RU2846388C1 - Conjugates of neo-destructors - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention relates to conjugates of neodestructors in which the neodestructor is conjugated with a binding moiety. There are also presented pharmaceutical compositions containing the conjugates, and a method for preparing the conjugate.

EFFECT: conjugates and pharmaceutical compositions containing them can be used for treating cancer, particularly breast cancer, stomach cancer, lymphoma, acute myeloid leukaemia, multiple myeloma, head and neck cancer, squamous cell carcinoma and/or hepatocellular carcinoma.

28 cl, 25 dwg, 17 tbl, 10 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention provides conjugates of neodegraders 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.

LEVEL OF TECHNOLOGY

[0002] Protein destruction has been validated as a therapeutic strategy by the efficacy of immunomodulatory imide drugs. These compounds have the ability to bind to cereblon (CRBN) and promote the recruitment and ubiquitination of substrate proteins mediated by the CRL4 ^CRBN E3 ubiquitin ligase. Immunomodulatory imides are thought to act as "molecular glues" by populating the binding surface as a hydrophobic patch that reprograms protein interactions between the ligase and neosubstrates.

[0003] Despite the interest in these compounds as new cancer treatments, their use has so far been limited to hematological malignancies such as multiple myeloma and myelodysplastic syndrome (MDS). Expanding the library of compounds that can function by destroying other oncoproteins, many of which have been considered “untreatable,” is an active area of drug development. Thus, there is an ongoing need for new compounds that can target these alternative oncoproteins and treat a broad range of cancers.

ESSENCE OF THE INVENTION

[0004] In certain aspects, the present invention provides a conjugate of formula (I):

or a pharmaceutically acceptable salt thereof, where

[0005] a is an integer from 1 to 10;

[0006] A represents a phenyl or _C4 - _C10 cycloalkyl ring;

[0007] U is selected from NH and CF ₂ ;

[0008] R ¹ is independently selected from hydrogen and halogen;

[0009] X is selected from -NR ² -, =C(CH ₃ )-, -Q-(CH ₂ ) _n - and -Q(CH ₂ ) _m Q'(CH ₂ ) _n -; where

[0010] each Q and Q' independently represent O, S, or N(R ² ) _v ;

[0011] v is 1 or 2;

[0012] each R ² is independently hydrogen or C ₁ -C ₆ alkyl;

[0013] n is an integer from 1 to 6;

[0014] m is an integer between 2 and 6;

[0015] where the left side of each group is attached to L and the right side is attached to A;

[0016] provided that when X is NH or -Q-(CH ₂ ) _n -, R ¹ is halogen;

[0017] L is a cleavable linker or a non-cleavable linker; and

[0018] Bm is a binding moiety that is capable of specifically binding to a protein.

[0019] In some aspects, the binding fragment is an antibody, an antibody fragment, or an antigen-binding fragment.

[0020] In some aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein a is an integer from 2 to 8.

[0021] In certain aspects, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein L is a non-cleavable linker. In some aspects, L is selected from the group consisting of

Where:

[0022] p is an integer between 1 and 10;

[0023] represents the attachment point to X; and

[0024] represents the attachment point to the linking fragment.

[0025] In some aspects, L is

[0026] In some aspects, p is equal to 5.

[0027] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is a cleavable linker. In some aspects, the cleavable linker is cleavable by a protease. In some aspects, L is selected from the group consisting of

Where:

[0028] q is an integer between 2 and 10;

[0029] Z ¹ , Z ² , Z ³ and Z ⁴ are each independently absent or represent a residue of a naturally occurring amino acid in the L- or D-configuration, provided that at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues;

[0030] represents the attachment point to X; and

[0031] represents the attachment point to the linking fragment.

[0032] In some aspects, Z ¹ , Z ² , Z ³ and Z ⁴ are independently absent or selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-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; provided that at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues.

[0033] In some aspects, Z ¹ is absent or is glycine; 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; and Z ⁴ is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalamine, D-phenylalanine, and glycine.

[0034] In certain aspects, L represents

[0035] In some aspects, q is equal to 5.

[0036] In certain aspects, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein L is a bioreducible linker. In some aspects, L is selected from the group consisting of

Where:

[0037] q is an integer between 2 and 10;

[0038] each R, R', R'' and R''' is independently selected from _hydrogen , _C1 -C6alkoxyC1 _{- C6alkyl} , ( _C1 - _C6 ) _{2NC1 - C6alkyl} and _C1 - _C6alkyl , or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring;

[0039] represents the attachment point to X; and

[0040] represents the attachment point to the linking fragment.

[0041] In certain aspects, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein L is an acid-cleavable linker. In some aspects, L is selected from the group consisting of

Where:

[0042] q is an integer between 2 and 10;

[0043] represents the attachment point to X; and

[0044] represents the attachment point to the linking fragment.

[0045] In certain aspects, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein L is a click-release drug linker. In some aspects, L is selected from

Where:

[0046] q is an integer between 2 and 10;

[0047] represents the attachment point to X; and

[0048] represents the attachment point to the linking fragment.

[0049] In certain aspects, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein L is a pyrophosphatase-cleavable linker. In some aspects, L is

Where:

[0050] q is an integer between 2 and 10;

[0051] represents the attachment point to X; and

[0052] represents the attachment point to the linking fragment.

[0053] In certain aspects, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof, wherein L is a beta-glucuronidase cleavable linker. In some embodiments, L is selected from

Where:

[0054] q is an integer between 2 and 10;

[0055] absent or represents a connection;

[0056] represents the attachment point to X; and

[0057] represents the attachment point to the linking fragment.

[0058] In certain aspects, the present invention 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 aspects, the protein to which the binding moiety binds is a surface antigen.

[0059] In some aspects, the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, growth factor Crypto 1, 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, gp100, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α ₄ , α _v β ₃ , α _v β ₅ , α _v β ₆ , α ₁ β ₄ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α _IIb β ₃ ), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumam, LewisY, LFA-1(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA/MARTI, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH ₂ , NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, mutant p53, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1/galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa, rabies virus gene, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, 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 their combinations.

[0060] In certain aspects, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1/CD248, TROP-2, and combinations thereof.

[0061] In some aspects, Bm is an antibody, wherein the antibody is selected from the group consisting of rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, balantamab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13/5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, and veltuzumab. In some aspects, the antibody is rituximab, trastuzumab, pertuzumab, OR000213 (huMy9-6 IgG4 S228P), lintuzumab, or gemtuzumab.

[0062] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0063] A is phenyl;

[0064] U is NH;

[0065] R ¹ is halogen; and

[0066] X is -n(r ² ) _v (CH ₂ ) _m O(CH ₂ ) _n -; where:

[0067] v is 1;

[0068] m and n are equal to 2; and

[0069] R ² is methyl.

[0070] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0071] A is phenyl;

[0072] U is NH;

[0073] R ¹ is halogen; and

[0074] X is -n(r ² ) _v (CH ₂ ) _m o(CH ₂ ) _n -; where:

[0075] v is 2;

[0076] m and n are equal to 2; and

[0077] each R ² is methyl.

[0078] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0079] A is phenyl;

[0080] U is NH;

[0081] R ¹ is halogen; and

[0082] X is -O(CH ₂ ) _n -; where:

[0083] n is 2.

[0084] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0085] A is phenyl;

[0086] U is NH;

[0087] R ¹ is halogen; and

[0088] X is -S(CH ₂ ) _n -; where:

[0089] n is 2.

[0090] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0091] A is phenyl;

[0092] U is NH;

[0093] R ¹ is hydrogen; and

[0094] X is --NR ² -; where:

[0095] R ² is methyl.

[0096] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0097] A is phenyl;

[0098] U is NH;

[0099] R ¹ is halogen; and

[0100] X is --NR ² -; where:

[0101] R ² is hydrogen.

[0102] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0103] A is phenyl;

[0104] U is NH;

[0105] R ¹ is hydrogen; and

[0106] X is -C(CH ₃ )=.

[0107] In certain aspects, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

[0108] A is a _C4 - _C10 cycloalkyl ring;

[0109] U is NH;

[0110] R ¹ is hydrogen; and

[0111] X is -n(r ² )(ch ₂ ) _m o(ch ₂ ) _n -; where:

[0112] n is 1;

[0113] m is 2; and

[0114] R ² is methyl.

[0115] In certain aspects, the present invention provides a compound of formula (II):

or a pharmaceutically acceptable salt thereof, where

[0116] A is a phenyl or C ₄ -C ₁₀ cycloalkyl ring;

[0117] R ¹ is independently selected from hydrogen and halogen;

[0118] U is selected from NH and CF ₂ ; and

[0119] R ² is selected from -C(O)R ³ , -n(r ⁴ ) ₂ , -(CH ₂ ) _n OH, -(CH ₂ ) _n SH, -(CH ₂ ) _n N(R ⁴ ) ₂ , -(CH ₂ ) _n Q'(CH ₂ ) _m OH, -(CH ₂ ) _n Q'(CH ₂ ) _m SH and -(CH ₂ ) _n Q'(CH ₂ ) _m N(R ⁴ ) ₂ ; where

[0120] R ³ is hydrogen or C ₁ -C ₆ alkyl;

[0121] each R ⁴ is independently hydrogen or C ₁ -C ₆ alkyl;

[0122] Q' is O, S or NR ⁴ ;

[0123] n is 1-6; and

[0124] m is 2-5;

[0125] provided that when R ² is NH ₂ , -(CH ₂ ) _n NH ₂ or -(CH ₂ ) _n OH, then R ¹ is halogen.

[0126] In certain aspects, the present invention provides a compound of formula (III):

or a pharmaceutically acceptable salt thereof;

[0127] In certain aspects, the present invention provides a compound of formula (IV):

or a pharmaceutically acceptable salt thereof;

[0128] In some aspects, the present invention provides a conjugate of formula (V):

or a pharmaceutically acceptable salt thereof, wherein Bm is a binding moiety that specifically binds to a protein. In some aspects, Bm is an antibody or an antigen-binding portion thereof. In some aspects, the protein to which the binding moiety specifically binds is a surface antigen.

[0129] In some aspects, the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, growth factor Crypto 1, 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, gp100, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI,24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α ₄ , α _v β ₃ , α _v β ₅ , α _v β ₆ , α ₁ β ₄ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α _IIb β ₃ ), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumain, LewisY, LFA-1(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA/MARTI, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH ₂ , NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, mutant p53, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1/galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa, rabies virus gene, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, 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 their combinations.

[0130] In some aspects, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1/CD248, TROP-2, and combinations thereof.

[0131] In some aspects, Bm is an antibody, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, balantamab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13/5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab, or veltuzumab. In some aspects, the antibody is rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, or gemtuzumab.

[0132] In certain aspects, the present invention provides a pharmaceutical composition comprising a conjugate or compound of any of the previous aspects or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

[0133] In some aspects, the present invention provides a method 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 following 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.

[0134] In some aspects, the method further comprises administering to the subject a pharmaceutically acceptable amount of an additional agent prior to, after, or simultaneously with the conjugate or compound of any of the previous aspects or a pharmaceutically acceptable salt thereof. In some aspects, the additional agent is a cytotoxic agent or an immune response modifier. In some aspects, the immune response modifier is a checkpoint inhibitor. In some aspects, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, and/or a LAG-3 inhibitor.

[0135] In certain aspects, the present invention provides a method for preparing a conjugate of formula (I) or a pharmaceutically acceptable salt thereof, comprising reacting a linking moiety with a compound of formula (I-1):

or a pharmaceutically acceptable salt thereof, where

[0136] a is an integer from 1 to 10;

[0137] A is a phenyl or C ₄ -C ₁₀ cycloalkyl ring;

[0138] R ¹ is independently selected from hydrogen and halogen;

[0139] U is selected from NH and CF ₂ ; and

[0140] X is selected from -N(R ² ) _v -, =C(CH ₃ )-, -Q-(CH ₂ ) _n - and -Q(CH ₂ ) _m Q'(CH ₂ ) _n -; where

[0141] v is 1 or 2;

[0142] each Q and Q' is independently O, S or KR ² ;

[0143] each R ² is independently hydrogen or C ₁ -C ₆ alkyl;

[0144] n is an integer from 1 to 6; and

[0145] m is an integer between 2 and 6;

[0146] where the left side of each group is attached to L' and the right side is attached to A;

[0147] provided that when X is NH or -Q-(CH ₂ ) _n -, R ¹ is halogen; and

[0148] L' is a cleavable or non-cleavable linker precursor that conjugates to the linking moiety.

[0149] In some aspects, the method further comprises reducing the linking moiety prior to reaction with the compound of formula (I-1).

[0150] In some aspects, a is an integer from 2 to 8.

[0151] In some aspects, L' is a non-cleavable linker precursor. In some aspects, L' is selected from the group consisting of

Where:

[0152] p is an integer between 1 and 10; and

[0153] represents the attachment point to X.

[0154] In some aspects, L' is

[0155] In some aspects, p is equal to 5.

[0156] In certain aspects, L' is a cleavable linker precursor. In some aspects, the cleavable linker precursor is cleavable by a protease. In some aspects, L' is selected from the group consisting of

Where:

[0157] q is an integer from 2 to 10;

[0158] each Z ¹ , Z ² , Z ³ and Z ⁴ is independently absent or is a naturally occurring amino acid residue in the L- or D-configuration, provided that at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues; and

[0159] represents the attachment point to X.

[0160] In some aspects, Z ¹ , Z ² , Z ³ and Z ⁴ are independently absent, selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-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 at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues.

[0161] In certain aspects, Z ¹ is absent or is glycine; 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; and Z ⁴ is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalamine, D-phenylalanine, and glycine.

[0162] In some aspects, L' is

[0163] In some aspects, q is equal to 5.

[0164] In certain aspects, L' is a precursor of a bioreducible linker. In some aspects, L' is selected from the group consisting of

Where:

[0165] q is an integer from 2 to 10;

[0166] each R, R', R'' and R''' is independently selected from hydrogen, _C1 - _{C6alkoxyC1 - C6alkyl} , ( _C1 - _C6 ) _{2NC1 - C6alkyl} and _C1 - _C6alkyl , or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cyclopropyl ring; and

[0167] represents the attachment point to X.

[0168] In certain aspects, L' is an acid cleavable linker precursor. In some aspects, L' is selected from the group consisting of

Where:

[0169] q is an integer from 2 to 10; and

[0170] represents the attachment point to X.

[0171] In certain aspects, L' is a precursor of a click drug release linker. In some aspects, L' is selected from

Where:

[0172] q is an integer from 2 to 10; and

[0173] represents the attachment point to X.

[0174] In certain aspects, L' is a precursor of a pyrophosphatase-cleavable linker. In some aspects, L' is

Where:

[0175] q is an integer between 2 and 10;

[0176] represents the attachment point to X.

[0177] In some aspects, L' is a precursor of a beta-glucuronidase cleavable linker. In certain aspects, L' is selected from

Where:

[0178] q is an integer between 2 and 10;

[0179] is absent or represents a connection; and

[0180] represents the attachment point to X.

[0181] In some aspects, a compound of formula (I-1) is reacted with a binding moiety that comprises an antibody or an antigen-binding portion thereof. In some aspects, the antibody or antigen-binding portion thereof binds to a surface antigen.

[0182] In certain aspects, the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-ab1, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, growth factor Crypto 1, 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, gp100, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI,24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor. IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α ₄ , α _v β ₃ , α _v β ₅ , α _v β ₆ , α ₁ β ₄ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α _IIb β ₃ ), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, Legumain, LewisY, LFA-1(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA/MARTI, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH ₂ , 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, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa, rabies virus gene, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, 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, TE, 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 combinations thereof.

[0183] In some aspects, the surface antigen comprises HER2, CD20, CD38, CD33, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1/CD248, TROP-2, or combinations thereof.

[0184] In some aspects, Bm is an antibody, wherein the antibody comprises rituximab, trastuzumab, gemtuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuximab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, lintuzumab, huMy9-6, balantamab, 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.

[0185] In some aspects, the present invention relates to a method for producing a conjugate of formula (I) from a compound of formula (I-1), wherein:

[0186] A is phenyl;

[0187] U is NH;

[0188] R ¹ is halogen; and

[0189] X is -n(r ² ) _v (ch ₂ ) _m o(ch ₂ ) _n -; where:

[0190] v is 1;

[0191] m and n are equal to 2; and

[0192] R ² is methyl.

[0193] In some aspects:

[0194] A is phenyl;

[0195] U is NH;

[0196] R ¹ is halogen; and

[0197] X is -n(r ² ) _v (CH ₂ ) _m O(CH ₂ ) _n -; where:

[0198] v is 2;

[0199] m and n are equal to 2; and

[0200] each R ² is methyl.

[0201] In certain aspects:

[0202] A is phenyl;

[0203] U is NH;

[0204] R ¹ is halogen; and

[0205] X is -O( _CH2 ) _n- ; where:

[0206] n is 2.

[0207] In certain aspects:

[0208] A is phenyl;

[0209] U is NH;

[0210] R ¹ is halogen; and

[0211] X is -S(CH ₂ ) _n -; where:

[0212] n is 2.

[0213] In some aspects:

[0214] A is phenyl;

[0215] U is NH;

[0216] R ¹ is hydrogen; and

[0217] X is -NR ² -; where:

[0218] R ² is methyl.

[0219] In some aspects:

[0220] A is phenyl;

[0221] U is NH;

[0222] R ¹ is halogen; and

[0223] X is -NR ² -; where:

[0224] R ² is hydrogen.

[0225] In certain respects:

[0226] A is phenyl;

[0227] U is NH;

[0228] R ¹ is hydrogen; and

[0229] X is -C( _CH3 )=.

[0230] In some aspects:

[0231] A is a _C4 - _C10 cycloalkyl ring;

[0232] U is NH;

[0233] R ¹ is hydrogen; and

[0234] X is -N(R ² )(CH ₂ ) _m O(CH ₂ ) _n -; where:

[0235] n is 1;

[0236] m is 2; and

[0237] R ² is methyl.

[0238] In some aspects, the compound of formula (I-1) is:

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0239] Fig. 1 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the BT-474 cell line. The x-axis is the logarithm of the antibody concentration. (M) The y-axis is the % viability of BT-474 cells upon treatment with trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (triangle, solid line); trastuzumab alone (triangle, dotted line), cadcyla (diamond), neodegradant P1 alone (cross), and rituximab-L-P1 (e.g., rituximab-Compound (Ia)) (circle). L is a linker.

[0240] Fig. 2 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the BT-474 cell line. The x-axis represents the logarithm of the antibody concentration. (M) The y-axis represents the % viability of BT-474 cells upon treatment with pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (triangle, solid line), pertuzumab alone (triangle, dotted line), cadcyla (diamond), neodegradant P1 alone (cross), and rituximab-L-P1 (e.g., rituximab-Compound (Ia)) (circle).

[0241] Fig. 3 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the BT-474 cancer cell line. The x-axis represents the logarithm of the antibody concentration. (M) The y-axis represents the % viability of BT-474 cells upon treatment with trastuzumab-L-P4 (e.g., trastuzumab-Compound (Ic)) (triangle, solid line), trastuzumab (triangle, dotted line), cadcyla (diamond), neodegradant P4 alone (cross), and rituximab-L-P4 (e.g., rituximab-Compound (Ic)) (circle).

[0242] Fig. 4 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the BT-474 cell line. The x-axis represents the logarithm of the antibody concentration. (M) The y-axis represents the % viability of BT-474 cells upon treatment with pertuzumab-L-P4 (e.g., pertuzumab-Compound (Ic)) (triangle, solid line), pertuzumab (triangle, dotted line), cadcyla (diamond), neodegradant P4 alone (cross), and rituximab-L-P4 (e.g., rituximab-Compound (Ic)) (circle).

[0243] Fig. 5 depicts the in vitro activity of illustrative examples of neodegrader conjugates with different drug:antibody (DAR) ratios against the BT-474 cell line. The X-axis represents the logarithm of the antibody concentration (M). The y-axis shows the % viability of BT-474 cells treated with trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) DAR 1.6 (upward triangle, solid line), trastuzumab-L-P3 (e.g., trastuzumab-Compound (Ib)) DAR 1.5 (downward triangle, solid line), trastuzumab-L-P4 (e.g., trastuzumab-Compound (Ic)) DAR 1.6 (circle, solid line), trastuzumab-L-P1 (e.g., trastuzumab-Compound (Id)) DAR 1.6 (square, solid line), trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) DAR 8 (triangle, dotted line), trastuzumab-L-P4 (e.g., trastuzumab-compound (Ic)) DAR 8 (shaded circle, dotted line), ENHERTU® (diamond, dotted line) and trastuzumab (circle, dotted line).

[0244] Fig. 6 depicts the in vitro activity of illustrative examples of neodegradants against the BT-474 cell line. The x-axis represents the logarithm of the antibody concentration. (M) The y-axis represents the % viability of BT-474 cells upon treatment with pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) DAR 8 (upward triangle, solid line), pertuzumab-L-P4 (e.g., pertuzumab-Compound (Ic)) DAR 8 (downward triangle, solid line), and ENHERTU® (diamond, dotted line).

[0245] Fig. 7 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the SK-BR-3 cell line. The x-axis represents the logarithm of the antibody concentration (M). The y-axis represents the % viability of SK-BR-3 cells upon treatment with trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia) (triangle, solid line), trastuzumab (triangle, dotted line), cadcyla (diamond), neodegradant P1 alone (circle), and rituximab-L-P1 (e.g., rituximab-Compound (Ia)) (cross).

[0246] Fig. 8 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the SK-BR-3 cell line. The x-axis represents the logarithm of the antibody concentration (M). The y-axis represents the % viability of SK-BR-3 cells upon treatment with pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (triangle, solid line), pertuzumab (triangle, dotted line), cadcyla (diamond), neodegradant P1 alone (circle), and rituximab-L-P1 (e.g., rituximab-Compound (Ia)) (cross).

[0247] Fig. 9 depicts the in vitro activity of illustrative examples of neodestructor conjugates against the HL-60 cell line. The x-axis represents the logarithm of the antibody concentration (M). The y-axis represents the % viability of HL-60 cells upon treatment with OR000213-L-P1 (e.g., OR000213-Compound (Ia)) (triangle), MYLOTARG® (diamond), and trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (circle).

[0248] Fig. 10 depicts the in vitro activity of illustrative examples of neodestructor conjugates against the HL-60 cell line. The X-axis represents the logarithm of the antibody concentration. (M) The y-axis represents the % viability of HL-60 cells treated with huMy9-6(IgG1)-L-P1 (e.g., huMy9-6(IgG1)-compound (Ia)) DAR 8 (upward shaded triangle, solid line), huMy9-6(IgG1)-L-P1 (e.g., huMy9-6(IgG1)-compound (Id)) DAR 8 (downward shaded triangle, solid line), lintuzumab IgG1-L-P1 (e.g., lintuzumab IgG1-compound (Ia)) DAR 8 (upward open triangle, dashed line), lintuzumab IgG1-L-P1 (e.g., lintuzumab IgG1-compound (Id)) DAR 8 (downward open triangle, dotted line), OR000213-L-P1 (eg, OR000213-compound (Ia)) DAR 8 (square) and rituximab-L-P4 (eg, rituximab - compound (Ic)) (circle, dotted line).

[0249] Fig. 11 depicts the in vitro activity of illustrative examples of conjugates of compound (Ia) with different drug:antibody (DAR) ratios against the HL60 cell line. The X-axis represents the logarithm of the antibody concentration (M). The y-axis shows the % viability of HL-60 cells treated with huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1-compound (Ia)) DAR 1.9 (upward triangle, solid line), huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1 - compound (Ia)) DAR 3.9 (downward triangle, solid line), huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1 - compound (Ia)) DAR 5.5 (diamond, solid line), huMy9-6 IgG1-L-P1 (e.g., huMy9-6 IgG1-compound (Ia)) DAR 8 (square, solid line), and rituximab-L-P4 (e.g., rituximab - compound (Ic)) (circle, dotted line).

[0250] Fig. 12 depicts the in vitro activity of illustrative examples of conjugates of compound (Ia) with different drug:antibody (DAR) ratios against the HL60 cell line. The X-axis represents the logarithm of the antibody concentration (M). The y-axis shows the % viability of HL-60 cells treated with OR000213-L-P1 (e.g., OR000213-compound (Ia)) DAR 1.2 (upward triangle, solid line), OR000213-L-P1 (e.g., OR000213 - compound (Ia)) DAR 1.8 (downward triangle, solid line), OR000213- L-P1 (e.g., OR000213-compound (Ia)) DAR 2.3 (diamond, solid line), OR000213-L-P1 (e.g., OR000213-compound (Ia)) DAR 8 (square, solid line) and rituximab-L-P4 (e.g., rituximab - compound (Ic)) (triangle, dotted line).

[0251] Fig. 13 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the Ramos cell line. The x-axis is the logarithm of the antibody concentration (M), and the y-axis is the % viability of Ramos cells upon treatment with rituximab-L-P4 (e.g., rituximab - Compound (Ic)) (upward triangle, solid line), rituximab-L-P1 (e.g., rituximab - Compound (Ia)) (downward triangle, solid line), rituximab (triangle, dotted line), neodegradant P1 alone (cross, dotted line), neodegradant P4 alone (star, dotted line), and trastuzumab-L-P1 (e.g., trastuzumab - Compound (Ia)) (circle, dotted line).

[0252] Fig. 14 depicts the in vitro activity of illustrative examples of neodestructor conjugates against the Daudi cell line. The x-axis is the logarithm of the antibody concentration (M), and the y-axis is the % viability of Daudi cells upon treatment with rituximab-L-P1 (e.g., rituximab - Compound (Ia)) (upward triangle, solid line), rituximab (triangle, dotted line), and trastuzumab-L-P1 (e.g., trastuzumab - Compound (Ia)) (circle, dotted line).

[0253] Fig. 15 depicts the in vitro activity of illustrative examples of neodestructor conjugates against the Ramos cell line. The x-axis is the logarithm of the antibody concentration (M), and the y-axis is the % viability of Ramos cells upon treatment with rituximab-L-P4 (e.g., rituximab - compound (Ic)) (upward triangle, solid line), rituximab-L-P1 (e.g., rituximab - compound (Ia)) (downward triangle, solid line), and neodestructor P1 alone.

[0254] Fig. 16 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the NCI-N87 cancer cell line. The x-axis represents the logarithm of the antibody concentration (M). The y-axis represents the % viability of NCI-N87 cells upon treatment with trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (triangle, solid line), trastuzumab (triangle, dotted line), cadcyla (diamond), neodegradant P4 alone (cross), and rituximab-L-P1 (e.g., rituximab-Compound (Ia)) (circle).

[0255] Fig. 17 depicts the in vitro activity of illustrative examples of neodegradant conjugates against the NCI-N87 cancer cell line. The x-axis represents the logarithm of the antibody concentration (M). The y-axis represents the % viability of NCI-N87 cells upon treatment with pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (triangle, solid line), pertuzumab (triangle, dotted line), cadcyla (diamond), neodegradant P1 alone (cross), and rituximab-L-P1 (e.g., rituximab-Compound (Ia)) (circle).

[0256] Fig. 18 depicts the in vitro activity of illustrative examples of neodestructor conjugates after 3-day incubation with human serum against the BT-474 cell line. The X-axis represents the logarithm of the antibody concentration (M). The y-axis shows the % viability of BT-474 cells treated with trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (human serum) (upward triangle, solid line), pertuzumab-L-P1 (e.g., pertuzumab - Compound (Ia)) (human serum) (downward triangle, solid line), OR000213-L-P1 (e.g., OR000213-Compound (Ia)) (human serum) (circle, solid line), human serum only (star), trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (upward triangle, dotted line), pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (downward triangle, dotted line), and OR000213-L-P1 (eg OR000213-compound (Ia)) (circle, dotted line).

[0257] Fig. 19 depicts the in vitro activity of illustrative examples of neodestructor conjugates after 3-day incubation with mouse serum against the BT-474 cell line. The X-axis represents the logarithm of the antibody concentration (M). The y-axis shows the % viability of BT-474 cells treated with trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (mouse serum) (upward triangle, solid line), pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (mouse serum) (downward triangle, solid line), OR000213-L-P1 (e.g., OR000213-Compound (Ia)) (mouse serum) (circle, solid line), mouse serum alone (star), trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (upward triangle, dotted line), pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (downward triangle, dotted line), and OR000213-L-P1 (eg OR000213-compound (Ia)) (circle, dotted line).

[0258] Fig. 20 depicts the in vivo activity of illustrative examples of neodestructor conjugates against BT-474 (Her2+) tumors in mice. The x-axis represents days post-dose. The y-axis represents tumor volume ( ^mm3 ) following administration of vehicle (solid circle), 5 mg/kg trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (square), 5 mg/kg rituximab - L-P1 (e.g., rituximab - Compound (Ia)) (triangle), and 5 mg/kg pertuzumab-L-P1 (e.g., pertuzumab-Compound (Ia)) (open circle).

[0259] Fig. 21 depicts the in vivo activity of illustrative examples of neodestructor conjugates against Daudi (CD20+) tumors. The x-axis represents days post-dose. The y-axis represents tumor volume ( ^mm3 ) following administration of vehicle (shaded circle), 5 mg/kg trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (square), 1 mg/kg rituximab-L-P1 (e.g., rituximab - Compound (Ia)) (triangle), and 5 mg/kg rituximab-L-P1 (e.g., rituximab - Compound (Ia)) (open circle).

[0260] Fig. 22 depicts the in vivo activity of illustrative examples of neodestructor conjugates against HL-60 (CD33+) tumors. The x-axis represents days post-dose. The y-axis represents tumor volume ( ^mm3 ) following administration of vehicle (shaded circle), 5 mg/kg trastuzumab-L-P1 (e.g., trastuzumab-Compound (Ia)) (square), 1 mg/kg OR000213-L-P1 (e.g., OR-000213-Compound (Ia)) (triangle), and 5 mg/kg OR-000213-L-P1 (e.g., OR000213-Compound (Ia)) (open circle).

[0261] Fig. 23 depicts the in vitro activity of the neodegrader conjugate against the HCC2157 cell line. The x-axis is the logarithm of the antibody concentration (M), and the y-axis is the % viability of HCC2157 cells when treated with sacituzumab-L-P1 (e.g., sacituzumab - compound (Ia)) (lane 1), sacituzumab alone (lane 2), and neodegrader P1 alone (lane 3).

[0262] Fig. 24 depicts the in vitro activity of the neodegrader conjugate against the LP1 cell line. The x-axis represents the logarithm of the antibody concentration (M), and the y-axis represents the % viability of LP1 cells upon treatment with HuAT 13/5-L-P1 (e.g., HuAT 13/5-compound (Ia)) (lane 1), HuAT 13/5 alone (lane 2), and neodegrader P1 alone (lane 3).

[0263] Fig. 25 depicts the in vivo activity of illustrative examples of neodestructor conjugates against NCI-H929 (CD38+) tumors. The x-axis represents days post-dose. The y-axis represents tumor volume ( ^mm3 ) following vehicle (circle), 5 mg/kg HuAT 13/5-L-P1 (e.g., HuAT13/5 - Compound (Ia)) (square).

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[0264] The present disclosure is directed to a conjugate of formula (I):

or a pharmaceutically acceptable salt thereof, where a is an integer from 1 to 10;

A represents a phenyl or _C4 - _C10 cycloalkyl ring;

R ¹ is independently selected from hydrogen and halogen;

U is selected from NH and CF ₂ ;

X is selected from -N( ^R2 ) _v , =C( _CH3 )-, -Q-( _CH2 ) _n- , and -Q( _CH2 ) _mQ '( _CH2 ) _n- ; where each Q and Q' is independently O, S, or N( ^R2 ) _v ;

v is equal to 1 or 2;

each R ² is independently hydrogen or C ₁ -C ₆ alkyl;

n is an integer between 1 and 6;

m is an integer between 2 and 6;

where the left side of each group is attached to L and the right side is attached to A;

provided that when X is NH or -Q-(CH ₂ ) _n -, R ¹ is halogen;

L is a cleavable linker or a non-cleavable linker; and

Bm is a binding fragment that is capable of specifically binding to a protein. In some aspects, the binding fragment is an antibody, an antibody fragment, or an antigen-binding fragment.

[0265] The present invention also provides the above-mentioned compound fused to a linking moiety, a composition comprising the compound or conjugate, or a method of using or producing the compound or conjugate.

I Definitions.

[0266] In order to make the present description more understandable, some terms are first defined. Further definitions are provided in the detailed description.

[0267] It should be noted that the singular terms include references to one or more; for example, "a nucleotide sequence" means one or more nucleotide sequences. Accordingly, the singular terms, as well as the expressions "one or more" and "at least one" may be used interchangeably herein. It should also be noted that the claims of the present invention may be amended to exclude any optional elements. Accordingly, this statement should serve as a preliminary basis for the use of such inclusive terminology as "solely", "only", etc. in connection with the indication of elements of the claims, or the use of a negative limitation.

[0268] Furthermore, the use of the conjunctions "and/or" herein is intended to include a specific disclosure of each of the two stated features or components, with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include the phrases "A and B," "A or B"; "A" (alone) and "B" (alone). Similarly, the term "and/or" as used in a phrase such as "A, B and/or C" is intended to include each of the following: 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 (only); B (only); and C (only).

[0269] It should be understood that where aspects are described herein with the term “comprising,” similar aspects are also otherwise provided when described in terms of “consisting of” and/or “consisting essentially of.”

[0270] Unless defined otherwise, 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. For example, the dictionaries 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 skilled in the art with a general explanation of many of the terms used in the disclosure of this invention.

[0271] Units, prefixes, and symbols are defined in the International System of Units (SI) format. Numerical ranges include the numbers defining the range. When a range of values is given, it is understood that each sub-integer value and each portion thereof between the stated upper and lower limits of the range are also specifically disclosed, along with each sub-range between such values. The upper and lower limits of any range may be independently included or excluded within the range, and each range that includes either limit, neither limit, or both limits is also encompassed by the present invention. Thus, the ranges given herein are intended to be shorthand for all values within the range, including the stated end points. For example, the range "1 to 10" means any number, combination of numbers, or subrange from the group consisting of the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0272] Where a value is explicitly stated, it is to be understood that values that have approximately the same number or quantity as the stated value are also within the scope of the present invention. When a combination is disclosed, each subcombination of elements of that combination is also specifically disclosed and is within the scope of the present invention. Conversely, where different elements or groups of elements are separately disclosed, combinations thereof are also disclosed. Where any element of the present invention is disclosed as having multiple alternatives, examples of the present invention in which each alternative is excluded, either individually or in any combination with other alternatives, are also disclosed herein; more than one element of the present invention may have such exceptions, and all combinations of elements having such exceptions are hereby disclosed.

[0273] The term "DAR" as used herein refers to the drug-to-antibody ratio of a conjugate, which is the average number of non-degrader-linker complexes associated with each antibody. In certain aspects, the DAR of the conjugates described herein is from 1 to 10. In some aspects, the DAR of the conjugates described herein is from 1 to 8. In some aspects, the DAR of the conjugates described herein is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 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.

[0274] The term "antibody" as used herein also 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 a target antigen of interest or a portion thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. An immunoglobulin described herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin molecule. Immunoglobulins can be obtained from any species. However, in one aspect, the immunoglobulin is of human, mouse, or rabbit origin.

[0275] The term "single-domain antibody," also known as a nanobody, is an antibody fragment consisting of a single monomeric variable domain of an antibody with a molecular weight of about 12 kDa to about 15 kDa. Single-body antibodies may be based on heavy chain or light chain variable domains.

Examples of single domain antibodies include, but are not limited to, V _H H fragments and v _nar fragments.

[0276] "Antibody fragments" include a portion of an intact antibody, typically 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 obtained from a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope-binding fragments of any of the foregoing fragments that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0277] An "intact antibody" is an antibody that comprises an antigen-binding variable region, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be constant domains of native sequences (e.g., constant domains of a native human sequence) or variant amino acid sequences.

[0278] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike 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 have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies 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 invention can be produced by the hybridoma method or can be produced by recombinant DNA methods. "Monoclonal antibodies" can also be isolated from antibody phage libraries.

[0279] Monoclonal antibodies as provided herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies obtained from another species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies as provided herein include "primitized" antibodies comprising antigen-binding sequences with a variable region derived from non-human primates (e.g., monkeys, apes, etc.) and human constant region sequences.

[0280] Various methods have been used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces one type of antibody, uses cells from different species, including mice (murine), hamsters, rats, and humans. Another method for producing MAbs uses genetic engineering, including recombinant DNA techniques. Monoclonal antibodies produced by these methods include, among others, chimeric antibodies and humanized antibodies. A chimeric antibody combines coding regions of DNA from more than one species. For example, the variable region of a chimeric antibody may be derived from a mouse and the constant region from a human. A humanized antibody is predominantly human, even if it contains non-human portions. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. However, unlike a chimeric antibody, the variable region may be partially derived from a human. The non-human synthetic portions of a humanized antibody are often derived from the CDRs of mouse antibodies. In either case, these regions are critical to allowing the antibody to recognize and bind to a specific antigen. Although mouse antibodies are useful for diagnostics and short-term therapy, they cannot be administered to humans over long periods of time without increasing the risk of a harmful immunogenic response. This response, called a human anti-mouse antibody (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death.

[0281] Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portions of the administered antibodies. In addition, chimeric and humanized antibodies may have the added benefit of activating secondary human immune responses such as antibody-dependent cellular cytotoxicity.

[0282] An intact antibody may have one or more "effector functions," which refer to biological activities inherent in the Fc region (either a native sequence Fc region or a variant amino acid sequence Fc region) of the antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., the B cell receptor, BCR), etc.

[0283] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be classified into different "classes." There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different antibody classes are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structure and three-dimensional configurations of the different immunoglobulin classes are well known.

[0284] As used herein, the term "about" is used to mean approximately, about, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above and below the stated value by a deviation of, for example, 10 percent, up or down (higher or lower).

[0285] The terms "administering," "administering," and grammatical variations thereof refer to administering a composition, such as an EV (e.g., an exosome) of the present invention, to a subject in a pharmaceutically acceptable manner. Administration of a composition, such as an EV (e.g., an exosome) of the present invention, to a subject is by any suitable route, including intratumorally, orally, pulmonarily, intranasally, parenterally (intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly, or topically. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by administering the composition or agent into a vein of the subject.

[0286] As used herein, the term "antibody" encompasses an immunoglobulin, whether natural or partially or wholly synthesized, and fragments thereof. The term also encompasses any protein; having a binding domain homologous to the binding domain of an immunoglobulin. "Antibody" further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. When the term "antibody" is used, it includes whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv) ₂ , Fab, Fab' and F(ab') ₂ , F(ab1) ₂ , Fv, dAb and Fd fragments, diabodies and antibody-linked polypeptides. Antibody includes bispecific antibodies and multispecific antibodies, provided that they exhibit the desired biological activity or function. In some aspects of the present invention, the biologically active molecule is an antibody or a molecule comprising an antigen-binding fragment thereof.

[0287] The terms "antibody-drug conjugate" and "ADC" are used interchangeably and refer to an antibody linked, for example, covalently, to a therapeutic agent (sometimes referred to herein as an agent, drug, or active pharmaceutical ingredient) or agents. In some aspects of the present invention, the biologically active molecule is an antibody-drug conjugate.

[0288] As used herein, the term "about" with respect to one or more values of interest refers to a value that is similar to a stated reference value. In certain aspects, the term "about" refers to a range of values that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or otherwise apparent from context (except where such number exceeds 100% of the possible value).

[0289] A "conservative amino acid substitution" is a conservative amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, an amino acid sequence may be conservatively replaced with a structurally similar sequence that differs in the order and/or composition of the side chain family members.

[0290] As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that occur unchanged at the same position in two or more compared sequences. Nucleotides or amino acids that are relatively conserved are those that are conserved in the most related sequences compared to nucleotides or amino acids that occur elsewhere in the sequence.

[0291] In some aspects, two or more sequences are "completely conserved" or "identical" if they are 100% identical to each other. In some aspects, two or more sequences are "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 each other. In some aspects, two or more sequences are "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 each other. Sequence conservation may apply to the entire length of a polynucleotide or polypeptide, or may apply to a portion, region, or feature thereof.

[0292] As used herein, the terms "linking" and "conjugation" are used interchangeably and each refers to the covalent or non-covalent attachment of two or more moieties comprising a non-degrader and a linking moiety. In some aspects, the linking or conjugation may comprise a linker.

[0293] The term "amino acid sequence variant" refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Typically, amino acid sequence variants have at least about 70% sequence identity to at least one receptor binding domain of a native antibody or at least one ligand binding domain of a native receptor, and will typically have at least about 80% sequence identity, more typically at least about 90% sequence homology, to such receptor or ligand binding domains. Amino acid sequence variants contain substitutions, deletions, and/or insertions at certain positions in the amino acid sequence of the native amino acid sequence. Amino acids are designated by conventional names, one-letter codes, and three-letter codes.

[0294] "Sequence identity" is defined as the percentage of residues in an amino acid sequence variant 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" by Genentech, Inc., which was filed with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, December 10, 1991.

[0295] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds the Fc region of an antibody. A typical FcR is a native sequence human FcR. Additionally, an FcR may be one that binds an IgG antibody (a gamma receptor), and includes the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII receptors, including allelic variants and alternatively spliced forms of these receptors. The Fc.gamma.RII receptors include Fc.gamma.RIIA (the "activating receptor") and Fc.gamma.RIIB (the "inhibitory receptor"), which contain similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are included in the term "FcR" in this document. The term also includes the neonatal receptor, FcRn, responsible for the transfer of maternal IgG to the fetus.

[0296] "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 the antigen it recognizes. A CDC assay can be used to assess complement activation.

[0297] "Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting 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 contains equally spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a series of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Specific amino acid residues are thought to form the interface between the light chain and heavy chain variable domains.

[0298] The term "variable" refers to the fact that certain portions of the variable domains vary widely among antibodies and contribute to the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed across the variable domains of antibodies. It is concentrated in three segments of the variable domains of both the light and heavy chains, called hypervariable regions. The more conserved portions of the variable domains are called framework regions (FRs). The variable domains of native light and heavy chains contain four FRs, predominantly adopting a beta-sheet configuration, and are connected by three hypervariable regions that form loops that connect, and in some cases are part of, the beta-type structures. The hypervariable regions in each chain are held together in close proximity by FRs and, with hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not directly involved in the binding of the antibody to the antigen, but they exert various effector functions, such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0299] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically comprises amino acid residues from the "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the variable light chain domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the variable heavy chain domain; Kabat et al. supra) and/or those residues from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the variable light chain domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the variable heavy chain domain). "Framework region" or "FR" residues, as defined herein, are hypervariable region residues other than variable domain residues.

[0300] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin digestion produces an F(ab')2 fragment that has two antigen-binding active sites and is still capable of cross-linking with antigen.

[0301] "Fv" is the minimum fragment of an antibody that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain 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 the 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 hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0302] A "Fab fragment" also comprises a light chain constant domain and the first constant domain (CH1) of a heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. As used herein, Fab'-SH is the designation 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 prepared as pairs of Fab' fragments that had hinge cysteines between them. Other methods of chemically linking antibody fragments are also known.

[0303] The "light chains" of antibodies from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0304] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that allows the scFv to form a desired structure for antigen binding.

[0305] The term "diabodies" refers to small antibody fragments with two antigen-binding sites that contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Due to the use of a linker that is too short to allow the two domains to be combined in one chain, the domains are forced to combine with complementary domains of another chain and create two antigen-binding sites.

[0306] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from an immunoglobulin of non-human origin. Humanization is a method of transferring the murine antigen-binding information to a non-immunogenic acceptor of a human antibody, which has led to the creation of many therapeutically useful drugs. The humanization method typically begins with the transfer of all six murine complementarity determining regions (CDRs) to a human antibody framework. These CDR-grafted antibodies typically do not retain their original antigen binding affinity, and in fact, this affinity is often severely compromised. In addition to the CDRs, selected residues of the non-human antibody framework must also be included to maintain proper CDR conformation. Transfer of key residues of the murine framework to a human acceptor to maintain the structural conformation of the grafted CDRs has been shown to restore antigen binding and affinity. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which hypervariable region residues of the recipient have been replaced by hypervariable region residues of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and potency. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues of non-human origin. In addition, humanized antibodies may contain residues that are not present in either the recipient antibody or the donor antibody. These modifications are made to further improve the characteristics of the antibody. In general, a humanized antibody will include substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to loops of a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin sequences. A humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0307] An "isolated" antibody is an antibody that has been identified and separated and/or recovered from a component of its natural environment. A contaminant component of the natural environment is a substance that interferes with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain aspects, the antibody will be purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method and greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a gas-phase protein sequencer, or (3) to homogeneity by SDS-PAGE electrophoresis under reducing or non-reducing conditions using Coomassie blue or a silver-based dye. An isolated antibody includes an antibody in situ in recombinant cells, since at least one component of the antibody's natural environment will be absent. Typically, however, an isolated antibody will be obtained by at least one purification step.

[0308] The term "cancer" refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors or cells that invade adjacent tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. As used herein, the term "cancer" refers to primary, metastatic, and recurrent cancer.

[0309] As used herein, the term "immune response" refers to a biological response within a vertebrate to foreign agents that protects the body from the foreign agents and the diseases they cause. An immune response is mediated by the action of a cell 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 by any of these cells or the liver (including antibodies, cytokines, and the complement system), which results in the selective targeting, binding, injury, destruction, and/or removal from the vertebrate body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. The immune response includes, for example, activation or inhibition of a T cell, such as an effector T cell or a Th cell, such as a CD4 ⁺ or CD8 ⁺ T cell, or inhibition of a Treg cell. As used herein, the terms "T cell" and "T lymphocytes" are used interchangeably and refer to any lymphocyte produced or processed by the thymus gland. In some aspects, the T cell is a CD4+ T cell. In some aspects, the T cell is a CD8+ T cell. In some aspects, the T cell is an NKT cell.

[0310] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0311] The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent (e.g., a neodisruptor or neodisruptor conjugate described herein) that provides a desired biological, therapeutic, and/or prophylactic result. This result may be a reduction, amelioration, palliation, reduction, delay, and/or alleviation of one or more manifestations, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause a tumor to shrink and/or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor progression. In some aspects, 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 a composition can, for example, (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, slow, slow to some extent, and may stop the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow to some extent and may stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and/or recurrence of the tumor; and/or (vii) relieve to some extent one or more symptoms associated with the cancer.

[0312] In some aspects, a "therapeutically effective amount" is an amount of a neodegradant or neodegradant conjugate that has been clinically shown to significantly reduce cancer or slow the progression (regression) of cancer, such as an advanced solid tumor. The ability of a therapeutic agent to promote disease regression can be assessed by a variety of methods known to a skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0313] As used herein, the term "standard of care" refers to a treatment that is recognized by medical experts as the appropriate treatment for a particular type of disease and that is widely used by health care professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard of care," and "standard therapy."

[0314] For example, an "anti-cancer agent" causes a cancer in a subject to regress or prevents further growth of a tumor. In certain aspects, a therapeutically effective amount of the drug causes a cancer to regress to the point of being eliminated.

[0315] The terms "effective" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects at the cellular, organ, and/or organismal level (side effects) that result from administration of a drug.

[0316] As used herein, the term "immune checkpoint inhibitor" refers to molecules that reduce, inhibit, interfere with, or modulate one or more checkpoint proteins, in whole or in part. Checkpoint proteins regulate the activation or function of T cells. Numerous 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, D. M., Nat Rev Cancer 12(4):252-64 (2012). These proteins are responsible for costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance, as well as the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0317] The terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures that aim to prevent or slow down (reduce) an undesirable physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of the progression of the disease, improvement or temporary relief of the disease state, and remission (partial or complete), detectable or undetectable. "Treatment" may also mean prolongation of survival compared to the expected survival if no treatment were administered. Persons in need of treatment include those who already have the condition or disorder, as well as those who are prone to the condition or disorder, or those in whom the condition or disorder is to be prevented.

II. Non-destructors

[0318] The present disclosure provides non-degraders of formula (II):

or their pharmaceutically acceptable salts, where:

[0319] A is a phenyl or C ₄ -C ₁₀ cycloalkyl ring;

[0320] U is selected from NH and CF ₂ ;

[0321] R ¹ is independently selected from hydrogen and halogen;

[0322] R ² is selected from -C(O)R ³ , -N(R ⁴ ) ₂ , -(CH ₂ ) _n OH, -(CH ₂ ) _n SH, -(CH ₂ ) _n N(R ⁴ ) ₂ , -(CH ₂ ) _n Q'(CH ₂ ) _m OH, -(CH ₂ ) _n Q'(CH ₂ ) _m SH and -(CH ₂ ) _n Q'(CH ₂ ) _m N(R ⁴ ) ₂ ; where

[0323] R ³ is hydrogen or C ₁ -C ₆ alkyl;

[0324] each R ⁴ is independently hydrogen or C ₁ -C ₆ alkyl;

[0325] Q' is O, S or NR ⁴ ;

[0326] n is 1-6; and

[0327] m is 2-5;

[0328] provided that when R ² is NH ₂ , -(CH ₂ ) _n NH ₂ or -(CH ₂ ) _n OH, then R ¹ is halogen.

[0329] In certain aspects, the present invention provides compounds of formula (II) or pharmaceutically acceptable salts thereof, wherein:

[0330] A represents a phenyl ring or a C ₄ -C ₁₀ cycloalkyl ring;

[0331] U is NH;

[0332] R ¹ is selected from hydrogen and halogen;

[0333] R ² is selected from -(CH ₂ ) _n Q'(CH ₂ ) _m N(R ⁴ ) ₂ , -(CH ₂ ) _n OH, -(CH ₂ ) _n SH, -N(R ⁴ ) ₂ and -C(O)R ³ ; where:

[0334] m is 2;

[0335] n is 2;

[0336] Q' represents -O-;

[0337] R ³ is methyl; and

[0338] each R ⁴ is independently selected from hydrogen and methyl;

provided that when R ² is NH ₂ or -(CH ₂ ) _n OH, then R ¹ is a halogen.

[0339] As used herein, the term "C ₁ -C ₆ alkoxy" refers to a C ₁ -C ₆ alkyl group attached to the parent molecular moiety through an oxygen atom.

[0340] As used herein _{, the term "C1-C6 alkoxyC1-C6 alkyl " refers} to a _C1 - _C6 alkoxy group appended to the parent molecular moiety through a _C1 - _C6 alkyl group.

[0341] As used herein, the term "C ₁ -C ₆ alkyl" refers to a group derived from a straight or branched chain saturated hydrocarbon containing from one to six carbon atoms.

[0342] As used herein, the term " _C4 - _C10 cycloalkyl" refers to a saturated monocyclic hydrocarbon ring system containing four to ten carbon atoms and zero heteroatoms. Illustrative examples of cycloalkyl groups include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups containing seven to ten atoms may be monocyclic or fused, spirocyclic, or bridged bicyclic structures.

[0343] As used herein, the term "halogen" refers to F, Cl, Br, or I.

[0344] In some aspects, the non-degrader of formula (II) is a compound selected from the group consisting of:

[0345] In some aspects, the non-degrader of formula (II) is

[0346] In some aspects, the non-degrader of formula (II) is

[0347] In some aspects, the non-degrader of formula (II) is

[0348] In some aspects, the non-destructor of formula (II) is

[0349] In some aspects, the non-degrader of formula (II) is

[0350] In some aspects, the non-destructor of formula (II) is

[0351] In some aspects, the non-destructor of formula (II) is

[0352] In some aspects, the non-destructor of formula (II) is

[0353] In some aspects, the present disclosure provides nondegraders of formula (II), or pharmaceutically acceptable salts thereof, wherein A is phenyl; U is NH; R ¹ is halogen; and R ² is -(CH ₂ ) _n Q'(CH ₂ ) _m N(R ⁴ ) ₂ , where m and n are 2, Q' is O, one R ⁴ is hydrogen, and the other is methyl.

[0354] In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is phenyl; U is NH; R ¹ is halogen; and R ² is -(CH ₂ ) _n Q'(CH ₂ ) _m N(R ⁴ ) ₂ , where m and n are 2, Q' is O, and each R ⁴ is methyl.

[0355] In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is phenyl; U is NH: R ¹ is halogen; and R ² is -(CH ₂ ) _n OH, where n is 2.

[0356] In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is phenyl; U is NH: R ¹ is halogen; and R ² is -(CH ₂ ) _n SH, where n is 2.

[0357] In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is phenyl; U is NH; R ¹ is hydrogen; and R ² is -N(R ⁴ ) ₂ , wherein one R ⁴ is hydrogen and the other is methyl.

[0358] In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is phenyl; U is NH; R ¹ is halogen; and R ² is -N(R ⁴ ) ₂ , wherein each R ⁴ is hydrogen. In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is phenyl; R ¹ is hydrogen; and R ² -C(O)R ³ , wherein R ³ is methyl.

[0359] In some aspects, the present disclosure provides non-degraders of formula (II), wherein A is a _C4 - _C10 cycloalkyl ring; U is NH; ^R1 is hydrogen; and ^R2 is -( _CH2 ) _nQ '( _CH2 ) _mN ( ^R4 ) ₂ , where m and n are 2, Q' is O, one ^R4 is hydrogen, and the other is methyl.

III. Neodestructor conjugates

[0360] The present invention provides conjugates of one or more neodegraders disclosed herein and a binding moiety. These conjugates can cleave proteins by binding to cereblon (CRBN), promoting the recruitment and ubiquitination of substrate proteins mediated by the CRL4 ^CRBN E3 ubiquitin ligase. These agents act as "molecular glues" by populating the binding surface as a hydrophobic patch that reprograms protein interactions between the ligase and neosubstrates.

[0361] In some aspects, the present invention provides a compound of formula (I),

or a pharmaceutically acceptable salt thereof, where

[0362] a is an integer from 1 to 10;

[0363] A is a phenyl or C ₄ -C ₁₀ cycloalkyl ring;

[0364] R ¹ is selected from hydrogen and halogen;

[0365] U is selected from NH and CF ₂ ;

[0366] X is selected from -NR ² -, =C(CH ₃ )-, -Q-(CH ₂ ) _n -, and -Q(CH ₂ ) _m Q'(CH ₂ ) _n -; where:

[0367] each Q and Q' is independently O, S, or NR ² ;

[0368] R ² is hydrogen or C ₁ -C ₆ alkyl;

[0369] n is an integer from 1 to 6;

[0370] m is an integer between 2 and 6;

[0371] where the left side of each group is attached to L and the right side is attached to A;

[0372] provided that when X is NH or -Q-(CH ₂ ) _n -, R ¹ is halogen;

[0373] L is a cleavable linker or a non-cleavable linker; and

[0374] Bm is a linking fragment.

[0375] In some aspects, U is NH.

[0376] In some aspects, a neodegradant conjugate described herein has in vitro antiproliferative activity against a tumor cell line. In some aspects, a neodegradant conjugate comprising a neodegradant and a binding moiety has an in vitro antiproliferative activity that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% greater than that of the neodegradant alone or the binding moiety alone. In some aspects, a neodegrader conjugate comprising a neodegrader and a binding moiety has an in vitro antiproliferative activity of 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, at least about 10-fold higher than the neodegrader alone or the binding moiety alone.

[0377] In some aspects, the neodisruptor conjugates described herein have in vitro antiproliferative activity against the BT-474 breast cancer cell line, such as greater antiproliferative activity against the BT-474 breast cancer cell line, compared to a single neodisruptor or a single binding moiety. In some aspects, the neodisruptor conjugates described herein have in vitro antiproliferative activity against the SK-BR-3 breast cancer cell line, such as greater antiproliferative activity against the SK-BR-3 breast cancer cell line, compared to a single neodisruptor or a single binding moiety. In some aspects, the neo-disruptor conjugates described herein have in vitro anti-proliferative activity against the NCI-N87 gastric cancer cell line, such as increased anti-proliferative activity against the NCI-N87 gastric cancer cell line, compared to a single neo-disruptor or a single binding moiety. In some aspects, the neo-disruptor conjugates described herein have in vitro anti-proliferative activity against the Daudi lymphoma cell line, such as increased anti-proliferative activity against the Daudi lymphoma cell line, compared to a single neo-disruptor or a single binding moiety. In some aspects, the neodestructor conjugates described herein have in vitro antiproliferative activity against the HL-60 acute myeloid leukemia cell line, such as greater antiproliferative activity against the HL-60 acute myeloid leukemia cell line, compared to the neodestructor or binding moiety alone. In some aspects, the neodestructor conjugates described herein have in vitro antiproliferative activity against the Ramos non-Hodgkin lymphoma cell line, such as increased antiproliferative activity against the Ramos non-Hodgkin lymphoma cell line, compared to the neodestructor or binding moiety alone. In some aspects, the neodestructor conjugates described herein are capable of maintaining their antiproliferative activity in the presence of human serum. The neodestructor conjugates described herein can be used to treat cancer.

III. A. Linker

[0378] The neodegrader of the present invention may be linked to the linking moiety via a linker. As used herein, the term "linker" refers to any chemical moiety capable of linking the linking moiety (Bm) to the group X in the compounds of formula (I).

[0379] In certain aspects, the linker may comprise a heterobifunctional group. As used herein, the term "heterobifunctional group" refers to a chemical moiety that connects the linker of which it is a part to the linking moiety. Heterobifunctional groups are characterized by the presence of different reactive groups at both ends of the chemical moiety. Attachment to "Bm" may be accomplished by chemical or enzymatic conjugation, or a combination thereof. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the linking moiety with a reactive handle on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysinamide coupling, cysteine coupling, and coupling via a non-natural amino acid introduced by genetic engineering, wherein the non-natural amino acid residues with the desired reactive handle are incorporated into "Bm". In enzymatic conjugation, an enzyme mediates the attachment of a linker to an accessible amino residue on the linker moiety. Examples of enzymatic conjugation include, but are not limited to, sortase-based transpeptidation, microbial transglutaminase-based transpeptidation, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can also be used to install unique "Bm" response markers for use in subsequent chemical conjugation.

[0380] In some aspects, the heterobifunctional group is selected from:

moreover

represents the point of attachment to the remainder of the linker; and

point of attachment to Bm.

[0381] In certain aspects, the linker "L" is non-cleavable. As used herein, the term "non-cleavable linker" is any chemical moiety that is capable of linking a linking moiety to a non-degrader in a stable covalent manner and does not fall into the categories defined herein as "cleavable linkers." Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. "Substantially resistant to cleavage" means that a chemical bond in or adjacent to a linker is 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 a non-degrader antibody conjugate that remains non-cleavable by an acid, a photolabile cleaving agent, a bioremediating agent, a peptidase, an esterase, or a chemical or physiological compound that cleaves a chemical bond (e.g., a disulfide bond) in a cleavable linker within several hours to several days after treatment with any of the agents described above. In some aspects, the linker is not susceptible to acid-induced cleavage, photo-induced cleavage, bioremediative cleavage, enzymatic cleavage, and the like. under conditions in which the non-cleavable linker and/or the linking moiety may remain active. ADC catabolites derived from non-cleavable linkers contain residual amino acid from the antibody. These catabolites may exhibit unique and unexpected properties in the target cells to which they are delivered.

[0382] One skilled in the art can easily distinguish between non-cleavable linkers and cleavable ones.

[0383] Examples of non-cleavable linkers include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linkers, succinimide thioester linkers, and linkers such as:

[0384] p is an integer between 1 and 10;

[0385] represents the attachment point to X; and

[0386] represents the attachment point to the linking fragment.

[0387] In some aspects, the linker is:

In some aspects p is equal to 5.

[0388] In certain aspects, the linker may be cleavable. In some aspects, the linker may be subject to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, etc. under conditions in which the non-degrader and/or linking moiety may remain active.

[0389] In some aspects, the cleavable linker can be enzymatically cleaved. In some aspects, the cleavable linker can be cleaved by a protease, peptidase, esterase, beta-glucuronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase.

[0390] In some aspects, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like.

[0391] In certain aspects, the cleavable linker comprises a peptide. In some aspects, the peptide is a 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 a particular enzyme, such as a tumor-associated protease, cathepsin B, C, and D, or plasmin protease. Examples of peptides comprising 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 containing 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 containing 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 amino acid combinations listed above can also be represented in reverse order (i.e., cit-val).

[0392] The peptides of the present invention may contain 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-" refers to an amino acid having a "D"- (dextrorotatory) configuration, as opposed to the configuration in naturally occurring ("L-") amino acids. The amino acids described herein can be purchased from commercial sources (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0393] In certain aspects, the linker ("L") is a protease cleavable linker selected from

Where:

[0394] q is an integer between 2 and 10;

[0395] Z ¹ , Z ² , Z ³ and Z ⁴ are each independently absent or represent a naturally occurring amino acid residue in the L- or D-configuration, provided that at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues;

[0396] represents the attachment point to X; and

[0397] represents the attachment point to the linking fragment.

[0398] In certain aspects, Z ¹ , Z ² , Z ³ and Z ⁴ are independently absent, 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; provided that at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues

[0399] In some aspects, Z ¹ is absent or is glycine, Z ² is absent or is 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; and Z ⁴ is selected from L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalamine, D-phenylalanine, and glycine.

[0400] In some aspects, L is

[0401] In some aspects, q is equal to 5.

[0402] In certain aspects, L is a pyrophosphatase cleavable linker.

[0403] In some aspects, L is a pyrophosphatase cleavable linker that is:

Where:

[0404] q is an integer between 2 and 10;

[0405] represents the attachment point to X; and

[0406] represents the attachment point to the linking fragment.

[0407] In certain aspects, L is a beta-glucuronidase cleavable linker.

[0408] In some aspects, L is a beta-glucuronidase cleavable linker selected from:

Where:

[0409] q is an integer between 2 and 10;

[0410] absent or represents a connection;

[0411] represents the point of attachment to X; and

[0412] represents the attachment point to the linking fragment.

[0413] In some aspects, the linker is bioreducible. Bioreducible linkers take advantage of the difference in reductive potential in the intracellular compartment compared to plasma. In tumor cell cytoplasm, glutathione is present at levels 1000 times higher than in normal cell cytoplasm, and tumor cells also contain enzymes that can promote reductive activity in cellular compartments. The linkers maintain the integrity of the conjugates during systemic circulation and are selectively cleaved by high intracellular glutathione concentrations, releasing active drugs from non-toxic prodrugs at tumor sites.

[0414] In some aspects, L is a bioreducible linker selected from:

Where:

[0415] q is an integer between 2 and 10;

[0416] each R, R', R'' and R''' is independently selected from _hydrogen , _C1 -C6alkoxyC1 _{- C6alkyl} , ( _C1 - _C6 ) _{2NC1 - C6alkyl} and _C1 - _C6alkyl , or two geminal R groups together with the carbon atom to which they are attached can form a cyclobutyl or cycloprohypinal ring;

[0417] represents the attachment point to X; and

[0418] represents the attachment point to the linking fragment.

[0419] In certain aspects, the linker is acid cleavable. Acid cleavable linkers are specifically designed to remain stable at the neutral pH of the bloodstream, but to undergo hydrolysis and release the cytotoxic drug in the acidic environment of cellular compartments.

[0420] In some aspects, L is an acid-cleavable linker selected from

Where:

[0421] q is an integer from 2 to 10;

[0422] represents the attachment point to X; and

[0423] represents the attachment point to the linking fragment.

[0424] In certain aspects, L is wherein L is a click-reactive drug release linker, wherein release of the neodegrader is chemically initiated by the tetrazine or related compound

[0425] In some aspects, L is a click drug release linker selected from

Where:

[0426] q is an integer between 2 and 10;

[0427] represents the attachment point to X; and

[0428] represents the attachment point to the linking fragment.

III.B. Linking fragment

[0429] The present invention provides neodegradants conjugated to binding moieties. The term "binding moiety" as used herein refers to any molecule that recognizes and binds to a cell surface marker or receptor. In certain aspects, the binding moiety binds to a protein, not limited to a polypeptide moiety. The binding moiety, in addition to targeting the neodegradant to a particular cell, tissue, or site, may also exert a particular therapeutic effect, such as antiproliferative (cytostatic and/or cytotoxic) activity against a target cell or pathway. In certain aspects, the binding moiety may comprise or be engineered to comprise at least one chemically reactive group, such as a carboxylic acid, amine, thiol, or chemically reactive amino acid moiety or side chain. In some aspects, the binding moiety may comprise a targeting moiety that binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. Upon specific binding or complex formation with the receptor, the cell becomes available for uptake of the target moiety or neodegrader conjugate, which is then internalized into the cell.

[0430] In some aspects, the "Bm" group may be a moiety that can specifically bind to a cell surface molecule. In some aspects, the "Bm" group may be a peptide or protein that binds to a cell surface receptor or antigen.

[0431] In certain aspects, the "Bm" moiety may be an antibody, an antibody fragment, or an antigen-binding fragment. An antibody is a protein produced by the immune system that is capable of recognizing and binding to a specific antigen. A target antigen typically has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to different epitopes 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, so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, hybrid, or derived from other species.

[0432] Monoclonal antibodies, which can be conjugated to a neodestructor, are homogeneous populations of antibodies to a specific antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, a nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen of interest can be produced using any method known in the art that involves the production of antibody molecules using continuous cell lines in culture. These include, but are not limited to, the hybridoma method, the human B-cell hybridoma method, and the EBV hybridoma method. Such antibodies can be of any class of immunoglobulins, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridoma producing the mAbs used in the present invention can be cultured in vitro or in vivo.

[0433] Suitable 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 produced by any of a variety of methods known in the art.

[0434] The antibody may also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies relies on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. Because of the random distribution of the immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the desired molecule, typically accomplished by affinity chromatography, is cumbersome and the yield of the product is low.

[0435] According to another approach, variable domains of antibodies with the desired binding specificity (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. The fusion may be with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge regions C.subregion.H2 and C.subregion.H3. The first heavy chain constant region (C.subregion.H1) may contain a site necessary for light chain binding present in at least one of the fusions. Nucleic acid sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression systems and co-transfected into a suitable host organism. This provides greater flexibility in adjusting the relative proportions of the three polypeptide fragments in aspects where unequal ratios of the three polypeptide chains used in the construct provide optimal yields. However, it is possible to insert 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 high yields or when the ratios are not particularly important.

[0436] 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 the separation of the desired bispecific compound from undesired immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy method of separation. Using such methods, bispecific antibodies can be prepared for conjugation with non-degradants for the treatment or prevention of diseases as defined herein.

[0437] Hybrid or bifunctional antibodies may be produced either biologically, i.e., by cell fusion techniques, or chemically, especially using cross-linking agents or reagents that form disulfide bridges, and may include whole antibodies or fragments thereof.

[0438] The antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens, or other antibodies associated with tumor cells or matrix. In this regard, "functionally active" means that the fragment, derivative, or analog is capable of eliciting anti-anti-idiotypic antibodies that recognize the same antigen as the antibody from which the fragment, derivative, or analog is derived. In particular, in an illustrative aspect, the antigenicity of the idiotype of an immunoglobulin molecule can be enhanced by deletion of the framework region and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in an antigen binding assay by any binding assay known in the art.

[0439] Other suitable antibodies include antibody fragments such as, but not limited to, F(ab')2 fragments that contain the variable region, the constant region of the light chain, and the CH1 domain of the heavy chain, which can be obtained by pepsin digestion of the antibody molecule, and Fab fragments that can be obtained by reducing the disulfide bridges of F(ab')2 fragments. Other suitable antibodies are dimers of the heavy and light chains of antibodies or any minimal fragments thereof, such as Fv or single chain antibodies (SCAs), or any other molecule with the same specificity as the antibody.

[0440] Also useful antibodies are recombinant antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, which can be produced using standard recombinant DNA techniques. A chimeric antibody is a molecule in which different portions are derived from different animal species, for example, having a variable region derived from a mouse monoclonal and a human immunoglobulin. Humanized antibodies are antibody molecules from a non-human species that have one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0441] Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, for example, all or part of a polypeptide of the present invention. Monoclonal antibodies directed against the antigen can be produced using conventional hybridoma technology. The human immunoglobulin transgenes contained in the transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutations. Thus, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced using this method. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). Other human antibodies are available commercially, for example from Abgenix, Inc. (Fremont, CA) and Genpharm (San Jose, CA).

[0442] Fully human antibodies that recognize a selected epitope can be produced using a technique called "directed selection." In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to direct the selection of a fully human antibody that recognizes the same epitope. Human antibodies can also be produced by a variety of methods known in the art, including phage display libraries.

[0443] The antibody may be a fusion protein of an antibody or a functionally active fragment thereof, for example, in which the antibody is fused via a covalent bond (e.g., a peptide bond) at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or a portion thereof, such as at least 10, 20, or 50 amino acid portion of the protein) that is not an antibody. The antibody or fragment thereof may be covalently linked to another protein at the N-terminus of a constant domain.

[0444] Antibodies include analogs and derivatives that have either been modified, i.e., by covalent attachment of any type of molecule, if such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not by way of limitation, derivatives and analogs of antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or another protein, etc. Any of a variety of chemical modifications can be accomplished by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, an analog or derivative can contain one or more unnatural amino acids.

[0445] Antibodies in the neodestructor conjugates may include antibodies having modifications (e.g., substitutions, deletions, or additions) at amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications at amino acid residues identified as participating in the interaction between the anti-Fc domain and the FcRn receptor. Antibodies immunospecific for a cancer cell antigen may be obtained commercially, for example, from Genentech (San Francisco, Calif.), or produced by any method known to one of skill in the art, such as, for example, chemical synthesis or recombinant expression methods. The nucleotide sequence encoding antibodies immunospecific for a cancer antigen may be obtained, for example, from the GenBank or similar database, from literature publications, or by routine cloning and sequencing.

[0446] In certain aspects, the antibody of the neodestructor conjugates may be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. In some aspects, the antibody may be an antibody fragment, such as a Fab fragment.

[0447] Known antibodies for treating or preventing cancer can be conjugated to the neodestructors described herein. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art, such as, for example, recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained commercially, for example, from the GenBank or similar database, from literature publications, or by routine cloning and sequencing. Examples of antibodies available for treating cancer include, but are not limited to, a humanized monoclonal antibody against HER2 for treating patients with metastatic breast cancer; RITUXAN.RTM. (rituximab; Genentech), which is a chimeric monoclonal antibody against CD20 for treating patients with non-Hodgkin's lymphoma; OvaRex (oregovomab; AltaRex Corporation, MA), a murine antibody for the treatment of ovarian cancer; Raporex (edrecolomab, Glaxo Wellcome, NC), a murine IgG 2a antibody for the treatment of colorectal cancer; Cetuximab erbitux (cetuximab, Imclone Systems Inc., NY), a chimeric IgG anti-EGFR antibody for the treatment of epidermal growth factor-positive cancers such as head and neck cancer; Vitaxin (etaracizumab, MedImmune, Inc., MD), a humanized antibody for the treatment of sarcoma; Campath I/H (alemtuzumab, Leukosite, MA), a humanized IgG subregion.1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., California), a humanized IgG anti-CD33 antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (epratuzumab, Immunomedics, Inc., New Jersey), a humanized IgG anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; Smart ID 10 (Protein Design Labs, Inc., California), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., California), a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, California), a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma; Avastin (bevacizumab, Genentech, Inc., California), a humanized anti-VEGF antibody for the treatment of lung and colorectal cancer; Epratuzamab (Immunomedics, Inc., New Jersey and Amgen, California), an anti-CD22 antibody for the treatment of non-Hodgkin lymphoma; and CEAcide (Immunomedics, New Jersey), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0448] Other antibodies useful for neodisruptor 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 aspects, the antibody is selected from the group consisting of rituximab, trastuzumab, pertuzumab, OR000213, lintuzumab, and gemtuzumab.

[0449] Other antibodies useful for neodestructor conjugates include, but are not limited to, antibodies against the following antigens: CA125 (ovary), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha-fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product (carcinomas). Some specific useful antibodies include, but are not limited to, mAb BR96 (Trail, P. A, et al. Science (1993) 261, 212-215), BR64 (Trail, P. A., et al. Cancer Research (1997) 57, 100-105), mAb against CD40 antigen such as mAb S2C6 (Francisco, J. A., et al. Cancer Res. (2000) 60:3225-3231), mAb against CD70 antigen such as mAb 1F6, and mAb against CD30 antigen such as AC10. Many other internalizing antibodies that bind to tumor-associated antigens can be used and have been considered.

[0450] Other antigens to which the present conjugates can bind include, but are not limited to, 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, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, cohesion factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cl13, c-MET, Crypto 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, EPHRB4, 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, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 1Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, αIIbβ3), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumain, LewisY, LFA-l(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA/MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-CD2, OR51E2, OY-TES1, p53, mutant p53, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA- 1/galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa, rabies virus gene, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, respiratory syncytial virus, Rhesus factor, RhoC, RON, ROR1, ROR2, RU1, RU2, translocation breakpoints sarcomas, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, sperm protein 17, sphingosine-1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1/CD248, TEM7R, tenascin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2, TIM-1, Tn Ag, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16,88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1 and/or XAGE1.

[0451] 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.

[0452] The neo destructor a conjugate antibodies may bind to either a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex may comprise a member of the immunoglobulin gene superfamily, a member of the TNF receptor superfamily, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members include CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD 152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable members of the TNF receptor superfamily include 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 include CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD 103, and CD 104. Non-limiting examples of suitable lectins include C-type, S-type, and I-type lectins.

[0453] In some aspects, antibodies that can be used for the present invention include, but are not limited to, 3F8, 8H9, abagovomab, abciximab (REOPRO ^® ), abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab (HUMIRA ^® ), adecatumumab, aducanumab, afacevicumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH ^® ), alirocumab (PRALUENT®), altumomab, amatuximab, anatumab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab (CEA-SCAN ^® ), ascrinvacumab, aselizumab, atidortoxumab, atlizumab (tocilizumab, ACTEMRA ^® , ROACTEMRA ^® ), atezolizumab (TECENTRIQ ^® ), atinumab, atorolimumab, avelumab (Bavencio), azintuxizumab, balantamab, bapineuzumab, basiliximab (SIMULECT ^® ), bavituximab, BCD-100, bectumomab (LYMPHOSCAN ^® ), begelomab, belantamab, belimumab (BENLYSTA ^® ), bemarituzumab, benralizumab (FASENRA ^® ), bermekimab, bersanlimab, bertilimumab, besilesomab (SCINITIMUN ^® ), bevacizumab (AVASTIN ^® ), bezlotoxumab (ZINPLAVA ^® ), biciromab (FIBRISCINT ^® ), bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blozozumab, bococizumab, brazicumab, brentuximab, briakinumab, brodalumab (SILIQ™), brolucumab (BEOVU ^® , brontictuzumab, burozumab (CRYSVITA ^® ), cabiralizumab, caplacizumab (CABLIVI ^® ), camidanlumab, camrelizumab, canakinumab (ILARIS ^® ), cantuzumab, capromab, carlumab, carotuximab, catumaxomab (REMOVAB ^® ), cBR96, CC49, cedelizumab, cemiplimab (LIBTAYO ^® ), cergutuzumab, certrelimab, certolizumab, cetuximab (ERBITUX ^® ), cibisatamab, cirmtuzumab, citutuzumab, cixutumumab, clazakizumab, clenoliximab, clivatuzumab, codrituzumab, coffetuzumab, coltuximab, conatumumab, concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab (ADAKVEO ^® ), crotedumb, cuzatuzumab, dacetuzumab, daclizumab (ZINBRYTA ^® ), dalotuzumab, dapirolizumab, daratumumab (DARZALEX ^® ), dextrecumab, demcizumab, denintuzumab, denosumab (PROLIA ^® ), depatuxumab, derlotuximab, detumomab, desamizumab, dinutuximab (UNITUXIN ^® ), diridavumab, domagrozumab, dostarlimab, dorlimomab, dorlixizumab, drositumab, DS-8201, duligotuzumab, dupilumab (DUPIXENT ^® ), durvalumab (IMFINZI ^® ), dusigitumab, ecomeximab, eculizumab (SOLIRIS ^® ), edobacomab, edrecolomab (PANOREX ^® ), efalizumab (RAPTIVA ^® ), efungumab (MYCOGRAB ^® ), eldelumab, elezanumab, elgemtumab, elotuzumab (EMPLICITI ^® ), elsilimomab, emactuzumab emapalumab (GAMIFANT ^® ), emibetuzumab, emicizumab (HEMLIBRA ^® ), enapotamab, enavatuzumab, enfortumab (PADCEV ^® ), enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, eptinezumab (VYEPTI ^® epratuzumab, erenumab (AIMOVIG ^® ), erlizumab, ertumaxomab (REXOMUN ^® ), etaracizumab (ABEGRIN ^® ), etigilimab, etrolizumab, evinacumab, evolocumab (REPATHA ^® ), exbivirumab, fanolesomab (NEUTROSPEC ^® ), faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibat ficlatuzumab, figitumumab, firivumab, flanvotumab, fleticumab, flotetuzumab, fontolizumab (HUZAF ^® ), foralumab, foravirumab, fremanezumab (AJOVY ^® ), frezolimumab, frovocimab, frunevetmab, fulranumab, futuximab, galcanezumab (EMGALITY ^® ), galiximab, ganitamamab, ganitumab gantenerumab, gavilimomab, gedivumab, gemtuzumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab, golimumab (SIMPONI ^® ), gomiliximab, guselkumab (TREMFYA ^® ), huMy9-6, OR000213, ianalumab, ibalizumab (TROGARZO ^® ), IBI308, ibritumomab, icrucumab, idarucizumab (PRAXBIND ^® ), ifabotuzumab, igovomab (INDIMACIS-125), iladatuzumab, IMAB362, imalumab, imaprelimab, imciromab (MYOSCINT ^® ), imgatuzumab, inclacumab, indatuximab, indusatumab, inebilizumab, infliximab ( ^REMICADE® ), intetumumab, inolimomab, inotuzumab, yomab-B, ipilimumab, iratumumab, isatuximab ( ^SARCLISA® ), iscalimab, istiratumab, itolizumab, ixekizumab ( ^TALTZ® ), keliximab, labetuzumab (CEA-CIDE™), lacnotuzumab, ladiratuzumab, lampalizumab, lanadelumab ( ^TAKHZYRO® ), landogrosumab, laprituximab, larkaviximab, lebrikizumab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab, ligelizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, loncastuximab, lorvotuzumab, losatuxizumab, lucatumumab, lulizumab, lumiliximab, lumretuzumab, lupartumab, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, matuzumab, mavrilimumab, mepolizumab (NUCALA ^® ), metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab, mitumomab, modotuximab, molalizumab, mogamulizumab (POTELIGEO ^® ), morolimumab, mosunetuzumab, motavizumab (NUMAX ^® ), moxetumomab (LUMOXITI ^® ), muromonab-CD3 (ORTHOCLONE OKT3 ^® ), nacolomab, namilumab, naptumomab, naratuximab, namatumab, natalizumab (TYSABRI ^® ), navicixizumab, navivumab, naxitamab, nebacumab, necitumumab (PORTRAZZA ^® ), nemolizumab, NEOD001, nerelimomab, nesvacumab, netakimab, nimotuzumab (THERACIM ^® ), nirsevimab, nivolumab, nofetumomab, obiltoxaximab (ANTHIM ^® ), obinutuzumab, ocaratuzumab, ocrelizumab (OCREVUS ^® ), odulimomab, ofatumumab (ARZERRA ^® ), olaratumab (LARTRUVO ^® ), oleclumab, olendalizumab, olokizumab, omalizumab (XOLAIR ^® ), OMS721, onartuzumab, ontecizumab, ontuxizumab, onvatilimab, opicinumab, opportuzumab, oregovomab (OVAREX), orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozogamicin, ozoralizumab, pagibaximab, palivizumab (SYNAGIS ^® ), pamrevlumab, panitumumab (VECTIBIX ^® ), pancomab, panobacumab, parsatuzinumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab (THERAGYN ^® ), perakizumab, pertuzumab (OMNITARG ^® ), pexelizumab, pidilizumab, pinatuzumab, pintumomab, placulumab, polatuzumab (Polivy), prezalumab, plozalizumab, pogalizumab, ponesumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ralpancezumab, ramucirumab, ranevetmab, ranibizumab (LUCENTIS ^® ), ravagalimab, ravulizumab (ULTOMIRIS ^® ), raxibacumab, refanezumab, regavirumab, REGN-EB3, renatlimab, remtolumab, reslizumab (CINQAIR ^® ), rilotumumab, rinucumab, risankizumab (SKYRIZI ^® ), rituximab (RITUXAN ^® ), rivabazumab, rmab, robatumumab, roledumab, romilkimab, romosozumab (EVENITY ^® ), rontalizumab, rozmantuzumab, rovalpituzumab, rovelizumab (LEUKARREST ^® ), rosanolyxizumab, ruplizumab (ANTOVA), SA237, sacituzumab, samalizumab, samrotamab, sarilumab (KEVZARA ^® ), satralizumab, satumomab pendetide, secukinumab (COSENTYX ^® ), selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, SGN-CD19A, SHP647, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, STI-6129, sulesomab (LEUKOSCAN ^® ), suptavumab, sotimlimab, suvizumab, suvratoxumab, tabalumab, takatuzumab (AFP- ^CIDE® ), tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tavolimab, tefibazumab (AUREXIS ^® ), telimomab, telistuzumab, tesidulumab, tetraxetane, tetulomab, tenatumomab, teneliximab, teprotumumab (TEPEZZA ^® ), teplizumab, tezepelumab, TGN1412, tibulizumab, ticilimumab (TREMELIMUMAB ^® ), tigatuzumab, timigutuzumab, timolumab, tiragolumab, tiragotumab, tislelizumab, tisotumab, tiuxetan, tildrakizumab (ILUMYA ^® ), TNX-650, tocilizumab (atlizumab, ACTEMRA ^® ), tomzotuximab, toralizumab, tosatoxumab, tositumomab (BEXXAR ^® ), tovetumab, tralokinumab, trastuzumab (HERCEPTIN ^® ), TRBS07, tregalizumab, tremelimumab, alarmrumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab (STELERA ^® ), ublituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vanalimab, vandortuzumab, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab (NUVION ^® ), vobarilizumab, volocisimab (HUMASPECT ^® ), vonlerolizumab, vopratelimab, vorsetuzumab, votumumab, vunakizumab, xentuzumab, HMAV-5574, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), zatuximab, zenokutuzumab, ziralimumab, zolbetuximab or zolimomab.

[0454] An antibody "that binds" a molecular target or antigen of interest is an antibody that is capable of binding that antigen with sufficient affinity such that the antibody can be used to target a cell expressing the antigen.

[0455] In the present disclosure, the "Bm" group may be conjugated to more than one neodegrader. In some aspects, "Bm" may be conjugated to 1 to 10 neodegraders. In some aspects, "Bm" may be conjugated to 1 to 9 neodegraders. In some aspects, "Bm" may be conjugated to 1 to 8 neodegraders. In some aspects, "Bm" may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 neodegraders. In some aspects, "Bm" may be conjugated to 7 or 8 neodegraders. In some aspects, "Bm" is conjugated to 5 neodegraders. In some aspects, "Bm" is conjugated to 6 neodegraders. In some aspects, "Bm" is conjugated to 7 neodegraders. In some aspects, "Bm" is conjugated to 8 neodegraders. In some aspects, "Bm" is conjugated to 9 neodegraders.

IV. Compositions and methods of application

[0456] The conjugates and/or compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some aspects, such salts are derived from inorganic or organic acids or bases.

[0457] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

[0458] Examples of suitable base addition salts include ammonium salts; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, and the like.

[0459] For example, Berge lists the following FDA-approved commercially sold salts: acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycollylarsanilate (glycollamidophenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (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, subacetate, succinate, sulfate, tannate, tartrate, theoclate (8-chlorotheophyllinate), and triethiodide; organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and metallic cations of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

[0460] Berge additionally lists the following non-FDA approved commercially sold (outside the United States) salts: adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), napadisylate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate, and undecanoate; organic cations benetamine (N-benzylphenethylamine), clemizole (1-n-chlorobenzyl-2-pyrrolyldin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations barium and bismuth.

[0461] Pharmaceutical compositions containing the neodegradant conjugates described herein may also include suitable carriers, excipients, and adjuvants, which may vary depending on the route of administration.

[0462] In some aspects, the pharmaceutical compositions can be formulated into a suitable dosage form for parenteral administration. These formulations can be prepared by a variety of methods known in the art. The pharmaceutical compositions 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 administration. Suitable devices for parenteral administration include needle injectors, needle-free injectors, and infusion techniques.

[0463] Compositions for parenteral administration are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents. However, the composition can also be prepared as a sterile non-aqueous solution or as a dried form for use in combination with a suitable vehicle such as sterile, pyrogen-free water.

[0464] The preparation of parenteral compositions under sterile conditions, for example by lyophilization, can readily be accomplished using standard techniques well known to those skilled in the art.

[0465] Compositions for parenteral administration may be prepared with immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release. Thus, the compositions may be prepared as a solid, semi-solid or thixotropic liquid for administration as an implanted depot providing modified release of the active agent.

[0466] Formulations for parenteral use may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

[0467] In another aspect, the pharmaceutical compositions can be prepared in suitable oral dosage forms such as tablets, capsules, powders, pellets, suspensions, solutions, emulsions, and the like. Other suitable carriers such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, excipients, anti-adhesives, and the like may be present.

[0468] Oral formulations may also contain other suitable pharmaceutical excipients such as sweeteners, carriers/wetting agents, colorants, flavors, preservatives, viscosity enhancing/thickening agents, etc.

[0469] The neodegradant conjugates described herein can be used to treat various types of cancer. Certain conjugates of the present invention can be superior in terms of expression efficacy, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interaction with other drugs (e.g., inhibitory effect on drug metabolization), safety (e.g., acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity), and stability (e.g., chemical stability, enzyme stability), and can be useful as a drug.

[0470] The neodegradant conjugates of the present invention can be used as agents, such as an agent for the prevention or treatment of diseases such as, for example, cancer such as various types of colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric/GI cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma), duodenal cancer colon cancer, small bowel cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian tumor of low malignant potential), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, non-hormone-dependent 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., 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, endometrial cancer, uterine sarcoma), gestational choriocarcinoma, brain tumors (eg, medulloblastoma, glioma, pineal gland tumors, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (eg, basal cell carcinoma, malignant melanoma), sarcomas (eg, rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, hematological/blood cancer (eg, multiple myeloma, leukemia (eg, acute myelogenous leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disease), primary cancer of unknown origin; cancer growth inhibitor; inhibitor of cancer metastasis; stimulator of apoptosis; an agent for the treatment of precancerous lesions (eg, myelodysplastic syndromes); etc.

[0471] In certain aspects, the neodestructor conjugates of the present invention can be used as a medicament for the treatment of breast cancer, gastric cancer, ovarian cancer, uterine cancer, lung cancer, pancreatic cancer, liver cancer, lymphoma, or hematological cancer.

[0472] In addition, the neodestructor conjugates of the present invention can be used simultaneously with non-drug therapy. To be specific, the conjugates can be combined with non-drug therapy such as (1) surgery, (2) hypertensive chemotherapy using angiotensin II, etc., (3) gene therapy, (4) thermotherapy, (5) cryotherapy, (6) laser cauterization, and (7) radiation therapy.

[0473] For example, by using the neodestructor conjugate of the present invention before or after the above-mentioned surgery and the like, effects such as preventing the occurrence of resistance, prolonging the period of disease-free survival, suppressing metastasis or recurrence of cancer, prolonging life and the like can be achieved.

[0474] In addition, it is possible to combine the treatment with the neodestructor conjugates of the present invention with supportive therapy by (i) administering an antibiotic (e.g., β-lactam type such as pansporin, etc., macrolide type such as clarithromycin, etc.) for complications of various infectious diseases, (ii) administering a high-calorie transfusion, an acid preparation, or a general vitamin preparation to improve malnutrition, (iii) administering morphine to reduce pain, (iv) administering a pharmaceutical agent to alleviate side effects such as nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, hepatopathy, renal disease, DIC syndrome, fever, etc. (v) administering a pharmaceutical agent to suppress multidrug resistance of cancer, etc.

[0475] In some aspects, a neodisruptor or neodisruptor conjugate of the present invention may be used in combination with standard medical therapy, such as one or more therapeutic agents (e.g., anti-cancer agents and/or immunomodulatory agents). Accordingly, in some aspects, the method of treating a tumor disclosed herein comprises administering a neodisruptor or neodisruptor conjugate of the present invention in combination with one or more additional therapeutic agents. In some aspects, a neodisruptor or neodisruptor conjugate of the present invention may be used in combination with one or more anti-cancer agents such that multiple elements of an immune pathway may be targeted. In some aspects, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a particular 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 combinations thereof. A complete and non-limiting list of combination therapies is detailed in the Combination Therapies section of this application.

[0476] In some aspects, a neodegradant or neodegradant conjugate of the present invention is administered to the subject prior to or subsequent to administration of an additional therapeutic agent. In other aspects, a neodegradant or neodegradant conjugate of the present invention is administered to the subject concurrently with an additional therapeutic agent. In certain aspects, a neodegradant or neodegradant conjugate of the present invention and an additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In other aspects, a neodegradant or neodegradant conjugate of the present invention and an additional therapeutic agent are administered concurrently as separate compositions.

[0477] In some aspects, the neodegradant or neodegradant conjugate of the present invention and the additional therapeutic agent are administered simultaneously as separate compositions. In some aspects, the subject to be treated is a human.

V. Methods for obtaining non-destructors and compositions

[0478] The present invention provides a method for producing conjugates of neodegraders, comprising reacting a linking moiety with a compound of formula (1-1):

or a pharmaceutically acceptable salt thereof, where

A is a phenyl or a _C4 - _C10 cycloalkyl ring; ^K1 is independently selected from hydrogen and halogen; U is selected from NH and _CF2 ;

X is selected from ^-NR2- , =C( _CH3 )-, -Q-( _CH2 ) _n- and -Q( _CH2 ) _mQ '( _CH2 ) _n- ; wherein each Q and Q' is independently O, S or ^NR2 ; ^R2 is hydrogen or _C1 - _C6 alkyl; n is an integer from 1 to 6; m is an integer from 2 to 6; and wherein the left side of each group is attached to L' and the right side is attached to A; provided that when X is NH or -Q-( _CH2 ) _n- , ^R1 is halogen;

L' is a cleavable or non-cleavable linker precursor that conjugates to the linking moiety.

[0479] As described herein, a linker precursor comprises a heterobifunctional group that is coupled to a linking moiety.

[0480] In some aspects, L' is a non-cleavable linker precursor. In some aspects, L' is selected from the group consisting of

Where:

p is an integer between 1 and 10; and

represents the attachment point to X;

[0481] In some aspects, L' is

[0482] In some aspects, p is equal to 5.

[0483] In certain aspects, L' is a cleavable precursor of a linker.

[0484] In some aspects, the linker precursor is cleaved by a protease. In some aspects, the linker precursor is selected from the group consisting of

Where:

q is an integer between 2 and 10;

each Z ¹ , Z ² , Z ³ and Z ⁴ is independently absent or represents the residue of a naturally occurring amino acid; in the L- or D-configuration; provided that at least two of Z ¹ , Z ² , Z ³ and Z ⁴ represent amino acid residues;

And

represents both the point of attachment to X;

[0485] In some aspects, Z ¹ , Z ² , Z ³ and Z ⁴ are independently absent, selected from the group consisting of L-valine, D-valine, L-citrulline, D-citrulline, L-alanine, D-alanine, L-glutamine, D-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 at least two of Z ¹ , Z ² , Z ³ and Z ⁴ are amino acid residues

[0486] In some aspects, Z ¹ is absent or is glycine; 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; and Z ⁴ is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalamine, D-phenylalanine, and glycine.

[0487] In some aspects, L' is

[0488] In some aspects, q is equal to 5.

[0489] In some aspects, L' is a bioregenerable linker precursor. In some aspects, the bioregenerable linker precursor is selected from the group consisting of

And

Where:

q is an integer between 2 and 10;

each R, R', R'' and R''' is independently selected from hydrogen, _C1 - _{C6alkoxyC1 - C6alkyl} , ( _C1 - _C6 ) _{2NC1 - C6alkyl} and _C1 - _C6alkyl , or two geminal R groups together with the carbon atom to which they are attached may form a cyclobutyl or cyclopropyl ring; and

represents the attachment point to X;

[0490] In certain aspects, L' is an acid cleavable linker precursor. In some aspects, L' is selected from the group consisting of

, ;

Where:

q is an integer between 2 and 10; and

represents the attachment point to X;

[0491] In certain aspects, L' is a precursor of a drug-release click reaction linker, in some aspects, L' is selected from

Where:

q is an integer between 2 and 10; and

represents the attachment point to X;

[0492] In certain aspects, L' is a precursor of a pyrophosphatase-cleavable linker. In certain aspects, L' is

Where:

q is an integer between 2 and 10;

represents the attachment point to X;

[0493] In certain aspects, L' is a precursor of a beta-glucuronidase cleavable linker. In some aspects, L' is selected from

Where:

q is an integer between 2 and 10;

is absent or represents a connection; and

represents the attachment point to X;

[0494] In some aspects, the compound of formula (I-1) is selected from

[0495] In some aspects, the binding moiety is pretreated prior to reacting it with a compound of formula (I-1). In certain aspects, the compound of formula (I-1) is reacted with a binding moiety that comprises an antibody or an antigen-binding portion thereof. In aspects where the binding moiety is an antibody, the antibody may be pretreated to reduce interchain disulfides prior to reacting with the compound of formula (I-1).

Examples

General synthetic routes and intermediates

[0496] The compounds of the present invention can be prepared by one skilled in the art in light of the present disclosure and knowledge in the art and/or in accordance with the schemes illustrated below and the synthetic examples. Illustrative synthetic routes are provided in the schemes below and in the Examples. It should be understood that variables (e.g., "R" groups) appearing in the following schemes and examples are to be read independently of those appearing elsewhere in the appendix. One skilled in the art will readily understand how the schemes and examples illustrated below illustrate the preparation of the compounds described herein.

[0497] The abbreviations used in the schemes generally follow the conventions accepted in the art. The chemical abbreviations used in the description and examples are defined as follows: "THF" for tetrahydrofuran; "DMF" for N,N-dimethylformamide; "Me" for methyl; "Bu" for butyl; "FA" for formic acid; "PE" for petroleum ether; "MeOH" for methanol; "EtOH" for ethanol; "DCM" for dichloromethane; "BOC" or "Boc""TFA" for trifluoroacetic acid; "DMSO" for dimethyl sulfoxide; "EtOAc" for ethyl acetate; "OAc" for acetate; "dppf" for 1,1'-bis(diphenylphosphino)ferrocene;"dba" for dibenzylideneacetone; "CDI" for 1,1'-carbonyldiimidazole;"TBAF" for tetrabutylammonium fluoride; "TBSC1" for tert-butyldimethylsilyl chloride; " _Et2O " for diethyl ether; "ACN" for acetonitrile; "hr" for hours; "min" for minutes; "rt" for room temperature or retention time (depending on context); "aq" for aqueous, "sat" for saturated; "min" for minutes; "HOBt" for 1-hydroxybenzotriazole hydrate; "HATU" for 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 N-oxide hexafluorophosphate; "DIEA" and "iPrNEt ₂ " for diisopropylethylamine; "Et ₃ N" and "TEA" for triethylamine.

Example 1: Synthesis of compound (Ia)

Step 1: Synthesis of compound 2

[0498] 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 BH ₃ -Me ₂ S (10 M in THF) (5.80 mL, 58.0 mmol, 2.50 equiv.) dropwise at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 70 °C under nitrogen atmosphere. 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, CDCl ₃ ) (8.26 (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).

Step 2: Synthesis of compound 3

[0499] 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 BU ₄ NHSO ₄ (6.74 g, 19.84 mmol, 0.80 equiv). To the above mixture was added NaOH (5M in H ₂ O) (500.00 mL) dropwise over 40 min at 0 °C. The resulting mixture was stirred for further 2 h at 25 °C. The resulting mixture was extracted with EtOAc (3 (500 mL). The combined organic layers were washed with brine (400 mL) and dried over anhydrous Na ₂ SO ₄ . After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc=4:1) to give tert-butyl 2-[2-(2-chloro-4-nitrophenyl)ethoxy]acetate (8 g, 65%) as a yellow oil. ¹ H NMR (400 MHz, CDCl ₃ ) (8.23 (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).

Step 3: Synthesis of compound 4

[0500] 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 for 1 h at room temperature.

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), dried over anhydrous Na ₂ SO ₄ . After filtration, the filtrate was concentrated under reduced pressure. This yielded [2-(2-chloro-4-nitrophenyl)ethoxy]acetic acid (6.5 g, crude) as a yellow oil. LCMS (ESI): 517 (2M-H)-

Step 4: Synthesis of compound 5

[0501] 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) were added CH ₃ NH ₂ .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 for 2 h at room temperature. 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), dried over anhydrous Na ₂ SO ₄ . 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] ⁺ .

Step 5: Synthesis of compound 6

[0502] 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 BH ₃ -THF (1 M in THF) (12.10 mL, 12.10 mmol, 1.00 equiv.) dropwise at room temperature under nitrogen. The resulting mixture was stirred for 2 h at 70 °C under nitrogen. The reaction mixture was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO ₃ (sat., aq.). The resulting mixture was extracted with EtOAc (3 x 100 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This afforded [2-[2-(2-chloro-4-nitrophenyl)ethoxy]ethyl](methyl)amine (2.5 g, 80%) as a yellow oil. LCMS (ESI) 259.26 [M+H] ⁺ .

Step 6. Synthesis of compound 7

[0503] 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 Boc ₂ O (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] ⁺ .

Step 7: Synthesis of compound 8

[0504] 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 NH ₄ Cl (750 mg, 14.2 mmol, 3.00 equiv) in EtOH (85 mL) and H ₂ O (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] ⁺ .

Step 8: Synthesis of compound 9

[0505] 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 the above solution dropwise 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 Na ₂ SO ₄ . After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give tert-butyl (2-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenethoxy)ethyl)(methyl)carbamate (670 mg, 70%) as a white solid. LCMS:(ESI):628.63 [M+H] ⁺

Stage 9: Synthesis of the P1 neodestructor

[0506] 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% Phase B to 60% over 30 min); detector, UV 220 nm. The collected fraction was 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-d ₄ ) (7.77 (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).

Step 10: Synthesis of compound (Ia)

[0507] 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 (Neodegrader P1, 200 mg, 0.38 mmol, 1.00 equiv) and lutidine (81 mg, 0.76 mmol, 2.00 equiv) in DMF (10 mL) were added portionwise 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) at room temperature. The reaction mixture was stirred for 12 h at 40 °C under nitrogen atmosphere. After cooling the reaction mixture to room temperature, the reaction mixture 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 (30 mL), brine (30 mL), dried over Na ₂ SO ₄ . After filtration, the filtrate was concentrated to dryness in vacuo. The residue was purified on a reversed-phase column (C18, mobile phase A: 0.1% FA in water, B: ACN). The collected fraction was concentrated to dryness in vacuo. 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: 33 V to 50 V in 7 min; 220 nm; RT1: 5.27 min). The collected fraction lyophilized to give [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl N-[2-(2-[2-chloro-4-[([[2-(2,6-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) ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) (10.99 (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).

Example 2: Synthesis of compound (Ib)

Step 1: Synthesis of compound 11

[0508] To a stirred mixture of methyl 4-bromo-2-(bromomethyl)benzoate (Compound 10, 20.0 g, 64.8 mmol, 1.00 equiv.) and 3-aminopiperidine-2,6-dione hydrochloride (10.64 g, 83.0 mmol, 1.28 equiv.) in DMF (80 mL) was added TEA (22.4 mL, 162.2 mmol, 2.50 equiv.) dropwise at 25 °C under nitrogen atmosphere. The mixture was stirred at 25 °C for 16 h. Then H ₂ O (60 mL), AcOH (23 mL), and Et ₂ O (60 mL) were added sequentially at 25 °C. The mixture was stirred at 25 °C for 2 h. The precipitated solids were collected by filtration and washed with Et ₂ O (60 mL). This afforded 3-(5-bromo-1-oxo-3-isoindol-2-yl)piperidine-2,6-dione (9.0 g, 42%) as a light blue solid. LCMS (ESI): 323.32 (M+H) ⁺

Step 2: Synthesis of compound 12

[0509] 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) were added Zn(OAc) ₂ (170 mg, 0.928 mmol, 0.30 equiv), Zn(CN) ₂ (545 mg, 4.64 mmol, 1.50 equiv) and _Pd2 (dba) ₃ (28 mg, 0.031 mmol, 0.01 equiv) at 25 °C under nitrogen atmosphere. The final reaction mixture was microwave irradiated for 2 h at 120 °C. The mixture was cooled to room temperature and filtered. The filter cake was washed with MeOH (3 x 30 mL). The filtrate was concentrated under reduced pressure. The residue was flash chromatographed (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] ⁺ .

Stage 3: INT1 Synthesis

[0510] 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 PtO2 (1.25 g, 5.5 mmol, 0.49 equiv) at 25 °C. The mixture was hydrogenated at 1 °C for 16 h under hydrogen atmosphere using a hydrogen balloon. The resulting mixture was filtered and the filter cake was washed with MeOH (2 x 30 mL). The filtrate was concentrated under reduced pressure. The resulting solid was washed with DCM:MeOH (3:1) (3 x 30 mL) and dried. This afforded 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-d ₆ ) (11.02 (s, 1H), 8.15 (s, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.89 (d, J=8.4 Hz, 1H), 5.16-5.11 (m, 1H), 4.52 (d, J=17.2 Hz, 1H), 4.40 (d, J=17.2 Hz, 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)

Step 4: Synthesis of compound 14

[0511] 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 BH ₃ -Me ₂ S (10 M in THF) (5.60 mL, 56 mmol, 2.50 equiv.) dropwise at 0 °C under 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, CDCl ₃ ) (8.26 (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)

Step 5: Synthesis of compound 15

[0512] 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 extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (3 x 100 mL), dried over anhydrous Na ₂ SO ₄ . 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, CDCl ₃ ) (8.24 (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).

Step 6: Synthesis of compound 16

[0513] 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 layer was 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] ⁺ .

Step 7: Synthesis of compound 17

[0514] 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) was added CDI (113 mg, 0.70 mmol, 1.00 equiv.) in DMF (1 mL) dropwise under nitrogen atmosphere at 0 °C. The resulting mixture was stirred at 25 °C for 1 hour. Then 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 h. The reaction mixture was diluted with water (20 mL), extracted with EtOAc (20 mL (3%). The combined organic layer was washed with water, brine, dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The residue was purified by 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)+

Stage 8: Synthesis of the P3 neodestructor

[0515] 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] ⁺ .

Step 9: Synthesis of compound 19

[0516] 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 solvent 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 portionwise. The resulting mixture was stirred at 15 °C for 15 min. Trace LCMS indicated that the reaction was complete. The reaction mixture 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 in water (0.1% NH ₄ HCO ₃ ), gradient from 10% to 100% in 30 min; detector, UV 254 nm. The collected fraction was 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] ⁺ .

Step 10: Synthesis of Compound 20

[0517] 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) were added MeSO ₂ Na (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; 0-35% EtOAc in petroleum ether) to give 2-(methanesulfonylsulfanyl)-2-methylpropan-1-ol (80 mg, 10%) as a yellow oil. ¹ H NMR (400 MHz, CD ₃ Cl): (3.50 (s, 2H), 3.33 (s, 3H), 2.16 (br s, 1H), 1.47 (s, 6H).

Step 11: Synthesis of compound (Ib)

[0518] 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 (non-degrader 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 h. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (20 mL (3). The combined organic layer was washed with water (20 mL (3), brine (20 mL), dried over sodium sulfate and evaporated to dryness in vacuo to give the crude product (2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethyl imidazole-1-carboxylate, 200 mg) as a pale yellow solid. To a solution of _the crude product (100.00 mg, 0.18 mmol, 1.00 equiv.) and _C2CO3 (115 mg, 0.35 mmol, 2.00 equiv.) in DMF (8 ml) 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 mixture was stirred at 15 °C for 22 h. The reaction mixture was diluted with EtOAc (50 ml) and ice water (100 ml). The organic layer was separated. The aqueous phase was extracted with EtOAc (30 ml (3). The combined organic layer was washed with brine (30 ml (3), dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to afford the crude product (150 mg) as a yellow solid. The crude product was purified by preparative HPLC (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: 38 V to 58 V over 7 min; 220 nm; RT1: 5.12 min). The collected fraction was 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-d ₆ ) (10.99 (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).

Example 3: Synthesis of compound (Ic)

Step 1: Synthesis of compound 23

[0519] 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) were added CDI (218 mg, 1.35 mmol, 1.00 equiv.) and TEA (68 mg, 1.35 mmol, 1.00 equiv.) dropwise at 0 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 2 h. To the above mixture was added 3-[5-(aminomethyl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (INT1, 368 mg, 1.35 mmol, 1.00 equiv.) The resulting mixture was stirred overnight at 75 °C. The reaction mixture was then cooled to room temperature. The resulting mixture was quenched with water (30 mL) and extracted with DCM (3 (30 mL). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (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] ⁺ .

Stage 2: Synthesis of the P4 neodestructor

[0520] 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 h. The resulting mixture was concentrated in vacuo. The crude product was purified by reverse phase under the following conditions (C18, mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL/min). The collected fraction was 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-d ₆ ) (10.99(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).

Step 3. Synthesis of compound (Ic)

[0521] To a stirred mixture 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-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl 4-nitrophenyl carbonate (394 mg, 0.53 mmol, 1.50 equiv.) at room temperature under nitrogen atmosphere. The reaction mixture was purified by reverse flash chromatography using 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 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: 24 V to 44 V in 7 min; 220 nm; RT1:6.33; RT2:). The collected fraction was lyophilized to give [4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanamido]pentanamido]phenyl]methyl N-[2-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]-N-methylcarbamate (18.1 mg, 5%) as a white solid. LCMS (ESI): 1020 (M+H) ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) (10.99 (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 (lat 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)

[0522] Scheme 4 illustrates how compound (Id) was prepared from the neodegrader P1.

Synthesis of compound (Id)

[0523] 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 DIEA (20.00 mg, 0.16 mmol, 2.04 equiv) dropwise at room temperature. The resulting mixture was stirred for 3 h at room temperature under nitrogen. 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 Na ₂ SO ₄ . After filtration, the filtrate was concentrated to dryness under vacuum. The residue was purified as follows: 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 V to 55 V over 8.5 min; 220 nm; RT1:8 min; the collected fraction was 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: (ESI, m/s): 721.723 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) (10.99 (s, 1H), 8.78 (s, 1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.41 (d, J=9.6 Hz, 1H), 7.18-7.16 (m, 2H), 7.00 (d, J=5.6 Hz, 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).

[0524] Schemes 5A and 5B illustrate how to prepare a complex of the P1 neodegrader with an alternative tripeptide linker.

[0525] Schemes 6A and 6B illustrate how to prepare a complex of the P1 neodegrader with a β-glucuronide linker.

Step 1. Synthesis of compound 25

[0526] 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 for 16 h at 80 °C. The desired product can be detected by LCMS (derivative with MeOH MS=326). LCMS showed the reaction to be complete. 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 that this was the desired product (MeOH derivative). ¹ H-NMR (300 MHz, CDCl ₃ ) (7.81-7.77 (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).

Step 2. Synthesis of compound 28

[0527] To a stirred solution of 4-formyl-2-nitrophenol (Compound 27, 4.21 g, 25.19 mmol, 1.00 equiv) and Ag ₂ O (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) portionwise at room temperature under N ₂ . The resulting mixture was stirred overnight at room temperature under N ₂ . LCMS indicated that 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 column chromatography on silica gel 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 showed that it was the desired product. LCMS (ESI, m/z): 484 [M+1] ⁺ . ¹ H-NMR (300 MHz, CDCl ₃ ) (10.00 (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).

Step 3. Synthesis of compound 29

[0528] To a stirred solution of methyl (2S,3S,4S,5R,6S)-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 ₄ (0.47 g, 12.42 mmol, 1.00 equiv) portionwise at rt under N ₂ . The resulting mixture was stirred for 2 h at rt under N ₂ . LCMS indicated that the reaction was complete. The reaction was quenched with water at rt. The resulting material was dried over Na ₂ SO ₄ . 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 (ESI, m/z): 486 [M+H] ⁺ .

Step 4. Synthesis of compound 30

[0529] To a stirred mixture of methyl (2S,3S,4S,5R,6S)-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%) portionwise at room temperature. The resulting mixture was stirred for 16 h at room temperature under H ₂ . LCMS showed the reaction to be 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 (ESI, m/z): 456 [M+H] ⁺ .

Step 5. Synthesis of compound 31

[0530] To a stirred solution of methyl (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 ₃ (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) portionwise at 0 °C under N ₂ . The resulting mixture was stirred for 6 h at 0 °C under N ₂ . LCMS indicated that 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 PEZEA (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 light yellow solid. LCMS (ESI, m/z): 749 [M+H] ⁺ .

Step 6. Synthesis of compound 33

[0531] To a stirred mixture of 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, 1.50 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 N ₂ . The resulting mixture was stirred overnight at room temperature under nitrogen. LCMS showed the reaction to be complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 90% over 40 min; detector, UV 254 nm. The collected fraction was 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 (ESI, m/z): 914 [M+H] ⁺ .

Step 7. Synthesis of compound 34

[0532] To a stirred mixture of 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, 1.00 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 (non-degrader P1, 0.58 g, 1.09 mmol, 1.00 equiv) in DMF (10 mL) were added HOVT (1.18 g, 8.72 mmol, 8.00 equiv) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv) in portions at room temperature under N ₂ . The resulting mixture was stirred for 16 h at room temperature under N ₂ . LCMS showed the reaction to be complete. The resulting mixture was used with additional purification. The residue was purified by reversed-phase flash chromatography with the following conditions: column, silica gel C18; mobile phase, ACN in water (0.1% FA), gradient from 10% to 80% over 40 min; detector, UV 254 nm. The collected fraction was 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 (ESI, m/z): 1302 [M+H] ⁺ .

Step 8. Synthesis of compound 35

[0533] To a stirred mixture of 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, 800.00 mg, 0.61 mmol, 1.00 equiv.) in THF (80 mL) was added HCl (6N, 80 mL) portionwise at room temperature under N ₂ atmosphere. The resulting mixture was stirred for 3 h at 50 °C under nitrogen atmosphere. LCMS showed that 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, silica gel C18; mobile phase, ACN in water (0.1% FA), gradient from 0% to 80% in 40 min; detector, UV 254 nm. The collected fraction was 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 (ESI, m/z) 1162 [M+H] ⁺ .

Step 9. Synthesis of compound 36

[0534] 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) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 10 min at room temperature under nitrogen. LCMS showed that the reaction was complete. The resulting mixture was used directly for further purification by preparative HPLC under the following conditions (column: XSelect CSH Prep 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 V to 40 V in 7 min; 220 nm; RT 1:5.78 min) to afford (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%) as a white solid. LCMS (ESI, m/z) 940 [M+H]+.

Step 10. Synthesis of compound (Ie)

[0535] 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) were 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 nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS showed that 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 V to 36 V in 10 min; 220 nm; RT1:11.15 min). The collected fraction was lyophilized to obtain (2S,3S,4S,5R,6S)-6-[4-[([[2-(2-[2-chloro-4-[([[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]carbamoyl)amino]phenyl]ethoxy)ethyl]-(methyl)carbamoyl]oxy)methyl]-2-[3-[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 (ESI, m/z): 1133 [M+H] ⁺ . ¹ h-nm (300 MHz, DMSO-d ₆ ) (10.9 (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).

[0536] Scheme 7 illustrates how to prepare a complex of the P6 neodegrader with a hydrazine linker.

Step 1. Synthesis of compound 38

[0537] 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 min at 0 °C. The resulting mixture was concentrated in vacuo. The resulting solid was redissolved in DMF (1.50 mL). To a stirred solution of 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) was added dropwise TEA (0.50 mL) at room temperature. The resulting mixture was stirred for 1 h at 0 °C. LCMS showed that the reaction was complete. Water (5 mL) was added to the mixture and extracted with CH ₂ Cl ₂ (3 x 10 mL). The organic layer was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography using the following conditions: column, silica gel C18; mobile phase, ACN in water (0.05% TFA), gradient from 10% to 50% over 35 min; detector, UV 254 nm. The collected fraction was 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 light yellow solid. LCMS: (IER.m/z):435[M+1] ⁺ .

Stage 2. Synthesis of the compound (If)

[0538] 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)hexane hydrazide; trifluoroacetic acid salt (75 mg, 1.20 equiv) in methanol (5.00 mL) was stirred overnight at 50 °C. The mixture was cooled to room temperature. LCMS indicated the reaction was complete. The precipitated solids were collected by filtration and washed with MeOH (2×5 mL). The crude solid was purified by reversed-phase flash chromatography using the following conditions: C18 column; mobile phase, ACN in water (0.1% FA), gradient 10% to 50% over 30 min; detector, UV 254 nm. The collected fraction was extracted with DCM (3×5 mL) and concentrated in vacuo. This afforded 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 a white metallic solid. LCMS: (ESI.m/z): 642[M+1] ⁺ , ¹ H NMR (400 MHz, DMSO-d ₆ ) (10.99 (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,6N)

[0539] Scheme 8 illustrates how to prepare a complex of the P2 neodegrader with a quaternary amine linker.

Step 1. Synthesis of compound 40

[0540] 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 ₂ (20 mg, 0.18 mmol, 1 equiv) in DCM (2 mL) dropwise under N ₂ at 0 °C. The resulting mixture was stirred at 0 °C for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was diluted with ice water (20 mL), extracted with DCM (10 mL*3), the combined organic layer was washed with water (10 mL), brine (10 mL), dried over anhydrous sodium sulfate and concentrated to dryness in vacuo to give 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 (ESI, m/z): 591.593 (M+H) ⁺

Step 2. Synthesis of compound 42

[0541] 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 dropwise BH ₃ -Me ₂ S (10.00 mL, 105.4 mmol, 2.64 equiv.) at 0 °C. The resulting mixture was stirred for 4 h at 70 °C under nitrogen atmosphere. TLC (PE:EA=1:2) showed that 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. ¹ H NMR (400 MHz, CDCl ₃ ) (8.27 (d, J=4.0 Hz, 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).

Step 3. Synthesis of compound 43

[0542] 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 Bu ₄ NHSO ₄ (10.37 g, 30.6 mmol, 0.80 equiv) portionwise at 0 °C. To the above mixture was added NaOH (15.00 g, 375.0 mmol, 9.82 equiv) in H ₂ O (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) showed that the reaction was complete. The resulting mixture was extracted with EtOAc (3 (200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na ₂ SO ₄ . 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, CDCl ₃ ) (8.20 (d, J = 4.0 Hz, 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).

Step 4. Synthesis of compound 44

[0543] 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 h at room temperature. LCMS showed the reaction to be 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: (ESI, m/s): 517 (2M-H) ^-1H NMR (400 MHz, DMSO-d ₆ ) (12.64 (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).

Step 5. Synthesis of compound 45

[0544] 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) were added CH ₃ NH ₂ .HCl (2.69 g, 39.79 mmol, 1.23 equiv) and DIEA (17.31 g, 133.93 mmol, 4.14 equiv) at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 4 h at room temperature under nitrogen atmosphere. LCMS indicated that the reaction was complete. The reaction mixture 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), dried over anhydrous Na ₂ SO ₄ . 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: (ESI, m/s): 273.275 (M+H) ⁺

Step 6. Synthesis of compound 46

[0545] 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 for 4 h at 70 °C. LCMS indicated that the reaction was complete. The mixture was allowed to cool to room temperature. The reaction mixture was quenched with MeOH. The residue was acidified to pH 6 with 1N HCl. The resulting mixture was extracted with EtOAc (20 mL). The aqueous phase was basified to pH 8 with saturated NaHCO ₃ (sat, aq). The resulting mixture was extracted with EtOAc (3 x 100 mL), washed with brine (50 mL) and dried over anhydrous Na ₂ SO ₄ . After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting (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: (ESI, m/s): 259.261 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) (8.26 (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).

Step 7. Synthesis of compound 47

[0546] 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 Boc ₂ O (3.80 g, 17.41 mmol, 1.13 equiv) in THF (20.00 mL) was added NaHCO ₃ (4.00 g, 47.61 mmol, 3.08 equiv) in H ₂ O (20.00 mL) dropwise at room temperature. The resulting mixture was stirred overnight at room temperature. LCMS indicated that the reaction was complete. The resulting mixture was extracted with EtOAc (3, 20 mL). The combined organic layers were washed with brine (20 mL ₎ , 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=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: (ESI, m/s): 359.36 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO- _d6 ) (8.24 (d, J=4.0 Hz, 1H), 8.13-8.10 (m, 1H), 7.67 (d, J=8.0 Hz, 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).

Step 8. Synthesis of compound 48

[0547] 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 EtOAc (112.00 mL) was added NH ₄ Cl (2.50 g, 46.74 mmol, 2.99 equiv) in H ₂ O (12.00 mL) and Fe (4.40 g, 78.79 mmol, 5.05 equiv) at room temperature. The resulting mixture was stirred for 3 h at 80 °C. LCMS indicated that 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), 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: (ESI, m/s): 329.331 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) (6.96 (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).

Step 9. Synthesis of compound 49

[0548] 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 LiA1H ₄ (92 mg, 2.43 mmol, 8.00 equiv) in THF (2 mL) at 0 °C under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 h. Five reactions were carried out in parallel. LCMS showed the reaction to be complete. The reaction mixture was then quenched with 1N NaOH (10 mL), filtered, concentrated to dryness in vacuo, and 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 0% to 60% over 30 min; detector, UV 254 nm. The collected fraction was concentrated to dryness to give 3-chloro-4-[2-[2-(dimethylamino)ethoxy]ethyl]aniline, 49 (180 mg, 44%) as a yellow oil. LCMS (ESI, m/z): 243.245 [M+H] ⁺ .

Step 10. Synthesis of compound 50

[0549] 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.) at 0 °C under nitrogen atmosphere. The resulting mixture was stirred at 0 °C for 1 h. Then, the reaction solution was concentrated to dryness in vacuo. The residue was redissolved in DMF (2 mL) and then 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 nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 h. LCMS showed the reaction to be complete. The reaction mixture was diluted with methanol and the resulting solution was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient 0% to 50% over 30 min; detector, UV 254 nm to afford 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, 19×250 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. The collected fraction was lyophilized to give 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 (ESI, m/z): 542.544 [M+H] ⁺

Stage 11. Synthesis of the compound (Ig)

[0550] To a solution of 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)ethoxy]ethyl]phenyl)-3-[[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) in DMF (1 mL) was added TBAI (4 mg, 0.01 mmol, 0.10 equiv) at room temperature in air. The resulting mixture was stirred at room temperature for 16 h. LCMS showed that the reaction was complete. The resulting mixture was purified by reversed-phase column chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 5% to 45% over 40 min; detector, UV 254 nm to afford 90 mg of crude product as a yellow oil. The crude product was then re-purified 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: 15 V to 35 V in 7 min; 220 nm; RT1: 6.00 min to afford ([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 (ESI, m/z): 1096 [M-FA] ⁺ , 549 [1/2(M-FA)] ⁺ ; ¹ H NMR (400 MHz, CD ₃ OD) (8.48 (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 (k, J=8.4 Hz, 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).

[0551] Schemes 9A and 9B illustrate how to prepare a complex of the P13 neodegrader with a peptide-containing linker.

[0552] Scheme 10 illustrates the synthesis of compounds of formula (Ih).

Step 1. Synthesis of compound 63

[0553] 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), SOCl ₂ (25 mL) was added at room temperature. The resulting mixture was stirred for 16 h at 80 °C. The desired product could be detected by LCMS (MeOH derivative MS=326). LCMS showed the reaction to be complete. 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 H NMR analysis showed that this was the desired product (MeOH derivative). ¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.81-7.77 (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).

Step 2. Synthesis of compound 66

[0554] To a stirred solution of 4-formyl-2-nitrophenol (Compound 65, 4.21 g, 25.19 mmol, 1.00 equiv.) and Ag ₂ O (7.00 g, 30.20 mmol, 1.20 equiv.) in ACN (100 mL, 190.24 mmol, 75.00 equiv.) was added methyl (2S,3S,4S,5R,6R)-3,4,5-tris(acetyloxy)-6-bromoxane-2-carboxylate (Compound 64, 10.00 g, 25.17 mmol, 1.00 equiv.) in portions at room temperature under N ₂ . The resulting mixture was stirred overnight at room temperature under N ₂ . LCMS showed the reaction was complete. The resulting mixture was filtered and the filter cake was washed with DCM (50 mL x 3). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography 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 that this was the desired product. LCMS (ESI, m/z): 484 [M+1] ⁺ . ¹ H-NMR (300 MHz, CDCl ₃ ) δ 10.00 (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).

Step 3. Synthesis of compound 67

[0555] To a stirred solution of methyl (2S,3S,4S,5R,6S)-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 ₄ (0.47 g, 12.42 mmol, 1.00 equiv) portionwise at room temperature under N ₂ . The resulting mixture was stirred for 2 h at room temperature under N ₂ . LCMS indicated that the reaction was complete. The reaction was quenched with water at room temperature. The resulting material was dried over Na ₂ SO ₄ . 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 (ESI, m/z): 486 [M+H] ⁺ .

Step 4. Synthesis of compound 68

[0556] To a stirred mixture of methyl (2S,3S,4S,5R,6S)-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%) portionwise at room temperature. The resulting mixture was stirred for 16 h at room temperature under H ₂ . LCMS showed the reaction to be 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 (ESI, m/z): 456 [M+H] ⁺ .

Step 5. Synthesis of compound 70

[0557] To a stirred solution of methyl (2S,3S,4S,5R,6S)-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 ₃ (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 N ₂ . The resulting mixture was stirred for 6 h at 0 °C under N ₂ . LCMS showed the reaction to be 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 light yellow solid. LCMS (ESI, m/z): 749 [M+H] ⁺ .

Step 6. Synthesis of compound 72

[0558] To a stirred mixture of 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, 1.50 g, 2.00 mmol, 1.00 equiv) and bis(4-nitrophenyl)carbonate (Compound 71, 0.68 g, 2.24 mmol, 1.12 equiv) in DMFA (15 mL) was added DIEA (0.52 g, 4.01 mmol, 2.00 equiv) portionwise at 0 °C under N ₂ . The resulting mixture was stirred overnight at room temperature under nitrogen. LCMS showed the reaction to be complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 10% to 90% over 40 min; detector, UV 254 nm. The collected fraction was 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 (ESI, m/z): 914[M+H] ⁺ .

Step 7. Synthesis of compound 73

[0559] To a stirred mixture of 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, 1.00 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 (non-degrader P1, 0.58 g, 1.09 mmol, 1.00 equiv) in DMF (10 mL) were added HOVT (1.18 g, 8.72 mmol, 8.00 equiv) and 2,4-dimethylpyridine (1.07 g, 8.72 mmol, 8.00 equiv) in portions at room temperature under N ₂ . The resulting mixture was stirred for 16 h at room temperature under N ₂ . LCMS showed the reaction to be complete. The resulting mixture was used with additional purification. The residue was purified by reversed-phase flash chromatography with the following conditions: column, silica gel C18; mobile phase, ACN in water (0.1% PA), gradient from 10% to 80% over 40 min; detector, UV 254 nm. The collected fraction was 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 73 (800 mg, 56%) as a solid. LCMS (ESI, m/z): 1302 [M+H] ⁺ .

Step 8. Synthesis of compound 74

[0560] To a stirred mixture of 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 (800.00 mg, 0.61 mmol, 1.00 equiv.) in THF (80 mL) was added HCl (6N, 80 mL) portionwise at room temperature under N ₂ . The resulting mixture was stirred for 3 h at 50 °C under nitrogen. LCMS indicated that 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, silica gel C18; mobile phase, ACN in water (0.1% PA), gradient from 0% to 80% over 40 min; detector, UV 254 nm. The collected fraction was 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 (ESI, m/z) 1162 [M+H] ⁺ .

Step 9. Synthesis of compound 75

[0561] 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) portionwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 10 min at room temperature under nitrogen. LCMS showed that the reaction was complete. The resulting mixture was used directly for further purification by preparative HPLC under the following conditions (column: XSelect CSH Prep C18 OBD column, 19x250 mm, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL/min; gradient: 20 V to 40 V in 7 min; 220 nm; RT1: 5.78 min) to afford (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%) as a white solid. LCMS (ESI, m/z) 940 [M+H] ⁺ .

Step 10. Synthesis of compound (Ih)

[0562] 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 75, 110 mg, 0.12 mmol, 1.00 equiv.) and bis(2,5-dioxopyrrolidin-1-yl)pentanedioate (Compound 76, 46 mg, 0.14 mmol, 1.2 equiv.) in DMF (2.0 mL) was added portionwise DIEA (30 mg, 0.23 mmol, 2.0 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 1 h at room temperature under nitrogen atmosphere. LCMS showed that the reaction was complete. The reaction mixture was purified by preparative HPLC under the following conditions (column: KinetexEVO prep C18, 30*150, 5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 mL/min; gradient: 21% B to 41% B over 7 min, 41% B; wavelength: 254 nm; RT1 (min): 5.8. The collected fraction was lyophilized to obtain (2S,3S,4S,5R,6S)-6-{4-[({[2-(2-{2-chloro-4-[({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl}carbamoyl)amino]phenyl}ethoxy)ethyl](methyl)carbamoyl}oxy)methyl]-2-(3-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanamido}propanamido)phenoxy}-3,4,5-trihydroxyoxane-2-carboxylic acid (Compound (Ih), 48 mg, 34% as a white solid LCMS (ESI, 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, 1H), 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).

Scheme 11: Synthesis of the P1 neodestructor-linker complex (Compound (Ii))

Step 1. Synthesis of compound 76

[0563] 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) were added TEA (104 mg, 1.02 mmol, 3.0 equiv) and 4-(chlorosulfonyl)-3-nitrobenzoic acid (181 mg, 0.68 mmol, 2.00 equiv) portionwise at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 4 h at 0 °C under nitrogen atmosphere. LCMS showed the reaction to be complete. The resulting mixture was used with further purification. The residue was purified by reversed-phase flash chromatography with the following conditions: column, silica gel C18; 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 light yellow solid. LCMS (ESI, m/z) 757 (M+1) ⁺ .

Step 2 Synthesis of compound (Ii)

[0564] 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) were 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) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 4 h at room temperature under nitrogen atmosphere. LCMS showed that the reaction was complete. The residue was purified by preparative HPLC (column: XBridge Prep Phenyl OBD column, 19x 150 mm 5 μm 13 nm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 25 mL/min; gradient: 25 V to 43 V in 10 min; 220 nm; RT1: 11.97 min). The collected fraction 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]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]-3-nitrobenzamide (Compound (I), 27 mg, 36%) as a white solid. LCMS (ESI, m/z): 879.881 [M+H]. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.00 (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).

Scheme 12: Synthesis of the P1 neodegrader-GGFG linker complex (Compound (Ij))

Step 1. Synthesis of compound 79

[0565] 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) ₄ (15.02 g, 33.86 mmol, 1.20 equiv) in THF (300 mL) and toluene (100 mL) was added pyridine (2.59 g, 32.74 mmol, 1.16 equiv) dropwise at room temperature under nitrogen. The resulting mixture was stirred overnight at 80 °C under nitrogen. LCMS indicated that 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 _{Na2SO4 .} 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 (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)methyl acetate (Compound 79, 6.5 g, 56%) as a white solid. LCMS (ESI, ms): 391[M+Na] ⁺ . ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.80(d, J=7.5 Hz, 2H), 7.62(d, J=7.5 Hz, 2H), 7.45(t, J=7.5 Hz, 2H), 7.36(d, J=7.5 Hz, 2H), 7.18(br s, 1H), 5.48(br s, 1H), 5.28(d, J=7.2 Hz, 2H), 4.48(d, J=6.6 Hz, 2H), 4.26(t, J=6.6 Hz, 1H), 3.93(d, 5.4 Hz, 2H), 2.08(s, 3H).

Step 2. Synthesis of compound 81

[0566] To a stirred mixture of (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)methyl 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) was added PPTS (400 mg, 1.59 mmol, 0.29 equiv) dropwise at 0 °C under nitrogen. The resulting mixture was stirred overnight at 45 °C under nitrogen. 40% of the desired product could be detected by LCMS. The mixture was allowed to cool to room temperature. The reaction mixture was quenched with water/ice. The resulting mixture was extracted with EtOEt (3 x 20 mL). The combined organic layers were washed with brine (30 mL), 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 (1:9) 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 H NMR (300 MHz, DMSO-d ₆ ): δ 8.58 (t, J=5.1 Hz, 1H), 8.22 (da, J=12, 2.4 Hz, 1H), 7.89 (d, J=7.5 Hz, 1H), 7.71-7.54 (m, 4H), 7.43-7.29 (m, 4H), 4.56 (d, J=6.9 Hz, 2H), 4.30-4.16 (m, 3H), 3.70-3.61(m, 4H), 3.04 (t, J=6.3 Hz, 2H).

Step 3. Synthesis of compound 82

[0567] 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) portionwise at 0 °C under nitrogen. The resulting mixture was stirred for 1 h at room temperature under nitrogen. LCMS showed the reaction to be complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient 0% to 50% over 40 min; detector, UV 254 nm. This afforded 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] ⁺

Step 4. Synthesis of compound 83

[0568] 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 Boc ₂ O (580 mg, 2.66 mmol, 1.02 equiv) in DMF (10.00 mL) was added NaHCO ₃ (477 mg, 5.68 mmol, 2.18 equiv) in H ₂ O (10.00 mL) dropwise at 0 °C. The resulting mixture was stirred for 3 h at room temperature. LCMS indicated that the reaction was complete. The reaction mixture was quenched by addition of water (20 mL). The resulting mixture was extracted with EtOEt (3 x 20 mL). The combined organic layers were washed with brine (20 mL), 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 (1:2) 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 H NMR (400 MHz, CDCl ₃ )) δ 8.21(d, J=2.4 Hz, 1H), 8.04(d, J=8.4 Hz, 2H), 7.46(d, J=8.4 Hz, 1H), 7.05(br s, 1H), 5.25(br s, 1H), 4.73(d, J=7.2 Hz, 2H), 3.81-3.73(m, 4H), 3.34-3.32(m, 2H), 3.08(t, J=6.8 Hz, 2H), 1.42(s, 9H).

Step 5. Synthesis of compound 84

[0569] 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 NH ₄ Cl (910 mg, 17.01 mmol, 10.1 equiv) in H ₂ O (3.00 mL) dropwise at room temperature. The resulting mixture was stirred for 4 h at 90 °C. LCMS indicated that 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 x 20 mL). The combined organic layers were washed with brine (20 mL), 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 H NMR (300 MHz, CDCl ₃ ) δ 7.02-6.96(m, 2H), 6.68(d, J=2.4 Hz, 1H), 6.52-6.49(m, 1H), 5.29(br s, 1H), 4.74(d, J=6.9 Hz, 2H), 3.80-3.78(m, 2H), 3.69-3.63(m, 2H), 2.88(t, J=7.2 Hz, 2H), 1.45(s, 9H).

Step 6. Synthesis of compound 86

[0570] 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) was added TEA (300 mg, 2.96 mmol, 2.12 equiv) at 0 °C. The resulting mixture was stirred for 2 h at room temperature. To the above mixture were added 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) portionwise. The resulting mixture was stirred overnight at 60 °C. LCMS showed that the reaction was complete. The mixture was allowed to cool to room temperature. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 50% over 30 min; detector, UV 254 nm. This afforded 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] ⁺ .

Step 7. Synthesis of compound 87

[0571] 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 min at 0 °C. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. This resulted in the formation of 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 salt (Compound 87, (510 mg, purity: 64%, yield: 60%) as a white metallic solid. LCMS (ESI, ms): 557 [M+H-TFA] ⁺

Step 8. Synthesis of compound 89

[0572] 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 ₃ (1.80 g, 21.41 mmol, 3.00 equiv) in H ₂ O (40.00 mL) was added Bco ₂ O (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 indicated that the reaction was complete. The reaction was quenched with water at room temperature. The resulting mixture was extracted with EtOEt (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous _Na2SO4 . After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography using the following conditions: column, silica gel _C18 ; mobile phase, ACN in water (0.05% TFA), gradient from 5% to 60% over 30 min; detector, UV 220 nm. This afforded (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 H NMR:(300 MHz, DMSO-d ₆ ) δ 8.17(d, J=8.1 Hz, 1H), 7.93(t, J=5.7 Hz, 1H), 7.31-7.20(m, 5H), 7.00(t, J=6.0 Hz, 1H), 4.46-4.39(m, 1H), 3.78-3.67(m, 2H), 3.56(d, J=5.7 Hz, 2H), 3.09-3.02(m, 1H), 2.92-2.73(m, 1H), 1.39(s, 9H).

Stage 9. Connection 90

[0573] 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) were added HOBT (102 mg, 0.75 mmol, 0.84 equiv.) in portions at 0 °C. The resulting mixture was stirred for 30 min at 0 °C. To the above mixture was added 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 salt; trifluoroacetic acid (Compound 87, 511 mg, purity: 64%, 0.48 mmol, 0.54 equiv) and DIEA (340 mg, 2.63 mmol, 2.94 equiv) at 0 °C. The resulting mixture was stirred for additional 2 h at room temperature. LCMS showed that the reaction was complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 50% over 30 min; detector, UV 220 nm. The collected fraction was concentrated in vacuo. This afforded 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]ethoxy)methyl]carbamoyl]methyl)carbamoyl]-2phenylethyl]carbamoyl]methyl)carbamoyl]methyl]carbamate (Compound 90, 210 mg, 48%) as a white metallic solid. LCMS (ESI, ms):918[M+H] ⁺ , 818[M+H-100] ⁺ , 1HNMR: (400 MHz, DMSO-d ₆ ): δ 10.97(s, 1H), 8.79(s, 1H), 8.50(t, J=6.4 Hz, 1H), 8.31(t, J=4.4 Hz, 1H), 8.15(d, J=9.6 Hz, 1H), 7.910(t, J=8.0 Hz, 1H), 7.68-7.64(m, 2H), 7.49(s, 1H), 7.43(d, J=9.6 Hz, 1H), 7.24-7.12(m, 7H), 7.00-6.95(m, 1H), 6.84(t, J=6.4 Hz, 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), 2.40-2.31(m, 1H), 2.01-1.94(m, 1H), 2.00-1.91(m, 1H), 1.35(s, 9H)

Step 10. Synthesis of compound 91

[0574] To a stirred mixture 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]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 TFA (1.00 mL) dropwise at 0 °C. The resulting mixture was stirred for 30 min at 0 °C. LCMS indicated that the reaction was complete. The resulting mixture was concentrated under reduced pressure. This afforded the (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 salt (Compound 91, 140 mg, 79%) as a white metallic solid. LCMS (ESI, ms): 818 [M+H-TFA] ⁺ .

Step 11. Synthesis of compound (Ij)

[0575] 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 salt; To a solution of 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) portionwise at 0 °C. The resulting mixture was stirred for 2 h at room temperature. LCMS showed the reaction to be complete. The reaction mixture was purified directly under the following conditions: Column: XSelect CSH Prep C18 OBD column, 19 x 250 mm, 5 μm; Mobile phase A: water (0.1% FA), Mobile phase B: ACN; Flow rate: 25 mL/min; Gradient: 25 V to 50 V in 7 min; 254 nm; RT1: 6.35 min; The collected fraction was lyophilized to obtain the crude product. The crude product was re-purified under 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: 20 V to 40 V in 7 min, 220 nm; RT1: 6.77 min; The collected fraction was lyophilized to obtain 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 metallic solid. LCMS (ESI, ms):1011[M+H] ⁺ . ^1H NMR:(400MHz, DMSO- _d6 ): δ 10.95(s, 1H), 8.79(s, 1H), 8.51(t, J=8.4 Hz, 1H), 8.29(t, J=8.0 Hz, 1H), 8.12-8.01(m, 3H), 7.70-7.66(m, 2H), 7.44(s, 1H), 7.42(d, J=8.0 Hz, 1H), 7.23-7.16(m, 7H), 6.99(s, 2H), 6.82(t, J=8.0 Hz, 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)

Scheme 13: Synthesis of the complex neodestructor P14-linker AAA (Compound (Ik))

Step 1. Synthesis of compound 94

[0576] 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 dropwise BH ₃ -Me ₂ S (28.00 mL, 295.23 mmol, 2.65 equiv.) under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 70 °C under nitrogen atmosphere. TLC (PE:EtOAc=3:1) showed the reaction to be complete. After cooling to room temperature, the resulting mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (3:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (Compound 94, 18.00 g, 80%) as a light yellow solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.27 (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).

Step 2. Synthesis of compound 95

[0577] 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) were added NBS (6.62 g, 1.50 equiv) and _PPh3 (9.76 g, 37.21 mmol, 1.50 equiv) portionwise at room temperature under _N2 . The resulting mixture was stirred overnight at room temperature under _N2 . TLC (PE:EtOAc=10:1) showed the reaction to be complete. The reaction mixture was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 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-d ₆ ) 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 = 6.8 Hz, 2H), 3.38 (t, J = 6.8 Hz, 2H).

Step 3. Synthesis of compound 96

[0578] 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 nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 h. TLC (PE:EtOAc=10:1) showed the reaction to be complete. The reaction mixture was diluted with water (600.00 mL) and extracted with EtOAc (2000 mL×3). The combined organic layer was 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]ethenone (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).

Step 4. Synthesis of compound 97

[0579] To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (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 N ₂ . The resulting mixture was stirred at 0 °C under N ₂ for 1 h. TLC (PE:EtOAc=10:1) showed the reaction to be complete. The reaction mixture was quenched with AcOH to pH 3-4. The resulting mixture was concentrated to dryness in vacuo. 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 light 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=8.0 Hz, 2H), 2.85 (dt, J=8.0, 7.2 Hz, 2H), 1.43 (t, J=7.2 Hz, 1H).

Step 5. Synthesis of compound 99

[0580] 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) were 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 air atmosphere. The resulting mixture was stirred for 1 h at room temperature. LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (200.00 mL), extracted with EtOAc (100.00 mL x 3). The combined organic layer was 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 (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 (ESI, m/z): 431[M+Na] ⁺ .

Step 6. Synthesis of compound 100

[0581] To a solution of D-alanine (1.09 g, 0.012 mmol, 1.00 equiv.) and NaHCO ₃ (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 h. LCMS indicated that the reaction was complete. The reaction was adjusted to pH 2-3 with 2 N HCl. The resulting mixture was extracted with EtOAc (100.00 mL x 3), and the combined organic layer was washed with brine (100.00 mL x 3), dried over anhydrous Na ₂ SO ₄ 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 (ESI, m/z: 383 [M+H] ⁺

Step 7. Synthesis of compound 101

[0582] To a solution of glycine (3.68 g, 48.97 mmol, 1.00 equiv.) and NaHCO ₃ (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 mixture was stirred at room temperature for 2 h. LCMS indicated that the reaction was complete. The reaction was adjusted to pH 2-3 with 2 N HCl. The resulting mixture was extracted with EtOAc (500.00 mL x 3), and the combined organic layer was 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 (ESI, m/z): 369 (M+H) ⁺

Step 8. Synthesis of compound 102

[0583] A solution of [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]acetic acid (Compound 101, 5.00 g, 13.57 mmol, 1.00 equiv), Pb(OAc) ₄ (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 _N2 was stirred at 80 °C for 16 h. After cooling to room temperature, the reaction mixture was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layer was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting (PE:EtOAc=1:2) to give [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]methyl acetate (Compound 102, 2.50 g, 45%) as a white solid. LCMS (ESI, 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).

Step 9. Synthesis of compound 103

[0584] To a stirred solution of [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]methyl 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) under N ₂ at room temperature. The resulting mixture was stirred at room temperature for 16 h. LCMS indicated that the reaction was complete. The reaction mixture was concentrated to dryness in vacuo. The residue was purified by silica gel column chromatography eluting with (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 (ESI, m/z): 540 (M+H) ⁺

Step 10. Synthesis of compound 104

[0585] 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 ₂ . The resulting mixture was stirred at 0 °C for 0.5 h under N ₂ . LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (600.00 mL). The resulting mixture was extracted with EtOAc (200.00 mL x 3). The combined organic layer was washed with brine (200.00 mL), dried over anhydrous _Na2SO4 and concentrated _to dryness in vacuo to afford 3.00 g of crude product. The crude product was repurified 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 (ESI, m/z) 318 [M+H] ⁺ .

Step 11. Synthesis of compound 105

[0586] 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 a solution of NaHCO ₃ (0.59 g, 7.08 mmol, 1.50 equiv) in H ₂ O (10.00 mL) and Boc ₂ O (1.03 g, 4.72 mmol, 1.00 equiv) at room temperature. The reaction mixture was stirred at room temperature for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (500.00 mL), extracted with EtOAc (200.00 mL x 3). The combined organic layer was washed with brine (200.00 mL x 3), dried over anhydrous Na ₂ SO ₄ 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 (ESI, m/z): 418 [M+H] ⁺ , 318 [M+H-100] ⁺

Step 12. Synthesis of compound 106

[0587] A suspension 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 NH ₄ Cl (2.33 g, 0.04 mmol, 10.00 equiv) in EtOH (100.00 mL)/H ₂ O (50.00 mL) was stirred at 70 °C for 2 h. LCMS indicated that the reaction was complete. The reaction mixture was filtered. The filtrate was concentrated to dryness in vacuo. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was concentrated to dryness and the residue was purified by column chromatography on silica gel 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 (ESI, m/z): 388 [M+H] ⁺

Stage 13. Compound 107

[0588] 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 were added CDI (209.00 mg, 1.29 mmol, 1 equiv.) and TEA (260 mg, 2.58 mmol, 2 equiv.). The resulting mixture was stirred at 0 °C for 2 h. Then 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 DM AP (472 mg, 3.87 mmol, 3.00 equiv) were added. The resulting mixture was stirred at 60 °C for 24 h. LCMS showed that the reaction was complete. After cooling to room temperature, the reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 60% over 30 min; detector, UV 254 nm to give 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 (ESI, m/z): 687 (M+H) ⁺

Stage 14. Compound 108

[0589] 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 indicated that the reaction was complete. The reaction mixture was concentrated to dryness in vacuo to give the (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 salt (Compound 108, 400.00 mg, crude) as a red oil. The residue was used in the next step without further purification. LCMS (ESI, m/z): 587 [M+H-TFA] ⁺

Step 15. Synthesis of compound 109

[0590] 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 at room temperature in air for 1 hour, 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 salt was added; trifluoroacetic acid (Compound 108, 400 mg, 0.57 mmol, 1.00 equiv) and DIEA (663 mg, 5.14 mmol, 9.00 equiv) at room temperature. The reaction mixture was stirred at room temperature for 2 h. LCMS indicated that the reaction was complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 50% over 30 min; detector, UV 254 nm to give 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, 480.00 mg, 75%) as a green solid. LCMS (ESI, m/z): 951 (M+H) ⁺

Stage 16. Compound 110

[0591] 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 h. LCMS indicated that the reaction was complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 60% over 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 (ESI, m/z): 729 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

Step 17. Synthesis of compound (Ik)

[0592] To a solution of (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, 63.00 mg, 0.09 mmol, 1.00 equiv) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (26 mg, 0.09 mmol, 1.00 equiv) in DMF (1.50 mL, 19.38 mmol, 224.36 equiv) was added DIEA (22.33 mg, 0.17 mmol, 2.00 equiv) at room temperature in air. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by reversed-phase flash chromatography as follows: Column: Kinetex EVO C18 column, 30x150.5 μm; mobile phase A: water (0.05% TFA), mobile phase B: ACN; flow rate: 60 ml/min; gradient: from 23 V to 43 V in 7 min, 254 nm; RT1: 6.58). The collected fraction was 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 (ESI, m/z): 922.924 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 8.80 (s, 1H), 8.44-8.41 (m, 1H), 8.15 (d, J=7.2 Hz, 1H), 8.03-8.00 (m, 2H), 7.7-7.65 (m, 2H), 7.51 (s, 1H), 7.44 (d, J=8.0 Hz,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, 12N).

Scheme 14: Synthesis of the neodestructor P 14-linker GGFG complex (Compound (II))

Step 1. Synthesis of compound 112

[0593] 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 BH ₃ -Me ₂ S (5.50 mL, 57.99 mmol, 2.50 equiv.) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 70 °C under nitrogen atmosphere. TLC (PE:EtOAc=3:1) showed the reaction to be complete. The residue was purified by column chromatography on silica gel eluting with PE/EtOAc (2:1) to give 2-(2-chloro-4-nitrophenyl)ethanol (Compound 112, 4.8 g, 92%) as a light 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).

Step 2. Synthesis of compound 113

[0594] 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) were 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 that the reaction was complete. The reaction mixture was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 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).

Step 3. Synthesis of compound 114

[0595] 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) was observed to show that the reaction was complete. The reaction mixture was diluted with water (600 mL). The resulting mixture was extracted with EA (200 mL*3). The combined organic layer was 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]ethenone (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).

Step 4. Synthesis of compound 115

[0596] To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (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 with N ₂ over 1 h. The reaction mixture was stirred at 0 °C for 1 h. TLC (PE:EA=10:1) showed the reaction to be complete. The reaction mixture was quenched with AcOH. The resulting mixture was concentrated to dryness in vacuo. The residue was diluted with DCM (100 mL) and filtered. The filtrate was purified by column chromatography on silica gel 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=8.0 Hz, 1H).

Step 5. Synthesis of compound 117

[0597] 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) ₄ (15 g, 33.86 mmol, 1.20 equiv) in TEF (300 mL) and toluene (100 mL) was added pyridine (2.59 g, 32.74 mmol, 1.16 equiv) dropwise at room temperature under nitrogen. The resulting mixture was stirred overnight at 80 °C under nitrogen. LCMS indicated that 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 Na2SO4} . 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 (2-[[(9EEfluoren-9-ylmethoxy)carbonyl]amino]acetamido)methyl acetate (Compound 117, 6.5 g, 56%) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.80(d, J=7.5 Hz, 2H), 7.62(d, J=7.5 Hz, 2H), 7.45(t, d=7.5 Hz, 2H), 7.36(d, d=7.5 Hz, 2H), 7.18(br s, 1H), 5.48(br s, 1H), 5.28(d, J=7.2 Hz, 2H), 4.48(d, J=6.6 Hz, 2H), 4.26(t, J=6.6 Hz, 1H), 3.93(d, 5.4 Hz, 2H), 2.08(s, 3H). LCMS (ESI, ms): 391[M+Na] ⁺

Step 6. Synthesis of compound 118

[0598] To a solution of (2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetamido)methyl 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 h. LCMS indicated that the reaction was complete. The reaction mixture was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel eluting with (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 a white metallic solid. LCMS (ESI, m/z): 526,528 (M+H) ⁺

Step 7. Synthesis of compound 119

[0599] 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 that the reaction was complete. The resulting mixture was diluted with water (400 mL), extracted with EA (200 mL x 3). The combined organic layer was washed with water (200 mL), brine (200 mL), dried over anhydrous sodium sulfate and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 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 (ESI, m/z): 304,306 (M+H) ⁺

Step 8. Synthesis of compound 120

[0600] 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 a solution of NaHCO ₃ (0.33 g, 3.92 mmol, 1.20 equiv) in water (10 mL), Boc ₂ O (0.72 g, 3.30 mmol, 1.00 equiv) at room temperature. The resulting mixture was stirred at room temperature for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (500 mL), extracted with EtOAc (200 mL x 3). The combined organic layer was 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 (PE:EtOAc=1:3) to give tert-butyl N-[[([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)carbamoyl]methyl]carbamate (Compound 120, 810 mg, 54%) as a white solid. LCMS (ESI, m/z): 404.406 [M+H] ⁺ , 304.306 [M+H-100] ⁺

Step 9. Synthesis of compound 121

[0601] 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) were added iron powder (1106 mg, 19.81 mmol, 10.00 equiv) and a solution of NH ₄ Cl (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 indicated that the reaction was complete. The reaction mixture was filtered. The filtrate was concentrated to dryness in vacuo. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was purified by column chromatography on silica gel eluting with 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 (ESI, m/z): 374.376 [M+H] ⁺ , 374.376 [M+H-100] ⁺

Step 10. Synthesis of compound 122

[0602] 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) were added CDI (130 mg, 0.80 mmol, 1.00 equiv.) and TEA (81 mg, 0.80 mmol, 1.00 equiv.) at 0°C in air. The resulting mixture was stirred at room temperature for 2 h. Then 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 DM AP (294 mg, 2.41 mmol, 3.00 equiv.) were added at room temperature in air. The resulting mixture was stirred at 60 °C for 48 h. LCMS showed that the reaction was complete. The resulting mixture was purified by reversed-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.05% TFA), gradient from 0% to 60% over 30 min; 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 (ESI, m/z): 673.675 [M+H] ⁺ , 573.575 [M+H-100] ⁺

Step 11. Synthesis of compound-123

[0603] 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 ₂ . The reaction mixture was stirred at room temperature for 2 h. LCMS indicated that 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 (ESI, m/z): 573.575 [M+H-HCl] ⁺

Step 12. Synthesis of compound-125

[0604] 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 in air for 16 h. The reaction was purified by reverse flash chromatography using the following conditions: column, silica gel C18; mobile phase, ACN in water (0.1% FA), gradient 0% to 60% over 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 (ESI, m/z): 473 (M+H) ⁺

Step 13. Synthesis of compound (II)

[0605] 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) were 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 h. 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 h. LCMS showed that 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 V to 60 V over 7 min, 254 nm; RT1: 6.67 min to afford 75 mg of crude product. The crude product was re-purified by Reverse 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 V to 44 V over 10 min; 254 nm; RT1: 10.52 min. The collected fraction was lyophilized to afford compound (II) (41.6 mg, 10%) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.99 (s, 1H), 8.79 (s, 1H), 8.38 (t, J=6.0 Hz, 1H), 8.31 (t, J=6.0 Hz, 1H), 8.12 (d, J=8.4 Hz, 1H), 8.06 (t, J=5.6 Hz, 1H), 8.01 (t, J=6.0 Hz, 1H), 7.70-7.66 (m, 2H), 7.51 (s, 1H), 7.44 (d, J=8.0 Hz, 1H), 7.25-7.21 (m, 5H), 7.19-7.14 (m, 2H), 6.99 (s, 2H), 6.82 (t, J=6.0 Hz, 1H), 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,4N), 1.20-1.16 (m, 2H). LCMS (ESI, m/z): 1027.1029 (M+H) ⁺

Scheme 15: Synthesis of the complex of neodestructor P14 - linker ALA (Compound (Im))

Step 1. Synthesis of compound 127

[0606] 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 dropwise BH ₃ -Me ₂ S (28.00 mL, 295.23 mmol, 2.65 equiv.) under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 70 °C under nitrogen atmosphere. TLC (PE:EtOAc=3:1) showed the reaction to be 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 light yellow solid. ¹ H NMR (300 MHz, CD ₃ Cl) δ 8.27 (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).

Step 2. Synthesis of compound 128

[0607] 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) were added NBS (6.62 g, 1.50 equiv) and _PPh3 (9.76 g, 37.21 mmol, 1.50 equiv) portionwise at room temperature under _N2 . The resulting mixture was stirred overnight at room temperature under _N2 . TLC (PE:EtOAc=10:1) showed the reaction to be complete. The reaction mixture was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 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-d ₆ ) 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).

Step 3. Synthesis of compound 129

[0608] 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 nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 h. TLC (PE:EtOAc=10:1) showed that the reaction was complete. The reaction mixture was diluted with water (600.00 mL). The resulting mixture was extracted with EtOAc (200.00 mL*3). The combined organic layer was washed with water (200.00 mL), brine (200.00 mL* ₃ ), dried over anhydrous _Na2SO4 and concentrated to dryness in vacuo to give 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (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).

Step 4. Synthesis of compound 130

[0609] To a stirred solution of 1-[[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]ethenone (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 N ₂ atmosphere. The resulting mixture was stirred at 0 °C under N ₂ atmosphere for 1 h. TLC (PE:EtOAc=10:1) showed the reaction to be complete. The reaction mixture was quenched with AcOH to pH 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 light 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 (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).

Step 5. Synthesis of compound 132

[0610] 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) were added TSTU (25.18 g, 83.52 mmol, 1.30 equiv.) and DIE A (16.60 g, 128.48 mmol, 2.00 equiv.) at room temperature under air atmosphere. The resulting mixture was stirred for 1 h at room temperature. LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (200.00 mL), and the resulting mixture was extracted with ETOAC (100.00 mL*3). The combined organic layer was 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 (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 (ESI, m/z): 431 [M+Na] ⁺

Step 6. Synthesis of compound 133

[0611] To a solution of glycine (3.68 g, 48.97 mmol, 1.00 equiv.) and NaHCO ₃ (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 mixture was stirred at room temperature for 2 h. LCMS indicated that the reaction was complete. The reaction was adjusted to pH 2-3 with 2 N HCl. The resulting mixture was extracted with EtOAc (500.00 mL*3), the combined organic layer was 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 (ESI, m/z): 369 [M+H] ⁺

Step 7. Synthesis of compound 134

[0612] 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) ₄ (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 _N2 was stirred at 80 °C for 16 h. After cooling to room temperature, the reaction mixture was filtered. The filter cake was washed with THF (100.00 mL). The combined organic layer was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel eluting (PE:ETOAC=1:2) to give [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]methyl acetate (Compound 134, 2.50 g, 45%) as a white solid. LCMS (ESI, m/z): 405 [M+Na] ⁺ . ¹ H 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).

Step 8. Synthesis of compound 135

[0613] To a stirred solution of [(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanamido]methyl 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) under N ₂ at room temperature. The resulting mixture was stirred at 40 °C for 16 h. LCMS indicated 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 (ESI, m/z): 540.542 (M+H) ⁺

Step 9. Synthesis of compound 136

[0614] 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 ₂ . The resulting mixture was stirred at 0 °C for 0.5 h under N ₂ . LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (600.00 mL). The resulting mixture was extracted with EA (200.00 mL x 3). The combined organic layer was washed with brine (200.00 mL), dried over anhydrous _Na2SO4 and concentrated to dryness in vacuo _to afford 3.00 g of crude product. The crude product was re-purified by silica gel column chromatography eluting with (DCM:MeOH=3:1) to afford (2S)-2-amino-N-([[2-(2-chloro-4-nitrophenyl)ethyl]sulfanyl]methyl)propenamide (Compound 136, 1.50 g, 78%) as a yellow oil. LCMS (ESI, m/z): 318.320 [M+H] ⁺ .

Step 10. Synthesis of compound 137

[0615] 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 NaHCO ₃ (0.59 g, 7.08 mmol, 1.50 equiv) in H ₂ O (10.00 mL) and Boc ₂ O (1.03 g, 4.72 mmol, 1.00 equiv) at room temperature in air. The reaction mixture was stirred at room temperature for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was diluted with water (500.00 mL), extracted with EtOAc (200.00 mL x 3). The combined organic layer was 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 137, 1.82 g, 83%) as a red oil. LCMS (ESI, m/z): 418-420 [M+H] ⁺ , 318-320 [M+H-100] ⁺

Step 11. Synthesis of compound 138

[0616] A suspension 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 NH ₄ Cl (2.33 g, 0.04 mmol, 10.00 equiv) in EtOH (100.00 mL)/H ₂ O (50.00 mL) was stirred at 70 °C for 2 h. LCMS indicated that the reaction was complete. The reaction mixture was filtered. The filtrate was concentrated to dryness in vacuo. The residue was dissolved in DCM (50.00 mL) and filtered. The filtrate was purified by column chromatography on silica gel 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 138, 1.20 g, 68%) as a yellow oil. LCMS (ESI, m/z): 388.390 [M+H] ⁺ , 288.290 [M+H-100] ⁺

Step 12. Synthesis of compound 139

[0617] 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 were added CDI (209.00 mg, 1.29 mmol, 1 equiv.) and TEA (260 mg, 2.58 mmol, 2 equiv.). The resulting mixture was stirred at 0 °C for 2 h. Then 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 DM AP (472 mg, 3.87 mmol, 3.00 equiv) were added. The resulting mixture was stirred at 60 °C for 24 h. LCMS showed that the reaction was complete. After cooling to room temperature, the reaction mixture was purified by reverse flash chromatography using the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1% FA), gradient from 0% to 60% over 30 min; detector, UV 254 nm to give 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 (ESI, m/z): 687.689 [M+H] ⁺ , 587.589 [M+H-100] ⁺

Step 13. Synthesis of Compound 140 [0618] 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 indicated the reaction was complete. The reaction mixture was concentrated to dryness in vacuo to give the (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 salt (Compound 140, 400.00 mg) as a red oil. LCMS (ESI, m/z): 578.589 [M+H-TFA] ⁺

Step 14. Synthesis of compound 142

[0619] To a suspension of L-valine (Compound 141, 0.50 g, 4.27 mmol, 1.00 equiv.) in DMSO (10 mL) were 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 h. LCMS showed the reaction to be complete. The reaction mixture was purified by reverse flash chromatography using the following conditions: column, silica gel C18; mobile phase, ACN in water (0.1% FA), gradient from 0% to 60% over 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 (ESI, m/z): 311 [M+H] ⁺

Step 15. Synthesis of compound (Im)

[0620] 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 at room temperature in air for 1 hour. Then, trifluoroacetic acid, (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 salt (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 h. LCMS showed that the reaction was complete. The resulting mixture was purified by reverse-phase flash chromatography under the following conditions: Column: YMC-Actus Triart C18, 30 mm X 150 mm, 5 μm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 60 mL/min; gradient: 28 V to 45 V in 10 min, 254 nm; RT1: 9.67 min. The collected fraction was 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 (ESI, m/z): 879.881 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.99 (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, 2H), 6.99 (s, 2H), 6.82 (t, J=6.0Hz, 1H), 5.13-5.10 (m, 1H), 4.47-4.40 (m, 3H), 4.33-4.29 (m, 3H), 4.24 (t, J=7.2 Hz, 1H), 4.14 (t, J=6.8 Hz, 1H), 3.38-3.36 (m, 1H), 2.97-2.90 (m, 1H), 2.86 (t, J=7.6 Hz, 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).

Example 4: General procedure for obtaining and characterizing a conjugated neodestructor

[0621] The antibody solution was treated with 30 equivalents of tris-(2-carboxyethyl)phosphine (TCEP) and incubated at 37°C for 1 hour to reduce interchain disulfides. The reduced antibody was purified in 50 mM EPPS, 5 mM EDTA pH 7.0 buffer using illustra NAP columns (GE Healthcare).

[0622] Conjugation was accomplished by treating a 2-5 mg/mL solution of reduced antibody in 50 mM EPPS, 5 mM EDTA pH 7.0 with 12 equiv of non-degrader linker 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 to 20 mM succinate, 8% sucrose, 0.01% Tween 20 pH 5.5 using illustra NAP columns (GE Healthcare) and concentrated using Amicon Ultra 50 kDa MWCO centrifugal concentrators (Millipore).

[0623] Concentration and monomer were determined by size exclusion chromatography using a 7.8 x 300 mM TSKGel 3000SWXL column with 5 μm particles (Tosoh Bioscience), eluting isocratically with 400 mM sodium perchlorate, 50 mM sodium phosphate, 5% (v/v) isopropanol mobile phase running at 0.5 mg/mL for 30 min. Neodegrader conjugates were quantified from antibody standard curves, detected at 214 nm.

[0624] The drug to antibody ratio (DAR) was determined by hydrophobic interaction chromatography using a TSKgel Butyl-NPR 4.6 x 35 mm column with 2.5 μm particles. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate at pH 7.0. Mobile phase B was 25 mM sodium phosphate pH 7.0, 25% (v/v) isopropanol. The analytes were eluted with a linear gradient of 0-100% B over 12 min at a flow rate of 0.6 mL/min. Detection was at 214 nm.

[0625] Free linker payload was determined by mixed chromatography using a HISEP 4.6 x 250 mm column with 2.5 μm particles (Supelco). Mobile phase A was 100 mM ammonium acetate. Mobile phase B was 100% acetonitrile. The analytes were eluted with a gradient of 25-40% B over 25 min, then 40-100% B over 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, detecting at 254 nm.

Example 5: General Procedure 1 for In Vitro Antiproliferation Assay for Neodegrader and Neodegrader Conjugate

[0626] The ability of the neodestructor conjugates to inhibit cell growth was measured using an in vitro antiproliferation assay. Target cells were seeded at 1500-5000 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). Conjugates were diluted in complete cell growth medium using 4-fold serial dilutions and added at 100 μl per well. The final concentration typically ranged from 1 x 10 ^-8 M to 1.53 x 10 ^-13 M or 1 x 10 ^-7 M to 1.53 x 10 ^-12 M. Cells were incubated at 37°C in a humidified 5% CO ₂ incubator for 5 days. The viability of the remaining cells was determined using the WST-8 colorimetric assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). WST-8 was added to 10% of the final volume and the plates were incubated at 37°C in a humidified 5% CO ₂ incubator for 2-4 hours. Plates were analyzed by measuring absorbance at 450 nm (A450) in a multiwell plate reader. Background absorbance A450 of wells with medium and WST-8 alone was subtracted from all values. Percent viability was calculated by dividing the value of each treated sample by the average value of wells with untreated cells. Percent viability was plotted as a semilogarithmic function of test sample concentration for each treatment. IC50 values were calculated automatically.

[0627] The antiproliferative activity of trastuzumab and pertuzumab conjugates of compounds (Ia) and (Ic) against the breast cancer cell line BT-474 is illustrated in Figs. 1-4 (drug:antibody ratio = 8 in each neodestructor conjugate). The antibody-drug conjugate Kadcyla and the unconjugated antibodies trastuzumab and pertuzumab were found to be >100-fold less active than the antibody-neodestructor conjugates, while the non-cell-binding control rituximab-neodestructor conjugate (Ia) and the released neodestructors P1 and P4 were found to be >1000-fold less active against BT-474 cells.

[0628] The antiproliferative activity of the trastuzumab and pertuzumab conjugates of compound (Ia) and compound (Ic) against the breast cancer cell line BT-474 is illustrated in Figs. 5-6 (drug:antibody ratios are indicated). The ENHERTU ^® antibody-drug conjugate and the unconjugated trastuzumab antibody were less active than the neodestructive antibody conjugates against BT-474 cells.

[0629] The antiproliferative activity of trastuzumab and pertuzumab Compound (Ia) conjugates against the SK-BR-3 breast cancer cell line is illustrated in Figs. 7 and 8 (drug:antibody ratio = 8 in each neodegrader conjugate). The conjugated neodegraders had similar activity to the Kadcyla antibody-drug conjugate, while the unconjugated trastuzumab and pertuzumab antibodies were significantly less active than the neodegrader conjugates. The non-cell-binding control rituximab-Compound (Ia) neodegrader conjugate and the released neodegraders P1 and P4a were found to be significantly less active against SK-BR-3 cells.

[0630] The antiproliferative activity of conjugates of compounds (Ia), (Id) OR000213, huMy9-6 and lintuzumab IgG1 against the HL-60 (acute myeloid leukemia) cell line is illustrated in Figs. 9-12 (drug:antibody ratio = 8, where not indicated). The neodegradant conjugate was shown to have similar activity as the approved agent MYLOTARG ^® against the cell line, while the non-cell binding control neodegradant conjugates trastuzumab-compound (Ia) and rituximab-compound (Id) were significantly less active.

[0631] The antiproliferative activity of the conjugate of compounds (Ia) and (Ic) rituximab against the Ramos cell line (non-Hodgkin's lymphoma) is illustrated in Fig. 13 (drug:antibody ratio = 8, where not indicated). Unconjugated rituximab, a control non-cell-binding neodegradant conjugate, trastuzumab-compound (Ia), and released neodegradants P1 and P4 were shown to be less active than the neodegradant conjugates against this cell line.

[0632] The antiproliferative activity of the rituximab conjugate of compounds (Ia) and (Ic) against the Daudi lymphoma cell line is illustrated in Figs. 14 and 15 (drug:antibody ratio = 8, unless otherwise noted). Unconjugated rituximab, a control non-cell-binding neodegradant conjugate, trastuzumab-compound (Ia), and released neodegradant P1 were shown to be less active than the neodegradant conjugates against this cell line.

[0633] The antiproliferative activity of trastuzumab and pertuzumab Compound (Ia) conjugates against the NCI-N87 gastric cancer cell line is illustrated in Figs. 16 and 17 (drug:antibody ratio = 8 in each neodegrader conjugate). The conjugated neodegraders had similar activity to the Kadcyla antibody-drug conjugate, while the unconjugated trastuzumab and pertuzumab antibodies were significantly less active than the neodegrader conjugates. The non-cell-binding control rituximab-Compound (Ia) neodegrader conjugate and the released neodegrader P1 were found to be significantly less active against NCI-N87 cells.

[0634] Figure 18 illustrates the antiproliferative activity of trastuzumab and pertuzumab Compound (Ia) conjugates following three days of incubation with human serum compared to the activity of the conjugates in the absence of serum in BT-464 breast cancer cells. As illustrated in the graph, the activity of the neodegrader conjugates was the same in the presence and absence of serum, indicating that human serum does not affect the activity. The non-cell-binding control neodegrader conjugate OR000213-Compound (Ia) was more than 1000-fold less active against this cell line.

[0635] Figure 19 illustrates the antiproliferative activity of trastuzumab and pertuzumab Compound (Ia) conjugates after three days of incubation with mouse serum compared to the activity of the conjugates in the absence of serum in BT-464 breast cancer cells. As illustrated in the graph, the activity of the neodegrader conjugates was the same in the presence and absence of serum, indicating that mouse serum does not affect the activity. The non-cell-binding control neodegrader conjugate OR000213-Compound (Ia) was more than 1000-fold less active against this cell line.

[0636] Tables 1 and 2 illustrate the IC50 values of trastuzumab conjugates of compounds (Ia), (Ib), (Ic), and (Id) and pertuzumab conjugates of compounds (Ia) and (1c) against various Her2 cell lines. As illustrated in Table 1, the neodegrader conjugates demonstrated improved activity in the BT-474 cell line compared to unconjugated antibodies, and also demonstrated improved activity against the released payloads and antibody-drug conjugates of Kadcyla and ENHERTU ^® . The neodegrader conjugates also demonstrated better activity against the SK-BR-3 breast cancer cell line and the NCI-N87 gastric cell line compared to unconjugated antibodies. As illustrated in Table 2, the non-destructor antibody conjugates also had enhanced activity against the SNU-182 liver cell line compared to unconjugated antibodies or Kadcyla.

[0637] Table 3 illustrates the activity of the non-degrader antibody conjugates in anti-CD20 cell lines. The non-degrader antibody conjugates had higher activity against the Daudi and Ramos lymphocyte cell lines compared to unconjugated antibodies, the non-cell-binding control trastuzumab-Compound I(a) non-degrader conjugate, and the released payload.

[0638] Table 4 shows the IC ₅₀ values of huMy9-6 and OR000213 conjugates of Compounds (Ia) and (Id) and IgG1 conjugates of lintuzumab and Compounds (Ia) and (Id) against HL-60 AML cell lines. As shown in Table 4, the non-disruptor conjugates demonstrated comparable activity in the HL-60 cell line compared to MYLOTARG® and improved activity compared to the non-binding rituximab-Compound (Ic) conjugate.

[0639] Table 5 illustrates the antiproliferative activity of trastuzumab and Compound (Ia) conjugates against the BT-474 breast cancer cell line after incubation with human or mouse serum compared to the non-cell-binding control OR000213 neodegrader-Compound (Ia) conjugate. As illustrated in the table, the activity of the neodegraders incubated in human or mouse serum was consistent with the activity when no serum was administered.

Example 6: General Procedure 2 for In Vitro Antiproliferation Assay for Neodegrader and Neodegrader Conjugates

[0640] 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 under culture conditions specified by the ATCC or DSMZ. Cells were thawed and maintained in culture for at least two passages before being subjected to experimental conditions.

[0641] Cytotoxicity assays. For adherent cell lines, cells were dissociated with enzyme-free PBS-based cell dissociation buffer (Gibco, USA) and seeded onto tissue culture-treated 96-well flat-bottomed polystyrene plates (Costar, Corning, USA) at appropriate well densities depending on cell doubling time. Eighteen hours after seeding, cells were treated with test samples at appropriate concentrations starting from 100 nM and diluted in 4-fold serial dilutions. For suspension cell lines, cells were seeded on the same day of treatment and cells were treated as described above. Adherent cells were treated for 5 days and suspension cells were treated for 3 days. Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) and measured 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 confirmed using three biological replicates.

[0642] Table 6 illustrates the activity of the neodegraders P1, P3, and P4 against various cancer cell lines. As illustrated in the table, the neodegraders have activity against each of the cell lines.

[0643] Table 7 illustrates the activity of pertuzumab-Compound (Ia) conjugate and the known antibody-drug conjugate ENHERTU® against various breast cancer cell lines. As illustrated in the table, pertuzumab-Compound (Ia) conjugate was more active in all cell lines described.

[0644] Table 8 illustrates the activity of the pertuzumab-Compound (Ia) conjugate and the known antibody-drug conjugate ENHERTU against three gastric cancer cell lines. As illustrated in the table, the pertuzumab-Compound (Ia) conjugate was more active in all of the cell lines described.

[0645] Table 9 illustrates the activity of OR000213-Compound (Ia) conjugate and the known antibody-drug conjugate MYLOTARG against various acute myeloid leukemia cell lines. As illustrated in the table, OR000213-Compound (Ia) conjugate had better activity in several cell lines.

[0646] Table 10 illustrates the activity of three anti-CD38 neodestructor conjugates against various multiple myeloma cell lines. As can be seen from the table, the conjugates had good activity in all cell lines.

[0647] Table 11 illustrates the activity of the anti-CD 138 neodestructor conjugate against various multiple myeloma cell lines. As can be seen from the table, the conjugate had good activity in all cell lines.

[0648] Table 12 illustrates the activity of the anti-BCMA neodestructor conjugate against various multiple myeloma cell lines. As can be seen from the table, the conjugate had good activity in all cell lines.

[0649] Table 13 illustrates the activity of the anti-Trop-2 neodestructor conjugate against various cancer cell lines. As can be seen from the table, the conjugate had good activity in all cell lines.

[0650] Table 14 illustrates the activity of the anti-FGFR4 neodegradant conjugate against two cancer cell lines. As illustrated in the table, the conjugate had good activity in both cell lines and had better activity than the US-1784 antibody and unconjugated neodegradant alone.

[0651] Table 15 illustrates the activity of the anti-EGFR neodegradant conjugate against two synovial sarcoma cell lines. As illustrated in the table, the conjugate had good activity in both cell lines and had better activity than cetuximab and unconjugated neodegradant alone.

[0652] Table 16 illustrates the activity of the anti-PDGF-R a neodestructor conjugate against two cancer cell lines. As can be seen from the table, the conjugate had good activity in both cell lines.

[0653] Table 17 illustrates the activity of the anti-TEMI/CD248 neodestructor conjugate against the rhabdomyosarcoma cell line. As can be seen from the table, the conjugate has good activity against the cell line.

Example 7: Treatment of breast cancer with an anti-Her2 antibody-neodestructor conjugate

[0654] Trastuzumab-Compound (Ia) and pertuzumab-Compound (Ia) conjugates were tested in immunodeficient mice (Fox Chase SCID ^® , CB17/Icr-Prkdc ^scid /IcrIcoCr1, Charles River). 1 ^mm3 BT474 human breast carcinoma fragments were implanted subcutaneously in the right flank of mice. Mice were injected with anti-Her2 antibody-neodestructor conjugates, non-targeting neodestructor conjugates, and vehicle control once tumors reached an average size of 100-150 ^mm3 .

[0655] Trastuzumab-Compound (Ia), rituximab-Compound (Ia), and pertuzumab-Compound (Ia) conjugate stock solutions were diluted with vehicle to obtain 0.5 mg/mL dosing solutions, providing 5 mg/kg in a dosing volume of 10 mL/kg (0.2 mL per 20 g mouse), adjusted for the body weight of each animal.

[0656] Mice were divided into 4 treatment groups (N=8/group) as follows: 1) vehicle; 2) trastuzumab-Compound (Ia) conjugate (5 mg/kg, i.v., daily x 1); 3) rituximab-Compound (Ia) conjugate (5 mg/kg, i.v., daily x 1); 4) pertuzumab-Compound (Ia) conjugate (5 mg/kg, i.v., daily x 1). All test drugs were administered intravenously (i.v.) as a single dose (daily x 1) in volumes adjusted for body weight (0.200 mL/20 g mouse).

[0657] Tumors were measured with calipers twice weekly, and each animal was euthanized when its tumor reached the endpoint volume (1000 mm ³ ) or on the last day (day 60) of the study, whichever came first. MTV(n) was defined as the mean tumor volume on the last day of the study among the remaining animals (n) whose tumor volume did not reach the endpoint.

[0658] As illustrated in Fig. 20, pertuzumab and rituximab conjugates provide slower tumor growth over time compared to vehicle and the non-cell-binding control neodegradant rituximab-Compound (Ia) conjugate.

Example 8: Treatment of non-Hodgkin's lymphoma (NHL) with anti-CD20-neodestructor antibody conjugates

[0659] The rituximab-compound (1a) conjugate was tested in immunodeficient mice (Fox Chase SCID ^® , CB17/Icr-Prkdc ^scid /IcrIcoCrl, Charles River). 1×10 ⁷ Daudi Burkitt's B-cell lymphoma cells (ATCC ^® CCL-213™) were injected subcutaneously into the right flank (0.1 ml of cell suspension) of the mice. Mice were injected with anti-CD20-neodestructor antibody conjugates, non-targeting neodestructor conjugates, and vehicle control once the tumors reached a mean size of 100-150 mm ³ .

[0660] Trastuzumab-Compound (Ia) and rituximab-Compound (Ia) stock solutions were diluted with vehicle to obtain 0.5 mg/mL and 0.1 mg/mL dosing solutions, providing 5 and 1 mg/kg in a dosing volume of 10 mL/kg (0.2 mL per 20 g mouse), adjusted for the body weight of each animal.

[0661] Mice were divided into 4 treatment groups (N=8/group) as follows: 1) vehicle; 2) trastuzumab-Compound (Ia) (5 mg/kg, IV, daily x 1); 3) rituximab-Compound (Ia) (1 mg/kg, IV, daily x 1); 4) rituximab-Compound (Ia) (5 mg/kg, IV, daily x 1). All test drugs were administered intravenously (IV) as a single dose (daily x 1) in volumes adjusted for body weight (0.200 mL/20 g mouse).

[0662] Tumors were measured with calipers twice weekly, and each animal was euthanized when its tumor reached the endpoint volume (1500 mm ³ ) or on the last day (day 45) of the study, whichever came first. MTV(n) was defined as the mean tumor volume on the last day of the study among the remaining animals (n) whose tumor volumes did not reach the endpoint.

[0663] As illustrated in Fig. 21, the 5 mg/kg dose of rituximab conjugate resulted in slower tumor growth over time compared to the 1 mg/kg dose, vehicle, and the non-cell-binding control trastuzumab-Compound (Ia) neodegradant conjugate.

Example 9. Treatment of acute myeloid leukemia (AML) with an anti-CD33 antibody-neodestructor conjugate

[0664] OR000213-compound (Ia) was tested in athymic nude mice (Crl:NU(NCr)-Foxnlnu, Charles River). 1 x 107 acute promyelocytic leukemia HL-60 cells (ATCC® CCL-240™) were injected subcutaneously into the right flank of the mice (0.1 ml of cell suspension). Mice were injected with anti-CD33 antibody-neodestructor conjugates, non-targeting neodestructor conjugates, and vehicle control once tumors reached a mean tumor size of 100-150 mm ³ .

[0665] Trastuzumab-Compound (Ia) and OR000213-Compound (Ia) stock solutions were diluted with vehicle to obtain 0.5 mg/mL and 0.1 mg/mL dosing solutions, providing 5 and 1 mg/kg in a dosing volume of 10 mL/kg (0.2 mL per 20 g mouse), adjusted for the body weight of each animal.

[0666] Mice were divided into 4 treatment groups (N=8/group) as follows: 1) vehicle; 2) trastuzumab-Compound (Ia) (5 mg/kg, IV, daily x 1); 3) rituximab-Compound (Ia) (1 mg/kg, IV, daily x 1); 4) rituximab-Compound (Ia) (5 mg/kg, IV, daily x 1). All test drugs were administered intravenously (IV) as a single dose (daily x 1) in volumes adjusted for body weight (0.200 mL/20 g mouse).

[0667] Tumors were measured with calipers twice weekly, and each animal was euthanized when its tumor reached the endpoint volume (2000 mm ³ ) or on the last day (day 45) of the study, whichever came first. MTV(n) was defined as the mean tumor volume on the last day of the study among the remaining animals (n) whose tumor volume did not reach the endpoint.

[0668] As illustrated in Fig. 22, all neodestructor conjugates provided slower tumor growth over time compared to vehicle.

Example 10. Treatment of multiple myeloma with an anti-CD38 antibody-neodestructor conjugate

[0669] The HuAT13/5 (Ia) compound was tested in CB.17 SCID mice (CB17/Icr-Prkdcscid/IcrlcoCrl, Charles River). 1 x 107 NCI-H929 myeloma cells (ATCC® CRL-9068™) in 50% Matrigel were injected subcutaneously into the axillary region of the mice (0.1 ml of cell suspension). Mice were injected with anti-CD38-neodestructor antibody conjugates and vehicle control once the tumors reached an average size of 100-150 mm ³ .

[0670] The HuAT 13/5-Compound (Ia) stock solution was diluted with vehicle to obtain a 0.5 mg/mL dosing solution, providing 5 mg/kg in a dosing volume of 10 mL/kg (0.2 mL per 20 g mouse), adjusted for the body weight of each animal.

[0671] Mice were divided into 2 treatment groups (N=10/group) as follows: 1) vehicle; 2) NIAT 13/5-compound (Ia) (5 mg/kg, IV, daily x 1). All test drugs were administered intravenously (IV) as a single dose (daily x 1) in volumes adjusted for body weight (0.200 mL/20 g mouse).

[0672] Tumors were measured with calipers twice weekly, and each animal was euthanized when its tumor reached the endpoint volume (2000 mm ³ ) or on the last day (day 45) of the study, whichever came first. MTV(n) was defined as the mean tumor volume on the last day of the study among the remaining animals (n) whose tumor volumes did not reach the endpoint.

[0673] As illustrated in Fig. 25, a 5 mg/kg dose of the HuAT13/5 neodestructor conjugate provides slower tumor growth over time compared to vehicle.

[0674] It should be appreciated that the Detailed Description of the Invention section, and not the Summary of the Invention and Abstract sections, is intended to interpret the claims. The Summary of the Invention and Abstract sections set forth one or more, but not all, illustrative aspects of the present invention as contemplated by the inventor(s), and thus are not intended to limit the present invention and the appended claims in any way.

[0675] The present invention has been described above using functional building blocks illustrating the implementation of said functions and their interrelations. The boundaries of these functional building blocks have been arbitrarily defined in this document for convenience of description. Alternative boundaries may be defined as long as the said functions and their relationships are properly performed.

[0676] The foregoing description of specific aspects fully reflects the general nature of the present invention, which others, using knowledge within the scope of the art, can easily modify and/or adapt to various ways of practicing such specific aspects, without undue experimentation, without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to fall within the meaning and range of equivalents of the disclosed aspects based on the teachings and instructions provided herein. It should be understood that the language or terminology herein is intended to be descriptive and not limiting, so that the terminology or language of the present description should be interpreted by one skilled in the art in light of the teachings and instructions.

[0677] The scope of the claims and the scope of the present invention should not be limited by any of the above-described illustrative aspects, but should be defined only in accordance with the following claims and their equivalents.

Claims (190)

1. Conjugate of formula (I) or a pharmaceutically acceptable salt thereof, where a is an integer from 1 to 10; A represents a phenyl or _C4 - _C10 cycloalkyl ring; U is selected from NH and CF ₂ ; R ¹ is independently selected from hydrogen and halogen; X is selected from -NR ² -, =C(CH ₃ )-, -Q-(CH ₂ ) _n - and -Q(CH ₂ ) _m Q'(CH ₂ ) _n -; where each Q and Q' independently represents O, S, or N(R ² ) _v ; v is equal to 1 or 2; each R ² is independently hydrogen or C ₁ -C ₆ alkyl; n is an integer between 1 and 6; and m is an integer between 2 and 6; where the left side of each group is attached to L and the right side is attached to A; provided that when X is NH or -Q-(CH ₂ ) _n -, R ¹ is halogen; L is a linker selected from: Where represents the attachment point to X; and represents the attachment point to the linking fragment; p is an integer between 1 and 10; q is an integer between 2 and 10; Z ¹ is absent or represents glycine; Z ² is absent or 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; and Z ⁴ is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine; each of R and R' is independently selected from hydrogen and C ₁ -C ₆ alkyl; and is absent; and Bm is a binding fragment, which is an antibody or antigen-binding fragment that specifically binds to a surface antigen, wherein said surface antigen is not CD33. 2. The conjugate of claim 1, wherein the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, growth factor Crypto 1, 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, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 IRa, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α ₄ , α _v β ₃ , α _v β ₅ , α _v β ₆ , α ₁ β ₄ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α _IIb β ₃ ), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, Legumain, LewisY, LFA-1(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA/MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, mutant p53, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1/galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa, rabies virus gene, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, respiratory syncytial virus, Rhesus 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 combinations thereof. 3. The conjugate of claim 2, wherein the surface antigen comprises HER2, CD20, CD38, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1/CD248, TROP-2, or combinations thereof. 4. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of rituximab, trastuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuxizumab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, balantamab, indatuximab, cetuximab, dinutuximab, CD38 A2 antibody, HuAT13/5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab and veltuzumab. 5. The conjugate of claim 4 or a pharmaceutically acceptable salt thereof, wherein the antibody is rituximab, trastuzumab or pertuzumab. 6. The conjugate according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein: (a) A is phenyl; U represents NH; R ¹ is halogen; and X is -N(R ² ) _v (CH ₂ ) _m O(CH ₂ ) _n -; where v is equal to 1; m and n are equal to 2; and R ² is methyl; (b) A is phenyl; U represents NH; R ¹ is halogen; and X is -N(R ² ) _v (CH ₂ ) _m O(CH ₂ ) _n -; where v is equal to 2; m and n are equal to 2; and each R ² is methyl; (c) A is phenyl; U represents NH; R ¹ is halogen; and X represents -O( _CH2 ) _n- ; where n is 2; (d) A is phenyl; U represents NH; R ¹ is halogen; and X represents -S(CH ₂ ) _n -; where n is 2; (e) A is phenyl; U represents NH; R ¹ is hydrogen; and X represents -NR ² -; where R ² is methyl; (f) A is phenyl; U represents NH; R ¹ is halogen; and X represents -NR ² -; where R ² is hydrogen; (g) A is phenyl; U represents NH; R ¹ is hydrogen; and X is -C( _CH3 )=; or (h) A represents a _C4 - _C10 cycloalkyl ring; U represents NH; R ¹ is hydrogen; and X is -N(R ² )(CH ₂ ) _m O(CH ₂ ) _n -; where n is equal to 1; m is equal to 2; and R ² is methyl. 7. Compound of formula (II) or a pharmaceutically acceptable salt thereof, where A is a phenyl or _C4 - _C10 cycloalkyl ring; R ¹ is independently selected from hydrogen and halogen; U represents NH; and R ² is selected from -C(O)R ³ , -N(R ⁴ ) ₂ , -(CH ₂ ) _n OH, -(CH ₂ ) _n SH, -(CH ₂ ) _n N(R ⁴ ) ₂ , -(CH ₂ ) _n Q'(CH ₂ ) _m OH, -(CH ₂ ) _n Q'(CH ₂ ) _m SH and -(CH ₂ ) _n Q'(CH ₂ ) _m N(R ⁴ ) ₂ ; where R ³ is hydrogen or C ₁ -C ₆ alkyl; each R ⁴ is independently hydrogen or C ₁ -C ₆ alkyl; Q' represents O, S or NR ⁴ ; n is 1-6; and m is 2-5; provided that when R ² is NH ₂ , -(CH ₂ ) _n NH ₂ or -(CH ₂ ) _n OH, then R ¹ is a halogen. 8. Compound of formula (III) or a pharmaceutically acceptable salt thereof. 9. Compound of formula (IV) or a pharmaceutically acceptable salt thereof. 10. Conjugate of formula (V) or a pharmaceutically acceptable salt thereof, wherein Bm is a binding moiety that is an antibody or antigen-binding fragment that specifically binds to a surface antigen, wherein said surface antigen is not CD33. 11. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, growth factor Crypto 1, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin A4, ephrin B2, EPHB4, ERBB2 (Her2/neu), ErbB3, ERG (TMPRSS2-ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 IRa, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α ₄ , α _v β ₃ , α _v β ₅ , α _v β ₆ , α ₁ β ₄ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α _IIb β ₃ ), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, Legumain, LewisY, LFA-1(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA/MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, mutant p53, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1/galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa , rabies virus gene, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, respiratory syncytial virus, Rhesus 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 their combinations. 12. The conjugate of claim 11 or a pharmaceutically acceptable salt thereof, wherein the surface antigen comprises HER2, CD20, CD38, BCMA, CD138, EGFR, FGFR, GD2, PDGFR, TEM1/CD248, TROP-2, or combinations thereof. 13. The conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein the antibody comprises rituximab, trastuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuxizumab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, balantamab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13/5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab or veltuzumab, wherein preferably the antibody is rituximab, trastuzumab or pertuzumab. 14. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of a conjugate according to any one of claims 1-6 or 10-13 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers. 15. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate according to any one of claims 1-6 or 10-13 or a pharmaceutically acceptable salt or composition thereof according to claim 14, wherein the cancer is breast cancer, gastric cancer, lymphoma, acute myeloid leukemia, multiple myeloma, head and neck cancer, squamous cell carcinoma and/or hepatocellular carcinoma. 16. The method of claim 15, further comprising administering to the subject a pharmaceutically acceptable amount of an additional agent before, after, or simultaneously with the conjugate or pharmaceutically acceptable salt or composition thereof. 17. The method of claim 16, wherein the additional agent is a cytotoxic agent or an immune response modifier. 18. The method of claim 17, wherein the immune response modifier is a checkpoint inhibitor. 19. The method of claim 18, wherein the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor and/or a LAG-3 inhibitor. 20. A method for producing a conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, comprising reacting a binding fragment, which is an antibody or antigen-binding fragment that specifically binds to a surface antigen, wherein said surface antigen is not CD33, with a compound of formula (I-1) or its pharmaceutically acceptable salt, where a is an integer from 1 to 10; A represents a phenyl or _C4 - _C10 cycloalkyl ring; R ¹ is independently selected from hydrogen and halogen; U is selected from NH and CF ₂ ; X is selected from -N(R ² ) _v -, =C(CH ₃ )-, -Q-(CH ₂ ) _n - and -Q(CH ₂ ) _m Q'(CH ₂ ) _n -; where v is 1 or 2; each of Q and Q' independently represents O, S, or NR ² ; each R ² is independently hydrogen or C ₁ -C ₆ alkyl; n is an integer between 1 and 6; and m is an integer between 2 and 6; where the left side of each group is attached to L' and the right side is attached to A; provided that when X is NH or -Q-(CH ₂ ) _n -, R ¹ is halogen; L' is selected from: where p is an integer from 1 to 10; q is an integer between 2 and 10; Z ¹ is absent or represents glycine; Z ² is absent or 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; and Z ⁴ is selected from the group consisting of L-alanine, D-alanine, L-citrulline, D-citrulline, L-asparagine, D-asparagine, L-lysine, D-lysine, L-phenylalanine, D-phenylalanine and glycine; each of R and R' is independently selected from hydrogen and C ₁ -C ₆ alkyl; and absent. 21. The method of claim 20, further comprising reducing the linking moiety prior to reacting with the compound of formula (I-1). 22. The method according to claim 20 or 21, wherein a is an integer from 2 to 8. 23. The method according to any of paragraphs. 20-22, characterized in that the surface antigen comprises 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, Axin1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, clumping factor, cKit, claudin 3, claudin 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, growth factor Crypto 1, CS1, CTLA-4, CXCR2, CXORF61, cyclin Bl, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, ephrin A4, ephrin B2, EPHB4, ERBB2 (Her2/neu), ErbB3, ERG (TMPRSS2-ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor alpha, folate receptor beta, FOLR1, Fos-related antigen 1, fucosyl GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-1 IRa, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including integrins α ₄ , α _v β ₃ , α _v β ₅ , α _v β ₆ , α ₁ β ₄ , α ₄ β ₁ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α _IIb β ₃ ), integrin alpha V, intestinal carboxylesterase, KIT, LAGE-la, LAIR1, LAMP-1, LCK, Legumain, LewisY, LFA-1(CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE Al, MelanA/MARTl, mesothelin, ML-IAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, nectin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, mutant p53, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1/galectin 8, PD-L1, PD-L2, PDGFR, PDGFR-beta, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostasis, prostate carcinoma cell, prostein, Pseudomonas aeruginosa , rabies virus gene, survivin and telomerase PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, mutant Ras, respiratory syncytial virus, Rhesus 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, TCRp, 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 combinations thereof. 24. The method of claim 23, wherein the surface antigen comprises FIER2, CD20, CD38, BCMA, CD138, EGFR, FGFR4, GD2, PDGFR, TEM1/CD248, Trop-2, or combinations thereof. 25. The method according to any one of claims 20-22, wherein the antibody comprises rituximab, trastuzumab, pertuzumab, obinutuzumab, ofatumumab, olaratumab, ontuxizumab, isatuximab, sacituzumab, U3-1784, daratumumab, STI-6129, balantamab, indatuximab, cetuximab, dinutuximab, anti-CD38 A2 antibody, HuAT13/5 antibody, alemtuzumab, ibritumomab, tositumomab, bevacizumab, panitumumab, tremelimumab, ticilimumab, catumaxomab, oregovomab or veltuzumab, wherein preferably the antibody is rituximab, trastuzumab or pertuzumab. 26. The method according to any of paragraphs 20-25, where A is phenyl; U represents NH; R ¹ is halogen; and X is -N(R ² ) _v (CH ₂ ) _m O(CH ₂ ) _n -; where v is equal to 1; m and n are equal to 2; and R ² is methyl; or where A is phenyl; U represents NH; R ¹ is halogen; and X is -N(R ² ) _v (CH ₂ ) _m O(CH ₂ ) _n -; where v is equal to 2; m and n are equal to 2; and each R ² is methyl; or where A is phenyl; U represents NH; R ¹ is halogen; and X represents -O( _CH2 ) _n- ; where n is 2; or where A is phenyl; U represents NH; R ¹ is halogen; and X represents -S(CH ₂ ) _n -; where n is 2; or where A is phenyl; U represents NH; R ¹ is hydrogen; and X represents -NR ² -; where R ² is methyl; or where A is phenyl; U represents NH; R ¹ is halogen; and X represents --NR ² -; where R ² is hydrogen; or where A is phenyl; U represents NH; R ¹ is hydrogen; and X is -C( _CH3 )=; or where A is a _C4 - _C10 cycloalkyl ring; U represents NH; R ¹ is hydrogen; and X is -N(R ² )(CH ₂ ) _m O(CH ₂ ) _n -; where n is equal to 1; m is equal to 2; and R ² is methyl. 27. The method according to claim 20, characterized in that the compound of formula (I-1) is 28. Use of a conjugate according to any one of claims 1-6 or 10-13 or a pharmaceutically acceptable salt thereof or a composition according to claim 14 for treating cancer in a subject in need thereof, wherein the cancer is breast cancer, gastric cancer, lymphoma, acute myeloid leukemia, multiple myeloma, head and neck cancer, squamous cell carcinoma and/or hepatocellular carcinoma.