Daudi cells when treated with rituximab-L-P1 (e.g., rituximab-compound (Ia)) (upward-pointing triangles, solid line), rituximab (triangles, dotted line), and trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (circles, dotted line). [Figure 15] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the Ramos cell line. The X-axis shows the antibody concentration log (M), and the Y-axis shows the % survival of Ramos cells when treated with rituximab-L-P4 (e.g., rituximab-compound (Ic)) (upward triangles, solid line), rituximab-L-P1 (e.g., rituximab-compound (Ia)) (downward triangles, solid line), and neoDegrader P1 alone. [Figure 16] 1 illustrates the in vitro performance of representative neoDegrader conjugates on the NCI-N87 cancer cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of NCI-N87 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (triangles, solid line), trastuzumab (triangles, dotted line), Kadcyla (diamonds), neoDegrader P4 alone (crosses), and rituximab-L-P1 (e.g., rituximab-compound (Ia)) (circles). [Figure 17]1 illustrates the in vitro activity of representative neoDegrader conjugates against the NCI-N87 cancer cell line. The X-axis shows the antibody concentration log (M). The Y-axis shows the % survival of NCI-N87 when treated with Pertuzumab-L-P1 (e.g., Pertuzumab-Compound (Ia)) (triangles, solid line), Pertuzumab (triangles, dotted line), Kadcyla (diamonds), neoDegrader P1 alone (crosses), and Rituximab-L-P1 (e.g., Rituximab-Compound (Ia)) (circles). [Figure 18] 1 illustrates the in vitro activity of representative neoDegrader conjugates against the BT-474 cell line after incubation with human serum for 3 days. The X-axis shows the antibody concentration log (M). The Y-axis shows the antibody concentration log (M) in human serum (upward triangles, solid line), pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (human serum) (downward triangles, solid line), OR000213-L-P1 (e.g., OR000213-compound (Ia)) (human serum) (circles, solid line), human serum only ( Figure 1 shows the % survival of BT-474 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (upward pointing triangle, dotted line), pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (downward pointing triangle, dotted line), and OR000213-L-P1 (e.g., OR000213-compound (Ia)) (circle, dotted line). [Figure 19]1 illustrates the in vitro activity of representative neoDegrader conjugates against the BT-474 cell line after incubation with mouse serum for 3 days. The X-axis shows the antibody concentration log (M). The Y-axis shows the antibody concentration log (M) of trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (mouse serum) (upward triangles, solid line), pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (mouse serum) (downward triangles, solid line), OR000213-L-P1 (e.g., OR000213-compound (Ia)) (mouse serum) (circles, solid line), and mouse serum. Figure 1 shows the % survival of BT-474 cells when treated with trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (upward pointing triangle, dotted line), pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (downward pointing triangle, dotted line), and OR000213-L-P1 (e.g., OR000213-compound (Ia)) (circle, dotted line). [Figure 20] 1 illustrates the in vitro activity of representative neoDegrader conjugates against BT-474 (Her2+) tumors in mice. The X-axis indicates the day after dosing. The Y-axis indicates tumor volume (mm3) after dosing with vehicle (filled circles), 5 mg / kg trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (squares), 5 mg / kg rituximab-L-P1 (e.g., rituximab-compound (Ia)) (triangles), and 5 mg / kg pertuzumab-L-P1 (e.g., pertuzumab-compound (Ia)) (open circles). [Figure 21] 1 illustrates the in vitro activity of representative neoDegrader conjugates against Daudi (CD20+) tumors. The X-axis represents the day after dosing. The Y-axis represents tumor volume (mm3) after dosing with vehicle (filled circles), 5 mg / kg trastuzumab-L-P1 (e.g., trastuzumab-compound (Ia)) (squares), 1 mg / kg rituximab-L-P1 (e.g., rituximab-compound (Ia)) (triangles), and 5 mg / kg rituximab-L-P1 (e.g., rituximab-compound (Ia)) (open circles). [Figure 22]1 illustrates the in vitro activity of representative neoDegrader conjugates against HL-60 (CD33+) tumors. The X-axis represents the day after dosing. The Y-axis represents tumor volume (mm3) after dosing with vehicle (filled circles), 5 mg / kg trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (squares), 1 mg / kg OR000213-L-P1 (e.g., OR000213-Compound (Ia)) (triangles), and 5 mg / kg OR000213-L-P1 (e.g., OR000213-Compound (Ia)) (open circles). [Figure 23] 1 illustrates the in vitro activity of neoDegrader conjugates against the HCC2157 cell line. The X-axis shows the antibody concentration log (M), and the Y-axis shows the % survival of HCC2157 cells when treated with sacituzumab-L-P1 (e.g., sacituzumab-compound (Ia)) (line 1), sacituzumab alone (line 2), and neoDegrader P1 alone (line 3). [Figure 24] 1 illustrates the in vitro activity of neoDegrader conjugates against the LP1 cell line. The X-axis shows the antibody concentration log (M), and the Y-axis shows the % survival of LP1 cells when treated with HuAT 13 / 5-L-P1 (e.g., HuAT 13 / 5-Compound (Ia)) (line 1), HuAT 13 / 5 alone (line 2), and neoDegrader P1 alone (line 3). [Figure 25] 1 illustrates the in vitro activity of representative neoDegrader conjugates against NCI-H929 (CD38+) tumors. The X-axis indicates the day after dosing. The Y-axis indicates tumor volume (mm3) after dosing with vehicle (circles) or 5 mg / kg of HuAT13 / 5-L-P1 (e.g., HuAT13 / 5-Compound (Ia)) (squares). DETAILED DESCRIPTION OF THE INVENTION

[0072] The present disclosure provides a conjugate of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 10, A is phenyl or C4-C 10 is a cycloalkyl ring, R 1 is independently selected from hydrogen and halo; U is selected from NH and CF; X is -N(R 2 ) V -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from: Q and Q' are each independently O, S, or N(R 2 ) V and v is 1 or 2, Each R 2 are independently hydrogen or C1-C6 alkyl; n is an integer from 1 to 6, m is an integer from 2 to 6, where the left side of each group is bonded to L and the right side is bonded to A, However, X is NH or -Q-(CH2) n -When R 1 is a halo, L is a cleavable or non-cleavable linker; Bm is a binding moiety capable of specifically binding to the protein. In some embodiments, the binding moiety is an antibody, an antibody fragment, or an antibody-binding fragment.

[0073] The present disclosure also provides the above compounds fused to a binding moiety, compositions comprising the compounds or conjugates, or methods of using or making the compounds or conjugates.

[0074] I definition In order that this specification may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0075] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a guidepost for the use of exclusive terms such as "solely," "only," or "negative" limitations with regard to the recitation of claim elements.

[0076] Furthermore, as used herein, "and / or" is understood to specifically disclose each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0077] Wherever embodiments are described herein using the word "comprising," it should be understood that otherwise similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, e.g., Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology,3rd ed.,1999,Academic Press, and Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide those of skill in the art with a general dictionary of many of the terms used in this disclosure.

[0079] Units, prefixes and symbols are defined in the Systeme International de Numerical ranges are expressed in the format accepted by SI Units. Numerical ranges are intended to be inclusive of the numbers defining the range. When a range of values ​​is recited, it is understood that each intermediate integer and fractional value between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may independently be included or excluded within the range, and ranges including either one of these limits, excluding any limit, or including both of these limits are included within the disclosure. Thus, ranges recited herein are understood to be shorthand notations for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0080] When a value is explicitly recited, it is understood that values ​​that are approximately the same quantity or amount as the recited value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded alone or in any combination with other alternatives are also disclosed herein. More than one element of the present disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0081] As used herein, the term "DAR" refers to the drug-antibody ratio of a conjugate, which is the average number of neoDegrader-linker complexes linked to each antibody. In certain embodiments, the DAR of the conjugates described herein is 1-10. In some embodiments, the DAR of the conjugates described herein is 1-8. In some embodiments, 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, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.

[0082] The term "antibody," as used herein, refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to a target antigen or portion thereof of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulin can be derived from any species. However, in one aspect, the immunoglobulin is of human, murine, or rabbit origin.

[0083] 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. Single body antibodies can be based on the heavy chain variable domain or the light chain. Examples of single domain antibodies include V H H fragment and V fragment NAR These include, but are not limited to, fragments.

[0084] An "antibody fragment" comprises a portion of an intact antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), and epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0085] An "intact antibody" is one which comprises an antigen-binding variable domain as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0086] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody within the population is identical, excluding the possibility of natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by hybridoma or recombinant DNA methods. "Monoclonal antibodies" may also be isolated from phage antibody libraries.

[0087] As used herein, monoclonal antibodies specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited, as well as fragments of such antibodies. Chimeric antibodies of interest include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0088] Various methods are used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a clonal cell line that produces a single type of antibody, uses cells from various species, including mice (murine), hamsters, rats, and humans. Another method for preparing MAbs uses genetic engineering, including recombinant DNA techniques. Monoclonal antibodies produced from these techniques include, among others, chimeric and humanized antibodies. Chimeric antibodies combine DNA encoding regions from more than one species. For example, a chimeric antibody may derive its variable region from a mouse and its constant region from a human. Humanized antibodies, despite containing nonhuman portions, are primarily human-derived. Like chimeric antibodies, humanized antibodies may contain fully human constant regions. However, unlike chimeric antibodies, the variable regions may be partially human-derived. The nonhuman synthetic portions of humanized antibodies are often derived from the CDRs of murine antibodies. In either case, these regions are important for enabling the antibody to recognize and bind to a specific antigen. While mouse antibodies are useful for diagnosis and short-term therapy, they cannot be administered to humans over the long term without increasing the risk of a harmful immunogenic response. This response, called human anti-mouse antibody (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and attacks it. The HAMA response can lead to toxic shock or even death.

[0089] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portion of the administered antibody. Furthermore, chimeric and humanized antibodies may have the added benefit of activating secondary human immune responses, such as antibody-dependent cellular cytotoxicity.

[0090] An intact antibody may possess one or more "effector functions," which refer to biological activities attributable to the Fc region of an antibody (a native-sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B-cell receptor, BCR), and the like.

[0091] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes." There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0092] The term "about" is used herein to mean approximately, roughly, in the region of, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify numerical values ​​above and below (high or low), for example, by a variance of 10%.

[0093] The terms "administration," "administering," and grammatical variations thereof refer to the introduction of a composition, such as an EV (e.g., exosome) of the present disclosure, into a subject via a pharmaceutically acceptable route. Introduction of a composition, such as an EV (e.g., exosome) of the present disclosure, into a subject may be by any suitable route, including intratumoral, oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical. Administration includes self-administration and administration by another person. A suitable route of administration enables 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 the subject's vein.

[0094] As used herein, the term "antibody" encompasses immunoglobulins and fragments thereof, whether natural or partially or wholly synthetically produced. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" further encompasses polypeptides comprising a framework region from an immunoglobulin gene or fragments thereof that specifically bind and recognize an antigen. The use of the term antibody is meant to encompass whole, polyclonal, monoclonal, and recombinant antibodies, fragments thereof, as well as single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, and 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, diabodies, and antibody-related polypeptides, so long as the antibody exhibits the desired biological activity or function. In some aspects of the present disclosure, the biologically active molecule is an antibody, or a molecule comprising an antigen-binding fragment thereof.

[0095] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to an antibody linked, e.g., covalently, to a therapeutic agent (sometimes referred to herein as a drug, medication, or active pharmaceutical ingredient) or agent. In some aspects of the present disclosure, the biologically active molecule is an antibody-drug conjugate.

[0096] As used herein, when applied to one or more values ​​of interest, the term "approximately" refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from the context, the term "approximately" refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of (greater than or less than) the stated reference value (except where such number exceeds 100% of possible values).

[0097] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid residue families with similar side chains have been defined in the art and include amino acids with 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0098] As used herein, the term "conserved" refers to nucleotide or amino acid residues of a polynucleotide or polypeptide sequence, respectively, that occur unaltered at the same position in two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are more conserved between related sequences than nucleotides or amino acids that appear elsewhere in the sequences.

[0099] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to one another. In some embodiments, two or more sequences are said to be "conserved" if they 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 one another. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, regions, or features thereof.

[0100] As used herein, the terms "linking" and "conjugating" are used interchangeably and refer to the covalent or non-covalent attachment of two or more moieties, including a neoDegrader and a binding moiety, respectively. In some embodiments, linking or conjugating can include a linker.

[0101] 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, an amino acid sequence variant will retain at least about 70% sequence identity with at least one receptor-binding domain of a native antibody or at least one ligand-binding domain of a native receptor, and will typically be at least about 80%, more typically at least about 90%, homologous in sequence to such receptor or ligand-binding domain. 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 designated by conventional names, one-letter, and three-letter codes.

[0102] "Sequence identity" is defined as the percentage of residues of amino acid sequence variants that are identical 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," developed by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991.

[0103] 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. Furthermore, FcRs may bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus.

[0104] "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 (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed.

[0105] "Native antibodies" are typically 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, while the number of disulfide bonds varies among the 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 several constant domains. Each light chain has a variable domain (VL) at one end followed by a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0106] The term "variable" refers to the fact that certain portions of the variable domains vary significantly in sequence among antibodies and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each adopt a primarily β-sheet configuration and contain four FRs connected by three hypervariable regions, forming loops that connect to, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not directly involved in binding the antibody to the antigen but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0107] The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody responsible for antigen binding. A hypervariable region generally comprises amino acid residues from the "complementarity-determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy-chain variable domain; Kabat et al., supra) and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chain variable domain and 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 as defined herein.

[0108] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and an "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0109] An "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0110] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 ​​residue(s) of the constant domains bear 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 couplings of antibody fragments are also known.

[0111] 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.

[0112] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0113] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a variable heavy domain (VH) connected to a variable light domain (VL) 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 can be paired with the complementary domains of another chain to create two antigen-binding sites.

[0114] "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 for transferring murine antigen-binding information into a non-immunogenic human antibody acceptor, resulting in numerous therapeutically useful drugs. The humanization process generally begins by transferring all six murine complementarity-determining regions (CDRs) into a human antibody framework. Antibodies with these CDRs grafted generally do not retain their original affinity for antigen binding; in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues must also be incorporated to maintain proper CDR configuration. Transferring key murine framework residues into the human acceptor to support the structural orientation of the grafted CDRs has been shown to restore antigen binding and affinity. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0115] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and these may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody is purified to (1) greater than 95% by weight, or greater than 99% by weight, as determined by the Lowry method; (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas-phase protein sequencer; or (3) homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0116] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream. As used herein, "cancer" refers to primary, metastatic, and recurrent cancers.

[0117] As used herein, the term "immune response" refers to a biological response in a vertebrate to foreign substances, which protects the organism from these substances and the diseases they cause. 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 produced either by these cells or the liver (including antibodies, cytokines, and complement), which result in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body, invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., CD4 + or CD8 + These include activation or inhibition of effector T cells, such as T cells, or Th cells, or inhibition of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are synonymous and refer to any lymphocyte produced or processed by the thymus. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a NKT cell.

[0118] A "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 embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0119] The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent (e.g., a neoDegrader or neoDegrader conjugate disclosed herein) that provides a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, amelioration, alleviation, reduction, delay, and / or alleviation of signs, symptoms, or pathogenesis of a disease, or any other desired alteration of a biological system. In the case of a solid tumor, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of the composition may (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, delay, slow, or stop, to some extent, the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow or stop, to some extent) the metastasis of tumors; (v) inhibit the growth of tumors; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) relieve, to some extent, one or more symptoms associated with cancer.

[0120] In some embodiments, a "therapeutically effective amount" is an amount of a neoDegrader or neoDegrader conjugate that has been clinically proven to affect a significant reduction in cancer, such as an advanced solid tumor, or a slowing of cancer progression (regression). The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0121] As used herein, the term "standard of care" refers to a treatment that is accepted by medical professionals as an appropriate treatment for a particular type of disease and is widely used by medical professionals. This term may be used interchangeably with any of the terms "best practice," "standard of care," and "standard therapy."

[0122] By way of example, an "anti-cancer drug" promotes the regression of cancer or prevents further tumor growth in a subject. In certain embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer.

[0123] The terms "effective" and "effectiveness" in reference to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organism level resulting from the administration of the drug.

[0124] 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 costimulatory or inhibitory interactions in T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0125] The term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) the development or spread of an undesirable physiological change or disorder, such as cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, palliation or remission of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0126] II. neoDegrader The present disclosure provides a neoDegrader of formula (II) [ka] or a pharmaceutically acceptable salt thereof, A is phenyl or C4-C 10 is a cycloalkyl ring, U is selected from NH and CF; R 1 is independently selected from hydrogen and halo; R 2 But -C(O)R 3 , -N(R 4 )2, -(CH2) n OH, -(CH2) n SH, -(CH2) n N(R 4 )2, -(CH2) n Q'(CH2) m OH, -(CH2) n Q'(CH2) m SH, and -(CH2) n Q'(CH2) m N(R 4)2, wherein: R 3 is hydrogen or C1-C6 alkyl; Each R 4 are independently hydrogen or C1-C6 alkyl; Q' is O, S, or NR 4 and n is 1 to 6; m is 2 to 5; However, R 2 NH2, -(CH2) n NH2, or -(CH2) n If OH, then R 1 is a halo.

[0127] In certain aspects, the present disclosure provides a compound of formula (II) or a pharmaceutically acceptable salt thereof: A is a phenyl ring or a C4-C 10 is a cycloalkyl ring, U is NH, R 1 is selected from hydrogen and halo; R 2 But -(CH2) n Q'(CH2) m N(R 4 )2, -(CH2) n OH, -(CH2) n SH, -N(R 4 )2, and -C(O)R 3 is selected from, where m is 2, n is 2, Q' is -O-; R 3 is methyl, Each R 4 is independently selected from hydrogen and halo; However, R 2 NH2 or -(CH2) n If OH, then R 1 is a halo.

[0128] As used herein, the term "C1-C6 alkoxy," as used herein, refers to a C1-C6 alkyl group attached to the parent molecular moiety through an oxygen atom.

[0129] The term "C1-C6 alkoxy C1-C6 alkyl," as used herein, refers to a C1-C6 alkoxy group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0130] As used herein, the term "C1-C6 alkyl" refers to a group derived from a straight or branched chain saturated hydrocarbon containing from 1 to 6 carbon atoms.

[0131] As used herein, "C4-C 10 The term "cycloalkyl" refers to a saturated monocyclic hydrocarbon ring system having from 4 to 10 carbon atoms and 0 heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups containing from 7 to 10 atoms can be monocyclic, fused, spirocyclic, or bridged bicyclic structures.

[0132] As used herein, the term "halo" refers to F, Cl, Br, or I.

[0133] In some embodiments, the neoDegrader of formula (II) is selected from the group consisting of: [ka]

[0134] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0135] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0136] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0137] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0138] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0139] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0140] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0141] In some embodiments, the neoDegrader of formula (II) is: [ka]

[0142] In some aspects, the disclosure provides a neoDegrader of formula (II) or a pharmaceutically acceptable salt thereof, wherein A is phenyl, U is NH, and R 1 is the halo, and R 2 Ga-(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is O, and one R 4 is hydrogen and the other is methyl.

[0143] In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is phenyl, U is NH, and R 1 is the halo, and R 2 Ga-(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is O, and each R 4 is methyl.

[0144] In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is phenyl, U is NH, and R 1 is the halo, and R 2 Ga-(CH2) n OH, where n is 2.

[0145] In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is phenyl, U is NH, and R 1 is the halo, and R 2 Ga-(CH2) n SH, where n is 2.

[0146] In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is phenyl, U is NH, and R 1 is hydrogen and R 2 -N(R 4 )2, where one R 4is hydrogen and the other is methyl.

[0147] In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is phenyl, U is NH, and R 1 is the halo, and R 2 -N(R 4 )2, where each R 4 In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is phenyl and R 1 is hydrogen and R 2 -C(O)R 3 where R 3 is methyl.

[0148] In some embodiments, the present disclosure provides a neoDegrader of formula (II), wherein A is C4-C 10 is a cycloalkyl ring, U is NH, and R 1 is hydrogen and R 2 Ga-(CH2) n Q'(CH2) m N(R 4 )2, where m and n are 2, Q' is O, and one R 4 is hydrogen and the other is methyl.

[0149] III. neoDegrader conjugates The present disclosure provides conjugates and binding moieties of one or more of the neoDegraders disclosed herein. These conjugates degrade proteins by binding to cereblon (CRBN) and CRL4. CRBN They can promote the recruitment and ubiquitination of substrate proteins mediated by E3 ubiquitin ligases. These agents act as "molecular glue" and fill the binding interface as a hydrophobic patch that reprograms protein interactions between the ligase and the new substrate.

[0150] In some aspects, the present disclosure provides a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein: a is an integer from 1 to 10, A is phenyl or C4-C 10 is a cycloalkyl ring, R 1 is selected from hydrogen and halo; U is selected from NH and CF; X is -NR 2 -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from: Q and Q' are each independently O, S, or NR 2 and R 2 is hydrogen or C1-C6 alkyl; n is an integer from 1 to 6, m is an integer from 2 to 6, where the left side of each group is bonded to L and the right side is bonded to A, However, X is NH or -Q-(CH2) n -When R 1 is a halo, L is a cleavable or non-cleavable linker; Bm is the binding moiety.

[0151] In some embodiments, U is NH.

[0152] In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against tumor cell lines. In some embodiments, a neoDegrader conjugate comprising a neoDegrader and a binding moiety has 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% greater in vitro antiproliferative activity than the neoDegrader alone or the binding moiety alone. In some embodiments, a neoDegrader conjugate comprising a neoDegrader and a binding moiety has at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater in vitro antiproliferative activity than the neoDegrader alone or the binding moiety alone.

[0153] In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against the BT-474 breast cancer cell line, e.g., greater antiproliferative activity against the BT-474 breast cancer cell line, compared to the neoDegrader alone or the binding moiety alone. In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against the SK-BR-3 breast cancer cell line, e.g., greater antiproliferative activity against the SK-BR-3 breast cancer cell line, compared to the neoDegrader alone or the binding moiety alone. In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against the NCI-N87 gastric cancer cell line, e.g., greater antiproliferative activity against the NCI-N87 gastric cancer cell line, compared to the neoDegrader alone or the binding moiety alone. In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against a Daudi lymphoma cell line, e.g., higher antiproliferative activity against a Daudi lymphoma cell line, compared to the neoDegrader alone or the binding moiety alone. In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against an HL-60 acute myeloid leukemia cell line, e.g., higher antiproliferative activity against the HL-60 acute myeloid leukemia cell line, compared to the neoDegrader alone or the binding moiety alone. In some embodiments, the neoDegrader conjugates described herein have in vitro antiproliferative activity against a Ramos non-Hodgkin's lymphoma cell line, e.g., higher antiproliferative activity against the Ramos non-Hodgkin's lymphoma cell line, compared to the neoDegrader alone or the binding moiety alone. In some embodiments, the neoDegrader conjugates described herein can maintain their antiproliferative activity in the presence of human serum. The neoDegrader conjugates described herein can be used to treat cancer.

[0154] III.A. Linkers The neoDegraders of the present disclosure can be linked to a binding moiety via a linker. As used herein, the term "linker" refers to any chemical moiety that can connect a binding moiety (Bm) to the X group in a compound of formula (I).

[0155] In certain embodiments, 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 moiety. A heterobifunctional group is characterized by having different reactive groups at both ends of the chemical moiety. Conjugation to "Bm" can be achieved by chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the binding moiety with reactive handles on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via unnatural amino acids incorporated by genetic engineering, where unnatural amino acid residues bearing the desired reactive handles are installed on "Bm." In enzymatic conjugation, an enzyme mediates the coupling of the linker to accessible amino residues on the binding moiety. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical and enzymatic conjugation may also be used sequentially, for example, enzymatic conjugation may be used to place a unique reactive handle on "B m" that is utilized in subsequent chemical conjugation.

[0156] In some aspects, the heterobifunctional group is selected from: [ka] During the ceremony, [ka] is the point of attachment to the remainder of the linker, [ka] It is the attachment point to Bm.

[0157] In certain embodiments, the linker "L" is not cleavable. As used herein, the term "non-cleavable linker" refers to any chemical moiety that is capable of linking a binding site to a neoDegrader in a stable, covalent manner and does not fall into the category defined herein as a "cleavable linker." Thus, a non-cleavable linker is substantially resistant to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. "Substantially resistant to cleavage" means that in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of a population of antibody-neoDegrader conjugates, the chemical bond within or adjacent to the linker remains non-cleavable by acids, photolabile cleavage agents, bioreductive agents, peptidases, esterases, or chemical or physiological compounds that cleave chemical bonds (e.g., disulfide bonds) within a cleavable linker within hours to days of treatment with any of the above-mentioned agents. In certain embodiments, the linker is not susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc., under conditions that allow the neoDegrader and / or binding moiety to remain active. ADC catabolic products generated from non-cleavable linkers contain residual amino acids from the antibody. These catabolic products may exert unique and unexpected properties in target cells to which they are delivered.

[0158] Those skilled in the art will readily distinguish between non-cleavable and cleavable linkers.

[0159] Examples of non-cleavable linkers include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linker, succinimide thioether linker, and linkers such as: [ka] p is an integer from 1 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0160] In some embodiments, the linker is [ka] is. In some embodiments, p is 5.

[0161] In certain embodiments, the linker may be cleavable. In some embodiments, the linker may be susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc., under conditions that allow the neoDegrader and / or binding moiety to remain active.

[0162] In some embodiments, the cleavable linker can be enzymatically cleaved by a protease, peptidase, esterase, beta-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase.

[0163] In some embodiments, the cleavable linker can be cleaved by a protease, examples of which include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like.

[0164] In certain embodiments, the cleavable linker contains a peptide. In some embodiments, the peptide is the cleavage site of the linker, thereby facilitating release of the drug upon exposure to intracellular proteases, such as lysosomal enzymes. Peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases. Examples of peptides having two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides having three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine-alanine-asparagine (ala-ala-asn), alanine-phenylalanine-lysine (ala-phe-lys), glycine-glycine-phenylalanine (gly-gly-phe), and glycine-glycine-glycine (gly-gly-gly). Examples of peptides having four amino acids include, but are not limited to, glycine-glycine-valine-citrulline (gly-gly-val-cit) and glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly). The above amino acid combinations can also occur in the reverse order (i.e., cit-val).

[0165] The peptides of the present disclosure may include L- or D-isomers of amino acid residues. The term "naturally occurring amino acid" refers to Ala, Asp, Asx, Cit, Cys, Glu, Phe, Glx, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr. "D-" denotes an amino acid having a "D" (dextrorotatory) configuration, as opposed to the configuration in naturally occurring ("L-") amino acids. The amino acids described herein may be obtained commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0166] In certain embodiments, the linker (“L”) is a protease-cleavable linker selected from: [ka] During the ceremony, q is an integer from 2 to 10, Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue in the L- or D-configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0167] In certain embodiments, Z 1 , Z 2 , Z 3 , and Z 4is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 At least two of the residues are amino acid residues.

[0168] In some embodiments, Z 1 is absent or is glycine, and Z 2 is absent or selected from L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 is selected from L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4 is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0169] In some embodiments, L is [ka] is.

[0170] In some embodiments, q is 5.

[0171] In certain embodiments, L is a pyrophosphatase-cleavable linker.

[0172] In some embodiments, L is a pyrophosphatase-cleavable linker; [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0173] In certain embodiments, L is a beta-glucorunidase cleavable linker.

[0174] In some embodiments, L is a beta-glucoronidase cleavable linker selected from: [ka] During the ceremony, q is an integer from 2 to 10, ---- is absent or is a bond, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0175] In some embodiments, the linker is bioreductive. Bioreductive linkers take advantage of the difference in reduction potential between intracellular compartments and plasma. The reduced glutathione present in the cytoplasm of tumor cells is up to 1000 times higher than that present in the cytoplasm of normal cells, and tumor cells also contain enzymes that can contribute to reduction in the cellular compartment. The linker keeps the conjugate intact in the systemic circulation and is selectively cleaved by the high intracellular concentration of glutathione, releasing the active drug from the non-toxic prodrug at the tumor site.

[0176] In some embodiments, L is a bioreducible linker selected from: [ka] During the ceremony, q is an integer from 2 to 10, R, R', R", and R'" are each independently selected from hydrogen, C-C alkoxyC-C alkyl, (C-C) NC-C alkyl, and C-C alkyl, or two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring; [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0177] In certain embodiments, the linker is acid-cleavable. Acid-cleavable linkers are specifically designed to remain stable at the neutral pH of the blood circulation, but undergo hydrolysis to release the cytotoxic drug in the acidic environment of the cellular compartment.

[0178] In some embodiments, L is an acid-cleavable linker selected from: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0179] In certain embodiments, L is a click-to-release linker, where release of the neoDegrader is chemically triggered by a tetrazine or related compound.

[0180] In some embodiments, L is a click-to-release linker selected from: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X, [ka] is the point of attachment to the binding moiety.

[0181] III.B. Joining part The present disclosure provides a neoDegrader conjugated to a binding moiety. As used herein, the term "binding moiety" refers to any molecule that recognizes and binds to a cell surface marker or receptor. In certain embodiments, the binding moiety binds to a protein, including but not limited to a polypeptide moiety. In addition to targeting the neoDegrader to a specific cell, tissue, or location, the binding moiety may also have a specific therapeutic effect, such as antiproliferative (cytostatic and / or cytotoxic) activity against the target cell or pathway. In certain embodiments, the binding moiety can include, or be engineered to include, at least one chemically reactive group, such as a carboxylic acid, amine, thiol, or chemically reactive amino acid site or side chain. In some embodiments, the binding moiety can include a targeting moiety that binds to or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. After specific binding or complexing with the receptor, the cell allows uptake of the targeting moiety or neoDegrader conjugate, which is then internalized by the cell.

[0182] In some embodiments, the "Bm" group can be a moiety that can specifically bind to a cell surface molecule. In some embodiments, the "Bm" group can be a peptide or protein that binds to a cell surface receptor or antigen.

[0183] In certain embodiments, the "Bm" group can be an antibody, antibody fragment, or antigen-binding fragment. Antibodies are proteins produced by the immune system that can recognize and bind to a specific antigen. A target antigen generally has multiple binding sites, also called epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one 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, provided they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0184] Monoclonal antibodies that can be conjugated to neoDegraders are homogeneous populations of antibodies against a particular antigenic determinant (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against an antigen of interest can be prepared by using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridoma techniques, human B-cell hybridoma techniques, and EBV-hybridoma techniques. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas producing mAbs for use in the present disclosure may be cultured in vitro or in vivo.

[0185] 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 made by any of a number of techniques known in the art.

[0186] The antibody can also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where these two chains have different specificities. Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually performed using affinity chromatography steps, is quite cumbersome and results in low product yields.

[0187] According to a different approach, antibody variable domains (antibody-antigen combining sites) with the desired binding specificities are fused to immunoglobulin constant domain sequences. Fusions may be with immunoglobulin heavy chain constant domains, including at least part of the hinge, C.sub.H2, and C.sub.H3 regions. The first heavy chain constant region (C.sub.H1) may contain the site necessary for light chain binding, present in at least one of the fusions. Nucleic acids encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when expression of at least two polypeptide chains in equal ratios results in higher yields or when the ratio is not particularly critical.

[0188] Bispecific antibodies may 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 facilitates separation of the desired bispecific compound from undesired immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy method of separation. Using such techniques, bispecific antibodies can be prepared for conjugation to neoDegraders in the treatment or prevention of diseases defined herein.

[0189] Hybrid or bifunctional antibodies can be derived biologically, i.e., by cell fusion techniques, or chemically, particularly by cross-linking or disulfide bridge-forming reagents, and may comprise whole antibodies or fragments thereof.

[0190] The antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen, or a microbial antigen, or to tumor cells, or other antibodies bound to tumor cells or a matrix. In this context, "functionally active" means that the fragment, derivative, or analog is capable of eliciting anti-anti-idiotypic antibodies that recognize the same antigen as that recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in exemplary embodiments, the idiotypic antigenicity of an immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences C-terminal to the CDR sequences that specifically recognize the antigen. To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen using any binding assay method known in the art.

[0191] Other useful antibodies include antibody fragments such as, but not limited to, F(ab')2 fragments containing the variable region, light chain constant region, and CH1 domain of the heavy chain, which can be produced by pepsin digestion of the antibody molecule, and Fab fragments which can be generated by reducing the disulfide bridges of F(ab')2 fragments. Other useful antibodies are antibody heavy and light chain dimers, or any minimal fragments thereof, such as Fv or single-chain antibodies (SCAs), or any other molecules having the same specificity as antibodies.

[0192] In addition, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which contain both human and non-human portions and can be produced using standard recombinant DNA techniques, are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from a murine monoclonal and a human immunoglobulin constant region. Humanized antibodies are antibody molecules from non-human species that have one or more complementarity-determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0193] Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but that can express human heavy and light chain genes. The transgenic mice are immunized in the normal manner with a selected antigen, e.g., all or a portion of a polypeptide of the present disclosure. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such techniques, 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 are commercially available from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0194] Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries.

[0195] An antibody can be, for example, a fusion protein of an antibody, or a functionally active fragment thereof, in which the antibody is fused at either the N- or C-terminus via a covalent bond (e.g., a peptide bond) to the amino acid sequence of another protein that is not an antibody (or a portion thereof, e.g., at least a 10, 20, or 50 amino acid portion of the protein). The antibody or fragment thereof may be covalently linked to the other protein at the N-terminus of the constant domain.

[0196] Antibodies include analogs and derivatives that are modified, i.e., by the covalent attachment of any type of molecule, so long as such covalent attachment enables the antibody to retain its antigen-binding immunospecificity. For example, without limitation, antibody derivatives and analogs include those that are further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular antibody entities or other proteins, etc. Any of a number of chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, analogs or derivatives can contain one or more unnatural amino acids.

[0197] The antibodies in the neoDegrader conjugates can 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 can be obtained commercially, for example, from Genentech (San Francisco, Calif.), or can be produced by any method known to those of skill in the art, such as, for example, chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, from literature publications, or by routine cloning and sequencing.

[0198] In certain embodiments, the antibody of the neoDegrader conjugate can be a monoclonal antibody, e.g., a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody can be an antibody fragment, e.g., a Fab fragment.

[0199] Known antibodies for the treatment or prevention of cancer can be conjugated to the neoDegraders described herein. Antibodies immunospecific for cancer cell antigens can be commercially obtained or produced by any method known to those skilled in the art, such as, for example, recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, from literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; RITUXAN™ (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (oregovomab; AltaRex Corporation, MA), a murine antibody for the treatment of ovarian cancer; Panorex (edrecolomab; GlaxoWellcome, NC), an IgG sub-2a antibody for the treatment of colorectal cancer; and cetuximab erbitux (cetuximab; Imclone Systems), an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor-positive cancers such as head and neck cancer. Inc., NY); Vitaxin (etaracizumab, MedImmune, Inc., MD), a humanized antibody for the treatment of sarcoma; Compass I / H (alemtuzumab, Leukosite, MA), a humanized IgG.sub.1 antibody for the treatment of chronic lymphocytic leukemia (CLL); and Smart (alemtuzumab, Leukosite, MA), a humanized anti-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML). MI95 (Protein Design Labs, Inc., CA); LymphoCide (epratuzumab, Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID 10 (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 murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; and humanized anti-CD2 for the treatment of Hodgkin's lymphoma or non-Hodgkin's lymphoma. These include, but are not limited to, the mAb Alloimmune (BioTransplant, CA); Avastin (bevacizumab, Genentech, Inc., CA), a humanized anti-VEGF antibody for the treatment of lung and colon cancer; epratuzumab (Immunomedics, Inc., NJ and Amgen, CA), an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colon cancer.

[0200] Other antibodies useful in neoDegrader conjugates include, but are not limited to, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, daratumumab, STI-6129, lintuzumab, huMy9-6, balantamab, indatuximab, dinutuximab, anti-CD38 A2 antibody, HuAT 13 / 5 H3s antibody, ibritumomab, tositumomab, panitumumab, tremelimumab, ticilimumab, catumaxomab, and veltuzumab. In certain embodiments, the antibody is selected from the group consisting of rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, and gemtuzumab.

[0201] Other antibodies useful in neoDegrader conjugates include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha fetoprotein (carcinoma), CA 242 (colon), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colon), 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 (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma), and Neu oncogene product (carcinoma). Some particularly 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.

[0202] Other antigens that can be bound by this conjugate include 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, and CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD17 9a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, D25, CD27L, CD28, CD3, CD30, CD31, CD 300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD 56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, C D138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto1 protein, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, 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, gp1OO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24、HMWMAA、HPV E6 hTERT was about to see photos of ICAM ICOS-L IFN-α IFN- γ、IGF-I merges with IGLL1 and IL-2 with IL-4 and IL-13Ra2 with IL-1 IRa、CH-1、CH-12、CH-23、CH-13、CH-22、CH- 4|IL-5、IL-6、and the α4α. v β3、α v β5、α v β6、α1β4、α4β1、α4β7、α5β1、α6β4、α IIbincluding β3 integrin), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, legumain, Lewis Y, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, 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-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostase, prostate cancer cells, prostein, Pseudomonas aeruginosa, rabies, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutants, respiratory syncytial virus, Rh factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoints, 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 These include, but are not limited to, 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.

[0203] 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 neoDegraders.

[0204] The antibodies of the neoDegrader conjugates can bind to both receptors or receptor complexes expressed on activated lymphocytes. The receptors or receptor complexes can include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, or complement regulatory proteins. Non-limiting examples of suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / 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, osteoprotegerin, 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, CD103, and CD104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectins.

[0205] In some embodiments, antibodies that may be useful in the present disclosure include 3F8, 8H9, abagovomab, abciximab (REOPRO®), abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab (HUMIRA®), adecatumumab, aducanumab, afacevicumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH®), alirocumab (PRALUENT®), altumomab, amatuximab, anatumomab, andecaliximab, anetuximab, and antuximab. Mab, anifrolumab, anrukinzumab, apolizumab, apultumab, arcitumomab (CEA-SCAN®), asclinbacumab, acelizumab, atidortoxumab, atlizumab (tocilizumab, ACTEMRA®, ROACTEMRA®), atezolizumab (TECENTRIQ®), atinumab, atorolimumab, avelumab (Bavencio), azintuximab, balantamab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, BCD-100, Bectumomab (LYMPHOSCAN®), begelomab, belantamab, belimumab (BENLYSTA®), bemarituzumab, benralizumab (FASENRA®), bermekimab, belanlimab, bertilimumab, besilesomab (SCINITIMUN®), bevacizumab (AVASTIN®), bezlotoxumab (ZINPLAVA®), biciromab (FIBRISCINT®), bimagrumab, bimekizumab, viltamimab, bivatuzumab, bleselumab, blinatumob Mab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab, briakinumab, brodalumab (SILIQ™), brolucizumab (BEOVU®), brontixutuzumab, burosumab (CRYSVITA®), cabilalizumab, caplacizumab (CABLIVI®), camidanlumab, camrelizumab, canakinumab (ILARIS®), cantuzumab, capromab, carlumab, carotuximab, catumaxomab (REMOVAB®), cBR96, CC49,Cedelizumab, cemiplimab (LIBTAYO®), sergituzumab, certolelimab, certolizumab, cetuximab (ERBITUX®), civisatamab, cirumtuzumab, sitatuzumab, cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, cofetuzumab, coltuximab, conatumumab, concizumab, cosfrobiximab, CR6261, crenezumab, crizanlizumab (ADAKVEO®), clotedumab, cusatuzumab, dacetuzumab, daclizumab (ZINBRYTA®), dalotuzumab, dapirolizumab, daratumumab (DARZALEX®), dectrecumab, demcizumab, denintuzumab, denosimumab (PROLIA®), depatuximab, dellotuximab, detunomab, dezamizumab, dinutuximab (UNITUXIN®), zilidamab, domagrozumab, dostarlimab, dorlimomab, dorlixizumab, drozitumab, DS-8201, durigotuzumab, dupilumab (DUPIXENT®), Durba lumab (IMFINZI®), dusigtumab, ecloneximab, eculizumab (SOLIRIS®), edovacomab, edrecolomab (PANOREX®), efalizumab (RAPTIVA®), efungumab (MYCOGRAB®), eldelumab, elezanumab, elgemtumab, elotuzumab (EMPLICITI®), elsilimomab, emactuzumab, emapalumab (GAMIFANT®), emibetuzumab, emicizumab (HEMLIBRA®), trademark), enapotamab, enavatuzumab, enfortumab (PADCEV®), enlimomab, enovolituzumab, enokizumab, enoticumab, encituximab, epitumomab, eptinezumab (VYEPTI®), epratuzumab, erenumab (AIMOVIG®), erlizumab, ertumaxomab (REXOMUN®), etracizumab (ABEGRIN®), etigilimab, etorolizumab, evinacumab, evolocumab (REPATHA®), exibirumab,Fanolesomab (NEUTROSPEC®), faramiromab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, favituzumab, ficartuzumab, figitumumab, filibumab, framvotumab, fretikumab, flotetuzumab, fontolizumab (HUZAF®), foralumab, foravirumab, fremanezumab (AJOVY®), fresolimumab, frobocimab, furunevetumab, furanumab, futuximab, galcanezumab (EMGALITY®) trademark), galiximab, gancotamab, ganitumab, gantenerumab, gavilimomab, gevivumab, gemtuzumab, gevokizumab, gilvetomab, gimsilumab, girentuximab, glembatumumab, golimumab (SIMPONI®), gomiliximab, guselkumab (TREMFYA®), huMy9-6, OR000213, inalumab, ibalizumab (TROGARZO®), IBI308, ibritumomab, icrucumab, idarucizumab (PRAXBIND®), ifavotuzumab, igovomab (INDIMACIS-125), iradatuzumab, IMAB362, imalumab, imaprelimab, imciromab (MYOSCINT®), imgatuzumab, inlacumab, indatuximab, indusatumab, inebilizumab, infliximab (REMICADE®), intetumumab, inolimomab, inotuzumab, iomab-B, ipilimumab, iratumumab, isatuximab (SARCLISA®), iscalimab, istiratumab, itolizumab, ixekizumab (TALTZ®), kelik cimab, labetuzumab (CEA-CIDE™), lacunotuzumab, radilatuzumab, lampalizumab, lanadelumab (TAKHZYRO®), landgrozumab, laprituximab, ralcabiximab, lebrikizumab, remaresomab, lendalizumab, lembervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifastuzumab, ligelizumab, rilotomab, lintuzumab, lirilumab, roderucizumab, lokivetmab, loncastuximab, lorvotuzumab,Rosatuximab, lucatumumab, lurizumab, lumiliximab, lumuletuzumab, rupartumab, rutuximab, mapatumumab, margetuximab, marstacimab, maslimomab, matuzumab, mavrilimumab, mepolizumab (NUCALA®), metelimumab, milatuzumab, minletunomab, mirikizumab, mirvetuximab, mitumomab, modutuximab, moralizumab, mogamulizumab (POTELIGEO®), morolimumab, mosunetuzumab, motavizumab (NUMAX®), moxetumomab (LUMOXITI®), muromonab-CD3 (ORTHOCLONE®), OKT3®), nacolomab, namilumab, naptumomab, naratuximab, namatumab, natalizumab (TYSABRI®), nabiciquizumab, navivumab, naxitamab, nebacumab, necitumumab (PORTRAZZA®), nemolizumab, NEOD001, nerelimomab, nesbacumab, netakimab, nimotuzumab (THERACIM®), niruse Bimab, nivolumab, nofetumomab, obilutoxaximab (ANTHIM®), obinutuzumab, ocaratuzumab, ocrelizumab (OCREVUS®), odulimomab, ofatumumab (ARZERRA®), olaratumab (LARTRUVO®), oleculumab, orendalizumab, olokizumab, omalizumab (XOLAIR®), Omburtamab, OMS721, onartuzumab, ontecizumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab, oregovomab (OVAREX), olticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozogamicin, ozoralizumab, pagibaximab, palivizumab (SYNAGIS®), pamrevlumab, panitut tuzumab (VECTIBIX®), pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuximab, pateclizumab, patritumab, PDR001, pembrolizumab, pentumomab (THERAGYN®), perakizumab, pertuzumab (OMNITARG®), pexelizumab, pidilizumab, pinatuzumab, pintumomab, placumab,Polatuzumab (Polivy), prezalumab, prozalizumab, pogalizumab, ponezumab, polgabiximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140, kilimumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevetomab, ranibizumab (LUCENTIS®), ravagalimab, ravulizumab (ULTOMIRIS®), raxibacumab, refanezumab, regavirumab, REGN-EB3, lenatolumab, lemtolumab, reslizumab (CINQAIR®), rilotumumab, linucumab, risankizumab (SKYRIZI®), rituximab (RI TUXAN®), rivavazumab, rmab, lobatumumab, lorezumab, romilukimab, romosozumab (EVENITY®), lontalizumab, rosmantuzumab, rovalpituzumab, rovelizumab (LEUKARREST®), rozanolixizumab, ruplizumab (ANTOVA), SA237, sacituzumab, samalizumab, samuraizumab, sarilumab (KEVZARA®), satralizumab, satumomab pendetide, secukinumab (COSENT YX®), cericlerumab, seribantumab, cetoxaximab, setursumab, cevirumab, SGN-CD19A, SHP647, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, siltratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, STI-6129, suresomab (LEUKOSCAN®), sputabumab, stimulimab, subizumab, sutuzumab Bratoxumab, tabalumab, taczuzu (AFP-CIDE®), tabocizumab, talaxuzumab, talizumab, tamtubetumab, tanezumab, taplitumomab paptox, talexuzumab, tabolitumomab, tefibazumab (AUREXIS®), terimomab, telisotuzumab, tesidolumab, tetraxetan, tetulomab, tenatumomab, teneliximab, teprotumumab (TEPEZZA®), teplizumab, tezepelumab, TGN1412, tiburizumab,Ticilimumab (TREMELIMUMAB®), tigatuzumab, timigtumab, timolubab, tiragolumab, tilagotumab, tislelizumab, tisotumab, tiuxetan, tildrakizumab (ILUMYA®), TNX-650, tocilizumab (atlizumab, ACTEMRA®), tomzutoxin, Mab, toralizumab, tosatoxumab, tositumomab (BEXXAR®), tobetumab, tralokinumab, trastuzumab (HERCEPTIN®), TRBS07, tregalizumab, tremelizumab, trevoglumab, tucotuzumab, tuvilumab, urtoxazumab, ustekinumab (STELERA®), ublituximab, urocuplumab, urelumab, utomilumab, vadastuximab, banalimab, bundletuzumab, vanticutuzumab, vanucizumab, bapaliximab, valisacumab, valiluril These include, but are not limited to, mab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab (NUVION®), bovalilizumab, volociximab (HUMASPECT®), bonlerolizumab, bopratelimab, borsetuzumab, votumumab, bunakizumab, xentuzumab, XMAB-5574, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), zatuximab, zenoctuzumab, diralimumab, zolbetuximab, or zolimomab.

[0206] An antibody that "binds with" a molecular target or antigen of interest is one that is able to bind to that antigen with sufficient affinity so that the antibody is useful in targeting cells that express the antigen.

[0207] In the present disclosure, a "Bm" group can be conjugated to more than one neoDegrader. In some embodiments, a "Bm" can be conjugated to 1 to 10 neoDegraders. In some embodiments, a "Bm" can be conjugated to 1 to 9 neoDegraders. In some embodiments, a "Bm" can be conjugated to 1 to 8 neoDegraders. In some embodiments, a "Bm" can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 neoDegraders. In some embodiments, a "Bm" can be conjugated to 7 or 8 neoDegraders. In some embodiments, a "Bm" is conjugated to 5 neoDegraders. In some embodiments, a "Bm" is conjugated to 6 neoDegraders. In some embodiments, a "Bm" is conjugated to 7 neoDegraders. In some embodiments, "Bm" is conjugated to 8 neoDegraders. In some embodiments, "Bm" is conjugated to 9 neoDegraders.

[0208] IV. Compositions and Methods of Use The conjugates and / or compounds described herein may be in the form of pharmaceutical or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases.

[0209] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

[0210] 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, dicyclohexylamine salts, salts with organic bases such as N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and the like.

[0211] For example, Berge lists the following FDA-approved commercially available salts: acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate), estradiol, and estradiol. Lauryl sulfate, esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyl arsanilate (glycolamidophenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isopropyl alcohol, methylparaben ... Thionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate (methanesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate (2-naphthalenesulfonate), nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, diacetate, succinate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), and triethiodide; 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.

[0212] Berge additionally lists the following commercially available (outside the United States) salts that are not FDA approved: the anions adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrogen fluoride, hydroiodide, methylene bis(salicylate), napadisilate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, phenylethanide, and phenylethanide. These include methylbarbiturates, picrates, propionates, thiocyanates, tosylates, and undecanoates, the organic cations benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane), and the metal cations barium and bismuth.

[0213] Pharmaceutical compositions containing the neoDegrader conjugates described herein may also contain suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0214] In some embodiments, pharmaceutical compositions can be formulated into suitable parenteral dosage forms.The formulation can be prepared by various methods known in the art.The pharmaceutical composition can be administered directly into the bloodstream, into muscle, or directly into an organ.Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous.Suitable devices for parenteral administration include needle-type injectors, needle-free injectors, and infusion techniques.

[0215] Parenteral compositions are typically 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 as a dry form for use in combination with a suitable vehicle such as sterile pyrogen-free water.

[0216] The preparation of parenteral compositions under sterile conditions, for example, by lyophilization, may be readily accomplished using standard techniques well known to those skilled in the art.

[0217] Formulations for parenteral administration can be formulated for immediate release and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, sustained-, targeted-, and programmed-release. Thus, the compositions can be formulated as solids, semisolids, or thixotropic liquids for administration as implanted depots that provide modified release of the active agent.

[0218] The parenteral preparation may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

[0219] In another aspect, the pharmaceutical composition can be formulated as a suitable oral dosage form, such as a tablet, capsule, powder, pellet, suspension, solution, emulsion, etc. Other suitable carriers can be present, such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, bulking agents, anti-adherents, etc.

[0220] The oral dosage formulations may also contain other suitable pharmaceutical excipients, such as sweeteners, vehicles / humectants, colorants, flavoring agents, preservatives, viscosity enhancing / thickening agents, and the like.

[0221] The neoDegrader conjugates described herein can be used to treat various cancers. Certain conjugates disclosed herein are useful as pharmaceuticals because they are superior in terms of efficacy, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interactions with other pharmaceuticals (e.g., drug-metabolizing enzyme inhibitory activity), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous system toxicity), and / or stability (e.g., chemical stability, enzymatic stability).

[0222] The neoDegrader conjugates of the present disclosure can be used to treat diseases, e.g., cancer, such as colorectal cancer (e.g., colon cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumors), 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, adenomatous adenocarcinoma, squamous cell carcinoma), duodenal cancer, small intestine cancer, breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, 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 carcinoma), kidney cancer (e.g., For example, 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 carcinoma, uterine sarcoma), gestational choriocarcinoma, brain tumors (e.g., medulloblastoma, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcomas (e.g., rhabdomyosarcoma, leiomyosarcoma, sarcoma of soft tissue, spinal cord tumor, thrombocytoma ... They can be used as pharmaceuticals such as drugs for the prevention or treatment of cancers of unknown primary origin (e.g., spine cell sarcoma), malignant bone tumors, bladder cancer, hematological / blood cancers (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), cancer of unknown primary origin; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; drugs for the treatment of precancerous lesions (e.g., myelodysplastic syndromes).

[0223] In certain embodiments, the neoDegrader conjugates of the present disclosure can be used as pharmaceuticals for breast cancer, gastric cancer, ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, liver cancer, lymphoma, or blood cancer.

[0224] Furthermore, the neoDegrader conjugates of the present disclosure can be used in conjunction with non-drug therapies, such as (1) surgery, (2) antihypertensive chemotherapy using angiotensin II or the like, (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser ablation, and (7) radiation therapy.

[0225] For example, the use of the neoDegrader conjugate of the present disclosure before or after the above-mentioned surgery may have effects such as preventing the development of resistance, extending disease-free survival, suppressing cancer metastasis or recurrence, and prolonging life.

[0226] In addition, treatment with the neoDegrader conjugates of the present disclosure can be combined with supportive care, such as: (i) administration of antibiotics (e.g., β-lactams such as pansporin, macrolides such as clarithromycin) for various intercurrent infections; (ii) administration of high-calorie infusions, amino acid preparations, or multivitamins to improve malnutrition; (iii) administration of morphine to relieve pain; (iv) administration of drugs to improve side effects such as nausea, vomiting, loss of appetite, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin levels, hair loss, liver damage, kidney damage, DIC, fever, etc.; and (v) administration of drugs to suppress multidrug resistance in cancer.

[0227] In some embodiments, the neoDegrader or neoDegrader conjugates of the present disclosure can be used in combination with standard of care therapy, e.g., one or more therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Thus, in certain embodiments, the methods of treating tumors disclosed herein comprise administering a neoDegrader or neoDegrader conjugate of the present disclosure in combination with one or more additional therapeutic agents. In some embodiments, the neoDegrader or neoDegrader conjugates of the present disclosure can be used in combination with one or more anti-cancer agents, such that multiple elements of the immune pathway can be targeted. In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods include a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof. A comprehensive, non-limiting list of combination therapies is disclosed in detail in the Combination Therapies section of this application.

[0228] In some embodiments, the neoDegrader or neoDegrader conjugate of the present disclosure is administered to a subject before or after administration of an additional therapeutic agent. In other embodiments, the neoDegrader or neoDegrader conjugate of the present disclosure is administered to a subject simultaneously with the additional therapeutic agent. In certain embodiments, the neoDegrader or neoDegrader conjugate of the present disclosure and the additional therapeutic agent can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other embodiments, the neoDegrader or neoDegrader conjugate of the present disclosure and the additional therapeutic agent are administered simultaneously as separate compositions.

[0229] In some embodiments, subjects that can be treated with a neoDegrader or neoDegrader conjugate of the present disclosure are non-human animals, such as rats or mice. In some embodiments, subjects that can be treated are humans.

[0230] V. Methods for Preparing NeoDegraders and Compositions The present disclosure provides a method for preparing a neoDegrader conjugate, the process comprising: combining a binding moiety with a compound of formula (I-1): [ka] or a pharmaceutically acceptable salt thereof, wherein A is phenyl or C4-C 10 is a cycloalkyl ring, R 1 is independently selected from hydrogen and halo; U is selected from NH and CF; X is -NR 2 -, =C(CH3)-, -Q-(CH2) n - and -Q(CH2) m Q'(CH2) n - selected from: Q and Q' are each independently O, S, or NR 2 and R 2 is hydrogen or C1-C6 alkyl; n is an integer from 1 to 6, m is an integer from 2 to 6, where the left side of each group is bonded to L and the right side is bonded to A, provided that X is NH or -Q-(CH2) n -When R 1 is a halo, L' is a precursor of a cleavable or non-cleavable linker that is conjugated to a binding moiety.

[0231] As described herein, the precursor of the linker comprises a heterobifunctional group that connects to the binding moiety.

[0232] In some embodiments, L' is a precursor of a non-cleavable linker. In some embodiments, L' is selected from the group consisting of: [ka] During the ceremony, p is an integer from 1 to 10, [ka] is the point of attachment to X.

[0233] In some embodiments, L' is [ka] is.

[0234] In some embodiments, p is 5.

[0235] In certain embodiments, L' is a precursor of a cleavable linker.

[0236] In some embodiments, the precursor of the linker is cleavable by a protease. In some embodiments, the precursor of the linker is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, Z 1 , Z 2 , Z 3 , and Z 4 are each independently absent or a naturally occurring amino acid residue in the L- or D-configuration, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 are amino acid residues, and [ka] is the point of attachment to X.

[0237] In some embodiments, Z 1 , Z 2 , Z 3 , and Z 4 is independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-phenylalanine, D-phenylalanine, L-lysine, D-lysine, and glycine, with the proviso that Z 1 , Z 2 , Z 3 , and Z 4 At least two of the residues are amino acid residues.

[0238] In some embodiments, Z 1 is absent or is glycine, and Z 2 is absent or is selected from the group consisting of L-glutamine, D-glutamine, L-glutamic acid, D-glutamic acid, L-aspartic acid, D-aspartic acid, L-alanine, D-alanine, and glycine; Z 3 is selected from the group consisting of L-valine, D-valine, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, and glycine; Z 4 is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine, and glycine.

[0239] In some embodiments, L' is [ka] is.

[0240] In some embodiments, q is 5.

[0241] In some embodiments, L' is a precursor of a bioreductive linker. In some embodiments, the precursor of a bioreductive linker is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, R, R', R", and R'" are each independently selected from hydrogen, C-C alkoxyC-C alkyl, (C-C) NC-C alkyl, and C-C alkyl, or two geminal R groups together with the carbon atoms to which they are attached can form a cyclobutyl or cyclopropyl ring; [ka] is the point of attachment to X.

[0242] In certain embodiments, L' is a precursor of an acid cleavable linker. In some embodiments, L' is selected from the group consisting of: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X.

[0243] In certain embodiments, L' is a precursor of a click-to-release linker. In some embodiments, L' is selected from: [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X.

[0244] In certain embodiments, L' is a precursor of a pyrophosphatase cleavable linker. [ka] During the ceremony, q is an integer from 2 to 10, [ka] is the point of attachment to X.

[0245] In certain embodiments, L' is a precursor of a beta-glucorunidase cleavable linker. In some embodiments, L' is selected from: [ka] During the ceremony, q is an integer from 2 to 10, ---- is absent or is a bond, [ka] is the point of attachment to X.

[0246] In some embodiments, the compound of formula (I-1) is [ka] is selected from.

[0247] In some embodiments, the binding moiety is pretreated before reacting with the compound of Formula (I-1). In certain embodiments, the compound of Formula (I-1) is reacted with a binding moiety comprising an antibody or an antigen-binding portion thereof. In embodiments in which the binding moiety is an antibody, the antibody can be pretreated to reduce interchain disulfides before reaction with the compound of Formula (I-1). [Example]

[0248] General Synthetic Methods and Intermediates The compounds of the present disclosure can be prepared by one of ordinary skill in the art in light of this disclosure and knowledge in the art, and / or by reference to the schemes and synthetic examples set forth below. Exemplary synthetic routes are shown in the schemes and examples below. It is understood that variables (e.g., "R" groups) appearing in the schemes and examples below should be read independently of their appearance elsewhere in this application. One of ordinary skill in the art will readily understand how the schemes and examples set forth below illustrate the preparation of the compounds described herein.

[0249] Abbreviations used in the schemes generally follow conventions used in the art. Chemical abbreviations used in the specification and examples are defined as follows: "THF" tetrahydrofuran; "DMF" N,N-dimethylformamide; "Me" methyl; "Bu" butyl; "FA" formic acid; "PE" petroleum ether; "MeOH" methanol; "EtOH" ethanol; "DCM" dichloromethane; "BOC" or "Boc", "TFA" trifluoroacetic acid; "DMSO" dimethyl sulfoxide; "EtOAc" ethyl acetate; "OAc" acetate; "dppf" 1,1'-bis(diphenylphosphino)ferrocene; "dba" dichloromethane. Benzylideneacetone; "CDI" 1,1'-carbonyldiimidazole; "TBAF" tetrabutylammonium fluoride; "TBSC1" tert-butyldimethylsilyl chloride; "Et2O" diethyl ether; "ACN" acetonitrile; "h" hour; "min" minute; "rt" room temperature or hold time (depending on context); "aq." aqueous; "sat." saturated; "min" minute; "HOBt" 1-hydroxybenzotriazole; "HATU" 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; "DIEA" and "iPrNEt2" diisopropylethylamine; "Et3N" and "TEA" triethylamine.

[0250] [ka] [ka] Example 1: Synthesis of compound (Ia) [ka] Step 1: Synthesis of Compound 2 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) was added BH3-Me2S (10 M in THF) (5.80 mL, 58.0 mmol, 2.50 equiv) dropwise at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a 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. 1 H NMR (400 MHz, 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).

[0251] [ka] Step 2: Synthesis of Compound 3 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 BuNHSO (6.74 g, 19.84 mmol, 0.80 equiv). To the above mixture, NaOH (5 M in HO) (500.00 mL) was added dropwise over 40 minutes at 0°C. The resulting mixture was stirred for an additional 2 hours at 25°C. The resulting mixture was extracted with EtOAc (3 x 500 mL). The combined organic layers were washed with brine (400 mL) and dried over anhydrous NaSO. 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. 1H NMR (400 MHz, CDCl3) δ8.23 (d, J = 4.0 Hz, 1H), 8.10-8.04 (m, 1H), 7.60 (d, J= 8.0 Hz, 1H), 4.09 (s, 2H), 3.83-3.80 (m, 2H), 3.17-3.14(m, 2H), 1.45(s, 9H).

[0252] [ka] Step 3: Synthesis of Compound 4 To a stirred solution of tert-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) was added TFA (16.00 mL) dropwise at room temperature. The resulting mixture was stirred at room temperature for 1 h. The resulting mixture was concentrated in vacuo. The resulting mixture was diluted with water (500 mL). The mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. This afforded [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (6.5 g, crude) as a yellow oil. LCMS (ESI): 517 (2M-H)-

[0253] [ka] Step 4: Synthesis of Compound 5 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) was added CH3NH2.HCl (1.77 g, 26.21 mmol, 1.20 equiv) and DIEA (15.20 g, 117.8 mmol, 4.00 equiv) dropwise at room temperature. The resulting mixture was stirred at room temperature for 2 hours. 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) +

[0254] [ka] Step 5: Synthesis of Compound 6 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 BH3-THF (1 M in THF) (12.10 mL, 12.10 mmol, 1.00 equiv) dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1 N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO3 (sat, aq). The resulting mixture was extracted with EtOAc (3 × 100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (2.5 g, 80%) as a yellow oil. LCMS (ESI): 259.26 (M+H)+

[0255] [ka] Step 6. Synthesis of Compound 7 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 BocO (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 in vacuo. 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). +

[0256] [ka] Step 7: Synthesis of Compound 8 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 NHCl (750 mg, 14.2 mmol, 3.00 equiv) in EtOH (85 mL) and HO (17 mL) was added Fe (1.3 g, 23.7 mmol, 5.00 equiv) at 25 °C. 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 × 50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EtOAc = 4: 1) to give 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) +

[0257] [ka] Step 8: Synthesis of Compound 9 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 in vacuo 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) was added dropwise the above solution at 25 °C. 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 × 40 mL). The combined organic layers were washed with brine (5 × 40 mL) and dried over anhydrous NaSO. 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)phenetoxy)ethyl)(methyl)carbamate (670 mg, 70%) as a white solid. LCMS: (ESI): 628.63 (M+H) +

[0258] [ka] Step 9: Synthesis of neoDegrader P1 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) was added TFA (2.5 mL) dropwise at 0° C. The mixture was stirred at 25° C. for 1 h. The resulting mixture was concentrated in vacuo. The crude product was purified by preparative HPLC using the following conditions: column, SunFire C18 OBD Prep Column, 100 μm, 19 × 250 mm; mobile phase, water (0.05% TFA) and ACN (5% of phase B up to 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 (400 MHz, Methanol-d4) δ7.77 (d, J= 8.0 Hz, 1H), 7.57-7.53 (m, 2H), 7.49 (d, J= 8.0 Hz, 1H), 7.21 (d, J= 4.0 Hz, 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, 1H), 2.83-2.73 (m, 2H), 2.69 (s, 3H), 2.55-2.42 (m, 1H), 2.21-2.12 (m, 1H).

[0259] [ka] Step 10: Synthesis of compound (Ia) 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 (neoDegrader To a stirred mixture of P1 (200 mg, 0.38 mmol, 1.00 equiv.) and lutidine (81 mg, 0.76 mmol, 2.00 equiv.), 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.) were added in portions at room temperature. The reaction mixture was stirred at 40 °C under a 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 × 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 reversed-phase column (C18, mobile phase A: 0.1% in water). The crude product (60 mg) was purified by preparative HPLC using the following conditions: Column: Xselect CSH OBD column 30 × 150 mm 5 μm, n; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 33 B to 50 B in 7 min; 220 nm; RT: 5.27 min). The collected fractions were lyophilized to give methyl [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]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 (23.8 mg, 5%) as a white solid. LCMS (ESI): 1126.11 (M+H) + . 1H NMR (400 MHz, 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).

[0260]

change

change

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[0261] [ka] Step 2: Synthesis of compound 12 To a stirred mixture of 3-(5-bromo-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione (compound 11, 1.00 g, 3.09 mmol, 1.00 equiv) and dppf (51 mg, 0.093 mmol, 0.03 equiv) in DMF (8 mL) was added Zn(OAc) (170 mg, 0.928 mmol, 0.30 equiv), Zn(CN) (545 mg, 4.64 mmol, 1.50 equiv), and Pd(dba) (28 mg, 0.031 mmol, 0.01 equiv) under a nitrogen atmosphere at 25 °C. The final reaction mixture was irradiated with microwave radiation at 120 °C for 2 h. The mixture was cooled to room temperature and filtered. The filter cake was washed with MeOH (3 × 30 mL). The filtrate was concentrated under reduced pressure. The residue was subjected to flash chromatography (silica gel, 80 g, DCM:MeOH=10:1) to give the desired product 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindole-5-carbonitrile (400 mg, 47%) as a brown solid. LCMS (ESI): 270 (M+H) +

[0262] [ka] Step 3: Composition of INT1 To a stirred mixture of 2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindole-5-carbonitrile (compound 12, 3.0 g, 11.14 mmol, 1.00 equiv.) and HCl (12 M) (3.6 mL) in MeOH (25 mL) was added PtO (1.25 g, 5.5 mmol, 0.49 equiv.) at 25 °C. The mixture was hydrogenated under a hydrogen atmosphere at room temperature using a hydrogen balloon for 16 h. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 30 mL). The filtrate was concentrated under reduced pressure. The resulting solid was washed with DCM:MeOH (3:1) (3 × 30 mL) and dried. This gave 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (2.5 g, 80%) as a grey solid. LCMS (ESI): 274 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ11.02 (s, 1H), 8.15 (s, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.89(d, J=8.4Hz, 1H),5.16-5.11 (m, 1H), 4.52 (d, J=17.2Hz, 1H), 4.40 (d, J=17.2Hz, 1H), 2.96-2.90 (m, 1H), 2.60-2.54 (m, 1H), 2.43-2.34 (m, 1H), 2.06-1.96 (m, 1H)

[0263] [ka] Step 4: Synthesis of compound 14 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 13, 5.00 g, 22.50 mmol, 1.00 equiv) in THF (75 mL) was added BH3-Me2S (10 M in THF) (5.60 mL, 56 mmol, 2.50 equiv) dropwise at 0 °C under a nitrogen atmosphere. The mixture was stirred at 70 °C for 2 h. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column and eluted with PE / EtOAc (5:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (4.44 g, 88%) as a yellow solid. 1 H NMR (400 MHz, 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)

[0264] [ka] Step 5: Synthesis of Compound 15 To a stirred mixture of 2-(2-chloro-4-nitrophenyl)ethanol (compound 14, 4.44 g, 22.02 mmol, 1.00 equiv) and imidazole (4.50 g, 66.06 mmol, 3.00 equiv) in DMF (50.00 mL) was added TBSC1 (6.97 g, 46.25 mmol, 2.10 equiv) at 25 °C. The mixture was stirred at 25 °C for 16 h. The resulting mixture was diluted with water (100 mL). The resulting mixture was diluted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with PE / EtOAc (10:1) to give tert-butyl[2-(2-chloro-4-nitrophenyl)ethoxy]dimethylsilane (6.6 g, 90%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ8.24(s, 1H), 8.06-8.04 (m, 1H), 7.46 (d, J = 8.4 Hz, 1H), 3.89-3.86 (m, 2H), 3.06-0.04 (m, 2H), 0.85(s, 9H), 0.04(s, 6H).

[0265] [ka] Step 6: Synthesis of Compound 16 To a mixture of tert-butyl[2-(2-chloro-4-nitrophenyl)ethoxy]dimethylsilane (compound 15, 5.70 g, 18.05 mmol, 1.00 equiv) and Fe (10.08 g, 180.45 mmol, 10.00 equiv) in EtOH (110 mL) / water (55 mL) was added NH4Cl (9.65 g, 180.45 mmol, 10 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 × 50 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to give 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chloroaniline (5.2 g, crude) as a pale brown oil. LCMS (ESI): 286.29 (M+H) +

[0266] [ka] Step 7: Synthesis of Compound 17 To a solution of 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chloroaniline (compound 16, 200.00 mg, 0.70 mmol, 1.00 equiv.) and TEA (141 mg, 1.40 mmol, 2.00 equiv.) in DMF (3 mL), CDI (113 mg, 0.70 mmol, 1.00 equiv.) in DMF (1 mL) was added dropwise under nitrogen at 0 °C. The resulting mixture was stirred at 25 °C for 1 h. Next, the above solution and TEA (141 mg, 1.40 mmol) were added dropwise to a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 192 mg, 0.70 mmol, 1.00 equiv.) in DMF (2 mL). The same reaction was repeated twice. The resulting mixture was stirred at 25° C. for 1 hour. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layers were washed with water, brine, dried over anhydrous sodium sulfate, and evaporated to dryness in vacuo. The residue was purified on a silica gel column (DCM:MeOH=10:1) to give 1-(4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chlorophenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (170 mg, 21%) as a white solid. LCMS (ESI): 585.59 (M+H)+

[0267] [ka] Step 8: Synthesis of neoDegrader P3 To a solution of 1-(4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-3-chlorophenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 17, 170.00 mg, 0.29 mmol, 1.00 equiv) in THF (2.00 mL) was added TBAF (1N in THF, 0.58 mL, 0.58 mmol, 2.00 equiv) at 0° C. The resulting mixture was stirred at 25° C. for 8 hours. The reaction was purified by preparative TLC (DCM:MeOH=10:1) to give 147 mg of crude 1-(3-chloro-4-(2-hydroxyethyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea as a white solid. LCMS (ESI): 471.47 (M+H) +

[0268] [ka] Step 9: Synthesis of Compound 19 2-Methyl-2-sulfanylpropan-1-ol (compound 18, 1.4 g, 13.2 mmol, 1.00 equiv) and 5-nitro-2-[(5-nitropyridin-2-yl)disulfanyl]pyridine (compound 120, 2.05 g, 6.67 mmol, 0.50 equiv) were added to a mixture of dichloromethane (3.50 mL) and MeOH (3.50 mL). The resulting mixture was stirred at 15° C. Manganese dioxide (2.29 g, 26.2 mmol, 2 equiv) was then added in portions. The resulting mixture was stirred at 15° C. for 15 minutes. The LCMS trace showed the reaction was complete. The reaction was evaporated to dryness and the residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN (0.1% NH4HCO3) in water, gradient from 10% to 100% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to dryness in vacuo to give 2-methyl-2-[(5-nitropyridin-2-yl)disulfanyl]propan-1-ol (2.2 g, 58%) as a yellow solid. LCMS (ESI): 261 (M+H)+ .

[0269] [ka] Step 10: Synthesis of Compound 20 To a solution of 2-methyl-2-[(5-nitropyridin-2-yl)disulfanyl]propan-1-ol (compound 20, 1.0 g, 3.84 mmol, 1.00 equiv) in anhydrous DCM (30 mL) was added MeSONa (1.57 g, 15.4 mmol, 4.00 equiv) and iodine (1.95 g, 7.68 mmol, 2.00 equiv) in portions. The reaction mixture was stirred at 45 °C for 24 h. The mixture was concentrated, and the residue was purified by column chromatography on silica gel (TLC: PE:EA = 3:1, Rf = 0.60; 0 to 35% EtOAc in petroleum ether) to give 2-(methanesulfonylsulfanyl)-2-methylpropan-1-ol (80 mg, 10%) as a yellow oil. 1 H NMR (400 MHz, CD3C1): δ3.50(s, 2H), 3.33(s, 3H), 2.16(br s, 1H), 1.47(s, 6H).

[0270] [ka] Step 11: Synthesis of compound (Ib) To a solution of 1-[3-chloro-4-(2-hydroxyethyl)phenyl]-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (neoDegrader P3, 200.00 mg, 0.42 mmol, 1.00 equiv) and TEA (129 mg, 1.26 mmol, 3.00 equiv) in DMF (4 mL) was added a solution of CDI (138 mg, 0.84 mmol, 2.00 equiv) in DMF (1 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with water (20 mL x 3), brine (20 mL), dried over sodium sulfate, and evaporated to dryness in vacuo to give the crude product (ethyl 2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]imidazole-1-carboxylate, 200 mg) as a pale yellow solid. The crude product (100.00 mg, 0.18 mmol, 1.00 equiv) and C in DMF (8 mL) were dissolved in 1 mL of HCl. S2To a solution of CO3 (115 mg, 0.35 mmol, 2.00 equiv) was added 2-(methanesulfonylsulfanyl)-2-methylpropan-1-ol (compound 20, 59 mg, 0.32 mmol, 1.80 equiv) in DMF (2 mL) dropwise at room temperature. The reaction was stirred at 15 °C for 22 h. The reaction was diluted with EtOAc (50 ml) and ice-cold water (100 mL). The organic layer was separated. The aqueous phase was extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous sodium sulfate, and evaporated to dryness in vacuo to give the crude product (150 mg) as a yellow solid. The crude product was purified by preparative HPLC (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: 38 B to 58 B in 7 min; 220 nm; RT1: 5.12 min). The collected fractions were lyophilized to give 1-[3-chloro-4-[2-([[2-(methanesulfonylsulfanyl)-2-methylpropoxy]carbonyl]-oxy)ethyl]phenyl]-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (15.7 mg, 11%) as a white solid. LCMS (ESI): 681.68 (M+H). + . 1 H NMR (400 MHz, DMSO-d6) δ10.99 (s, 1H), 8.86 (s, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J= 8.0 Hz, 1H), 7.24-7.17 (m, 1H), 6.87-6.84 (m, 1H), 5.76 (s, 2H), 5.13-5.11 (m, 1H), 4.42-4.40 (m, 2H), 4.32-4.28 (m, 4H), 3.54 (s, 3H), 3.00-2.87 (m, 3H), 2.62-2.58 (m, 1H), 2.44-2.34 (m, 1H), 2.01-1.95 (m, 1H), 1.45 (s, 6H).

[0271] [ka] [ka] Example 3: Synthesis of compound (Ic) [ka] Step 1: Synthesis of compound 23 To a stirred solution of tert-butyl (2-aminophenyl)(methyl)carbamate (compound 22, 300 mg, 1.35 mmol, 1.00 equiv) in DMF (20 mL), CDI (218 mg, 1.35 mmol, 1.00 equiv) and TEA (68 mg, 1.35 mmol, 1.00 equiv) were added dropwise at 0°C under a nitrogen atmosphere. The mixture was stirred at 0°C for 2 hours. To the above mixture, 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 368 mg, 1.35 mmol, 1.00 equiv) was added in portions. The resulting mixture was stirred at 75°C overnight. The reaction mixture was then cooled to room temperature. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=10:1) to give tert-butyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate (300 mg, 42%) as a white solid. LCMS (ESI): 522 (M+H) +

[0272] [ka] Step 2. Synthesis of neoDegrader P4 To a stirred solution of tert-butyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate (compound 23, 300 mg, 1.00 equiv.) in DCM (20 mL) was added TFA (5 mL) at 0° C. The mixture was stirred at 0° C. for 2 hours. The resulting mixture was concentrated in vacuo. The crude product was purified by reverse phase (C18, mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min). The collected fractions were concentrated in vacuo to give 3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[2-(methylamino)phenyl]urea (210 mg, 87%) as a white solid. LCMS (ESI): 422 (M+H) + . 1 H NMR (300 MHz, DMSO-d6) δ10.99(s, 1H), 7.69 (d, J= 7.8 Hz, 1H), 7.60(s, 1H), 7.53(s, 1H), 7.45 (d, J= 8.4 Hz, 1H), 7.26-7.24(m, 1H), 6.99-6.93(m, 1H), 6.76-6.72(m, 1H), 6.60-6.55(m, 2H), 5.14-5.08(m, 1H), 5.00-4.85(br s, 1H), 4.48-4.28(m, 4H), 2.92-2.82(m, 1H), 2.70(s, 3H), 2.62-2.57(m, 1H), 2.49-2.41(m, 1H), 2.02-1.95(m, 1H).

[0273] [ka] Step 3. Synthesis of compound (Ic) To a stirred solution of 3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[2-(methylamino)phenyl]urea (P4, 150.00 mg, 0.36 mmol, 1.00 equiv), 2,6-lutidine (76 mg, 0.71 mmol, 2.00 equiv), and HOBT (96 mg, 0.71 mmol, 2.00 equiv) in DMF (3.00 mL) was added 4-nitrophenyl methyl [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]carbonate (394 mg, 0.53 mmol, 1.50 equiv) under a nitrogen atmosphere at room temperature. The reaction mixture was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, mobile phase A: water (0.1% FA), mobile phase B: ACN; to give the crude product (60 mg) as a white solid. The crude product (60 mg) was purified by preparative HPLC under the following conditions: column: Xselect CSH OBD column 30 × 150 mm 5 μm, n; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 24 B to 44 B in 7 min; 220 nm; RT1: 6.33; RT2: ). The collected fractions were lyophilized to give methyl [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-carbamate (18.1 mg, 5%) as a white solid. LCMS (ESI): 1020 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ10.99 (s, 1H), 9.96(s, 1H), 8.19-8.06 (m, 3H), 7.79 (d, J= 8.8 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.53-7.41 (m, 5H), 7.20-7.05 (m, 4H), 7.00(s, 2H), 6.95-6.90(m, 1H), 5.95(br s, 1H), 5.41(s, 2H), 5.18-4.89(m, 3H), 4.44-4.20(m, 5H), 4.19-4.17(m, 1H), 3.09(s, 3H), 3.07-2.85(m, 3H), 2.22-2.02(m, 2H), 2.00-1.85(m, 2H), 1.71-1.25(m, 10H), 1.20-1.12(m, 3H), 0.84-0.80(m, 6H)

[0274] Scheme 4 shows how compound (Id) was prepared from neoDegrader P1. [ka]

[0275] Synthesis of compound (Id) To a stirred mixture 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 (P1, 40.00 mg, 0.076 mmol, 1.00 equiv) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (25.00 mg, 0.081 mmol, 1.07 equiv) in DMF (2.00 mL) was added dropwise DIEA (20.00 mg, 0.16 mmol, 2.04 equiv) at room temperature. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3 × 30 mL). The combined organic layers were washed with water (30 mL), brine (30 mL), and dried over Na2SO4. After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified using the following conditions: Column: SunFire C18 OBD Prep Column, 100 μm, 19 mm × 250 mm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow Rate: 25 mL / min; Gradient: 25 B to 55 B in 8.5 min; 220 nm; RT: 8 min. The collected fractions were lyophilized to give N-[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethoxy)ethyl]-6-(2,5-dioxopyrrol-1-yl)-N-methylhexanamide (compound (Id), 24 mg, 43%) as a white solid. LCMS: (ES, m / s): 721,723 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ10.99 (s, 1H), 8.78 (s, 1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.41 (d, J=9.6Hz, 1H), 7.18-7.16 (m, 2H), 7.00(d, J= 5.6Hz, 2H), 6.85-6.80 (m, 1H), 5.12-5.05 (m, 1H), 4.42-4.33 (m, 5H), 3.39-3.36 (m, 3H), 2.91-2.76 (m, 7H), 2.68-2.52 (m, 1H), 2.48-2.35 (m, 1H), 2.33-2.20 (m, 3H), 2.05-1.95 (m, 1H), 1.48-1.44 (m, 5H), 1.28-1.12 (m, 3H).

[0276] Schemes 5A and 5B show how to prepare conjugates of neoDegrader P1 with alternative tripeptide linkers. [ka] [ka]

[0277] Schemes 6A and 6B show how to prepare conjugates of neoDegrader P1 with a β-glucuronide linker. [ka]

[0278] Step 1. Synthesis of compound 25 To a stirred mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanoic 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 could be detected by LCMS (derivatized with MeOH, MS = 326). LCMS showed the completion of the reaction. The resulting mixture was concentrated in vacuo 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 this to be the desired product (derivatized with MeOH). 1H-NMR (300 MHz, 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=3.0 Hz, 2H), 4.24 (t, J=6.0 Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J=3.0 Hz, 2H), 2.59 (t, J=6.0 Hz, 2H).

[0279] Step 2. Synthesis of compound 28 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 a N atmosphere. The resulting mixture was stirred overnight at room temperature under a N atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered, and the filter cake was washed with DCM (50 mL × 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (PE:EA=1:2) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed this to be the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300 MHz, 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).

[0280] Step 3. Synthesis of compound 29 To a stirred solution of (2S,3S,4S,5R,6S)-methyl 3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 28, 6.00 g, 12.41 mmol, 1.00 equiv) in MeOH (50 mL) was added NaBH (0.47 g, 12.42 mmol, 1.00 equiv) in portions at RT under a N atmosphere. The resulting mixture was stirred at room temperature under a N atmosphere for 2 hours. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The product was dried over NaSO. The resulting mixture was filtered, and the filter cake was washed with DCM. The resulting mixture was concentrated in vacuo to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 29, 5.5 g, 91%) as a solid. LCMS (ES, m / z): 486 [M+H]+.

[0281] Step 4. Synthesis of compound 30 To a stirred mixture of (2S,3S,4S,5R,6S)-methyl 3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (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 an H atmosphere for 16 hours. LCMS showed the reaction was complete. The resulting mixture was filtered, and the filter cake was washed with DCM and MeOH. The filtrate was concentrated in vacuo to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (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] + .

[0282] Step 5. Synthesis of compound 31 To a stirred solution of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (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 a N atmosphere. The resulting mixture was stirred at 0 °C under a N atmosphere for 6 h. LCMS showed 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 eluting with PE / EA (EA=100%) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (compound 31, 1.1 g, 66%) as a pale yellow solid. LCMS (ES, m / z): 749 [M+H] + .

[0283] Step 6. Synthesis of compound 33 To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (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) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv) in portions at 0° C. under a N atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere overnight. LCMS showed the reaction was complete. The reaction mixture was purified by reverse-phase flash chromatography using 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 methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 33, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] + . [ka]

[0284] Step 7. Synthesis of compound 34 (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (Compound 33, 1.00 g, 1.09 mmol, 1.00 equiv.) and 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 (neodegrader To a stirred mixture of P1 (0.58 g, 1.09 mmol, 1.00 equiv.), HOBT (1.18 g, 8.72 mmol, 8.00 equiv.) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv.) were added portionwise at room temperature under a N atmosphere. The resulting mixture was stirred at room temperature for 16 hours under a N atmosphere. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 80% in 40 min; detector, UV 254 nm. The collected fractions were concentrated in vacuo to give methyl (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]propanamido)phenoxy]oxane-2-carboxylate (compound 34, 800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] + .

[0285] Step 8. Synthesis of Compound 35 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]propanamido)phenoxy]oxane-2-carboxylate (Compound 34, 800.00 mg, 0.61 mmol, 1.00 equiv) in THF (80 mL) was added HCl (6N, 80 mL) in portions at room temperature under a N atmosphere. The resulting mixture was stirred at 50° C. under a nitrogen atmosphere for 3 hours. LCMS showed the reaction was complete. The resulting mixture was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography using 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]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 35, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] + .

[0286] Step 9. Synthesis of Compound 36 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]propanamido)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) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 10 minutes. LCMS indicated the reaction was complete. The resulting mixture was directly used for further purification by preparative HPLC under the following conditions (Column: XSelect CSH Preparative C18 OBD Column, 19 × 250 mm, 5 μm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow Rate: 25 mL / min; Gradient: 20 B to 40 B in 7 min; 220 nm; RT 1:5.78 min), (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-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%) was obtained as a white solid. LCMS (ES, m / z): 940 [M+H]+.

[0287] Step 10. Synthesis of compound (Ie) To a stirred solution of (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-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) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. LCMS showed the reaction was complete. The resulting mixture was purified by preparative HPLC under the following conditions: Column: Xselect CSH OBD column 30 × 150 mm 5 μm, Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 21 B to 36 B in 10 min; 220 nm; RT: 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]propanamido]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] + . 1 H-NMR (300 MHz, 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=3,0 Hz, 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=6 Hz, 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).

[0288] Scheme 7 shows how to prepare a conjugate of neoDegrader P6 with a hydrazine linker. [ka]

[0289] Step 1. Synthesis of compound 38 To a stirred solution of 4-aminoacetophenone (compound 37, 100 mg, 0.73 mmol, 1.00 equiv) in THF (2.00 mL) was added diphosgene (0.40 mL) dropwise at room temperature. The resulting mixture was stirred for 30 minutes at 0 °C. The resulting mixture was concentrated in vacuo. The resulting solid was redissolved in DMF (1.50 mL). To the stirred solution was added 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 200 mg, 0.73 mmol, 1.00 equiv) in DMF (3.00 mL) and TEA (0.50 mL) dropwise at room temperature. The resulting mixture was stirred for 1 hour at 0 °C. LCMS indicated the reaction was complete. Water (5 mL) was added to the mixture, which was then extracted with CHCl (3 × 10 mL). The organic layer was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 10% to 50% gradient in 35 min; detector, UV 254 nm. The collected fractions were concentrated to dryness to give 1-(4-acetylphenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 38, 80 mg, 25%) as a pale yellow solid. LCMS: (ES m / z): 435 [M+1] + .

[0290] Step 2. Synthesis of compound (If) A mixture of 1-(4-acetylphenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 38, 80.00 mg, 0.18 mmol, 1.00 equiv) and 6-(2,5-dioxopyrrol-1-yl)hexanehydrazide; trifluoroacetic acid (75 mg, 1.20 equiv) in methanol (5.00 mL) was stirred overnight at 50 °C. The mixture was cooled to room temperature. LCMS showed the reaction was complete. The precipitated solid was collected by filtration and washed with MeOH (2 × 5 mL). The crude solid was purified by reverse-phase flash chromatography using the following conditions: C18 column; mobile phase, ACN in water (0.1% FA), 10% to 50% gradient in 30 min; detector, UV 254 nm. The collected fractions were extracted with DCM (3 x 5 mL) and concentrated in vacuo to give 3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-1-[4-[(1E)-1-[[6-(2,5-dioxopyrrol-1-yl)hexanamido]imino]ethyl]phenyl]urea (compound (If), 4.4 mg, 3.7%)) as an off-white solid. LCMS: (ES m / z): 642 [M+1] + . 1 H NMR (400 MHz, DMSO-d6) δ10.99 (s, 1H), 10.26-10.15 (m, 1H), 8.82 (s, 1H),7.69-7.62(m, 3H), 7.52-7.43 (m, 4H), 7.01-6.99 (m, 2H), 5.13-5.09 (m, 1H), 4.42-4.33 (m, 4H), 2.98-2.82 (m, 1H), 2.62-2.58 (m, 2H), 2.20-2.12 (m, 2H), 1.58-1.51 (m, 6H),1.26-1.09 (m,6H)

[0291] Scheme 8 shows how to prepare a conjugate of neoDegrader P2 with a quaternary amine linker. [ka]

[0292] Step 1. Synthesis of compound 40 To a stirred solution of N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound 39, 100 mg, 0.18 mmol, 1.00 equiv) in DMF (2 mL) was added SOCl (20 mg, 0.18 mmol, 1 equiv) in DCM (2 mL) dropwise at 0 °C under N. The resulting mixture was stirred at 0 °C for 1 h. LCMS indicated the reaction was complete. The reaction mixture was diluted with ice-cold water (20 mL) and extracted with DCM (10 mL*3). The combined organic layers were washed with water (10 mL), brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to dryness in vacuo to give the product N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(chloromethyl)phenyl]-carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound 40, 80 mg, 53%) as a white solid. LCMS (ES, m / z): 591,593 [M+H] +

[0293] Step 2. Synthesis of compound 42 To a stirred mixture of (2-chloro-4-nitrophenyl)acetic acid (compound 41, 8.60 g, 39.9 mmol, 1.00 equiv) in THF (130 mL) was added BH3-Me2S (10.00 mL, 105.4 mmol, 2.64 equiv) dropwise at 0 °C. The resulting mixture was stirred at 70 °C for 4 h under a nitrogen atmosphere. TLC (PE:EA = 1:2) showed the reaction was complete. The mixture was allowed to cool to room temperature. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 42, 7.7 g, 96%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ8.27 (d, J=4.0Hz, 1H), 8.11-8.07 (m, 1H), 7.53 (d, J= 8.0 Hz, 1H), 3.99 (t, J = 8.0 Hz, 2H), 3.15 (t, J = 8.0 Hz, 2H).

[0294] Step 3. Synthesis of compound 43 To a stirred mixture of 2-(2-chloro-4-nitrophenyl)ethanol (compound 42, 7.70 g, 38.2 mmol, 1.00 equiv) and tert-butyl 2-bromoacetate (57.74 g, 296.0 mmol, 7.75 equiv) in toluene (70 mL) was added BuNHSO (10.37 g, 30.6 mmol, 0.80 equiv) in portions at 0 °C. To the above mixture was added NaOH (15.00 g, 375.0 mmol, 9.82 equiv) in HO (90 mL) dropwise over 30 h at 0 °C. The resulting mixture was stirred for an additional 4 h at room temperature. TLC (PE:EA = 3:1) indicated completion of the reaction. The resulting mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (5:1) to give tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (Compound 43, 12.2 g, 91%) as a yellow oil. 1 H NMR (300 MHz, CDCl3) δ8.20 (d, J = 4.0Hz, 1H), 8.07-8.03 (m, 1H), 7.61 (d, J = 8.1 Hz, 1H), 4.11 (s, 2H), 3.83 (t, J= 8.1 Hz, 2H), 3.16(t, J= 8.1 Hz, 2H), 1.45(s, 9H).

[0295] Step 4. Synthesis of compound 44 To a stirred mixture of tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (compound 43, 12.20 g, 38.6 mmol, 1.00 equiv) in DCM (120 mL) was added TFA (20 mL) dropwise at 0° C. The resulting mixture was stirred for 4 hours at room temperature. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. This afforded [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 44, 8.4 g, 83%) as a yellow solid. LCMS: (ES, m / s): 517 (2M-H) - 1 H NMR (400 MHz, DMSO-d6) δ12.64(s, 1H), 8.20 (d, J= 4.0 Hz, 1H), 8.11-8.08 (m, 1H), 7.72 (d, J = 8.0 Hz, 1H), 4.06 (s, 2H), 3.74 (t, J = 8.0 Hz, 2H), 3.06(t, J= 8.0 Hz, 2H).

[0296] Step 5. Synthesis of compound 45 To a stirred mixture of [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (compound 44, 8.40 g, 32.35 mmol, 1.00 equiv) and HATU (19.19 g, 50.47 mmol, 1.56 equiv) in DMF (80 mL) was added CHNHHCl (2.69 g, 39.79 mmol, 1.23 equiv) and DIEA (17.31 g, 133.93 mmol, 4.14 equiv) under a nitrogen atmosphere at 0 °C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 4 h. LCMS showed the reaction was complete. The reaction was quenched with water / ice. The resulting mixture was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=10:1) to give 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 45, 7.2 g, 81%) as a yellow oil. LCMS: (ES, m / s): 273,275 (M+H) +

[0297] Step 6. Synthesis of compound 46 To a stirred mixture of 2-[2-(2-chloro-4-nitrophenyl)ethoxy]-N-methylacetamide (compound 45, 7.20 g, 26.40 mmol, 1.00 equiv) in THF (70 mL) was added BH3-THF (10 M in THF, 52.0 mL, 520.0 mmol, 20 equiv) dropwise at room temperature. The resulting mixture was stirred at 70 °C for 4 hours. LCMS showed the reaction was complete. The mixture was allowed to cool to room temperature. The reaction was quenched with MeOH. The residue was acidified to pH 6 with 1 N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO3 (sat, aq). The resulting mixture was extracted with EtOAc (3 × 100 mL), 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 eluting with (DCM:MeOH=8:1) to give [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 46, 5.4 g, 79%) as a yellow solid. LCMS: (ES, m / s): 259,261 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ8.26 (d, J= 4.0 Hz, 1H), 8.15-8.12 (m, 1H), 7.73 (d, J= 8.0 Hz, 1H), 3.72 (t, J= 8.0 Hz, 2H), 3.61(t, J= 8.0 Hz, 2H), 3.10 (t, J= 8.0 Hz, 2H), 2.87 (t, J= 8.0 Hz, 2H), 2.40 (s, 3H).

[0298] Step 7. Synthesis of compound 47 To a stirred mixture of [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (compound 46, 4.00 g, 15.46 mmol, 1.00 equiv) and BocO (3.80 g, 17.41 mmol, 1.13 equiv) in THF (20.00 mL) was added NaHCO (4.00 g, 47.61 mmol, 3.08 equiv) in HO (20.00 mL) dropwise at room temperature. The resulting mixture was stirred overnight at room temperature. LCMS showed the reaction was complete. The resulting mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=12:1) to give tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 47, 4.8 g, 77%) as a yellow solid. LCMS: (ES, m / s): 359,361 (M+H). + ; 1 H NMR (400 MHz, DMSO-d6) δ8.24 (d, J= 4.0 Hz, 1H), 8.13-8.10 (m, 1H), 7.67 (d, J=8.0Hz, 1H), 4.05-4.00(m, 1H), 3.69 (t, J= 8.0 Hz, 2H), 3.50(t, J= 8.0 Hz, 2H), 3.28 (t, J= 8.0 Hz, 2H), 3.07(t, J= 8.0 Hz, 2H), 2.75(s, 3H), 1.36(s, 9H).

[0299] Step 8. Synthesis of compound 48 To a stirred mixture of tert-butyl N-[2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 47, 5.60 g, 15.6 mmol, 1.00 equiv) in EtOH (112.00 mL) was added NH4Cl (2.50 g, 46.74 mmol, 2.99 equiv) and Fe (4.40 g, 78.79 mmol, 5.05 equiv) in HO (12.00 mL) at room temperature. The resulting mixture was stirred at 80 °C for 3 h. LCMS showed the reaction was complete. The mixture was allowed to cool to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with DCM (3 × 30 mL). The combined organic layers were washed with brine (30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=10:1) to give tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 48, 4.2 g, 81%) as a yellow oil. LCMS: (ES, m / s): 329,331 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ6.96 (d, J= 8.0 Hz, 1H), 6.59(d, J= 4.0 Hz, 1H), 6.46-6.43 (m, 1H), 5.18(br s, 2H), 3.50-3.45(m, 4H), 3.29-3.26(m, 2H), 2.75-2.71(m, 5H), 1.38(s, 9H).

[0300] Step 9. Synthesis of Compound 49 To a solution of tert-butyl N-[2-[2-(4-amino-2-chlorophenyl)ethoxy]ethyl]-N-methylcarbamate (compound 48, 100 mg, 0.30 mmol, 1.00 equiv.) in THF (3 mL) was added LiAlH (92 mg, 2.43 mmol, 8.00 equiv.) in THF (2 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 h. Five reactions were run in parallel. LCMS indicated the reactions were complete. The reactions were then quenched with 1 N NaOH (10 mL), filtered, and concentrated to dryness in vacuo. The residue was then purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 60% in 30 min; detector, UV 254 nm. The collected fractions were concentrated to dryness to give 3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]aniline, 49 (180 mg, 44%) as a yellow oil. LCMS (ES, m / z): 243,245 [M+H] +

[0301] Step 10. Synthesis of Compound 50 To a solution of 3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]aniline (compound 49, 140 mg, 0.58 mmol, 1.00 equiv) in THF (9 mL) was added diphosgene (137 mg, 0.69 mmol, 1.20 equiv) under a nitrogen atmosphere at 0°C. The resulting mixture was stirred at 0°C for 1 hour. The reaction solution was then concentrated to dryness under vacuum. The residue was dissolved in DMF (2 mL) and added dropwise to a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (158 mg, 0.58 mmol, 1.00 equiv) and TEA (117 mg, 1.15 mmol, 2.00 equiv) in DMF (4 mL) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was diluted with methanol, and the resulting solution was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN (0.1% FA) in water, gradient 0% to 50% in 30 min; detector, UV 254 nm to give 100 mg of product as a colorless solid. The crude product was purified by preparative HPLC under the following conditions: column: XBridge Shield RP18 OBD column, 19 x 250 mm, 10 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 14% to 32% in 7 min; 220 nm; RT1: 5.25 min. Collected fractions were lyophilized to afford 1-(3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]phenyl)-3-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]urea (compound 50, 60 mg, 18%) as a colorless solid. LCMS (ES, m / z): 542,544 [M+H]+

[0302] Step 11. Synthesis of compound (Ig) N-[(1S)-1-[[(1S)-4-(carbamoylamino)-1-[[4-(chloromethyl)phenyl]-carbamoyl]butyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (Compound 40, 66 mg, 0.11 mmol, 1.00 equiv.), 1-(3-chloro-4-[2-[2-(dimethylamino)phenyl]-2-methylpropyl)-hexanamide) in DMF (1 mL) To a solution of (2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl)methyl)urea (compound 50, 60 mg, 0.11 mmol, 1.00 equiv.) and DIEA (29 mg, 0.22 mmol, 2.00 equiv.), TBAI (4 mg, 0.01 mmol, 0.10 equiv.) was added in air at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The LCMS trace indicated completion of the reaction. The resulting mixture was purified by reverse-phase column chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), 5% to 45% gradient in 40 minutes; detector, UV 254 nm, to give 90 mg of crude product as a yellow oil. The crude product was then purified under the following conditions: Column: Xselect CSH OBD column 30*150mm 5um, n; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60mL / min; Gradient: 15 B to 35 B in 7 min B; 220 nm; RT1:6.00 min to give ([4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl)[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]dimethylazanium, compound (Ig) (19 mg, 14.8%) as a white solid. LCMS (ES, m / z): 1096 [M-FA] + , 549 [1 / 2(M-FA)] + ; 1 H NMR (400 MHz, CD3OD) δ8.48 (s, 1H), 7.77-7.72 (m, 3H), 7.55 - 7.47 (m, 3H), 7.37-7.35 (d, J = 8.4 Hz, 2H), 7.18 - 7.14 (m, 2H), 6.77 (s, 2H), 5.17-5.13 (q, J = 8, 4Hz, 1H), 4.51 -4.46 (m, 5H), 4.35 (s, 2H), 4.12 (d, J = 8.0 Hz, 1H), 3.90 (s, 2H), 3.79 (t, J = 5.6 Hz, 2H), 3.45 (t, J = 7.2 Hz, 4H), 3.22-3.15 (m, 1H), 3.11-3.05 (m, 1H), 3.00 (t, J = 6.0 Hz, 2H), 2.92 (s, 6H), 2.89 - 2.84 (m, 1H), 2.81 - 2.73 (m, 1H), 2.54-2.43 (m, 1H), 2.27 (t, J = 7.2 Hz, 2H), 2.21 - 2.12 (m, 1H), 2.10 - 2.02 (m, 1H), 1.95 - 1.82 (m, 1H), 1.78-1.69 (m, 1H), 1.64-1.59 (m, 7H), 1.32-1.25 (m, 2H), 0.98-0.96 (m, 6H).

[0303] Schemes 9A and 9B show how to prepare conjugates of neoDegrader P13 with peptide-containing linkers. [ka] [ka]

[0304] Scheme 10 illustrates the synthesis of compounds of formula (Ih). [ka]

[0305] Step 1. Synthesis of compound 63 To a stirred mixture of 3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (Compound 62, 5.00 g, 16.06 mmol, 1.00 equiv.), SOCl2 (25 mL) was added at room temperature. The resulting mixture was stirred at 80 °C for 16 hours. The desired product could be detected by LCMS (derivatized with MeOH, MS = 326). LCMS showed the completion of the reaction. The resulting mixture was concentrated in vacuo 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 1 H NMR analysis showed this to be the desired product (derivatized with MeOH). 1 H-NMR (300 MHz, 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=3.0 Hz, 2H), 4.24 (t, J=6.0 Hz, 1H), 3.74-3.67 (m, 3H), 3.50 (d, J=3.0 Hz, 2H), 2.59 (t, J=6.0 Hz, 2H).

[0306] Step 2. Synthesis of compound 66 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.), methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate (compound 64, 10.00 g, 25.17 mmol, 1.00 equiv.) was added portionwise at room temperature under a N atmosphere. The resulting mixture was stirred overnight at room temperature under a N atmosphere. LCMS showed the reaction was complete. The resulting mixture was filtered, and the filter cake was washed with DCM (50 mL × 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (PE:EA=1:2) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (Compound 66, 10.5 g, 86%) as a white solid. 1 H-NMR analysis showed this to be the desired product. LCMS (ES, m / z): 484 [M+1] + . 1 H-NMR (300 MHz, 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).

[0307] Step 3. Synthesis of compound 67 To a stirred solution of (2S,3S,4S,5R,6S)-methyl 3,4,5-tris(acetyloxy)-6-(4-formyl-2-nitrophenoxy)oxane-2-carboxylate (compound 66, 6.00 g, 12.41 mmol, 1.00 equiv) in MeOH (50 mL) was added NaBH (0.47 g, 12.42 mmol, 1.00 equiv) in portions at room temperature under a N atmosphere. The resulting mixture was stirred at room temperature for 2 hours under a N atmosphere. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The product was dried over NaSO. The resulting mixture was filtered, and the filter cake was washed with DCM. The resulting mixture was concentrated in vacuo to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (compound 67, 5.5 g, 91%) as a solid. LCMS (ES, m / z): 486 [M+H] + .

[0308] Step 4. Synthesis of compound 68 To a stirred mixture of (2S,3S,4S,5R,6S)-methyl 3,4,5-tris(acetyloxy)-6-[4-(hydroxymethyl)-2-nitrophenoxy]oxane-2-carboxylate (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 an H atmosphere for 16 h. LCMS showed the reaction was complete. The resulting mixture was filtered, and the filter cake was washed with DCM and MeOH. The filtrate was concentrated in vacuo to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (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] + .

[0309] Step 5. Synthesis of Compound 70 To a stirred solution of (2S,3S,4S,5R,6S)-methyl 3,4,5-tris(acetyloxy)-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (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 9H-fluoren-9-ylmethyl N-(3-chloro-3-oxopropyl)carbamate (compound 69, 0.87 g, 2.62 mmol, 1.20 equiv.) in portions at 0 °C under a N atmosphere. The resulting mixture was stirred at 0 °C for 6 h under a N atmosphere. 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 eluting with PE / EA (EA=100%) to give methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (Compound 70, 1.1 g, 66%) as a pale yellow solid. LCMS (ES, m / z): 749 [M+H] + .

[0310] Step 6. Synthesis of Compound 72 To a stirred mixture of (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (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) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv) in portions at 0 °C under a N atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere overnight. LCMS showed the reaction was complete. The reaction mixture was purified by reverse-phase flash chromatography using 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 methyl (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (compound 72, 1.4 g, 48%) as a yellow solid. LCMS (ES, m / z): 914 [M+H] + .

[0311] Step 7. Synthesis of Compound 73 (2S,3S,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-[2-(3-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido)-4-[[(4-nitrophenoxycarbonyl)oxy]methyl]phenoxy]oxane-2-carboxylate (Compound 72, 1.00 g, 1.09 mmol, 1.00 equiv.) and 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 (neoDegrader To a stirred mixture of P1 (0.58 g, 1.09 mmol, 1.00 equiv.), HOBT (1.18 g, 8.72 mmol, 8.00 equiv.) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv.) were added portionwise at room temperature under a N atmosphere. The resulting mixture was stirred at room temperature for 16 hours under a N atmosphere. LCMS showed the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 80% in 40 min; detector, UV 254 nm. The collected fractions were concentrated under vacuum to give methyl (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]propanamido)phenoxy]oxane-2-carboxylate (compound 73, 800 mg, 56%) as a solid. LCMS (ES, m / z): 1302 [M+H] + .

[0312] Step 8. Synthesis of Compound 74 (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]propanamide) in THF (80 mL) To a stirred mixture of methyl phenoxyoxane-2-carboxylate (compound 73 (800.00 mg, 0.61 mmol, 1.00 equiv.) was added HCl (6N, 80 mL) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 50° C. for 3 hours under a nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN (0.1% in water) FA), 0% to 80% gradient in 40 min; detector, UV 254 nm. 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]propanamido)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (compound 74, 230 mg, 32%) as a white solid. LCMS (ES, m / z): 1162 [M+H] + .

[0313] Step 9. Synthesis of Compound 75 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]propanamido)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) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 10 minutes under a nitrogen atmosphere. LCMS showed the reaction was complete. The resulting mixture was directly used for further purification by preparative HPLC under the following conditions (Column: XSelect CSH Preparative C18 OBD Column, 19 × 250 mm, 5 μm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow Rate: 25 mL / min; Gradient: 20 B to 40 B in 7 min). B; 220 nm; RT1: 5.78 min), (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-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%) was obtained as a white solid. LCMS (ES, m / z): 940 [M+H] + .

[0314] Step 10. Synthesis of compound (Ih) (2S,3S,4S,5R,6S)-6-[2-(3-aminopropanamido)-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]phenyl in DMF (2.0 mL) To a stirred solution of [[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[()])])]) ... B; wavelength: 254 nm; RT1 (min): 5.8. 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-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanamido}propanamido)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=8.1 Hz,1H), 7.25-7.00 (m, 4H), 6.82-6.80 (m, 1H), 5.86 (s, IH), 5.39-5.30 (m, 2H), 5.14-5.07 (m, 1H), 4.97 (s, 2H), 4.84 (d, J=7.2 Hz,1H), 4.47-4.27 (m, 4H), 3.90 (d, J=9.6 Hz, 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).

change

[0315] ステップ1. Synthesis of compound 76 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 (Compound P1, 180 mg, 0.34 mmol, 1.00 equiv) in DMF (8 mL), TEA (104 mg, 1.02 mmol, 3.0 equiv) and 4-(chlorosulfonyl)-3-nitrobenzoic acid (181 mg, 0.68 mmol, 2.00 equiv) were added portionwise under a nitrogen atmosphere at 0° C. The resulting mixture was stirred at 0° C. under a nitrogen atmosphere for 4 hours. LCMS indicated the reaction was complete. The resulting mixture was used for further purification. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 60% in 10 min; detector, UV 254 nm. The mixture was lyophilized to give 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)sulfamoyl]-3-nitrobenzoic acid (compound 76, 70 mg, 27%) as a pale yellow solid. LCMS (ES, m / z): 757 [M+1] + .

[0316] Step 2. Synthesis of compound (Ii) To a stirred mixture of 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)sulfamoyl]-3-nitrobenzoic acid (Compound 76, 60 mg, 0.08 mmol, 1.00 equiv) in DMF (6 mL) was added HATU (45 mg, 0.12 mmol, 1.5 equiv), 1-(2-aminoethyl)pyrrole-2,5-dione hydrochloride (Compound 77, 17 mg, 0.10 mmol, 1.20 equiv), and DIEA (31 mg, 0.24 mmol, 3.0 equiv) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 4 hours under a nitrogen atmosphere. LCMS showed the reaction was complete. The residue was purified by preparative HPLC (Column: XBridge Preparative Phenyl OBD Column, 19 x 150 mm 5 µm 13 nm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow Rate: 25 mL / min; Gradient: 25 B to 43 B in 10 min; 220 nm; RT: 11.97 min). The collected fractions were lyophilized to give 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)sulfamoyl]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]-3-nitrobenzamide (Compound (li), 27 mg, 36%) as a white solid. LCMS (ES, m / z): 879,881 [M+H]. 1 H NMR (300 MHz, DMSO-d6) δ11.00 (s, 1H), 9.01 (t, J=6.0 Hz, 1H), 8.82 (s, 1H), 8.20 (s, 1H), 8.11 (s, 2H), 7.71-7.67 (m, 2H), 7.52 (s, 1H), 7.44 (d, J=3.0 Hz, 1H), 7.21-7.12 (m, 2H), 7.02 (s, 2H), 6.84 (t, J=6.0 Hz, 1H), 5.14-5.08 (m, 1H), 4.48-4.28 (m, 4H), 3.62-3.50(m, 6H), 3.40-3.28 (m, 2H), 2.95-2.85(m, 4H), 2.80-2.73 (m, 2H), 2.65-2.60 (s, 1H), 2.41-2.27 (m, 1H), 2.05-1.95 (m, 1H). [ka]

[0317] Step 1. Synthesis of Compound 79 To a stirred mixture of (2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-acetamido)acetic acid (compound 78, 10.00 g, 28.22 mmol, 1.00 equiv) and Pb(OAc) (15.02 g, 33.86 mmol, 1.20 equiv) in THF (300 mL) and toluene (100 mL), pyridine (2.59 g, 32.74 mmol, 1.16 equiv) was added dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 80 °C overnight under a nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was allowed to cool to room temperature. The resulting mixture was filtered, and the filter cake was washed with ethyl acetate (20 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (20 mL), washed with water, brine, and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:4) to give methyl (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)acetate (compound 79, 6.5 g, 56%) as a white solid. LCMS (ESI, ms): 391 [M+Na] + . 1HNMR (300MHz, CDCl3) δ7.80(d, J=7.5Hz, 2H), 7.62(d, J=7.5Hz, 2H), 7.45(t, J =7.5Hz, 2H), 7.36(d, J =7.5Hz, 2H), 7.18(br s, 1H),5.48(br s, 1H), 5.28(d, J=7.2Hz, 2H), 4.48(d, J=6.6Hz, 2H), 4.26(t, J=6.6Hz, 1H), 3.93(d, 5.4Hz, 2H), 2.08(s, 3H).

[0318] Step 2. Synthesis of Compound 81 To a stirred mixture of methyl (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]acetamido)acetate, 79 (2.00 g, 5.43 mmol, 1.00 equiv.), and 2-(2-chloro-4-nitrophenyl)ethanol (compound 3, 3.20 g, 15.85 mmol, 2.92 equiv.) in DCM (40 mL), PPTS (400 mg, 1.59 mmol, 0.29 equiv.) was added dropwise at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred overnight at 45 °C under a nitrogen atmosphere. 40% of the desired product could be detected by LCMS. The mixture was allowed to cool to room temperature. The reaction was quenched with water / ice. The resulting mixture was extracted with EtOEt (3 × 20 mL). The combined organic layers were washed with brine (30 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:9) to give 9H-fluoren-9-ylmethyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 81, 1.7 g, 55%) as a white solid. LCMS (ESI, ms): 510, 512 [M+H] + . 1 HNMR (300MHz, DMSO-d6): δ8.58 (t, J=5.1Hz, 1H), 8.22 (dd, J = 12, 2.4Hz, 1H), 7.89 (d, J=7.5Hz, 1H), 7.71-7.54 (m, 4H), 7.43-7.29 (m, 4H), 4.56 (d, J=6.9Hz, 2H), 4.30-4.16 (m, 3H), 3.70-3.61(m, 4H), 3.04 (t, J=6.3Hz, 2H).

[0319] Step 3. Synthesis of Compound 82 To a stirred mixture of 9H-fluoren-9-ylmethyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 81, 1.60 g, 3.14 mmol, 1.00 equiv) in DMF (5.0 mL) was added piperidine (1.0 mL) in portions at 0° C. under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. LCMS showed the reaction was complete. The reaction mixture was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 50% in 40 minutes; detector, UV 254 nm. This gave 2-amino-N-[[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]acetamide (compound 82, 750 mg, 76%) as a yellow oil. LCMS (ESI, ms) 288 [M+H] + ,329[M+H+ACN] +

[0320] Step 4. Synthesis of Compound 83 To a stirred mixture of 2-amino-N-[[2-(2-chloro-4-nitrophenyl)ethoxy]-methyl]acetamide (compound 82, 750 mg, 2.61 mmol, 1.00 equiv) and BocO (580 mg, 2.66 mmol, 1.02 equiv) in DMF (10.00 mL) was added NaHCO (477 mg, 5.68 mmol, 2.18 equiv) in HO (10.00 mL) dropwise at 0 °C. The resulting mixture was stirred at room temperature for 3 h. LCMS showed the reaction was complete. The reaction was quenched by adding water (20 mL). The resulting mixture was extracted with EtOEt (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:2) to give tert-butyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (compound 83, 650 mg, 58%) as a yellow oil. LCMS (ESI, ms), 388 [M+H] + , 332[M+H-56] + . 1 HNMR (400MHz, CDCl3) δ8.21(d, J=2.4Hz, 1H), 8.04(d, J=8.4Hz, 2H), 7.46(d, J=8.4Hz, 1H), 7.05(br s, 1H), 5.25(br s, 1H), 4.73(d, J=7.2Hz, 2H), 3.81-3.73(m, 4H), 3.34-3.32(m, 2H), 3.08(t, J=6.8Hz, 2H), 1.42(s, 9H).

[0321] Step 5. Synthesis of Compound 84 To a stirred mixture of tert-butyl N-[([[2-(2-chloro-4-nitrophenyl)ethoxy]methyl]-carbamoyl)methyl]carbamate (compound 83, 650 mg, 1.68 mmol, 1.00 equiv) and Fe (260 mg, 4.66 mmol, 2.78 equiv) in EtOH (9.00 mL) was added NH4Cl (910 mg, 17.01 mmol, 10.1 equiv) in HO (3.00 mL) dropwise at room temperature. The resulting mixture was stirred at 90 °C for 4 h. LCMS showed the reaction was complete. The mixture was allowed to cool to room temperature. The resulting mixture was concentrated in vacuo. The resulting mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:1) to give tert-butyl N-[([[2-(4-amino-2-chlorophenyl)ethoxy]methyl]-carbamoyl)methyl]carbamate (compound 84, 500 mg, 83%) as a yellow solid. LCMS (ESI, ms): 358 [M+H] + , 380[M+Na] + . 1 HNMR (300MHz, CDCl3) δ7.02-6.96(m, 2H), 6.68(d, J=2.4Hz, 1H), 6.52-6.49(m, 1H), 5.29(br s, 1H), 4.74(d, J=6.9Hz, 2H), 3.80-3.78(m, 2H), 3.69-3.63(m, 2H), 2.88(t, J=7.2Hz, 2H), 1.45(s, 9H).

[0322] Step 6. Synthesis of Compound 86 To a stirred mixture of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione hydrochloride (Compound 85, 398 mg, 1.28 mmol, 0.92 equiv.) and CDI (450 mg, 2.78 mmol, 1.99 equiv.) in DMF (5.00 mL), TEA (300 mg, 2.96 mmol, 2.12 equiv.) was added at 0 °C. The resulting mixture was stirred at room temperature for 2 h. To the above mixture, tert-butyl N-[([[2-(4-amino-2-chlorophenyl)ethoxy]methyl]carbamoyl)methyl]carbamate (Compound 84, 500 mg, 1.40 mmol, 1.00 equiv.) and DMAP (550 mg, 4.50 mmol, 3.22 equiv.) were added in portions. The resulting mixture was further stirred overnight at 60° C. LCMS indicated the reaction was complete. The mixture was allowed to cool to room temperature. The reaction mixture was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 50% in 30 min; detector, UV 254 nm. This gave tert-butyl N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)carbamate (compound 86, 550 mg, 60%) as a light brown solid. LCMS (ESI, ms): 657 [M+H] + , 601[M+H-56] + ,557[M+H-100] + .

[0323] Step 7. Synthesis of Compound 87 To a stirred mixture of tert-butyl N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)-carbamate (Compound 86, 530 mg, 0.80 mmol, 1.00 equiv) in DCM (5.00 mL) was added TFA (1.00 mL) at 0° C. The resulting mixture was stirred for 30 minutes at 0° C. LCMS showed the reaction was complete. The resulting mixture was concentrated under reduced pressure. This gave 2-amino-N-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]acetamide; trifluoroacetic acid (compound 87, (510 mg, purity: 64%, yield: 60%) as an off-white solid. LCMS (ESI, ms): 557 [M+H-TFA] +

[0324] Step 8. Synthesis of Compound 89 To a stirred mixture of (2S)-2-[2-(2-aminoacetamido)acetamido]-3-phenylpropanoic acid (compound 88, 2.00 g, 7.16 mmol, 1.00 equiv) and NaHCO (1.80 g, 21.41 mmol, 3.00 equiv) in HO (40.00 mL) was added BocO (1.86 g, 8.52 mmol, 1.20 equiv) in DMF (40.00 mL) dropwise at 0 °C. The resulting mixture was stirred overnight at room temperature. LCMS showed the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with EtOEt (3 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 5% to 60% in 30 min; detector, UV 220 nm. This gave (2S)-2-(2-[2-[(tert-butoxycarbonyl)amino]acetamido]acetamido)-3-phenylpropanoic acid (compound 89, 1.8 g, 60%) as a white semi-solid. LCMS (ESI, ms): 380 [M+H] + ,324[M+H-56] + . 1 HNMR:(300MHz, DMSO-d6) δ8.17(d, J=8.1Hz, 1H), 7.93(t, J=5.7Hz, 1H), 7.31-7.20(m, 5H), 7.00(t, J=6.0Hz, 1H), 4.46-4.39(m, 1H),3.78-3.67(m, 2H), 3.56(d, J =5.7Hz, 2H), 3.09-3.02(m, 1H), 2.92-2.73(m, 1H), 1.39(s, 9H).

[0325] Step 9. Compound 90. To a stirred mixture of (2S)-2-(2-[2-[(tert-butoxycarbonyl)amino]acetamido]-acetamido)-3-phenylpropanoic acid (Compound 89, 340 mg, 0.90 mmol, 1.00 equiv) and HATU (340 mg, 0.90 mmol, 1.00 equiv) in DMF (5.00 mL) was added HOBT (102 mg, 0.75 mmol, 0.84 equiv) in portions at 0° C. The resulting mixture was stirred at 0° C. for 30 minutes. To the above mixture, 2-amino-N-[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]acetamide; trifluoroacetic acid (compound 87, 511 mg, purity: 64%, 0.48 mmol, 0.54 equiv) and DIEA (340 mg, 2.63 mmol, 2.94 equiv) were added at 0° C. The resulting mixture was stirred for another 2 hours at room temperature. LCMS showed that 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), gradient from 0% to 50% in 30 minutes; detector, UV 220 nm. The collected fractions were concentrated under vacuum. This gave tert-butyl N-[[([[(lS)-1-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)carbamoyl]-2-phenylethyl]carbamoyl]methyl)carbamoyl]methyl]carbamate (compound 90, 210 mg, 48%) as an off-white solid. LCMS (ESI, ms): 918 [M+H] + , 818[M+H-100] + . 1HNMR: (400MHz, DMSO-d6): δ10.97(s, 1H), 8.79(s, 1H), 8.50(t, J=6.4Hz, 1H), 8.31(t, J=4,4Hz, 1H), 8.15(d, J =9.6Hz, 1H), 7.910(t, J=8,0Hz, 1H), 7.68-7.64(m, 2H), 7.49(s, 1H), 7.43(d, J=9.6Hz, 1H), 7.24-7.12(m, 7H), 7.00-6.95(m, 1H), 6.84(t, J=6.4Hz, 1H), 5.13-5.06(m, 1H), 4.55-4.27(m, 7H), 3.72-3.60(m, 6H), 3.75-3.67(m, 3H), 3.59-3.49(m, 5H), 3.07-3.01(m, 1H), 2.94-2.73(m, 4H), 2.62-2.54(m, 1H), 2.40-2.3l(m, 1H), 2.01-1.94(m, 1H), 2.00-1.91(m, 1H), 1.35(s, 9H)

[0326] ステップ10. Synthesis of Compound 91 To a stirred mixture of N-[[([[(1S)-l-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)-methyl]carbamoyl]methyl)carbamoyl]-2-phenylethyl]carbamoyl]methyl)carbamoyl]-methyl]carbamate (Compound 90, 140 mg, 0.15 mmol, 1.00 equiv) in DCM (5.00 mL) was added dropwise at 0° C. The resulting mixture was stirred for 30 minutes at 0° C. LCMS indicated the reaction was complete. The resulting mixture was concentrated under reduced pressure. This gave (2S)-2-[2-(2-aminoacetamido)acetamido]-N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]-carbamoyl]methyl)-3-phenylpropanamide; trifluoroacetic acid (compound 91, 140 mg, 79%) as an off-white solid. LCMS (ESI, ms): 818 [M+H-TFA] + .

[0327] Step 11. Synthesis of compound (Ij) To a stirred mixture of (2S)-2-[2-(2-aminoacetamido)acetamido]-N-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethoxy)methyl]carbamoyl]methyl)-3-phenylpropanamide; trifluoroacetic acid (Compound 91, 140 mg, 0.15 mmol, 1.00 equiv) and DIEA (70 mg, 0.54 mmol, 3.61 equiv) in DMF (2.00 mL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (Compound 92, 70 mg, 0.23 mmol, 1.50 equiv) in portions at 0° C. The resulting mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction mixture was directly purified using the following conditions: Column: XSelect CSH Prep C18 OBD column, 19 x 250 mm, 5 µm; Mobile phase A: water (0.1% TFA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 25 B to 50 B in 7 min; 254 nm; RT: 6.35 min. The collected fractions were lyophilized to give the crude product. The crude product was repurified using the following conditions: Column: Kinetex EVO C18 column, 30 x 150 mm, 5 µm; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 20 B to 40 B in 7 min, 220 nm; RT: 6.77 min. The collected fractions were lyophilized to give N-[[([[(1S)-1-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)methyl]carbamoyl]methyl)carbamoyl]-2-phenylethyl]carbamoyl]methyl)carbamoyl]methyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound (Ik), 22.8 mg, 14%) as a white solid. LCMS (ESI, ms): 1011 [M+H] + . 1HNMR:(400MHz,DMSO-d6): δ10.95(s, 1H), 8.79(s, 1H), 8.51(t, J=8,4Hz, 1H), 8.29(t, J=8.0Hz, 1H), 8.12-8.01(m, 3H), 7.70-7.66(m, 2H), 7.44(s, 1H), 7.42(d, J=8.0Hz, 1H), 7.23-7.16(m, 7H), 6.99(s, 2H), 6.82(t, J=8.0Hz, 1H), 5.13-5.09(m, 1H), 4.55-4.28(m, 7H), 3.72-3.60(m, 6H), 3.55-3.51(m, 2H), 3.36-3.34(m, 2H), 3.05-3.00(m, 1H), 2.94-2.72(m, 4H), 2.62-2.54(m, 1H), 2.40-2.32(m, 1H), 2.12-2.05(m, 2H), 2.00-1.91(m, 1H), 1.50-1.38(m, 4H), 1.19-1.10(m, 2H) [ka] [ka]

[0328] Step 1. Synthesis of Compound 94 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 93, 24.00 g, 111.32 mmol, 1.00 equiv) in THF (240.00 mL) was added BH3-Me2S (28.00 mL, 295.23 mmol, 2.65 equiv) dropwise under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. TLC (PE: EtOAc = 3:1) showed the reaction was complete. After cooling to room temperature, the resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (3:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 94, 18.00 g, 80%) as a pale yellow solid. 1 H NMR (300 MHz, CDCl3) δ8.27 (s, 1H), 8.10-8.07 (m, 1 H), 7.52 (d, J = 3 Hz, 1H), 3.96 (t, J= 6 Hz, 2H), 3.13 (t, J=6Hz, 2H).

[0329] Step 2. Synthesis of Compound 95 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 94, 5.00 g, 24.80 mmol, 1.00 equiv) in DCM (100.00 mL) was added NBS (6.62 g, 1.50 equiv) and PPh3 (9.76 g, 37.21 mmol, 1.50 equiv) in portions at room temperature under N2. The resulting mixture was stirred overnight at room temperature under N2. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (4:1) to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 95, 5.10 g, 72%) as a red oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.4, 2.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 3.79 4(t, J = 6.8 Hz, 2H), 3.38 (t, J = 6.8 Hz, 2H).

[0330] Step 3. Synthesis of Compound 96 To a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 95, 5.00 g, 18.90 mmol, 1.00 equiv) in DMF (50.00 mL) was added potassium thioacetate (2.16 g, 18.90 mmol, 1.00 equiv) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 hours. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was diluted with water (600.00 mL) and extracted with EtOAc (2000 mL × 3). The combined organic layers were washed with water (200.00 mL), brine (200.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethanone (compound 96, 4.50 g, 85%) as a red oil. 1 H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.4 Hz, 1H), 8.07 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.20 -3.05 (m, 4H), 2.34 (s, 3H).

[0331] Step 4. Synthesis of Compound 97 To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethanone (compound 96, 2.00 g, 7.70 mmol, 1.00 equiv) in MeOH (300.00 mL) was added MeONa (6.93 mL, 37.33 mmol, 5.00 equiv, 30% in MeOH) at 0 °C under N2. The resulting mixture was stirred at 0 °C for 1 h under N2. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was quenched with AcOH to a pH value of 3-4. The resulting mixture was concentrated to dryness under vacuum. The residue was diluted with DCM (50.00 mL) and filtered. The filtrate was purified by preparative TLC (PE: EtOAc = 10:1) to give 2-(2-chloro-4-nitrophenyl) ethanethiol (compound 97, 1.35 g, 72%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.4 Hz, 1H), 8.09 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.14 (t, J = 8.0Hz, 2H), 2.85 (dt, J = 8.0, 7.2 Hz, 2H), 1.43 (t, J = 7.2 Hz, 1H).

[0332] Step 5. Synthesis of Compound 99 To a stirred solution of (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (compound 98, 20.00 g, 64.24 mmol, 1.00 equiv) in DMF (200.00 mL) was added TSTU (25.18 g, 83.52 mmol, 1.30 equiv) and DIEA (16.60 g, 128.48 mmol, 2.00 equiv) at room temperature under an air atmosphere. The resulting mixture was stirred at room temperature for 1 hour. LCMS showed the reaction was complete. The reaction was diluted with water (200.00 mL) and extracted with EtOAc (100.00 mL × 3). The combined organic layers were washed with water (100.00 mL), brine (100.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting with (PE: EtOAc = 1:2) to give 2,5-dioxopyrrolidin-1-yl (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 99, 25.00 g, 83%) as a white solid. LCMS (ES, m / z): 431 [M+Na] + .

[0333] Step 6. Synthesis of Compound 100 To a solution of D-alanine (1.09 g, 0.012 mmol, 1.00 equiv) and NaHCO3 (3.09 g, 0.04 mmol, 3.00 equiv) in water (50.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 99, 5.00 g, 12.24 mmol, 1.00 equiv) in DMF (50.00 mL). The resulting mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction was adjusted to a pH value of 2-3 with 2N HCl. The resulting mixture was extracted with EtOAc (100.00 mL × 3), and the combined organic layers were washed with brine (100.00 mL × 3), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo to give (2R)-2-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido]propanoic acid (compound 100, 4.00 g, 71%) as a white solid. LCMS (ES, m / z: 383 [M+H] +

[0334] Step 7. Synthesis of Compound 101 To a solution of glycine (3.68 g, 48.97 mmol, 1.00 equiv) and NaHCO3 (12.34 g, 146.89 mmol, 3.00 equiv) in water (200.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (compound 99, 20.00 g, 48.97 mmol, 1.00 equiv) in DMF (200.00 mL). The reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction was adjusted to a pH value of 2-3 with 2N HCl. The resulting mixture was extracted with EtOAc (500.00 mL × 3), and the combined organic layers were washed with brine (500.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo to give [(2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetic acid (compound 101, 15.00 g, 71%) as a white solid. LCMS (ES, m / z): 369 [M+H] +

[0335] Step 8. Synthesis of Compound 102 A solution of [(2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]propanamide]-acetic acid (compound 101, 5.00 g, 13.57 mmol, 1.00 equiv), Pb(OAc) (7.22 g, 16.28 mmol, 1.20 equiv), and pyridine (1.29 g, 16.31 mmol, 1.20 equiv) in THF (300.00 mL) / toluene (100.00 mL) under N was stirred at 80 °C for 16 h. LCMS indicated the reaction was complete. After cooling to room temperature, the reaction was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layers were concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography eluting with (PE: EtOAc = 1:2) to give methyl [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetate (compound 102, 2.50 g, 45%) as a white solid. LCMS (ES, m / z): 405 [M+Na] + . 1 H NMR (400 MHz, chloroform-d) δ 7.77 (t, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.43 - 7.37 (m, 2H), 7.36 - 7.29 (m, 2H), 7.10 (s, 1H), 5.24 (d, J = 7.6 Hz, 2H), 4.51 - 4.35 (m, 2H), 4.23-4.09 (m, 2H), 2.04 (s, 3H), 1.39 (d, J = 6.8 Hz, 3H).

[0336] Step 9. Synthesis of Compound 103 To a stirred solution of methyl [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido]acetate (compound 102, 2.25 g, 5.88 mmol, 1.00 equiv) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 97, 1.28 g, 5.88 mmol, 1.00 equiv) in DCM (120 mL) was added TFA (0.27 mL, 2.37 mmol, 0.62 equiv) at room temperature under N. The resulting mixture was stirred at room temperature for 16 hours. LCMS indicated the reaction was complete. The reaction was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (PE: EtOAc = 1:4) to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl) ethyl] sulfanyl] methyl) carbamoyl] ethyl] carbamate (compound 103, 3.10 g, 90%) as a yellow solid. LCMS (ES, m / z): 540 [M+H] +

[0337] Step 10. Synthesis of Compound 104 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 103, 3.10 g, 5.74 mmol, 1.00 equiv) in DMF (155.00 mL) was added piperidine (31.00 mL) at 0° C. under N. The resulting mixture was stirred at 0° C. under N for 0.5 h. LCMS showed the reaction was complete. The reaction was diluted with water (600.00 ml). The resulting mixture was extracted with EtOAc (200.00 mL × 3). The combined organic layers were washed with brine (200.00 ml), dried over anhydrous NaSO, and concentrated to dryness under vacuum to give 3.00 g of crude product. The crude product was re-purified by silica gel column chromatography eluting with (DCM:MeOH=3:1) to give (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)propenamide, 104 (1.50 g, 78%) as a yellow oil. LCMS (ES, m / z): 318 [M+H] +.

[0338] Step 11. Synthesis of Compound 105 To a solution of (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)-propenamide (Compound 104, 1.50 g, 4.72 mmol, 1.00 equiv) in DMF (75.00 mL) was added NaHCO (0.59 g, 7.08 mmol, 1.50 equiv) and B in HO (10.00 mL). OC2 A solution of 1.03 g (4.72 mmol, 1.00 equiv) was added at room temperature. The reaction was stirred at room temperature for 1 hour. LCMS showed the reaction was complete. The reaction was diluted with water (500.00 mL) and extracted with EtOAc (200.00 mL × 3). The combined organic layers were washed with brine (200.00 mL × 3), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo to give tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 105, (1.82 g, 83)) as a red oil. LCMS (ES, m / z): 418 [M+H] + , 318 [M+H-100] +

[0339] Step 12. Synthesis of Compound 106 A slurry of tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)carbamoyl]ethyl]carbamate (compound 105, 1.82 g, 4.36 mmol, 1.00 equiv), iron powder (2.43 g, 0.04 mmol, 10.00 equiv), and NH4Cl (2.33 g, 0.04 mmol, 10.00 equiv) in EtOH (100.00 mL) / HO (50.00 mL) was stirred at 70 °C for 2 h. LCMS showed the reaction was complete. The reaction was filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was concentrated to dryness, and the residue was purified by silica gel column chromatography eluting with (DCM:MeOH=13:1) to give tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]-carbamate (compound 106, 1.20 g, 68%) as a yellow oil. LCMS (ES, m / z): 388 [M+H] +

[0340] Step 13. Compound 107 To a stirred solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT 1, 352 mg, 1.29 mmol, 1.00 equiv) in DMF (5.00 mL) at 0 °C, CDI (209.00 mg, 1.29 mmol, 1 equiv) and TEA (260 mg, 2.58 mmol, 2 equiv) were added. The resulting mixture was stirred at 0 °C for 2 h. Next, tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]-methyl)-carbamoyl]ethyl]carbamate (Compound 106, 500.00 mg, 1.29 mmol, 1.00 equiv) and DMAP (472 mg, 3.87 mmol, 3.00 equiv) were added. The resulting mixture was stirred at 60° C. for 24 hours. LCMS indicated the reaction was complete. After cooling to room temperature, 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), gradient from 0% to 60% in 30 minutes; detector, UV 254 nm to afford tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamate (compound 107, 450.00 mg, 48%) as a yellow solid. LCMS (ES, m / z):687 [M+H] +

[0341] Step 14. Compound 108 To a stirred solution of tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]-methyl]carbamoyl)ethyl]carbamate (Compound 107, 440.00 mg, 0.64 mmol, 1.00 equiv) in DCM (22.00 mL) was added TFA (2.20 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h. LCMS showed the reaction was complete. The reaction was concentrated to dryness in vacuo to afford (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]-methyl]propanamide; trifluoroacetic acid (compound 108, 400.00 mg, crude) as a red oil. The residue was used in the next step without further purification. LCMS (ES, m / z): 587 [M+H-TFA] +

[0342] Step 15. Synthesis of Compound 109 A solution of (2R)-2-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido]propanoic acid (218 mg, 0.57 mmol, 1.00 equiv), HOBT (77 mg, 0.57 mmol, 1.00 equiv), and HATU (216 mg, 0.01 mmol, 1.00 equiv) was stirred in air at room temperature for 1 hour, followed by (2S)-2-amino-N-[[(2 -[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propanamide; trifluoroacetic acid (compound 108, 400 mg, 0.57 mmol, 1.00 equiv) and DIEA (663 mg, 5.14 mmol, 9.00 equiv) were added at room temperature. The reaction was stirred at room temperature for 2 hours. LCMS showed 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 (0.05% TFA), gradient 0% to 50% in 30 min; detector, UV 254 nm to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(lR)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamate (compound 109, 480.00 mg, 75%) as a green solid. LCMS (ES, m / z): 951 [M+H] +

[0343] Step 16. Compound 110 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1R)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]carbamate (Compound 109, 110.00 mg) in DMF (5.00 mL) was added piperidine (1.00 mL) at 0° C. The resulting mixture was stirred at 0° C. for 0.5 hours. LCMS showed 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 (0.05% TFA), gradient 0% to 60% in 40 min; detector, UV 254 nm to give (2S)-2-[(2R)-2-[(2S)-2-aminopropanamido]propanamido]-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]-phenyl]ethyl)sulfanyl]methyl]propenamide (compound 110, 80.00 mg, 60%) as a red solid. LCMS (ES, m / z): 729 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.00 (br s, 1H), 8.53 br (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.10 (br s, 1H), 7.69 - 7.62 (m, 2H), 7.49 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.26-7.13 (m, 3H), 7.00 (br s, 1H), 5.11-5.06 (m, 1H), 4.45 - 4.36 (m, 3H), 4.35 - 4.13 (m, 6H), 2.90-2.83 (m, 3H), 2.73-2.71 (m, 2H), 2.05-1.90 (m, 1H), 1.70-1.53 ​​(m, 4H), 1.22-1.17(m, 6H), 1.14 - 1.05 (m, 3H).

[0344] ステップ17.Synthesis of compound (Ik) (2S)-2-[(2R)-2-[(2S)-2-aminopropanamido]propanamido]-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl in DMF (1.50 mL, 19.38 mmol, 224.36 equiv) To a solution of [2,5-dioxopyrrolidin-1-yl]-6-(2,5-dioxopyrrol-1-yl)hexanoate (26 mg, 0.09 mmol, 1.00 equiv.) and DIEA (22.33 mg, 0.17 mmol, 2.00 equiv.) was added in air at room temperature. The reaction was stirred at room temperature for 1 hour. The reaction mixture was purified by reverse-phase flash chromatography using the following conditions: Column: Kinetex EVO C18 column, 30 x 150, 5 μm; Mobile phase A: water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 23 B to 43 B in 7 min, 254 nm; RT: 6.58). The collected fractions were lyophilized to give N-[(1S)-1-[[(1R)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]ethyl]carbamoyl]ethyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (compound (Ik), 16.10 mg, 20%) as a white solid. LCMS (ES, m / z): 922,924 [M+H] + . 1 HNMR (400 MHz, DMSO- d6) δ 11.00 (s, 1H), 8.80 (s, 1H), 8.44-8.41 (m, 1H), 8.15 (d, J =7.2Hz, 1H), 8.03-8.00 (m, 2H), 7.7-7.65 (m, 2H), 7.51 (s, 1H), 7.44 (d, J =8.0Hz,1H), 7.22-7.14(m, 2H), 6.98 (s, 2H), 6.83-6.81 (m, 1H), 5.13-5.08 (m, 1H), 4.48-4.40 (m, 3H), 4.29-4.17 (m, 6H), 2.96-2.85 (m, 3H), 2.75-2.70 (m, 2H), 2.67-2.57 (m, 1H), 2.40-2.33 (m, 1H), 2.09-1.98 (m, 3H), 1.52-1.45 (m, 5H), 1.26-1.16 (m, 12H). [ka] [ka]

[0345] Step 1. Synthesis of Compound 112 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 111, 5.00 g, 23.19 mmol, 1.00 equiv) in THF (50 mL) was added BH3-Me2S (5.50 mL, 57.99 mmol, 2.50 equiv) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. TLC (PE: EtOAc = 3:1) showed the reaction was complete. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (2:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 112, 4.8 g, 92%) as a pale yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 8.27 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.4, 2.4 Hz, 1H), 7.46 (s, 1H), 3.20 -3.09 (m, 4H).

[0346] Step 2. Synthesis of compound 113 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 112, 4.80 g, 23.81 mmol, 1.00 equiv) in DCM (100 mL) was added NBS (6.36 g, 35.71 mmol, 1.50 equiv) and PPh3 (9.37 g, 35.72 mmol, 1.50 equiv) in portions at room temperature under air atmosphere. The resulting mixture was stirred overnight at room temperature under air atmosphere. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (4:1) to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 113, 3.9 g, 57%) as a red oil. 1 H NMR (400 MHz, chloroform-d) δ 8.29 (d, J = 2.4 Hz, 1H), 8.13 (dd, J = 8.4, 2.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 3.67 (t, J = 7.2 Hz, 2H), 3.42 (t, J = 7.2 Hz, 2H).

[0347] Step 3. Synthesis of compound 114 To a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 113, 3.90 g, 14.75 mmol, 1.00 equiv) in DMF (39 mL) was added potassium thioacetate (1.68 g, 14.75 mmol, 1.00 equiv) at room temperature. The resulting mixture was stirred at room temperature for 2 h. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was diluted with water (600 mL). The resulting mixture was extracted with EA (200 mL * 3). The combined organic layers were washed with water (200 mL), brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to dryness in vacuo to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethanone (compound 114, 3.7 g, 85%) as a red oil. 1H NMR (400 MHz, chloroform-d) δ 8.27 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.4, 2.4 Hz, 1H), 7.46 (s, 1H), 3.21–3.02 (m, 4H), 2.37 (s, 3H).

[0348] Step 4. Synthesis of Compound 115 To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethanone (Compound 114, 4.00 g, 15.40 mmol, 1.00 equiv) in MeOH (600 mL) was added MeONa (14.31 mL, 77.00 mmol, 5.00 equiv, 30%) at 0 °C under N2 for 1 h. The reaction mixture was stirred at 0 °C for 1 h. TLC showed the reaction was complete (PE:EA = 10:1). The reaction was quenched with AcOH. The resulting mixture was concentrated to dryness under vacuum. The residue was diluted with DCM (100 mL) and filtered. The filtrate was purified by silica gel column chromatography eluting with (PE:EtOAc = 10:1) to give 2-(2-chloro-4-nitrophenyl)ethanethiol (Compound 115, 3 g, 80%) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ 8.28 (d, J = 2.4 Hz, 1H), 8.11 (dd, J = 8.4, 2.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 3.17 (t, J = 7.2 Hz, 2H), 2.87 (dt, J = 8.0, 7.2 Hz, 2H), 1.46 (t, J = 8.0Hz, 1H).

[0349] Step 5. Synthesis of Compound 117 To a stirred mixture of (2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-acetamido)acetic acid (compound 116, 10 g, 28.22 mmol, 1.00 equiv) and Pb(OAc) (15 g, 33.86 mmol, 1.20 equiv) in THF (300 mL) and toluene (100 mL), pyridine (2.59 g, 32.74 mmol, 1.16 equiv) was added dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 80° C. overnight under a nitrogen atmosphere. LCMS showed the reaction was complete. The mixture was allowed to cool to room temperature. The resulting mixture was filtered, and the filter cake was washed with EA (20 mL). The filtrate was concentrated in vacuo. The residue was dissolved in EA (20 mL). The resulting mixture was washed with water, brine, and dried over anhydrous NaSO. After filtration, the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (1:4) to give methyl (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)acetate (Compound 117, 6.5 g, 56%) as a white solid. 1 HNMR(300MHz, CDCl3) δ7.80(d, J =7.5Hz, 2H), 7.62(d, J =7.5Hz, 2H), 7.45(t, d =7.5Hz, 2H), 7.36(d, d =7.5Hz, 2H), 7.18(br s, 1H), 5.48(br s, 1H), 5.28(d, J =7.2Hz, 2H), 4.48(d, J =6.6Hz, 2H), 4.26(t, J =6.6Hz, 1H), 3.93(d, 5.4Hz, 2H), 2.08(s, 3H).LCMS (ESI, ms):391[M+Na] +

[0350] Step 6. Synthesis of Compound 118 To a solution of methyl (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)acetate (compound 117, 3.00 g, 8.14 mmol, 1.00 equiv) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 115, 1.77 g, 8.13 mmol, 1.00 equiv) in DCM (300 mL) was added TFA (0.56 g, 4.91 mmol, 0.60 equiv) at room temperature. The resulting mixture was stirred at 60° C. for 16 hours. LCMS showed the reaction was complete. The reaction was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography (PE: EtOAc = 2:3) to give 9H-fluoren-9-ylmethyl N-[[([[2-(2-chloro-4-nitrophenyl) ethyl] sulfanyl] methyl) carbamoyl] -methyl] carbamate (compound 118, 3.7 g, 67%) as an off-white solid. LCMS (ES, m / z): 526, 528 [M+H] +

[0351] Step 7. Synthesis of Compound 119 To a solution of 9H-fluoren-9-ylmethyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]methyl]carbamate (compound 118, 3.70 g, 7.03 mmol, 1.00 equiv) in DMF (40 mL) was added piperidine (8 mL) at 0° C. The resulting mixture was stirred at 0° C. for 0.5 h. LCMS showed the reaction was complete. The resulting mixture was diluted with water (400 mL) and extracted with EA (200 mL×3). The combined organic layers were washed with water (200 mL), brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting with (DCM:MeOH=10:1) to give 2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)acetamide (Compound 119, 1.01 g, 40%) as a yellow oil. LCMS (ES, m / z): 304, 306 [M+H] +

[0352] Step 8. Synthesis of Compound 120 To a solution of 2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)-acetamide (Compound 119, 1.00 g, 3.29 mmol, 1.00 equiv) in DMF (50 mL) was added NaHCO3 (0.33 g, 3.92 mmol, 1.20 equiv) and B in water (10 mL). OC2 A solution of 2H2O (0.72 g, 3.30 mmol, 1.00 equiv) was added at room temperature. The resulting mixture was stirred at room temperature for 1 hour. LCMS showed the reaction was complete. The reaction was diluted with water (500 mL) and extracted with EtOAc (200 mL x 3). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting with (PE: EtOAc = 1:3) to afford tert-butyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]methyl]carbamate (compound 120, 810 mg, 54%) as a white solid. LCMS (ES, m / z): 404, 406 [M+H] + , 304,306 [M+H-100] +

[0353] Step 9. Synthesis of Compound 121 To a solution of tert-butyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)carbamoyl]methyl]carbamate (compound 120, 800.00 mg, 1.98 mmol, 1.00 equiv) in EtOH (40) was added iron powder (1106 mg, 19.81 mmol, 10.00 equiv) and a solution of NH4Cl (1059 mg, 19.81 mmol, 10.00 equiv) in water (10 mL) at room temperature. The resulting mixture was stirred at 70 °C for 2 h. LCMS showed the reaction was complete. The reaction was filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was purified by silica gel column chromatography (DCM:MeOH=13:1) to give tert-butyl N-[[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]methyl)-carbamoyl]methyl]carbamate (compound 121, 610 mg, 74%) as a yellow oil. LCMS (ES, m / z): 374, 376 [M+H] + , 374,376 [M+H-100] +

[0354] Step 10. Synthesis of Compound 122 To a solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT 1, 219 mg, 0.80 mmol, 1.00 equiv.) in DMF (10 mL), CDI (130 mg, 0.80 mmol, 1.00 equiv.) and TEA (81 mg, 0.80 mmol, 1.00 equiv.) were added in air at 0 °C. The resulting mixture was stirred at room temperature for 2 h. Next, tert-butyl N-[[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]-methyl)carbamoyl]methyl]carbamate (Compound 121, 300 mg, 0.80 mmol, 1.00 equiv.) and DMAP (294 mg, 2.41 mmol, 3.00 equiv.) were added in air at room temperature. The resulting mixture was stirred at 60° C. for 48 hours. LCMS showed the reaction was complete. The resulting mixture was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 60% in 30 minutes; detector, UV 254 nm to give tert-butyl N-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)methyl]carbamate (compound 122, 270 mg, 49%) as a yellow solid. LCMS (ES, m / z): 673,675 [M+H] + , 573,575 [M+H-100] +

[0355] Step 11. Synthesis of Compound-123 To a solution of tert-butyl N-[([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)methyl]-carbamate (Compound 122, 250 mg, 0.37 mmol) in 1,4-dioxane (12 mL) was added HCl (4N in 1,4-dioxane, 6 mL) at 0° C. under N. The reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness in vacuo to give 2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]acetamide (compound 123, 260 mg, crude) as a brown solid. LCMS (ES, m / z): 573,575 [M+H-HC1] +

[0356] Step 12. Synthesis of Compound-125 A solution of (2S)-2-[2-(2-aminoacetamido)acetamido]-3-phenylpropanoic acid (compound 124, 500 mg, 1.79 mmol, 1.00 equiv) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (552 mg, 1.79 mmol, 1.00 equiv) in DMSO (5.00 mL) was stirred at room temperature under air for 16 hours. LCMS showed 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), gradient 0% to 60% in 30 min; detector, UV 220 nm to give (2S)-2-(2-[2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]acetamido]acetamido)-3-phenylpropanoic acid (compound 125, 760 mg, 83%) as a white solid. LCMS (ES, m / z): 473 [M+H] +

[0357] Step 13. Synthesis of Compound (II) To a solution of (2S)-2-(2-[2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]acetamido]-acetamido)-3-phenylpropanoic acid (Compound 125, 175 mg, 0.37 mmol, 1.00 equiv) in DMF (5.00 mL) was added HATU (141 mg, 0.37 mmol, 1.00 equiv) and HOBT (50 mg, 0.37 mmol, 1.00 equiv) at room temperature in air. The resulting mixture was stirred at room temperature for 1 hour. Then, 2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]acetamide (Compound 123, 250 mg, 0.37 mmol, 1.00 equiv, 85%) and DIEA (240 mg, 1.85 mmol, 5.00 equiv) were added. The resulting mixture was stirred at room temperature for 1 hour. LCMS showed the reaction was complete. The reaction mixture was purified under the following conditions: Column: XSelect CSH Prep C18 OBD column, 19*250 mm, 5 μm; Mobile phase A: water (0.05% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 30 B to 60 B in 7 min, 254 nm; RT: 6.67 min to give 75 mg of crude product. The crude product was repurified by reverse-phase flash chromatography under the following conditions: Column: XBridge Shield RP18 OBD column, 19*250 mm, 10 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL / min; Gradient: 25 B to 44 B in 10 min, 254 nm; RT: 10.52 min. The collected fractions were lyophilized to give Compound (II) (41.6 mg, 10%) as a white solid. 1 HNMR(400MHz, DMSO-d6) δ10.99 (s, 1H), 8.79 (s, 1H), 8.38 (t, J =6.0Hz, 1H), 8.31 (t, J =6.0Hz, 1H), 8.12 (d, J =8.4Hz, 1H), 8.06 (t, J =5.6Hz, 1H), 8.01 (t, J =6.0Hz, 1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J =8.0Hz, 1H), 7.25-7.21 (m, 5H), 7.19-7.14 (m, 2H), 6.99 (s, 2H), 6.82 (t, J =6.0Hz,lH), 5.13-5.08 (m, 1H), 4.47-4.40 (m, 4H), 4.33-4.29 (m, 3H), 3.76-3.70 (m, 3H), 3.67-3.55 (m, 3H), 3.38-3.36 (m, 2H), 3.06-3.02 (m, 1H), 2.91-2.86 (m,3H), 2.82-2.70 (m, 3H), 2.62-2.57 (m, 1H), 2.50-2.45 (m, 1H), 2.10 (m, 2H), 2.05-1.95 (m,1H), 1.50-1.44 (m,4H), 1.20-1.16 (m, 2H).LCMS (ES, m / z):1027,1029 [M+H] + [ka] [ka]

[0358] Step 1. Synthesis of Compound 127 To a stirred solution of (2-chloro-4-nitrophenyl)acetic acid (compound 126, 24.00 g, 111.32 mmol, 1.00 equiv) in THF (240.00 mL) was added BH3-Me2S (28.00 mL, 295.23 mmol, 2.65 equiv) dropwise under a nitrogen atmosphere. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 2 h. TLC (PE: EtOAc = 3:1) showed the reaction was complete. After cooling to room temperature, the resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (3:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (compound 127, 18.00 g, 80%) as a pale yellow solid. 1 H NMR (300 MHz, CD3C1) δ8.27 (s, 1H), 8.10-8.07 (m, 1 H), 7.52 (d, J = 3 Hz, 1H), 3.96 (t, J = 6 Hz, 2H), 3.13 (t, J = 6 Hz, 2H).

[0359] Step 2. Synthesis of Compound 128 To a stirred solution of 2-(2-chloro-4-nitrophenyl)ethanol (compound 127, 5.00 g, 24.80 mmol, 1.00 equiv) in DCM (100.00 mL) was added NBS (6.62 g, 1.50 equiv) and PPh3 (9.76 g, 37.21 mmol, 1.50 equiv) in portions at room temperature under N2. The resulting mixture was stirred overnight at room temperature under N2. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (4:1) to give 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 128, 5.10 g, 72.31%) as a red oil. 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.4, 2.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 3.79 (t, J = 7.2 Hz, 2H), 3.38 (t, J = 7.2 Hz, 2H).

[0360] Step 3. Synthesis of Compound 129 To a solution of 1-(2-bromoethyl)-2-chloro-4-nitrobenzene (compound 128, 5.00 g, 18.90 mmol, 1.00 equiv) in DMF (50.00 mL) was added potassium thioacetate (2.16 g, 18.91 mmol, 1.00 equiv) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 hours. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was diluted with water (600.00 mL). The resulting mixture was extracted with EtOAc (200.00 mL*3). The combined organic layers were washed with water (200.00 mL), brine (200.00 mL*3), dried over anhydrous NaSO and concentrated to dryness under vacuum to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethanone (compound 129, 4.50 g, 85%) as a red oil. 1 H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.4 Hz, 1H), 8.07 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.20 - 3.05 (m, 4H), 2.34 (s, 3H).

[0361] Step 4. Synthesis of Compound 130 To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethanone (compound 129, 2.00 g, 7.70 mmol, 1.00 equiv) in MeOH (300.00 mL) was added MeONa (6.93 mL, 37.33 mmol, 5.00 equiv, 30%) at 0 °C under N2. The resulting mixture was stirred at 0 °C under N2 for 1 h. TLC (PE: EtOAc = 10:1) showed the reaction was complete. The reaction was quenched with AcOH to a pH value of 3-4. The resulting mixture was concentrated to dryness under vacuum. The residue was diluted with DCM (50.00 mL) and filtered. The filtrate was purified by preparative TLC (PE: EtOAc = 10:1) to give 2-(2-chloro-4-nitrophenyl) ethanethiol (compound 130, 1.35 g, 72%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.4 Hz, 1H), 8.09 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 3.14 (dd, J = 8.0, 6.8 Hz, 2H), 2.85 (dt, J = 8.0, 7.2 Hz, 2H), 1.43 (t, J = 8.0 Hz, 1H).

[0362] Step 5. Synthesis of Compound 132 To a stirred solution of (2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]propanoic acid (compound 131, 20.00 g, 64.24 mmol, 1.00 equiv) in DMF (200.00 mL) was added TSTU (25.18 g, 83.52 mmol, 1.30 equiv) and DIEA (16.60 g, 128.48 mmol, 2.00 equiv) at room temperature under an air atmosphere. The resulting mixture was stirred at room temperature for 1 h. LCMS showed the reaction was complete. The reaction was diluted with water (200.00 mL), and the resulting mixture was extracted with ETOAC (100.00 mL*3). The combined organic layers were washed with water (100.00 mL), brine (100.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting with (PE: EtOAc = 1:2) to give 2,5-dioxopyrrolidin-1-yl (2S)-2-[[(9H-fluoren-9-ylmethoxy) carbonyl] amino] propanoate (compound 132, 25.00 g, 83%) as a white solid. LCMS (ES, m / z): 431 [M+Na] +

[0363] Step 6. Synthesis of Compound 133 To a solution of glycine (3.68 g, 48.97 mmol, 1.00 equiv) and NaHCO3 (12.34 g, 146.89 mmol, 3.00 equiv) in water (200.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (Compound 132, 20.00 g, 48.97 mmol, 1.00 equiv) in DMF (200.00 mL). The reaction was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The pH of the reaction was adjusted to 2-3 with 2N HCl. The resulting mixture was extracted with EtOAc (500.00 mL*3), and the combined organic layers were washed with brine (500.00 mL), dried over anhydrous Na2SO4, and concentrated to dryness in vacuo to give [(2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetic acid (compound 133, 15.00 g, 71%) as a white solid. LCMS (ES, m / z): 369 [M+H] +

[0364] Step 7. Synthesis of Compound 134 A solution of [(2S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-propanamido]acetic acid (compound 133, 5.00 g, 13.57 mmol, 1.00 equiv), Pb(OAc) (7.22 g, 16.28 mmol, 1.20 equiv), and pyridine (1.29 g, 16.31 mmol, 1.20 equiv) in THF (300.00 mL) / toluene (100.00 mL) under N was stirred at 80 °C for 16 h. LCMS showed the reaction was complete. After cooling to room temperature, the reaction was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layers were concentrated to dryness under vacuum. The residue was purified by silica gel column chromatography eluting with (PE:ETOAC=1:2) to give methyl [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetate (compound 134, 2.50 g, 45%) as a white solid. LCMS (ES, m / z): 405 [M+Na] + . 1H NMR (400 MHz, chloroform-d) δ 7.77-7.73 (m, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.43 - 7.37 (m, 2H), 7.36 - 7.29 (m, 2H), 7.10 (s, 1H), 5.24 (d, J = 7.6 Hz, 2H), 4.51 - 4.35 (m, 2H), 4.22 (t, J = 6.8 Hz, 2H), 2.04 (s, 3H), 1.39 (d, J = 6.8 Hz, 3H).

[0365] Step 8. Synthesis of Compound 135 To a stirred solution of methyl [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-propanamido]acetate (compound 134, 2.25 g, 5.88 mmol, 1.00 equiv) and 2-(2-chloro-4-nitrophenyl)ethanethiol (compound 500, 1.28 g, 5.88 mmol, 1.00 equiv) in DCM (120 mL) was added TFA (0.27 mL, 2.376 mmol, 0.62 equiv) at room temperature under N. The resulting mixture was stirred at 40 °C for 16 h. LCMS showed the reaction was complete. The reaction was concentrated to dryness in vacuo to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 135, 3.10 g, 90%) as a yellow solid. LCMS (ES, m / z): 540,542 [M+H] +.

[0366] Step 9. Synthesis of Compound 136 To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (Compound 135, 3.10 g, 5.74 mmol, 1.00 equiv) in DMF (155.00 mL) was added piperidine (31.00 mL) at 0° C. under N. The resulting mixture was stirred at 0° C. under N for 0.5 h. LCMS showed the reaction was complete. The reaction was diluted with water (600.00 ml). The resulting mixture was extracted with EA (200.00 mL × 3). The combined organic layers were washed with brine (200.00 ml), dried over anhydrous NaSO, and concentrated to dryness in vacuo to give 3.00 g of crude product. The crude product was re-purified by silica gel column chromatography eluting with (DCM:MeOH=3:1) to give (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)propenamide (compound 136, 1.50 g, 78%) as a yellow oil. LCMS (ES, m / z): 318, 320 [M+H] +.

[0367] Step 10. Synthesis of Compound 137 To a solution of (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]-methyl)propenamide (compound 136, 1.50 g, 4.72 mmol, 1.00 equiv) in DMF (75.00 mL) was added a solution of NaHCO3 (0.59 g, 7.08 mmol, 1.50 equiv) and Boc2O (1.03 g, 4.72 mmol, 1.00 equiv) in HO (10.00 mL) at room temperature under air. The reaction was stirred at room temperature for 1 hour. LCMS showed the reaction was complete. The reaction was diluted with water (500.00 mL) and extracted with EtOAc (200.00 mL × 3). The combined organic layers were washed with brine (200.00 mL*3), dried over anhydrous Na2SO4, and concentrated to dryness under vacuum to give tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)-carbamoyl]ethyl]carbamate (compound 137, 1.82 g, 83%) as a red oil. LCMS (ES, m / z): 418,420 [M+H] + , 318,320 [M+H-100] +

[0368] Step 11. Synthesis of Compound 138 A slurry of tert-butyl N-[(1S)-1-[([[2-(2-chloro-4-nitrophenyl)ethyl]-sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 137, 1.82 g, 4.36 mmol, 1.00 equiv), iron powder (2.43 g, 0.04 mmol, 10.00 equiv), and NH4Cl (2.33 g, 0.04 mmol, 10.00 equiv) in EtOH (100.00 mL) / HO (50.00 mL) was stirred at 70 °C for 2 h. LCMS showed the reaction was complete. The reaction was filtered. The filtrate was concentrated to dryness under vacuum. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was purified by silica gel column chromatography (DCM:MeOH=13:1) to give tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]methyl)carbamoyl]ethyl]carbamate (compound 138, 1.20 g, 68%) as a yellow oil. LCMS (ES, m / z): 388, 390 [M+H] + , 288,290 [M+H-100] +

[0369] Step 12. Synthesis of Compound 139 To a stirred solution of 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT 1, 352 mg, 1.29 mmol, 1.00 equiv) in DMF (5.00 mL) at 0 °C, CDI (209.00 mg, 1.29 mmol, 1 equiv) and TEA (260 mg, 2.58 mmol, 2 equiv) were added. The resulting mixture was stirred at 0 °C for 2 h. Next, tert-butyl N-[(1S)-1-[([[2-(4-amino-2-chlorophenyl)ethyl]sulfanyl]-methyl)carbamoyl]-ethyl]carbamate (Compound 138, 500.00 mg, 1.29 mmol, 1.00 equiv) and DMAP (472 mg, 3.87 mmol, 3.00 equiv) were added. The resulting mixture was stirred at 60° C. for 24 hours. LCMS indicated the reaction was complete. After cooling to room temperature, 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), gradient from 0% to 60% in 30 minutes; detector, UV 254 nm to afford tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamate (compound 139, 450.00 mg, 48%) as a yellow solid. LCMS (ES, m / z): 687,689 [M+H] + , 587,589 [M+H-100] +

[0370] Step 13. Synthesis of Compound 140 To a stirred solution of tert-butyl N-[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]-methyl]carbamoyl)ethyl]carbamate (Compound 139, 440.00 mg, 0.64 mmol, 1.00 equiv) in DCM (22.00 mL) was added TFA (2.20 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h. LCMS showed the reaction was complete. The reaction was concentrated to dryness in vacuo to give (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propanamide; trifluoroacetic acid (compound 140, 400.00 mg) as a red oil. LCMS (ES, m / z): 578,589 [M+H-TFA] +

[0371] Step 14. Synthesis of Compound 142 To a slurry of L-valine (compound 141, 0.50 g, 4.27 mmol, 1.00 equiv) in DMSO (10 mL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (1.32 g, 4.28 mmol, 1.00 equiv) and DIEA (1103 mg, 8.54 mmol, 2.00 equiv). The resulting mixture was stirred at room temperature for 4 hours. LCMS showed 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), gradient 0% to 60% in 30 min; detector, UV 220 nm to give (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanoic acid (compound 142, 1.2 g, 72%) as a brown solid. LCMS (ES, m / z): 311 [M+H] +

[0372] Step 15. Synthesis of compound (Im) A solution of (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanoic acid (compound 142, 59 mg, 0.19 mmol, 1.00 equiv.), HOBT (26 mg, 0.19 mmol, 1.00 equiv.), and HATU (72 mg, 0.19 mmol, 1.00 equiv.) in DMF (2 mL) was stirred in air at room temperature for 1 h. Then, (2S)-2-amino-N-[[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl)sulfanyl]methyl]propanamide trifluoroacetic acid (compound 140, 200 mg, 0.19 mmol, 1.00 equiv, 66.70%) and DIEA (197 mg, 1.52 mmol, 8.00 equiv) were added at room temperature. The reaction mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The resulting mixture was purified by reversed-phase flash chromatography under the following conditions: Column: YMC-Actus Triart C18, 30 mm × 150 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient: 28 B to 45 B in 10 min, 254 nm; RT1: 9.67 min. The collected fractions were lyophilized to give N-[(1S)-1-[[(1S)-1-([[(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]-phenyl]ethyl)sulfanyl]methyl]carbamoyl)ethyl]carbamoyl]-2-methylpropyl]-6-(2,5-dioxopyrrol-1-yl)hexanamide (Compound (Im), 27.8 mg, 16%) as a white solid. LCMS (ES, m / z): 879,881 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ10.99 (s, 1H), 8.80 (s, 1H), 8.47 (t, J=6.0Hz, 1H), 8.03 (d, J =7.2Hz, 1H), 7.78 (d, J =8.8Hz,1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J =8.0Hz, 1H), 7.21-7.14 (m, 2 H), 6.99 (s, 2H), 6.82 (t, J=6.0Hz, 1H), 5.13-5.10 (m,1 H), 4.47-4.40 (m, 3H), 4.33-4.29 (m, 3H), 4.24 (t, J=7.2 Hz, 1H), 4.14 (t, J =6.8Hz, 1H), 3.38-3.36 (m, 1H), 2.97-2.90 (m, 1H), 2.86 (t, J=7.6Hz, 2H), 2.73-2.67 (m, 2H), 2.62-2.57 (m, 1H), 2.40-2.35 (m, 1H), 2.20-2.05 (m, 2H), 2.02-1.96 (m, 1H), 1.95-1.88 (m, 1H), 1.48-1.46 (m, 4H), 1.23-1.16 (m, 6H), 0.83-0.78 (m, 6H).

[0373] Example 4. General procedure for preparation and characterization of NeoDegrader conjugates The antibody solution was treated with 30 equivalents of tris-(2-carboxyethyl)phosphine (TCEP) and incubated for 1 hour at 37°C to reduce interchain disulfides. The reduced antibody was purified using an illustra NAP column (GE Healthcare) into a buffer of 50 mM EPPS, 5 mM EDTA, pH 7.0.

[0374] Conjugation occurred by treating a 2-5 mg / mL solution of reduced antibody in 50 mM EPPS, 5 mM EDTA pH 7.0 with 12 equivalents of linker-neoDegrader, added as a stock solution in N,N-dimethylacetamide (DMA) such that the final DMA concentration was 15% (v / v). The resulting reaction mixture was left overnight at 4°C. The resulting newDegrader conjugate was purified using an illustra NAP column (GE Healthcare) into 20 mM succinate, 8% sucrose, 0.01% Tween®-20 pH 5.5 and concentrated using Amicon Ultra centrifugal concentrators with a 50 kD molecular weight cutoff (Millipore).

[0375] Concentration and monomer were determined by size-exclusion chromatography using a 7.8 × 300 mm TSKGel 3000SWXL column (Tosoh Bioscience) with 5 μm particles, run at 0.5 mg / mL for 30 min, and eluted isocratically with a mobile phase of 400 mM sodium perchlorate, 50 mM sodium phosphate, and 5% (v / v) isopropanol. NeoDegrader conjugates were quantified from an antibody standard curve and detected at 214 nm.

[0376] The drug-to-antibody ratio (DAR) was determined by hydrophobic interaction chromatography using a 4.6 x 35 mm TSKgel Butyl-NPR column with 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 from 0 to 100% B in 12 min at a flow rate of 0.6 mL / min. Detection was at 214 nm.

[0377] Free linker-payload was determined by mixed-mode chromatography using a 4.6 x 250 mm HISEP column (Supelco) with 2.5 μm particles. Mobile phase A was 100 mM ammonium acetate. Mobile phase B was 100% acetonitrile. Analytes were eluted with a gradient of 25 to 40% B in 25 min, followed by 40 to 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 and detected at 254 nm.

[0378] Example 5. General Procedure 1 for In Vitro Antiproliferative Assay of NeoDegrader and NeoDegrader Conjugates The ability of NeoDegrader conjugates to inhibit cell growth was measured using an in vitro proliferation prevention assay. Target cells were plated at 1,500–5,000 cells per well in 100 μL of complete cell growth medium (RPMI 1640, 10% fetal bovine serum, and 1% penicillin-streptomycin for most cell lines; Hybri-care medium, 1.5 g / L sodium bicarbonate, 10% fetal bovine serum, and 1% penicillin-streptomycin for BT-474; RPMI 1640, 20% fetal bovine serum, and 1% penicillin-streptomycin for HL-60). The conjugates were diluted using a four-fold serial dilution in complete cell growth medium, and 100 μL was added per well. The final concentration was typically 1 × 10 -8 M~1.53×10 -13 M, or 1 x 10 -7 M~1.53×10 -12The concentrations ranged from 100 to 1000 M. Cells were incubated at 37°C in a humidified 5% CO2 incubator for 5 days. The remaining cell viability was determined by a colorimetric WST-8 assay (Dojindo Molecular Technologies, Inc., Rockville, MD, US). WST-8 was added to 10% of the final volume, and the plates were incubated at 37°C in a humidified 5% CO2 incubator for 2 to 4 hours. The plates were analyzed by measuring absorbance at 450 nm (A450) in a multiwell plate reader. The background A450 absorbance of wells containing medium and WST-8 alone was subtracted from all values. Percent survival was calculated by dividing each treated sample value by the average of wells containing untreated cells. Percent survival values ​​were plotted against test sample concentration on a semi-logarithmic plot for each treatment. IC50 values ​​were calculated automatically.

[0379] The antiproliferative activity of trastuzumab and pertuzumab conjugates of compounds (Ia) and (Ic) against the BT-474 breast cancer cell line is shown in Figures 1-4 (drug:antibody ratio = 8 for each neoDegrader conjugate). The antibody-drug conjugate Kadcyla and the unconjugated antibodies trastuzumab and pertuzumab were found to be over 100-fold less active than the antibody neoDegrade conjugates, while the non-cell-bound control neoDegrade conjugates rituximab-compound (Ia) and released neoDegraders P1 and P4a were found to be over 1000-fold less active against BT-474 cells.

[0380] The antiproliferative activity of trastuzumab and pertuzumab conjugates of Compound (Ia) and Compound (Ic) against the BT-474 breast cancer cell line is shown in Figures 5-6 (drug:antibody ratios are indicated). The antibody-drug conjugate Enhertz® and the unconjugated antibody trastuzumab were found to be less active than the antibody neoDegrader conjugate against BT-474 cells.

[0381] The antiproliferative activity of trastuzumab and pertuzumab conjugates of compound (Ia) against the SK-BR-3 breast cancer cell line is shown in Figures 7 and 8 (drug:antibody ratio = 8 for each neoDegrader conjugate). The conjugated neoDegraders had activity similar to that of the antibody-drug conjugate Kadcyla, while the unconjugated antibodies trastuzumab and pertuzumab were found to be significantly less active than the neoDegrader conjugates. The non-cell-bound control NeoDegrader conjugate rituximab-compound (Ia) and the released neoDegraders P1 and P4a were found to be significantly less active against SK-BR-3 cells.

[0382] The antiproliferative activity of OR000213, huMy9-6, and lintuzumab IgG1 conjugates of compounds (Ia) and (Id) against the HL-60 (acute myeloid leukemia) cell line is shown in Figures 9-12 (drug:antibody ratio = 8 unless otherwise specified). The neoDegrader conjugates were shown to have activity similar to the approved drug Mylotarg® against the cell line, while the non-cell-bound control neoDegrader conjugates trastuzumab-compound (Ia) and rituximab-compound (Id) were significantly less active.

[0383] The antiproliferative activity of rituximab conjugates of compounds (Ia) and (Ic) against the Ramos (non-Hodgkin's lymphoma) cell line is shown in Figure 13 (drug:antibody ratio = 8 unless otherwise specified). Unconjugated rituximab, the non-cell-bound control neoDegrader conjugate trastuzumab-compound (Ia), and released neoDegraders P1 and P4 were shown to be less active against this cell line than the neoDegrader conjugates.

[0384] The antiproliferative activity of rituximab conjugates of compounds (Ia) and (Ic) against the Daudi lymphoma cell line is shown in Figures 14 and 15 (drug:antibody ratio = 8 unless otherwise specified). Unconjugated rituximab, the non-cell-bound control neoDegrader conjugate trastuzumab-compound (Ia), and released neoDegrader P1 were shown to be less active against this cell line than the neoDegrader conjugates.

[0385] The antiproliferative activity of trastuzumab and pertuzumab conjugates of compound (Ia) against the NCI-N87 gastric cancer cell line is shown in Figures 16 and 17 (drug:antibody ratio = 8 for each neoDegrader conjugate). The conjugated neoDegraders had activity similar to that of the antibody-drug conjugate Kadcyla, while the unconjugated antibodies trastuzumab and pertuzumab were found to be significantly less active than the neoDegrader conjugates. The non-cell-bound control NeoDegrader conjugate rituximab-compound (Ia) and the released neoDegrader P1 were found to be significantly less active against NCI-N87 cells.

[0386] Figure 18 shows the antiproliferative activity of trastuzumab and pertuzumab conjugates of Compound (Ia) in BT-464 breast cancer cells after 3 days of incubation with human serum, compared to the activity of the conjugates in the absence of serum. As shown in the graph, the activity of the neoDegrader conjugates was similar in the presence and absence of serum, indicating that human serum does not affect activity. The non-cell-bound control neoDegrader conjugate, OR000213-Compound (Ia), was over 1000-fold less active against this cell line.

[0387] Figure 19 shows the antiproliferative activity of trastuzumab and pertuzumab conjugates of Compound (Ia) in BT-464 breast cancer cells after 3 days of incubation with mouse serum, compared to the activity of the conjugates in the absence of serum. As shown in the graph, the activity of the neoDegrader conjugates was similar in the presence and absence of serum, indicating that mouse serum did not affect activity. The non-cell-bound control neoDegrader conjugate, OR000213-Compound (Ia), was over 1000-fold less active against this cell line.

[0388] Tables 1 and 2 show the IC50 values ​​of trastuzumab conjugates of compounds (Ia), (Ib), (Ic), and (Id) and pertuzumab conjugates of compounds (Ia) and (Ic) against various Her2 cell lines. As shown in Table 1, the neoDegrader conjugates exhibited improved activity in the BT-474 cell line compared to the unconjugated antibody, and also exhibited improved activity against the released payload and antibody-drug conjugates Kadcyla and Enhertz®. The neoDegrader conjugates also exhibited better activity against the SK-BR-3 breast cancer cell line and the NCI-N87 gastric cell line compared to the unconjugated antibody. As shown in Table 2, the antibody-neoDegrader conjugates also had improved activity against the SNU-182 liver cell line compared to the unconjugated antibody or Kadcyla. [Table 1] [Table 2]

[0389] Table 3 shows the activity of antibody neoDegrader conjugates in anti-CD20 cell lines. The antibody neoDegrader conjugates had superior activity against Daudi and Ramos lymphocyte cell lines compared to unconjugated antibody, the non-cell-binding control neoDegrade conjugate trastuzumab-compound I(a), and the released payload. [Table 3]

[0390] Table 4 shows the IC50 values ​​of huMy9-6 and OR000213 conjugates of compounds (Ia) and (Id), and lintuzumab IgG1 conjugates of compounds (Ia) and (Id), against the AML HL-60 cell line. As shown in Table 4, the neoDegrader conjugates showed comparable activity compared to MYLOTARG® and improved activity over the non-binding conjugate, rituximab-compound (Ic), in the HL-60 cell line. [Table 4]

[0391] Table 5 shows the antiproliferative activity of trastuzumab and pertuzumab Compound (Ia) conjugates against the BT-474 breast cancer cell line after incubation with human or mouse serum, compared to the non-cell-bound control neoDegrader conjugate OR000213-Compound (Ia). As shown in the table, the activity of neoDegrader incubated in human or mouse serum was consistent with the activity in the absence of serum. [Table 5]

[0392] Example 6. General Procedure 2 for In Vitro Antiproliferative Assay of NeoDegrader and NeoDegrader Conjugates Cell culture: Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and maintained according to the culture conditions specified by the ATCC or DSMZ. Cells were thawed and maintained in culture for at least two passages before proceeding with experimental conditions.

[0393] Cytotoxicity assay: For adherent cell lines, cells were dissociated with enzyme-free PBS-based cell dissociation buffer (Gibco, USA) and plated into tissue culture-treated 96-well flat-bottom polystyrene plates (Costar, Corning, USA) at an appropriate cell density depending on the cell doubling time. 18 hours after plating, cells were treated with the appropriate concentration of test article, starting at 100 nM, followed by four-fold serial dilutions. For suspension cell lines, cells were seeded on the day of treatment and treated as described above. Adherent cells were treated for 5 days, and suspension cells were treated for 3 days. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan), and measurements were acquired using a Promega GloMax Discover plate reader (Promega, USA). Data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). All data points were obtained from three technical replicates, and experiments were validated using three biological replicates.

[0394] Table 6 shows the activity of neoDegraders P1, P3, and P4 against various cancer cell lines. As shown in the table, the neoDegraders have activity against each of the cell lines. [Table 6]

[0395] Table 7 shows the activity of the pertuzumab-compound (Ia) conjugate and known antibody-drug conjugates, Enhertz®, against various breast cancer cell lines. As shown in the table, the pertuzumab-compound (Ia) conjugate was more active in all reported cell lines. [Table 7]

[0396] Table 8 shows the activity of the pertuzumab-compound (Ia) conjugate and known antibody-drug conjugates against three gastric cancer cell lines. As shown in the table, the pertuzumab-compound (Ia) conjugate was more active in all reported cell lines. [Table 8]

[0397] Table 9 shows the activity of the OR000213-Compound (Ia) conjugate and known antibody-drug conjugate Mylotarg against various acute myeloid leukemia cell lines. As shown in the table, the OR000213-Compound (Ia) conjugate had better activity in some of the cell lines. [Table 9]

[0398] Table 10 shows the activity of three anti-CD38 neoDegrader conjugates against various multiple myeloma cell lines. As shown in the table, the conjugates had good activity in all of the cell lines. [Table 10]

[0399] Table 11 shows the activity of anti-CD138 neoDegrader conjugates against various multiple myeloma cell lines. As shown in the table, the conjugates had good activity in all of the cell lines. [Table 11]

[0400] Table 12 shows the activity of the anti-BCMA neoDegrader conjugates against various multiple myeloma cell lines. As shown in the table, the conjugates had good activity in all of the cell lines. [Table 12]

[0401] Table 13 shows the activity of the anti-Trop-2 neoDegrader conjugate against various cancer cell lines. As shown in the table, the conjugate had good activity in all of the cell lines. [Table 13]

[0402] Table 14 shows the activity of the anti-FGFR4 neoDegrader conjugate against two cancer cell lines. As shown in the table, the conjugate had good activity in both cell lines and had better activity than the US-1784 antibody and unconjugated neoDegrader alone. [Table 14]

[0403] Table 15 shows the activity of the anti-EGFR neoDegrader conjugates against two synovial sarcoma cell lines. As shown in the table, the conjugates had good activity in both cell lines and had better activity than cetuximab and unconjugated neoDegrader alone. [Table 15]

[0404] Table 16 shows the activity of the anti-PDGF-R alpha neoDegrader conjugate against two cancer cell lines. As shown in the table, the conjugate had good activity in both cell lines. [Table 16]

[0405] Table 17 shows the activity of anti-TEM1 / CD248 neoDegrader conjugates against rhabdomyosarcoma cell lines. As shown in the table, the conjugates had good activity against the cell lines. [Table 17]

[0406] Example 7: Treatment of breast cancer with anti-Her2 antibody-neoDegrader conjugate Trastuzumab-compound (Ia) conjugate and pertuzumab-compound (Ia) conjugate were injected into immunodeficient mice (Fox Chase SCID®, CB17 / Icr-Prkdc scid Tested in IcrIcoCrl (Charles River). 3 BT474 human breast cancer fragments were implanted subcutaneously into the right flank of mice. Tumors were 100–150 mm 3 Once the average size of the neodegrader conjugates reached 100%, the mice were dosed with anti-Her2 antibody-neoDegrader conjugates, non-targeting neoDegrader conjugates, and vehicle control.

[0407] Stock solutions of trastuzumab-compound (Ia) conjugate, rituximab-compound (Ia) conjugate, and pertuzumab-compound (Ia) conjugate were diluted with vehicle to obtain a 0.5 mg / mL dosing solution providing 5 mg / kg at a dosing volume of 10 mL / kg adjusted to each animal's body weight (0.2 mL per 20 g mouse).

[0408] Mice were divided into four treatment groups (N=8 / group) as follows: 1) vehicle, 2) trastuzumab-compound (Ia) conjugate (5 mg / kg, iv, qd x 1), 3) rituximab-compound (Ia) conjugate (5 mg / kg, iv, qd x 1), 4) pertuzumab-compound (Ia) conjugate (5 mg / kg, iv, qd x 1). All test articles were administered intravenously (iv) as a single dose (qd x 1) in a volume adjusted to body weight (0.200 mL / 20 g mouse).

[0409] Tumors were measured twice weekly using calipers and each animal was monitored until its tumor reached its endpoint volume (1000 mm 3 ) or on the final day of the study (day 60), whichever came first. MTV (n) was defined as the median tumor volume on the final day of the study for the remaining number (n) of animals whose tumors had not reached the endpoint volume.

[0410] As shown in Figure 20, pertuzumab and rituximab conjugates resulted in a delay in tumor growth over time compared to vehicle and the non-cell bound control neoDegrader conjugate rituximab-compound (Ia).

[0411] Example 8: Treatment of non-Hodgkin's lymphoma (NHL) with anti-CD20 antibody-neoDegrader conjugates The rituximab-compound (Ia) conjugate was injected into immunodeficient mice (Fox Chase SCID®, CB17 / Icr-Prkdc scid Tested in 1 × 10 7 Daudi Burkitt's B-cell lymphoma cells (ATCC® CCL-213™) were injected subcutaneously into the right flank of mice (0.1 mL of cell suspension). Tumors grew to 100–150 mm 3 When the tumor reached an average size of 1000 mg / kg, mice were started to be dosed with anti-CD20 antibody-neoDegrader conjugate, non-targeting neoDegrader conjugate, and vehicle control.

[0412] Trastuzumab-Compound (Ia) and rituximab-Compound (Ia) stock solutions were diluted with vehicle to yield 0.5 mg / mL and 0.1 mg / mL dosing solutions providing 5 and 1 mg / kg at a dosing volume of 10 mL / kg adjusted to each animal's body weight (0.2 mL per 20 g mouse).

[0413] Mice were divided into four treatment groups (N=8 / group) as follows: 1) vehicle, 2) trastuzumab-compound (Ia) (5 mg / kg, iv, qd x 1), 3) rituximab-compound (Ia) (5 mg / kg, iv, qd x 1), 4) pertuzumab-compound (Ia) (5 mg / kg, iv, qd x 1). All test articles were administered intravenously (iv) as a single dose (qd x 1) in a volume adjusted to body weight (0.200 mL / 20 g mouse).

[0414] Tumors were measured twice weekly using calipers and each animal was monitored until its tumor reached the endpoint volume (1500 mm 3 ) or on the final day of the study (day 45), whichever came first. MTV (n) was defined as the median tumor volume on the final day of the study for the remaining number (n) of animals whose tumors had not reached the endpoint volume.

[0415] As shown in Figure 21, the 5 mg / kg dose of rituximab conjugate resulted in a delay in tumor growth over time compared to the 1 mg / kg dose, vehicle, and the non-cell-bound control neoDegrader conjugate trastuzumab-compound (Ia).

[0416] Example 9. Treatment of acute myeloid leukemia (AML) with anti-CD33 antibody-neoDegrader conjugate OR000213 - Compound (Ia) was tested in athymic nude mice (Crl:NU(NCr)-Foxnlnu, Charles River). 1 x 107 HL-60 acute promyelocytic leukemia cells (ATCC® CCL-240™) were injected subcutaneously into the right flank of the mice (0.1 mL of cell suspension). Tumors were 100-150 mm 3 Once the average size of the neodegrader was reached, mice were dosed with anti-CD33 antibody-neoDegrader conjugate, non-targeting neodegrader conjugate, and vehicle control.

[0417] Trastuzumab-Compound (Ia) and OR000213-Compound (Ia) stock solutions were diluted with vehicle to yield 0.5 mg / mL and 0.1 mg / mL dosing solutions providing 5 and 1 mg / kg at a dosing volume of 10 mL / kg adjusted to each animal's body weight (0.2 mL per 20 g mouse).

[0418] Mice were divided into four treatment groups (N=8 / group) as follows: 1) vehicle, 2) trastuzumab-compound (Ia) (5 mg / kg, iv, qd x 1), 3) rituximab-compound (Ia) (1 mg / kg, iv, qd x 1), 4) pertuzumab-compound (Ia) (5 mg / kg, iv, qd x 1). All test articles were administered intravenously (iv) as a single dose (qd x 1) in a volume adjusted to body weight (0.200 mL / 20 g mouse).

[0419] Tumors were measured twice weekly using calipers, and each animal was euthanized when its tumor reached the endpoint volume (2,000 mm3) or on the final day of the study (day 45), whichever came first. MTV (n) was defined as the median tumor volume on the final day of the study among the remaining number (n) of animals whose tumors had not yet reached the endpoint volume.

[0420] As shown in Figure 22, all neoDegrader conjugates resulted in a delay in tumor growth over time compared to vehicle.

[0421] Example 10. Treatment of multiple myeloma with anti-CD38 antibody-neoDegrader conjugate HuAT 13 / 5-Compound (Ia) was tested in CB.17 SCID mice (CB17 / Icr-Prkdcscid / IcrlcoCrl, Charles River). 1 x 107 NCI-H929 mye...

Claims

[Claim 1] The invention described in this specification.

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